Method of cell population discrimination and application thereof

FIELD: medicine.

SUBSTANCE: there is offered a method of discrimination and calculation of at least two populations of biological elements - carriers of specific signs, probably presented in a sample. The method provides the use of three different probes, each of which is specifically fixed with one of the populations of biological elements which are required to be detected. Each probe itself becomes detectable due to its proper marker, and two different markers specified have two emission spectra containing at least one common part (overlapping emission spectra), and the third one has the emission spectrum which essentially contain no common parts with two others (nonoverlapping spectrum).

EFFECT: invention allows definitely detecting three populations of biological elements by using only two detection devices that assumes that two populations of biological elements will be detected by the same detection device.

9 cl, 2 ex, 1 tbl, 4 dwg

The present invention relates to a method of detection, discrimination and counting of biological elements present in the liquid, using the principles of flow cytometry, adapted to the devices commonly used in Hematology.

Commercially available automated analyzers Hematology (Hematology machines) give more and more opportunities for analysis and classification of the analyzed elements.

Measurement of fluorescence, already widespread in flow cytometry, is mainly used to classify elements by immunophenotyping. In commonly used Hematology machines it serves mainly to identify electron microscope dyes used as molecular probes for the quantitative determination of nucleic acids or other cellular components.

The use of immunological probes in standard Hematology has not become generally accepted, although some trials with embryonic cells have already been carried out by different manufacturers.

For example, BAYER (Bayer Diagnostics, Tarrytown, New York, USA), through the BAYER-TECHNICON H*1 was first proposed in the classification of lymphocytes to use a mixture of antibodies to identify different types of lymphocytes. In this case, the measurement of the expression of antigens is carried out n the fluorescence, and by measuring the absorption of light generated by the connection avicenniaceae having a greater affinity to Biotin, which is itself conjugated with the antibody. The specified antibody is specific for antigens of the target surface molecules specific for the characterized cell types (CD4, CD8, CD2, CD19).

System ABBOTT CD4000 manufactured by ABBOTT (Abbott Laboratories, Abbott Park, Illinois, USA) offers an analysis using two wavelengths of fluorescence to implement, among other things, immunophenotypically. In patent WO 98/02727 owned by Abbott Laboratories, described apparatus of this type, allowing the tagging of antibodies in the sample is whole blood. ABBOTT describes in detail in the patent devices intended for carrying out reactions of the antibody - antigen in the sample of whole blood.

Simple, fast, effective and specific way phenotyping should be applied in a very simple automatic type hematological machines, allowing to work with a limited number of detectors. Even if these methods are highly sensitive, conventional machines used in Hematology, have different characteristics from flowing cytometers, particularly in regard to the measurement parameters, the number of which is reduced to the most necessary, especially in routine measurements. In addition, as it is a very important limiting factor is the cost, simplification of devices and tests that they can perform, can be performed only in the direction of the General economy.

Flow cytometry is a method that allows the simultaneous measurement of several parameters, corresponding to different physical characteristics of the biological element, such as, for example, the cell or cell organelle. Biological elements are addicted to liquid flow, and the device registers the properties of each of them, when they are in the measuring cell facing the light source. This method is actually a combination of three systems:

1) Liquid system: laminar flow, which allows the biological elements in suspension to pass each other before the light in the measuring cell.

2) Optical system: the laser beam or other light source and various filters that allow you to choose the appropriate wavelengths as at excitation and emission.

3) the Electronic system: PMT (photomultiplier tube or photodiode, which intercepts the emitted light, allowing you to convert it to electric, and then into a digital signal.

Thus, the light source can generate light that will pass through the lens and illuminate the biological elements moving in the measuring cell. Upon contact with the biological element frequent the light is scattered. This light passes through multiple lenses and other diaphragms and focuses on sensor type photodiode to generate the measurement FSC (Forward Scatter). This measurement in the selected range of angles gives information about the size of a biological element.

Another part of the light is deflected at a right angle and passes through another set of lenses and a set of partially reflecting mirrors to be measured at the level of the sensor and to generate a signal SSC (Side Scatter). This measurement side light gives information about the density of the biological element, and its granularity (structure).

Finally, the light intensity measurements can be performed through both photomultiplier tubes or photodiodes, and the wavelengths and analyze optical signals.

The passage of biological cells through cytometer requires a preliminary step in which the biological element is doing detectable, i.e. labeled probe specific to the structure or function of the specified biological element, and the specified probe was made findable in advance.

