119-P

• Published in: Human immunology Vol. 73; p. 124
• Main Authors: Souza, Stephanie, Ching, Patrick, Endres, Robert O, Su, Leon L, Valdez, Riccardo
• Format: Journal Article
• Language: English
• Published: 01-10-2012
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2012.07.245

Aim Rituximab (RIT) is currently used to treat and prevent allograft rejection in transplant recipients, and is known to interfere with the flow cytometric crossmatch (FCXM). Pre-FCXM pronase treatment reduces, but does not eliminate, background interference. In this study, we employed common red cell serology techniques to reduce interference in the FCXM for five patients on RIT therapy. Methods Pre- (ACD) and post-administration (CLOT) blood samples were collected from five subjects eligible to receive RIT therapy. Buffy coats were harvested from the ACD samples and cryopreserved. Sera from the CLOT samples was filtered and frozen at <−20 °C until testing. A 3-color FCXM was performed as follows: 1) NEAT–All samples were tested without modification. 2) Autologous Adsorption (AA)–Subject serum was adsorbed against a double volume autologous leukocyte-rich buffy coat aliquot. 3) Competitive Binding (CD20)–Donor cells were incubated with non-humanized monocolonal anti-CD20. 4) Inhibition–Donor cells were incubated with heat-inactivated rabbit serum (HIRS). 5) Enzyme Treatment (PRO)–Donor cells were treated with pronase prior to testing. Median fluorescence intensity (MFI) data from each modification were compared against the NEAT data, within run, for each subject. The results were analyzed for significance using the student’s t-test for unequal variance. Results All post-RIT samples resulted in strongly positive B cell FCXMs, (mean MFI NEAT = 895). CD20 and PRO significantly reduced mean MFI (835.2, p < 0.001; 608.2, p < 0.001), while AA and HIRS showed no significant difference from the neat FCXM (896.2, p = 0.867; 891.2, p = 0.558). The T cell FCXM was unaffected by RIT, however, an artifact was observed during HIRS that resulted in strongly positive T cell FCXMs for all samples (529.6, p < 0.001). Conclusions Two modifications to the FCXM protocol showed promise in reducing RIT interference: 1) competitive binding using a non-humanized monoclonal anti-CD20, and 2) pronase treatment of the donor cells.