RU2347224C2 - Method of blood analyses and blood analyser - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to methods of laboratory diagnostics. According to the method of blood test and automated technical blood tester. Blood sample is mixed with diluent and/or solvent reagent. Blood sample flow is formed and exposed to coherent polarised radiation of wavelength 330-680 nm directed along the flow axis. Named blood sample is analysed for polyangular light dispersion with blood cells detected and counted by polyangular light dispersion. Blood analyser applied for method implementation contains flow-type chamber, blood sample flow former in specified flow-type chamber, coherent radiation source, photodetector, and data control and processing unit connected to specified photodetector. Flou-type chamber represents scanning dish with the case made of optically transparent material and internal straight canal, and spherical mirror axis of which matches with that of internal channel of scanning dish. Coherent radiation source is polarised coherent radiation source of wavelength 330-680 nm mounted so that emitting radiation is directed along the axis of specified internal channel of scanning dish. The internal surface of spherical mirror of scanning dish is optically connected with specified photodetector.

EFFECT: possibility receive complex data referring to qualitative and quantitative blood structure providing minimum modification of measured blood corpuscles (cells) and high accuracy, besides, complete express blood analysis within no more than two stages.

26 cl, 11 dwg

Description

The invention relates to methods for determining the composition of blood and automated technical means that determine the composition of blood, and can be used in medicine to determine the qualitative and quantitative composition of blood.

One of the most important factors determining the physical condition of humans and animals is the composition of the blood, the ratio of the formed elements included in it, which include immature red blood cells-reticulocytes, erythroblasts, mature red blood cells-red blood cells, white blood cells, white blood cells, platelets, T- and B-lymphocytes, granulocytes, etc., their morphological characteristics: shape, size, refractive index, structure, as well as their absolute number and other indicators. For example, an increase in the number of only one type of reticulocyte cell is associated with hemolytic and sideroblastic anemia, thalassemia, and other acute and chronic diseases. In general, the clinical need for hematological control exists in the diagnosis and / or treatment of most diseases.

Known methods for conducting blood tests can be divided into manual and automated.

Manual methods are based on the study under a microscope of blood smears prepared using special technologies or obtained using a microscope slides with the image of these smears. For example, a method is known that allows one to simultaneously visualize erythrocytes, reticulocytes, basophils, eosinophils, pseudo-eosinophils, lymphocytes, monocytes and platelets on a single smear, for which a sample of unstabilized blood is stained with supravital stain with diamond cresyl blue, after drying, fix it and fix it and then repainted with a dilute buffer solution with a pH of 6.8-7.2, consisting of sodium phosphate disubstituted and potassium phosphate monosubstituted dye-fixative dye-fixative for L Eishman [RF patent No. 222425, IPC G01N 1/30]. This method, like other manual methods for determining the formed elements (cells) of blood, is time-consuming and not accurate enough.

Automated methods for conducting blood tests involve the use of specialized equipment that increases the speed and information content of the analysis. This equipment, as a rule, is able to identify some of the formed elements of the blood and determine their concentration. Also, in some methods, the cells are characterized by one or more inherent parameters, and the data obtained are systematized and generalized.

For example, an automated method is known for determining the diameter of red blood cells and other blood parameters, according to which a blood sample is placed in the gap between horizontally mounted plates, the lower of which is a receiver-transducer of ultrasonic vibrations, and is applied to the said sample with ultrasound, measuring the resonant frequency peak using the minimum shift which determine the diameter of red blood cells and the broadening band of the resonance curve, which determine the dynamic modulus of blood elasticity [RF patent 2206091, IPC G01N 33/49].

A known method for detecting reticulocytes, including adding a sample of blood cells to a reagent system that allows you to dissolve mature blood cells, then analyzing the named sample in a cell stream by measuring one or two lower signals from the angle of light scattering, defined less than 10 degrees relative to the direction of the original radiation to differentiate reticulocytes from other types of cells [US patent No. 5874311]. This method is suitable only for the detection of one type of blood cells - reticulocytes, and does not allow complex blood tests.

There is a method for identifying five subpopulations of leukocytes [US patent No. 5389549], according to which three different dissolving reagents are added to three separate blood samples — one for each sample, and measurements are taken to identify eosinophils, neutrophils and basophils, except for subpopulations of lymphocytes and monocytes in automatic mode . This method allows you to get only a complete picture of the content of leukocytes in the blood and does not provide for complex blood tests.

A larger number of types of blood cells can be determined by a known automated method for conducting blood tests, allowing you to simultaneously determine the presence of red, white blood cells and platelets in a blood sample [US patent No. 5891734]. In accordance with this method, a blood sample is divided into at least three parts and placed in separate vessels. The first part of the blood sample is mixed with diluent, the second part with solvent, and the third part with fluorescent reagents. Next, the first diluted portion of the blood sample is passed through a cuvette — an electrical resistance analyzer, through which red cells and platelets are detected and counted. The second part of the blood sample is passed through an optical flow transducer and multi-angle light scattering by blood cells is determined, according to which white blood cells are counted and differentiated. Then, multi-angle light scattering and fluorescence from the second dissolved part of the blood sample or from the first diluted part of the blood sample are determined, and red blood cells containing the nucleus or reticulocytes, or both, are counted and differentiated by means of the said optical flow transducer. Next, a third part of the blood sample is passed through the said optical flow transducer, multi-angle light scattering and fluorescence are determined, and platelets or their clots are calculated and differentiated by these indicators. Then, the data obtained from the described tests are combined, processed and compiled according to them, a report on the composition of the blood in automatic mode.

This method is the closest analogue of the proposed and adopted as a prototype of the invention. The disadvantages of the prototype include its multi-stage (analysis includes four successive stages), and, as a consequence, the duration and complexity, high labor costs, and the need to use expensive reagents, such as monoclonal antibodies CD4, CD19, CD14, CD3 and others labeled with fluorescent substances. In the prototype of the invention, as in other known methods, the accuracy of determining the parameters is limited for the following reasons. Modification procedures used, such as immunofluorescence labeling, although traditional in modern cytometry, lead to ambiguous changes in the formed elements of a blood sample. These modifications lead in most cases to the appearance of statistical and systematic errors in the measured parameters and make it impossible to increase the information content of the measurement, except by immunofenotyping with limited accuracy. As a result, in the prototype, as in other existing methods of automated analysis, in addition to phenotyping, the population of shaped elements is characterized by no more than three physical parameters: the distribution by volume of red blood cells, platelets and the concentration of hemoglobin in the red blood cell. Improving the measurement accuracy of even these parameters would be of great clinical significance [McLaren C.E., Brittenham G.M. and Hasselblad V. Analysis of the volume of red blood cells: application of the expectation-maximization algorithm to grouped data from the doubly-truncated lognormal distribution. // Biometrics. - 1986. - V.42 - P.143-158] and fundamental research value [Lew V.L., Raftos J.E., Sorette M., Bookchin R.M. and Mohandas N. Generation of normal human red cell volume, hemoglobin content, and membrane area distributions by "birth" or regulation? // Blood. - 1995. - V.86 - P.334-341].

