Indian Journal of Pathology and Microbiology

: 2020  |  Volume : 63  |  Issue : 2  |  Page : 241--246

Frequency and clinical significance of red cell antibodies in pregnancy – A prospective study from India

Soumya Das1, Shamee Shastry1, Lavanya Rai2, Poornima B Baliga1,  
1 Department of Immunohematology and Blood Transfusion, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Obstetrics and Gynaecology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India

Correspondence Address:
Shamee Shastry
Department of Immunohematology and Blood Transfusion, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka


Background: For appropriate management of hemolytic disease of the fetus and newborn (HDFN), it is important to detect irregular red cell antibody in the antenatal period. Though it is a simple one-step method, it is not part of routine antenatal screening in many developing countries. To reiterate the importance of antenatal antibody screening, we have assessed the frequency and clinical significance of irregular red cell antibodies in our patient population. Materials and Methods: A prospective study was carried out from October 2013 to May 2015 at a tertiary care center from south India. All antenatal samples received by the laboratory for red cell antibody screening were screened using a commercial three-cell screening panel. Antibody identification along with further Immunohematological techniques as required were performed for cases with positive screening results. Neonates of the alloimmunized cases were followed up to determine the clinical significance of the antibody. Results: A total of 2336 antenatal mothers were screened for red cell antibodies. The overall rate of alloimmunization in the study group was 2.27%. Alloimmunization rate among RhD-negative pregnancies was 6.9%. Other than anti-D (49%), we identified anti-D + anti-C (5%), anti-G (5%), anti-c (5%), anti-E (2%), anti-e (2%), anti-H (Bombay phenotype) (7%), anti-M (2%), anti-Lea (2%), anti-Leb (12%), and autoantibodies (9%) in the maternal serum. Anti-D, anti-D + anti-C, anti-G, anti-c, and anti-H were found to be clinically significant in this study. Conclusion: This study showed that 1 in 125 RhD-positive pregnancies can develop red cell alloantibodies. Hence, implementing routine antenatal antibody screening irrespective of RhD status is essential.

How to cite this article:
Das S, Shastry S, Rai L, Baliga PB. Frequency and clinical significance of red cell antibodies in pregnancy – A prospective study from India.Indian J Pathol Microbiol 2020;63:241-246

How to cite this URL:
Das S, Shastry S, Rai L, Baliga PB. Frequency and clinical significance of red cell antibodies in pregnancy – A prospective study from India. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Jul 15 ];63:241-246
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Full Text


Immune-mediated hemolysis of fetal and newborn's red cells due to blood group incompatibility is one of the main causes of hemolytic disease of fetus and newborn (HDFN).[1] Managing red cell alloimmunized pregnancies is still a challenge for obstetricians and transfusion specialists. More than 60 different red blood cell (RBC) antigens are capable of eliciting an antibody response, and anti-D is the commonly reported one.[2] Since the advent and routine administration of RhIg in RhD-negative pregnancies, the prevalence of alloimmunization to D antigen is reduced.[3],[4] However, it is still a common practice in India to screen and monitor only RhD-negative antenatal mothers for alloimmunization. The overall prevalence of antibodies to red cell antigens other than “D” antigen varies from 0.5% to 2.2%. Some of these red cell alloantibodies do not have a significant impact during pregnancy, while others may cause HDFN and its incidence is about 1 in 500 pregnancies.[4],[5],[6] Management of an alloimmunized pregnancy is still a challenge to the obstetricians and immunohematologists. It is crucial to identify such at-risk pregnancies as early as possible. And such cases can be referred to a tertiary care center for appropriate diagnosis and treatment.[7] Proper periodic screening of antenatal mothers for irregular antibodies will make the clinician observant for the development of HDFN. This strategy will also help the transfusion center to ensure timely availability of antigen negative blood, if needed, especially in antenatal mothers with rare antibodies.

The prevalence of red cell antibodies varies from population to population, and there are very limited data on the subject from the southern part of India. The purpose of this study is to evaluate the frequency of maternal alloimmunization to red cell antigens in our population and to study their clinical significance during the antenatal period and in the newborn.

 Materials and Methods

Study participants

We conducted a prospective study in the Department of Immunohaematology and Blood Transfusion, from October 2013 to May 2015 at our tertiary care center from south India. Ethical clearance was obtained from the Kasturba Hospital, Manipal Institutional Ethical Committee No. IEC 353/2013. The study included 2336 antenatal mothers. An informed written consent was obtained from study participants. The patients' medical history, obstetric history [including any stillbirths, abortions, medical termination of pregnancy (MTP), and cases of HDFN among siblings], blood group, and history of blood transfusions were recorded before taking the blood samples.

