CN114252633A

CN114252633A - Sample analysis system, sample detection method, composition and application thereof

Info

Publication number: CN114252633A
Application number: CN202010998037.3A
Authority: CN
Inventors: 张子千
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2022-03-29

Abstract

The present invention relates to a sample analysis system comprising: a sampling device; a sample preparation device having a reaction cell for receiving a sample aspirated by a sampling device and a reagent supply portion for supplying a reagent to the reaction cell, so that the sample aspirated by the sampling device and the reagent supplied by the reagent supply portion are mixed in the reaction cell to prepare a sample to be tested, wherein the reagent includes a hemolytic agent, a first staining agent, and a second staining agent; an optical detection device; and a data processing device electrically connected to the optical detection device and comprising a processor and a computer readable storage medium storing a computer program, wherein the data processing device is configured to perform the following steps when the computer program is executed by the processor: and acquiring parameters of the white blood cells in the sample to be detected according to the at least two optical signals of the sample to be detected. In addition, the invention also relates to a sample detection method, a composition for analyzing the leucocyte and application thereof.

Description

Sample analysis system, sample detection method, composition and application thereof

Technical Field

The invention belongs to the field of biochemical analysis, and particularly relates to classification and counting of leukocytes.

Background

Blood cells, also called "blood cells", are cells present in blood and flow through the blood vessels of a living body along with the blood. In mammals, blood cells are mainly composed of three parts, red blood cells, white blood cells, and platelets. Leukocytes are a very important blood cell group in human blood, and have the ability to resist pathogen invasion, immune resistance to diseases, antibody production by phagocytosis of foreign bodies, injury healing ability of human injuries and diseases, and the like. When the human body is untimely, the white blood cells are usually expressed by the remarkable changes of the number, the morphology and the group proportion of the white blood cells, so the white blood cells are very special and important cells, and the accurate determination of the white blood cells has great and crucial significance for clinical diagnosis and pathological analysis.

Currently, several ways of measuring leukocytes have been investigated. In one example, leukocyte cell counting and classification is achieved using a hypotonic solution to disrupt leukocyte cell membranes and stain cellular nucleic acids with a fluorescent dye. In one example, differentiation of different groups of leukocytes is achieved by the intensity of the fluorescent signal using a hemolysis reagent that alters leukocyte morphology and disrupts cell membrane structure by adjusting the pH of the hemolysis reagent or adding a surfactant component, in combination with a fluorescent dye that stains nucleic acid species. In one example, a reagent is employed that causes differences in the volume and internal structure of different groups of leukocytes by detecting the conductance signal (DC) and high frequency current signal (RF) of the reagent-processed sample to effect classification of the leukocytes. In one example, a combination of hemolytic and chemical staining reagents is used, which can form a colored precipitate at a specific location within the leukocytes and achieve differentiation between different leukocytes by collecting light scattering signals. And in one example, leukocyte classification is achieved by fluorescence detection methods using specific monoclonal antibodies that are fluorescently labeled to bind to leukocyte surface antigens. However, these methods of leukocyte analysis are still limited in their ability to improve the accuracy and precision of leukocyte clustering.

The accuracy and precision of the leucocyte determination are improved, more definite data support can be provided for clinical diagnosis and pathological analysis, and the method has great significance for popularization and improvement of medical equipment.

Therefore, there is a strong need in the art for a leukocyte classification method with high accuracy and high precision.

Disclosure of Invention

In view of the above, the present invention provides a sample analysis system, comprising:

a sampling device comprising a sampler for drawing a sample;

a sample preparation device having a reaction cell for receiving a sample aspirated by a sampling device and a reagent supply portion for supplying a reagent to the reaction cell so that the sample aspirated by the sampling device is mixed with the reagent supplied by the reagent supply portion in the reaction cell to prepare a sample to be tested, wherein the reagent includes a hemolytic agent capable of maintaining physiological morphology and structural integrity of leukocytes while lysing erythrocytes, a first staining agent containing a nucleic acid dye capable of staining nucleic acid substances of leukocytes, and a second staining agent containing a reductive staining agent capable of forming a colored precipitate at a position of peroxidase present in the leukocytes;

an optical detection device comprising a light source, a flow chamber and at least two detectors, wherein particles of the sample to be detected can flow in the flow chamber, the light emitted by the light source irradiates the particles in the flow chamber to generate an optical signal, and the detectors are used for collecting the optical signal; and

a data processing apparatus electrically connected with the optical detection apparatus and comprising a processor and a computer readable storage medium storing a computer program, wherein the data processing apparatus is configured to perform the following steps when the computer program is executed by the processor: and acquiring parameters of the white blood cells in the sample to be detected according to the at least two optical signals of the sample to be detected.

