Occurrence of erythrocyte alloimmunization in women attending antenatal care clinic

Heeya Gupta; Anuradha Kusum; Manish Raturi

doi:10.4103/gjtm.gjtm_42_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 7 | Issue : 2 | Page : 164-168

Occurrence of erythrocyte alloimmunization in women attending antenatal care clinic

Heeya Gupta¹, Anuradha Kusum¹, Manish Raturi²
¹ Department of Pathology, Himalayan Institute of Medical Sciences, Swami Rama Himalayan University, Jolly Grant, Dehradun, Uttarakhand, India
² Department of Immunohematology and Blood Transfusion, Himalayan Institute of Medical Sciences, Swami Rama Himalayan University, Jolly Grant, Dehradun, Uttarakhand, India

Date of Submission	18-May-2022
Date of Decision	18-Aug-2022
Date of Acceptance	02-Sep-2022
Date of Web Publication	5-Nov-2022

Correspondence Address:
Manish Raturi
Department of Immunohematology and Blood Transfusion, Himalayan Institute of Medical Sciences, Swami Rama Himalayan University, Jolly Grant, Dehradun, Uttarakhand
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/gjtm.gjtm_42_22

Abstract

Background and Objectives: There is limited literature available on the alloimmunization rates among pregnant women in India. Our primary aim was to estimate the occurrence of irregular erythrocyte alloantibody (IEA) immunization amongst women attending our antenatal care (ANC) clinic and to guide our obstetricians for improved ANC as well as their fetal outcomes. Methods: A cross-sectional study was performed at the blood center in North India over 12 months. We recruited a total of 212 study participants. Antibody screening and identification were performed by using semi-automated column agglutination technology and commercially available 3-red cell antibody screen panel with reagent cards having anti-human globulin (AHG) (Ortho Clinical Diagnostics Inc., USA) and 11-red cell antibody identification panel with reagent cards having AHG (Resolve Panel A; Ortho Clinical Diagnostics Inc., USA) respectively. Once identified, the semi-quantitative doubling dilutions, i.e., serial titrations of the respective IEA were performed by the conventional tube technique to assess their potency. The association of various antibodies identified was analyzed using the Pearson's Chi-square test. Results: Of the 7248 pregnant women attending our ANC clinics, the antibody screening was requested for 212 cases. Out of these, 10.8% (n = 23/212) of women were positive and were sensitized both by active and passive immunization. Their average age (in years) was 27.6 ± 3.52 (20 to 34). The majority belonged to the Dehradun district of Uttarakhand. ABO system-wise, the most common type amongst the included subjects was A as recorded in 34.4%, followed by B (30.18%), O (24.12%) and AB (11.3%), respectively. Rh system-wise, nearly 47% (n=100/212) of subjects had Rh (D) negative blood group. Interestingly, amongst these 23 ANC cases with positive IAT, nearly 13% had a history of having received a blood transfusion. Conclusion: To sum up, the screening of majority of ANC in our hospital is restricted to Rh (D) negative pregnancies. Perhaps, more awareness among obstetricians regarding the universal screening of all ANC cases irrespective of their Rh (D) status is the need of the hour.

Keywords: Antenatal care, Maternally derived antibodies, Blood group systems, Blood transfusion center, Red cell alloimmunization, RhD, Uttarakhand

How to cite this article:
Gupta H, Kusum A, Raturi M. Occurrence of erythrocyte alloimmunization in women attending antenatal care clinic. Glob J Transfus Med 2022;7:164-8

How to cite this URL:
Gupta H, Kusum A, Raturi M. Occurrence of erythrocyte alloimmunization in women attending antenatal care clinic. Glob J Transfus Med [serial online] 2022 [cited 2023 Mar 28];7:164-8. Available from: https://www.gjtmonline.com/text.asp?2022/7/2/164/360485

