Falsely Incompatible B-cell Flow Cytometry Crossmatch After Pronase Treatment: A Case Report

Hart, John; Lutz, Cathleen; Jennings, C. Darrell; May, Jan‐Niklas; Nelson, Kim L.; Jacobs, Sandra; Hoopes, Charles W.

doi:10.1016/j.transproceed.2014.12.022

Cited by 10 publications

(5 citation statements)

References 6 publications

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“…Pronase treatment has been reported to "improve flow cytometric detection of HLA antibodies" 25 and "improve flow cytometry crossmatch results" 26 . However, pronase may also unmask cryptic epitopes on T cells 27 , elicit new reactivity to donor B cells and autologous B cells 28 , reduce HLA density on target cells leading to incorrect FCXM results 29 including false positive T cell FCXM tests 30 .…”

Section: Discussionmentioning

confidence: 99%

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests

Putheti¹,

Sharma²,

Friedlander³

et al. 2022

Exp Clin Transplant

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Section: Discussionmentioning

confidence: 99%

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests

Putheti¹,

Sharma²,

Friedlander³

et al. 2022

Exp Clin Transplant

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“…The implementation of pronase pretreatment of lymphocytes in the FCXM has been shown to decrease the false positive rate 50 as well as increase sensitivity of the B-cell crossmatch without disrupting MHC expression. 51 Interestingly, however, cases of false positives B-cell and T-cell crossmatches following pronase treatment have been reported 52,53 and are likely due to non-HLA antibodies, autoantibodies that recognize exposed epitopes upon pronase treatment, or an artifact resulting from the use of extremely high concentrations of pronase. In the scenario outlined above in which the FCXM is positive and VXM is negative, transplantation does not represent an increased risk to the patient.…”

Section: Future Of Crossmatchingmentioning

confidence: 99%

Out with the old, in with the new: Virtual versus physical crossmatching in the modern era

Morris

Sullivan

Krummey

et al. 2019

HLA

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The virtual crossmatch (VXM) is gaining acceptance as an alternative approach to assess donor:recipient compatibility prior to transplantation. In contrast to a physical crossmatch, the virtual crossmatch does not require viable donor cells but rather relies on complete HLA typing of the donor and current antibody assessment of the recipient. Thus, the VXM can be performed in minutes which allows for faster transplant decisions thereby increasing the likelihood that organs can be shipped across significant distances yet safely transplanted. Here, we present a brief review of the past 50 years of histocompatibility testing; from the original complement‐dependent cytotoxicity crossmatch in 1969 to the new era of molecular HLA typing, solid‐phase antibody testing and virtual crossmatching. These advancements have shaped a paradigm shift in our approach to transplantation. That is, foregoing a prospective physical crossmatch in favor of a VXM. In this review, we undertake an in‐depth analysis of the pros‐ and cons‐ of physical and virtual crossmatching.Finally, we provide objective data on the selected use of the VXM which demonstrate the value of a VXM in lieu of the traditional physical crossmatch for safe and efficient organ transplantation.

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“…24 Auto-XM (using recipient lymphocytes and serum) can show these phenomena. 23 In an effort to increase the specificity and sensitivity of FC-XM, researchers have treated cells with pronase (a proteolytic enzyme that can remove Fc receptors from the cell surface) in a large panel of concentrations and incubation times [25][26][27][28][29][30][31] (Table 1). However, pronase treatment may affect other cell surface molecules (such as HLA), which in turn may affect reactivity in an XM assay.…”

Section: Improved Fc-xm By Avoiding False-positive Reactions On B-cmentioning

confidence: 99%

“…Higher serum-to-cell suspension volume ratio 20 Higher median fluorescence channel shift Improves lymphocyte purity and lack of interference of myeloid cells Assay to avoid false positive reactions on B-cells Pronase treatment [25][26][27][28][29][30][31] Reduce fc receptor, surface immunoglobulin and CD20 expression levels Heterogeneity in published protocol (incubation time, pronase concentration). May affect others targets on B-cells.…”

Section: Conflicts Of Interestmentioning

confidence: 99%

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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Falsely Incompatible B-cell Flow Cytometry Crossmatch After Pronase Treatment: A Case Report

Cited by 10 publications

References 6 publications

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests

Out with the old, in with the new: Virtual versus physical crossmatching in the modern era

Improved flow cytometry crossmatching in kidney transplantation

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