Rhesus alloantibody specificity and titers among alloimmunized pregnant women

INTRODUCTION

All antibodies to red cell antigens other than naturally occurring antibody A (anti-A) and anti-B are considered “irregular,” and they can either be alloantibodies directed toward non-ABO antigens or autoantibodies directed towards other red cell antigens.^[1] Pregnancy and blood transfusion are two major and most common risk factors for developing irregular antibodies (alloimmunization) to red blood cells (RBCs) in affected individuals. Irregular antibody screening in antenatal women contributes to the detection and monitoring of patients who are at the risk of delivering infants with hemolytic disease of the fetus and newborn (HDFN)^.[2] The most common irregular antibodies are of the Rhesus (Rh) origin.

HDFN, also called erythroblastosis fetalis, is a clinical condition that occurs as a result of hemolysis of fetal or neonatal RBCs due to the presence of maternal red cell immunoglobulin G antibodies that can cross the placenta.^[3] HDFN is characterized by fetal or neonatal anemia, hyperbilirubinemia, and sometimes, fatal hydrops fetalis or death and can occur in case of antigenic difference between maternal and fetal RBCs.^[4] Clinically significant red cell alloantibodies are those that have the potential to cause hemolysis of red cells bearing the corresponding antigen.

Hemolysis in newborn can be immune or nonimmune mediated. The nonimmune causes include α-thalassemia, RBC membrane, or enzyme defects. Immunomediated hemolysis of fetal red cells which is mainly due to blood group incompatibility, occurs when there is transplacental passage of maternal antibody active against paternal red cell antigen of the newborn and is the main cause of HDFN in them.^[5] More than 60 different RBC antigens are capable of eliciting an antibody response but significant morbidity is associated primarily with D antigen of Rh group. In routine antenatal visits, in our study environment, Rh antigen typing is rarely done and restricted to only D phenotype screening.^[1] This is partly due to unavailability of antisera: C, c, E, and e for specific identification of the corresponding involved antibody and lack of policy to this effect. This practice leaves women of childbearing age in our study environment, and similar settings are at higher risk of developing hemolytic disease of the newborn due to antibodies against these non-screened Rh antigens, which is at times fatal during their reproductive age.^[6]

Rh antibody detection and quantification can play a key role in pregnancy management during antenatal and mainly aid in the detection and monitoring of pregnancies at risk of delivering infants with HDFN.^[7] Investigations for the presence of irregular antibodies and its titers during pregnancy are important to calculate the risk of developing HDFN and deduce on the outcomes of the pregnancy.^[8] This study was thus undertaken to look for the prevalence of alloantibodies, their specificity, and titers in pregnant women during the antenatal period.

MATERIAL AND METHODS

A cross-sectional study was carried out on antenatal pregnant women attending antenatal clinics at Kagando Hospital and Mbarara Regional Referral Hospital from August 2020 to September 2021 where 1350 pregnant women were recruited. During this antenatal visit, ABO and RhD grouping were performed together with additional screening for alloantibodies. An informed consent was taken from the pregnant women/their husbands before the blood draw. Blood samples, 2 mL collected in ethylenediaminetetraacetic acid tube and 5 mL in the plain glass test tube, were collected for performing Rh grouping and indirect antiglobulin tests (IAT) for antibody screening. Titration of RhD alloantibody was also done on those found having the alloantibodies.

The Rh grouping, antibody screening, and titration were performed using the agglutination technology comprised of glass beads and reagent contained in a column of 75 percent packed gel to capture agglutinates in a semisolid medium of the Ortho Biovue ID-Micro Typing System (Ortho clinical diagnostics, 1001 US Highway 202, Raritan, NJ 08869 USA)^[9,10] together with commercial three red cells panel (Immucor, Inc. Norcross, USA) in IAT test. Samples which were screened positive for alloantibodies were stored at −80°C for further alloantibody identification performed by using a commercial 11 RBC panel (Panoscreen-10, a 12 vial set including an autocontrol, a buffered preservative diluents, Immucor, Inc. Norcross, USA) Titration of RhD alloantibody was also done by serial dilutions of patient’s serum till the agglutination is negative on the semisolid medium of the Ortho Biovue ID Micro Typing System.^[10] All the data were analyzed using Stata Ver.17; frequencies of different antibodies, specificity, and titers were summarized and reported as percentages in frequency tables.

RESULTS

A total of 1350 fulfilled the recruitment requirements of the study, out of which, 117(8.7%) were alloimmunized. Several antibodies of the Rh system were identified including anti-E 39(37.6%), anti-D 32(27.3%), anti-C 16(13.7%) [Table 1]. Most of the participants had no history of transfusion before the pregnancy and the RhD negative (RhD –ve) pregnant women report having not been given anti-D prophylaxis during previous pregnancies. All the husbands were RhD positive (RhD +ve).

