4-Color Flow Cytometric Cytotoxicity Crossmatch Using Whole Blood Lysis

The conventional lymphocyte crossmatch (XM) assay necessitates mononuclear cell isolation. In our previous work, we introduced a novel 3-laser, 4-color flow cytometric (FC) XM protocol utilizing whole blood lysis (WBL) and CD45 fluorescence-triggered acquisition to detect human leukocyte antigen (HLA) antibody binding. Building on this, we aimed to adapt these advancements for complement-dependent cytotoxicity (CDC) XM using FC (FCCDC).

A total of 164 donor/recipient pairs undergoing transplantation were stratified into 2 groups based on donor-specific HLA alloantibody presence: DSA-positive (DSA+, n = 73) and DSA-negative (DSA-, n = 91). The DSA- group was further subdivided by ABO compatibility into ABO-incompatible (ABOi, n = 52) and ABO-compatible (n = 39) subgroups. Protocol optimization for the WBL FCCDC with CD45 V500-C was conducted using a FACSLyric cytometer (BD Biosciences). T and B cell indices were calculated as delta (test minus control) percentages of dead cells (Δ%DC). WBL FCCDC results were compared with those of conventional FCCDC in each group.

WBL FCCDC showed no significant quantitative difference from conventional FCCDC. In the DSA+ group, B cell Δ%DC values were 28.00 ± 27.14 and 19.16 ± 27.74 for WBL FCCDC and conventional FCCDC, respectively (P = .0777). No ABO antibody interference was observed in the ABOi subgroup. Qualitatively, B cell WBL FCCDC sensitivity in the DSA+ group was 69.9%, comparable to conventional FCCDC sensitivity (65.8%, P = .5078). Additionally, WBL FCCDC reduced the turnaround time by 50 minutes relative to conventional FCCDC.

WBL FCCDC achieved performance equivalent to conventional FCCDC, similar to WBL FCXM. The absence of adverse effects from the lysis step supports its integration into XM assays. Given its simplicity and maintained sensitivity, the WBL FCCDC protocol presents a viable alternative to conventional methods in histocompatibility laboratories.