ICCS/ESCCA Consensus Guidelines to detect GPI‐deficient cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) and related Disorders Part 2 – Reagent Selection and Assay Optimization for High‐Sensitivity Testing

Sutherland, D. Robert; Illingworth, Andrea; Marinov, Iuri; Ortíz, Fernando; Andreasen, John; Payne, Daniel; Wallace, Paul K.; Keeney, Michael

doi:10.1002/cyto.b.21610

Cited by 50 publications

(117 citation statements)

References 38 publications

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“…Details of how individual clones/ conjugates were selected and titrated will be published elsewhere (29). Details of how individual clones/ conjugates were selected and titrated will be published elsewhere (29).…”

Section: Materials and Methods Antibody Clone/conjugate Selectionmentioning

confidence: 99%

“…However, 80H5 is not available in either of these formulations. This conjugate can be detected efficiently on both Navios and Canto II instruments using the FL4 and FL3 PMTs respectively (29). Earlier evaluation of the PerCPCy5.5 conjugate of the HI98 clone (BD Biosciences) had shown this reagent to be sub-optimal at delineating neutrophils from monocytes ( Fig.…”

Section: Assay Developmentmentioning

confidence: 99%

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High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

Sutherland

Ortíz

Quest

et al. 2018

Cytometry Part B Clinical

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CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R = 1) and monocytes (R = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

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Section: Materials and Methods Antibody Clone/conjugate Selectionmentioning

confidence: 99%

Section: Assay Developmentmentioning

confidence: 99%

High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

Sutherland

Ortíz

Quest

et al. 2018

Cytometry Part B Clinical

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“…Users should be aware that most antibodies that preform perfectly for techniques, such as Western blot (WB) or immunohistochemistry analyses do not work for flow cytometry. Antibodies to other highly O-glycosylated structures such as CD235a (glycophorin A) can behave in unpredictable ways as well, with PE conjugated forms sometimes causing significantly greater aggregation of RBCs than their negatively-charged FITC counterparts (45). Flow cytometry analyses whole cells and their proteins in their native form.…”

Section: What Issues Do We Face With Antibody Reagents?mentioning

confidence: 99%

“…Moreover, using the recommended dilution of the antibody by the vendor is not always a guarantee of good performance under the specific conditions of our assay. In high concentrations, they can aggregate target cells (45,48). 2).…”

Section: Antibody Dilution-why Is Determining the Dilution So Important?mentioning

confidence: 99%

Relevance of Antibody Validation for Flow Cytometry

2019

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Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance.Flow cytometry has developed into an indispensable technique in the research and clinical investigation of immune and hematologic systems, with increasing applications in other cell biology disciplines. There are three main pillars in the practical application of this technology, namely the instrumentation, the analytical methods for large data sets, and the reagents used to design biological experiments. Despite dramatic developments in instrumentation (polychromatic (1), mass (2,3), and spectral (4,5) cytometry) and the significant developments in data analysis techniques for high content analyses (6-8), the last pillar of this trio remains consistent across time in its importance to the application: the fluorescent antibody conjugate.Our and others' experiences indicate that nearly half of antibodies, sold by companies or described by academic groups, do not function for the recommended application. They present staining patterns that conflict with those reported in the literature, show unexpected cross-reactivity, or have even failed the most basic specificity tests (9-11). There is growing alarm about results that cannot be reproduced by other research groups, including data published in high-impact journals. Antibodies are believed to be, in part, responsible for inconsistent experimental results and the publication of inaccurate data in the scientific literature (12,13).During recent years, both industry and academic groups have increased their efforts to increase the quality of t...

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“…Since over 40% of samples positive for the presence of PNH cells contain GPI‐deficient cells at a level of 1% or less (Figure ), the development and validation of sensitive, standardized methodologies are essential to reliably detect small populations of GPI‐deficient PNH phenotypes. To successfully develop highly sensitive, accurate, and reproducible assays, careful selection and titration of antibody clones/conjugates for lineage‐specific gating (RBCs, neutrophils, and monocytes) and specific GPI‐antigen detection within each cell lineage is required . The detection of PNH phenotypes by flow cytometry represents a unique challenge in that the assays are designed to detect “negatives,” that is, PNH phenotypes have “lost” the antigens of interest, namely GPI‐linked structures.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and accurate identification of PNH clones based on ICCS/ESCCA PNH Consensus Guidelines—A summary

Illingworth¹,

Marinov

Sutherland

2019

Int J Lab Hematology

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Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X‐linked phosphatidyl‐inositol glycan complementation Class A (PIG‐A) gene. Depending on the severity of the mutation in the PIG‐A gene, there is a partial or absolute inability to make glycosylphosphatidyl‐inositol (GPI)‐anchored proteins including complement‐defense structures such as CD55 and CD59 on RBCs and WBCs. Flow cytometric detection of PNH clones has become the gold standard and has played an increasingly important role in the diagnosis, monitoring, and clinical management of patients with PNH. Recently, a 4‐part set of Consensus Guidelines have been published by flow experts in the field to address the key assay‐specific considerations for the identification of PNH clones in RBC and WBC, how to report such data and a full validation document for the assays described. Below, we have summarized the most significant aspects of this International effort.

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ICCS/ESCCA Consensus Guidelines to detect GPI‐deficient cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) and related Disorders Part 2 – Reagent Selection and Assay Optimization for High‐Sensitivity Testing

Cited by 50 publications

References 38 publications

High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

Relevance of Antibody Validation for Flow Cytometry

Sensitive and accurate identification of PNH clones based on ICCS/ESCCA PNH Consensus Guidelines—A summary

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