Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry

Abstract

We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii) relative percentage of yeast cells that become associated with phagocytes, and (iii) percentage of killing of C. albicans. The first two parameters are obtained through the additional use of a phycoerythrin-conjugated monoclonal antibody that selectively labels monocytes and polymorphonuclear granulocytes in peripheral blood. Killing is assessed by solubilizing blood cells with deoxycholate to liberate yeast cells from the phagocytes. Viable yeast cells retain carboxyfluorescein, but nonviable cells lose the fluorescent marker; thus, the reduction in number of fluorescent particles directly reflects phagocytic killing. Results obtained by the present method correlated excellently with parallel enumerations by colony counting. Test results with seven healthy individuals revealed a marked dissociation between the process of opsonophagocytosis, which was essentially complete after 20 min at 37°C, and killing rates, which were 48% 11% and 63% 9% (standard deviation) after 1 and 2 h, respectively, when yeast cell-to-phagocyte ratios were in the range of 0.5:1 to 2:1. The described assay is unrivaled in simplicity, rapidity, and reproducibility and generates results for a large number of samples within hours.

Phagocytosis, oxidative burst, and killing of Candida dubliniensis and Candida albicans by human neutrophils

“…This also holds true for the percentage level of oxidative burst after 30 min of incubation (67.3 vs. 60%) [21]. Comparison of the percentage levels of killing from the present study with those of another previous study [20] reveals comparable results for C. albicans: 31.4 þ 19% and 54.5 þ 24% compared to 48 þ 11% and 63 þ 9% after 1 and 2 h, respectively. Note that isolated leukocytes were used in this previous study and percentage levels of killing were estimated by the lysis of leukocytes with desoxycholate followed by analysis of BCECF-mediated £uorescence in liberated fungal cells [20].…”

“…Comparison of the percentage levels of killing from the present study with those of another previous study [20] reveals comparable results for C. albicans: 31.4 þ 19% and 54.5 þ 24% compared to 48 þ 11% and 63 þ 9% after 1 and 2 h, respectively. Note that isolated leukocytes were used in this previous study and percentage levels of killing were estimated by the lysis of leukocytes with desoxycholate followed by analysis of BCECF-mediated £uorescence in liberated fungal cells [20]. To avoid the previously reported arti¢cial activation of neutrophils via enhanced Mac-1 expression by dextran sedimentation of leukocytes [14], a process known to potentiate neutrophil phagocytosis [14], heparinized whole blood was used throughout the present study.…”

“…This methodological di¡erence may also account for the further increase in the killing rate of up to 68.6 þ 14.3% and 72.7 þ 14.2% (¢nal killing rate) after 3 and 4 h, respectively. Therefore in comparison with the previous study, killing was not completed within 2 h and the ¢nal killing rates were slightly higher [20].…”

Flow cytometric quantitation of opsonophagocytosis and intracellular killing of Candida albicans using a whole blood microassay

“…Neutrophils, also called polymorphonuclear leucocytes (PMNs), are first‐line defenses against invading microorganisms. Various flow cytometric or whole blood fractionation methods exist to assess PMN function (1–8), but most require relatively large numbers of PMNs, making them impractical for pediatric blood samples. In addition, the assays perform poorly on samples from patients with hematological conditions such as thalassemia.…”

“…This procedure is based on an assay described earlier (2, 9), which exploits the use of a vital fluorescent membrane‐permeable marker, bis‐carboxyethyl‐carboxyfluorescein (BCECF), for staining bacteria or yeast cells, and the labeling of PMNs by a fluorochrome‐labeled monoclonal antibody (CD13) with specificity for this hematopoietic cell lineage.…”