Blood sample detection reagent, detection method and analyzer

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly discloses a blood sample detection reagent, a detection method and an analyzer. The reagents comprise a first group of reagents and a second group of reagents; the first group of reagents comprises a first hemolytic agent and a first fluorescent dye; the second group of reagents comprises a second hemolytic agent and a second fluorescent dye. According to the method, the second hemolytic agent is added into the sample treated by the first group of reagents for secondary hemolysis, so that white blood cells and nucleated red blood cells except basophilic granulocytes are shrunk to a semi-naked nucleus state which still maintains a certain volume, and fluorescence is quenched and / or fluorescence intensity is weakened; then a second fluorescent dye is added for secondary dyeing, and then the sample is pushed to the same detection area to complete secondary detection, so that white blood cell five-classification and nucleated red blood cell classification counting are realized in a single channel. Therefore, the nucleated red blood cells can be counted in a classified manner, one channel is reduced, the detection efficiency can be improved, and the production cost of the blood analyzer is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a blood sample detection reagent, a detection method and an analyzer. Background Art

[0002] Nucleated red blood cells (NRBC) are immature bone marrow erythroid cells. There are no NRBCs in the blood of healthy adults, but they can be seen in the blood of newborns or infants. Once NRBCs appear in the peripheral blood of adults, it is mostly a pathological state, which is common in blood system diseases such as hemolytic anemia, acute and chronic leukemia, etc. A large number of studies have reported that an increase in NRBCs is closely related to increased mortality and poor prognosis of the disease, so the detection of NRBCs has an important clinical role. Since peripheral blood NRBCs have nuclei, blood analyzers may treat them as white blood cells and interfere with the white blood cell count, so detecting NRBCs to correct the white blood cell count is also critical to ensure the reliability of blood analysis results.

[0003] Hematology analyzers are commonly used equipment for blood analysis. During the measurement process, the sample needle and the quantitative pump are used to quantitatively draw blood samples and distribute them to the reaction pool. After the blood sample and the reagent react fully, the test sample is obtained, and then the sample is transported to the counting detection room for measurement, thereby obtaining different measurement parameter results. Most fluorescent hematology analyzers use the first set of reagents (the first hemolytic agent and the first fluorescent dye) in the DIFF channel to achieve the four classifications of white blood cells (WBC), namely lymphocytes (LYM), monocytes (MON), neutrophil (NEU) and eosinophil (EOS); in the WNB channel, the second set of reagents (the second hemolytic agent and the second fluorescent dye) are used to achieve the classification and counting of basophils (BASO) and NRBC, and output different parameter measurement results. When this detection method is carried out in two channels (reaction pools), cumbersome operations such as sample suction and sample transfer are required, which prolongs the detection time; at the same time, the addition of reaction pools will also increase costs and increase the size of the equipment, which is not conducive to the overall layout of the machine.

[0004] At present, time-division multiplexing technology is generally used to detect four types of white blood cell classification, BASO, hemoglobin (HGB), C-reactive protein (CRP), serum amyloid A (SAA), etc. Patent CN104297497A discloses a blood cell analyzer and its blood analysis method and control device. After the first group of reagents are added to the blood sample to obtain the first sample liquid, the second group of reagents are added to continue the treatment to obtain a test solution for BASO classification and / or HGB detection. In this way, the measurement of four types of white blood cells and BASO can be achieved on a single channel in a reaction pool through time-division multiplexing technology. Although this method can obtain the measurement results of five types of white blood cells and HGB, simplify the structure of the instrument, and improve the cost performance of the instrument, it cannot detect NRBC.

[0005] Patent CN114689893A discloses a blood analyzer, a blood analysis method, a blood sample collection and distribution method, and a blood analysis method. The blood analyzer includes a reaction pool group, an adding component for adding corresponding reagents to each reaction pool, and a blood sample collection and distribution component for collecting and distributing blood samples. The reaction pool group includes multiple reaction pools (such as RET pool, DIFF pool, RBC pool, CRP pool and SAA pool), wherein at least one reaction pool is a shared reaction pool, and the shared reaction pool is used to react to produce products for measuring HGB, WBC, and BASO in time-sharing periods. By sharing the reaction pool, the number of reaction pools is reduced, so that when distributing blood samples, the number of reaction pools that need to be injected with blood samples is reduced, the time required for blood sample distribution is shortened, thereby improving the measurement efficiency and reducing the amount of blood used; furthermore, the reduction in the number of reaction pools is conducive to reducing the volume of the blood analyzer and reducing the cost of the blood analyzer. Although the above method can improve the detection efficiency and reduce the cost of the blood analyzer by means of sharing reaction pools and time-sharing multiplexing, it still cannot detect NRBC, and there are certain limitations.

[0006] Patent CN114317673A discloses a reagent for classifying nucleated red blood cells, basophils and other white blood cells, which can distinguish nucleated red blood cells, basophils and other white blood cells, and then accurately count nucleated red blood cells, basophils and other white blood cells. In the prior art, in the process of five-category counting of white blood cells, when there is interference from nucleated red blood cells, the total count of other white blood cells in the sample containing nucleated red blood cells will be biased. This invention distinguishes nucleated red blood cells and Baso through the use of a hemolytic agent and a dye solution, which not only reduces the interference of nucleated red blood cells on the white blood cell count, but also accurately classifies and counts nucleated red blood cells. However, this method cannot classify and count the remaining four types of white blood cells, is independent of the DIFF channel detection, has low measurement efficiency and high cost. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a blood sample detection reagent, a detection method and an analyzer, which can realize the classification and counting of five types of white blood cells and nucleated red blood cells in a single channel.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides a blood sample detection reagent, including a first set of reagents and a second set of reagents;

[0009] The first set of reagents includes a first hemolytic agent and a first fluorescent dye, wherein the first hemolytic agent is selected from any one of the hemolytic agents used to lyse red blood cells and process white blood cells so that the white blood cells can be stained with the fluorescent dye;

[0010] The second set of reagents includes a second hemolytic agent and a second fluorescent dye, wherein the second hemolytic agent is used to dissolve nucleated red blood cells and white blood cells other than basophils to a semi-naked nucleus state, and to quench the fluorescence of the first fluorescent dye and / or weaken the fluorescence intensity;

[0011] The first fluorescent dye is used for staining nucleic acid substances in white blood cells, and the second fluorescent dye is used for differentially staining nucleic acid substances in nucleated red blood cells.

