Clinical evaluation of an aerolysin-based screening test for paroxysmal nocturnal haemoglobinuria.
BACKGROUND
Recently, a toxin produced by Aeromonas hydrophila was demonstrated to bind directly to the glycosyl-phosphatidyl-inositol (GPI) anchor. After coupling it to a fluorescent dye and applying it in fluorescence-activated cell scanning (FACS), this property was exploited to detect GPI-negative cells in the diagnosis of paroxysmal nocturnal haemoglobinuria (PNH).


METHODS
We used this reagent according to a very simple staining protocol followed by single-colour FACS and compared the results in patients with PNH and normal controls with those obtained with antibody-mediated detection of cells lacking GPI-anchored proteins.


RESULTS
We observed very good concordance between the two methods, with correlation coefficients (R2) of quantified GPI-deficient cell populations ranging from 0.952 to 0.969. The lower limit of detection was determined at 0.50% GPI-negative cells, which was in the range obtained with double-colour staining with antibodies (0.20-1.00%, depending on the antibody). A significant correlation was observed between the fraction of GPI-negative granulocytes and laboratory parameters of haemolysis, with the erythrocyte creatine having the best correlation (R2 = 0.671, P < 0.0001).


CONCLUSIONS
Using this protocol, we were able to reliably diagnose PNH with a high sensitivity. The test allows the identification of GPI-negative granulocyte populations as small as 0.5%.
Peghini
Pietro Edmondo
PE
Division of Haematology, Department of Internal Medicine, University Hospital Zurich, Zurich, Switzerland. pietro.peghini@usz.ch
Fehr
Jörg
J
D001427:Bacterial Toxins / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D019937:Diagnostic Techniques and Procedures; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis*; D006801:Humans; D052899:Pore Forming Cytotoxic Proteins; D012680:Sensitivity and Specificity; D013223:Statistics as Topic
D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin