1

Current Diagnostic Techniques in Food Allergy

Matthew Greenhawt MD MBA MScProfessor of Pediatrics

Director Food Challenge and Research UnitSection of Allergy and Immunology

Childrenrsquos Hospital ColoradoUniversity of Colorado School of Medicine

2

Objectives

Describe the indications for when to test for diagnosing food allergy and what

test to run

Upon completion of this session the clinician should be able tohelliphellip

Review the evidence

surrounding whether

diagnostic testing can be used to

infer future reaction severity

Discuss the potential role for

component testing epitope

mapping and the basophil

activation test

3

Testing Model and Outcomes

Perform Diagnostic

Test

True PositiveDiagnosis confirmed

False PositiveOverdiagnosis

True NegativeAllergy excluded

False NegativeMissed diagnosis

bull Testing supports a clinical suspicion of a diagnosis but doesnrsquot automatically make a diagnosisbull All tests have an errormisclassification rate we must be aware ofbull We want to maximize the true positivenegative and minimize the false positivenegativebull What is the worst for our patientsmdashthe false positive or the false negative bull No matter what I tell you today no current test outperforms an OFC The rest are surrogates with an error rate

4

Trying To Find a Test To Prevent OFCshellip

5

Stop Chasing Predictive Valueshellip

bull From the analysis of ANY sample EVERY statistical package will provide SnSp PPVNPV

minus These are not special because they predict behavior ONLY in that sample and nowhere else

minus Predictive values require valid population prevalence NPV is dependent on how good a test ishellip

bull All tests are surrogates of some gold standard and by definition will have an error rate

minus OFC surrogates trade error rates and misclassification against tiny risks of severe reactions one canrsquot control

minus This instills our values in patients and forcing them to accept the health and economic detriments of misclassification

bull ldquoPredictiverdquo values from retrospective banked samplescohorts are highly biased and overspecify the accuracy of a test vs real world performancemdashselection bias timingflow are very significant issues

bull If you are avoiding doing an OFC because you are afraid of provoking a reaction you are in the wrong line of work and should also not be giving either OIT or AIT This is part of our jobs like it or nothellip

bull No test can or should ethically be marketed to ldquoreduce the necessityrdquo for a gold standard test

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

2

Objectives

Describe the indications for when to test for diagnosing food allergy and what

test to run

Upon completion of this session the clinician should be able tohelliphellip

Review the evidence

surrounding whether

diagnostic testing can be used to

infer future reaction severity

Discuss the potential role for

component testing epitope

mapping and the basophil

activation test

3

Testing Model and Outcomes

Perform Diagnostic

Test

True PositiveDiagnosis confirmed

False PositiveOverdiagnosis

True NegativeAllergy excluded

False NegativeMissed diagnosis

bull Testing supports a clinical suspicion of a diagnosis but doesnrsquot automatically make a diagnosisbull All tests have an errormisclassification rate we must be aware ofbull We want to maximize the true positivenegative and minimize the false positivenegativebull What is the worst for our patientsmdashthe false positive or the false negative bull No matter what I tell you today no current test outperforms an OFC The rest are surrogates with an error rate

4

Trying To Find a Test To Prevent OFCshellip

5

Stop Chasing Predictive Valueshellip

bull From the analysis of ANY sample EVERY statistical package will provide SnSp PPVNPV

minus These are not special because they predict behavior ONLY in that sample and nowhere else

minus Predictive values require valid population prevalence NPV is dependent on how good a test ishellip

bull All tests are surrogates of some gold standard and by definition will have an error rate

minus OFC surrogates trade error rates and misclassification against tiny risks of severe reactions one canrsquot control

minus This instills our values in patients and forcing them to accept the health and economic detriments of misclassification

bull ldquoPredictiverdquo values from retrospective banked samplescohorts are highly biased and overspecify the accuracy of a test vs real world performancemdashselection bias timingflow are very significant issues

bull If you are avoiding doing an OFC because you are afraid of provoking a reaction you are in the wrong line of work and should also not be giving either OIT or AIT This is part of our jobs like it or nothellip

bull No test can or should ethically be marketed to ldquoreduce the necessityrdquo for a gold standard test

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

3

Testing Model and Outcomes

Perform Diagnostic

Test

True PositiveDiagnosis confirmed

False PositiveOverdiagnosis

True NegativeAllergy excluded

False NegativeMissed diagnosis

bull Testing supports a clinical suspicion of a diagnosis but doesnrsquot automatically make a diagnosisbull All tests have an errormisclassification rate we must be aware ofbull We want to maximize the true positivenegative and minimize the false positivenegativebull What is the worst for our patientsmdashthe false positive or the false negative bull No matter what I tell you today no current test outperforms an OFC The rest are surrogates with an error rate

4

Trying To Find a Test To Prevent OFCshellip

5

Stop Chasing Predictive Valueshellip

bull From the analysis of ANY sample EVERY statistical package will provide SnSp PPVNPV

minus These are not special because they predict behavior ONLY in that sample and nowhere else

minus Predictive values require valid population prevalence NPV is dependent on how good a test ishellip

bull All tests are surrogates of some gold standard and by definition will have an error rate

minus OFC surrogates trade error rates and misclassification against tiny risks of severe reactions one canrsquot control

minus This instills our values in patients and forcing them to accept the health and economic detriments of misclassification

bull ldquoPredictiverdquo values from retrospective banked samplescohorts are highly biased and overspecify the accuracy of a test vs real world performancemdashselection bias timingflow are very significant issues

bull If you are avoiding doing an OFC because you are afraid of provoking a reaction you are in the wrong line of work and should also not be giving either OIT or AIT This is part of our jobs like it or nothellip

bull No test can or should ethically be marketed to ldquoreduce the necessityrdquo for a gold standard test

