Sample detection method and sample analyzer

Abstract

The application provides a sample detection method and a sample analyzer. The method comprises: obtaining a sample to be detected; providing reagents, the reagents comprising at least two fluorescent dyes for staining particles in the sample to be detected, wherein the absolute value of the difference between the wavelengths corresponding to the peak values of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%; mixing the sample to be detected with the reagents to form a sample to be tested; making the sample to be tested flow in a flow chamber in a single test, irradiating the particles flowing in the flow chamber with light of a single wavelength, so that the particles generate optical signals, and detecting at least the fluorescent signals in the optical signals; and obtaining the particle detection result in the sample to be detected according to at least the fluorescent signals. The scheme of the application can realize the detection of multiple parameters for a sample to be detected in a single test.

Description

Technical Field

[0001] This application relates to the field of sample detection technology, and more specifically to a sample detection method and a sample analyzer. Background Technology

[0002] Currently, there are many methods for analyzing blood cell samples, including the use of combinations of radio frequency, high-angle scattering, low-angle scattering, impedance, and side-scattering fluorescence to analyze red and white blood cells. Among these, the technique of collecting fluorescence signals to achieve cell clustering is currently a leading analytical technique internationally.

[0003] Current known methods for analyzing fluorescent blood cell samples require setting up multiple reaction channels in the blood cell analysis to obtain parameters such as red blood cells, white blood cells, reticulocytes, and nucleated red blood cells. Each channel can only analyze one parameter. In different reaction channels, different fluorescent dyes are used to fluoresce the blood cells. Then, a single exciter is used to excite the blood cells and collect a single side-scattered fluorescence signal. In this process, each dye can only specifically stain a single parameter, thereby obtaining cell information.

[0004] Existing blood cell analyzers based on fluorescence detection have some drawbacks: (1) white blood cells and red blood cells need to be measured in different channels; (2) the dyes and reagents used in each channel are different, resulting in a complex liquid path structure for the instrument. Therefore, reducing the types of dyes and reagents has become an urgent need for the next generation of blood cell analyzers. Summary of the Invention

[0005] This application is made to address the aforementioned problems. According to one aspect of this application, a sample detection method is provided, the method comprising: acquiring a sample to be tested; providing a reagent comprising at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the wavelength difference corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%; mixing the sample to be tested with the reagent to form a test sample solution; in a single test, flowing the test sample solution in a flow chamber, irradiating the particles flowing in the flow chamber with light of a single wavelength to generate an optical signal from the particles, and detecting at least a fluorescence signal in the optical signal; and acquiring a particle detection result in the sample to be tested based at least on the fluorescence signal.

[0006] According to another aspect of this application, a sample analyzer is provided, the sample analyzer comprising: a sampling unit for acquiring a sample to be tested and conveying the sample to a reaction unit; a reagent supply unit for storing reagents and supplying the reagents to the reaction unit as needed; wherein the reagents include at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%; the reaction unit includes a mixing chamber for mixing the sample to be tested with the reagents. The reagents are mixed and reacted to form a test sample solution; an optical detection system includes a first light source, a flow chamber, and an optical signal detector, wherein particles in the test sample solution are capable of flowing in the flow chamber in a single test, the first light source is used to irradiate the particles flowing in the flow chamber with light of a single wavelength so that the particles generate an optical signal, and the optical signal detector is used to detect at least a fluorescence signal in the optical signal; a delivery device is used to deliver the test sample solution treated with the reagents in the reaction section to the flow chamber of the optical detection system; and a processor is configured to acquire particle detection results in the test sample based at least on the fluorescence signal.

[0007] According to the sample detection method and sample analyzer of the embodiments of this application, a mixed dye including two fluorescent dyes is used to detect particles in the sample to be tested. Since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes and the overlap of their emission spectra are both within a preset range, different cells in the sample to be tested emit different fluorescence signals after being stained with different dyes in the mixed dye and irradiated by a single light source. This enables the detection of multiple parameters in a single test (single reaction channel) of a sample to be tested. Attached Figure Description

[0008] The above and other objects, features, and advantages of this application will become more apparent from the more detailed description of the embodiments of this application in conjunction with the accompanying drawings. The accompanying drawings are used to provide a further understanding of the embodiments of this application and form part of the specification. They are used together with the embodiments of this application to explain this application and do not constitute a limitation thereof. In the accompanying drawings, the same reference numerals generally represent the same components or steps.

[0009] Figure 1 A schematic flowchart illustrating a sample detection method according to an embodiment of this application is shown.

[0010] Figure 2 An exemplary schematic diagram shows the emission spectra of two fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of this application.

[0011] Figure 3 An exemplary schematic diagram showing the emission and excitation spectra of one of the fluorescent dyes in the mixed dyes used in the sample detection method according to an embodiment of this application is shown.

[0012] Figure 4 Show Figure 3 An exemplary schematic diagram of the parent structure of the fluorescent dye is shown.

[0013] Figure 5 An exemplary schematic diagram shows the parent structure of another fluorescent dye in the mixed dyes used in the sample detection method according to an embodiment of this application.

[0014] Figure 6 and Figure 7 An exemplary schematic diagram shows the structures of two dyes in a mixed dye used in a sample detection method according to an embodiment of this application.

[0015] Figure 8 and Figure 9 An exemplary schematic diagram shows particle detection results obtained using a sample detection method according to an embodiment of this application.

[0016] Figure 10 This diagram illustrates the correlation between the percentage of reticulocyte fragments detected using the sample detection method according to an embodiment of this application and the percentage of reticulocytes measured on a blood cell analyzer.

[0017] Figure 11 and Figure 12 An exemplary schematic diagram shows particle detection results obtained using a sample detection method according to another embodiment of this application.

[0018] Figure 13 This diagram illustrates the correlation between the percentage of reticulocytes detected using the sample detection method according to an embodiment of this application and the percentage of reticulocytes tested on a blood cell analyzer.

[0019] Figure 14 An exemplary schematic diagram shows the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of this application.

[0020] Figure 15 An exemplary schematic diagram shows the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of this application.

[0021] Figure 16 An exemplary schematic diagram shows the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of this application.

[0022] Figure 17 An exemplary schematic diagram showing the structure of the third fluorescent dye in the mixed dyes used in the sample detection method according to an embodiment of this application is provided.

[0023] Figure 18 , Figure 19 and Figure 20 An exemplary schematic diagram shows the particle detection results obtained by a sample detection method according to another embodiment of this application.

[0024] Figure 21 A schematic structural block diagram of a sample analyzer according to an embodiment of this application is shown. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application more apparent, exemplary embodiments according to this application will be described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments of this application. It should be understood that this application is not limited to the exemplary embodiments described herein. Based on the embodiments of this application described herein, all other embodiments obtained by those skilled in the art without inventive effort should fall within the protection scope of this application.

[0026] First, refer to Figure 1 This describes a sample detection method according to embodiments of the present application. Figure 1 A schematic flowchart of a sample detection method 100 according to an embodiment of this application is shown. Figure 1 As shown, the sample detection method 100 according to an embodiment of this application may include the following steps:

[0027] In step S110, the sample to be tested is obtained.

[0028] In step S120, a reagent is provided, the reagent comprising at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%.

[0029] In step S130, the sample to be tested is mixed with the reagent to form a test sample solution.

[0030] In step S140, the test sample liquid is flowed in a flow chamber during a single test, and the particles flowing in the flow chamber are irradiated with light of a single wavelength so that the particles generate an optical signal, and at least the fluorescence signal in the optical signal is detected.

[0031] In step S150, at least the particle detection result in the sample to be tested is obtained based on the fluorescence signal.

[0032] In the embodiments of this application, a mixed dye comprising two fluorescent dyes is used to stain the sample to be tested (such as a blood sample) in a single reaction channel (single test). A single light source (light with a single wavelength) is used to irradiate the stained particles flowing in the flow chamber. Because the emission spectra of the two fluorescent dyes in the mixed dye meet certain conditions, different particles (such as different cells) stained by the two fluorescent dyes in the same reaction channel can emit different fluorescence signals, thereby enabling different particles to be distinguished and detected in a single test. In the embodiments of this application, in order to achieve the above objective, the applicant has determined that the emission spectra of the two fluorescent dyes included in the mixed dye should meet the following conditions: the absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nanometers (nm) and less than 80 nanometers, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%. The following is in conjunction with... Figure 2 This describes the emission spectra of the two fluorescent dyes included in the mixed dye according to embodiments of this application.

