TO STUDY THE PREVALENCE OF ABO INCOMPATIBILITY ...

TO STUDY THE PREVALENCE OF ABO INCOMPATIBILITY IN O BLOOD

GROUP MOTHERS AND ASSESSMENT OF IgG SUBCLASSES (IgG1 AND

IgG3) IN THEM AND ITS CORRELATION WITH OCCURRENCE AND

SEVERITY OF ABO HEMOLYTIC DISEASE OF NEWBORN (ABO-HDN) IN

A TERTIARY HEALTH CARE CENTRE IN SOUTH INDIA

A DISSERTATION SUBMITTED IN PART FULFILLMENT OF THE REGULATION

FOR THE AWARD OF THE DEGREE OF M.D. PATHOLOGY BRANCH III

THE TAMIL NADU DR.M.G.R.UNIVERSITY, CHENNAI, TAMIL NADU

MAY - 2020

REGISTRATION NUMBER: 201713358

To study the prevalence of ABO

incompatibility in O blood group mothers

and assessment of IgG subclasses (IgG1 and

IgG3) in them and its correlation with

occurrence and severity of ABO Hemolytic

Disease of Newborn (ABO HDN) in a

tertiary health care centre in South India

A dissertation submitted in part fulfillment of the regulation

for the award of the degree of M.D. Pathology Branch III

CERTIFICATE

This is to certify that the dissertation “To study the prevalence of ABO

incompatibility in O blood group mothers and assessment of IgG subclasses

(IgG1 and IgG3) in them and its correlation with occurrence and severity of ABO

Hemolytic disease of newborn (ABO HDN) in a tertiary health care centre in

south India” is the bonafide work done by Dr. Reshma Kurian, in part fulfillment of

the rules and regulations for the M.D. Branch III (Pathology) Degree Examination of

Tamil Nadu Dr. M.G.R. Medical University, to be held in May 2020.

Dr. Geeta Chacko, MD, PhD.

Professor and Head,

Department of General Pathology,

Christian Medical College,

Vellore.

Dr. Anna B Pulimood, MD, PhD.

Principal,


Vellore.

CERTIFICATE





south India” is the bonafide work done by Dr. Reshma Kurian, in part fulfillment of

the rules and regulations for the M.D. Branch III (Pathology) Degree Examination of

Tamil Nadu Dr. M.G.R. Medical University, to be held in May 2020.

The candidate has independently reviewed the literature, standardized the data

collection methodology and carried out the evaluation towards completion of the

thesis.

Dr. Dolly Daniel, M.B.B.S, MD

Professor, Thesis Guide & Supervisor

Department of Transfusion Medicine and Immunohematology,

Christian Medical College, Vellore.

CERTIFICATE





south India” is the bonafide work done by me, under the guidance of Dr. Dolly

Daniel in part fulfillment of the rules and regulations for the M.D. Branch III

(Pathology) Degree Examination of Tamil Nadu Dr. M.G.R. Medical University, to be

held in May 2020.

.

I have independently reviewed the literature, performed the data collection, analyzed

the data and carried out the evaluation towards completion of the thesis.

Dr. Reshma Kurian,

Postgraduate Registrar,

Department of General Pathology,


Vellore.

ANTI PLAGIARISM CERTIFICATE

CERTIFICATE

This is to certify that this dissertation work titled “To study the prevalence of ABO




south India” of the candidate Reshma Kurian with registration number 201713358 for

the award of Degree of MD Pathology in the branch III. I personally verified the

urkund.com website for the purpose of plagiarism Check. I found that the uploaded

thesis file contains from introduction to conclusion pages and result shows 4% of

plagiarism in the dissertation.

Guide & Supervisor sign with Seal.

ACKNOWLEDGEMENT

First of all I would like to thank God the almighty for me the strength and grace and

for providing me with this opportunity. This journey would have been impossible

without his grace and blessings.

I express my deepest gratitude to my mentor, Dr. Dolly Daniel for being the never

ending support, the encourager and the ever cheering guide who led the way to the

finish line of this dissertation.

I am indebted to my professor Dr. Geeta Chacko for her immense care, concern and

support.

I extend my warmest regards to Dr. Joy Mammen, for his support and encouragement.

I would like to specially thank Mrs. Sheela Kayalvezhi and Mr. Amal Raj,

Department of Transfusion Medicine and Immunohematology for helping me in

executing this project.

I extend my sincere thanks to Dr. Santhanam Sridhar, Dr. Annie Regi, Dr. Manisha

Mathai Beck and Dr. Swathi Rathore for their invaluable help in the clinical aspects of

my study.

I extend my sincere thanks to Miss. Dona Maria, for her patience and input in

statistical analysis.

I wish to thank my spouse Dr. Habie Thomas, for being my pillar of strength and

source of constant motivation and my family, friends and colleagues for their

relentless love, care, help and guidance.

LIST OF ABBREVIATIONS USED

AAP American Academy Of Paediatrics

ADCC Antibody Mediated Cell Dependent Cytotoxicity

AHG Anti Human Globulin

CAT Column Agglutination

CNS Central Nervous System

CH

Constant Heavy chain

CL

Constant Light chain

CLIA

Chemiluminescence Immuno Assay

CMV

Cytomegalo Virus

COHb Carboxy haemoglobin

DAT Direct Antiglobulin Test

DTT

Dithiothreitol

Fab

Fragment, antigen binding

Fc

Fragment, crystallisable

FC

Flow Cytometry

GCT

Gel Centrifugation Test

Hb Haemoglobin

HDN Haemolytic Disease of the Fetus and Newborn

HDFN Haemolytic Disease of the Fetus and Newborn

IgA Immunoglobulin A

IgD Immunoglobulin D

IgE

Immunoglobulin E

IgG Immunoglobulin G

IVIG

Intravenously administered Immunoglobulin

LISS

Low Ionic Strength Saline

LSCS

Lower Segment Caesarian Section

MMA

Monocyte monolayer assay

MTP

Medical Termination of Pregnancy

NICU

Neonatal Intensive Care Unit

NND

Neonatal death

NVD

Normal Vaginal Delivery

PBS

Phosphate buffered saline

RBC

Red Blood Cell

RhD

Rhesus D

RIA

Radio Immuno Assay

TcB

Transcutaneous bilirubinometry

TSB

Total Serum Bilirubin

VH

Variable Heavy chain

VL

Variable Light chain

TABLE OF CONTENTS

INTRODUCTION ................................................................................... 1

AIMS AND OBJECTIVES ........................................................................ 2

MATERIALS AND METHODS ............................................................... 42

DISCUSSION ....................................................................................... 82

CONCLUSIONS ................................................................................... 94

LIMITATIONS ..................................................................................... 96

REFERENCES ...................................................................................... 97

ANNEXURES ..................................................................................... 106

INTRODUCTION

1

INTRODUCTION

ABO Hemolytic disease of the newborn(ABO HDN) is an immune reaction that

occurs most commonly in newborns of A, B or AB blood group born to mothers of O

blood group(1). Following the introduction of Rh immunoprophylaxis, the prevalence

of Rh haemolytic disease has come down drastically and now ABO-HDN is emerging

as the most common cause of HDN in the developed countries (2).

ABO HDN occurs due to transplacental transfer of maternal anti-A and anti-B IgG

antibodies leading to immune haemolysis of fetal erythrocytes. IgG has 4 subtypes –

IgG1, IgG2, IgG3 and IgG4, of which IgG1 and IgG3 are effectively transported

across placenta via an Fc receptor (3).

There is very limited literature on the prevalence and factors that impact on ABO-

HDN from India. Against the background of extremely variable health care facilities in

our country, we thought it is critical to study factors that may predict the severity of

ABO-HDN in mothers with O blood group. Early identification, diagnosis and

intervention can reduce morbidity and mortality due to ABO hemolytic disease of

newborn.

2

AIMS AND OBJECTIVES

3

AIMS

1) To study the prevalence of ABO incompatible pregnancies in O blood group mothers in a

tertiary health care centre in south India.

2) To correlate the maternal IgG1and IgG3 subclasses with occurrence of ABO HDN.

OBJECTIVES

1) To study the prevalence of ABO incompatible pregnancies in O blood group mothers in a

tertiary health care centre in south India.

2) To study the incidence of ABO-HDN.

3) To correlate the maternal IgG anti-A and anti-B titers with occurrence and severity of

ABO HDN.

4) To correlate the maternal IgG1and IgG3 subclasses with occurrence and severity of ABO

HDN.

4

REVIEW OF LITERATURE

5

REVIEW OF LITERATURE

1. History

2. Causes of hyperbilirubinemia in term newborns

3. Spectrum of hemolytic disease of newborn

4. ABO-Haemolytic Disease of Newborn

5. Epidemiology of ABO-HDN

6. Etiology and disease mechanism

7. Pathogenesis of ABO-HDN

8. Factors determining ABO-HDN

9. Grading the severity of ABO-HDN

10. Serological testing in ABO-HDN

11. Hematologic findings in ABO-HDN

12. Prediction of severity of AB0-HDN

13. Therapeutic measures in ABO-HDN

14. Recent advances

15. Conclusion

6

HAEMOLYTIC DISEASE OF NEWBORN

Hemolytic disease of the newborn (HDN) is an immune reaction characterized by the

shortened lifespan of fetal red cells due to transplacental transfer of specific maternal

antibodies that are directed against the paternally derived antigens that are present in

the fetal RBCs. The severe cases manifests as ascites, hydrops, heart failure and even

death in the fetus. The less severe cases present postnatally as neonatal anemia,

hyperbilirubinemia and suppression of erythropoiesis(1,2). The antibodies are

produced in the mother following the previous pregnancy or prior transfusions(3).

ABO hemolytic disease of the newborn (ABO-HDN) more commonly occurs in a

newborn with blood group A, B or AB who are born to O blood group mothers(4).

This is because; the antibodies to A and B formed in individuals with O blood group

are of IgG type while that found in the serum of individuals of blood group A and B is

of IgM type. IgG antibodies cross the placenta effectively.

Following the introduction of anti-D prophylaxis, the incidence of Rh-HDN has

drastically reduced and ABO incompatibility has now emerged as the major cause of

hemolytic disease of the newborn. It is now the largest cause of HDN in the developed

countries. Transfusion services have an imperative role in the timely detection and

providing transfusion support to such cases (5).

7

I.HISTORY:

Hemolytic disease of fetus and newborn as an important cause of fetal loss and death

of newborn was first described by a French midwife named Louis Bourgeois who

worked at the royal court of King Henry IV and Queen Marie de Medicis in 1906. She

noted that while assisting in the delivery of twins, one baby was swollen and died soon

after birth while the other baby developed jaundice and died several days later(5).

The term “erythroblastosis foetalis” was coined by Diamond et al and was identified as

a syndrome in 1932 which encompasses congenital anemia, severe neonatal jaundice

and hydrops foetalis. The pathogenesis of the disease was attributed to a defect in the

erythron (4).

The immunological nature of HDN was described in 1938 by Ruth Darrow and was

proposed that fetal hemoglobin plays a crucial role in the pathogenesis of hemolytic

disease by inducing a maternal antibody response after crossing the placenta. The

Rhesus blood group was discovered by Landsteiner and Wiener in 1940 (6) and

subsequently the Rh(D) antigen was discovered in 1941 by Levine el(7). The RhD

antigen was identified in the D negative mothers following the transplacental transfer

of foetal D positive RBCs. The final clinical event was found to be caused due to the

transfer of maternal anti-D immunoglobulin IgG across the placenta(8). The

contributions of all these scientists have been the stepping stones that have led to our

current knowledge of HDN.

8

II. CAUSES OF HYPERBILIRUBINEMIA IN TERM NEWBORNS:

The common risk factors involved in neonatal hyperbilirubinemia are fetal-maternal

blood group incompatibility, delayed breastfeeding, ethnicity, birth trauma,

prematurity and neonatal infections(9–11).

Neonatal hyperbilirubinemia is classified based on the etiology as follows(12):

1. Increased bilirubin load

A. Hemolytic causes:

Rh incompatibility

ABO and other minor antigen incompatibility

RBC membrane defects

B. Non-hemolytic causes:

Extra vascular sources - Cephalhaematoma, CNS hemorrhage

Delayed cord clamping, twin to twin transfusion

2. Decreased conjugation of bilirubin

Physiologic jaundice

Breast milk jaundice

Crigler Najjar syndrome

Gilbert syndrome

3. Impaired excretion of bilirubin

Biliary obstruction

Dubin Johnson syndrome

Rotor syndrome

9

III. SPECTRUM OF HEMOLYTIC DISEASE OF NEWBORN:

At present, more than fifty different red cell antibodies have been identified which can

lead to hemolytic disease(13). Anti-D is associated with greatest severity of hemolytic

disease. The other frequent alloantibodies are Rh antigens (anti-c, anti-C, anti-e, and

anti-E), Kell antigens, Duffy antigens, Kidd and MNS antigens.

Among the non D (Rh) antibodies, anti-E and anti-c are the most commonly involved

in the pathogenesis of the hemolytic disease(14,15). Absorption of antibodies by fetal

antigen in placenta (17) and lack of the corresponding antigens on fetal red cells (16)

lead to failure of a few antibodies of other blood group systems to cause hemolytic

disease.

Table 1: Alloantibodies reported to cause HDN (1):

Within the Rh system Anti-D, -c, -C – Cw, -Cx, -e, -E, - Ew, -c,

Ce, -Rh32, -Goa ,-Bea, -Evans, -L, W

Outside the Rh system Anti-K, -k, -Ku -Kp8 ,-Kpb ,-Js8, -Jsb, -

Fy8, -Fy3, -Jk8, -Jkb, -M,-N, -S, -s ,-U, -

Vw, -Far, -Mv ,- Mit, -Mta, -M,u r, –Hil, -

Hut, -

Antibodies to high-incidence antigens Anti -At8 ,-J ra, -Lan, -Ge

Antibodies to low incidence antigens - Anti-Bi, -By, -Fr8, -Good, -Rd, -Re8, Zd

10

Rh-HDFN:

Rh alloimmunization leading to HDFN occurs when an Rh- negative mother carries an

Rh- positive fetus(16). Most of the time, the first pregnancy is the sensitizing event,

which goes uneventful and hemolytic disease occurs in subsequent pregnancies.

Following sensitization, re-exposure to the antigen leads to an anamnestic response

increasing the maternal anti-D titers(17). The initial anti-D formed at time of

sensitization is IgM type. In subsequent pregnancies, IgG anti-D is formed which can

be transported across the placenta leading to destruction of fetal RBCs.

As per the studies, Rh hemolytic disease does not occur in primies, but there is

literature to support that it can occur rarely in 1% of first pregnancy(18). Rh

Hemolytic disease is very common in developing countries including which can be

attributed to improper antenatal care and failure to administer Rh IG(19).

Factors influencing Rh (D) HDFN include:(20)

A. Size of feto-maternal hemorrhage: Only a small amount of fetal red cells cross the

placenta into the maternal circulation during the third trimester which is insufficient to

stimulate antibody production in the mother. Transplacental hemorrhage occurs during

delivery which leads to the transfer of fetal red cells into the maternal circulation

leading to sensitization. After the first delivery, mothers form antibodies up to 6

months after delivery. The fetal red cells cross the placenta as early as 24 th week of

gestation in the second pregnancy with Rh (D) positive fetus which further increases

the sensitization and antibody titers leading to HDFN.

