title: Evaluation and comparison of different approaches for the detection of PNH clones by flow cytometry following the ICCS guidelines.
abstract: 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.
authors.forename: I
authors.initials: I
authors.lastname: Marinov
authors.forename: M
authors.initials: M
authors.lastname: Kohoutová
authors.forename: V
authors.initials: V
authors.lastname: Tkácová
authors.forename: A
authors.initials: A
authors.lastname: Pesek
authors.forename: J
authors.initials: J
authors.lastname: Cermák
mesh_terms: D002999:Clone Cells; D005434:Flow Cytometry / Q000379:methods*; D017408:Guidelines as Topic; D006457:Hemoglobinuria, Paroxysmal / Q000473:pathology*; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012958:Societies, Scientific