[0012] Furthermore, under acidic conditions, the fluorescence quenching intensity and fluorescence intensity of the first fluorescent dye are both weaker than the fluorescence quenching intensity and fluorescence intensity of the second fluorescent dye.

[0013] Furthermore, the first fluorescent dye and the second fluorescent dye are both cyanine dyes.

[0014] Further, the first hemolytic agent is selected from known hemolytic agents, and the known hemolytic agent is preferably a commercially available hemolytic agent, and more preferably a commercially available hemolytic agent produced by Mindray or Sysmex.

[0015] Furthermore, the second hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt.

[0016] Furthermore, the second hemolytic agent includes 0.5-1.2 g / L of an osmotic pressure regulator, 0.3-0.6 g / L of a surfactant, and 0.5-3.0 g / L of an organic acid and / or an organic acid salt.

[0017] Furthermore, the second hemolytic agent further comprises at least one of a buffer, a chelating agent or a preservative.

[0018] Furthermore, the second hemolytic agent further comprises at least one of 5-15 mmol / L buffer, 0.05-1.0 g / L chelating agent or 0.05-0.5 g / L preservative.

[0019] Furthermore, the osmotic pressure regulator is selected from at least one of sodium chloride, glucose or mannitol.

[0020] Furthermore, the surfactant is selected from quaternary ammonium salt cationic surfactants or betaine zwitterionic surfactants, the quaternary ammonium salt cationic surfactant is selected from at least one of dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride or dodecyltrimethylammonium bromide, and the betaine zwitterionic surfactant is selected from at least one of dodecyldimethylbetaine, dodecylsulfopropylbetaine or dodecylethoxysulfobetaine.

[0021] Furthermore, the organic acid and / or organic acid salt is selected from at least one of citric acid, malic acid, sodium salicylate, maleic acid, tartaric acid, phthalic acid or succinic acid.

[0022] Furthermore, the buffer is selected from at least one of phosphate buffer, citrate buffer, 2-Morpholinoethanesulphonic acid (MES) buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer or acetic acid-sodium acetate buffer.

[0023] Furthermore, the chelating agent is selected from disodium ethylenediaminetetraacetate (EDTA-2Na) and / or dipotassium ethylenediaminetetraacetate (EDTA-2K).

[0024] Furthermore, the preservative is selected from at least one of the Proclin series, sodium azide, potassium hydrogen phthalate or isothiazolinone.

[0025] Furthermore, the pH of the second hemolytic agent is 2.5-4.0.

[0026] Furthermore, the second hemolytic agent also includes a solvent, and the solvent is selected from water.

[0027] The present invention also provides another application of a hemolytic agent in blood sample detection, wherein the hemolytic agent is used for a second hemolytic treatment of blood cells, and the hemolytic agent comprises an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt.

[0028] Furthermore, the hemolytic agent includes 0.5-1.2 g / L of an osmotic pressure regulator, 0.3-0.6 g / L of a surfactant, and 0.5-3.0 g / L of an organic acid and / or an organic acid salt.

[0029] Furthermore, the hemolytic agent further comprises at least one of a buffer, a chelating agent or a preservative.

[0030] Furthermore, the hemolytic agent further comprises at least one of 5 to 15 mmol / L buffer, 0.05 to 1.0 g / L chelating agent, and 0.05 to 0.5 g / L preservative.

[0031] Furthermore, the osmotic pressure regulator is selected from at least one of sodium chloride, glucose or mannitol.

[0032] Furthermore, the surfactant is selected from at least one of quaternary ammonium salt cationic surfactants or betaine zwitterionic surfactants.

[0033] Furthermore, the organic acid and / or organic acid salt is selected from at least one of citric acid, malic acid, sodium salicylate, maleic acid, tartaric acid, phthalic acid or succinic acid.

[0034] Furthermore, the buffer is selected from at least one of phosphate buffer, citrate buffer, 2-morpholineethanesulfonic acid buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer or acetic acid-sodium acetate buffer.

[0035] Furthermore, the chelating agent is selected from at least one of disodium ethylenediaminetetraacetate or dipotassium ethylenediaminetetraacetate.

[0036] Furthermore, the preservative is selected from at least one of the Proclin series, sodium azide, potassium hydrogen phthalate or isothiazolinone.

[0037] Furthermore, the pH of the hemolytic agent is 2.5 to 4.0.

[0038] Furthermore, the hemolytic agent also includes a solvent, and the solvent is selected from water.

[0039] The present invention also provides a blood sample detection method, comprising the following steps:

[0040] (1) mixing the blood sample to be tested with a first hemolytic agent to dissolve the red blood cells in the sample to be tested, and treating the white blood cells so that they can be stained with a first fluorescent dye;

[0041] (2) mixing the reaction solution of step (1) with a first fluorescent dye to stain the leukocytes to obtain a first test solution, and performing a test on the first test solution to obtain the four-category classification and / or counting results of the leukocytes of the test blood sample;

[0042] (3) mixing part or all of the first test solution with the second hemolytic agent to dissolve the nucleated red blood cells and the white blood cells other than the basophils to a semi-naked nucleus state, and quenching the fluorescence of the first fluorescent dye and / or weakening the fluorescence intensity;

[0043] (4) Mixing the reaction solution of step (3) with a second fluorescent dye to differentially stain the nucleic acid substance in the nucleated red blood cells to obtain a second test solution, and testing the second test solution to obtain the classification and / or counting results of the nucleated red blood cells and basophils in the test blood sample.

[0044] Furthermore, step (4) is to add a second fluorescent dye under acidic conditions.

[0045] The present invention also provides a blood sample analyzer, comprising:

[0046] A sampling device, used for drawing a blood sample to be tested;

[0047] A test solution preparation device, comprising a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive part / all of the blood sample to be tested sucked by the sampling device, the reagent supply unit provides a first hemolytic agent and a first fluorescent dye to the reaction pool and mixes and reacts with the blood sample to be tested to form a first test solution, and provides a second hemolytic agent and a second fluorescent dye to the reaction pool and mixes and reacts with the first test solution to form a second test solution;

[0048] An optical detection device having a detection area for detecting a first liquid to be tested and generating a first signal, and detecting a second liquid to be tested and generating a second signal;

[0049] The processor obtains four classification and / or counting results of white blood cells of the blood sample to be tested based on the first signal, and obtains classification and / or counting results of nucleated red blood cells and basophils of the blood sample to be tested based on the second signal.