[0033] Figure 2 An exemplary schematic diagram of the emission spectra of two fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of this application is shown. For ease of description, the two fluorescent dyes included in the mixed dye are referred to as the first fluorescent dye and the second fluorescent dye, respectively. Figure 2 As shown, the emission spectrum of the first fluorescent dye is emission spectrum 210 shown by the dashed line, and the emission spectrum of the second fluorescent dye is emission spectrum 220 shown by the solid line.

[0034] In this embodiment, the peak point of the emission spectrum 210 of the first fluorescent dye is D, and the peak point of the emission spectrum 220 of the second fluorescent dye is A. In this embodiment, the difference in the abscissas of points D and A (i.e., the difference in wavelength λ corresponding to the peaks) is greater than 30 nm and less than 80 nm. Furthermore, the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye is 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; 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 fluorescent dye) and the baseline 230. When calculating the percentage of the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye relative to the emission spectrum 210 of the first fluorescent dye, the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 210 of the first fluorescent dye and the baseline 230; when calculating the percentage of the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye relative to the emission spectrum 220 of the second fluorescent dye, the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 220 of the second fluorescent dye and the baseline 230; that is, the percentage of the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye relative to their respective emission spectra should not exceed 50%. For simplicity, this is described in this document as the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye not exceeding 50%. The baseline 230 is as follows: Figure 2 The dashed horizontal line parallel to the horizontal axis is located at 5% of the normalized peak value of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye. Points E and F are the left and right intersections of the emission spectrum 210 of the first fluorescent dye and the baseline 230, respectively, and points B and C are the left and right intersections of the emission spectrum 220 of the second fluorescent dye and the baseline 230, respectively. In the embodiments of this application, the overlap between the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye is no more than 50%.

[0035] In a further embodiment of this application, in order to achieve the detection of different particles in a single test under a single light source, the applicant has determined that the absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes included in the aforementioned mixed dye can be in the range of 30 nm to 80 nm, or greater than 40 nm and less than 80 nm; or greater than 50 nm and less than 80 nm; or greater than 50 nm and less than 70 nm. Similarly, the overlap of the emission spectra of the two fluorescent dyes included in the aforementioned mixed dye can be not greater than 50%, or not greater than 35%, or even not greater than 15%. Under any combination of the above conditions, the detection of different particles in a single test under a single light source can be achieved, which will be described later in conjunction with specific embodiments.

[0036] In embodiments of this application, at least one of the two fluorescent dyes used in step S120 can be a large Stokes shift dye. In embodiments of this application, a large Stokes shift dye can be a dye whose wavelength difference between the peak values ​​of its emission and excitation spectra is greater than a predetermined threshold. The following is in conjunction with... Figures 3 to 4 To describe.

[0037] Figure 3 An exemplary schematic diagram shows the emission and excitation spectra of one fluorescent dye from a mixed dye used in the sample detection method according to an embodiment of this application. Figure 3 As shown, the excitation spectrum (also known as the absorption spectrum) of the fluorescent dye is the dashed excitation spectrum 310, and the emission spectrum of the fluorescent dye is the solid emission spectrum 320. The peak point of the excitation spectrum 310 is A1, and the peak point of the emission spectrum 320 is A2. In the embodiments of this application, the difference between the abscissas of 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. In one example, the predetermined threshold is 30 nm. In another example, the predetermined threshold is 50 nm. In yet another example, the difference between the abscissas of points A2 and A1 (i.e., the difference in wavelengths corresponding to the peaks of the emission and excitation spectra) can be greater than 30 nm and less than 150 nm. In yet another example, the difference between the abscissas of points A2 and A1 (i.e., the difference in wavelengths corresponding to the peaks of the emission and excitation spectra) can be greater than 50 nm and less than 100 nm.

[0038] In the embodiments of this application, a large Stokes shift dye whose wavelength difference between the peak values ​​of its emission and excitation spectra is greater than a predetermined threshold can be mixed with a common dye (non-large Stokes shift dye) to form a combined dye used in step S120. Alternatively, a large Stokes shift dye whose wavelength difference between the peak values ​​of its emission and excitation spectra is greater than a predetermined threshold can be mixed with another large Stokes shift dye to form a combined dye used in step S120. Because the wavelength difference between the peak values ​​of the emission and excitation spectra of a large Stokes shift dye is greater than a predetermined threshold, two fluorescent dyes in a mixed dye comprising at least one large Stokes shift dye are more likely to satisfy the aforementioned conditions regarding the wavelength difference between the peak values ​​of the emission spectra and the overlap of the emission spectra, thereby making it easier to detect different particles in a single test under a single light source.

[0039] In the embodiments of this application, the aforementioned large Stokes shift dye has leukocyte clustering properties in blood samples, which will be discussed below. Figure 4 Describe its parent structure. Figure 4 It shows Figure 3 An exemplary schematic diagram of the parent structure of the fluorescent dye is shown. Figure 4 As shown, the parent structure of large Stokes shift dyes can be a mesoamino-substituted cyanine dye (or a dye with other structures). Meanwhile, dye parent structures such as carbazole and coumarin, which have typical electron push-pull systems, can be used as parent structures for performance modification and for leukocyte differentiation. Figure 4 In this context, R1, R2, and R3 are substituents, which can be any elements. Figure 6 A schematic diagram of the dye structure obtained when all elements are hydrogen is shown.

[0040] Figure 5 An exemplary schematic diagram of the parent structure of another fluorescent dye in the mixed dyes used in the sample detection method according to an embodiment of this application is shown. Figure 5 As shown, this fluorescent dye can be a nucleic acid dye, which can be used to detect red blood cells in blood samples. Figure 5 In this context, R and R2 are substituents, which can be any elements. Figure 7 A schematic diagram of the dye structure obtained when all elements are hydrogen is shown.

[0041] In one embodiment of this application, it can be Figure 6 The large Stokes shift dye shown is Figure 7 Another fluorescent dye phase shown is used for cell detection in blood samples. The following is combined with... Figures 6 to 13 To describe including Figure 6 and Figure 7An example of using a mixture of fluorescent dyes to detect cells in blood samples.

[0042] In one embodiment, the sample to be tested obtained in step S110 may be a blood sample; the reagents provided in step S120 may include a first reagent and a second reagent, wherein the first reagent includes a first fluorescent dye and a second fluorescent dye (their respective structures may be as follows...). Figure 6 and Figure 7 As shown in the figure, in this embodiment, the wavelength difference corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye is equal to 62 nanometers, and the overlap of the emission spectra of the first fluorescent dye and the second fluorescent dye is 31%. Furthermore, the first reagent may also include a solvent (e.g., ethylene glycol), and the first and second fluorescent dyes can be prepared in this solvent at corresponding concentrations to obtain the first reagent. The second reagent can be a hemolysin. After the first reagent, the second reagent, and the blood sample are mixed evenly, they are incubated at a predetermined temperature for a predetermined time to obtain a test sample solution. This test sample solution is then flowed in a flow chamber, and a single wavelength of light is used to irradiate the particles flowing in the flow chamber so that the particles generate an optical signal. The fluorescence signal in the optical signal is detected, and the cell detection result in the blood sample is obtained based on the fluorescence signal.

[0043] In one example, the concentration of the first fluorescent dye in the first reagent is 0.05 g / L, the concentration of the second fluorescent dye is 0.025 g / L, the solvent is ethylene glycol, and it also includes methanol at a concentration of 10 g / L; the blood sample to be tested is 4 μL, the first reagent is 20 μL, and the second reagent is 1 mL. After mixing thoroughly, the mixture is incubated at 42°C for 40 seconds to obtain the test sample solution. The test sample solution is then placed in a flow cytometer (Mindray BriCyte E6), and the fluorescein isothiocyanate (FITC) channel and the peridinin chlorophyll protein (PerCP) channel are activated for counting detection. Specifically, firstly, a first detection threshold of 500 was set to primarily detect pre-scattering of blood cell debris and fluorescence signals from the FITC channel, resulting in a particle count of 5000. Then, a second detection threshold of 20000 was set to continue detecting lateral scattering of leukocytes and fluorescence signals from the PerCP channel, again resulting in a particle count of 2000. The resulting counting graph is shown below. Figure 8 and Figure 9 shown.

