PREVALENCE OF RED CELL ANTIBODIES IN ANTENATAL MOTHERS
Dissertation submitted to
THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY In partial fulfillment of the regulations
for the award of the degree of
M.D. BRANCH - XXI
IMMUNOHAEMATOLOGY &
BLOOD TRANSFUSION
DEPARTMENT OF TRANSFUSION MEDICINE
THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI, INDIA
ABSTRACT
Background & Objectives: Hemolytic disease of the newborn can be caused by incompatibility of maternal and fetal erythrocyte for Rh (D) or other blood type antigens. Routine antibody screening is advocated in all antenatal mothers, irrespective of whether they are Rh positive or Rh negative, to look for clinically significant antibodies other than Rh (D) that might cause hemolytic disease of the newborn. The purpose of this study was to assess the Prevalence of Red Blood Cell Antibodies in Antenatal Mothers.
METHODS: A prospective analysis of red cell antibody screening test in antenatal mothers was undertaken over a period of one year.
RESULTS: There were 1225 antenatal mothers included in the study.
The prevalence of irregular antibodies in antenatal mothers was 1.1 %. In the overall prevalence the percentages of anti-D and non-anti-D antibodies were 76% in Rh D Negative mothers and 24% in Rh D positive mothers respectively.
CONCLUSION: Prevalence of Red blood cell alloantibody in antenatal mother was 1.1% with Anti-D still being the most common antibody in Rh D negative mothers similar to the findings in others studies conducted
in India. This study has also detected 3 cases of non-anti-D antibodies, one each of Anti-c, Anti-Lea and an inconclusive antibody in Rh D Positive mothers. This study also included one inconclusive antibody, which needs to be further elucidated with indigenously developed cell panel for our population.
(Keywords: Red cell Alloantibody, hemolytic disease of the new born,)
TABLE OF CONTENTS
SL. No TITLE PAGE No.
1 INTRODUCTION 1
2 RATIONALE 4
3 REVIEW OF LITERATURE 5
4 OBJECTIVES 17
5 METHODOLOGY 18
6 RESULTS 32
7 DISCUSSIONS 48
8 SUMMARY 53
9 CONCLUSIONS 54
10 BIBLIOGRAPHY i-vii
11 ANNEXURE i-v
INTRODUCTION
1 1. INTRODUCTION
The antigens on the surfaces of the red blood cells determine the blood group based on inherited differences. The human ABO blood groups were discovered by Austrian-born American biologist Karl Landsteiner in 1901. Landsteiner found that there are substances in the blood like antigens and antibodies that induce clumping of red cells, when red cells of one type are added to those of another type. He recognized three groups A, B, and O based on their reactions to each other. A fourth group, AB was identified a year later by another research team.
Landsteiner and Alexander Wiener in 1940 tested human red cells with antisera from rabbits and guinea pigs immunized with the red cells of the rhesus monkey leading to the discovery of the Rh blood group system.1
The International society of blood transfusion now recognizes 302 blood group antigens, most of which belong to one of 29 genetically discrete blood group system. Antibodies to, many of these 302 antigens have the potential to cause Haemolytic Disease of the Fetus and Newborn (HDFN) and they are, therefore, clinically significant. To name a few are Rh MNS, Kell, Duffy and Kidd. These blood group systems were first
described after antibodies were identified in patients. Frequently, such discoveries resulted from the search for the explanation of an unexpected unfavorable reaction in a recipient after a transfusion with formerly compatible blood.2
Antibodies are part of the circulating plasma proteins known as immunoglobulins, which are classified by molecular size, weight and by several other biochemical properties. Most blood group antibodies are either immunoglobulin G (IgG) or immunoglobulin M (IgM) type;
Immune antibodies are evoked by exposure to the corresponding red cell antigen. Antigens vary in their antigenic potential. Maternal alloimmunization, also known as isoimmunization, occurs when a woman's immune system is sensitized to foreign erythrocyte surface antigens, stimulating the production of immunoglobulin G (IgG) antibodies.
Hemolytic disease of the fetus and newborn (HDFN) remains a serious concern, despite the introduction of Rh prophylaxis in 1968 in almost all countries in the world. Accelerated destruction of red blood cells (RBCs) in the fetus and newborn is a direct consequence of mother’s RBC alloantibodies. The development of RBC alloantibodies is triggered by previous incompatible blood transfusions and fetomaternal hemorrhage during pregnancy.3 Although anti-D is the most common
3
cause of HDFN, more than sixty other RBC alloantibodies have been implicated. The reported overall incidence of HDFN is 10.6 cases per 10,000 deliveries, with a wide geographic variation. Atypical antibodies, other than anti-D can also cross the placenta and cause HDFN.4
The Federation of Obstetric and Gynecological Societies of India (FOGSI) recommends the use of anti D immunoglobulin for Rh (D) prophylaxis in all Rh negative mothers undergoing sensitizing events.5 The majority of the transfusion centers in India perform antenatal antibody test in Rh (D) negative mothers to prevent hemolytic disease of new born.
It is of utmost importance that the blood bank performs an adequate screen on antenatal patients, irrespective of whether they are Rh (D) positive or Rh (D) negative, to look for clinically significant alloantibodies other than Rh (D) that might cause hemolytic disease of the newborn.6
Proper periodic screening of Red blood cell antibodies in antenatal mothers can alert the doctor to identify potential association with HDFN and plan accordingly. Later in cases of postpartum hemorrhage it alerts the laboratory to possible difficulty in obtaining blood for the obstetric patients, who is always a potential transfusion recipient so that the time taken to find acceptable donor is minimized.
RATIONALE
4 2. RATIONALE
The purpose of this study was to assess the prevalence of red cell antibodies in antenatal mothers. Since there are only few such studies conducted in India, it was decided to screen antenatal mothers in Chennai over the period of one year prospectively.
