CZ297482B6 - Use of macarpine for classification of cells by flow cytometry - Google Patents

Abstract

The present invention relates to the use of macarpine as a supravital probe for measuring presence and/or content of nucleic acids in cell suspensions analyzed by flow cytometers and the like analyzers. Intracellular bond of macarpine to nucleic acid enables distinction of erythrocytes, reticulocytes and leukocytes of marrow and peripheral blood in both animals and humans without detection of expression of differentiating marks. It enables distinction of individual cycle stages of cells with intact cytoplasmatic membrane. The distinction can be also made on simple flow cytometers provided with excitation source in blue-green spectrum region. Macarpine can be also used as a supravital fluorescent probe in combination with common as well as less traditional fluorochromes at simultaneous multicolor immune phenotypization and quantification of DNA by the flow cytometer. The invented method represents a quick, non-destructive diagnostic and separation process enabling objective distinction and classification of individual types of both animal and vegetable cells.

Description

Technical field

The invention relates to the use of macarpine to classify cells by flow cytometry based on supravital identification and quantification of nucleic acids (DNA and / or RNA) by their visibility in non-permeabilized cells, with the possibility of parallel immunophenotyping. Binding of macarpine to nucleic acids in intact cells allows resolution of erythrocytes, reticulocytes and leukocytes of bone marrow and peripheral blood within seconds after its addition without detecting the expression of surface features when measured on standard flow cytometers equipped with an excitation source in the blue-green spectrum (488 standard wavelength) nm) and three fluorescence detectors with a common set of emission filters. The emission spectral characterization of macarpine bound to nucleic acids allows simultaneous detection of fluorescence of macarpine in addition to the fluorescence of some common fluorochromes, eg fluorescein isothiocyanate (FITC) on single source cytometers and allophycocyanin (APC) and its tandem with Cy7 (APC-Cy7) two sources (488, 633 or 635 nm by default), possibly Pacific Blue and Cascade Yellow with the possibility of excitation in the violet region of the spectrum (eg lasers with a wavelength of 405 nm). Furthermore, the binding and emission properties of macarpine allow semi-quantitative determination of DNA content in intact cells.

State of the art

Flow cytometry uses the principle of light scattering, excitation and emission of fluorochromic molecules to obtain multi-parameter data from microscopic particles and cells. In this method, the cells are hydrodynamically concentrated into a thin current in the capillary, which flows through at high speed, irradiated with monochromatic coherent radiation, preferably from a laser. The uniqueness of flow cytometry lies in the fact that, unlike spectrophotometry, which measures total absorption or transmission, it can measure the fluorescence of each particle or cell separately. Prior to measurement, a fluorescent dye, referred to as a fluorescent probe or fluorescent label, may be attached to the object to be studied. Flow cytometer optical systems and its functions are known and are described, for example, in US 6,248,590 or US 2004/145725. If we irradiate the fluorescent dye of the studied object, which passes through the cytometer, with light of a suitable wavelength, its excitation occurs, ie the transition of electrons to a higher energy level. The excited state, however, is unstable and the electrons immediately return to their original, basic state. This transition is accompanied by the release of thermal and light energy - fluorescence, the wavelength of which depends on the level of energy jump. Since some of the energy is lost in the form of heat, the light emitted has a longer wavelength than the original excitation radiation. By suitably selected combination of filters, both radiation can be separated and the fluorescence measured by a flow cytometer. The fluorescence intensity of the individual cells then corresponds to the number of structures that bind the fluorescent dye. Light scattering in a direction parallel to the direction of the beam coming from the light source makes it possible to assess the cell size, light scattering in the direction perpendicular to the beam is characteristic of the granularity (internal structure) of the cells.

The use of the method is very versatile, eg in the field of nuclear DNA determination, ploidy determination, cell cycle analysis, gene expression study, blood cell counting and determination, detection and characterization of microorganisms, sorting of required particles, etc.

The main advantages of flow cytometry are: simple sample preparation, high speed analysis of large sets of single cells or particles, non-destructivity, easy detection of particle subpopulations, as well as relatively low cost of single sample analysis.

In addition to the selection and preparation of the observed object and optical system, the quality of the result is fundamentally influenced by the choice of fluorescent dye. It is desirable that the selected substance binds specifically (does not stain other objects) and quantitatively (i.e. the amount of bound dye is proportional to the number of objects). Many types of fluorescent dyes are currently known. As fluorescent dyes, molecules containing conjugated double bonds and free electron pairs are used which can be excited easily and in a coordinated manner and which also quickly return to a lower energy level. They are aromatic compounds and aromatic heterocycles, usually with multiple condensed rings, whose π-electrons are delocalized in orbitals above and below the plane of the plane of the molecule.

Fluoresceins, rhodamines, coumarins, pyrenes, etc. are used as fluorescent dyes. The use of ethidium bromide in DNA research is described, for example, in U.S. Pat. No. 6,143,151, for use in the detection of intracellular bacteria in blood samples of U.S. Pat. No. 4,508,821. Hoechst 33258 dye was used in DNA research. Combinations of known or new fluorescent dyes are also preferably used in cytometry. A tri-color reagent containing 7-aminoactinomycin D (7-AAD), fluorescein isothiocyanate (FITC), and phycoerythrin (PE) used in 15-color cytometry to measure the number of CD4 positive lymphocytes is disclosed in US 2004/110122. The use of a pair of Cy7 dyes and allophycocyanin is described in US 5,714,386.

Supravital probes are fluorochromes that spontaneously pass through the intact cytoplasmic membrane of cells and, upon binding to specific structures, such as nucleic acids, change their fluorescence yield and spectral characteristics in the intracellular space. Supravital probes can be used to qualitatively and / or quantitatively detect, e.g., the nucleic acid content of cells without the need to treat the cells with fixation and permeabilizing agents. Examples of supravital DNA probes are the previously mentioned Hoechst 33258 or LDS-751. To date, a supravital probe that can be used on flow cytometers with an excitation wavelength of approximately 488 nm (in the blue-green region of the spectrum) and 25 that allows (semi) quantitative determination of the nucleic acid content of individual cells has not been described.

Makarpin (I) is a benzo [c] phenanthridine alkaloid (5,7-dimethoxy-13-methyl [1,3] benzodioxolo [5,6-c] -1,3-dioxolo [4,5-i] phenanthridine), which was first isolated by Nightingale from the root of the plant species Macleaya microcarpa in 1955 (Nightingale, J., Slavikova, L., Collect. Czech. Chem. 30 Commun. 20, 356 (1955). Later, it was found in other Papaveraceae (Nightingale,

J., Slavikova, L., Appelt, J. Collect. Czech. Chem. Commun. 30, 887 (1965), Slavik, J. Collect. Czech. Chem. Commun. 26, 2933 (1961), Slavik, J., Hanus, V., Slavikova, L. Collect. Czech Chem. Commun. 56, 1116 (1991). A complete synthesis of this alkaloid has also been described (Ishikawa T., Saito T., Ishii H. Tetrahedron 51, 8447 (1995). In German patent DE 3835632, the use of macarpine for labeling nucleic acids in fluorescence spectroscopy and microscopy has been described.

and ₃ (I)

Benzophenanthridine alkaloids are a large group of substances which find application in many pharmaceutical fields. Their antibacterial and antifungal effects are described, for example, in US 5,395,615. A method for separating individual benzophenanthridine alkaloids from an extract is described in US 5,133,981.

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Object of the invention

It has been found that macarpine allows for the differentiation of erythrocytes from leukocytes and other nuclear cells, while allowing multicolour surface immunophenotyping on flow cytometers in single-cell flow cytometers, in mixtures of nuclear and nuclear cells, such as peripheral blood, bone marrow biopsy, and cell suspensions prepared from highly perfused organs. or more excitation sources. In addition, the spectral characteristics of macarpine bound to RNA and / or DNA make it possible to distinguish erythrocytes, reticulocytes and leukocytes of bone marrow and peripheral blood without detecting the expression of surface features even on simple flow cytometers equipped with a single excitation source of about 488 nm.