In a particular embodiment, the data processing apparatus is configured such that when the computer program is executed by the processor, the following steps are performed:

and obtaining parameters of the white blood cells in the sample to be detected according to the fluorescence signal and the side scattering light signal of the sample to be detected.

The system of the invention collects different optical signals in one detection to detect in two dimensions of nucleic acid and peroxidase contents, and can reflect cell signals close to physiological states. Meanwhile, the hemolytic agent for maintaining the physiological morphology and the structural integrity of the white blood cells provides better resolution capability for two kinds of staining, further improves the accuracy and precision of white blood cell classification, and provides more definite data support for clinical diagnosis and pathological analysis.

In some embodiments, the data processing apparatus is configured such that when the computer program is executed by the processor, the following steps are performed:

and obtaining parameters of the white blood cells in the sample to be detected according to the fluorescence signal, the side scattering light signal and the forward scattering light signal of the sample to be detected.

In a specific embodiment, obtaining the parameters of the leukocytes in the test sample comprises: classification and/or counting of leukocytes.

As understood by those skilled in the art, the side scattered light signal typically reflects the complex length of the particle, while the forward scattered light signal typically reflects the volume size of the particle.

In particular embodiments, the physiological morphology and structural integrity of the white blood cells can now be maintained while the red blood cells are lysed by adding a surfactant and adjusting osmotic compaction.

Therefore, in the present invention, the hemolytic agent may include an osmotic pressure regulator and one or more surfactants selected from the group consisting of a cationic surfactant, a nonionic surfactant, and a zwitterionic surfactant.

Specifically, the cationic surfactant may be selected from one or more of dodecyltrimethylammonium chloride, ammonium chloride, and the like. The nonionic surfactant (e.g., fatty acid glycerides, polyols, polyoxyethylene type) may be selected from one or more of the group consisting of fatty acid monoglycerides, fatty acid diglycerides, sucrose fatty acid esters, sorbitol fatty acid esters, polysorbates, polyoxyethylene fatty acid esters, polyoxyethylene (40) stearates, polyoxyethylene (20) oleates, polyoxyethylene (23) lauryl alcohol ethers, polyoxyethylene (20) cetyl alcohol ethers, polyoxyethylene fatty alcohol ethers, polyoxyethylene phenol ethers, polyoxyethylene polyoxypropylene copolymers, nonylphenol polyoxyethylene ethers, pluronic 68, Spans (Spans), Tweens (Tweens), tritons (e.g., Triton X-100), sells (Myri), brizes (Brij), and saponins. The zwitterionic surfactant may be, for example, an amino acid type zwitterionic surfactant and/or a betaine type zwitterionic surfactant.

In a preferred embodiment, the surfactant of the present invention is a nonionic surfactant.

The present invention further improves the accuracy of leukocyte detection by selecting a nonionic surfactant as a component of the hemolytic agent. Without wishing to be bound by theory, this is because the selection of the nonionic surfactant reduces the effect on the nucleic acid dye compared to other surfactants, thereby improving the degree of development of the optical signal from the nucleic acid dye.

In the present invention, the surfactant concentration is formulated so as not to cause a change in the morphological structure of the leukocytes. Such a concentration can be confirmed by observing the structural morphology of leukocytes under a microscope under bright field after adding a hemolytic agent containing a surfactant. For example, the anionic and cationic surfactants of the present invention may be present at a final concentration of 0.1 to 1g/L during leukocyte detection; the nonionic surfactants of the invention can be present at a final concentration of 0.5-2g/L during leukocyte detection.

In the present invention, there is no particular limitation on the kind of the osmotic pressure adjusting agent, which may be selected, for example, from one or more of organic alcohols (e.g., ethylene glycol, xylitol, etc.), inorganic salts (e.g., potassium hydrogencarbonate, sodium chloride, etc.), organic acid alkali metal salts (e.g., tetrasodium ethylenediaminetetraacetate, citric acid, and sodium citrate), saccharides (e.g., glucose, mannose, etc.), and amino acids (e.g., glycine, tryptophan, etc.).

In a specific embodiment, the osmolality adjusting agent of the present invention is formulated to provide an osmolality range of 150mOsm/kg to 700mOsm/kg during the leukocyte detection process.

In the present invention, the nucleic acid dye refers to a dye that specifically stains nucleic acid substances of cells. In some embodiments, the nucleic acid dye is giemsa staining solution. In other embodiments, the nucleic acid dye is a fluorescent nucleic acid dye.