When the marker probe is a fluorescent molecule, it can absorb a photon in a characteristic range of wavelengths (excitation spectrum). After excitation of the fluorescent molecule will return to its ground state and give f the tone with less energy. Thus, the fluorescent molecule has its own range of excitation wavelengths, in which it will absorb energy to re-emit it in the form of fluorescence (emitted fluorescence), in accordance with a characteristic emission spectrum. The wavelength of the emitted fluorescence is always greater (i.e. frequency less)than the wavelength of excitation.

Signals from scattering (FSC, SSC) and fluorescence will be converted into electrical signals by suitable detectors, and then analyzed by the computing system.

In flow cytometry is also generally accepted everyone used to probe or a mixture of probes to match the band of wavelengths of fluorescence. Thus, each probe is connected to fluorochromes (fluorescent marker), the signals from which are measured on only one channel fluorescence, and the specified fluorochrome grafted on one or more types of probes.

Costs could be reduced by standardizing parameter immunophenotyping with a limited number of channels for measuring in addition to classic physical parameters selected from the scattering axis (Forward Scatter or FSC), side scatter (Side Scatter or SSC), volume of impedancometry etc.

The ability to summarize responses fluorescence would Express the synchronize several different labels on the same measuring channel and, thus, to increase the analytical capabilities of the system and thus its relation price/quality, without increasing the complexity of the device due to the addition of measurement channels.

The difficulty when multiple probes are conjugated to the same fluorochrome, is that the expression of biological traits that can be recognized respectively by different probes, will directly affect the total number of the emitted fluorescence.

For example, if one biological characteristic strongly expressed (for example, the antigen on the cell surface), a fixed number of labeled probes is proportional to the number specified biological characteristic. As a consequence, the amount of fluorescence emitted by the specified probe, being proportional to the number of biological characteristics, fixed specified labeled probe, will be high.

At the same time, if another biological characteristic of the same sample is presented poorly, the amount of fluorescence emitted by the corresponding probe, conjugated with the same fluorochrome will be too weak and may go unnoticed, as will be hidden by the fluorescence of the first probe.

Of course, these same difficulties are encountered when using other types of tagging.

One of the purposes of the present invention is to allow a clear and not vymyslene to detect at least three probe using only two means of detection. This suggests that at least two probes will be detected by one and the same means of detection. This can be done if you use three different probes, each of which recognizes and is fixed with one of the biological elements to be detected, or one of the biological characteristics of the element or elements, each probe itself is detectable using a different marker, and two of these marker are two of the emission spectrum of containing at least one common part (overlapping emission spectra), and the third has a spectrum of emission, essentially no common parts with the other two (non-overlapping range). These two (or n) token having overlapping emission spectrum, measured by the first detection means, and the token with non-overlapping emission spectrum, measured by the second means of detection.

Thus, the present invention is that:

(1) to equalize the difference in antigenic expression or cellular characteristics through a quart release issue n probes associated with n markers measured in the same band detection;

(2) to be the ratio between the signals emitted by the second detection means on the one hand, and the signals emitted by the first detection means, on the other hand, to use his ka is the main axis of the matrix;

(3) to combine in two dimensions the relation, obtained in paragraph (2), on the one hand, and the signals emitted by the first detection means, on the other hand, to improve the graphical representation and thereby improve the characterization and quantification of biological interest elements.

Under "biological element, according to the invention, it is understood, for example, eukaryotic or prokaryotic cell, group of cells, a fragment of a cell.

Under "biological characteristic" means a special component of the investigated biological element, such as a biological component of a cell, such as cell organelle, protein, lipid, carbohydrate or nucleic acid (RNA or DNA).

By "probe"according to the invention, is defined as any tool that allows you to specifically identify biological or chemical characteristic that is present in or on the studied biological elements. These products suitable for the invention, well-known, in particular, in the field of cell biology, molecular biology, immunology and flow cytometry. Can be called, without limitation, antibodies, nucleic acids (DNA or RNA), chemical compounds, in particular intercalating chemicals, compounds, active ion medium (H⁺Ca^+4"etc), lectins, whether the Andes or other dyes to assess the viability of the cells.

Under "marker probe" refers to any compound which, by their nature, be directly detected, either visually or by using a suitable device, or becomes detectable after stimulation. Such a connection, once you join the probe, makes it findable. Can be called fluorescent molecules, whether, for example, chemical compounds or biological molecules, like proteins. All these products are commercially available and are offered by different suppliers of laboratory chemicals, such as, for example, from Sigma, Aldrich, Fluka, Riedel de Haen, etc.

