Blood analyzer, blood analysis method, cell analysis reagent and application

Abstract

The invention provides a blood analyzer, a blood analysis method, a cell analysis reagent and application. The blood analyzer comprises: a sample suction device for sucking a blood sample to be detected; the sample preparation device is used for preparing a first sample and comprises a reaction tank for mixing at least one part of a blood sample to be detected with a first reagent comprising a first hemolytic agent and a lysosome staining agent containing a lysosome dye; the optical detector is used for detecting the first sample to obtain a first optical signal. The processor executes the following steps: processing the first optical signal to obtain first leukocyte information; extracting at least one first leukocyte parameter of the first leukocyte information; and obtaining an infection marker parameter based on the at least one first leukocyte parameter.

Description

Technical Field

[0001] The present disclosure relates to the field of in vitro diagnosis, and in particular to a blood analyzer, a blood analysis method, a cell analysis reagent and uses thereof for infection-related use in a subject. Background Art

[0002] Infectious diseases are common clinical diseases. Among them, sepsis is a serious infectious disease. The incidence of sepsis is high, with more than 18 million cases of severe sepsis each year worldwide. In addition, sepsis is a serious disease with a high mortality rate. About 14,000 people die from its complications every day worldwide. According to foreign epidemiological surveys, the mortality rate of sepsis has exceeded that of myocardial infarction and has become the main cause of death in non-heart disease patients in intensive care units. In recent years, despite advances in anti-infection treatment and organ function support technology, the mortality rate of sepsis is still as high as 30% to 70%. Sepsis treatment is expensive, consumes a lot of medical resources, seriously affects the quality of human life, and has posed a huge threat to human health.

[0003] For the rapid identification of infectious diseases, the industry's existing solutions include: microbial culture, examination of inflammatory markers such as C-reactive protein (CRP), procalcitonin (PCT) and serum amyloid A (SAA), serum antigen and antibody testing and routine blood tests.

[0004] Microbial culture is considered the most reliable gold standard. However, this method has a long reporting cycle, specimens are easily contaminated, and the false negative rate is high, which cannot meet the clinical requirements for rapid and accurate results.

[0005] Tests for inflammatory factors such as CRP, PCT, and SAA have good sensitivity and are widely used. However, these tests have weak specificity for infectious diseases, and usually require a combined test of CRP, PCT, and SAA, which increases the economic burden on patients. In addition, CRP and PCT are interfered with by specific diseases, and therefore sometimes cannot accurately reflect the patient's infection status. For example, CRP is produced in the liver, and infected patients with liver damage have normal CRP levels, which can result in false negative results in the diagnosis of infectious diseases.

[0006] Serum antigen-antibody testing can confirm specific virus types, but its role is limited when the pathogen type is unclear. In addition, the testing cost is high, which increases the financial burden on patients.

[0007] Routine blood tests can indicate the occurrence and type of infection to a certain extent. However, the proportion of white blood cells (WBC) and neutrophils (Neu) in routine blood tests is easily affected by other non-infectious inflammatory reactions and normal physiological fluctuations of the body.

[0008] At present, dyes are often used to fluorescently stain blood cells, and then blood analyzers are used to distinguish different blood cells based on the difference in fluorescence intensity. Commonly used fluorescent dyes are nucleic acid dyes, which can specifically bind to DNA and RNA in cells, thereby changing the fluorescence intensity of the dye. Using nucleic acid dyes for blood analysis has high sensitivity and fast response, so it is widely used.

[0009] However, most analysis methods based on staining nucleic acid substances have certain limitations. For example, many abnormal blood cells undergo changes in the state of organelles, and nucleic acid dyes are difficult to identify such changes. Summary of the invention

[0010] The purpose of the present disclosure is to provide a blood analyzer. The blood analyzer utilizes the changes in lysosomes in blood cells caused by infection, especially severe infection, and uses a reagent containing a lysosomal dye to treat the sample, so as to quickly obtain parameters related to the infection of the subject, and then quickly evaluate the infection status of the subject. The present disclosure also provides a blood analysis method, the use of a cell analysis reagent containing a lysosomal dye, and the cell analysis reagent.

[0011] To this end, the first aspect of the present disclosure provides a blood analyzer, including a sample suction device, a sample preparation device, a light source, a flow chamber, an optical detector, a processor, and a non-transitory computer-readable storage medium programmed with a computer application, wherein:

[0012] The sample aspirating device is configured to aspirate at least a portion of the blood sample to be tested and transport it to the sample preparation device;

[0013] The sample preparation device comprises a reagent supply part and at least one reaction pool, wherein the reagent supply part is configured to provide a first reagent to a first reaction pool, the first reaction pool is configured to mix the first reagent with at least a portion of the blood sample to be tested therein to prepare a first sample, and transport the first sample to the flow chamber, wherein the first reagent comprises a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on lysosomes of cells in the blood sample to be tested;

[0014] The light source is configured to direct a light beam toward a detection hole of the flow chamber that is in communication with the reaction cell;

[0015] The flow chamber is configured to allow particles in the first sample to pass through the flow chamber one by one;

[0016] The optical detector is mounted around the flow cell and configured to detect a first light signal of particles in the first sample passing through a detection hole of the flow cell;

[0017] The processor is operably connected to the optical detector, and when the computer application in the non-transitory computer readable storage medium is executed by the processor, the processor is caused to perform the following steps:

[0018] Processing the first light signal to obtain first white blood cell information;

[0019] extracting at least one first white blood cell parameter of the first white blood cell information;

[0020] An infection marker parameter is obtained based on the at least one first white blood cell parameter.

[0021] A second aspect provides a blood analysis method, the method comprising:

[0022] Obtaining a blood sample to be tested from a subject;

[0023] preparing a first sample, wherein the first sample contains at least a portion of the blood sample to be tested and a first reagent, wherein the first reagent includes a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on the lysosomes of cells in the blood sample to be tested;

[0024] Allowing particles in the first sample to pass through an optical detection area illuminated by light one by one to obtain a first optical signal generated by the particles in the first sample after being illuminated by light;

[0025] Processing the first light signal to obtain first white blood cell information;

[0026] extracting at least one first white blood cell parameter of the first white blood cell information;

[0027] An infection marker parameter is obtained based on the at least one first white blood cell parameter.

[0028] A third aspect provides a use of a cell analysis reagent in analyzing infection marker parameters of a blood sample from a subject, wherein the cell analysis reagent comprises a hemolytic agent and a lysosomal stain, wherein the lysosomal stain comprises a lysosomal dye targeting a cell lysosome.

[0029] The fourth aspect provides a cell analysis reagent.

[0030] The cell analysis reagent is the same as the first reagent described above.

[0031] According to various embodiments of the present disclosure, the blood analyzer uses infection, especially severe infection such as sepsis, to cause changes in lysosomes in blood cells, and uses lysosomal dyes to stain and detect cell lysosomes, thereby obtaining cell optical parameters of blood cell changes relative to blood samples of normal subjects, and thereby obtaining infection marker parameters, so as to be able to quickly and accurately evaluate the infection status of the subject. In addition, the blood analyzer can process and detect the same sample using traditional nucleic acid dyes, combine the obtained optical parameters with the cell optical parameters obtained after the sample is treated with lysosomal dyes, and thereby obtain infection marker parameters with further improved reliability and sensitivity, thereby more accurately evaluating the infection status. While obtaining infection marker parameters, the blood analyzer does not affect the detection of traditional blood analysis items in routine blood tests, saving detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic structural diagram of a blood analyzer according to an embodiment;

[0033] Figure 2 is a block diagram of an optical detector according to one embodiment;

[0034] Figure 3 is a schematic diagram of a blood analysis method according to one embodiment;

[0035] Figure 4 is a schematic flow chart of a blood analysis method according to another embodiment;

[0036] Figure 5 Confocal laser scanning microscopic images of cells stained with compound 1 and commercially available lysosomal dye according to Example 1 and their superimposed comparison images;

[0037] Figure 6 is a SS-FL scatter plot obtained according to Example 2, wherein the left figure is the test result of the blood sample of a healthy subject, and the right figure is the test result of the blood sample of a sepsis patient;

[0038] Figure 7 is a SS-FL scatter plot obtained according to Example 3, wherein the left figure is the test result of the blood sample of a healthy subject, and the right figure is the test result of the blood sample of a sepsis patient;

[0039] Figure 8 is a SS-FL scatter plot obtained according to Example 4, wherein the figures from left to right correspond to the test results of treating blood samples of sepsis patients using reagents A1, A2 and A3 respectively;

[0040] Fig. 9is a SS-FL scatter plot obtained according to Example 5, wherein the left figure is the test result of the blood sample of a healthy subject, and the right figure is the test result of the blood sample of a sepsis patient;

[0041] Fig.10 is the infection marker parameter correlation curve (ROC graph) obtained according to Example 6;

[0042] Fig.11 is a SS-FL scatter plot obtained according to Example 7, wherein the left figure is the test result of the blood sample of a healthy subject, and the right figure is the test result of the abnormal blood sample containing nucleated red blood cells; and

[0043] Fig.12 SS-FL scatter plot obtained according to Example 8, wherein the left figure is the test result of the blood sample of a healthy subject, and the right figure is the test result of the abnormal blood sample containing nucleated red blood cells. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the implementation methods of the present invention, not all of the implementation methods. Based on the implementation methods of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] It should be noted that the terms "first\second\third" involved in the embodiments of the present application are merely used to distinguish similar objects and do not represent a specific ordering of the objects. It can be understood that "first\second\third" can be interchanged in a specific order or sequence where permitted.

[0046] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0047] The term "lysosomal dye" used in this article refers to a class of fluorescent dyes that can be targeted and bound to cell lysosomes. After the dye binds to the lysosome, it will produce different fluorescence intensities due to changes in the internal environment of the lysosomes of cells of different types (such as different leukocyte subsets) and / or states (such as whether they are infected).

[0048] The term "scatter plot" used herein is a two-dimensional or three-dimensional graph generated by a blood cell analyzer, on which two-dimensional or three-dimensional characteristic information of multiple particles is distributed. The X-axis, Y-axis, and Z-axis of the scatter plot all represent a characteristic of each particle. For example, in an illustrative scatter plot, the X-axis represents the forward scattered light intensity, the Y-axis represents the fluorescence intensity, and the Z-axis represents the side scattered light intensity. The term "scatter plot" not only refers to a distribution diagram of at least two sets of data in the form of data points in a rectangular coordinate system, but also includes a data array, that is, it is not limited to its graphical presentation form.

