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.
Department of Pathology and Oncology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA. mborowit@jhmi.edu
Michael J
MJ
Borowitz
Fiona E
FE
Craig
Joseph A
JA
Digiuseppe
Andrea J
AJ
Illingworth
Wendell
W
Rosse
D Robert
DR
Sutherland
Carl T
CT
Wittwer
Stephen J
SJ
Richards
D015703:Antigens, CD / Q000378:metabolism; D032921:Consensus; D004912:Erythrocytes / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis*; D006801:Humans; D016130:Immunophenotyping; D007962:Leukocytes / Q000378:metabolism
D015703:Antigens, CD