REVIEW OF LITERATURE
5 3. REVIEW OF LITERATURE
3.1 Historical aspects of Alloimmunization
Hydrops fetalis or erythroblastosis fetalis is a dreaded event in the life of a woman filled with disappointment and despondency for future outcome. The twentieth century witnessed the identification of HDFN, as a clinical entity, identifying its pathogenesis and introduction of an effective strategy for preventing its severe form. Numerous case reports in the 1900 and earlier described still born, edematous infant with hydrops fetalis, and anemic infants with prominent jaundice. In 1932, Diamond et al described the combination of varying degrees of neonatal anemia, jaundice and edema as erythroblastosis fetalis.7
In 1939, Lewis and Stetson linked a woman’s severe transfusion reaction to her husband’s blood with her recent delivery of a hydrops still born. She was found to have an antibody that agglutinated her husband’s red cell. Lewis postulated that she became sensitized to an antigen that the fetus had inherited from his/her father. In 1940, Landsteiner and Wiener proposed the identity of the antigen by generating antibody to Rhesus monkey Red Blood Cells in guinea pigs and rabbits. The antisera agglutinated RBC from 85% of Whites. These people were designated as Rh positive. The 15% whose RBC did not agglutinate were Rh negative.8
Subsequent years saw rapid and dramatic progress in this field, which includes the recognition of IgG transplacental crossing and development of Coomb’s test. This test uses antihuman IgG antibodies to agglutinate IgG labeled RBC’s and remains important in the detection and management of HDFN.9 The inciting stimulus for red cell alloimmunization in pregnancy, the passage of fetal red cell in to maternal circulation, or fetomaternal hemorrhage was directly demonstrated by Chown in 1954.1
The most common routes of maternal sensitization are via blood transfusion or fetomaternal hemorrhage associated with delivery, trauma, spontaneous or induced abortion, ectopic pregnancy, or invasive obstetric procedures. These antibodies can cross the placenta during pregnancies in alloimmunized women and, if the fetus is positive for erythrocyte surface antigens, result in hemolysis of fetal erythrocytes and anemia. This, in turn, can lead to potentially disastrous consequences for the fetus, such as hydrops fetalis, a high-output cardiac failure syndrome.10
In 1961, Lilley described the relationship of the concentration of bilirubin in amniotic fluid and degrees of destruction of fetal RBC, providing a tool to decrease the severity of intra uterine hemolytic disease.11 Two years later Lilley introduced intraperitoneal transfusion of
7
anemic fetuses which were used for more than 20 years and it was largely replaced by intravascular transfusion technique.12, 13
The middle cerebral artery peak systolic velocity (MCV-PSV) has recently been found to be an accurate and noninvasive means of diagnosing fetal anemia in patients with Rh alloimmunization. A cut off point of 1.5 times the median MCA-PSV is found to correlate with moderate to severe anemia, with a sensitivity of 100% and a false positive rate of 12%.14
Introduction and utilization of Rh D Immunoglobulin (Anti D) for prophylaxis against Rh immunization-initially postpartum and subsequently antepartum is one of the major medical achievements and successful application of preventive obstetric medicine in the last century.
However in developing countries like India such prophylaxis is under achievable because of major percentage of home deliveries, Rh typing being not done routinely, high cost of anti-D immunoglobulins and sheer negligence.
There are several classes of antibodies. The two of interest are IgM and IgG. If the antibody is identified as IgM, then it won’t cross the placenta and there is no risk of hemolysis to the fetus.
According to the British Committee for Standards in Hematology Guidelines, Blood Grouping and Antibody Testing during Pregnancy and their likelihood of Causing HDFN have been grouped as follows: 6
Group 1 Anti -D, -c, -E, -e, -C, -K, -k, -Fya
These antibodies are commonly associated with clinical HDFN, demands determination of titre / quantitation of the antibody which should be repeated every 4 weeks until 32 weeks gestation and then grouping of red cell antibodies according to their likelihood of causing HDFN every 2 weeks until delivery. When a clinically significant rise in titre / quantitation is noted, fetal monitoring should be initiated by ultrasound, amniocentesis or cordocentesis.
Group 2 Anti, -Cw, -Fyb, -Jka, -Jkb, Jk3, -S, -s, -M, Gea
These are antibodies that are known to cause a positive direct antiglobulin test, but treatment is likely to be limited to phototherapy.
Group 3 Anti-P1, -N, -H, -Lea, -Leb, -Lea+b, -Lua, -Lub, -Sda, -HLA
These antibodies are not documented to cause clinical HDFN.