[0049] The term "particle cluster" or "cell cluster" used in this article is a particle group distributed in a certain area of ​​the scatter plot, which is formed by multiple particles with the same cell characteristics. For example, a leukocyte particle cluster refers to a group including all types of leukocytes, and a leukocyte cluster particle cluster refers to a group including at least one leukocyte subpopulation among neutrophil particle clusters, lymphocyte particle clusters, monocyte particle clusters, eosinophil particle clusters or basophil particle clusters.

[0050] The term "ROC curve (receiver operator characteristic curve)" used in this article is a receiver operating characteristic curve, which is a curve drawn according to a series of different binary classification methods (cutoff thresholds) with the true positive rate as the vertical axis and the false positive rate as the horizontal axis. ROC_AUC (area under the curve) represents the area enclosed by the ROC curve and the horizontal axis.

[0051] The principle of making ROC curve is to set multiple different critical values ​​for continuous variables, calculate the corresponding sensitivity and specificity at each critical value, and then draw a curve with sensitivity as the vertical axis and 1-specificity as the horizontal axis.

[0052] Since the ROC curve is composed of multiple critical values ​​representing their respective sensitivities and specificities, the ROC curve can be used to select the best diagnostic limit value for a diagnostic method. The closer the ROC curve is to the upper left corner, the higher the test sensitivity, the lower the false positive rate, and the better the performance of the diagnostic method. It can be seen that the point on the ROC curve closest to the upper left corner has the largest sum of sensitivity and specificity. This point or the value corresponding to its adjacent points is often used as a diagnostic reference value (also called a diagnostic threshold or judgment threshold or preset condition or preset range).

[0053] The blood analysis method used in traditional blood routine tests generally uses hemolytic agents and nucleic acid dyes to treat blood samples to break red blood cells, while other blood cells, such as white blood cells, still maintain their cell morphology. The nucleic acid substances therein are stained with nucleic acid dyes, and blood cells such as white blood cells are counted and classified through the DIFF channel and / or WNB channel of the blood analyzer. White blood cells can be identified and counted in four categories (lymphocytes (Lym), monocytes (Mon), neutrophils (Neu), and eosinophils (Eos)) through the DIFF channel. White blood cell counts and basophil counts can be obtained through the WNB channel, and nucleated red blood cells can also be identified and counted.

[0054] It is known that lysosomes are closely related to the occurrence of many diseases such as metabolic diseases, inflammation, etc. When a subject has inflammation or infection, enzymes, bactericidal substances and lysosomal membrane proteins in the lysosomes are upregulated. The inventors have found that blood cells can be distinguished based on lysosomal staining, and that rapid detection of infection can be achieved by fluorescently staining the lysosomes of blood cells with a lysosomal staining agent under the action of a hemolytic agent.

[0055] Thus, a blood analyzer is provided.

[0056] According to one embodiment of the present disclosure, the blood analyzer includes a sample aspirating device, a sample preparing device, a light source, a flow chamber, an optical detector, a processor, and a non-transitory computer-readable storage medium programmed with a computer application.

[0057] The sample aspirating device is configured to aspirate at least a portion of the blood sample to be tested and transport it to the sample preparation device.

[0058] The sample preparation device includes a reagent supply part and at least one reaction pool, wherein the reagent supply part is configured to provide a first reagent to a first reaction pool, the first reaction pool is configured to mix the first reagent with at least a portion of the blood sample to be tested therein to prepare a first test sample, and transport the first test sample to the flow chamber, wherein the first reagent includes a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye fluorescently stains the lysosomes of cells in the blood sample to be tested.

[0059] The light source is configured to direct a light beam toward a detection hole of the flow chamber connected to the reaction pool.

[0060] The flow cell is configured to allow particles in the first sample to pass through the flow cell one by one.

[0061] The optical detector is mounted around the flow cell and is configured to detect a first light signal of particles in the first sample passing through a detection hole of the flow cell.

[0062] The processor is operably connected to the optical detector, and when the computer application in the non-transitory computer-readable storage medium is executed by the processor, the processor performs the following steps: processing the first light signal to obtain first white blood cell information; extracting at least one first white blood cell parameter of the first white blood cell information; and obtaining an infection marker parameter based on the at least one first white blood cell parameter.

[0063] The following combination Figure 1 and Figure 2 The blood analyzer is described in detail. Figure 1 It is a three-dimensional schematic diagram of a blood analyzer according to one embodiment. Figure 2 The figure is a structural block diagram of an optical detector in a blood analyzer according to one embodiment.

[0064] like Figure 1 As shown, the blood analyzer includes a sample aspirating device 10, a sample preparation device 30, a light source (not shown), a flow chamber (not shown) and an optical detector 50, a processor 70 and a non-transitory computer-readable storage medium (not shown) programmed with a computer application.

[0065] The sample suction device 10 is configured to suck at least a portion of the sample to be tested and transport it to the sample preparation device. Part or all of the blood sample to be tested can be sucked for testing as needed. The sample suction device 10 can suck part of the sample to be tested multiple times, which are used for tests including the above-mentioned blood analysis method to obtain infection marker parameters and white blood cell, reticulocyte and other test results, as well as other test items, such as platelet detection, hemoglobin detection, etc., but not limited to this. The sample suction device can also suck a certain amount of sample at one time, divide it into several sub-samples, and process and test different samples separately.

[0066] The sample preparation device 30 includes a reagent supply unit and a reaction pool. The sample suction device 10 and the reagent supply unit are respectively connected to the reaction pool through pipelines. As needed, the reagent supply unit can transport a specific reagent to a specific reaction pool to mix with the blood sample to be tested provided by the sample suction device to prepare a sample for a predetermined detection purpose.

[0067] In the above embodiment, the reagent supply unit is configured to provide a first reagent to the first reaction pool. The first reagent, as described in detail below, includes a first hemolytic agent and a lysosomal stain containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on the lysosomes of cells in the blood sample to be tested. The reagent supply unit includes a plurality of reagent storage containers, and the first hemolytic agent and the lysosomal stain in the first reagent can be stored in different storage containers respectively; or the first hemolytic agent and the lysosomal stain are stored in a mixed form in one storage container.

[0068] The reaction pool is configured to mix the first reagent with at least a portion of the blood sample to be tested to obtain a first sample, and to deliver the first sample to the flow chamber. The reaction pool can be heated to a suitable reaction temperature (such as 42° C.) to obtain the best processing effect for the sample.

[0069] The blood sample to be tested in the reaction pool is treated with a first reagent to prepare a first sample, which is transported to the optical detector 50 through a pipeline assembly for detection to obtain a first optical signal of each particle in the first sample.

[0070] The optical detector 50 includes a light source, a flow chamber, and at least one (usually multiple) optical detectors. The light source is configured to direct a light beam toward a detection hole of the flow chamber connected to the reaction pool. The flow chamber is configured to allow particles in the first sample to pass through the flow chamber one by one. The optical detector is mounted around the flow chamber to detect the first light signal of the particles in the first sample passing through the detection hole of the flow chamber.

[0071] See also Figure 6 , which shows a schematic structural block diagram of multiple optical detectors. Figure 6 As shown, the optical system 1 and the flow chamber 2 are arranged on the optical axis, and the optical system 1 includes a light source and a lens group, which are used to align the light emitted by the light source with the detection hole on the flow chamber 2. There are multiple optical detectors, which are arranged around the flow chamber 2, and are used to detect the light scattering signals and fluorescence signals of the particles passing through the detection holes one by one. The optical detector generally includes a first detector 3, a second detector 4 and a fluorescence detector 5. Among them, the light scattering signal includes a forward light scattering intensity signal emitted from the optical system 1 and passing through the flow chamber 2 and collected by the first detector 3 arranged on the optical axis, and a side light scattering intensity signal emitted by the flow chamber 2 and collected by the second detector 4 arranged on the side of the optical axis. In addition, the optical detector also includes a fluorescence detector 5 arranged on the side of the optical axis for collecting the fluorescence intensity signal emitted by the flow chamber 2. It should be understood that Figure 6 Only one arrangement of the optical detectors is shown. The optical detectors may also be arranged in other ways. For example, the second detector 4 and the fluorescence detector 5 may be arranged relative to the optical axis as follows: Figure 6 The first detector 3 may be arranged vertically as shown, or at other angles, for example, at an angle of about 65° to about 115° to the incident light beam. Figure 6 The arrangements shown are in a straight line or at an angle of about 1° to about 10° to the incident light beam.

[0072] According to different treatments of the samples, each detector can collect scattered light intensity signals and fluorescence intensity signals from each particle in different samples. In the embodiment, the fluorescence detector 5 is used to collect lysosomal fluorescence intensity signals emitted by lysosomal dyes bound to lysosomes from particles.

[0073] The processor 70 is operably connected to the optical detector 50, and the optical signal of the particle detected by the optical detector is transmitted to the processor 70 for analysis. The signal can be amplified by an amplifier and then transmitted to the processor 70.

[0074] When the computer application in the non-temporary computer-readable storage medium is executed by the processor 70, the processor 70 obtains corresponding particle information based on the received light signal and performs the steps of obtaining infection marker parameters according to the blood analysis method in the above-mentioned embodiments.

[0075] In this embodiment, when the computer application in the non-transitory computer-readable storage medium is executed by the processor 70, the processor is caused to perform the following steps: processing the first light signal to obtain first white blood cell information; extracting at least one first white blood cell parameter of the first white blood cell information; obtaining an infection marker parameter based on the at least one first white blood cell parameter. As in each embodiment of the aforementioned blood analysis method, the first white blood cell information includes at least one of first white blood cell cluster information and first white blood cell cluster information. The first white blood cell cluster information includes first lymphocyte cluster information and first neutrophil cluster information.

[0076] In some embodiments, the white blood cell clusters WBC (including all types of white blood cells) in the sample can be identified based on the forward scattered light signal (or forward scattered light intensity) FS, the side scattered light signal (or side scattered light intensity) SS and the lysosomal fluorescence signal (or lysosomal fluorescence intensity) FL in the optical signal, and the neutrophil cluster Neu and the lymphocyte cluster Lym in the white blood cells in the first sample can be identified.

[0077] The first white blood cell information includes at least one of the first white blood cell particle cluster information and the first white blood cell subgroup particle cluster information. The first white blood cell particle cluster information refers to the information of all white blood cell particles in the first sample. The first white blood cell subgroup particle cluster information includes the first lymphocyte particle cluster information and the first neutrophil particle cluster information. According to a preferred embodiment, the first white blood cell information includes the first white blood cell particle cluster information and the first neutrophil particle cluster information.

[0078] It should be understood that the first leukocyte parameter does not include parameters for counting or classifying leukocytes, but rather includes characteristic parameters reflecting cell characteristics such as volume, internal granularity, and internal lysosome content of cells in the target particle cluster.