The present invention solves the problem of creating such a method of blood tests, which would allow to obtain complex data on the qualitative and quantitative composition of blood without the use of expensive fluorescent reagents, with minimal modifications of the measured blood cells (cells) and high accuracy, allowing, in addition, conduct a complete rapid blood test in no more than two stages.

The problem is solved in that a method for conducting blood tests is proposed, according to which the sample is mixed with a diluting and / or dissolving reagent, then a blood sample stream is formed, thereby determining multi-angle light scattering by cells in the blood stream, blood cells are detected and counted by multi-angle light scattering moreover, the flow of blood samples is affected by coherent polarized radiation in the visible wavelength range, directing it along the axis of the stream.

To perform a complete blood test, it is necessary to divide the blood sample into two parts, the first of which is mixed with diluent, and the second with dissolving reagents.

It is optimal to mix a blood sample with a diluting reagent and a dissolving reagent to a concentration of blood cells in it of 10 ⁵ -10 ⁶ cells / ml.

The flow rate of the blood sample should be maintained at a level of 1-10 m / s.

As a diluting reagent, for example, isotonic buffer solution of sodium chloride can be used.

As a solvent reagent, for example, a solution of ammonium chloride can be used.

It is most effective to use coherent polarized radiation with a wavelength lying in the range of 330-680 nm.

As a rule, coherent polarized radiation is laser radiation.

For greater accuracy of the analysis results, it is advisable to change the polarization state of coherent radiation when determining the scattering of light at different angles.

To ensure high accuracy of the analysis of multi-angle light scattering by blood cells in the blood sample stream, a two-dimensional light scattering indicatrix of blood cells is determined.

To ensure high accuracy of the analysis of light scattering by blood cells at different angles in each of the blood sample streams, one to nine angular dependences of the light scattering intensity of blood cells on the observation angle are determined.

The proposed method for conducting blood tests is based on measuring scattered light by single blood cells at different angles. In this case, the main analyzed data are the measured light scattering intensities from a single particle, called here “light scattering indicatrixes".

The interaction of coherent polarized radiation with a blood cell in the optical system can be visually described using the formalism of Mueller matrices. The polarization state of the incident and scattered radiation is calculated by multiplying the matrices, and each element of the optical system is represented in the form of a matrix. The intensity and polarization state of the radiation is described by the Stokes vector [Collett E. Polarized Light: Fundamentals and Applications. - New York: Marcel Dekker, 1993 - 581 p.]. For example, radiation linearly polarized at an angle φ relative to the laboratory coordinate system is described by the Stokes vector:

When the basis vectors of the laboratory system are rotated through an angle φ, the Stokes vector is transformed as:

The Muller matrix of a particle of arbitrary shape has the form [K. Boren and D. Hafman. Absorption and scattering of light by small particles: trans. from English - M .: Mir, 1986. - 664 p.]:

опущен. Матрица Мюллера для идеального линейного поляризатора, повернутого осью пропускания под углом ξ к лабораторной системе координат, описывается так:The elements S _ij depend on the polar and azimuthal scattering angles: S _ij = S _ij (θ, φ), and θ varies from 0 to 180 °, and φ varies from 0 to 360 °, k is the wave number, r is the distance to the point observations. Next factor

omitted. The Mueller matrix for an ideal linear polarizer, rotated by the axis of transmission at an angle ξ to the laboratory coordinate system, is described as follows:

The phase holding element, which shifts the horizontal component of the field by δ _x = Θ / 2, and the vertical one by δ _y = -Θ / 2, is described as follows:

.The photo detector recording the intensity is described by the vector:

.

All of the above matrices are given in the laboratory coordinate system, while the Muller matrix is given in the coordinate system related to the blood cell, therefore, for calculations, you must first go to the coordinate system of the blood cell by turning it at an angle φ, and after converting the incident light to the blood cell, go back into the laboratory system by turning it at an angle -φ. Thus, for arbitrary positions or states of optical elements in the means of photodetection of scattered light and the state of incident radiation, determined by the vector V, the measured signal has the form:

In the Mueller Matrix of 16 elements, there are only 7 independent ones that fully characterize the electromagnetic field scattered by the blood cell. Any measured intensities (light scattering indicatrixes) are combinations of the Mueller matrix of the analyzed cell. To restore the elements of the Mueller matrix of blood cells, it is enough to measure 7 independent combinations of these elements. In the present invention, the measurement of 9 combinations of the Mueller matrix can be achieved, for example, by alternately changing the state of polarization of the incident radiation in the form of three states and alternately changing the states of the phase-holding optical element located in front of the photodetector in the form of three states or dividing the scattered light through three channels having different states of polarization elements.

Along with the point photodetector described above, it is possible to measure the electromagnetic field of scattering using a two-dimensional photosensitive matrix. The photosensitive matrix is located orthogonally to the optical axis of the system. In this configuration, a two-dimensional intensity function S (x, y) is measured, where x and y are the coordinates of the photosensitive matrix element. The sought light scattering indicatrix S _ij (θ, φ) is reconstructed from the solution of the integral equation

where t is the position of the analyzed cell within the detection zone, extending from L ₁ to L ₂ . The functions g (x, y, t) and f (x, y, t) determine the angles θ and φ at which the light emerging from the point t hits the point with coordinates (x, y).

The main data obtained in this measurement method are signals of light scattering indicatrixes. The class of mathematical problems arising in the analysis of data can be divided into two types: the first type is the task of classifying blood cells and the second type is the task of characterizing blood cells.

The tasks of the first type, namely the classification of blood cells, include the tasks of dividing the measured signals into classes and determining whether each measured signal belongs to any class. In this case, the establishment of the relationship between the light scattering signal and the physico-morphological characteristics of the blood cell is not required. Training is carried out according to signals belonging to known classes of blood cells (for example, a cell line), or the division into classes occurs empirically in the space of parameters of the measured signal.

