HLA-antibody testing: The immune phagocytosis inhibition test is superior to the PRA-STAT and NIH lymphocytotoxic test with respect to specificity

We compared the specificity and sensitivity of four different methods for t he detection of antibodies specific for HLA antigens. The NIH version of th e complement-dependent cytotoxic test (CDC) was used as the gold standard t o which we compared two Fc gamma receptor (Fc gammaR)-dependent immune phag ocytosis inhibition tests (IPI) and one commercial enzyme-labelled immunoso rbent assay (ELISA) with soluble HLA class I-antigen preparations bound to the plate (PRA-STAT). Both IPI tests are based on the fact that HLA-antibod ies specifically bind to antigens on the monocyte surface via their Fab por tion, and in so doing block a neighbouring Fc gammaR with their Fc region. This blockade prevents phagocytosis of IgG-coated red blood cells (RBCs), w hich can be measured either microscopically (IPIm) or photometrically (IPIp ). The four assays were used in blind tests on 20 human alloantisera or mon oclonal antibodies with known HLA-antigen reactivities. Additionally, two m onoclonal antibodies and one human serum were titrated to elucidate the sen sitivity of each test. After all tests were completed, the identities of th e samples were disclosed. Both IPI methods detected and identified all clin ically relevant HLA class I and class II specific antibodies. In contrast, the CDC was not able to detect noncytotoxic HLA-antibodies and HLA class II specific antibodies; however, it detected clinically insignificant IgM Iym phocytotoxins. The PRASTAT assay enabled identification of all cytotoxic an d noncytotoxic IgG antibodies with specificity for HLA-class I antigens. Wi th respect to sensitivity, the CDC and the IPI methods were superior to the PRA-STAT These facts demonstrate the advantage of IPI methods in the detec tion of clinically relevant HLA-antibodies. J. Clin. Lab. Anal. 15:334-341, 2001. (C) 2001 Wiley-Liss, Inc.