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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.