, где N - число параметров индикатрисы, q - индекс принадлежности клетки к кластеру или подклассу, j - номер клетки в кластере или подклассе. Кластеризация возможна в случае, когда между типами клеток есть явные и существенные различия, например, как в случае разделения лейкоцитов на три основных типа: лимфоциты, моноциты и гранулоциты. Границы отдельных кластеров производится там, где плотности точек

в N-мерном пространстве минимальны. При кластеризации нет алгоритма обучения, т.е. нет необходимости использовать независимые от светорассеяния методы выявления типов клеток, например окрашивания иммунофлуоресцентными маркерами для создания обучающей выборки или, другими словами, выделения существенных признаков. В случае когда различия между клетками крови слабо выражены, необходимо применять классификацию. Первый шаг в классификации - это создание обучающей выборки. Обучающую выборку создают путем одновременного измерения флуоресценции от исследуемых клеток, окрашенных иммунофлуоресцентными маркерами, или специфичными флуохромами, и индикатрис светорассеяния. Тип каждой клетки, с одной стороны, однозначно определен с помощью флуоресценции, а с другой стороны, имеет вполне определенный набор параметров индикатрис

. Далее, измеряя индикатрисы светорассеяния и определяя вектор параметров

для произвольной клетки, можно судить, к какому подклассу относится тестируемая клетка, уже не применяя окрашивания иммунофлуоресцентными маркерами или специфичными флуорохромами.To identify and count blood cells, it is necessary to use clustering or classification of an array of measured cells. From the measured light scattering indicatrixes of blood cells, a set of parameters of light scattering indicatrixes is determined, for example, the indicatrix integrals in the given angular intervals, the positions and amplitudes of the maxima in the Fourier spectrum of the indicatrix, parameters can also be taken directly from the solution of the inverse light scattering problem from the measured indicatrixes, total N parameters. Each cell has a well-defined set of parameters

where N is the number of indicatrix parameters, q is the index of the cell belonging to the cluster or subclass, j is the number of the cell in the cluster or subclass. Clustering is possible when there are obvious and significant differences between cell types, for example, as in the case of the division of leukocytes into three main types: lymphocytes, monocytes and granulocytes. The boundaries of individual clusters are made where the density of points

in N-dimensional space are minimal. With clustering, there is no learning algorithm, i.e. there is no need to use methods independent of light scattering for detecting cell types, for example, staining with immunofluorescence markers to create a training sample or, in other words, highlight significant features. In the case where the differences between blood cells are weak, it is necessary to apply the classification. The first step in the classification is to create a training sample. A training sample is created by simultaneously measuring fluorescence from the studied cells stained with immunofluorescence markers, or specific fluochromes, and light scattering indicatrixes. The type of each cell, on the one hand, is uniquely determined using fluorescence, and on the other hand, it has a well-defined set of indicatrix parameters

. Further, by measuring the light scattering indicatrixes and determining the vector of parameters

for an arbitrary cell, it can be judged to which subclass the test cell belongs, no longer using immunofluorescence markers or specific fluorochromes.

, строят разделяющие поверхности второго порядка в N-мерном пространстве, каждая поверхность разделяет два граничащих подкласса.Classification can be carried out by different methods, for example, by the Bayes method, the method of k nearest neighbors, etc. In each case, the selection of essential features is based on various assumptions about the property of the system and can vary greatly. In the Bayesian method, based on the assumption that the distribution of cells by parameters is normal

, build dividing surfaces of the second order in N-dimensional space, each surface separates two adjacent subclasses.

The most common classification method is the k nearest neighbors method. This method is based on the assumption of local compactness, from which it follows that the similarity of two objects by l characteristics is usually accompanied by their similarity and by the (l + 1) th attribute. It follows that next to a small ε-neighborhood of the existing implementations of the i-th image of the training sample, only realizations of the same i-th image can appear. Moreover, the closer the control implementation j is to the existing implementation of the image q, the more likely the assignment of the point j to the qth image will be correct. Based on this, the following procedure can be proposed as a decisive rule: to leave in the computer memory all the implementations of the training set and the control point j to relate to the image whose implementation was closest to point j. This rule is really used in the practice of solving some problems, and it is called the "rule of the nearest neighbor." However, it should be borne in mind that real measurements of signs are often accompanied by interference and errors, so it is dangerous to trust the evidence of one precedent. It is advisable to take into account evidence and other objects of the training sample. To this end, they pay attention not to one, but to several nearby points. Such rules are called the rules of k nearest neighbors. If more than half of the k neighbors belongs to the image q, then the point j belongs to the qth image.

The tasks of the second type, namely the tasks of characterizing blood cells, include the determination of various parameters of blood cells. To solve them, in the general case, it is necessary to solve the inverse problem of light scattering. For the correct construction of the latter with controlled accuracy, it is necessary to calculate the direct problem of light scattering. The solution to the direct problem of light scattering is possible, for example, by constructing optical models of blood cells in which the cells are modeled in some form with a finite set of parameters based on data from microscopic measurements of blood cells. In this case, it is possible to theoretically calculate the light scattering from optical cell models for a set of parameters of the optical model of a blood cell. The calculation methods can be different, for example, the method of multilayer concentric spheres, the T-matrix method, discrete dipole approximation (DDA), etc.

Further, the methods for solving the inverse problem can be different, for example, the method of direct comparison of the minimum standard deviation, various neural networks, etc. The method of direct comparison and search for the most appropriate indicatrix is the simplest and most universal method for solving the inverse problem and is a special case of the well-known method of nodal space. It compares theoretically calculated light scattering indicatrixes with those measured during analysis and enumeration and finds a model cell whose indicatrix gives the minimum standard deviation when compared with the experimentally measured indicatrix. In this case, the characteristics of the analyzed blood cell are taken equal to the characteristics of the found model.

The method is as follows. The blood sample to be tested is divided into two, for example, equal parts and placed in separate containers. A diluent reagent, for example, an isotonic sodium chloride solution, is added to the first container to a concentration of 10 ⁵ -10 ⁶ cells / ml in the resulting mixture. A dissolving reagent is added to the second vessel - this is a red blood cell dissolving reagent, also called a lyzing reagent [Tiirikainen MI Evaluation of red blood cell lysing solutions for the detection of intracellular antigens by flow cytometry. // Cytometry. - 1995. - V.20 - P.341-348], for example, a solution of ammonium chloride, also to a concentration of blood cells in the resulting mixture of 10 ⁵ -10 ⁶ cells / ml. After lysing red blood cells, a reagent can be added that acts on white blood cells while maintaining the indicated concentration.

From the first part of the blood sample, its flow is formed at a speed of 1-10 m / s and at the same time this flow is irradiated with coherent radiation with a wavelength of 330-680 nm and a sequential change in its polarization state. The light scattered at different angles and its polarization state are determined for each blood cell in the blood sample stream. According to light scattering indicatrixes, using classification methods, for example, the method of k nearest neighbors, and clustering methods, for example, the method of k averages, determine and count the number of red blood cells, reticulocytes, platelets and their clots, and also, by solving the inverse light scattering problem, for example, they use the nodal space method characterization task, determining the parameters of these blood cells: volume, hemoglobin concentration, sphericity index, diameter, membrane area for red blood cells; volume, diameter, refractive index, semiaxis ratio for platelets and reticulocytes. Temporary changes in light scattering indicatrixes, solving the inverse problem of light scattering, for example, by the nodal space method, also determine temporary changes in the parameters of red blood cells, reticulocytes, platelets or platelet agglutination time, and also determine the kinetic characteristics of the processes occurring with these blood cells.

