Prevalence of ABO antigens for “A” blood group in Indian population is 21.77% and 9.09% for “AB” blood group, making chances of prevalence of A₂/A₂B subgroup rare. Among the five Rh antigens, phenotyped serologically, the highest prevalence in Indian population is of “e” antigen followed by “D,” “C,” “c,” and “E” being the lowest prevalent Rh antigen [1]. There are various studies from India and across the world reporting more frequent prevalence of subgroup A₂B than A₂. Anti-A₁ antibodies appear as cold agglutinins, present in 0.4% of A₂ subgroup and 25% of A₂B subgroup individuals, making it clinically significant when reactive at 37 °C, causing hemolysis of A₁ red blood cells (RBCs) [2]. Individuals with an A₂B phenotype are more likely to produce anti-A₁ antibody than A₂ phenotype because of the relative reduction of A antigens on A₂B cells [3]. Development of clinically significant anti-A₁ antibody occurs either from an exposure by transfusion with non-A₂ red cells or a pregnancy [4]. Once an A₂/A₂B individual develops an anti-A₁ antibody, they can only be transfused with antihuman globulin (AHG) cross match compatible red cell concentrates (RCC) of group A₂B, B, A₂, or O. Cases are reported from all over the globe of A₂/A₂B subgroup patients having clinically significant anti-A₁ antibody with implicated hemolytic transfusion reactions (HTR) and death; and no transfusion could be given to such patients with rare Rh phenotypes due to non-availability of blood and database of A₂/A₂B donors with rare Rh phenotypes who can be asked for directed donation. Therefore, this study was undertaken with an objective of creating donor registry of A₂/A₂B blood donors with Rh and Kell phenotype, to provide transfusion to all A₂/A₂B patients, to prevent adverse transfusion reaction due to immunological stimulation by pre-existing anti-A₁ antibodies having enhanced titer and thermal amplitude.

A retrospective data analysis was done in a tertiary care hospital-based blood center at capital city of India from January 2016 to December 2020. As a part of routine serological testing, all donors were subjected to ABO Rh grouping, Rh and Kell phenotyping by solid phase red cell adherence (SPRCA) method (Immucor NEO/Galileo). As a routine practice of our department, for all A/AB group samples, conventional tube technique was used to distinguish A₂/A₂B subgroup by agglutination reaction with anti-A₁ lectin antisera (Tulip Diagnostics). Data was analyzed by Excel sheet.

A total 40,051 donors were analyzed during study period, of which, A and AB groups constituted 21.26% (n=8517) and 9.01% (n=3611) of donor population respectively, out of which only 0.47% (n=189) belonged to A₂ subgroup while A₂B subgroup was more frequent; 1.09% (n=440). Out of total 629 donors having A₂/A₂B subgroup, blood samples of 15 donors were found to be reactive on mandatory transfusion transmissible infections (TTI) testing, and hence were excluded from the study (Figure 1). The detailed sex-based distribution of A₂/A₂B subgroup blood donors for registry (n=614) is shown in Table 1. Out of 614 blood donors included in the study, 186 (30.3%) males and 3 (0.48%) females had A₂ subgroup and 420 (68.4%) males and 5 (0.81%) females had A₂B subgroup. The detailed distribution of Rh phenotype among A₂/A₂B subgroup donors is shown in Table 2. R1R1, R1r, R1R2, and R2r, in decreasing order, were found to be the commonest Rh phenotype in A₂/A₂B subgroup blood donors. There was complete absence of R2Rz, RzRz, r’r’, and r’’r’’ in both A₂ and A₂B group donors (highlighted green in Table 2). In A₂B subgroup donors, the least prevalent phenotypes found, were r’r, R1Rz, r”r and r’r” (n=5 and 1 case each) (highlighted yellow in Table 2) while they were absent in A₂ subgroup blood donors (Table 2). Out of total 614 A₂/A₂B donors, only 3.42% donors of A₂/A₂B subgroup were found to be Kell positive (K+), while majority (96.58%) donors were Kell negative.

Differences between A₁ and A₂ are quantitative and qualitative. Qualitative differences of A₁ and A₂ subgroup lie in their chemical structures. Individuals with A₁ phenotype express Aa, Ab, Ac, and Ad determinants while A₂ subgroup has only Aa and Ab antigenic determinants. Absence of Ac and Ad determinants is assumed to be the cause of development of anti-A₁ in A₂ and A₂B subgroup individuals [5]. The genetic basis of high prevalence of anti-A₁ antibodies in A₂B is due to the *R101 allele. This allele is present in 41% of A₂B subgroup as compared to A₂ subgroup individuals (1%) [6]. Usually anti-A₁ antibodies exist as naturally occurring IgM with thermal amplitude of less than 25°C. However, cases of anti-A₁ reacting at 37°C have also been reported in the literature [7],[8]. Anti-A₁ antibody is one of the causes of ABO discrepancies, can develop HTR and its clinical manifestations have also been reported in hematopoietic stem cell and organ transplantation. Identification of A₂ subgroup is important for identification of prospective donor for renal transplant in O and B group patients and for managing platelet inventory of A₂/A₂B subgroup platelet, as it can be safely transfused with good recovery and corrected count increment (CCI) in O and B group patients [9].

