Sample detection method and sample analyzer

Abstract

The invention provides a sample detection method and a sample analyzer. The method comprises the following steps: acquiring a to-be-detected sample; providing a reagent, wherein the reagent comprises a hemolytic agent and comprises a first fluorescent dye and a third fluorescent dye; mixing and reacting a to-be-tested sample with a reagent to form a to-be-tested sample solution; the to-be-tested sample liquid flows in the flow chamber in a single test, particles flowing in the flow chamber are irradiated by a first light source and a second light source so that the particles generate optical signals, at least scattered light signals and fluorescence signals in the optical signals are detected, and the fluorescence signals comprise a first fluorescence signal and a third fluorescence signal; obtaining a classification result and / or a counting result of the leukocytes based on the at least one scattered light signal and the first fluorescence signal; and acquiring a counting result of the nucleated red blood cells based on the at least one scattered light signal and the third fluorescence signal. According to the scheme provided by the invention, the detection of leukocyte parameters and nucleated red blood cell parameters of one to-be-detected sample in a single test can be realized.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number "202011008754.3". The application date of the original application is September 23, 2020, and the name of the invention is "Sample detection method and sample analyzer". Technical Field

[0002] The present application relates to the technical field of sample detection, and more specifically to a sample detection method and a sample analyzer. Background Art

[0003] There are many methods for analyzing blood cell samples, including the use of a combination of radio frequency, high-angle scattering, low-angle scattering, impedance, side scatter fluorescence, etc. to analyze red and white blood cells. Among them, the technology of collecting fluorescence signals to achieve cell clustering is currently the most advanced analysis technology in the world.

[0004] The currently known fluorescent blood cell sample analysis method requires setting up multiple reaction channels in the blood cell analysis in order to obtain parameters such as red blood cells, white blood cells, reticulocytes, and nucleated red blood cells. Each channel can only analyze one of the parameters. In different reaction channels, different fluorescent dyes are used to fluorescently stain the blood cells, and then the blood cells are excited with a single exciter to collect a single side scattered fluorescence signal. In this process, each dye can only specifically stain a single parameter to obtain cell information.

[0005] Existing blood cell analyzers based on the principle of fluorescence detection have some disadvantages: (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 fluid path structure of the instrument. Therefore, reducing the types of dyes and reagents has become an urgent need for a new generation of blood cell analyzers. Summary of the invention

[0006] The present application is made in order to solve the above-mentioned problems.

[0007] According to one aspect of the present application, a sample detection method is provided, the method comprising: obtaining a sample to be tested; providing a reagent, the reagent comprising a hemolytic agent and comprising a first fluorescent dye and a third fluorescent dye for dyeing particles in the sample to be tested; mixing the sample to be tested with the reagent to react to form a sample liquid to be tested; in a single test, the sample liquid to be tested flows in a flow chamber, using a first light source and a second light source to irradiate the particles flowing in the flow chamber so that the particles generate an optical signal, and at least detecting a scattered light signal and a fluorescent signal in the optical signal, the fluorescent signal comprising a first fluorescent signal emitted by the particles of the sample liquid to be tested after being dyed with the first fluorescent dye and irradiated by the first light source, and a fluorescent signal emitted by the particles of the sample liquid to be tested after being irradiated by the third fluorescent dye. A third fluorescent signal is emitted after being dyed with a fluorescent dye under the irradiation of a second light source; based on at least one of the scattered light signals and the first fluorescent signal, a classification result and / or a counting result of white blood cells in the sample to be tested is obtained, wherein the classification result includes distinguishing the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, and the counting result includes distinguishing the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, and then counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively; and a counting result of nucleated red blood cells in the sample to be tested is obtained based on at least one of the scattered light signals and the third fluorescent signal.

[0008] According to another aspect of the present application, a sample analyzer is provided, the sample analyzer comprising: a sampling part for obtaining a sample to be tested and conveying the sample to be tested to a reaction part; a reagent supply part for storing reagents and supplying the reagents to the reaction part as needed, wherein the reagents comprise a hemolytic agent and comprise a first fluorescent dye and a third fluorescent dye for dyeing particles in the sample to be tested; the reaction part comprises a mixing chamber for the sample to be tested and the reagents to be mixed and reacted to form a sample liquid to be tested; an optical detection system comprising a first light source, a second light source, a flow chamber, and an optical signal detector, the particles in the sample liquid to be tested can 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 first wavelength and the second light source is used to irradiate the particles flowing in the flow chamber with light of a second wavelength so that the particles generate optical signals, the optical signal detector comprises a scattered light signal detector, a first fluorescence detector and a third fluorescence detector, the scattered light signal detector is used to detect a scattered light signal in the optical signal, the first fluorescence detector The detector is used to detect a first fluorescent signal emitted by the particles of the test sample liquid after being dyed with the first fluorescent dye under the irradiation of the first light source, and the third fluorescent detector is used to detect a third fluorescent signal emitted by the particles of the test sample liquid after being dyed with the third fluorescent dye under the irradiation of the second light source; a conveying device is used to convey the test sample liquid treated with the reagent in the reaction part to the flow chamber of the optical detection system; and a processor is configured to: obtain the classification result and / or counting result of the white blood cells in the test sample based on at least one of the scattered light signals and the first fluorescent signal, obtaining the classification result includes distinguishing the white blood cells in the test sample into lymphocytes, monocytes, neutrophils and eosinophils, and obtaining the counting result includes distinguishing the white blood cells in the test sample into lymphocytes, monocytes, neutrophils and eosinophils and then counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively; and obtain the counting result of the nucleated red blood cells in the test sample based on at least one of the scattered light signals and the third fluorescent signal.

[0009] According to the sample detection method and sample analyzer of the embodiments of the present application, a mixed dye including two fluorescent dyes and two light sources are used to perform particle detection on the sample to be tested, which can realize the detection of white blood cell parameters and nucleated red blood cell parameters of a sample to be tested in a single test (single reaction channel). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] By describing the embodiments of the present application in more detail in conjunction with the accompanying drawings, the above and other purposes, features and advantages of the present application will become more apparent. The accompanying drawings are used to provide a further understanding of the embodiments of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application and do not constitute a limitation of the present application. In the accompanying drawings, the same reference numerals generally represent the same components or steps.

[0011] Figure 1 A schematic flow chart of a sample detection method according to an embodiment of the present application is shown.

[0012] Figure 2 An exemplary schematic diagram showing emission spectra of two fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of the present application.

[0013] Figure 3 An exemplary schematic diagram showing the emission spectrum and excitation spectrum of a fluorescent dye in a mixed dye used in a sample detection method according to an embodiment of the present application.

[0014] Figure 4 Show Figure 3 Exemplary schematic diagrams of the parent structures of the fluorescent dyes shown.

[0015] Figure 5 An exemplary schematic diagram showing the parent structure of another fluorescent dye in the mixed dye used in the sample detection method according to an embodiment of the present application.

[0016] Figure 6 and Figure 7 An exemplary schematic diagram showing the structures of two dyes in a mixed dye used in a sample detection method according to an embodiment of the present application.

[0017] Figure 8 and Fig. 9 An exemplary schematic diagram showing a particle detection result obtained by using a sample detection method according to an embodiment of the present application.

[0018] Fig.10 A schematic diagram showing the correlation between the percentage of reticulocyte fragment particles detected by the sample detection method according to an embodiment of the present application and the percentage of reticulocytes tested on a hematology analyzer.

[0019] Fig.11 and Fig.12 An exemplary schematic diagram showing particle detection results obtained by using a sample detection method according to another embodiment of the present application.

