It's About Everything: Validation and Optimization of 96-Well Tray Flow Cytometry Crossmatch

Area of Science:

• Transplantation immunology
• Clinical chemistry
• Immunogenetics

Background:

• Flow cytometric crossmatch (FCXM) is crucial for pre-transplant risk assessment to detect donor-specific anti-HLA antibodies.
• The standard tube FCXM method's limited throughput hinders testing of highly sensitized patients, often leading to organ allocation to non-sensitized recipients.

Purpose of the Study:

• To optimize a 96-well tray FCXM protocol for increased throughput without compromising assay quality or sensitivity.
• To enhance the probability of highly sensitized patients receiving transplants by expanding crossmatch capabilities.

Main Methods:

• Validation of a novel 96-well tray FCXM protocol against the standard tube method.
• Simultaneous testing of 63 crossmatches using 20 donors' cells, patient sera, pooled positive controls, and negative controls.
• Optimization of assay parameters including incubation time, reagent volumes, and cell concentrations.

Main Results:

• Achieved 98% overall concordance between the 96-well tray and standard tube methods.
• Demonstrated slight improvement in sensitivity with the tray method, detecting weak donor-specific antibodies and mild T-cell reactivity.
• Median channel fluorescence values were equivalent for both T and B cell crossmatches (r² of 0.98 and 0.97, respectively).

Conclusions:

• The 96-well tray FCXM assay significantly increases crossmatch capacity, reducing turnaround time and assay costs.
• Enhanced sensitivity provides more accurate patient sensitization data, aiding therapeutic decisions.
• The optimized protocol has the potential to increase transplant rates for highly sensitized patients.