[0044] in, Figure 8The image shows a scatter plot of particles detected by the FITC channel, including reticulocyte (RET) fragments and white blood cell (WBC) particles. Figure 9 This shows a scatter plot of particles detected by the PerCP channel, including various cell types, including white blood cells. Based on Figure 9 The scatter plot yielded the following white blood cell count results: lymphocyte percentage 29.8%; monocyte percentage 5.7%; neutrophil percentage 60.6%; eosinophil percentage 3.1%. Therefore, the sample detection method according to the above embodiments of this application can perform multi-parameter (reticulocyte fragments, white blood cells, etc.) detection on a blood sample in a single test (single reaction channel) under hemolytic conditions; this detection can be completed using the instrument's existing white blood cell detection channel.

[0045] The cell detection results obtained after testing the above-mentioned blood sample on a hematology analyzer (Mindray BC-6800) are as follows: reticulocyte percentage 3.2%; lymphocyte percentage 31.1%; monocyte percentage 5.5%; neutrophil percentage 60%; eosinophil percentage 3.2%; and basophil percentage 0.2%. This comparison shows that the sample detection method according to the above embodiments of this application can accurately classify and count white blood cells in blood samples.

[0046] Furthermore, in the above embodiments, reticulocytes formed reticulocyte fragments due to treatment with a hemolytic agent, such as... Figure 8 As shown, it contains a small amount of nucleic acid material that can be stained by dye, thus exhibiting particles in the fluorescent direction. Using the WBC particle count as a benchmark, the percentage of RET particles can be calculated, which is 2.5%. Since these RET particles are not complete RET cells, an absolute count cannot be performed, but it has a good correlation with the reticulocyte percentage measured on the aforementioned hematology analyzer. Figure 10 shown.

[0047] In the above combination Figures 6 to 10 In the described embodiments, under hemolytic conditions, a first fluorescence signal and a second fluorescence signal can be detected. The first fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a first fluorescent dye and then illuminated by a light source. The second fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a second fluorescent dye and then illuminated by a light source. The classification results and / or counting results of white blood cells in the blood sample can be obtained based on the first fluorescence signal and the scattered light signal, and the information of reticulocytes in the blood sample can be obtained based on the second fluorescence signal.

[0048] Obtaining information about reticulocytes in a blood sample can include counting reticulocyte fragments in the blood sample. This counting result can be obtained solely based on the second fluorescence signal, or it can be obtained by combining the second fluorescence signal and the scattered light signal. The scattered light signal can include forward scattered light signals and / or side scattered light signals. Combining the second fluorescence signal and the scattered light signal allows for more accurate differentiation between leukocytes and reticulocyte fragments, thus enabling a more accurate counting result of reticulocyte fragments. Based on this counting result, the proportion of reticulocytes among all red blood cells in the blood sample can be determined. For example, the proportion of reticulocytes to all red blood cells in a blood sample can be calculated as follows: Calculate a first ratio, which is the ratio of the count of reticulocyte fragments to the count of white blood cells in the blood sample; calculate a third ratio based on the first ratio and a pre-measured second ratio, where the second ratio is the ratio of the count of white blood cells to the count of red blood cells in the blood sample, and the third ratio is the proportion of reticulocyte fragments to all red blood cells in the blood sample; calculate the proportion of reticulocytes to all red blood cells in the blood sample based on the third ratio and a preset standard curve, where the preset standard curve reflects the relationship between the proportion of reticulocyte fragments to all red blood cells and the proportion of reticulocytes to all red blood cells.

[0049] The process of obtaining the classification results of white blood cells in a blood sample may include: classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils; and obtaining the counting results of white blood cells in a blood sample may include: counting the white blood cells in the blood sample, or, after classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils, counting the lymphocytes, monocytes, neutrophils, and eosinophils separately. The classification results and / or counting results of white blood cells can be obtained based on the first fluorescence signal and the scattered light signal. Furthermore, the white blood cell counting results can also be obtained based on the aforementioned second fluorescence signal.

[0050] The following is combined Figures 11 to 13 Description to include Figure 6 and Figure 7 Another embodiment of using a mixture of fluorescent dyes to perform cell detection on blood samples.

[0051] In another embodiment, the sample to be tested obtained in step S110 may be a blood sample; the reagents provided in step S120 may include a first reagent and a second reagent, wherein the first reagent includes a first fluorescent dye and a second fluorescent dye (their respective parent structures may be as follows). Figure 6 and Figure 7As shown in the figure, in this embodiment, the wavelength difference corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye is equal to 62 nanometers, and the overlap of the emission spectra of the first fluorescent dye and the second fluorescent dye is 31%. Furthermore, the first reagent may also include a solvent (e.g., ethylene glycol), in which the first fluorescent dye and the second fluorescent dye can be prepared at corresponding concentrations. The second reagent can be a diluent. After the first reagent, the second reagent, and the blood sample are mixed evenly, they are incubated at a predetermined temperature for a predetermined time to obtain a test sample solution. This test sample solution is then flowed in a flow chamber, and a single wavelength of light is used to irradiate the particles flowing in the flow chamber so that the particles generate an optical signal. The fluorescence signal in the optical signal is detected, and the cell detection result in the blood sample is obtained based on the fluorescence signal.

[0052] In one example, the concentration of the first fluorescent dye in the first reagent is 0.05 g / L, the concentration of the second fluorescent dye is 0.025 g / L, the solvent is ethylene glycol, and it also includes methanol at a concentration of 10 g / L; the blood sample to be tested is 4 μL, the first reagent is 20 μL, and the second reagent is 1 mL. After mixing thoroughly, the mixture is incubated at 42°C for 40 seconds to obtain the test sample solution. This test sample solution is then placed in a flow cytometer (Mindray BriCyte E6), and the FITC and PerCP channels are activated for counting detection. Specifically, the first detection threshold is first set to 500, mainly detecting pre-scattering of blood cell debris and the fluorescence signal of the FITC channel, resulting in a particle count of 5000; then the second detection threshold is set to 20000, and white blood cells are isolated from red blood cells by fluorescence signal, calculating the percentage of each white blood cell cluster separately. The resulting count graph is shown below. Figure 11 and Figure 12 shown.

[0053] in, Figure 11 The image shows a scatter plot of particles detected by the FITC channel, including mature erythrocytes and reticulocytes. Figure 12 This shows a scatter plot of particles detected by the PerCP channel, including various cell types, including white blood cells. Based on Figure 11 and Figure 12 The scatter plot showed that the percentage of reticulocytes was 5.14%, and the differential count of white blood cells was as follows: lymphocytes 34.9%; monocytes 3.7%; neutrophils 57.8%; and eosinophils 3.6%. Therefore, the sample detection method according to the above embodiments of this application can perform multi-parameter (reticulocytes, mature red blood cells, white blood cells, etc.) detection on a blood sample in a single test (single reaction channel) under non-hemolytic conditions; this detection can be completed using the instrument's existing white blood cell detection channel.

[0054] The cell detection results obtained after testing the above-mentioned blood sample on a hematology analyzer (Mindray BC-6800) are as follows: reticulocyte percentage 4.98%; lymphocyte percentage 34.5%; monocyte percentage 3.6%; neutrophil percentage 58.0%; eosinophil percentage 3.4%; and basophil percentage 0.5%. This comparison shows that the sample detection method according to the above embodiments of this application, under non-hemolytic conditions, can simultaneously count reticulocytes and white blood cells in a single test, and can effectively distinguish between different types of white blood cells; this detection can be completed using the existing RET channel in the instrument.

