Evaluation and comparison of different approaches for the detection of PNH clones by flow cytometry following the ICCS guidelines.

BACKGROUND: Comparison between consensual approaches for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) following the international clinical cytometry society (ICCS) guidelines has not been widely reported. METHODS: We determined the performance characteristics of 4, 5, and 6-color protocols for white blood cell (WBC) and one and two-color protocols for red blood cell (RBC) evaluation for different PNH target clones and compared results from PNH patient analysis. RESULTS: Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.01%/0.12% to 2.56%/ 3.59% for granulocytes, from 0.07%/0.08% to 3.87%/11.61% for monocytes and from 0.4%/1.02% to 6.53%/ 5.1% for RBCs within different approaches and target PNH clones. Comparison of individual protocols revealed excellent correlation (r = 0.99), Wilcoxon rank tests found no statistically significant differences (p > 0.05), Bland-Altman analysis proved agreement for all PNH clones (mean bias ranging from 0.02 to 2.2). CONCLUSIONS: Our results confirm good intralaboratory characteristics for precision and reproducibility analysis, excellent correlation and agreement between approaches underlining the primary role of optimally selected glycophosphatidylinositol (GPI)-specific reagents and secondary role of number, type of gating reagents and gating strategy.

Clinical relevance of CD157 for rapid and cost-effective simultaneous evaluation of PNH granulocytes and monocytes by flow cytometry.

INTRODUCTION: CD157 has been reported as a potentially useful marker for paroxysmal nocturnal hemoglobinuria (PNH) testing by flow cytometry (FCM). METHODS: We determined the performance characteristics of a CD157-based five-color assay and compared results from patient analysis with results obtained with a previously validated CD14/CD24-based six-color protocol. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analysis of granulocytes ranged from 0.88/0.09% to 3.02/3.71% and from 0.20/0.08% to 8.83/4.04% for the five- and six-color protocol, respectively. For monocyte, CV ranged from 0.42/0.49% to 8.13/4.80% for the five-color protocol and from 0.28/0.70% to 5.41/3.19% for the six-color protocol within various PNH clones. Coefficient of determination (r(2) ) for linear regression analysis of PNH clones ranging from 0.3 to 99.8% was >0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), and Bland-Altman analysis demonstrated agreement with mean bias ranging from -0.02 to 0.38. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, excellent correlation, and agreement between approaches. In agreement with recently published data, our experience supports the clinical relevance of CD157 for a rapid and cost-effective simultaneous evaluation of PNH leukocytes by flow cytometry.