Commercial and research laboratories are working on methods to improve HLA typing, antibody screening and crossmatch methods. I n a traditional cell cytotoxicity assay, anti-HLA serum, or monoclonal antibody, is mixed with live lymphocytes. Specific Ig binds to the polymorphic protein moiety of the HLA molecule expressed on the cell surface. Exogenous complement is added to the well which will result in lysis of cells to which antibody has been bound. Cell death is determined by ethidium bromide vital stain exclusion.
Specific binding is detected by vital dye stain.
Alloantisera are limited in quantity and are often not specific for a single allele. Production of monoclonal antibodies is also difficult since most react with all alleles, i.e., framework specificity. Vital dye exclusion techniques, which are commonly used, often require millions of viable lymphocytes that express HLA antigens which makes them difficult to perform in small children or patients with low white cell counts. Molecular methods for detection of HLA antigens K nowledge of the genes that encode HLA molecules bypasses the requirement for viable lymphocytes that express the protein. The polymorphism in class II genes is limited to a single exon which has allowed DR and DQ typing by various molecular methods to become routine in most labs worldwide.