Novel aspects of systems biology and clinical cytomics


  • Part of this work was presented at the 10th Leipziger Workshop “Systems Biology and Clinical Cytomics”, April 7–9, 2005, Leipzig, Germany.


The area of Cytomics and Systems Biology became of great impact during the last years. In some fields of the leading cytometric techniques it represents the cutting edge today. Many different applications/variations of multicolor staining were developed for flow- or slide-based cytometric analysis of suspensions and sections to whole animal analysis. Multispectral optical imaging can be used for studying immunological and tumorigenic processes. New methods resulted in the establishment of lipidomics as the systemic research of lipids and their behavior. All of these development push the systemic approach of the analysis of biological specimens to enhance the outcome in the clinic and in drug discovery programs. © 2006 International Society for Analytical Cytology


The current development of quantitative cell analysis and morphological documentation in Cytomics and Systems Biology increases the interest of multiple characterizations of biological samples (1–5). Besides the continuous development of new or enhanced cytometers by manufactures, novel cytometric methods and algorithms are one of the major research fields too. Thus, to further increase the number of simultaneously measurable parameters is the most challenging task. Another focus of interest is to obtain more information within a shorter period of time using less analyte or to characterize the examined material in more detail (4, 5). In some cases, a limiting factor is the availability of samples of some biological sources resulting in a low cell number, e.g. in neonates, children, and small animals. Another very effective driving force to develop new staining or measuring techniques are the relatively low costs of multicolor analyses. For example a new nine parameter protocol for a standard four-color flow cytometer allows to detect as many cell populations as possible in a minute sample volume, using only one staining step (6). This increased power of analysis is of particular importance in high-throughput high-content drug-discovery (7, 8).

Besides flow cytometry, slide-based cytometry offers a great variety of possibilities for qualitative and quantitative determination of cytological parameters not only for cell suspensions, but also for tissue sections to determine the relation and the morphology of the analyzed cells (5, 9–14). With features such as reanalysis and merging, slide-based cytometry is the key technology for polychromatic cytometric investigations. Iterative restaining and the method of sequential photobleaching result in an increased information density on a single cell level and represent important components to perform polychromatic cytometry (9, 15). Currently, fluorescence microscopic systems are characterized by strong technical and methodological development. Four-color fluorescent labeling was developed for automated scanning fluorescence microscope analysis of peripheral blood samples (11). Microscopy-based multicolor tissue cytometry constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers. Novel algorithms in focussing accelerate image acquisition (16) and push hereby further development of automated cell-based serological test methods (17). The quantitative data analysis improves visual discrimination of subpopulations (10, 14) and is superior to manual scoring.

Currently, the development of the concept of cytomics takes place in parallel with the development in areas such as location proteomics, high-content and high-throughput methodologies in flow and tissue cytometry, screening assays as well as cell and tissue arrays toward a broad, systematic collection of information to cluster and catalogue cells according to their molecular, organelle, and morphometric phenotype (18).

The aim of Systems Biology is to understand the integral functionality of single cells, organs, or organisms by molecular analysis and mathematical modeling (1, 8, 19). Combining data streams collected in different levels of biological organization such as molecular, cellular, and physiological responses offers the chance to a system-wide view in biology. Recently, an unbiased analysis of tissues that provides data- dense descriptors of tissue architecture, cell types, and cell states has become available (18). Concepts to statistically identify, correlate, and model relationships across scales are introduced that rely on a state-space matrix derived from multiomics data aggregation (13). A photonic microscopic robot technology is capable of tagging and imaging hundreds, and possibly thousands, of different molecular components, e.g. proteins, of morphologically intact fixed cells and tissue. Data, assembled in a toponome (all-protein network on a single cell level) dictionary of the cell, led to the development of a new concept for target and drug lead discovery (18, 20). Cell-based disease models in which the molecular diversity of the human cytome could be taken into account will improve the predictive value of drug discovery (7).


Originally, flow cytometric systems in life sciences were created for measurements of different particles, e.g. cells, cell nuclei, or other cell components such as mitochondria. Using plastic beads with activated surface, serum or plasma components became measurable in flow. After solid and fluid phase analysis, the vapor phase (exhaled breath condensate) from patients can be used as an examination material for further cytometric analyses (21). This procedure could be shown to facilitate diagnostics in chronic lung diseases (22). By metabolomics a fluorescent visualization of substrate uptake of individual cells is possible together with the simultaneous analysis of proliferative activity and the proportion of dead cells. This allows reliable and quasi-online process optimization (23).

