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, optical signals generated when particles in a to-be-detected sample solution are irradiated by exciting light when passing through an optical detection area one by one are obtained, the to-be-detected sample solution is obtained after a blood sample is treated with a hemolytic agent, a first dye and a second dye, the first dye can dye white blood cells, and the second dye can dye white blood cells. The second dye can dye infected red blood cells, and the optical signal comprises a scattered light signal, a first fluorescence signal corresponding to the first dye and a second fluorescence signal corresponding to the second dye; identifying nucleated red blood cells based on the scattered light signal and the first fluorescence signal; erythrocyte optical information is obtained based on the scattered light signal and the second fluorescence signal. According to the method, the optical information of the nucleated red blood cells and the optical information of the infected red blood cells can be simultaneously obtained in the same detection channel or the same test, so that the blood volume and the cost for detection are reduced.

Description

[0002] This application is a divisional application of Chinese invention patent application number "202080107626.6". The original application was filed on December 1, 2020, and the invention was entitled "Sample Analysis Method, Sample Analyzer and Computer-Readable Storage Medium". Technical Field

[0003] This application relates to the field of blood testing, and in particular to a sample analysis method, a sample analyzer, and a computer-readable storage medium. Background Technology

[0004] Malaria is a serious disease that threatens human health, caused by Plasmodium parasites. Currently, blood smear microscopy is commonly used to examine for Plasmodium parasites, but this method is highly dependent on the operator's experience, requires a high level of professional skills, and is time-consuming.

[0005] With the development of blood cell analysis technology, there are now several known methods for rapidly detecting red blood cells infected with malaria parasites using blood cell analyzers.

[0006] European patent application EP0613003B1 discloses a method for staining infected red blood cells using a variety of fluorescent dyes under non-hemolytic conditions, in order to better distinguish between reticulocytes and infected red blood cells.

[0007] European patent application EP1406088A2 discloses a method for detecting malaria parasites by combining fluorescent dyes under hemolytic conditions, which can classify and count malaria parasites, but cannot classify and count white blood cells at the same time.

[0008] US Patent Application US2006 / 0223137 discloses a reagent that can partially dissolve the cell membrane of red blood cells infected with malaria parasites, allowing the parasites to remain within the red blood cells while a fluorescent dye can pass through the cell membrane. However, this reagent cannot accurately detect malaria-infected red blood cells when the sample has a high reticulocyte count.

[0009] Chinese patent application CN106483278B discloses a method for detecting red blood cells infected with malaria parasites. This method uses a specific concentration of a specific fluorescent dye to treat the sample, thereby enabling more accurate detection of red blood cells infected with malaria parasites than the method disclosed in US patent application US2006 / 0223137.

[0010] Chinese patent application CN102016573B discloses a blood analysis device and method that can classify white blood cells in a 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 hemolysing agent is used, two blood samples need to be provided for different treatments, and white blood cell classification and malaria-infected red blood cell detection are performed separately in two tests, thus increasing the test time, blood volume, and hemolysing agent cost. Summary of the Invention

[0011] The objective of this application is to provide an improved method for detecting malaria, which enables the simultaneous detection of nucleated red blood cell parameters and infected red blood cell parameters in a single test, particularly in an existing white blood cell detection channel. Compared to existing technologies, this method can obtain multiple detection parameters in a single test, saving blood volume and reducing testing costs.

[0012] To achieve the objectives of this application, the first aspect of this application relates to a sample analysis method for analyzing blood samples, comprising:

[0013] In a single test, the optical signal generated by each particle in the test sample solution being irradiated by excitation light, especially single-wavelength excitation light, as it passes through the optical detection area of ​​an optical detection device, is obtained. The test sample solution is obtained by treating the blood sample with a hemolytic agent, a first dye, and a second dye. The first dye can stain leukocytes, and the second dye can stain infected erythrocytes. The optical signal includes a scattered light signal, a first fluorescence signal corresponding to the first dye, and a second fluorescence signal corresponding to the second dye.