9
3.2. GLOBAL SCENARIO OF ATYPICAL ANTIBODIES:
The epidemiology of HDFN in different ethnic and racial populations directly reflects the frequency of blood group alleles in the population and the likelihood of incompatibility and consequent maternal alloimmunization.15 Complex genetic composition influences the ability of individuals responding to red cell antigen. Rh D is the most potent immunogen and as little as 0.1 to 1ml of D positive red cell can stimulate antibody production. Beside this, the severity of HDFN is determined by the immunoglobulin subclass, amount of antibody and number of antigenic sites on the red cell.16
According to Ossie Geifman et al study (1997, In New York) approximately 400 red blood cell antigens have been identified. These RBC antigens and alloantibodies differ significantly among human population and ethnic groups. Hence, alloimmunization after exposure to red cell antigens depend on genetic and acquired patient related factors, dose and immunogenecity of the antigens. The exact kinetic of alloimmunization are not clear. The development of alloantibodies can significantly complicate transfusion therapy and also capable of causing mild or severe hemolytic disease of the new born.17
There have been studies which have reported the significance and incidence of atypical antibodies in antenatal mothers. The First antibody to be described as a cause of HDFN was anti–D. Until the use of Rh immunoprophylaxis in 1968 anti-D was the most common antibody to cause HDFN. Deka D et al also observed that failure to administer post natal anti-D prophylaxis was responsible for Rh D alloimmunization in more than 50% of cases, followed by failure to administer Anti D after MTP (10%).5
Hemolytic disease of the newborn can also be caused by less common atypical antibodies, which can also cross the placenta and affect the fetus in a similar fashion. A combined incidence of 0.1 to 2% of severe fetal disease due to anti-D and non-anti-D antibodies has been reported. The incidence of atypical antibodies in Rh positive antenatal mother is low, with a reported incidence between 0.01% and 1.3%.18
In 2009, Naje Awad Al-Ibrahim et al from Saudi Arabia in their study over 2 year period on antenatal mothers found 2.0%
alloimmunization.19 While Howard et al, in 2008 detected clinically significant antibodies among 1.0% of all pregnant women.20 In contrast, Gottvall et al. from Sweden in 2010 found 0.4% of alloimmunization in all pregnancies, out of 0.4% only 0.16% of antibodies were found to be clinically significant.21
11
Giblett et al reported that 93% of antibodies detected in sera of pregnant women were anti D and (7%) were of the other specificities.22 In 1967, Polesky screened 43,000 women and found that 2460 (5.7%) were isoimmunized, 2024(4.7%) with Rh antibodies and 436(1.0%) with atypical antibodies.23 In 1969, Queenan et al reported that 3.1% pregnant were alloimmunized, 68% of these were associated with anti D and 1.3%
with specificities other than Rh. Since Rh prophylaxis become widespread the picture has changed and anti-D is much less common. The comparative incidence of alloantibodies other than anti-D has also been affected by the increased transfusion in pregnant women. Queenan at al reported that history of prior transfusion was 9 times more frequent among alloimmunized pregnant women.24, 25
In France, P. Moncharmont et al done a study for a period of 30 years showed that number of HDFN’s with an anti-D alloimmunization in the mother has significantly decreased particularly since 1970, when the anit-D immunoglobulins have been used. Furthermore, during this period, an increase of the number of HDFN other than anti-D detected is observed.26
Kornstead reported that in Norway, the occurrence of new cases of anti D has fallen from 57.6 cases per ten thousand (0.6%) women during
the period 1967-1969 to 16.5 per ten thousand (0.2%) during the period 1981 – 1983 after the introduction of Rh Ig Immunoprophlyaxis.27
P.J Bowell et al, in a 2 year study period of 667 sera from approximately 70,000 (0.95%) antenatal mothers, found to contain 726 atypical red cell antibodies. Overall, 66% of the immunized mothers were Rh (D) positive and 29% had previous blood transfusion.28
In the year 1983, Leif Kornstad encountered new cases alloimmunization with Rh antibodies other than anti-D in the Rh positive women, especially anti-E and anti-C. The other group systems which cause HDFN are Kell and Duffy.29
Chandrasekar et al, in Northern Ireland during the study period of 1999 -2000, found 186 women to have clinically significant non-Rh D antibodies among 34,913 antenatal mothers. Out of 186 women 99 were found to have Rh group and 87 have non-Rh groups. There was also a definitive history of transfusion in 46 (61.3%) women, and in 10 (13.3%) women the antibody was induced by pregnancy.30
In 2004, Reem Ameen et al study in Kuwait showed 0.49%
clinically significant antibodies. Anti-D, anti-E and anti-K were the most clinically significant antibodies among Kuwait females. Immunization
13
through pregnancy is the main reason for the greater RBC alloimmunization in females.31
Anti-c is usually described as the next most common cause of severe HDFN after anti-D. About half of anti-c antibodies are attributable to transfusion and they are often present at low titre. P.J Bowell et al in 8 years study reported 177 of 280,000 pregnancies complicated by maternal anti c alloimmunization.32 The study by Tomas Gottvall et al showed anti-c antibodies in 11% (33/287) of antenatal mothers.21
Anti-E is relatively common but seldom causes HDFN. Saju D et al in his retrospective study found 283 pregnancies with anti E.33 P. Moran et al over the 29 years of retrospective study period identified Anti-E as a sole maternal antibody in 122 pregnancies among 118 antenatal mothers.34
The frequency of perinatal death in pregnancy with anti Kell is debatable although poor fetal outcome is reported between 1.5 and 3.9 percentage. Management of these pregnancies remains controversial. But the incidence of anti-Kell immunization is low in the obstetrics population at 0.1 to 0.2% and it is reported to be mainly transfusion induced. Anti-Kell antibodies appear to cause significant suppression of erythropoiesis rather than red cell destruction.35