11

B. Parity: Parity has been found to correlate significantly with alloimmunization and

risk of HDFN especially associated with second and third pregnancy.

C. Effect of ABO incompatibility between mother and fetus: ABO incompatibility has

been shown to provide some protection from Rh HDFN. It is suggested that an

existing ABO incompatibility between mother and fetus may destroy the Rh (D)

positive fetal red cells as soon as they enter the maternal circulation before they can

initiate any sensitization in mother.

D. Medical termination of pregnancy/abortion: Abortion or any interventional

procedure during pregnancy can also be the sensitizing event leading to

alloimmunization in women.

In cases of ABO incompatible pregnancies, there is reduced risk of sensitization to Rh-

D antigen. This can be attributed to the rapid destruction of fetal cells by anti-A and

anti-B antibodies.

IV. ABO HAEMOLYTIC DISEASE

ABO hemolytic disease is usually considered as a mild form of the disease, with only

less than 1% of affected infants requiring exchange transfusion. It is defined as a

newborn with positive DAT and/ or laboratory evidence of hemolysis of blood group

A, B or AB who are born to O blood group mothers without Rhesus factor

compatibility(21) .

The severe manifestations of ABO HDN are very rare and disease manifests as the

onset of hyperbilirubinemia within 12-48 hours of birth.

12

IV.A. Factors affecting incidence and severity:

Following are the four stages involved in the pathogenesis of hemolytic disease;

1) Alloimmunization of the mother

2) Transplacental transfer of antibodies to the fetus

3) Destruction of the sensitized red cells

4) Secondary clinical manifestations (22)

There are several factors that affect the severity of the disease major ones including the

quantity of maternal antibody(anti-A/ anti-B) that crosses the placenta, antigenic

strength on RBCs of infants, subclasses of immunoglobulins, inhibitory effect of fetal

secretions, absorption capacity of fetal tissues, liver maturity and efficiency of

phagocytosis(23).

ABO HDN usually occurs in A,B or AB blood group infants who are born to mothers

of O group as ABO antibodies occur as high-titer IgG antibodies in individuals with O

blood group than in those with group A or B. The incidence and severity of the disease

is unrelated to the history of prior transfusions or pregnancies in the mother.

Therefore, ABO HDN can occur in the first as well as consequent pregnancies.

13

Table 2: Comparison of ABO v/s Rh HDN (3):

Characteristic feature ABO Rh

First pregnancy

Yes

Rare

IgG antibody Present Present

Severity

DCT

Bilirubin at birth

Mild

Weakly positive/ negative

Normal range

Severe

Strongly positive

Elevated

Anemia at birth No Yes

Phototherapy Yes Yes

Exchange transfusion Rare Sometimes

Intrauterine transfusion None Sometimes

Spherocytosis

Prevention

Yes

Not available

Rare

Rh immunoglobulin

V. EPIDEMIOLOGY:

About 95% cases of the hemolytic disease were caused in the past by maternal

antibodies directed against Rh antigen D. The incidence of hemolytic disease due to

anti D has tremendously decreased since 1968 after the introduction of Rh D

14

immunoprophylaxis (24). Now, hemolytic disease due to ABO incompatibility and

other alloantibodies has emerged as the major cause of hemolytic disease.

It is estimated that there are 3/100000 to 80/10000 cases of hemolytic disease in the

United States per year(25).

It was found that ABO incompatibility between mother and baby occurs in about 15-

20% of all pregnancies, which produces HDN in 10% of these cases(26).

VA. Previous studies on the prevalence of ABO incompatibility:

Incidence of ABO HDN depends on incidence of ABO incompatibility. It

should be noted however that ABO incompatibility is not synonymous with ABO

HDN. In other words, ABO HDN is the extreme end of the spectrum of ABO

incompatibility.

Table 3: Previous studies on prevalence of ABO incompatibility and ABO-HDN

Author Place Year Prevalence of Prevalence of

ABO-incompatibility ABO-HDN

Cariani et al Venezuela 1995 27.7 % 31%

Bhat and Kumar India 1998 17.3% 16.5%

Sarici et al Turkey 2001 14.8% 21.3%

Bekkeheim et al Norway 2009 15-25% 1-4%

Heydarian et al Iran 2012 19.7% 27.6%

Shilpa et al India 2017 40.8% 36%

15

The low rate of clinically significant severe hemolytic disease due to ABO

incompatibility is due to the following reasons:

1. ABO antigens are expressed by variety of fetal tissue, thereby reducing the

chances of anti-A/B binding to fetal red cells.

2. A and B antigens are not well developed and expressed in the fetal RBCs.

The lower expression of ABH antigens on the fetal RBCs is due to high sialylation,

low 1, 2 a fucosylation, and its unbranched lactosaminyl backbone. This minimizes the

level of feto-maternal incompatibilities following the transplacental transfer of

antibodies(27).

Hemolytic disease due to ABO antibodies can occur in the first pregnancy and can

recur in about 87% cases as maternal ABO antibodies are present without previous

sensitization(25).

3. There is a weaker binding affinity for alloantibodies reacting with A and B

antigens than those reacting with protein antigens.

V B. Racial factors and prevalence of ABO HDN:

There were several studies in the past on race as a vital risk factor in the occurrence of

ABO HDN.

ABO HDN is usually seen in group O mothers with group B infants (African ancestry)

or group A infants (European ancestry)(28). It was proved that as compared to

Caucasians, Asians and black tend to have a higher prevalence of DAT positive ABO

HDN(23).

16

As per studies, ABO incompatibility occurs in one out of every five pregnancies in

Caucasians(29). ABO HDN occurs at a higher rate in blacks as compared to

Caucasians, which is attributed to the higher prevalence of immune anti-A and anti-B

antibody titers in the black population (30).

Neonates with A blood group especially A1 group have a more severe disease as

compared to neonates with B or A2 blood group.

V C. Familial incidence:

An affected infant with severe hemolytic disease is often followed by similar severe

disease in the family; while in families with mild ABO hemolytic disease; affected

infant may be followed by an unaffected infant.

VI . ETIOLOGY AND DISEASE MECHANISM

Hemolytic disease is caused by the destruction of fetal RBCs by maternal allo

antibodies. Transfer of antibodies from mother to fetus takes place only via placenta in

human beings. It is an active process and takes place only in one direction, i.e. from

mother to fetus.

Only small amounts of IgG are transferred in the first 12 weeks of gestation. The mean

IgG concentration is around 1.8 g/dl in 24th week of pregnancy and the concentration

thereafter rises exponentially until term. At term, the IgG level of the fetus is higher

than that of the mother, around 15.12 g/dl in infants and 12.60 g/dl in mothers.

Antibody transfer becomes more significant with the increase in gestational age

leading to more positive DAT and greater incidence of neonatal

hyperbilirubinemia(32). Only immunoglobulin G antibodies are actively transported

17

across the placenta by Fc receptor (FcRn) while IgA and IgM are not. The rate of

transfer is relatively slow at term.

Transfer of IgG antibodies is a physiologic event and is mediated by FcRn which is

similar to class I MHC molecule(1). It is beneficial because it provides passive

immunity to the newborn in the first few months of life as they are targeted against

bacterial, fungal and viral antigens. However, in hemolytic disease, maternal

antibodies are directed against fetal RBC antigens that were inherited from father.

Transport of IgG (IgG1 and IgG3 which causes more effective hemolysis) by the Fc

receptor starts in the second trimester of gestation and continues till the birth of the

baby(32, 36).

FcRn:

The structure of the Fc receptor varies from other receptors. FcRn belongs to MHC

class I protein family and dimerises with beta 2 microglobulin for its function. It is

found in monocytes, dendritic cells and macrophages in humans. The C γ2 and C γ 3

domains of IgG bind with α 2 and β 2 microglobulin domains of FcRn. Following are

the functions of FcRn:

1) Transfer of IgG from mother to fetus thereby providing passive immunity

2) Protection of IgG from normal catabolism.

IgG is transported in vesicles by FcRn which prevents lysosomal degradation. Due to

the above reasons, half-life of IgG is longer than other immunoglobulin classes (22-23

days) in humans(1).

18

VII. PATHOGENESIS OF ABO HDN

VII A. Hemolysis and anemia:

The maternal IgG attaches to fetal red blood cells. The antibody coated red cells are

cleared by the macrophages of the fetal spleen. The antibody titers, specificity as well

as the number of antigenic sites on fetal RBCs determine the rate of destruction of the

fetal RBCs which in turn leads to anemia. Anemia accelerates the RBC production by

the fetal marrow and immature RBCs called erythroblasts are released into the fetal

circulation. This finding is termed as “erythroblastosis foetalis.”

Extramedullary haematopoeisis occurs when the bone marrow fails to keep up the

production of RBCs as compared to the rate of destruction. This leads to enlargement

of the liver and spleen resulting in portal hypertension and hepatocellular damage.

Decreased production of plasma proteins by liver along with severe anemia leads to a

condition termed as hydrops foetalis (24). Hydrops foetalis is characterized by

widespread effusion, high output cardiac failure and death(10).

RBC destruction continues as long as maternal antibodies persist in the fetal

circulation with the rate decreasing after birth as no longer maternal antibodies can

enter the fetal circulation. Antibody binding and hemolysis continue for several weeks

to days after delivery because IgG has a half-life of 25 days and is distributed both in

extra vascular and intravascular compartments.

VII B. Bilirubin:

Hemoglobin which is released following the destruction of RBCs is metabolized into

indirect bilirubin. Indirect bilirubin is conjugated into direct bilirubin in the maternal

19

liver which is then excreted by the mother. In cases of moderate to severe hemolysis,

indirect bilirubin more than 18-20 mg/dl cause kernicterus and permanent damage to

the fetal brain (24).

ABO hemolytic disease leads to early onset of hyperbilirubinemia and may present

with a rapid increase in serum bilirubin values(33).

.VII C. IgG subclasses and allotypes:

There are five classes of immunoglobulins in human beings: IgM, IgD, IgG, IgA and

IgE of which IgG is the most abundant protein (10-20%) plasma. IgG is further sub-

classified in the order of decreasing abundance as IgG1,IgG2,IgG3 and IgG4(33).

Each subclass has a unique property in regards to binding of antigen, activation of

complement, half-life, transplacental transport and immune complex formation(34).

The concentration of IgG subclasses vary depending on the fetal age and are

transported actively across the placenta. IgG1 rises earlier than IgG3 in the gestational

period. There is an acceleration of ratios of IgG1, IgG3 and IgG4 in the third

trimester(35). As per the study conducted by Morell et al in 1971 and Schur eta l in

1973(33,36), there is a relative deficiency of IgG2 in cord serum. This is because Fc

γreceptor in the syncytiotrophoblast cells in the placental tissue binds with IgG1 with a

higher affinity than IgG2(1). IgG4 cannot activate the classic pathway of complement

system and therefore cannot destroy the RBCs.(37)

IgG subclass typing can be done by various methods like Flow Cytometry, ELISA test

or gel test.

20

Table 4: Properties of human IgG subclasses:(38)

Properties IgG1 IgG2 IgG3 IgG4

Abundance (% of total IgG) 70 20 6 4

Half-life in serum (days) 23 23 7 23

Placental passage +++ + +++ +++

Complement fixation + + +++ -

Binding to Fc receptor +++ + ++ -

VII D. Structure of immunoglobulin:

Antibodies are immunoglobulins which belong to the family of proteins and they

combine with antigens and mediate biological effects. The immunoglobulin molecules

are made up of four polypeptide chains - two heavy chains of molecular weight of

50,000-75,000 Daltons and two light chains of molecular weight 22,500 Daltons which

are interconnected by covalent and non-covalent bonds. The covalent bonds are mainly

found in the hinge region. There are five different classes of Ig: IgG, IgM, IgA, IgD

and IgE due to five types of heavy chains termed as γ, α, μ, δ and ε. The light chains

are the same in the five types of Ig, either κ or λ type. It is the heavy chain that varies

for each type. Immunoglobulin has an amino (-NH2) and a carboxyl (-COOH) end.

On treatment with papain enzyme, an immunoglobulin is divided to form one

crystalline Fc fragment and two antigen binding Fab segments. Fc region extends from

21

carboxyl-terminal to the hinge region and is primarily responsible for monocyte

binding and complement fixation, and in IgG subtype, it is the Fc fragment that crosses

the placenta leading to the pathophysiology of hemolytic disease. The Fab fragment

extends from the amino- terminal to the hinge region of the molecule.

The domains of an immunoglobulin molecule are made up of regions of both heavy

and light chains folded into globular structure or loops, and these are made up of

approximately 110-120 amino acids. The domains consist of a variable (V) and

constant (C) regions made up of heavy and light chains. The number of domains

depends upon the immunoglobulin isotype and IgG molecules have three constant

heavy chain regions (CH1, CH2 and CH3). The hinge region of the immunoglobulin

structure exists between CH1 and CH2 domains of the heavy chain. Minor differences

in these hinge regions are used to categorize the four subclasses of IgG.

In IgG molecule, there is a specific constant heavy chain region which allows the

attachment of Fc receptors of monocytes and macrophages. IgG molecules occur as

monomers, IgM as pentamers and IgA as monomers or dimers. IgG is the most

common Ig in plasma with a concentration of 12g/dl.

IgG molecules can be subdivided on the basis of minor differences in the hinge region

of the IgG structure that can be separated by electrophoresis as four subclasses

(IgG1,IgG2,IgG3 and IgG4) in the order of decreasing abundance as follows:

IgG1,6.63; IgG2,3.22; IgG3, 0.58 and IgG4, 0.46(1).

The structures of the four immunoglobulin subclasses are very similar to each other

with over 90% homology in their amino acid sequence and the difference between

22

them affects their ability to bind to receptors and accessory molecules which in turn

affect their function. Most of the variations are found in the hinge region and N-

terminal CH2 domain with few differences in other domains(34).

The number of inter H chain disulfide bonds vary in different subclasses with IgG3

having a much longer hinge region compared to other subclasses.

The IgG immunoglobulin contains four polypeptide chains which are further

composed of two 50kDa heavy chains that are identical to each other and two 25 kDa

light chains.

The light chain consists of kappa and lambda chains which are linked together by

disulfide bonds. The heavy chain comprises of three constant domains (CH1, CH2 and

CH3) and N terminal variable domain (VH). A hinge region is present between CH1

and CH2. The light chain comprises a constant domain (CL) and an N terminal

variable domain (VL). The effector functions of the antibodies are largely due to the

residues in the CH2 domain of Fc part proximal to the hinge region due to the

overlapping in the binding site for C1q complement and IgG-Fc receptor on the

effector cells.