[0014] Nucleated red blood cells in the blood sample are identified based on at least one of the scattered light signals and the first fluorescence signal;

[0015] Optical information of infected red blood cells in the blood sample is obtained based on at least one of the scattered light signals and the second fluorescence signal.

[0016] The second aspect of this 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 first aspect of this application.

[0017] The third aspect of this application relates to a sample analyzer, comprising:

[0018] A sampling device having a suction tube with a suction nozzle and a driving device for driving the suction tube to quantitatively aspirate blood samples through the suction nozzle;

[0019] A sample preparation apparatus includes a reaction chamber and a reagent supply unit, wherein the at least one reaction chamber is used to receive a blood sample drawn by a sampling device, and the reagent supply unit provides a hemolysin, a first dye, and a second dye to the at least one reaction chamber, thereby mixing the blood sample drawn by the sampling device with the hemolysin, the first dye, and the second dye provided by the reagent supply unit in the reaction chamber to prepare a sample solution to be tested, wherein the first dye is capable of staining leukocytes and the second dye is capable of staining infected erythrocytes;

[0020] An optical detection device includes a light source, a flow chamber, a scattered light detector, a first fluorescence detector, and a second fluorescence detector. The light source emits a light beam to illuminate the flow chamber. The flow chamber is connected to a reaction cell, and each particle in the sample solution to be tested can pass through the flow chamber one by one. The scattered light detector detects the scattered light signal generated by the particles passing through the flow chamber after being illuminated. The first fluorescence detector detects a first fluorescence signal corresponding to a first dye generated by the particles passing through the flow chamber after being illuminated. The second fluorescence detector detects a second fluorescence signal corresponding to a second dye generated by the particles passing through the flow chamber after being illuminated.

[0021] A processor configured to perform the following steps: acquiring a scattered light signal, a first fluorescence signal, and a second fluorescence signal of the sample solution to be tested in a single test from the optical detection device; identifying nucleated red blood cells of the blood sample based on at least one of the scattered light signals and the first fluorescence signal; and obtaining optical information of infected red blood cells of the blood sample based on at least one of the scattered light signals and the second fluorescence signal.

[0022] The processor of the sample analyzer according to the third aspect of this application is specifically configured to implement the sample analysis method according to the first aspect of this application.

[0023] In the technical solutions provided in this application, in a single test, especially in a leukocyte test, the same blood sample is treated with a hemolytic agent, a first dye capable of staining leukocytes, and a second dye capable of staining infected erythrocytes to obtain a test sample solution. Then, an optical detection device is used to detect the scattered light signal, the first fluorescence signal, and the first fluorescence signal generated by each particle in the test sample solution after being irradiated by excitation light, especially excitation light of a single wavelength. Thus, optical information of nucleated erythrocytes is obtained based on at least one scattered light signal and the first fluorescence signal, and optical information of infected erythrocytes is obtained based on at least one scattered light signal and the second fluorescence signal, or based on the first fluorescence signal and the second fluorescence signal. This allows for the simultaneous acquisition of optical information of nucleated erythrocytes and infected erythrocytes without increasing the amount of blood used, greatly reducing the detection cost. Attached Figure Description

[0024] Figure 1 This is a schematic appearance diagram of a sample analyzer according to an embodiment of this application;

[0025] Figure 2 This is a schematic block diagram of an optical detection apparatus according to an embodiment of this application;

[0026] Figure 3 This is a schematic block diagram of an optical detection apparatus according to another embodiment of this application;

[0027] Figure 4 This is a schematic flowchart of a sample analysis method according to an embodiment of this application;

[0028] Figure 5 This is a schematic flowchart of a sample analysis method according to another embodiment of this application;

[0029] Figure 6 This is a schematic diagram of the emission spectra of two dyes according to an embodiment of this application;

[0030] Figure 7 This is a schematic diagram of the emission and excitation spectra of a large Stokes shift dye according to an embodiment of this application;

[0031] Figure 8A This is the first scatter plot of Example 1. Figure 8B This is the second scatter plot of Example 1;

[0032] Figure 9A This is the first scatter plot of Example 2. Figure 9B This is the second scatter plot of Example 2;

[0033] Figure 10A This is the first scatter plot of Example 3. Figure 10B This is the second scatter plot of Example 3;

[0034] Figure 11A This is the first scatter plot of Example 4. Figure 11B This is the second scatter plot of Example 4;

[0035] Figure 12A This is the first scatter plot of Example 5. Figure 12B This is the second scatter plot of Example 5;

[0036] Figure 13 This is the second scatter plot of Example 6. Detailed Implementation

[0037] The embodiments of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0038] The serial numbers assigned to components in this document, such as "first" and "second," are used only to distinguish the described objects and have no sequential or technical meaning. The terms "connection" and "linkage" used in this application, unless otherwise specified, include both direct and indirect connections (linkages).

[0039] The blood cell analyzer used in this application classifies and counts particles in blood samples using flow cytometry combined with laser scattering and fluorescent staining. The detection principle of the blood cell analyzer is as follows: First, a blood sample is drawn and treated with a hemolysin and fluorescent dye. Red blood cells are destroyed and dissolved by the hemolysin, while white blood cells are not dissolved. However, the fluorescent dye can enter the nucleus of white blood cells with the help of the hemolysin and bind to nucleic acid substances in the nucleus. Next, the particles in the sample pass one by one through a detection aperture illuminated by a laser beam. When the laser beam illuminates the particles, the characteristics of the particles themselves (such as volume, staining degree, size and content of cell contents, and nuclear density) can block or change the direction of the laser beam, thereby generating scattered light at various angles corresponding to their characteristics. This scattered light is received by a signal detector to obtain information about the particle structure and composition. Specifically, forward scatter (FS) reflects the number and volume of particles, side scatter (SS) reflects the complexity of the internal structure of cells (such as intracellular particles or the cell nucleus), and fluorescence (FL) reflects the content of nucleic acid substances in the cells. This optical information can be used to classify and count particles in blood samples.

[0040] Figure 1 This is a schematic diagram of one embodiment of the blood cell analyzer used in this application. The blood cell analyzer 100 includes 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 liquid path system (not shown) for connecting the sampling device 110, the sample preparation device 120, and the optical detection device 130 to facilitate liquid transfer between these devices.

[0041] The sampling device 110 has a pipette with a suction nozzle and a driving device for driving the pipette to quantitatively aspirate the blood sample to be tested through the suction nozzle. The sampling device can transport the collected blood sample to the sample preparation device 120.

[0042] The sample preparation apparatus 120 has at least one reaction chamber and a reagent supply unit. The at least one reaction chamber receives the blood sample to be tested aspirated by the sampling device 110. The reagent supply unit provides a hemolysin and a fluorescent dye (including a first dye capable of staining leukocytes and a second dye capable of staining infected erythrocytes) to the at least one reaction chamber. The blood sample to be tested aspirated by the sampling device is mixed with the hemolysin and fluorescent dye provided by the reagent supply unit in the reaction chamber to prepare a sample solution for testing. The hemolysin can be any existing hemolysin used for leukocyte classification in automated blood analyzers, and can be any one or a combination of several of cationic surfactants, nonionic surfactants, anionic surfactants, and amphiphilic surfactants. Details regarding the first and second dyes will be further described below.

[0043] The optical detection device 130 includes a light source, a flow chamber, at least one scattered light detector, and at least two fluorescence detectors. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction cell, and each particle in the sample solution to be tested can pass through the flow chamber one by one. The scattered light detector is used to detect the scattered light signal generated by the particles passing through the flow chamber after being illuminated by light. The fluorescence detector is used to detect the fluorescence signal generated by the particles passing through the flow chamber after being illuminated by light.

[0044] In some embodiments, the optical detection device 130 includes a forward-scattering detector for detecting forward-scattered light or a side-scattering detector for detecting side-scattered light. Preferably, the optical detection device 130 includes both a forward-scattering detector and a side-scattering detector.

[0045] 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.

[0046] 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.

