Sample analyzer and sample analysis method

Abstract

The embodiment of the invention relates to a sample analysis method of a sample analyzer. The sample analysis method comprises the following steps: sucking a to-be-detected blood sample; mixing at least one part of the blood sample to be detected, the hemolytic agent, the first fluorescent dye and the second fluorescent dye to prepare a detection sample at a time; in a single test, each particle in a measurement sample is passed through an optical detection zone irradiated by excitation light to acquire optical information of the measurement sample, the excitation light including light of a first wavelength capable of exciting a first fluorescent dye and light of a second wavelength capable of exciting a second fluorescent dye, the second wavelength being greater than the first wavelength, the optical information comprises a first fluorescent signal corresponding to the first fluorescent dye and a second fluorescent signal corresponding to the second fluorescent dye, which are obtained in a single test; and obtaining infected red blood cell information of the measurement sample based on the first fluorescence signal and the second fluorescence signal. Therefore, the infected red blood cell information can be quickly and accurately obtained to assist malaria diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of blood cell detection, and in particular to a sample analyzer and a sample analysis method. BACKGROUND

[0002] Malaria is a serious parasitic disease that harms human health caused by Plasmodium parasitizing in human body, which is mainly transmitted in humans and anopheles by female anopheles biting human body infected with Plasmodium, and is one of the most serious tropical insect-borne infectious diseases in the world so far, with a high mortality rate. The main cause of clinical symptoms is the red inner stage Plasmodium. In the process of malaria infection, patients will have obvious fever, and different degrees of organ involvement, leading to local or systemic damage, and also causing hematological changes. If not timely and other febrile diseases are differentiated and treated, malaria may develop into a serious disease within 24 hours, causing delayed treatment, leading to the occurrence of severe and death cases. Therefore, accurate and rapid diagnosis of malaria is of great significance for effective treatment of malaria, reduction of malaria mortality and control of malaria transmission.

[0003] Currently, blood smear microscopy is usually used to check Plasmodium, but this method is highly dependent on the experience of the operator, requires high professional ability of the operator, and takes a long time.

[0004] In recent years, with the rapid development of blood cell analysis technology, blood cell analyzers have developed from cell counting detection to multi-functional combined detection instruments that can simultaneously analyze tens of thousands of cells at high speed and obtain multiple parameters describing the morphological characteristics of cells. Studies have found that the use of digital and image results in blood cell analyzers helps to screen malaria, but the application of blood cell analyzers in malaria diagnosis still has many limitations so far. SUMMARY

[0005] The task of the present application is to provide an improved scheme for detecting malaria, in which infected red blood cell parameters can be detected under hemolytic conditions using two different fluorescent dyes.

[0006] The first aspect of the present application relates to a sample analyzer, comprising:

[0007] a sample suction device for sucking a blood sample to be tested;

[0008] a sample preparation device for mixing at least a portion of the blood sample to be tested, a hemolytic agent, a first fluorescent dye and a second fluorescent dye in a reaction cell to prepare a test sample;

[0009] An optical detection device comprises a light emitting device for emitting excitation light to irradiate a flow chamber, the flow chamber being in communication with the reaction cell and for passing each particle in the assay sample therethrough, and a light detector for detecting optical information generated by each particle in the assay sample after being irradiated by the excitation light when passing through the flow chamber in a single test, the light emitting device comprising a first light source for emitting light of a first wavelength capable of exciting the first fluorescent dye and a second light source for emitting light of a second wavelength capable of exciting the second fluorescent dye, wherein the second wavelength is greater than the first wavelength, and the optical information comprises a first fluorescent signal corresponding to the first fluorescent dye and a second fluorescent signal corresponding to the second fluorescent dye detected in the single test; and

[0010] a processor configured to obtain the infected red blood cell information of the assay sample based on the first fluorescent signal and the second fluorescent signal.

[0011] The second aspect of the present application relates to a corresponding sample analysis method, comprising:

[0012] aspirating a blood sample to be tested;

[0013] mixing at least a portion of the blood sample to be tested, a hemolytic agent, a first fluorescent dye and a second fluorescent dye to prepare one assay sample in a single test;

[0014] passing each particle in the assay sample through an optical detection area irradiated by excitation light in a single test to obtain optical information generated by each particle in the assay sample after being irradiated by the excitation light, the excitation light comprising light of a first wavelength capable of exciting the first fluorescent dye and light of a second wavelength capable of exciting the second fluorescent dye, wherein the second wavelength is greater than the first wavelength, and the optical information comprises a first fluorescent signal corresponding to the first fluorescent dye and a second fluorescent signal corresponding to the second fluorescent dye obtained in the single test; and

[0015] obtaining the infected red blood cell information of the assay sample based on the first fluorescent signal and the second fluorescent signal. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a structural schematic diagram of an embodiment of a sample analyzer according to the present application.