[0050] Furthermore, the first liquid to be tested and the second liquid to be tested are both generated in the same reaction pool.

[0051] Furthermore, the first liquid to be tested and the second liquid to be tested are generated in different reaction pools.

[0052] Furthermore, the detecting of the first test liquid and generating the first signal and the detecting of the second test liquid and generating the second signal are both performed in the same detection zone.

[0053] As described above, the blood sample detection reagent, detection method and analyzer of the present invention, the detection principle and beneficial effects are as follows:

[0054] The present invention adds another hemolytic agent (i.e., a second hemolytic agent) to the first test liquid that has been treated with the first hemolytic agent and the first fluorescent dye in the same reaction pool or transferred to another reaction pool, and performs a secondary hemolytic treatment, so that the white blood cells and nucleated red blood cells other than basophils shrink to a semi-naked nucleus state that still maintains a certain volume, and the fluorescence of the first fluorescent dye is quenched and / or the fluorescence intensity is weakened, and then the second fluorescent dye is added for secondary staining treatment to obtain the second test liquid, and then the second test liquid is pushed to the same detection area (i.e., the same detection channel) to complete the second detection, thereby achieving the five classifications of white blood cells and the classification and counting of nucleated red blood cells in a single channel. In this way, the classification and counting of nucleated red blood cells are completed, and the problem that nucleated red blood cells cannot be detected by sharing reaction pools and time-sharing multiplexing in the prior art is solved, and one channel can be reduced, thereby improving the detection efficiency and reducing the production cost of the blood analyzer.

[0055] Compared with the technology disclosed in CN114317673A, the present invention uses time-based two-dimensional fluorescence technology and adds a new reagent to achieve the fusion of the DIFF channel and the WNB channel. In the same channel, it can not only realize the five-classification of white blood cells, but also accurately classify and count nucleated red blood cells, and improve the detection efficiency and reduce the cost of the blood analyzer. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shown is a flow scatter plot of samples 1 to 3 in Example 4 of the present invention obtained by detecting using the DIFF channel and the WNB channel according to the above operations.

[0057] Figure 2 Shown are flow cytometry scatter plots obtained by detecting normal samples 1 to 3 and nucleated red samples 1 to 3 according to method 1 and method 2, respectively, in Example 4 of the present invention.

[0058] Figure 3 The results are shown as the correlation analysis results of the white blood cell classification count results obtained by testing 10 different blood samples using the traditional method and the method of the present invention in Example 4 of the present invention.

[0059] Figure 4 The results are shown as the correlation analysis results of the basophil classification and counting results obtained by testing 10 different blood samples using the traditional method and the method of the present invention in Example 4 of the present invention.

[0060] Figure 5 The results are shown as the correlation analysis results of the nucleated red blood cell classification and counting results obtained by testing 10 different blood samples using the traditional method and the method of the present invention in Example 4 of the present invention. DETAILED DESCRIPTION

[0061] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] The terms "first", "second", etc. in the description and claims of the embodiments of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchanged where appropriate, so that the embodiments of the present disclosure described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions.

[0063] Unless otherwise stated, the term "plurality" means two or more.

[0064] In the embodiment of the present invention, the character " / " indicates that the preceding and following objects are in an "or" relationship. For example, A / B indicates: A or B.

[0065] The term "and / or" is a description of the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, A and B.

[0066] One embodiment of the present invention provides a blood sample detection reagent, including a first set of reagents and a second set of reagents;

[0067] The first set of reagents includes a first hemolytic agent and a first fluorescent dye, wherein the first hemolytic agent is selected from any one of the hemolytic agents used to lyse red blood cells and process white blood cells so that the white blood cells can be stained with the fluorescent dye;

[0068] The second set of reagents includes a second hemolytic agent and a second fluorescent dye, wherein the second hemolytic agent is used to dissolve nucleated red blood cells and white blood cells other than basophils to a semi-naked nucleus state, and to quench the fluorescence of the first fluorescent dye and / or weaken the fluorescence intensity;

[0069] The first fluorescent dye is used for staining nucleic acid substances in white blood cells, and the second fluorescent dye is used for differentially staining nucleic acid substances in nucleated red blood cells.

[0070] The blood sample detection reagent of the present invention comprises two groups of reagents. When in use, a first hemolytic agent is first used to perform hemolysis treatment on the blood sample to be tested, and then a first fluorescent dye is used to perform staining treatment to obtain a first test liquid. The first test liquid is used to perform a first test to obtain the four classifications and counting results of the white blood cells of the blood sample to be tested.

[0071] Next, the second hemolytic agent is added to the first test liquid that has been treated with the first group of reagents for secondary hemolysis, which can shrink the white blood cells and nucleated red blood cells except basophils to a semi-naked nucleus state that still maintains a certain volume, and at the same time quench the fluorescence of the first fluorescent dye or weaken the fluorescence intensity of the first fluorescent dye; after the secondary hemolysis, the second fluorescent dye is added for secondary staining to obtain the first test liquid, and the second test liquid is used for a second test to obtain the classification and / or counting results of the nucleated red and basophilic granulocytes of the blood sample to be tested. Based on the characteristics of the first fluorescent dye and the second fluorescent dye, the second fluorescent dye occupies a core position and plays a leading role, binds more to the cells and exhibits a higher fluorescence quantum yield, achieving the original WNB classification effect, thereby merging the two channels, and still being able to accurately classify and count basophils, nucleated red blood cells, and other subtypes of white blood cells. Through coordination with the system, a single channel can be used to complete the detection of five types of white blood cells and nucleated red blood cells, reducing one channel, reducing instrument costs, and improving instrument detection speed.

[0072] In some embodiments, under acidic conditions, the fluorescence quenching intensity and fluorescence intensity of the first fluorescent dye are both weaker than the fluorescence quenching intensity and fluorescence intensity of the second fluorescent dye. In this way, during the secondary dyeing process, the second fluorescent dye can occupy a core position and play a leading role under acidic conditions, thereby playing the above role.