It should be noted that in one and the same experience of discrimination of populations of biological elements, which uses the method according to the invention, it will be possible to use probes of different nature (for example, conjugated antibodies and labeled nucleic acids or dyes to assess the viability of the cells,if their detection can be performed according to the criteria defined according to the invention (at least two overlapping of the emission spectrum of and one non-overlapping range).

Under the "detection means"according to the invention, is defined as any tool to detect and even determine the number of the marker probe. Of course, this tool detection is characteristic for the method used to make the substance of the detectable probe. In all cases, the specified tool detection consists of a set of physical components, capable of detecting a marker of a specific probe. For example, if we are talking about the marker that emits fluorescence, the detection means is a system consisting of an optical lens control samples, spatial filters (aperture, "pin hole" and so on), the stereoscopic filter (dichroic, interference), specially designed to skip to the sensor only part of the spectrum, called the "lane detection", a specific marker or markers that will be measured, and the optoelectronic sensor (photodiode, photomultiplier tube and the like). Then this sensor will be connected electronically with e-channel data collection, which will carry out the signal processing in the analog and/or digital mode and, finally, the computational processing of data by computer.

Can also be used, for example, a fluorescence detector, if the probe Machen fluorescent marker or if the probe itself fluoresce, or spectrophotometer, if the probe is a marker that absorbs a certain part of the color spectrum of the exciting light.

Under the "emission spectrum", according to the invention, refers to the distribution of fluorescence intensity depending on the wavelength in characteristic p is the elk marker or the probe. Usually this spectrum has a peak intensity corresponding to the maximum emission. Detection can be done in the band, consisting of between two wavelengths, usually covering the intermediate wavelength corresponding to the maximum peak of the emitted fluorescence (maximum peak detection).

Under the "lane detection" means the range of wavelengths selected in the spectrum of emission of the considered marker or markers (for example, if we are talking about the fluorescent marker, lane detection will be a band, preferably selected around the maximum peak of the emission fluorescence). This band detection is determined by spectral filtering, built-in vehicle detection, via at least one interference filter, or a bandpass filter, if necessary, collected for one or more dichroic mirrors.

By "overlapping emission spectra", it is understood that in the same vehicle detection at two different markers of their emission spectra have a common area. For example, if you use two fluorescent marker, their emission spectra will be referred to as overlapping emission spectra, when they have a common band emission, which is contained between two wavelengths that are common to their emission spectra. Typically, these spectra have different the peaks of maximum emission.

By "essentially non-overlapping spectrum emission" means that in the case of three different markers, two of which have overlapping emission spectra, the emission spectrum that does not contain significant areas of overlap with any of the spectra of the other two markers.

Thus, from the above it is clear that if there are three probes that recognize three different biological characteristic, labeled three markers with three different emission spectrum, and the two are overlapping, and one non-overlapping, the method uses only two means of detection in the dimension in which the markers with overlapping spectra have a common band detection.

This distinguishes the method which is the object of the invention, methods known in the prior art, in which for the detection of three biological characteristics are used in the three probe having three independent emission spectrum, which are analyzed by three different means of detection.

As for the overlapping markers, a reasonable choice of the markers with respect to their discovery forces us to choose two markers, which can give a response, one more effective than another in the same lane detection.

According to one variant of the invention the quantum yield for emission in the band is lekcii can be selected to be inversely proportional to the expression of the considered biological characteristics. The lane detection will also be determined from this expression, so as not to hide a weak positive expression (this is the first phase adjustment in the invention).

For example, for weakly expressed antigen should be selected marker, which has increased the quantum yield for emission in the lane detection, and for highly expressed antigen choose the marker with very high quantum yield of emission in the same lane detection. This allows you to control the number of measured markers to equalize emission in a ratio inverse to the level of expression of the examined antigens or probes.

The method according to the invention allows simple, fast and effectively to discriminate and count biological elements. In addition, it allows you to use a simple device that has the consequence of reducing the cost of manufacturing and maintenance. In addition, facilitates automated analysis.

