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

Sutherland, D. Robert; Ortíz, Fernando; Quest, Graeme; Illingworth, Andrea; Benko, Miroslav; Nayyar, Rakesh; Marinov, Iuri

doi:10.1002/cyto.b.21626

Cited by 26 publications

(38 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In this way, the FLAER‐based analysis plots can be directly compared with the non‐FLAER plots in each lineage within the same stained sample. Analysis of >40 PNH samples has shown that the FLAER‐based plots derive virtually identical data to the non‐FLAER plot for both neutrophil and monocyte lineages .…”

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 95%

“…Subsequently, 5‐color single tube assays capable of simultaneous detection of both neutrophils and monocytes based on FLAER, CD157, CD15, CD64, and CD45 were developed for a variety of clinical cytometers with 5 or more PMTs , and again independently validated . Most recently, 6‐color assays have been developed for clinical instruments with 6 or more PMTs.…”

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 99%

“…This assay comprised of FLAER, CD157‐PE, CD64‐ECD, CD15‐PC5, and CD45‐PC7 (FC500 and Navios) or FLAER, CD157‐PE, CD15‐PerCPeFluor710, CD64‐APC, and CD45‐eFluor450 (Canto II). The latter can also be used on a Navios cytometer as it employs reagents that can be used across instrument platforms without modification . The 5‐C assays had closely similar sensitivities to the original 4‐color assays with similar very low background rates on 10 normal samples .…”

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 99%

See 2 more Smart Citations

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

Illingworth²,

Marinov

et al. 2018

Cytometry Part B Clinical

Self Cite

116

View full text Add to dashboard Cite

Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. V C 2017 International Clinical Cytometry Society

show abstract

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 95%

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 99%

Section: Reagent Selection: White Blood Cell Assaysmentioning

confidence: 99%

See 1 more Smart Citation

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

Illingworth²,

Marinov

et al. 2018

Cytometry Part B Clinical

Self Cite

116

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

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...

show abstract

“…CD157 is a GPI‐linked protein that is simultaneously expressed on both neutrophils and monocytes (Hernández‐Campo et al, ; Hernández‐Campo, Almeida, Sánchez, Malvezzi, & Orfao, ). Recent work has suggested that CD157 is a potentially useful reagent in the diagnosis of PNH neutrophils and monocytes (Marinov et al, ; Sutherland, Acton, Keeney, Davis, & Illingworth, ; Sutherland et al, ). The latest work of DR Sutherland et al showed that five‐, six‐, and seven‐color PNH panels that included CD157 and were implemented on both 10‐color Navios or 8‐color FACSCanto II platforms had excellent diagnostic performance (Sutherland, Ortiz, et al, ).…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of paroxysmal nocturnal hemoglobinuria with flowcytometry panels including CD157: Data from the real world

Zhang

Ding

et al. 2019

Cytometry Part B Clinical

View full text Add to dashboard Cite

Background Several studies have used CD157 in white blood cells with or without proaerolysin (fluorescein‐labeled proaerolysin [FLAER])‐based flow cytometry assays in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Methods We designed a seven‐color CD marker panel comprising FLAER, CD15, CD64, CD24, CD14, CD157, and CD45 to verify CD157's clinical applicability and diagnostic performance in a clinical setting. Results A total of 356 samples were tested. These included 43 PNH‐positive samples and 313 PNH‐negative samples. PNH clones confirmed by the CD157/FLAER combination were almost identical in size to the clones detected by the CD24/CD14/FLAER combination, and the accuracy of the CD157/FLAER combination was 100% in granulocytes and 99.7% in monocytes. Substitution of FLAER with CD157 resulted in 1.9% and 3.5% false‐positives in granulocytes and monocytes, respectively. The accuracy was 98.3% and 96.9% in granulocytes and monocytes, respectively. Moreover, the loss of CD157 expression in granulocytes and monocytes was commonly observed in non‐PNH patients. Some monocytes in non‐PNH patients had weak expression of CD14 but normal expression of FLAER. In this study, PNH clones in granulocytes were always lower than those in matched monocytes. Conclusions We performed the first prospective exploration of the clinical usefulness of FLAER and CD157 in simultaneously recognizing PNH clones in granulocytes and monocytes and verified the applicability of CD157 in substitute for both CD14 and CD24. In the conditions where FLAER is not available, substitution of FLAER with CD157 is acceptable for the identification of PNH clones under the premise of giving full attention to the potential for false‐positives.

show abstract

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

Cited by 26 publications

References 32 publications

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

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

Relevance of Antibody Validation for Flow Cytometry

Diagnosis of paroxysmal nocturnal hemoglobinuria with flowcytometry panels including CD157: Data from the real world

Contact Info

Product

Resources

About