[0020] Fig.13A schematic diagram showing the correlation between the reticulocyte percentage detected by the sample detection method according to an embodiment of the present application and the reticulocyte percentage tested on a hematology analyzer.

[0021] Fig.14 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of the present application.

[0022] Fig.15 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of the present application.

[0023] Fig.16 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to yet another embodiment of the present application.

[0024] Fig.17 An exemplary schematic diagram showing the structure of a third fluorescent dye in a mixed dye used in a sample detection method according to an embodiment of the present application.

[0025] Fig.18 , Fig.19 and Fig. 20 An exemplary schematic diagram showing particle detection results obtained by a sample detection method according to yet another embodiment of the present application.

[0026] Fig.21 A schematic structural block diagram of a sample analyzer according to an embodiment of the present application is shown. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present application more obvious, the example embodiments according to the present application will be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application, and it should be understood that the present application is not limited to the example embodiments described herein. Based on the embodiments of the present application described in the present application, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present application.

[0028] First, refer to Figure 1 To describe the sample detection method according to an embodiment of the present application. Figure 1 FIG. 1 is a schematic flow chart of a sample detection method 100 according to an embodiment of the present application. Figure 1 As shown, the sample detection method 100 according to the embodiment of the present application may include the following steps:

[0029] In step S110, a sample to be tested is obtained.

[0030] In step S120, a reagent is provided, wherein the reagent includes at least two fluorescent dyes for dyeing the 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 no more than 50%.

[0031] In step S130 , the sample to be tested is mixed with the reagent to react to form a sample solution to be tested.

[0032] In step S140, the test sample liquid is made to flow in a flow chamber in a single test, light of a single wavelength is used to illuminate particles flowing in the flow chamber so that the particles generate optical signals, and at least a fluorescent signal in the optical signal is detected.

[0033] In step S150, a particle detection result in the sample to be tested is obtained at least according to the fluorescence signal.

[0034] In an embodiment of the present application, a mixed dye including two fluorescent dyes is used to dye a sample to be tested (such as a blood sample) in a single reaction channel (single test), and a single light source (light with a single wavelength) is used to irradiate the particles flowing in the flow chamber after dyeing. Since the emission spectra of the two fluorescent dyes in the mixed dye meet certain conditions, different particles (such as different cells) dyed by the two fluorescent dyes in the same reaction channel can emit different fluorescent signals, so that different particles can be distinguished and detected in one test. In an embodiment of the present application, in order to achieve the above purpose, according to the applicant's research, 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 combined with Figure 2 The emission spectra of the two fluorescent dyes included in the mixed dye according to the embodiment of the present application are described.

[0035] 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 the present application is shown. For ease of description, the two fluorescent dyes included in the mixed dye are referred to as a first fluorescent dye and a second fluorescent dye, respectively. Figure 2 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 210 shown by the dotted line, and the emission spectrum of the second fluorescent dye is the emission spectrum 220 shown by the solid line.

[0036] 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 the embodiment of the present application, the difference between the horizontal coordinates of point D and point A (i.e., the difference in wavelength λ corresponding to the peak value) is greater than 30 nanometers and less than 80 nanometers. In addition, the overlap amount of 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 surrounded by the three points E, G and C; the area of ​​the second polygon is equal to the area of ​​the curved polygon surrounded by the emission spectrum 210 of the first fluorescent dye (or the emission spectrum 220 of the second fluorescent dye) and the reference line 230. When calculating the ratio of the overlapping part of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye 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 reference line 230; when calculating the ratio of the overlapping part of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye 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 reference line 230; that is, the ratio of the overlapping part of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye to their respective emission spectra should not be greater than 50%. For the sake of simplicity, it is described in this article that the overlapping amount of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye is not greater than 50%. Wherein, the reference line 230 is as follows: Figure 2 The dashed horizontal line parallel to the horizontal axis is shown, and the horizontal line is 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. Point E and point F are the left intersection point and the right intersection point of the emission spectrum 210 of the first fluorescent dye and the reference line 230, respectively, and point B and point C are the left intersection point and the right intersection point of the emission spectrum 220 of the second fluorescent dye and the reference line 230, respectively. In the embodiment of the present application, the overlap amount of the emission spectrum 210 of the first fluorescent dye and the emission spectrum 220 of the second fluorescent dye is not greater than 50%.

[0037] In a further embodiment of the present application, in order to detect different particles in a single test under the illumination of a single light source, the applicant has studied 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 within the range of 30 nanometers to 80 nanometers, or can be greater than 40 and less than 80 nanometers; or greater than 50 nanometers and less than 80 nanometers; or greater than 50 nanometers and less than 70 nanometers. Similarly, the overlap of the emission spectra of the two fluorescent dyes included in the aforementioned mixed dye can be no more than 50%, or can be no more than 35%, or even no more than 15%. Under any combination of the above conditions, it is possible to detect different particles in a single test under the illumination of a single light source, which will be described later in conjunction with specific embodiments.

[0038] In an embodiment of the present application, at least one of the two fluorescent dyes used in step S120 may be a large Stokes shift dye. In an embodiment of the present application, the large Stokes shift dye may be a dye whose wavelength difference corresponding to the peak values ​​of the emission spectrum and the excitation spectrum is greater than a predetermined threshold. Figure 3 to Figure 4 to describe.

[0039] Figure 3 FIG. 1 is an exemplary schematic diagram showing the emission spectrum and excitation spectrum of a fluorescent dye in a mixed dye used in a sample detection method according to an embodiment of the present application. Figure 3 As shown, the excitation spectrum (also called absorption spectrum) of the fluorescent dye is the excitation spectrum 310 shown by the dotted line, and the emission spectrum of the fluorescent dye is the emission spectrum 320 shown by the solid line. Among them, the peak point of the excitation spectrum 310 is A1, and the peak point of the emission spectrum 320 is A2. In an embodiment of the present application, the difference between the horizontal coordinates of point A2 and point A1 (i.e., the difference in wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum) is greater than a predetermined threshold. In one example, the predetermined threshold is 30 nanometers. In another example, the predetermined threshold is 50 nanometers. In another example, the difference between the horizontal coordinates of point A2 and point A1 (i.e., the difference in wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum) can be greater than 30 nanometers and less than 150 nanometers. In another example, the difference between the horizontal coordinates of point A2 and point A1 (i.e., the difference in wavelengths corresponding to the peak values ​​of the emission spectrum and the excitation spectrum) can be greater than 50 nanometers and less than 100 nanometers.

[0040] In the embodiment of the present application, the aforementioned large Stokes shift dye, whose difference in wavelengths corresponding to the peaks of the emission spectrum and the excitation spectrum 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, or the aforementioned large Stokes shift dye, whose difference in wavelengths corresponding to the peaks of the emission spectrum and the excitation spectrum is greater than a predetermined threshold, can be mixed with another large Stokes shift dye to form a combined dye used in step S120. Since the difference in wavelengths corresponding to the peaks of the emission spectrum and the excitation spectrum of the large Stokes shift dye is greater than the predetermined threshold, the two fluorescent dyes in the mixed dye including at least one large Stokes shift dye are more likely to meet the aforementioned conditions regarding the difference in wavelengths corresponding to the peaks of the emission spectrum and the amount of overlap of the emission spectrum, thereby making it easier to detect different particles in a single test under the irradiation of a single light source.