Laboratory investigations and clinical significance of detected alloantibodies

Initial screening was performed during the visit in the first trimester and a repeat test during the 28th week of gestation. Blood samples in a plain vial (5 mL) and EDTA vial (2 mL) were collected in the antenatal clinic for immunohematological workup as shown in [Figure 1]. The blood grouping was performed using automated column agglutination technology (Ortho Auto-Vue® Innova System – Ortho Clinical Diagnostics) using the EDTA sample. The plain samples were used for red cell antibody screening, using a commercial three-cell panel (BioRad ID, Micro Typing System, Switzerland). Antibody identification along with further immunohematological techniques as required were performed on the samples with positive screening results. The quantification of the antibody was performed by the double dilution technique during the follow-up period. The noninvasive methods for monitoring HDFN in the fetus were also performed by the obstetricians during the follow-up period, which included ultrasound and middle cerebral artery doppler whenever needed. We have adopted the existing definition of “clinically significant antibody,” where an antibody is considered clinically significant when it is capable of causing shortened red cell survival, leading to hemolytic disease of fetus or newborn.[8] All the cases were followed up to know the outcome and determine the clinical significance of the antibody whenever the patient is isoimmunized.{Figure 1}

The immunohematological workup on the newborn samples of the mother with an alloantibody included blood group (cell grouping) along with direct antiglobulin test (DAT) using column agglutination technology (CAT) (Bio-Rad LISS/Coombs anti-IgG + C3d ID cards, Switzerland). Heat elution studies were performed to confirm the specificity of the antibody. All laboratory investigations were carried out as per the standard guidelines and departmental standard operating procedures (SOP). All the methods carried out as part of this prospective study were performed by following the relevant guidelines and regulations. The newborns were evaluated for HDFN by the clinical and laboratory parameters.

Statistical methods

Data were analyzed using SPSS statistical software data sheet (version 14.0, USA). Data were expressed using descriptive statistics such as frequencies and percentages. The association between the presence of antibodies and other study parameters such as abortions, gravidity status, gestational age, and blood transfusion was carried out using the Chi-square test. All the statistical analyses were carried out at 5% level of significance and a P value <0.05 was considered significant.


During the study period, a total of 2336 samples from antenatal cases were screened for red cell antibodies. The median age of the mothers 28 years (range 18–42 years). The majority (72.2%) of mothers were in the age group of 21–30 years and 24.1% of mothers were above 31 years. The gravida status among antenatal mothers ranged from 1 to 13, which included pregnancies with full-term deliveries and abortions. Among them, 1305 (56%) mothers were multigravida and 1031 (44%) were primigravida. Among 2336 antenatal mothers, 1693 (72.5%) had no abortions, while 643 (27.5%) had history of abortion and 60 patients (2.6%) gave a history of blood transfusion. Out of total 2336 cases, 67.3% cases were booked cases in the hospital, while the rest were referred from other hospitals.

The most common blood group in the study group was O (40.5%), followed by B (28%), A (26.6%), and AB (4.7%), and four mothers (0.2%) had Bombay phenotype (Oh). Among the 2336 samples received from the antenatal clinic, 1826 (78.2%) were RhD-positive and 510 (21.8%) were RhD-negative. RhD immunoprophylaxis was given in the antenatal period to 66 (13%) of the RhD-negative cases in our study group. Antibody screening was positive in 86 mothers (3.7%) as shown in [Table 1].{Table 1}

Active immunization was seen in 53 (2.27%) antenatal mothers, while the rest were passively acquired anti-D. These 33 cases of passive immunization were excluded while calculating the frequency of red cell antibodies. Anti-D (49.1%) was the most common antibody identified followed by anti-Leb (12.3%). The antibody profile of the study group and its clinical significance is shown in [Table 2]. Alloimmunization rate was significantly higher among patients with Rh D negative phenotype (6.9%) compared with the group with Rh D positive blood group (1.1%). Similarly, gravida status had significantly influenced the alloimmunization rate. The prevalence of antibodies among multigravida was 2.8% compared with 1.6% in primigravida. The rate of alloimmunization to RBC antigens were significantly higher (P < 0.05) in case of mothers with a history of invasive procedures and blood transfusion (0.18% and 0.36%), respectively. We had also noted a higher rate of alloimmunization (0.85%) among mothers with a history of abortion; however, the difference was statistically not significant.{Table 2}


