DE19935047A1 - Flow-cytometric analysis of leukocyte subpopulations, comprises combining fluorescence-labeled components with a cell system and detecting in a single batch through fluorescence-induced label reactions - Google Patents

Abstract

A process (I) in which fluorescence-labeled components, such as antibodies, mixtures, microorganisms, parts of microorganisms, cells or parts of cells, are combined with a cell system and detected in a single batch through fluorescence-induced label reactions in the cell system or in a subpopulation, is new. Independent claims are also included for the following: (1) a process (II) in which a comprehensive representation and measurement of even complex reactions patterns of cell systems in direct and indirect relation to influencing factors (multidimensional dynamic cytofluorophoresis) can be achieved by a combination of multiple fluorescence-labeled biologically active components, a combination of multiple fluorescence-induced reaction-dependent labels and/or a combination of fluorescence-induced subpopulation labels, certain fluorescent dyes can be used repeatedly as a result of different signal intensities; and (2) software within which the cytometric listmode data from processes (I) and (II) are stored in a database together with all data characterizing the cell preparation and measurement, vector-graphic representations are generated from the data and analysis and comparison of the data is possible, even on a complex level, by tiltable multidata table lattices, hierarchical maskable or gateable fields, individual dataset representations and cross-tables of the parameters and diagram-overlapping discriminator lines.

Description

PRIOR ART - BACKGROUND AND PREREQUISITES OF THE METHOD

Through the development of cytometry (flow cytometry, scanning cytometry) in connection with Fluorescence technology and monoclonal antibody production is a biological measurement technique Single cell level has become possible. The specialty of flow cytometry when analyzing cells and biological systems lies in the rapid, multiparametric, quantitative representation of characteristics on a large number of single cells in heterogeneous cell suspension (e.g. human whole blood). On the methodological and technical side, there are four, partly on top of each other, for the new process to emphasize advanced measurement and preparation techniques according to the state of the art:

A. Static-descriptive measurement of single cells

(1) Antibodies bind to their associated antigen in a highly specific manner. Monoclonal antibodies can be used to selectively recognize and bind defined antigens on the cell surface and inside the cell. Many of the cell surface markers known today can be found in the international CD nomenclature (Clusters of Differentiation).
(2) Different cell types differ in the type and strength of expression of different antigens. Since almost no antigen alone is specific for a specific cell population, ie it can occur on different cell populations, and an antigen can theoretically occur in a wide variety of expression levels, theoretically unmanageably large numbers of combinations of antigens or their different expressions are possible. In fact, there are far fewer, often discretely delimitable combinations of antigen expressions that can be assigned to biologically characterizable cell populations. Here it is necessary to differentiate more precisely between the differentiation and the activation state of a cell. With antigens that are typical for a differentiation state of cells (e.g. CD3 for T lymphocytes), hardly any fluctuations in intensity are to be expected (static), while with activation and function markers (e.g. CD69, cytokine expression) Short-term changes (e.g. within hours) occur in response to substances, stimulators or microorganisms (dynamic).
(3) By coupling the antibodies with different fluorescent dyes, several fluorescent colors and thus antigens can be measured simultaneously in flow cytometry / scanning cytometry at the individual cell level.
(4) Based on quantitative measurement of the fluorescence intensity, the expression level of the antigens is additionally measured. With constant measurement conditions, this allows a direct comparison of antigen expression levels, at least relative to one another. When using calibration techniques (fluorescence beads), constant antibody-fluorochrome coupling ratio and saturation of all antigen binding sites with antibodies, it is also possible to determine the absolute number of antigen molecules per cell surface or per cell.
(5) Since, especially with flow cytometry, thousands of cells from the same sample can be analyzed within minutes, measurements of the antigens are possible with a high degree of statistical certainty and also on very poorly represented cell types.

B. binding studies, intemalization, phagocytosis

(6) In addition to antibodies, other molecules of known or possible biological relevance (ligands) up to larger particles and entire microorganisms can also be fluorescence-labeled and their binding behavior towards cells can be quantitatively measured in flow cytometry / scanning cytometry. It should be noted here that the biological properties of the substances can be changed by the fluorescent labeling. This must be checked or checked in individual cases.
(7) Based on the multiparametric measurement, the binding behavior can also be analyzed on a subpopulation basis.
(8) If receptors for the labeled substances are known or are postulated, the distribution and expression level of these receptors can be determined on different cell populations.
(9) In addition to the purely static binding behavior, functional reactions of the cells can also be recorded, particularly as a function of temperature and time (further increase in the fluorescence intensity during internalization and intracellular enrichment of the labeled substances or phagocytosis of the labeled microorganisms). Changes in the fluorescent markings (e.g. quenching) must be controlled by biological processes.