[0041] In the embodiment of the present application, the aforementioned large Stokes shift dye has the performance of leukocyte grouping for blood samples. Figure 4 Describe its parent structure. Figure 4 Shows Figure 3 Schematic diagram of the parent structure of the fluorescent dye shown. Figure 4 As shown, the matrix of the large Stokes shift dye can be a meso-amino-substituted cyanine dye (or a dye of other structures). Meanwhile, dye matrices with typical electron push-pull systems such as carbazole and coumarin can be used as matrix structures for performance modification and used for leukocyte grouping. Figure 4 In the formula, R1, R2, and R3 are substituents, which can be any element. Figure 6 A schematic diagram showing the dye structure obtained when both of them are hydrogen elements.

[0042] Figure 5 FIG. 1 is an exemplary schematic diagram showing the parent structure of another fluorescent dye in the mixed dye used in the sample detection method according to an embodiment of the present application. Figure 5 As shown, the fluorescent dye can be a nucleic acid dye, which can be used to detect red blood cells in a blood sample. Figure 5 In the formula, R and R2 are substituents, which can be any element. Figure 7 A schematic diagram showing the dye structure obtained when both of them are hydrogen elements.

[0043] In one embodiment of the present application, Figure 6 The large Stokes shift dyes shown are Figure 7 Another fluorescent dye shown is mixed for cell detection in blood samples. Figures 6 to 13 To describe to include Figure 6 and Figure 7An example of using a mixed dye of fluorescent dyes to perform cell detection on a blood sample is shown.

[0044] In one embodiment, the sample to be tested obtained in step S110 may be a blood sample; the reagent 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 (the structures of which may be respectively as shown in FIG. Figure 6 and Figure 7 As shown), in this embodiment, the difference between the wavelengths corresponding to the peaks 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%; in addition, the first reagent may also include a solvent (such as ethylene glycol), and the first fluorescent dye and the second fluorescent dye may be prepared in the solvent according to corresponding concentrations to obtain the first reagent; the second reagent may be a hemolytic agent. After the first reagent, the second reagent and the blood sample are evenly mixed, they are incubated at a predetermined temperature for a predetermined time to obtain a sample liquid to be tested, and the sample liquid to be tested is made to flow in the flow chamber, and a single wavelength of light is used to irradiate the particles flowing in the flow chamber so that the particles generate optical signals, and the fluorescent signal in the optical signal is detected, and the cell detection result in the blood sample is obtained according to the fluorescent signal.

[0045] In one example, the concentration of the first fluorescent dye in the first reagent is 0.05 g / L (g / L), the concentration of the second fluorescent dye is 0.025 g / L, the solvent is ethylene glycol, and also includes methanol with a concentration of 10 g / L; the blood sample to be tested is 4 μL, the first reagent provided is 20 μL, and the second reagent is 1 ml. After these are mixed evenly and incubated at 42 degrees Celsius for 40 seconds, the sample to be tested is obtained. The sample to be tested is placed in a flow cytometer (Mindray BriCyte E6), and the fluorescein isothiocyanate (FITC) channel and the peridinin chlorophyll protein (PerCP) channel are opened for counting and detection. Specifically, the first detection threshold is set to 500, mainly detecting the front scatter of blood cell fragments and the fluorescence signal of the FITC channel, and the number of particles detected is 5000; then the second detection threshold is set to 20000, and the side scatter of white blood cells and the fluorescence signal of the PerCP channel are continuously detected, and the number of particles detected is 2000. The counting graph obtained is as follows: Figure 8 and Fig. 9 shown.

[0046] in, Figure 8The scatter plot of particles detected by the FITC channel is shown, including reticulocyte (RET) debris particles and white blood cells (WBC). Fig. 9 A scatter plot of particles detected by the PerCP channel, including various cells of leukocytes. Fig. 9 The scatter plot of the white blood cell classification count results is as follows: the percentage of lymphocytes is 29.8%; the percentage of monocytes is 5.7%; the percentage of neutrophils is 60.6%; the percentage of eosinophils is 3.1%. It can be seen that the sample detection method according to the above embodiment of the present application can detect multiple parameters (reticulocyte fragments, white blood cells, etc.) of a blood sample in a single test (single reaction channel) under hemolysis conditions; the detection can be completed using the existing white blood cell detection channel of the instrument.

[0047] The cell detection results of the above blood sample after testing on the blood cell analyzer (Mindray BC-6800) are as follows: the percentage of reticulocytes is 3.2%; the percentage of lymphocytes is 31.1%; the percentage of monocytes is 5.5%; the percentage of neutrophils is 60%; the percentage of eosinophils is 3.2%; the percentage of basophils is 0.2%. Through this comparison, it can be seen that the sample detection method according to the above embodiment of the present application can accurately classify and count the white blood cells in the blood sample.

[0048] Furthermore, in the above-mentioned embodiment, the reticulocytes are treated with a hemolytic agent to form reticulocyte fragment particles, such as Figure 8 As shown, it contains a small amount of nucleic acid material that can be stained by dyes, thereby exhibiting particles in the direction of fluorescence. The percentage of RET particles can be calculated based on the number of WBC particles, and its content is 2.5%. Since these RET particles are not complete RET cells, they cannot be absolutely counted, but they have a good correlation with the reticulocyte percentage value measured on the above-mentioned blood cell analyzer, as shown in Figure 1. Fig.10 shown.

[0049] In the above combination Figures 6 to 10 In the described embodiments, under hemolytic conditions, a first fluorescent signal and a second fluorescent signal can be detected, wherein the first fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a first fluorescent dye and under illumination of a light source, and the second fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a second fluorescent dye and under illumination of a light source; classification results and / or counting results of white blood cells in the blood sample can be obtained based on the first fluorescent signal and the scattered light signal, and information on reticulocytes in the blood sample can be obtained based on the second fluorescent signal.

[0050] Wherein, obtaining the information of reticulocytes in the blood sample may include obtaining the counting result of reticulocyte fragments in the blood sample, and the counting result may be obtained only based on the second fluorescent signal, or may be obtained in combination with the second fluorescent signal and the scattered light signal. Wherein, the scattered light signal may include a forward scattered light signal and / or a side scattered light signal. Combining the second fluorescent signal and the scattered light signal may more accurately distinguish white blood cells from reticulocyte fragments, thereby obtaining a more accurate counting result of reticulocyte fragments. Based on the counting result, the proportion of reticulocytes in the blood sample to all red blood cells may be obtained. For example, the ratio of reticulocytes to all red blood cells in a blood sample can be calculated in the following manner: a first ratio is calculated, the first ratio being the ratio of the counting result of reticulocyte fragments in the blood sample to the counting result of white blood cells; a third ratio is calculated based on the first ratio and a pre-measured second ratio, the second ratio being the ratio of the counting result of white blood cells in the blood sample to the counting result of red blood cells, and the third ratio being the ratio of reticulocyte fragments in the blood sample to all red blood cells; the ratio of reticulocytes in the blood sample to all red blood cells is calculated based on the third ratio and a preset standard curve, the preset standard curve reflecting the relationship between the ratio of reticulocyte fragments to all red blood cells and the ratio of reticulocytes to all red blood cells.

[0051] Wherein, obtaining the classification result of the white blood cells in the blood sample may include: distinguishing the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils; obtaining the counting result of the white blood cells in the blood sample may include: counting the white blood cells in the blood sample, or, after distinguishing the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively. The classification result and / or the counting result of the white blood cells can be based on the first fluorescent signal and the scattered light signal. In addition, the counting result of the white blood cells can also be obtained based on the aforementioned second fluorescent signal.

[0052] Combine the following Figure 11 to Figure 13 Description to include Figure 6 and Figure 7 Another embodiment of cell detection in a blood sample is shown by using a mixed dye of fluorescent dyes.

