Improved Flow Cytometric Detection of Hla Alloantibodies Using Pronase

Vaidya, Smita; Cooper, Todd Y.; Avandsalehi, Jeanne; Barnes, Titus; Brooks, Karl; Hymel, Phoumymala; Noor, Maryam; Sellers, Rachel; Thomas, Alice J.; Stewart, Dod; Daller, John A.; Fish, Jay C.; Gugliuzza, Kristene; Bray, Robert A.

doi:10.1097/00007890-200102150-00015

Cited by 91 publications

(46 citation statements)

References 25 publications

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“…Several studies have been published that report improved sensitivity and specificity of B-cell crossmatches when the lymphocytes are pretreated with pronase [5][6][7]. It appears that nonspecific binding of human immunoglobulin (Ig)G to target F c receptors on B cells is reduced on removal of F c receptors.…”

Section: Introductionmentioning

confidence: 99%

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

et al. 2004

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Section: Introductionmentioning

confidence: 99%

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

et al. 2004

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“…B-cell false positive FC CM results may erroneously preclude transplants that should have favorable outcomes. The non-specific antibody binding may be reduced by the incubation of the lymphocytes with pronase, enzyme destroying Fc receptors, and other members of the Ig superfamily [79][80][81][82] . FC cell-based assays can also be used for PRA analysis.…”

Section: Flow Cytometry Methodsmentioning

confidence: 99%

Methodological aspects of anti-human leukocyte antigen antibody analysis in solid organ transplantation

Lobashevsky¹

2014

WJT

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Donor human leukocyte antigen (HLA)-specific antibodies (DSA) play an important role in solid organ transplantation. Preexisting IgG isotype DSA are considered a risk factor for antibody mediated rejection, graft failure or graft loss. The post-transplant development of DSA depends on multiple factors including immunogenicity of mismatched antigens, HLA class Ⅱ typing of the recipient, cytokine gene polymorphisms, and cellular immunoregulatory mechanisms. De novo developed antibodies require special attention because not all DSA have equal clinical significance. Therefore, it is important for transplant clinicians and transplant immunologists to accurately characterize DSA. In this review, the contemporary immunological techniques for detection and characterization of anti-HLA antibodies and their pitfalls are described.

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“…Cells were pronase digested to remove Fc receptors (15). Using the same cell:serum combinations as described for the AHG-CDC studies, 1 3 10 5 cells and 50 ll of serum were incubated in the dark for 40 min at room temperature.…”

Section: Cytotoxic Flow Cytometric Crossmatchmentioning

confidence: 99%

“…We found that decreasing the complement incubation time to 10 min optimized the assay conditions. Additionally, we reduced inter and intra-assay variability by removing Fc receptors which nonspecifically bind immunoglobulin and contribute to high background values by enzymatic digestion with pronase (15). Finally, only sera from renal transplant candidates with well-defined HLA specificities identified by flow cytometry-based solid phase detecting assays were selected for study.…”

Section: The Cfcxm Assaymentioning

confidence: 99%

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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Improved Flow Cytometric Detection of Hla Alloantibodies Using Pronase

Abstract: Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.

Cited by 91 publications

References 25 publications

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

Methodological aspects of anti-human leukocyte antigen antibody analysis in solid organ transplantation

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

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