Thus, the object of the invention is a method of discrimination of at least two populations of biological elements - media specific characteristics, may present in a sample, including:

- simultaneous tagging of these biological populations the ski elements in three different probes, you can find, or which become detectable through three different markers, and two of these marker (overlapping markers, or MC) have their range of emission, the emission spectra overlap, and the third (non-overlapping marker, or MnC) has an emission spectrum that do not overlap with the spectra of the other two markers (non-overlapping range of emission);

- measurement by any suitable means the total number of non-overlapping marker (qMnC) in-band detection, selected in the spectrum of emission of the specified non-overlapping marker;

- measurement by any suitable means the total number of overlapping markers (qMC) in-band detection, common to the spectra of emission of these overlapping markers;

establishing for each of the analyzed biological element ratios (R) of the full number of non-overlapping tokens to the total number of overlapping markers (R= (qMnC)/(qMC));

- build any tool diagrams showing the ratio (R) depending on the number of biological elements, labeled overlapping markers (R=f(qMC)), and/or

- quantitative determination by any suitable means, usually by computer, biological elements that are identified on the chart or charts and comply with the adequate statistical data.

The method according to the invention, is a method that allows us to distinguish and count biological elements responsible positively or not on a given criterion. The method is not intended to determine the number of probes fixed biological element. The method allows to unambiguously identify the sample, in particular, in a biological sample, populations of biological elements that have the desired trait or traits.

In accordance with the final stage of the method according to the invention the analysis of the two-parameter matrix with the ordinate R and the abscissa qMC allows you to improve graphics performance and thereby improve the characterization and quantification of biological interest elements.

According to the invention the sample may be a natural biological sample, in particular a liquid natural biological breakdown. It can also be unnatural cell suspension, such as the cultural environment. As a natural biological fluids include, without limitation, blood, urine, dissociating tissue, bone marrow, cerebrospinal fluid, pleural fluid, or synovial fluid, the product of the process of apheresis, and as synthesized cell suspension - cultural environment of cells or organisms.

According to the invention the biological elements contained in the robe, can be eukaryotic or prokaryotic cells, or their mixture, or fragments of these cells, Granelli. Probes that are applicable according to the method according to the invention, may be the same or different antibodies, nucleic acids (DNA or RNA), as well as any molecular probes, such as, for example, dyes, specific recognition of nucleic acids, enzyme substrates, as well as specific markers of proteins, ligands, receptors, molecules that are sensitive to the ionic environment (probes for pH, Ca⁴⁺etc), or any other molecule that is specific for the desired biological characteristic.

When the probes are antibodies, they can be monoclonal or polyclonal, natural or recombinant antibodies, human or animal.

If the probes are not detectable by nature, to make them discoverable, they need to be konjugierte with a marker that can be detected by means of detection in the selected band detection.

According to the invention are markers that serve to make the probe detectable, may be detectable chemical compounds able to get a probe, intercalating or deintercalated markers of nucleic acids, as well as specific markers of protein or other molecule that is specific for ISCO is wow biological characteristic. In this connection we may refer to such fluorescent dyes, as well as absorbents, such as: Alexa Fluor^®350, Alexa Fluor^®488, Alexa Fluor^®532, Alexa Fluor^®633, Alexa Fluor^®647, Alexa Fluor^®660, Alexa Fluor^®680, allophycocyanin, aminomethylation in acetic acid, Cy2^®, Cy5.1^®, Cy5^®, Cy5.5^®dichlorofluorescein (DCFH), dihydrorhodamine (DHR), "enhanced GFP" (EGFP), Fluo-3, FluorX^®, fluorescein, 5-maleimid the fluorescein, isothiocyanate fluorescein (FITC), PerCP, r-phycoerythrin (PE), tandem r-phycoerythrin-Cyanin 5 or Spectral red^®or CyChrome^®r-phycoerythrin-Cyanin 5.5 PE-CY 5.5^®), r-phycoerythrin-Cyanin 7 (PE-CY 7^®), r-phycoerythrin-Texas red-x^®, Red 613^®, rhodamine 110, rhodamine 123, S65L, S65T, isothiocyanate of tetramethylrhodamine, Texas red-x^®Real red^®, Indo-1, nanocrystals (Quantum Dots), Fura 2, Fura 3, Queen, DS red, as well as specific markers, in particular, nucleic acids (whether DNA or RNA), such as, for example, intercalant or other dyes to assess cell viability, such as ethidium bromide and thiazole orange, thiazole blue, and their derivatives, tioflavin S, tioflavin T, tioflavin TCN^®, diethyl-finalisation iodide (DEQTC), TOTO-l^®TO-PRO-1^®and YOYO-1^®, Hoechst^®33258, Hoechst^®33342, Hoechst^®34580, diaminophenyl the Dole (DAPI), iodide of propecia, pyronin Y, 7-aminooctanoic D (7 AAD), acridine orange, auramine O, calcein, new methylene blue, alamin-O, oxazin 750, blue Aster, SYTOX^®Green, SYTO 11^®, SYTO 12^®, SYTO 13^®, SYTO 16^®, SYTO 18^®, SYTO 80^®, SYTO 81^®etc.

