Sample analysis method, sample analyzer, and computer-readable storage medium

Abstract

A sample analysis method, a sample analyzer and a computer readable storage medium for analyzing a blood sample. In one test, the optical signals generated by each particle in the sample liquid to be tested when irradiated by the excitation light when passing through the optical detection area one by one are obtained, the sample liquid to be tested is obtained by treating the blood sample with a hemolytic agent, a first dye and a second dye, the first dye can stain white blood cells, and the second dye can stain infected red blood cells, the optical signals include a scattering light signal, a first fluorescent signal corresponding to the first dye and a second fluorescent signal corresponding to the second dye; obtain white blood cell optical information based on the scattering light signal and the first fluorescent signal; obtain red blood cell optical information based on the scattering light signal and the second fluorescent signal. Through the method, white blood cell optical information and infected red blood cell optical information can be obtained simultaneously in the same detection channel or in the same test, thereby reducing the blood volume and cost for detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of blood detection, and in particular to a sample analysis method, a sample analysis instrument and a computer readable storage medium. BACKGROUND

[0002] Malaria is one of the diseases that seriously endanger human health, which is caused by Plasmodium. Currently, blood smear microscopy is usually used to detect Plasmodium, but this method is highly dependent on the experience of the operator and requires high professional ability of the operator, and it takes a long time.

[0003] With the development of blood cell analysis technology, currently there are many methods known to quickly detect Plasmodium-infected red blood cells by using a blood cell analyzer.

[0004] European Patent Application EP0613003B1 discloses a method of using multiple fluorescent dyes to stain infected red blood cells under non-hemolytic conditions, so as to better distinguish reticulocytes and infected red blood cells.

[0005] European Patent Application EP1406088A2 discloses a method of detecting Plasmodium by combining fluorescent dyes under hemolytic conditions, which can achieve the classification and counting of Plasmodium, but cannot simultaneously achieve the classification and counting of white blood cells.

[0006] U.S. Patent Application US2006 / 0223137 discloses a reagent that can partially dissolve the cell membrane of Plasmodium-infected red blood cells, so that the Plasmodium remains in the red blood cells, and the fluorescent dye can penetrate the cell membrane. However, when the sample has a high value of reticulocytes, it cannot accurately detect Plasmodium-infected red blood cells.

[0007] Chinese Patent Application CN106483278B discloses a method of detecting Plasmodium-infected red blood cells. In this method, a specific fluorescent dye at a specific concentration is used to process the sample to be tested, so that Plasmodium-infected red blood cells can be detected more accurately than the scheme disclosed in U.S. Patent Application US2006 / 0223137.

[0008] Chinese Patent Application CN102016573B discloses a blood analysis device and a blood analysis method that can classify white blood cells in a test sample into four categories and detect malaria-infected red blood cells while reducing the burden on users caused by reagent development. However, in this method, although the same hemolytic agent is used, two blood samples need to be provided for different processing, and white blood cell classification detection and malaria-infected red blood cell detection are performed in two tests, respectively, thus increasing the test time, blood volume, and cost of hemolytic agent. SUMMARY

[0009] It is an object of the present application to provide an improved solution for detecting malaria, in which solution it is possible to detect both white blood cell parameters and infected red blood cell parameters in one test, especially in an existing white blood cell detection channel, to obtain a plurality of detection parameters in one test compared to the prior art, to save blood volume for detection, and to reduce detection costs.

[0010] It is another object of the present application to provide an improved solution for detecting malaria, in which solution it is possible to detect infected red blood cell parameters using two fluorescent dyes under hemolytic conditions.

[0011] To achieve the objects of the present application, a first aspect of the present application relates to a sample analysis method for analyzing a blood sample, comprising:

[0012] obtaining optical signals generated by each particle in a sample liquid to be tested when passing through an optical detection zone of an optical detection device one by one under irradiation of excitation light, especially single-wavelength excitation light, in one test, wherein the sample liquid to be tested is obtained by processing the blood sample using a hemolytic agent, a first dye and a second dye, the first dye being capable of staining white blood cells, and the second dye being capable of staining infected red blood cells, wherein the optical signals include a scattering light signal, a first fluorescent signal corresponding to the first dye, and a second fluorescent signal corresponding to the second dye;

[0013] obtaining white blood cell optical information of the blood sample based on at least one of the scattering light signals and the first fluorescent signal;

[0014] obtaining infected red blood cell optical information of the blood sample based on at least one of the scattering light signals and the second fluorescent signal.

[0015] A second aspect of the present application relates to a computer-readable storage medium having instructions stored thereon, which instructions, when executed by a processor, cause the processor to implement the sample analysis method according to the first aspect of the present application.

[0016] A third aspect of the present application relates to a sample analyzer, comprising:

[0017] a sampling device having a pipette with a pipette tip and having a drive device for driving the pipette to quantitatively aspirate a blood sample through the pipette tip;

[0018] A sample preparation device has a reaction cell for receiving a blood sample drawn by a sampling device and a reagent supply for providing a hemolytic agent, a first dye and a second dye to the reaction cell, so that the blood sample drawn by the sampling device is mixed with the hemolytic agent, the first dye and the second dye provided by the reagent supply in the reaction cell to prepare a sample liquid to be tested, wherein the first dye is capable of staining white blood cells and the second dye is capable of staining infected red blood cells.

[0019] An optical detection device includes a light source for emitting a light beam to illuminate a flow cell, the flow cell being in communication with the reaction cell and through which each particle in the sample liquid to be tested can pass one by one, a scattered light detector for detecting a scattered light signal generated by a particle passing through the flow cell after being illuminated by light, a first fluorescence detector for detecting a first fluorescence signal corresponding to the first dye generated by a particle passing through the flow cell after being illuminated by light, and a second fluorescence detector for detecting a second fluorescence signal corresponding to the second dye generated by a particle passing through the flow cell after being illuminated by light.

[0020] A processor is configured to perform the following steps: acquiring the scattered light signal, the first fluorescence signal and the second fluorescence signal of the sample liquid to be tested in one test from the optical detection device, obtaining white blood cell optical information of the blood sample based on at least one of the scattered light signal and the first fluorescence signal, and obtaining infected red blood cell optical information of the blood sample based on at least one of the scattered light signal and the second fluorescence signal.