[0055] Figure 13 This diagram illustrates the correlation between the percentage of reticulocytes detected using the sample detection method according to embodiments of this application and the percentage of reticulocytes measured on a blood cell analyzer. Figure 13 It can be seen that the percentage of reticulocytes detected by the sample detection method according to the embodiments of this application has a good correlation with the percentage of reticulocytes tested on the blood cell analyzer. This indicates that the sample detection method according to the embodiments of this application has good accuracy when used to test the percentage of reticulocytes.

[0056] In the above combination Figures 11 to 13 In the described embodiments, under non-hemolytic conditions, a first fluorescence signal and a second fluorescence signal can be detected. The first fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a first fluorescent dye and then illuminated by a light source. The second fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a second fluorescent dye and then illuminated by a light source. The classification and / or counting results of white blood cells in the blood sample are obtained based on the first fluorescence signal, and the classification and / or counting results of red blood cells in the blood sample are obtained based on the second fluorescence signal.

[0057] The process of obtaining the classification and / or counting results of red blood cells in a blood sample based on the second fluorescence signal may further include: obtaining the counting results of reticulocytes in the blood sample based on the second fluorescence signal; or obtaining the classification and / or counting results of red blood cells in the blood sample based on the second fluorescence signal and the scattered light signal. Obtaining the classification results of red blood cells in the blood sample may include: distinguishing the red blood cells in the blood sample into mature red blood cells and reticulocytes. Obtaining the counting results of red blood cells in the blood sample may include: counting the red blood cells in the blood sample, or distinguishing the red blood cells in the blood sample into mature red blood cells and reticulocytes, and then counting the mature red blood cells and reticulocytes separately. Combining the second fluorescence signal and the scattered light signal can more accurately distinguish between white blood cells, reticulocytes, and mature red blood cells, thus enabling the acquisition of more accurate red blood cell classification and counting results.

[0058] The process of obtaining the classification results of white blood cells in a blood sample may include: classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils; and obtaining the counting results of white blood cells in a blood sample may include: counting the white blood cells in the blood sample, or, after classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils, counting the lymphocytes, monocytes, neutrophils, and eosinophils separately. The classification results and / or counting results of white blood cells can be obtained based on the first fluorescence signal and the scattered light signal. Furthermore, the white blood cell counting results can also be obtained based on the aforementioned second fluorescence signal.

[0059] The above two embodiments describe a scenario in which a mixed dye comprising two fluorescent dyes is used to perform multi-parameter detection on a blood sample in a single test in the sample detection method according to the embodiments of this application. In a further embodiment of this application, the sample detection method according to the embodiments of this application can also use a mixed dye comprising two or more fluorescent dyes to perform multi-parameter detection on a blood sample in a single test. The following describes the process in conjunction with... Figures 14 to 20 The following description uses three fluorescent dyes as examples.

[0060] Figure 14 This diagram illustrates an exemplary schematic of the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of this application. For ease of description, the three fluorescent dyes included in the mixed dye are referred to as the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye, respectively. Figure 14 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1410 shown by the dashed line, the emission spectrum of the second fluorescent dye is the emission spectrum 1420 shown by the solid line, and the emission spectrum of the third fluorescent dye is the emission spectrum 1430 shown by the solid line.

[0061] In this embodiment, the peak point of the emission spectrum 1410 of the first fluorescent dye is D, the peak point of the emission spectrum 1420 of the second fluorescent dye is A, and the peak point of the emission spectrum 1430 of the third fluorescent dye is H. In this embodiment, the difference in the abscissas of points D and A (i.e., the difference in wavelengths corresponding to their peaks) is greater than 30 nm and less than 80 nm, and the difference in the abscissas of points H and D (i.e., the difference in wavelengths corresponding to their peaks) is greater than 30 nm and less than 80 nm. That is, the difference in wavelengths corresponding to the peaks of the emission spectra of the first and second fluorescent dyes is greater than 30 nm and less than 80 nm, and the difference in wavelengths corresponding to the peaks of the emission spectra of the third and first fluorescent dyes is greater than 30 nm and less than 80 nm.

[0062] Furthermore, the overlap between the emission spectrum 1410 of the first fluorescent dye and the emission spectrum 1420 of the second fluorescent dye is 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; the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 1410 of the first fluorescent dye (or the emission spectrum 1420 of the second fluorescent dye) and the reference line 1440. The reference line 1440 is as follows... Figure 14 The dashed horizontal line parallel to the horizontal axis is located at 5% of the normalized peak height of the emission spectra of the first, second, and third fluorescent dyes. Points E and F are the left and right intersections of the emission spectrum 1410 of the first fluorescent dye with the baseline 1440, respectively. Points B and C are the left and right intersections of the emission spectrum 1420 of the second fluorescent dye with the baseline 1440, respectively. In the embodiments of this application, the overlap between the emission spectrum 1410 of the first fluorescent dye and the emission spectrum 1420 of the second fluorescent dye is no more than 50%.

[0063] Furthermore, the overlap between the emission spectrum 1410 of the first fluorescent dye and the emission spectrum 1430 of the third fluorescent dye is the ratio of the area of ​​the third polygon to the area of ​​the fourth polygon. The area of ​​the third polygon is equal to the area of ​​the curved polygon enclosed by points I, K, and F; the area of ​​the fourth polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 1410 of the first fluorescent dye (or the emission spectrum 1430 of the third fluorescent dye) and the baseline 1440. Points I and J are the left and right intersection points of the emission spectrum 1430 of the third fluorescent dye and the baseline 1440, respectively. In the embodiments of this application, the overlap between the emission spectrum 1420 of the first fluorescent dye and the emission spectrum 1430 of the third fluorescent dye is no greater than 50%.

[0064] In this embodiment, since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye, as well as the overlap in their emission spectra, are all within a preset range, and the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the third fluorescent dye and the first fluorescent dye, as well as the overlap in their emission spectra, are also within a preset range, different cells in the blood sample emit different fluorescence signals after being stained with different dyes in a mixed dye containing these three fluorescent dyes and then irradiated by the same light source. This enables the detection of multiple parameters in a single blood sample in a single test (single reaction channel).

[0065] Figure 15 This diagram illustrates an exemplary schematic of the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of this application. For ease of description, the three fluorescent dyes included in the mixed dye are referred to as the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye, respectively. Figure 15 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1510 shown by the dashed line, the emission spectrum of the second fluorescent dye is the emission spectrum 1520 shown by the solid line, and the emission spectrum of the third fluorescent dye is the emission spectrum 1530 shown by the solid line.

[0066] In this embodiment, the peak point of the emission spectrum 1510 of the first fluorescent dye is D, the peak point of the emission spectrum 1520 of the second fluorescent dye is A, and the peak point of the emission spectrum 1530 of the third fluorescent dye is H. In this embodiment, the difference between the abscissas of points D and A (i.e., the difference in wavelengths corresponding to their peaks) is greater than 30 nm and less than 80 nm, and the abscissa of point H is greater than the abscissa of point D. That is, the difference in wavelengths corresponding to the peaks of the emission spectra of the first and second fluorescent dyes is greater than 30 nm and less than 80 nm, and the wavelength corresponding to the peak of the emission spectrum 1530 of the third fluorescent dye is greater than the wavelength corresponding to the peak of the emission spectrum of the first fluorescent dye 1510.

[0067] Furthermore, the overlap between the emission spectrum 1510 of the first fluorescent dye and the emission spectrum 1520 of the second fluorescent dye is 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; the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 1510 of the first fluorescent dye (or the emission spectrum 1520 of the second fluorescent dye) and the reference line 1540. The reference line 1540 is as follows... Figure 15The dashed horizontal line shown is parallel to the horizontal axis and is located at 5% of the normalized peak height of the emission spectra of the first, second, and third fluorescent dyes. Points E and F are the left and right intersections of the emission spectrum 1510 of the first fluorescent dye with the baseline 1540, respectively. Points B and C are the left and right intersections of the emission spectrum 1520 of the second fluorescent dye with the baseline 1540, respectively. In the embodiments of this application, the overlap between the emission spectrum 1510 of the first fluorescent dye and the emission spectrum 1520 of the second fluorescent dye is no greater than 50%.