23

Figure 1: Crystal structure of IgG1 molecule viewed from two different

angles

VII E. Structural variation in the hinge region:

The Fab arms and Fc parts are linked together by the hinge region. The hinge exon of

IgG1 encompasses 15 amino acids, IgG2 has 12 amino acids and IgG4 has 14 amino

acids. IgG2 has the shortest hinge of all the IgG subclasses and are rigid and stabilized

by extra interheavy chain disulfide bonds. IgG3 has a much longer hinge region than

all other subtypes containing 62 amino acids. The long hinge region of IgG3 is due to

the duplication of the hinge exon which is encoded by one exon each in IgG1, IgG2

and IgG4 and up to 4 exons in IgG3. The elongated hinge attributes to the high

molecular weight of IgG3. The orientation and movement of the Fab arms and Fc tail

of the IgG antibody is influenced by the difference in the hinge flexibility.

The Fab arms shield the binding site of C1qand/or FcγR which in turn affects the

binding sites of IgG(34). The flexibility of IgG subclasses is in the order:

24

lgG3>lgG1>lgG4>lgG2 which in turn affects their binding to Fc γr and C1q and

antigen binding capacity and immune complex formation(38).

Interchain disulfide bonds:

The IgG subclasses differ from each other with regard to the number of inter heavy

chain disulfide bonds in the hinge region. IgG2 and IgG4 are found as several isomers

due to variations in the interconnections of hinge disulfide bonds.

Hinge isomers in IgG2 and IgG4:

Structural hinge isomers are which are commonly found in IgG2 antibodies with κ

light chains than λ chains. These isomers are formed due to alternative disulfide bond

formation between cysteine in the heavy chain hinge region and those involved in the

formation of disulfide bonds between the heavy and light chains. Two forms of

isomers are described – Classical A form, the most common form, characterized by

four disulfide bridges between the two IgG2 heavy chains and the less common B

form in which disulfide bond is formed between one hinge cysteine and light chain.

The FcRn binding site is common for all the isoforms.

IgG4 Fab arm exchange:

The random combining of half molecules of IgG4 with other half molecules of IgG4 in

vivo, leads to the formation of monovalent bispecific antibodies which prevents

effective cross-linking of the target antigen. In addition to this, non specific

multivalent target binding results in low avidity. IgG4 has low affinity to activate

FcγR and retains high affinity to inhibit FcγIIb.

Allotypes:

25

Allotypes are discovered by serology and are defined as polymorphic immunoglobulin

epitopes that vary between individuals and ethnic groups.

G1m (z, a), G1m (f, a) and G1m (f) are the most common allelic forms of IgG1 of

which G1m (f) is found in Caucasians and G1m (f, a) allele is common in Orientals.

There are no serologically important allotypes for IgG4.

The overall ratio of κ to λ in human IgG is2:1, but the ratio is 1:1 and 8:1 for

individual IgG2 and IgG4 subclasses respectively. The disulfide bonds linking the

heavy chains also act as factors for structural variation among the different subclasses.

While IgG1 and IgG4 have two bonds each, IgG2 and IgG3 have four and five bonds

respectively. The disulfide bonds lend flexibility to the hinge regions of the subclasses

of IgG molecule and the distance the Fab fragment can cover determines the antigen it

can accommodate(39).

Each of the subclass exhibits different properties including placental transfer and

complement fixation. While IgG1 and IgG3 bind to complement C1q molecule more

strongly than IgG2. IgG4 doesn’t bind at all and cannot activate the complement

cascade(1).

Fc region of antibodies is recognized by several membrane-bound receptors. The

lower hinge region of IgG is recognized by FcRI, FcRII and FcRIII. Monomeric IgG1

and IgG3 bind to FcRI, polymeric IgG1 and IgG3 bind to FcRII and FcRIII also. There

are three different types of FcRII- FcRIIa, FcRIIb and FcRIIc. FcRIIa is of two types,

one which binds weakly to IgG2 and other which binds strongly. IgG4 binds to both

forms of FcRIIa

26

Figure 2: The schematic representation of the IgG subclasses and its isomers

IgG in serum of infants is predominantly derived via placental transfer from mother

and a small amount is fetal origin. As per the studies Zak and Good et al, IgG level are

negligible at birth, starts to increase by 3-6 weeks and becomes demonstrable when it

reaches 2g/L by 2 months of age. IgG levels reach about 60% of adult value by 10-18

months of age(1).

VIII. FACTORS DETERMINING ABO HDN

As per the literature, there had been many studies in the past to identify the predictors

of the severity of ABO HDN. Various tests like the effect of maternal antibodies in

ADCC assay, maternal IgG anti-A and anti-B antibody titers, DAT on fetal red blood

cells and titer of IgG anti-A and anti-B antibodies in serum of infants were compared

and was concluded that ADCC assay with maternal serum was the most sensitive

assay(42).

27

VIII A. The role of IgG subclasses in ABO HDN:

The role of anti-Rh (D) IgG subclasses on the severity of hemolytic disease was

studied in the past and was proved that IgG subclasses have a significant correlation

with hemolytic disease, of which IgG1 or the combination of IgG1and IgG3 was

significantly associated with the severity of the hemolytic disease. The onset of

hydrops was earliest with a combination of IgG1+IgG3 than with IgG1 alone(43–45).

But there is very limited literature on the correlation of IgG subclasses and ABO

hemolytic disease. As per the study conducted in 2011, by Yi-qiang et al in China, it

was concluded that IgG1 and IgG3 titer levels, especially that of IgG1, had a

significant correlation with ABO hemolytic disease(46). This was contradicting the

findings of the study by Kaplan et al in 2009 in umbilical cord blood samples, that IgG

subclass 1, was not predictive of ABO HDN(47).

VIII B. The role of maternal titers in predicting ABO HDN:

In addition to blood grouping and direct antiglobulin testing, maternal anti-A/-B titers

contribute to the prediction of risk of severe hyperbilirubinemia in neonates. They can

be used as a guide in the management of maternal-fetal ABO incompatibility(1).

Owa J A et al, in their study conducted on 50 mothers with O blood group, suggested

treatment and exchange transfusion is indicated when the maternal anti-A and anti-B

titer was 1:64 and more(48). As per the study conducted by Yi-qiang et al in China, it

was suggested that neonates with maternal IgG titers more than 64 were prone to

develop ABO-HDN(46). As per some other studies, 1:512 was the cut off for initiation

of phototherapy, immunoglobulin treatment and exchange transfusion(49–51). A

28

study by Kadri et al, with the titer > 256, the risk of neonatal jaundice becomes 18

times higher than those with low antibody titers <256(52).On the other hand, a few

trials proved that there was no association between maternal anti-A and anti-B titers

with ABO-HDN(29).

Therefore, the role of maternal anti-A and anti-B titers and IgG subclasses on

predicting the risk of ABO-HDN remains controversial.

VIII C. OA and OB incompatibility:

In the western population, hemolysis due to anti-A is more common than anti-B(49).

As per a few studies, ABO incompatibility due to anti-B is more severe(53). and there

are reported cases of hydrops foetalis due to the same(54).

As per a few reports, there is an increase in trend of exchange transfusion in first 24

hours of life in cases of O-B incompatibility as compared to O- A incompatibility(55–

57) while a few studies established that there is no relationship between the clinical

course and infant’s blood group(58). As per the study by Bekkeheim et al and a few

others, there is a considerably increased rate of invasive treatments in O-B infants than

O-A(50). As per the studies by Sisson and Kaplan, there is no significant difference in

response to therapy in O-B neonates compared to O-A(59).

As per the studies, OA and OB incompatibility between mother and fetus renders 90%

and 50% protection respectively to immunization to D antigen. The theory proposed

behind this is the protective ability of anti-A and anti-B to destroy fetal RBCs in the

maternal circulation.(22)

29

IX. GRADING THE SEVERITY OF ABO-HDN:

ABO-HDN is usually considered as a sub clinical condition and less severe than Rh-HDN.

Nonetheless, there are reported cases from the Indian subcontinent on unusually severe cases

of ABO-HDN necessitating multiple exchange transfusions(60,61).

It is described in literature that the severity of HDN is determined by the severity of

haemolysis. Mild haemolytic disease of newborn presents as mild jaundice and can be

managed with phototherapy alone and severe HDN causes kernicterus and brain damage.

Following are the different guidelines used worldwide available till date to classify HDN.

The major grading system used to grade the disease severity in HDN was put forward by

Walker in 1971(62):

1. Mild HDN : DAT positive cases requiring no transfusion

2. Moderate HDN: DAT positive cases with cord blood Hb >11 g/dL and requiring

transfusion

3. Severe HDN: DAT-positive cases with cord blood Hb <11 g/dL and requiring

transfusion;

4. Very severe HDN: DAT-positive cases with cord blood Hb <7.5 g/dL, or hydropic.

Another grading system proposed by Andrew et al is as follows(63):

1. Mild - Grade 0; Hb > 12.5 g/dL, no transfusions

2. Moderate - Grade 1: Hb > 12.5 g/dL + top-up or exchange transfusion

30

3. Severe- Grade 2: Hb < 12.5 g/dL + exchange transfusion

4. Very Severe- Grade 3: intrauterine transfusions and/or Hb < 10.0 g/dL ± exchange

transfusions or foetal death.

Choudhuri et al has proposed the following way of classifying Rh-HDN(45):

1. Mild: Positive DAT alongside some evidence of haemolysis but requiring no

intervention.

2. Moderate: Cases with evidence of haemolysis and intervention was limited to

phototherapy

3. Severe: When the baby was hydropic or intrauterine/ exchange or top up transfusion

was required.

In spite of a few grading systems offered, there is no consensus on grading of HDN. In our

study, a combination of DAT positivity, hyperbilirubinemia, anemia and intervention required

defined the HDN secondary to ABO incompatibility and also in grading its severity.

X. SEROLOGICAL TESTING IN NEWBORN:

Investigation of a newborn suspected of ABO HDN broadly includes the following:

1. ABO grouping

2. Rh typing

3. DAT

4. Hemoglobin estimation

5. Serum bilirubin

31

6. Hematological parameters

CO-oximeters can be used to measure COHb which can confirm hemolysis in infants

with ABO incompatibility(64).

X A. Direct Antiglobulin Testing:

DAT is an important screening tool for ABO hemolytic disease. It can detect

immunoglobulin or complement bound to the RBCs using anti-human reagents.

There are different platforms for performing DAT like tube method, micro column or

gel method and solid phase. The gel and solid phase are automated methods with

greater sensitivity than conventional tube testing(65).

A positive DAT indicates that there is a sensitization of fetal red cells. DAT can be

negative in ABO hemolytic disease. This is because A and B antigenic sites are

weak(66) and there is very little anti-A or anti-B antibody in the newborn RBC

membrane, as a result of which cord blood DAT can be weakly positive or often

reported as negative unless a sensitive platform like gel test is used(67). A positive

DAT indicated that at least 150 molecules of the maternal antibody are adsorbed into

the fetal red cell(68). Using more sensitive platforms like autoanalyzer with low ionic

strength solution with enhancer, anti-A and anti-B can be demonstrated in all ABO

incompatible cases.

As compared to Rh hemolytic disease, DAT tends to be weakly positive or negative in

ABO incompatibility.

32

The infant’s red cells do not react with anti-C3d owing to low levels of complement in

the serum of newborns and weak expression A and B antigens on the fetal red cell

surface.

X B. Elution of antibody from infant’s red cells:

In the newborn with positive DAT and clinical features of HDN, red cell eluate

confirms the antibody specificity and the diagnosis of HDN can be confirmed by the

presence of the corresponding antigen on the red cells(19). Eluates from infant’s red

cells give stronger IAT even in cases of weak positive or negative DAT due to a

considerable increase in antibody concentration through elution procedure.

X C. Spontaneous agglutination of red cells:

Red cells taken from ABO incompatible infants tend to clump more spontaneously;

also washed red cells from ABO incompatible infants tend to sediment more rapidly in

PVP when compared to similarly treated red cells which prove that there is some

alteration in charges on those red cells.

X D. Secretor status of infant:

It is proved that there is no role of secretor status in protecting against ABO hemolytic

disease(1).

XI. HAEMATOLOGICAL FINDINGS:

There is no single diagnostic test for ABO-HDN.

33

ABO-HDN is one among the causes to be investigated in a newborn infant developing

jaundice. As compared to the Rh hemolytic disease, immunological findings of ABO

HDN do not correlate well with the severity of the clinical manifestations.

Following are the diagnostic criteria:

a) Varying degrees of jaundice and anemia

b) Microspherocytosis

c) Polychromasia and reticulocytosis (> 4.6%)

d) Positive DAT(69)

Positive DAT with a family history of neonatal jaundice and high reticulocyte count

are considered as the strong predictors of hyperbilirubinemia in ABO

incompatibility(70).

Anemia is usually mild and rarely occurs in ABO incompatibility. In cases of the

severe hemolytic disease, the cord blood hemoglobin may be low but it is very rare to

have persistent anemia beyond two weeks as compared to Rh hemolytic disease. The

normal range of reticulin in term neonate is between 1.8-4.6% and reticulocytosis is to

compensate for the hemolysis.

Microspherocytes is one of the most common peripheral smear finding in ABO

incompatibility. It occurs due to the reduction in erythrocyte surface area due to

phagocytosis of the RBC membrane by macrophages(69). Similarly, red cells osmotic

fragility is mostly above the normal limits as compared to Rh hemolytic disease.

34

Neonates with blood group B tend to receive higher invasive treatment than those with

blood group A(53) and reports indicate that ABO-HDN due to anti-B is more severe

and there are reported cases of antenatal hydrops due to IgG anti-B(54).

In contrast to Rh HDN, there are no preventable measures for ABO-HDN. Hence

ABO incompatibility is the single most common cause of neonatal jaundice(58).

XII. PREDICTION OF SEVERITY OF HDN

The management of HDN relies on screening and detection of the antibody and

predicting its significance in the development of hemolytic disease. Several methods

are used for predicting the severity of hemolytic disease including serological,

quantitative and cellular assays.

XII A. Serological assay

Indirect Antiglobulin Test: Titration studies facilitate in detecting the critical titer

which plays a vital role in fetal monitoring(71). It is a semi-quantitative technique of

measuring an antibody in the serum employing a serial double dilution of the serum

against selected red cells. Titer is the reciprocal of the highest serum dilution at which

macroscopic agglutination occurs after the addition of antiglobulin reagent (68).

XII B. Quantitative assay: Enzyme Linked Immunosorbent Assay (73), Flow

cytometry and RIA (74)have been developed to accurately measure the level of red

cell binding IgG anti-D in maternal serum. Auto analyzers can process a large number

of samples with a greater reproducibility than manual antibody titration method by

IAT (75).

XII C. Cellular assay

35

K cell antibody-dependent cell-mediated cytotoxicity Assay: In addition to antibody

concentration, variations in functional and biological activities of antibodies in

different individuals help in predicting the severity of the hemolytic disease. Monocyte

mediated and K cell mediated assays are based on the differences in their interaction

with IgG sensitized red cells.

K cell mediated ADCC assay was the first cellular assay that was developed to predict

the severity of the hemolytic disease. As per the study conducted by Urbaniak et al,

this assay was able to predict the babies with mild HDN from moderate HDN(76)

while the study by Hadley et al failed to prove the correlation between KADCC and

the severity of HDN(77).

The monocyte-mediated ADCC assay: MADCC assay is a better predictor of disease

severity when compared with the Indirect Antiglobulin Test. It is similar to KADCC

except that peripheral blood mononuclear cells are used as effector cells(78).