S.R. Grant et al in his retrospective review identified 65 pregnancies in 52 Kell sensitized women with Kell positive partners.36 Bowman et al studied in their study over the period of 47 years from 1944 to 1990 identified 31 anti-E affected pregnancies.18
There is a marked international variation in frequency and distribution of alloantibodies for example in Asian countries Rh D negativity is rare so is anti-D. K.H.Wu et al identified 0.01 % of HDFN caused by maternal atypical antibodies over a period of 10 years in Taiwan. They also identified a rare antibody Anti-Mi, twice common in their population which recognizes MNS blood group system. Although it caused no cases of severe HDFN in their study, its potential to do so has been reported.37 K.F.Wong et al screened 1997 pregnant women for irregular antibodies. He found 17 women (0.85%) had irregular antibodies of which four where of potential clinical significance. He suggested that antibody screening may not be necessary for Chinese populations except for those are Rh negative or who have history of HDFN.38
The Duffy RBC system includes Fya & Fyb. The prevalence of anti Fyaduring pregnancy is between 0.01% and 5.4%. Fyb antibodies are less common and have been found to cause mild HDFN.39
15
Following table shows the prevalence of Red Cell Alloantibodies in various studies carried out in different population worldwide:
Table 1 - Prevalence of Red Cell Alloantibodies - Global Scenario Study Carried
out by
Polesky23 (1967)
Queenan et al24 (1969)
Pepperell et al40 (1977)
Filbey et al41 (1995)
Geifman- Holtzman17
(1996) Place of Study Minnesota New York Australia Sweden Central New
York Period of study 1960-66
(7yrs)
1960-67 (8 yrs)
1965-75 (10yrs)
1980-91 (12yrs)
1993-95 (2.5 yrs) No. of blood
samples studied 43,000 18,378 72,138 1,10,765 37,506
Before/After the discovery of RhIg
Before Before After After After
Alloantibodies Prevalence
D E C Cw c e Kell Duffy MNS Kidd Lutheran P1 Lea, Leb I
Others
1864 (63.1%) 80 (2.7%) 448 (15.2%) 4 (0.14%) 68 (2.3%) 2 (0.07%) 93 (3.1%) 17 (0.6%) 45 (1.5%) 7 (0.2%) -
27 (0.9%) 94 (3.2%) 13 (0.4%) 194 (6.6%)
304 (48.3%) 34 (5.3%) 34 (5.3%) -
12 (1.9%) 3 (0.4%) 30 (4.7%) 12 (1.9%) 20 (3.1%) 7 (1.1%) 3 (0.4%) 15 (2.3%) 51 (8.1%) 15 (2.3%) 90 (14.3%)
958 (65.3%) 69 (4.7%) 9 (0.6%) -
59 (4.0%) 6 (0.04%) 34 (2.3%) 8 (0.5%) 18 (1.2%) 2 (1.14%) -
129 (8.8%) 174 (11.9%) -
1 (0.07%)
159 (19.0%) 51 (6.1%) 36 (4.3%) 10 (1.2%) 38 (4.5%) 1 (0.1%) 48 (5.7%) 26 (3.1%) 35 (4.2%) 10 (1.2%) 13 (1.6%) 48 (5.7%) 241 (28.8%) -
120 (14.4%)
101 (18.4%) 77 (14.0%) 26 (4.7%) 1 (0.2%) 32 (5.8%) -
121 (22.0%) 31 (5.6%) 26 (4.7%) 8 (1.5%) 7 (1.3%) 1 (0.2%) 113 (20.5%) 5 (0.9%) 1 (0.2%) Total No. of
Alloantibodies Identified
2956 630 1467 836 550
3.3. INDIAN SCENARIO:
In 2009, Daniel.D et al in her study among antenatal mothers in a tertiary care hospital at Vellore in South India reported the prevalence of 1.48% Red Cell Alloimmunization.42
In a study by Sangeetha Pahuja et al in New Delhi on 3577 multigravida antenatal mothers, the overall prevalence of Red Cell Alloantibodies was found to be 1.25%.43
Shanthala A.M. et al from Bangalore did red blood cell antibody screening on 624 antenatal mothers and found 9 cases positive for the presence of irregular Alloantibodies with prevalence of 1.4%.44
In 2007, Thakaral Bet al reported a case of Haemolytic Disease of Newborn by Anti-c and Anti-E in Rh (D) positive mother.45 In 2010 Shilpa Singla in New Delhi reported a case of severe Hydrops in an infant born to an Rh (D) positive mother due to anti-c antibodies detected antenatally.46
OBJECTIVES
4. OBJECTIVE
To find out the prevalence of Red Blood Cell antibodies in the antenatal mothers in Chennai.
.
METHODOLOGY
5. METHODOLOGY
This study was performed on 1225 Multigravida women who had attended the antenatal clinic in the outpatient department of Government and corporation Hospital in Chennai, over a period of one year (2010- 2011). The Blood group and Rh (D) type along with antibody screening was performed at the first antenatal visit. The pre-tested pro-forma was used to collect the data.
5.1. MATERIALS AND METHODS
Study design: Cross sectional study.
Study area: Obstetrics & Gynecology Hospitals (Corporation &
Government) in Chennai.
Study population: Antenatal mothers.
Inclusion Criteria: Multigravida Women in their first trimester Antenatal Visit.
Exclusion Criteria: Primi and Antenatal mothers who are not willing to participate and if their first antenatal visit bypasses first trimester.
Sampling Technique: Simple random sampling
Study Period: One year (2010-2011).
19 Sample size:
Prevalence: 2%
Confidence Interval: 95%
q = (1- Prevalence)
Allowable error (d) = (0.8%) Sample size (n) = 4pq/d2
= (4 x 0.02 x 0.98) / (0.008 x 0.008) Therefore Sample size n = 1225
5.3. DATA COLLECTION PROCEDURE:
A semi-structured questionnaire has been developed to collect the data.
Data was collected from antenatal mothers in Obstetrics &
Gynecology Hospitals (Corporation & Government) in Chennai during their first trimester antenatal visit.
Informed Consent: The details of the study were written in consent form both in English and Tamil. Study details were explained to the participant and after obtaining their consent, the individuals were included for the study.
For each patient name, age, sex, obstetric history, blood group, husband’s blood group, history of Rh anti-D immunoprophylaxis and history of blood transfusions and associated medical illness were recorded prior to taking the blood samples.
5.4. ETHICAL COMMITTEE CLEARANCE:
Ethical clearance was obtained from the Institutional Ethical Committee of The Tamilnadu Dr. M.G.R Medical University, Chennai-32.
21 5.5. SAMPLING PROCEDURE:
The study involved 1225 multigravida antenatal mothers during their first trimester antenatal visit. Cases were selected randomly from both Government and Corporation Hospitals in Chennai over a period of one year. Data were acquired by standardized methodology using proformas.
5.6 DATA ANALYSIS:
Data entry was done using SPSS version 10 and p value <
0.05 is considered to be significant. Demographic & clinical variables were given in frequencies with their percentage. Incidence was given in proportion with 95% confidence interval. Statistical
differences were found using one sample chi square test < 0.05 considered significant.