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

4

Trying To Find a Test To Prevent OFCshellip

5

Stop Chasing Predictive Valueshellip

bull From the analysis of ANY sample EVERY statistical package will provide SnSp PPVNPV

minus These are not special because they predict behavior ONLY in that sample and nowhere else

minus Predictive values require valid population prevalence NPV is dependent on how good a test ishellip

bull All tests are surrogates of some gold standard and by definition will have an error rate

minus OFC surrogates trade error rates and misclassification against tiny risks of severe reactions one canrsquot control

minus This instills our values in patients and forcing them to accept the health and economic detriments of misclassification

bull ldquoPredictiverdquo values from retrospective banked samplescohorts are highly biased and overspecify the accuracy of a test vs real world performancemdashselection bias timingflow are very significant issues

bull If you are avoiding doing an OFC because you are afraid of provoking a reaction you are in the wrong line of work and should also not be giving either OIT or AIT This is part of our jobs like it or nothellip

bull No test can or should ethically be marketed to ldquoreduce the necessityrdquo for a gold standard test

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

5

Stop Chasing Predictive Valueshellip

bull From the analysis of ANY sample EVERY statistical package will provide SnSp PPVNPV

minus These are not special because they predict behavior ONLY in that sample and nowhere else

minus Predictive values require valid population prevalence NPV is dependent on how good a test ishellip

bull All tests are surrogates of some gold standard and by definition will have an error rate

minus OFC surrogates trade error rates and misclassification against tiny risks of severe reactions one canrsquot control

minus This instills our values in patients and forcing them to accept the health and economic detriments of misclassification

bull ldquoPredictiverdquo values from retrospective banked samplescohorts are highly biased and overspecify the accuracy of a test vs real world performancemdashselection bias timingflow are very significant issues

bull If you are avoiding doing an OFC because you are afraid of provoking a reaction you are in the wrong line of work and should also not be giving either OIT or AIT This is part of our jobs like it or nothellip

bull No test can or should ethically be marketed to ldquoreduce the necessityrdquo for a gold standard test

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

6

A Primer on Testing Peanut as a Case Study

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

7

Peanut as a Component Model

Diagram schematic courtesy Thermo Fisher 2010

bull Peanut containsbull Major allergensbull Cross-reactive allergensbull Other compounds

bull Patients can recognize multiple epitopes simultaneously

bull A ldquopositiverdquo test is a sum of all recognized parts

bull Same scenario exists for any food allergen but the issue is particularly of concern for plant-based foods such as legumes seeds nuts grains

bull Could component patter infer greater sensitivityspecificity in a diagnosis

Ara h 1

Ara h 2

Ara h 3

Ara h 6

Ara h 9 CCDAra h 8

-

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

8

Peanut Model of Theoretical Risk

Slide courtesy Anna-Nowak-Wegryzn Jaffe Food Allergy Institute Mount Sinai School of Medicine with permission

rAra h 1rAra h 2rAra h 3rAra h 8rAra h 9CCD

Ara h 8 Ara h 1

Ara h 26

Ara h 3

ProfilinCCD PR-10 LTP Storage Proteins

Ara h 9

Risk

Ara h 5

Pollen cross-reactive

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

9

Peanut Allergy Parameter Questions

1) Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

2a) In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 --would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

2b) In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

3) In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

10

GRADE Quality of Evidence

http gdtguidelinedevelopmentorgapp

Strength of RecommendationFor the Patient For the Clinician

StrongMost individuals in this situation would prefer the recommended course of action and only a small proportion would not

The attending provider should strongly consider the recommended course of action as a first-line management Formal decision aids may have less of a role to help individuals make decisions consistent with their values and preferences

ConditionalThe majority of individuals in this situation would prefer the suggested course of action but many would not

Different choices may be appropriate for different patients Decision aids may be useful in helping individuals in making decisions consistent with their values and preferences Clinicians should expect to spend more time with patients when working towards a decision

Quality of RecommendationsHigh There is high confidence that the true effect lies close to that of the estimate of the effect

Moderate There is moderate confidence in the effect estimate The true effect is likely to be close to the estimate of the effect but there is a possibility that it is substantially different

LowThere is limited confidence in the effect estimate The true effect may be substantially different from the estimate of the effect

Very Low There is very little confidence in the effect estimate The true effect is likely to be substantially different from the estimate of effect

11

InclusionExclusion Criteria

bull Literature search of Pubmed Embase and Medline for articles (1946-present) pertaining to the use of peanut allergy diagnostic testing borrowing from three recent searches and expanding these terms (Klemans et al 2015 Chaffen et al 2009 Flores Kim et al 2018)

minus Population patients with known or suspected peanut allergy

minus Intervention testing for serologic peanut specific IgE against Ara h 1-3 6 8 and whole peanut extract

minus Comparator testing for whole extract peanut specific IgE via prick skin testing and serum-specific IgE

minus Outcome peanut allergy proven through use of an oral food challenge (open single-blinded or double blinded oral food challenge) in at least 50 of the study population

bull 1314 studies identified 127 selected for full text review with 89 selected for extraction (sensspec TPFP TNFN PPVNPV) at cut-offs of 3mm035 KUL for diagnosis 2 KUL 50 KUL 10mm for severity

bull Meta-analysis of diagnostic testing run in Stata 15Rev Man (peto method random effects) with QUADAS-2 used for assessing riskapplicability of bias and GRADEpro to construct evidence profiles

bull A total of 42 studies included in the final analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

12

Search LimitsmdashWhy Only Ara h 2

bull 89 articles selected for final evidence synthesis 41 directly pertained to this question 24 had data available for extraction Had to have 50 having OFC as a criteria minimum

bull We were unable to find sufficient number of studies to analyze any other individual peanut components or pool the use of component panels

bull We can offer no comment regarding the role or significance of evaluating these other components individually or in aggregate or what the clinical implications of their use may be

bull There ware no studies identified comparing the value of the use Ara h 2 or any components used reflexively after assessment of whole peanut SPT or sIgE

bull We know that there are studies out there beginning to suggesting value of other components such as Ara h 6 or 8 but they didnrsquot meet the inclusion criteria and this is a significant limitation of data related to those tests