[0047] The processor 140 processes the optical signals collected by the optical detection device 130 to obtain the desired results. For example, it can generate two-dimensional or three-dimensional scatter plots based on the collected optical signals and perform particle analysis on the scatter plots using a gating method. The processor 140 can also visualize intermediate or final calculation results and then display them through the display device 150. In this embodiment, the processor 140 is configured to implement the methods described in further detail below. The processor 140 includes, but is not limited to, devices such as a central processing unit (CPU), a microcontroller unit (MCU), a field-programmable gate array (FPGA), and a digital signal processor (DSP) used to interpret computer instructions and process data in computer software. For example, the processor 140 executes various computer applications in a computer-readable storage medium, thereby enabling the blood cell analyzer 100 to perform corresponding detection procedures and analyze the optical signals detected by the optical detection device 130 in real time.

[0048] In addition, the blood cell analyzer 100 also includes a first housing 160 and a second housing 170. The display device 150 may be, for example, a user interface. An optical detection device 130 and a processor 140 are disposed inside the second housing 170. A sample preparation device 120 is disposed, for example, inside the first housing 160, and the display device 150 is disposed, for example, 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, for example, installed in a location far from the laboratory where the blood cell analyzer is located, such as in a doctor's examination room.

[0049] The detection method proposed in this application will then be described in detail. The method and its various embodiments proposed in this application are particularly applicable to the blood cell analyzer 100 described above, and are implemented by the processor 140 of the blood cell analyzer 100.

[0050] To achieve simultaneous detection of infected red blood cells and white blood cells in a single test, this application proposes for the first time to treat and detect the same blood sample under hemolytic conditions using at least two fluorescent dyes, and then simultaneously identify white blood cells and infected red blood cells based on the optical signals obtained from the same test on the same treated blood sample. In this application, one dye can stain white blood cells, while the other can stain infected red blood cells.

[0051] Figure 4This is a schematic flowchart of a sample analysis method 200 according to an embodiment of the present application. The sample analysis method 200 includes the following steps.

[0052] Step S210 involves acquiring the optical signals generated when each particle in the test sample solution is irradiated by excitation light as it passes through the optical detection area of ​​the optical detection device in a single test. In this step, the test sample solution is obtained by treating a 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 scattered light signal, a first fluorescence signal corresponding to the first dye, and a second fluorescence signal corresponding to the second dye.

[0053] Specifically, a blood sample from a subject is first provided. The blood sample is typically stored in a test tube. A portion of the blood sample is drawn from the test tube by the sampling device 110 via a pipette and transported to the sample preparation device 120. This portion of the blood sample is mixed with a hemolysin, a first dye, and a second dye in the reaction chamber 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 membranes are destroyed by the hemolysin and the cells are stained, thereby forming the test sample solution. The test sample solution is transported to the flow chamber 133 of the optical detection device 130 via a liquid system, allowing each particle in the test sample solution to pass through the detection aperture of the flow chamber one by one. Then, the scattered light detectors 134 and 136, the first fluorescence detector 138, and the second fluorescence detector 139 detect the scattered light signal, the first fluorescence signal, and the second fluorescence signal generated by the particles passing through the flow chamber after being irradiated with light, respectively.

[0054] In step S210, the hemolysin, the first dye, and the second dye may be added to the blood sample sequentially, or simultaneously. Alternatively, the first dye and the second dye may be added to the blood sample in a mixed form.

[0055] Step S220: Obtain leukocyte optical information of the blood sample based on at least one of the scattered light signals and the first fluorescence signal. Here, leukocyte optical information refers to optical information related to leukocytes.

[0056] For example, the optical information of white blood cells can be a first scatter plot. In this step, a first scatter plot of the blood sample is generated based on at least one scattered light signal and the first fluorescence signal, and then the white blood cells in the sample solution are classified and / or counted based on the first scatter plot. The first scatter plot can be a two-dimensional scatter plot generated by the forward scattered light signal and the first fluorescence signal, or a two-dimensional scatter plot generated by the side scattered light signal and the first fluorescence signal, or preferably a three-dimensional scatter plot composed of the forward scattered light signal, the side scattered light signal, and the first fluorescence signal. It should be noted that the scatter plot in this document is not limited to a graphical form and can also be in data form, such as a table or list with the same or similar resolution as the scatter plot, or presented in any other suitable manner known in the art.