[0053] In another embodiment, the sample to be tested obtained in step S110 may be a blood sample; the reagent 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 this embodiment, the difference between the wavelengths corresponding to the peaks 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%; in addition, the first reagent may also include a solvent (such as ethylene glycol), and the first fluorescent dye and the second fluorescent dye may be prepared in the solvent according to corresponding concentrations to obtain the first reagent; the second reagent may be a diluent. After the first reagent, the second reagent and the blood sample are evenly mixed, they are incubated at a predetermined temperature for a predetermined time to obtain a sample liquid to be tested, and the sample liquid to be tested is made to flow in the flow chamber, and a single wavelength of light is used to irradiate the particles flowing in the flow chamber so that the particles generate optical signals, and the fluorescent signals in the optical signals are detected, and the cell detection results in the blood sample are obtained according to the fluorescent signals.

[0054] In one example, the concentration of the first fluorescent dye in the above-mentioned first reagent is 0.05 grams per liter (g / L), the concentration of the second fluorescent dye is 0.025 grams per liter, the solvent is ethylene glycol, and also includes methanol at a concentration of 10 grams per liter; the blood sample to be tested is 4 microliters, the first reagent provided is 20 microliters, and the second reagent is 1 milliliter. After these are evenly mixed and incubated at 42 degrees Celsius for 40 seconds, the sample liquid to be tested is obtained. After the sample liquid to be tested is placed in a flow cytometer (Mindray BriCyte E6), the FITC channel and the PerCP channel are opened for counting detection. Specifically, the first detection threshold of 500 is first set, and the front dispersion of blood cell fragments and the fluorescence signal of the FITC channel are mainly detected, and the number of particles detected is 5000; then the second detection threshold of 20000 is set, and the white blood cells and red blood cells are isolated by the fluorescence signal, and the percentage of each group of white blood cells is calculated separately. The counting graph obtained is as shown Fig.11 and Fig.12 shown.

[0055] in, Fig.11 A scatter plot of particles detected by the FITC channel is shown, including mature erythrocytes and reticulocytes. Fig.12 A scatter plot of particles detected by the PerCP channel, including various cells of leukocytes. Fig.11 and Fig.12 The scatter plot shows that the percentage of reticulocytes is 5.14%, and the classification and counting results of white blood cells are as follows: the percentage of lymphocytes is 34.9%; the percentage of monocytes is 3.7%; the percentage of neutrophils is 57.8%; and the percentage of eosinophils is 3.6%. It can be seen that the sample detection method according to the above embodiment of the present application can detect multiple parameters (reticulocytes, mature red blood cells, white blood cells, etc.) of a blood sample in a single test (single reaction channel) under non-hemolytic conditions; the detection can be completed using the existing white blood cell detection channel of the instrument.

[0056] The cell detection results obtained after the above blood samples were tested on the blood cell analyzer (Mindray BC-6800) are as follows: the percentage of reticulocytes is 4.98%; the percentage of lymphocytes is 34.5%; the percentage of monocytes is 3.6%; the percentage of neutrophils is 58.0%; the percentage of eosinophils is 3.4%; the percentage of basophils is 0.5%. Through this comparison, it can be seen that according to the sample detection method of the above embodiment of the present application, in a single test, under the condition of non-hemolysis, reticulocytes and white blood cells are counted simultaneously, and different types of white blood cells can be effectively distinguished; this detection can be completed using the existing RET channel in the instrument.

[0057] Fig.13 A schematic diagram showing the correlation between the reticulocyte percentage detected by the sample detection method according to an embodiment of the present application and the reticulocyte percentage tested on a blood cell analyzer is shown. Fig.13 It can be seen that the reticulocyte percentage detected by the sample detection method according to the embodiment of the present application has a good correlation with the reticulocyte percentage tested on the blood cell analyzer, which shows that the sample detection method according to the embodiment of the present application has good accuracy when used to test the reticulocyte percentage.

[0058] In the above combination Figure 11 to Figure 13 In the described embodiments, under non-hemolytic conditions, a first fluorescent signal and a second fluorescent signal can be detected, wherein the first fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a first fluorescent dye under illumination of a light source, and the second fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a second fluorescent dye under illumination of a light source; the classification result and / or counting result of white blood cells in the blood sample is obtained based on the first fluorescent signal, and the classification result and / or counting result of red blood cells in the blood sample is obtained based on the second fluorescent signal.

[0059] Wherein, obtaining the classification result and / or counting result of the red blood cells in the blood sample according to the second fluorescent signal may further include: obtaining the counting result of the reticulocytes in the blood sample according to the second fluorescent signal; or obtaining the classification result and / or counting result of the red blood cells in the blood sample according to the second fluorescent signal and the scattered light signal. Wherein, obtaining the classification result of the 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 result of the red blood cells in the blood sample may include: counting the red blood cells in the blood sample, or after distinguishing the red blood cells in the blood sample into mature red blood cells and reticulocytes, counting the mature red blood cells and reticulocytes respectively. Combining the second fluorescent signal and the scattered light signal can more accurately distinguish white blood cells, reticulocytes and mature red blood cells, thereby obtaining more accurate classification and counting results of red blood cells.

[0060] Wherein, obtaining the classification result of the white blood cells in the blood sample may include: distinguishing the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils; obtaining the counting result of the white blood cells in the blood sample may include: counting the white blood cells in the blood sample, or, after distinguishing the white blood cells in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively. The classification result and / or the counting result of the white blood cells can be based on the first fluorescent signal and the scattered light signal. In addition, the counting result of the white blood cells can also be obtained based on the aforementioned second fluorescent signal.

[0061] The above two embodiments describe the scenario in which a mixed dye including two fluorescent dyes is used to perform multi-parameter detection on a blood sample in a single test according to the sample detection method of the embodiment of the present application. In a further embodiment of the present application, the sample detection method according to the embodiment of the present application can also use a mixed dye including two or more fluorescent dyes to perform multi-parameter detection on a blood sample in a single test. Figures 14 to 20 Three fluorescent dyes are used as examples for description.

[0062] Fig.14 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to an embodiment of the present application is shown. For ease of description, the three fluorescent dyes included in the mixed dye are respectively referred to as a first fluorescent dye, a second fluorescent dye, and a third fluorescent dye. Fig.14 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1410 shown by the dotted 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.

[0063] 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 the embodiment of the present application, the difference between the horizontal coordinates of point D and point A (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers, and the difference between the horizontal coordinates of point H and point D (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers. That is, the difference in wavelengths corresponding to the peaks of the emission spectra of the first fluorescent dye and the second fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the difference in wavelengths corresponding to the peaks of the emission spectra of the third fluorescent dye and the first fluorescent dye is greater than 30 nanometers and less than 80 nanometers.

[0064] In addition, the overlap amount of the emission spectrum 1410 of the first fluorescent dye and the emission spectrum 1420 of the second fluorescent dye is the ratio of the first polygon area to the second polygon area. The first polygon area is equal to the area of ​​the curved polygon surrounded by the three points E, G and C; the second polygon area is equal to the area of ​​the curved polygon surrounded 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: Fig.14 The dashed horizontal line parallel to the horizontal axis is shown, and the horizontal line is at 5% of the normalized peak height of the emission spectra of the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye. Point E and point F are the left and right intersection points of the emission spectrum 1410 of the first fluorescent dye and the reference line 1440, respectively, and point B and point C are the left and right intersection points of the emission spectrum 1420 of the second fluorescent dye and the reference line 1440, respectively. In the embodiment of the present application, the overlap amount of the emission spectrum 1410 of the first fluorescent dye and the emission spectrum 1420 of the second fluorescent dye is not greater than 50%.