Introduction

According to the guidelines the British Committee for Standards in Hematology, all pregnant women attending antenatal care (ANC) clinics should be ABO and Rh (D) antigen typed and screened for the presence of irregular erythrocyte alloantibodies (IEA) either early in pregnancy or at 28 weeks of gestation.^[1] However, such a guideline is rarely followed in developing countries such as India. Therefore, limited published literature is available on the occurrence of these IEA (1.3%1.5%) from India.^[2],[3] Further, immune-mediated hemolysis of fetal and newborn's red cells, due to blood group isoimmunization is the leading cause of hemolytic disease of fetuses and newborn (HDFN).^[4] Various studies on HDFN have shown that the prevalence of red cell alloantibodies other than Rhesus (Rh)-D is 0.15%2.2%.^[5],[6] Some of these red cell alloantibodies do not have a significant impact during pregnancy, while others may cause HDFN varying from clinically insignificance to intrauterine fetal death as early as 17 weeks of gestation.^[7],[8] Hence, it is crucial to identify such at-risk pregnancies as early as possible.

Aims and objectives

The role of transfusion medicine specialists (TMS) and/or immunohematologists is to detect and identify these IEA and provide clinical guidance to obstetricians regarding their impact during pregnancy.^[9] This remains a major challenge both for obstetricians and the TMS to tackle these IEA-immunized pregnancies.^[10] With this background, we aimed to estimate the occurrence of maternal IEA immunization to red blood cell (RBC) antigens for our population and consequently guide our obstetricians for better ANC and fetal outcomes.

Materials and Methods

Study design

A cross-sectional study was designed to determine the frequency of IEA immunization among the women attending our ANC clinic. It was planned by the department of pathology in association with the department of immunohematology and blood transfusion with effect from January 2020 to December 2020 at our tertiary care blood transfusion center (BTC) located in the Dehradun District of Uttarakhand, North India.

Ethics

Ethical clearance was sought and obtained from our Institutional Ethical Committee (IEC No: SRHU/HIMS/ETHICS/2021/21 dated 14/10/21) before commencing the study.

Study participants

The study was conducted on all the ANC mothers irrespective of their period of gestation and obstetric history and written consent was obtained from all the study participants. For each ANC mother, a thorough review was conducted regarding their medical history, obstetric history (including any stillbirths, abortions, medical termination of pregnancy, and cases of HDFN among siblings), blood group and their husband's blood group. Any history of blood transfusion/s was also recorded before taking their blood samples.

Laboratory investigations

A 3-5mL plain clotted blood sample was received for each ANC mother whose antibody screening (AS) was requested. A 2mL blood sample in EDTA was also collected to determine the blood group. All the immunohematological (IH) work-up had been performed as per the flowchart [Figure 1]. All the samples were centrifuged at 3000 rpm for 3 min before the tests. The cell and serum blood grouping was performed in the Ortho working station (Ortho Clinical Diagnostics, New Jersey, USA) using the EDTA sample. In case of any discrepancies, it was resolved with the conventional tube technique (CTT) and other IH tests by the standard operating procedure (SOP) followed in our department. AS and identification were performed using semi-automated column agglutination technology (CAT) and commercially available 3-red cell antibody screen panel with reagent cards having anti-human globulin ([AHG] Ortho Clinical Diagnostics Inc., USA) and 11-red cell antibody identification panel with reagent cards having AHG (Resolve Panel A; Ortho Clinical Diagnostics Inc., USA) respectively. Laboratory monitoring for the severity of HDFN was determined by antibody titration at regular intervals by CTT per our SOP, while clinical monitoring was done by Doppler ultrasound estimating the middle cerebral artery peak systolic velocity. Further, the “clinically significant” antibodies to RBC antigens were usually adjudged by their capacity to either shorten the red cell survival by causing hemolytic transfusion reactions (HTR) or through their association with HDFN.^[11] In the laboratory, these clinically significant IEA were identified as those active at 37°C (in AHG phase) in the form of a positive indirect anti-globulin test (IAT). The newborn's group along with a direct anti-globulin test (DAT) was performed on the cord blood or neonatal venous blood samples. Only cell blood typing was done for the newborn using the CAT method. In case of a positive DAT result, elution studies were used to determine the specificity of the antibody. The newborns were evaluated for HDFN both by clinical and laboratory parameters.

Figure 1: Flowchart for ANC workup. ANC: Antenatal care

Click here to view

Statistical methods

Data were tabulated into an SPSS statistical software data sheet version 25 (IBM Statistics, Armonk, New York, 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 gravida status, gestational age and blood transfusion were carried out using a chi-square test. All the statistical analyses were carried out at a 5% level of significance and a p-value <0.05 was considered as statistically significant.

