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 (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 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 (CL2,CL3) 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
(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 exhaustive optimization of the classification process for the same group of septically admitted IC patients showed (CL3) 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 (CL3).
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
3. Fully 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.
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