Detection of RBC-bound antibodies using flow cytometry and their correlation with HLA polymorphism.

BACKGROUND: The immune response in elderly individuals is influenced by genetic factors, particularly polymorphisms in major histocompatibility complex (MHC), such as human leukocyte antigen (HLA). Age-related changes in immune function, referred to as immunosenescence, can lead to the formation of RBC-bound antibodies, which may pose risks in transfusion medicine. OBJECTIVE: This study aims to investigate the association between HLA-DQB1 alleles and the presence of RBC-bound antibodies in elderly individuals. MATERIALS AND METHODS: A total of 83 healthy elderly (aged 60-70) samples were collected. Blood samples were processed by centrifugation at 3500 rpm for 10 min to obtain serum. RBCs were isolated through centrifugation at 3000 rpm, washed with saline, and 2% type O RBC was prepared. Serum (100 µL) was mixed with saline (100 µL) and 2% type O RBCs (50 µL), incubated at 37 °C for 30 min, and analyzed via flow cytometry. At the same time, control sample was also processed with equal volume. DNA was extracted using the sequence-based typing kit, and specific genes at the HLA DQB1 locus were amplified by PCR, confirmed through agarose gel electrophoresis and sequencing for allele identification. RESULTS: RBC-bound antibodies were detected in 42 of 83 participants, indicating a higher index of agglutination (IAG), while 41 samples showed no RBC-bound antibodies, indicating negative IAG. The mean IAG was -1.88 (SD = 25.943), reflecting variability in immune reactions. Sequence based typing (SBT) analysis revealed a significant association between the HLA-DQB1*04 allele and IAG (p < 0.034), while no significant associations were found for alleles DQB1*02, *03, *05, and *06. CONCLUSION: This study highlights the role of HLA-DQB1*04 in the presence of RBC-bound antibodies in elderly individuals, emphasizing the complexities of immunosenescence and variability in immune responses within this population.