To the best of our knowledge, this is the largest population study in which 40,051 donors were included to find A₂/A₂B subgroups with extended Rh and Kell phenotyping. Comparison of various studies showing A₂/A₂B subgroup donors is analyzed (Table 3) but could not find literature categorizing A₂/A₂B subgroup donors with Rh phenotyping. Prevalence of A₂ and A₂B donors vary with different ethnic, regional, and geographic differences.

Cases have been reported from India as well as from other parts of world when lot of time and resources were consumed in finding an appropriate cross match compatible unit of A₂ subgroup and the patient was given non-group specific RCC transfusion due to nonavailability of blood or database of A₂ donors in their local donor pool.

In a case reported from Southern state of India of a 31-year-old obstetrics patient of central placenta previa with antepartum hemorrhage (APH), serological testing of patient’s samples indicated presence of rare subgroup of A₂ (RhD positive). The blood samples were found to be incompatible with all ABO groups on major cross matching. However, due to non-availability of database of blood donors having A₂ subgroup or RCC units of A₂ subgroup in the local major blood banks, nearly 600 units of “A” positive were screened to find four units of compatible A2 subgroup (RhD positive) [4]. Generally an A₂ subgroup is not identified until they have developed an antibody to A₁ cells. This occurs either from an exposure by transfusion or a pregnancy. Once an A₂ group individual has developed an anti-A₁ antibody, the A₂ subgroup individual can only be transfused with A₂ subgroup or with O group RCC that is compatible [10].

In another case reported from India of a 42-year-old male patient diagnosed as acute on chronic pancreatitis, the blood group of patient was confirmed, on serologically testing, as “A₂B with anti-A₁ antibodies, RhD positive.” Due to non-availability of A₂B group RCC unit, compatibility test with two units each of O RhD positive and B RhD positive was undertaken. O group RCC units had minor match problems due to donor anti-B antibody. Hence, the patient was transfused with eight units of compatible B RhD positive RCC over 36 hours [11].

One more case of acute hemolytic transfusion reaction (AHTR) is reported in a 96-year-old female who died shortly after transfusion due to a warm reactive anti-A₁ antibody. She was non-alloimmunized, having blood group “A₂ with an anti-A₁.” The patient was transfused electronically cross matched “A” group RCC, shortly after which the patient expired. Investigations confirmed intravascular hemolysis caused by existent anti-A₁ antibody [12].

Rh phenotyping is associated in this study which will help in building database of donors having rare subtypes as well as rare Rh phenotype (e.g., R1Rz, r”r, r’r”). In our analysis, we could find only one A₂B donor each of Rh phenotype R1Rz, r”r, and r’r” in donor population of five years, making their prevalence of 0.0025% each. There are several cases reported from India where due to non-availability of database of donors having rare Rh phenotype, the patient could not be transfused at all. One such case reported is of a 25-year-old male patient, known case of β-thalassemia intermedia, whose blood group was determined to be “O” RhD positive and on Rh phenotyping, it was found to have R2R2 (DccEE). Since the patient underwent multiple transfusions during his lifetime, he had alloimmunization to multiple antibodies and was advised R2R2 phenotype matched RCC transfusion. Since R2R2 is a rare phenotype, even after screening more than 100 donor units, not a single Rh phenotype matched RCC unit could be found. This rare phenotype R2R2 could not be availed and transfusion was not given to this patient [13]. With all these problematic cases in literature, an algorithm is planned in our institute (Figure 2) and staff is trained accordingly to help patients serve safe blood as per requirements.

The establishment of a state, national, and international register of donors of rare blood groups and their alleles would aid in creating awareness of their existence. Maintenance of rare group registry will help to overcome such difficulties. This study is our small contribution toward preparing rare donor registry of India and we will try to incorporate this registry with other donor registries at various levels, wherever possible.

Life-threatening transfusion reactions occurring due to minor incompatibilities can be avoided by knowing the prevalence of these subgroups and Rh phenotypes. The transfusion safety could be further enhanced by doing molecular characterization of the subtypes; however, it needs further studies with large sample size.

There is a need to create rare donor registry for A₂/A₂B subgroups and clinically significant blood group antigens and share at different levels. This is a first small step to create a rare donor registry of A₂/A₂B subgroups with Rh and Kell phenotype, to provide transfusion to all A₂/A₂B patients to prevent adverse transfusion reaction by pre-existing anti-A₁ antibodies having enhanced titer or thermal amplitude to ensure improved quality of transfusion therapy.