Techniques used to define human MHC antigens: serology

Area of Science:

• Immunology
• Genetics
• Clinical Diagnostics

Background:

• The complement-mediated cytotoxicity assay, developed in the 1960s, is the classical method for human leukocyte antigen (HLA) tissue typing.
• This assay defines HLA antigens on lymphocytes from peripheral blood, spleen, or lymph nodes, crucial for transplantation and disease studies.

Purpose of the Study:

• To describe the established complement-mediated cytotoxicity assay for HLA antigen definition.
• To highlight the application of this assay in various clinical and research settings, including cell lines and prenatal diagnostics.

Main Methods:

• Target cells (lymphocytes) are incubated with typing antisera, followed by the addition of rabbit serum as a complement source.
• Incubation at 22°C in Terasaki trays, followed by staining to detect target cell death, indicates a positive reaction.
• Automation and antibody-coated magnetisable microspheres are employed for enhanced efficiency and pure B lymphocyte separation for class II typing.

Main Results:

• The assay accurately defines HLA-A, B, Cw (class I) and HLA-DR, DQ (class II) antigens.
• Successful HLA typing has been demonstrated using various cell sources, including cultured cells and cell lines.
• The method allows for the definition of antibodies to HLA antigens using cells with known HLA phenotypes.

Conclusions:

• The complement-mediated cytotoxicity assay is a robust and adaptable method for HLA antigen definition.
• Modern adaptations, including automation and microsphere technology, improve the efficiency and precision of HLA typing.
• This assay remains fundamental for immunological and clinical applications requiring precise HLA characterization.