Management of alloimmunized pregnancies is still a challenge for obstetricians and transfusion medicine specialists. Periodic screening of antenatal mothers for irregular antibodies will make the physician observant for the development of HDFN and would help in an early referral of such women to a tertiary care center for appropriate management.[7] This strategy will also help the transfusion center to ensure timely availability of antigen-negative blood, if needed, especially in antenatal mothers with rare antibodies. There are limited data on alloimmunization rates among pregnant women in India, where the routine antenatal screening for red cell antibodies is not implemented universally.[9],[10] Antenatal antibody screening in India is mainly focused on detection of anti-D in RhD-negative mothers and there are limited data in the literature on the prevalence of antibodies other than anti-D in the antenatal period.[9],[11] This study showed that 1 in 125 RhD-positive pregnancies were alloimmunized, requiring special intervention for the management of HDFN. Hence, formulating guidelines for universal antibody screening for pregnant women in India is the need of the hour.

In this study, 21.8% of the cases had a Rh D negative blood group, unlike the general population in south India, where the frequency of RhD-negative group is 5%.[12] This discrepancy in the prevalence of RhD-negative phenotype between the study population and the general population is mainly because of our hospital being a tertiary care referral center receives a majority of the RhD-negative pregnancies from peripheral centers. We have noted that obstetricians refer all the cases of RhD-negative pregnancy for antibody screening, whereas it is considered as an optional test in patients with the Rh D positive blood group. Similar to our study, Al-Ibrahim andAlSaeed reported a higher prevalence of RhD-negative (14%) cases in their study.[13] In Riyadh region in Saudi Arabia especially the central province, 14.14% of the population is RhD-negative.

Overall, the alloimmunization rate in this study was 2.27%, and 6.9% among Rh-negative pregnancies, which is higher than the other studies mentioned in [Table 3]. The reason for this higher rate of alloimmunization could be due to selection bias that exists in our study as the setup is a tertiary care referral center.{Table 3}

The published literature gives a very diverse rate of alloimmunization among RhD-negative pregnancies ranging from 6.9% to 12.8%.[5],[11],[14] The U.S. National Center for Health Statistics in 2003 reported 6.8 cases of Rh alloimmunization per 1000 live births.[15] In this study, anti-D (49%), either alone or in combination, was the most common antibody identified among the antenatal mothers. Among the total number of alloimmunized anti-D pregnancies, 50% of the newborns have features of moderate HDFN managed with phototherapy and had a successful outcome. Administration of RhIg to RhD-negative pregnant women is the cornerstone for prevention of alloimmunization to D antigen and subsequent prevention of HDFN. It is a very safe and effective therapy. Revised guidelines of BCSH in 2014 summarize “administration of anti-D Ig, following potentially sensitizing events, as soon as possible and always within 72 h of the sensitizing event.”[16] This regimen is recommended by the obstetric professional societies and serves as the standard of care in many countries.[16],[17] In our study, only 12.9% of the RhD-negative antenatal mothers had received Rh immunoprophylaxis. Despite established guideline by regulatory bodies like the Federation of Obstetric and Gynaecological Societies of India (FOGSI), the use of anti-D prophylaxis in all enlisted sensitizing events such as abortions, ectopic pregnancy, and MTP, as low as 10%–20%.[18] The lack of uniform and universal anti-D immunoprophylaxis is the reason for this higher rate of alloimmunization in India.[5],[11],[14] While in the developed countries, even with immunoprophylaxis, anti-D is still a leading offender, as shown by Gottvall et al. (60%) and Howard et al. (42%).[19] There are several reasons why anti-D still is a common cause of HDFN.[20],[21] D antigen typing errors, inaccessibility to anti-D prophylaxis, the insufficient dose of Rh Ig, or immunization secondary to transfusion of blood products are some of the causes. The need of the hour is to focus on the importance of anti-D immunoprophylaxis.

The rate of alloimmunization among RhD-positive patients was 1.1% in this study. This rate of alloimmunization was higher when compared with previous studies [Table 4]. Whereas the general profile of clinically significant antibodies is in concordance with other studies from India.[5],[11],[14] As discussed earlier, our patient population consists of a significant number of high-risk cases, which explains the higher rate of alloimmunization even among Rh-positive pregnancies.{Table 4}

