A Numerical Analysis Model for Interpretation of Flow Cytometric Studies of Ex Vivo Phagocytosis

Abstract

The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m), the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha), and the probability of phagocytosis per adsorbed target (p). The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+) and [adsorption(−), phagocytosis(+)] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

Increased uptake by splenic red pulp macrophages contributes to rapid platelet turnover in WASP(–) mice

“…Human WASP(−) platelets, in contrast, show increased ex vivo uptake by a macrophage cell line (THP-1 cells) even without opsonization [14, 15]. These contrasting findings suggests that an intrinsic effect of human, but not murine, platelet WASP deficiency on uptake by splenic macrophages could contribute to the more severe thrombocytopenia seen in clinical vs. murine WAS.…”

“…Such a mechanism is also supported by the increased susceptibility of antibody-opsonized murine WASP(−) platelets to ex vivo phagocytosis by bone marrow derive macrophages[9]. Platelets from WAS patients show a similar increased susceptibility to ex vivo phagocytosis by activated THP-1 cells[14, 15], although in this case the effect is evident in the absence of opsonization. While WAS patients are prone toward a number of autoimmune conditions, including (after splenectomy) episodic thrombocytopenias indistinguishable from ITP[12], a purely autoimmune etiology for their baseline thrombocytopenia seems unlikely.…”

Platelets from WAS patients show an increased susceptibility toex vivophagocytosis

“…Procedures were as previously described [14]. All studies were approved by the Institutional Review Boards of the Memphis VA Medical Center and the University of Tennessee Health Science Center.…”

“…Procedures were adapted from those used by Semple et al [15] and Hoffmeister et al [16], as described previously [14]. Briefly, 1 × 106 THP-1 cells per ml, grown in an RPMI medium [supplemented with 10% ifbs, penicillin/streptomycin, l-glutamine, and 5 μM beta mecraptoethanol], were activated for 3 hours with PMA (50 ng/ml).…”

“…This type of study with fluorescently labeled platelets presents analytic difficulties due to (A) the problem of quantitatively distinguishing the rates of the sequential processes of platelet adsorption (adhesion to the cell surface) and platelet uptake and (B) the problem of distinguishing an increased rate of phagocytosis from reduced quenching of fluorescence after uptake. We recently described a numerical analysis method which resolves these issues [14]. Here we employ it to study platelets from a series of WAS patients.…”