[0068] In this embodiment, since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the first and second fluorescent dyes and the overlap of their emission spectra are both within a preset range, and the wavelength corresponding to the peak value of the third fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye, particles in the blood sample stained with the first and second fluorescent dyes can emit first and second fluorescent signals under the same light source (light of the same wavelength), and particles in the blood sample stained with the third fluorescent dye can emit a third fluorescent signal under the illumination of another light source (light of another wavelength), thereby enabling the detection of multiple parameters in a single test on a blood sample.

[0069] Figure 16 This diagram illustrates an exemplary schematic of the emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of this application. For ease of description, the three fluorescent dyes included in the mixed dye are referred to as the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye, respectively. Figure 16 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1610 shown by the dashed line, the emission spectrum of the second fluorescent dye is the emission spectrum 1620 shown by the solid line, and the emission spectrum of the third fluorescent dye is the emission spectrum 1630 shown by the solid line.

[0070] In this embodiment, the peak point of the emission spectrum 1610 of the first fluorescent dye is D, the peak point of the emission spectrum 1620 of the second fluorescent dye is A, and the peak point of the emission spectrum 1630 of the third fluorescent dye is H. In this embodiment, the difference between the abscissas of points D and A (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nm and less than 80 nm, and the abscissa of point H is less than the abscissa of point A. That is, the difference in wavelengths corresponding to the peaks of the emission spectra of the first and second fluorescent dyes is greater than 30 nm and less than 80 nm, and the wavelength corresponding to the peak of the emission spectrum 1630 of the third fluorescent dye is less than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye 1620.

[0071] Furthermore, the overlap between the emission spectrum 1610 of the first fluorescent dye and the emission spectrum 1620 of the second fluorescent dye is 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; the area of ​​the second polygon is equal to the area of ​​the curved polygon enclosed by the emission spectrum 1610 of the first fluorescent dye (or the emission spectrum 1620 of the second fluorescent dye) and the reference line 1640. The reference line 1640 is as follows... Figure 16 The dashed line parallel to the horizontal axis is located at 5% of the normalized peak height of the emission spectra of the first, second, and third fluorescent dyes. Points E and F are the left and right intersections of the emission spectrum 1610 of the first fluorescent dye with the baseline 1640, respectively. Points B and C are the left and right intersections of the emission spectrum 1620 of the second fluorescent dye with the baseline 1640, respectively. In the embodiments of this application, the overlap between the emission spectrum 1610 of the first fluorescent dye and the emission spectrum 1620 of the second fluorescent dye is no more than 50%.

[0072] In this embodiment, since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the first and second fluorescent dyes and the overlap of their emission spectra are both within a preset range, and the wavelength corresponding to the peak value of the emission spectrum 1630 of the third fluorescent dye is smaller than the wavelength corresponding to the peak value of the emission spectrum 1620 of the second fluorescent dye, particles in the blood sample stained with the first and second fluorescent dyes can emit first and second fluorescent signals under the same light source (light of the same wavelength), and particles in the blood sample stained with the third fluorescent dye can emit a third fluorescent signal under the illumination of another light source (light of another wavelength), thereby enabling the detection of multiple parameters in a single test of a blood sample.

[0073] The following is combined Figures 17 to 20 Description Figure 15 and Figure 16 An example of using a mixture of fluorescent dyes to detect cells in blood samples.

[0074] In this embodiment, the sample to be tested obtained in step S110 can be a blood sample; the reagents provided in step S120 can include a first reagent and a second reagent, wherein the first reagent includes a first fluorescent dye, a second fluorescent dye, and a third fluorescent dye (the structures of which can be as follows respectively). Figure 6 , Figure 7 and Figure 17As shown in the figure, in this embodiment, the wavelength difference corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye is equal to 62 nanometers, and the overlap of the emission spectra of the first fluorescent dye and the second fluorescent dye is 31%. The wavelength corresponding to the peak value of the emission spectrum of the third fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. In addition, the first reagent may also include a solvent (e.g., ethylene glycol), and the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye can be prepared in the solvent at corresponding concentrations to obtain the first reagent. The second reagent can be a hemolytic agent. After the first reagent, the second reagent, and the blood sample are mixed evenly, they are incubated at a predetermined temperature for a predetermined time to obtain a test sample solution. The test sample solution is then flowed in a flow chamber, and two wavelengths of light are used to irradiate the particles flowing in the flow chamber so that the particles generate optical signals. The fluorescence signal in the optical signal is detected, and the cell detection result in the blood sample is obtained based on the fluorescence signal.

[0075] In one example, the concentrations of the first fluorescent dye in the first reagent were 0.05 g / L, the second fluorescent dye was 0.025 g / L, and the third fluorescent dye was 0.025 g / L. The solvent was ethylene glycol, and it also included methanol at a concentration of 10 g / L. The blood sample to be tested was 4 μL, the first reagent was 20 μL, and the second reagent was 1 mL. After mixing thoroughly, the mixture was incubated at 42°C for 40 seconds to obtain the test sample solution. The test sample solution was placed in a flow cytometer (Mindray BriCyte E6), and the FITC channel (488 nm excitation light), PerCP channel (488 nm excitation light), and Allophycocyanin (APC) channel (633 nm excitation light) were activated for counting and detection. The fluorescence signals collected by these three channels corresponded to the emission fluorescence signals of the second, first, and third fluorescent dyes, respectively. Specifically, the detection threshold was first set to 500, primarily collecting pre-spreading fluorescence signals of blood cell debris and FITC channel fluorescence signals (reticulin fragment signal at the blood shadow). After collecting 5000 particles, the threshold was set to 20000, and leukocyte lateral dispersion, PerCP channel (leukocyte clustering signal), and APC channel (nucleated clustering signal) fluorescence signals were collected, with a particle count of 2000. The resulting counting graph is shown below. Figures 18 to 20 shown.

[0076] in, Figure 18 The image shows a scatter plot of particles detected by the FITC channel, including reticulated red fragments. Figure 19 The image shows a scatter plot of particles detected by the APC channel, including nucleated red blood cells and white blood cells. Figure 20 The image shows a scatter plot of particles detected by the PerCP channel, including various types of white blood cells.

[0077] based on Figure 20 The scatter plot yielded the following white blood cell differential count results: lymphocytes 23.5%; monocytes 6.4%; neutrophils 68.9%; and eosinophils 1.2%. Figure 19 The scatter plot shows that nucleated erythrocytes are effectively displayed in the fluorescence direction, which is consistent with the above. Figure 19 By dividing the scatter plot of white blood cells, the percentage of nucleated red blood cells can be obtained as 2%. Based on Figure 18 The scatter plot can be used to obtain the counting results of reticulocyte fragments. Therefore, it can be seen that the sample detection method according to the above embodiments of this application can perform multi-parameter (reticulocyte fragments, nucleated red blood cells, white blood cells, etc.) detection on a blood sample in a single test (single reaction channel) under hemolytic conditions.

[0078] The cell detection results obtained after testing the above-mentioned blood sample on a hematology analyzer (Mindray BC-6800) are as follows: reticulocyte percentage 2.2%; lymphocyte percentage 23.2%; monocyte percentage 6.5%; neutrophil percentage 68%; eosinophil percentage 1.1%; and basophil percentage 2.1%. This comparison shows that the sample detection method according to the above embodiment of this application, through two excitation light excitations, can simultaneously collect three signals to classify and count white blood cells, nucleated red blood cells, and reticulocyte fragments.

[0079] In the above combination Figures 17 to 20 In the described embodiments, under hemolytic conditions, a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal can be detected. The first fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a first fluorescent dye and irradiated by a first light source. The second fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a second fluorescent dye and irradiated by a first light source. The third fluorescence signal may include the fluorescence signal emitted by cells in the blood sample after being stained with a third fluorescent dye and irradiated by a second light source. The classification and / or counting results of white blood cells in the blood sample are obtained based on the first fluorescence signal and the scattered light signal. The information of reticulocytes in the blood sample is obtained based on the second fluorescence signal. The counting results of nucleated red blood cells in the blood sample are obtained based on the third fluorescence signal and the scattered light signal.

