Unmasking alloimmunization: A prevalence study in modern transfusion practice

 Corresponding author: Anubhuti Jain Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Unmasking alloimmunization: A prevalence study in modern transfusion practice Anubhuti Jain 1,  and Joyce Regi 2 1 Clinical assistant, Blood Centre, Sir HN Reliance Hospital, Mumbai, Maharashtra, India. 2 Consultant & Head, Blood Centre, Sir HN Reliance Hospital, Mumbai, Maharashtra, India. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 118–122 Publication history: Received on 26 July 2025; revised on 01 September 2025; accepted on 04 September 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.23.3.0805 Abstract Background: Alloimmunization against non-ABO red cell antigens remains a significant clinical concern, potentially leading to hemolytic transfusion reactions and complications in pregnant women. Aim: To assess and compare the prevalence and types of irregular red cell antibodies in transfusion recipients. Study Design and Setting: This was a prospective study conducted over one year at a tertiary care hospital blood center in Mumbai. A total of 9756 blood samples were analyzed—3243 from patients and 6513 from healthy donors. Materials and Methods: Antibody screening was performed using a commercially available three-cell panel. Samples testing positive underwent further identification using a 14-cell panel in low-ionic strength saline, with and without enzyme treatment. Statistical Analysis: Chi-square test was applied to evaluate the association between antigen exposure and antibody formation. Results: Irregular antibodies were detected in 65 of 3243 patient samples (2%). Anti-c (21%) was the most prevalent, followed by pan-reactive antibodies (20%) and anti-D (16%). Among males, pan-reactive antibodies were most common (36%), while anti-D (28%) was predominant among females. The prevalence among healthy donors was 0.72%. Conclusion: Alloimmunization is significantly higher in transfusion-exposed patients, particularly females. Routine antibody screening should be prioritized in patients requiring multiple transfusions. Keywords: Alloimmunization; Antibody Screening; Irregular Red Cell Antibodies; Indirect Coombs Test; Transfusion Medicine 1. Introduction Unexpected antibodies, also known as irregular or non-ABO antibodies, are immune responses typically formed following exposure to foreign red blood cell (RBC) antigens through transfusion or pregnancy. These antibodies can lead to haemolytic transfusion reactions and complicate crossmatching procedures, posing significant risks to patient safety. The prevalence of such antibodies varies across populations, with studies reporting rates ranging from 1% to 10%, and even higher in patients with conditions who undergo frequent transfusions [1]. Gender differences have been observed in the formation of unexpected antibodies. Women, particularly those of childbearing age, are at increased risk due to alloimmunization events during pregnancy and childbirth. A study by Evers et al. found that women over 45 World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 118–122 119 years had a higher risk of post-transfusion antibody formation compared to men [2]. Additionally, the specificity of antibodies can differ between genders, influenced by factors such as hormonal differences and exposure histories [3]. Understanding the distribution and specificity of unexpected antibodies is crucial for improving transfusion practices and patient outcomes. This study aims to analyse the prevalence and gender-specific distribution of unexpected antibodies in a cohort of 65 patients, providing insights into potential risk factors and informing strategies for safer transfusion protocols. Usually, ABOand Rh (D) antigen-matched blood is provided by the blood banks, so the risk of alloimmunization to minor blood group antigens is very high. The most important irregular RBC alloantibodies are directed toward Rh (antiD, -C, -E, -c, and -e), Kell (anti-K), Duffy (anti-Fya and-Fyb), Kidd (anti-Jka and-Jkb), and MNS (anti-M, -S, and-s) blood group systems [4] 2. Materials and methods This study was conducted in a Blood Centre of a tertiary care hospital in Mumbai over a period of 1 year from January 2024 to December 2024. A total of 9756 samples were analyzed, 6513 samples were of healthy blood donors and 3243 samples were of patients during the course of the study. Distribution is summarized in table 1. Table 1 Sample Distribution (n=9756) Healthy Donors 6513 Men 4256 Women 2257 Total Patients 3243 Men 1933 (59%) Women 1310 (41%) Positive for irregular antibody 65 (2% of all the patients requiring transfusion) Men 27 (42% of all patients with irregular antibody) Women 38 (58% of all patients with irregular antibody) Among the patients, 1865 did not have any exposure to antigen previously. The remaining patients had a previous history of antigenic exposure, i.e had a history of transfusion, pregnancy, or both. Ethylenediaminetetraacetic acid blood samples were used for direct Coombs test, while plain samples were used for antibody screening and identification. Serum of recipients was tested for unexpected antibodies with pooled O cells at RT and using a 3-cell panel by IAT 2.1. Statistical analysis Chi-square test was done to calculate the association of exposures to the risk of alloimmunization. 3. Results Among the total population studied, 9756, 63.4% were men and 36.6% were women. Among 3243 patient samples studied, males were 59% and female were 41%. Rest were donor samples. Irregular antibodies were detected in 65 patient samples comprising 2% of the total samples. In healthy donors, the percentage of antigen positivity was 0.72% of all the donors. Way lesser than the patient group. 