P014 : THE EFFECTS OF ANTIGEN TARGET NUMBER ON ACCURATE FCXM INTERPRETATION

Abstract

Aim We examined the relationship between Luminex single antigen bead (LSAB) MFI values and flow cytometry crossmatch (FCXM) interpretation according to variable number of target antigens on the cells tested. Methods Sera ( n = 4) with select high reactivity to Aw4/Bw4, Bw6, or A2 CREG specificities alone (no class II reactivity) were selected. Sera were serially diluted (8-fold) up to 1:4096. Dilutions were analyzed by LSAB and FCXMs using PBL from individuals ( n = 14) with variable number of targets pertaining to the specificity of each serum (⩾3 PBL/serum). PBL class I antigen density was measured using W6/32. Results LSAB MFI values from serum dilutions highly correlated to the median channel shift (MCS) obtained against all cells tested ( r > 0.955). Comparing MCS from PBL with 1 target vs 2, we consistently observed an increase in MCS for each of the 4 sera ( Fig. 1 ). The % increase in MCS was higher using a 1:8 dilution when compared to neat. Only one serum was tested against pbl with 3 targets and another serum against PBL with 4 targets. In general, the trend was with increased MCS with increased number of targets. The number of target antigens on the PBL tested affected the overall FCXM interpretation. At high dilution ratios (1:64), the top bead MFI for the epitope (Bw4) was 2633 (median 1483 for all Bw4 containing beads) and interpretation varied between T and B neg results to T and B pos results with increasing number of targets (Table 1). Although class I antigen density also translated into increased MCS values, the increases were less than those observed with increased number of targets. Conclusion Our data suggest that accurate FCXM prediction requires knowledge of the epitope reactivities in a serum and the number of targets on the cells tested.