Blood group A_int causing uncertainty during organ donor work-up for incompatible (A₂-O) liver transplantation

Dear Sir,

The ABO blood group system, discovered more than a century ago, still often raises uncertainty among immunohaematologists during subtyping or detection of weak variants. A₁ and A₂ are the major subgroups of blood group A which are differentiated on the basis of reactivity of A₁ cells but not A₂ cells with anti-A₁ lectin (Dolichos biflorus)¹. Routine subgrouping of all A and AB blood groups in India is limited to a few blood centres because of economic constraints and this subgrouping is usually performed only when there is a group discrepancy, a problem during compatibility testing or a history of a transfusion reaction. The role of subtyping group A is, however, critically important when it is being done for (A₂-O) incompatible organ transplantation.

There are a few studies on weak subgroups of the ABO system but the prevalence and occurrence of the "A-intermediate" (A_int) subgroup in India are underreported and have not been published in recent decades. We report here the case of A_int subgroup encountered in two siblings during the pre-transplant work-up of an incompatible live donor liver transplant.

A 34-year old female patient, resident in Kerala, was admitted to the Liver Transplant and Hepato-Biliary Department with features of acute on chronic liver disease due to Wilson's disease; a liver transplant was planned. Samples from the patient and two siblings were subjected to urgent blood grouping and subtyping in the Immunohaematology Laboratory of our Blood Bank. During blood grouping, forward grouping was done using gel technology (Diaclon, Diamed ID, Switzerland) while reverse grouping was performed using in-house prepared pooled A, B and O cells. All the laboratory techniques were carried out according to the manufacturer's instructions. Blood groups were interpreted based on the agglutination pattern with forward and reverse grouping. The recipient's blood group was O-positive and both siblings were A-positive. Considering the possibility of incompatible (A₂-O) liver transplantation, the siblings were further subtyped using anti-A₁ lectin (Diamed AG, Cressier sur Morat, Switzerland) to classify them into A₁ or A₂.

Using a tube method with A₁ lectin, both siblings showed 1− 2+ positive agglutination. A provisional report A₁ of subtype was given to both siblings. Due to the weak reaction with A₁ lectin, the sample was further tested by H lectin (Tulip Diagnostics, Goa, India) which gave a strong 4+ reaction favouring towards an A₂ subtype. Bearing in mind the possibility of false positive results, we checked the potency and specificity of the A₁ and H lectins using control A₁, A₂ and O cells. The titres of A₁ lectin and H lectin were 1:16 with standard A₁ and O cells, respectively. Saliva inhibition studies performed on both the siblings showed the presence of A and H substances. The blood groups of both siblings were reported as A_int and the previous reports of A₁ subtype were withdrawn. As the antigenicity of this subgroup was likely to be higher than that of A₂, both the siblings were cancelled from the potential liver donor list. Blood samples from the patient's parents were also tested, given the possibility of A_int in other family members. In this family, the parents' blood groups were A₁ and O group; A_int was present only in the two siblings.

A₁ and A₂ differ from each other both qualitatively and quantitatively, with A₁ cells carrying 8.1 to 11.7×10⁵ antigenic sites as compared to 2.4 to 2.9×10⁵ sites on A₂ red blood cells. The prevalences of A₁, A₂ and weak subgroups in South India were recently reported to be 98.4%, 1.85% and 0.01%, respectively².

There are reports of safe and successful liver transplants from A₂ blood group donors to O blood group recipients, without plasmapheresis or rejection. The role of subtyping the A blood group has gained importance since the beginning of ABO incompatible organ transplantation. There are, however, cases reports of accelerated acute rejection of A₂ renal allografts in O recipients which might have resulted from misapplication of the results of standard lectin agglutination to the transplant setting, highlighting the importance of subtyping the A group. More sophisticated methods of ABO phenotyping, such as erythrocyte flow cytometric analysis, have been suggested in the transplant setting³.

The existence of an additional subtype of A, which exhibited characteristics intermediate between A₁ and A₂ (A_int), was first recognised by Landsteiner and Levine¹. While Brain's classification was based on weaker reactions (+ or ++) with Dolichos, with H (Ulex) reactivity stronger than A₂⁴, the "A_int" designated by BIRD⁵ suggested stronger reactivity with both Dolichos and Ulex.

A_int is a heterogeneous subgroup which is more common in black people than in white ones. Of group A African-Americans, 8.5% were found to be A_int compared with about 1% of group A white Americans and 2.5% of group A Caucasians. Of group A black South Africans 13.7% were A_int⁴. The incidence of A_int in India was reported to be 2% in group A Maharastrians⁵. It is likely that the incidence may vary in different endogamous caste groups of India and needs extensive study.

No detailed study has so far been reported to elucidate the nature of inheritance of this subgroup. Some studies reported A_int phenotype in two generations during family studies and concluded that A_int is likely to be inherited by a separate allele. However in other family studies, A_intz was present in siblings but not in parents. Transmission of at least one strong H gene from parents to their children partnered by an A₁ gene could give the serological picture of A_int blood group with large H antigen. This is compatible with our case in which both the siblings were A_int while the father was A₁ group and the mother O group.

The expression of different A subtypes in red blood cells is the consequence of diverse formations of the A substances by the action of three types of blood group N-acetyl-galactosaminyl-transferases controlled by A₁, A₂, and A_int genes. The A subtypes are, therefore, more directly identified by examining the kinetic characteristics of A-enzymes in the plasma. A molecular characterization or enzyme analysis would have been useful but could not be performed in this case. Our report points to the need to confirm some cases by molecular tests or by flow cytometric analysis, especially in the organ transplant setting in India. We also recommend the mandatory use of H lectin when subtyping for A to avoid mistyping and errors of interpretation.