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

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.

This study aims to investigate the association between HLA-DQB1 alleles and the presence of RBC-bound antibodies in elderly individuals.

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.

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.

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.