Diagnosing PNH with FLAER and multiparameter flow cytometry.
BACKGROUND
PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders.


METHODS
In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages.


RESULTS
Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER.


CONCLUSIONS
FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders.
Sutherland
D Robert
DR
Clinical Flow Cytometry Laboratory, University Health Network, Toronto, Canada. rob.southerland@utoronto.ca
Kuek
Nancy
N
Davidson
Jeff
J
Barth
David
D
Chang
Hong
H
Yeo
Erik
E
Bamford
Sylvia
S
Chin-Yee
Ian
I
Keeney
Michael
M
D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001427:Bacterial Toxins; D018957:CD59 Antigens / Q000378:metabolism; D004355:Drug Stability; D004912:Erythrocytes / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006402:Hematologic Diseases / Q000175:diagnosis; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D063268:Sialic Acid Binding Ig-like Lectin 3
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D001427:Bacterial Toxins; C571163:CD33 protein, human; D018957:CD59 Antigens; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D052899:Pore Forming Cytotoxic Proteins; D011933:Reagent Kits, Diagnostic; D063268:Sialic Acid Binding Ig-like Lectin 3; C107096:proaerolysin; C051590:CD59 protein, human; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human