After that, from the second part of the blood sample, its flow is formed at a speed of 1-10 m / s and at the same time this flow is irradiated with coherent radiation with a wavelength of 330-680 nm and a sequential change in its polarization state, and the light scattered at different angles and its polarization state each blood cell in the stream of blood samples. According to light scattering indicatrixes, using classification methods, for example, the k method of nearest neighbors, and clustering methods, for example, the k average method, determine and calculate the absolute and relative number of white blood cells, and also, solving the inverse light scattering problem, for example, by the nodal space method, determine the parameters of these blood cells: size, size of the nucleus, refractive indices of the cytoplasm and nucleus.

Additional biophysical parameters of blood cells are determined by measuring light scattering indicatrixes as a result of a response to a change in the composition of a solution with an in vitro sample or in dynamics - during the reaction of a reagent with cells, for example, as part of a surrounding solution. Due to the relatively complex design of the experiment and the need to develop mathematical models for its interpretation, the direction of hematological analysis with kinetic analysis remains practically undeveloped. Only the application of the effect of platelet activation and agglutination is known, as well as the use of automated devices in the clinic [Danon D.E. The fragiligraph and its applications. // Br. J. Haematol. - 1967. - V.13 - P.61-66 and Terskov I.A., Gitelzon I.I. The method of chemical (acid) erythrograms. // Biophysics. - 1957. - T.2, N2. - S.259-266].

A complete blood test is described above. In the event that a complete analysis is not required, a blood sample can be mixed, for example, only with a diluting reagent and determining the relevant indicators of interest. In the same way, you can mix a blood sample only with a solvent reagent and get data only on the relevant indicators.

To implement this method, a device for conducting blood tests in an automatic mode is proposed. As a prototype of the proposed device, a device for measuring the concentration of blood cells and hemoglobin is selected, which contains a flow chamber for a blood sample, the axis of the optical axis of the first optical and second optical channels, the coherent radiation source, the light intensity modulator of the coherent radiation source, the first and second photodetectors, control and indexing unit [RF patent No. 2196989, IPC G01N 33/48]. The disadvantage of this device is the impossibility of using it to conduct a comprehensive blood test.

The invention solves the problem of creating a blood analyzer that allows for complex blood tests in automatic mode.

The problem is solved in that a blood analyzer is proposed that includes a flow chamber, a means forming a blood sample stream in said flow chamber, a coherent radiation source, photo detection means, and also data management and processing means associated with said photo detection means, in which the flow chamber made in the form of a scanning cell containing a body of optically transparent material with an internal rectilinear channel and a spherical mirror, the axis of which coincides with the axis of the above the morning channel, and the coherent radiation source is made in the form of a coherent polarized radiation source in the visible wavelength range and is set in such a way that its radiation is directed along the axis of the named internal channel of the scanning cell and the inner surface of the spherical mirror of the scanning cell is optically connected with the said photodetection means.

The inner surface of the spherical mirror of the scanning cell may be optically coupled to the photodetection means by means of a mirror mounted at an angle to the axis of the scanning cell.

The tool that forms the flow of blood samples can be made in the form of a hydrofocusing head.

A coherent polarized radiation source may have a wavelength in the range of 330-680 nm.

A computer can serve as a control and data processing unit.

The scanning cuvette may be equipped with a diffractive optical element, and the photodetection means is configured to detect fluorescence.

The source of coherent polarized radiation can be equipped with a λ / 4 phase-holding element.

The source of coherent polarized radiation can be equipped with a controlled phase-holding element made in the form of an electro-optical crystal.

A coherent polarized radiation source may contain one laser and one polarizer.

A coherent polarized radiation source can contain two lasers and is equipped with a beam splitting element, two polarizers and a λ / 4 phase-holding element.

A coherent polarized radiation source can contain three lasers and is equipped with two beam splitting elements, three polarizers and two λ / 4 phase-holding elements.

The photodetection means may include a controlled phase-holding element made in the form of an electro-optical crystal, a polarizer and a photodetector.

The photodetection means may comprise a beam splitter, two polarizers, a λ / 4 phase retaining element, and two photodetectors.

The photodetection means may comprise two beam splitting elements, three polarizers, two λ / 4 phase retaining elements and three photodetectors.

The photodetection means may be a two-dimensional photosensitive matrix.

The photodetection means may be associated with a means for controlling and processing data through an analog-to-digital converter.

Figure 1 shows the proposed device in its basic form.

Figure 2 shows the principle of action of a scanning cell when measuring light scattering by a blood cell at various angles.

Figure 3 shows embodiments of a coherent polarized radiation source.

Figure 4 shows embodiments of the photodetection means.

Figure 5 shows the principle of operation of a scanning cell with a diffractive optical element (DOE) when measuring the fluorescence spectrum.

Figure 6 shows an embodiment of a scanning cell for measuring scattered light in the range of polar angles from 5 to 175 °.

Figure 7 shows examples of light scattering indicatrixes for the main types of blood cells - lymphocytes, platelets, monocytes, erythrocytes, granulocytes.

On Fig depicts examples of comparison of measured and calculated light scattering indicatrix for platelets.

Figure 9 shows examples of comparison of measured and calculated light scattering indicatrixes for red blood cells.

Figure 10 shows an example of differentiation of the main types of leukocytes according to the integrals of the indicatrix of light scattering I ₇ .

11 shows the experimentally measured kinetics of spherization and hemolysis, and also shows the result of processing by the model.

In the drawings, the following numbering was used: 1 — a source of coherent polarized radiation, 2 — a lens, 3 — a mirror with a hole or a beam splitter, 4 — an optical window, 5 — a focused beam of coherent polarized radiation, 6 — a focused trigger beam, 7 — scanning cuvette, 8 - crimping fluid of the hydrofocusing head, 9 - blood sample flow, 10 - hydrofocusing head, 11 - photodetection means, 12 - analog-to-digital converter, 13 - data management and processing tool, 14 - spherical the fifth mirror, 15, 16, 60, 61 — the position point of the blood cell, 17, 18 — radiation scattered by the blood cell, 19, 59 — measurement zone, 20, 25, 26, 27 — coherent radiation source (laser), 22, 39 - controlled phase-holding element, 23, 42 - control means of the phase-holding element, 24 - control input, 21, 28, 30, 31, 40, 52, 53, 54 - polarizer, 29, 50, 51 - λ / 4 phase-holding element, 32 , 33 - beam splitting element, 34 - means for controlling the power supply of a coherent radiation source, 35, 43 - control input, 36 - means for receiving fluorescence, 37 - means for receiving scattered light a, 38, 45, 47 — diaphragm, 44 — dichroic mirror, 41, 46, 48, 49 — beam splitter, 55, 56, 57 — photodetector, 58 — diffractive optical element (DOE), 62.63 — blood cell fluorescence .

