Cytotoxic Antibody Detection by Means of Flow-Cytometric Cross-Match

Bilgen, Türker; Ata, Pinar; Tozkır, Jülide Duymaz; Tozkır, Hilmi; Titiz, M.I.

doi:10.1016/j.transproceed.2017.01.016

Cited by 4 publications

(8 citation statements)

References 12 publications

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“…In order to evaluate the cytotoxicity parameter, this assay was compared with CDC-XM and was performed using sera with and without DSA C1q-fixing antibodies. Unfortunately, no clear evidence of a higher sensitivity for FCtox assay-XM compared with CDC was observed, 39,43 as no significant difference was observed between FCtox assay-XM and CDC in terms of their ability to detect serum containing C1q-fixing antibodies. Moreover, a number of crossmatches with C1q+ sera did not induce lysis of T-and/or B-cells in the FCtox assay-XM.…”

Section: Improved Fc-xm By Means Of a Functional Assaymentioning

confidence: 99%

“…Efforts have therefore been made to combine determination of Ab binding and cytotoxicity using flow cytometry-based assays (FCtox assay). [37][38][39][40][41][42][43][44][45] Briefly, after incubation between patient serum and donor lymphocytes, rabbit complement was added, followed by 7 aminoactinomycin-D (7-AAD), a fluorescent intercalator that undergoes a spectral shift upon association with DNA. Cytotoxicity was determined by the percentages of 7-AADpositive cells (ie, nonviable) and Ab binding was determined by the median fluorescence intensity of the fluorochromeconjugated secondary Ab.…”

Section: Improved Fc-xm By Means Of a Functional Assaymentioning

confidence: 99%

Section: • Use Of Donor Epcs As Target Cells For Detection Of Non-mentioning

confidence: 99%

“…46 Differences were also observed in terms of DSA detection based on the anti-IgG-fluorochrome MFI values detected on FCtox assay-XM and conventional FC-XM assays. Bilgen et al 43 Another approach consists of directly detecting C4dfixing alloantibodies on T-and B-cells by using a fluorochrome-conjugated C4d Ab after incubation with a complement source. Jain et al 44 tested this assay in a small series of clinical cases.…”

Section: Donor Recipientmentioning

confidence: 99%

“…Use of anti-rituximab antibody 35 Neutralizes rituximab present in the serum High cost FC-XM as a functional assay Use of a fluorescent intercalator (7-AAD) [37][38][39][40][41][42][43] FC-XM becomes a complement dependent cytotoxicity assay No clear evidence of a higher sensitivity for FCtox assay-XM compared with CDC The negativity of this assay does not imply that the DSAs are not cytotoxic Use of a fluorochrome-conjugated C4d antibody 44,45 Detection of antibody-binding endothelial cells Use of donor endothelial precursor cells (EPCs) as target cells [47][48][49][50][51] Can detect a non-HLA specific antibody population not detected by lymphocyte crossmatches…”

Section: Conflicts Of Interestmentioning

confidence: 99%

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Improved flow cytometry crossmatching in kidney transplantation

Guillaume

2018

HLA

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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.

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Section: Improved Fc-xm By Means Of a Functional Assaymentioning

confidence: 99%