[0017] Figure 2 is a structural schematic diagram of an embodiment of an optical detection device according to the present application.

[0018] Figure 3A schematic flow chart of one embodiment of the sample analysis method according to the present application.

[0019] Figure 4 A first scatter plot obtained from testing a blood sample to be tested that is not infected with Plasmodium according to Example 1 of the present application.

[0020] Figure 5 A second scatter plot obtained from testing a blood sample to be tested that is not infected with Plasmodium according to Example 1 of the present application.

[0021] Figure 6 A first scatter plot obtained from testing a blood sample to be tested that is infected with Plasmodium according to Example 1 of the present application.

[0022] Figure 7 A second scatter plot obtained from testing a blood sample to be tested that is infected with Plasmodium according to Example 1 of the present application.

[0023] Figure 8 A first scatter plot obtained from testing another blood sample to be tested that is infected with Plasmodium according to Example 2 of the present application.

[0024] Figure 9 A second scatter plot obtained from testing another blood sample to be tested that is infected with Plasmodium according to Example 2 of the present application. DETAILED DESCRIPTION

[0025] The embodiments of the present application will be described in detail below with reference to the drawings. It is obvious that the described embodiments are only a part of embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0026] In the present application, the serial numbers of components, such as "first", "second", etc., are used only to distinguish the described objects, and do not have any sequence or technical meaning. The "connection" and "coupling" in the present application, if not specially stated, include direct and indirect connection (coupling).

[0027] For the convenience of subsequent description, some terms related to the present application are first described herein:

[0028] 1) Scatter plot: a two-dimensional or three-dimensional graph generated by a blood cell analyzer, on which a plurality of particles are distributed with two-dimensional or three-dimensional characteristic information. The X-axis, Y-axis and Z-axis of the scatter plot all represent a characteristic of each particle. For example, in a scatter plot, the X-axis represents the forward scattering light intensity, the Y-axis represents the fluorescence intensity, and the Z-axis represents the side scattering light intensity.

[0029] The term "scatter plot" used in the present application not only refers to the distribution of at least two groups of data in the form of data points in a rectangular coordinate system, but also includes data arrays, i.e. is not limited by the form of their graphical presentation.

[0030] 2) Cell population: a group of particles distributed in a certain area of the scatter plot and formed by a plurality of particles with the same characteristics, also known as "particle group" or "cell group", such as a group of white blood cells (including all types of white blood cells), and subgroups of white blood cells, such as a group of neutrophils, a group of lymphocytes, a group of monocytes, a group of eosinophils, or a group of basophils, etc.

[0031] 3) Blood shadow: a fragment particle obtained by lysing red blood cells and platelets in blood with a hemolytic agent.

[0032] The blood cell analyzer used in the present application classifies and counts particles in a blood sample by combining laser scattering method and fluorescence staining method of flow cytometry. Here, the principle of detecting the blood sample by the blood cell analyzer can be, for example: first, draw the blood sample, treat the blood sample with a hemolytic agent and a fluorescent dye, wherein the red blood cells are destroyed and lysed by the hemolytic agent, and the white blood cells are not lysed, but the fluorescent dye can enter the cell nucleus of the white blood cells with the help of the hemolytic agent and bind to the nucleic acid substances in the cell nucleus; then the particles in the sample pass through the detection hole irradiated by the laser beam one by one, when the laser beam irradiates the particles, the characteristics of the particles themselves (such as volume, staining degree, cell content size and content, nuclear density, etc.) can block or change the direction of the laser beam, thereby generating scattered light of various angles corresponding to the characteristics of the particles. After the scattered light is received by the signal detector, information related to the structure and composition of the particles can be obtained. Among them, the forward scattering light (Forward scatter, FS) reflects the number and volume of the particles, the side scattering light (Side scatter, SS) reflects the complexity of the internal structure of the cells (such as intracellular particles or cell nucleus), and the fluorescence (Fluorescence, FL) reflects the content of nucleic acid substances in the cells. Using these light information, the cells in the blood sample can be classified and counted.

[0033] Figure 1A schematic diagram of a sample analyzer, in this case a blood cell analyzer, according to some embodiments of the present application. The sample analyzer 100 comprises at least a sample suction device 110, a sample preparation device 120, an optical detection device 130, and a processor 140. The sample suction device 110 is configured to suction a blood sample to be tested. The sample preparation device 120 is configured to mix at least a portion of the blood sample to be tested, a hemolytic agent, a first fluorescent dye, and a second fluorescent dye in a reaction cell to prepare a test sample. The optical detection device 130 comprises a light emitting device, a flow chamber, and a light detector. The light emitting device is configured to emit a light beam to illuminate the flow chamber, which is in communication with the reaction cell and configured to pass each particle in the test sample therethrough. The light detector is configured to detect optical information generated by a particle in the test sample when illuminated by the light beam in a single test. The light emitting device comprises a first light source configured to emit a light beam of a first wavelength capable of exciting the first fluorescent dye, and a second light source configured to emit a light beam of a second wavelength capable of exciting the second fluorescent dye, wherein the second wavelength is greater than the first wavelength. The optical information comprises a first fluorescent signal corresponding to the first fluorescent dye and a second fluorescent signal corresponding to the second fluorescent dye.