5.7 LABORATORY INVESTIGATIONS
5.7.1 STEP- I
Five ml of blood was collected in a plain test tube from antenatal mothers. The samples were then centrifuged at 3000 rpm for 5 minutes and serum was separated. Samples were processed on the same day or frozen at -20 C and assayed later.47
5.7.2 STEP-II
ABO blood grouping and Rh typing were performed for each cases and their husbands (wherever possible) by standardized tube technique method. All RhD negative samples by tube method were confirmed for weak D by an indirect anti-globulin test. Interpretation was done based on various grades of agglutination ranging from + to ++++, whereby the result was recorded as positive, or negative when no sign of agglutination was present.
23 ABO BLOOD GROUPING
ABO blood grouping was done by Tube Technique as per the following steps given in the DGHS Technical Manual.48
Preparation of 2% to 5% red cell suspension:
Wash red cells two times in saline. The wash supernatant should be clear.
Add 0.2 ml of washed red cell to 10 ml of saline.
TUBE TEST FOR DETERMINING ABO GROUP OF RED CELLS AND SERUM
Testing Red cells:
Place one drop of anti A, anti B and anti AB in a clean labeled test tubes.
To each test tube, add one drop of 2% to 5% red cell suspension.
Gently mix the content of the test tube; then centrifuge at 1000 rpm for 1 minute.
Gently resuspend the cell buttons and examine them for agglutination.
Read, interpret and record the test results.
Testing Serum:
Use tube technique to test patients’/donors’ serum against 3-4% saline suspensions of pooled 2-3 samples of group A cells, B cells and O cells.
Label three tubes A cells, B cells and O cells.
Place two volumes (2 drops) of the test serum, in each tube.
Add one volume (1 drop) of A cells to tube labeled A, one volume (1 drop) of B cells to tube labeled B and one volume (1 drop) of O cells to tube labeled O.
Mix the contents of each tube by gentle shaking and leave at R.T for 30-45 minutes.
Observe the supernatant fluid for the presence of haemolysis against well lighted background.
Gently disperse cell button and see for agglutination.
All negative results must be examined under microscope.
Read, interpret and record the test results.
25
TUBE TEST FOR DETERMINATION OF RH TYPING
It is important to test for D antigen on red cells using anti-D from two sources i.e., IgM – anti-D and IgM+IgG (blend).
Prepare 2-5% washed red cell suspension of test sample.
Place 1 drop of anti-D (D1) in cleaned tube labeled D1 and place 1 drop of anti-D (D2) from a different manufacturer in a clean tube labeled D2.
Place 1 drop of 22% bovine albumin/control reagent in another tube labeled C.
Add 1 drop of 2-5% test cell suspension to each tube.
Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D, incubate at 37 deg C for 10 minutes and centrifuge
Re-suspend the cell button and look for agglutination. All negative results must be confirmed under microscope.
Interpretation
Agglutination in tubes D1 and D2 indicated that the person is Rh positive. No agglutination in either tube indicated that the person is Rh
negative. All Rh negatives are confirmed by AHG test to rule out Weak D.
DU TESTING
If negative, wash the cells 3-4 times with saline and decant the last washing.
Add 1-2 drop of anti-human globulin reagent (AHG-Coombs’
reagent). Mix gently and centrifuge at 1000 rpm for 1 minute.
Re-suspend the cell button gently, examine for agglutination and record the results.
All negative reactions should be confirmed by adding known IgG sensitized cells, re-centrifuge and re-examine for agglutination.
The presence of agglutination confirms the test result.
5.7.4 STEP-III
Red Cell Alloantibodies were screened using commercially available three cell antigen panel (Asia -ID-DiaCell I, II, III) by Coombs gel card. Whenever antibody screening was positive, extended eleven cell panel was used for antibody identification. Antibody screening and identification was done by Gel Technique as per the steps given in the Kit Literature.
27
Methodology for antibody screening and identification
Use the ready-to-use test cell reagents DiaCell I-II-III with the Liss/Coombs Card or Coombs Anti-IgG cards.
Allow the reagent cells & /serum to come to room temperature before use.
Identify the appropriate microtubes of the Cards with the patient’s name/number.
Take off the aluminium foil.
Add 50 L of each DiaCell test cells to the appropriate microtubes of appropriate cards.
Add 25 L of the patient’s serum/plasma to all the microtubes.
Incubate for 15 mins at 37°C. in the incubator.
Centrifuge the cards 10 minutes.
Record the results.
Interpretation is done according to the charts provided.
Antibody Identification:-
Use the ready-to-use test cell reagents Dia Panel with the Coombs Anti- IgG cards.
The panels contain 11 vials of ready-to-use reagent cells.
The last micro tube (no.12) can be used as auto control.
Allow the reagent cells & serum to come to room temperature before use.
Identify the appropriate micro tubes and the Cards with the patient’s name/number. Take off the aluminum foil.
Add 50 L of each DiaPanel test cell to the appropriate micro tubes of appropriate cards.i.e. DiaPanel to the Liss/Coombs cards.
29
Add 50 L of the patient’s own red cell suspension (0.8%) to microtube no. 12 in both sets as autocontrol.
Add 25 L of the patient’s serum/plasma to all the microtubes.
Incubate the cards for 15 mins at 37°C. in the incubator.
Centrifuge the cards in the Centrifuge for 10 minutes.
Record the results.
Interpretation is done according to the antigram provided.
GRADING OF REACTION IN GEL TECHNIQUE
For all card tests, a positive result means agglutinated cells form a red line on the surface of the gel in the microtube or agglutinates dispersed in the gel. A negative result means the presence of a compact button of cells on the bottom.
5.7.5. EXCLUSION OR CROSSING OUT
The first approach to the interpretation of panel results was to exclude specificities based on non-reactivity with the serum tested. Such a system is sometimes referred to as a “cross-out” or “rule-out” method.
Once results had been recorded on the worksheet, the antigen profile of the first non-reactive cell was examined. If an antigen was present on the cell and the serum did not react, the presence of the corresponding antibody could be at least tentatively ruled out. After all antigens present on that cell had been crossed off, interpretation proceeded with the other non-reactive cells and additional specifications were excluded.