[0021] The processor of the sample analyzer according to the third aspect of the present application is particularly configured to implement the sample analysis method according to the first aspect of the present application.

[0022] To achieve another object of the present application, the fourth aspect of the present application relates to another sample analysis method for analyzing a blood sample, comprising:

[0023] In one test, each particle in the sample liquid to be tested is illuminated by excitation light, especially single-wavelength excitation light, when passing through the optical detection area of the optical detection device one by one, wherein the sample liquid to be tested is obtained by processing the blood sample with a hemolytic agent, a first dye and a second dye, the first dye being capable of staining white blood cells and the second dye being capable of staining infected red blood cells, wherein the optical signal includes a first fluorescence signal corresponding to the first dye and a second fluorescence signal corresponding to the second dye;

[0024] obtain the infected red blood cell optical information of the blood sample based on the first fluorescent signal and the second fluorescent signal.

[0025] The fifth aspect of the present application relates to a computer readable storage medium having instructions stored thereon, which when executed by a processor, cause the processor to implement the sample analysis method according to the fourth aspect of the present application.

[0026] The sixth aspect of the present application relates to a sample analyzer, comprising:

[0027] a sampling device having a pipette with a pipette nozzle and having a drive device for driving the pipette to quantitatively aspirate a blood sample through the pipette nozzle;

[0028] a sample preparation device having a reaction cell and a reagent supply, wherein the at least one reaction cell is configured to receive the blood sample aspirated by the sampling device, and the reagent supply is configured to supply a hemolytic agent, a first dye and a second dye to the at least one reaction cell, so that the blood sample aspirated by the sampling device is mixed with the hemolytic agent, the first dye and the second dye provided by the reagent supply in the reaction cell to prepare a to-be-tested sample liquid, wherein the first dye is capable of staining white blood cells, and the second dye is capable of staining infected red blood cells;

[0029] an optical detection device comprising a light source, a flow chamber, a first fluorescent detector and a second fluorescent detector, the light source is configured to emit a light beam to irradiate the flow chamber, the flow chamber is in communication with the reaction cell and each particle in the to-be-tested sample liquid can pass through the flow chamber one by one, the first fluorescent detector is configured to detect a first fluorescent signal corresponding to the first dye generated by the particle passing through the flow chamber after being irradiated by light, and the second fluorescent detector is configured to detect a second fluorescent signal corresponding to the second dye generated by the particle passing through the flow chamber after being irradiated by light; and

[0030] a processor configured to perform the following steps: acquiring the first fluorescent signal and the second fluorescent signal of the to-be-tested sample liquid in one test from the optical detection device, and obtaining the infected red blood cell optical information of the blood sample based on the first fluorescent signal and the second fluorescent signal.

[0031] In the technical solutions provided in the aspects of the present application, in one test, especially in a white blood cell test, the same blood sample is treated with a hemolytic agent, a first dye capable of staining white blood cells and a second dye capable of staining infected red blood cells to obtain a sample liquid to be tested, and then an optical detection device is used to detect the scattering light signals, the first fluorescent signals and the second fluorescent signals generated by each particle in the sample liquid to be tested after being irradiated by an excitation light, especially a single-wavelength excitation light, so as to obtain white blood cell optical information according to at least one scattering light signal and the first fluorescent signal, and to obtain infected red blood cell optical information according to at least one scattering light signal and the second fluorescent signal or according to the first fluorescent signal and the second fluorescent signal, thereby being capable of obtaining the white blood cell optical information and the infected red blood cell optical information at the same time without increasing the blood volume, and greatly reducing the detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A schematic appearance diagram of a sample analyzer according to an embodiment of the present application;

[0033] Figure 2 A schematic block diagram of an optical detection device according to an embodiment of the present application;

[0034] Figure 3 A schematic block diagram of an optical detection device according to another embodiment of the present application;

[0035] Figure 4 A schematic flow chart of a sample analysis method according to an embodiment of the present application;

[0036] Figure 5 A schematic flow chart of a sample analysis method according to another embodiment of the present application;

[0037] Figure 6 A schematic diagram of emission spectra of two dyes according to an embodiment of the present application;

[0038] Figure 7 A schematic diagram of emission spectra and excitation spectra of a large Stokes shift dye according to an embodiment of the present application;

[0039] Figure 8A A first scatter plot of Example 1, Figure 8B A second scatter plot of Example 1;

[0040] Figure 9A A first scatter plot of Example 2, Figure 9B A second scatter plot of Example 2;

[0041] Figure 10A A first scatter plot of Example 3, Figure 10B A second scatter plot of Example 3;

[0042] Figure 11A a first scatter plot for Example 4, Figure 11B a second scatter plot for Example 4;

[0043] Figure 12A a first scatter plot for Example 5, Figure 12B a second scatter plot for Example 5;

[0044] Figure 13 a second scatter plot for Example 6. DETAILED DESCRIPTION

[0045] The embodiments of the present application will be described below in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0046] The serial numbers of components in the present application, such as "first", "second", etc., are used only to distinguish the described objects, and have no technical meaning. The "connection" and "coupling" in the present application include direct and indirect connection (coupling) unless otherwise specified.

[0047] The blood cell analyzer used in the present application classifies and counts particles in a blood sample by combining laser scattering method and fluorescence staining flow cytometry technology. The detection principle of the blood cell analyzer is as follows: first, the blood sample is taken and treated with hemolytic agent and fluorescent dye. The red blood cells are destroyed and dissolved by the hemolytic agent, and the white blood cells are not dissolved, but the fluorescent dye can enter the cell nucleus of the white blood cells with the help of the hemolytic agent and combine with the nucleic acid substances in the cell nucleus; then the particles in the sample pass through the detection hole irradiated by the laser beam one by one. When the laser beam irradiates the particles, the characteristics of the particles themselves (such as volume, staining degree, cell content size and content, nuclear density, etc.) can block or change the direction of the laser beam, thereby generating various angle scattering lights corresponding to their characteristics. After these scattering lights are received by the signal detector, the relevant information of the particle structure and composition can be obtained. Among them, the forward scattering light (Forward scatter, FS) reflects the number and volume of the particles, the side scattering light (Side scatter, SS) reflects the complexity of the internal structure of the cells (such as intracellular particles or cell nucleus), and the fluorescence (Fluorescence, FL) reflects the content of nucleic acid substances in the cells. Using these optical information, the particles in the blood sample can be classified and counted.

