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.

High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

“…CD15 (for neutrophils) and CD64 conjugates (for monocytes) are preferred over CD33 conjugates for a variety of reasons as detailed previously (19,20,22,25). CD15 (for neutrophils) and CD64 conjugates (for monocytes) are preferred over CD33 conjugates for a variety of reasons as detailed previously (19,20,22,25).…”

“…This excluded the use of the 80H5 clone (Beckman Coulter) that performs well in PECy5 format (19,22,25), as it is not available in this tandem combination. IgM subclass reagents are difficult to reliably conjugate with large tandem dyes such as PECy5.5, and so such conjugates are not generally commercially available (M. Benko, unpublished observations).…”

“…For gating neutrophils, earlier versions (for FC500 and Navios cytometers) of our 4-color neutrophil and 5-color combined (neutrophil/monocyte) assay used a CD15-PECy5 conjugate of clone 80H5 (Beckman Coulter) (19,20,22,25). To minimize spillover for the 7-color assay that includes an APC conjugate of CD24 required a change to either a PECy5.5 or PerCPCy5.5 or similar tandem conjugate of CD15.…”

“…Regardless, as part of inherent robust assay design and in keeping with guideline recommendations to detect the loss of at least two GPI-linked structures per lineage assessed (18)(19)(20)(21)(22)(23)(24)(25)(26), in each CD157-case uncovered, FLAER, the second reagent in our assay was found to be normally expressed. Regardless, as part of inherent robust assay design and in keeping with guideline recommendations to detect the loss of at least two GPI-linked structures per lineage assessed (18)(19)(20)(21)(22)(23)(24)(25)(26), in each CD157-case uncovered, FLAER, the second reagent in our assay was found to be normally expressed.…”

“…The importance of addressing both neutrophil and monocyte lineages has been underscored by the observations of Illingworth et al, who showed that it is not uncommon for monocytes to more accurately reflect PNH clone size than neutrophils (10). The 4-color assays, specific derivatives of which were developed for Becton Dickinson and Beckman Coulter cytometers were proven to be robust and reproducible (21) and suitable for deployment in International studies performed under the auspices of the UK NEQAS organization using whole blood stabilized samples (22). CD157 (23) is a GPI-linked structure brightly expressed on both neutrophils and monocytes (11,12) and can be used in 5-color combination with FLAER (FLAER, CD157, CD15, CD64, and CD45) instead of CD24 and CD14 for single tube high-sensitivity assessment of GPI-deficient neutrophils and monocytes (24)(25)(26)(27).…”