In a private form of the invention, when the cellular characteristics, for example, antigens can be detected by probes, which became detectable due to overlapping markers, you can select the first cell condition called familial, that is present in all and is characteristic for all the considered biological elements. Because the expression of this cell trait in biological items are usually high, the token that will be grafted on recognition of its probe, can be a token giving a weaker response in the band detection, common to two overlapping markers (MC). Such a probe can be sure that it is still of interest to a good cell populations.

The second cell characteristic, recognizable by the probe, which became detectable due to overlapping marker may be a cellular characteristic weaker expressed by only one population studied biological elements. The token that will be grafted on the specified probe must be a token that gives a stronger response in the band detects and, common to two overlapping markers (MC).

The third investigated the cellular basis corresponds to the probe, labeled with a third marker, which is non-overlapping (MnC) and which will be detected in the second lane detection, different from the first.

The method according to the invention can be implemented in any device that can detect the markers. In this regard, we call for example, detection of fluorescence, which is emitted by the dye, conjugated with a molecular probe, such as an antibody, or directly electron microscope fluorescent dye, such as intercalating or neandertalensis dye that is specific for nucleic acids, and detection of absorption or scattering of light, in particular, in a wavelength range of the spectrum, are there these measurements through extinction or scattering in the wavelength range or by spectrometry in the band, selected from UV to infrared radiation. This absorption can be absorption, caused by the dye, conjugated with a molecular probe, such as an antibody, or directly with electron microscope dye, such as intercalating or neandertalensis dye specific for nucleic acids.

The object of the invention is also the use of the above ways is for discrimination of at least two cell populations - media special features, possibly present in the sample. The method can be used in diagnostic medicine, for example in the area of chronic lymphoid leukemia (LLC) and acute leukemia (LA), using antibodies anti-CD45, anti-CD19 and anti-CD5; in detecting residual disease (Minimum Residual Desease - minimal residual disease, MRD) and leukocyte differentiation using antibodies anti-CD45, anti-CD16 and anti-CD11b; hematopoietic precursor cells using antibodies anti-CD45, anti-CD34, anti-CD33 and 7-AAD, or DAPI or other marker to assess viability; in the field of inflammation and cellular activation using antibodies anti-CD45, anti-CD64 and anti-CD163; in the detection or monitoring of infection by the HIV virus using antibodies anti-CD45, anti-CD8 or anti-CD4 and anti-CD3; acute lymphoblastic leukemia type b (LALb) in children; examination of bone marrow using antibodies anti-CD45, anti-CD19 and anti-CD10, and differentiation of precursor B-lymphocytes (hematogenous) using antibodies anti-CD19, anti-CD10 and anti-CD38. This list is neither exhaustive nor limiting, and reflects only the current state of medical knowledge.

Other features of the invention will appear upon consideration of the figures and the following examples, which are given only as illustration and not limiting the Ute of the invention.

Thus, figure 1 gives a schematic representation of the phenotypes of biological elements, and A and B are overlapping markers, and C is non-overlapping marker, which correspond respectively to the probes (SA, SB and SC), which became detectable and that recognize specific signs (CPA, CPB and CPC) of the biological elements.

Figa shows a classic presentation of the measured flow cytometry fluorescence A+B (qMC) depending on the fluorescence of C (qMnC).

Figv gives a graphical representation obtained according to the invention, as defined by flow cytometry relations fluorescence C (qMnC), referred to the full fluorescence A+B (qMC) (qMnC/qMC) depending on the total fluorescence of A+B (qMC) (qMnC/qMC=f(qMC)).

The implementation of the invention allows the same axis fluorescence (x-axis) to distinguish between biological elements expressing (A+ B+) on one side (A - B-) on the other hand, and (A+ B-) or (A - B+) with a third party.

Figure 2 shows the application of the method according to the invention, for the analysis of blood leukocytes in a sample of normal blood (2a) and in the blood sample of a patient suffering from acute leukemia (2b), after labeling with antibodies anti-CD45, conjugated with r-phycoerythrin (PE)antibodies, anti-CD5, conjugated with phycoerythrin-tsianina 5 (PC5), and the antibodies, anti-CD19, conjugated with FITC. The expression of ar is wow CD45 allows you to identify the blasts, present in this pathology.

Figure 3 shows samples of normal blood and the blood of a patient suffering from chronic lymphoid leukemia (A2 and B2). Figa and 3A2 show the results of analysis by flow cytometry, and figv and 3B2 show the same results after applying the method according to the invention.

