Pronase treatment improves flow cytometry crossmatching results

Area of Science:

• Immunology
• Transplantation Science
• Cell Biology

Background:

• Flow cytometry crossmatching (FC-XM) is crucial for detecting donor-specific antibodies in organ transplantation.
• Elevated background reactivity in B-cell FC-XM assays is caused by nonspecific immunoglobulin binding via Fc receptors and B-cell surface immunoglobulins.

Purpose of the Study:

• To reduce background reactivity in B-cell FC-XM assays.
• To investigate the effect of pronase treatment on B-cell surface markers and assay performance.

Main Methods:

• Lymphocytes were treated with pronase (1 mg/mL for 30 minutes).
• Expression of kappa light chains, Fc receptors (CD32b), CD19, CD20, and MHC I/II on B-cells was analyzed.
• Prospective experiments evaluated assay sensitivity and specificity with pronase-treated cells.

Main Results:

• Pronase treatment significantly reduced kappa light chains and Fc receptors (CD32b).
• Acceptable decreases in CD19, CD20, and MHC I/II expression were observed.
• Higher pronase concentrations (>2 mg/mL) caused significant reductions in key B-cell markers.
• Pronase treatment led to a relative increase in sensitivity and specificity in B-cell FC-XM assays.

Conclusions:

• Pronase treatment is an effective method for reducing background reactivity in B-cell FC-XM.
• This approach improves the sensitivity and specificity of FC-XM for detecting donor-specific antibodies in organ transplantation.