Macarpine in an aqueous solution, when added to the cell suspension, binds at very low concentrations to DNA and RNA contained in a wide range of living cells. The fluorescence of macarpine is approximately proportional to the amount of DNA or RNA, and therefore allows their approximate quantitative determination in living intact cells. It does not require cell permeabilization, its penetration into cells is very fast, in a matter of seconds and does not damage cells morphologically during the measurement. When added to the cell suspension, macarpine binds to nucleic acids, which can be used to make them visible in microscopic or flow cytometric analysis. It can be used alone as a dye for DNA and / or RNA or in combination with other dye-fluorescent labels having a suitable absorption and / or emission spectrum. It can advantageously be combined with, for example, Cascade Yellow, Pacific Blue, fluorescein isothiocyanate (FITC) or allophycocyanine (APC) and its tandem with Cy7 (APC-Cy7).

Makarpin emits bright fluorescence in the yellow to red region of the visible spectrum, allowing the detection of individual cells containing DNA and RNA nucleic acids. Preferably, it can be used to distinguish intact cells containing DNA or RNA from intact cells that do not contain significant amounts of nucleic acids, such as platelets and mammalian erythrocytes. The advantages of macarpine in flow cytometry are particularly evident in the labeling of DNA and RNA in mixed populations of erythrocytes and leukocytes. Unlike most DNA probes used to date, cell permeabilization is not necessary because the plasma membrane of a living cell is fully permeable to macarpine.

By means of macarpine, three phases of the cell cycle can be distinguished by flow cytometry based on the DNA content of the cells. In the first stage (G0 / G1) the cell contains the basic amount of DNA (2 sets of chromosomes in diploid cells, one in haploid cells), in the second stage (S) the genetic information is duplicated (DNA synthesis) without changing the number of cells. at the end of this phase, the cells contain twice the amount of DNA (4 sets of chromosomes in diploid cells, 2 sets in haploid cells). In the third phase (G2 / M), the amount of DNA is initially kept at twice the amount and then the genetic material is split - mitosis, with the amount of DNA in the cell decreasing to baseline (2 sets of chromosomes in diploid cells, one in haploid cells). Makarpin, thanks to its spectral properties, enables these three phases of the cell cycle to be distinguished and determined qualitatively (microscopically) and quantitatively (flow cytometry) in a simple way. The situation can be graphically represented by the number of points corresponding to the individual cells (y-axis) that have passed through the cytometer measuring zone depending on the fluorescence intensity in each cell (x-axis).

Makarpin can be advantageously used in automated testing and evaluation of a large number of samples in the pharmaceutical industry for the effects of biologically active substances on the cell cycle course.

Combined with the visibility of common leukocyte and erythrocyte antigens, macarpine fluorescence allows the classification and quantification of leukocytes, immature erythrocytes and mature erythrocytes in both anemia models and in anemic patients. For example, the method according to the invention can be distinguished

CD45-positive leukocytes with clear fluorescence of macarpine from subgroups of CD45 negative

B6 blood and hematopoietic cells. In the CD45 negative cell group, macarpine fluorescence distinguishes between macarpine positive cells containing RNA (reticulocytes) and macarpine negative cells (mature erythrocytes). Due to the difference in the emission spectra of RNA-bound macarpine and DNA-bound macarpine, it allows the distinction between DNA and RNA-containing cells and RNA-only cells when measured on standard single-laser flow cytometers. The presence of the nucleus (DNA) results in an overall higher fluorescence intensity and its shift to the shorter wavelength range, which is reflected in the shift in the FL2 / FL3 diagram when measured on the instrument. DNA-free cells can be distinguished because the absence of DNA results in an overall lower emission and relatively higher fluorescence (FL) of macarpine in channel 3 (FL3) and thus a higher ratio of FL3 to FL2 intensity.

For flow cytometry using instruments with two or more excitation sources, it is possible to utilize the combined fluorescence of macarpine in addition to the fluorescence of other fluorophores with suitable spectral properties, such as the fluorescent labels Pacific Blue, Cascade Yellow or APC and its APC-Cy7 tandem.

In the simultaneous immunofluorescence method (visualization with fluorochrome-bound antibodies such as fluorescein isothiocyanate and allophycocyanin), excess antibody is removed by washing and then macarpine at a concentration of 1 µg / 10 ⁶ cells is added at least 10 seconds prior to measurement.

The method is suitable for its speed and simplicity of the working protocol as a very fast, non-destructive diagnostic and separation method capable of objectively distinguishing and sorting individual types of animal and plant cells.