[0048] Figure 1A schematic diagram of an embodiment of a blood cell analyzer for use in the present application. The blood cell analyzer 100 comprises a sampling device 110, a sample preparation device 120, an optical detection device 130, and a processor 140. The blood cell analyzer 100 has a fluidic system (not shown) for connecting the sampling device 110, the sample preparation device 120, and the optical detection device 130 for fluid transfer between these devices.

[0049] The sampling device 110 has a pipette with a pipette nozzle and a drive device for driving the pipette to quantitatively aspirate a blood sample to be tested through the pipette nozzle. The sampling device can deliver the collected blood sample to be tested to the sample preparation device 120.

[0050] The sample preparation device 120 has at least one reaction cell for receiving the blood sample to be tested aspirated by the sampling device 110 and a reagent supply for providing a hemolytic agent and fluorescent dyes (including a first dye capable of staining white blood cells and a second dye capable of staining infected red blood cells) to the at least one reaction cell, so that the blood sample to be tested aspirated by the sampling device and the hemolytic agent and fluorescent dyes provided by the reagent supply are mixed in the reaction cell to prepare a sample liquid to be tested. The hemolytic agent can be any existing hemolytic agent used to achieve white blood cell classification in an automated blood analyzer, which can be any one or a combination of cationic surfactants, non-ionic surfactants, anionic surfactants, and amphiphilic surfactants. Details of the first dye and the second dye will be further described below.

[0051] The optical detection device 130 includes a light source for emitting a light beam to irradiate a flow cell in communication with the reaction cell and through which each particle in the sample liquid to be tested can pass one by one, at least one scattered light detector for detecting a scattered light signal generated by the particle passing through the flow cell after being irradiated by light, and at least two fluorescence detectors for detecting a fluorescence signal generated by the particle passing through the flow cell after being irradiated by light.

[0052] In some embodiments, the optical detection device 130 includes a forward scattered light detector for detecting forward scattered light or a side scattered light detector for detecting side scattered light. The optical detection device 130 preferably includes both a forward scattered light detector and a side scattered light detector.

[0053] Figure 2A specific example of an optical detection device 130 is shown. This optical detection device 130 includes a laser 131, a front light assembly 132, a flow chamber 133, a forward scattering detector 134, a first dichroic mirror 135, a side scattering detector 136, a second dichroic mirror 137, a first fluorescence detector 138, and a second fluorescence detector 139. The first fluorescence detector 138 detects a first fluorescence signal corresponding to a first dye generated by particles passing through the flow chamber 133 after being irradiated with light, and the second fluorescence detector 139 detects a second fluorescence signal corresponding to a second dye generated by particles passing through the flow chamber 133 after being irradiated with light. Here, the laser 131, the front light assembly 132, the flow chamber 133, and the forward scattering detector 134 are arranged sequentially along the optical axis. The front light assembly is configured to converge the excitation light emitted by the laser 131 into the detection area of ​​the flow chamber 133 in the particle flow direction, so that particles flowing through the detection area of ​​the flow chamber 133 can generate scattered light. On one side of the flow chamber 133, a first dichroic mirror 135 is arranged at a 45° angle to the optical axis. A portion of the lateral light generated when particles flow through the detection area of ​​the flow chamber 133 is reflected by the first dichroic mirror 135 and captured by the side-scattering light detector 136, while another portion of the lateral light passes through the first dichroic mirror 135 and reaches a second dichroic mirror 137, which is also arranged downstream of the first dichroic mirror 135 at a 45° angle to the optical axis. A portion of the lateral light passing through the first dichroic mirror 135 is reflected by the second dichroic mirror 137 and captured by the first fluorescence detector 138, while another portion passes through the second dichroic mirror 137 and is captured by the second fluorescence detector 139.

[0054] In other embodiments, such as Figure 3 As shown, with Figure 2 Unlike the optical detection device shown, the forward-scattering light detector 134 can also be arranged tilted to the optical axis. A reflector 1341 is arranged downstream of the flow chamber along the optical axis, which reflects the forward-scattered light of the particles into the forward-scattering light detector 134, which is tilted to the optical axis.

[0055] The processor 140 is configured to process the optical signals collected by the optical detection device 130 to obtain the desired results. For example, a two-dimensional scatter plot or a three-dimensional scatter plot can be generated based on the collected optical signals, and particle analysis can be performed on the scatter plot based on a gating method. The processor 140 can also perform visualization processing on intermediate operation results or final operation results, and then display the results through the display device 150. In the embodiments of the present application, the processor 140 is configured to implement the methods described in detail below. The processor 140 includes, but is not limited to, a central processing unit (CPU), a micro controller unit (MCU), a field-programmable gate array (FPGA), a digital signal processor (DSP), and other devices for interpreting computer instructions and processing data in computer software. For example, the processor 140 is configured to execute various computer applications in the computer readable storage medium, so that the blood cell analyzer 100 performs corresponding detection processes and analyzes the optical signals detected by the optical detection device 130 in real time.

[0056] In addition, the blood cell analyzer 100 further includes a first housing 160 and a second housing 170. The display device 150 can be a user interface, for example. The optical detection device 130 and the processor 140 are arranged inside the second housing 170. The sample preparation device 120 is arranged inside the first housing 160, for example, and the display device 150 is arranged on the outer surface of the first housing 160 and is used to display the detection results of the blood cell analyzer. In other embodiments, a computer with a display can be remotely connected to the blood cell analyzer 100. The computer can be installed in a place away from the laboratory where the blood cell analyzer is located, such as a doctor's office, for example.

[0057] Next, the detection method proposed in the present application will be described in detail. The method proposed in the present application and each embodiment thereof is particularly applied to the blood cell analyzer 100 described above, and is particularly implemented by the processor 140 of the blood cell analyzer 100 described above.

