Flow Cytometry PRA Using Pooled Lymphocytes for both HLA Class I and II Antibodies

It is well known that human leukocyte antigen (HLA) class I antibodies can be detected by flow cytometry (FC) using T cells in pooled lymphocytes from randomly selected donors of a sufficiently large number. However, this methodology cannot detect class II antibodies. In this study, a new panel reactive antibody (PRA) assay for HLA antibodies of both classes was developed. This new assay, which pooled FC PRA, also uses pooled lymphocytes. However, the lymphocytes are treated beforehand with pronase, and both T and B cells are analyzed. When performing this assay, lysis of highly fragile lymphocytes should be avoided throughout each step. An optimal protocol was investigated for the preparation of a pooled lymphocyte panel and for the assay procedure. Performance of the established protocol was compared with that of the enzyme-linked immunosorbent assay (ELISA) or Luminex PRA assay. Using ELISA PRA-proven sera, pooled FC PRA determined 17 allosera as positive among 18 patient sera with HLA class II antibodies only (sensitivity, 94%), and determined 68 sera as negative among 73 patient sera without detected HLA antibodies (specificity 93%). In the endpoint titration, however, the sensitivity of this assay was lower than that of ELISA PRA, particularly for HLA antibodies against low frequency antigens. Pooled FC PRA can simultaneously detect HLA class I and II antibodies, reducing the shortcomings of the previous FC PRA which uses T cells only. This new assay is suggested as another complementary approach for a more comprehensive PRA assay using pooled lymphocytes.