BACKGROUND: Flow cytometry crossmatch (FCXM) results are used to assess the safety of a transplant across pre-existing DSA. The number of targets on donor cells dictates how much DSA may be bound yet this variable is not currently considered in FCXM interpretation. We examined the relationship between Luminex single antigen bead (LSAB) MFI values and FCXM interpretation according to the number of target antigens on the cells tested.

METHODS: Sera (n=4) with select high reactivity to either Aw4/Bw4, Bw6, or A2 CREG specificities (no class II reactivity) were selected and diluted (8-fold) to 1:4096. Dilutions were analyzed by LSAB and FCXMs using PBL (n=14) containing variable number of targets pertaining to the specificity of each serum (≥3 PBL/ serum).

RESULTS: LSAB MFI values from dilutions correlated to the median channel shift (MCS) for all cells tested (r>0.955). Comparing MCS from PBL with 1 target vs 2, we consistently observed an increase (>120%) in MCS in all 4 sera (Fig.1). The increase in MCS was higher using a 1:8 dilution when compared to neat. One serum was tested against PBL with 1, 2, or 3 targets and the MCS increased with increased number of targets (Table 1). At high dilutions (1:64), the top bead MFI for the DSA was 2633 and interpretation varied between T and B neg to T and B pos results depending on the number of targets.

CONCLUSION: Our data suggest that FCXM results can vary depending on the number of targets on the cells tested particularly with weak levels of DSA and is a variable that must be accounted for to accurately interpret FCXMs.

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