13

Question 1

bull Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

bull Recommendation 1a We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy for both of whom shared decision-making has been employed to arrive at the final decision Conditional recommendation Certainty of evidence very low

bull Recommendation 1b We suggest against diagnostic testing in patients where there is low or very low Conditional recommendation Certainty of evidence very low

bull Not searchable no GRADE assessment

bull Comment this is a message that has been part of multiple past guidelines

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

14

Question 2

bull Question 2a In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

bull Question 2b In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

bull Recommendation 2a We suggest in favor of Ara h2 diagnostic testing (over SPT or sIgE to whole peanut) in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios Conditional recommendation Certainty of evidence moderate

bull Recommendation 2b We suggest against component testing in addition to either to skin prick test or sIgEto whole peanut to increase diagnostic accuracy Conditional recommendation Certainty of evidence moderate

bull Comment there is a knowledge gap in understanding how to use these tests in tandem with one another

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

15Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 3mm

Sensitivity 097

Specificity 046

Positive likelihood 182

Negative likelihood 005

SENSITIVITY (95 CI)

Q =17186 df = 1700 p = 000

I2 = 9011 [8660 - 9361]

097[093 - 099]

095 [085 - 099]

088 [047 - 100]

100 [095 - 100]

100 [097 - 100]

092 [062 - 100]

093 [088 - 097]

087 [076 - 094]

100 [086 - 100]

066 [051 - 079]

100 [092 - 100]

100 [083 - 100]

090 [055 - 100]

096 [078 - 100]

064 [048 - 078]

100 [098 - 100]

091 [071 - 099]

098 [087 - 100]

100 [088 - 100]100 [088 - 100]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

05 10SENSITIVITY

SPECIFICITY (95 CI)

Q =24286 df = 1700 p = 000

I2 = 9300 [9076 - 9524]

046[029 - 065]

049 [035 - 063]

099 [096 - 100]

071 [059 - 082]

008 [000 - 036]

026 [009 - 051]

063 [058 - 069]

067 [053 - 079]

044 [024 - 065]

052 [034 - 069]

040 [026 - 054]

000 [000 - 031]

027 [006 - 061]

020 [008 - 039]

081 [071 - 088]

066 [059 - 073]

030 [015 - 049]

000 [000 - 084]

044 [030 - 059]044 [030 - 059]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

00 10SPECIFICITY

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

12

Search LimitsmdashWhy Only Ara h 2

bull 89 articles selected for final evidence synthesis 41 directly pertained to this question 24 had data available for extraction Had to have 50 having OFC as a criteria minimum

bull We were unable to find sufficient number of studies to analyze any other individual peanut components or pool the use of component panels

bull We can offer no comment regarding the role or significance of evaluating these other components individually or in aggregate or what the clinical implications of their use may be

bull There ware no studies identified comparing the value of the use Ara h 2 or any components used reflexively after assessment of whole peanut SPT or sIgE

bull We know that there are studies out there beginning to suggesting value of other components such as Ara h 6 or 8 but they didnrsquot meet the inclusion criteria and this is a significant limitation of data related to those tests

13

Question 1

bull Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

bull Recommendation 1a We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy for both of whom shared decision-making has been employed to arrive at the final decision Conditional recommendation Certainty of evidence very low

bull Recommendation 1b We suggest against diagnostic testing in patients where there is low or very low Conditional recommendation Certainty of evidence very low

bull Not searchable no GRADE assessment

bull Comment this is a message that has been part of multiple past guidelines

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

14

Question 2

bull Question 2a In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

bull Question 2b In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

bull Recommendation 2a We suggest in favor of Ara h2 diagnostic testing (over SPT or sIgE to whole peanut) in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios Conditional recommendation Certainty of evidence moderate

bull Recommendation 2b We suggest against component testing in addition to either to skin prick test or sIgEto whole peanut to increase diagnostic accuracy Conditional recommendation Certainty of evidence moderate

bull Comment there is a knowledge gap in understanding how to use these tests in tandem with one another

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

15Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 3mm

Sensitivity 097

Specificity 046

Positive likelihood 182

Negative likelihood 005

SENSITIVITY (95 CI)

Q =17186 df = 1700 p = 000

I2 = 9011 [8660 - 9361]

097[093 - 099]

095 [085 - 099]

088 [047 - 100]

100 [095 - 100]

100 [097 - 100]

092 [062 - 100]

093 [088 - 097]

087 [076 - 094]

100 [086 - 100]

066 [051 - 079]

100 [092 - 100]

100 [083 - 100]

090 [055 - 100]

096 [078 - 100]

064 [048 - 078]

100 [098 - 100]

091 [071 - 099]

098 [087 - 100]

100 [088 - 100]100 [088 - 100]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

05 10SENSITIVITY

SPECIFICITY (95 CI)

Q =24286 df = 1700 p = 000

I2 = 9300 [9076 - 9524]

046[029 - 065]

049 [035 - 063]

099 [096 - 100]

071 [059 - 082]

008 [000 - 036]

026 [009 - 051]

063 [058 - 069]

067 [053 - 079]

044 [024 - 065]

052 [034 - 069]

040 [026 - 054]

000 [000 - 031]

027 [006 - 061]

020 [008 - 039]

081 [071 - 088]

066 [059 - 073]

030 [015 - 049]

000 [000 - 084]

044 [030 - 059]044 [030 - 059]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

00 10SPECIFICITY

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

13

Question 1

bull Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

bull Recommendation 1a We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy for both of whom shared decision-making has been employed to arrive at the final decision Conditional recommendation Certainty of evidence very low

bull Recommendation 1b We suggest against diagnostic testing in patients where there is low or very low Conditional recommendation Certainty of evidence very low

bull Not searchable no GRADE assessment

bull Comment this is a message that has been part of multiple past guidelines

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

14

Question 2

bull Question 2a In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

bull Question 2b In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

bull Recommendation 2a We suggest in favor of Ara h2 diagnostic testing (over SPT or sIgE to whole peanut) in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios Conditional recommendation Certainty of evidence moderate

bull Recommendation 2b We suggest against component testing in addition to either to skin prick test or sIgEto whole peanut to increase diagnostic accuracy Conditional recommendation Certainty of evidence moderate

bull Comment there is a knowledge gap in understanding how to use these tests in tandem with one another