[0057] Step S230: Obtain the optical information of infected red blood cells in the blood sample based on at least one of the scattered light signals and the second fluorescence signal, or at least based on the first fluorescence signal and the second fluorescence signal. That is, obtain the optical information of infected red blood cells based on the second fluorescence signal and one of the other optical signals besides the second fluorescence signal. Here, the optical information of infected red blood cells refers to the optical information related to infected red blood cells.

[0058] Similarly, the optical information of infected red blood cells can be a second scatter plot. For example, this second scatter plot can be a two-dimensional scatter plot generated by forward-scattered light signals or side-scattered light signals and a second fluorescence signal, or a two-dimensional scatter plot composed of a first fluorescence signal and a second fluorescence signal.

[0059] In some embodiments, the first dye is a non-nucleic acid-specific dye, while the second dye is a deoxyribonucleic acid (DNA)-specific fluorescent dye. The first fluorescent signal is the fluorescence produced after the non-nucleic acid-specific dye binds to leukocytes, and the second fluorescent signal is the fluorescence emitted after the nucleic acid-specific dye binds to malaria-infected cells. The nucleic acid dye can specifically stain infected erythrocytes, and because the nucleic acid content varies among different types and / or developmental stages of infected erythrocytes, this application can distinguish between different types and / or developmental stages of infected erythrocytes by the degree of staining of the second dye while simultaneously counting infected erythrocytes.

[0060] A particularly advantageous feature is that in the optical detection device 130 of this application, the sample liquid to be tested in the flow chamber is irradiated with excitation light of a single wavelength. That is, the optical signal is generated by each particle in the sample liquid being irradiated by the excitation light of a single wavelength as it passes through the optical detection area 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 may be a laser that emits blue-green or red light, for example, a laser that emits light with a wavelength of 488 or 520 nanometers.

[0061] In some embodiments, such as Figure 5 As shown, the sample analysis method 200 may further include step 221: classifying and / or counting the white blood cells in the sample solution to be tested based on the white blood cell optical information.

[0062] For example, step S221 may include: classifying the white blood cells in the sample solution to be tested into neutrophils, lymphocytes, monocytes, and eosinophils based on the white blood cell optical information. Specifically, a first scatter plot is generated based on the side-scattered light signal and the first fluorescence signal, or based on the forward-scattered light signal, the side-scattered light signal, and the first fluorescence signal. On the first scatter plot, the white blood cells in the sample solution to be tested are classified into neutrophils, lymphocytes, monocytes, and eosinophils according to gating technology, and these cell groups are counted.

[0063] In an alternative embodiment, step S221 may include: identifying basophils in the test sample solution based on the leukocyte optical information and counting the leukocytes in the test sample solution. Specifically, a first scatter plot is generated based on the forward scattered light signal and the first fluorescence signal, and basophils in the test sample solution are identified and counted based on the first scatter plot. Further, in this embodiment, nucleated red blood cells in the test sample solution can also be identified simultaneously with basophils.

[0064] In some embodiments, the sample analysis method 200 may further include identifying immature leukocytes in the sample solution to be tested based on at least one of the scattered light signals and the first fluorescence signal.

[0065] In some embodiments, such as Figure 5As shown, the sample analysis method 200 may further include steps 231 and 232. In step 231, infected red blood cells are counted based on the optical information of the infected red blood cells to obtain a count value. For example, a second scatter plot is generated based on the forward scattered light signal and the second fluorescence signal, or based on the first fluorescence signal and the second fluorescence signal. A region representing infected red blood cells is obtained from this second scatter plot using gating technology, and the scatter points falling into this region are counted to obtain the count value of the infected red blood cells. In step 232, if the count value of the infected red blood cells is greater than a predetermined threshold, an alarm is output (determining that the blood sample is a malaria-positive sample). Furthermore, different types of infected red blood cells and / or infected red blood cells at different developmental stages can be classified and counted based on the optical information of the infected red blood cells. For example, infected red blood cells can be classified at least as ring bodies, such as ring bodies, trophozoites, and schizonts.

