Flow cytometric detection of complement‐activating HLA antibodies

Schönemann, Constanze; Lachmann, Nils; Kiesewetter, H.; Salama, Abdulgabar

doi:10.1002/cyto.b.20027

Cited by 14 publications

(10 citation statements)

References 36 publications

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“…Compared with the conventional methods, the advantages we documented are: (1) HLA alloantibodies, complement-fixing, and nonfixing can be detected simultaneously, and the mechanisms of cell death can be identified, which is Ab-dependent or not; (2) the determination of positivity is objective; (3) a high number of lymphocytes is assessed; (4) detection of weak positive reaction is enhanced; (5) 7-AAD has minimal spectral overlap with FITC and PE fluorescence emissions than that of propidium iodide or ethidium bromide used in other studies (12,(15)(16)(17) to detect the dead cells. This allows other fluorescence to be measured more accurately in three-color FC.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

Won

Jeong

Kim

et al. 2006

Cytometry Part B Clinical

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The simultaneous detection of Ab binding and cytotoxicity was possible by the cytotoxic FCXM with the test efficiencies similar to the conventional counterparts. If this new assay is improved through the further studies to optimize the critical assay variables, this may be used as an alternative to the conventional assays to acquire more information on the characteristics of the recipient's HLA alloantibodies.

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Section: Discussionmentioning

confidence: 99%

Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

Won

Jeong

Kim

et al. 2006

Cytometry Part B Clinical

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“…Other investigators previously explored the possibility of a cFCXM for HLA antibodies (8,9). However, those studies were limited in scope.…”

Section: The Cfcxm Assaymentioning

confidence: 99%

“…However, those studies were limited in scope. For example, Schönemann et al (8) restricted their experiments to sera that were positive by conventional cytotoxicity assays and did not compare their flow cytotoxicity results to conventional FCXM data using the same cell:serum combinations. More recently, Won et al (9) compared cytotoxicity by flow to both AHG-CDC and FCXM.…”

Section: The Cfcxm Assaymentioning

confidence: 99%

“…In their system, patient sera (as a source of both HLA antibodies and complement) were incubated with HLA antigen coated microparticles and interrogated for complement fixation using flourochrome-labeled antibodies to human C1q, C3d, or C4d. Schönemann et al (8) then developed a cell-based complement dependent flow cytometric assay that correlated with the results of conventional cytotoxicity. Subsequently, Won et al (9) made the first attempt to combine antibody binding and cytotoxicity into a single flow cytometric assay.…”

mentioning

confidence: 99%

“…In both of those studies, analysis was restricted to sera that fixed complement by conventional cytotoxicity. Furthermore, the assays from both studies (8,9) were not optimized for routine clinical application. In this study, sera selected for analysis were based on two criteria, such as (1) antibody specificity as determined by solid phase methodology and (2) a positive FCXM with cells expressing the corresponding HLA antigen(s).…”

mentioning

confidence: 99%

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Cytotoxicity and antibody binding by flow cytometry: A single assay to simultaneously assess two parameters

Saw¹,

Bray²,

Gebel³

2008

Cytometry Part B Clinical

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Background: Cell death, as determined by complement-dependent cytotoxicity or antibody binding, as assessed by flow cytometry, are the two main methods histocompatibility laboratories use to identify HLA antibodies. Conventional cytotoxicity assays, even anti-human globulin (AHG) enhanced, are relatively insensitive, subjective and require complement fixation. In contrast, antibody binding assays (e.g.: flow cytometry crossmatch, FCXM) are sensitive, objective, and complement independent. When sera from potential transplant recipients test positive by both methods, most centers would not proceed to transplant unless patients undergo desensitization. However, when the CDC crossmatch is negative but the FCXM positive, how or whether to proceed is controversial. Frequently, both assays are performed.Methods: In this study, we developed a comprehensive procedure that determines cell death, and antibody binding in a single assay: the cytotoxic flow crossmatch (cFCXM). The assay was validated in parallel studies with two other conventional methodologies, namely AHG and FCXM.Results: Using a combination of eight cells and 15 sera, we have determined an optimal method for the simultaneous detection of HLA antibody binding and cytotoxicity using a single platform. Additionally, we have identified three distinct patterns of HLA antibody reactivity: (1) AHG

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Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay

Alheim

2013

CP Cytometry

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Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody‐mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement‐fixing HLA antibodies is commonly performed via the complement‐dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read‐out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry–based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead–enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells. Curr. Protoc. Cytom. 66:6.34.1‐6.34.11. © 2013 by John Wiley & Sons, Inc.

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Flow cytometric detection of complement‐activating HLA antibodies

Cited by 14 publications

References 36 publications

Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

Cytotoxicity and antibody binding by flow cytometry: A single assay to simultaneously assess two parameters

Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay

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