The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.