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

  1. Michael J. Borowitz1,*,
  2. Fiona E. Craig2,
  3. Joseph A. DiGiuseppe3,
  4. Andrea J. Illingworth4,
  5. Wendell Rosse5,
  6. D. Robert Sutherland6,
  7. Carl T. Wittwer7,
  8. Stephen J. Richards8

Article first published online: 28 APR 2010

DOI: 10.1002/cyto.b.20525

Issue

Cytometry Part B: Clinical Cytometry

Cytometry Part B: Clinical Cytometry

Volume 78B, Issue 4, pages 211–230, July 2010

Additional Information

How to Cite

Borowitz, M. J., Craig, F. E., DiGiuseppe, J. A., Illingworth, A. J., Rosse, W., Sutherland, D. R., Wittwer, C. T. and Richards, S. J. (2010), Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry, 78B: 211–230. doi: 10.1002/cyto.b.20525

Author Information

  1. 1

    Department of Pathology and Oncology, Johns Hopkins Medical Institutions, Baltimore, Maryland

  2. 2

    Division of Hematopathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

  3. 3

    Department of Pathology and Laboratory Medicine, Hartford Hospital, Hartford, Connecticut

  4. 4

    Flow Cytometry Laboratory, Dahl-Chase Diagnostic Services, Bangor, Maine

  5. 5

    Department of Medicine, Duke University School of Medicine, Durham, North Carolina

  6. 6

    Department of Medicine, Toronto General Hospital, Toronto, Ontario, Canada

  7. 7

    Department of Pathology, University of Utah, Salt Lake City, Utah

  8. 8

    Haematology Malignancy Diagnostic Service, Department of Clinical Haematology, St. James University Hospital, Leeds, United Kingdom

*Professor of Pathology and Oncology, Department of Pathology, Johns Hopkins Medical Institutions, Weinberg 2335, 401 N Broadway, Baltimore, MD 21231, USA

  1. †

    How to cite this article: Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. Cytometry Part B 2010; 78B: 211–230.

  2. ‡

    Disclosures: MJB, AJI, WR and SJR have consulting agreements with Alexion Pharmaceuticals; MJB and SJR have research support from BD Biosciences.

Publication History

  1. Issue published online: 21 JUN 2010
  2. Article first published online: 28 APR 2010
  3. Manuscript Accepted: 22 MAR 2010
  4. Manuscript Revised: 13 MAR 2010
  5. Manuscript Received: 25 NOV 2009

Funded by

  • Alexion Pharmaceuticals

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Keywords:

  • flow cytometry;
  • paroxysmal nocturnal hemoglobinuria;
  • practice guidelines

Abstract

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. © 2010 Clinical Cytometry Society

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