[0079] According to some embodiments, at least one first white blood cell parameter includes one of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, lysosomal fluorescence intensity distribution width, lysosomal fluorescence intensity distribution center of gravity, lysosomal fluorescence intensity distribution coefficient of variation of at least one of the first white blood cell grouping particle cluster and the first white blood cell particle cluster, the area of ​​the distribution area of ​​at least one of the first white blood cell grouping particle cluster and the first white blood cell particle cluster in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of the distribution area of ​​at least one of the first white blood cell grouping particle cluster and the first white blood cell particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity.

[0080] According to a preferred embodiment, the first white blood cell information includes first neutrophil particle cluster information and first white blood cell particle cluster information.

[0081] Specifically, the at least one first white blood cell parameter includes one of the following parameters:

[0082] the forward scattered light intensity distribution width (N_WBC_FS_W), the forward scattered light intensity distribution center of gravity (N_WBC_FS_P), the forward scattered light intensity distribution coefficient of variation (N_WBC_FS_CV), the side scattered light intensity distribution width (N_WBC_SS_W), the side scattered light intensity distribution center of gravity (N_WBC_SS_P), the side scattered light intensity distribution coefficient of variation (N_WBC_SS_CV), the lysosomal fluorescence intensity distribution width (N_WBC_FL_W), the lysosomal fluorescence intensity distribution center of gravity (N_WBC_FL_P), the lysosomal fluorescence intensity distribution coefficient of variation (N_WBC_FL_CV),

[0083] The area of ​​the distribution region of the first leukocyte particle group in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of the distribution region of the first leukocyte particle group in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity (for example: the area of ​​the distribution region of the first leukocyte particle group in the two-dimensional scatter plot generated by the side scattered light intensity and the forward scattered light intensity (N_WBC_SSFS_Area), the area of ​​the distribution region of the first leukocyte particle group in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the forward scattered light intensity (N_WBC_FLFS_Area), the area of ​​the distribution region of the first leukocyte particle group in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the side scattered light intensity (N_WBC_FLSS_Area));

[0084] The forward scattered light intensity distribution center of the first neutrophil cluster (N_NEU_FS_P), forward scattered light intensity distribution width (N_NEU_FS_W), forward scattered light intensity distribution coefficient of variation (N_NEU_FS_CV), side scattered light intensity distribution center of gravity (N_NEU_SS_P), side scattered light intensity distribution width (N_NEU_SS_W), side scattered light intensity distribution coefficient of variation (N_NEU_SS_CV), lysosomal fluorescence intensity distribution center of gravity (N_NEU_FL_P), lysosomal fluorescence intensity distribution width (N_NEU_FL_W), lysosomal fluorescence intensity distribution coefficient of variation (N_NEU_FL_CV),

[0085] the area of ​​the distribution region of the first neutrophil cluster in a two-dimensional scatter plot generated based on two light intensities among the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of the distribution region of the first neutrophil cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity (for example: the area of ​​the distribution region of the first neutrophil cluster in the two-dimensional scatter plot generated by the side scattered light intensity and the forward scattered light intensity (N_NEU_SSFS_Area), the area of ​​the distribution region of the first neutrophil cluster in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the forward scattered light intensity (N_NEU_FLFS_Area), the area of ​​the distribution region of the first neutrophil cluster in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the side scattered light intensity (N_NEU_FLSS_Area)); and

[0086] The forward scattered light intensity distribution center of the first lymphocyte cluster (N_LYM_FS_P), forward scattered light intensity distribution width (N_LYM_FS_W), forward scattered light intensity distribution coefficient of variation (N_LYM_FS_CV), side scattered light intensity distribution center of gravity (N_LYM_SS_P), side scattered light intensity distribution coefficient of variation (N_LYM_SS_CV), side scattered light intensity distribution width (N_LYM_SS_W), lysosomal fluorescence intensity distribution center of gravity (N_LYM_FL_P), lysosomal fluorescence intensity distribution width (N_LYM_FL_W), lysosomal fluorescence intensity distribution coefficient of variation (N_LYM_FL_CV),

[0087] The area of ​​the distribution region of the first lymphocyte cluster in a two-dimensional scatter plot generated based on two light intensities among the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of the distribution region of the first lymphocyte cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity (for example: the area of ​​the distribution region of the first lymphocyte cluster in the two-dimensional scatter plot generated by the side scattered light intensity and the forward scattered light intensity (N_LYM_SSFS_Area), the area of ​​the distribution region of the first lymphocyte cluster in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the forward scattered light intensity (N_LYM_FLFS_Area), the area of ​​the distribution region of the first lymphocyte cluster in the two-dimensional scatter plot generated by the lysosomal fluorescence intensity and the side scattered light intensity (N_LYM_FLSS_Area)).

[0088] Preferably, at least one first leukocyte parameter includes each cell parameter of the first leukocyte particle group. Specifically, the at least one first leukocyte parameter includes one or more of the following parameters: the forward scattered light intensity distribution width, the forward scattered light intensity distribution center of gravity, the forward scattered light intensity distribution coefficient of variation, the side scattered light intensity distribution width, the side scattered light intensity distribution center of gravity, the side scattered light intensity distribution coefficient of variation, the lysosomal fluorescence intensity distribution width, the lysosomal fluorescence intensity distribution center of gravity, the lysosomal fluorescence intensity distribution coefficient of variation, the area of ​​the distribution area of ​​the first leukocyte particle group in a two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity, and the lysosomal fluorescence intensity, and the volume of the distribution area of ​​the first leukocyte particle group in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity, and the lysosomal fluorescence intensity.

[0089] More preferably, the at least one leukocyte parameter includes one or more of a lysosomal fluorescence intensity distribution width, a lysosomal fluorescence intensity distribution center, and a forward scattered light intensity distribution width of the first leukocyte particle group.

[0090] Using the cell parameters of the white blood cell mass WBC and / or the neutrophil mass Neu in the first sample is advantageous for effectively assessing the infection state. More preferably, combining the cell characteristic parameters of the neutrophil mass Neu and the white blood cell mass WBC can provide infection marker parameters that can more truly reflect the infection state.

[0091] In some embodiments, the infection marker parameter is composed of a single first leukocyte parameter, such as one of the first leukocyte parameters listed above. Alternatively, the infection marker parameter is a linear function or a nonlinear function of the single first leukocyte parameter.

[0092] In other embodiments, the infection marker parameter is calculated by combining the at least one first white blood cell parameter and another first white blood cell parameter different from the first white blood cell parameter obtained from the optical information, for example, by combining multiple first white blood cell parameters among the parameters listed above, especially by combining through a linear function.

[0093] According to some embodiments, an infection marker parameter may be obtained by comparing a single first leukocyte parameter to a threshold value.

[0094] The first reagent is described in detail below: The first reagent includes a first hemolytic agent and a lysosomal dye containing a lysosomal dye, and the lysosomal dye performs fluorescent dyeing on the lysosomes of cells in the blood sample to be tested.

[0095] Lysosomal stains are used to fluorescently stain the lysosomes of blood cells. Lysosomal stains include a lysosomal dye dissolved in a solvent.

[0096] The present disclosure does not particularly limit the types of lysosomal dyes, and any lysosomal dye that has no adverse effect on the method can be used. According to some embodiments, the lysosomal dye is a lysosomal dye that can be excited by red light. The term "red light excitable" refers to a lysosomal dye that can be excited by light in the red spectrum, such as a wavelength of 600nm to 800nm, and then emits fluorescence. For example, the compound of the general formula I described in detail below can be excited by a 633nm laser, but is not limited thereto. According to other embodiments, the lysosomal dye is a lysosomal dye that can be excited by blue light. The term "blue light excitable" refers to a dye that can be excited by light in the blue spectrum, such as a wavelength of 400nm to 500nm, and then emits fluorescence. For example, the compound of the general formula II described in detail below can be excited by a 450nm laser, but is not limited thereto. Those skilled in the art can select a suitable lysosomal dye according to the light source equipped with a specific detection device (such as a blood analyzer described in detail below). For example, suitable lysosomal dyes may be commercially available Lyso-Tracker red (structural formula shown below), etc., but are not limited thereto.

[0097]

[0098] According to a preferred embodiment, the lysosomal dye is at least one compound selected from the group consisting of the compounds represented by the following general formula I and general formula II.

[0099]

[0100] wherein X is selected from —O—, —S— and —CR′R″—, wherein R′ and R″ are C1-C4 alkyl groups; and R1 and R2 are each independently selected from C3-C20 alkenyl groups and C3-C20 alkynes.

[0101] Preferably, R1 and R2 are each independently selected from C3-C10 alkenyl and C3-C10 alkyne.

[0102]

[0103] Wherein, R3 and R4 are each independently selected from H, C1-C12 straight chain alkane, and R5 is selected from hydrogen, and C1-C12 straight chain alkanes.

[0104] Preferably, R3 and R4 are each independently selected from H, C1-C6 straight chain alkane, and R5 is selected from hydrogen, and C1-C6 straight chain alkanes.

[0105] The compound of the general formula I is a lysosomal dye that can be excited by red light (such as light of about 633 nm). The compound of the general formula II is a lysosomal dye that can be excited by blue light (such as light of about 450 nm).

[0106] More specifically, the lysosomal dye is at least one selected from the following compounds 1 to 8:

[0107]

[0108]

[0109]

[0110] The solvent in the lysosomal stain is an organic solvent. The present disclosure has no particular restrictions on the type of solvent. Any solvent that does not affect the detection and is conducive to the dissolution of the lysosomal dye can be used. The preferred solvent is one or more selected from methanol, ethanol, glycerol, glycol and diethylene glycol.

[0111] The concentration of the lysosomal dye in the lysosomal stain is 10-100 mg / L, preferably 40-60 mg / L. For example, the concentration of the lysosomal dye can be 20, 30, 40, 50, 60, 70, 80, 90 mg / L.

[0112] The first hemolytic agent is used to lyse the red blood cells in the test sample into fragments, thereby eliminating the influence of the red blood cells on the test. At the same time, the first hemolytic agent also acts on the cell membrane of other blood cells, such as white blood cells, to increase the permeability of the blood cell membrane in a short period of time, making it easier for the lysosomal dye to enter the cell and combine with the lysosome, but it does not destroy the complete cell morphology of other blood cells.

[0113] The first hemolytic agent includes a surfactant and a buffer for lysing red blood cells.

[0114] The surfactant is at least one selected from a cationic surfactant, an anionic surfactant and a nonionic surfactant.

[0115] According to some embodiments, the cationic surfactant is selected from quaternary ammonium salts represented by the following general formula III.