The blood analyzer works as follows.

The hydrofocusing head of the analyzer 10 (Fig. 1) generates a flow of blood cells moving one after another at a certain speed in the center of the channel of the scanning cell 7. The accuracy of the delivery of cells to the center of the scanning cell is ensured by the difference in the flow rate of the squeezing fluid 8 and the blood sample 9. The speed of the particles is regulated squeezing liquid flow 8. For illumination of blood cells, polarized radiation with a strictly defined state of polarization is used, created by a source of coherent polarized radiation 1. Os broadcasting is coaxial with the flow of blood samples. Lighting can be made both in the direction of flow of the blood sample and towards it. Lens 2 coherent polarized radiation is focused inside the channel of the scanning flow cell 7, while in the place of measurement of light scattering and fluorescence creates a zone of constant illumination. The light scattered by the cell is reflected from a spherical mirror located at the end of the scanning flow cell 7, exits through an optical window 4, is reflected from a mirror with an aperture, or a beam splitter element 3 and enters the photodetection means 11. The multi-channel ADC 12 converts the signals received by the photodetectors that enter the means management and data processing 13, which is essentially a computer. The computer processes the data obtained, which performs a report on the composition of the blood.

The principle of operation of the scanning cell is shown in figure 2. A focused beam of coherent polarized radiation in the visible wavelength range 5 and rays 17, 18 scattered by the blood cell are shown by a dashed line. For each position of the cell in the measurement zone 19, the scattered light, reflected from the spherical mirror, enters the photodetection means 11 only at a certain angle θ. When a particle passes through point 15, only light scattered at an angle θ _{1 is} received. When the cell moves from point 15 to point 16, the angle of light reception continuously changes from θ ₁ to θ ₂ . The measured signal is a function of time and is directly related to the angular dependence of the intensity of the scattered light. A focused trigger beam 6, directed perpendicular to the cell flow, serves to create the reference signal necessary for transforming the measured time of flight of the cell in the cell position in the cell.

The source of coherent polarized radiation 1 is used to form a controlled state of polarization of the input radiation. The analyzer implements three necessary polarization states: two independent planar polarizations and circular polarization. In the device, several versions of the source of polarized coherent radiation are possible, two of which are shown in FIG. The source of polarized coherent radiation 1 consists of:

- option "a" - the source of coherent radiation 20, the polarizer 21, the controlled phase-retaining element 22, the control means of the phase-retaining element 23, the control input 24;

- option "b" - from sources of coherent radiation 25, 26, 27, polarizers 28, 30, 31, λ / 4 of the phase-holding element 29, beam splitting elements 32, 33, means for controlling the power of radiation sources 34, control input 35.

The principle of operation of the formation of polarized coherent radiation in option "a" is as follows: the laser 20 creates coherent radiation; the polarizer 21 converts the polarization state of the laser radiation to an ellipticity of not more than 10 ^-3 ; the controlled phase-holding element 22 creates three successive phase delay values between two polarization states - 0, π / 2, π, thereby forming three independent states of radiation polarization. The controlled phase-holding element can be made, for example, of an electro-optical crystal, the optical properties of which depend on the magnitude of the electric field. The control means of the phase-holding element 23 serves to create an electric field and control its magnitude.

The principle of operation of the formation of polarization radiation in option "b" is as follows: coherent radiation sources, for example, lasers 25, 26, 27 create coherent radiation, polarizers 28, 30, 31 transform each polarization state of laser radiation to an ellipticity of not more than 10 ^-3 moreover, each laser and each polarizer are installed so that at the exit from the source of polarized coherent radiation provide three independent states of polarization, λ / 4 phase-retaining element forms a circular state tion of polarization, power control means alternately the radiation source 34 comprises a source of coherent radiation, thereby forming three independent states of polarization of the radiation.

The photodetection means 11 is used for receiving scattered light, fluorescence and analysis of the polarized state of the scattered light. In the blood analyzer, several versions of the photodetection means are possible, two of which are shown in Fig. 4. The photodetection means 11 consists of a fluorescence receiving means 36 and the scattered light receiving means 37. The fluorescence receiving means 36 is shown in detail in Fig. 4, b and consists of a dichroic mirror 44, an aperture 45, a photodetector 46. The scattered light receiving means 37 in embodiment a "Consists of a diaphragm 38, a controlled phase-retaining element 39, a polarizer 40, a photodetector 41, a means for controlling a phase-holding element 42, a control input 43. The scattered light receiving means 37 in embodiment" b "consists of a diaphragm 47, a beam splitter Yelnia elements 48, 49, λ / 4 phase-shifting elements 50, 51, the polarizers 52, 53, 54, photodetectors 55, 56, 57.

The measured intensities are combinations of the Mueller matrix. To restore the elements of the Mueller matrix of a cell, it is enough to measure 7 independent combinations of these elements. In the invention, the measurement of 9 combinations of the Muller matrix is achieved by alternately changing the state of polarization of the incident radiation in the form of three states and alternating the states of the phase-holding optical element in front of the photodetector in the form of three states - see option "a" in the scattered light receiving means 37 (Fig. 4) , or dividing the scattered light into three channels having different states of polarizing elements - see option "b" in the scattered light receiving means 37 (Fig. 4).

The principle of operation of the scattered light receiving means 37 in embodiment "a" is as follows: the diaphragm 38 cuts off unwanted scattered light, leaving for this cell position only rays traveling at a certain angle to the optical axis of the scanning cell; the controlled phase-holding element 39 creates three successive phase delay values between two polarization states - 0, π / 2, π, thereby creating three independent polarization states of the received radiation.

The principle of operation of the scattered light receiving means 37 in embodiment “b” is as follows: the diaphragm 38 operates in the same manner as in embodiment “a”; Further, the scattered radiation is divided into three parts by beam-splitting elements 48, 49 that do not change the polarization state of each part, one part of the radiation passes through the λ / 4 phase-holding element 50 and the polarizer 54 to the photodetector 57, the second part of the radiation passes through the λ / 4 phase-holding element 51 and a polarizer 53 to the photodetector 56, a third part of the radiation passes the polarizer 52 to the photodetector 55; polarization optical elements for each part of the scattered radiation are installed so that each photodetector receives and measures three independent polarization states of the received radiation.

