Performance Characteristics of a Non-Fluorescent Aerolysin-Based Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay for Simultaneous Evaluation of PNH Neutrophils and PNH Monocytes by Flow Cytometry, Following Published PNH Guidelines.
CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available.



We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach.



Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2.



Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.
Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
Iuri
I
Marinov
Dahl-Chase Diagnostic Services, Bangor, Maine.
Andrea J
AJ
Illingworth
Exbio Praha, Prague, Czech Republic.
Miroslav
M
Benko
Department of Laboratory Medicine, Toronto General Hospital, Toronto, Ontario, Canada.
D Robert
DR
Sutherland
D036541:ADP-ribosyl Cyclase / Q000276:immunology / Q000378:metabolism; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D001427:Bacterial Toxins; D015415:Biomarkers / Q000378:metabolism; D051927:CD24 Antigen / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D058851:GPI-Linked Proteins / Q000276:immunology / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins
D015703:Antigens, CD; D001427:Bacterial Toxins; D015415:Biomarkers; D051927:CD24 Antigen; D058851:GPI-Linked Proteins; D018950:Lipopolysaccharide Receptors; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D036541:ADP-ribosyl Cyclase; C087521:ADP-ribosyl cyclase 2
CD157; PNH; flow cytometry; non-FLAER-based approach