Prevalence of “weak B” phenotypes and their evaluation and differentiation in healthy blood donor population in Eastern India

Abstract

BACKGROUND:

Examples of group B red cells that react weakly or not at all with anti-B have been described. Subgroups of B such as B₃, B_x, B_m, and B_el are rare and are less frequently reported. We studied the frequency of subgroups of B in our healthy blood donor population and serologically characterized and differentiated these subgroups.

MATERIALS AND METHODS:

The 9-year prospective study included 84,534 healthy blood donors. Initial blood grouping and antibody screening of all donor samples were performed using automated solid-phase assay. Any sample showing blood group discrepancy or weaker agglutination was subjected to further immunohematological investigations.

RESULTS:

Among 84,534 healthy donors, “B” blood group was found in 29,190 (34.53%). Weak B phenotypes were demonstrated in 9 (0.031%) B donors. Among the 9 weak B phenotypes, B₃ was the most common followed by B_m. The frequency of B₃, B_m, B_x, and B_el in our blood donor population was found to be 1 in 21,133, 1 in 28,178, 1 in 84,534, and 1 in 84,534, respectively. Red cell agglutination with anti-B and anti-AB varied from Wk+ to 2+ with or without mixed-field agglutination in the B₃ and B_x phenotypes. Naturally occurring anti-B of immunoglobulin M type was detected in the B_x donor. Two (22.2%) of the 9 donors were found to be nonsecretor. Adsorption-elution demonstrated “B” antigen specificity in different strengths in B_m, B_x, and B_el phenotypes.

CONCLUSION:

We conclude that differentiating weak subgroups of “B” by serological assays is possible to a great extent with technical expertise. Mistyping weak subgroups of B as “O” group may lead to reporting errors and wrong blood transfusion. Therefore, blood centers in developing countries including India should establish simple techniques to detect and differentiate weak subgroups and develop procedures to ensure safe blood transfusion and transplantation.

Introduction

Karl Landsteiner discovered the ABO blood group system and this led to the beginning of modern transfusion medicine.[1] The ABO system is the most clinically significant antigen system in blood banking and is the most frequently performed test in a blood center.[2] ABO blood groups have subgroups that are often distinguished by decreased amounts of A or B antigens on red blood cells (RBCs). Blood group A appears to have more subgroup variations than group B.[3] Unlike A subgroup, there are no comparable B1 and B2 subgroups, but examples of group B RBC that reacts weakly or not at all with anti-B have been described. Subgroups of B are rare and are less frequently reported.[4] Most subgroups of B are discovered as a result of discrepancy between cell grouping and serum grouping. These subgroups are difficult to classify and mostly differentiated by combination of basic and advanced immunohematological investigations and molecular studies.[1,3] The International Society of Blood Transfusion included B₃, B_x, B_m, and B_el phenotypes as the main subgroups of B.[5,6] These weak B phenotypes mainly result from weaker expression of an alternate weak allele present at the B locus.[7] Definitive identification of ABO subgroups is usually performed at a reference laboratory using molecular methods, which is unavailable in many developing countries including India and often has long turnaround times.

Here, we studied the frequency of subgroups of B in the healthy blood donor population of Eastern India and serologically characterized and differentiated the weak subgroups based on various immunohematological investigations.

Materials and Methods

The prospective study conducted from January 2013 to December 2021 in a tertiary care hospital blood center in Eastern India included 84,534 healthy blood donors. Due ethical clearance was obtained from the hospital Ethics Committee to conduct the study. All donors were selected for blood donations after proper screening as mandated in the Drugs and Cosmetics Act, India.[8] As per departmental standard operating procedure (SOP), blood samples both in ethylenediamine tetraacetic acid (EDTA) and plain vials were collected for mandatory testing to determine the suitability of the blood units.