[0034] In some embodiments, the sample suction device 110 comprises a sampling needle (not shown) configured to suction the blood sample to be tested. In addition, the sample suction device 110 can further comprise a driving device configured to drive the sampling needle to quantitatively suction the blood sample to be tested from a test tube through a needle nozzle of the sampling needle. Furthermore, the sample suction device 110 can further deliver the suctioned blood sample to be tested to the sample preparation device 120.

[0035] In some embodiments, the sample preparation device 120 can comprise at least one reaction cell and a reagent supply device (not shown). The at least one reaction cell is configured to receive the blood sample to be tested suctioned by the sample suction device 110, and the reagent supply device is configured to provide processing reagents (including a hemolytic agent, a first fluorescent dye, a second fluorescent dye, etc.) to the at least one reaction cell, so that the blood sample to be tested suctioned by the sample suction device 110 is mixed with the processing reagents provided by the reagent supply device in the reaction cell to prepare a test sample.

[0036] For example, the sample preparation device 120 is configured to mix and incubate the blood sample, the hemolytic agent, the first fluorescent dye, and the second fluorescent dye, and the reaction time is 10s-1min, and the reaction temperature is 25-50℃.

[0037] In the embodiments of the present application, the hemolytic agent can dissolve red blood cells and platelets in the blood sample, but can keep the morphology of white blood cells substantially unchanged. The fragments of red blood cells and platelets in the blood sample after being dissolved form blood shadows.

[0038] In some embodiments, the hemolytic agent can comprise any one or a combination of a cationic surfactant, a non-ionic surfactant, an anionic surfactant, an amphiphilic surfactant, a buffer pair. The cationic surfactant is for example selected from at least one or a combination of dodecyltrimethylammonium chloride, octyltrimethylammonium bromide, tetradecyltrimethylammonium chloride. The non-ionic surfactant is for example selected from at least one or a combination of long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, fatty amine polyoxyethylene ether. The buffer pair is for example selected from at least one or a combination of phosphate, citrate, Tris-HCl. The anionic surfactant is for example selected from at least one or a combination of dodecylbenzenesulfonic acid, fatty alcohol acylsulfuric acid sodium, ethoxylated fatty acid methyl ester sulfonic acid sodium, secondary alkyl sulfonic acid sodium, alcohol ether carboxylate.

[0039] In other embodiments, the hemolytic agent can comprise at least one of an alkyl glycoside, a triterpenoid saponin, a steroidal saponin.

[0040] In some embodiments, the first fluorescent dye can be a fluorescent dye capable of specifically labeling Plasmodium, which comprises a compound of the following general structure

[0041]

[0042] In the general structure:

[0043] R1is selected from substituted or unsubstituted phenyl;

[0044] R2is selected from C 1-8 nitrogen-containing alkyl, substituted or unsubstituted nitrogen-containing or oxygen-containing heterocycle; the nitrogen-containing or oxygen-containing heterocycle is substituted with substituted or unsubstituted phenyl;

[0045] X is halogen or PF6 - ;

[0046] each of the phenyl groups of R1and R2is optionally substituted with substituents selected from the group consisting of CN, COOH, NH2, NO2, OH, SH, C 1-6 alkoxy, C 1-6 alkylamino, C 1-6 amide, halogen or C 1-6 haloalkyl.

[0047] Further, R1is selected from hydrogen, phenyl or biphenyl. Still further, R1is selected from biphenyl.

[0048] Further, R2is selected from N, N dimethyl, piperazinyl or 2,6 dimethylpiperazinyl. Still further, R2is selected from 2,6 dimethylpiperazinyl.

[0049] The concentration of the first fluorescent dye is 0.1-1000 mg / L.

[0050] As some embodiments, the compound for the first fluorescent dye has one of the following structures:

[0051]

[0052] For more embodiments of the first fluorescent dye of the present application, please refer to the Chinese application CN202310028120.1 filed by the applicant on January 9, 2023, the entire disclosure of which is incorporated herein by reference.

[0053] In some embodiments, the second fluorescent dye can be a fluorescent dye capable of being used for specifically labeling Plasmodium, which includes a compound with the following general structure

[0054]

[0055] In the general structure:

[0056] wherein R1and R2are each independently selected from C2-C8alkyl, wherein R1and R2are each independently selected from C2-C8alkyl;

[0057] The concentration of the second fluorescent dye is 0.1-1000 mg / L.

[0058] As some embodiments, the compound for the second fluorescent dye has one of the following structures A, B:

[0059]

[0060] A

[0061]

[0062] B

[0063] The second fluorescent dye can also be used for white blood cell classification.

