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Improved flow cytometry crossmatching in kidney transplantation
Author(s) -
Guillaume Nicolas
Publication year - 2018
Publication title -
hla
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.347
H-Index - 99
eISSN - 2059-2310
pISSN - 2059-2302
DOI - 10.1111/tan.13403
Subject(s) - antibody , pronase , flow cytometry , rituximab , transplantation , monoclonal antibody , complement dependent cytotoxicity , microbiology and biotechnology , immunology , chemistry , medicine , biology , biochemistry , antibody dependent cell mediated cytotoxicity , trypsin , enzyme
Flow cytometry crossmatching (FC‐XM) assay is the most sensitive cell‐based method for detecting donor‐specific antibodies (DSAs). However, the use of FC‐XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement‐fixing and noncomplement‐fixing antibodies. FC‐XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20 + B‐cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC‐XM. Pronase treatment (0.5‐1 mg/mL) prevents false‐positive B‐cell FC‐XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B‐cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti‐rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin‐D (7‐AAD) or fluorochrome‐conjugated C4d Ab, after complement incubation, allows complement‐fixing antibodies to be distinguished from noncomplement‐fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab‐binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.