The factors adversely affecting the alloimmunization were multigravida status, previous abortions, blood transfusion, and invasive procedures done during the antenatal period. And this finding is in concordance with published literature.[9],[11] Koelewijn et al. showed that parity is one of the independent risk factors for alloimmunization during pregnancy. They also described that blood transfusion especially the RBC component is the most important independent risk factor for non-D alloimmunization during pregnancy.[22] In this study, a significant number (13%) of antenatal mothers with a history of blood transfusion were alloimmunized. Non-D alloantibodies accounted for 75% of cases. The guidelines recommend transfusion of RhD-negative blood components (PRBC, platelet concentrate) for all RhD-negative women in childbearing age. In case D-positive blood products have been transfused, the appropriate dose of Rh- immunoglobulin should be administered.[23] Use of RhcDE and K-compatible blood in premenopausal women is also mentioned, as a preventive measure for severe HDFN caused by anti-c and anti-K and to a lesser extent anti-E.[4] A study from the Netherlands has shown that K-antigen matched blood transfusion in premenopausal women has reduced the incidence of K-alloimmunized pregnancy.[24]

Apart from anti-D, the other clinically significant antibody causing HDFN included anti-C in combination with anti-D, anti-G, anti-c, and anti-H, respectively. In our study, mothers, with dual antibodies specificity, that is, anti-D + anti-C, showed features of moderate to severe HDFN. Alloimmunization with a combination of three or four antibodies is very rare. A study by Nordvall et al. showed 27% of cases with multiple antibodies, among which the majority (67%) had anti-D as one among the many antibodies.[25] It is relevant to assess whether only anti-D is present or another antibody is also present with it. We also noted similar finding during the study period. Severely affected newborn, in spite of receiving exchange transfusion, failed to survive. While the newborns with moderate HDFN could be managed conservatively with phototherapy.

Identification of anti-G helped us in administering RhIg in the mothers to prevent further isoimmunization to D, which causes severe hemolytic disease in newborns. The clinical significance of anti-G alone remains controversial.[26] We observed that one newborn required both exchange transfusion and phototherapy, while another could be managed with phototherapy only.

Anti-C is the next most common antibody belonging to the Rh family to cause severe HDFN. In this study, one of the pregnancies alloimmunized to C antigen, showed no features of HDFN, whereas moderate features of HDFN were seen in other pregnancies which were managed conservatively with phototherapy. A study done by Rath et al. showed that the postnatal outcome of neonates with severe HDFN due to C antigen is similar to HDFN due to RhD.[27] BCSH guidelines recommend to retest women with allo-anti-C in a similar manner as followed for anti-D, that is, at least monthly till 28 weeks of gestation and every 2 weeks thereafter. Quantification of anti-C is useful for monitoring and predicting the severity of HDFN.[28] All the patients were managed in accordance with the guidelines as mentioned above.

Bombay phenotype is a rare blood group with a prevalence of 0.004%–0.005% in the south Indian population. The anti-H is present in both IgM and IgG forms. There are very few reports on Oh phenotype mother, with hemolytic disease of the newborn.[29],[30] In our study, four antenatal mothers with Bombay blood group were identified. However, one newborn had features of the moderate hemolytic disease, due to anti-A, as described by Shastry et al., whereas anti-H was implicated in causing HDFN in a newborn with an 'O' blood group.[30] HDFN due to ABO and H antigen generally have a mild course of disease due to the weak expression of the antigens on RBCs in utero and in neonates. Another major factor is neutralization or absorption of anti-H antibodies by the soluble substances in plasma on to the surface of all the cell of the body expressing H antigen.[30]

A timely diagnosis of alloimmunization in an antenatal case will ensure better patient care and outcome. This study supports the existing evidence on the importance of implementing routine antenatal antibody screening in all the cases irrespective of the Rh status.

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Conflicts of interest

There are no conflicts of interest.


