4-Color Flow Cytometric Cytotoxicity Crossmatch Using Whole Blood Lysis.

Area of Science:

• Transplantation immunology
• Histocompatibility testing
• Immunosuppression monitoring

Background:

• Conventional lymphocyte crossmatch assays require laborious mononuclear cell isolation.
• Previous work introduced whole blood lysis (WBL) for flow cytometric (FC) crossmatch (XM) using CD45 triggering.
• This study adapts WBL FCXM advancements for complement-dependent cytotoxicity (CDC) XM (FCCDC).

Purpose of the Study:

• To adapt and validate a novel WBL flow cytometry crossmatch for complement-dependent cytotoxicity (FCCDC).
• To evaluate the performance and efficiency of WBL FCCDC compared to conventional FCCDC.
• To assess the impact of WBL FCCDC on turnaround time and sensitivity in detecting HLA antibodies.

Main Methods:

• 164 donor/recipient pairs were stratified into DSA-positive (n=73) and DSA-negative (n=91) groups.
• DSA-negative group further divided into ABO-incompatible (n=52) and ABO-compatible (n=39) subgroups.
• WBL FCCDC protocol optimized using CD45 V500-C on a FACSLyric cytometer; T and B cell indices calculated as delta (% dead cells).

Main Results:

• WBL FCCDC demonstrated no significant quantitative difference compared to conventional FCCDC.
• B cell delta (% dead cells) in DSA+ group: 28.00 ± 27.14 (WBL) vs. 19.16 ± 27.74 (conventional), P = .0777.
• WBL FCCDC sensitivity (69.9%) comparable to conventional FCCDC (65.8%, P = .5078) in DSA+ group; no ABO antibody interference observed.

Conclusions:

• WBL FCCDC achieves performance equivalent to conventional FCCDC, mirroring WBL FCXM findings.
• The lysis step in WBL FCCDC shows no adverse effects, supporting its integration into crossmatch assays.
• WBL FCCDC offers a simpler, sensitive, and viable alternative for histocompatibility laboratories.