Establishing a New Stem Cell Crossmatch Assay to Assess the Impact of HLA ‐Specific Antibodies on CD34 + Stem Cells

Mismatched allogeneic haematopoietic stem cell transplants (HSCT) are complicated by the presence of pre-existing HLA-specific antibodies targeting the mismatched HLA antigens (DSAs). Data indicates that the presence of DSAs pre-transplant, termed HLA incompatible HSCT, is associated with inferior outcomes and graft failure. Whilst studies acknowledge that DSAs should be avoided there is limited information regarding the impact of specific DSAs and their levels on HSCT outcomes. There is also a recognised lack of knowledge and methods which allow the immunological risk of HLA incompatible HSCT to be defined, with a large volume of data being extrapolated from solid organ transplant data. We have established a stem cell specific flow cytometry crossmatch (SC-FCXM) for the in vitro assessment of the binding capacity of HLA-specific antibodies to CD34+ stem cells. A library of HLA typed expanded human CD34+ progenitor cells from cord blood (CB-CD34+) was generated and used to assess HLA-specific antibody binding of 86 serum samples from the National External Quality Assurance Scheme (NEQAS) Histocompatibility and Immunogenetics crossmatching 2B external proficiency testing scheme issued between 2023 and 2025. Sera were analysed using LABScreen Single Antigen (LSSAg) Class I and II and donor specific HLA antibodies (DSAs) were identified for each CB-CD34+ and serum combination. A range of DSAs targeting single HLA antigens, plus multiple HLA-specific Class I, Class II or a combination of Class I and II antibodies based on mean fluorescence intensity (MFI) was examined. The impact of increasing DSA MFI on SC-FCXM reactivity was determined by correlation and regression studies comparing cumulative peak LSSAg MFI versus median fluorescence intensity of anti-human IgG APC as determined by the SC-FCXM (APC Median SC-FCXM). As LSSAg MFI of DSAs targeting HLA-A (p = 0.0001), -B (p = 0.0001) and -DRB1 (p = 0.0155) increased, there was significant correlation with APC Median SC-FCXM, but no correlation (p > 0.05) between LSSAg MFI and APC Median SC-FCXM for HLA-C, -DQB1, -DQA1 or -DPB1. A significant correlation (p ≤ 0.05) was observed for multiple HLA Class I (p = 0.0005), Class II (p = 0.0001), plus combinations of Class I and II DSAs (p = 0.0013) between combined LSSAg MFI and APC Median SC-FCXM. The extrapolation of this preliminary data has provided an indication of which DSAs and their levels (as determined by LSSAg MFI) cause FCXM reactivity against CD34+ stem cells, which could prove to be relevant in HLA incompatible HSCT. However, the assay currently remains a proof-of-concept and further work is required to establish clinical correlation of the results to patient outcomes.