A real-time high resolution intrasurgical diagnostics, and their tight spatio-temporal coupling into intervention could be the key to the complex analysis of whole organism imaging, as a synthesis of cytometry. Newly developed or modified instrumentation for optical imaging based on reflectance, two photon, and multispectral imaging, can detect and localize cellular signatures of cancer in vivo, without the use of contrast agents or extrinsic dyes (2, 24).


On the background of macromorphological alterations, flow cytometric analysis could play some crucial role in the research of leukocyte function and interaction. One of these fields is atherosclerosis, where the macrophage–monocyte system shows characteristic changes. Studies give new understanding of mechanisms responsible for this process. Besides the cytometric analysis of the cell surface expression of sugar residues, lipids, and proteins, the behavior of lipids and their interaction with peptides on the cell surface was discovered in the last few years, contributing to the systemic concept of lipidomics (25).

Cholesterol- and glycosphingolipid-enriched plasma membrane microdomains (rafts), involved in various cellular processes, contain lipid anchored membrane antigens and transmembrane proteins, and show detergent insolubility at 4°C (26). Formation of different rafts may have consequences for raft-associated signaling in cholesterol homeostasis and apoptosis in human macrophages. Changes in membrane microdomains were directly related to gene expression of sphingolipid and sterol metabolism. Distinct abnormal regulations of the cellular cholesterol and phospholipids influx/efflux were demonstrated with implications on, e.g., apoptosis and cholesterol homeostasis in macrophages during atherogenesis (27). High density lipoprotein (HDL) modulates platelet and neutrophil function, and its vesicular transport mechanisms is involved in cholesterol efflux and activation associated degranulation, which might be linked to HDL-induced signal transduction (28). Cholesterol loading promotes the confluence of relatively small rafts into larger domains modulating the lipid microdomains at the plasma membrane in vivo (29). Microfluorometry with total internal reflection fluorescence microscopy can be used to characterize membrane stiffness, which decreases with temperature and increases with the amount of cholesterol (30). Within arteriosclerotic lesions, uptake of hemoglobin–haptoglobin complexes could also in a concentration dependent manner modulate the phenotype of lesion-associated macrophages with regard to oxidative burst activity and pro- and anti-inflammatory cytokine secretion, thereby contributing to the progression of the arteriosclerotic plaques (31). Ezetimib, a ligand for aminopeptidase N (CD13), modifies the raft assembly in macrophages, likely through a CD13-dependent dysclustering mechanism, as detected by flow cytometry or by confocal microscopy (32).


Th1/Th2 cytokine analysis with the multiplex array technique in mitogen-stimulated whole blood provides the possibility to predict immunosuppression in heart-transplant recipients (33) or other immunology-related complications (34). Novel flow and image cytometric assays were developed to assess the effects of immunosupressants or chemotherapy on leukocyte or tumor-cell apoptosis (35–37) or cyclooxygenase mRNA expression by quantitative “in situ” hybridization (38). The Th1/Th2 reaction monitoring shows a transient immunosupression with an increased proportion of Th2 cells induced by cardiac surgery with cardiopulmonary bypass. The shift of the immune system to the Th2 immune response correlates with postoperative morbidity (39). In addition, special cytometric methods are now available to characterize phagocytosis, immune-surveillance of the central nervous system, and antinuclear auto-antibodies (40–42). Improved disease characterization combined with individualized disease course prediction are supportive of emerging therapeutic approaches such as immune or stem cell therapy (43, 44), regenerative medicine, and tissue engineering (45, 46).


Research in biomedicine and medicine in the past was limited because of the biological hierarchy within a narrow range of pathological changes in the diseases. The possibility to analyze the whole-cell suspension instead of only a few cells, which is important to unravel the complexity of the immune system for example, opens the way to better understand complex clinical processes in patients. Cytomics and Systems Biology can show information of the present status and diagnosis, and consequently allows an individualized therapy as a general practice of medicine (1, 2, 19). The general applicability of differential molecular cell phenotype screening in disease favors accurate diagnostics and individualized disease course predictions for optimized patient therapy (personalized medicine) and the search for new drug targets (7, 34, 47) (Fig. 1). The proposed top–down differential molecular cell phenotype screening has the potential to improve the efficacy of the general health system through more specific diagnostics and individualized patient therapy within stratified patient groups. Considering this potential, the establishment of a dedicated research effort (48) such as a specifically allocated Human Cytome Project has been proposed (4). The Human Cytome Project is conceived as a joint cross-disciplinary effort of cytomics, systems biology, and high-throughput-oriented research (with a steadily improved conceptual framework over the last years) involving basic, clinical, and industry scientists (1, 3, 4, 8, 48, 49).

Figure 1.

Flow chart how in a generalized way studies to achieve predictive medicine are performed.