Next, the cells reactive with the serum were evaluated. The pattern of reactivity of the non-excluded specificity was compared to the pattern of reactivity obtained with the test serum. If there was a pattern that matched exactly, that was most likely the specificity of the antibody in the serum however, if there were remaining specificities that had not been excluded, additional testing was needed to eliminate remaining possibilities and to confirm the specificity identified. This requires testing the serum against additional cells.
31
RESULTS
32 6x RESULTS
During the study period, 1225 multigravida women were screened for the presence of alloantibodies. The descriptive statistics are as follows.
Table 2 - Age Distribution among Antenatal Mothers
Age (yrs) Frequency Percentage
18- 20 51 4.2%
21-30 1127 92.0%
31-40 45 3.7%
>40 2 0.2%
Total 1225 100.0%
Majority of the antenatal mothers in the study were in the age group of 21 to 30 yrs (92%).
Table 3 - Distribution of Blood Groups among Antenatal Mothers in the study
Blood Group Frequency Percentage
A+ 173 14.12%
A- 26 2.12%
B+ 423 34.53%
B- 35 2.86%
O+ 421 34.37%
O- 66 5.39%
AB+ 78 6.37%
AB- 2 0.16%
Oh+ 0 0.00%
Oh- 1 0.08%
Most of the study population belongs to the B positive blood group (39.8%) and one rare Bombay negative blood group (oh-) was identified.
34
Figure 1 -Distribution of Rh Typing among Antenatal Mothers in the study
Among the total population of the study1095 antenatal mothers were Rh D positive (89%) and 130 were Rh D negative (11%).
Figure 2 - Distribution of the Gravid status in the study
Most of the antenatal mothers were in gravida 2 and only one was in gravida 6.
Table 4 – Frequencies of Abortions among the antenatal mothers
No. of
Abortions Frequency Percentage
0 1051 85.80%
1 127 10.37%
2 42 3.43%
3 2 0.16%
4 2 0.16%
5 1 0.08%
Total 1225 100.0%
Among the study, 86% of them did not have any abortion.
10% had one time abortion and less than 1% had 3 to 5 times abortions.
36
Table 5 – Frequencies of Blood Transfusion among study population
Transfusion Frequency Percentage
YES 4 0.3%
NO 1221 99.7%
Total 1225 100.0%
Out of 1225 antenatal mothers only 0.3% had history of blood transfusion and 99.7% did not have transfusion.
Table 6 - Antibody screening among the study population
Antibody Presence Frequency Percentage
YES 13 1.1%
NO 1212 98.9%
Total 1225 100%
Figure 3 – Prevalence of Red cell antibody
The prevalence of red cell alloantibody in antenatal mothers was 1.1% and 98.9% did not have any red cell antibody.
38
Table 7 - Antibody identification among the study population
Antibody Frequency Percentage
Anti-D 10 76%
Anti-c 1 8%
Anti-Lea 1 8%
Unknown 1 8%
Total 13 100%
Figure 4 – Antibody Identification in the study
Antibody Identification in the study
0 1 2 3 4 5 6 7 8 9 10
Anti-D Anti-c Anti-Lea Unknown
Number of Cases
Anti D was 76%, the most common antibody identified in the study group.
Figure 5 - History of RhIg Immunoprophylaxis in the study
Out of 130 Rh negative ante natal mothers, anti D was given for 78(58%) and not given for 52 (42%).
40
Table 8 - Association of Rh D Antigen with Alloimmunization Antibodies not
detected
Antibodies
detected p-value
Rh D Positive 1092 3
Rh D Negative 120 10 < 0.01
Among the 130 women with Rh D negative group, 10 developed antibodies, so the prevalence of alloimmunization in this group was 7.69%.
Figure 6 - Anti-D alloimmunization in the study group
Among the Rh D negative antenatal mothers 10 of them developed anti-D Alloimmunization. Out of 10 positives, nine of them had not been given RhIg Immunoprophylaxis. Only one had developed anti D antibody in spite of RhIg Immunoprophylaxis , which was found to be significant (p<0.01)
Table 9 - Cross Tabulation: Antibody Identification Vs Blood Group:
Antibody A+ A- B+ B- O+ O- AB+ AB- Oh+ Oh- Total
Anti-D 0 4 0 1 0 3 0 2 0 0 10
Anti-c 0 0 0 0 1 0 0 0 0 0 1
Anti-Lea 0 0 1 0 0 0 0 0 0 0 1
Unknown 1 0 0 0 0 0 0 0 0 0 1
Total 1 4 1 1 1 3 0 2 0 0 13
p-value 0.000
P value < 0.01 is accepted as statistically significant.
10 of the Rh D Negative mothers developed only Anti D antibodies and 3 of the Rh D Positive mothers developed antibodies such as Anti-c, Anti-Lea and an Unknown antibody. Most of the alloantibodies were detected in A negative (n=4) 30.7% blood group followed by O negative (n=3) 23.07% blood group.
42
Table 10 - Cross Tabulation: Antibody Identification Vs Gravida
Gravida Antibody
2nd 3rd 4th 5th 6th
Total
Anti-D 3 3 2 2 0 10
Anti-c 0 1 0 0 0 1
Anti- Lea 1 0 0 0 0 1
Unknown 0 0 1 0 0 1
Total 4 4 3 2 0 13
p-value 0.000
p- value < 0.01 is accepted as statistically significant
The Gravida status of women showed a statistically significant correlation with alloantibody formation.
.
Table 11 – Cross Tabulation: Antibody Identification Vs Abortion:
Abortion Antibody
0 1st 2nd 3rd 4th Total
Anti-D 0 7 1 1 1 10
Anti-c 0 1 0 0 0 1
Anti-Le a 1 0 0 0 0 1
Unknown 0 0 1 0 0 1
Total 1 8 2 1 1 13
p-value 0.00
p-value < 0.01is accepted as statistically significant.
The study found a statistically significant correlation between frequency of alloimmunization and adverse obstetric history.