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

15Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 3mm

Sensitivity 097

Specificity 046

Positive likelihood 182

Negative likelihood 005

SENSITIVITY (95 CI)

Q =17186 df = 1700 p = 000

I2 = 9011 [8660 - 9361]

097[093 - 099]

095 [085 - 099]

088 [047 - 100]

100 [095 - 100]

100 [097 - 100]

092 [062 - 100]

093 [088 - 097]

087 [076 - 094]

100 [086 - 100]

066 [051 - 079]

100 [092 - 100]

100 [083 - 100]

090 [055 - 100]

096 [078 - 100]

064 [048 - 078]

100 [098 - 100]

091 [071 - 099]

098 [087 - 100]

100 [088 - 100]100 [088 - 100]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

05 10SENSITIVITY

SPECIFICITY (95 CI)

Q =24286 df = 1700 p = 000

I2 = 9300 [9076 - 9524]

046[029 - 065]

049 [035 - 063]

099 [096 - 100]

071 [059 - 082]

008 [000 - 036]

026 [009 - 051]

063 [058 - 069]

067 [053 - 079]

044 [024 - 065]

052 [034 - 069]

040 [026 - 054]

000 [000 - 031]

027 [006 - 061]

020 [008 - 039]

081 [071 - 088]

066 [059 - 073]

030 [015 - 049]

000 [000 - 084]

044 [030 - 059]044 [030 - 059]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

00 10SPECIFICITY

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

14

Question 2

bull Question 2a In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

bull Question 2b In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

bull Recommendation 2a We suggest in favor of Ara h2 diagnostic testing (over SPT or sIgE to whole peanut) in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios Conditional recommendation Certainty of evidence moderate

bull Recommendation 2b We suggest against component testing in addition to either to skin prick test or sIgEto whole peanut to increase diagnostic accuracy Conditional recommendation Certainty of evidence moderate

bull Comment there is a knowledge gap in understanding how to use these tests in tandem with one another

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

15Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 3mm

Sensitivity 097

Specificity 046

Positive likelihood 182

Negative likelihood 005

SENSITIVITY (95 CI)

Q =17186 df = 1700 p = 000

I2 = 9011 [8660 - 9361]

097[093 - 099]

095 [085 - 099]

088 [047 - 100]

100 [095 - 100]

100 [097 - 100]

092 [062 - 100]

093 [088 - 097]

087 [076 - 094]

100 [086 - 100]

066 [051 - 079]

100 [092 - 100]

100 [083 - 100]

090 [055 - 100]

096 [078 - 100]

064 [048 - 078]

100 [098 - 100]

091 [071 - 099]

098 [087 - 100]

100 [088 - 100]100 [088 - 100]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

05 10SENSITIVITY

SPECIFICITY (95 CI)

Q =24286 df = 1700 p = 000

I2 = 9300 [9076 - 9524]

046[029 - 065]

049 [035 - 063]

099 [096 - 100]

071 [059 - 082]

008 [000 - 036]

026 [009 - 051]

063 [058 - 069]

067 [053 - 079]

044 [024 - 065]

052 [034 - 069]

040 [026 - 054]

000 [000 - 031]

027 [006 - 061]

020 [008 - 039]

081 [071 - 088]

066 [059 - 073]

030 [015 - 049]

000 [000 - 084]

044 [030 - 059]044 [030 - 059]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

00 10SPECIFICITY

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

15Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 3mm

Sensitivity 097

Specificity 046

Positive likelihood 182

Negative likelihood 005

SENSITIVITY (95 CI)

Q =17186 df = 1700 p = 000

I2 = 9011 [8660 - 9361]

097[093 - 099]

095 [085 - 099]

088 [047 - 100]

100 [095 - 100]

100 [097 - 100]

092 [062 - 100]

093 [088 - 097]

087 [076 - 094]

100 [086 - 100]

066 [051 - 079]

100 [092 - 100]

100 [083 - 100]

090 [055 - 100]

096 [078 - 100]

064 [048 - 078]

100 [098 - 100]

091 [071 - 099]

098 [087 - 100]

100 [088 - 100]100 [088 - 100]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

05 10SENSITIVITY

SPECIFICITY (95 CI)

Q =24286 df = 1700 p = 000

I2 = 9300 [9076 - 9524]

046[029 - 065]

049 [035 - 063]

099 [096 - 100]

071 [059 - 082]

008 [000 - 036]

026 [009 - 051]

063 [058 - 069]

067 [053 - 079]

044 [024 - 065]

052 [034 - 069]

040 [026 - 054]

000 [000 - 031]

027 [006 - 061]

020 [008 - 039]

081 [071 - 088]

066 [059 - 073]

030 [015 - 049]

000 [000 - 084]

044 [030 - 059]044 [030 - 059]

StudyId

COMBINED

Abrahms 2017

Begin 2017

Bernard2003

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo2015

Ludman 2013

Preece 2014

Rajput 2018

Rance2003

Sampson1997

Song 2015

Van Erp2013

00 10SPECIFICITY

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

16

Fagan Nomogram SPT 3mm

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

17Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 035 KUL

Sensitivity 095

Specificity 038

Positive likelihood 152

Negative likelihood 014

SENSITIVITY (95 CI)

Q =70858 df = 2900 p = 000

I2 = 9591 [9506 - 9676]

095[091 - 097]

082 [066 - 092]

078 [067 - 087]

100 [063 - 100]

039 [030 - 048]

093 [085 - 098]

095 [088 - 098]

099 [096 - 100]

092 [062 - 100]

093 [087 - 097]

075 [063 - 084]

100 [087 - 100]

100 [095 - 100]

095 [090 - 098]

100 [086 - 100]