The fluorescent nucleic acid dye of the present invention may be selected from, for example, one or more of Acridine Orange (AO), thioflavin (T), chrysiline (chrysaniline), Thiazole Orange (TO), Ethidium Bromide (EB), Propidium Iodide (PI), 4, 6-diamidino diphenyl indole (DAPI), hoechst (ho), auramine O (AuO), SYBR Green and cyanine compounds.

It is understood that the nucleic acid dye is formulated to be present at a supersaturated final concentration during the detection of leukocytes. For example, in the case of acridine orange, it can be present at a concentration of > 50mg/L during the leukocyte detection process; for another example, in the case of thiazole orange, it may be present at a concentration of ≧ 35mg/L during the leukocyte detection; as another example, in the case of using a cyanine compound, it may be present at a concentration of 10mg/L or more during the leukocyte detection.

In a specific embodiment, the reducing dye of the present invention may be selected from the group consisting of 4-chloro-1-naphthol, 3-Diaminobenzidine (DAB), and combinations thereof. The present invention is not particularly limited with respect to the final concentration of the reductive dye in the leukocyte detection process, as long as it is present in excess with respect to the peroxidase present in the cells, for example, 100-1000 mg/L.

In addition, the present invention provides a sample detection method comprising:

obtaining a sample to be detected;

providing a reagent, wherein the reagent comprises a hemolytic agent, a first staining agent and a second staining agent, and a sample to be tested is treated by using the reagent to obtain a sample solution to be tested; wherein the hemolytic agent is capable of lysing erythrocytes while maintaining the physiological morphology and structural integrity of leukocytes; the first staining agent comprises a nucleic acid dye capable of staining nucleic acid material of leukocytes; the second stain comprises a reductive dye capable of forming a colored precipitate at the site of peroxidase present within leukocytes;

the particles in the sample liquid to be detected pass through a detection area of an optical detection device one by one and are irradiated by a light source of the optical detection device so as to acquire an optical signal of the sample to be detected;

classifying and/or counting leukocytes according to the optical signal.

In a specific embodiment, the optical signals comprise fluorescence signals and side scattered light signals, from which the white blood cells are sorted and/or counted.

In some embodiments, the optical signals further comprise forward scattered light signals, and the white blood cells are classified and/or counted based on the fluorescent signals, the side scattered light signals, and the forward scattered light signals.

In some embodiments, the method further comprises the steps of obtaining the sample, transferring the sample to a suitable container, and aspirating the sample from the container prior to processing the sample; mixing the aspirated sample with the hemolysis agent, nucleic acid dye, and reducing dye while processing the sample; subsequently passing particles in the processed sample through a detection zone one by one, illuminating the particles with a light source and collecting fluorescent signals by a fluorescent detector and side scattered light signals by a scattered light detector; the white blood cell region is divided from the scatter diagram of the fluorescence signal and the side scattered light signal, and then counting of the white blood cells is realized based on the number of scatter points in the white blood cell region.

In some embodiments, the leukocyte region is selected from one or more of the group consisting of: primitive leukocyte region, lymphocyte region, monocyte region, neutrophil region and eosinophil region.

In some embodiments, the leukocyte region comprises a primitive leukocyte region.

The order of addition of the hemolytic agent, nucleic acid dye and reductive dye is not particularly limited in the present invention. For example, the hemolytic agent may be added first, and the nucleic acid dye and the reducing dye may be added; for another example, a hemolytic agent, a nucleic acid dye and a reductive dye may be added simultaneously, but the present invention is not limited thereto.

In some embodiments, the time of treatment is 30-150 s.

In a preferred embodiment, the time of treatment is 50-70 s.

In the present invention, the kind of the optical signal to be collected can be determined according to the kind of the nucleic acid dye. For example, in the case of using giemsa staining solution as a nucleic acid dye, a reflected light angle or a light absorption signal may be collected; in the case of using a fluorescent dye as the nucleic acid dye, a fluorescent signal can be collected.

In the present invention, the sample may be a blood sample, preferably a peripheral blood sample.

In addition, the present invention provides a composition for analyzing leukocytes, comprising:

a hemolytic agent capable of lysing erythrocytes while maintaining the physiological morphological and structural integrity of leukocytes;

a first staining agent comprising a nucleic acid dye capable of staining nucleic acids in a cell; and

a second stain comprising a reductive dye and hydrogen peroxide, the reductive dye being capable of forming a colored precipitate at a location within the leukocytes where peroxidase is present.

In the present invention, analyzing leukocytes means classifying leukocytes and optionally counting the classified leukocytes.