[0073] In some embodiments, the first fluorescent dye and the second fluorescent dye are both cyanine dyes. The first fluorescent dye is preferably a cyanine dye whose polymethylenic part does not contain a rigid structure, including but not limited to Cy3, Cy5, Cy7, etc. The second fluorescent dye is preferably a cyanine dye whose polymethylenic part is a planar structure, such as the cyanine compounds disclosed in invention patent CN102115456B, but not limited to this.

[0074] The first fluorescent dye is a cyanine compound, which can stain intracellular nucleic acids and proteins to different degrees based on the characteristics of white blood cells themselves, and at least realize the four classifications of white blood cells. In addition, the cyanine compound is a pH-sensitive dye, containing tertiary amino groups and ammonium ions, forming a D-π-A electron push-pull system. When in a strong acidic environment, these amino groups will undergo a protonation reaction, thereby changing their electronic structure and charge distribution within the molecule, affecting the fluorescence characteristics, and quenching the fluorescence or weakening the fluorescence intensity under acidic conditions; in addition, the first fluorescent dye uses a cyanine dye that does not contain a rigid structure in the polymethylenic part, such as Cy 3. Cy5, Cy7, etc. have rotatable characteristics. The electrons in the dye molecules mainly reach a stable state through the non-radiative transition method of vibration relaxation. When it is electrostatically adsorbed, groove-bound or embedded in the nucleic acid substance, its rotation is inhibited, thereby maintaining a stable state through radiative transition, and fluorescence is generated at this time; nucleic acid substances are negatively charged under normal circumstances, and such cyanine dye molecules are easily electrostatically adsorbed with them and further embedded to emit light. Under strong acidic conditions, nucleic acid molecules are protonated and positively charged, resulting in a greatly reduced binding efficiency of such cyanine dye molecules with them, and the fluorescence almost disappears.

[0075] The second fluorescent dye is also a cyanine compound, but it can maintain good fluorescence properties under acidic conditions, and its fluorescence quantum yield is significantly higher than the first fluorescent dye. The main reason is that the second fluorescent dye uses a cyanine dye with a planar structure of the polymethylene part, which has strong rigidity, increases the molecular planarity, can weaken the vibration of the molecule, thereby reducing its non-radiative transition, and is conducive to the delocalization of π electrons in the molecule. Under acidic conditions, even if a chimeric structure is not formed, it has a good fluorescence quantum yield, so it is less affected by pH. The second fluorescent dye can differentially stain nucleated red blood cells and white blood cells based on the difference in the degree of membrane rupture between nucleated red blood cells and white blood cells under acidic conditions and the difference in the ability to bind to the nucleic acid of each cell, so that nucleated red blood cells can be distinguished from white blood cells in terms of fluorescence intensity. Therefore, in the present invention, the remaining samples are reused after the first detection, and a second hemolysis treatment is performed by adding a new hemolytic agent (i.e., the second hemolytic agent), and the overall pH and related ion environment of the reaction system are changed, and then the second fluorescent dye is added to the sample after the second hemolysis treatment for secondary staining. Based on the characteristics of the two cyanine dyes, the second fluorescent dye occupies a core position and plays a leading role under the above conditions, thereby playing the above-mentioned role.

[0076] In some embodiments, the first set of reagents includes a first staining solution, the first staining solution includes a first fluorescent dye and a solvent; the first set of reagents includes a second staining solution, the second staining solution includes a second fluorescent dye and a solvent. Generally, the first fluorescent dye and the second fluorescent dye are first formulated into a staining solution and then added to the reaction system. Further, in some embodiments, the first fluorescent dye is preferably a Mindray M-60FD staining solution or a Mindray M-68FD staining solution, and the second fluorescent dye is preferably a Mindray M-60FN staining solution, a Mindray M-68FN staining solution, etc. These staining solutions are all commercially available staining solutions. Compared with homemade or self-developed staining solutions, the use of commercially available staining solutions is more conducive to improving detection efficiency, reducing research and development costs, and ensuring the accuracy of detection results. In some embodiments, the first hemolytic agent is selected from known hemolytic agents, and the first hemolytic agent is preferably a commercially available hemolytic agent produced by Mindray or Sysmex, such as Mindray M-60LD hemolytic agent, Mindray M-68LD hemolytic agent, Sysmex XN-1000WDF hemolytic agent, etc., which can not only ensure the detection accuracy but also reduce the research and development cost.

[0077] In some embodiments, the second hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt. The second hemolytic agent used in the present invention is a new type of acidic hemolytic agent independently developed. After the second hemolytic agent is added, it can change the overall pH of the reaction system to make it acidic, thereby quenching the fluorescence of the first fluorescent dye and / or weakening the fluorescence intensity, allowing the second fluorescent dye to occupy a core position and play a leading role under acidic conditions. Among them,

[0078] The osmotic pressure regulator is used to adjust the osmotic pressure of the system. In some implementations, the osmotic pressure of the second hemolytic agent is maintained in the range of 30 to 100 mOsm / kg, preferably in the range of 40 to 80 mOsm / kg.

[0079] Surfactants can quickly dissolve red blood cells. The surface of red blood cells carries a negative charge. Surfactants are adsorbed on the surface through electrostatic effects, act on phospholipids, and change the conformation of membrane proteins, thereby causing the dissolution of the cell membrane.

[0080] On the one hand, organic acids / organic acid salts are used to adjust the pH of the system, and on the other hand, they affect the composition and arrangement of lipids in the cell membrane, thereby changing the physical properties and conductivity of the cell membrane. This change will also cause changes in the charge distribution on the cell membrane and accelerate the dissolution of the cell membrane. After the cell membrane has dissolved to a certain extent, the organic acid is electrostatically adsorbed to the negatively charged surface of the cell membrane to form a protective layer, which protects the cell membrane from further dissolution to a certain extent.

[0081] In some embodiments, the second hemolytic agent includes the following components: 5-15 mmol / L buffer, 0.5-1.2 g / L osmotic pressure regulator, 0.3-0.6 g / L surfactant, 0.5-3.0 g / L organic acid and / or organic acid salt.