[0066] Preferably, in order to more accurately distinguish between leukocytes and infected erythrocytes under hemolytic conditions using two dyes, especially when using the same excitation light source, the first dye and the second dye are selected such that the absolute value of the wavelength difference 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. Alternatively or additionally, the first dye and the second dye are selected such that the overlap of the emission spectra of the first dye and the second dye is no greater than 50%. By selecting the first dye and the second dye in this way, not only can the mutual detection interference between the first fluorescence signal and the second fluorescence signal be greatly reduced, i.e., the distinction between the first fluorescence signal and the second fluorescence signal can be greatly increased, but the size and complexity of the optical detection device will not be increased.

[0067] Figure 6 A schematic diagram of the emission spectra of the first dye and the second dye is shown. The solid line represents the emission spectrum 210 of the first dye, and the dashed line represents the emission spectrum 220 of the second dye. 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 in the abscissas of peak points D and A (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nm and less than 80 nm. Furthermore, the overlap between the emission spectra 210 and 220 of the first dye can be expressed as the ratio of the area of ​​the first polygon to the area of ​​the second polygon. The area of ​​the first polygon is equal to the area of ​​the curved polygon enclosed by points E, G, and C, while the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 210 (or the emission spectrum 220 of the second dye) and the baseline 230. The baseline 230 is as follows: Figure 6The dashed horizontal line shown is parallel to the horizontal axis and is located at 5% of the normalized peak value 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 intersections of the emission spectrum 210 of the first dye with the baseline 230, respectively, and points B and C are the left and right intersections of the emission spectrum 220 of the second dye with the baseline 230, respectively. Here, the overlap between the emission spectra 210 of the first dye and the emission spectrum 220 of the second dye is no more than 50%.

[0068] Furthermore, especially when using a single light source for illumination, the absolute value of the wavelength difference 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, and more preferably greater than 50 nanometers and less than 70 nanometers. This allows for further reduction of the mutual detection interference between the first fluorescence signal and the second fluorescence signal without increasing the size and complexity of the optical detection device.

[0069] Furthermore, it is advantageous that the overlap of the emission spectra of the first dye and the second dye is no more than 35%, preferably no more than 15%, which can further reduce the detection interference between the first fluorescence signal and the second fluorescence signal.

[0070] In some embodiments, at least one of the first dye and the second dye, particularly the first dye, may be a large Stokes shift dye. Here, a large Stokes shift dye refers to a dye in which the difference between the wavelengths corresponding to the peaks of the emission spectrum and the excitation spectrum is greater than a predetermined threshold.

[0071] Figure 7 This is a schematic diagram of the spectrum of a large Stokes shift dye. The excitation spectrum (also known as the absorption spectrum) 310 of the large Stokes shift dye is shown by a dashed line, and the emission spectrum 320 is shown by a solid line. 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 abscissas of peak points A2 and A1 (i.e., the difference in wavelengths corresponding to the peaks of the emission and excitation spectra) is greater than a predetermined threshold. This predetermined threshold can be, for example, greater than 30 nm and less than 150 nm, preferably greater than 50 nm and less than 100 nm.

[0072] By using at least one large Stokes shift dye, the detection interference between the first fluorescence signal and the second fluorescence signal can be reduced.

[0073] In some embodiments, the parent material of the first dye may be a mesoamino-substituted cyanine dye or a dye parent material with a typical electron push-pull system, such as carbazole or coumarin. For example, the first dye may have a parent structure of general formula I:

[0074] (I)

[0075] R1, R2, and R3 are substituents, which can be any element, such as hydrogen.

[0076] Further details regarding the first and second dyes of this application can be found in Chinese patent application No. 202011008754.3, the entire disclosure of which is incorporated herein by reference.

[0077] In addition, this application also provides a computer-readable storage medium having instructions stored thereon, which, when executed by a processor, cause the processor to implement the above-described sample analysis method 200 and one of its embodiments.

