title: Flow cytometric quantitation of opsonophagocytosis and intracellular killing of Candida albicans using a whole blood microassay.
abstract: Flow cytometric assays for quantifying polymorphonuclear leucocyte (PMN) function involve peripheral blood fractionation methods. However, most are of limited use for conditions like pediatrics and thalassemia, as they may require large blood volumes or complicated by red blood cell contamination. In whole blood assay, 500-mul aliquots of normal or thalassemic whole blood were incubated with bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester (BCECF-AM)-labeled Candida albicans cells and phycoerythrin-conjugated anti-CD13 monoclonal antibody for PMNs. Phagocytosis was quantitated by gating on CD13 label PMNs and determining the frequency of phagocytosed yeast using flow cytometry. The killing activity was quantitated by estimating the number of viable fluorescence retaining yeast cells liberated following lysis of PMNs. In normal control or thalassemia samples, nonphagocytosed yeast and PMNs with or without phagocytosed yeast were readily distinguished by two-color dot plot, permitting phagocytosis estimates. Viable and nonviable yeast killed by whole blood PMNs were distinguished by side scatter and fluorescence, permitting killing estimates. The patterns seen using whole blood was similar to that seen with fractionated PMNs. Data from thalassemia patients showed similar opsonophagocytosis but slightly decreased intracellular killing of yeast cells when compared with normal subjects. This assay provides an alternative method to assess PMN function in small blood samples, which could be especially useful in conditions like thalassemia and pediatric patients.
authors.affiliation: Center of Excellence for Flow Cytometry, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. grkpy@mahidol.ac.th
authors.forename: Kovit
authors.initials: K
authors.lastname: Pattanapanyasat
authors.forename: Kasama
authors.initials: K
authors.lastname: Sukapirom
authors.forename: Kalaya
authors.initials: K
authors.lastname: Tachavanich
authors.forename: Sakorn
authors.initials: S
authors.lastname: Kaewmoon
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D018826:CD13 Antigens / Q000276:immunology; D002176:Candida albicans / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D009504:Neutrophils / Q000276:immunology*; D009895:Opsonin Proteins / Q000097:blood; D010587:Phagocytosis / Q000502:physiology; D017086:beta-Thalassemia / Q000097:blood
chemical_list: D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009895:Opsonin Proteins; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D018826:CD13 Antigens