[0065] In addition, 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 surrounded by the three points I, K and F; the area of ​​the fourth polygon is equal to the area of ​​the curved polygon surrounded by the emission spectrum 1410 of the first fluorescent dye (or the emission spectrum 1430 of the third fluorescent dye) and the reference line 1440. Point I and point J are the left intersection and the right intersection of the emission spectrum 1430 of the third fluorescent dye and the reference line 1440, respectively. In the embodiment of the present application, the overlap between the emission spectrum 1420 of the first fluorescent dye and the emission spectrum 1430 of the third fluorescent dye is not greater than 50%.

[0066] 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 among the three fluorescent dyes and the overlap of the emission spectra of the two are both within the 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 and the overlap of the emission spectra of the two are both within the preset range, different cells in the blood sample emit different fluorescent signals after being stained with different dyes in the mixed dye including the three fluorescent dyes and irradiated by the same light source, thereby realizing multi-parameter detection of a blood sample in a single test (single reaction channel).

[0067] Fig.15 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of the present application is shown. For ease of description, the three fluorescent dyes included in the mixed dye are respectively referred to as a first fluorescent dye, a second fluorescent dye, and a third fluorescent dye. Fig.15 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1510 shown by the dotted 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.

[0068] 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 the embodiment of the present application, the difference between the abscissas of point D and point A (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers, 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 fluorescent dye and the second fluorescent dye is greater than 30 nanometers and less than 80 nanometers, 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.

[0069] In addition, the overlap amount of the emission spectrum 1510 of the first fluorescent dye and the emission spectrum 1520 of the second fluorescent dye is the ratio of the first polygon area to the second polygon area. The first polygon area is equal to the area of ​​the curved polygon surrounded by the three points E, G and C; the second polygon area is equal to the area of ​​the curved polygon surrounded 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: Fig.15The dashed horizontal line parallel to the horizontal axis is shown, and the horizontal line is at 5% of the normalized peak height of the emission spectra of the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye. Point E and point F are the left and right intersection points of the emission spectrum 1510 of the first fluorescent dye and the reference line 1540, respectively, and point B and point C are the left and right intersection points of the emission spectrum 1520 of the second fluorescent dye and the reference line 1540, respectively. In the embodiment of the present application, the overlap amount of the emission spectrum 1510 of the first fluorescent dye and the emission spectrum 1520 of the second fluorescent dye is not greater than 50%.

[0070] In this embodiment, since the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye among the three fluorescent dyes and the overlap of the emission spectra of the two 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, the particles in the blood sample dyed with the first fluorescent dye and the second fluorescent dye can emit the first fluorescent signal and the second fluorescent signal under the irradiation of the same light source (light of the same wavelength), and the particles in the blood sample dyed with the third fluorescent dye can emit the third fluorescent signal under the irradiation of another light source (light of another wavelength), thereby realizing multi-parameter detection of a blood sample in a single test.

[0071] Fig.16 An exemplary schematic diagram showing emission spectra of three fluorescent dyes in a mixed dye used in a sample detection method according to another embodiment of the present application is shown. For ease of description, the three fluorescent dyes included in the mixed dye are respectively referred to as a first fluorescent dye, a second fluorescent dye, and a third fluorescent dye. Fig.16 As shown, the emission spectrum of the first fluorescent dye is the emission spectrum 1610 shown by the dotted 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.

[0072] 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 the embodiment of the present application, the difference between the abscissas of point D and point A (i.e., the difference in wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers, 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 fluorescent dye and the second fluorescent dye is greater than 30 nanometers and less than 80 nanometers, 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.

[0073] In addition, the overlap amount of the emission spectrum 1610 of the first fluorescent dye and the emission spectrum 1620 of the second fluorescent dye is the ratio of the first polygon area to the second polygon area. The first polygon area is equal to the area of ​​the curved polygon surrounded by the three points E, G and C; the second polygon area is equal to the area of ​​the curved polygon surrounded 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: Fig.16 The dashed horizontal line parallel to the horizontal axis is shown, and the horizontal line is at 5% of the normalized peak height of the emission spectra of the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye. Point E and point F are the left and right intersection points of the emission spectrum 1610 of the first fluorescent dye and the reference line 1640, respectively, and point B and point C are the left and right intersection points of the emission spectrum 1620 of the second fluorescent dye and the reference line 1640, respectively. In the embodiment of the present application, the overlap amount of the emission spectrum 1610 of the first fluorescent dye and the emission spectrum 1620 of the second fluorescent dye is not greater than 50%.

[0074] In this embodiment, since the difference between the wavelengths corresponding to the peak values ​​of the emission spectra of the first fluorescent dye and the second fluorescent dye among the three fluorescent dyes and the overlap of the emission spectra of the two 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, the particles in the blood sample dyed with the first fluorescent dye and the second fluorescent dye can emit the first fluorescent signal and the second fluorescent signal under the irradiation of the same light source (light of the same wavelength), and the particles in the blood sample dyed with the third fluorescent dye can emit the third fluorescent signal under the irradiation of another light source (light of another wavelength), thereby realizing multi-parameter detection of a blood sample in a single test.

[0075] Combine the following Figures 17 to 20 Description Fig.15 and Fig.16 An example of using a mixed dye of fluorescent dyes to perform cell detection on a blood sample is shown.

[0076] In this embodiment, the sample to be tested obtained in step S110 may be a blood sample; the reagent provided in step S120 may 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 may be respectively as follows: Figure 6 , Figure 7 and Fig.17As shown), in this embodiment, the difference between the wavelengths 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 amount of the emission spectra of the first fluorescent dye and the second fluorescent dye is 31%, and 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 (such as ethylene glycol), and the first fluorescent dye, the second fluorescent dye and the third fluorescent dye may be prepared in the solvent according to corresponding concentrations to obtain the first reagent; the second reagent may be a hemolytic agent. After the first reagent, the second reagent and the blood sample are evenly mixed, they are incubated at a predetermined temperature for a predetermined time to obtain a sample liquid to be tested, and the sample liquid to be tested is made to flow in the 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, and the fluorescent signals in the optical signals are detected, and the cell detection results in the blood sample are obtained according to the fluorescent signals.

[0077] In one example, the concentration of the first fluorescent dye in the first reagent is 0.05 g / L (g / L), the concentration of the second fluorescent dye is 0.025 g / L, the concentration of the third fluorescent dye is 0.025 g / L, the solvent is ethylene glycol, and also includes methanol with a concentration of 10 g / L; the blood sample to be tested is 4 microliters, the first reagent provided is 20 microliters, and the second reagent is 1 ml. After these are mixed evenly, they are incubated at 42 degrees Celsius for 40 seconds to obtain the sample liquid to be tested. After the sample liquid to be tested is placed in a flow cytometer (Mindray BriCyte E6), the FITC channel (488nm excitation light) and the PerCP channel (488nm excitation light) and the Allophycocyanin (APC) channel (633nm excitation light) are opened for counting and detection, and the fluorescence signals collected by these three channels correspond to the emission fluorescence signals of the second fluorescent dye, the first fluorescent dye and the third fluorescent dye, respectively. Specifically, the detection threshold was first set to 500, and the front scatter of blood cell fragments and the fluorescence signal of the FITC channel (the signal of reticulum fragments at the blood shadow) were mainly collected. After collecting 5000 particles, the threshold was set to 20000, and the fluorescence signals of the lateral scatter of white blood cells, the PerCP channel (white blood cell clustering signal) and the APC channel (nuclear clustering signal) were continued to be collected. The number of collected particles was 2000, and the counting graph obtained was as follows: Figure 18 to Figure 20 shown.

[0078] in, Fig.18 A scatter plot of particles detected in the FITC channel is shown, including reticulin debris. Fig.19 A scatter plot of particles detected by the APC channel is shown, including nucleated erythrocytes and leukocytes. Fig. 20 A scatter plot of particles detected by the PerCP channel is shown, showing various cells including leukocytes.

[0079] based on Fig. 20 The scatter plot of the white blood cell classification results is as follows: lymphocyte percentage is 23.5%; monocyte percentage is 6.4%; neutrophil percentage is 68.9%; eosinophil percentage is 1.2%. Fig.19 From the scatter plot, we can see that the nucleated red blood cells can effectively display in the fluorescence direction. Fig.19 By dividing the white blood cell scatter plot in the figure, we can get the percentage of nucleated red blood cells to be 2%. Fig.18 The scatter plot of the reticulocyte fragments can be used to obtain the counting result of the reticulocyte fragments. It can be seen that the sample detection method according to the above embodiment of the present application can detect multiple parameters (reticulocyte fragments, nucleated red blood cells, white blood cells, etc.) of a blood sample in a single test (single reaction channel) under hemolytic conditions.

[0080] The cell detection results obtained after the above blood sample was tested on the blood cell analyzer (Mindray BC-6800) are as follows: the percentage of reticulocytes is 2.2%; the percentage of lymphocytes is 23.2%; the percentage of monocytes is 6.5%; the percentage of neutrophils is 68%; the percentage of eosinophils is 1.1%; the percentage of basophils is 2.1%. Through this comparison, it can be seen that the sample detection method according to the above embodiment of the present application can collect three signals at the same time through two excitation light excitations, and classify and count white blood cells, nucleated red blood cells, and reticulocyte fragments.

[0081] In the above combination Figures 17 to 20 In the described embodiment, under hemolytic conditions, a first fluorescent signal, a second fluorescent signal and a third fluorescent signal can be detected, wherein the first fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a first fluorescent dye and under irradiation with a first light source, the second fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a second fluorescent dye and under irradiation with a first light source, and the third fluorescent signal may include a fluorescent signal emitted by cells in a blood sample after being stained with a third fluorescent dye and under irradiation with a second light source; the classification result and / or counting result of white blood cells in the blood sample are obtained based on the first fluorescent signal and the scattered light signal, the information of reticulocytes in the blood sample is obtained based on the second fluorescent signal, and the counting result of nucleated red blood cells in the blood sample is obtained based on the third fluorescent signal and the scattered light signal.

[0082] Wherein, obtaining the information of reticulocytes in the blood sample may include obtaining the counting result of reticulocyte fragments in the blood sample. The counting result may be obtained only based on the second fluorescent signal, or may be obtained in combination with the second fluorescent signal and the scattered light signal. Wherein, the scattered light signal may include a forward scattered light signal and / or a side scattered light signal. Combining the second fluorescent signal and the scattered light signal may more accurately distinguish white blood cells from reticulocyte fragments, thereby obtaining a more accurate counting result of reticulocyte fragments. Based on the counting result, the proportion of reticulocytes in the blood sample to all red blood cells may be obtained. For example, the ratio of reticulocytes to all red blood cells in a blood sample can be calculated in the following manner: a first ratio is calculated, the first ratio being the ratio of the counting result of reticulocyte fragments in the blood sample to the counting result of white blood cells; a third ratio is calculated based on the first ratio and a pre-measured second ratio, the second ratio being the ratio of the counting result of white blood cells in the blood sample to the counting result of red blood cells, and the third ratio being the ratio of reticulocyte fragments in the blood sample to all red blood cells; the ratio of reticulocytes in the blood sample to all red blood cells is calculated based on the third ratio and a preset standard curve, the preset standard curve reflecting the relationship between the ratio of reticulocyte fragments to all red blood cells and the ratio of reticulocytes to all red blood cells.

[0083] Wherein, obtaining the classification result of the leukocytes in the blood sample may include: distinguishing the leukocytes in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils; obtaining the counting result of the leukocytes in the blood sample may include: counting the leukocytes in the blood sample, or, after distinguishing the leukocytes in the blood sample into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively. The classification result and / or the counting result of the leukocytes may be based on the first fluorescent signal and the scattered light signal.

[0084] In addition, the white blood cell count result can also be obtained based on the aforementioned second fluorescent signal. In addition, the white blood cell count result can also be obtained based on the aforementioned third fluorescent signal. In addition, white blood cells and nucleated red blood cells can be distinguished based on the aforementioned third fluorescent signal and the scattered light signal.

[0085] In addition, the ratio of nucleated red blood cells to all red blood cells in the blood sample can be obtained based on the counting result of the nucleated red blood cells in the blood sample.

[0086] The above is an exemplary description of the sample detection method according to the embodiment of the present application. Based on the above description, the sample detection method according to the embodiment of the present application uses a mixed dye including 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 the emission spectra of the two are both within a preset range, different cells in the sample to be tested emit different fluorescent signals after being stained with different dyes in the mixed dye and irradiated by a light source, thereby enabling multi-parameter detection of a sample to be tested in a single test (single reaction channel).

[0087] Combine the following Fig.21 A sample analyzer provided according to another aspect of the present application is described. Fig.21 A schematic block diagram of a sample analyzer 2100 according to an embodiment of the present application is shown. Fig.21 As shown, the sample analyzer 2100 includes: a sampling unit 2110, used to obtain a sample to be tested and transport the sample to be tested to a reaction unit 2130; a reagent supply unit 2120, used to store reagents and supply the reagents to the reaction unit 2130 as needed; wherein the reagents include at least two fluorescent dyes for dyeing 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 nanometers and less than 80 nanometers, 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 the sample to be tested and the reagent to mix and react to form a sample solution to be tested; an optical detection system 2 140, including a first light source 2141, a flow chamber 2142, and an optical signal detector 2143, the particles in the sample liquid to be tested can flow in 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, and the optical signal detector 2143 is used to detect at least the fluorescent signal in the optical signal; a conveying device 2150 is used to convey the sample liquid to be tested after being treated with the reagent in the reaction part 2130 to the flow chamber 2142 of the optical detection system 2140; a processor 2160 is configured to obtain the particle detection result in the sample to be tested at least according to the fluorescent signal. The sample analyzer 2100 can be used to perform the sample detection method according to the embodiment of the present application described above. Those skilled in the art can understand the structure and operation of the sample analyzer 2100 in combination with the description of the sample detection method according to the embodiment of the present application in the above text. For the sake of brevity, the specific detailed operation of each component of the sample analyzer 2100 will not be repeated here, and only their main operation steps will be briefly described.

[0088] In an embodiment of the present 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, and the processor 2160 is further configured to obtain a first particle detection result in the sample to be tested based on at least the first fluorescence signal, and to obtain a second particle detection result in the sample to be tested based on at least the second fluorescence signal.

[0089] In an embodiment of the present application, the optical signal detector 2143 also includes a scattered light signal detector (not shown), which is used to detect the scattered light signal in the optical signal, and the processor 2160 is further configured to obtain a first particle detection result in the sample to be tested based on the first fluorescence signal and the scattered light signal.

[0090] In an embodiment of the present application, the processor 2160 is further configured to obtain a second particle detection result in the sample to be tested according to the second fluorescence signal and the scattered light signal.