[0058] In order to simultaneously detect infected red blood cells and white blood cells in one test, the present application proposes for the first time to process and detect the same blood sample using at least two fluorescent dyes under hemolytic conditions, and then to simultaneously identify white blood cells and infected red blood cells based on the optical signals obtained from the same test on the processed same blood sample. In the present application, one dye can stain white blood cells, and the other can stain infected red blood cells.

[0059] Figure 4A schematic flowchart of a sample analysis method 200 according to an embodiment of the present application. The sample analysis method 200 comprises the following steps.

[0060] In step S210, optical signals generated by each particle in a sample liquid to be tested when passing through an optical detection zone of an optical detection device one by one are acquired in one test. In this step, the sample liquid to be tested is obtained by processing a blood sample by using a hemolytic agent, a first dye and a second dye, the first dye being capable of staining white blood cells, and the second dye being capable of staining infected red blood cells, wherein the optical signals include a scattering light signal, a first fluorescent signal corresponding to the first dye and a second fluorescent signal corresponding to the second dye.

[0061] Specifically, first, a blood sample of a subject is provided, which is usually stored in a test tube. A portion of the blood sample in the test tube is taken by a sampling device 110 through a pipette and delivered to a sample preparation device 120. The portion of the blood sample is mixed with a hemolytic agent, a first dye and a second dye in a reaction cell of the sample preparation device 120 and incubated for a period of time, for example, 10 to 30 seconds, to ensure that the red blood cell membrane is destroyed by the hemolytic agent and the cells are stained, thereby forming a sample liquid to be tested. The sample liquid to be tested is delivered to a flow chamber 133 of an optical detection device 130 through a liquid channel system, and each particle in the sample liquid to be tested passes through the detection hole of the flow chamber one by one, and then the scattering light signal, the first fluorescent signal and the second fluorescent signal generated by the particles passing through the flow chamber after being irradiated by light are detected by a scattering light detector 134, 136, a first fluorescent detector 138 and a second fluorescent detector 139, respectively.

[0062] In step S210, the hemolytic agent, the first dye and the second dye can be added to the blood sample successively or simultaneously. It is also possible that the first dye and the second dye are added to the blood sample in a mixed form.

[0063] In step S220, white blood cell optical information of the blood sample is obtained based on at least one of the scattering light signals and the first fluorescent signal. Here, the white blood cell optical information refers to optical information related to white blood cells.

[0064] For example, the white blood cell optical information can be a first scatter plot. In this step, a first scatter plot of the blood sample is generated based on at least one of the scattered light signals and the first fluorescent signal, and then the white blood cells in the sample liquid under test are classified and / or counted based on the first scatter plot. The first scatter plot can be a two-dimensional scatter plot generated from the forward scattered light signal and the first fluorescent signal, a two-dimensional scatter plot generated from the side scattered light signal and the first fluorescent signal, or preferably a three-dimensional scatter plot composed of the forward scattered light signal, the side scattered light signal and the first fluorescent signal. It should be noted that the scatter plot herein is not limited in form, and can also be in the form of data, such as a table or a list of numbers with equivalent or similar resolution to the scatter plot, or in any other suitable form known in the art.

[0065] In step S230, the infected red blood cell optical information of the blood sample is obtained based on at least one of the scattered light signals and the second fluorescent signal, or based on at least the first fluorescent signal and the second fluorescent signal, that is, the infected red blood cell optical information is obtained based on the second fluorescent signal and one of the remaining optical signals other than the second fluorescent signal. Here, the infected red blood cell optical information refers to the optical information related to the infected red blood cells.

[0066] Similarly, the infected red blood cell optical information can be a second scatter plot. For example, the second scatter plot can be a two-dimensional scatter plot generated from the forward scattered light signal or the side scattered light signal and the second fluorescent signal, or a two-dimensional scatter plot composed of the first fluorescent signal and the second fluorescent signal.

[0067] In some embodiments, the first dye is a non-nucleic acid specific dye, and the second dye is a deoxyribonucleic acid (DNA) specific fluorescent dye. The first fluorescent signal is the fluorescence generated after the non-nucleic acid specific dye binds to the white blood cells, and the second fluorescent signal is the fluorescence emitted after the nucleic acid specific dye binds to the malaria infected cells. The nucleic acid dye can specifically stain the infected red blood cells, and because the nucleic acid content of infected red blood cells of different species and / or at different developmental stages differs, the present application can also distinguish infected red blood cells of different species and / or at different developmental stages by the degree of staining by the second dye while counting the infected red blood cells.

[0068] It is particularly advantageous that, in the optical detection device 130 of the present application, the sample liquid to be measured in the flow chamber is irradiated with excitation light of a single wavelength, i.e. the optical signals are generated by individual particles in the sample liquid to be measured being irradiated with excitation light of a single wavelength as they pass through the optical detection zone of the optical detection device one by one. In other words, the light source 131 of the optical detection device 130 is configured as a laser that emits excitation light of a single wavelength. In some embodiments, the light source 131 can be a laser that emits blue-green light or red light, e.g. a laser that emits light at a wavelength of 488 or 520 nanometers.

[0069] In some embodiments, as shown in FIG. 2, the sample analysis method 200 can further comprise a step S221 of classifying and / or counting the white blood cells in the sample liquid to be measured based on the white blood cell optical information. Figure 5

[0070] For example, the step S221 can comprise classifying the white blood cells in the sample liquid to be measured into a neutrophil group, a lymphocyte group, a monocyte group and an eosinophil group based on the white blood cell optical information. Specifically, a first scatter plot is generated based on the side scatter light signal and the first fluorescence signal or based on the forward scatter light signal, the side scatter light signal and the first fluorescence signal, and the white blood cells in the sample liquid to be measured are classified into the neutrophil group, the lymphocyte group, the monocyte group and the eosinophil group and counted based on a gating technique on the first scatter plot.

[0071] In an alternative embodiment, the step S221 can comprise identifying basophilic granulocytes in the sample liquid to be measured and counting the white blood cells in the sample liquid to be measured based on the white blood cell optical information. Specifically, a first scatter plot is generated based on the forward scatter light signal and the first fluorescence signal, and the basophilic granulocytes in the sample liquid to be measured are identified and the white blood cells in the sample liquid to be measured are counted based on the first scatter plot. Further, in this embodiment, nucleated red blood cells in the sample liquid to be measured can also be identified at the same time as the basophilic granulocytes.