100 [094 - 100]

069 [039 - 091]

096 [079 - 100]

078 [065 - 088]

096 [085 - 099]

095 [075 - 100]

093 [087 - 097]

098 [089 - 100]

097 [082 - 100]

085 [074 - 092]

096 [078 - 100]

095 [084 - 099]

097 [093 - 099]

097 [093 - 099]

097 [092 - 099]

098 [090 - 100]098 [090 - 100]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q =40397 df = 2900 p = 000

I2 = 9282 [9105 - 9460]

038[028 - 048]

068 [055 - 080]

046 [027 - 067]

097 [092 - 099]

035 [024 - 047]

040 [019 - 064]

026 [018 - 035]

010 [000 - 045]

011 [001 - 033]

049 [043 - 056]

046 [032 - 059]

023 [010 - 041]

007 [003 - 015]

000 [000 - 012]

031 [009 - 061]

046 [031 - 063]

047 [030 - 065]

025 [010 - 047]

046 [030 - 063]

045 [032 - 060]

030 [007 - 065]

016 [008 - 028]

073 [054 - 087]

027 [016 - 041]

045 [035 - 055]

043 [025 - 063]

062 [051 - 072]

062 [055 - 069]

038 [028 - 049]

034 [026 - 044]

033 [018 - 052]033 [018 - 052]

StudyId

COMBINED

Abrahms 2017

Balmer weber 2015

Begin 2017

Beigelman 2012

Bernard2003

Beyer2015

Chinthrajah 2018

Comberiati 2016

Dang2012

DunnGalvin 2011

Ebisawa 2012

Ebisawa 2015

Eller 2013

Glaumann 2012

Guilloux 2009

Gupta2014

Johannsen 2016

Klemans Broekman2013

Klemans Otte 2013

Leo 2015

Lieberman 2013

Martinet 2016

Nicolaou 2011

Perry 2004

Preece 2014

Rajput 2018

Rance2003

Sampson 1997

Van Erp2013

Wainstein 2007

00 10SPECIFICITY

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

18

Fagan Nomogram sIgE 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 2Post_Prob_Pos () = 3LR_Negative = 014Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 2Post_Prob_Pos () = 39LR_Negative = 014Post_Prob_Neg () = 6

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 2Post_Prob_Pos () = 78LR_Negative = 014Post_Prob_Neg () = 25

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

19Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 035 KUL

Sensitivity 086

Specificity 084

Positive likelihood 55

Negative likelihood 017

SENSITIVITY (95 CI)

Q =12356 df = 2300 p = 000

I2 = 8139 [7458 - 8819]

086[081 - 089]

094 [083 - 099]

071 [060 - 081]

100 [088 - 100]

057 [044 - 068]

087 [070 - 096]

095 [084 - 099]

084 [074 - 092]

058 [028 - 085]

095 [086 - 099]

060 [046 - 074]

080 [071 - 087]

080 [056 - 094]

093 [088 - 097]

086 [077 - 092]

089 [083 - 093]

091 [078 - 097]

091 [071 - 099]

091 [080 - 098]

082 [063 - 094]

068 [043 - 087]

096 [080 - 100]

074 [052 - 090]

088 [070 - 098]

081 [071 - 088]081 [071 - 088]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 7598 df = 2300 p = 000

I2 = 6973 [5711 - 8235]

084[079 - 089]

100 [090 - 100]

085 [062 - 097]

096 [087 - 100]

100 [087 - 100]

067 [043 - 085]

073 [063 - 082]

078 [068 - 086]

089 [067 - 099]

073 [057 - 086]

085 [068 - 095]

092 [082 - 097]

070 [035 - 093]

100 [048 - 100]

086 [078 - 092]

060 [041 - 077]

073 [058 - 085]

100 [029 - 100]

072 [058 - 083]

090 [073 - 098]

095 [076 - 100]

085 [055 - 098]

063 [044 - 080]

071 [054 - 085]

090 [080 - 096]090 [080 - 096]

StudyId

COMBINED

Martinet 2016

Bernard2003

Nicolaou 2011

Balmer weber 2015

Schots 2016

Rajput 2018

Ebisawa 2015

Comberiati 2016

Kukkonen 2015

Klemans Broekman2013

Lieberman 2013

Leo 2015

Chinthrajah 2018

Beyer 2015

Eller 2013

Van Erp2013

Peeters2007

Klemans Otte 2013

Ebisawa 2012

Suratannon2013

Glaumann 2012

Preece 2014

Keet2013

Dang 2012

03 10SPECIFICITY

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

20

Fagan Nomogram Ara h 2 035

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 2LR_Positive = 5Post_Prob_Pos () = 10LR_Negative = 017Post_Prob_Neg () = 0

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 30LR_Positive = 5Post_Prob_Pos () = 70LR_Negative = 017Post_Prob_Neg () = 7

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 70LR_Positive = 4Post_Prob_Pos () = 89LR_Negative = 025Post_Prob_Neg () = 36

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

21

Question 3

bull Question 3 In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

bull Recommendation We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a reaction andor allergy phenotype or to predict the severity of a future reaction Conditional recommendation Certainty of evidence very low

bull Comment there is a knowledge gap in studies that have uniform severity criteria to evaluate the relationship between sensitization and severity

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

22Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Ara h 2 sIgE 2 KUL

Sensitivity 078

Specificity 045

SENSITIVITY (95 CI)

Q = 2875 df = 900 p = 000

I2 = 6870 [4811 - 8929]

078[069 - 085]

065 [048 - 079]

093 [082 - 098]

067 [009 - 099]

092 [064 - 100]

081 [070 - 089]

066 [055 - 076]

040 [012 - 074]

070 [035 - 093]

086 [042 - 100]

082 [048 - 098]082 [048 - 098]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

01 10SENSITIVITY

SPECIFICITY (95 CI)

Q =10703 df = 900 p = 000

I2 = 9159 [8771 - 9547]

045[028 - 063]

076 [063 - 087]

025 [015 - 036]