[0064] In some embodiments, the first fluorescent dye and / or the second fluorescent dye are preserved in water-soluble organic phases such as glycerol, glycol, ethylene glycol, etc.

[0065] In some embodiments, the first fluorescent dye and / or the second fluorescent dye can be preserved alone or mixed with hemolytic agents.

[0066] In some embodiments, the flow cell in the optical detection device 130 refers to a chamber adapted to detect a focused fluid flow of light scatter signals and fluorescence signals. When a particle, such as a blood cell, passes through a detection aperture of the flow cell, the particle scatters the incident light beam directed at the detection aperture from the light source in all directions. Light detectors can be positioned at one or more different angles relative to the incident light beam to detect the light scattered by the particle, resulting in light scatter pulse signals (also referred to as light scatter signals). Since different particles have different light scattering properties, light scatter signals can be used to distinguish different populations of particles.

[0067] In particular, light scatter signals detected near the incident light beam are often referred to as forward light scatter signals or small angle light scatter signals. In some embodiments, the forward light scatter signals can be detected at an angle of about 1° to about 10° from the incident light beam. In other embodiments, the forward light scatter signals can be detected at an angle of about 2° to about 6° from the incident light beam. Light scatter signals detected at an angle of about 90° from the incident light beam are often referred to as side light scatter signals. In some embodiments, the side light scatter signals can be detected at an angle of about 65° to about 115° from the incident light beam. Typically, fluorescence signals emitted from blood cells stained with a fluorescent dye are also detected at an angle of about 90° from the incident light beam.

[0068] In some embodiments, the light detectors in the optical detection device 130 can include a scatter light detector, such as a side scatter light detector, for detecting scatter light pulse signals (also referred to as scatter light signals), such as side scatter light pulse signals (also referred to as side scatter light signals), and a first fluorescence detector for detecting first fluorescence pulse signals (also referred to as first fluorescence signals) and a second fluorescence detector for detecting second fluorescence pulse signals (also referred to as second fluorescence signals).

[0069] In some embodiments, the optical detection device 130 includes a forward scatter light detector for detecting forward scatter light signals or a side scatter light detector for detecting side scatter light signals. Preferably, the optical detection device 130 includes both a forward scatter light detector and a side scatter light detector.

[0070] Figure 2A specific example of an optical detection device 130 is shown. This optical detection device 130 includes a light emitting device 131, a front light assembly 132, a flow chamber 133, a forward scattering light detector 134, a first dichroic mirror 135, a side scattering light detector 136, a second dichroic mirror 137, a first fluorescence detector 138, and a second fluorescence detector 139. The first fluorescence detector 138 detects a first fluorescence signal corresponding to a first fluorescent dye generated by particles passing through the flow chamber 133 after being irradiated by a light beam. The second fluorescence detector 139 detects a second fluorescence signal corresponding to a second fluorescent dye generated by particles passing through the flow chamber 133 after being irradiated by a light beam. Here, the light emitting device 131, the front light assembly 132, the flow chamber 133, and the forward scattering light detector 134 are arranged sequentially along the optical axis. The front light assembly is configured to converge the light beam emitted by the light emitting device 131 into the detection area of ​​the flow chamber 133 in the particle flow direction, so that particles flowing through the detection area of ​​the flow chamber 133 can generate scattered light. On one side of the flow chamber 133, a first dichroic mirror 135 is arranged at a 45° angle to the optical axis. A portion of the lateral light generated when particles flow through the detection area of ​​the flow chamber 133 is reflected by the first dichroic mirror 135 and captured by the side-scattering light detector 136, while another portion of the lateral light passes through the first dichroic mirror 135 and reaches a second dichroic mirror 137, which is also arranged downstream of the first dichroic mirror 135 at a 45° angle to the optical axis. A portion of the lateral light passing through the first dichroic mirror 135 is reflected by the second dichroic mirror 137 and captured by the first fluorescence detector 138, while another portion passes through the second dichroic mirror 137 and is captured by the second fluorescence detector 139.

[0071] In some embodiments, the first wavelength is between 315 nm and 490 nm, and the second wavelength is between 610 nm and 750 nm. Preferably, the first wavelength is between 400 nm and 450 nm, and the second wavelength is between 620 nm and 700 nm.

[0072] In some embodiments, the processor 140 is used to process and perform calculations on the data to obtain the desired results, such as generating a two-dimensional or three-dimensional scatter plot based on the collected optical information, and performing particle analysis on the scatter plot according to the gating method.

[0073] In some embodiments, the processor 140 may visualize intermediate or final processing results and then display them through the display device 150. For example, the display device 150 may include a user interface, and the processor 140 may display the processing results on the user interface of the display device 150.