[0080] Obtaining information about reticulocytes in a blood sample can include counting reticulocyte fragments. This counting can be based solely on the second fluorescence signal, or it can be obtained by combining the second fluorescence signal and a scattered light signal. The scattered light signal can include forward scattered light and / or side scattered light signals. Combining the second fluorescence signal and the scattered light signal allows for more accurate differentiation between leukocytes and reticulocyte fragments, thus enabling a more accurate count of reticulocyte fragments. Based on this count, the proportion of reticulocytes among all red blood cells in the blood sample can be determined. For example, the proportion of reticulocytes to all red blood cells in a blood sample can be calculated as follows: Calculate a first ratio, which is the ratio of the count of reticulocyte fragments to the count of white blood cells in the blood sample; calculate a third ratio based on the first ratio and a pre-measured second ratio, where the second ratio is the ratio of the count of white blood cells to the count of red blood cells in the blood sample, and the third ratio is the proportion of reticulocyte fragments to all red blood cells in the blood sample; calculate the proportion of reticulocytes to all red blood cells in the blood sample based on the third ratio and a preset standard curve, where the preset standard curve reflects the relationship between the proportion of reticulocyte fragments to all red blood cells and the proportion of reticulocytes to all red blood cells.

[0081] The process of obtaining the classification results of white blood cells in a blood sample may include: classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils; and obtaining the counting results of white blood cells in a blood sample may include: counting the white blood cells in the blood sample, or, after classifying the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils, and eosinophils, counting the lymphocytes, monocytes, neutrophils, and eosinophils separately. The classification results and / or counting results of white blood cells can be based on the first fluorescence signal and the scattered light signal.

[0082] Furthermore, the white blood cell count can also be obtained based on the aforementioned second fluorescence signal. Additionally, the white blood cell count can also be obtained based on the aforementioned third fluorescence signal. Furthermore, white blood cells and nucleated red blood cells can be distinguished based on the aforementioned third fluorescence signal and scattered light signal.

[0083] In addition, the proportion of nucleated red blood cells in a blood sample can be obtained based on the count of nucleated red blood cells in the blood sample.

[0084] The above description exemplarily illustrates a sample detection method according to embodiments of this application. Based on the above description, the sample detection method according to embodiments of this application uses a mixed dye comprising at least two fluorescent dyes to perform particle detection on the sample to be tested. Since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes and the overlap of their emission spectra are both within a preset range, different cells in the sample to be tested emit different fluorescence signals after being stained with different dyes in the mixed dye and irradiated by a light source. This enables the detection of multiple parameters in a single test (single reaction channel) of a sample to be tested.

[0085] The following is combined Figure 21 Describes a sample analyzer provided according to another aspect of this application. Figure 21 A schematic block diagram of a sample analyzer 2100 according to an embodiment of this application is shown, such as Figure 21 As shown, the sample analyzer 2100 includes: a sampling unit 2110 for acquiring a sample to be tested and conveying the sample to be tested to a reaction unit 2130; a reagent supply unit 2120 for storing reagents and supplying the reagents to the reaction unit 2130 as needed; wherein the reagents include at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%; the reaction unit 2130 includes a mixing chamber for mixing the sample to be tested with the reagents to form a test sample solution; and an optical detection system 2. The optical detection system 2140 includes a first light source 2141, a flow chamber 2142, and an optical signal detector 2143. Particles in the sample solution to be tested are capable of flowing within the flow chamber 2142 in a single test. The first light source 2141 is used to irradiate the particles flowing in the flow chamber 2142 with light of a single wavelength so that the particles generate an optical signal. The optical signal detector 2143 is used to detect at least a fluorescence signal in the optical signal. A delivery device 2150 is used to deliver the sample solution to be tested, after being treated with the reagent in the reaction section 2130, to the flow chamber 2142 of the optical detection system 2140. A processor 2160 is configured to acquire the particle detection result in the sample to be tested based at least on the fluorescence signal. The sample analyzer 2100 can be used to execute the sample detection method according to the embodiments of this application described above. Those skilled in the art can understand the structure and operation of the sample analyzer 2100 in conjunction with the foregoing description of the sample detection method according to the embodiments of this application. For simplicity, the specific details of the operation of each component of the sample analyzer 2100 will not be repeated here; only their main operating steps will be briefly described.

[0086] In an embodiment of this application, the optical signal detector 2143 includes a first fluorescence detector and a second fluorescence detector (not shown). The first fluorescence detector is used to detect a first fluorescence signal in the optical signal, and the second fluorescence detector is used to detect a second fluorescence signal in the optical signal. The processor 2160 is further configured to obtain a first particle detection result in the sample to be tested based at least on the first fluorescence signal and a second particle detection result in the sample to be tested based at least on the second fluorescence signal.

[0087] In an embodiment of this application, the optical signal detector 2143 further includes a scattered light signal detector (not shown), which is used to detect the scattered light signal in the optical signal. The processor 2160 is further configured to obtain the first particle detection result in the sample to be tested based on the first fluorescence signal and the scattered light signal.

[0088] In an embodiment of this application, the processor 2160 is further configured to acquire a second particle detection result in the sample to be tested based on the second fluorescence signal and the scattered light signal.

[0089] In the embodiments of this application, the absolute value of the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies any one of the following: greater than 40 nm and less than 80 nm; greater than 50 nm and less than 80 nm; greater than 50 nm and less than 70 nm.

[0090] In the embodiments of this application, the overlap of the emission spectra of the two fluorescent dyes is no more than 35%.

[0091] In the embodiments of this application, the overlap of the emission spectra of the two fluorescent dyes is no more than 15%.

[0092] In the embodiments of this application, the difference between the wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes is greater than a predetermined threshold.

[0093] In embodiments of this application, the reagent further includes a hemolytic agent; the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes information on reticulocytes.

[0094] In embodiments of this application, the information of the reticulocytes includes the counting results of reticulocyte fragments.

[0095] In embodiments of this application, the processor 2160 is further configured to: obtain the proportion of reticulocytes to all red blood cells in the test sample based on the counting results of reticulocyte fragments in the test sample.

[0096] In embodiments of this application, the reagent further includes a diluent: the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the counting results of reticulocytes.

[0097] In embodiments of this application, the reagent further includes a diluent: the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the classification and / or counting results of red blood cells.

[0098] In an embodiment of this application, the processor 2160 obtains the classification result of red blood cells in the sample to be tested, including: distinguishing the red blood cells in the sample to be tested into mature red blood cells and reticulocytes; the processor 2160 obtains the counting result of red blood cells in the sample to be tested, including: counting the red blood cells in the sample to be tested, or distinguishing the red blood cells in the sample to be tested into mature red blood cells and reticulocytes, and then counting the mature red blood cells and the reticulocytes respectively.

[0099] In embodiments of this application, the scattered light signal includes a forward scattered light signal and / or a side scattered light signal.

[0100] In embodiments of this application, the processor 2160 acquires the classification results of white blood cells in the sample to be tested, including: classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils, and eosinophils; the processor 2160 acquires the counting results of white blood cells in the sample to be tested, including: counting the white blood cells in the sample to be tested, or, after classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils, and eosinophils, counting the lymphocytes, monocytes, neutrophils, and eosinophils respectively.

[0101] In an embodiment of this application, the predetermined threshold is 30 nanometers.

[0102] In the embodiments of this application, the difference between the wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies any one of the following: greater than 50 nm; greater than 30 nm and less than 150 nm; greater than 50 nm and less than 100 nm.

[0103] In embodiments of this application, the reagent further includes a third fluorescent dye, and the optical signal detector 2143 further includes a third fluorescence detector (not shown), which is used to detect a third fluorescence signal in the optical signal. The processor 3260 can also be configured to obtain a third particle detection result in the sample to be tested based at least on the third fluorescence signal.

[0104] In an embodiment of this application, the optical signal detector 2143 further includes a scattered light signal detector (the same scattered light signal detector mentioned above), which is used to detect the scattered light signal in the optical signal. The processor 2160 is further configured to obtain the detection result of the third particle in the sample to be tested based on the third fluorescence signal and the scattered light signal.

[0105] In embodiments of this application, the two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: the wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye; the wavelength difference between the peak value of the emission spectrum of the third fluorescent dye and the first fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the overlap of the emission spectra of the third fluorescent dye and the first fluorescent dye is not greater than 50%.