Figure 4 shows the application of the method according to the invention for analysis of blood T lymphocytes after labeling with antibodies anti-CD45, conjugated with PE antibodies anti-CD3 conjugated with PC5, and antibodies anti-CD8, conjugated with FITC. Figa shows the results of analysis by flow cytometry, and figv shows the same results after applying the method according to the invention.

Example 1:

The most frequent abnormalities in Hematology are different types of acute leukemia (LA) and lymphoid hemopathy B, and the incidence of chronic lymphoid leukemia (LLC) reveals a steady growth (30/10⁶).

Acute leukemia (LA) can be characterized by bone marrow failure resulting from the proliferation of hemopoietic cells.

Chronic lymphoid leukemia (LLC) can be characterized by the proliferation of monoclonal lymphocytic cells, lymphoid cells atypical line B.

Leukemic blasts LA are characterized by moderate expression of CD45 compared to normal limpot is Tami and blood monocytes.

Abnormal lymphocytes LLC type B differ incorrect simultaneous expression of antigens CD5 and CD19 on the same cells, whereas normal lymphocytes Express or CD5 (lymphocytes line T)or CD19 (b lymphocytes line B).

The phenotypes of these cells can be summarized in the following table:

To date, the analysis and identification of these different populations in the blood sample by flow cytometry required the use of three different antibodies directed against the three antigens CD5, CD19 and CD45 and side scatter SSC. For this analysis, each antibody is associated with different floorof the Ohm and each expression is measured at different wavelengths with different photomultiplier tubes.

Application of the method according to the invention allows to offer a system of tagging and cytometrical analysis, which allows to detect and quantify in the same blood sample and the same measurement of the actual presence of leukemic cells with just two photomultiplier tubes and side scatter SSC.

On a flow cytometer is optimized optical filtering light signals for this fluorochrome to measure fluorescence in the band of wavelengths centered in the maximum emission: fluorescein (FITC) are usually analyzed at 525 nm, r-phycoerythrin (PE) at 575 nm and the tandem r-phycoerythrin-cyanin 5 (PC5) at 675 nm, bandwidth of about 30 nm.

The spectra of emission of PE and PC5 are overlapping. Common to them is, for example, a wavelength of 675 nm, where PE is much weaker quantum yield of fluorescence than the PC5. One of the differences of the method according to the invention consists in the measurement of the fluorescence of one of the fluorochromes (PE) at a wavelength other than the peak emission (here the dimension PE at 675 nm). Thus, at this wavelength, it becomes possible, by playing on the different efficiency of the measuring system with respect to the two fluorochromes selected with this purpose, to compensate for the significant difference in the expression of the analyzed antigens.

This principle is PI used for the analysis of blood leukocytes after labeling with antibodies anti-CD45, conjugated with PE (λmax=575 nm), antibodies, anti-CD5, conjugated with PC5 (λmax=675 nm), and the antibodies, anti-CD19, conjugated with FITC (λmax=525 nm).

This tagging is analyzed in the first lane detection centered at 675 nm, which represents the wavelength of maximum emission PC5. Range of PE overlaps the range of PC5. Thus, analysis at 675 nm will be determined by the full amount of fluorescence emitted PC5 and PE (qMC).

On the other hand, analyzes the amount of fluorescence emitted by antibodies, anti-CD19, conjugated with FITC at 525 nm (qMnC).

Granulocytes and monocytes are excluded from the analysis due to the impact on their light scattering properties.

Protocol:

1) From the sample peripheral blood taken the sample of blood in a few milliliters of (5-50), which is mixed with an aliquot in a few milliliters (3-50) solution of the three above-described conjugated antibodies.

2) the Mixture is incubated at ambient temperature or at a controlled temperature, protected from light, in a few minutes (1 to 30).

3) After the incubation period, the mixture was added a reagent lysis of red cells, to obtain a final solution of the blood with the particular desired concentration (1/40, 1/80, 1/100, and so on).

4) Obtained a solution of lysed blood leave to Mature for a few seconds, depending on the lysis reagent and the incubation temperature (typically 15 to 30 seconds).

5) Then the solution is injected into the measuring complex type flow cytometer to measure all cells that intersect the light beam (usually a laser 488 nm for a specific dyes), the parameters of the scattering axis FSC, side scatter SSC, green fluorescence at 525 nm (FL1) and red fluorescence at 675 nm (FL2). Steps 3-5 can be performed automatically on a semi-automatic device flow cytometry or on a specially designed Hematology machine, and steps 1-5 can be automated on a specially designed automatic device flow cytometry.

