Exploring trends of red blood cell alloimmunization among transfusion recipients and healthy blood donors at a tertiary care hospital centre: A comprehensive analysis

Antibody screening and identification

The primary technique involved the use of the low ionic strength solution coombs card, along with screening and identification panels, which included a 3-cell screening and 11-cell identification panels (BIORAD). The indirect antiglobulin test using the ID-card “Coombs anti-immunoglobulin G (IgG)” was employed to demonstrate in vitro reactionships between red cells and antibodies that sensitize but do not agglutinate cells expressing the corresponding antigen. This is critical for detecting and identifying antibodies, blood grouping, and compatibility testing.^[7] An ID-card containing six microtubes, each with anti-human globulin (AHG) (anti-IgG rabbit) and C3d, was utilized. This method eliminates the need for extensive washing steps, as the red cell suspension forms a barrier over the gel suspension, preventing the neutralization of AHG by immunoglobulins in the patient’s plasma/serum.

Specimen collection

Patients did not require special preparation before giving a specimen but had to be correctly identified at bedside, with the specimen collected in plain and ethylenediamine tetra acetic acid (EDTA) vacutainers labeled with the patient’s full name, collection date, hospital ID, collection time, and the doctor’s initials on the requisition form. The sample for blood donors was also obtained in EDTA and plain vacutainers from the diversion pouch of the blood bag, and all the blood bags had inline leucodepletion filters in them. Samples were centrifuged at 2500 rpm for 5 min before use for obtaining plasma/serum and RBCs for testing. 5% RBC suspension was prepared for testing from the patients’/donors’ blood samples. Hemolyzed samples were deemed unsuitable.

Procedure

The procedure of antibody screening and identification began with identifying the appropriate microtube on the card and labeling it with the patient’s name and hospital ID. Next, 50 μL of the provided red cell suspension from a three-cell panel was transferred into three labeled microtubes marked as I, II, and III. Subsequently, 25 μL of the test serum or plasma was added to each microtube. The card was then incubated in the column agglutination technology (CAT) incubator for 15 min at 37°C. After incubation, the card was centrifuged in the CAT centrifuge for 10 min. The results were then interpreted and recorded in a designated register and the patient’s report. A negative Indirect Coombs Test result was indicated by the settling of all RBCs at the bottom of the column, while a positive result was indicated by visible trapping of RBCs in the column or at the top of the microtube. The reactions were graded from 0 to 4+, with 0 being negative and 4 being RBC button at the top of the microtube, and 1–3 being intermediate between 0 and 4. Any antibody detected as a positive reaction (Grade 1–4) in any of the cell panels, I, II, and III, was identified using the 11-cell panel. The antigram charts provided with the 3-cell and 11-cell panels are used for antibody screening and identification according to the reactions observed after testing.

The detection of antibodies in serum can be compromised if the ratio of serum to cells in the test, or the test, or the length of incubation, is incorrect. Failure to detect all antibodies in a serum can result from:

• Low-titer antibodies that are too weak to be detected by the methods and/or media being used

• An antibody that may be exhibiting a dosage effect

• The lack of an antigen on the screening cells to bind to an antibody in the serum.

Ethical considerations

Ethical approval was obtained from the Institutional Ethical Committee (IEC) Review Board of the medical college before the start of the study, dated December 26, 2023.

Sample size calculation

The sample size was calculated from the study of Shastry et al., who had found the prevalence of rate of alloimmunization as 0.5% for blood donors and 4.8% for patients receiving transfusion,^[8] in a systematic meta-analysis of all the original articles published in English on RBC alloimmunization among transfusion recipients from India in MEDLINE, SCOPUS, CINAHL, and Google Scholar bibliographic databases.

So, using the Formula: -Sample size (n) = (Z × 1-α/2)² × PQ/d²

1. Where, for the donor group: -

   • P (Prevalence) = 0.5%, Q (100-prevalence) = 99.5%,

   • d (Absolute Precision) = 0.3%,

   • α (Standard Error) = 5%.

   n (Sample Size) = 2119 blood donors.

2. For patient group: -

   • P (Prevalence) = 4.8%, Q (100-Prevalence) = 95.2%

   • d (Absolute Precision) = 1%

   • α (Standard Error) = 5%

   n (sample size) = 1753 blood recipient patients.

Convenient sampling was done with an estimate of around 2500 patients and 2500 blood donors in 2 month duration (which is more than the Estimated Sample Size). Since it was a routine screening study; thus, all the available data were sampled for a period of 2 months duration.

Statistical analysis

The analysis revealed a statistically significant association between female gender and higher rates of alloimmunization among blood recipients and patients, with a Chi-square value of 6.98 and P = 0.0082, confirming significance at P < 0.05. In contrast, no significant association was observed between gender and alloimmunization in the blood donors’ group. Here, the Chi-square statistic with Yates’ correction was 0.41, and P = 0.51, indicating no significant difference (P > 0.05).

