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Sutherland D.R.,Laboratory Medicine Program | Keeney M.,London Health Sciences Center | Illingworth A.,Dahl Chase Diagnostic Services
Cytometry Part B - Clinical Cytometry | Year: 2012

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. Copyright © 2012 International Clinical Cytometry Society.


Sutherland D.R.,University of Toronto | Illingworth A.,Dahl Chase Diagnostic Services | Keeney M.,London Health Sciences Center | Richards S.J.,University of Leeds
Current Protocols in Cytometry | Year: 2015

Flow cytometry is the method of choice to 'diagnose' paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and interlaboratory performance characteristics even in laboratories with little prior experience performing PNH testing. © 2015 by John Wiley & Sons, Inc.


Sutherland D.R.,University of Toronto | Acton E.,University of Toronto | Keeney M.,London Health Sciences Center | Davis B.H.,Trillium Diagnostics LLC | Illingworth A.,Dahl Chase Diagnostic Services
Cytometry Part B - Clinical Cytometry | Year: 2014

Background Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusions While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 International Clinical Cytometry Society © 2013 Clinical Cytometry Society.


Fletcher M.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Sutherland D.R.,Laboratory Medicine Program | Whitby L.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Whitby A.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | And 7 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2014

Background Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol-deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published. Methods UK NEQAS LI issued three stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific Standard Operating Procedures (SOPs) and pretitered 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 External Quality Assessment (EQA) programs. 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 interquartile range of 1.7% 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 among the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 International Clinical Cytometry Society.


Sipol A.A.,Saint Petersburg State University | Babenko E.V.,Saint Petersburg State University | Borisov V.I.,Alexion Pharma | Naumova E.V.,Moscow Medical Academy | And 9 more authors.
Hematology | Year: 2015

Objectives: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia.Methods: PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent ‘practical guidelines’. Follow-up measurements were compared with each other and with the values determined at diagnosis.Results: PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41–9.7% of granulocytes) and high in patients with severe symptoms (58–99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis.Conclusions: The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories. © 2015 W. S. Maney & Son Ltd.

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