[0091] In an embodiment of the present application, the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the two fluorescent dyes satisfies any one of the following: greater than 40 nanometers and less than 80 nanometers; greater than 50 nanometers and less than 80 nanometers; greater than 50 nanometers and less than 70 nanometers.

[0092] In an embodiment of the present application, the overlap of the emission spectra of the two fluorescent dyes is no more than 35%.

[0093] In an embodiment of the present application, the overlap of the emission spectra of the two fluorescent dyes is no more than 15%.

[0094] In an embodiment of the present application, a difference between wavelengths corresponding to peak values ​​of an emission spectrum and an excitation spectrum of at least one of the two fluorescent dyes is greater than a predetermined threshold.

[0095] In an embodiment of the present application, the reagent further includes a hemolytic agent; the first particle detection result includes a classification result and / or a counting result of white blood cells, and the second particle detection result includes information of reticulocytes.

[0096] In an embodiment of the present application, the reticulocyte information includes the counting result of reticulocyte fragments.

[0097] In an embodiment of the present application, the processor 2160 is further configured to obtain the ratio of reticulocytes to all red blood cells in the sample to be tested based on the counting result of the reticulocyte fragments in the sample to be tested.

[0098] In an embodiment of the present application, the reagent further includes a diluent: the first particle detection result includes a classification result and / or a counting result of white blood cells, and the second particle detection result includes a counting result of reticulocytes.

[0099] In an embodiment of the present application, the reagent also includes a diluent: the first particle detection result includes a classification result and / or a counting result of white blood cells, and the second particle detection result includes a classification result and / or a counting result of red blood cells.

[0100] In an embodiment of the present application, the processor 2160 obtains the classification result of the 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 the red blood cells in the sample to be tested, including: counting the red blood cells in the sample to be tested, or after distinguishing the red blood cells in the sample to be tested into mature red blood cells and reticulocytes, counting the mature red blood cells and the reticulocytes respectively.

[0101] In an embodiment of the present application, the scattered light signal includes a forward scattered light signal and / or a side scattered light signal.

[0102] In an embodiment of the present application, the processor 2160 obtains the classification result of the white blood cells in the sample to be tested, including: distinguishing the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils; the processor 2160 obtains the counting result of the white blood cells in the sample to be tested, including: counting the white blood cells in the sample to be tested, or, after distinguishing the white blood cells in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively.

[0103] In an embodiment of the present application, the predetermined threshold is 30 nanometers.

[0104] In an embodiment of the present application, the difference between the wavelengths corresponding to the peaks 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 nanometers; greater than 30 nanometers and less than 150 nanometers; greater than 50 nanometers and less than 100 nanometers.

[0105] In an embodiment of the present application, the reagent also includes a third fluorescent dye, and the optical signal detector 2143 also includes a third fluorescence detector (not shown), and the third fluorescence detector is used to detect a third fluorescent 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 on at least the third fluorescent signal.

[0106] In an embodiment of the present application, the optical signal detector 2143 also includes a scattered light signal detector (the same scattered light signal detector as the scattered light signal detector mentioned above), which is used to detect the scattered light signal in the optical signal, and the processor 2160 is further configured to obtain a third particle detection result in the sample to be tested based on the third fluorescence signal and the scattered light signal.

[0107] In an embodiment of the present application, the two fluorescent dyes include a first fluorescent dye and a second fluorescent dye, wherein: 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 difference between the wavelengths corresponding to the peaks 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 of the emission spectra of the third fluorescent dye and the first fluorescent dye is no more than 50%.

[0108] In an embodiment of the present 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 is smaller than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye; the sample analyzer 2100 also includes a second light (not shown), and the second light source is used to use light of another wavelength to irradiate the particles flowing in the flow chamber 2142, so that the particles generate an optical signal including the third fluorescent signal.

[0109] In an embodiment of the present application, the reagent further includes a hemolytic agent; and the third particle detection result includes a nucleated red blood cell counting result.

[0110] In an embodiment of the present application, the processor 2160 is further configured to obtain the ratio of nucleated red blood cells in the sample to be tested to all red blood cells based on the counting result of the nucleated red blood cells in the sample to be tested.

[0111] Based on the above description, the sample analyzer according to the embodiment of the present application uses a mixed dye including two fluorescent dyes to perform particle detection on the sample to be tested. Since the difference in wavelengths corresponding to the peaks of the emission spectra of the two fluorescent dyes and the overlap of the emission spectra of the two are both within a preset range, different cells in the sample to be tested emit different fluorescent signals after being stained with different dyes in the mixed dye and irradiated by a single light source, thereby enabling multi-parameter detection of a sample to be tested in a single test (single reaction channel) to improve detection efficiency. In addition, since multi-parameter detection of a sample to be tested can be achieved in a single reaction channel, the complexity of the liquid path structure of the sample analyzer can be reduced, thereby reducing costs.

[0112] Although example embodiments have been described herein with reference to the accompanying drawings, it should be understood that the above example embodiments are merely exemplary and are not intended to limit the scope of the present application to this. Those of ordinary skill in the art may make various changes and modifications therein without departing from the scope and spirit of the present application. All these changes and modifications are intended to be included within the scope of the present application as required by the appended claims.

[0113] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example 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 performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0114] 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 only schematic, for example, the division of the units is only a logical function division, and there may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another device, or some features can be ignored or not executed.

[0115] In the description provided herein, a large number of specific details are described. However, it is understood that the embodiments of the present application can be practiced without these specific details. In some instances, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this description.

[0116] Similarly, it should be understood that in order to streamline the present application and help understand one or more of the various inventive aspects, in the description of the exemplary embodiments of the present application, the various features of the present application are sometimes grouped together into a single embodiment, figure, or description thereof. However, the method of the present application should not be interpreted as reflecting the following intention: the claimed application requires more features than the features clearly stated in each claim. More specifically, as reflected in the corresponding claims, the inventive point is that the corresponding technical problem can be solved with features less than all the features of a single disclosed embodiment. Therefore, the claims following the specific embodiment are hereby explicitly incorporated into the specific embodiment, wherein each claim itself serves as a separate embodiment of the present application.

[0117] It will be understood by those skilled in the art that, except for mutually exclusive features, all features disclosed in this specification (including the accompanying claims, abstracts and drawings) and all processes or units of any method or device disclosed in this specification may be combined in any combination. Unless otherwise expressly stated, each feature disclosed in this specification (including the accompanying claims, abstracts and drawings) may be replaced by an alternative feature that provides the same, equivalent or similar purpose.

[0118] In addition, those skilled in the art will appreciate that, although some embodiments described herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the claims, any one of the claimed embodiments can be used in any combination.

[0119] The various component embodiments of the present application can be implemented in hardware, or in software modules running on one or more processors, or in a combination thereof. It should be understood by those skilled in the art that a microprocessor or digital signal processor (DSP) can be used in practice to implement some or all of the functions of some modules in the article analysis device according to the embodiment of the present application. The present application can also be implemented as a device program (e.g., computer program and computer program product) for executing part or all of the methods described herein. Such a program implementing the present application can be stored on a computer-readable medium, or can have the form of one or more signals. Such a signal can be downloaded from an Internet website, or provided on a carrier signal, or provided in any other form.

[0120] It should be noted that the above embodiments illustrate the present application rather than limit the present application, and that those skilled in the art may design alternative embodiments without departing from the scope of the appended claims. In the claims, any reference symbol between brackets should not be constructed as a limitation to the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "one" or "an" preceding an element does not exclude the presence of multiple such elements. The present application may be implemented by means of hardware including several different elements and by means of a suitably programmed computer. In a unit claim that lists several devices, several of these devices may be embodied by the same hardware item. The use of the words first, second, and third, etc. does not indicate any order. These words may be interpreted as names.