Monocyte monolayer assay: MMA aims at microscopic evaluation of adherence and

phagocytosis of red cells by adherent monolayers of macrophages. It has been

ha proved to have a significant correlation with severity of HDN when compared with

manual titration(79).

Chemiluminescence test: CLIA has a better correlation with the fetal outcome when

compared to other auto analyzers. It is based on the principle of incubation of

monocytes and the sensitized red cells in the presence of luminol. Luminol helps in the

conversion of metabolic byproducts into light which is then measured by a

luminometer(63).

36

Cellular assays have a greater additive value when used in conjunction with serologic

and quantitative assays.

XIII. THERAPEUTIC MEASURES IN ABO-HDN:

The spectrum of hemolytic disease of the newborn has changed over time and was

almost synonymous with Rh-D allo-immunization around forty years back. In the past,

it led to overt hemolysis, marked hyperbilirubinemia and severe anemia in neonates

which led to their mortality and morbidity. Early diagnosis and adequate care are

necessary to prevent complications such as kernicterus. With the advancements in the

field of transfusion and neonatology services, the clinical burden of HDN has

drastically reduced over the years.

The spectrum of hemolytic disease also got altered following the introduction of new

treatment modalities like phototherapy, intravenous immunoglobulin and exchange

transfusion(80). The earliest and timely intervention helps to prevent bilirubin

encephalopathy and kernicterus with subsequent development of neurological sequelae

and death(81).

Guidelines for the management of neonatal hyperbilirubinemia in newborns > 35

weeks of gestation has been published by the American Academy Of Paediatrics

(AAP)(82) and also in guidelines of other counties like Canada, Israel, Norway, South

Africa, the Netherlands and the United Kingdom(83–88). In view of no similar

guidelines for the treatment of infants < 35 weeks of gestation, NICU s have

37

established their own criteria based on birth weight and gestational age for the use of

phototherapy and exchange transfusion(89).

XIII A. Phototherapy:

Phototherapy aims to convert unconjugated bilirubin into biliverdin. It can prevent the

need for exchange transfusion in cases of mild to moderate hemolysis(24). The gold

standard treatment for severe hyperbilirubinemia is Exchange transfusion(90).

As per the AAP guidelines, the four important risk factors for severe

hyperbilirubinemia are: blood group incompatibility with positive DAT, high-risk zone

TSB values predischarge, preterm babies and jaundice in the first 24 hours of life(91).

There were many studies in the past regarding the level of bilirubin and the risk of

kernicterus. As per the study conducted by Hsia et al in 1952, it was concluded that

risk of kernicterus increased significantly when serum bilirubin exceeded 340 pmol/L

and the risk rose to 50% when the serum bilirubin levels were above 510 pmol/L.

The use of phototherapy was well established by 1979. Cockinton et al developed

different thresholds as per the birth weight and age in hours for the initiation of

exchange transfusion in addition to phototherapy(92). Bhutani et al further modified

the approach to jaundiced near term and term infants based on the age in hours(93).

Based on the various intensities of light, phototherapy can be classified as close

phototherapy, double conventional and triple therapy. The conventional phototherapy

exposes the newborn to 7-10Uw/CM2/nm which helps in decline in serum bilirubin to

6-20% in the initial 24 hours(45). Double conventional phototherapy is more effective

than single phototherapy in reducing the serum bilirubin levels and duration of

38

treatment in term infants of birth weight over 2500g. But the role of double surface

phototherapy in abating the role of exchange transfusion is not well studied(90).

Efficacy of prophylactic phototherapy in preventing neonatal hyperbilirubinemia in

newborns with ABO incompatibility and positive DAT were studied in the past with

inconclusive results(94,95). A few studies concluded that prophylactic phototherapy

administered on the first day of life leads to a significant reduction in TSB in first 48

hours but not later on(70).

XIII B. Exchange transfusion:

Exchange transfusion is employed in conditions wherever there is a significant threat

of kernicterus being caused by hyperbilirubinemia or when phototherapy has not been

effective. It contributes to get rid of the maternal antibodies in the circulation in

addition to removing excess bilirubin, thereby reducing the further probabilities of

ongoing hemolysis.

It is performed with double volume irradiated (160 ml/kg), leukocyte depleted,

compatible blood, negative for CMV via an intravenous catheter inserted in the

umbilical vein. This procedure has its own independent risk hazards and is associated

with a high risk of procedure related morbidity(92, 92, 93).

Intravenous immunoglobulin has become an alternative therapy to exchange

transfusion. Even though, multiple doses of IVIG may be required to prevent the

ongoing hemolysis, even a single dose of IVIG has been well tried to be very

effective(94, 95).

39

Large evidence based studies have been limited due to the low prevalence of disease

which has led to the dearth of literature to compare the transfusion needs and outcome

measures. It is not possible to avert the incompatibility reaction. Prenatal counseling

is not a practical solution as ABO incompatibility neither leads t o significant mortality

nor lifelong morbidity. There have been significant changes made in the antenatal and

postnatal management strategies. New diagnostic techniques, monitoring tools and

management options are constantly under investigation and can go a long way to

reduce the factors that increase the severity and morbidity of hyperbilirubinemia of

newborn.

XIII C. Alternative to exchange transfusion:

Treatment with oral D pencillamine and A or B trisaccharides were also described in

ABO incompatible infants in the literature(1).

XIV. RECENT ADVANCES:

There is emerging literature on the use of Flow cytometry (FC) to estimate the number

of soluble or RBC bound IgG as it has a higher exactitude and reliability when

compared to standard serological procedures. It can be used to study the IgG subclass

and amount on the red cells. The downside of FC is that it is cost and unavailability in

most of the laboratories(100).

A Gel Centrifugation Test (GCT) has been recently developed for the quantification of

the RBC bound antibodies and IgG1 and IgG3 subclasses. It has been proved to be

40

extra sensitive in the demonstration of RBC allo-antibodies and conjointly the

determination of IgG subclasses and was found to be in agreement with the Flow

Cytometry results(101).

The degree of RBC destruction is determined not only by the IgG subclasses and

density of red cell bound antibodies but also conjointly by numerous other factors like

the effector cell type and its Fc receptor, complement activation, factors affecting the

actions of the monocyte-macrophage system and the type of hemolytic disease(78).

There were a few studies in the past, which concluded that severe forms of Rh-HDN

were associated with IgG1 carrying G1M (3) allotypes as compared to G1M (1)

allotypes. GM allotypes are located on the heavy chains of Ig. GM typing can be done

on slides or microtiter plates or by using gel test(102).

A few studies were conducted to investigate the polymorphism of FcγRIIa (CD32) in

cases of ABO-HDN. FcγRIIa is a member of FcRn which mediates the placental

transfer of IgG by binding to the subclasses(35). Polymorphism within the gene can

affect the binding of IgG subclass and was concluded that HR131 genotype was

associated with susceptibility to ABO-HDN.

XV. CONCLUSION:

ABO incompatibility is now emerging as the main cause of hemolytic disease. Early

blood grouping and DAT-testing in neonates with early jaundice are recognized as risk

appraisals. The current study aims to assess the prevalence of ABO incompatible

pregnancies and to see if high maternal IgG anti-A or anti-B titers and IgG subclasses

41

are consistent with increased risk of developing ABO hemolytic disease of newborn.

Against the backdrop of a country such as ours, where neonatal assessment and care

may be limited in large areas, it may be prudent to utilize these tools to guide mothers

at high risk to centers where appropriate care is available. Timely diagnosis and

interventions can reduce the incidence of ABO haemolytic disease of newborn and

improve the population quality.

This study was performed to determine the prevalence of IgG subclasses in ABO

hemolytic disease of the newborn using DAT IgG1/IgG3 card test on the column

agglutination platform to see if it helps in the prognostication of HDN. It is a simple

and low cost technique using small quantity of serum. This might allow early

intervention and proactive management of the newborn. If this is found to be clinically

relevant, it can be enforced within the routine prenatal investigation.

42

MATERIALS AND METHODS

43

MATERIALS AND METHODS

SETTING:

This study was conducted in Christian Medical College, Vellore, Tamil Nadu. It is a

teaching hospital providing tertiary medical care to residents of Vellore and

surrounding districts of Tamil Nadu, Andhra Pradesh and Kerala. It also serves as a

referral centre for patients from rest of India and South East Asia. This observational

cohort study was carried out in Department of Transfusion Medicine and

Immunohematology (Blood Bank) in coordination with departments of Obstetrics and

Neonatology.

The study was approved by Institutional Review Board of Institute. Informed consent

was obtained from the mothers who were recruited for study and venous samples were

collected from them.

BIAS:

All efforts were made to eliminate any bias in selection of patients. Despite this the

prevalence of ABO incompatible pregnancies may show a skewed result since CMC is

a tertiary hospital and cases at risk are often referred here. Instrumental bias was likely

to be negligible as variables were measured using calibrated and automated analyzers

in the clinical pathology department and blood bank and all serological tests done in

blood bank were run in duplicates. Investigator bias in interpretation of severity of

HDN was avoided as the investigator was not involved in the treatment directly.

44

SAMPLE SIZE:

Sample size was estimated to be approximately 575.

According to the study conducted by Heydarian F et al in 355 cases of ABO

incompatibility, the prevalence of ABO-HDN was detected to be 27%. This

prevalence was taken into consideration and the following formula was used for

statistical analysis.

N=4xpq/d2= 4x27x73/49 =161

p=percentage of ABO incompatibility; q=1-p; d=precision=7%

Using the precision of 75 and 95% confidence interval, at least 161 cases of ABO

incompatible pregnancies were to be considered to obtain 27% prevalence of ABO-

HDN.

The prevalence of ABO incompatible pregnancies among O blood group mothers was

28% as per the study by Cernadas et al. Therefore to detect 161 cases of ABO

incompatible pregnancies, 575 mothers with O blood group were recruited for the

study.

Screening number=161/0.28=575

Note: There was no proper literature available for prevalence of IgG subtypes among

ABOHDN. So the prevalence of hemolytic disease among ABO incompatibility was

used for sample size calculation.

45

References for calculation of sample size:

1. Farhad Heydarian, Seyedeh Fatemeh Khatami, Pouya Parvaresh, Masumeh

Reanjad et al. ABO Hemolytic Disease Leading to Hyperbilirubinemia in term

Newborns: Value of Immuno hematological Tests. Iranian Journal of

Neonatology Vol.3, No.2, 2012

2. JM ceriani Cernadas, C Fustinana, M Bujas et al. 23 Epidemiology of the ABO

incompatibility. Pediatric Research Volume 15,February 1981

PARTICIPANTS:

Inclusion criteria: : All O blood group mothers who were negative for alloantibodies

on a red cell antibody screen, and who gave birth to babies with A, B or AB blood

group were included in this study.

Exclusion criteria: Neonates with other known causes of jaundice and hemolysis other

than ABO incompatibility.

STUDY IMPLEMENTATION:

All antenatal women during their antenatal visit had a blood grouping and typing and

antibody screen performed as per the routine protocol. Mothers with O blood group

with a negative antibody screen and who delivered at term (after 37 weeks of

gestation) were recruited for our study. The proforma was explained to the mothers or

care givers and informed consent regarding participation in the study was obtained in

the regional language. 8 ml venous samples were collected from those mothers who

46

fulfilled the above inclusion criteria. The plasma was separated and frozen for

maternal IgG anti-A /anti-B titer analysis and IgG1/ IgG3 subclass typing.

The babies were then followed up with help of Department of Neonatology and

medical records. Clinical details and relevant laboratory investigations were accessed

from the clinical workstation of the hospital, both for the mother and subsequently for

the baby. Data was collected as per the pre-structured clinical research form designed

at the beginning of the study.

The newborn was evaluated for the presence or absence of HDN. Unlike Rh-HDN,

there were no clear guidelines to grade the severity of ABO-HDN.

SAMPLE PROCESSING:

8ml venous samples were collected from the mothers using EDTA vaccutainers. The

samples were centrifuged at 3000 rpm for 10 minutes, plasma was separated and

stored in eppendorf tubes at -70 degree C. IgG anti-A and anti-B titer analysis was

done using tube method and double dilution technique. IgG1 and IgG3 subclass typing

was done by column agglutination technique using ID card DAT IgG1/IgG3 DiaMed

GmbH 1785Cressier FR Switzerland (BIO-RAD).

47

575 antenatal women with O blood group

and negative antibody screen who

presented to the labour room at term were

recruited for the study

Pregnancies were followed up. 8 ml

venous samples were collected from

mothers who delivered babies with non

O group

DAT was performed in all newborns and

they were monitored as per the

guidelines of neonatology department

Plasma was

separated and

frozen

Cases were classified based on the

presence or absence of ABO HDN.

Maternal IgG1/IgG3 subtypes and IgG

anti-A and anti- B titers were correlated

with the occurrence of ABO-HDN

48

DATA COLLECTION AND SOURCE OF DATA:

Data of mother

1. Demographic

2. Personal

3. Blood group and Rh typing

4. Period of gestation

5. Obstetric history

6. Indirect antiglobulin test

7. Outcome of pregnancy

Baby’s data

1. Live/ still birth/intrauterine death

2. Preterm/term/post-term

3. Gender

4. Birth weight

5. Blood group

6. Direct Antiglobulin test

7. Haemoglobin (monitored over hospital stay; lowest level documented)

8. Total serum bilirubin (monitored over hospital stay; highest level documented)

9. Direct serum bilirubin

10. Transcutaneous bilirubin (if done)

11. Reticulocyte count (monitored over hospital stay; highest level documented)

49

12. Peripheral smear

13. Treatment required – including admission to Neonatal intensive care unit

14. Treatment details:

15. Phototherapy – single/ double surface

16. Exchange transfusion

17. Intra- uterine transfusion

18. None

Tests done in the blood bank:

On mother’s sample:

1. Blood grouping and Rh typing

2. Antibody screen

3. Maternal IgG anti-A and anti-B titer

4. IgG subclass typing (IgG1 and IgG3) using Column agglutination technique,

finding high(1:100) and low titers(1:1) for each respectively

On newborns’ sample:

1. Blood grouping and Rh typing

2. Direct Antiglobulin Test

50

COLUMN AGGUTINATION TECHNOLOGY:

It is a commonly used platform in blood banks presently, and it uses gel or glass beads

to trap agglutinated red cells following an antigen antibody reaction. Test can be

performed on this platform manually or by automated means, which allow for

processing multiple samples at the same time. CAT as it is commonly known as, can

be used for tests like blood grouping, antibody screening, DAT, IAT and cross-

matching. Antigens expressed on the red cells interact with antibodies in the chambers

at the top of a column, and after proper incubation and centrifugation as recommended

by the manufacturer, the test can be interpreted. The agglutinated red cells remain at

the top, and are read as a positive reaction, while the free red cells are forced through

the column to the bottom and read as negative. This forms the principle of many tests

carried out via this platform in our blood bank.

STATISTICAL ANALYSIS:

Categorical variables were summarized using counts and percentages. Quantitative

variables were summarized using mean and standard deviation or median and IQR.