For example, option "b" means of generating polarized radiation 1 and the reception of scattered light 37 can be shown which combinations of the elements of the Mueller matrix will be measured for a specific position of the polarizing elements. The coherent radiation source 27 and the polarizer 31 are set so that the radiation polarization direction is directed at 45 ° to the laboratory system, the coherent radiation source 26 and the polarizer 30 are installed so as to provide the radiation polarization direction parallel to the laboratory system, coherent radiation source 25, polarizer 28, a λ / 4 phase retaining element 29 is arranged so that the radiation has circular polarization. In this case, the Stokes vectors of these three positions will have the form:

where the factors in front of the vectors mean loss of intensity when passing through the beam splitting elements. Next, the reception of scattered light in the conversion of the polarization elements P _ξ × C _Θ set λ / 4 phase-shifting elements 50, 51 such that Θ = π / 2, a polarizer 54 at an angle of ξ = 45 ° to the laboratory system, the polarizer 53 by angle ξ = -45 ° to the laboratory system, polarizer 52 at an angle ξ = 0 ° to the laboratory system. Then the Muller matrices of polarization transformations at the reception of scattered light have the form:

Now we record the measured intensities of the scattered light for the above states of polarization of the incident light and the states of the polarization elements at the reception:

The photodetection means 11 may be a two-dimensional array of photosensitive elements. The principle of operation of the scattered light photodetection means is as follows: each matrix element accumulates a photo signal when the cell moves in the measurement zone 19. In this case, the resulting signal value is described by the expression

where (x, y) are the coordinates of the photosensitive matrix element, S _ij (θ, φ) is the desired light scattering indicatrix, t is the position of the analyzed cell inside the detection zone, extending from L ₁ to L ₂ . The functions g (x, y, t) and f (x, y, t) determine the angles θ and φ at which the light emerging from the point t hits the point with coordinates (x, y).

Figure 5 shows in detail the principle of measuring the fluorescence spectrum of the analyzed cells. A focused beam of coherent polarized radiation 5 and fluorescence from a blood cell for its two positions 62, 63 are shown by a dashed line. Fluorescence changes when the blood cell moves in the center of the scanning cell 7. The measurement of the fluorescence spectrum is based on large chromatic aberrations of the diffractive optical element DOE. DOE 58 is made in the form of an optically transparent window 4 with strokes applied on one side in the form of concentric rings. The size and shape of the strokes are specifically calculated for the specific geometry of the analyzer. DOE works like a lens, transferring fluorescence from a blood cell to a diaphragm 45 on a fluorescence receiving means. For a specific wavelength λ, the fluorescence emitted by the blood cell will focus the DOE into the diaphragm 45 only at a specific cell position. For a blood cell at position 60, only fluorescence emitted with a wavelength of λ ₁ will reach the photodetector. The wavelength of the received fluorescence varies from λ ₁ to λ ₂ when the blood cell moves in the measurement zone 59 from position 60 to position 61.

An integral part of the invention is, firstly, the presence of a scanning cuvette having a spherical mirror, the coincidence of the axes of the said mirror, the internal channel (blood sample stream) and coherent polarized radiation, and secondly, a photodetector that receives light scattered by blood cells, allowing aggregates measure 9 combinations of the scattered light Mueller matrix. The use of DOE, which allows you to measure the fluorescence spectrum of blood cells, is also extremely useful.

The optical system described above makes it possible to measure 9 combinations of the elements of the Mueller matrix depending on the scattering angle of a single cell in the range of angles from 5 to 120 °.

Figure 6 shows a scanning cuvette that combines two spherical mirrors arranged in series, but in opposite directions, and allows one to measure light scattering indicatrixes in an extended range of angles from 5 to 175 °.

Example 1

Determination of red blood cell characteristics by light scattering indicatrix

For the analysis of light scattering at wavelengths of the visible range of red blood cells, it can be considered as a homogeneous object [Tycko D.H., Metz M.H., Epstein E.A., Grinbaum A. Flow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentration. // Applied Optics. - 1985. - V.24. - P.1355-1365], having the shape of a biconcave disc. From the analysis of literature data [Fimg Y.C., Tsang W.C. and Patitucci P. High-resolution data on the geometry of red blood cells. // Biorheology. - 1981. - V.18 - P.369-385] to calculate the direct problems of light scattering, the following parameterization was chosen:

where S, P, Q, R are the parameters that are associated with the geometry parameters of the blood cell as follows:

where d is the diameter of the cell, h is the maximum thickness of the cell, b is the minimum thickness, c is the radial coordinate of the position of the maximum thickness of the cell relative to the axis of symmetry of the cell.

The calculation of the direct problem of light scattering is carried out by the method of approximation by discrete dipoles [Draine V.T. The discrete-dipole approximation and its application to interstellar graphite grains. // Astrophys. J. - 1988 - V.333, 848-872]. The distribution of cells by parameters is considered uniform in the ranges: for d from 5.8 to 10.2 micrometers, for h from 0.85 to 4 micrometers, for b from 0.1 to 2.1 micrometers, and c was set in the range 0.33 * d ± 0.05 micrometers. The hemoglobin concentration varies from 25 to 42 grams / deciliter. The rotation angle of the longest coordinate of the cell relative to the incident radiation varies in a narrow range from 0 to 15 °, because the hydrofocusing system of the device has good orienting characteristics for non-spherical objects (in the case of red blood cells, the spread in orientation is several degrees).

The values of all parameters vary independently according to the Monte Carlo method in the indicated ranges. Thus, the base of light scattering characteristics from 10 ⁵ model cells is calculated.

Measurements and calculations are carried out for indicatrix I ₇ (see (17)). For each measured erythrocyte indicatrix, the closest model indicatrix is found in the model database, for example, by the least squares method with a certain weight function depending on the scattering angle. The values of the parameters corresponding to the model indicatrix (volume, surface area) are taken equal to those of the measured cell.

Example 2

Determination of platelet characteristics by light scattering indicatrix

An analysis of the literature on platelet morphology in various anticoagulants and a preliminary detailed study of the measured light scattering indicatrixes allow us to conclude that the platelet in EDTA is a flattened spheroid with several processes, pseudopodia. Next, the light scattering is calculated by the discrete dipole method (DDA) for a flattened spheroid with pseudopodia. Analysis of the calculated data shows that the presence of pseudopodia makes an insignificant contribution to the light scattering indicatrix, which greatly simplifies the platelet model. A preliminary calculation of light scattering indicatrixes for flattened spheroids is carried out with a variation of the parameters characteristic of platelets in EDTA. The dimensional parameter, defined as α = 2πr · n ₀ / λ, the phase shift defined by ρ = 2α (m-1), the ratio of the spheroid semiaxes, where m = n / n ₀ is the relative refractive index, λ is the wavelength, is varied incident radiation, r is the radius of the sphere of equivalent volume. Measurements and calculations are carried out for indicatrix I ₇ (see (17)). By comparing the calculated indicatrices with the measured ones, the above platelet parameters are found. Next, for a known wavelength and refractive index of a medium, a volume defined as V = 4 / 3πr ³ and a refractive index defined as n = (ρ / 2α + 1) · n ₀ are calculated.