Examples

Example 1

Determination of DNA content in intact cells of a cell line

2x10 ⁴ cells in 100 µl of labeling medium (phosphate buffer with 0.2 wt% gelatin and 0.1 wt% sodium azide, hereinafter WSB) were used to determine the DNA content of intact cells of the MOLT-1 line in exponential growth phase, to which 10 µl of macarpine (100 µg / ml) was added for 30 sec. Then, the sample was measured on a standard FACSCalibur flow cytometer (Becton Dickinson, La Jolla, USA) equipped with a blue argon laser (488 nm 15 mW) and a red semiconductor laser (633 nm 20 mW) at low (LOW) acquisition rate.

Giant. 1- Two-dimensional diagram of 488 nm excitation beam scattering in straight direction (FSC) and in perpendicular direction (SSC) for all cells,

Giant. 2 - FSC versus fluorescence peak area in FL2 channel (FL2-A) on a linear scale. Populations in the individual phases of the cell cycle are indicated by phase abbreviations.

Giant. 3 - FSC versus FL3-A on a linear scale. A comparison of the differentiation in FL2 and FL3 channels indicates that the FL2 channel is preferable for more accurate DNA content determination because the FL3 channel is more sensitive to RNA content. The position of the populations in the individual phases of the cell cycle is shown in FIG. 2.

Giant. 4 - Diagram of FL2-A versus peak width (FL2-W) allowing to distinguish individual cells in different parts of the cell cycle (R1 region) from apoptotic cells (R2 region) and multicellular events (multiplets, R3 region). Objects in the R1 region have the character of intact cells (FIG.

5), while the objects in R2 are smaller, corresponding to cells in apoptosis (Fig. 6).

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Example 2

Determination of cell proportions by nucleic acid content, cell cycle phases and surface antigens presence in anemic piglet blood model - identification of nuclear cells and reticulocytes. (In symbolic designations, the abbreviation MA is used for brevity, unless otherwise possible).

100 μΙ of heparinized (20 U / ml) blood from piglets with severe anemia (10-fold red blood cell reduction) was labeled with fluorescein-labeled mouse anti-porcine CD45 monoclonal antibody followed by 10 μΐ of macarpine (100 pg / ml) for 30 sec. The sample was measured on a FACSCalibur analyzer.

Giant. 7 - Size (FSC) versus logarithm (log) fluorescence of macarpine in FL2 channel (FL2-H). In addition to negative cells (population 1), cells with weak fluorescence (population II) and cells clearly labeled with macarpine (population III) can be distinguished.

Giant. 8 - Diagram of CD45 (FL1-H) versus macarpine (FL-2H) expression shows the possibility to distinguish leukocytes (R1 region; CD45 ⁺ MA ⁺ ) from non-white hematopoiesis cells. Regions R4 and R5 contain erythroid precursors with high (R4) and lower (R5) RNA content (CD45 MA ⁺ ), erythrocytes without nucleic acids are found in the R6 region (CD45 MA '). The population in the R1 region can be further divided into mononuclear leukocytes with higher CD45 expression and higher fluorescence of macarpine (R3) and granulocytes (R2).

Giant. 9 - A comparison of the fluorescence intensity of macarpine in FL2 and FL3 channels shows that the presence of DNA increases the relative intensity in the FL2 channel, as shown by shifting the leukocyte population to the right. Therefore, it is preferable to measure the DNA content of the FL2 channel on a FACSCalibur and devices with similar equipment. For other cytometric analyzers, the optimum channel may vary depending on the optical and optoelectric elements used. The diagrams at the bottom of the figure (FSC versus SSC) show the scattering characteristics of individual populations discernible by anti-CD45 and macarpine binding. The regions R2 and R3 in FIG. 8 correspond to mononuclear leukocytes (monocytes and lymphocytes, R3) with higher CD45 expression and more intense MA fluorescence than granulocytes (R2). The R4, R5 and R6 regions contain reticulocytes with different RNA content, while the macarpine negative cells in the R6 region are zero nucleic acid erythrocytes.

Example 3

Determination of cell proportions by number of nucleic acids, cell cycle phases and presence of surface antigens in human bone marrow model - identification of nuclear cells and reticulocytes.