[0116]

[0117] Wherein, R is selected from C8-C16 alkyl, X - It is a chloride ion or a bromide ion.

[0118] Examples of quaternary ammonium salts include octyltrimethylammonium chloride, octyltrimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, etc., but are not limited thereto.

[0119] The concentration of the cationic surfactant in the hemolytic agent is 300-800 mg / L.

[0120] Examples of the anionic surfactant include dodecylbenzenesulfonic acid, sodium dodecyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, and alcohol ether carboxylates, but are not limited thereto.

[0121] The concentration of the anionic surfactant in the hemolytic agent is 0.1-2 g / L.

[0122] According to some embodiments, the nonionic surfactant is selected from polyoxyethylene nonionic surfactants. Preferably, examples of the polyoxyethylene nonionic surfactants include: long-chain fatty alcohol polyoxyethylene ethers (such as dodecyl alcohol polyoxyethylene (23) ether, hexadecyl alcohol polyoxyethylene (25) ether, hexadecyl alcohol polyoxyethylene (30) ether, etc.), alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene esters and fatty amine polyoxyethylene ethers, but are not limited thereto.

[0123] The concentration of the nonionic surfactant in the hemolytic agent is 1-5 g / L.

[0124] According to a preferred embodiment, the first hemolytic agent contains a cationic surfactant, especially a quaternary ammonium salt cationic surfactant as a red blood cell lysing agent.

[0125] The present disclosure has no particular restrictions on the buffer in the first hemolytic agent, and any commonly used buffer pair can be used, as long as the pH value of the first hemolytic agent can be stabilized at 3.0 to 3.5. The buffer is also used to stabilize the pH of the mixed system within the above range when the first reagent is mixed with the sample to be tested to prepare the first test sample. When the pH value of the mixed system is within the range of 3.0 to 3.5, it is beneficial for the surfactant to play a role and for the lysosomal dye to pass through the cell membrane into the cell and bind to the lysosome.

[0126] Examples of buffer pairs include, but are not limited to, phosphoric acid-phosphate, citric acid-citrate, Tris-hydrochloric acid, salicylic acid-salicylate, and the like.

[0127] The concentration of the buffer is not particularly limited and can be adjusted according to the selected buffer. Exemplarily, the concentration of the buffer is in the range of 0.5-5 g / L.

[0128] The first hemolytic agent also includes a preservative. The present disclosure has no particular limitation on the preservative in the first hemolytic agent, and any commonly used preservative can be used. For example, the preservative can be 4-methoxyphenol, sodium azide, ProClin series, etc. The concentration of the preservative is 0.1 to 0.5 g / L.

[0129] The first hemolytic agent and the lysosomal staining agent in the first reagent can be separate reagents, packaged and stored separately, and mixed only when used. Alternatively, the first hemolytic agent and the lysosomal staining agent are mixed into one reagent, packaged and stored.

[0130] According to an embodiment of the present disclosure, the first reagent consists only of the first hemolytic agent and the lysosomal staining agent. That is, when the first reagent is used to process the blood sample, only the red blood cells are lysed and the lysosomes of other blood cells are stained, and no other effects are produced on the blood sample. For example, the first reagent does not contain reagents or substances that can stain or fluorescently label other organelles or cell substances outside the lysosome.

[0131] According to some embodiments, the processor of the blood analyzer, when the computer application in the non-transitory computer-readable storage medium is executed by the processor, further performs the following steps: further identifying the nucleated red blood cells and basophils in the first sample according to the first light signal. Further, the counts of nucleated red blood cells, white blood cells, and basophils are obtained. That is to say, using the first reagent containing a lysosomal stain to treat the blood sample does not affect the identification and classification of white blood cells, and can also identify nucleated red blood cells. This is the same as the traditional analysis items obtained by using a nucleic acid stain and a hemolytic agent to treat the blood sample and WNB channel detection.

[0132] Therefore, the blood analyzer disclosed in the present invention uses lysosomal stains instead of nucleic acid stains for blood analysis, which not only does not affect the acquisition of traditional blood routine test items but also can obtain infection marker parameters to evaluate the infection status of the subject. The test is fast and saves test costs.

[0133] According to another embodiment, the sample aspirating device of the blood analyzer is further configured to aspirate at least another portion of the blood sample to be tested and transfer it to the sample preparation device.

[0134] The sample preparation device further includes a second reaction pool. The reagent supply unit is further configured to provide a second reagent to the second reaction pool. The second reaction pool is configured to mix the second reagent with at least another portion of the blood sample to obtain a second sample, and to transport the second sample to the flow chamber.

[0135] The second reaction pool is different from the first reaction pool. The second reaction pool can also be heated to promote the reaction between the second reagent and the blood sample to be tested. Alternatively, the first reaction pool and the second reaction pool are the same reaction pool. After preparing one sample, the reaction pool is rinsed and then another sample is prepared.

[0136] The second reagent includes a second hemolytic agent and a nucleic acid staining agent containing a nucleic acid dye, and the nucleic acid dye performs fluorescent staining on nucleic acid substances in cells of the blood sample to be tested.

[0137] The flow cell is further configured to allow particles in the second sample to pass through the flow cell one by one. The optical detector is further configured to detect a second optical signal of each particle of the second sample passing through the detection hole of the flow cell.

[0138] When the computer application in the non-transitory computer-readable storage medium is executed by the processor, the processor is caused to further perform the following steps: processing the second light signal to obtain second white blood cell information; extracting at least one second white blood cell parameter of the second white blood cell information; and obtaining the infection marker parameter based on the at least one first white blood cell parameter and the at least one second white blood cell parameter.

[0139] In some embodiments, the leukocyte clustering particle clusters in the second sample can be identified based on the forward scattered light signal (or forward scattered light intensity) FS, the side scattered light signal (or side scattered light intensity) SS, and the nucleic acid fluorescence signal (or nucleic acid fluorescence intensity) FL in the optical signal. According to some embodiments, the leukocyte clustering particle clusters in the second sample include at least one or more of the single cell particle clusters Mon, the neutrophil particle clusters Neu, and the lymphocyte particle clusters Lym. According to other embodiments, the leukocyte clustering particle clusters in the second sample include one or more of the single cell particle clusters Mon, the neutrophil particle clusters Neu, the lymphocyte particle clusters Lym, and the eosinophil particle clusters Eos.

[0140] The second leukocyte information includes at least one second leukocyte grouping particle cluster information. Preferably, the at least one second leukocyte grouping particle cluster information includes one or more of second monocyte group information, second neutrophil group information, and second lymphocyte group information.

[0141] According to some embodiments, the at least one second white blood cell parameter includes one or more of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, nucleic acid fluorescence intensity distribution width, nucleic acid fluorescence intensity distribution center of gravity, nucleic acid fluorescence intensity distribution coefficient of variation of at least one second white blood cell clustering particle group; the area of ​​the distribution area of ​​the at least one second white blood cell clustering particle group in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity; and the volume of the distribution area of ​​the at least one second white blood cell clustering particle group in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity.

[0142] Specifically, the at least one second white blood cell parameter includes one or more of the following parameters:

[0143] The second monocyte particle cluster has the following components: forward scattered light intensity distribution width (D_MON_FS_W), forward scattered light intensity distribution center of gravity (D_MON_FS_P), forward scattered light intensity distribution coefficient of variation (D_MON_FS_CV), side scattered light intensity distribution width (D_MON_SS_W), side scattered light intensity distribution center of gravity (D_MON_SS_P), side scattered light intensity distribution coefficient of variation (D_MON_SS_CV), nucleic acid fluorescence intensity distribution width (D_MON_FL_W), nucleic acid fluorescence intensity distribution center of gravity (D_MON_FL_P), nucleic acid fluorescence intensity distribution coefficient of variation (D_MON_FL_CV),

[0144] the area of ​​the distribution region of the second monocyte particle cluster in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity, the volume of the distribution region of the second monocyte particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity (for example, the area of ​​the distribution region of the second monocyte particle cluster in the two-dimensional scatter plot generated by the forward scattered light intensity and the nucleic acid fluorescence intensity (D_MON_FLFS_Area), the area of ​​the distribution region of the second monocyte particle cluster in the two-dimensional scatter plot generated by the side scattered light intensity and the nucleic acid fluorescence intensity (D_MON_FLSS_Area), the area of ​​the distribution region of the second monocyte particles in the two-dimensional scatter plot generated by the forward scattered light intensity and the side scattered intensity (D_MON_SSFS_Area);

[0145] The forward scattered light intensity distribution width (D_NEU_FS_W), forward scattered light intensity distribution center of gravity (D_NEU_FS_P), forward scattered light intensity distribution coefficient of variation (D_NEU_FS_CV), side scattered light intensity distribution width (D_NEU_SS_W), side scattered light intensity distribution center of gravity (D_NEU_SS_P), side scattered light intensity distribution coefficient of variation (D_NEU_SS_CV), nucleic acid fluorescence intensity distribution width (D_NEU_FL_W), nucleic acid fluorescence intensity distribution center of gravity (D_NEU_FL_P), nucleic acid fluorescence intensity distribution coefficient of variation (D_NEU_FL_CV) of the second neutrophil particle group,

[0146] the area of ​​the distribution region of the second neutrophil particle group in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the fluorescence intensity, and the volume of the distribution region of the neutrophil population in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the fluorescence intensity (for example: the area of ​​the distribution region of the neutrophil population in the two-dimensional scatter plot generated by the forward scattered light intensity and the nucleic acid fluorescence intensity (D_NEU_FLFS_Area), the area of ​​the distribution region of the neutrophil population in the two-dimensional scatter plot generated by the side scattered light intensity and the nucleic acid fluorescence intensity (D_NEU_FLSS_Area), the area of ​​the distribution region of the neutrophil population in the two-dimensional scatter plot generated by the forward scattered light intensity and the side scattered light intensity (D_NEU_SSFS_Area)); and

[0147] The forward scattered light intensity distribution width (D_LYM_FS_W), forward scattered light intensity distribution center (D_LYM_FS_P), forward scattered light intensity distribution coefficient of variation (D_LYM_FS_CV), side scattered light intensity distribution width (D_LYM_SS_W), side scattered light intensity distribution center (D_LYM_SS_P), side scattered light intensity distribution coefficient of variation (D_LYM_SS_CV), nucleic acid fluorescence intensity distribution width (D_LYM_FL_W), nucleic acid fluorescence intensity distribution center (D_LYM_FL_P), nucleic acid fluorescence intensity distribution coefficient of variation (D_LYM_FL_CV),

[0148] The area of ​​the distribution region of the second lymphocyte particle cluster in the two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the fluorescence intensity, and the volume of the distribution region of the lymphocyte population in the three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the fluorescence intensity (for example: the area of ​​the distribution region of the lymphocyte population in the two-dimensional scatter plot generated by the forward scattered light intensity and the fluorescence intensity (D_LYM_FLFS_Area), the area of ​​the distribution region of the lymphocyte population in the two-dimensional scatter plot generated by the side scattered light intensity and the fluorescence intensity (D_LYM_FLSS_Area), the area of ​​the distribution region of the lymphocyte population in the two-dimensional scatter plot generated by the forward scattered light intensity and the side scatter intensity (D_LYM_SSFS_Area)).