[0074] In some embodiments, the processor 140 includes, but is not limited to, devices such as a central processing unit (CPU), a microcontroller unit (MCU), a field-programmable gate array (FPGA), and a digital signal processor (DSP) for interpreting computer instructions and processing data in computer software. For example, the processor is used to execute various computer applications in a computer-readable storage medium, thereby enabling the sample analyzer 100 to execute corresponding detection procedures and analyze the scattered light signal and fluorescence signal detected by the optical detection device 130 in real time.

[0075] In addition, the sample analyzer 100 may also include a liquid path system (not shown) for connecting the sample aspiration device 110, the sample preparation device 120 and the optical detection device 130 to facilitate liquid transfer between these devices.

[0076] Furthermore, the sample analyzer 100 may also include a first housing 160 and a second housing 170. An optical detection device 130 and a processor 140 are disposed inside the second housing 170. A sample preparation device 120 is disposed, for example, inside the first housing 160, and a display device 150 is disposed, for example, on the outer surface of the first housing 160 and is used to display the detection results of the sample analyzer 100.

[0077] In this embodiment of the application, to assist doctors in determining whether a subject is infected with malaria, the processor 140 is configured to obtain information on infected red blood cells in the test sample based on a first fluorescence signal and a second fluorescence signal. Here, the information on infected red blood cells refers to optical information related to infected red blood cells.

[0078] In some embodiments, the infected red blood cell information may include a first scatter plot, that is, the processor 140 obtains the infected red blood cell information of the test sample based on a first fluorescence signal and a second fluorescence signal, including: generating a first scatter plot based on the first fluorescence signal and the second fluorescence signal, and obtaining the infected red blood cell information of the test sample based on the first scatter plot.

[0079] In some embodiments, the infected red blood cell information may include an infected red blood cell count, that is, the processor 140 obtains the infected red blood cell information of the test sample based on a first fluorescence signal and a second fluorescence signal, which may include: obtaining the count result of infected red blood cells in the test sample based on the first fluorescence signal and the second fluorescence signal.

[0080] Obtaining quantitative results from infected red blood cells can more accurately assist doctors in diagnosing malaria.

[0081] In a specific example, a first scatter plot is generated based on a first fluorescence signal and a second fluorescence signal. Using a gating technique, a region representing infected red blood cells is obtained from this first scatter plot. The scatter points falling into this region are counted to obtain the count result of infected red blood cells, i.e., the count value. If the count value of infected red blood cells is greater than a predetermined threshold, an alarm is output (indicating that the blood sample is a malaria-positive sample).

[0082] In some alternative or additional embodiments, the processor 140 obtains information on infected red blood cells in the test sample based on a first fluorescence signal and a second fluorescence signal, which may include: identifying infected red blood cells containing multiple Plasmodium rings based on the first fluorescence signal and the second fluorescence signal. This allows for more accurate acquisition of the count value of infected red blood cells, avoiding the misidentification of a single Plasmodium ring within multiple Plasmodium rings in an infected red blood cell as a single infected red blood cell.

[0083] In a specific example, a first scatter plot is generated based on a first fluorescence signal and a second fluorescence signal. Using a gating technique, a region representing infected erythrocytes is obtained from this first scatter plot. From this region, a first infected erythrocyte population and a second infected erythrocyte population are identified. Each infected erythrocyte in the second infected erythrocyte population contains multiple Plasmodium rings. The centroids of the first and second fluorescence signals of the second infected erythrocyte population are correspondingly larger than those of the first and second infected erythrocyte populations.

[0084] In some embodiments, the optical information further includes at least one scattered light signal detected in a single test. Here, the processor 140 is also configured to obtain a four-part differential white blood cell count of the test sample based on at least one scattered light signal and a second fluorescence signal, wherein the four-part differential white blood cell count includes the counts of lymphocytes, monocytes, neutrophils, and eosinophils in the test sample.

[0085] This enables the processing of a single blood sample, particularly in leukocyte testing, with a hemolytic agent, a first dye capable of staining infected erythrocytes, and a second dye capable of staining both leukocytes and infected erythrocytes to obtain a test sample solution. An optical detection device then detects the scattered light signal, the first fluorescence signal, and the first fluorescence signal generated by each particle in the test sample solution after being irradiated with excitation light of two wavelengths. Information on infected erythrocytes is obtained based on the first and second fluorescence signals, and further information is obtained based on at least one scattered light signal and the second fluorescence signal. This allows for the simultaneous acquisition of infected erythrocyte information and leukocyte four-part differential results without increasing the amount of blood used, significantly reducing testing costs.

[0086] In a specific example, the scattered light signal includes a first lateral scattered light signal generated by particles passing through the flow chamber after being irradiated with light of a first wavelength, detected in a single test, and / or a second lateral scattered light signal generated by particles passing through the flow chamber after being irradiated with light of a second wavelength, detected in a single test. Here, the processor 140 obtains the white blood cell four-part differential result of the test sample based on at least one scattered light signal and a second fluorescence signal, including: generating a second scatter plot based on the first lateral scattered light signal and the second fluorescence signal, or based on the second lateral scattered light signal and the second fluorescence signal, and obtaining the white blood cell four-part differential result of the test sample based on the second scatter plot.