[0082] The present invention optimizes and screens the content of each component in the second hemolytic agent and finds that the concentration of the above components is appropriately controlled within the above range. The reason for this is that:

[0083] If the concentration of the osmotic pressure regulator is lower than 0.5g / L, the osmotic pressure of the hemolytic agent will be low, and the white blood cells and nucleated red blood cells will be in a hypotonic state, affecting cell clustering; if the concentration of the osmotic pressure regulator is higher than 1.2g / L, the ion concentration is too high, which is not conducive to the binding of the dye to the target molecule and will also affect cell clustering.

[0084] If the concentration of the surfactant is too low, the cells will not be completely broken and the blood ghost fragments will be larger; if the concentration of the surfactant is too high, the white blood cells will be excessively destroyed, affecting the classification and also not conducive to the function of the fluorescent dye.

[0085] If the concentration of organic acid and / or organic acid salt is too low, it will affect the hemolysis process and thus affect cell clustering, while if it is too high, the pH of the entire system will be too low, causing irreversible corrosive damage to instrument pipelines and devices.

[0086] In some embodiments, the osmotic pressure regulator is selected from at least one of sodium chloride, glucose or mannitol, but is not limited thereto.

[0087] In some embodiments, the surfactant is selected from quaternary ammonium salt cationic surfactants and / or betaine zwitterionic surfactants; the quaternary ammonium salt cationic surfactant is selected from at least one of dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride or dodecyltrimethylammonium bromide, but is not limited thereto; the betaine zwitterionic surfactant is selected from at least one of dodecyldimethylbetaine, dodecylsulfopropylbetaine or dodecylethoxysulfobetaine, but is not limited thereto.

[0088] The principle of surfactant dissolving cell membrane is mainly divided into membrane dissolution and osmotic dissolution. In principle, any surfactant that can dissolve red blood cells, present naked nuclei in nucleated red blood cells, damage white blood cell membranes but still maintain a certain cell structure and allow fluorescent dyes to enter the cell and bind to nucleic acid substances can be used. The surface of red blood cells carries a negative charge. Cationic surfactants can be adsorbed on their surface through electrostatic action, directly act on phospholipids, separate them from proteins, and cause the dissolution of cell membranes. The dissolution ability of cationic surfactants on phospholipids is ten times that of anionic surfactants. In addition, quaternary ammonium salts can provide a strong antibacterial effect and can also be used for the preservation of reagents. Anionic surfactants mainly dissolve through osmotic dissolution. Surfactants with long-chain hydrophobic groups can antagonize the electrostatic repulsion between them and the cell membrane and adsorb on its surface. Their mechanism of action is to form a micellar mixture with the cell membrane, causing the original cell membrane to dissolve. Zwitterionic surfactant molecules contain both positively and negatively charged groups, which can achieve the characteristics of positive or negative ions at different pH values, and both hemolysis methods are available. Therefore, considering the hemolytic efficiency and the antibacterial effect, the surfactant in the second hemolytic agent of the present invention is preferably a quaternary ammonium salt cationic surfactant and / or a betaine zwitterionic surfactant.

[0089] In some embodiments, the organic acid and / or organic acid salt is selected from at least one of citric acid, malic acid, sodium salicylate, maleic acid, tartaric acid, phthalic acid or succinic acid, but is not limited thereto. Other organic acids or organic acid salts that have the function of adjusting the pH of the hemolytic agent without affecting other properties of the hemolytic agent may also be used.

[0090] In some embodiments, the second hemolytic agent further includes a buffer solution, preferably at a content of 5 to 15 mmol / L. The buffer solution can ensure a stable pH environment for the system, and mainly includes a weak acid and its conjugate base. When the pH changes during the reaction, the acid-generating reaction causes the weak acid to ionize more H + ions, these H + The ion combines with the conjugate base, thereby reducing the H + concentration, preventing the pH from dropping significantly. On the contrary, the alkalinity reaction causes the conjugate base to release more OH - ions, these OH - The ions combine with the weak acid, thereby reducing the OH in the solution. -Concentration, prevent the pH value from rising significantly, and finally maintain the acid-base balance of the reaction system. If the buffer concentration is lower than 5mmol / L, the buffer effect will be poor. If it is higher than 15mmol / L, the osmotic pressure of the whole system will be high, and the osmotic pressure regulator will be difficult to reconcile. Further, in some embodiments, the buffer is selected from at least one of phosphate buffer, citrate buffer, MES buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer or acetic acid-sodium acetate buffer, but is not limited thereto.

[0091] In some embodiments, the second hemolytic agent also includes a chelating agent, and the content is preferably 0.05 to 1.0 g / L. The chelating agent can prevent blood cells or platelets from aggregating, and can chelate the multivalent ions present in the hemolytic agent, thereby preventing the multivalent ions from binding to other components in the hemolytic agent. If the concentration of the chelating agent is too low, the role of chelating metal ions cannot be effectively exerted, and if the concentration of the chelating agent is too high, it may be over-anticoagulated, and may combine with components such as proteins in the blood to form precipitation, increasing the risk of clogging the instrument pipeline. Further, in some embodiments, the chelating agent is selected from EDTA-2Na and / or EDTA-2K, but is not limited thereto. Using these compounds as chelating agents can not only prevent blood cells or platelets from aggregating, but also chelate the multivalent ions present in the hemolytic agent, thereby preventing the multivalent ions from binding to other components in the hemolytic agent, and also avoid affecting the performance of the hemolytic agent. Therefore, other chemical substances having the above-mentioned functional effects and not affecting the performance of the second hemolytic agent can also be used as chelating agents that can be used in the present invention.

[0092] In some embodiments, the second hemolytic agent also includes a preservative, and the content is preferably 0.05-0.5 g / L. The preservative is used for preservation, to prevent the hemolytic agent from deteriorating, and to extend its shelf life. If the concentration of the preservative is too low, it cannot effectively inhibit the growth of microorganisms; and if the concentration of the preservative is too high, on the one hand, it may interfere with the hemolysis process, and on the other hand, it may cause potential harm to the operator, such as skin irritation, respiratory tract irritation, etc. Further, in some embodiments, the preservative is selected from at least one of the Proclin series, sodium azide, potassium hydrogen phthalate or isothiazolinone, but is not limited thereto.