Results

During the study period of 12 months, a total of 7248 women attended the ANC clinic and red-cell AS was requested for 212 subjects. Of these 212 cases, 23 women were found to have been sensitized either with active or passive immunization. The mean age of the mothers (± standard deviation; range) was 27.6 years (± 3.52; 20 to 34). The majority were from the Dehradun district of Uttarakhand. The majority (47.6%) of mothers were in the age group of 21-30 years and 18.3% of mothers were above 30 years. ABO blood group-wise, Group A was the most common blood type observed in the mothers, 34.4%. Among the 212 mothers, 112 (53%) were Rh (D) positive and 100 (47%) were Rh (D) negative, which depicted that Rh (D) negative cases were screened more regularly and frequently than Rh (D)-positive ones. Of all the study subjects with a positive IAT (n = 23), 13% (n = 3/23) of the study subjects had a history of transfusion. On the other hand, around 2% (n = 4/189) of the study subjects with negative IAT had a history of having received a blood transfusion in the past. Of the total study subjects with identified alloantibodies, 82.7% (n = 19/23) subjects had allo-anti Rh (D) antibody followed by 13% (n = 3/23) and 4.3% (n = 1/23) of clinically insignificant allo-anti Le^a and allo-anti Le^b, respectively. Among the 19 anti-Rh (D) alloimmunized cases, barring the rest, there was one interesting case of an Rh (D) negative ANC mother whose minimum and maximum allo-anti-(D) antibody titers were reported as 1:8 and 1:16 with the progression of her pregnancy from 28 to 30 weeks, respectively. Since allo-anti-(D) titer of 32 or more is usually considered significant; despite rising titers, these were adjudged as clinically insignificant titers. Furthermore, the fetal outcome was good in this case. Our study reported a statistically insignificant difference between the age-group-wise distribution and identified IEA immunization [P = 0.49; [Table 1]]. The gravida status among antenatal mothers ranged from 1 to 6. Among them 73.9% (n = 17/23) mothers were multigravida and 26.1% (n = 6/23) were primigravida. Among 212 cases, around 15% (n = 32/212) developed fetal complications.

Table 1: Irregular erythrocyte alloantibody immunization identified among the ANC cases in an age-wise manner

Click here to view

Discussion

ANC services in India are fragmented and lack uniformity. In fact, there is a limited published data on the IEA immunization rates among pregnant women in India. Although guidelines for screening have been laid down by the Drug Controller General, India,^[1],[12] screening for alloantibodies is usually done largely for Rh(D) negative women or those presenting with an adverse obstetric history. With an aim to determine the frequency of IEA among antenatal women, the present cross-sectional study recruited a total of 212 study subjects. Proper periodic screening of antenatal mothers for IEA will make the physicians observant of the development of HDFN. This clinical strategy will also help the BTCs, to ensure timely availability of antigen-negative blood, if needed, especially in ANC mothers who are already sensitized with rare allo-antibodies.^[10]

De Vrijer et al.^[13] included 2392 subjects, whereas Pahuja et al.^[3] and Bondagji^[14] included 3577 and 24005 subjects, respectively. In more recent studies by Varghese et al.,^[15] Dholakiya et al.,^[16] and Al-Dughaishi, et al.^[17] recruited 5347, 8920, and 1251 subjects, respectively, in their studies. These studies included a large number of study subjects as compared to the 212 subjects only in the current study. According to the age-wise distribution of subjects in the present study, the majority were in the age group between 25 and 30 years (47.6%). However, the least number of the study subjects were noticed in the age group of more than 30 years (18.3%). In fact, triggers for IEA during pregnancy are not well-understood beyond the presence of a feto-maternal bleed. Apart from extrinsic ones, an important inherent biological factor could rather possibly be the age of ANC mothers which could make them either responders or nonresponders. In fact, none of the other published literature mentions a correlation of age distribution with the presence of IEA unlike our current research, wherein, an age-wise distribution and correlation become justifiably relevant for understanding the probable isoimmunisation due to age in ANC mothers. Further, about the major blood group systems (ABO and Rh), the most common phenotype in our study was A Rh (D) positive. In comparing our study, Pahuja et al., had B Rh (D) positive blood group as the most common phenotype.^[3] Moreover, there were 53% of the study subjects which are Rh (D) positive women, whereas almost 47% of women were Rh (D) negative in the present study. We found a significantly higher percentage of Rh (D)-negative blood types were sent to us for work-up and this was mainly due to the biased selection of Rh (D) negative pregnancies by our obstetricians. Furthermore, in the study by Pahuja et al., of the total subjects (n = 3,577), 88.9% of the women were Rh (D) positive and 11% of women were Rh (D) negative. On the other hand, the findings by Bondagji^[14] reported that the most common ABO blood group encountered was O followed by blood groups A, B, and AB type. Moreover, in the study carried out by Varghese et al.,^[15] screening was performed on 5347 women, of whom 6.34% were Rh (D) negative.

