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About the Authors:
Ted S. Strom
* E-mail: [email protected]
Affiliations Department of Pathology and Laboratory Medicine, University of Tennessee Health Sciences Center, Memphis, Tennessee, United States of America, Department of Pathology and Laboratory Medicine, Memphis Veterans Administration Medical Center, Memphis, Tennessee, United States of America
Praveen Anur
Affiliation: Oregon Health Sciences University, Portland, Oregon, United States of America
Amanda Prislovsky
Affiliation: Department of Pathology and Laboratory Medicine, Memphis Veterans Administration Medical Center, Memphis, Tennessee, United States of America
Introduction
Ex vivo studies of the phagocytosis of platelets, red cells, and microorganisms are useful for the study of disease states such as autoimmune thrombocytopenia, hemolytic anemia, immunodeficiency, and a number of infectious diseases. While phagocytosis can be reliably distinguished from adsorption by confocal microscopy, that method is not well suited to the analysis of large numbers of events. In flow cytometric studies of phagocytosis of fluorescent targets, “quenching” of adsorbed fluorescent markers with agents like ammonium acetate [1], Trypan blue [2], [3], or proprietary kit reagents [4], has been used to distinguish between uptake and adsorption. However, studies which use these methods rarely show control data demonstrating the effectiveness of quenching. Alternatively, a second fluorescent marker able to quantify cells showing adsorption of the targets is sometimes used to make this distinction [5], [6], [7], [8]. This method in fact distinguishes (1) cells showing adsorption OR (adsorption+phagocytosis) from (2) cells showing phagocytosis only. Because the relative proportions of these two groups will be affected both by the ratio of targets to macrophages and the probability of phagocytosis per adsorbed target, simply ignoring the first group excludes relevant data from the analysis of such experiments. Also, quenching of the fluorescence of internalized targets is often accelerated in the low-pH, protease-rich environment they encounter after phagocytosis. This can result in a phagocytosis(+) population evident only as a ‘bulge’ on the negative population [7], making its quantification problematic. In that context, the distinction between an experimental effect on phagocytosis and an effect on quenching efficiency is not immediately evident. The issue is made more difficult to address by the frequent omission of raw data in published studies utilizing this method.
Here we describe a numerical analysis model which resolves these issues. The model evaluates the concurrent contributions of variation...