Gender differences and alloimmunization

Our study included 3315 male (66.3%) and 1685 female (33.7%) participants. Of these, 22 female patients and five male patients, along with four male donors, were alloimmunized. The seropositivity rate in female patients was 1.30%, significantly higher than the 0.27% rate observed in males (P < 0.05). This finding is consistent with Pimpaldara et al.^[12] who identified a significant association between gender and alloantibody formation. They noted that females, particularly those receiving multiple transfusions, are more prone to alloimmunization. Factors such as higher anemia rates among females, increased likelihood of pregnancy-related transfusions, and Rh +ve pregnancy in Rh −ve females contribute to this gender disparity.^[13]

Alloimmunization in thalassemia patients

In our study, alloimmunization in chronically transfused thalassemia patients was notably lower than in other studies. We observed a 1.33% alloimmunization rate among 150 thalassemia patients, with only two patients developing Anti-D and Anti-C_w (Weak C) antibodies individually. This contrasts with higher rates reported by Dhawan et al. (5.64%)^[14] and Yadav et al. (6.6%).^[15] The lower incidence in our study may be attributable to our practice of providing leukoreduced and Rh-Kell phenotyped matched blood to thalassemic patients for the last many years, which helps reduce alloimmunization rates.

Antibody types and clinical significance

In our donor population, we identified four antibodies: AntiLe^a (Lewis), Anti-M (MNS), Anti-C (Rh-hr), and Anti-c (Rh-hr). Anti-M and Anti-Le^a are often naturally occurring or immune-stimulated, predominantly immunoglobulin M antibodies with some IgG component. Anti-C and Anti-c, on the other hand, are typically not naturally occurring and may result from prior blood transfusions or pregnancy.

Since all patients were male, the presence of anti-C and anti-c antibodies is particularly notable, as these antibodies are not typically naturally occurring in the absence of transfusion or pregnancy-related exposure to RBCs. The formation of these antibodies may be attributed to a previous history of multiple blood transfusions following trauma and major surgery in our two male blood donors aged 25 and 35 years, which was evident on proper history taking. Whereas specific exposures in females, such as feto-maternal hemorrhage, abruptio placentae, spontaneous or therapeutic abortion, cesarean delivery, ectopic pregnancy, or transfusion, can result in the rise of these unexpected antibodies.^[16] It’s crucial to investigate the underlying cause of antibody formation, especially considering their potential to induce acute or delayed hemolytic reactions. Among non-D Rh antibodies, anti-c is the most encountered and can lead to severe hemolytic disease of the fetus and newborn, while anti-E is less frequent, and anti-C is rare in the absence of anti-D. Therefore, understanding the etiology of antibody formation in these cases is essential for effective clinical management.

Among patients, the most common antibodies were anti-D and anti-E from the Rh-hr system, comprising 45.16% of the total antibody pool. Anti-D was particularly prevalent, found in 19.4% of cases, all in females. This aligns with findings by Ameen et al., who also reported anti-D as the most common antibody. The high prevalence of anti-D antibodies in our study may be due to a significant proportion of antenatal patients from areas with limited awareness of anti-D prophylaxis.^[17]

Anti-E antibodies, found in 12.90% of cases, were exclusively in females, mostly in the 25–30 age range. Anti-Kell antibodies, present in 12.90% of cases, are significant due to their potential to cause severe reactions and fetal anemia. Anti-Duffy (Anti-Fy^b) antibodies (6.45%) and Kidd (Anti-Jk^b) antibodies (6.45%) were also identified. Anti-Fyb antibodies are clinically significant, causing hemolytic transfusion reactions and hemolytic disease of the newborn. Kidd antibodies rarely cause severe hemolytic disease but can affect blood transfusion outcomes.^[18]

The emergence of alloantibodies complicates transfusion therapy, making blood compatibility challenging and increasing adverse reaction risks. The direct antiglobulin test, developed by Coombs in 1945, significantly advanced transfusion safety by detecting antibodies causing reactions.^[19] This study emphasizes the need for comprehensive screening, including antibody screening, identification, and donor phenotyping, to enhance patient safety and transfusion outcomes. Incorporating leucodepletion has also shown promise in reducing alloimmunization and autoimmunization.^[20] A multifaceted approach, combining these strategies, can improve transfusion safety and efficacy, advocating for an evidence-based assessment approach that could influence transfusion practices both in India and globally. However, the limited sample size of transfusion recipients and blood donors from a single hospital may limit the generalizability of these results. In addition, not all risk factors for alloimmunization could be fully explored due to the lack of comprehensive data for certain patient populations.