In the present invention, the nucleic acid dye may be present in a form dissolved in an organic solvent. Suitable organic solvents may be selected according to the particular kind of nucleic acid dye, such as ethanol, methanol, isopropanol, diethyl ether, acetone, acetonitrile, phenol, dimethyl sulfoxide, or dimethylformamide, and the like.

In the present invention, the reducing dye may be present in the form of being dissolved in a buffer. The buffer is not particularly limited as long as it is suitable for maintaining the morphology of leukocytes and for staining. Suitable buffers may have a pH of between 5 and 11, for example between 7 and 10.

In specific embodiments, the buffer may be selected from the group consisting of PBS buffer, borate buffer, citrate buffer, acetate buffer, phthalate, glycine-sodium hydroxide buffer, Tris (Tris) -HCl buffer, N- (2-acetamide) iminodiacetic acid (ADA) buffer, piperazine-1, 4-diethylsulfonate (PIPES) buffer, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES) buffer, N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) buffer, 3- (N-morpholino) ethanesulfonic acid (MOPS) buffer, 2-hydroxy-3- (N-morpholino) ethanesulfonic acid (MOPSO) buffer, and mixtures thereof, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.

The terms "first" and "second," and the like, in the present invention are used only for distinguishing between similar elements and not intended to imply any difference in importance or order between the elements unless otherwise specified.

In the present invention, the hemolytic agent, the first coloring agent, and the second coloring agent may be packaged individually or in combination. For example, the packaging may be in the form of a combination of a hemolytic agent and a second staining agent, as well as the first staining agent alone; or may be packaged in the form of a hemolytic agent, a first staining agent, and a second staining agent.

In addition, the invention also provides the application of the composition in preparing a leukocyte classification reagent.

Drawings

Fig. 1 shows a two-dimensional scattergram of fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 1, Blast: primitive leukocyte, Lym: lymphocytes, Mon: monocytes, Neu: neutrophils, Eos: eosinophils;

FIG. 2 is a two-dimensional scattergram showing a fluorescence signal and a side scatter signal when leukocytes are detected using the kit prepared in example 2;

FIG. 3 shows a two-dimensional scattergram of fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 3;

FIG. 4 is a two-dimensional scattergram showing fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 4;

FIG. 5 is a two-dimensional scattergram showing fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 5;

FIG. 6 is a two-dimensional scattergram showing fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 6;

FIG. 7 is a two-dimensional scattergram showing fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in example 7;

FIG. 8 is a two-dimensional scattergram showing a fluorescence signal and a side scatter signal when leukocytes are detected using the kit prepared in example 8;

FIG. 9 shows a two-dimensional scattergram of forward scatter signals and side scatter signals for leukocyte detection and a two-dimensional scattergram of forward scatter signals and fluorescence signals for 40 seconds, 60 seconds, and 120 seconds of processing the sample;

FIG. 10 shows a two-dimensional scattergram of fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in comparative example 1;

FIG. 11 shows a two-dimensional scattergram of fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in comparative example 2;

FIG. 12 shows a two-dimensional scattergram of fluorescence signals and side scatter signals when leukocytes are detected using the kit prepared in comparative example 3;

FIG. 13 shows an image of a sample under a microscope bright field after treatment with a hemolysing agent and a first stain of the present invention;

FIG. 14 shows an image of a sample treated with a hemolysing agent and a first stain of the present invention under microscope fluorescence;

FIG. 15 shows two-dimensional scatter plots of the detected fluorescence signal and forward scatter signal before and after treatment with a hemolysing agent and a first stain of the present invention;

FIG. 16 shows an image of an untreated sample under a microscope bright field;

FIG. 17 shows an image of a sample treated with a hemolysing agent and a second stain of the present invention under a bright field microscope;

FIG. 18 shows two-dimensional scatter plots of the side scatter signal and the forward scatter signal detected before and after treatment with a hemolysing agent and a second stain of the present invention;

fig. 19 shows a schematic view of the composition of an exemplary blood cell analysis system of the present invention.

Detailed Description

The embodiments of the present application will be described more fully hereinafter, and it is to be understood that the embodiments described are merely exemplary of some, but not all, embodiments of the present application. All other embodiments obtained by a person of ordinary skill in the art without any inventive work based on the embodiments in the present application are within the scope of protection of the present application.

As described above, in order to improve the accuracy and precision of leukocyte detection, the present invention provides a method for classifying leukocytes by using the difference in staining of different leukocyte populations in both dimensions of nucleic acid and peroxidase, while maintaining the physiological morphology and structural integrity of leukocytes.