[0072] In some embodiments, the sample analysis method 200 can further comprise identifying immature white blood cells in the sample liquid to be measured based on at least one of the scatter light signals and the first fluorescence signal.

[0073] In some embodiments, as shown in FIG. 2, the sample analysis method 200 can further comprise a step S221 of classifying and / or counting the white blood cells in the sample liquid to be measured based on the white blood cell optical information. Figure 5 ​As shown, the sample analysis method 200 can further comprise a step 231 and a step 232. In the step 231, the infected red blood cells are counted according to the infected red blood cell optical information to obtain a count value. For example, a second scatter plot is generated according to the forward scattering light signal and the second fluorescence signal or according to the first fluorescence signal and the second fluorescence signal, and a region representing the infected red blood cells is obtained according to the second scatter plot in the case of using a gating technique, and the scatter points falling into the region are counted to obtain the count value of the infected red blood cells. In the step 232, if the count value of the infected red blood cells is greater than a predetermined threshold value, an alarm prompt is output (judging that the blood sample is a malaria positive sample). Further, different kinds of infected red blood cells and / or infected red blood cells at different developmental stages can also be classified and counted based on the infected red blood cell optical information, for example, the infected red blood cells are classified into at least ring forms, for example, can be classified into ring forms, trophozoites and merozoites.

[0074] Preferably, in order to be able to more accurately distinguish white blood cells and infected red blood cells by two dyes under hemolytic conditions, especially in the case of using the same excitation light source, the first dye and the second dye are selected such that the absolute value of the difference between the wavelengths corresponding to the peak values of the emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers. Alternatively or additionally, the first dye and the second dye are selected such that the amount of overlap of the emission spectra of the first dye and the second dye is not greater than 50%. By selecting such a first dye and a second dye, not only can the detection interference between the first fluorescence signal and the second fluorescence signal be greatly reduced, that is, the discrimination degree of the first fluorescence signal and the second fluorescence signal can be greatly increased, but also the volume and complexity of the optical detection device will not be increased.

[0075] Figure 6 A schematic diagram showing the emission spectra of the first dye and the second dye is shown, the curve shown by the solid line is the emission spectrum 210 of the first dye, and the curve shown by the dashed line is the emission spectrum 220 of the second dye. Among them, the peak point of the emission spectrum 210 of the first dye is D, and the peak point of the emission spectrum 220 of the second dye is A. Here, the difference between the abscissa of the peak point D and the peak point A (that is, the difference between the wavelengths corresponding to the peak values) is greater than 30 nanometers and less than 80 nanometers. In addition, the amount of overlap of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye can be the ratio of the first polygonal area to the second polygonal area, wherein the first polygonal area is equal to the curved polygonal area surrounded by the three points E, G and C, and the second polygonal area is equal to the curved polygonal area surrounded by the emission spectrum 210 of the first dye (or the emission spectrum 220 of the second dye) and the reference line 230, wherein the reference line 230 is as Figure 6The dotted horizontal line parallel to the horizontal axis is shown at 5% of the normalized peak of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye. Points E and F are the left and right intersection points of the emission spectrum 210 of the first dye and the reference line 230, respectively, and points B and C are the left and right intersection points of the emission spectrum 220 of the second dye and the reference line 230, respectively. Here, the overlap of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye is not greater than 50%.

[0076] Further advantageously, especially in the case of illumination with a single light source, the absolute value of the difference between the wavelengths corresponding to the peak values of the emission spectra of the first dye and the second dye is greater than 40 and less than 80 nanometers, preferably greater than 50 nanometers and less than 80 nanometers, more preferably greater than 50 nanometers and less than 70 nanometers, so that the detection interference between the first fluorescent signal and the second fluorescent signal can be further reduced without increasing the volume and complexity of the optical detection device.

[0077] Furthermore advantageously, the overlap of the emission spectra of the first dye and the second dye is not greater than 35%, preferably not greater than 15%, so that the detection interference between the first fluorescent signal and the second fluorescent signal can also be further reduced.

[0078] In some embodiments, at least one of the first dye and the second dye, especially the first dye, can be a large Stokes shift dye. Here, the large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum each have a peak value corresponding to a wavelength difference greater than a predetermined threshold value.

[0079] Figure 7 A schematic diagram of the spectrum of a large Stokes shift dye is shown, in which the excitation spectrum (also referred to as the absorption spectrum) 310 is shown by a dotted line and the emission spectrum 320 is shown by a solid line. Here, the peak point of the excitation spectrum 310 is A1 and the peak point of the emission spectrum 320 is A2. The difference between the respective abscissas of the peak points A2 and A1 (i.e., the wavelength difference corresponding to the respective peak values of the emission spectrum and the excitation spectrum) is greater than a predetermined threshold value. The predetermined threshold value may, for example, be greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.

[0080] By using at least one large Stokes shift dye, the detection interference between the first fluorescent signal and the second fluorescent signal can be reduced.

[0081] In some embodiments, the parent of the first dye can be a meso-amino-substituted cyanine dye or a dye parent having a typical electron push-pull system such as carbazole, coumarin, etc. For example, the first dye can have a parent structure of general formula I:

[0082]

[0083] wherein R1, R2, R3 are substituents, which can be any element, for example a hydrogen element.

[0084] Further details regarding the first dye and the second dye of the present application can be found in the Chinese patent application No. 202011008754.3, the entire disclosure of which is incorporated herein by reference.

[0085] In addition, the present application also provides a computer readable storage medium having instructions stored thereon, which, when executed by a processor, cause the processor to implement the sample analysis method 200 and one of the embodiments thereof.

[0086] The computer readable storage medium described above can be a volatile memory or a non-volatile memory, and can also include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory, a programmable read-only memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a magnetic random access memory, a flash memory, a magnetic surface memory, an optical disk, or a read-only optical disk; the magnetic surface memory can be a disk memory or a tape memory. The volatile memory can be a random access memory, which is used as an external cache. In addition, many forms of RAM can be applied to the present application, such as static random access memory, synchronous static random access memory, dynamic random access memory, synchronous dynamic random access memory, double data rate synchronous dynamic random access memory, enhanced synchronous dynamic random access memory, synchronous connection dynamic random access memory, direct memory bus random access memory.