Initial blood grouping and antibody screening of all donor samples were performed using automated solid-phase assay (NEO Iris, Immucor, USA). Any sample showing blood group discrepancy or weaker agglutination was subjected to further investigation as per departmental SOP using conventional tube technique (CTT). Samples showing weaker (≤2+), mixed field (mf), or no agglutination with anti-B were further evaluated through immunohematological techniques. The criteria used for differentiation of weak B phenotypes included:[3,7,9]

1. Type of agglutination with anti-B, anti-AB, and anti-H

2. Presence or absence of antibodies in the serum

3. Adsorption-elution studies with anti-B

4. Presence of B and H substances in saliva.

Forward grouping and reverse grouping on discrepant samples were performed by CTT using commercial monoclonal anti-sera (Tulip Diagnostics, Goa, India) and in-house prepared pooled reagent A, B, and O red cells, respectively.[9] mf appearances obtained by CTT were further confirmed by gel-based column agglutination technique (BIO-RAD, Cressier sur/Morat, Switzerland). Heat elution using 6% bovine albumin was performed to conduct the adsorption-elution studies. Water-soluble B and H substances have been demonstrated in saliva by inhibition tests with corresponding antisera.[9]

Results

In 84,534 healthy donors under study, A, B, O, and AB blood group was found in 20,332 (24.05%), 29,190 (34.53%), 26,913 (31.84%), and 8099 (9.58%), respectively. Total Rh-positive donors were 80,476 (95.2%). Among the 29,190 B group donors, weak B phenotypes were demonstrated in 9 (0.031%) donors [Figure 1]. Among the 9 weak B phenotypes, B₃ was the most common (n = 4, 44.4%) followed by B_m (n = 3, 33.3%). B_x and B_el were found in each donor. Figure 2 depicts the detailed investigation process flow of weak B phenotypes. Investigations comprising testing red cells with anti-B, anti-H, and anti-AB followed by saliva inhibition test and adsorption-elution studies formed the basis of subgroup characterization and differentiation. Table 1 describes the serological evaluation of weak “B” subgroups in the 9 donors. Red cell agglutination with anti-B and anti-AB varied from Wk+ to 2+ with or without mf agglutination in the B₃ and B_x phenotypes. The naturally occurring anti-B found in the B_x donor was of immunoglobulin M type showing weak to 1+ reactivity at 22°C and 4°C, respectively. Saliva test could demonstrate specific blood group substances, and 2 (22.2%) of the 9 donors were found to be nonsecretors with the absence of any substances. Adsorption-elution demonstrated “B” antigen specificity in different strengths in B_m, B_x, and B_el phenotypes.

Figure 1.

Frequency of “weak B” phenotypes

Figure 2.

Investigation process flow of “weak B” phenotypes

Table 1.

Serological evaluation of weak “B” subgroups (n=9)

Test on red cells				Test on serum				Substance present in saliva	Adsorption-elution with anti-B	Serologic weak B subgroup
Anti-A	Anti-B	Anti-AB	Anti-H	A-cell	B-cell	O-cell	Antibody screen (3 cell panel)
0	2+ with mf	1+ with mf	3+	4+	0	0	0	B (2+), H (1+)	NA	B3
0	0	0	4+	4+	0	0	0	B (1+), H (2+)	3+	Bm
0	2+ with mf	2+ with mf	4+	4+	0	0	0	Not found (nonsecretor probably)	NA	B3
0	2+ with mf	1+ with mf	4+	4+	0	0	0	B (2+), H (1+)	NA	B3
0	0	0	3+	4+	0	0	0	B (2+), H (2+)	2+	Bm
0	Weak	Weak	3+	3+	Weak	0	0	H (2+)	3+	Bx
0	0	0	4+	3+	0	0	0	Not found (nonsecretor probably)	2+	Bm
0	2+ with mf	2+ with mf	4+	3+	0	0	0	B (2+), H (2+)	NA	B3
0	0	0	3+	4+	0	0	0	H (1+)	2+	Bel

mf=Mixed field agglutination, NA=Not applicable

Discussion

Subgroups of B are very rare, less frequent than those of A, and are classified as B₃, B_X, B_m, and B_el in decreasing order of the amount of the B antigen.[3,10] The B₃ phenotype occurs with a frequency of 1 in 900 B donors and 1 in 1800 A₁B donors in the Chinese population.[11] The frequency of B₃ phenotype in the French population is 1:116,667.[12] Reports from India showed the frequency of weak B phenotype to be 1:86,687 among the donors of Northern India and 1 in 24,000 from Bombay.[10,13] Infrequent reporting of findings on weak B phenotypes could be due to the lack of technical expertise and the complex behavior and classification of ABO subgroups. In the present study, the frequency of weak B phenotype was observed to be high. The frequency was 1 in 9393 in the blood donor population and 1 in 3243 in the B group population. This may be due to the increased number of B group donors in this population. The frequency of B₃, B_m, B_x, and B_el in our blood donor population was found to be 1 in 21,133, 1 in 28,178, 1 in 84,534, and 1 in 84,534, respectively.

