Introduction: The crossmatch assay is used to determine sensitization of solid organ transplant recipients to donor-expressed HLA molecules, a potential contraindication to transplant. Multicolor flow cytometry-based crossmatch (FCXM), the current state of the art, involves incubation of recipient serum with donor leukocytes, with detection of donor-bound recipient antibodies by anti-human IgG. A known interfering factor of this assay is the detection of irrelevant IgG bound to donor B cells by the Fc receptor CD32. Pronase treatment of donor leukocytes can be used to digest CD32, and reliably eliminates false-positive B cell FCXM results. However, pronase treatment adds time to the FCXM assay, and can induce other false-positive results, notably when assaying serum from antiretroviral-treated patients. Here we tested Fc blocking reagents as a replacement for pronase treatment.

Methods: Leukocytes were isolated from peripheral blood, spleens, or lymph nodes of either deceased donors or normal, living individuals. Pronase treatment (0.5 mg/ml) was carried out for 15 minutes at 37[deg]C, followed by 3 minutes of treatment with DNase (2.75 mg/ml). CD32 monoclonal antibodies 6C4 (eBioscience) and IV.3 (Stemcell Technologies), and a recombinant, immunoglobulin-derived protein (Human BD Fc block; BD Biosciences) were incubated with donor cells for 10 minutes before serum was added. To induce Fc receptor-mediated false positives, heat aggregated gamma globulin (Quidel) or heat-treated serum (20 minutes at 56[deg]C) were used.

Results: Of the three Fc receptor-blocking reagents, only CD32 monoclonal antibody 6C4 was able to prevent aggregated immunoglobulin binding to untreated donor B cells, producing mean channel shift values consistent with a negative FCXM. Additionally, serum from an antiretroviral-treated HIV+ patient resulted in false-positive T cell FCXM when tested against pronase-treated cells, but not with 6C4-incubated cells.

Conclusion: The nonspecific enzyme cocktail pronase can be used to abrogate false-positive B cell FCXM results, due to aggregated or antigen-bound IgG binding to B cell-expressed Fc receptor. Our results demonstrate that a more specific reagent, CD32 monoclonal antibody 6C4, can replace pronase treatment. Importantly, 6C4 incubation is faster than pronase treatment, and does not introduce other false positives. Blocking the Fc receptor increases the specificity of the FCXM assay, reduces test time, and is expected to improve patient management.

CITATION INFORMATION: Brown N, Wang J, Marino S. Blocking Fc Receptor in Flow Cytometric Crossmatch: Refining an Old Assay to Improve Patient Management. Am J Transplant. 2016;16 (suppl 3).

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