051 [040 - 061]

004 [000 - 021]

028 [021 - 035]

079 [049 - 095]

040 [012 - 074]

062 [032 - 086]

023 [010 - 041]

070 [059 - 080]070 [059 - 080]

StudyId

COMBINED

Balmer weber 2015

Chinthrajah 2018

Dang2012

Glaumann 2012

Klemans Blom 2015

Kukkonen 2015

Leo 2015

Preece 2014

Rajput 2018

Van Erp2013

00 10SPECIFICITY

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

23Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

sIgE 50 KUL

Sensitivity 039

Specificity 089

SENSITIVITY (95 CI)

Q = 4943 df = 1200 p = 000

I2 = 7572 [6263 - 8881]

039[026 - 053]

072 [058 - 084]

000 [000 - 071]

027 [006 - 061]

069 [039 - 091]

028 [018 - 039]

100 [016 - 100]

027 [008 - 055]

020 [003 - 056]

040 [005 - 085]

050 [019 - 081]

021 [007 - 042]

035 [015 - 059]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q =13253 df = 1200 p = 000

I2 = 9095 [8723 - 9466]

089[075 - 095]

052 [040 - 064]

096 [091 - 099]

100 [089 - 100]

073 [052 - 088]

068 [061 - 075]

057 [034 - 077]

100 [077 - 100]

100 [075 - 100]

094 [079 - 099]

053 [035 - 071]

085 [079 - 090]

100 [090 - 100]

085 [062 - 097]085 [062 - 097]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Glaumann 2012

Klemans Blom 2015

Lewis2005

Peeters 2007

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

03 10SPECIFICITY

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

24Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

SPT 10 mm

Sensitivity 037

Specificity 062

SENSITIVITY (95 CI)

Q = 3058 df = 1100 p = 000

I2 = 6403 [4177 - 8629]

037[022 - 055]

061 [047 - 074]

017 [000 - 064]

000 [000 - 037]

059 [039 - 076]

050 [019 - 081]

050 [007 - 093]

000 [000 - 031]

029 [004 - 071]

050 [019 - 081]

083 [036 - 100]

038 [015 - 065]

067 [022 - 096]067 [022 - 096]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

00 10SENSITIVITY

SPECIFICITY (95 CI)

Q = 9134 df = 1100 p = 000

I2 = 8796 [8236 - 9355]

062[044 - 077]

035 [024 - 048]

067 [059 - 075]

100 [090 - 100]

026 [016 - 037]

055 [023 - 083]

057 [041 - 073]

085 [055 - 098]

075 [053 - 090]

041 [024 - 059]

055 [043 - 067]

072 [055 - 085]

035 [015 - 059]035 [015 - 059]

StudyId

COMBINED

Chinthrajah 2018

Dang2012

DunnGalvin 2011

Klemans Blom 2015

Leo 2015

Lewis2005

Preece 2014

Rajput 2018

Song 2015

Van Erp2013

Wainstein 2010

Wensing 2002

02 10SPECIFICITY

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

25

Risk of Bias Assessment

High

Unclear

Low

bull Mild to moderate risk of bias overall bull Most comes from studies where small samples

were chosen large clinic populations without an explained rationale or sample was not at random or not consecutive

bull Additional risk of bias from studies where the reference diagnostic challenge and the index diagnostic peanut test were obtained at different times

bull A lot of this is not necessarily avoidable in these studies but it does help weaken the certainty in the evidence

bull Sensitivity analysis removing studies at risk for bias for both patient selection and flowtiming revealed no significant differences in estimates

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

26

Which Test is Most Cost-effective

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

bull Diagnosis compared using each of the 3 tests at a presumed prevalence of 3 14 or 75 bull At each respective prevalence assessment use of Ara h 2 vs either SPT or peanut sIgE was associated

with larger QALY gain and lower net cost (dominated analysis)bull While we make no recommendation for or against the use of any component testing this simulation does

suggest superior health and economic benefits would be associated with preferential use of Ara h 2 as a stand-alone diagnostic test assuming these are used in populations similar to those pooled for analysis

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

27

Sensitivity Analysis

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

28

Bottom Line Test Responsibly

SPT 3mm sIgE 035 KL Ara h 2 sIgE 035 KL

bull Testing someone with no history or risk is absolutely worthlessmdashthis means the milk allergic kid you want to also test for peanut as a common allergen has no positive value no matter which test you use

bull If this is not understood then anything in this parameter is not going to matter

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 65LR_Negative = 005Post_Prob_Neg () = 5

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 2Post_Prob_Pos () = 60LR_Negative = 014Post_Prob_Neg () = 12

0001000200050010020050102051251020501002005001000

Likelihood Ratio

01020305071235710

20304050607080

909395979899993995997998999

Post

-test

Pro

babi

lity

()

0102030507

12357

10

20304050607080

909395979899

993995997998999

Pre-

test

Pro

babi

lity

()

Prior Prob () = 50LR_Positive = 5Post_Prob_Pos () = 85LR_Negative = 017Post_Prob_Neg () = 15

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

29

Summary Recommendations

Greenhawt M Shaker M Wang J et al Peanut Allergy Diagnosis- a 2020 Practice Parameter Update Systematic Review and GRADE Analysis [published online ahead of print 2020 Aug 15] J Allergy Clin Immunol 2020S0091-6749(20)31137-4 doi101016jjaci202007031

Question Recommendation Evidence Certainty Risk of Bias

Should diagnostic testing for peanut allergy be performed in adults and children with a history of suspected peanut allergy who are requesting evaluation for peanut allergy

We suggest in favor of diagnostic (skin prick or serum sIgE) testing for peanut allergy in patients with a 1) physician-judged high pre-test probability of peanut allergy or 2) prior to an oral food challenge for patients with moderate pre-test probability of peanut allergy with whom shared decision-making has been employed to arrive at the final decision

We suggest against diagnostic testing in patients where there is low or very low pre-test probability of peanut allergy