An adverse obstetric history (abortion or medical termination of pregnancy) was present in 76% of patients with anti-D (10/13).
44
Table 12 – Red Cell Antibody Identification Vs History of blood transfusion
History of Blood Transfusion Antibody
YES NO
Total
Anti-D 0 10 10
Anti-c 1 0 1
Anti-Lea 0 1 1
Unknown 0 1 1
Total 1 12 13
p-value 0.000
p-value < 0.01is accepted as statistically significant.
A history of blood transfusion was present in 7.6% (1/13) women with Red Cell alloantibodies.
Table 13 - Cross Tabulation: Antibody Identification Vs Spouse Blood Group:
Antibody A+ A- B+ B- O+ O- AB+ AB- Total
Anti-D 2 0 3 0 5 0 0 0 10
Anti-c 0 0 0 0 1 0 0 0 1
Anti-Lea 0 0 1 0 0 0 0 0 1
Unknown 0 0 0 0 1 0 0 0 1
Total 2 0 4 0 7 0 0 0 13
p-value 0.000
p-value < 0.01 is accepted as statistically significant.
Out of 10 Rh D negative women with anti-D, the husband’s blood group in all cases was found to be Rh D positive. Out of three Rh D Positive women who had developed Alloimmunization to Non-Rh D Antigens, their husbands were also belong to Rh D Positive group, which was found to be significant with p value of less than 0.01 (p<0.01).
46
Table 14 - The clinical data of antenatal mothers who were Alloimmunized
Sl.
No Age Blood
group Antibody Previous History
H/O of RhIg Immuno- prophylaxis
Husband Blood group
1 28 A- Anti-D G2P0L0 A1 No O +
2 24 AB - Anti-D G2P0L0 A1 No A+
3 26 A- Anti-D G4P1L1 A2 No O+
4 23 O - Anti-D G2P0L0 A1 No B +
5 26 A- Anti-D G3P1L1 A1 No B+
6 27 A- Anti-D G3P0L0 A2 No O+
7 30 AB- Anti-D G4P1L1 A2 No O+
8 32 O + Anti- c transfusion No O +
9 34 O- Anti-D G5P0L0 A4 Yes A+
10 28 O- Anti-D G5P1L1 A3 No B+
11 27 A+ Unknown G4P1L1 A2 No O+
12 27 B- Anti-D G3P1L1 A1 No O+
13 23 B+ Anti-Lea G2P1L1 A0 No B+
Among the study group13 red cell alloantibodies were detected with prevalence of 1.1%.
Table 15 – Comparison of our study with other studies worldwide
Comparison of our study with other studies worldwide Study Carried out by: Polesky
(1967)
Queenan et al (1969)
Pepperell et al (1977)
Filbey et al (1995)
Geifman-Holtzman (1996)
Sangeeta Pahuja (2009)
Our Study (2011)
Place of Study Minnesota New York Australia Sweden Central New York New Delhi Chennai
Period of study 1960-66 (7yrs) 1960-67 1965-75 1980-91 1993-95 2008-09 2010-11
No. of samples studied 43,000 18,378 72,138 1,10,765 37,506 3577 1225
Before/After the discovery
of RhIg Before Before After After After After After
D 1864 (63.1%) 304 (48.3%) 958 (65.3%) 159 (19.0%) 101 (18.4%) 40 (78.43%) 10 (76%)
E 80 (2.7%) 34 (5.3%) 69 (4.7%) 51 (6.1%) 77 (14.0%) - -
C 448 (15.2%) 34 (5.3%) 9 (0.6%) 36 (4.3%) 26 (4.7%) 6 (11.76%) -
Cw 4 (0.14%) - - 10 (1.2%) 1 (0.2%) -
c 68 (2.3%) 12 (1.9%) 59 (4.0%) 38 (4.5%) 32 (5.8%) 1 (1.96%) 1 (8%)
e 2 (0.07%) 3 (0.4%) 6 (0.04%) 1 (0.1%) - - -
Kell 93 (3.1%) 30 (4.7%) 34 (2.3%) 48 (5.7%) 121 (22.0%) 1 (1.96%) -
Duffy 17 (0.6%) 12 (1.9%) 8 (0.5%) 26 (3.1%) 31 (5.6%) - -
MNS 45 (1.5%) 20 (3.1%) 18 (1.2%) 35 (4.2%) 26 (4.7%) 3 (5.88%) -
Kidd 7 (0.2%) 7 (1.1%) 2 (1.14%) 10 (1.2%) 8 (1.5%) - -
Lutheran - 3 (0.4%) - 13 (1.6%) 7 (1.3%) - -
P1 27 (0.9%) 15 (2.3%) 129 (8.8%) 48 (5.7%) 1 (0.2%) - -
Lea, Leb 94 (3.2%) 51 (8.1%) 174 (11.9%) 241 (28.8%) 113 (20.5%) - 1 (8%)
I 13 (0.4%) 15 (2.3%) - - 5 (0.9%) - -
Others 194 (6.6%) 90 (14.3%) 1 (0.07%) 120 (14.4%) 1 (0.2%) - 1 (8%)
Total 2956 630 1467 836 550 51 13
DISCUSSIONS
7. DISCUSSION
Antenatal services in India are fragmented and not uniform and there is a limited amount of published data on red cell alloimmunization among pregnant women in India. Although guidelines for screening have been laid down by Directorate General of Health Services, India48 for screening alloantibodies primarily for Rh D negative women presenting with an adverse obstetric history, it has not been carried out as a routine in many of the centres.
Blood grouping and Red cell alloantibody screening though is heavily biased towards the prevention and management of the continuing problems of HDFN, it also gives an advanced warning for adequate compatible blood supply for emergency. This latter point is especially relevant to the urgency of blood demands late in pregnancy and the fact that 16 to 22% of women have blood cross matched during pregnancy.28
The overall prevalence of red cell alloantibodies among antenatal mothers in our study was 1.1%. Screening and identification of red cell alloantibodies showed 10 cases of Rh D, 1 case each of anti-c, anti-Lea and inconclusive antibodies in 1225 mothers during first trimester antenatal visit. This is in accordance with Queenan et al,12 Helen Howard
49
et al20 and Sangeeta Pahuja et al43 studies, which had reported prevalence of 1 to 2% red cell alloimmunization in antenatal mothers.