[0149] Preferably, the at least one second leukocyte parameter comprises one or more of the following parameters:

[0150] the forward scattered light intensity distribution width, the forward scattered light intensity distribution center of gravity, the forward scattered light intensity distribution coefficient of variation, the side scattered light intensity distribution width, the side scattered light intensity distribution center of gravity, the side scattered light intensity distribution coefficient of variation, the nucleic acid fluorescence intensity distribution width, the nucleic acid fluorescence intensity distribution center of gravity, the nucleic acid fluorescence intensity distribution coefficient of variation of the second monocyte particle cluster, the area of ​​the distribution region of the second monocyte particle cluster in a two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity, and the nucleic acid fluorescence intensity, and the volume of the distribution region of the second monocyte particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity, and the nucleic acid fluorescence intensity; and

[0151] The forward scattered light intensity distribution width, the forward scattered light intensity distribution center, the forward scattered light intensity distribution variation coefficient, the side scattered light intensity distribution width, the side scattered light intensity distribution center, the side scattered light intensity distribution variation coefficient, the nucleic acid fluorescence intensity distribution width, the nucleic acid fluorescence intensity distribution center, the nucleic acid fluorescence intensity distribution variation coefficient of the second neutrophil particle cluster, the area of ​​the distribution area of ​​the second neutrophil particle cluster in the two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity, and the volume of the distribution area of ​​the second neutrophil particle cluster in the three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity.

[0152] According to this embodiment, at least one first white blood cell parameter and at least one second white blood cell parameter are combined, preferably by a linear function combination to obtain the infection marker parameter, which can further improve the reliability of the detection.

[0153] The infection marker parameter is obtained by combining the first white blood cell parameter and the second white blood cell parameter through a linear function, that is, the infection marker parameter is calculated by the following formula:

[0154] Y=A×X1+B×X2+C

[0155] Among them, Y represents the infection marker parameter, X1 represents the first white blood cell parameter, X2 represents the second white blood cell parameter, and A, B, and C are constants.

[0156] In other embodiments, the first white blood cell parameter and the second white blood cell parameter may be combined by a nonlinear function to obtain an infection marker parameter, which is not specifically limited in the present disclosure. A person skilled in the art will appreciate that in other embodiments, the two white blood cell parameters may not be calculated by a function, but the first white blood cell parameter and the second white blood cell parameter may be used in combination to compare with their respective thresholds to obtain an infection marker parameter.

[0157] According to a preferred embodiment, the processor 70 performs the following steps: obtaining infection marker parameters based on the lysosomal fluorescence intensity distribution width of the first leukocyte particle group and the side scattered light intensity distribution width of the second monocyte particle group.

[0158] The meanings of the terms distribution width, distribution center of gravity, coefficient of variation, and area or volume of the distribution region mentioned in the present disclosure, their calculation methods, the method of combining multiple white blood cell parameters through functions, and the method of obtaining infection marker parameters based on one or more white blood cell parameters are recorded in the applicant's prior international patent applications No.: PCT / CN2022 / 143965 and PCT / CN2022 / 144177. The entire contents of these patent applications are incorporated herein by reference and will not be repeated here.

[0159] The second reagent is described in detail below. The second reagent includes a second hemolytic agent and a nucleic acid stain containing a nucleic acid dye, and the nucleic acid dye performs fluorescent staining on the nucleic acid substances of the cells in the blood sample to be tested. The present disclosure has no particular restrictions on the second hemolytic agent and the nucleic acid stain in the second reagent, and any reagent conventionally used for DIFF channel detection in blood analysis can be used.

[0160] In some embodiments, the second hemolytic agent is the same as or different from the first hemolytic agent, and can be any one or a combination of a cationic surfactant, a nonionic surfactant, an anionic surfactant, or an amphiphilic surfactant. In other embodiments, the hemolytic agent can include at least one of an alkyl glycoside, a triterpenoid saponin, and a steroidal saponin.

[0161] The second hemolytic agent also includes a buffer, a preservative, etc., and compounds known to be used for DIFF channel detection can be used.

[0162] In an embodiment of the present application, the nucleic acid stain is a fluorescent dye used to achieve leukocyte classification, for example, it can be a fluorescent dye that can classify leukocytes in a blood sample into at least three leukocyte subsets (monocytes, lymphocytes and neutrophils).

[0163] In some embodiments, the nucleic acid stain is a solution of a nucleic acid dye dissolved in an organic solvent. The nucleic acid dye may include a cationic cyanine compound, and more details thereof may be found in Chinese patent application CN101750274A filed by the applicant on September 28, 2019, the entire disclosure of which is incorporated herein by reference. Organic solvents may use those suitable for lysosomal stains.

[0164] According to other implementations, the processor 70 further performs the following steps.

[0165] The first light signal is processed to further obtain information of nucleated red blood cells and / or basophils. Further, the first light signal is processed to obtain the count of white blood cells, nucleated red blood cells and / or basophils.

[0166] According to other implementations, the processor 70 further performs the following steps.

[0167] The second light signal is processed to obtain information of at least one of the eosinophils, monocytes, neutrophils and lymphocytes. Further, the second light signal is processed to obtain counts of at least one of the eosinophils, monocytes, neutrophils and lymphocytes.

[0168] That is, the blood analyzer can be used to obtain infection marker parameters and results of traditional blood routine test items while performing the above tests.

[0169] The obtained infection marker parameters can be used to evaluate the infection status of the subject. The infection status evaluation includes one or more of early prediction of sepsis, diagnosis of sepsis, differentiation of common infection and severe infection, infection condition monitoring, sepsis prognosis analysis, differentiation of bacterial infection and viral infection, differentiation of non-infectious inflammation and infectious inflammation, and evaluation of sepsis efficacy.

[0170] Sepsis is a serious infectious disease with a high incidence and mortality rate. For every hour of delayed treatment, the patient's mortality rate increases by 7%. Therefore, early warning of sepsis is particularly important. Early identification and warning of sepsis can increase precious diagnosis and treatment time for patients and greatly improve survival rate.

[0171] Exemplarily, in an application scenario of early prediction of sepsis, that is, the infection marker parameter is used for early prediction of sepsis. When the infection marker parameter meets a first preset condition, it indicates that the subject may develop sepsis within a certain period of time after the blood sample is collected.

[0172] Here, the first preset condition may be, for example, that the value of the infection marker parameter is greater than a preset threshold value. The preset threshold value may be determined according to a specific parameter combination and a blood cell analyzer.

[0173] Further references Figure 1 , the blood analyzer of each embodiment of the present disclosure further includes an output device 90. The output device 90 includes a user interaction interface for human-computer interaction. The output device 90 including the user interaction interface is operably connected to the processor 70. The user interaction interface is configured to display the test results and / or input instructions to the blood analyzer. For example, according to another specific embodiment, the user interface displays the infection marker parameter to the user and / or outputs a prompt message indicating the infection status of the subject based on the infection marker parameter.

[0174] In addition, the blood analyzer in each of the above embodiments may also include a first housing 100 and a second housing 200 (see Figure 5 The sample preparation device 30 is disposed inside the first housing 100 , and the output device 90 and the sample suction device 10 are arranged on the outer surface of the first housing 100 . The optical detector 50 and the processor 70 are disposed inside the second housing 200 .

[0175] The embodiments of the present disclosure also provide a blood analysis method. Different from the conventional blood analysis method, the blood analysis method uses lysosomal dyes to stain the lysosomes of blood cells such as white blood cells to obtain optical parameters of target particle clusters.

[0176] Specifically, refer to Figure 3 , which shows a schematic flow chart of a blood analysis method according to an embodiment. The blood analysis method comprises the following steps.

[0177] S110, obtaining a blood sample to be tested from a subject.

[0178] S120, preparing a first test sample, wherein the first test sample contains at least a portion of the blood sample to be tested and a first reagent, wherein the first reagent includes a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on the lysosomes of cells in the blood sample to be tested.

[0179] S130, allowing particles in the first sample to pass through an optical detection area irradiated with light one by one to obtain first optical signals generated by the particles in the first sample after being irradiated with light.

[0180] S140, processing the first light signal to obtain first white blood cell information.

[0181] S150, extracting at least one first white blood cell parameter of the first white blood cell information.

[0182] S160: Obtain an infection marker parameter based on the at least one first white blood cell parameter.

[0183] In S110, a sample to be tested is obtained from a subject. The sample to be tested may be blood from the subject, especially peripheral blood. The subject is not particularly limited, and may be a healthy subject or a patient, such as an infected patient or a patient with inflammation.

[0184] In S120, a first sample is prepared. Specifically, at least a portion of the acquired sample to be tested is mixed with a first reagent to process the sample to be tested, thereby preparing the first sample.

[0185] The first reagent is as described above and will not be described again here.

[0186] In S120, the first reagent is mixed with at least a portion of the blood sample to be tested and incubated at, for example, 42° C. for about 30 seconds to complete the processing of the blood sample and obtain a first test sample. The incubation temperature and time are not limited to the above-mentioned temperature and time, and can be specifically determined according to the selected surfactant type and lysosomal dye type.

[0187] After the first sample is prepared, S130 is performed to make the particles in the first sample pass through the optical detection area irradiated with light one by one to obtain the first optical signal generated by the particles in the first sample after being irradiated with light.

[0188] The first optical signal may include scattered light signals at different angles generated after each particle passing through the optical detection area is irradiated by a light source, such as side scattered light, forward scattered light, etc., and lysosomal fluorescence signals emitted by lysosomal dyes bound to lysosomes.

[0189] At S140, the first light signal is processed to obtain first white blood cell information.

[0190] The first white blood cell information includes at least one of the first white blood cell particle cluster information and the first white blood cell subgroup particle cluster information. As mentioned above, the first white blood cell particle cluster information refers to the information of all white blood cell particles in the first sample. The first white blood cell subgroup particle cluster information includes the first lymphocyte particle cluster information and the first neutrophil particle cluster information. According to a preferred embodiment, the first white blood cell information includes the first white blood cell particle cluster information and the first neutrophil particle cluster information.