Analysis of fluorescence at 675 nm (FL1) based on side scatter SSC (figa) allows to distinguish between:

- very weakly fluorescent in the population, corresponding to the blasts LA, expressing little CD45,

- population with intermediate fluorescence corresponding to lymphocytes expressing CD45, but not expressing CD5 (B-lymphocytes and NK cells), and

- highly fluorescent population corresponding to lymphocytes, also expressing CD5 (T-lymphocytes and cells of the LLC).

In this last population analysis of fluorescence at 525 nm allows the identification of LLC cells expressing CD19 also.

The application of the method according to the invention allows to distinguish lymphocytes non-T lymphocytes T cells and LLC, takes into account the I for each cell the ratio of fluorescence at 525 nm for fluorescence at 675 nm (qMnC/qMC). This relationship is expressed in the following truth table:

Distribution histogram obtained according to the invention ((qMnC)/(qMC)=f(qMQ)) (figs)allows to identify, separate and quantify three very distinctly different populations.

- Normal T lymphocytes (L.T.) does not fluoresce at 525 nm, but strongly fluoresce at 675 nm, thus, they have a very low regard.

Normal B lymphocytes (L.B.) fluoresce at 525 nm with intermediate fluorescence at 675 nm, thus, they have high regards.

- LLC cells simultaneously Express CD5, CD45 and CD19, fluoresce at 525 nm and 675 nm and, therefore, their attitude is intermediate (figure 2).

- NK cells that do not Express any CD5 or CD19, were excluded from the analysis because of bytheir fluorescence at 675 nm (CD45+, CD5 and CD19-).

So, based on blood samples, after ringing three fluorescent antibodies and red blood cell lysis in one assay to discriminate and count the T lymphocytes, B lymphocytes and NK cells by measuring only two wavelengths of fluorescence.

The application of the method according to the invention, allows to discriminate the T lymphocytes and cells LLC (pigv)that the diagram obtained by conventional analysis (figa), seen as a single cloud. The same is true for B lymphocytes and NK cells.

In addition, it may be found possible presence of blasts in acute leukemia or cell chronic lymphoid leukemia, and these cells can be converted.

The method according to the invention can find application in the diagnosis and monitoring of chronic lymphoid B pathologies, acute leukemia and in monitoring residual disease during treatment.

Example 2:

The method according to the invention can also be used for analysis of T-lymphocytes after labeling with anti-CD45 conjugated with PE, anti-CD3 conjugated with PC5, and anti-CD8 conjugated to FITC (figure 3).

1) From the sample peripheral blood taken the sample of blood in a few milliliters of (5-50), which is mixed with an aliquot in a few milliliters (3-50) solution of the three above-described conjugated antibodies.

2) the Mixture is incubated at which the temperature of the environment or at a controlled temperature, protect from light, in a few minutes (1 to 30).

3) After the incubation period, the mixture was added a reagent lysis of red cells, to obtain a final solution of the blood with the particular desired concentration (1/40, 1/80, 1/100, and so on).

4) the Obtained solution is left to Mature for a few seconds, depending on the reagent lysis and incubation temperature (typically 15 to 30 seconds).

5) Then the solution is injected into the measuring complex type flow cytometer to all cells that intersect the light beam (usually a laser 488 nm for a specific dyes), to measure the scattering parameters along the axis of the FSC, side scatter SSC, green fluorescence at 525 nm (FL1) and red fluorescence at 675 nm (FL2).

Steps 3-5 can be performed automatically on a semi-automatic device flow cytometry or on a specially designed Hematology machine, and steps 1-5 can be automated on a specially designed automatic device flow cytometry.

Samples labeled PE and PC5, analyze at 675 nm (qMC). Samples labeled with FITC, analyze at 525 nm (qMnC).

Granulocytes and monocytes are excluded from the analysis due to their properties, side scatter SSC.

Obtained by classical methods (figa) chart fluorescence at 675 nm depending on side scatter SSC-ru is et to identify the cloud representing populations of lymphocytes with an average intensity of fluorescence expressing CD45, but not expressing CD3 (B lymphocytes and NK cells) and highly fluorescent population corresponding to lymphocytes, also expressing CD3 (T lymphocytes).

Application of the method according to the invention allows to discriminate different populations (pigv).

In particular, this allows to identify in highly fluorescent populations corresponding to lymphocytes expressing, along with CD3, CD8 lymphocytes+.