[0087] This application also provides a sample analysis method 200, such as... Figure 3 As shown, it includes S210, S220, S230 and S240.

[0088] In step S210, a blood sample to be tested is drawn. For example, a portion of the blood sample to be tested is drawn from a test tube containing the blood sample to be tested.

[0089] In step S220, at least a portion of the blood sample to be tested, a hemolysin, a first fluorescent dye, and a second fluorescent dye are mixed to prepare a single test sample.

[0090] For example, in step S220, the hemolysin, the first fluorescent dye, and the second fluorescent dye can be added sequentially to the same blood sample to obtain a test sample. The test sample is then incubated to allow the dyes to fully stain the particles in the test sample. Alternatively, in step S220, the first and second fluorescent dyes can be pre-mixed with the hemolysin to obtain a mixed reagent. This mixed reagent is then mixed with the blood sample to be tested at a volume ratio of 250:1 to 1000:1. After thorough mixing, the resulting test sample is incubated at 25°C to 50°C for 10 seconds to 1 minute, preferably 20 seconds to 40 seconds.

[0091] Examples of the hemolytic agent and fluorescent dye used in step S220 can be found in the above description and will not be repeated here.

[0092] In step S230, each particle in the test sample is passed through an optical detection area irradiated by excitation light in a single test to obtain the optical information generated by each particle in the test sample after being irradiated by excitation light. The excitation light includes light of a first wavelength that can excite the first fluorescent dye and light of a second wavelength that can excite the second fluorescent dye, wherein the second wavelength is greater than the first wavelength. The optical information includes a first fluorescence signal corresponding to the first fluorescent dye and a second fluorescence signal corresponding to the second fluorescent dye obtained in a single test.

[0093] Here, the first fluorescence signal is the fluorescence signal generated by the binding of particles in the sample with the first fluorescent dye under the excitation of light at the first wavelength, while the second fluorescence signal is the fluorescence signal generated by the binding of particles in the sample with the second dye under the excitation of light at the second wavelength.

[0094] In other words, in step S230, the fluorescence signal or fluorescence signal intensity of the sample is obtained based on the principle of flow cytometry.

[0095] In step S240, the information on infected red blood cells in the test sample is obtained based on the first fluorescence signal and the second fluorescence signal.

[0096] In some embodiments, obtaining the infected red blood cell information of the test sample based on the first fluorescence signal and the second fluorescence signal in step S240 may include: generating a first scatter plot based on the first fluorescence signal and the second fluorescence signal, and obtaining the infected red blood cell information of the test sample based on the first scatter plot.

[0097] In some embodiments, obtaining the infected red blood cell information of the test sample based on the first fluorescence signal and the second fluorescence signal in step S240 may include: obtaining the count result of infected red blood cells in the test sample based on the first fluorescence signal and the second fluorescence signal, for example, based on the first scatter plot.

[0098] Optionally or additionally, in step S240, obtaining the information of infected red blood cells in the test sample based on the first fluorescence signal and the second fluorescence signal may include: identifying infected red blood cells containing multiple malaria parasite rings based on the first fluorescence signal and the second fluorescence signal, for example, based on a first scatter plot.

[0099] In some embodiments, the optical information further includes at least one scattered light signal detected in a single test. The sample analysis method 200 further includes:

[0100] The white blood cell four-part differential result of the test sample is obtained based on at least one scattered light signal and a second fluorescence signal, wherein the white blood cell four-part differential result includes the count results of lymphocyte population, monocyte population, neutrophil population and eosinophil population in the test sample.

[0101] Further, the scattered light signal includes a first lateral scattered light signal generated by particles passing through the flow chamber after being irradiated with light of a first wavelength, detected in a single test, and / or a second lateral scattered light signal generated by particles passing through the flow chamber after being irradiated with light of a second wavelength, detected in a single test. Obtaining the white blood cell four-part differential result of the test sample based on at least one scattered light signal and a second fluorescence signal may include: generating a second scatter plot based on the first lateral scattered light signal and the second fluorescence signal, or based on the second lateral scattered light signal and the second fluorescence signal, and obtaining the white blood cell four-part differential result of the test sample based on the second scatter plot.

[0102] In some embodiments, the first wavelength is between 315 nm and 490 nm and the second wavelength is between 610 nm and 750 nm.