[0106] In embodiments of this application, the wavelength corresponding to the peak of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye; the wavelength corresponding to the peak of the emission spectrum of the third fluorescent dye is greater than the wavelength corresponding to the peak of the emission spectrum of the first fluorescent dye or less than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye; the sample analyzer 2100 further includes a second light (not shown), the second light source being used to irradiate the particles flowing in the flow chamber 2142 with light of another wavelength, so that the particles generate an optical signal including the third fluorescent signal.

[0107] In embodiments of this application, the reagent further includes a hemolytic agent; the third particle detection result includes the count of nucleated red blood cells.

[0108] In embodiments of this application, the processor 2160 is further configured to obtain the proportion of nucleated red blood cells in the test sample relative to all red blood cells based on the count of nucleated red blood cells in the test sample.

[0109] Based on the above description, the sample analyzer according to the embodiments of this application uses a mixed dye comprising two fluorescent dyes to detect particles in the sample to be tested. Since the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes and the overlap of their emission spectra are both within a preset range, different cells in the sample to be tested emit different fluorescence signals after being stained with different dyes in the mixed dye and irradiated by a single light source. This enables the detection of multiple parameters in a single test (single reaction channel), improving detection efficiency. Furthermore, since multiple parameters can be detected in a single reaction channel, the complexity of the sample analyzer's liquid path structure can be reduced, thereby lowering costs.

[0110] Although exemplary embodiments have been described herein with reference to the accompanying drawings, it should be understood that the above exemplary embodiments are merely illustrative and are not intended to limit the scope of this application. Various changes and modifications can be made therein by those skilled in the art without departing from the scope and spirit of this application. All such changes and modifications are intended to be included within the scope of this application as claimed in the appended claims.

[0111] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0112] In the several embodiments provided in this application, it should be understood that the disclosed devices and methods can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another device, or some features may be ignored or not executed.

[0113] Numerous specific details are set forth in the specification provided herein. However, it will be understood that embodiments of this application may be practiced without these specific details. In some instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0114] Similarly, it should be understood that, in order to streamline this application and aid in understanding one or more of the various inventive aspects, features of this application may sometimes be grouped together in a single embodiment, figure, or description thereof in the description of exemplary embodiments of this application. However, this approach should not be construed as reflecting an intention that the claimed application requires more features than are expressly recited in each claim. Rather, as reflected in the corresponding claims, its inventive point lies in solving the corresponding technical problem with features fewer than all features of a single disclosed embodiment. Therefore, the claims following the detailed description are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of this application.

[0115] Those skilled in the art will understand that, apart from the mutual exclusion of features, all features disclosed in this specification (including the accompanying claims, abstract, and drawings) and all processes or units of any method or apparatus so disclosed can be combined in any combination. Unless otherwise expressly stated, each feature disclosed in this specification (including the accompanying claims, abstract, and drawings) may be replaced by an alternative feature that serves the same, equivalent, or similar purpose.

[0116] Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features but not others included in other embodiments, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the claims, any one of the claimed embodiments can be used in any combination.

[0117] The various component embodiments of this application can be implemented in hardware, or as software modules running on one or more processors, or a combination thereof. Those skilled in the art will understand that microprocessors or digital signal processors (DSPs) can be used in practice to implement some or all of the functions of some modules in the article analysis device according to the embodiments of this application. This application can also be implemented as an apparatus program (e.g., a computer program and computer program product) for performing part or all of the methods described herein. Such an implementation of this application can be stored on a computer-readable medium, or can be in the form of one or more signals. Such signals can be downloaded from an Internet website, provided on a carrier signal, or provided in any other form.

[0118] It should be noted that the above embodiments are illustrative of this application and not restrictive, and that those skilled in the art can devise alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses should not be construed as limiting the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. This application can be implemented by means of hardware comprising several different elements and by means of a suitably programmed computer. In the unit claims enumerating several means, several of these means may be embodied by the same item of hardware. The use of the words first, second, and third, etc., does not indicate any order. These words can be interpreted as names.

[0119] The above description is merely a specific embodiment or illustration of the embodiments of this application. The scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. The scope of protection of this application shall be determined by the scope of the claims.

Claims

1. A sample detection method, characterized in that, The method includes: Obtain the sample to be tested; The reagent includes at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nm and less than 80 nm, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%, and at least one of the two fluorescent dyes is a large Stokes shift dye. The sample to be tested is mixed with the reagent to form a test sample solution; In a single test, the test sample liquid is flowed in a flow chamber, and the particles flowing in the flow chamber are irradiated with light of a single wavelength so that the particles generate an optical signal, and at least the fluorescence signal in the optical signal is detected. At least the particle detection results in the sample to be tested are obtained based on the fluorescence signal; The fluorescence signal includes a first fluorescence signal and a second fluorescence signal, and the step of obtaining the particle detection result in the sample to be tested based at least on the fluorescence signal includes: At least the detection result of the first particle in the sample to be tested is obtained based on the first fluorescence signal; At least the detection result of the second particle in the sample to be tested is obtained based on the second fluorescence signal.

2. The method according to claim 1, characterized in that, The method further includes: detecting the scattered light signal in the optical signal. The step of obtaining the first particle detection result in the sample to be tested based at least on the first fluorescence signal further includes: obtaining the first particle detection result in the sample to be tested based on the first fluorescence signal and the scattered light signal.

3. The method according to claim 2, characterized in that, The step of obtaining the second particle detection result in the sample to be tested based at least on the second fluorescence signal further includes: obtaining the second particle detection result in the sample to be tested based on the second fluorescence signal and the scattered light signal.

4. The method according to any one of claims 1-3, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies the condition that it is greater than 40 nm and less than 80 nm.

5. The method according to any one of claims 1-3, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies: Greater than 50 nanometers and less than 80 nanometers.

6. The method according to any one of claims 1-3, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies: Greater than 50 nanometers and less than 70 nanometers.

7. The method according to any one of claims 1-3, characterized in that, The overlap of the emission spectra of the two fluorescent dyes is no more than 35%.

8. The method according to claim 7, characterized in that, The overlap of the emission spectra of the two fluorescent dyes is no more than 15%.

9. The method according to any one of claims 1-3, characterized in that, The difference between the wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes is greater than a predetermined threshold.

10. The method according to claim 2 or 3, characterized in that, The reagent also includes a hemolytic agent; the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes information on reticulocytes.

11. The method according to claim 10, characterized in that, The information about the reticulocytes includes the count of reticulocyte fragments.

12. The method according to claim 11, characterized in that, The method further includes: Based on the counting results of reticulocyte fragments in the test sample, the proportion of reticulocytes to all red blood cells in the test sample is obtained.

13. The method according to claim 2, characterized in that, The reagent also includes a diluent; the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the counting results of reticulocytes.

14. The method according to claim 3, characterized in that, The reagent also includes a diluent; the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the classification and / or counting results of red blood cells.

15. The method according to claim 14, characterized in that, Obtaining the classification results of red blood cells in the test sample includes: distinguishing the red blood cells in the test sample into mature red blood cells and reticulocytes; Obtaining the red blood cell count result in the sample to be tested includes: counting the red blood cells in the sample to be tested, or distinguishing the red blood cells in the sample to be tested into mature red blood cells and reticulocytes, and then counting the mature red blood cells and the reticulocytes separately.

16. The method according to claim 2, characterized in that, The scattered light signal includes forward scattered light signal and / or side scattered light signal.

17. The method according to claim 13 or 14, characterized in that, Obtaining the classification results of white blood cells in the sample to be tested includes: classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils; Obtaining the white blood cell count result in the sample to be tested includes: counting the white blood cells in the sample to be tested, or, after classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, monocytes, neutrophils and eosinophils respectively.

18. The method according to claim 9, characterized in that, The predetermined threshold is 30 nanometers.

19. The method according to claim 18, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 50 nanometers.

20. The method according to claim 18, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 30 nm and less than 150 nm.

21. The method according to claim 18, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 50 nm and less than 100 nm.

22. The method according to any one of claims 1-3, characterized in that, The reagent further includes a third fluorescent dye, and the fluorescence signal further includes a third fluorescent signal. The step of obtaining particle detection results in the sample to be tested based at least on the fluorescence signal further includes: At least the detection result of the third particle in the sample to be tested is obtained based on the third fluorescence signal.