The identified antibodies in this study were at different concentrations (titers) ranging between 4 and 128 as presented in Table 2. Specific anti-E was the highest antibody present in 41 (42.3%) of the RhD +ve, Specific anti-D was in 18(18.6%) and C was in 14 (14.4%) as indicated in Table 3. Among the alloimmunized RhD −ve, specific anti-D was the most common 14 (70%) with 1 (5.0%) of the RhD −ve mothers, having the highest titer of 1:128, while the highest titer in the RhD +ve was 1:64 found in 24 (24.7%) [Table 4].

DISCUSSION

The use of protective anti-D immunoglobulin against RhD alloimmunization has greatly reduced alloimmunization due to anti-D and unmasking the capabilities of minor RBC antibodies towards alloimmunization. In this study, anti-E was the most prevalent antibody in the alloimmunized Rh+ve pregnant women, while anti-D was the most common in the Rh−ve alloimmunized pregnant women.^[11]

The rate of alloimmunization among the Rh−ve (26.3%) pregnant women was high compared to that in the Rh+ve (7.6%) pregnant women which is closely comparable with a study by Dholakiya et al.^[12] and Varghese et al.^[13] who obtained the same trend.

The reason for the higher rate may be due to unassisted home deliveries and poor attendance to antenatal care which are common in many rural areas. Other underlying factors like population obtained information cannot be ruled out.^[4] The population in this study lives in hilly areas and utilization of healthcare facilities is poor; this is coupled with ignorance about the importance of alloimmunization and its implications. So many of these pregnant women do not receive full antenatal checkup during their pregnancy periods, deliveries/abortions when necessary, and come to hospital later with repeated fetal loss or stillbirths. The observed variations may also be due to both genetic and racial differences.

The most frequently identified Rh alloantibody in this study was anti-E (36.5%) followed by anti-D (27.9%) among the RhD −ve, anti-D while anti-E was more frequent in the RhD +ve. This is similar to what was observed in the Malaya population by Hassan et al where anti-E (33.3%) was the most frequent followed by anti-D (10%).^[14] Smith et al, observed the two most frequently identified anti-bodies were in the order of anti-E (31%) and ant-D (18.7%) similar to what was in this study.^[15] In several studies, the prevalence of these antibodies is commonly followed by non-Rh antibodies, mostly the anti-K. Other studies have observed to the contrary with anti-C being the most observed alloantibody.^[16] These variations could be due to genetic variations of the different study locations.

The trends of the titers for the Rh alloantibodies in this study are seen to increase in the RhD−ve pregnant women with most appearing above 1:32 as the critical point, whereas in the RhD+ve pregnant women, few appearing above the critical value. Once a mother has been identified as having a clinically significant RBC alloantibody, further monitoring and evaluation are required.^[17] As the pregnancy progresses, serial RBC antibody titers may be used. Once a critical titer threshold (1:32) is reached, and the fetus is found to be at risk of carrying the antigen by paternal zygosity testing or more direct measurements, or the fetal result is unknown, the fetus must be assessed for clinical anemia.^[17] This trend is similar to a study by Sánchez-Durán et al., where of the 194 high-risk pregnancies, 38 had titers <1:32 and 156 had titers ≥1:32 which was a rising trend.^[18]

Although anti-D hemolytic disease of the fetus and newborn (HDFN) is a rare complication of Rh+ve pregnancies, the presence of alloimmunization in this group indicates potential for its existence. This suggests that in Rh+ve pregnancies with anti-D alloimmunization, clinical HDFN could be present and thus need for further study within this population. Its therefore recommended that Rh+ve pregnant women with anti-D should be monitored for potentially significant HDFN.

CONCLUSION

The presence of clinically significant alloantibodies is high (8.6%) and significant in Rh+ve pregnant mothers. Alloantibody E is the most prevalent antibody, especially in the Rh-positive pregnant women with a critical titer of 1:32 and these were comparable with other studies, but the difference could be due to factors like genetic difference, racial variations, and testing methodologies. RhD+ve pregnant women are alloimmunized with anti-E being the most common antibody.

In addition, to sensitizing pregnant women about the alloimmunization outcome, there is need to improve on the detection mechanisms for these antibodies at the time of antenatal visits and establish the alloantibody relation to HDFN in the affected pregnant women. The presence of other antibodies apart from anti-D necessitates a universal protocol formulated for the screening of all the antibodies and their clinical significancy with respect to obstetric importance established alongside efficient anti-D monoprophylaxis measures during antenatal visits.