Improved flow cytometry crossmatching in kidney transplantation

Area of Science:

• Transplantation immunology
• Flow cytometry
• Immunoserology

Background:

• Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based assay for detecting donor-specific antibodies (DSAs).
• Current FC-XM methods face limitations due to methodological variations and inability to distinguish antibody types.
• Pre-test clinical factors can significantly impact FC-XM results, affecting assay reliability.

Purpose of the Study:

• To enhance the sensitivity and specificity of the FC-XM assay.
• To address limitations in distinguishing complement-fixing from non-complement-fixing antibodies.
• To mitigate interferences from pre-test factors and therapies like rituximab.

Main Methods:

• Implementing pronase treatment to prevent nonspecific immunoglobulin binding.
• Utilizing 7-aminoactinomycin-D (7-AAD) or C4d antibody staining to differentiate complement-fixing antibodies.
• Employing donor endothelial precursor cells for detecting non-HLA antibodies.
• Incorporating anti-rituximab antibody incubation to neutralize rituximab interference.

Main Results:

• Pronase treatment effectively prevents false-positive B-cell FC-XM results.
• Distinguishing complement-fixing antibodies is achievable with 7-AAD or C4d staining.
• Endothelial precursor cells allow detection of non-MHC antibodies.
• Rituximab interference can be prevented with specific blocking strategies.

Conclusions:

• Assay adjustments significantly improve FC-XM sensitivity and specificity.
• FC-XM, when optimized, provides more reliable DSA detection.
• Interpretation of FC-XM results must consider virtual crossmatch for comprehensive assessment.