The expression of IgG subclasses were presented as numbers and frequencies. Pearson

and Fischer’s exact chi square was used for comparison of categorical data. Univariate

and binary logistic regression analysis was used for comparing the categorical data and

to calculate the odds ratio. P value <0.05 was considered significant. SPSS 16.0 was

used for statistical analysis.

51

RESULTS AND ANALYSES

52

RESULTS AND ANALYSIS

A total of 575 mothers with O blood group and negative antibody screen who

delivered during the period June 2018 to August 2019 were considered for our study.

I.PREVALENCE OF ABO INCOMPATIBLE PREGNANCIES:

Out of the 575 mothers, 203 mothers delivered babies of A or B blood group.

Therefore the prevalence of ABO incompatible pregnancies was found to be 35.30 %

in O blood group mothers.

II. DEMOGRAPHIC DETAILS:

II 1. Geographic distribution:

Our cohort of patients included 165 (81.28%) mothers from Tamil Nadu, 32 (15.77%)

from Andhra Pradesh, 4(1.97%) from Karnataka and 1(0.49%) each from West Bengal

and Kerala.

Figure 3: Pie chart depicting geographic distribution of study population (n=203)

165 (81.28%)

32 (15.77%)

4(1.97%)

1 (0.49%)

1 (0.49%)

Tamil Nadu

Andhra Pradesh

Karnataka

West Bengal

Kerala

Geographic distribution

53

I.2 Age of mothers:

The mean age of the study population was 28 years (Maximum 37 years and minimum

19 years).

I.3 Parity of mothers:

With regards to parity, 102 (50.25 %) of these patients were primiparous and

101(49.76 %) were multiparous.

Among the multiparous mothers, 63 (31.04%), 25(12.31%), 11(5.42%), 1(0.49%) and

1(0.49%) belonged to gravida G2, G3, G4, G5 and G7 respectively.

Figure 4: Pie chart depicting parity of mothers in study population (n=203)

I.4 Previous obstetric history:

Among the multiparous mothers, 54 out of 101 (53.46 %) mothers had an uneventful

obstetric history. However a bad obstetric history (BOH) defined by events such as

101 (49.76%)

63(31.04%)

25 (12.31%)11(5.42%)

1 (0.49%)

1(0.49%)G1

G2

G3

G4

G5

G7

G= Gravida

Parity of mothers

54

previous 1st or 2nd trimester loss, still birth, neonatal death or medical termination of

pregnancy were recorded for 47(46.54 %) mothers among the multiparous group. 36

mothers had a previous history of abortion, 5 had history of neonatal death, 4 abortion

and 2 medical termination of pregnancy.

* NND: Neonatal death, MTP: Medical Termination of Pregnancy

Figure 5: Bar graph depicting BOH among multiparous mothers (n=101)

I.5 Mode of delivery:

Analysis of the 203 mothers under consideration, showed that 95(46.79%) delivered

by normal vaginal delivery, 56(27.59%) by LSCS and rest 52(25.62%) by instrumental

delivery.

0

5

10

15

20

25

30

35

40

Abortion NND Still birth MTP

36

5 42

Nu

mb

er

of

case

s

Bad obstetric history

Bad obstetric history in multiparous mothers

55

*NVD: Normal Vaginal Delivery, LSCS: Lower Segment Caesarian Section

Figure 6: Pie chart illustrating mode of delivery of the study population (n=203)

I.6. Gender of the newborn:

Among the 203 babies delivered, 110(54.19%) were males and 93(45.82%) were

females.

Figure 7: Pie chart depicting gender distribution of newborns (n=203)

95( 46.79%)

56(27.59%)

52(25.62%)

NVD

LSCS

Instrumentaldelivery

Mode of delivery

110 (54.19%)

93 (45.81%) Male

Female

Gender distribution of newborn

56

I.7. Birth weight of the newborn:

Among the 203 babies, 23 (11.33%) were found to be of low birth weight (birth

weight <2.5 kg) and the rest 180 (88.67%) were of normal weight.

Figure 8: Pie chart illustrating birth weight of newborns (n=203)

II. BLOOD GROUP DISTRIBUTION IN BABIES:

81(39.90 %) out of the 203 newborns were A blood group and 122 (60.10%) were of B

blood group.

Figure 9: Pie chart showing prevalence of blood groups in newborns (n=203)

23 (11.33%)

180 (88.67%)

Low birth weight

Normal birth weight

Birth weight of newborns

81( 39.90%)

122( 60.10%)A group

B group

Distribution of blood groups in newborns

57

III. IMMUNO-HAEMATOLOGY PARAMETERS:

III.1 Antibody titration: Antibody titration was performed using tube method and

double dilution technique in the 203 plasma samples.

Table 5. Distribution of titers in mothers included in study:

Titer level Number of cases (n=203) Percentage of cases

2 10 4.93 %

4 8 3.94 %

8 34 16.75%

16 33. 16.26%

32 49 24.13%

64 44 21.68 %

128 22 10.83 %

256 3 1.48 %

The most frequently observed titer was 32 (24.13 % cases) followed by 64 in 44 cases

(21.68%). The least frequently observed titer was 256 in 3 cases (1.48%)

III.2 IgG subclasses identified (IgG1 and IgG3):

IgG subclass typing was performed using a DAT IgG1/IgG3 ID-Card from DiaMed

GmbH, using CAT in total 203 samples.

58

Our study showed the overall prevalence for IgG1 and /or IgG3 subclasses in our

population of antenatal women to be 46.79 %.

Table 6. Prevalence of IgG1/IgG3 subclasses in mothers (n=203)

IgG1/IgG3 results in mothers(n=203):

IgG1/IgG3 positive IgG1/IgG3 negative

95 (46.79%) 108(53.20%)

Analyzing it within subclasses showed the prevalence of IgG1/IgG3 as follows:

Table 7. Distribution of IgG1/IgG3 subclasses in mothers (n=203)

IgG subclasses No. of cases Percentage

Only IgG1(1:1) 34 16.75 %

Only IgG1 (1:100) 13 6.40 %

Both IgG1(1:100) and IgG3(1:100) 42 20.69 %

Both IgG1(1:100) and IgG3(1:1) 6 2.96 %

Negative for both 108 53.20 %

On analysis, we concluded that 108 cases( 53.20) % of our study group did not have

either IgG1 or IgG3, while in 42 cases (20.69%), both these subclasses co-existed in

high titers. It was interesting to note that all patients who had IgG3, also had IgG1 in

high titers i.e, IgG3 was not detected in isolation.

59

IV. LABORATORY PARAMETERS OF THE NEWBORNS:

IV 1. Anaemia:

Anaemia is defined as serum Hb levels below 14.6 g/dl in a term neonate. Clinical

assessment formed the initial mode of assessment. In suspected cases of haemolysis, in

which newborns presented with jaundice; haemoglobin and total bilirubin levels were

estimated. Haemoglobin levels were available for 170 out of the total 203 newborns,

of which 49(28.82%) newborns had anemia.

Figure 10: Pie chart depicting prevalence of anemia in ABO incompatible cases

1V. 2 Hyperbilirubinemia

Neonatal hyperbilirubinemia is defined as any PTB values above ≥ 95th percentile on

the hour of life specific normogram. In our study, serum bilirubin levels or

transcutaneous bilirubin (TcB) levels were estimated. Majority of neonates developed

jaundice on second day (46%) followed by third day (24%). The mean age of

49(28.82%)

121( 71.17%)

<14.6 g/dl

≥14.6 g/dl

Anaemia in newborn

60

presentation of jaundice was 2.35± 1.15 days, earliest cases of hyperbilirubinemia

reported within 24 hours. The mean initial indirect bilirubin was 14.64 ± 3.92 mg%

with the range from 7.46 mg% to 27.51 mg%.

1V. 3. Reticulocytosis

Reticulocytosis in newborn is defined as reticulocyte count >4.6%. Reticulocyte count

was available for 106 newborns and a high reticulocyte count was found in

51(48.11%) cases.

Figure 11: Pie chart showing prevalence of reticulocytosis in ABO incompatible cases

V. MODE OF TREATMENT:

Treatment details were available for all cases of ABO incompatibility. Single Light

Phototherapy (SLPT) was the most common treatment administered in around 65%

cases followed by Double Surface Phototherapy (DSPT) in 6.3% cases and Exchange

Transfusion (ET) in 1 case. In 7 cases, DSPT was administered following SLPT.

51( 48.11%)55( 51.89%)

≥4.6%

<4.6%

Reticulocyte count in study population

61

Table 8: Mode and average duration of treatment administered (n=203)

VI. Follow up of pregnancy and diagnosis of ABO-HDN

ABO-HDN is defined as a newborn with blood group A, B or AB with positive DCT

and laboratory evidence of haemolysis, born to a mother with blood group O.

Among the 203 cases of ABO incompatibility analyzed, 76 newborns were found to

have early onset jaundice, positive DCT and evidence of haemolysis, and therefore,

the prevalence of ABO-HDN was estimated to be 37.43%.

Figure 12: Pie chart illustrating the prevalence of ABO-HDN (n=203)

76(37.43%)

127(62.57%)ABO HDN

No HDN

Prevalence of ABO-Haemolytic disease of newborn

Mode of treatment Number of cases(n=203) Average duration

Single phototherapy (hrs) 133 32.5 hours

Double phototherapy (hrs) 13 46 hours

Exchange Transfusion 1 Once

62

Figure 13: Flow chart to illustrate patient follow up and results

Mothers who delivered babies of either A, B or

AB blood group (n=203). 8 ml maternal

venous samples were collected. Plasma was

separated and frozen.

Cases with ABO-HDN

(N=76).

IgG anti-A and anti-B

titers and IgG1/IgG3

subtyping was done on

maternal samples.

Cases without HDN

(N=127).

Maternal titers and subclasses were correlated with occurrence of ABO-HDN

Newborns were monitored with the help of

neonatology department to see if they

developed ABO-HDN.

Total number of antenatal mothers with O

blood group and negative antibody screen who

were recruited for the study (N=575)

63

VII. DEMOGRAPHIC DETAILS IN ABO- HDN:

VII. 1 Geographic distribution:

Among the 76 cases of ABO-HDN, 59 (77.64%) of their mothers were from Tamil

Nadu, 16 (21.05%) from Andhra Pradesh and 1(1.31%) from Karnataka.

Figure 14: Pie chart depicting geographic distribution of mothers of ABO-HDN

VII. 2. Age of the mother:

The mean age of the study population of mothers was 28 years (Maximum 38 years

and minimum 19 years).

VII. 3. Parity of mother:

Among 76 cases of ABO-HDN, 39(51.31%) of the mothers were primiparous and

37(48.69%) were multiparous.

59(77.64%)

16(21.05%)

1( 1.31%)

Tamil Nadu

Andhra Pradesh

Karnataka

Geographic distribution of mothers of ABO-HDN group

64

Figure 15: Pie chart illustrating the parity of mothers of ABO-HDN (n=76)

Among the multiparous mothers, 22 (28.95 %), 9 (11.84 %), 5 (6.58%) and 1(1.32%)

were G2, G3, G4 and G5 gravida respectively.

Figure 16: Pie chart illustrating overall distribution of parity among mothers

39( 51.31%)

37( 48.69%) Primiparous

Multiparous

Parity of mothers in ABO-HDN group

39(51.31%)

22(28.95%)

9(11.84)%)

5(6.58%) 1(1.32%)

G1

G2

G3

G4

G5

Distribution of parity in mothers in ABO-HDN group

65

Table 9. Correlation between parity of mother and occurrence of ABO-HDN

Parity ABO-HDN No HDN p value

Primiparous 39 63 0.885

Multiparous 37 64

Occurrence of ABO haemolytic disease of newborn was independent of the parity of

mother.

VII. 4. Mode of delivery:

Out of the total 76 cases of ABO HDN, 38(50%), 18 (23.68%) and 20(26.32%) were

by normal vaginal delivery, LSCS and instrumental delivery respectively.

*NVD: Normal vaginal delivery, LSCS: Lower Segment Caesarian Section

Figure 15: Pie chart showing mode of delivery of mothers in cases with ABO-HDN

38 (50%)

18 (23.68%)

20 (26.32%)

NVD

LSCS

Instrumental

Mode of delivery in mothers of ABO-HDN group

66

Table 10: Correlation between mode of delivery and occurrence of ABO-HDN

ABO-HDN Vaginal delivery LSCS Instrumental p value

Present 38 18 20 0.61

Absent 57 38 32

There was no association between occurrence of ABO-HDN and mode of delivery.

VII. 5. Gender of the newborn:

39(51.31%) out of 76 newborns were males and 37(48.69%) were females.

Figure 16: Pie chart depicting gender distribution in cases of ABO-HDN

Table 11: Correlation between gender of newborn and occurrence of ABO-HDN

Gender of

newborn

ABO-HDN No HDN p value

Males 39 71 0.56

Females 37 56

39 (51.31%) 37 (48.69%)

Males

Females

Gender distribution of newborns in ABO-HDN group

67

There was no association between gender of the newborn and occurrence of ABO-

HDN.

VII. 6. Birth weight of the newborns:

Among the 76 cases of ABO-HDN analyzed, 8(10.52%) babies were of low birth

weight (<2.5kg) and rest 68 (88.48%) were of normal birth weight.

Figure 17: Prevalence of low birth weight in ABO-HDN group (n=76)

Table:12: Correlation between birth weight and occurrence of ABO-HDN

Birth weight ABO-HDN No HDN p value

< 2.5 kg 68 112 0.823

≥ 2.5 kg 8 15

There was no association between birth weight of newborn and occurrence of ABO-

HDN.

8(10.52%)

68 (88.48%)

Low birth weight

Normal weight

Prevalence of low birth weight in ABO-HDN cases

68

VII.7. Blood group distribution in ABO HDN cases:

33 out of 76 newborns (43.42%) were A blood group and 43 (56.58%) were B blood

group

Figure 18: Blood group distribution in ABO-HDN (n=76)

Table 13 : Correlation between blood group and occurrence of ABO-HDN

Blood group ABO-HDN No HDN p value

A group 33 48 0.428

B group 43 79

Occurrence of ABO-HDN was independent of the blood group of the newborn.

VII. IMMUNO-HAEMATOLOGY PARAMETERS IN ABO-HDN GROUP

VII 1. Antibody titration: Antibody titration was performed using tube method and

double dilution technique.

33( 43.42%)

43( 56.58%) A group

B group

Blood group distribution in newborns

69

Table 14: Comparison of titer levels in ABO-HDN and no HDN group:

Titer level ABO-HDN (n=76) No HDN (n=127)

Number Percentage Number Percentage

2 0 0 % 10 4.93 %

4 0 0 % 8 3.94 %

8 0 0 % 34 16.75 %

16 2 0.99 % 31 15.27 %

32 12 5.91 % 37 18.23 %

64 37 18.23 % 7 3.44 %

128 22 10.83 % 0 0 %

256 3 1.48 % 0 0 %

It was observed that the study group without ABO-HDN had a lower titer levels

ranging from 2 to a maximum of 64 when compared to the ABO-HDN group. The

latter group had a higher range of titers ranging from 16 to a maximum of 256. The

most frequently observed titer level in ABO-HDN group was 64 (48.68%) and in no

ABO-HDN group was 32 (15.79%). It was observed that, with increase in the maternal

IgG titer levels >64, there was an increase in trend of newborns developing ABO-

HDN.