[0121] The above is only a specific implementation or description of a specific implementation of the present application, and the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. The protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A sample detection method, characterized in that: The method comprises: Obtaining samples to be tested; Providing a reagent, wherein the reagent includes a hemolytic agent and includes a first fluorescent dye and a third fluorescent dye for staining particles in the sample to be tested; Mixing the sample to be tested with the reagent to form a sample solution to be tested; In a single test, the sample liquid to be tested is made to flow in a flow chamber, and a first light source and a second light source are used to irradiate particles flowing in the flow chamber so that the particles generate optical signals, and at least a scattered light signal and a fluorescent signal in the optical signal are detected, wherein the fluorescent signal includes a first fluorescent signal emitted by the particles of the sample liquid to be tested after being dyed with a first fluorescent dye under the irradiation of the first light source, and a third fluorescent signal emitted by the particles of the sample liquid to be tested after being dyed with a third fluorescent dye under the irradiation of the second light source; Based on at least one of the scattered light signals and the first fluorescent signal, a classification result and / or a counting result of the leukocytes in the sample to be tested is obtained, wherein the classification result includes distinguishing the leukocytes in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, and the counting result includes distinguishing the leukocytes in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils and then counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively; and A counting result of nucleated red blood cells in the sample to be tested is obtained based on at least one of the scattered light signals and the third fluorescent signal.

2. The method according to claim 1, characterized in that Obtaining a classification result and / or a counting result of white blood cells in the sample to be tested based on at least one of the scattered light signals and the first fluorescent signal, comprising: generating a first scatter plot based on the side scattered light signal and the first fluorescent signal, and obtaining a classification result and / or a counting result of the white blood cells based on the first scatter plot; Obtaining the counting result of the nucleated red blood cells in the sample to be tested based on at least one of the scattered light signals and the third fluorescent signal includes: generating a second scatter plot based on the forward scattered light signal and the third fluorescent signal, and obtaining the counting result of the nucleated red blood cells based on the second scatter plot.

3. The method according to claim 1 or 2, characterized in that: The difference between the wavelengths corresponding to the peaks 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 of the emission spectra of the third fluorescent dye and the first fluorescent dye is no more than 50%.

4. The method according to any one of claims 1 to 3, characterized in that The reagent further includes a second fluorescent dye, and the fluorescent signal further includes a second fluorescent signal emitted by particles of the test sample liquid after being dyed by the second fluorescent dye under the irradiation of the first light source; The method further includes: acquiring information of reticulocytes in the sample to be tested based on at least one of the scattered light and the second fluorescent signal.

5. The method according to claim 4, characterized in that Acquiring information about reticulocytes in the sample to be tested based on at least one of the scattered light and the second fluorescent signal includes: A third scattergram is generated based on the forward scattered light signal and the second fluorescent signal, and the information of the reticulocytes is acquired based on the third scattergram.

6. The method according to claim 4 or 5, characterized in that: The reticulocyte information includes the counting result of reticulocyte fragments.

7. The method according to any one of claims 4 to 6, characterized in that The absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first fluorescent dye and the second fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the overlap of the emission spectra of the first fluorescent dye and the second fluorescent dye is no more than 50%.

8. The method according to claim 7, characterized in that 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, and 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 smaller than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye.

9. A sample analyzer, characterized in that: The sample analyzer comprises: The sampling part is used to obtain the sample to be tested and transport the sample to be tested to the reaction part; a reagent supplying part, used for storing reagents and supplying the reagents to the reaction part as needed, wherein the reagents include a hemolytic agent and include a first fluorescent dye and a third fluorescent dye for dyeing particles in the sample to be tested; The reaction part includes a mixing chamber for the sample to be tested to mix and react with the reagent to form a sample liquid to be tested; An optical detection system, comprising a first light source, a second light source, a flow chamber, and an optical signal detector, wherein the particles in the sample liquid to be tested can 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 first wavelength and the second light source is used to irradiate the particles flowing in the flow chamber with light of a second wavelength, so that the particles generate optical signals, the optical signal detector comprises a scattered light signal detector, a first fluorescence detector and a third fluorescence detector, the scattered light signal detector is used to detect the scattered light signal in the optical signal, the first fluorescence detector is used to detect a first fluorescence signal emitted by the particles of the sample liquid to be tested after being dyed with a first fluorescent dye under the irradiation of the first light source, and the third fluorescence detector is used to detect a third fluorescence signal emitted by the particles of the sample liquid to be tested after being dyed with a third fluorescent dye under the irradiation of the second light source; A conveying device, used for conveying the sample liquid to be tested in the reaction part after being treated by the reagent to the flow chamber of the optical detection system; and A processor configured to: Based on at least one of the scattered light signals and the first fluorescent signal, a classification result and / or a counting result of the leukocytes in the sample to be tested is obtained, wherein the classification result includes distinguishing the leukocytes in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils, and the counting result includes distinguishing the leukocytes in the sample to be tested into lymphocytes, monocytes, neutrophils and eosinophils and then counting the lymphocytes, the monocytes, the neutrophils and the eosinophils respectively; and A counting result of nucleated red blood cells in the sample to be tested is obtained based on at least one of the scattered light signals and the third fluorescent signal.

10. The sample analyzer according to claim 9, wherein the processor obtains a classification result and / or a counting result of white blood cells in the sample to be tested based on at least one of the scattered light signal and the first fluorescent signal, comprising: The processor generates a first scattergram based on the side scattered light signal and the first fluorescent signal, and obtains a classification result and / or a counting result of the white blood cells based on the first scattergram; The processor obtains the counting result of the nucleated red blood cells in the sample to be tested based on at least one of the scattered light signals and the third fluorescence signal, including: the processor generates a second scatter plot based on the forward scattered light signal and the second fluorescence signal, and obtains the counting result of the nucleated red blood cells based on the second scatter plot.

11. The sample analyzer according to claim 9 or 10, characterized in that: The difference between the wavelengths corresponding to the peaks 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 of the emission spectra of the third fluorescent dye and the first fluorescent dye is no more than 50%.

12. The sample analyzer according to any one of claims 9 to 11, characterized in that: The reagent further includes a second fluorescent dye, and the optical signal detector further includes a second fluorescent detector, which is used to detect a second fluorescent signal emitted by particles of the test sample liquid after being dyed with the second fluorescent dye under the irradiation of the first light source; The processor is further configured to obtain information of reticulocytes in the sample to be tested based on at least one of the scattered light and the second fluorescent signal.

13. The sample analyzer according to claim 12, characterized in that: The processor acquires information of reticulocytes in the sample to be tested based on at least one of the scattered light and the second fluorescent signal, including: The processor generates a third scattergram based on the forward scattered light signal and the second fluorescent signal, and acquires the reticulocyte information based on the third scattergram.

14. The sample analyzer according to claim 12 or 13, characterized in that: The reticulocyte information includes the counting result of reticulocyte fragments.

15. The sample analyzer according to any one of claims 12 to 14, characterized in that: The absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first fluorescent dye and the second fluorescent dye is greater than 30 nanometers and less than 80 nanometers, and the overlap of the emission spectra of the first fluorescent dye and the second fluorescent dye is no more than 50%.

16. The sample analyzer according to claim 15, characterized in that: 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, and 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 smaller than the wavelength corresponding to the peak of the emission spectrum of the second fluorescent dye.

Images