Method for the quantitative simultaneous determination of cells, and reagent therefor.

The invention relates to a method for the simultaneous quantitative determination of the cells and a reagent suitable for its implementation.

Blood cell counting is one of the most frequently arranged laboratory tests in the clinic and practice area. It is estimated that ten thousand such determinations are carried out in the Federal Republic of Germany alone. The determination is of particular importance in the context of accident medicine, intensive care medicine and for the operating room.

For a complete blood count, six separate tests are usually performed on each blood sample:<tables id="tabl0001" num="0001"><img file="EP0106339A2_D0001.tif" /></tables>

Reticulocyte and platelet counting as well as the differential blood count are particularly time-consuming or methodologically difficult. The time required for a quantitative determination of the blood cells can be measured in hours. The same also applies to the determination of other cells that are present as individual cells, for example by mechanical or chemical disintegration of tissue. There is therefore a need for the development of fast and automatable processes which make it possible to significantly reduce the large amount of time and manpower previously required.

This object is achieved according to the invention by a method for the simultaneous quantitative determination of cells, which is characterized in that it incubates the sample containing the cells to be determined with a fluorescent dye which stains at least one property of the blood cells, and then the volume and the fluorescence of the cells measures at least one wavelength simultaneously.

In principle, the method of the invention is based on the fact that properties of the cells are stained by dyes and measured simultaneously with the cell volume, for example in a flow cytometer. By simultaneously determining the volume and fluorescence of the stained cells, which is carried out in the machine, each individual cell is detected by type and quantity and can be assigned to a specific blood cell type depending on the determined cell volume and fluorescence, since each of the cell types is determined by specific values ​​of Fluorescence and cell volume is characterized. In this way it will be e.g. B. possible to create a complete blood count within a few minutes with minimal human resources.

The properties of the cells stained by the dyes used according to the invention are inventory or functional properties. Stand properties are those that arise from cellular synthesis, such as_B. DNA, RNA, proteins and lipids, while functional properties are the result of metabolic processes such as B. transmembrane potential and intracellular pH.

The simultaneous staining of the cells with a DNA / RNA dye and a membrane potential-sensitive dye has proven to be particularly suitable for the method according to the invention and is therefore preferred. DNS / RNA dyes which are particularly suitable for this preferred embodiment of the invention are acridine orange (AO), quinacrine (quinacrine) and pyronine Y, the former being particularly preferred. From the group of membrane potential-sensitive dyes, 3,3'-dihexyl-oxa-carboxyanin (DiOC6 (3)) is preferred. The preferred substances can be excited in the same spectral range with the same light source and are therefore suitable for producing a premixed reagent which can be added to the cell samples.

Examples of other dyes from the group mentioned above which are suitable in the context of the invention are fluororeszamine (Fluram), l-anilinonaphtaline-8-sulfonic acid (ANS) and o-phthalaldehyde for staining the cell protein, 4-aminoacridine for staining the lipids, N. - (3-fluo-anthyl) maleimide for coloring free SH groups, Fluorescein diacetate (FDA) for staining enzyme activities (in the mentioned case of ester activity) and 1; 4-di-acetoxy-2,3-dicyano-benzene (ADB) for staining the intracellular pH.

The volume or the size distribution of the cells is determined either by light scattering (J. Histochem. Cytochem. 27, 359-365 (1979)) or by measuring the change in electrical resistance when passing through a narrow passage (Coulter method, cf. e.g. "Flow cytometry and sorting, by Melamed, Mullaney and Mendelsohn, John Wiley and sons, Inc. 1979, pages 61 to 101). The Coulter method is preferred. It is based on running the blood sample through a short opening of small diameter and measuring the change in electrical resistance at this point, the particle resistance being different from that of the electrolyte. The change in voltage that occurs when a cell passes through the opening through which a constant electric current is applied between two electrodes is directly proportional to the particle volume.

Incubation with the dye can be carried out conveniently at room temperature within a few minutes. In general, 1 to 10, preferably 2 to 6, minutes at room temperature are sufficient, calculated from the addition of a solution of the dyes to the blood sample to be examined, which has been suitably diluted with isotonic saline. After the incubation, the sample to be examined is placed in a suitable apparatus, for example a commercially available flow cytometer, which must be set up in such a way that the cell volume and the fluorescence can be determined at the same time and thus a corresponding fluorescence measurement can also be assigned to the volume measured for each particle .