23. The method according to claim 22, characterized in that, The method further includes: detecting the scattered light signal in the optical signal. The step of obtaining the detection result of the third particle in the sample to be tested based at least on the third fluorescence signal further includes: obtaining the detection result of the third particle in the sample to be tested based on the third fluorescence signal and the scattered light signal.

24. The method according to claim 22, characterized in that, The two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: The wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The wavelength difference between the peak values ​​of the emission spectra of the third fluorescent dye and the first fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the overlap between the emission spectra of the third fluorescent dye and the first fluorescent dye is not greater than 50%.

25. The method according to claim 23, characterized in that, The two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: The wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The wavelength corresponding to the peak value of the emission spectrum of the third fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye or less than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The method further includes irradiating the particles flowing in the flow chamber with light of another wavelength so that the particles generate an optical signal including the third fluorescence signal.

26. The method according to claim 25, characterized in that, The reagent also includes a hemolytic agent; the third particle detection result includes the count of nucleated red blood cells.

27. The method according to claim 26, characterized in that, The method further includes: Based on the count of nucleated red blood cells in the sample to be tested, the proportion of nucleated red blood cells in the sample to all red blood cells is obtained.

28. A sample analyzer, characterized in that, The sample analyzer includes: A sampling unit is used to acquire a sample to be tested and to transport the sample to be tested to a reaction unit; A reagent supply unit is used to store reagents and supply the reagents to the reaction unit as needed; wherein the reagents include at least two fluorescent dyes for staining particles in the sample to be tested, wherein the absolute value of the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes is greater than 30 nanometers and less than 80 nanometers, and the overlap of the emission spectra of the two fluorescent dyes is not greater than 50%, and at least one of the two fluorescent dyes is a large Stokes shift dye. The reaction section includes a mixing chamber for mixing the sample to be tested with the reagent to form a test sample solution; An optical detection system includes a first light source, a flow chamber, and an optical signal detector. Particles in the sample liquid to be tested are able to flow in the flow chamber in a single test. The first light source is used to irradiate the particles flowing in the flow chamber with light of a single wavelength so that the particles generate an optical signal. The optical signal detector is used to detect at least a fluorescence signal in the optical signal. A delivery device is used to deliver the test sample solution, after being treated with the reagent in the reaction section, to the flow chamber of the optical detection system; A processor configured to acquire particle detection results in the sample to be tested based at least on the fluorescence signal; The optical signal detector includes a first fluorescence detector and a second fluorescence detector. The first fluorescence detector is used to detect a first fluorescence signal in the optical signal, and the second fluorescence detector is used to detect a second fluorescence signal in the optical signal. The processor is further configured to obtain a first particle detection result in the sample to be tested based at least on the first fluorescence signal, and to obtain a second particle detection result in the sample to be tested based at least on the second fluorescence signal.

29. The sample analyzer according to claim 28, characterized in that, The optical signal detector further includes a scattered light signal detector, which is used to detect the scattered light signal in the optical signal. The processor is further configured to obtain the first particle detection result in the sample to be tested based on the first fluorescence signal and the scattered light signal.

30. The sample analyzer according to claim 29, characterized in that, The processor is further configured to acquire the detection result of the second particle in the sample to be tested based on the second fluorescence signal and the scattered light signal.

31. The sample analyzer according to any one of claims 28-30, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies the condition that it is greater than 40 nm and less than 80 nm.

32. The sample analyzer according to any one of claims 28-30, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies the condition that it is greater than 50 nanometers and less than 80 nanometers.

33. The sample analyzer according to any one of claims 28-30, characterized in that, The absolute value of the difference in wavelengths corresponding to the peak values ​​of the emission spectra of the two fluorescent dyes satisfies the condition that it is greater than 50 nanometers and less than 70 nanometers.

34. The sample analyzer according to any one of claims 28-30, characterized in that, The overlap of the emission spectra of the two fluorescent dyes is no more than 35%.

35. The sample analyzer according to claim 34, characterized in that, The overlap of the emission spectra of the two fluorescent dyes is no more than 15%.

36. The sample analyzer according to any one of claims 28-30, characterized in that, The difference between the wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes is greater than a predetermined threshold.

37. The sample analyzer according to claim 29 or 30, characterized in that, The reagent also includes a hemolytic agent; the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes information on reticulocytes.

38. The sample analyzer according to claim 37, characterized in that, The information about the reticulocytes includes the count of reticulocyte fragments.

39. The sample analyzer according to claim 38, characterized in that, The processor is also configured to: Based on the counting results of reticulocyte fragments in the test sample, the proportion of reticulocytes to all red blood cells in the test sample is obtained.

40. The sample analyzer according to claim 29, characterized in that, The reagent also includes a diluent: the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the counting results of reticulocytes.

41. The sample analyzer according to claim 30, characterized in that, The reagent also includes a diluent: the first particle detection result includes the classification and / or counting results of white blood cells, and the second particle detection result includes the classification and / or counting results of red blood cells.

42. The sample analyzer according to claim 41, characterized in that, The processor acquires the classification result of red blood cells in the sample to be tested, including: classifying the red blood cells in the sample to be tested into mature red blood cells and reticulocytes; The processor acquires the red blood cell count results in the sample to be tested, including: counting the red blood cells in the sample to be tested, or distinguishing the red blood cells in the sample to be tested into mature red blood cells and reticulocytes, and then counting the mature red blood cells and the reticulocytes respectively.

43. The sample analyzer according to claim 29, characterized in that, The scattered light signal includes forward scattered light signal and / or side scattered light signal.

44. The sample analyzer according to claim 40 or 41, characterized in that, The processor obtains the classification results of white blood cells in the sample to be tested, including: classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils; The processor acquires the white blood cell count results in the sample to be tested, including: counting the white blood cells in the sample to be tested, or, after classifying the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, monocytes, neutrophils and eosinophils respectively.

45. The sample analyzer according to claim 36, characterized in that, The predetermined threshold is 30 nanometers.

46. ​​The sample analyzer according to claim 45, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 50 nanometers.

47. The sample analyzer according to claim 45, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 30 nm and less than 150 nm.

48. The sample analyzer according to claim 45, characterized in that, The difference in wavelength between the peak values ​​of the emission spectrum and the excitation spectrum of at least one of the two fluorescent dyes satisfies the condition that it is greater than 50 nm and less than 100 nm.

49. The sample analyzer according to any one of claims 28-30, characterized in that, The reagent also includes a third fluorescent dye, and the optical signal detector also includes a third fluorescence detector, which is used to detect a third fluorescence signal in the optical signal. The processor is also configured to obtain a third particle detection result in the test sample based at least on the third fluorescence signal.

50. The sample analyzer according to claim 49, characterized in that, The optical signal detector also includes a scattered light signal detector, which is used to detect the scattered light signal in the optical signal. The processor is further configured to obtain the detection result of the third particle in the sample to be tested based on the third fluorescence signal and the scattered light signal.

51. The sample analyzer according to claim 50, characterized in that, The two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: The wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The wavelength difference between the peak values ​​of the emission spectra of the third fluorescent dye and the first fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the overlap between the emission spectra of the third fluorescent dye and the first fluorescent dye is not greater than 50%.

52. The sample analyzer according to claim 50, characterized in that, The two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: The wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The wavelength corresponding to the peak value of the emission spectrum of the third fluorescent dye is greater than the wavelength corresponding to the peak value of the emission spectrum of the first fluorescent dye or less than the wavelength corresponding to the peak value of the emission spectrum of the second fluorescent dye. The sample analyzer also includes a second light source for illuminating particles flowing in the flow chamber with light of a different wavelength, so that the particles generate an optical signal including the third fluorescence signal.

53. The sample analyzer according to claim 52, characterized in that, The reagent also includes a hemolytic agent; the third particle detection result includes the count of nucleated red blood cells.

54. The sample analyzer according to claim 53, characterized in that, The processor is also configured to obtain the proportion of nucleated red blood cells in the test sample relative to all red blood cells based on the count of nucleated red blood cells in the test sample.

Images