EVALUATION OF HLA CLASS II ANTIBODY DETECTION BY FLOW PRA II.
P Warner and E Klohe, HLA Laboratory, Inland Northwest Blood Center, Spokane, WA.


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The FlowPRA I assay has been shown to be more specific and sensitive than complement dependent cytotoxicity (CDC)-based T-cell PRA assays and is a better predictor of flow cytometric (FC) T-cell crossmatch (XM) results. We evaluated the FlowPRA II assay to determine whether it's specificity, sensitivity and utility in predicting FC XM results is superior to CDC-based B-cell PRA assays. The unabsorbed sera of 65 patients and 3 reagent-grade typing sera were analyzed by FlowPRA II methodology. PRAs were compared to those obtained using platelet micro-absorbed sera via our in-house CDC B-cell PRA assay. The summarized results are:
                               ______CDC______

		POS	NEG
FLOWPRA II	POS	21	6
	NEG	8	33

The initial concordance rate is 80% (31% +/+, 49% -/-). Of the 8 CDC+/FlowPRA II-, 4 were due to unabsorbed Class I reactivity, 3 were due to autoantibodies, and 1 was due to IgM antibodies. If those sera with unabsorbed Class I reactivity and autoantibodies are deleted from the analysis, the concordance rate is 88%. Of the 6 CDC-/FlowPRA II+, 5 exhibited weak positive FL1 channel values, below the threshold for CDC detection (<750-800, linear 1024 scale), when compared to channel values of known Class II antibodies that have been diluted past CDC titration endpoints. The single serum that showed intermediate FlowPRA II activity demonstrates reactivity by unabsorbed B-cell CDC and FC XMs. Class II antibody identification for these six sera was accomplished through a combination of retrospective initial CDC B-cell XM results and FlowPRA II specific assays. Antibody specificities correlated well with FlowPRA II results when analyzed in the context of Class II antigen frequencies in the FlowPRA II bead pool. In conclusion, the primary disadvantage of the FlowPRA II assay is it's inability to detect IgM HLA Class II antibodies. We have found the FlowPRA II assay to be more sensitive and specific than CDC. In addition, it is an excellent predictor of FC XM results. Finally, the FlowPRA II values correlate well with expected values of specific antibodies.