The electrical method is preferred because the experimentally determined dye contents of the individual cells of each blood cell type can be expressed as dye concentrations, since absolute volumes are measured. As a result, the measured properties of the cell types can be directly compared with one another in a normalized manner. However, this is not possible with the light scattering method, since the light scattering depends not only on the cell volume and shape, but also on the nature of the cell surface and interior.

In the preferred embodiment of the invention using acridine orange (AO) in combination with DiOC6, use is made of the fact that all blood cells, except the erythrocytes, are well stained by AO, that the erythrocytes are also well stained by DiOC6, but at the same time the coloration of the remaining cells is improved.

According to a further preferred embodiment of the invention, the measurement is carried out in the presence of a fluorescent, in particular a monodisperse calibration phase colored with a fluorescent dye. If the concentration of the added fluorescent calibration phase is known, it is possible to determine the absolute concentrations of the various cells in the blood. Monodisperse latex particles with a diameter of 1 to 10 μm, particularly preferably between 4 and 6 μm, are preferably used as the calibration phase. However, other fluorescent particles of known uniform size in the blood cell range and concentration can also be used.

The implementation of the method according to the invention is extremely simple. Blood is mixed with physiological saline and the dye as well as with the particles of the calibration phase. After, for example, three to five minutes of staining, the stained blood cells are measured in a suitable device, for example using a commercially available flow cytometer at a rate of about 2000 cells per second for a period of about 5 to 15 minutes. In this way, all types of blood cells can be quantitatively recorded in sufficient quantities.

If tissue cells are to be determined, they are first zoned by disintegrating the corresponding tissue. B. released by cutting with a "tissue chopper"! The further procedure then corresponds to that described above for blood cells.

The method of the invention also makes it possible to measure not just one but several fluorescences. For example, in the preferred embodiment of the method using AO and DiOC6, both the yellow fluorescence of native DNA / RNS (spiraled form) and the red fluorescence of the relaxed form can be used! be determined. This makes it possible to additionally make a statement about the functional state of the blood cells. The method of the invention therefore enables in this embodiment not only a quantitative. tive blood cell counting, but also to make statements about the functional state of the specific individual cell types, which is not possible with known methods.

The dyes used according to the invention can be determined both with optical systems which are equipped with mercury or xenon lamps and with those which are provided with laser devices.

Another object of the invention is a reagent for performing the method according to the invention, which is characterized in that it is a fluorescent dye from the group DNA / RNA dyes, cell protein dyes, lipid dyes, enzyme dyes, membrane potential-sensitive dyes, intracellular Contains pH dyes, SH group dyes and additionally a monodisperse calibration phase.

A preferred reagent according to the invention contains a DNA / RNA dye and a membrane potential sensitive dye.

A reagent which contains acridine orange, 3,3'-dihexyl-oxy-carbocyanine, monodisperse latex particles with a diameter of 1 to 10 μm and a solvent is particularly preferred.

Suitable solvents for the reagent according to the invention are those which can bring the chosen dye and the monodisperse phase into solution in sufficient concentration without attacking, for example dissolving, the particles of the calibration phase. Preferred solvents are dimethyl sulfoxide, dimethylformamide and alkanols.

With the invention it is achieved that all cell types can be determined quantitatively in a few minutes, the rapid staining time and the full automatability of the process, namely staining, measurement and evaluation, having a decisive part in this. In addition to cell counting, it is also possible to obtain information about the functional state of the individual cell types.

In addition, the method not only enables the measuring times to be shortened and the significance to be expanded, but can also be used in a particularly large measuring range which is approximately 2.5 decades log, ie approximately 1: 500.

The importance of this advantage can be seen from the following: If one starts from the normal concentrations of the various blood cells, that of erythrocytes at 5 x 10⁶, for platelets at 3 x 105, for leukocytes at 5 x 10³ per mm³ lie, it can be seen that a wide measuring range is absolutely necessary if all these blood cells are to be determined simultaneously. So far, it has been difficult to keep platelet concentrations below 5 to 7 x 10⁵ per mm² to determine. According to the invention, this lower limit is 1 x 10³ per mm³ lowered. This is important because it is in the range of 100,000 and 30,000 per mm³ the particularly critical areas are. In the former case this is the pathological area, in the latter case the acutely dangerous area.

Another possible application of the method according to the invention is to investigate the effect of drugs on individual cells, for example the effect of cytostatic drugs on tumor cells. This enables preliminary testing, in particular for drugs with high toxicity, to determine whether their effectiveness in a particular individual justifies the acceptance of the toxic side effects or not. For example, individual cells can be obtained mechanically from tumor tissue, tested in a suitable nutrient medium, for example in haparinized patient blood plasma, in the presence of the drug to be examined and then quantified by the method according to the invention as to what proportion of the tumor cells by; the drug was killed and what percentage is still alive. The method of the invention can thus be used to find out the most effective of a number of possible medicaments.