Example 3

White blood cell differentiation according to light scattering indicatrixes

To separate the main three leukocyte populations into lymphocytes, monocytes, and granulocytes, the light scattering indicatrix parameters I _{7 are used} . The parameters of the indicatrix are taken to be the integrals of the indicatrix of light scattering at two defined solid angles. For these purposes, the integrals were selected in the intervals of 7.5 ± 2.5 ° and at 45 ± 10 ° (see Fig. 10).

Example 4

Measurement of additional characteristics of red blood cells during lysis

A more detailed determination of the characteristics of blood cells by the present method is performed during the analysis of the kinetic process with the participation of changes in morphological characteristics. In the process of the kinetic process, the determination of the morphological parameters of the cell population is simplified due to the adoption by the measured objects of the form for which the accuracy of solving the inverse light scattering problem increases (for example, spherical). An example of such a process is the lysis of red blood cells in an aqueous solution of a salt of ammonium chloride in an isotonic concentration. As shown in FIG. 11, in this process, it is possible to identify the spherization stage (gray area).

Measurement of the time evolution of the distribution function over the morphological parameters of the cell population in combination with the kinetic model of the process allows us to determine the “dynamic” parameters that are not available for measurement in statics. To determine the "dynamic" parameters according to the measurement of hemolysis kinetics at the TWS, anion transport models, membrane stretching, and cell pore formation were used. Table 1 shows the blood parameters determined in this way and their distribution in the population.

Example 5

Construction of a training sample for the division of lymphocytes into subclasses CD3 (T-lymphocytes), CD19 (B-lymphocytes), CD4 (T-helpers), CD8 (T-killers)

The blood of a healthy donor is placed in a tube filled with EDTA as an anticoagulant in a ratio of 1.5 mg EDTA to 1 ml of blood. To create a training sample for the division of lymphocytes into classes using the following sample preparation procedure [Ormerod MG. Preparing suspensions of single cells. Flow Cytometry: A Practical Approach (3rd edition). (Ormerod MG., Ed) Oxford Univ Press, 2000]: 2 ml of blood diluted with an anticoagulant is diluted with 6 ml of saline phosphate buffer solution and centrifuged at ≈450 g for 20 minutes in a Ficoll®-sodium metrizonate solution, whose density is 1.077 mg / l Then a thin layer of mononuclear cells is selected from the plasma / Ficoll phase interface, washed in phosphate buffer and stained with immunofluorescence markers CD3, CD19, CD4, CD8, each of which bears its own fluorescent dye: FITC, PE, TRPE, Cy5PE, respectively. After 20 minutes of staining, the sample is again washed and adjusted by dilution in phosphate buffer to a concentration of 10 ⁶ cells / ml.

, где N - число параметров индикатрисы, q - индекс принадлежности клетки подклассу CD3, CD19, CD4 или CD8, j - номер клетки в подклассе.For each cell from the prepared sample, the fluorescence spectrum and light scattering indicatrix are simultaneously measured. From the measured fluorescence spectrum, the cell belongs to one or another type: CD3, CD19, CD4 or CD8. From the measured indicatrixes, a set of parameters is determined, for example, the integrals of indicatrixes in the given angular intervals, the positions and amplitudes of the maxima in the Fourier spectrum of indicatrixes, also the parameters can be taken directly from solving the inverse light scattering problem from the measured indicatrixes, total N parameters. The type of each cell, on the one hand, is uniquely determined using fluorescence, on the other hand, it has a well-defined set of parameters

where N is the number of indicatrix parameters, q is the index of the cell belonging to the subclass CD3, CD19, CD4 or CD8, j is the cell number in the subclass.

Example 6

Complete blood count

For analysis, 100 microliters of whole anticoagulated blood are taken. This blood sample is divided into two parts: 25 microliters and 75 microliters, and placed in two test tubes. A dilution reagent in a ratio of 500: 1 is added to the first tube. 1,500 microliters of a dissolving (lysing) solution is added to the second test tube — a solution of ammonium chloride in isotonic concentration with a concentration of HCO ₃ ions of 8 ± 2 mmol. Red blood cells are lysed for 3 minutes at room temperature 23 ± 3 ° C.

Next, the first and second parts of the blood sample are sequentially passed through a scanning flow cell of the blood analyzer shown in FIG. 1 at a speed of 5 m / s, irradiating them with circularly polarized radiation with a wavelength of 660 μm. The light scattering indicatrixes I ₇ and I _{9 of} single blood cells are measured (see expressions (17) and (19) and Fig. 7). The determination of red blood cells, platelets and reticulocytes occurs when measuring the first part of the sample, the determination of the remaining blood cells occurs when measuring the second part of the sample.

и

Measurement data of red blood cells and platelets form clusters in the space of two parameters. These cells are separated by the method of k means for the following parameters of the indicatrix I ₇ : integrals

and

The inverse light scattering problem is solved by the nodular space method using the base of indicatrixes I ₇ calculated from model cells with randomly varying geometry within the range of literary values. Examples of solutions for several measured indicatrixes are shown in Fig. 8 and Fig. 9 for red blood cells and platelets, respectively. Platelets are modeled by flattened ellipsoids, erythrocytes are modeled by a biconcave disc with known parameterizations. The size of the base used is at least 10 ⁵ and 10 ⁴ calculated indicatrixes for red blood cells and platelets, respectively.

i=(1, 2…10), всего 10 параметров.The identification of reticulocytes is carried out according to a pre-trained algorithm by classification by the method of k nearest neighbors. An algorithm is used, trained by labeling with a thiazole orange dye capable of staining reticulocytes. In this case, the integrals are taken as parameters for training and identification

i = (1, 2 ... 10), a total of 10 parameters.

(см. фиг.10).The differentiation of lymphocytes, monocytes and granulocytes (many of them combine neutrophils, basophils, eosinophils) is carried out by the method of k averages in space according to the measured parameters J ₁ and

(see figure 10).

i=(1, 2…10), всего 11 параметров.Differentiation of granulocytes into subclasses - neutrophils, basophils, eosinophils, is carried out according to a pre-trained algorithm by classification by the k method of nearest neighbors. An algorithm trained by labeling with specific monoclonal antibodies (CD63, CD203c, CD244) associated with fluorescent dyes is used. Moreover, the integrals are taken as parameters for training and differentiation

i = (1, 2 ... 10), a total of 11 parameters.

всего 20 параметров.In the obtained set of lymphocytes, a subset of T-lymphocytes and B-lymphocytes is isolated by the method of k closest neighbors according to a pre-trained algorithm. An algorithm trained by labeling with specific monoclonal antibodies CD3 and CD19, respectively, is used. As parameters for training take integrals

Only 20 parameters.