After the hemolytic agent, the nucleic acid dye and the reductive dye are mixed with the blood sample, red blood cells in the blood sample are dissolved and destroyed, partial red blood cells which are not completely destroyed also form a ghost, the influence on subsequent optical signals is small, and meanwhile, leukocyte nucleic acid substances which keep the physiological shape and the structure of the cells complete are combined with the nucleic acid dye and the reductive dye.

Without wishing to be bound by theory, since different leukocytes have different cellular characteristics, such as granulocytes, lymphocytes, etc., and the extent of damage to their membrane structure is different after treatment, the amount of dye that enters varies from cell to cell, and the size of nuclei varies from cell to cell, the amount of nucleic acid dye that binds intracellular nucleic acid species varies from leukocyte to leukocyte, resulting in different optical signals produced by different leukocytes after staining with nucleic acid dyes. In addition, the reductive dye enters the white blood cells which keep the physiological shape and the structure of the cells intact, and generates colored precipitates at the positions where peroxidase exists in the cells, so that the complexity degree of the interior of the cells is changed. Accurate differentiation of different leukocyte populations is achieved by measuring the optical signal from the nucleic acid dye and the side scattered light from the reducing dye for each leukocyte.

It should be noted that the number of red blood cells in the whole blood is about 1000 times of that of white blood cells on average, and the red blood cells include nucleated red blood cells and reticulocytes, which have a great influence, so that it is necessary to dissolve the red blood cells in the sample with a hemolytic agent while detecting the white blood cells in order to eliminate interference of the red blood cells with their accurate classification and quantification. However, the inventor finds that the physiological shape and structure of the white blood cells are kept intact in the hemolysis process, which is an important factor for realizing the subsequent staining differentiation, and if the hemolysis of the hemolytic agent is not limited, the morphological structure of the white blood cells is changed, so that the nucleic acid staining and the peroxidase staining are generated with errors.

Accordingly, the present invention provides a sample analysis system for implementing the above method, comprising:

a sampling device comprising a sampler for drawing a sample;

The processor may be a central processing unit, or may be other general-purpose processor, a digital signal processor, an application specific integrated circuit, an off-the-shelf programmable gate array or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or the like. A general purpose processor may be a microprocessor or the processor may be any conventional processor or the like.

The computer readable storage medium may be either volatile memory or nonvolatile memory, and may include both volatile and nonvolatile memory. The nonvolatile memory can be a read-only memory, a programmable read-only memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a magnetic random access memory, a flash memory, a magnetic surface memory, an optical disc, or a read-only optical disc; the magnetic surface storage may be disk storage or tape storage. Volatile memory may be random access memory, which acts as external cache memory. By way of illustration and not limitation, many forms of RAM are available, such as SRAM, SDRAM, DRAM, SDRAM, DDR SDRAM, SSRAM, SDRAM, and DMA bus RAM. The memory described in connection with the embodiments of the invention is intended to comprise these and any other suitable types of memory.

FIG. 19 illustrates a particular sample analysis system according to the present invention. The sample analysis system includes a first housing 100, a second housing 200, a sampling device 10, a sample preparation device 30, an optical detection device 50, a data processing device 70, and an output 90. In practical applications, the output 90 may be a user interface. In the present embodiment, the optical detection device 50 and the data processing device 70 are disposed inside the second housing 200, and are disposed on both sides of the second housing 200. The sample preparation device 30 is disposed inside the first housing 100, and the output 90 and the sampling device 10 are disposed on an outer surface of the first housing 100.

The sampling device 10 has a sampling needle for collecting a blood sample and delivering the collected blood sample to the sample preparation device 30. According to various embodiments, the sampling device may collect multiple blood samples, provide different chambers of the sample preparation device for different processing, and then perform different tests.

The sample preparation device 30 has a reaction cell and a reagent supply portion that stores reagents for reacting with the blood sample (e.g., at least the aforementioned hemolytic agent, first staining agent, and second staining agent) and supplies the respective reagents to the reaction cell as necessary.

The sample preparation device 30 may comprise at least one reaction cell, wherein at least one reaction cell may be configured to allow the blood sample from the sampling portion and the reagent from the reagent supply portion to react to obtain a test solution containing a plurality of leukocyte particles, such that the leukocyte particles flow through the flow chamber of the optical detection device one by one.

The optical detection device 50 may include: the optical subsystem having a light source, a flow cell, and at least two detectors described above. The detector is used to obtain optical signals generated by the first and second stains, respectively. The flow chamber through which blood particles, such as white blood cells, are queued. At least two detectors are used to collect optical signals, in particular light intensity signals, of blood particles passing through the flow chamber.