C. Ex vivo stimulation of cells and measurement of reactions (activation markers, Cytokine expression)

(10) Using cell culture techniques, cells, particularly in their physiological milieu and in a heterogeneous mixture (human whole blood), can be cultivated for hours or days and stimuli such as lipopolysaccharides, lectins, microorganisms or parts thereof can be exposed.
(11) Since flow cytometry / scanning cytometry enables specific changes to be measured on a subpopulation basis, the cells can be cultured in their full physiological context (whole blood). In this way, even more complex overall reactions involving several cell populations can be recorded ex vivo, which is closer to the in vivo situation than with a cell separation required for other techniques.
(12) In addition to morphological changes and cell proliferation, activation markers, which are expressed on the cell surface, and intracellular cytokines are measured. This not only allows the behavior of individual cell populations to be measured in relation to specific stimuli, but also further cell sub-populations can be characterized at the functional level, for example pro- and anti-inflammatory cells (TH1 / TH2) etc.

D. Specific stimulation of cells / signaling by antibodies

(13) In addition to many other biologically active molecules, antibodies can also be used trigger biological signals on cells. The particular advantage here is again the high Specificity of antibodies. For example, a physiological ligand can have several Receptors with different downstream transduction pathways different at the same time trigger individual biological reactions, cross-reactivity of an antibody is much rarer or avoidable by appropriate selection of the antibody. Thus, antibodies can Study more complex biological activation and regulation processes on cells in sub-processes and Analyze or influence the overall reactions using step-by-step combinations (costimulation).

The importance of stimulation with antibodies, especially on immune cells, ranges from Basic research (molecular biological questions, signal transduction) about Autoimmune diseases (e.g. autoimmune threoditis, being pathologically stimulating Autoantibodies against the thyroid hormone receptor can occur) up to current therapeutic approaches for targeted immune stimulation e.g. B. in tumor diseases.

PROBLEM POSITION

With highly specialized and highly developed methods of molecular biology, many Individual aspects of biological systems are precisely analyzed and identified down to the molecular level. Despite the rapidly growing number of genes and molecules identified and the establishment of the "Molecular medicine" is always clear that the variety of determinable parameters alone often only can contribute little to the overall understanding of biological systems and diseases. Numerous Approaches, especially in the context of diagnosis, therapy and prognosis of diseases, diversity reducing the parameters to a few key markers and active ingredients has proven to be limited and depend on the other heterogeneity of the group examined (example: diagnostics,  Prognosis and therapy in sepsis). On the other hand, the technical, time and cost Effort has long been exceeded that all new parameters are comprehensively shared with all others may still determine associated in individual cases.

The biological behavior of cell systems is still not simply the sum of the behavior of the classifiable subpopulations, just as the behavior of a single cell is not easy can be derived from the sum of their molecular processes, even if you measure all individual processes and could describe.

Thus new methods are required that allow cells in their context and entire cell systems to analyze and compare their properties and their functionality on a more complex level and to come as close as possible to the in vivo conditions.

Current techniques and measurement methods in cytometry focus, despite multiparametric Measurement on a single cell level, but often again on partial aspects of the examined biological system. For example, only certain cell populations are separated according to highly specific characteristics (Cell sorting) and performed with these stimulations and measurements. However, since the cell is off their physiological / pathological system context, the results cannot basically applied to the in vivo conditions. The same applies to biological activity of substances in cell systems. The sum of the individual effects cannot simply be based on Overall effect on the system can be concluded, especially since concentration ratios, target population, The time and duration of the individual substances can have completely different influences. Also these aspects of synergistic effects, agonism and / or antagonism, costimulation and Modulation, i. H. ultimately complex patterns of action from substances or substance mixtures up to Microorganisms are only little detected with the usual cytometric measurement techniques.

Current flow cytometers measure 5-6 parameters per single cell (forward scattered light, Side scattered light, 3-4 fluorescent colors). Devices that can measure even more fluorescence are already in use or in development. The published state of the art is 11 Parameters (forward scattered light, side scattered light, 9 fluorescent colors), but this is already 3 requires different excitation lasers. With each additional fluorescent color increases significantly the expense of the measuring equipment, additional new fluorescent labels, their biological behavior is not predictable, and last but not least the cost. Also from physical principle of fluorescence forth the spectrum with as few stimulating as possible Light sources and at the same time as many different, little overlapping emitting Fluorescent dyes are already quite exhausted.