The results of a comprehensive blood test performed by the described method are summarized in table 2.

Table 1 Lognormal Distribution Parameters Average Width Volume, V ₀ , µm ³ 87.5 ± 0.3 (1.22 ± 0.03) · 10 ^-1 Hemoglobin, [Hb], mM 4.910 ± 0.005 (6.46 ± 0.10) 10 ^-2 Sphericity Index, V _s / V ₀ 1.74 ± 0.02 (4.60 ± 0.07) · 10 ^-2 Membrane area, S, µm ² (1.38 ± 0.01) · 10 ² (8.0 ± 0.2) · 10 ^-2 The amount of protein N _band3 molecules (1.3 ± 0.2) · 10 ⁶ -

Membrane elasticity

(1.5 ± 0.6) · 10 ^-2 -

table 2 Value Value Hemoglobin, medium cell concentration 32.8 (gram / dl) Width of distribution of intracellular hemoglobin concentration 4.2% Red blood cells 5.1 × 10 ¹² (l- ^l ) Reticulocytes, (% of the concentration of red blood cells) 0.9% White blood cells 8.3 × 10 ⁹ (L ^-1 ) Neutrophils 5.1 × 10 ⁹ (l ^-1 ) Eosinophils 0.2 × 10 ⁹ (l ^-1 ) Basophils 0.05 × 10 ⁹ (L ^-1 ) Monocytes 0.4 × 10 ⁹ (L ^-1 ) Lymphocytes 2.5 × 10 ⁹ (l ^-1 ) T lymphocytes (CD3 +) 1.6 × 10 ⁹ (L ^-1 ) B lymphocytes (CD19 +) 0.45 × 10 ⁹ (l ^-1 ) The average diameter of red blood cells 7.7 μm The width of the distribution of red blood cells in diameter 12.3% The average surface area of the membrane of red blood cells 137.1 μm ³ The width of the distribution of red blood cells by surface area,% 9.5% The average volume of red blood cells 87 (femtoliters) The width of the distribution of red blood cells by volume,% 15.1% Average platelet volume 9.3 (femtoliters) The width of the distribution of platelets by volume,% 17.8% Platelet Average Refractive Index 1.39 The width of the distribution of platelets by refractive index,% fifteen%

Claims (26)

1. The method of conducting blood tests, in which a blood sample is mixed with a diluting and / or dissolving reagent, forming a blood sample stream and determining multi-angle light scattering by blood cells in the said blood sample stream, detecting and counting blood cells by multi-angle light scattering, characterized in that the flow of blood samples is affected by coherent polarized radiation with a wavelength of 330-680 nm, directing it along the axis of the named stream, and the polarization state of the radiation is successively changed. 2. The method according to claim 1, characterized in that the blood sample is divided into two parts, the first of which is mixed with a diluent, and the second with a solvent reagent. 3. The method according to claim 1, characterized in that the blood sample is mixed with a diluting and / or dissolving reagent to a concentration of blood cells in it of 10 ⁵ -10 ⁶ cells / ml. 4. The method according to claim 1, characterized in that the flow rate of the blood sample is maintained at a level of 1-10 m / s. 5. The method according to claim 1, characterized in that the diluting reagent is a buffer isotonic sodium chloride solution. 6. The method according to claim 1, characterized in that the solvent reagent is a solution of ammonium chloride. 7. The method according to claim 1, characterized in that the coherent polarized radiation is laser radiation. 8. The method according to claim 1, characterized in that according to multi-angle light scattering by blood cells in the flow of blood samples, N indicatrixes of light scattering of blood cells are determined at N equal to 1-9. 9. The method according to claim 1, characterized in that according to multi-angle light scattering by blood cells in the blood sample stream, a two-dimensional light scattering indicatrix of blood cells is determined. 10. A blood analyzer comprising a flow chamber, a means for generating a blood sample stream in said flow chamber, a coherent radiation source, photo detection means, and also data management and processing means associated with said photo detection means, wherein the flow chamber is made in the form a scanning cuvette having a body of optically transparent material with an internal rectilinear channel, and a spherical mirror, the axis of which coincides with the axis of the internal channel of the scanning cuvette, and the source X-ray radiation is made in the form of a source of polarized coherent radiation with a wavelength of 330-680 nm, which is installed in such a way that its radiation is directed along the axis of the named internal channel of the scanning cell, and the inner surface of the spherical mirror of the scanning cell is optically connected with the said photodetection means. 11. The analyzer of claim 10, characterized in that the inner surface of the spherical mirror of the scanning cell is optically connected to the photodetection means by means of a mirror mounted at an angle to the axis of the internal channel of the scanning cell. 12. The analyzer of claim 10, wherein the means forming the flow of blood samples is made in the form of a hydrofocusing head. 13. The analyzer according to claim 10, characterized in that the means of control and data processing is a computer. 14. The analyzer of claim 10, wherein the scanning cuvette is equipped with a diffractive optical element. 15. The analyzer of claim 10, wherein the photodetection means is arranged to detect fluorescence. 16. The analyzer of claim 10, characterized in that the source of coherent polarized radiation is equipped with a λ / 4 phase-holding element. 17. The analyzer of claim 10, characterized in that the source of coherent polarized radiation is equipped with a controlled phase-holding element made in the form of an electro-optical crystal. 18. The analyzer of claim 10, characterized in that the source of coherent polarized radiation contains one laser and a polarizer. 19. The analyzer of claim 10, wherein the coherent polarized radiation source contains two lasers and is equipped with a beam splitting element, two polarizers and a λ / 4 phase-holding element. 20. The analyzer of claim 10, characterized in that the source of coherent polarized radiation contains three lasers and is equipped with two beam splitting elements, three polarizers and two λ / 4 phase-retaining elements. 21. The analyzer of claim 10, wherein the photodetection means comprises a controllable phase-holding element made in the form of an electro-optical crystal, a polarizer and a photodetector. 22. The analyzer according to claim 10, characterized in that the photodetection means comprises a beam splitter element, two polarizers, a λ / 4 phase retaining element and two photodetectors. 23. The analyzer according to claim 10, characterized in that the photodetection means comprises two beam splitting elements, three polarizers, two λ / 4 phase-retaining elements and three photodetectors. 24. The analyzer of claim 10, wherein the photodetection means is a two-dimensional matrix of photosensitive elements. 25. The analyzer of claim 10, wherein the scanning cell is equipped with an additional spherical mirror, and the spherical surfaces of the mirrors are directed in opposite directions. 26. The analyzer according to claim 10, characterized in that the control and data processing means is connected to the photodetection means through an analog-to-digital converter.

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