Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.
BACKGROUND
Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.


METHODS
CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages.


RESULTS
Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples.


CONCLUSIONS
While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.
Laboratory Medicine Program, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada.
D R
DR
Sutherland
E
E
Acton
M
M
Keeney
B H
BH
Davis
A
A
Illingworth
D036541:ADP-ribosyl Cyclase / Q000032:analysis; D015703:Antigens, CD / Q000032:analysis; D001427:Bacterial Toxins; D051927:CD24 Antigen / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005647:Fucosyltransferases / Q000032:analysis; D058851:GPI-Linked Proteins / Q000032:analysis; D006098:Granulocytes / Q000166:cytology*; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D016256:Lewis X Antigen / Q000032:analysis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D009000:Monocytes / Q000166:cytology*; D052899:Pore Forming Cytotoxic Proteins; D017452:Receptors, IgG / Q000032:analysis
D015703:Antigens, CD; D001427:Bacterial Toxins; D051927:CD24 Antigen; C497889:CD24 protein, human; D058851:GPI-Linked Proteins; D016256:Lewis X Antigen; D018950:Lipopolysaccharide Receptors; D052899:Pore Forming Cytotoxic Proteins; D017452:Receptors, IgG; C032676:aerolysin; C066693:FUT4 protein, human; D005647:Fucosyltransferases; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D036541:ADP-ribosyl Cyclase; C087521:ADP-ribosyl cyclase 2
CD157; high sensitivity 5-color flow; paroxysmal nocturnal hemoglobinuria