[0087] The specific implementation methods and corresponding results of the present application are then described with the help of the following specific examples.

[0088] Example 1

[0089] Staining reagent formula:

[0090] First dye 50mg

[0091] Second dye 50mg

[0092] Ethylene glycol 1000g

[0093] wherein the first dye has the following general formula:

[0094] The second dye has the following general formula:

[0095] The BC-6800 supporting 68LN hemolysis agent of Mindray Biomedical Electronics Co., Ltd. is used.

[0096] Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added into 1 ml of hemolytic reagent at the same time, incubated for 30 seconds, and then the sample to be tested is detected by using a flow cytometer to collect forward scattering signal, first fluorescence signal and second fluorescence signal. The first scatter plot shown in FIG. 1 is generated according to the forward scattering light and the first fluorescence signal, according to which leukocytes are identified and counted, and in particular, basophils in the leukocytes are identified. Figure 8A The second scatter plot shown in FIG. 2 is generated according to the forward scattering light and the second fluorescence signal, according to which infected red blood cells are identified and classified into ring bodies, trophozoites and schizonts. Figure 8B The second scatter plot shown in FIG. 2 is generated according to the forward scattering light and the second fluorescence signal, according to which infected red blood cells are identified and classified into ring bodies, trophozoites and schizonts.

[0097] Example 2

[0098] Staining reagent formula:

[0099] First dye 50 mg

[0100] Second dye 50 mg

[0101] Ethylene glycol 1000 g

[0102] The first dye has the following general formula:

[0103] The second dye has the following general formula:

[0104] BC-6800 from Meiya Biomedical Electronics Co., Ltd. is used.

[0105] Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added into 1 ml of hemolytic reagent at the same time, incubated for 30 seconds, and then the sample to be tested is detected by using a flow cytometer to collect forward scattering signal, first fluorescence signal and second fluorescence signal. The first scatter plot shown in FIG. 1 is generated according to the forward scattering light and the first fluorescence signal, according to which leukocytes are identified and counted, and in particular, basophils in the leukocytes are identified. Figure 9A The second scatter plot shown in FIG. 2 is generated according to the forward scattering light and the second fluorescence signal, according to which infected red blood cells are identified and classified into ring bodies, trophozoites and schizonts. Figure 9B The second scatter plot shown in FIG. 2 is generated according to the forward scattering light and the second fluorescence signal, according to which infected red blood cells are identified and classified into ring bodies, trophozoites and schizonts.

[0106] Example 3

[0107] Staining reagent formula:

[0108] First dye 50 mg

[0109] Second dye 50 mg

[0110] Ethylene glycol 1000 g

[0111] wherein the first dye has the following general formula:

[0112] The second dye has the following general formula:

[0113] A 68LN hemolytic agent from Mindray Biomedical Electronics Co., Ltd. is used.

[0114] Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added to 1 ml of hemolytic agent at the same time, incubated for 30 seconds, and after incubation, the test sample is detected by using a flow cytometer, and forward scattering signals, first fluorescence signals and second fluorescence signals are collected. The first scatter plot shown in Figure 10A is generated according to the forward scattering light and the first fluorescence signal, and white blood cells are identified according to the first scatter plot and counted accordingly. The second scatter plot shown in Figure 9B is generated according to the forward scattering light and the second fluorescence signal, and infected red blood cells are identified according to the second scatter plot and classified into ring bodies, trophozoites and schizonts.

[0115] Example 4

[0116] Staining reagent formula:

[0117] First dye 50 mg

[0118] Second dye 50 mg

[0119] Ethylene glycol 1000 g

[0120] wherein the first dye has the following general formula:

[0121] The second dye has the following general formula:

[0122] A 68LN hemolytic agent from Mindray Biomedical Electronics Co., Ltd. is used.

[0123] Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added to 1 ml of hemolytic agent at the same time, incubated for 30 seconds, and after incubation, the test sample is detected by using a flow cytometer, and forward scattering signals, first fluorescence signals and second fluorescence signals are collected. The first scatter plot shown in Figure 11A is generated according to the forward scattering light and the first fluorescence signal, and white blood cells are classified into neutrophil group, lymphocyte group, monocyte group and eosinophil group according to the first scatter plot. The second scatter plot shown in Figure 11B is generated according to the forward scattering light and the second fluorescence signal, and infected red blood cells are identified according to the second scatter plot.

[0124] Example 5

[0125] Staining reagent formulation:

[0126] First dye 50mg

[0127] Second dye 50mg

[0128] Ethylene glycol 1000g

[0129] wherein the first dye has the following general formula:

[0130] The second dye has the following general formula:

[0131] A 68LD hemolytic agent from Mindray Biomedical Electronics Co., Ltd. BC-6800 is used.

[0132] Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added to 1 ml of hemolytic agent at the same time, incubated for 30 seconds, and after incubation, the test sample is detected by flow cytometry, and the forward scattering signal, side light scattering signal, first fluorescence signal and second fluorescence signal are collected. According to the side light scattering and the first fluorescence signal, the first scatter plot shown in FIG. 1 is generated, and according to the first scatter plot, the white blood cells are classified into neutrophil group, lymphocyte group, monocyte group and eosinophil group. According to the forward scattering light and the second fluorescence signal, the second scatter plot shown in FIG. 2 is generated, and according to the second scatter plot, the infected red blood cells are identified. Figure 12A Figure 12B

[0133] Example 6

[0134] Staining reagent formulation:

[0135] First dye 50mg

[0136] Second dye 50mg

[0137] Ethylene glycol 1000g

[0138] wherein the first dye has the following general formula:

[0139] The second dye has the following general formula:

[0140] A 68LN hemolytic agent from Mindray Biomedical Electronics Co., Ltd. BC-6800 is used.

