3.333333
#31
VALIDATION OF INCONCLUSIVE RESULTS OF BD HLA-B27 FLOW CYTOMETRY TEST USING BD-CellQuest Pro.
Dora Ninova MD,PhD 1, Steven R. DeGoey 1 and S. Breanndan Moore MD 1. 1 Tissue Typing Laboratory, Division of Transfusion Medicine, Mayo Clinic, Rochester, MN, USA .
A number of clinical laboratories in the US and worldwide perform HLA-B27 typing by BD HLA-B27 kit, a flow cytometry test using CD3 cell population for analysis. However, the test may give inconclusive results and samples are retested by complement dependent cytotoxicity (CDC) using HLA typing trays. The aim of this study was to introduce analysis of data files of inconclusive B27 samples using BDCellQuestPro (CQP) software and validate the procedure by comparing the results with CDC. Methods: BD HLA-B27 test, CQP analysis, CDC using NIH protocol. Results: CQP analysis template was created and used to validate results when B27 software could not position the gate properly as: 1/ CD3 cells <2% of all acquired events; 2/ CD3 population not well separated from the other cells; 3/ gate set incorrectly on cells other than CD3. Out of 26 samples with low events CQP analysis showed 23 (-) and 3 (+) results. Out of 29 samples with low separatrion 19 were (+) and 10(-). All 55 samples showed the same result by CDC. Analysis of samples with wrong gate demonstrated that if the gate was set on granulocytes instead of CD3 cells, in 70% the result was false negative according to the kit preset cut-off marker of 147 median fluorescent intensity (MFI). In 4 patients with deficient CD3 population granulocytes were utilized for gating using a cut-off of 122 MFI, which was calculated as mean+2SD of the MFI of B27 on granulocytes in 100 samples. Conclusion: analysis of inconclusive results with CQP showed 100% confirmation by CDC and 1/ saved technologist time and money by eliminating re-testing by CDC; 2/ prevented false negative/false positive results by correcting wrong gates; 3/ provided analysis of HLA-B27 in CD3 deficient patients.