Very Low Not Rated

In the patient presenting for evaluation of suspected peanut allergy which of the three testsmdashSPT sIgE to whole peanut or Ara h2 would provide the highest diagnostic accuracy as determined by the more optimal positivenegative likelihood ratio

We suggest in favor of Ara h2 diagnostic testing in a patient presenting for evaluation of suspected peanut allergy for which a single diagnostic test is to be used as Ara h2 would provide the best diagnostic accuracy as determined by virtue of more optimal positivenegative likelihood ratios

Moderate High

In a patient presenting for evaluation of suspected peanut allergy does testing for peanut components in addition to either SPT or sIgE to whole peanut increase the diagnostic accuracy

We suggest against component testing in addition to either to skin prick test or sIgE to whole peanut to increase diagnostic accuracy

Very Low High

In the patient presenting for evaluation of suspected peanut allergy can the results of a diagnostic test be used to predict the severity of a future allergic reaction

We suggest against the clinician using the results of a SPT sIgE to whole peanut extract or sIgE to peanut components to determine the severity of a previous reaction andor allergy phenotype or to predict the severity of a future reaction

Very Low High

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

30

What About an Ara h 2 Cut-off Point

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

31

Is There an Optimal Ara h 2 Cut Off Point

bull Multiple studies suggest Ara h 2 as best discriminator but cut-off points have varied and there probably is no absolute ldquopredictiverdquo level for general use

bull Generating a PPV for the parameter was a non-starter without knowing the accurate prevalence

bull We felt likelihood ratios were a better assessment which we provided instead

Beyer et al Allergy 2015 70 90-98Klemans et al Clin Exp Allergy 2015 45 720-30

Study 95 PPV Cutoff (kuL) Sensitivity Specificity

Sampson 2001 15 (peanut) 57 100

Nicolaou 2011 035 (optimal cut point) 100 96

Codreanu 2011 023 (optimal cut point) 93 97

Eller 2013 128 (not a PPV) 76 97

Dang 2012 196 96

Keet 2013 2 (75 PPV 623 NPV) 23 94

Klemans 2013 035 (74 PPV ) 91 72

Beyer 2015 42 [035 (50)144 (90)]

Kukkonen 2015 035 (PPV 91NPV 84 for severe rxn)

Leo 2015 2 (91 PPV 47 NPV) 50 90

Ballmer-Weber 2015 2 (97 prob in sample for severe rxn)

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

32

95 PPV 422 KUL80 PPV 45 KUL

Cut-Off Values Are Population Specific

Beyer et al Allergy 2015 70 90-98

20 PPV 063 KUL 80 PPV 879 KUL

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

33

Beyond Peanuthellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

34

Summary Slide

bull Components have been developed for a number of allergens but best studied for peanut

bull There is no evidence supporting the necessity or added utility of components for diagnosing egg milk tree nut seed or wheat allergy

bull There are emerging data from single center studies that are trying to demonstrate particular key indicator components exist for cashew walnut sesame and wheat akin to Ara h 2

bull Limitations of these data are the same as for peanut if not worse for these

bull These tests are not at the point where they are reliable and usable surrogates that approach the potential utility of Ara h 2

bull These will not ldquobeatrdquo a good history appropriate use of first order testing and an OFC

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

35

Different Epitopes Different Phenotypes

Flinterman et al J Allergy Clin Immunol 2008 121737-43Jarvinen KM et al J Allergy Clin Immunol 2002110293-7

bull Persistent milk egg peanut allergy related to increased sequential epitope recognition

bull Epitope mapping can elucidate clinical phenotypes

bull Shows that α-s1 α-s2 κ-casein associated with persistence of milk allergy and β-lactabglobulin with transient allergy

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

36

Components for Milk

bull Milk proteins are casein (80) and whey

minus α-lactalbumin (bos d 4) β-lactabglobulin (bos d 6) casein (bos d 8) whey

minus 5 caseins (κ αs1-2 γ1-3) most with αs1 and κ (likely sequential)

minus 5 proteins (α-lactalb β-lactabglob BSA immunoglobulin lactoferrin)

bull Persistent allergy associated w higher number of sequential epitopes

bull Casein gt078 kUL α-lact gt 34 kUL β-lact gt 99 kUL are proposed 95 predictive decision points (single study)

minus Baked milk proposed 95 predictive decision point 5 kUL (single study)

minus These are defined in single samples and likely do not generalizemdashuse cautiously

bull No clear predictor of persistent milk allergy or who are the ~20 that donrsquot tolerate baked milk

Fiochi et al Curr Opin Allergy Clin Immunol 11216ndash221Drsquourbano et al Clin Exp Allergy 2010 401561 ndash 1570Nowak-Wegryzn et al J Allergy Clin Immunol 2008 122342 ndash 347

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

37

Components for Egg

bull Egg white has gt 20 proteins

minus Ovomucoid heatdigestion stable dominant allergen (gal d 1)

minus Ovalbumin ovotransferrin lysozyme (gal d 2 3 4) all heatacid labile

minus Egg yolk (gal d 5)mdashseen in bird-egg syndrome

bull Ovomucoid associated with persistent allergy

minus 95 PDPrsquos of 11 30 50 and kUL for baked egg reactivity

minus 1 kUL reported as ldquosaferdquo level for bakedcooked egg

minus Persistent egg allergy seen with increased number of sequential epitopes

bull HealthNuts 80 of children failing egg challenge were baked egg TOLERANT

bull Decision points have same cautions as with milkAndo et al J Allergy Clin Immunol 2008 122 583ndash8Caubet et al Curr Opinion Allergy Immunol 2011 11210ndash215Lemon Mule J Allergy Clin Immunol 2008 122977ndash983e1Osborne et al J Allergy Clin Immunol 2011 127 668-76

