Potential for quick diagnosis:
Cell Biochemistry Martinsried
Risk Assessment for Sepsis and Shock
in Intensive Care Unit (ICU) Patients
G.Valet, W.Kellermann1)
1) Abt.An„sthesiologie, Krankenhaus-Schwabing der TUM, Munich, Germany
SMDC in Research and Medicine
Intensive care patients are in life threatening conditions when affected by sepsis or non infectious shock.
Granulocytes represent a major defense barrier against these affections. They phagocytose microorganisms or tissue
breakdown products, but they also release enzymes or pharmacologically active mediators. In this way they can represent
a danger for the organism if their potent functionalities escape inhibitory control mechanisms.
It is clinically of utmost importance to recognize the danger of sepsis or shock as early as possible. Unfortunately, this
is not readily possible by the determination of humoral biochemical markers in the vascular or other body
compartments.
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 (2). 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 very sensitive oxidative burst indicator dye (8,10,11,14) dihydrorhodamine123 (DHR) and of
the highly 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.
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 (4,6) multiparameter data classification program.
The analysis of the entire data set by the CLASSIF1 program system 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 (6) 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 exhaustive optimization of the classification process for the same group of septically admitted IC patients showed
(6) that the most discriminant predictive information was contained in only two assays which were the FMLP and
TNF-alpha stimulated oxidative burst (DHR123) assays rather than in the unstimulated ex-vivo assay or in the
maximum (PMA), in the combined (FMLP+TNF-alpha) stimulation assays or in the protease activity 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 (6).
Conclusion:
1. functional parameters of granulocytes and monocytes contain predictive information for disease outcome in
septically admitted IC patients already on the day of admission
2. Optimization of multiparameter data classification by CLASSIF1 analysis shows that the most discriminant
information is contained in two (FMLP/TNF-alpha) physiologically stimulated oxidative burst (DHR123) assays out of
seven cytometrically determined oxidative burst and protease activity assays on vital peripheral blood leukocytes
On-line classification of sepsis and shock cases (in preparation)
Cell Biochemistry
For problems or comments, please contact:
G.Valet E-mail: valet@biochem.mpg.de, Max-Planck-Institut fr Biochemie, Am Klopferspitz 18a, D-82152
Martinsried, Germany, Tel: +49/89/8578-2518, -2525, Fax: +49/89/8578-2563, INTERNET address:
biochem.mpg.de
Last Update: Jun.29, 1998 |