[0103] The present application is described below with reference to embodiments intended to illustrate the application and not limit it. Unless otherwise specified, the experiments and methods described in the embodiments are generally performed according to conventional methods well known in the art and described in various references. Furthermore, where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0104] Example 1

[0105] The reagent formula is as follows:

[0106] A: Fluorescent dye solution

[0107] 30mg of the first fluorescent dye

[0108] 200mg of the second fluorescent dye

[0109] 1000g of ethylene glycol

[0110] The first fluorescent dye has the following general formula:

[0111]

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

[0113]

[0114] B: Hemolytic agent

[0115] 0.5g dodecyltrimethylammonium chloride

[0116] TRIS 18g

[0117] HCl 20ml

[0118] 1L of water

[0119] Brij 35 1.5g

[0120] pH 7.0

[0121] The sample analyzer according to this application, equipped with a red laser (emitting a beam with a wavelength of approximately 633 nm) and a blue laser (emitting a beam with a wavelength of approximately 488 nm), was used to process and detect uninfected and infected blood samples, respectively: 20 μL of reagent A and 20 μL of the blood sample were added to 1 mL of reagent B to form a test sample; the side-scattered light intensity corresponding to blue light excitation, the first fluorescence intensity corresponding to blue light excitation, and the second fluorescence intensity corresponding to red light excitation were detected; based on the first fluorescence intensity and the side-scattered light intensity... A two-dimensional first scatter plot is generated, and the region Infect_Area representing infected red blood cells is obtained based on the first scatter plot. The scatter points falling into this region are counted to obtain the count value of infected red blood cells. A two-dimensional second scatter plot is generated based on the intensity of side-scattered light and the intensity of second fluorescence, and the white blood cell four-part differential result is obtained based on the second scatter plot. The white blood cell four-part differential result includes the count results of neutrophils (neu), lymphocytes (lym), monocytes (mon), and eosinophils (eos) in the test sample.

[0122] Figure 4 and 5 The first and second scatter plots are shown respectively, obtained by the sample analyzer after processing and detecting blood samples that are not infected with malaria parasites. In the first scatter plot, there are basically no scatter points in the infected red blood cell area Infect_Area, indicating that the blood sample is not infected with malaria parasites.

[0123] Figure 6 and 7 The first scatter plot and the second scatter plot are shown respectively, obtained by the sample analyzer after processing and detecting the blood sample infected with malaria parasites. In the first scatter plot, there are a certain number of scatter points in the infected red blood cell area Infect_Area, indicating that the blood sample is infected with malaria parasites.

[0124] Example 2

[0125] The sample analyzer according to this application, equipped with a red laser (emitting a beam with a wavelength of approximately 633 nm) and a blue laser (emitting a beam with a wavelength of approximately 488 nm), was used to process and detect blood samples infected with Plasmodium parasites: 20 μL of reagent A and 20 μL of the blood sample were added to 1 mL of reagent B to form a test sample; the intensity of side-scattered light corresponding to blue light excitation, the first fluorescence intensity corresponding to blue light excitation, and the second fluorescence intensity corresponding to red light excitation were detected; a two-dimensional first scatter plot was generated based on the first fluorescence intensity and the side-scattered light intensity, as shown below. Figure 8 As shown, based on the first scatter plot, the region Infect_Area representing infected red blood cells is obtained. Scatter points falling into this region are counted to obtain the count value of infected red blood cells. A two-dimensional second scatter plot is generated based on the side-scatter light intensity and the second fluorescence intensity, as shown below. Figure 9 As shown, the white blood cell four-part differential results are obtained based on the second scatter plot. The white blood cell four-part differential results include the count results of neutrophils (neu), lymphocytes (lym), monocytes (mon), and eosinophils (eos) in the test sample.

[0126] Here, based on Figure 8 The first scatter plot shown delineates the region Infect_Area, representing infected red blood cells. From this region Infect_Area, the first infected red blood cell group Infect_CellPop1 and the second infected red blood cell group Infect_CellPop2 are identified. Each infected red blood cell in the second infected red blood cell group Infect_CellPop2 contains multiple malaria parasite rings.

[0127] All features or combinations of features mentioned above in the specification, drawings, and claims may be used in any combination or individually, provided they are meaningful within the scope of this application and do not contradict each other. The advantages and features described with reference to the sample analyzer provided in this application shall be applied accordingly to the sample analysis method provided in this application, and vice versa.

[0128] The above description is merely a preferred embodiment of this application and does not limit the scope of patent protection of this application. All equivalent modifications made based on the content of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the scope of patent protection of this application.

Claims

1. A sample analyzer, comprising: A sampling device is used to collect blood samples for testing. A sample preparation apparatus for mixing at least a portion of the blood sample to be tested, a hemolysin, a first fluorescent dye, and a second fluorescent dye in a reaction cell to prepare a test sample; An optical detection device includes a light emitting device, a flow chamber, and a photodetector. The light emitting device emits excitation light to irradiate the flow chamber, which is connected to a reaction cell and allows individual particles in the test sample to pass through. The photodetector detects the optical information generated when individual particles in the test sample are irradiated by the excitation light as they pass through the flow chamber in a single test. The light emitting device includes a first light source and a second light source. The first light source emits light of a first wavelength capable of exciting a first fluorescent dye, and the second light source emits light of a second wavelength capable of exciting a second fluorescent dye, wherein the second wavelength is greater than the first wavelength. The optical information includes a first fluorescence signal corresponding to the first fluorescent dye and a second fluorescence signal corresponding to the second fluorescent dye detected in the single test. as well as The processor is configured to obtain information about infected red blood cells in the test sample based on the first fluorescence signal and the second fluorescence signal.

