Validation of a single tube 3-colour immature red blood cell screening assay for the detection and enumeration of small, medium and large paroxysmal nocturnal haemoglobinuria clones by flow cytometry.

INTRODUCTION: The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and monocytic lineages. In this study, we have evaluated the performance characteristics of a recently published immature red blood cell (iRBC) assay as a potential screening test for PNH by flow cytometry. METHODS: Intra- and inter-assay imprecision were determined in five replicates of small, medium and large PNH iRBC clones. Analytical and functional sensitivity was assessed by performing spiking tests for five replicates. Thirty healthy donors and 441 PNH patients were tested for evaluation of clinical specificity, sensitivity, positive and negative predictive values. RESULTS: Coefficients of variation (CV) for intra-/inter-assay imprecision analyses were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes, respectively. Absolute values (100%) were found for both clinical specificity and sensitivity as well as for both positive and negative predictive values. The CV from 5 replicate results for 10 clustered events was 15.7%. The coefficient of determination (r2 ), Pearson's correlation coefficient (r) and Bland-Altman mean bias were 0.9436/0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for PNH iRBCs compared to PNH monocytes. CONCLUSION: Our results confirm very good performance characteristics, high analytical and functional sensitivity, absolute clinical specificity and sensitivity as well as favourable correlation between PNH iRBCs and both PNH neutrophils and monocytes, suggesting that this cost-effective 3-colour iRBC assay can be used as a reliable screening test for evaluation of small, medium and large PNH clones by flow cytometry.

Flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: International guidelines for assay characteristics.

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported.

Immunophenotyping of Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Detection of PNH and assessment of PNH clone size in RBC and WBC lineages by flow cytometric analysis has increased in importance due to the availability of novel therapies. These therapies typically block the hemolysis of red blood cells and thus significantly lower the morbidities and mortality associated with this disease. This chapter describes validated, state-of-the-art, high-sensitivity flow cytometric methodologies based on latest published testing guidelines for PNH.

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

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.

Performance Characteristics of a Non-Fluorescent Aerolysin-Based Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay for Simultaneous Evaluation of PNH Neutrophils and PNH Monocytes by Flow Cytometry, Following Published PNH Guidelines.

BACKGROUND: CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available. METHODS: We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2 ) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

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.

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI-linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells. METHODS: In this document, we present a consensus effort that describes flow cytometric procedures for detecting PNH cells. RESULTS: We discuss clinical indications and offer recommendations on data interpretation and reporting but mostly focus on analytical procedures important for analysis. We distinguish between routine analysis (defined as identifying an abnormal population of 1% or more) and high-sensitivity analysis (in which as few as 0.01% PNH cells are detected). Antibody panels and gating strategies necessary for both procedures are presented in detail. We discuss methods for assessing PNH populations in both white blood cells and red blood cells and the relative advantages of measuring each. We present steps needed to validate the more elaborate high-sensitivity techniques, including the need for careful titration of reagents and determination of background rates in normal populations, and discuss technical pitfalls that might affect interpretation. CONCLUSIONS: This document should both enable laboratories interested in beginning PNH testing to establish a valid procedure and allow experienced laboratories to improve their techniques.