70

Table 15: Association of maternal titer levels with occurrence of ABO-HDN:

Titer levels ABO HDN No ABO-HDN Odds ratio CI for odds ratio

< 64 14 120

64-128 37 7 45.306 17.01, 0.63

≥ 128 25 0

The odds of developing HDN at a titer of 64-128 is 45.31(95% CI 17.01-0.63) times as

compared to cases with titers < 64.

Table 16: Correlation between maternal titer levels and occurrence of ABO-HDN:

Titer levels ABO HDN No ABO-HDN p value

< 64 14 120 < 0.001

≥ 64 62 7

VII.2 IgG subclasses identified (IgG1 and IgG3):

IgG1 and / or IgG3 subclasses were present in all the cases of ABO-HDN i.e. the

prevalence of IgG1 and / or IgG3 subclasses was 100% in the ABO-HDN group

71

Table 17: Analyzing within IgG subclass:

IgG subclasses No. of cases Percentage

Only IgG1(1:1) 18 23.68 %

Only IgG1 (1:100) 11 14.47 %

Both IgG1(1:100) and IgG3(1:100) 45 52.27 %

Both IgG1(1:100) and IgG3(1:1) 5 6.58 %

Table 18: Correlation between presence of IgG1/ IgG3 and occurrence of HDN:

ABO - HDN

IgG1 and / or IgG3

present

IgG1 and / or IgG3

absent

p value

HDN present

76 0 <0.001

HDN absent

19 108

Presence of IgG1/IgG3 subclasses was significantly associated with occurrence of

ABO-HDN. This was independent of the maternal IgG anti-A or anti-B titers.

72

Comparison of distribution of IgG1/G3 subclasses in ABO-HDN and no HDN

groups:

Figure 19: Bar graph showing distribution of IgG1/IgG3 subclasses

Table 19: Presence of IgG1/IgG3 subclass at various titers and occurrence of

ABO-HDN:

Titer ABO-HDN IgG1/IgG3

present

IgG1/IgG3

absent

2

HDN Present 0 0

HDN Absent 0 10

4

HDN Present 0 0

HDN Absent 0 8

8

HDN Present 0 0

HDN Absent 6 28

16

HDN Present 2 0

HDN Absent 3 28

0

5

10

15

20

25

30

35

40

45

IgG1 low titeronly

IgG1 high titeronly

Both IgG1 andIgG3 high

IgG1 high andIgG3 low

18

11

42

5

16

20 1

ABO-HDN

No HDN

73

32

HDN Present 12 0

HDN Absent 8 29

64

HDN Present 37 0

HDN Absent 2 5

128

HDN Present 22 0

HDN Absent 0 0

256

HDN Present 3 0

HDN Absent 0 0

It was observed that, ABO-HDN was not present at lower maternal titers. With the

gradual increase in maternal IgG titers, there was a gradual increase in the incidence of

ABO-HDN also, with a maximum of 37 cases at a titer of 64. At titers > 64, all the

cases developed ABO-HDN.

Correlation between presence IgG subclasses with occurrence of ABO-HDN at

various titers:

Correlation between the presence of IgG1/IgG3 subclasses with occurrence of ABO-

HDN were analyzed at various titers ranging from ≥ 16 to ≤ 64. Titers < 16 and > 64

were excluded for this comparison, as there were no cases of HDN reported at titer <

16 and at a titer of > 64, all the cases had HDN.

74

Table 20: Correlation between presence IgG subclasses with occurrence of ABO-

HDN at titer 16 (n=33)

ABO-HDN

IgG1 and / or IgG3

present

IgG1 and / or IgG3

absent

p value

HDN present

2 0 0.040

HDN absent

3 28

At a titer of 16, presence of IgG1/IgG3 subclasses is significantly associated with the

occurrence of ABO-HDN.



ABO-HDN

IgG1 and / or IgG3

present

IgG1 and / or IgG3

absent

p value

HDN present

12 0 < 0.001

HDN absent

8 29





ABO-HDN

IgG1 and / or IgG3

present

IgG1 and / or IgG3

absent

p value

HDN present

37 0 < 0.001

HDN absent

2 5

75


occurrence of ABO-HDN

VIII. LABORATORY PARAMETERS IN THE NEWBORN:

Serum haemoglobin, bilirubin levels and reticulocyte count were available in all the 76

cases of ABO-HDN.

VIII. 1. Anaemia:

Anemia (Hb <14.6 gm %) was documented in 12 (15.78%) out of 76 cases of ABO-

HDN.

Figure 20: Prevalence of anaemia in ABO-HDN (n=76).

VIII. 2 Hyperbilirubinemia

Neonatal hyperbilirubinemia was detected in all the cases of ABO-HDN. Majority of

neonates developed jaundice on second day (61%) followed by first day (18%), third

15.78%

84.22%

Anaemia

Prevalence of anaemia in newborns

76

day (16%) and fourth day (5%). The mean initial indirect bilirubin was 16.24 ± 4.04

mg% ranging from 7.46 mg% to 27.51 mg%.

VIII. 3. Reticulocytosis:

Reticulocyte count was documented in all cases of ABO-HDN. Reticulocytosis

(reticulocyte count >4.6%) was observed in 42(55.26%) out of 76 cases of ABO-HDN.

Figure 21: Pie chart depicting prevalence of reticulocytosis in ABO-HDN.

42(55.26%)

34(44.74%)

Reticulocytosis

Reticulocyte count

77

Table 23: Comparison of parameters of newborns in ABO-HDN and without

ABO-HDN

Parameters ABO-HDN No HDN

Male/ Female 42/34 68/59

Birth weight(kg) 3.06 ± 0.60 2.99 ± 0.50

A blood group 33 48

B blood group 43 79

Haemoglobin (gm %)

Total bilirubin (mg %)

Retic count (%)

Polychromasia and

microspherocytes

16.68 ± 1.8

16.24 ± 4.04

5.63 ± 3.05

37%

15.38 ± 3.32

13.30 ± 3.30

3.74 ± 1.68

4%

DCT negativ

78

Table 24: Comparison of parameters of newborns in OA-HDN and OB-HDN.

Characteristic Total OA HDN (n=33) OB HDN (n=43)

Gender:

Male 42 20 22

Female 34 13 21

Birth weight:

< 2.5 kg 8 5 3

≥ 2.5 kg 68 28 40

Mode of delivery:

Normal vaginal 38 17 21

Instrumental 20 5 15

LSCS 18 11 7

Parity:

Primigravida 39 15 24

Multigravida 37 18 19

Titer level

< 64 14 5 9

≥ 64 62 28 34

79

IX. TREATMENT DETAILS IN ABO-HDN:

Treatment details were available for all 76 cases of ABO-HDN. Single Light

phototherapy (SLPT) was initiated in 93.4% cases. Double Surface Phototherapy was

administered in 15.7% cases of which 5 of the cases were given both SLPT and DSPT.

Exchange Transfusion was performed in one case.

Table 25: Mode and average duration of treatment in ABO-HDN

Treatment modality Number of cases Average duration

Single phototherapy 64 (84.21%) 40.50 hrs

Double phototherapy 12 (15.78%) 8.84 hrs

ExchangeTransfusion 1 (1.31%) Once

X. GRADING OF ABO-HDN:

We used the grading system proposed by Choudhuri et al classification of Rh-HDN to

grade the severity of cases in our study, which was as follows:

1. Mild: Positive DAT alongside some evidence of haemolysis but requiring no

intervention.

2. Moderate: Cases with evidence of haemolysis and intervention was limited to

phototherapy

3. Severe: When the baby was hydropic or intrauterine/ exchange or top up transfusion

was required.

80

Using the above guidelines of grading ABO-HDN, there were 75 cases of mild/

moderate ABO-HDN and one case of severe ABO-HDN in our study.

XI. FAMILIAL INCIDENCE OF ABO-HDN:

Among the 203 cases of ABO incompatible cases, 33 (16.2%) had a prior family

history of the same. 9 (11.84%) out of 76 ABO-HDN cases and 5 (3.9%) out of 127

non HDN cases had history of ABO-HDN in their siblings.

Table 26: Correlation between family history and occurrence of ABO-HDN

ABO-HDN

Family history of ABO- HDN

p value Present Absent

Present 9 68 0.043

Absent 5 122

Family history of ABO-HDN was significantly associated with occurrence of ABO-

HDN. Newborns with siblings who had history of ABO-HDN were at higher risk of

developing ABO-HDN when compared to newborns with a negative family history.

81

DISCUSSION

82

DISCUSSION

ABO haemolytic disease of newborn (ABO-HDN) is emerging as the most common

cause of haemolytic disease of newborn in the developed countries. There have been

constant attempts to develop tests which can predict the onset of HDN in Rh-HDN,

and also to assess its severity, in order to put therapeutic interventions in place on

time. But there is a dearth of literature on the prevalence and factors that impact ABO-

HDN from overseas and India. Against the background of extremely variable health

care facilities in our country, we thought it is critical to study factors that may predict

the severity of ABO-HDN in mothers with O blood group. At present, there are no

satisfactory methods to predict the occurrence of ABO-HDN.

Early blood grouping and DAT in newborns with jaundice are a well recognized risk

appraisal in ABO incompatible newborns. Our study aimed to find the prevalence of

ABO incompatible pregnancies in O blood group mothers and investigated the role of

maternal IgG anti-A and anti-B titers and IgG1/IgG3 subclasses as a powerful

predictor of occurrence of ABO-HDN. Various maternal and fetal parameters and their

association with ABO-HDN were also analyzed.

The study included 575 O blood group mothers who were recruited during the study

period July 2018 to September 2019.

83

Geographical distribution:

According to previous studies, race was a risk factor in ABO-HDN and the incidence

of ABO HDN was higher in black population than caucasians. This could be attributed

to the higher prevalence of anti-A and anti-B titers in the black population(103).

Our cohort of patients included more than 80% mothers from Tamil Nadu, around

15% from Andhra Pradesh and rest from Karnataka, West Bengal and Kerala.

Age of mother:

The median age of the study population of mothers was 28 years and ranged from 19

years to 37 years. There were no studies available till date which proved the effect of

maternal age on occurrence of ABO-HDN.

Prevalence of ABO incompatible pregnancies:

Among the total 575 mothers, 203 mothers delivered babies of either A or B blood

group (non-O group) and the prevalence of ABO incompatibility was thus estimated to

be 35.3% i.e, one third of the pregnancies in O blood group mothers were found to be

ABO incompatible. None of the babies had AB blood group. All previous literature

has reported a varying range of prevalence of ABO incompatibility, which differed

from geographical area and population. The prevalence in our study was similar to that

observed in the studies done by Christensen et al in which a prevalence 32.4 % was

reported in a cohort of 42,529 neonates (104) and 40.8% as reported by Shilpa et al in

a cohort of 250 O blood group mothers (105). But this was higher than the prevalence

of 28 % as reported in the studies by Cernadas et al(106) , 17.3 % by Bhat and Kumar

(21)and 14.8% by Sarici et al(107).

84

ABO-Haemolytic Disease of Newborn:

ABO-HDN is defined in the literature as a condition which occurs in newborns with

blood group A, B or AB having positive DAT and laboratory evidence of haemolysis,

born to a mother with blood group O (22). Evidence of haemolysis included

reticulocytosis and spherocytes in the peripheral smear (108). In our study, 76 out of

the 203 newborns with ABO incompatibility developed ABO haemolytic disease as

per these criteria. Therefore, the prevalence of ABO-HDN was estimated to 37.4%.

That is one out of 3 newborns were affected by ABO-HDN. This was similar to the

observations of Cariani et al (29)who reported 31% prevalence of ABO HDN in 68

ABO incompatible pregnancies and Heydarian et al (Iran), who reported 27.6%

prevalence of ABO-HDN in ABO incompatible pregnancies(109). There are only a

few studies from India which has looked into the prevalence of hyperbilirubinemia and

haemolysis in ABO incompatible newborns. As per the study by Bhat and Kumar, in

which they studied 151 cases of ABO incompatible pregnancies, 30.4% had significant

hyperbilirubinemia and 16.5% had laboratory evidence of hemolysis which was much

lower prevalence when compared to our study. Studies by Bekkeheim et al and Alani

et al concluded that the prevalence of ABO-HDN was 1-4% and 4.3% respectively in

ABO incompatible pregnancies. The prevalence of ABO-HDN as estimated in our

study was much higher than that observed in studies from India and overseas. This

could be attributed to the lesser awareness of ABO incompatibility compared to Rh

incompatibility, ineffective neonatal screening and absence of well developed hospital

protocols in the management of ABO-HDN worldwide.

85

Parity of mother

The number of primiparous and multiparous mothers were almost equal in the total

study population as well as in cases of ABO-HDN. As per our study, there was no

association between occurrence of ABO-HDN and parity of mother (p value= 0.88).

This was similar to the observation made in studies by Kattimani et al, Shah A et al

and Kalakheti et al, that parity had no effect on ABO-HDN(81,110,111). Our study

also concluded that ABO-HDN can occur in first pregnancy unlike in Rh-HDN and

can recur in about 87% cases as maternal ABO antibodies are present without previous

sensitization.

Mode of delivery:

The mode of delivery in all 203 cases were analyzed. About half of the mothers

delivered by normal vaginal delivery (46.7%) and the rest by LSCS (27 %) and

instrumental delivery (26.5%). In our study, occurrence of ABO haemolytic disease

was independent of the mode of delivery. This was similar to the observations made in

the study by Kattimani VS et al(81).

Gender of newborn:

Among the total 203 babies delivered, the number of male babies (54.1%) were

slightly higher than females(45.9%). But, there was a uniform sex wise distribution in

the HDN group (48.7% males and 51.3% females). In our study there was no

significant association between the gender of newborn and occurrence of ABO-HDN

86

(p value=0.56). This observation was comparable with that of the studies by Kattimani

et al, Kumar A et al, Akgul Set al(81,112,113). A few studies in the past postulated

that male newborns had an increased risk of developing jaundice(114,115).

Birth weight of newborn:

Low birth weight is defined as < 2.5 kg at birth in India. In our study, 23 (11.3%) out

of 203 babies were found to be of low birth weight. Among the 76 cases with ABO

HDN, 10.5 % were of low birth weight. There was no correlation with birth weight

and occurrence of ABO-HDN (p value=0.82).

OA and OB HDN:

In our study, though OB incompatibility was found to be more common (56.6%) than

OA incompatibility, there was no significant difference in occurrence of ABO-HDN in

both groups (p value=0.42). This was similar to the observations made in study by

Bhat and Kumar et al(21). As per the western literature, there is conflicting evidence

regarding occurrence of ABO-HDN and blood group of newborn. In a few studies, it

was observed that greater hemolytic activity was seen associated with anti-B

antibodies with an increase in trend of transfusion in first 24 hours of life in O-B

incompatibility as compared to O- A incompatibility(55–57) while a few studies

established that there was no relationship between the clinical course and infant’s

blood group (58).