Lymphocytes T CD4+ not fluoresce at 525 nm, but strongly fluoresce at 675 nm and have only a very weak relationship 525/675.

Lymphocytes T CD8+ fluoresce at 525 nm and 675 nm, thus they have a higher ratio.

The NK cells and B cells that do not Express CD3, identified on the basis of their moderate fluorescence at 675 nm (T).

1. How discrimination and counting at least two populations of biological elements selected from eukaryotic cells or prokaryotic cells, or a mixture thereof, or fragments of these cells, and are carriers of a particular biological characteristics selected from the cell organelles, protein, lipid, carbohydrate or nucleic acid possibly present in the liquid sample, including
simultaneous tagging of these populations b the ideological elements in three different probes, you can find, or which become detectable through three different markers, and two of these markers (overlapping markers, or MS) have their range of emission, the emission spectra overlap, and the third (non-overlapping marker, or MnC) has an emission spectrum that do not overlap with the spectra of the other two markers (non-overlapping range of emission);
measurement by any suitable means the total number of non-overlapping marker (qMnC) in-band detection, selected in the spectrum of emission of the specified non-overlapping marker;
measuring any suitable means full amount of overlapping markers (qMC) in-band detection, common to the spectra of emission of these overlapping markers;
the establishment of relations (R) total number of non-overlapping tokens to the total number of overlapping markers (R=(qMnC)/(qMC));
build any tool diagrams showing the ratio (R) depending on the total number of biological elements, labeled overlapping marker (R=f(qMC)); and/or
quantitative determination by any means of populations of biological elements present on the chart.

2. The method according to claim 1, characterized in that the liquid sample is a blood, urine, dissociatively tissue,bone marrow, cerebrospinal fluid, pleural fluid, synovial fluid, the end product of the process of apheresis.

3. The method according to claim 1 or 2, characterized in that the probes are the same or different, are antibodies or nucleic acids (DNA or RNA), or specific dyes, nucleic acid, or enzyme substrates or specific marker proteins, ligands, receptors or markers that are sensitive to the ionic environment.

4. The method according to claim 3, characterized in that the antibodies are monoclonal or polyclonal, natural or recombinant antibodies, human or animal.

5. The method according to claim 1, characterized in that the markers that are used to make the detectable probe are intercalating or deintercalation marker nucleic acid or a specific marker proteins or other molecules, characteristic for the studied biological element.

6. The method according to claim 5, characterized in that the markers are fluorescent or afluorescent markers.

7. The method according to claim 5 or 6, characterized in that the marker is selected from the Alexa Fluor^®350, Alexa Fluor^®488, Alexa Fluor^®532, Alexa Fluor^®633, Alexa Fluor^®647, Alexa Fluor^®660, Alexa Fluor^®680, allophycocyanin, aminoethylamino in acetic acid, the 2nd^®Su-5.1^®, Cu^®Su-5.5^®dihl is fluoroscein (DCFH), dihydrorhodamine (DHR), enhanced GFP (EGFP), Fluo-3, FluorX^®, fluorescein, 5-maleimide fluorescein, isothiocyanate fluorescein (FITC), PerCP, r-phycoerythrin (RE), tandem r-phycoerythrin-cyanin 5 or Spectral red®, or CyChrome^®r-phycoerythrin-cyanin 5.5 PE-CY 5.5^®), r-phycoerythrin-cyanin 7 (PE-CY 7^®), r-phycoerythrin-Texas red-x^®, Red 613^®, rhodamine 110, rhodamine 123, S65L, S65T, isothiocyanate of tetramethylrhodamine, Texas red-x^®, Real red^®, Indo-1, nanocrystals (Quantum Dots), Fura-2, Fura-3, quina, DS red, intercalating compounds such as ethidium bromide or the thiazole orange, thiazole blue, tioflavin S, tioflavin T, tioflavin TCN^®, iodide, diethyl-finalisation (DEQTC), TOTO-1^®TO-PRO-1^®or YOYO-1^®, Hoechst^®33258, Hoechst^®33342, Hoechst^®34580, diaminonaphthalene (DAPI), iodide of propecia, pyronin Y, 7-aminooctanoic D (7AAD), acridine orange, auramine O, calcein, new methylene blue, alamin-O, oxazin 750, Blue Aster, SYTOX^®green, SYTO 11^®, SYTO 12^®, SYTO 13^®, SYTO 16^®, SYTO 18^®, SYTO 80^®, SYTO 81^®.

8. Application of the method according to any one of claims 1 to 7 for discrimination of at least two populations of biological elements - media specific characteristics, possibly present in the sample.