[Common, standardized and recommended approaches in the diagnosis and monitoring of paroxysmal nocturnal haemoglobinuria using flow cytometry]. / Bezné, standardizované a doporucené postupy v diagnostice a monitorování paroxysmální nocní hemoglobinurie prutokovou cytometrií

Paroxysmal nocturnal hemoglobinuria is an acquired clonal disease characterized by proliferation of stem cells, deficient of proteins linked to the membrane via glycophosphatidylinositol (GPI) anchors. PNH cell characterization by flow cytometry was introduced in 1986, since 1996 is considered as method of choice for PNH diagnosis. Flow cytometry PNH analysis is nowadays crucial for disease monitoring in terms of progression, regression, remission or response to therapy and screening for small PNH clones (< 1.0%) in patients with aplastic anemia or myelodysplastic syndrome. Flow cytometry is unfortunately still poorly standardized, there is a variety of different methodological approaches for PNH evaluation and results from external quality assurances schemes reveal heterogeneous results. The aim of this work is to review the applicability of flow cytometry for the diagnosis and monitoring of PNH with respect to our experience and in the context of the recent trends and guidelines for PNH evaluation by flow cytometry.

Comparison of three single platform methods for CD34+ hematopoietic stem cell enumeration by flow cytometry.

In the present study, we compared three single platform methods for CD34+ hematopoietic stem cell (HSC) enumeration by flow cytometry. For this purpose, we analyzed the performance characteristics and results obtained from different HSC sources. Interlaboratory coefficients of variation (CV) for precision/reproducibility analysis varied from 4.0% to 6.7% / 6.7% to 9.2% for the low and 3.2% to 4.1% / 4.3% to 6.7%, respectively, for the high stem cell control. Correlation between methods ranged from 0.92% to 0.99%; Wilcoxon test showed no significant differences (p > 0.05); Bland-Altman analysis confirmed good agreement between assays (mean bias ranging from -0.48 to 6.91). Our results demonstrate very good intralaboratory correlation and agreement between methods, confirm the major impact of single platform strategy for accurate and reproducible HSC enumeration and suggest that high interlaboratory variability could be influenced by incorrect performance of validated methods.