[0078] The aforementioned computer-readable storage medium can be volatile memory or non-volatile memory, or both. Non-volatile memory can be read-only memory, programmable read-only memory, erasable programmable read-only memory, electrically erasable programmable read-only memory, magnetic random access memory, flash memory, magnetic surface memory, optical disk, or read-only optical disk; magnetic surface memory can be disk storage or magnetic tape storage. Volatile memory can be random access memory used as an external cache. Furthermore, many forms of RAM can be used in this 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 linked dynamic random access memory, and direct memory bus random access memory.

[0079] The following specific examples will be used to describe the specific implementation method and corresponding results of this application.

[0080] Example 1

[0081] Staining reagent formula:

[0082] First dye 50mg

[0083] Second dye 50mg

[0084] 1000g of ethylene glycol

[0085] The first dye has the following general formula: ,

[0086] The second dye has the following general formula: .

[0087] The 68LN hemolytic agent, which is compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0088] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, and incubate for 30 seconds. After incubation, use flow cytometry to analyze the sample, collecting the forward scattering signal, first fluorescence signal, and second fluorescence signal. The results are based on the forward scattering light and the first fluorescence signal. Figure 8A The first scatter plot shown is used to identify and count white blood cells, particularly basophils. It generates data based on forward scattered light and a second fluorescence signal. Figure 8B The second scatter plot shown identifies infected red blood cells and classifies them into ring bodies, trophozoites, and schizonts.

[0089] Example 2

[0090] Staining reagent formula:

[0091] First dye 50mg

[0092] Second dye 50mg

[0093] 1000g of ethylene glycol

[0094] The first dye has the following general formula: ,

[0095] The second dye has the following general formula: .

[0096] The 68LN hemolytic agent, which is compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0097] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, and incubate for 30 seconds. After incubation, use flow cytometry to analyze the sample, collecting the forward scattering signal, first fluorescence signal, and second fluorescence signal. The results are based on the forward scattering light and the first fluorescence signal. Figure 9A The first scatter plot shown is used to identify and count white blood cells, specifically nucleated red blood cells and basophils. The data is generated based on forward scattered light and a second fluorescence signal. Figure 9B The second scatter plot shown identifies infected red blood cells and classifies them into ring bodies, trophozoites, and schizonts.

[0098] Example 3

[0099] Staining reagent formula:

[0100] First dye 50mg

[0101] Second dye 50mg

[0102] 1000g of ethylene glycol

[0103] The first dye has the following general formula: ,

[0104] The second dye has the following general formula: .

[0105] The 68LN hemolytic agent, which is compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0106] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, and incubate for 30 seconds. After incubation, use flow cytometry to analyze the sample, collecting the forward scattering signal, first fluorescence signal, and second fluorescence signal. The results are based on the forward scattering light and the first fluorescence signal. Figure 10A The first scatter plot shown is used to identify and count white blood cells. White blood cells are generated based on the forward scattered light and the second fluorescence signal. Figure 9B The second scatter plot shown identifies infected red blood cells and classifies them into ring bodies, trophozoites, and schizonts.

[0107] Example 4

[0108] Staining reagent formula:

[0109] First dye 50mg

[0110] Second dye 50mg

[0111] 1000g of ethylene glycol

[0112] The first dye has the following general formula: ,

[0113] The second dye has the following general formula: .

[0114] The 68LN hemolytic agent, which is compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0115] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, and incubate for 30 seconds. After incubation, use flow cytometry to analyze the sample, collecting the forward scattering signal, first fluorescence signal, and second fluorescence signal. The results are based on the forward scattering light and the first fluorescence signal. Figure 11A The first scatter plot shown classifies white blood cells into neutrophils, lymphocytes, monocytes, and eosinophils. The results are generated based on forward scattered light and the second fluorescence signal. Figure 11BThe second scatter plot shown is used to identify infected red blood cells.

[0116] Example 5

[0117] Staining reagent formula:

[0118] First dye 50mg

[0119] Second dye 50mg

[0120] 1000g of ethylene glycol

[0121] The first dye has the following general formula: ,

[0122] The second dye has the following general formula: .

