Cell Biochemistry Martinsried
The concept of this work was to determine cell function parameters
of granulo- and monocytes at a molecular level to obtain predictive
(prognostic) indicators of imminent danger to patients.
- Our earlier flow cytometric work ( 1, 2) using bacterial phagocytosis ( 6, 7), ADB intracellular pH and esterase ( 1, 2) measurements as well as acridine orange as indicator of cellular and bacterial RNA and DNA( 7) had shown for the first time that the prediction of imminent danger of sepsis and non infectious shock in intensive care (IC) patients was already possible two to three days in advance to the appearence of clinical symptoms (CL1). These findings provide a significantly increased therapeutic lead time for the clinician.
- Although conceptually promising, the use of bacteria in a phagocytosis assay is comparatively complicated e.g. in automatically operated flow cytometers. The concept was therefore to facilitate the practical approach to cell function assays. This was achieved by:
1. the use of humoral stimulators like e.g. cytokines and
2. the development of the sensitive oxidative burst indicator dye dihydrorhodamine123 (DHR) ( 8, 10, 11, 14) and of the specific rhodamine110 substrates for the determination of protease activity ( 12, 13, 17-22, 24) in vital cells. These developments have substantially simplified the determination of blood cell functions in infection, sepsis or non infectious shock.
Data Pattern Analysis
Flow cytometric data of such measurements are typically collected
as list mode files. They are then evaluated in a
standardized and automated way by the CLASSIF1
multiparameter data classification program.
- The analysis of the entire data set in this way reveiled that the incubation of ex-vivo leukocyte preparations:
- alone (ex-vivo)
- with physiological stimulators such as: suboptimal concentrations of FMLP (formyl-methionyl-leucyl-phenylalanyl bacterial peptide), TNF-alpha (tumor necrosis factor-alpha), FMLP+TNF-alpha and
- with phorbol ester (PMA, phorbol-myristate-acetate) as maximum stimulator
provides a sufficient amount of predictive information (CL3) for the early risk determination in septically admitted IC patients e.g. already on the day of admission, similarly as the cytometric determination of proteolytic enzyme activities like:
- cysteine proteinases or
- serine proteinases
- The optimization of the classification process for the same group of septically admitted IC patients showed (CL3) that the most discriminatory predictive information was contained in the FMLP and TNF-alpha stimulated oxidative burst (DHR123) assays.
- As a practical consequence of the CLASSIF1 multiparameter data analysis, only two out of the seven performed assays were really required for survival prediction in this group of septically admitted IC patients (CL3).
1. Functional parameters of granulocytes and monocytes contain predictive information for individualized risk assessment in septically admitted IC patients already on the day of admission
2. Automated operation with assay preparation, flow cytometric measurement, on-line data analysis in combination with standardized result classification is possible in principle either with laboratory flow cytometer + cell staining robot station but conceptually also with larger hematology analyzers equipped with fluorescence channels such as increasingly available for clinical routine laboratories in the context of medical cytomics or clinical cytomics.
CL1. G.Rothe, W.Kellermann, G.Valet: Flow cytometric parameters of neutrophil function as early indicators of sepsis or trauma-related pulmonary or cardiovascular organ failure. J.Lab.Clin.Invest.11552-61(1990)
CL2. G.Rothe, W.Kellermann, J.Briegel, B.Schaerer, G.Valet: Activation of neutrophils by tumor necrosis factor-alpha during sepsis, in: Immune Consequences of Trauma, Shock and Sepsis Vol.II, Ed: E.Faist, J.Ninnemann, D.Green, Springer Verlag, Berlin 1993, p.727-733
CL3. G.Valet, G.Roth, W.Kellermann: Risk assessment for intensive care patients by automated classification of flow cytometric oxidative burst, serine and cysteine proteinase measurements using CLASSIF1 triple matrix analysis, in: Cytometric Cellular Analysis, Eds: J.P.Robinson, G.Babcock, Wiley-Liss, New York 1998, p.289-306
© 2023 G.Valet
Last update: Apr.3,2003
First display: Feb 26,1996