The following examples further illustrate the invention in conjunction with the drawing. In this represent:<ul id="ul0001" list-style="none"><li>1 shows a schematic representation of a flow cytometer suitable for carrying out the invention,</li><li>2 shows the graphical representation of cell volume against fluorescence of a stained (a) and an unstained (b) blood sample diluted 1: 250, (Th = platelets, Er = erythrocytes, Re = reticulocytes, St =_Ei_chpa_rti_kel, Ly = lymphocytes, Gr =_Gra_nulo- Cytes, Ba =_Basi_sli_nie)</li><li>3 is a graphical representation as in FIG. 2 using other dyes,</li><li>4 is a graphical representation analogous to FIG. 2, using different dyes,</li><li>5 shows a graphical representation with simultaneous measurement of 3 parameters, 2 fluorescences being plotted in addition to the cell volume.</li><li>FIG. 6 shows a graphical plot of the two fluorescence measurements obtained in the context of the 3-parameter measurement of FIG. 5 against one another, so that the metabolic states of the cells can be derived therefrom.</li><li>7 shows a graphical representation of the results of a drug test in cells of integrated lymph node metastases of a breast cancer, the type of drug being plotted against the number of tumor cells or inflammatory cells.</li></ul>

Example 1

5 µl of blood are removed from the fingertip and diluted 1/250 with isotonic saline (0.15 M NaCl with 10 mM TRIS / HCL pH 7.4, (TBS)). 500 µl of the cell suspension are mixed with 5 µl reagent (AO 0.4 mg / ml, DiOC6_0.02 µg / ml and 4 µm monodisperse latex particles with a concentration of 5 x 10 stained with FITC⁷/ ml) incubated for 3 to 5 minutes at room temperature. Dimethyl sulfoxide (DMSO) is used as the solution and suspension medium for the reagent. The suspension is mixed well by shaking and introduced into a flow cytometer for measurement, the construction principle of which is shown in Fig. 1 of the accompanying drawing. The device is commercially available under the name Fluvo-Metricell. In the case of staining with AO / DiOC6, the following filters and mirrors are used:<ul id="ul0002" list-style="none"><li>1 Short pass (KP) 500 nm with long pass (LP) 418 nm filter;</li><li>2nd Dichroic splitter mirror (D) 500 nm;</li><li>3rd Reflection mirror for two-parameter measurement or D 530 nm for three-parameter measurement;</li><li>4th LP 500 nm;</li><li>5 Reflection mirror;</li><li>6 LP 550 nm.</li></ul>

AO / DiOC6 are excited between 418 and 500 nm. The emitted fluorescence light is collected in two-parameter measurements (volume against fluorescence 1) between 500 and 700 nm through the phototube 1 (PM1). In three-parameter measurements, the yellow light between 500 and 530 nm and the red light between 550 and 700 nm are measured by PM1 and PM2.

For the results shown in FIGS. 2 to 6 of the drawing, a cylindrical measuring opening with a diameter of 50 μm and a length of 50 μm was used. The fluid system of the flow cytometer was filled with TBS buffer at 25 ° C. The cell volume was measured at an electrical current of 0.385 mA.

The fluorescence was excited with an HBO-100 Hg high pressure lamp. The logaritmically amplified fluorescence and volume signals of the cells were either stored in a multi-channel analyzer as two-parameter histograms or written ON-LINE on a magnetic tape. The curves were evaluated graphically and mathematically by magnetic tape. When using a microprocessor-controlled data module, both dye addition and measurement, as well as the evaluation, can in principle be completely automated.

Fig. 2a shows a graphical representation of cell volume against fluorescence.

The calculation by integrating the cell clusters in Fig. 2a gives the following values<tables id="tabl0002" num="0002"><img file="EP0106339A2_D0002.tif" /></tables>

2b shows the result of an analog measurement without the addition of dye. No cell-bound fluorescence is detectable.

The volume and fluorescence pulses of the FLUVO-METRICELL flow cytometer were logarithmized with the help of 2.5 decades of logarithmic amplifiers and then counted according to their maximum amplitude in the 64 x 64 matrix of a multi-channel analyzer. For the graphical representation, the channel contents of the matrix were logarithmic (3 decades amplitude log) and normalized to the value denoted by (M). The maximum channel content (M) was divided into 20 equal parts (5% steps). Each channel content received a number between 1 and 10 according to its relative frequency. Channel contents with an amplitude greater than 50% of the maximum value were identified by (*).