In a particular sample analysis system, the at least two detectors comprise, for example, a first detector that detects a fluorescent signal of a particle flowing in the flow cell, i.e., a fluorescence detector. The at least two detectors further comprise a second detector. The second detector is arranged at an angle to a line in which the light source and the flow cell are located to detect laterally scattered light from particles flowing in the flow cell. Optionally, a third detector may be further included, the third detector being disposed generally on a line on which the light source and the flow cell are located, and the light source being disposed on both sides of the flow cell, respectively, to detect forward scattered light of the particles flowing in the flow cell.

The data processing device 70 is configured to detect blood particles flowing through the flow chamber based on at least the optical signal to obtain a detection result corresponding to the white blood cells.

The output unit 90 is configured to output a detection result corresponding to the blood (e.g., leukocyte) particles.

According to the aforementioned method of the present invention, the at least two detectors include a fluorescence detector and a side scatter detector, and the data processing device is configured to further perform the above-mentioned steps of the sample detection method of the present invention when the computer program is executed by the processor.

According to the method of the present invention, the data processing device of the sample analysis system can further classify the white blood cells and accurately count the classified white blood cells by using the fluorescence signal and the side scattered light signal.

The data processing device of the sample analysis system can also obtain a three-dimensional scatter diagram by simultaneously using fluorescence, forward scattered light and side scattered light, so that the white blood cells can be better classified, and the classified white blood cells can be accurately counted.

In another aspect, the present invention relates to a composition for analyzing leukocytes, comprising:

a hemolytic agent comprising no surfactant;

a first staining agent comprising a nucleic acid dye capable of staining nucleic acids in cells; and

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

after adding 4. mu.L of fresh anticoagulated blood to 1mL of The above-mentioned reagent (of which, 900. mu.L of hemolytic agent; 20. mu.L of first staining agent; 80. mu.L of second staining agent), The osmolality was 642mOsm/kg as measured by The Advanced Osmometer Model 3250.

And adding 4 mu L of fresh anticoagulation blood into the 1mL of reagent, mixing and processing for 60 seconds under the condition of keeping the temperature at 42 ℃, and detecting cells by using a laser flow method (the excitation wavelength is 633-635 nm). The fluorescence signal of the cells is measured with a fluorescence detector and the side scattered light signal of the cells is measured with a side scattered light detector. Two-dimensional scattergrams of the side scattered light and the fluorescence are shown in FIG. 1. As can be seen from fig. 1, the leukocyte detection kit provided in this embodiment can accurately classify leukocytes.

Example 2

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 213 mOsm/kg.

The two-dimensional scattergram formed by reacting and generating side scattered light and fluorescence according to the method of example 1 is shown in fig. 2. As can be seen from fig. 2, the leukocyte detection kit provided in this embodiment can accurately classify leukocytes.

Example 3

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and the result was 158 mOsm/kg.

The two-dimensional scattergram formed by reacting and generating side scattered light and fluorescence according to the method of example 1 is shown in fig. 3. As can be seen from fig. 3, the leukocyte detection kit provided in this embodiment can accurately classify leukocytes.

Example 4

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and was 247 mOsm/kg.

The two-dimensional scattergram formed by reacting and generating side scattered light and fluorescence according to the method of example 1 is shown in fig. 4. As can be seen from fig. 4, the leukocyte detection kit provided in this example can accurately classify leukocytes.

Example 5

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

acridine orange 0.6mg

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 251 mOsm/kg.

The two-dimensional scattergram formed by the side scattered light and the fluorescence was generated by reacting and generating the same according to the method of example 1, as shown in fig. 5. As can be seen from fig. 5, the leukocyte detection kit provided in this example can accurately classify leukocytes.

Example 6

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

thiazole orange 0.6mg

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 316 mOsm/kg.

The two-dimensional scattergram formed by reacting and generating the side scattered light and the fluorescence according to the method of example 1 is shown in fig. 6. As can be seen from fig. 6, the leukocyte detection kit provided in this example can accurately classify leukocytes.

Example 7

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 152 mOsm/kg.

The two-dimensional scattergram formed by the side scattered light and the fluorescence was generated by reaction according to the method of example 1, as shown in fig. 7. As can be seen from fig. 7, the leukocyte detection kit provided in this example can accurately classify leukocytes.

Example 8

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 379 mOsm/kg.

The two-dimensional scattergram formed by the side scattered light and the fluorescence was generated by reaction according to the method of example 1, as shown in fig. 8. As can be seen from fig. 8, the leukocyte detection kit provided in this example can accurately classify leukocytes.