1Murray NA, Roberts IA. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007;92:83-8.
2Maheshwari A, Carlo WA. Hemolytic disease of the newborn (Erythroblastosis fetalis). In: Kliegman RM, Stanton BF, Schor NF, Geme III JW St., Behrman RE, editors. Nelson Textbook of Pediatrics. New Delhi: Thomas Press India Ltd; 2012. p. 615-9.
3Klein HG, Anstee DJ. Haemolytic transfusion reactions. In: Mollison's Blood Transfusion in Clinical Medicine. West Sussex, UK: John Wiley & Sons, Ltd.; 2014. p. 458-98.
4Haas M de, Thurik FF, Koelewijn JM, van der Schoot CE. Haemolytic disease of the fetus and the newborn. Vox Sang 2015;109:99-113.
5Basu S, Kaur R, Kaur G. Hemolytic disease of the fetus and newborn: Current trends and perspectives. Asian J Transfus Sci 2011;5:3-7.
6Poole J, Daniels G. Blood group antibodies and their significance in transfusion medicine. Transfus Med Rev 2007;21:58-71.
7Shanwell A, Sallander S, Bremme K, Westgren M. Clinical evaluation of a solid-phase test for red cell antibody screening of pregnant women. Transfusion 1999;39:26-31.
8Meny G. Determining the clinical signi fi cance of alloantibodies. ISBT Sci Ser 2015;10:39-43.
9Pahuja S, Gupta SK, Pujani M, Jain M. The prevalence of irregular erythrocyte antibodies among antenatal women in Delhi. Blood Transfus 2011;9:388-93.
10Wong KF, Tse KT, Lee AW, Mak CS, So CC. Is antenatal antibody screening worthwhile in Chinese? Br J Haematol 1997;97:917-9.
11Suresh B, Babu KVS, Arun R, Jothibai DS, Bharathi T. Prevalence of “unexpected antibodies” in the antenatal women attending the Government maternity hospital, Tirupati. J Clin Sci Res 2014;4:22-30.
12Periyavan S, Sangeetha SK, Marimuthu P, Manjunath BK, Seema DM. Distribution of ABO and rhesus-D blood groups in and around Bangalore. Asian J Transfus Sci 2010;4:41.
13Al-Ibrahim N, AlSaeed AH. Red blood cell alloimmunization among Saudi pregnant women in the central province of Saudi Arabia. Kuwait Med J 2008;40:116-23.
14Varghese J, Chacko M, Rajaiah M, Daniel D. Red cell alloimmunization among antenatal women attending a Tertiary care hospital in South India. Indian J Med Res 2013;138:68-71.
15Moise KJJ. Management of rhesus alloimmunization in pregnancy. Clin Expert Ser 2008;112:164-76.
16Qureshi H, Massey E, Kirwan D, Davies T, Robson S, White J, et al. BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn. Transfus Med 2014;24:8-20.
17Royal T, Zealand N. Guidelines for the use of Rh (D) immunoglobulin (Anti-D) in obstetrics in Australia. Blood 2010;1-3.
18The Federation of Obstetric and Gynaecological Societies of India [Internet]. Available from: [Last accessed on 2020 Jan 08].
19Gottvall T, Filbey D. Alloimmunization in pregnancy during the years 1992-2005 in the Central West region of Sweden. Acta Obstet Gynecol Scand 2008;87:843-8.
20Bennardello F, Curciarello G. Survey on the prevention and incidence of haemolytic disease of the newborn in Italy. Blood Transfus 2013;11:518-27.
21Koelewijn JM, Haas M De, Vrijkotte TGM, Schoot CE Van Der. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. Br J Obs Gynaecol 2009;116:1307-14.
22Koelewijn JM, Vrijkotte TG, de Haas M, van der Schoot CE, Bonsel GJ. Risk factors for the presence of non-rhesus D red blood cell antibodies in pregnancy. An Int J Obstet Gynaecol 2009;655-64.
23Parker J, Wray J, Gooch A, Robson S, Qureshi H. Guidelines for the use of prophylactic anti-D immunoglobulin British Committee for Standards in Haematology. Br Comm Stand Hematol [Internet] 2006;1-33. Available from: [Last accessed on 2020 Jan 08].
24Luken J, Schonewille H, Ligthart P, van der Schoot C, van der Bom J, Koopman M, et al. Preventive matching for K-antigen in female blood recipients under 45 years of age reduces the incidence of K-alloimmunisation in pregnancy. Vox Sang 2015;109:316.
25Nordvall M, Dziegiel M, Hegaard HK, Bidstrup M, Jonsbo F. Red blood cell antibodies in pregnancy and their clinical consequences: Synergistic effects of multiple specificities. Transfusion 2009;49:2070-5.
26Hadley AG, Poole GD, Poole J, Anderson NA, Robson M. Haemolytic disease of the newborn due to anti-G. Vox Sang 1996;71:108-1.
27Rath ME, Smits-Wintjens VEH, Lindenburg ITM, Folman CC, Brand A, Van Kamp IL, et al. Postnatal outcome in neonates with severe Rhesus c compared to Rhesus D hemolytic disease. Tranfusion 2013;53:1580-5.
28Gooch A, Parker J, Wray J, Qureshi H. Guideline for blood grouping and antibody testing in pregnancy. Transfus Med [Internet] 2007;17:252-62. Available from: [Last accessed on 2020 Jan 08].
29Bhattacharya S, Makar Y, Laycock RA, Gooch A, Poole J, Hadley A. Outcome of consecutive pregnancies in a patient with bombay (OH) blood group. Transfus Med 2002;12:379-81.
30Shastry S, Lewis L, Bhat SS. A rare case of haemolytic disease of newborn with Bombay phenotype mother. Asian J Transfus Sci 2013;7:141-3.