[0191] At S150, at least one first white blood cell parameter of the first white blood cell information is extracted.

[0192] According to some embodiments, the at least one first leukocyte parameter comprises one of the first leukocyte population particle mass and the first leukocyte particle mass.

[0193] The specific first white blood cell parameter that can be used is as described above and will not be repeated here.

[0194] Preferably, the at least one first leukocyte parameter comprises one or more of the cell parameters of the first leukocyte particle group.

[0195] More preferably, the at least one first leukocyte parameter includes one or more of a lysosomal fluorescence intensity distribution width, a lysosomal fluorescence intensity distribution center, and a forward scattered light intensity distribution width of the first leukocyte particle group.

[0196] In S160, an infection marker parameter is obtained based on the at least one first white blood cell parameter.

[0197] In some embodiments, the infection marker parameter is composed of a single first white blood cell parameter; or, the infection marker parameter is a linear function or a nonlinear function of the single first white blood cell parameter.

[0198] In other embodiments, the infection marker parameter is calculated by combining the at least one first white blood cell parameter and another first white blood cell parameter different from the first white blood cell parameter obtained from the optical information.

[0199] According to some embodiments, an infection marker parameter may be obtained by comparing a single first leukocyte parameter to a threshold value.

[0200] The method also includes further identifying nucleated red blood cells and basophils in the first sample according to the first light signal. Furthermore, the counts of nucleated red blood cells, white blood cells, and basophils can be obtained. That is to say, using the first reagent containing a lysosomal stain to process the blood sample does not affect the identification and classification of white blood cells, and can also identify nucleated red blood cells. This is the same as the traditional use of nucleic acid stains and hemolytic agents to process blood samples and obtain analysis items through WNB channel detection. Therefore, the present invention uses lysosomal stains instead of nucleic acid stains for blood analysis, which can not only obtain traditional blood routine test items but also infection marker parameters, and the detection is fast and saves detection costs.

[0201] Another embodiment of the present disclosure also provides a blood analysis method. Figure 4 , which shows a flow chart of a blood analysis method according to another embodiment. The blood analysis method in this embodiment includes steps S110-S150 of the above embodiment, which are not repeated here, and further includes the following steps.

[0202] S220, preparing a second sample, wherein the second sample contains at least a portion of the blood sample to be tested and a second reagent, wherein the second reagent includes a second hemolytic agent and a nucleic acid staining agent containing a nucleic acid dye, and the nucleic acid dye fluorescently stains nucleic acid substances in cells of the blood sample to be tested.

[0203] S230, allowing the particles in the second sample to pass through the optical detection area irradiated with light one by one to obtain a second optical signal generated by the particles in the second sample after being irradiated with light.

[0204] S240, processing the second light signal to obtain second white blood cell information.

[0205] S250: Extract at least one second white blood cell parameter from the second white blood cell information.

[0206] S260: Obtain an infection marker parameter based on the at least one first white blood cell parameter and the at least one second white blood cell parameter.

[0207] The above steps S220 to S250 can be performed sequentially with steps S120 to S150. For example, they can be performed in the order of S110-(S120 to S150)-(S220 to S250)-S260, or in the order of S110-(S220 to S250)-(S120 to S150)-S260; or if the equipment conditions permit, steps S220 to S250 can be performed simultaneously with steps S120 to S150.

[0208] In S220, a second sample is prepared. Specifically, at least a portion of the acquired sample to be tested is mixed with a second reagent to process the sample to be tested, thereby preparing the second sample.

[0209] The second reagent is as described above and will not be described again here.

[0210] The second reagent is mixed with at least a portion of the blood sample to be tested and incubated at, for example, 42° C. for about 30 seconds to complete the processing of the blood sample and obtain a second test sample. The incubation temperature and time are not limited to the temperature and time exemplified above, and can be specifically determined according to the selected surfactant type and nucleic acid dye type.

[0211] In S230 , particles in the second sample are made to pass through an optical detection area illuminated by light one by one to obtain second optical signals generated by the particles in the second sample after being illuminated by light.

[0212] The second optical signal may include scattered light signals at different angles generated after each particle passing through the optical detection area is irradiated by a light source, such as side scattered light, forward scattered light, etc., and nucleic acid fluorescence signals emitted by nucleic acid dyes bound to nucleic acid substances.

[0213] At S240, the second light signal is processed to obtain second white blood cell information.

[0214] Similar to the above-mentioned embodiments of the blood analyzer, the second white blood cell information includes at least one second white blood cell grouping particle cluster information.

[0215] Preferably, the at least one second leukocyte clustering particle information includes one or more of second monocyte cluster information, second neutrophil cluster information, and second lymphocyte cluster information.

[0216] The available second white blood cell parameters are the same as those described above and will not be described in detail here.

[0217] In S250, at least one second white blood cell parameter in the second white blood cell information is extracted.

[0218] According to some embodiments, the at least one second leukocyte parameter comprises one or more of the parameters of at least one second leukocyte population particle mass.

[0219] The specific second white blood cell parameters that can be used are as described above and will not be described in detail here.

[0220] Preferably, the at least one second leukocyte parameter comprises

[0221] One or more of the parameters of the second monocyte particle group and the second neutrophil particle group.

[0222] In S260, an infection marker parameter is obtained based on the at least one first white blood cell parameter and the at least one second white blood cell parameter.

[0223] According to this embodiment, at least one first leukocyte parameter and at least one second leukocyte parameter are combined, preferably by combining the linear functions as described above to obtain the infection marker parameter, thereby further improving the reliability of detection.

[0224] In other embodiments, the first white blood cell parameter and the second white blood cell parameter can be combined through a nonlinear function to obtain an infection marker parameter; or the first white blood cell parameter and the second white blood cell parameter can be used together and compared with their respective thresholds to obtain an infection marker parameter, which is not specifically limited in the present disclosure.

[0225] Preferably, the infection marker parameter is obtained based on the lysosomal fluorescence intensity distribution width of the first leukocyte particle group and the side scattered light intensity distribution width of the second monocyte particle group.

[0226] The method further includes identifying eosinophils, lymphocytes, neutrophils, and monocytes (four types of white blood cells) in the second sample according to the second light signal, and further counting these cells in groups. That is, the blood analysis method disclosed in the present invention can also obtain analysis items obtained by DIFF channel detection in traditional detection.

[0227] The embodiment of the present disclosure also provides a use of a cell analysis reagent in analyzing infection marker parameters of a blood sample. The reagent comprises a hemolytic agent and a lysosomal staining agent, wherein the lysosomal staining agent comprises a lysosomal dye targeting a cell lysosome.

[0228] Unlike conventional reagents of the same type that contain nucleic acid dyes, the cell analysis reagent contains lysosomal dyes, which are used to perform lysosomal staining on blood cells such as white blood cells when red blood cells are lysed. After a blood sample is processed by the cell analysis reagent to obtain a test sample, the optical information of the target particle group in the test sample can be obtained through the detection of the above-mentioned blood analyzer, and at least one white blood cell parameter can be extracted and obtained, and an infection marker parameter can be obtained based on the white blood cell parameter.

[0229] The infection marker parameters are used to evaluate the infection status of the subject. As mentioned above, the infection status evaluation includes one or more of the following: early prediction of sepsis, diagnosis of sepsis, differentiation of common infection from severe infection, infection condition monitoring, sepsis prognosis analysis, differentiation of bacterial infection from viral infection, differentiation of non-infectious inflammation from infectious inflammation, and evaluation of the efficacy of sepsis, but is not limited thereto.

[0230] The hemolytic agent of the reagent is the same as the first hemolytic agent mentioned above, and the lysosome staining agent is also the same as mentioned above, which will not be repeated here.

[0231] In addition to being used to obtain infection marker parameters in blood analysis, the above-mentioned cell analysis reagent can replace the analysis reagent used in the traditional WNB channel, can distinguish white blood cells, and basophils, lymphocytes and neutrophil subpopulations in white blood cells, and can also identify reticulocytes.

[0232] The present disclosure also provides the above-mentioned cell analysis reagent.

[0233] According to a preferred embodiment, the dye in the reagent is a solution containing at least one lysosomal dye of the above general formula II.

[0234] According to some embodiments, the hemolytic agent and the staining agent are separate reagents, or according to other embodiments, the hemolytic agent and the staining agent are mixed into one reagent.

[0235] In some embodiments, the reagent consists only of the hemolytic agent and the lysosomal stain.

[0236] The various aspects and advantages of the present invention are further illustrated by specific embodiments below.

[0237] Example 1

[0238] The staining sites of compound 1 and commercially available lysosomal dye (Lyso-Tracker red) on living cells were observed under a laser microscope.

[0239] 10 μL of PBS buffer containing compound 1 at a concentration of 1 mM was added to the six-well plate of cultured HepG2 cells and incubated in a cell culture incubator at 37°C and 5% CO2 for 30 min. Then, the cells were washed three times with PBS buffer and then the cell culture medium was added. The cell morphology was observed with a confocal laser scanning microscope (FV1000IX81). Figure 5 shown. Figure 5 The upper left picture is a fluorescence micrograph of compound 1 staining HepG2 live cells, and the upper right picture is a fluorescence micrograph of commercially available lysosomal dye staining HepG2 live cells. The lower left picture is an overlapping fluorescence micrograph, and the lower right picture is the ratio of the fluorescence overlap between the two. As shown in the lower right picture, the Pierce coefficient of the fluorescence correlation between the two is 0.89, indicating that the staining sites of the two dyes are highly similar, both of which are lysosomes.

[0240] Example 2

[0241] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0242] A: Lysosomal stain

[0243] Compound 1 50mg

[0244] Ethylene glycol 950ml

[0245] Methanol 50ml

[0246] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0247]

[0248] Add 20 microliters of anticoagulated blood sample and 20 microliters of reagent A to 1 mL of reagent B, and incubate at 42°C for 30 seconds to obtain the first sample. Use Mindray 6800plus blood analyzer, in which the light source is a red laser light source (wavelength 633nm) for detection, and measure the forward low-angle scattered light intensity (FS), the side high-angle scattered light intensity (SS) and the side fluorescence intensity (FL, i.e., lysosomal fluorescence intensity). According to the above method, blood samples of healthy subjects and blood samples of sepsis patients were tested respectively to obtain the corresponding light signals, and a scatter plot of the side scattered light intensity and the side fluorescence intensity SS-FL of the two samples was obtained, as shown in FIG. Figure 6 As shown in the figure, it can be seen that the blood samples of sepsis patients have higher side fluorescence intensity than normal blood samples.

