Flow cytometry crossmatch reactivity with pronase-treated T cells induced by non-HLA autoantibodies in human immunodeficiency virus-infected patients

Szewczyk, Katarzyna; Barrios, Kelly; Magas, Daniel; Sieg, Kristin; Labuda, Bozena; Jendrisak, Martin D.; Jaramillo, Andrés

doi:10.1016/j.humimm.2016.04.014

Cited by 22 publications

(13 citation statements)

References 25 publications

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“…Pronase treatment of donor cells is thought to enhance the specificity of FC‐XM. However, some limitations of pronase treatment have been reported in the literature, such as false negative results due to a reduction of MHC expression on lymphoid cells, and false positive T‐cell FC‐XM results as example in HIV‐positive patients due to the exposure of cryptic epitopes . We studied the impact of treatment with a range of pronase concentrations (from 0 to 3 mg/mL) on CD3, CD19, CD20, kappa light chain, and MHC‐I and ‐II expression.…”

Section: Discussionmentioning

confidence: 99%

“…However, some limitations of pronase treatment have been reported in the literature, such as false negative results due to a reduction of MHC expression on lymphoid cells, and false positive T-cell FC-XM results as example in HIV-positive patients due to the exposure of cryptic epitopes. 9,[12][13][14] We studied the impact of treatment with a range of pronase concentrations (from 0 to 3 mg/mL) on CD3, CD19, CD20, kappa light chain, and MHC-I and -II expression. Treatment pronase (1 mg/mL for 30 minutes at 37 C) had a moderate (and therefore acceptable) impact on MHC expression and our ability to interpret the FC-XM results.…”

Section: Discussionmentioning

confidence: 99%

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Pronase treatment improves flow cytometry crossmatching results

Apithy

Desoutter

Gicquel

et al. 2017

HLA

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Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Pronase treatment improves flow cytometry crossmatching results

Apithy

Desoutter

Gicquel

et al. 2017

HLA

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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%

“…Moreover, some limitations of pronase treatment have been reported in the literature, such as false-negative results due to a reduction of HLA expression on lymphoid cells, 26 and false-positive T-cell FC-XM results, for example, in HIV-positive patients due to exposure of cryptic epitopes. 28 However, Apithy et al 27 studied the impact of treatment with a range of pronase concentrations (from 0 to 3 mg/mL) on CD3, CD19, CD20, kappa light chain and HLA-I and -II expression and temperate this risk of false negativity. Pronase treatment (1 mg/mL for 30 minutes at 37 C) had a moderate (and therefore acceptable) impact on HLA expression and the ability to interpret FC-XM results.…”

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

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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“…This strongly suggested that FC-XM reactivity was due to specific autoantibodies recognizing cryptic epitopes exposed by the pronase treatment of T cells. 25,26 Interference by therapeutic antibodies and other treatments The therapeutic antibodies used to treat acute rejection or to desensitize patients (such as rituximab [an anti-CD20 antibody], daclizumab [anti-CD25], and alemtuzumab [anti-CD52]) can interfere with crossmatching assays. 27 In a recent study, CDC-XM was performed after the addition of various concentrations of therapeutic antibodies (intravenous immunoglobulins, rituximab, basiliximab, eculizumab, and antithymocyte globulin) to negative and positive control sera.…”

Section: Human Immunodeficiency Virus-positive Casesmentioning

confidence: 99%

Untitled

Desoutter

Apithy²,

Guillaume³

2017

Exp Clin Transplant

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Preformed donor-specific antibodies against human leukocyte antigen can induce antibody-mediated rejection after organ transplant. Hence, future transplant recipients undergo pretransplant screening for preformed antibodies (ie, virtual crossmatch). Subsequently, prospective (analytic) crossmatching is performed using conventional, complementdependent cytotoxicity assays and/or flow cytometrybased methods. The present article reviews factors that must be considered when unexpected, positive, prospective crossmatches are observed. First, the prozone effect caused by the interference of complement or immunoglobulin M must be abrogated by treating the serum with moderate heat, dilution, hypotonic dialysis, EDTA, or dithiothreitol. Second, the physician must check for the presence of potentially interfering autoantibodies (in a context of autoimmune disease or human immunodeficiency virus infection) or therapeutic antibodies (such as rituximab and antithymocyte globulin). In conclusion, knowledge of each assay's technical characteristics will enable the physician to reliably interpret any discrepancies. The reasons for an unexpected, positive, prospective crossmatch must be elucidated before transplant to ensure efficient organ allocation and optimize patient outcomes.

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Flow cytometry crossmatch reactivity with pronase-treated T cells induced by non-HLA autoantibodies in human immunodeficiency virus-infected patients

Cited by 22 publications

References 25 publications

Pronase treatment improves flow cytometry crossmatching results

Pronase treatment improves flow cytometry crossmatching results

Improved flow cytometry crossmatching in kidney transplantation

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