Furthermore, in the study by Pahuja et al.,^[3] around 51 cases were allo-immunized, whereas, the studies by Al-Dughaishi et al.^[17] and Dholakiya et al.^[16] showed 35 and 39 cases with alloimmunization, respectively. Our study had 23 allo-immunized subjects, of whom 95.7% (n = 22/23) showed active immunization. Of the total subjects in our study, the history of anti-D immunoglobulin injections (passive immunization) was recorded in only 4.3% (n = 1/23) of the mothers. Further Bondagji^[14] have stated in their study that there was a dramatic reduction in the IEA rate to merely 2% with routine postpartum administration of a single dose of anti-D immune globulin and further reduction to as low as 0.1% with the addition of routine ANC administration, particularly in the third trimester. A majority of the study subjects in our population had healthy fetal outcomes accounting for 85% (180/212). Only 15% (32/212) cases had IUD and abortion with no causal association with IEA iso-immunization. However, multiple congenital anomalies were recorded in one of the subjects. In the study by Pahuja et al.,^[3] an adverse obstetric history (any history of stillbirth, abortion, or medical termination of pregnancy) was present in 65% of patients with anti-D (26/40) and in 66.7% of patients with combined anti-C and anti-D (4/6). Further in their study, Pahuja et al. showed a statistically significant correlation between the frequency of IEA and adverse obstetric history (P < 0.001, odds ratio = 11.10, 95% confidence interval = 5.75-21.64), which means that the odds of an antibody-positive woman having an adverse obstetric history was more than 10 times higher than women who were antibody negative, whereas Al-Ibrahim and Al Saeed found an alloimmunization rate of 7.1% among pregnant women in Saudi Arabia.^[18] In comparison to our overall alloimmunization rate of 10.8%, various similar studies from India^[2],[3],[15] have shown a comparative rate of alloimmunization, i.e., 9.43 – 12.8% among Rh (D) negative pregnancies [Table 2]. The gravida status of women also showed a statistically significant, positive correlation with alloantibody formation. However, no such correlation between gravida status and fetal outcome was noted in our study. The study done by Bondagji^[14] showed a medical history of the 424 antibodies-positive cases, the vast majority of them were multiparous women (92.6%) and only 8.4% were primigravida. Nineteen patients out of the 424 (4.5%) reported a history of blood transfusion, one patient had a previous ectopic pregnancy, and 170 (40%) had a history of previous miscarriages. The present study also reported an interesting case of an Rh (D) negative ANC mother whose minimum and maximum allo-anti-(D) antibody titers were reported as 1:8 and 1:16 with the progression of her pregnancy from 28 to 30 weeks, respectively. Since allo-anti-(D) titer of 32 or more is usually considered significant; despite rising titers, they were adjudged as clinically insignificant titers. Furthermore, the fetal outcome was good in this case. On the other hand, the findings by Al-Dughaishi et al.^[17] reported that despite the maximum titers being 1:256, in the actively sensitized women, there was no fetus with hydrops in their study. Our study supports the existing evidence on the importance of implementing routine AS in all ANC cases irrespective of their Rh (D) status. Two major limitations of our research were that out of 7248 women only 212 ANC subjects underwent AS, and therefore, few IEA may have been missed especially in Rh (D) positive women. Furthermore, the ABO and Rh (D) blood group distribution may have been skewed for the same reasons.