Haneda et al J Allergy Clin Immunol 2012 1291681-2

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

38

Hazelnut Components

Datema MR et al Allergy 201873549ndash559

bull Multicenter Europreval study looking at CRD use in hazelnut allergy to predict reaction severity

bull From n=731 patients with reported hazelnut allergy n=423 available sera were analyzed for hazelnut CRD with n=124 consenting for DBPCFC (~17 total sample)

bull Cor a 9 and 14 had OR 105 and 101 to predict reaction severity and Cor a 1 OR 014 for predicting reaction severity at gt035 KUAL

bull When Cor a 14 combined in a multivariate model with eczema walnut sensitization and pollen sensitization this had 92 specificity and 763 specificity but misclassified severity in a ldquolarge number of patientsrdquo per authors given a low prevalence of severe hazelnut allergy (16)

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

39

CashewPistachio Cross-Reactivity

Savvatianos et al J Allergy Clin Immunol 2015 136 192-4

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

40

Practical Limitations of Components

bull Necessity In a patient you clearly suspect is either allergic or suspect is non-allergic any test will

suffice and there is no proven advantage for components

bull Uncertainty Where there is low to moderate pretest probability components have higher

specificity which reduces the chances of false positive tests but the positive likelihood is not

strong enough to translate to very high post test odds and a challenge is still needed This may

be the best choice in these situations but not as good as the OFC

bull Evidence Certainty Many of the component studies have issues with selection bias flow and

timing and degree of use of OFC These have tended to characterize known allergic individuals

and have not robustly characterized non-allergic individuals or community samples

bull Use there are limited data about how to best use components in the diagnostic algorithm

highlighting the necessity and uncertainty issues

bull Consider the harm that results from a false positive diagnosismdashwhich is worse the false positive

or the false negative in the grander scheme

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

41

Basophil Activation Testing

bull Growing importance of the role of the basophil in mediating allergic responses in mice

bull Growing body of evidence for utility of measuring basophil activation as a marker in humans as well

bull Basophils release histamine from preformed granules similar to mast cells

bull Basophils have a half-life of only one week

bull Markers

minus Advances in flow cytometry has made it possible to measure surface markers as opposed to histaminemediator release

minus Method more accessible than auto-analyzers for mediator measurement

minus Can use heparanized whole blood

minus Common markers include CD63 CD203c and CD69

MacGlashan J Allergy Clin Immunol 2013132 777-87

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

42

Basophil Histamine Release

bull Basophils release varying degrees of histamine but is a highly individualized response

--Spontaneous release has been used in foodeczema studies

--Release can be triggered by FcER1 binding

--However other molecules can trigger histamine release such as C5a so the process can be non-specific

bull Measurement is very technical--Involves lymphocyte enrichment requires careful platelet removal

--Measurement through RIA or ELISA

--1ml blood (20000 basophils) generally sufficient

--Release from antigenFcER1 cross-linking is susceptible to dose-response effects surface receptor density and receptor reductions

MacGlashan J Allergy Clin Immunol 2013132 777-87

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

43

Basophil Activation Test

bull CD63 (BAT) is most commonly used--Most closely associated with anaphylactic vs piecemeal degranulation

--Because degranulation processes are distinct CD63 only appears with anaphylaxis and can distinguish histamine release mechanisms

--Commercial assay available

--Assay sensitivity enhanced by IL-3

bull CD203c and CD69 expression can also be quantified--Evidence that CD203c expression is not related to histamine degranulation and can be triggered by multiple stimuli including IL-3

--CD69 is expressed by basophil cytokine expression and occurs slowly

--Expression of both is highly distinct from CD63MacGlashan J Allergy Clin Immunol 2013132 777-87

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

44

BAT Clinical Applications

bull CD63 may have diagnostic properties--Expression not altered by anti-histamines

--Can run on blood within 1 year of exposure

bull Sensitivity for food allergy diagnosis is 77-98 and sensitivity 75-100

--Has shown enhanced accuracy vs SPT and sIgE

bull Has shown potential ability to determine --if peanut OFC is needed and how severe a reaction in an OFC was

--if baked milkegg tolerance and if have outgrown milk allergy

--if OIT subject achieved clinical desensitization

--if Omalizumab treatment had effect on food tolerance

MacGlashan J Allergy Clin Immunol 2013132 777-87Hoffmna et al Allergy 2015 70 1393-1405Santos et al J Allergy Clin Immunol 2015 134 645-52Glaumann et al Allergy 2012 67242-47Jones et al J Allergy Clin Immunol 2009 124292-300Burks et al N Engl J Med 2012 367 292-300

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

45

BAT Limitations

bull Still mainly a research tool

bull Accessibility

bull Price (cost-effectiveness)

bull Limited data showing effectiveness

bull Is a very promising and potentially useful tool that should play a larger role going forward

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

46

Mast Cell Activation Test

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

47

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

48

What is an Epitope

bull 3-D allergen binding site

--No universally common structure

bull Can bind IgE and IgG

--Can elicit cross-reactivity

--T cell epitopes exist as well

bull Conformational tertiary

--Heat labile subject to hydrolysis

bull Linear sequential

--Heat stable not alterable

Sampson HA J Allergy Clin Immunol 2004 113 805-819

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

49

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

50

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

51All taken from Twitter FoodAllergy Some tweets have since been deleted by FoodAllergy

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

52

Practical Take Home Points

1 Positive tests detect sensitization not allergymdashthese are not the same concept

minus People can have a rdquopositiverdquo test to peanut and not be allergic

2 When there is a very high or very low pre-test probability choice of test really doesnrsquot mattermdasharguably you donrsquot need testing though we do so for several reasons

minus Where there is indeterminant pre-test probability Ara h 2 offers lowest false positive risk

3 Chose to test carefully and within the most narrow contextsmdashdo not run panels or test for common allergens looking for peanut if the history does not indicate it

minus Recommend referral to specialist if considering testing due to eczema a sibling history other food allergy or the patient has never eaten peanut previously

4 Please consider the harm that results from a false positive diagnosismdashthese are hard to de-label and result in significant health and economic detriments

minus Is the false negative or the false positive the worse scenario

53

A Glimpse of Colorado Livinghellip

53

A Glimpse of Colorado Livinghellip