In our study, the percentage of red cell alloimmunization in RhD negative antenatal mother was 7.6%. There is a wide variation in alloimmunization rates among Rh-negative women. Al-Ibrahimet al,19 in their study in Saudi Arabia observed 7.1%. red cell alloimmunization.
Out of 130 Rh D Negative antenatal mothers 78 had RhIg Immunoprophylaxis and 52 had no RhIg Immunoprophylaxis. Out of 52 antenatal mothers who had no history of RhIg Immunoprophylaxis 9 (17.31%) developed red cell alloimmunization. This is in line with a study done by Deka D et al who also observed that failure to administer postnatal anti D prophylaxis was responsible for RhD alloimmunization in more than 50% of cases, followed by failure to administer Anti D after MTP (10%).5 Another factor also has to be taken seriously as previous studies have shown that there is a complex genetic factor which plays an important role in production of antibodies. The risk of immunization is only about 6 to 9% for an RhD negative mother after an RhD positive pregnancy if RhIg is not administered.3,9
In this study out of 78 RhD Negative antenatal mothers who had RhIg Immunoprophylaxis 1 (1.2%) developed red cell alloimmunization.
This is similar to the study done by J.M. Koelewijnet al49 and Shanthala A.M et al, which states that despite use of anti–D immunization, 1to2%
of the cases are still sensitized, indicates that transplacental or fetomaternal hemorrhage may occur during pregnancy or at delivery and can lead to alloimmunization. Half of the failures of anti D immunoglobulin prophylaxis were due to increased fetomaternal hemorrhage.44,51
In our study, out of 1195 RhD Positive antenatal mothers 3(0.25%) developed red cell alloimmunization against c, Le (a+b) and Unknown antigen.
The development of anti-c antibody in one of the 3 patients in this study was due to the previous history of multiple blood transfusions for anaemia in earlier pregnancy. This is in an agreement with other studies such as those of Leif Kornstad,29 Smith et al,25 and P.J Bowell et al 32 who showed a significant proportion of mothers had previously received blood transfusion developed anti-c. In India, Thakral B et al 45 and Shilpa Singla et al 46 reported severe hydrops in an infant born to Rh D positive mother due to anti c antibodies who have received multiple blood transfusions. The percentage of red cell alloimmunized antenatal mothers developed anti-c in this study was 8% (1/13). This is in accordance with
51
the study by Tomas Gottvall et al 21 who had reported anti-c antibodies in 11% (33/287) of antenatal mothers.
The percentage of anti-Lea antibody in this study was 8 %( 1/13).
This is in line with the studies by Al Ibrahim and Daniel D et al who showed 2.38% and 18.98% prevalence of anti-Lea antibodies respectively in antenatal mothers. The development of anti-Lea antibody in these studies could be explained by the overexpression of autoantibodies in pregnancies. 50,52
The percentage of an Unknown antibody after 11 cell panel identification in our study was 8 % (1/13). This is the fact which has to be taken seriously as in previous studies Daniel Det al identified about 36%
of inconclusive antibodies.42 In another study K.H.Hu et al found different specificity of antibodies compared to those reported for western countries and anti Mi being the most frequently encountered antibody in their population.37 In Indian scenario, the antibody screening by using imported Asian cell panel may fail to detect antibodies against un- representing antigens.
The current study found a statistically significant correlation between frequency of alloimmunization with adverse obstetrics history and gravid status of the women. Bowman et al and Sangeeta Phauja et al
in their study stated that gravid women are at great risk for blood group sensitization due to transplacental immunization of incompatible pregnancy.18,43
SUMMARY
8. SUMMARY
The prevalence of Red Cell Alloantibodies in antenatal mothers is 1.1% (13/1225).
Prevalence of Red Cell Alloantibodies in Rh D negative antenatal mothers is 7.6% (10/130).
Prevalence of Red Cell Alloantibodies in Rh positive women is 0.27% (3/1095).
Out of 13 cases with red cell alloimmunization Anti-D is the most common Antibody identified, accounting for 76%
(10/13).
Anti c, Anti Leaand inconclusive antibodies were the non-Rh D antibodies found in each of the 3 non-Rh D alloimmunized antenatal mothers [8 % (1/13) each]. All these three antibodies were found in Rh D Positive mothers.
History of blood transfusion in previous pregnancies found significant in red cell alloimmunization.
CONCLUSSIONS
9. CONCLUSSION
In our study the prevalence of red cell antibodies in antenatal mothers in Chennai was 1.1%, which is similar to other studies conducted in India. However, large numbers of samples have to be studied to arrive at a conclusive number and type of the antibody prevalent in our population.
This study also included one inconclusive antibody, which needs to be further elucidated with indigenously developed cell panel for our population.
Our study on red cell alloimmunization in antenatal mothers showed definite source of antigenicity such as husband’s incompatible blood group, gravid status, bad obstetric history and previous history of blood transfusion.
It has been observed that anti-D is the most common antibody in Rh D negative mothers who had not been given RhIg Immunoprophylaxis. Also, it has been found that even Rh D positive mothers developed red cell alloimmunization. Hence, as a routine it is essential to type and screen antibodies against red cells antigens in all antenatal mothers irrespective of Rh D status.
55
With the advent of advanced IUT (Intrauterine transfusion) technologies and non-invasive investigative procedures like middle cerebral artery peak systolic velocity to predict intrauterine hemolysis, it is imperative to do antenatal red cell antibody screening to save not only fetal/newborn but also alloimmunized mothers who may be in dire need of suitable compatible blood during delivery.
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