[0141] ​​Test method: 20 microliters of blood sample and 20 microliters of staining reagent are added into 1ml hemolytic agent at the same time, incubated for 30 seconds, after incubation, the first fluorescence signal and the second fluorescence signal are collected by flow cytometry. According to the first fluorescence signal and the second fluorescence signal, the second scatter diagram shown in the formula (2) is generated, according to the second scatter diagram, the infected red blood cells are identified and classified into ring bodies, trophozoites and schizonts. Figure 13

[0142] The features or combinations of features mentioned in the specification, drawings and claims can be used or implemented alone or in any combination, as long as they are meaningful and do not contradict each other within the scope of the application. The advantages and features described with reference to the sample analysis method provided by the application are applicable to the sample analysis instrument and computer readable storage medium provided by the application in a corresponding manner, and vice versa.

[0143] The above description is only the preferred embodiment of the application, and does not limit the patent protection scope of the application. Any equivalent transformation or direct / indirect application in other related technical fields based on the application concept, the content of the specification and drawings of the application are included in the patent protection scope of the application.​

Claims

1. A sample analysis method for analyzing a blood sample, characterized by, comprising: acquiring, in one test, optical signals generated by each particle in a sample liquid to be tested when the particle passes through an optical detection zone of an optical detection device one by one, wherein the sample liquid to be tested is obtained by treating the blood sample with a hemolytic agent, a first dye and a second dye, the first dye being capable of staining white blood cells, and the second dye being capable of staining infected red blood cells, and wherein the optical signals include a scattering light signal, a first fluorescent signal corresponding to the first dye, and a second fluorescent signal corresponding to the second dye; obtaining white blood cell optical information of the blood sample based on at least one of the scattering light signal and the first fluorescent signal; obtaining infected red blood cell optical information of the blood sample based on at least one of the scattering light signal and the second fluorescent signal.

2. The sample analysis method of claim 1, wherein, The optical signals are generated by each particle in the sample liquid to be tested when the particle passes through the optical detection zone of the optical detection device one by one under irradiation of excitation light of a single wavelength.

3. The sample analysis method according to claim 1 or 2, characterized by further comprising: classifying and / or counting white blood cells in the sample liquid to be tested based on the white blood cell optical information.

4. The sample analysis method of claim 3, wherein, The step of classifying and / or counting white blood cells in the sample liquid to be tested based on the white blood cell optical information comprises: classifying white blood cells in the sample liquid to be tested into a neutrophil group, a lymphocyte group, a monocyte group and an eosinophil group based on the white blood cell optical information; or identifying basophilic granulocytes in the sample liquid to be tested and counting white blood cells in the sample liquid to be tested based on the white blood cell optical information.

5. The sample analysis method according to claim 1 or 2, characterized by further comprising: identifying nucleated red blood cells and / or immature white blood cells in the sample liquid to be tested based on at least one of the scattering light signal and the first fluorescent signal.

6. The sample analysis method of claim 1 or 2, wherein further comprising: counting infected red blood cells in the sample liquid to be tested and optionally classifying and counting infected red blood cells of different kinds and / or infected red blood cells of different developmental stages based on the infected red blood cell optical information.

7. The sample analysis method according to claim 1 or 2, characterized by, The absolute value of the difference between the wavelengths corresponding to the peak values of the emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers, and / or the overlap amount of the emission spectra of the first dye and the second dye is not greater than 50%.

8. The sample analysis method according to claim 1 or 2, characterized by, The difference between the wavelengths corresponding to the peak values of the emission spectrum and the excitation spectrum of at least one of the first dye and the second dye is greater than a predetermined threshold value.

9. A computer-readable storage medium having instructions stored thereon, which, when executed by a processor, cause the processor to implement a sample analysis method according to any one of claims 1 to 8.

10. A sample analyzer characterized by, comprising: a sampling device having a pipette with a pipette tip and having a drive device for driving the pipette to quantitatively take up a blood sample through the pipette tip; A sample preparation device has a reaction cell for receiving a blood sample drawn by a sampling device and a reagent supply for providing a hemolytic agent, a first dye and a second dye to the reaction cell, so that the blood sample drawn by the sampling device is mixed with the hemolytic agent, the first dye and the second dye provided by the reagent supply in the reaction cell to prepare a test sample liquid, wherein the first dye is capable of staining leukocytes and the second dye is capable of staining infected red blood cells; An optical detection device includes a light source for emitting a light beam to illuminate a flow cell, the flow cell being in communication with the reaction cell and through which each particle in the test sample liquid can pass one by one, a scattered light detector for detecting a scattered light signal generated by a particle passing through the flow cell after being illuminated by the light, a first fluorescence detector for detecting a first fluorescence signal corresponding to the first dye generated by a particle passing through the flow cell after being illuminated by the light, and a second fluorescence detector for detecting a second fluorescence signal corresponding to the second dye generated by a particle passing through the flow cell after being illuminated by the light; and A processor configured to perform the following steps: acquiring the scattered light signal, the first fluorescence signal and the second fluorescence signal of the test sample liquid in one test from the optical detection device, obtaining leukocyte optical information of the blood sample based on at least one of the scattered light signal and the first fluorescence signal, and obtaining infected red blood cell optical information of the blood sample based on at least one of the scattered light signal and the second fluorescence signal.

11. The sample analyzer of claim 10, wherein, The light source is configured to emit excitation light of a single wavelength.

12. The sample analyzer of claim 10 or 11, wherein, The processor is further configured to classify and / or count leukocytes in the test sample liquid based on the leukocyte optical information.

13. The sample analyzer of claim 12, wherein, When performing the step of classifying and / or counting leukocytes in the test sample liquid based on the leukocyte optical information, the processor is further configured to: classify leukocytes in the test sample liquid into a neutrophil group, a lymphocyte group, a monocyte group and an eosinophil group based on the leukocyte optical information; or identify basophils in the test sample liquid and count leukocytes in the test sample liquid based on the leukocyte optical information. The processor is further configured to identify nucleated red blood cells and / or immature leukocytes in the test sample liquid based on at least one of the scattered light signal and the first fluorescence signal.

14. The sample analyzer of claim 10 or 11, wherein, The processor is further configured to count infected red blood cells in the test sample liquid based on the infected red blood cell optical information, and optionally classify and count infected red blood cells of different kinds and / or infected red blood cells at different developmental stages.