[0093] In some embodiments, the pH of the second hemolytic agent is controlled between 2.5 and 4.0. If the pH is lower than 2.5, the blood cell structure will almost completely disappear due to excessive acidity, making it impossible to classify the blood cells, and irreversible corrosive damage will be caused to instrument pipelines and devices. If the pH is higher than 4.0, the cell nucleus will be incompletely exposed, making it impossible to distinguish the nucleated red blood cells from the rest of the white blood cells, making it impossible to accurately classify the blood cells.

[0094] In some embodiments, the second hemolytic agent further includes a solvent, and the solvent is selected from water, such as purified water, pure water, ultrapure water, etc.

[0095] Another embodiment of the present invention provides the use of the second hemolytic agent as described in the above embodiment / example in blood sample detection, wherein the second hemolytic agent is used for a second hemolysis treatment of blood cells.

[0096] In some embodiments, the second hemolysis treatment refers to dissolving the nucleated red blood cells and white blood cells other than basophils in the red blood cell lysed blood sample to a semi-naked nucleus state. Exemplarily, the red blood cell lysed blood sample or blood cell sample is obtained by mixing and incubating the blood sample with a hemolytic agent A, and the hemolytic agent A is selected from the first hemolytic agent or other hemolytic agents that can dissolve red blood cells, preferably the first hemolytic agent.

[0097] In some embodiments, the second hemolytic agent is also used to quench the fluorescence and / or weaken the fluorescence intensity of the fluorescent dye A. An exemplary method is that during the blood sample detection process, the pH of the reaction system containing blood cells stained with the fluorescent dye A is adjusted to acidic using the second hemolytic agent, and the fluorescent dye B is added under acidic conditions to perform a secondary staining treatment on the blood cells. Wherein, the reaction system containing blood cells stained with the fluorescent dye A is obtained by mixing and incubating the blood sample with the fluorescent dye A, or by mixing and incubating the blood sample, the hemolytic agent A and the fluorescent dye A. Wherein, the fluorescent dye A is selected from the first fluorescent dye or other fluorescent dyes used to stain nucleic acid substances in white blood cells, preferably the first fluorescent dye; the fluorescent dye B is selected from the second fluorescent dye or other fluorescent dyes used to differentially stain nucleic acid substances in nucleated red blood cells, preferably the second fluorescent dye.

[0098] Another embodiment of the present invention provides a blood sample detection method, comprising the following steps:

[0099] (1) mixing the blood sample to be tested with a first hemolytic agent to dissolve the red blood cells in the sample to be tested, and treating the white blood cells so that they can be stained with a first fluorescent dye;

[0100] (2) mixing the reaction solution of step (1) with a first fluorescent dye to stain the leukocytes to obtain a first test solution, and performing a test on the first test solution to obtain the four-category classification and / or counting results of the leukocytes of the test blood sample;

[0101] (3) mixing part or all of the first test solution with the second hemolytic agent to dissolve the nucleated red blood cells and the white blood cells other than the basophils to a semi-naked nucleus state, and quenching the fluorescence of the first fluorescent dye and / or weakening the fluorescence intensity;

[0102] (4) Mixing the reaction solution of step (3) with a second fluorescent dye to differentially stain the nucleic acid substance in the nucleated red blood cells to obtain a second test solution, and testing the second test solution to obtain the classification and / or counting results of the nucleated red blood cells and basophils in the test blood sample.

[0103] In some embodiments, step (4) is adding a second fluorescent dye under acidic conditions.

[0104] In some embodiments, the first test liquid and the second test liquid are prepared using the blood sample detection reagent as described in the above embodiment / example. In the above embodiment / example, step (2) is the first detection, and the specific operation process is as follows: a portion of the first test liquid is drawn into the flow chamber for the first detection, so that the particles in the first test liquid pass through the detection area of ​​the optical detection device one by one and the light source of the optical detection device is used to irradiate the particles in the first test liquid to obtain optical information of the first test liquid, and the white blood cells are classified and / or counted based on the optical information of the first test liquid to obtain the four-category classification result of the white blood cells.

[0105] Step (4) is a second test. Exemplarily, the specific operation process is as follows: aspirate the second test liquid into the flow chamber for a second test, allow the particles in the second test liquid to pass through the detection area of ​​the optical detection device one by one, and use the light source of the optical detection device to irradiate the particles in the second test liquid to obtain optical information of the second test liquid, and classify and / or count the nucleated red blood cells, basophils, and other white blood cells (i.e., white blood cells other than basophils) based on the optical information of the second test liquid to obtain the results of nucleated red blood cells and basophils.

[0106] The optical information includes forward scattered light information, side scattered light information and fluorescence information, and the white blood cells and nucleated red blood cells are classified and / or counted according to the optical information.

[0107] In some embodiments, the detection process of steps (2) and (4) is completed in the same detection area, that is, the detection uses the same detection channel, which is a channel for four-classification of white blood cells, or a channel for detecting white blood cells, basophils and nucleated red blood cells. Exemplarily, if a Mindray blood cell analyzer is used to detect a blood sample, the detection channel is selected from the DIFF channel or the WNB channel; if a Sysmex blood cell analyzer is used to detect a blood sample, the detection channel is selected from the WDF channel or the WNR channel.

[0108] Another embodiment of the present invention provides a blood sample analyzer, comprising:

[0109] A sampling device, used for drawing a blood sample to be tested;

[0110] A test solution preparation device, comprising a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive part / all of the blood sample to be tested sucked by the sampling device, and the reagent supply unit provides the first hemolytic agent and the first fluorescent dye as described above to the reaction pool and mixes and reacts with the blood sample to be tested to form a first test solution, and provides the second hemolytic agent and the second fluorescent dye as described above to the reaction pool and mixes and reacts with the first test solution to form a second test solution;

[0111] An optical detection device having a detection area for detecting a first liquid to be tested and generating a first signal, and detecting a second liquid to be tested and generating a second signal;

[0112] The processor obtains four classification and / or counting results of white blood cells of the blood sample to be tested based on the first signal, and obtains classification and / or counting results of nucleated red blood cells and basophils of the blood sample to be tested based on the second signal.

