Rare B(A)02/O01 was found in Sichuan again: a case report

INTRODUCTION

As a rare ABO blood subgroup, B(A) normally showed inconsistent forward and reverse blood group-ing results (nearly normal B antigen and very little A antigen on the RBC but obviously with anti-A in the serum). B(A) patients might be misjudged as B or AB and have the risk of hemolytic transfusion reaction if they accepted either AB RBC or B plasma transfusion. Therefore, to precisely type the blood groups makes great sense for B(A) patients. In this study, we reported one B(A)02/O01 case identified by serology testing and genotyping methods.

CASE REPORT

A 25-year-old lady with unbearable pain in the right lower abdomen was sent to the emergency department of the Affiliated Hospital of Southwest Medical University. She was diagnosed with acute appendicitis, which required emergent operation. For preoperative preparation, blood typing was conducted by microcolumn gel agglutination assay (201909001, Jiangsu LIBO Medicine, China). Interestingly, both A and B antigens were present (normal B antigen reactivity and slightly weak A antigen reactivity) in the forward grouping test, but anti-A against A₁ and A₂ RBC was also detected in the patient's serum during the reverse grouping test (Table 1). As the patient had no blood transfusion or pregnancy history and had a negative result for Coomb's test (Table 2), we speculated that the blood type of this patient might belong to an ABO subgroup. To investigate, DNA was extracted and genotyping was performed by Tianjin Super Biotechnology Developing Co., Ltd. By using normal ABO PCR-SSP Kit (SUPER007-002-060, BIOSUPER, China), the patient showed a BO1 genotype, with B in phenotype (Fig. 1 and Fig. 2), which was unable to explain the inconsistency of the forward and reverse blood grouping results of serology tests. To investigate further, a Cis-AB / B(A) PCR-SSP Kit (SUPER-007-007-060, SUPER007-007-012, BIOSUPER, China) was used. The result showed a B(A)02/O01 heterozygous panel (Fig. 3 and Fig. 4). To further confirm the genotyping result, exons 1 to 7 of the ABO gene were amplificated by PCR and sequenced by Tianjin Super Biotechnology Developing Co., Ltd (ABI 3130, ABI, USA). Compared with ABO*A1.01, we found one deletion at c. 261G of exon 6, and 8 heterozygous point mutations, c.297A > G, 526C > G, 657C > T, 700C > G, 703G > A, 796C > A, 803G > C, and 930G > A in the 6 and 7 exons (Fig. 5 and Fig. 6). By referencing the results with the Blood Group Antigen Gene Mutation Database (BGMUT), the genotype was finally confirmed as B(A)02/O01.

Table  1.  Serology test results

Reagent	Agglutination reactions
Anti A	3+
Anti B	4+
Anti A1	0
Anti H	4+
Human Anti A	0
A1 RBC	3+
A2 RBC	3+
B RBC	0
O RBC	0
Ctrl RBC	0
Serological phenotype	B(A) or AwB
Note: 0 indicates no agglutination.

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Table  2.  Coomb's test results

Test	Agglutination reactions
DAT	0
IAT	0
Note: 0 indicates no agglutination.

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Figure  1.  Electropherogram of PCR-SSP products (normal ABO PCR-SSP Kit)

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Figure  2.  Interpret panel for ABO genotyping (normal ABO PCR-SSP Kit)

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Figure  3.  Electropherogram of PCR-SSP products (cis-AB / B(A) PCR-SSP Kit)

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Figure  4.  Interpret panel for cis-AB and B(A) genotyping (cis-AB / B(A) PCR-SSP Kit)

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Figure  5.  Mutations in Exon 6

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Figure  6.  Mutations in Exon 7

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DISSCUSION

The blood subgroup B(A) was first identified in 1993 by Yamamoto and his colleagues^[1]. They found one ABO allele, named as B(A)01, which could encode glycosyltransferases with both glycosyltransferase B activity and glycosyltransferase A activity^[1]. Until now, six B(A) alleles have been indexed in the BGMUT^[2]. The distribution of B(A) subgroups have special characteristics. B(A)03 allele is mainly distributed in Europe, while B(A)04 allele (16 per million) and B(A)02 allele (7.8 per million) have been the most two frequent B(A) subgroups in China^[3]. B(A)04 allele was first reported in China^[4], and is distributed in multiple provinces of China, such as Hebei^[5] and Hubei^[6]. B(A)02 allele, resulting from c.700 > G substitution which predicts the alteration of Pro234 to Ala, was first reported in 1999 by Yu and his colleagues^[7]. B(A)02 allele was previously discovered in Sichuan^[8], and the B(A)02 case in this study come from Sichuan too.

The serological manifestations of B(A) subgroups are diverse. The agglutination reactions of B(A) erythrocytes to anti-B and anti-H are strong (4+), but the agglutination reaction of B(A) erythrocytes to anti-A ranges from 1+ to 3+ in different individuals^[9]. This is mainly due to the quality of reagents, such as the equipment manufacturer and the batches. B(A) erythrocytes normally react with both mice and human anti-A reagent, but in this individual, they reacted with the mice antibody (20190700704, KINGHAWK, China), but not with the human antibody. Thus, it is critical to use standardized reagents for blood type serology tests, especially for ABO subgroup typing.

Since serology tests have intrinsic limitations, genotyping is the gold standard for B(A) subgroup typing. PCR-SSP and PCR-SBT are the two most commonly used genotyping methods. PCR-SSP has simple equipment requirements, which is easier to be implemented in ordinary transfusion departments or blood centers. However, we must pay attention to the choice of kits. It is impossible to identify B(A) subgroups with normal ABO PCR-SSP Kit (SUPER007-002-060, BIOSUPER, China). This study found the cis-AB / B(A) PCR-SSP Kit (SUPER-007-007-060, SUPER007-007-012, BIOSUPER, China) was suitable for B(A) subgroup typing. In comparison, PCR-SBT can type the B(A) subgroup more accurately. In addition, it is able to identify new ABO genotypes. Although sequencing instruments are currently not available in ordinary transfusion departments or blood centers, specimens can be sent to sequencing companies for PCR-SBT analysis. Thus, PCR-SBT will become more prevalent for the blood typing of rare ABO subtypes in the future.