100 μΐ of a human bone marrow sample was labeled with a combination of monoclonal antibodies; anti-human CD45 conjugated with allophycocyanin (anti-CD45-APC) and anti-human glycophorin-A conjugated with fluorescein (anti-GlphA-FITC). Then 10 μΐ of macarpine (100 pg / ml) was added for 30 sec and the sample was analyzed for FACSCalibur with two lasers (488nm and 633 nm).

Giant. 10 - FSC / SSC diagram showing the representation of all cells analyzed with a predominant population with light scattering characteristic of the erythroid series (only 2000 objects are shown for clarity).

Giant. 11 - Expression of GlphA (FL1-H) and CD45 (FL4-H) distinguishes CD45 GlphA ⁺ erythroid cells from white hematopoiesis cells (CD45 ⁺ GlphA). CD45 ⁺ population with poor channel fluorescence

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FL1 are cells with light scattering corresponding to eosinophilic granulocytes. They are marked with an asterisk and without macarpine labeling, this population is not distinguishable from other GlphA cells.

From this, we conclude that co-labeling with anti-CD45 and MA allows the relative numbers of eosinophils to be read.

Giant. 12 - Diagram of CD45 expression and fluorescence of macarpine in FL2 channel (peak area shown). Clearly, both CD45 ⁺ leukocytes and CD45 ^low precursors and erythroid lineage CD45 cells can find a majority population with lower macarpine fluorescence and a smaller proportion of cells emitting more light into the FL2 channel. Together with cell line findings (see Example 1), we interpret this difference as a difference in the content of nucleic acids, especially DNA. Obviously, the baseline level of fluorescence in erythrocyte-free cells is much lower than that of leukocytes containing normal amounts of DNA.

Giant. 13 - Histogram of FL2 channel fluorescence intensity peak area on a linear scale. R regions are defined by populations with different emission intensities: R1 - very low fluorescence indicating no or very low nucleic acid content, R2 - medium fluorescence intensity roughly corresponding to diploid DNA content in the nucleus (N), R3 - high fluorescence intensity corresponding to DNA content in cells synthesizing DNA and cells in mitosis.

Giant. 14-16 - CD45 versus Glph A expression on cells from regions R1 (Fig. 14), R2 (Fig. 15) and R3 (Fig. 16) of the histogram (Fig. 13). Obviously, the R1 region comprises mostly erythroid cells (Fig. 14), while the majority of leukocytes are located in the R2 region (Fig. 15). Mitotically active cells (both leukocytes and erythroid precursors) are located in the R3 region (Fig. 16). The scattering characteristics in FIG. 17 to FIG. Figures 19 confirm the affinity of cells from the R1 region to the erythroid row (cf. Figure 17 with Figure 10), the cells from the R2 region to the white hematopoiesis row (Figure 18 shows a typical scattering profile of blood or bone marrow leukocytes). Giant. 19 shows where the cells in the active phase of the cell cycle (S-phase, M-phase) are located in the two-dimensional diagram.

Industrial use

Use of macarpine as a supravital probe to quickly measure the presence and content of nucleic acids and other multi-parameter data on simple flow cytometers in bulk samples containing suspended cells.

In particular, macarpine can be advantageously used in the automated testing and evaluation of a large number of samples in the pharmaceutical industry for the effects of biologically active substances on the course of the cell cycle.

The use of macarpine is advantageous for determining the relative proportion of cells according to the amount of nucleic acids, for determining the individual phases of the cell cycle, and for identifying nuclear cells and reticulocytes in bone marrow or blood without the need for surface antigen detection.

Macarpine is also useful as a supravital fluorescent probe also in combination with other fluorochromes in simultaneous multicolour immunophenotyping of cells.

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PATENT CLAIMS

Claims (4)

PATENT CLAIMS Use of macarpine to classify cells by flow cytometry by supravital identification and quantification of nucleic acids on cytometers with an excitation source radiating in the blue-green region of the spectrum from 480 to 490 nm. Use of macarpine according to claim 1, wherein the cells are further labeled with at least one further fluorochrome. The use of macarpine according to claim 2, wherein the fluorochrome is a compound selected from the group consisting of fluorescein isothiocyanate, allophycocyanin, Cascade Yellow, Pacific Blue. 4 drawings