[0249] For healthy subject samples and sepsis patient samples, for example, the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) parameter can be extracted according to the aforementioned method, and compared with the threshold, a value higher than the threshold indicates a sepsis patient, thereby helping to assess the patient's infection status. The experimental results of the applicant's clinical statistics show that the diagnostic efficacy of assessing whether a subject has sepsis based on the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) can reach 0.915.

[0250] Example 3

[0251] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0252] A: Lysosomal stain

[0253] Compound 6 50mg

[0254] Ethylene glycol 950ml

[0255] Methanol 50ml

[0256] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0257]

[0258] Add 20 microliters of anticoagulated blood sample and 20 microliters of reagent A to 1 mL of reagent B, and incubate at 42°C for 30 seconds to obtain the first sample. Use Mindray 6800plus blood analyzer, in which the light source is replaced with a blue laser light source (wavelength 450nm) for detection, and measure the forward low-angle scattered light intensity (FS), the side high-angle scattered light intensity (SS) and the side fluorescence intensity (FL, i.e., lysosomal fluorescence intensity). According to the above method, blood samples of healthy subjects and blood samples of sepsis patients were tested respectively to obtain the corresponding light signals, and a scatter plot of the side scattered light intensity and the side fluorescence intensity SS-FL of the two samples was obtained, as shown in Figure 2. Figure 7 As shown in the figure, it can be seen that the blood samples of sepsis patients have higher side fluorescence intensity than normal blood samples.

[0259] For healthy subject samples and sepsis patient samples, for example, the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) parameter can be extracted according to the aforementioned method, and compared with the threshold, a value higher than the threshold indicates that the patient is a sepsis patient, thereby helping to assess the patient's infection status. The experimental results of the applicant's clinical statistics show that the diagnostic efficacy of assessing whether a subject has sepsis based on the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) can reach 0.910.

[0260] Example 4

[0261] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0262] A1~A3:Lysosome staining agent

[0263] Compound 3 15mg (A1), 30mg (A2), 50mg (A3)

[0264] Ethylene glycol 950ml

[0265] Methanol 50ml

[0266] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0267]

[0268] Add 20 microliters of blood samples from sepsis patients treated with anticoagulation and reagent A1 to 1 mL of reagent B, and incubate at 42°C for 30 seconds to obtain the first sample. Use Mindray 6800plus blood analyzer, in which the light source is a red laser light source (wavelength 633nm) for detection, and measure the forward low-angle scattered light intensity (FS), the side high-angle scattered light intensity (SS) and the side fluorescence intensity (FL, i.e., lysosomal fluorescence intensity). According to the above method, replace reagent A1 with reagent A2 and A3 for the same blood sample, obtain the corresponding light signal, and obtain the scatter plot of the side scattered light intensity and the side fluorescence intensity SS-FL of each detection, as shown in Figure 2. Figure 8 shown.

[0269] Depend on Figure 8 The results shown show that after the samples were treated with staining agents containing different concentrations of lysosomal dyes, the difference in lateral fluorescence of sepsis blood samples was small. In comparison, the boundaries of the 50 mg / L cell scatter clusters were clearer and the staining was more sufficient.

[0270] Example 5

[0271] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0272] A: Lysosomal dyes

[0273] Compound 6 50mg

[0274] Ethylene glycol 950ml

[0275] Methanol 50ml

[0276] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0277]

[0278] Add 20 microliters of anticoagulated blood sample and 20 microliters of reagent A to 1 mL of reagent B, and incubate at 42°C for 30 seconds to obtain the first sample. Use Mindray 6800plus blood analyzer, in which the light source is replaced with a blue laser light source (wavelength 450nm) for detection, and measure the forward low-angle scattered light intensity (FS), the side high-angle scattered light intensity (SS) and the side fluorescence intensity (FL, i.e., lysosomal fluorescence intensity). According to the above method, blood samples of healthy subjects and blood samples of sepsis patients were tested respectively to obtain the corresponding light signals, and a scatter plot of the side scattered light intensity and the side fluorescence intensity SS-FL of the two samples was obtained, as shown in Figure 2. Fig. 9 As shown in the figure, it can be seen that the blood samples of sepsis patients have higher side fluorescence intensity than normal blood samples.

[0279] Depend on Fig. 9The results show that after the blood sample is treated with the first hemolytic agent containing anionic surfactant, the difference in lateral fluorescence between sepsis blood samples and healthy blood samples is small. However, relatively reliable infection marker parameters can still be obtained. For healthy subject samples and sepsis patient samples, for example, the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) parameter can be extracted separately according to the aforementioned method, and by comparing with the threshold, a value higher than this value is indicated as a sepsis patient, thereby helping to assess the patient's infection status; the applicant's clinical statistical experimental results show that the diagnostic efficacy of assessing whether a subject has sepsis based on the lysosomal fluorescence intensity distribution width (N_WBC_SFL_W) can reach 0.910.

[0280] Example 6

[0281] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0282] A: Lysosomal stain

[0283] Compound 3 50mg

[0284] Ethylene glycol 950ml

[0285] Methanol 50ml

[0286] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0287]

[0288]

[0289] A large number of blood samples collected from sepsis patients and healthy subjects were tested according to the same detection method as in Example 2. At the same time, commercial M-6LD was used as the second hemolytic agent, and M-6FD was used as the nucleic acid staining agent. The above samples were tested simultaneously in the DIFF channel according to the regulations of the Mindray 6800plus blood analyzer. The first white blood cell parameter and / or the second white blood cell parameter were extracted according to the above blood analysis method, and the following correlation curve (i.e., ROC curve) about the infection marker parameter was drawn, as shown in FIG. Fig.10 The corresponding parameters are shown in Table 1 below. Fig.10As shown in Table 1 below, the first leukocyte particle group lysosome fluorescence signal (lysosome fluorescence intensity distribution width (N_WBC_SFL_W)) obtained by lysosomal dye staining has high specificity and sensitivity to infection, with an AUC coefficient of up to 0.927. In addition, the parameters (D_MON_SSC_W&N_WBC_SFL_W) obtained by combining the first leukocyte particle group lysosome fluorescence signal obtained by the WNB channel with the second monocyte side scattered light intensity distribution width obtained by the DIFF channel have higher specificity and sensitivity to infection, with an AUC coefficient of up to 0.960.

[0290] Table 1 Statistical results of sensitivity and specificity of each cell parameter and infection

[0291]

[0292] Example 7

[0293] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0294] A: Lysosomal lytic agent

[0295] Compound 3 50mg

[0296] Ethylene glycol 950ml

[0297] Methanol 50ml

[0298] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0299]

[0300] The same detection method as in Example 2 was used, except that the reagent A and reagent B of this example were used to detect the blood samples of healthy subjects and the abnormal blood samples containing nucleated red blood cells, respectively. The obtained SS-FL scatter plots are shown in FIG. Fig.11 As shown in the figure, compared with normal blood samples, nucleated red blood cells appear in the low fluorescence area (circled in the right figure). It can be seen that the use of the lysosomal staining agent of the present invention can further identify nucleated red blood cells.

[0301] Example 8

[0302] Prepare the lysosomal stain and first lysing reagent according to the following recipe:

[0303] A: Lysosomal stain

[0304] Compound 7 50mg

[0305] Ethylene glycol 950ml

[0306] Methanol 50ml

[0307] B: First hemolytic agent, adjust the pH value of the reagent to 3.0 with HCl

[0308]

[0309] The same detection method as in Example 5 was used, except that the reagent A and reagent B of this example were used to detect the blood samples of healthy subjects and the abnormal blood samples containing nucleated red blood cells, respectively. The obtained SS-FL scatter plots are shown in FIG. Fig.12 As shown in the figure, compared with normal blood samples, nucleated red blood cells appear in the low fluorescence area (circled in the right figure). It can be seen that the use of the lysosomal staining agent of the present invention can further identify nucleated red blood cells.

[0310] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A blood analyzer comprising a sample aspirating device, a sample preparation device, a light source, a flow chamber, an optical detector, a processor, and a non-transitory computer-readable storage medium programmed with a computer application, in, The sample aspirating device is configured to aspirate at least a portion of the blood sample to be tested and transport it to the sample preparation device; The sample preparation device comprises a reagent supply part and at least one reaction pool, wherein the reagent supply part is configured to provide a first reagent to a first reaction pool, the first reaction pool is configured to mix the first reagent with at least a portion of the blood sample to be tested therein to prepare a first sample, and transport the first sample to the flow chamber, wherein the first reagent comprises a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on lysosomes of cells in the blood sample to be tested; The light source is configured to direct a light beam toward a detection hole of the flow chamber that is in communication with the reaction cell; The flow chamber is configured to allow particles in the first sample to pass through the flow chamber one by one; The optical detector is mounted around the flow cell and configured to detect a first light signal of particles in the first sample passing through a detection hole of the flow cell; The processor is operably connected to the optical detector, and when the computer application in the non-transitory computer readable storage medium is executed by the processor, the processor is caused to perform the following steps: Processing the first light signal to obtain first white blood cell information; extracting at least one first white blood cell parameter of the first white blood cell information; An infection marker parameter is obtained based on the at least one first white blood cell parameter.

2. The blood analyzer according to claim 1, wherein: The first leukocyte information includes at least one of first leukocyte grouping particle cluster information and first leukocyte particle cluster information, wherein the first leukocyte grouping particle cluster information includes first lymphocyte particle cluster information and first neutrophil particle cluster information; Preferably, the first white blood cell information includes the first neutrophil particle cluster information and the first white blood cell particle cluster information.

3. The blood analyzer according to claim 2, wherein: The at least one leukocyte parameter includes one or more of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, lysosomal fluorescence intensity distribution width, lysosomal fluorescence intensity distribution center of gravity, lysosomal fluorescence intensity distribution coefficient of variation of at least one of the first leukocyte grouping particle cluster and the first leukocyte particle cluster, the area of ​​a distribution region of at least one of the first leukocyte grouping particle cluster and the first leukocyte particle cluster in a two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of a distribution region of at least one of the first leukocyte grouping particle cluster and the first leukocyte particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity; Preferably, the at least one first leukocyte parameter includes one or more of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, lysosomal fluorescence intensity distribution width, lysosomal fluorescence intensity distribution center of gravity, lysosomal fluorescence intensity distribution coefficient of variation, the area of ​​the distribution region of the first leukocyte particle cluster in a two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity, and the volume of the distribution region of the first leukocyte particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the lysosomal fluorescence intensity; More preferably, the at least one leukocyte parameter includes one or more of the following parameters: a lysosomal fluorescence intensity distribution width, a lysosomal fluorescence intensity distribution center of gravity, and a forward scattered light intensity distribution width of the first leukocyte particle group.

4. The blood analyzer according to any one of claims 1 to 3, wherein: The sample aspirating device is further configured to aspirate at least another portion of the blood sample to be tested and deliver it to the sample preparation device; The sample preparation device further comprises a second reaction pool, the second reaction pool and the first reaction pool are the same reaction pool or different reaction pools, wherein the reagent supply unit is further configured to provide a second reagent to the second reaction pool, the second reaction pool is configured to mix the second reagent with at least another part of the blood sample therein to obtain a second sample, and transport the second sample to the flow chamber, wherein the second reagent comprises a second hemolytic agent and a nucleic acid staining agent containing a nucleic acid dye, and the nucleic acid dye performs fluorescent staining on nucleic acid substances of cells in the blood sample to be tested; The flow chamber is further configured to allow particles in the second sample to pass through the flow chamber one by one; The optical detector is further configured to detect a second optical signal of each particle of the second sample passing through the detection hole of the flow chamber; When the computer application in the non-transitory computer-readable storage medium is executed by the processor, the processor is caused to further perform the following steps: processing the second light signal to obtain second white blood cell information; extracting at least one second white blood cell parameter of the second white blood cell information; The infection marker parameter is obtained based on the at least one first white blood cell parameter and the at least one second white blood cell parameter.

5. The blood analyzer according to claim 4, wherein: The second leukocyte information includes at least one second leukocyte grouping particle cluster information. Preferably, the at least one second leukocyte clustering particle information includes one or more of second monocyte cluster information, second neutrophil cluster information and second lymphocyte cluster information.

6. The blood analyzer according to claim 5, wherein the at least one second leukocyte parameter comprises one or more of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, nucleic acid fluorescence intensity distribution width, nucleic acid fluorescence intensity distribution center of gravity, nucleic acid fluorescence intensity distribution coefficient of variation of at least one second leukocyte clustering particle group, the area of ​​the distribution region of the at least one second leukocyte clustering particle group in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity, and the volume of the distribution region of the at least one second leukocyte clustering particle group in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity; Preferably, the at least one second leukocyte parameter includes one or more of the following parameters: forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, nucleic acid fluorescence intensity distribution width, nucleic acid fluorescence intensity distribution center of gravity, nucleic acid fluorescence intensity distribution coefficient of variation, the area of ​​the distribution region of the second monocyte particle cluster in a two-dimensional scatter plot generated by two light intensities among the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity, and the area of ​​the distribution region of the second monocyte particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity. The volume of the distribution area in the dot plot; as well as the forward scattered light intensity distribution width, forward scattered light intensity distribution center of gravity, forward scattered light intensity distribution coefficient of variation, side scattered light intensity distribution width, side scattered light intensity distribution center of gravity, side scattered light intensity distribution coefficient of variation, nucleic acid fluorescence intensity distribution width, nucleic acid fluorescence intensity distribution center of gravity, nucleic acid fluorescence intensity distribution coefficient of variation of the second neutrophil particle cluster, the area of ​​the distribution area of ​​the second neutrophil particle cluster in a two-dimensional scatter plot generated by two light intensities of the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity, and the volume of the distribution area of ​​the second neutrophil particle cluster in a three-dimensional scatter plot generated by the forward scattered light intensity, the side scattered light intensity and the nucleic acid fluorescence intensity.

7. The blood analyzer according to claim 6, wherein: The obtaining of the infection marker parameter based on the at least one first white blood cell parameter and the at least one second white blood cell parameter comprises: The infection marker parameter is obtained based on the lysosomal fluorescence intensity distribution width of the first leukocyte particle group and the side scattered light intensity distribution width of the second monocyte particle group.

8. The blood analyzer according to claim 1, wherein: When the computer application in the non-transitory computer-readable storage medium is executed by the processor, the processor is caused to further perform the following steps: The first optical signal is processed to further obtain information of nucleated red blood cells and / or basophils.

9. The blood analyzer according to claim 4, wherein: When the computer application in the non-transitory computer-readable storage medium is executed by the processor, the processor is caused to further perform the following steps: The second optical signal is processed to obtain information of at least one of the eosinophils, monocytes, neutrophils and lymphocytes.

10. The blood analyzer according to claim 1, wherein: The lysosomal dye is selected from a lysosomal dye that can be excited by red light or a lysosomal dye that can be excited by blue light.

11. The blood analyzer according to claim 10, wherein: The lysosomal dye is at least one compound selected from the following general formula I and general formula II: wherein X is selected from -O-, -S- and -CR'R"-, wherein R' and R" are C1-C4 alkyl, and R1 and R2 are each independently selected from C3-C20 alkenyl and C3-C20 alkyne; Preferably, R1 and R2 are each independently selected from C3-C10 alkenyl and C3-C10 alkyne; as well as Wherein, R3 and R4 are each independently selected from H, C1-C12 straight chain alkane, and R5 is selected from hydrogen, and C1-C12 straight chain alkanes; Preferably, R3 and R4 are each independently selected from H, C1-C6 straight chain alkane, and R5 is selected from hydrogen, and C1-C6 straight chain alkanes.

12. The blood analyzer according to claim 11, wherein: The lysosomal fluorescent dye is at least one selected from the following compounds 1 to 8:

13. The blood analyzer according to any one of claims 10 to 12, wherein: The lysosomal staining agent is a solution containing 10-100 mg / L, preferably 40-60 mg / L, of the lysosomal fluorescent dye, wherein the solvent is one or more selected from methanol, ethanol, glycerol, glycol and diethylene glycol.

14. The blood analyzer according to claim 1, wherein: The first hemolytic agent comprises a surfactant and a buffer, wherein the surfactant is used to lyse red blood cells and is selected from at least one of a cationic surfactant, an anionic surfactant and a nonionic surfactant. Preferably, the pH value of the first hemolytic agent is 3.0-3.

5.

15. The blood analyzer according to any one of claims 1 to 14, further comprising a user interaction interface, wherein the user interaction interface is configured to display test results and / or input instructions to the blood analyzer.

16. The blood analyzer according to claim 15, wherein: The user interaction interface is configured to output the infection marker parameter and / or output prompt information indicating the infection status of the subject based on the infection marker parameter.

17. The blood analyzer according to claim 1, wherein: The infection marker parameters are used to assess the infection status of the subject.

18. The blood analyzer according to claim 17, wherein: The infection status assessment includes one or more of early prediction of sepsis, diagnosis of sepsis, differentiation of common infection from severe infection, infection condition monitoring, sepsis prognosis analysis, differentiation of bacterial infection from viral infection, differentiation of non-infectious inflammation from infectious inflammation, and evaluation of sepsis efficacy.

19. A blood analysis method, the method comprising: Obtaining a blood sample to be tested from a subject; preparing a first sample, wherein the first sample contains at least a portion of the blood sample to be tested and a first reagent, wherein the first reagent includes a first hemolytic agent and a lysosomal staining agent containing a lysosomal dye, and the lysosomal dye performs fluorescent staining on the lysosomes of cells in the blood sample to be tested; Allowing particles in the first sample to pass through an optical detection area illuminated by light one by one to obtain a first optical signal generated by the particles in the first sample after being illuminated by light; Processing the first light signal to obtain first white blood cell information; extracting at least one first white blood cell parameter of the first white blood cell information; An infection marker parameter is obtained based on the at least one first white blood cell parameter.

20. Use of a cell analysis reagent in analyzing infection marker parameters of a blood sample from a subject, wherein: The cell analysis reagent comprises a hemolytic agent and a lysosomal staining agent, wherein the lysosomal staining agent comprises a lysosomal dye targeting a cell lysosome.

21. The use according to claim 20, wherein the lysosomal dye is selected from a lysosomal dye that can be excited by red light or a lysosomal dye that can be excited by blue light, Preferably, the lysosomal dye is at least one compound selected from the group consisting of the following general formula I and general formula II: in, X is selected from -O-, -S- and -CR'R"-, wherein R' and R" are C1-C4 alkyl, and R1 and R2 are each independently selected from C3-C20 alkenyl and C3-C20 alkyne; Preferably, R1 and R2 are each independently selected from C3-C10 alkenyl and C3-C10 alkyne; as well as Wherein, R3 and R4 are each independently selected from H, C1-C12 straight chain alkane, and R5 is selected from hydrogen, and C1-C12 straight chain alkanes; Preferably, R3 and R4 are each independently selected from H, C1-C6 straight chain alkane, and R5 is selected from hydrogen, and C1-C6 straight chain alkanes.

22. The use according to claim 21, wherein The lysosomal dye is at least one selected from the following compounds 1 to 8:

23. The use according to claim 21 or 22, wherein The dye is a solution containing 10-100 mg / L of the lysosomal dye, wherein the solvent is one or more selected from methanol, ethanol, glycerol, glycol and diethylene glycol.

24. The use according to claim 23, wherein The hemolytic agent includes a surfactant, wherein the surfactant is used to lyse red blood cells and is selected from at least one of a cationic surfactant, an anionic surfactant, and a nonionic surfactant.

25. The use according to claim 24, wherein the cationic surfactant is selected from the quaternary ammonium salt shown in the following general formula III: in, R is selected from C8-C16 alkyl, X - is chloride ion or bromide ion; preferably, the concentration of the cationic surfactant in the hemolytic agent is 300-800 mg / L; Wherein, the anionic surfactant is selected from dodecylbenzenesulfonic acid, sodium dodecyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate and alcohol ether carboxylate; preferably, the concentration of the anionic surfactant in the hemolytic agent is 0.1-2g / L; Wherein, the nonionic surfactant is selected from polyoxyethylene nonionic surfactants, preferably, the polyoxyethylene nonionic surfactant is selected from long-chain fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester and fatty amine polyoxyethylene ether; preferably, the concentration of the nonionic surfactant in the hemolytic agent is 1-5g / L.

26. The use according to claim 24 or 25, wherein the hemolytic agent further comprises a buffer, and the pH value of the hemolytic agent is 3.0 to 3.

5.

27. The use according to claim 20, wherein The infection marker parameter is used to evaluate the infection status of the subject. Preferably, the infection status assessment includes one or more of early prediction of sepsis, diagnosis of sepsis, differentiation of common infection from severe infection, infection condition monitoring, sepsis prognosis analysis, differentiation of bacterial infection from viral infection, differentiation of non-infectious inflammation from infectious inflammation, and evaluation of sepsis efficacy.

28. A cell analysis reagent, wherein: The cell analysis reagent is defined according to any one of claims 20-27.

29. The reagent according to claim 28, wherein the hemolytic agent and the staining agent are separate reagents, or the hemolytic agent and the staining agent are mixed into one reagent.

30. The reagent according to claim 29, wherein the reagent consists of a hemolytic agent and a lysosomal staining agent.

Images