Genotyping of blood groups in alloimmunized patients with β-thalassemia major by T-ARMS-PCR and multiplex-aso-pcr

Introduction: Beta-thalassemia major is a severe hemolytic anemia requiring life-long blood transfusion. Planned random donor blood transfusion is associated with alloimmunization against incompatible antigens. Determination of the minor blood group systems phenotype or genotype, and administration of the compatible blood components can significantly reduce the rate of alloimmunization. The present study aimed to determine the prevalence of alloimmunization, and genotype/phenotype characteristics of the minor blood groups systems in patients with β-thalassemia major.

Material and methods: This study was conducted on 1147 β-thalassemia major patients. Initially, antibody screening and antibody identification were performed. Then, phenotyping and genotyping for the Rh, Kell, Kidd, and Duffy blood groups were done in alloimmunized patients using monoclonal antibodies and Multiplex-Allele Specific Oligonucleotide-Polymerase Chain Reaction (Multiplex-ASO-PCR) and Tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR), respectively. Any phenotype/genotype discrepancy was assessed by direct sequencing.

Results: Ninety-seven (8.5 %) out of 1147 patients had alloantibodies against the minor blood group antigens (44 males, 45.4 %, and 53 female, 54.6 %). The most common alloantibodies were against the RH (n: 47, 48.5 %), and the Kell (n: 23, 23.7 %) blood groups systems. Twenty-three (2.1 %) genotype/phenotype discrepancies out of 1067 tests, including 9 in the Rh (9.3 %), 8 in Duffy (34.8 %), and 6 in Kidd (26.1 %) blood groups were detected. No discrepancy was found in the Kell blood group system. Direct sequencing revealed that the results of molecular methods were correct.

Conclusion: Multiplex-ASO-PCR and T-ARMS-PCR molecular methods are fast, reliable and cost-benefit molecular methods for the minor blood group genotyping in multi-transfused β-thalassemia major patients.