Family history:

87

In our study, presence of a sibling with ABO haemolytic disease was found to be

significantly associated with occurrence of ABO-HDN in current pregnancy. Similar

trend was also observed in studies by Heydarian et al and Sarici et al (109,107).

Laboratory parameters:

Anaemia:

Anaemia is defined as Hb levels below 14.6 g/dl in term neonates(116). Clinical

assessment of newborns was done and in suspected cases of haemolysis, haemoglobin,

bilirubin levels and reticulocyte counts were estimated. Haemoglobin levels were

available for 170 out of the total 203 newborns, of which 50 (29.4%) newborns had

anemia. In the 76 cases of ABO-HDN, anaemia was documented in 13 cases (17.1%).

The lowest level of haemoglobin documented in our study was 10 gm/dl.

Hyperbilirubinemia:

Neonatal hyperbilirubinemia is defined as any PTB values above ≥ 95th percentile on

the hour of life specific normogram. In our study it was estimated by serum bilirubin

levels or by measuring transcutaneous serum bilirubin (TSB). Neonatal

hyperbilirubinemia was detected in all the cases of ABO-HDN. Majority of neonates

developed jaundice after 24 hours (61% cases) followed by those within 24 hours of

birth (18% cases). The mean initial indirect bilirubin was 16.24 ± 4.04 mg%.

Reticulocytosis:

88

Reticulocytosis in newborn is defined as reticulocyte count >4.6%. Reticulocyte count

was available for 106 newborns and a high reticulocyte count was found in 51(48.1%)

cases. Reticulocyte count was documented in all cases of ABO-HDN and was

observed in 42(55%) out of 76 cases of ABO-HDN.

Treatment of ABO-HDN:

Treatment details were available for all cases of ABO incompatibility. Single Light

Phototherapy (SLPT) was the most common treatment modality in about 65% cases,

followed by Double Surface Phototherapy (DSPT) in about 6.5% cases. In cases with

ABO-HDN, Single Light phototherapy (SLPT) was initiated in 93.4% cases. Double

Surface Phototherapy was administered in 15.7% cases of which 5 of the cases were

given both SLPT and DSPT. Exchange Transfusion was performed in one case, in

which the blood group of the newborn was group B. The initial haemoglobin level was

12.8 g%, indirect serum bilirubin was as high as 26.91 mg% and a reticulocyte count

was 10.65%. Both IgG1 and IgG3 were detected to be in high titers and maternal IgG

anti-B titer was recorded to be 256. Following exchange transfusion, the newborn’s

clinical and laboratory conditions improved significantly.

Maternal IgG anti-A and anti-B titers:

The maternal IgG anti-A and anti-B titers have been studied by many groups

worldwide as they tend to impact the occurrence of ABO-HDN. It was concluded in

our study that titer level > 64 was significantly associated with occurrence of ABO-

89

HDN (p value <0.001). The odds of developing HDN at a titer of 64-128 is 45.31(95%

CI 17.01-0.63) times as compared to cases with titers < 64. This was independent of

the presence of IgG subclasses. This range of titer levels were also in concordance

with the studies conducted by Owa J et al(48) on 50 ABO incompatible pregnancies

in Nigeria and Yi-qiang et al (46)on 625 ABO incompatible pregnancies in China.

According to the study conducted by Usha and Sulochana in India, an IgG titer levels

between 32 and 1024 were found to be critical(26).

Other studies by Li et al in China on 5246 mothers (51) and by Kadri et al (52) in

Indonesia on 268 mothers observed significantly high titers than our study. Li et al, in

their study concluded that antibody titers ≥ 512 was significantly associated with

neonatal jaundice. Kadri el al suggested that titer ≥ 256 had a significant risk

associated with neonatal hyperbilirubinemia. Our study differed from a few studies in

the past which opined that there was no correlation between maternal IgG titers and

occurrence of HDN(29,108).

Maternal IgG titers tend to rise rapidly in the third trimester. Therefore, antenatal

monitoring of maternal IgG anti-A and anti-B titers can significantly predict the

occurrence of ABO-HDN(52), though it was also dependent on factors like placenta

density, red cell surface antigen and IgG antibody subclass(117).

IgG subclasses:

The predictive diagnosis of ABO-HDN was further improved by studying the IgG

subclasses. In our study, it was IgG1 and IgG3 which were looked at to prognosticate

ABO-HDN, in view of all previous literature, which stated that these were the

90

predominant subclasses involved in the causation of Haemolytic Disease of Newborn.

In contrast, IgG2 was the least efficient among all the IgG subclasses in crossing the

placenta, and also the contribution of IgG2 and IgG4 in HDN is limited due to absence

of receptors for these on the monocytes. The effect of IgG subclasses on Rh-HDN is

quite clear but their role in ABO-HDN is uncertain. There was no major study from

India which explored the prognostic value of IgG subclasses on ABO-HDN. We

initiated our study in this background.

IgG subclass typing was performed using a DAT IgG1/IgG3 ID-Card from DiaMed

GmbH, using CAT in total 203 samples. This test contains dilutions of IgG1 and IgG3

in 1:1 and 1:100 strengths alongside a positive and negative control. The strength of

each of the subclass IgG1 and IgG3 could therefore be interpreted according to

positivity recorded on the relevant micro tubes representing strengths of either 1:1(low

titre) or 1:100(high titre). This is a relatively easy test and can be used for rapid

identification of IgG1 and IgG3 subclasses.

We found that the overall prevalence of IgG1/IgG3 subclasses in the study group to be

46.7%. IgG1/IgG3 subclasses were detected in all the cases with ABO-HDN. In our

study, IgG3 subclass was never detected in isolation, but always in association with

high titer of IgG1. The most frequently observed subclass was a combination of high

titer IgG1and low titer IgG3 in 42 cases (20.6%). In our study, the presence of IgG1

and IgG3 subclasses was significantly associated with the occurrence of ABO-HDN

and was independent of the maternal IgG anti-A and anti-B titer levels. It was

91

interesting to compare our results to those published by Zhou Yi-qiang et al in their

study from China. They found that maternal titer more than 64 and maternal

concentration of IgG1 and IgG3 subclasses, especially IgG1 subclass were

significantly associated with ABO-HDN.

This result was very similar to that observed in our study. Our study differed from the

findings by Kaplan et al in a study conducted in California in umbilical cord blood

samples which postulated that identification of IgG subclass in umbilical cord blood

was not predictive of hemolysis in ABO-incompatible neonates(47).

We also studied the correlation between the presence of IgG1/IgG3 subclasses at

various maternal IgG anti-A/ anti-B titers. It was concluded that at titer of 16, presence

of IgG1 and/ or IgG3 subclasses were significantly associated with occurrence of

ABO-HDN ( p value= 0.040). Titers higher than or equal to 32 along with presence of

IgG1 and/ or IgG3 subclasses had a highly significant association with occurrence of

ABO-HDN (p value <0.01).

Our results helped us identify, that 35.3% in O blood group mothers can be ABO

incompatible and among them, almost 37.4% of ABO can develop ABO-Haemolytic

disease of newborn. Occurrence of ABO-HDN was independent of the factors like

parity of mother, birth weight and blood group of newborn.

We could conclude that presence of IgG1 and /or IgG3 subclasses in maternal plasma

was significantly associated with the occurrence of ABO-HDN, and this was

independent of the maternal IgG anti-A and anti-B titer levels. We also inferred that

the odds of developing HDN progressively increased when the maternal IgG anti-A/B

92

titer ≥64. Presence of either of these subclasses at maternal IgG titers ≥16 had highly

significant association with occurrence of ABO-HDN.

The spectrum of heamolytic disease of newborn has changed over the last few

decades. The incidence of Rh D alloimmunization in pregnancy has decreased

drastically and now ABO HDN has emerged as the major cause of HDN in the

developed countries. Early blood grouping and DAT testing in newborns are already

recognized as risk appraisals in ABO-HDN, but our study clearly indicates that high

maternal IgG anti-A/B titers and presence of IgG1/G3 subclasses are consistent with

an increased risk of developing ABO-HDN. Therefore, it will be prudent to use these

parameters in the prediction of occurrence of ABO-HDN and take active preventive

and treatment measures which is critical in preventing the long-term sequelae. Against

the backdrop of a country such as ours, where neonatal assessment and care may be

limited in large areas, it may be prudent to utilize these tools to guide mothers at high

risk to centers where appropriate timely management can be initiated. This high-risk

approach should be adopted if there is a history of ABO-HDN in previous pregnancies

or if there is ABO incompatibility between the partners. Early identification, diagnosis

and intervention can reduce morbidity and mortality due to HDN due to ABO

Incompatibility.

93

CONCLUSIONS

94

CONCLUSIONS

1. The prevalence of ABO incompatible pregnancies in O blood group mothers was

35.30% and among them 37.4% cases developed ABO-HDN.

2. The overall prevalence of IgG1 and / or IgG3 subclasses in O blood group mothers

was 46.79%.

3. IgG1/ IgG3 subclasses was present in all mothers whose newborns developed ABO-

HDN.

4. There was an independent and strong association between occurrence of ABO-HDN

and presence of IgG1/IgG3 subclasses (p< 0.001).

5. There was an independent and strong association between occurrence of ABO-HDN

and IgG anti-A/B titers ≥ 64 when comparing mothers with titers < 64 (p< 0.001).

6. Presence of IgG1/IgG3 subclasses at titers ≥16 , was significantly associated with


7. IgG1/IgG3 subclass typing using the CAT is a simple test, which if implemented

can contribute significantly to clinical decision making algorithms and appropriate

timely interventions in the context of ABO-HDN.

95

LIMITATIONS

96

LIMITATIONS

1. Most of our patients (81.6%) are from Tamil Nadu, and hence it may not be able to

extrapolate our findings regarding IgG sub class as representative of the South Indian

population, as we had hoped it to be.

2. Laboratory investigations were not available for a few of the newborns in no ABO –

HDN group. Therefore, it was difficult to assess the degree of haemolysis, if present in

them

IgG1 and IgG3 subclass typing

Figure 22: IgG1 low titer (1:1) positive n=34

Figure 23: IgG1 high titer (1:100) positive n=13

Figure 24: IgG1 high titer and IgG3 low titer positive n=6

Figure 25: Both IgG1 and IgG3 high titer positive n=42

Figure 26: Both IgG1 and IgG3 negative n=127

97

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ANNEXURES

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STANDARD OPERATING PROCEDURE:

A. IgG1 and IgG3 subtyping:

The risk of hemolysis in HDN depends upon the amount of IgG coated on the red cells

as well as the class of IgG involved. To induce phagocytosis, it is suggested that

approximately 1000-4000 IgG1 molecules or 135-500 IgG3 molecules must coat the

red cells. Therefore, it is important to identify the presence of these subclasses.

Principle: The ID-card “DAT IgG1/IgG3” has two dilutions (1:1 and 1:100) of both

the subclasses and works on the principle of CAT.

Materials: ID card with 6 columns as in fig (having four micro tubes for IgG1 and

IgG3 in two dilutions each, a negative control well, a positive control well), ID-Dia

cell (0.8%) suspension, pipettes, tips, incubator and centrifuge.

Procedure:

1. Previously stored serum or plasma at -70 degree C is thawed to room

temperature and centrifuged at 1500 g for 10 minutes to avoid fibrin residues

which can interfere with reaction pattern

2. Label the ID card with appropriate patient hospital number.

3. Remove the aluminium foil, holding the card in upright position.

4. Pipette 50 μl of ID-Dia cell into each microtube.

5. Add 25 μl of patient’s plasma to each microtube

6. Incubate the card for 15 minutes at 37 degrees C in the incubator.

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7. Centrifuge the card at 910 rpm for ten minutes.

8. Interpret and record the agglutination reaction

If the positive or negative controls failed, the test was considered un-interpretable.

Interpretation:

A. Positive: Agglutinated cells form a red line on the surface of the gel (strong

reaction) or the agglutinates tend to disperse in gel in a weaker reaction.

B. Negative: Compact button of cells at the bottom of microtube.

A positive reaction with 1:1 dilution has a sensitivity of approximately 1000 Ig1

molecules and 125 anti- Ig3 molecules respectively. A positive reaction in

1:100 dilution is indicative of a higher strength of antibodies in the plasma.

Figure: The DAT IgG1/IgG3 card

DAT IgG1/IgG3 card consists of six micro tubes as depicted in the figure.

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The first two tubes are for the detection of IgG1 in titres of 1:1 and 1:100 respectively.

The next two wells are coated for IgG3 in 1:1 and 1:100 titers. The fifth tube acts as

negative control for the test, and should be negative for the test to be interpretable. The

last well is for overall IgG (1:10) which acts as the positive control for the test.

Fig : Test results on the card

Fig : Test results on the card

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Table 2: Result interpretation on the DAT IgG1/IgG3 card

IgG1

IgG3 Control IgG

1:1 1:100 1:1 1:100 1:10

Reaction 2+ - - - - 3+

Interpretation Moderate risk of hemolysis (Subclass IgG1; low concentration)

Reaction 3+ 2+ - - - 3+

Interpretation High risk of hemolysis ( Subclass IgG1; high concentration)

Reaction 3+ 2+ 2+ - - 3+

Interpretation High risk of hemolysis ( IgG1 high concentration; low concentration

IgG3)

Reaction - - 3+ 2+ - 3+

Interpretation High risk of hemolysis (Subclass IgG3; high concentration)

Reaction - - 2+ - - 3+

Interpretation Moderate risk of hemolysis (Subclass IgG3; low concentration)

Reaction 2+ - 3+ 2+ - 3+

Interpretation High risk of hemolysis (IgG1, low concentration, IgG3 in high

concentration)

Reaction 2+ 2+ - - - 3+

Interpretation Moderate risk of hemolysis (Subclasses IgG1 and IgG3, low

concentration)

Reaction 3+ 2+ 3+ 3+ 2+ - 3+

Interpretation High risk of hemolysis (Subclasses IgG1 and IgG3, high

concentration)

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Note: The first two micro tubes contain Anti-IgG1 (cell line M345/795) in two

dilutions and third and fourth micro tubes contains Anti-IgG3 (cell line M346/805) in

two dilutions

respectively.

Reference: DAT IgG1/IgG3 ID-card, (Diamed GmbH 1785 Cressier FR Switzerland)

BIO-RAD manual

STANDARD OPERATING PROCEDURE FOR ALL OTHER TESTS:

A. ANTIBODY SCREENING:

Antibody screening is routinely done in blood bank for all donor and patient’s samples

to detect irregular antibodies. It is based on the indirect antiglobulin test. This test uses

patient’s plasma/ serum against red cells to detect unexpected antibodies.

Screen cells used for this test are commercially prepared 2-5% suspension of group O

cells obtained from individual donors and phenotyped for the most commonly

encountered and clinically important red blood cell antigens, which is in contrast to

IAT which utilizes inhouse preparation of O pooled cells from three phenotyped

donors. Screen cells require the expression of the antigens as per the BCSH guidelines

for antibody screening in antenatal women: D,C,E, c,e,M,N,S,s,Lea ,K,k,Jka, JKb,

Fya, Fy b. Group O red cells are preferred since it does not contain A and B antigens

on the red cell membrane, minimizing the chances of reacting anti-A and anti-B

antibodies, and making it insensitive to the “regular or expected antibodies”

Material required:

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1. Coombs cassette

2. Biorad system centrifuge

3. Micropipette and tips

4. Low ionic strength solution(LISS)

5. Dia Cel –I-II-III Asia cells(Three panel reagent cells)

6. Incubator

Procedure:

Antibody screening is done using commercial 3 cell panel DiaCel –I-II-III Asia.