Also by sequential staining and measurements as is possible with scanning cytometry, the number of parameters that can be measured simultaneously can be increased somewhat.  

In principle, however, there are already hundreds of individual markers, many of which are fluorescence-labeled monoclonal antibodies, measurable at cell level. In contrast, the number of in cytometry measurable parameters at the same time relatively small. For the comprehensive characterization of biological Systems and functions also make sense, however, in which as many as possible at the same time Enter parameters, even if the measurement and data technology no longer provides a complete individual breakdown is possible, but the complex overall reaction can be analyzed and compared.

The problem can be illustrated using an analogy from the field of molecular biology: If you separate serum proteins using the classic technique of electrophoresis, you get 5 "Main populations" of molecules: albumin, α1-globulins, α2-globulins, β-globulins, γ-globulins. If you use refined methods such as immunoelectrophoresis, the resolution can be broken down into more Increase "subpopulations" of molecules to single proteins. Although many now individual serum proteins are exactly identified and it is known in which serum electrophoresis area these can be found, is still used today as such for such serum electrophoresis. B. at the Comparison of certain sera and in the diagnosis of diseases. On the one hand, this is faster and cheaper, on the other hand, a single analysis of all known today Serum proteins still have gaps and can be a holistic overall analysis with one do not replace the typical pattern of the parliamentary groups, only supplement them. Through the technology of 2D Electrophoresis makes it possible to transform a complete heterogeneous mixture of molecules into a 2-dimensional one To separate patterns, with a direct comparison of these with constant execution conditions Pattern is possible even before molecular identification of individual spots, i. H. "Molecular populations" takes place.

TROUBLESHOOTING

Most of the antigens primarily described as pure cell markers (cf. CD; clusters of Differentiation) shows in further investigations that these also have functional significance, since cells particularly have differentiated surface structures with their surroundings and their environment communicate (signal processing etc.)

Decisive for the Multidimensional Dynamic Cytofluorophoresis (MDCFP) are 2 steps that include these aspects and the parameter-dependent distribution of cells in the multidimensional Clearly expand the measuring room qualitatively and quantitatively.  

1. The use of fluorescence-labeled components to represent biological effects already in the cell culture phase (dynamic staining)

Fluorescence-labeled molecules, especially antibodies, up to entire microorganisms and In contrast to the purely labeling staining that is customary in cytometry, cells are already in the Cell culture used to trigger biological effects. Due to the stable marking, the binding The components are tracked to specific cells and directly related to biological activities or measured in these cells (combination of binding analysis and simultaneous measurement of function or activation parameters).

This will be possible if

• a) the binding of the fluorescence-labeled component to the target cell is stable via the cell culture remains,
• b) the fluorescent label remains largely stable in the cell culture,
• c) the fluorescent label does not affect the biological activity of the molecules or microorganisms significantly impaired
• d) the sodium azide content found in fluorescence-labeled antibodies for conservation, has no significant influence on the biological response of the target cells.

Approaches and measurements with commercially available for the usual use in flow cytometry Suitable substances, especially antibodies, clearly show in this dynamic application and reproducible effects. Due to targeted control of the critical requirements a) to d) if necessary, a fine optimization of the biological activity is possible.

The spectrum of components to be used ranges from molecules and substances to Microorganisms and target cells (e.g. tumor cells), components that are already in unlabelled form are widely used in ex vivo approaches.

At typical ligand-receptor ratios, fluorescent-labeled antibodies have stimulators and / or blockers compared to the physiological, usually smaller ligand molecules have the following advantages: The loading with fluorescent dye molecules is higher, which results in a stronger and therefore Intensity signal that can be better distinguished from the background noise. Furthermore, the measuring range can make better use of it, since even more and higher dilutions of the ligand can be prepared. The binding of the antibody is highly specific, usually against a certain previously known one or antigen analyzed by other methods, so that specific signaling pathways and Signal processing in the heterogeneous cell system can be analyzed.

When used in multidimensional dynamic cytofluorophoresis, this is not the case with antibodies only the antigen specificity, e.g. B. emerges from the CD designation (Cluster of Differentiation),  crucial, but also the functional specificity, d. H. whether the antibody is at the binding site additional activation, inhibition / displacement of a natural ligand with or without your own Activation effect shows.

