title: Standardizing Leucocyte PNH clone detection: An international study.
abstract: Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.
authors.affiliation: UK NEQAS for Leucocyte Immunophenotyping (UK NEQAS LI), Department of Haematology, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Fletcher
authors.forename: D Robert
authors.initials: DR
authors.lastname: Sutherland
authors.forename: Liam
authors.initials: L
authors.lastname: Whitby
authors.forename: Alison
authors.initials: A
authors.lastname: Whitby
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Richards
authors.forename: Erica
authors.initials: E
authors.lastname: Acton
authors.forename: Michael
authors.initials: M
authors.lastname: Keeney
authors.forename: Michael
authors.initials: M
authors.lastname: Borowitz
authors.forename: Andrea
authors.initials: A
authors.lastname: Illingworth
authors.forename: John T
authors.initials: JT
authors.lastname: Reilly
authors.forename: David
authors.initials: D
authors.lastname: Barnett
keywords: EQA; PNH; Quality Assessment; Quality Control; Standardization