[0113] In some embodiments, the first test liquid and the second test liquid are both generated in the same reaction pool. Exemplarily, the test liquid preparation device may be provided with only one reaction pool. If the reaction pool is defined as the first reaction pool, in this case, the detection operation process is as follows: the blood sample to be tested is mixed with the first group of reagents in the first reaction pool, and the first test liquid is obtained through the first incubation reaction; a portion of the first test liquid is drawn into the flow chamber, and the first detection is performed to obtain the four classifications and / or counting results of the white blood cells of the blood sample to be tested; the second group of reagents is added to the first reaction pool, mixed with the remaining first test liquid in the first reaction pool, and the second test liquid is obtained through the second incubation reaction, and then the second detection is performed.

[0114] In some embodiments, the first test liquid and the second test liquid are generated in different reaction pools. Exemplarily, the test liquid preparation device is provided with at least two reaction pools, one of which provides a reaction place for the preparation of the first test liquid, defined as the first reaction pool, and the other of which provides a reaction place for the preparation of the second test liquid, defined as the second reaction pool. In this case, the detection operation process is as follows: the blood sample to be tested is mixed with the first group of reagents in the first reaction pool, and the first test liquid is obtained after the first incubation reaction; part of the first test liquid is drawn into the flow chamber, and the first detection is performed to obtain the four classifications and / or counting results of the white blood cells of the blood sample to be tested; the reagent supply unit draws part or all of the first test liquid remaining in the first reaction pool into the second reaction pool, and adds the second group of reagents, obtains the second test liquid after the second incubation reaction, and then performs the second detection.

[0115] In some embodiments, the detection of the first test liquid and generation of the first signal and the detection of the second test liquid and generation of the second signal are both performed in the same detection zone, that is, the detection uses the same detection channel.

[0116] In some embodiments, the optical detection device is provided with a detection channel, which is a channel for four-classification of white blood cells, or a channel for detecting white blood cells, basophils and nucleated red blood cells.

[0117] In some embodiments, the optical detection device includes a light source and a fluorescent signal receiver. The light source of the optical detection device is a single laser, and the excitation wavelength matches the first fluorescent dye and the second fluorescent dye in the present invention; the detection area of ​​the optical detection device uses a fluorescent signal receiver to collect the fluorescent signal of the cell to obtain fluorescent information, and uses a scattered light signal receiver to collect the forward scattered light signal and the side scattered light signal of the cell; further, the fluorescent signal receiver can use a long-pass filter to filter out stray light below the emission wavelength of the fluorescent dye and then amplify the signal through a photomultiplier tube to collect the fluorescent signal.

[0118] In the above-mentioned embodiment mode / embodiment, the four types of white blood cells refer to lymphocytes, monocytes, neutrophils and eosinophils, and the five types of white blood cells refer to lymphocytes, monocytes, neutrophils, eosinophils and basophils.

[0119] It should be noted that the blood sample analyzer provided in the above implementation manner / example and the blood sample detection method provided in the above implementation manner / example belong to the same concept, and the detection mechanism and technical effects involved have been described in detail in the method implementation manner / example, and will not be repeated here.

[0120] The following further examples are given to explain the present invention in detail. It should also be understood that the following examples are only used to explain the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples in a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description of this article, and are not limited to the specific values ​​​​exemplified below.

[0121] Example 1

[0122] This embodiment provides a second hemolytic agent, which is prepared according to the following formula, stirred and mixed thoroughly, and then stored at 2-8°C.

[0123] The second hemolytic agent formula in this embodiment is as follows:

[0124]

[0125] Add purified water to 1.0 L and adjust the pH to 3.0.

[0126] Example 2

[0127] This embodiment provides a second hemolytic agent, which is prepared according to the following formula, stirred and mixed thoroughly, and then stored at 2-8°C.

[0128] The second hemolytic agent formula in this embodiment is as follows:

[0129]

[0130] Add purified water to 1.0 L and adjust the pH to 3.2.

[0131] Example 3

[0132] This embodiment provides a second hemolytic agent, which is prepared according to the following formula, stirred and mixed thoroughly, and then stored at 2-8°C.

[0133] The second hemolytic agent formula in this embodiment is as follows:

[0134]

[0135] Add purified water to 1.0 L and adjust the pH to 3.0.

[0136] Example 4

[0137] This example uses the Mindray blood cell analyzer, and the specific implementation process is as follows:

[0138] 1. First, the blood sample to be tested is mixed with the first hemolytic agent (Mindray M-60LD hemolytic agent) and the first fluorescent dye (Mindray M-60FD staining solution) in a reaction pool, and incubated to obtain a first test solution; a portion of the first test solution is taken to a flow chamber, and the blood sample is tested for the first time using a DIFF channel; a second hemolytic agent (prepared in Example 1) and a first fluorescent dye (Mindray M-60FN staining solution) are added to the remaining first test solution in the same reaction pool, and the mixture is incubated to obtain a second test solution; the second test solution is taken to a flow chamber, and the second test is performed using a DIFF channel.

[0139] Use the same method as above to perform detection using the WNB channel.

[0140] Figure 1The flow cytometric scatter plots obtained by using the DIFF channel and the WNB channel to detect samples 1 to 3 in this embodiment according to the above operation are shown. Among them, samples 1 to 3 are nucleated red blood cell simulants, which are close to the position of nucleated red blood cells on the current instrument. Due to the lack of clinical samples of nucleated red blood cells, the simulants are used as a substitute.

[0141] Depend on Figure 1 It can be seen that after adding the second hemolytic agent, FD faded.

[0142] 2. Take normal samples 1 to 3 and nucleated red samples 1 to 3, and test them according to the following method 1 and method 2 respectively.

[0143] Method 1: First, mix the blood sample to be tested with Mindray M-60LN hemolytic agent and Mindray M-60FN staining solution in the reaction pool and incubate to obtain the test solution; draw part of the test solution into the flow chamber and use the WNB channel to detect the blood sample.

[0144] Method 2: First, the blood sample to be tested is mixed with the first hemolytic agent (Mindray M-60LD hemolytic agent) and the first fluorescent dye (Mindray M-60FD staining solution) in a reaction pool, and incubated to obtain a first test solution; a portion of the first test solution is drawn into the flow chamber, and the blood sample is tested for the first time using the WNB channel; the second hemolytic agent (prepared in Example 1) and the first fluorescent dye (Mindray M-60FN staining solution) are added to the remaining first test solution in the same reaction pool, mixed and incubated to obtain a second test solution; the second test solution is drawn into the flow chamber, and the second test is performed using the WNB channel.