2. Presentation and inclusion of more parameters than by the number of different ones Colors / measuring channels are limited (characteristic signal overlay pattern of components with the same color marking)

The flow cytometry / scanning cytometry can be viewed as an image of each cell based on its individual parameter values as a point in n-dimensional space. Corresponding markers using fluorescence-labeled antibodies are usually used in a saturating concentration and then the position of distinguishable cell populations is determined with evaluation of the percentage and statistical parameters. If one generalizes this principle and reverses it, the position of this point can be influenced by

• a) the selection of the biological marker / parameter,
• b) the fluorochrome used in relation to the signal amplification,
• c) the concentration or dilution of the marker molecule, when bound in non-saturating Concentration or mixture of labeled and unlabeled marker molecules can only achieved an overall weaker signal intensity than under optimal conditions of a) and b) become. As a result, z. B. with two approximately equally expressed antigens one weaker are displayed and thus a distinction between the two can be made on the same color.
• d) the use of reinforcement systems, e.g. B. indirect immunofluorescence and sandwich techniques; in this way, weakly expressed antigens can only be displayed, low and medium-strength antigens can be moved into the upper measuring range and thus "space" for others Make parameters.

So the primary goal of the method is to have as many different characteristics and Functional states of heterogeneous cell systems in a measurement side by side (i.e. in the measuring range from 1 to 1024 measuring channels) and thus make it directly comparable. First of all also basically regardless of whether a single area in the multidimensional space is one can be assigned to a specific, biologically clearly definable population. If the Preparation and measuring conditions is e.g. B. also a direct comparison of 2-parameter correlated Dot charts possible. A calibration of the fluorescence intensity can also be used during the measurement may be dispensed with in one approach, since signal intensities (activations etc.) at different Cell populations can be compared directly.  

However, it must be taken into account that especially in the case of dynamic coloring through activation / Changing the cells can also change the intensity of population markers so that the Recovery of certain subpopulations in comparison with static coloring may be more difficult can.

Methodical details using the example of whole human blood

Whole blood anticoagulated with heparin is diluted 2: 1 with RPMI medium without further additives. By the still relatively high proportion of cells here also results from whole blood material with low cell counts and measurement of proportionately low represented cell populations such as B. monocytes at the end of culture still good cell yield for measurement in flow cytometry. The lower medium share has proven to be sufficient for whole blood culture with culture times of at least 24 h.

The whole blood cultures are in sterile, customary flow cytometer tubes with loosely attached Incubated lid at 37 ° C. In the case of a shorter culture time (3-6 h), polystyrene tubes can be used with longer culture times or multiple sampling from the same stock culture (Kinetic measurements) are preferable to polypropylene tubes for cell adherence and to reduce activation phenomena through the plastic surface.

The fluorescence-labeled components used in cell culture are basically also in Individual approaches and individual staining as well as in comparison with the corresponding unmarked Components (stability, binding behavior, biological effects etc.) to be tested.

The cell culture duration depends on the times (hours to days) customary for cell reactions. Also Here, a period should be selected in which the most varied of reactions are possible in one Measurement can be recorded side by side (e.g. duration of 8-9 hours for simultaneous Measurement of early pro-inflammatory and later anti-inflammatory reactions).

After the end of the culture, the cells are cooled on ice, shaken and cold EDTA added brought back into suspension well and according to usual protocols for surface coloring and / or prepared intracellular staining. When preparing whole blood, the intracellular staining of the Permeabilization step also cause sufficient lysis of the erythrocytes, so that no additional lysis steps are required. In the entire staining preparation, only 2 up to 3 washing steps to avoid cell loss.

A lysis / permeabilization process should be chosen which clearly delimits the Main populations already allowed in forward-sideways scattered light, with leukocytes in whole blood should especially the monocytes can be well delineated from lymphocytes and granulocytes, so as not yet for that Main population demarcation to require additional fluorescent markers.

To the surface staining customary in flow cytometry / scanning cytometry and the intracellular / cytoplasmic staining after permeabilization of cells thus comes a third Type of "dynamic" coloring, which also includes aspects of marking, binding behavior  as well as biological reaction such. B. Activation on one or more cell populations makes measurable.

If the components used are an antibody, the following three aspects result from the dynamic staining:

• 1. 1.) The antibodies mark by binding to their specific antigen as in the usual Surface staining is a corresponding cell population (e.g. CD3 + cells → T cells).
• 2. 2.) The fluorescence intensity shows the corresponding expression on one or more Cell subpopulations. Since the antibodies were used dynamically, the condition shows up against End of cell culture phase, d. H. possible uplift caused by the binding or stimulation itself or down-regulation of the target molecules or receptors is included in the fluorescence intensity.
• 3. 3.) Appropriate biological can be counter-stained on the cell populations marked in this way Reactions (activation markers, cytokines) can be detected.

As a control for each dynamic coloring, the analog surface coloring is carried out, d. H. Add all fluorescently labeled antibodies or components as usual after End of cell culture. Through fluorescence-labeled isotype controls in dynamic or normal use non-specific reactions can also be checked in advance. Overall, with the new Process also for otherwise poorly characterized components, substances, antibodies or Microorganisms quickly determine the binding behavior in one batch and the associated control heterogeneous cell populations and at the same time direct and indirect biological effects on the binding or other cell populations are shown.

Through periodic removal of partial material from the same dynamically colored cell culture or control approach and usual coloring of the other parameters can dynamic effects on Cell system can also be represented kinetically.

By combining the dynamic staining of the cells (abbreviation: st) with the two usual ones Process of "static" membrane-related staining (abbreviation: m) and cytoplasmic Staining (abbreviation: cy) results in a large number of further cell population violations.

Explanation using an example

• a) Conventional methodology: mCD14 ⁺ cyTNFα ⁺ cyIL10 ^- : cell population that is caused by the appearance of CD14 (m = membrane-bound) on the cell surface and cytoplasmic detection (cy = cytoplasmic) of tumor necrosis factor-α, but not of IL-10 is marked. Since cytokines such as TNF-α and IL-10 are practically undetectable in resting cells, this cell population is only after stimulation with an activator such. B. Lipopolysaccharide (LPS) representable and corresponds biologically pro inflammatory monocytes. LPS is added unlabeled to the whole blood culture for stimulation, and in principle it is not possible to distinguish with the labeling and measurement of the population whether the stimulator has bound directly to the cell population in question or has been indirectly activated via mediation by other cell populations.
• b) Dynamic staining with labeled ligand: stLPS ⁺ cyTNFα ⁺ cylL10 ^- : This cell population is characterized by the binding of LPS to the cell membrane beyond the cell culture period (st = stimulatory), with simultaneous intracellular detection of TNF-α, but not IL10. Here the direct detection and reference to the ligand becomes clear.
• c) Dynamic staining with labeled antibody: stCD14 ⁺ cyTNFα ⁺ cylL10 ^- : This cell population is characterized by the appearance of CD14, a marker especially for monocytes. Since the labeled antibody was used dynamically (st = stimulatory) and at the same time biological effects can be triggered via the CD-14 molecule as LPS receptor, static and dynamic properties of the target cell population are recorded in a complex manner.

Databasing, data analysis and data comparison

The multiparametrically colored cells are measured on the flow cytometer or by means of scanning Cytometry, with the data being stored in binary form in list mode files (standard format FCS). Because the data from multidimensional dynamic cytofluorophoresis is very complex can and should also be analyzable and comparable on a complex level also requires software-related processing special requirements. The binary data are stored in so-called BLOB Fields (binary large objects) of a database read in and saved. Every record of the The database corresponds to a test sample and contains additional text and numerical fields Information about the sample (material, color, test conditions, device settings). The The data is viewed on the one hand in a zoomable multi-data set grid, the text data are displayed as text and the list mode data in usual histogram and dot plots. The However, graphic representations are not saved as actual images, but are instead only when data is accessed from the binary data as scalable vector graphics (Windows metafile format) generated. Multi-dimensional cursors / discriminator values to delimit certain zones or Subpopulations are also stored separately and graphically when accessing each data set reconstructed.

Since multidimensional data is fundamentally no longer represented in a diagram alone multiple histograms or dot plots must be displayed. At a The specific implementation of multidimensional dynamic cytofluorophoresis is its number and type then also specify exactly.

In the multi-data record representation as a table grid, which is common in database programs, the Data fields are shown as columns and the data records as cells. The software for the new process additionally allows a change of orientation of the table grid (swapping grid), d. H. the the samples corresponding data records are shown as columns and the fields as cells. Can continue especially in the columnar representation of the data records by the hierarchical structure  Field structure (representation as a tree diagram / treeview) specific diagram representations and other information can be shown or hidden and thereby direct or visual Facilitate data comparison.

When analyzing data in flow cytometry, specific cell populations are usually deliberately delimited, described by statistical parameters (percentage, mean and median of parameters, etc.) and
By storing the listmode data in the database, the complex distribution pattern of the cell parameters is preserved over the 1024 measurement channels in n-dimensions and can be compared on a more complex level between the data records that correspond to the individual samples.

In addition to the usual overlay histograms are overlay and subtraction dot plots representable, which is a direct comparison of the cytofluorophoreograms even at a more complex level enable.

In conventional density plots, frequencies of cells are color-coded in two-parametric dot plots shown. With the software in the new process, the color coding can also be used for display of activation and functional markers. For those for the multidimensional Dynamic cytofluorophoresis typical of dynamic staining is thereby created even more characteristic image of the complex data pattern.

The storage of all measurement data in a database offers further possibilities and advantages. In addition to the usual search options in databases for specific characters and strings, i.e. H. According to alphanumeric information (ASCII), the new software is also supposed to be based on cytometric Data patterns such as those used in multidimensional dynamic cytofluorophoresis are expected, search or compare at different levels. In analogy to Complex text queries from databases using their own query languages (SQL) can be used here based on biological-medical questions, an area of a cytofluorophoreogram or links thereof like a filter over data records and so certain Filtered, compared and comparing cell events and parameter constellations via the database be evaluated.

This enables the gradual expansion to an expert system.

EXAMPLES

The following exemplary embodiments relate to flow cytometric measurements with 3 Fluorescent colors in whole human blood.  

Fig. 1: Multidimensional static cytoflurophoresis

This example is based on the stains for the classic leukocyte subpopulations in the human whole blood (immune status) including more parameters than available colors demonstrate. In the usual determination, individual subpopulations are in several colors and measurements determined by flow cytometry in order to be clearly distinguishable from each other (e.g. CD3 / CD19: T / B cells, CD3 / CD4 / CD8: T4 / T8 cells, CD3 / CD16-56: T / NK cells). In the Multidimensional cytofluorophoresis are all used individually on different colors Markers used simultaneously in one approach. Since as many parameters as possible and thus more than Available colors are to be included, antibody mixtures are used. At The composition of the antibody mixtures can have known expression differences in individual Parameters are taken into account in order to differentiate the signal intensities as much as possible to achieve, but the selection can also be made purely empirically. The main goal is to get one if possible to achieve a wide and varied distribution of the cells in the n-dimensional space, so that a complex, but typical display image characterized by almost any number of parameters results. In principle, it is also not necessary for certain areas and / or Point cloud densifications in the cell populations defined in the cytofluorogram (T cells, B cells etc.) correspond. For each specific antibody mixture, standard cytofluorophoreograms e.g. B. on healthy normal subjects are created. In comparison with these you can then investigating samples e.g. B. by cross-diagram discriminators, as in the figures are shown, analyzed zone by zone or it can also down to the measurement channel resolution from currently 1024 point-by-point overlay and / or difference plots (point-by-point subtraction) and be analyzed. If the screening-like comparison shows that there are certain samples Areas or zones can be tried, secondary to certain cell populations in these identify or confirm by usual individual staining.

Figure 2: CD14 dynamic cytofluorophoreogram of the monocyte region

Each column shows the measurement of a sample in different 2-dimensional dot plot representations. in the the top diagram (sub-figures 2.1, 2.5., 2.9) are in the typical forward versus Sideways display of the leukocytes the 3 main populations (lymphocytes, monocytes, granulocytes) limited by regions. The region R2 at the bottom right corresponds to the monocytes and all other diagrams only show cells from this area (gate on R2). The sample of the left Gap serves as a control, with the CD14 antibody used in the usual surface staining has been. The sub-figures 2.2. until 2.4. show almost no cytokine detection of TNF-αlpha and IL-10 in the tents to which the CD14 antibody has bound. In the samples of the 2nd and 3rd column the same CD14 antibodies used in dynamic staining, d. H. already added to whole blood culture 8 h. Sub-figures 2.6., 2.7., 2.10. and 2.11. show the clear evidence of specific pro- and anti inflammatory cytokines TNF-alpha, IL-10, IL-6 and IL-12 in direct dependence on the binding of the  CD14 antibody to the cells of the monocyte region. The sub-figures 2.4, 2.8 and 2.12 each show the Representation of the cytokines measured in parallel against each other.

Combinations of different ones are further developments of this exemplary embodiment Cytokine staining possible on the same color.

On the one hand, the pro-inflammatory cytokine TNF-alpha shows a significantly higher expression than the anti inflammatory cytokine IL-10. If both cytokines are displayed in the same color, arrange themselves Cells with predominant TNF-alpha expression (pro-inflammatory) more in the upper area, cells with predominant IL-10 expression more in the middle range, and cells with little expression of both cytokines in the lower area. As a result, a rough division of Cell populations take place according to functional criteria.

On the other hand, functionally similar cytokines can be displayed with the same color (e.g. typical pro-inflammatory cytokines such as TNF-alpha and IL-6) and thereby the on one color Detectability, intensity and thus discriminatory power compared to other cell populations in the Cytofluorophoreogram increased.

FIGS. 3 and 4: Dynamic Cytofluoreogramm of pokeweed mitogen in the lymphocyte region

Pokeweed Mitogen (PWM) is a classic substance used to stimulate lymphocytes. Corresponding receptors on cell populations can be displayed using fluorescence-labeled PWM. FIG. 3 shows a dynamic cytofluorophoreogram of PWM on the whole leukocytes of human whole blood, FIG. 4 with gating the lymphocyte region (sub-figure 4.1.). In addition to dynamic staining with PWM with the first fluorescent color (st-PWM-FL1, partial figures 3.4., 3.5., 3.6. And 4.2., 4.5., 4.6., 4.7.), A membrane-related staining of the activation marker CD69 with the second fluorescent color (m-CD69-FL2, sub-figures 3.2., 3.6., 3.8. and 4.3., 4.6., 4.8.). By means of the same-colored antibody mixture against predominantly very strongly expressed CD8, moderately strongly expressed CD4 and by dilution in under-saturation weakly marked CD19 (marked with *), a division into 4-5 essential subpopulation areas is achieved on the third fluorescent color, which largely corresponds to the essential lymphocyte subpopulations T8- Cells, T4 cells, B cells and NK cells can be assigned. By digrammübergreifende Diskriminatorensetzung a division in up to 49, in Figure 4 is in Fig. 3. In up to 40 different 2-dimensional areas in which the cells are arranged is shown.

Fig. 5: Dynamic CD3 staining and effects on T cells and other leukocytes

This exemplary embodiment shows a dynamic cytofluorophoreogram from the T lymphocyte region with a CD3 antibody. The first and third columns of FIG. 5 show the controls with the usual membrane-related staining of CD3. In the samples of the 2nd and 4th column, the CD3 antibody was used dynamically and cell reactions were counterstained via surface markers (CD69) and intracellularly (interferon-gamma). A subpopulation separation was carried out on the third color as in the example of FIGS. 3 and 4.

Fig. 6: Combinations of dynamic and static colorations using the example of CD3-CD28 costimulation

As increasing studies show, the complex interaction of cellular and / or molecular signals in cell systems, especially the immune system, plays a decisive role and cannot be derived from the individual effects of individual components alone. The exemplary embodiment in FIG. 6 uses the example of a stimulation with antibodies against CD3 and CD28 to represent the step-by-step representation of the costimulation in multidimensional dynamic cytofluorophoresis. In all 4 approaches, a gate was placed on the lymphocyte region and is the basis for all diagrams. The diagrams in the first column (sub-figures 6.1., 6.2., 6.3.) Show the approach in which both the CD3 and CD28 antibodies were used in the usual way for membrane-related staining. In the sample of the 2nd column (sub-figures 6.4., 6.5., 6.6.) Only the CD28 antibody was dynamic, in the sample of the 3rd column (sub-figures 6.7., 6.8., 6.9.) Only the CD3 antibody Coloring used. The fourth column (figures 6.10., 6.11., 6.12.) Shows the sample in which both the CD28 and CD3 antibodies were used dynamically (costimulation). As from the sub-figures 6.1., 6.2., 6.5., 6.7., And 6.10., 6.11. corresponding cell subpopulations can be displayed at the same time via the binding of the antibodies, and the expression strength and activation (CD69 expression) can be assessed and compared as a function of the antibody binding.

Fig. 7: Dynamic representation of the labeled bacterium E. coli on leukocytes

Fluorescence-labeled bacteria are usually used to display and measure the phagocytosis of leukocytes. However, the phagocytosis process is only a partial process in the defense reaction of bacteria. In the multidimensional dynamic cytofluorophoresis, further reactions to the bacteria on cell populations that interact with the bacteria (adhesion and / or phagocytosis) can be detected (activation, Cytokine formation). However, indirect, cross-population reactions to populations that are not associated with the labeled bacteria are also recorded by the multiparametric measurement (indirect activation via messenger substances / signals at the cell system level). Fig. 7 shows the dynamic staining with fluorescent-labeled E. coli bacteria which a significant association to monocytes and granulocytes; comprise (phagocytosis partial figure 7.6.). When the activation marker CD69 is measured at the same time, however, there is also a clear reaction on lymphocytes and subpopulations (sub-figure 7.7; cross-population reaction, which can only be shown in context by including the entire cell system).

Other possible uses of multidimensional dynamic cytoflurophoresis

• - Specific cell function tests with verification of specific activation pathways in the heterogeneous Cell system (whole blood and other body fluids, cells from organs) and influence on it System through certain substances (toxicological, pharmacological)
• - Simultaneous subpopulation-related binding and activation studies of substances and / or Microorganisms and / or tumor cells (imaging and measurement of e.g. bacterial host Interactions, cross-talking reactions)
• - Dynamic-functional clustering of cell populations
• - Rapid, material, effort and cost-saving screening methods of drugs / Pollutants and yet incorporating extensive, even complex, effects patterns
• - Differential diagnostic and / or differential therapeutic, especially combined cytometric tests, also individual or patient-related

Claims (10)

1. Method in which fluorescence-labeled components (molecules, in particular antibodies, Substances and substance mixtures, microorganisms or parts thereof, cells or parts thereof) with be brought together in a cell system and in one approach, via likewise fluorescence-related marker reactions on this cell system or on subpopulations be detected. 2. Method in which by combining several fluorescence-labeled biologically active Components and / or combination of several fluorescence-related reaction-dependent markers and / or combination of fluorescence-related subpopulation markers, whereby due to different Signal intensities of certain fluorescent colors can also be used several times, one comprehensive representation and measurement of even complex reaction patterns of cell systems in direct and indirect relationship to influencing factors can be achieved (multidimensional Dynamic cytofluorophoresis). 3. Software with which the cytometric listmode data from procedures as described in 1 and 2, in A database can be stored, along with all of the cell preparation and measurement characterizing data, vector graphics being generated when data is accessed and by tilting multi-data record table grid, hierarchically showing or hiding fields, Individual data record representation also as a crosstab of the parameters and with cross-diagram Discriminator lines analysis and comparison of data is also possible on a more complex level. 4. Software as described under 3, in which the cytometric data are additionally determined Areas, parameter constellations and complex patterns are filtered, compared and analyzed can be. 5. Procedure as described under 2, in which one approach may involve diseases associated etiological components (pollutants, allergens, microorganisms, tumor cells) that Therapeutic components possibly influencing the disease (drugs, Microorganisms, cells) and evidence of possibly resulting therapeutic Effects on a cell system takes place. 6. The method as described under 2, in which fluorescence-labeled bacteria / fungi in one batch, together with or without fluorescence-labeled substances that act against the bacteria / fungi are directed and / or influence the defense response of the cell system and via fluorescence-related marker reactions on the bacteria and / or on the cell system of Body fluids or organs are measured.   7. The method as described under 2, in which fluorescence-labeled viruses or parts in one batch of these, together with or without fluorescence-labeled substances, which act against the Viruses are directed and / or affect the defense response of the cell system, are used and changes in the viruses and / or in the cell system of Body fluids or organs are measured. 8. The method as described under 2, in which fluorescence-labeled tumor cells or Parts of it, together with or without fluorescence-labeled substances, which are effective against the tumor cells are directed and / or influence the defense response of the cell system and changes in the viruses and / or the cell system via fluorescence-related markers of body fluids or organs. 9. The method as described under 2, in which on a fluorescent color by the same color fluorescence-labeled antibody mixtures against highly expressed, moderately expressed and weakly expressed antigens and / or by selective reduction of the fluorescent label (Dilution, mixtures with unlabelled antibody) and / or selective increase in Fluorescence labeling (higher antibody loading with fluorescent molecules, indirect Immunofluorescence), separation into several subpopulation areas is achieved. 10. Method described under 2, in which on a fluorescent color by mixing antibodies of the same color fluorescence marked against activation markers and / or cytokines functionally different subpopulation areas are shown.