Example 1 e 2 to 9

As described in Example 1, using the same apparatus, the simultaneous quantitative determination of the blood cells was carried out with 500 μl of human blood diluted 1: 250, which had been stained with 5 μl of reagent according to the table below:<tables id="tabl0003" num="0003"><img file="EP0106339A2_D0003.tif" /></tables>

The results are shown graphically in FIGS. 3 and 4, Example 10

As described in Example 1, a determination was carried out on a blood sample stained with AO / DiOC6, but recording two different fluorescences, namely the yellow (fluorescence 1; transmembrane potential in the AO / DiOC6 staining) and the red (fluorescence 2; Relaxed RNA / DNS). The acquisition of these three parameters enables the measurement data shown in FIG. 5 to be displayed in cloud form.

Each scale in Figures 5 and 6 spans 2.5 logarithmic decades. The three measuring pulses of each individual cell were logarithmized for the representation in FIG. 5 and transferred ON-LINE to magnetic tape. For evaluation, the values ​​were classified in a 32 x 32 x 32 matrix and displayed using a cloud program (. Cytometry f 1,222 -228 (1980)). The presence of cells and calibration particles is shown by a contour line at 1% of the maximum channel content. The individual cell types and the calibration particles are well distinguished from one another.

Figure 6 shows the yellow (Fluor. 1) versus red (Fluor. 2) fluorescence from 3-parameter measurements in which 1/500 diluted human blood was stained with DiOC6 (a) and with AO / DiOC6 simultaneously (b) was. As can be seen, the three-parameter measurement, RNA content and RNA concentration of the different cell types makes it possible to determine. The additional AO staining gives the part of curve 6b marked with Th + Re a clear red shift. If the cell volumes are drawn against fluorescence 1 histograms of the red-shifted (I) and the unchanged (II) particles (c, d), the comparison with FIG. 1a shows that the RNA-containing red cells contain the thrombocytes (Th), the reticulocytes ( Re) and the granulocytes (Gr) correspond, while the erythrocytes (Er), the calibration particles (St) and the lymphocytes (Ly) contain no RNA and therefore have retained their yellow fluorescence. As a side result, the double staining also shows that the reticulocytes have a higher membrane potential than the erythrocytes. This can be seen from FIGS. 6a and 6b. The cells lying directly above the erythrocyte cluster correspond to the zone labeled Th + Re in FIG. 6a. This zone becomes quantitative through the additional AO staining (6b). shifted to red, which means that the cells colored yellow by DiOC6 contain RNA. In the volume / fluorescence histogram, these cells correspond to thrombocytes and reticulocytes. This shows that the cells overlying the erythrocytes with DiOC6 stained are reticulocytes.

Example 11

Fresh sterile material of a lymph node metastasis from a breast cancer is mechanically cut into pieces using a tissue chopper, which are sieved through a sieve with a 60 μm mesh size. The cells obtained are cultivated for seven days in heparinized patient blood plasma as a culture medium in microtiter plates in the presence or absence of various cytostatics. The cell suspension is then washed and stained with 1,4-diacetoxy-2,3-dicyano-benzene (ADB) and propidium iodide (PI) for five minutes. ADB shows the activity of cytoplasmic esterase and the intracellular pH of the living cells, PI stains the DNA of dead cells. The cell volume and the blue and green fluorescence of the stained cells are measured simultaneously in a flow cytometer as described in Example 1. Before the measurement, fluorescent monodisperse latex particles of 6 μm are added to the cell suspension as a concentration and fluorescence standard. Cell volume and the fluorescence signals of each cell, which correspond to pH, esterase activity and DNA, are measured simultaneously.

The number of tumor cells or inflammatory cells living or surviving in a culture is then calculated from their ratio to the standardized number of calibration particles. The results of the various cytostatics examined and the untreated controls are summarized in the resistance diagram shown in FIG. 7. The abscissa of the diagram identifies the individual drugs and the controls; the ordinate shows the respective number of tumor cells or Inflammatory cells, based on 100% of the controls, applied. It can be seen that a reduction in tumor cells was achieved with cytostatics Nos. 1, 4, 5 and 7, while the other medications had no effect. The effect on inflammatory cells, which makes the less toxic medication easier to recognize, is recorded separately. The ratio of remaining inflammatory cells to tumor cells after incubation is also shown in the figure as a therapeutic index.