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G.Valet, W.Kellermann1)
Intensive care patients are in life threatening conditions when affected by septic or non infectious shock. Granulocytes as cellular elements for phagocytosis of microorganisms, release of enzymes and pharmacologically active mediators represent a major defense mechanisms against these affections.
It is clinically of utmost importance to recognize the danger of sepsis or shock as early as possible. This is not readily possible by biochemical markers in the vascular or other body compartments accessible to biochemical analysis. The concept of this work was to determine functional parameters in granulocytes at a molecular level to obtain indicators of imminent danger.
Our earlier flow cytometric work (1,2) using bacterial phagocytosis (6,7), ADB intracellular pH and esterase (1,2) measurements as well as of acridine orange as indicator of cellular and bacterial RNA and DNA(7) had shown for the first time that the prediction of imminent sepsis and shock was possible two to three days in advance to the appearence of clinical symptoms. These findings will provide a significantly increased therapeutic lead time for the clinician.
Although conceptually promising, the use of bacteria and of a phagocytosis assay is more complicated than the stimulation of blood by humoral means e.g. cytokines. Following the development of the sensitive oxidative burst indicator (8,10,11,14) dihydrorhodamine123 (DHR) and of highly specific rhodamine110 substrates for protease activity (12,13,17-22,24) determinations in our group, the concept for the determination of blood cell function was substantially simplified.
Flow cytometric data are collected as list mode files and evaluated in a standardized and automated way by the multiparameter data classification program CLASSIF1 (2) (Ann.NY Acad.Sci 677,233-251(1993))
The incubation of viable ex-vivo leukocyte preparations either alone or with suboptimal concentrations of FMLP, TNF-alpha, FMLP+TNF-alpha as physiologic stimulators and phorbol ester (PMA) as maximum stimulator provides now a very similar prognostic information (Valet, Kellermann: Methods in Cell Biology 1996, in press) as the earlier phagocytosis assay.
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