The ABO alleles are listed in the open-access database dbRBC on the National Center for Biotechnology Information website.[14] The allele listing currently includes more than 40 different alleles associated with the expression of B antigens. Twenty-eight of these alleles were found to be related to weak B phenotypes such as B₃, B_el, B_m, or B_weak and are characterized by different single nucleotide exchanges predominantly in exon 7.[15,16,17] Such mutations in the B allele confer differences in the specificity and activity of transferases that add low levels of B immunodominant sugars to the precursor H antigen.[7,18]

Due to the lack of advanced resources and molecular methods of blood grouping, we characterized and differentiated the weak B phenotypes solely by serological methods as discussed in the literature.[3,7,9] We also observed that immunohematological laboratories having facilities to perform advanced techniques such as adsorption-elution and saliva inhibition test can differentiate weak A or B phenotypes with more ease. As demonstrated by the saliva inhibition test, two of the nine (22.2%) donors with weak B phenotypes were found to be nonsecretors. This is in accordance with the previous findings that approximately 78% of all individuals possess the “Se” gene that governs the secretion of water-soluble ABH antigens into all body fluids with the exception of cerebrospinal fluid.[7,9] The demonstration of B substances in the saliva of our B subgroup donors is evidence for the inheritance of B gene and Se gene.[3]

The frequency of B₃ in our “B” group blood donor population was found to be 1 in 7297. The immunohematological investigations typically demonstrated a mf pattern and rapid agglutination with anti-B and anti-AB reagents similar to serological characteristics described by previous authors.[19] Both B and H substances were present in adequate amounts (1+ to 2+) in the saliva of our B₃ secretors.[20]

The frequency of B_m in our “B” group blood donor population was found to be 1 in 9730. Characteristically, the red cells failed to react with anti-B or anti-AB and they easily adsorbed and eluted anti-B.[3] The eluted anti-B significantly agglutinated (≥2+) with the corresponding red cell antigens. Both B and H substances were demonstrated in our B_m secretors (1+ to 2+). The frequency of B_m has been reported more in Japan.[20]

The frequency of B_x in our “B” group donor population was found to be 1 in 29,190. The red cells demonstrated weak agglutination with anti-B and anti-AB antisera without any mf reactions. As mentioned by previous authors, some examples of B_x red cells may not agglutinate at all while others may agglutinate stronger with anti-AB than with anti-B.[19,21] The red cells readily adsorbed and eluted anti-B with high grade of reaction (3+) with corresponding red cell antigens. The young donor was a secretor and showed H substances (2+) in the saliva.[20]

The frequency of B_el in our “B” group blood donor population was found to be 1 in 29,190. This phenotype is extremely rare and is serologically determined by adsorption-elution of anti-B. The red cells failed to agglutinate with anti-B and anti-AB antisera [Table 1]. Although previous workers demonstrated weak naturally occurring anti-B antibodies in the serum, we could not detect anti-B despite extended incubation and cell-serum ratio alteration. The donor was a secretor and showed H substances (1+) in his saliva.[3]

Other weak B phenotypes have been reported, which do not possess the appropriate characteristics for classification into any of the groups already discussed. These may represent new classification and new representation of ABO polymorphism.[3,22] Chaurasia et al. described a case of B_w that could not be serologically classified into any of the subgroups of B.[23]

Detection and differentiation of weaker subgroups of B such as B_m and B_el are of prime importance, as these may be wrongly typed as blood group “O” in the donors. These subgroups, if wrongly transfused with “O” group blood, can result in an adverse hemolytic transfusion reaction.[7,23]

Conclusion

We conclude that differentiating weak subgroups of “B” by serological assays is possible to a great extent with technical expertise; however, molecular genotyping is essential because it confirms the result. Mistyping weak subgroups of B as “O” group leads to erroneous blood group reporting, incorrect blood labeling, and wrong blood transfusion. Differentiating subgroup of B also enables transfusion and transplant facilities to plan organ transplantations appropriately. Therefore, blood centers in developing countries including India should establish simple procedures to detect and differentiate weak subgroups and ensure safe blood transfusion and transplantation.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil