CURICULUM VITAE

Nama : DR Dr Purwanto AP, SpPK (K)Tempat/Tgl Lahir : Solo, 5 April 1953Alamat Pekerjaan : Bagian Patologi Klinik F.K.UNDIPAlamat Rumah : Jl Diponegoro II / 18, Semarang, 50264Kepangkatan : Lektor Kepala / IV D

Riwayat Pendidikan :Dokter FK UNDIP 1981Sp 1. Patologi Klinik 1997SP 2. Konsultan Patologi Klinik 2007Doktor 2015

Organisasi :Ketua I IDI Wilayah JatengKetua Program Studi Spesialis 2 PATOLOGI KLINIK UNDIPAnggauta Komisi Ujian Nasional Kolegium Patologi KlinikPenatar Nasional Kompetensi Analis

Quality Control in Imunnoserology Test

Purwanto AP

Introduction

Immunodiagnostics

ImmunologicCompetence

Test

ImmunoSerologic

Test

TumorMarker

Test

ImmunologicCompetence

Test

NonSpecificImmuneResponse

Test

LeukocyteFunction

TestChemotactic Fs

TestIngestion Fs

Test

IntracellularKilling Fs Test

SpecificImmuneResponse

Test

Quantitative

LymphocyteT & B count

Lymphocyte TSubset count

Qualitative

*Lym.Transfor T.*DNA Syn.rate T.*Mixed cult. reac.*Cytotoxicity.T*Lym. Produc.T.

To analysesReduction

NBT To count bacteriain leukocyte withFlowcytometry

Ingestionand

metabolismFunction

Test

LeukocyteFunctionTest

Syntesis andSecretioncytokine

With RIA orELISA

To analyses Reactiveoxygen species with:Spectrofotometry,Flourometry orChemiluminescen

ImmunoserologicTest

•Typhoid• Streptococcus•Gonococcus•Syphilis•Hepatitis etc

Tumor Marker Test

• CEA• AFP• PSA• Ca 19-9• Ca 125• MCA• Cyfra 21• HCG• etc

Definitions

– Quality Control - QC refers to the measures that must be included during eachassay run to verify that the test is working properly.

– Quality Assurance - QA is defined as the overall program that ensures that thefinal results reported by the laboratory are correct.

– “The aim of quality control is simply to ensure that the results generated by thetest are correct. However, quality assurance is concerned with much more: thatthe right test is carried out on the right specimen, and that the right result andright interpretation is delivered to the right person at the right time”

Definitions

– Quality Assessment - quality assessment (also known as proficiencytesting) is a means to determine the quality of the results generated by thelaboratory. Quality assessment is a challenge to the effectiveness of theQA and QC programs.

– Quality Assessment may be external or internal, examples of externalprograms include National External Quality Assurance Scheme (NEQAS-Philiphines), Hong Kong Model Technique Association (HKMTA),and Q-probes (College American Pathologist).

Variables in Quality Control

Variables that affect the qualityof results

– The educational background and training of the laboratory personnel

– The condition of the specimens

– The controls used in the test runs

– Reagents

– Equipment

– The interpretation of the results

– The transcription of results

– The reporting of results

Error in quality control

Errors in measurement

– True value - this is an ideal concept which cannot be achieved.

– Accepted true value - the value approximating the true value, thedifference between the two values is negligible.

– Error - the discrepancy between the result of a measurement and thetrue (or accepted true value).

Sources of error

– Input data required - such as standards used, calibration values, and values ofphysical constants.

– Inherent characteristics of the quantity being measured - e.g. CFT and HAItitre.

– Instruments used - accuracy, repeatability.

– Observer fallibility - reading errors, blunders, equipment selection, analysisand computation errors.

– Environment - any external influences affecting the measurement.

– Theory assumed - validity of mathematical methods and approximations.

SEROLOGY ANALYTICMETHOD

A. Precipitation Reaction in Gelsa. Immuno-double diffusionb. Immunoelectrophoresisc. Single Radialimmunodiffusiond. Countercurrent electrophoresise. Rocket electrophoresis

B. Aglutination &HaemaglutinationC. Complement FixationD. Immunofluorescence

a. Directb. Indirectc. Indirect Complement Amplified

E. Radioimmunoassay(RIA)

F. Enzyme-Linked ImmunosorbentAssay (ELISA)

G. Radioallergosorbent test(RAST)

H. Radioimmunosorbent test(RIST)

I. Immunoblotting

RESULT

Ag - Latex Antibody

Aglutination

– INCUBATIONhomogenization, temperature and time

– WASHINGvolume, frequency and time

– ADDED REAGENSIAvolume, pipetation

– RESULT INTERPRETATIONwave length, validity test

EAg

EAg Directlabeled Antigen

E

EAg Indirect

EAg DirectSandwich

EE

Ag

Basic Elisa Method

1. DIRECT ELISA :* Direct labeled antibody* Direct labeled antigen

2. INDIRECT ELISA3. SANDWICH ELISA

* Direct Sandwich* Indirect Sandwich

4. COMPETITION ELISA* Direct Antibody Competition* Direct Antigen Competition* Indirect Antibody Competition* Indirect Antigen Competition

Internal Quality ControlIn serology testing

Internal Quality Control Programfor Serological Testing

An internal quality control program depend on the use of internal quality control(IQC) specimens, Shewhart Control Charts, and the use of statistical methods forinterpretation.

Internal Quality Control Specimens

IQC specimens comprises either (1) in-house patient sera (single or pooled clinicalsamples), or (2) international serum standards with values within each clinicallysignificant ranges.

Shewhart Control Charts

A Shewhart Control Chart depend on the use of IQC specimens and is developed in the followingmanner:-

– Put up the IQC specimen for at least 20 or more assay runs and record down the O.D./cut-off valueor antibody titre (whichever is applicable).

– Calculate the mean and standard deviations (s.d.)

– Make a plot with the assay run on the x-axis, and O.D./cut-off or antibody titre on the y axis.

– Draw the following lines across the y-axis: mean, -3, -2, -2, 1, 2, and 3 s.d.

– Plot the O.D./cut-off obtained for the IQC specimen for subsequent assay runs

– Major events such as changes in the batch no. of the kit and instruments used should be recorded onthe chart.

Westgard rules

– The formulation of Westgard rules were based on statistical methods. Westgard rules arecommonly used to analyse data in Shewhart control charts.

– Westgard rules are used to define specific performance limits for a particular assay and canbe use to detect both random and systematic errors.

– There are six commonly used Westgard rules of which three are warning rules and the otherthree mandatory rules.

– The violation of warning rules should trigger a review of test procedures, reagentperformance and equipment calibration.

– The violation of mandatory rules should result in the rejection of the results obtained withpatients’ serum samples in that assay.

0102030405060708090100

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Shewhart Chart

+3 sd

-3 sd

+2 sd

-2 sd-1 sd

+1 sd

VZV IgG ELISA: Target Value = 49 U/ml

Antibody Units

Target value

Assay Run

Warning rules

– Warning 12SD : It is violated if the IQC value exceeds the mean by 2SD. It is anevent likely to occur normally in less than 5% of cases.

– Warning 22SD : It detects systematic errors and is violated when two consecutive IQCvalues exceed the mean on the same side of the mean by 2SD.

– Warning 41SD : It is violated if four consecutive IQC values exceed the same limit(mean 1SD) and this may indicate the need to perform instrument maintenance orreagent calibration.

Mandatory rules

– Mandatory 13SD : It is violated when the IQC value exceeds the mean by 3SD. Theassay run is regarded as out of control.

– Mandatory R4SD : It is only applied when the IQC is tested in duplicate. This rule isviolated when the difference in SD between the duplicates exceeds 4SD.

– Mandatory 10x : This rule is violated when the last 10 consecutive IQC values areon the same side of the mean or target value.

0102030405060708090100

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Westgard Rules: 1 3SD

+3 sd

-3 sd

+2 sd

-2 sd-1 sd

+1 sd

VZV IgG ELISA: Target Value = 49 U/ml

Antibody Units

Target value

Assay Run

0102030405060708090100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Westgard Rules: 10X

+3 sd

-3 sd

+2 sd

-2 sd-1 sd

+1 sd

VZV IgG ELISA: Target Value = 49 U/ml

Antibody Units

Target value

Assay Run

Follow-up action in the event of a violation

There are three options as to the action to be taken in the event of a violation of a Westgardrule:

– Accept the test run in its entirety - this usually applies when only a warning rule isviolated.

– Reject the whole test run - this applies only when a mandatory rule is violated.

– Enlarge the greyzone and thus re-test range for that particular assay run - thisoption can be considered in the event of a violation of either a warning ormandatory rule.

External control samples

33

Frequency of Use: When Should You Test ExternalControl Samples?

– Minimum once a week, beginning of the week

– New shipment of test kits

– Beginning a new lot number

– Environmental conditions exceed range needed for stability of kits

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Invalid Results – What Do YouDo?– Repeat test– If repeatedly invalid:

• assume problem with test product or procedure• continue with alternative testing algorithm– Identify cause of problem– Inform supervisor– Take corrective actions

Jika QC “out of control”

Apa yang harus dilakukan ?

Jika QC “out of control”– STOP pemeriksaan– Identifikasi dan koreksi masalah– Ulang pemeriksaan kontrol dan sampel setelah

koreksi– Jangan melaporkan hasil pasien hingga masalah

diatasi dan kontrol mengindikasikan performa yangbaik

Mengatasi masalah out-of-control

– Identifikasi masalah

– Rujuk pada kebijakan / prosedur SOP aksipengulangan yang berlaku

Mencari Kemungkinan Penyebab Masalah

– Kerusakan reagen– Kerusakan bahan kontrol– Operator error– Kegagalan mengikuti instruksi pabrik– Prosedur tidak update– Kegagalan alat– Kegagalan kalibrasi

UPAYA PERBAIKAN

1. Jika hasil menyimpang jauh dari nilai target periksa:(a) kesalahan dalam penulisan hasil(b) kesalahan perhitungan(c) sampel yang diperiksa adalah benar(d) sampel tidak terkontaminasi pada saat diperiksa

2. Jika hasil tersebar pada kedua sisi nilai target, tetapi tidak melebihi 3 SD menandakanadanya impresisi. Periksa apakah

(a) cara kerja sudah benar(b) keterampilan personil laboratorium(c) perawatan alat antara lain pipet, fotometer sudah baik

3. Jika hasilnya menyimpang dari target, bila nilai target ditentukan tanpamembedakan metoda, kemungkinan karena perbedaan metoda.Pertimbangkan penggantian metoda untuk memperbaiki kesesuaian antarlaboratorium

4. Jika hasil menyimpang dari nilai rata-rata untuk metoda yang sama, periksaapakah

(a)standar dan kalibrator baik(b) pipet dan alat lain baik

……………UPAYA PERBAIKAN

QualityAssurance

Serology testing

1- Serological Testing

– The technologist must be well trained,– there should be continuing education programs,– well developed standard operating procedures,– preventive maintenance of instruments,– effective quality control programs– Participation in proficiency surveys– and inventory control

Quality assurance practices in serological testing are similar to those in otherareas of the laboratory (the clinical chemistry Lab.):

2- Appropriate Control ProceduresThree levels of control samples for qualitative and semiquantitaive serologicaltesting should be obtained– Negative control:

– This controls monitors the reaction for specificity, that is, the reaction will not occurin absence of specific antigen

– Weak positive control:

– This control checks the reaction for sensitivity, the concentration of this controlshould be at the lowest level the procedure is able to detect

– Failure at this concentration indicates that the method has lost sensitivity

– Positive control:

– Used to check the reactivity of the weak positive control to ensure that the observedreaction is the result of the expected antigen -antibody reaction

3- Appropriate Control Procedures

– All of these controls can be purchased or made frompooled patient sera– Commercially prepared controls are usually available in

lyophilized form to be reconstituted when needed– After reconstitution, the control will remain stable for

weeks or months

Short Message

Program QC serologi sangat esensial,

• harus memonitor semua pemeriksaan– punya kebijakan dan prosedur tertulis yang dipatuhi staf laboratorium– punya manajer QC untuk monitoring dan reviewing data QC– gunakan analisis statistik dan tercatat dengan baik (recording)– sediakan prosedur untuk troubleshooting / koreksi– Di penghujung : quality assurance

TERIMAKASIH