Evaluation of the PRA-STATTM system for HLA antibody testing
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文摘
Despite early reports that a positive lymphocytotoxicity crossmatch(XM) predicted hyperacute or acute rejection of a kidney graft, there have been multiple reports of successful transplantation in the face of a positive XM. It is becoming increasingly clear that the antibodies relevant to transplant outcome are those that are HLA-specific. However, antibody screening by cytotoxicity is costly and laborious and cannot be readily performed at the time of transplant. We have evaluated the PRA-STATTM (PS) kit (Nextran,McGaw Park, IL), an ELISA assay that utilizes soluble HLA class I molecules for its ability to detect and define HLA-specific antibodies. 219 tests were performed on sera from 118 subjects and, of these, 128 specimens were tested by both cytotoxicity (CYT) and PS. There was a significant correlation (r = 0.78, p<0.001) between PS and CYT for the detection of IgG antibodies. PS uses an anti-IgG conjugate and does not detect IgM antibodies. Of 66 sera reactive in both assays, 12 had identical specificities defined in both; 18 were more reactive in PS; 5 were more reactive in CYT; and 31 had different specificities in the 2 assays, with overlap in some cases. Heat inactivation of the sera revealed that some of the difference in reactivity was due to the presence of IgM antibodies. However, even after heat inactivation, we found that some sera reacted with all cells bearing a specific antigen in CYT but did not react with all wells containing the antigen in PS. Of 14 specimens reactive only in PS, 3 were from healthy, non-transfused, non-transplanted males. These sera have been tested extensively by antiglobulin and flow cytometry and found not to be reactive with lymphocytes. Studies to characterize the antibodies in these sera are underway. Since the PS system does not detect IgM antibodies, we tested in blocking experiments if HLA-specific, IgM antibodies would interfere with the detection of IgG antibodies of the same specificity. IgM blocking antibody altered the O.D. in 82 % of test wells, but did not always reduce IgG binding. Because many of the IgG antibodies were oligospecific, clear interpretation of blocking specificity was not possible. Duplicate tests of several sera revealed that reproducibility was very good within a lot but not between lots of kits. The composition of the panel makes it difficult to clearly determine antibody specificity. We have found the PS test to be a rapid system for antibody testing that can be automated, in part. Improvements in the cell panel, resolution of the basis of false positive reactions, the opportunity to detect IgM as well as IgG antibodies, and improved reproducibility between lots will make this a valuable tool for clinical testing.

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