15. The sample analyzer of claim 10 or 11, wherein, The absolute value of the difference between the wavelengths corresponding to the peak values of the emission spectra of the first dye and the second dye is greater than 30 nm and less than 80 nm, and / or the overlap of the emission spectra of the first dye and the second dye is not greater than 50%.

16. The sample analyzer of claim 10 or 11, wherein, ​ 17. The sample analyzer of claim 10 or 11, wherein, The difference between the wavelengths corresponding to the peak values of the emission spectrum and the excitation spectrum of at least one of the first dye and the second dye is greater than a predetermined threshold value.

18. A sample analysis method for analyzing a blood sample, characterized by, Comprising: obtaining, in one test, optical signals generated by each particle in a sample liquid to be tested when passing through an optical detection region of an optical detection device one by one under irradiation of excitation light, wherein the sample liquid to be tested is obtained by treating the blood sample with a hemolytic agent, a first dye and a second dye, the first dye being capable of staining leukocytes, and the second dye being capable of staining infected red blood cells, wherein the optical signals include a first fluorescent signal corresponding to the first dye and a second fluorescent signal corresponding to the second dye; obtaining infected red blood cell optical information of the blood sample based on the first fluorescent signal and the second fluorescent signal.

19. The sample analysis method of claim 18, wherein, The optical signals are generated by each particle in the sample liquid to be tested when passing through the optical detection region of the optical detection device one by one under irradiation of excitation light of a single wavelength.

20. The sample analysis method of claim 18 or 19, wherein, The optical signals further include scattering light signals, and the sample analysis method further comprises: obtaining leukocyte optical information of the blood sample based on at least one of the scattering light signals and the first fluorescent signal; classifying and / or counting leukocytes in the sample liquid to be tested based on the leukocyte optical information.

21. The sample analysis method of claim 20, wherein, The step of classifying and / or counting leukocytes in the sample liquid to be tested based on the leukocyte optical information comprises: classifying leukocytes in the sample liquid to be tested into a neutrophil group, a lymphocyte group, a monocyte group and an eosinophil group based on the leukocyte optical information; or identifying basophilic granulocytes in the sample liquid to be tested based on the leukocyte optical information and counting leukocytes in the sample liquid to be tested.

22. The sample analysis method of claim 20, wherein Further comprising: identifying nucleated red blood cells and / or immature leukocytes in the sample liquid to be tested based on at least one of the scattering light signals and the first fluorescent signal.

23. The sample analysis method of claim 18 or 19, wherein Further comprising: counting infected red blood cells in the sample liquid to be tested based on the infected red blood cell optical information and optionally classifying and counting infected red blood cells of different kinds and / or infected red blood cells at different developmental stages.

24. The sample analysis method of claim 18 or 19, wherein, The difference between the wavelengths corresponding to the peak values of the emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers, and / or the overlap amount of the emission spectra of the first dye and the second dye is not greater than 50%.

25. A computer-readable storage medium having instructions stored thereon, which, when executed by a processor, cause the processor to implement the sample analysis method according to any one of claims 18 to 24.

26. A sample analyzer comprising: Comprising: a sampling device having a pipette with a pipette tip and having a drive device for driving the pipette to quantitatively take up a blood sample through the pipette tip; A sample preparation device has a reaction cell for receiving a blood sample drawn by a sampling device and a reagent supply for providing a hemolytic agent, a first dye and a second dye to the reaction cell, so that the blood sample drawn by the sampling device is mixed with the hemolytic agent, the first dye and the second dye provided by the reagent supply in the reaction cell to prepare a sample liquid to be tested, wherein the first dye is capable of staining white blood cells and the second dye is capable of staining infected red blood cells; An optical detection device includes a light source for emitting a light beam to illuminate a flow cell, the flow cell being in communication with the reaction cell and each particle in the sample liquid to be tested being able to pass through the flow cell one by one, a first fluorescence detector for detecting a first fluorescence signal corresponding to the first dye generated by a particle passing through the flow cell after being illuminated by light, and a second fluorescence detector for detecting a second fluorescence signal corresponding to the second dye generated by a particle passing through the flow cell after being illuminated by light; and A processor configured to perform the following steps: obtaining the first fluorescence signal and the second fluorescence signal of the sample liquid to be tested in a test from the optical detection device, and obtaining infected red blood cell optical information of the blood sample based on the first fluorescence signal and the second fluorescence signal.

27. The sample analyzer of claim 26, wherein, The light source is configured to emit excitation light of a single wavelength.

28. The sample analyzer of claim 26 or 27, wherein, The optical detection device further includes a scattered light detector for detecting a scattered light signal generated by a particle passing through the flow cell after being illuminated by light; The processor is further configured to: obtain the scattered light signal in the test from the optical detection device, obtain white blood cell optical information of the sample liquid to be tested based on at least one of the scattered light signal and the first fluorescence signal, classify and / or count white blood cells in the sample liquid to be tested based on the white blood cell optical information.

29. The sample analyzer of claim 28, wherein, The processor is further configured to, when performing the step of classifying and / or counting white blood cells in the sample liquid to be tested based on the white blood cell optical information: classify white blood cells in the sample liquid to be tested into a neutrophil group, a lymphocyte group, a monocyte group and an eosinophil group based on the white blood cell optical information; or identify basophilic granulocytes in the sample liquid to be tested and count white blood cells in the sample liquid to be tested based on the white blood cell optical information. The processor is further configured to identify nucleated red blood cells and / or immature white blood cells in the sample liquid to be tested based on at least one of the scattered light signal and the first fluorescence signal.

30. The sample analyzer of claim 28, wherein, The processor is further configured to count infected red blood cells in the sample liquid to be tested based on the infected red blood cell optical information, and optionally classify and count infected red blood cells of different kinds and / or infected red blood cells at different developmental stages.

31. The sample analyzer of claim 26 or 27, wherein, ​ 32. The sample analyzer of claim 26 or 27, wherein, an absolute value of a difference between wavelengths corresponding to peak values of emission spectra of the first dye and the second dye is greater than 30 nanometers and less than 80 nanometers, and / or an amount of overlap of the emission spectra of the first dye and the second dye is no greater than 50%.

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