PNH testing guidelines

Consensus guidelines

Although to date there is no standardised methodology for paroxysmal nocturnal haemoglobinuria (PNH) testing, published consensus guidelines offer recommendations on the diagnosis and management of patients with PNH and analytical procedures for routine and high-sensitivity PNH testing.

Background

The first guidelines for the diagnosis and management of PNH were published by the International PNH Interest Group in 2005. This document provides a classification scheme for diagnosing PNH in patients with variable presenting features, clinical manifestations and natural history.

  • Parker C et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005; 106: 3699-3709.

Specific guidelines on the accurate identification and monitoring of PNH clones using flow cytometry were then published by the International Clinical Cytometry Society (ICCS) in 2010. The goal of this document was not to mandate a single approach but rather to provide guidelines regarding best practice in PNH testing based on combined knowledge from technical and clinical experts.

The main criterion for PNH testing reported in these guidelines is the selection of suitable gating antibodies with at least two different antibodies against two or more different surface glycosylphosphatidylinositol (GPI)-linked proteins. Testing should be performed on at least two different cell populations (not lymphocytes) and performed using fresh peripheral blood samples with no signs of degranulation.

  • Borowitz MJ et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 2010; 78B: 211-230.

The 2010 ICCS guidelines were followed in 2012 by a supporting document outlining concise, practical protocols for the high-sensitivity detection of PNH clones. These guidelines describe in detail the proper selection of antibody clones and fluorochromes, sample preparation and set-up of protocols, including diagnostic dot plots and control dot plots. Common problems are also highlighted, along with recommendations on instrument optimisation.

  • Sutherland DR et al. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom 2012; 82B: 195-208.

Current guidelines

Updates to the 2010 ICCS consensus guidelines were recently developed and published as the 2018 ICCS/European Society for Clinical Cell Analysis (ESCCA) consensus guidelines. These guidelines summarise a large volume of work completed in recent years to develop, optimise and validate several high-sensitivity flow cytometry assays for reliable detection of PNH clones. Part 1 of the updated guidelines outlines the clinical utility of flow cytometry for the diagnosis, classification and monitoring of patients with PNH:

  • Dezern AE, Borowitz MJ. ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 1 – clinical utility. Cytometry B Clin Cytom 2018; 94: 16-22.

Part 2 of the 2018 ICCS/ESCCA guidelines outlines high-sensitivity PNH assay design, with detailed guidance on specific instrument set-up and the identification and optimisation of appropriate antibody/conjugate combinations:

  • Sutherland DR et al. 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. Cytometry B Clin Cytom 2018; 94: 23-48.

Part 3 of the 2018 ICCS/ESCCA consensus guidelines outlines best practices for the analysis and reporting of PNH results and provides guidance on how to avoid the most common challenges preventing the accurate reporting of PNH results:

  • Illingworth A et al. ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 3 – data analysis, reporting and case studies. Cytometry B Clin Cytom 2018; 94: 49-66.

Part 4 of the 2018 ICCS/ESCCA consensus guidelines provides assay-specific guidance for the validation of high-sensitivity flow cytometric PNH assays and provides recommendations for quality assurance:

  • Oldaker T et al. ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 – assay validation and quality assurance. Cytometry B Clin Cytom 2018; 94: 67-81.

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