Staphylococcus aureus phagocytosis. A new cytofluorometric method using FITC and paraformaldehyde.

A new method for measuring the uptake of Staphylococcus aureus by human polymorphonuclear neutrophils (PMN) using flow cytometry (FCM) is described. Bacteria were labeled with fluorescein isothiocyanate (FITC) and incubated with PMN in a suspension assay. At the end of the assay, phagocytosis was arrested by the addition of cold paraformaldehyde. Thereafter, phagocytosis was quantitated by FCM, using crystal violet to distinguish adherent versus ingested bacteria. The method was validated in multiple samples by reference to our microscopic technique; correlations of phagocytic indices were in good agreement. The FCM method was also found to be reproducible and provided a mean to detect low phagocyte values. Another feature of the approach is that FCM readings can be delayed without any appreciable alterations in the results.

Applications of the monoclonal antibody PC10 assessment of proliferative grade in cell smears.

In view of the increasing need to assess the proliferative activity of hematologic malignancies, slide-based methods to quantify the proliferating cell nuclear antigen (PCNA) were developed and evaluated. Two techniques to evaluate this antigen were adapted to the infiltration level of the disease. The first one is particularly appropriate to massive invasion and is based on the alkaline phosphatase-antialkaline phosphatase complex (APAAP)method. The second one is reversed for reduced infiltration and uses a double immunofluorescence labeling. One is specific for the target cell to be identified and the other one is specific for the PCNA. These techniques permit an easy and accurate routine evaluation of cell cycle marker expression in hematologic malignancies.