[0123] The 68LD hemolytic agent, compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0124] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, incubate for 30 seconds, and after incubation, use flow cytometry to detect the test sample, collecting the forward scatter signal, side scatter signal, first fluorescence signal, and second fluorescence signal. Generate fluorescence based on the side scatter signal and the first fluorescence signal. Figure 12A The first scatter plot shown classifies white blood cells into neutrophils, lymphocytes, monocytes, and eosinophils. The results are generated based on forward scattered light and the second fluorescence signal. Figure 12B The second scatter plot shown is used to identify infected red blood cells.

[0125] Example 6

[0126] Staining reagent formula:

[0127] First dye 50mg

[0128] Second dye 50mg

[0129] 1000g of ethylene glycol

[0130] The first dye has the following general formula: ,

[0131] The second dye has the following general formula: .

[0132] The 68LN hemolytic agent, which is compatible with Mindray Bio-Medical Electronics Co., Ltd.'s BC-6800, was used.

[0133] Test method: Take 20 μL of blood sample and 20 μL of staining reagent, add 1 ml of hemolysin, incubate for 30 seconds, and after incubation, use flow cytometry to detect the test sample, collecting the first fluorescence signal and the second fluorescence signal. Genes are generated based on the first and second fluorescence signals. Figure 13 The second scatter plot shown identifies infected red blood cells and classifies them into ring bodies, trophozoites, and schizonts.

[0134] All features or combinations thereof mentioned in the specification, drawings, and claims are freely combined or used individually, provided they are meaningful within the scope of this application and do not contradict each other. The advantages and features described with reference to the sample analysis method provided in this application are accordingly applied to the sample analyzer and computer-readable storage medium provided in this application, and vice versa.

[0135] The above description is merely a preferred embodiment of this application and does not limit the scope of patent protection of this application. All equivalent modifications made based on the content of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the scope of patent protection of this 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 passing 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 leukocytes, 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; identifying nucleated red blood cells 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 passing 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: identifying basophilic granulocytes in the sample liquid to be tested and counting leukocytes in the sample liquid to be tested based on at least one of the scattering light signal and the first fluorescent signal.

4. The sample analysis method according to any one of claims 1 to 3, characterized by, identifying nucleated red blood cells of the blood sample based on at least one of the scattering light signal and the first fluorescent signal, comprising: generating a first scatter plot according to the forward scattering light signal and the first fluorescent signal; identifying nucleated red blood cells in the sample liquid to be tested, and preferably also identifying basophilic granulocytes in the sample liquid to be tested and counting leukocytes in the sample liquid to be tested, according to the first scatter plot.

5. The sample analysis method according to any one of claims 1 to 4, characterized by further comprising: identifying immature leukocytes 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 any one of claims 1 to 5, 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 any one of claims 1 to 6, 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 any one of claims 1 to 7, 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 the 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 each particle in the test sample liquid being able to pass through the flow cell 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; 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, identifying the nucleated red blood cells of the blood sample based on at least one of the scattered light signal and the first fluorescence signal, and obtaining the 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 identify basophils in the test sample liquid and count leukocytes in the test sample liquid based on at least one of the scattered light signal and the first fluorescence signal.

13. The sample analyzer of any one of claims 10 to 12, wherein, The processor is further configured to: generate a first scatter plot according to the forward scattered light signal and the first fluorescence signal; identify the nucleated red blood cells in the test sample liquid according to the first scatter plot, and preferably identify basophils in the test sample liquid and count leukocytes in the test sample liquid.

14. The sample analyzer of any one of claims 10 to 13, wherein, The processor is further configured to identify immature leukocytes in the test sample liquid based on at least one of the scattered light signal and the first fluorescence signal.

15. The sample analyzer of any one of claims 10 to 14, 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.

16. The sample analyzer of any one of claims 10 to 15, 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 nanometers and less than 80 nanometers, and / or the overlap of the emission spectra of the first dye and the second dye is not greater than 50%.

17. The sample analyzer of any one of claims 10 to 16, wherein, The difference between the wavelength corresponding to the peak 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.

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