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

Michael J Borowitz, Fiona E. Craig, Joseph A. DiGiuseppe, Andrea J. Illingworth, Wendell Rosse, D. Robert Sutherland, Carl T. Wittwer, Stephen J. Richards

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)211-230
Number of pages20
JournalCytometry Part B - Clinical Cytometry
Volume78
Issue number4
DOIs
StatePublished - 2010

Fingerprint

Paroxysmal Hemoglobinuria
Flow Cytometry
Guidelines
Population
Protein Deficiency
Hematopoietic Stem Cells
Leukocytes
Research Design
Erythrocytes
Cell Membrane
Mutation
Antibodies

Keywords

  • Flow cytometry
  • Paroxysmal nocturnal hemoglobinuria
  • Practice guidelines

ASJC Scopus subject areas

  • Cell Biology
  • Histology
  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. / Borowitz, Michael J; Craig, Fiona E.; DiGiuseppe, Joseph A.; Illingworth, Andrea J.; Rosse, Wendell; Sutherland, D. Robert; Wittwer, Carl T.; Richards, Stephen J.

In: Cytometry Part B - Clinical Cytometry, Vol. 78, No. 4, 2010, p. 211-230.

Research output: Contribution to journalArticle

Borowitz, MJ, Craig, FE, DiGiuseppe, JA, Illingworth, AJ, Rosse, W, Sutherland, DR, Wittwer, CT & Richards, SJ 2010, 'Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry', Cytometry Part B - Clinical Cytometry, vol. 78, no. 4, pp. 211-230. https://doi.org/10.1002/cyto.b.20525
Borowitz, Michael J ; Craig, Fiona E. ; DiGiuseppe, Joseph A. ; Illingworth, Andrea J. ; Rosse, Wendell ; Sutherland, D. Robert ; Wittwer, Carl T. ; Richards, Stephen J. / Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. In: Cytometry Part B - Clinical Cytometry. 2010 ; Vol. 78, No. 4. pp. 211-230.
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