3.1. Overall Trends • Most common antibodies overall: World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 118–122 120 • Anti-c: 21% • Anti-D: 20% • Anti-E: 14% 3.2. Gender-specific trends Females • Anti-D (34%) and Anti-c (23%) were the most common. • Anti E antibody was 15% Males • Antibodies identified commonly were anti E (14%) and Anti c 13% • The antibodies that remained undetermined were 36% • Autoantibodies were more common in males (7%) compared to females (2.6%). • Multiple antibodies, detected in 6% overall, fairly equally in both genders. The distribution of the antibodies was studied. Majority of unexpected antibodies detected overall was “Anti-c” seen in 21% of the total. In women, anti-D was the most common antibody found in 34% followed by Anti-c in 23%. In males, majority of the antibodies identified were anti-E (14%). Distribution shown in the Table 2 and depicted graphically in Figure 1. Table 2 Distribution of unexpected antibodies in patients Antibody Percentage Over all % (n=65) Female % (n=38) Male % (n=27) c 21 23 13 D 20 34 7 E 14 15 14 M 07 5.6 7 Jka 03 6 3 Jkb 03 2.6 0 N 01 2.6 0 Lea 01 0 3 Kpa 01 0 3 Autoantibody 03 2.6 7 Undetermined 20 2.6 36 Multiple 06 6 7 4. Discussion This study highlights the distribution of unexpected antibodies among a cohort of 65 patients undergoing antibody screening. The findings reveal significant gender-based differences in alloantibody profiles, which may have important implications for transfusion safety and immunohematology practices. The most frequently detected antibody overall was anti-c (21%), consistent with prior studies that have shown a high immunogenic potential of Rh system antigens, particularly anti-c and anti-D. Notably, anti-D was the most prevalent antibody among female patients (34%), likely attributable to sensitization events such as pregnancy and transfusion, which are well-established risk factors for Rh alloimmunization in women of childbearing age [2]. Anti-c was also notably higher in females (23%) than in males (13%). World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 118–122 121 Antibodies against antigen E, antigen M and Kidd antigens (Jka and Jkb) were observed with relatively balanced distribution. Interestingly, rare antibodies such as anti-Lea and anti-Kpa were detected only in male patients, though their overall prevalence remained low (≤3%). However, larger data needs to be analysed to establish this fact. Undetermined (Pan-reactive antibodies), which indicate broad reactivity, were markedly more prevalent in males (36%) compared to females (2.6%). This striking disparity may reflect underlying clinical conditions such as autoimmune hemolytic anemia, chronic inflammatory states, or polyclonal activation associated with transfusion history in male patients. The relatively higher incidence of autoantibodies in males (7%) versus females (2.6%) also supports this possibility. These findings underscore the importance of routine antibody screening, particularly in transfusion-dependent populations for analysis in transfusion risk assessment. The observed trends also support the implementation of extended antigen matching, especially in females of reproductive age, to reduce the risk of alloimmunization. Further studies with larger cohorts and correlation with clinical history—including transfusion exposure, pregnancy, and autoimmune disorders—are warranted to elucidate the underlying causes of these patterns and to refine transfusion practices accordingly. In our study, the seropositivity rate among healthy blood donors was 0.2%. Pahuja et al. showed the prevalence of 0.05% among 7756 whole blood donors. [5] Garg et al. reported a prevalence of 0.09% among 47,450 whole blood donors. [6] On the contrary, Giblett had reported a 0.32% incidence of irregular RBC antibodies in blood donors.[4] In our study, the incidence of irregular antibodies in healthy donors is comparable to the study by Gilbett. In our study, the incidence of irregular antibodies in the exposed group was 2% which can be compared to the study done by Agarwal et al. on multi-transfused patients, they documented a prevalence of 2.71% (7/258).[8] A similar study by Patel et al. done on multi-transfused patients also reported a prevalence of 7.0% (14/200).[9] Koelewijn et al., in their study to assess the efficacy of a universal antibody screening program for pregnant females, found a total alloimmunization rate of 1.2%.[10] Al-Ibrahim et al. found a 2.0% alloimmunization rate, while Howard et al. detected clinically significant antibodies among 1.0% of all pregnant women. [11,12] In our study, the overall seropositivity of females is 2.8%. Autoantibodies (DAT positive) were detected in 0.7% of our nonexposed subjects. The DAT-positive bags were discarded as per the institutional policy. 5. Conclusion Routine irregular antibody screening should be incorporated into pre-transfusion testing, particularly for patients at higher risk of alloimmunization. Extended antigen matching, especially for Rh and Kell antigen, is advisable for repeated transfusion patients. Limitations Cost constraints may limit universal antibody screening. Establishing regional center for antigen negative blood supply could help address this issue. Compliance with ethical standards Acknowledgments I gratefully acknowledge everyone who has been there constantly throughout this work. Disclosure of conflict of interest There are no conflicts of interest. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 118–122 122 References [1] Prevalence of “unexpected antibodies” in patients requiring blood transfusion at National Referral Hospital Thimphu from 2015 to 2017: Retrospective study. [Internet]. 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