Example 9 examination of the Effect of reaction time on discrimination Effect

The reagents were prepared in triplicate as described in example 1, and cells were detected by laser flow method (excitation wavelength 633-635 nm) after treatment for 40 seconds, 60 seconds and 120 seconds, respectively. The fluorescence signal of the cell is measured by a fluorescence detector, and the forward scattered light signal and the side scattered light signal of the cell are measured by a forward scattered light detector and a side scattered light detector, respectively. Two-dimensional scattergrams of the forward scattered light and the side scattered light, and the forward scattered light and the fluorescence are shown in FIG. 9, respectively.

As can be seen from fig. 9, as the incubation time is prolonged, the fluorescence of the leukocyte population is gradually increased, the reaction time is 120 seconds, the leukocyte population can still be effectively distinguished, but the reaction time gradually exceeds the detection range, and when the incubation time is 60 seconds, different leukocytes have a more ideal grouping effect.

Comparative example 1 use of hemolytic agent that disrupts the morphological and structural integrity of leukocytes

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

the osmotic pressure was measured according to the method in example 1, and found to be 85 mOsm/kg.

The two-dimensional scattergram formed by reacting and generating side scattered light and fluorescence according to the method of example 1 is shown in fig. 10.

As can be seen from fig. 10, when the hemolytic agent used has too strong hemolytic ability to maintain the morphology and structural integrity of leukocytes, leukocytes cannot be distinguished accurately even when stained with nucleic acid dye and reductive dye.

Comparative example 2

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

cy5.5 Carboxylic acid 0.6mg

Ethylene glycol 20mL

A second coloring agent:

the two-dimensional scattergram formed by reacting and generating side scattered light and fluorescence according to the method of example 1 is shown in fig. 11.

As can be seen from fig. 11, when using the cy5.5 carboxylic acid stain, it is difficult to accurately distinguish leukocytes from both dimensions of fluorescent and reductive dye staining, even though the morphological and structural integrity of the leukocytes is maintained.

Comparative example 3

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

A second coloring agent:

basic Red 200mg

10mL of ethanol

Tris-hydrochloric acid buffer (0.05M) 190mL

The two-dimensional scattergram formed by reacting and generating the side scattered light and the fluorescence according to the method of example 1 is shown in fig. 12.

As can be seen from fig. 12, when a basic red stain is used for the cell particles, it is difficult to accurately distinguish white blood cells from the two dimensions of lateral scattering of the nucleic acid dye, even though the morphology and structural integrity of the white blood cells are maintained.

Example 10 detection Using a hemolytic agent and a first staining agent of the present invention

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a first coloring agent:

0.6mg of cyanine compound

Ethylene glycol 20mL

The blood sample was first treated with the hemolytic agent and the first staining agent according to the method described in example 1, and then the treated leukocytes were observed in a bright field, and as a result, as shown in fig. 13, after the treatment with the hemolytic agent and the first staining agent, the morphological structure of the leukocytes was intact in a microscopic bright field, and clear cell membrane, cytoplasm, and nucleus structure were observed. The treated blood samples were then observed under microscopic fluorescence, and as a result, clear fluorescence from the nuclei was observed as shown in FIG. 14.

Then, before and after the treatment with the hemolytic agent and the first staining agent, cells were detected by a laser flow method (excitation wavelength of 633 to 635 nm). The fluorescence signal of the cells was measured by forward fluorescence at a measurement angle of 90 degrees, and the forward scattered light signal of the cells was measured by forward low-angle scattered light, and as a result, as shown in FIG. 15, the difference in fluorescence signal was caused by the difference in the degree of fluorescent staining between the different types of cells.

Example 11 detection Using a hemolytic agent of the present invention and a second staining agent

Preparing a leukocyte detection kit consisting of:

hemolytic agent:

a second coloring agent:

first, untreated fresh anticoagulated blood samples were taken and observed under a microscope under bright field, and the results are shown in FIG. 16. The blood sample was then treated with a hemolytic agent and a second staining agent according to the method described in example 1, and the treated leukocytes were observed under a bright field, with the results shown in fig. 17. As can be seen from a comparison of fig. 16 and 17, before the hemolytic agent and the second staining agent are treated, the white blood cells are blurred in outline under the microscope bright field, and the whole cells are transparent and colorless; after being treated by hemolytic agent and chemical color reagent, the leucocyte has intact morphological structure under microscope bright field, and clear colored precipitate granule can be seen in some leucocyte cytoplasm.

And (3) detecting the cells by using a laser flow method (with the excitation wavelength of 633-635 nm) before and after the treatment of the hemolytic agent and the second staining agent. The forward and side scattered light signals of the cells were measured using forward low angle scattered light, and the results are shown in FIG. 18. It can be seen that the difference in peroxidase content between different types of leukocytes after treatment with hemolytic agents and chemochromogenic agents causes a difference in the amount of pellet formed in the cells, i.e., the degree of change in the complexity of the interior of the cells varies, resulting in a difference in the laterally scattered light.