Antibody screening is done using a Coombs cassette

1. Label the coombs cassette with patient/donor ID

2. Label three columns D1, D2 and D3

3. Pipette 50 μl of reagent cells into columns labelled as D1, D2 and D3.

4. Pipette 25 μl of patient’s plasma into the 3 wells.

5. Incubate for 15 minutes at 37 degrees.

6. Centrifuge the card in Diamed card centrifuge for 10 minutes at 910 rpm.

7. Read the cassette from both sides.

8. Read and record the results.

Interpretation of column agglutination testing:

Negative - All cells settle at the bottom

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1+ reaction -A button of cells at the bottom with most aggregates remaining at the

lower half of the beaded column.

2+ reaction- A small button of cells may also be visible at the bottom while

aggregates are observed throughout the length of the beaded column.

3+ reaction -Most aggregates remain in the upper half of beaded column.

+/- reaction – Indeterminate

Weak reaction-Granular suspension

MF reaction-Mixed field agglutination/double population

Reporting of results:

Antibody screen-positive or negative–mentioning which screen cells are positive and

strength of positivity. Entry of results to be made on the patient card filed in blood

bank.

Quality control:

To check the proficiency of screen cells and the coombs card, antibody screening is

done with diluted anti –D IgG (1 in 10 dilution) as positive control and LISS as

negative control.

COOMBS TEST (DIRECT ANTIGLOBULIN TEST)

Aim: To detect the presence of incomplete antibodies and complement binding

antibodies in serum /plasma or those coated on patients red cells.

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Principle: Washed red cells which are coated with immune antibodies (commonly

IgG) and complement components (generally C3) will show agglutination with broad

spectrum AHG reagent. The coating (sensitization) of red cells can occur in vivo or in

vitro following incubation at 37 deg C with serum containing antibody.

Methodology:

Coombs test can be done by the following techniques:

A) Tube technique

B) CAT

Sample: Properly labeled 4 ml EDTA samples/ 6 ml clotted serum samples are

required. Hemolysed samples are not acceptable.

Materials required for Tube method for DAT:

Test tube 10x75 mm

Test tube rack

Marker

Centrifuge

Pasteur pipette

Slide

Normal saline

Microscope

Water bath at 37 degree C

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Reagents for DAT:

1. Coombs serum

2. Negative control for DAT- 3-5% suspension of saline washed pooled O group cells.

3. Positive control for DAT- Sensitized O pooled cells – (made by adding 0.5 ml of

IAT positive control to 1 ml of washed and packed O pooled cells and incubating at 37

degree for half an hour )

Procedure for DAT:

Tube technique:

1. Take 3 tubes and label as patient ID, positive control and negative control.

2. Add one drop of patient cells, positive control, negative control to the respective

tubes.

3. Wash 3 times with normal saline.

4. After a final wash, decant the supernatant completely.

5. Add one drop of coombs serum to all the 3 tubes, mix and centrifuge at 3000 rpm

for 15 seconds.

6. The dissolve the button gently and read both macroscopically and microscopically

and grade the reactions.

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GRADING OF THE REACTIONS FOR ALL TESTS DONE BY TUBE

TECHNIQUE: (Reference AABB Manual)

No agglutination Negative

Small aggregation 1+ Positive

Medium size aggregation 2+ Positive

Several large aggregates 3+ Positive

One solid aggregate 4+ Positive

The presence of haemolysis in the supernatant also indicates a positive reaction.

Column Agglutination Technique:

Material required: Coombs card

ID centrifuge

Normal saline

Micropipette and tips

Biovue system work rack/ID holder

LISS

Heat block(Incubator)/ID card Incubator

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Procedure for DAT using Bio-Rad card

1. Ensure that the cassette is at room temperature before use.

2. Label the cassette appropriately with patient ID and control ID.

3. Add 50μl of 0.8% suspension of patient, positive and negative control cells

respectively to all the 3 wells.

4. Centrifuge the card in ID card centrifuge for 10 minutes at 910 rpm.

5. Read and record the reactions (read the card from both sides)

Grading of results by CAT as mentioned previously.

Precautions:

1. Adequate washing of sensitized cells is the critical part of the test. A trace

amount of residual globulin, due to improper washing causes neutralization of

the AHG serum

2. A false negative reaction can occur if testing is interrupted or delayed. Washing

of sensitized cells, addition of AHG serum and centrifugation must be quick

and uninterrupted to avoid elution of antibodies attached to red cells and

neutralization of AHG serum.

3. Inappropriate serum to cell ratio.

4. Failure to add AHG reagent may give false negative results.

5. Under centrifugation may not provide optimal conditions for agglutination.

6. Failure to check the negative reaction microscopically.

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7. Over centrifugation packs red cells tightly that they cannot be dispersed

completely.

8. Red cells may be agglutinated before washing and this may persist through the

washing phase i.e. adsorption of complement during storage. In such cases, a

positive antiglobulin test may be incorrectly interpreted as a result of the IgG or

complement coating.

Documentation and Result entry: All reactions to be entered in the grouping

worksheet. All results to be entered with strength of reactivity on patient grouping

card.

ANTIBODY TITRATION (Coombs with DTT):

Principle: Titration is a semi quantitative technique of measuring the concentration of

an antibody in a serum. The titer of an antibody is usually determined by testing the

serial two fold dilution of the serum against selected red cells. Dithiothreitol (DTT) is

used to abolish IgM antibody activity to permit detection of coexisting IgG antibodies.

Specimen required: Properly labeled 4 ml EDTA sample/ 6ml clotted serum sample

is required. Haemolysed samples are not accepted.

Materials:

1. 100 μl automatic pipette and pipette tips

2. Test red blood cells 3-5%

3. Sterile 12x75 ml test tubes

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4. Sterile saline 0.9%

5. Serofuge

6. Glass slides

7. Water bath

8. Microscope

Reagents Required:

0.01M Dithiothreitol (DTT)

Phosphate buffered saline (PBS) at pH 7.3

Procedure:

DTT treated plasma/serum is used for coombs antibody titration

To prepare DTT treated serum:

1. Dispense 200μl of serum or plasma into each of two test tubes

2. To one tube (labeled dilution control), add 200μl of PBS

3. To the other tube (labeled test), add 200μl of 0.01M DTT

4. Mix and incubate at 37degC for 30 to 60 minutes

5. One set label as Serum+DTT and another set as Serum+PBS

Coombs (AHG) Titre:

1. Label the tubes according to the serial dilution using 1:2 through 1:1024

2. Deliver one volume of saline(100 μl) of saline into all tubes

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3. Add one volume (100 μl) of the DTT treated serum/plasma to tube 1:2

4. Mix the contents of the tube several times and transfer one volume (100 μl) of

the mixture from 1:2 to 1:4 and continue through the remaining tubes.

5. Discard 100 μl from the tenth tube i.e. 1:1024

6. Add on volume of (100 μl) of 3-5% saline cell suspension of appropriate red

cells to each tube.

7. Mix well and incubate the serum mixture at 37 deg C for one hour.

8. After one hour, wash three times with normal saline.

9. After the third wash, decant the supernatant and add AHG serum

10. Spin at 3000rpm for 15 seconds

11. Gently dislodge the button. Examine the test result macroscopically and grade

the reaction

12. If there is agglutination in the tube containing the most diluted serum, an end

point has not been reached. Prepare and test additional dilutions.

Grading of the Reaction: As for all test done by tube technique.

Interpretation:

1. The titre is the reciprocal of the highest serum dilution at which macroscopic

agglutination is observed.

2. Reactivity in the dilution control serum and no reactivity in the DTT treated

indicate an IgM antibody.

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3. Reactivity in the dilution control serum and in the DTT treated serum indicates

an IgG and IgM mixture.

4. No reactivity in the dilution control serum indicates dilution of weak antibody

reactivity and an invalid test.

Quality Control for DTT treated serum:

A serum or plasma sample known to contain an IgM antibody should be treated in

parallel.

Test Sample 1:2 1:4 1:8 1:16 1:32 Interpretation

Serum+DTT 3+ 2+ 2+ 1+ 0 IgG

Serum+PBS 3+ 2+ 2+ 1+ 0

Serum+DTT 0 0 0 0 0 IgM

Serum+PBS 3+ 2+ 2+ 1+ 0

Serum+DTT 2+ 1+ 0 0 0 IgG + IgM

Serum+PBS 3+ 2+ 2+ 1+ 0

Prozone reaction: In a serum or plasma with a very high antibody concentration, the

prozone phenomenon may cause a weaker reaction in the lower dilution than in higher

dilutions.

Results: The titre of the antibody should be reported as- Coombs titre done with DTT

treated serum

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BLOOD GROUPING BY COLUMN AGGLUTINATION TECHNIQUE:

Using the ABD card:

1. Label the ABD card with patient/ donor ID.

2. Insert the liner

3. Pipette 10 μl of 3% cell suspension of patient/ donor cells to the wells labelled

Anti-A, Anti-B and Anti-D (3% saline suspension)

4. Centrifuge the card in Bio-Vue centrifuge for 5 minutes which will spin at 800

rpm for the first 2 minutes followed by 1500 rpm for the next 3 minutes

(biphasic centrifugation)

5. Read and record the reactions

Using the ABD reverse card:

1. Label the ABD reverse column agglutination card with patient/ donor ID.

2. Insert the liner

3. Pipette 10 μl of 3% cell suspension of patient/ donor cells to the wells labelled

Anti-A, Anti-B and Anti-D (3% saline suspension)

4. To the wells labelled as O,A and B cells add 40 μl of patient/ donor serum

5. Pipette 10 μl of reverse typing A,B and O cells into the respective columns

6. Centrifuge the card in Bio-Vue centrifuge for 5 minutes which has a

programme 800 rpm for the first 2 minutes followed by 1500 rpm for the next 3

minutes (biphasic centrifugation)


Blood grouping by Column Agglutination Technique

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Using the ABD card: Two types of ABD cards are used-Donor grouping is done

using DVI+ cards and patient grouping is done using DVI- cards

1. Label the ABD card with patient ID/ donor ID

2. Pipette 12.5 μl of 5% cell suspension of patient/ donor cells to the wells labelled

Anti-A, Anti-B and Anti-D (5% cell suspension)

3. Centrifuge the card in Diamed centrifuge for 10 minutes at 910 rpm


Interpretation:

Group FRONT TYPING BACK TYPING

Anti-A Anti-B Anti AB A cell B cell O cell

A + - + - + -

B - + + + - -

AB + + + - - -

O - - - + + -

Bombay - - - + + +

Quality control:

1. Before starting the technical procedure everyday, every shift, known AB

positive and O negative group samples have to be processed to check the

working proficiency of both reverse typing cells and the card

2. If QC fails, a new batch of cards need to be obtained and the tests repeated

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3. If it clears, then the failed batch of cards to be discarded, reported to the

supplier

4. Every new group is performed by 2 different technologists on 2 different

platforms

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Proforma

To study the prevalence of ABO incompatibility in O blood group mothers and assessment of IgG

subclasses (IgG1 and IgG3) in them and its correlation with occurrence and severity of ABO

Hemolytic disease of the newborn (ABO HDN) in a tertiary health care centre in south India.

Name of mother Study number

Hospital number Telephone number

Age/ DOB Address

Husband’s name

Mother’s information

Blood group and Rh typing

IgG anti A and anti B titer

Period of gestation

IgG subclass:

IgG1 1:1 1:100 IgG3 1:1 1:100

Obstetric formula

Antibody screen

Newborn’s information

Hospital number Hemoglobin Live/still born/IUD Total Bilirubin Pre/Term/Post Direct Bilirubin Normal/Instrumental/LSCS Retic count Gender Peripheral smear Birth weight Treatment required Yes/ No

Blood grouping and Rh typing

Single phototherapy

DCT Double phototherapy Exchange transfusion

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Informed Consent form to participate in a research study

Study Title:

To study the prevalence of ABO incompatibility in O blood group mothers and assessment of

IgG subclasses (IgG1 and IgG3) in them and its correlation with occurrence and severity of

ABO Hemolytic disease of Newborn (ABO HDN) in a tertiary health care centre in south

India.

Study Number: ____________

Subject’s Initials: __________________

Subject’s Name: _________________________________________

Date of Birth / Age: ___________________________

(Subject)

(i) I confirm that I have read and understood the information sheet dated

____________ for the above study and have had the opportunity to ask

questions. [ ]

(ii) I understand that my participation in the study is voluntary and that I am free to

withdraw at any time, without giving any reason, without my medical care or

legal rights being affected. [ ]

(iii) I understand that the Sponsor of the clinical trial, others working on the

Sponsor’s behalf, the Ethics Committee and the regulatory authorities will not

need my permission to look at my health records both in respect of the current

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study and any further research that may be conducted in relation to it, even if I

withdraw from the trial. I agree to this access. However, I understand that my

identity will not be revealed in any information released to third parties or

published. [ ]

(iv) I agree not to restrict the use of any data or results that arise from this study

provided such a use is only for scientific purpose(s). [ ]

(v) I agree to take part in the above study. [ ]

Signature (or Thumb impression) of the Subject/Legally Acceptable

Date: _____/_____/______

Signatory’s Name: _________________________________ Signature:

Or

Representative: _________________

Date: _____/_____/______

Signatory’s Name: _________________________________

Signature of the Investigator: ________________________

Date: _____/_____/______

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Study Investigator’s Name: _________________________

Signature (or) thumb impression of the Witness: ___________________________

Date: _____/_____/_______

Name and Address of the Witness: ______________________________

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Information sheet

What is this study about? ABO Hemolytic Disease Of Newborn (ABO HDN) is the

most common materno-fetal blood group incompatibility. It is seen in group A, B or

AB babies born to O blood group mothers. It is caused by maternal immunoglobulin

IgG, mostly by IgG1 and IgG3 as they can cross the. Through this study, we aim to

assess the prevalence of ABO incompatible pregnancies in O blood group mothers and

correlation of IgG1 and IgG3with occurrence and severity of ABO Hemolytic Disease

Of Newborn.

What do you need to do if you decide to participate? You will be asked to fill the

informed consent sheet. 8 ml venous sample will be collected from you for the study

purpose and frozen. We will proceed as per our set protocol and will not incur any

extra expense from you.

What are the drawbacks of participation? There are no major drawbacks other than

having to undergo extra blood collection. If you have other blood tests that need to be

done, blood for these can be drawn at the same time. You will also have to spend a few

minutes of your time to fill out the questionnaire.

What are the benefits of participation? There are no direct benefits in the study.

Do you need to pay for participation in the study? No.

Can you refuse to participate in the study? Yes. Participation in the study is entirely

a matter of your choice. The tests ordered by your doctor will be done even if you do

not want to participate in the study. This choice will not affect how you are are looked

after in our hospital.

TO STUDY THE PREVALENCE OF ABO INCOMPATIBILITY ...

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