2. The sample analyzer according to claim 1, characterized in that, The processor obtains the infected red blood cell information of the test sample based on the first fluorescence signal and the second fluorescence signal, including: The count of infected red blood cells in the test sample is obtained based on the first fluorescence signal and the second fluorescence signal; and / or Infected red blood cells containing multiple malaria parasite rings were identified based on the first and second fluorescence signals.

3. The sample analyzer according to claim 1 or 2, characterized in that, The optical information also includes at least one scattered light signal detected in the single test; The processor is further configured to obtain a four-part differential white blood cell count of the test sample based on at least one of the scattered light signals and the second fluorescence signal, wherein the four-part differential white blood cell count includes the counts of lymphocytes, monocytes, neutrophils and eosinophils in the test sample.

4. The sample analyzer according to claim 3, characterized in that, The scattered light signal includes a first side-scattered light signal generated by a particle passing through the flow chamber after being irradiated by light of the first wavelength, which is detected in the single test, and / or a second side-scattered light signal generated by a particle passing through the flow chamber after being irradiated by light of the second wavelength, which is detected in the single test. The processor obtains the infected red blood cell information of the test sample based on the first fluorescence signal and the second fluorescence signal, including: generating a first scatter plot based on the first fluorescence signal and the second fluorescence signal, and obtaining the infected red blood cell information of the test sample based on the first scatter plot; The processor obtains the white blood cell four-part differential result of the test sample based on at least one of the scattered light signals and the second fluorescence signal, including: generating a second scatter plot based on the first side-scattered light signal and the second fluorescence signal or based on the second side-scattered light signal and the second fluorescence signal, and obtaining the white blood cell four-part differential result of the test sample based on the second scatter plot.

5. The sample analyzer according to claim 1, characterized in that, The first wavelength is between 315nm and 490nm and the second wavelength is between 610nm and 750nm.

6. A sample analysis method, comprising: Collect the blood sample to be tested; At least a portion of the blood sample to be tested, a hemolytic agent, a first fluorescent dye, and a second fluorescent dye are mixed to prepare a single test sample. In a single test, each particle in the test sample is passed through an optical detection area irradiated by excitation light to obtain optical information generated by each particle in the test sample after being irradiated by the excitation light. The excitation light includes light of a first wavelength capable of exciting the first fluorescent dye and light of a second wavelength capable of exciting the second fluorescent dye, wherein the second wavelength is greater than the first wavelength. The optical information includes a first fluorescence signal corresponding to the first fluorescent dye and a second fluorescence signal corresponding to the second fluorescent dye obtained in the single test. as well as The information on infected red blood cells in the test sample is obtained based on the first fluorescence signal and the second fluorescence signal.

7. The sample analysis method according to claim 6, characterized in that, The information on infected red blood cells in the test sample is obtained based on the first scatter plot, including: The count of infected red blood cells in the test sample is obtained based on the first fluorescence signal and the second fluorescence signal; and / or Infected red blood cells containing multiple malaria parasite rings were identified based on the first and second fluorescence signals.

8. The sample analysis method according to claim 6 or 7, characterized in that, The optical information also includes at least one scattered light signal detected in the single test. The sample analysis method further includes: The white blood cell four-part differential result of the test sample is obtained based on at least one of the scattered light signals and the second fluorescence signal, wherein the white blood cell four-part differential result includes the count results of lymphocytes, monocytes, neutrophils and eosinophils in the test sample.

9. The sample analysis method according to claim 8, characterized in that, The scattered light signal includes a first side-scattered light signal detected in the single test, generated by particles passing through the flow chamber after being irradiated with light of the first wavelength, and / or a second side-scattered light signal detected in the single test, generated by particles passing through the flow chamber after being irradiated with light of the second wavelength. Obtaining the infected erythrocyte information of the test sample based on the first fluorescence signal and the second fluorescence signal includes: generating a first scatter plot based on the first fluorescence signal and the second fluorescence signal, and obtaining the infected erythrocyte information of the test sample based on the first scatter plot; and Obtaining the white blood cell four-part differential result of the test sample based on at least one of the scattered light signals and the second fluorescence signal includes: generating a second scatter plot based on the first side-scattered light signal and the second fluorescence signal or based on the second side-scattered light signal and the second fluorescence signal, and obtaining the white blood cell four-part differential result of the test sample based on the second scatter plot.

10. The sample analysis method according to any one of claims 6 to 9, characterized in that, The first wavelength is between 315nm and 490nm and the second wavelength is between 610nm and 750nm.

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