Multiparameter flow cytometry for the diagnosis and monitoring of small GPI-deficient cellular populations.
BACKGROUND
Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection.


METHODS
We performed serial quantification of the PNH clone size in 38 samples using multiparameter flow cytometry. Granulocytes, monocytes, and RBCs were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers fluorescent aerolysin (FLAER), CD55, and CD59 were comparatively evaluated. We also evaluated CD16 on granulocytes and CD14 on monocytes. The sensitivity of detection by each marker was further defined by serial dilution experiments on a flow-sorted sample. Two patients had quantification of their GPI-negative clones before and after allogeneic BMT.


RESULTS
FLAER was the most discriminant marker and allowed identification of 0.1% of GPI-negative cells despite other markers having superior signal-to-noise characteristics. CD14 and CD16 were inferior to CD55 at lower concentrations and in clinical application.


CONCLUSIONS
Multiparameter flow cytometry permits quantification of small GPI-negative clones with a sensitivity limit of about 0.1%. The single most reliable marker to monitor small granulocyte or monocyte PNH clones is FLAER, especially in conditions such as myelodysplastic syndromes or BMT, when traditional GPI-linked surface marker expression can be significantly altered. © 2010 International Clinical Cytometry Society.
Battiwalla
Minoo
M
Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York, USA. kunashni@yahoo.com
Hepgur
Mehmet
M
Pan
Dalin
D
McCarthy
Philip L
PL
Ahluwalia
Manmeet S
MS
Camacho
Susan H
SH
Starostik
Petr
P
Wallace
Paul K
PK
D000293:Adolescent; D000328:Adult; D000368:Aged; D000741:Anemia, Aplastic; D001427:Bacterial Toxins / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D001773:Blood Cells / Q000737:chemistry* / Q000276:immunology; D001855:Bone Marrow Diseases; D000080983:Bone Marrow Failure Disorders; D016026:Bone Marrow Transplantation / Q000276:immunology; D018958:CD55 Antigens / Q000032:analysis / Q000276:immunology; D018957:CD59 Antigens / Q000032:analysis / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D058851:GPI-Linked Proteins / Q000032:analysis / Q000276:immunology; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000172:deficiency* / Q000276:immunology; D006098:Granulocytes / Q000276:immunology; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis / Q000276:immunology; D008297:Male; D008875:Middle Aged; D008991:Monitoring, Physiologic / Q000379:methods*; D009000:Monocytes / Q000276:immunology; D052899:Pore Forming Cytotoxic Proteins / Q000032:analysis; D017452:Receptors, IgG / Q000032:analysis / Q000276:immunology; D015203:Reproducibility of Results; D055815:Young Adult
D001427:Bacterial Toxins; D015415:Biomarkers; D018958:CD55 Antigens; D018957:CD59 Antigens; C428200:FCGR3B protein, human; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D018950:Lipopolysaccharide Receptors; D052899:Pore Forming Cytotoxic Proteins; D017452:Receptors, IgG; C051590:CD59 protein, human; C032676:aerolysin