The foregoing is merely an exemplary embodiment and is not to be construed as limiting the scope of the disclosure. Obviously, any modifications and variations made within the scope of the present disclosure are within the scope of the disclosure as disclosed in the specification and defined in the claims of the present disclosure.

Claims

1. A sample analysis system, the sample analysis system comprising:

a sampling device comprising a sampler for drawing a sample;

2. The sample analysis system of claim 1, wherein the data processing arrangement is configured to perform the following steps when the computer program is executed by the processor:

3. The sample analysis system of claim 1, wherein the data processing arrangement is configured to perform the following steps when the computer program is executed by the processor:

4. The sample analysis system of any of claims 1-3, the obtaining parameters of leukocytes in a test sample comprising: classification and/or counting of leukocytes.

5. The sample analysis system of claim 4, wherein the obtaining a classification of the white blood cells comprises: distinguishing the white blood cells in the sample to be tested into at least: lymphocytes, monocytes, neutrophils and eosinophils, and/or blasts; and the counting of the white blood cells comprises the step of counting various white blood cells obtained by classification after the white blood cell classification is obtained.

6. A sample analysis system as claimed in any one of claims 1 to 3, wherein the haemolysing agent comprises an osmolality adjusting agent and a surfactant selected from one or more of a cationic surfactant, a non-ionic surfactant and a zwitterionic surfactant, preferably the osmolality adjusting agent is capable of maintaining an osmolality of 150 to 700mOsm/kg during treatment, preferably the surfactant is a non-ionic surfactant.

7. A method of sample detection, comprising:

obtaining a sample to be detected;

classifying and/or counting leukocytes according to the optical signal.

8. The method of claim 7, wherein the optical signals comprise fluorescent signals and side-scattered light signals from which leukocytes are classified and/or counted.

9. The method of claim 8, wherein the optical signals further comprise forward scattered light signals, and the white blood cells are classified and/or counted based on the fluorescent signals, side scattered light signals, and forward scattered light signals.

10. The method of any one of claims 7-9, wherein the classifying the white blood cells comprises: distinguishing the white blood cells in the sample to be tested into at least: lymphocytes, monocytes, neutrophils and eosinophils, and/or blasts; and the counting of the white blood cells comprises counting various white blood cells obtained by classification after the white blood cells are classified.

11. The method according to any one of claims 7 to 9, wherein the nucleic acid dye and the reductive dye are present in excess relative to the substance to be stained in the sample.

12. The method according to any one of claims 7 to 9, wherein the haemolysing agent comprises an osmolality adjusting agent and a surfactant selected from one or more of a cationic surfactant, a non-ionic surfactant and a zwitterionic surfactant, preferably the osmolality adjusting agent is capable of maintaining an osmolality of 150 to 700mOsm/kg during the treatment, preferably the surfactant is a non-ionic surfactant.

13. The method of any one of claims 7-9, wherein the nucleic acid dye is a fluorescent nucleic acid dye; and/or the reducing dye is selected from the group consisting of 4-chloro-1-naphthol, 3-diaminobenzidine, and combinations thereof.

14. The method according to any one of claims 7 to 9, wherein the treatment time is between 30 and 150s, preferably between 50 and 70 s.

15. A composition for analyzing leukocytes, comprising:

a second stain comprising a reductive dye and hydrogen peroxide, the reductive dye being capable of forming a colored precipitate at a location within the leukocytes where peroxidase is present,

preferably, the nucleic acid dye and the reductive dye are present in excess, so that saturation staining can be achieved at the time of use.

16. The composition according to claim 15, wherein the hemolytic agent comprises an osmolality adjusting agent and a surfactant selected from one or more of a cationic surfactant, a non-ionic surfactant and a zwitterionic surfactant, preferably the osmolality adjusting agent is capable of maintaining the osmolality during the treatment between 150 and 700mOsm/kg, preferably the surfactant is a non-ionic surfactant.

17. The composition of claim 15 or 16, wherein the nucleic acid dye is a fluorescent nucleic acid dye; and/or the reducing dye is selected from the group consisting of 4-chloro-1-naphthol, 3-diaminobenzidine, and combinations thereof.

18. The composition of claim 15 or 16, wherein the composition is capable of classifying and/or enumerating one or more selected from the group consisting of primitive leukocytes, lymphocytes, monocytes, neutrophils, and eosinophils.

19. Use of a composition according to any one of claims 15 to 18 in the preparation of a leukocyte classification reagent.