Microdroplet testing for HLA-A, -B, -C, and -D antigens. The Phillip Levine Award Lecture.

TLDR: It is now necessary to distinguish antibodies against T and B lymphocytes and those that react in cold and in warm conditions, previously thought to be detectable only by lymphocyte-determined (LD) tests.

Abstract: The microdroplet lymphocyte cytotoxicity test is universally accepted as the standard test for HLA antigen determination. An update of the technical details of the test is given, based on the authors’ testing 160,000 persons. Methods for quality control of the test as well as reproducibility data are provided. International standardization of the specificities has been accomplished by seven international workshops and a continuous cell exchange involving testing of 108 cells since 1974 by as many as 180 laboratories. The test has recently become applicable to the detection of HLA-D determinants, previously thought to be detectable only by lymphocyte-determined (LD) tests. Purified peripheral blood lymphocytes are reacted with antisera from which HLA-A, -B, and -C antibodies have been removed. B lymphocytes were found to be smaller than T lymphocytes by Coulter counter sizing. The purity of cell suspensions enriched for B lymphocytes can be individually monitored, as shown by the reactions produced by 126 test samples. HLA-D antigens have a linkage disequilibrium with certain HLA-A and -B specificities as demonstrated by population and family studies. Haplotypes found in 34 parents of 18 families demonstrate the new haplotypes, which now consist of four antigens per haplotype. Studies of HLA-D frequencies in Caucasians, Negroes, and Orientals show a distinctive distribution in the races. B lymphocytes also appear to have an autoantigen against which autoantibodies are readily produced. The autoantibodies are more active against B than T lymphocytes and act most effectively at 5 C. Although they appear in many diseases, most notably in systemic lupus erythematosus, they also occur in 10% of normal males and females. In patients awaiting kidney transplants, antibodies against B lymphocytes are often found. Patients with cold B-cell antibodies (autoantibodies) were shown to have higher transplant survival rates than those with warm B-cell antibodies (allogeneic). Thus, in performing crossmatch tests it is now necessary to distinguish antibodies against T and B lymphocytes and those that react in cold and in warm conditions.