Table 2: List of various studies depicting the rate of irregular erythrocyte alloantibody immunization in antenatal care cases

Click here to view

Conclusion

To sum up, in a developing country like India, extra efforts should be put by the respective BTCs in collaboration with the hospital transfusion committee to sensitize the obstetricians about the need and obvious benefits of performing AS in all antenatal cases irrespective of the Rh (D) antigen status.

Financial support and sponsorship

This study was supported by a postgraduate student research grant received from Swami Rama Himalayan University, Dehradun Uttarakhand (Grant number: SRHU/Reg/Int/2020-80(4) dated February 22, 2020).

Conflicts of interest

There are no conflicts of interest.

References

1.	British Committee for Standards in Haematology Blood Transfusion Task Force, Gooch A, Parker J, Wray J, Qureshi H. Guideline for blood grouping and antibody testing in pregnancy. Transfus Med 2007;17:252-62.
2.	Suresh B, Babu KV, 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.
3.	Pahuja S, Gupta SK, Pujani M, Jain M. The prevalence of irregular erythrocyte antibodies among antenatal women in Delhi. Blood Transfus 2011;9:388-93.
4.	Maheshwari A, Carlo WA. Hemolytic disease of the newborn (Erythroblastosis fetalis). In: Kliegman RM, Stanton BF, Schor NF, St. Geme JW 3^rd, Behrman RE, editors. Nelson Textbook of Pediatrics. 19^th ed. New Delhi: Thomas Press India Ltd.; 2012. p. 615-9.
5.	Natukunda B, Mugyenyi G, Brand A, Schonewille H. Maternal red blood cell alloimmunisation in south western Uganda. Transfus Med 2011;21:262-6.
6.	Gottvall 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.
7.	Poole J, Daniels G. Blood group antibodies and their significance in transfusion medicine. Transfus Med Rev 2007;21:58-71.
8.	Rai D, Raturi M. Hemolytic disease of the newborn due to anti-rhesus (E) alloimmunization. J Clin Anat Pathol 2020;5:1-3.
9.	Jeremiah ZA, Mordi A, Harcourt P. Evaluation of the clinical utility of maternal alloantibody screening as a surrogate to antiglobulin crossmatch procedures in resource limited settings. Int J Blood Transfus Immunohematol 2011;1:1-6.
10.	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;63:241-6. [PUBMED] [Full text]
11.	Makroo RN, Arora B, Bhatia A, Chowdhry M, Luka RN. Clinical significance of antibody specificities to M, N and Lewis blood group system. Asian J Transfus Sci 2014;8:96-9. [PUBMED] [Full text]
12.	Saran RK. Hemolytic Disease of the New Born (HDN). In: Transfusion medicine technical manual DGHS. 2nd edition. Ministry of Health and Family Welfare; 2003. Govt. of India, New Delhi. p. 179.
13.	de Vrijer B, Harthoorn-Lasthuizen EJ, Oosterbaan HP. The incidence of irregular antibodies in pregnancy: A prospective study in the region of the 's-Hertogenbosch. Ned Tijdschr Geneeskd 1999;143:2523-7.
14.	Bondagji NS. Rhesus alloimmunization in pregnancy. A tertiary care center experience in the Western region of Saudi Arabia. Saudi Med J 2011;32:1039-45.
15.	Varghese J, Chacko MP, 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. [PUBMED] [Full text]
16.	Dholakiya SK, Bharadva S, Vachhani JH, Upadhyay BS. Red cell alloimmunization among antenatal women attending tertiary care center in Jamnagar, Gujarat, India. Asian J Transfus Sci 2021;15:52-6. [Full text]
17.	Al-Dughaishi T, Al Harrasi Y, Al-Duhli M, Al-Rubkhi I, Al-Riyami N, Al Riyami A, et al. Red cell alloimmunization to rhesus antigen among pregnant women attending a tertiary care hospital in Oman. Oman Med J 2016;31:77-80.
18.	Al-Ibrahim N, Al Saeed AH. Red blood cell alloimmunization among Saudi pregnant women in the central province of Saudi Arabia. Kuwait Med J 2008;40:116-23.