Multiparameter flow cytometry for the diagnosis and monitoring of small GPI‐deficient cellular populations

Battiwalla, Minoo; Hepgur, Mehmet; Pan, Dalin; McCarthy, Philip L.; Ahluwalia, Manmeet; Camacho, Susan H.; Starostik, Petr; Wallace, Paul K.

doi:10.1002/cyto.b.20519

Cited by 26 publications

(21 citation statements)

References 23 publications

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“…With respect to WBCs, while a variety of GPI-specific reagents such as CD55, CD59, CD16, CD66b, CD24, and CD14 have been widely used to detect PNH phenotypes (18)(19)(20)(21), FLAER-based reagent cocktails that include lineage-specific gating reagents have become more widely deployed in recent years (22)(23)(24)(25)(26)32,(34)(35)(36). We, and others have previously designed CD33-based assays to identify granulocyte and monocyte lineages in which the gated subsets can subsequently be assessed for FLAER and individual GPI-linked antigen expression (22,25).…”

Section: Discussionmentioning

confidence: 99%

Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

Sutherland

Keeney

Illingworth³

2012

Cytometry Part B Clinical

123

284

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Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a life‐threatening disorder caused by an inability to make glyco‐phosphatidyl‐inositol (GPI) anchors. While flow cytometry is the method of choice to detect the loss of GPI‐linked proteins, the development and validation of sensitive, standardized, methodologies have been hampered by the rarity of this disease and by technical difficulties in the accurate identification of PNH cells.Methods:Guidelines for the diagnosis and monitoring of PNH by flow cytometry were recently published by the International Clinical Cytometry Society (ICCS). However, specific reagent cocktails, and associated detailed analytic strategies were not directly addressed therein. In this supporting document based on the ICCS guidelines, we provide concise practical protocols for the high‐sensitivity detection of PNH RBCs and WBCs (both granulocytes and monocytes).Results:The CD235aFITC/CD59PE assay described was capable of detecting as few as 20 Type III PNH RBCs per million cells. Frequencies of Type III PNH cells in 10 normal samples were in the 0–6 per million RBCs. The high‐resolution granulocyte/neutrophil assays described in this study could detect PNH phenotypes consistently at a level of 0.01% sensitivity. Frequencies of PNH phenotypes in normal individuals were in the 0–10 per million granulocytes/neutrophils range.Conclusions:The careful screening and selection of specific antibody conjugates has allowed the development of reagent cocktails suitable for high‐sensitivity flow cytometric detection of PNH RBCs and PNH WBCs. The reagent cocktails described herein can be used on a variety of clinical flow cytometers equipped with four or more photo multiplier tubes. © 2012 International Clinical Cytometry Society

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Section: Discussionmentioning

confidence: 99%

Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

Sutherland

Keeney

Illingworth³

2012

Cytometry Part B Clinical

123

284

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“…It allows evident discrimination between normal and altered cells (4,17,23) , and, as it detects the GPI molecule, it is superior in situations in which one or more GPI-linked surface markers present reduced expression, erroneously suggesting the presence of cells with a PNH phenotype (2) . This study permitted us to confirm the good performance of the reagent, described in the literature (3,4,10,17,21,23) .…”

Section: The Black Bar Represents the Cut-off Point (01%) Chosen Formentioning

confidence: 99%

Detection of PNH cells by flow cytometry, using multiparameter analysis

Rocha¹,

Silva²,

Souza³

et al. 2014

Jornal Brasileiro De Patologia E Medicina Laboratorial

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Introduction: The laboratory diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), disease that is categorized by reduced synthesis of glycosylphosphatidylinositol (GPI) anchor, is based on the detection of blood cells deficient in GPI-anchored proteins by flow cytometry. PNH clones have been detected in patients with aplastic anaemia (AA) and myelodysplastic syndrome (MDS), with therapeutic implications. Objectives: To validate a sensitive assay for detection of GPI-anchored protein-deficient cells, by flow cytometry, and to analyze the clone frequency in AA and MDS patients. Methods: Samples from 20 AA patients, 30 MDS patients and 20 adult volunteers (control group) were analyzed using monoclonal antibodies to CD16, CD24, CD55 and CD59 (neutrophils); CD14 and CD55 (monocytes); CD55 and CD59 (erythrocytes); besides fluorescent aerolysin reagent (FLAER) (neutrophils and monocytes) and lineage markers. The proportions of PNH cells detected in neutrophils and monocytes, using different reagent combinations, were compared by analysis of variance (ANOVA) and Pearson's correlation. Results: PNH cells were detected in five (25%) AA patients, and the proportions of PNH cells varied from 0.14% to 94.84% of the analyzed events. PNH cells were not detected in the MDS patients. However, by the analysis of these samples, it was possible to identify the technical challenges caused by the presence of immature and dysplastic circulating cells. FLAER showed clear distinction of GPI-deficient cells. Conclusion: Multiparameter flow cytometry analysis offers high sensitivity and accuracy in the detection of subclinical PNH clones. FLAER shows excellent performance in detection of PNH neutrophils and monocytes.

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“…For PNH WBC detection, the ICCS Guidelines recommended the use of one antibody for lineage gating together with two GPI-linked reagents, one of which should be FLAER. This reagent has proven to be well suited to detect GPI-deficient cells in peripheral blood samples as it has an excellent signal to noise ratio (21)(22)(23)(24)(25)(26) SOPs utilizing specific assay cocktails were not identified in the ICCS Guidelines. In developing more detailed 'Practical Guidelines', a 4-color combination using FLAER, CD24, CD15 and CD45 was proposed for highsensitivity detection of PNH neutrophils that could be deployed on a variety of clinical cytometers (29).…”

Section: Introductionmentioning

confidence: 98%

“…While a variety of more sensitive approaches have subsequently been developed for PNH, WBC detection based on the fluorescent derivative of the bacterial lysin Proaerolysin (FLAER) (21)(22)(23)(24)(25)(26), FLAER-based assays are still not widely deployed. Recent data from the UK NEQAS PNH proficiency testing programs has highlighted wide variations in reagent selection, testing protocols for neutrophils and variable capabilities of laboratories to accurately detect WBC PNH clones in stabilized whole blood samples (27).…”

Section: Introductionmentioning

confidence: 99%

Standardizing Leucocyte PNH clone detection: An international study

et al. 2014

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Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.

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Multiparameter flow cytometry for the diagnosis and monitoring of small GPI‐deficient cellular populations

Cited by 26 publications

References 23 publications

Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

Detection of PNH cells by flow cytometry, using multiparameter analysis

Standardizing Leucocyte PNH clone detection: An international study

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