[0145] Figure 2 Shown are flow cytometry scatter plots of normal samples 1 to 3 and nucleated red samples 1 to 3 in this embodiment detected according to the above method 1 and method 2, respectively.

[0146] Depend on Figure 2 It can be seen that the FN staining solution plays a leading role, and the cells that faded after being treated with the second hemolytic agent reappear. However, since FD occupies some dye binding sites, the fluorescence (FL) display in the second detection is lower than that of the original WNB test alone, but it has little effect on cell division.

[0147] Tables 1 to 5 below show the WBC, Baso and NRBC test results of normal samples 1 to 3 and nucleated red blood cell samples 1 to 3, as well as detailed parameters of forward projection (FSCP) and fluorescence projection (FLP).

[0148] Combination Figure 1 , Figure 2Tables 1 to 5 further show that the normal samples and the nucleated red samples were tested normally in the WNB channel and re-stained after adding the second hemolytic agent of the present invention. The most important parameter FSCP affecting the division was close, and the FLP was lower than the original WNB display as a whole. This is because the FN staining solution played a leading role, and the cells that faded after being treated with the first hemolytic agent were re-displayed. However, since the FD staining solution occupied some dye binding sites, the FL display in the second test was lower than that of the original WNB test alone, but it had little effect on cell division.

[0149] Table 1 Normal sample WBC test results

[0150]

[0151]

[0152] Table 2 Baso test results of normal samples

[0153]

[0154] Table 3 WBC test results of nucleated red samples

[0155]

[0156] Table 4 Baso test results of nuclear red samples

[0157]

[0158] Table 5 NRBC test results of nucleated red blood cell samples

[0159]

[0160]

[0161] 3. The white blood cells, basophils and nucleated red blood cells in 10 different blood samples were classified and counted using a commercially available kit according to the traditional method (specifically referring to the above method 1) and using the reagent of the present invention according to the method of the present invention (specifically referring to the above method 2) to obtain Figures 3 to 5 .

[0162] according to Figures 3 to 5 The results of correlation analysis of the classification counting results shown show that the correlations between the classification counting results of WBC, Baso, and NRBC obtained by the method of the present invention and the test results obtained by the WNB channel of the traditional method are 0.99412, 0.97300, and 0.99717, respectively, and the correlation coefficient is greater than 0.95, indicating that the test results obtained by using the reagents and methods of the present invention are highly correlated with those obtained by using commercially available kits and traditional methods, which shows that the reagents and methods of the present invention are feasible.

[0163] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A blood sample detection reagent, characterized in that: including a first group of reagents and a second group of reagents; The first set of reagents includes a first hemolytic agent and a first fluorescent dye, wherein the first hemolytic agent is used to dissolve red blood cells and process white blood cells so that the white blood cells can be stained by the first fluorescent dye; The second set of reagents includes a second hemolytic agent and a second fluorescent dye, wherein the second hemolytic agent is used to dissolve nucleated red blood cells and white blood cells other than basophils to a semi-naked nucleus state, and to quench the fluorescence of the first fluorescent dye and / or weaken the fluorescence intensity; The first fluorescent dye is used for staining nucleic acid substances in white blood cells, and the second fluorescent dye is used for differentially staining nucleic acid substances in nucleated red blood cells.

2. The detection reagent according to claim 1, characterized in that Under acidic conditions, the fluorescence quenching intensity and / or fluorescence intensity of the first fluorescent dye is weaker than that of the second fluorescent dye.

3. The detection reagent according to claim 1 or 2, characterized in that The first fluorescent dye and the second fluorescent dye are both cyanine dyes.

4. The detection reagent according to claim 1, characterized in that The second hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt; Alternatively, the second hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt, and also includes at least one of a buffer, a chelating agent, or a preservative.

5. The detection reagent according to claim 4, characterized in that The second hemolytic agent comprises 0.5-1.2 g / L of an osmotic pressure regulator, 0.3-0.6 g / L of a surfactant, and 0.5-3.0 g / L of an organic acid and / or an organic acid salt; Alternatively, the second hemolytic agent includes 0.5-1.2 g / L osmotic pressure regulator, 0.3-0.6 g / L surfactant, 0.5-3.0 g / L organic acid and / or organic acid salt, and also includes at least one of 5-15 mmol / L buffer, 0.05-1.0 g / L chelating agent, and 0.05-0.5 g / L preservative.

6. The detection reagent according to claim 4, characterized in that The osmotic pressure regulator is selected from at least one of sodium chloride, glucose or mannitol; And / or, the surfactant is selected from at least one of a quaternary ammonium salt cationic surfactant or a betaine zwitterionic surfactant; and / or, the organic acid and / or organic acid salt is selected from at least one of citric acid, malic acid, sodium salicylate, maleic acid, tartaric acid, phthalic acid or succinic acid; And / or, the buffer is selected from at least one of a phosphate buffer, a citrate buffer, a 2-morpholineethanesulfonic acid buffer, a glycine-hydrochloric acid buffer, a phthalic acid-hydrochloric acid buffer or an acetic acid-sodium acetate buffer; And / or, the chelating agent is selected from at least one of disodium ethylenediaminetetraacetate or dipotassium ethylenediaminetetraacetate; And / or, the preservative is selected from at least one of the Proclin series, sodium azide, potassium hydrogen phthalate or isothiazolinone.

7. The detection reagent according to any one of claims 1 to 6, characterized in that The pH of the second hemolytic agent is 2.5-4.

0.

8. Use of a hemolytic agent in blood sample detection, characterized in that: The hemolytic agent is used for the second hemolytic treatment of blood cells, and the hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt; Alternatively, the hemolytic agent includes an osmotic pressure regulator, a surfactant, and an organic acid and / or an organic acid salt, and also includes at least one of a buffer, a chelating agent, or a preservative.

9. The use of the hemolytic agent according to claim 8 in blood sample detection, characterized in that: The hemolytic agent includes 0.5-1.2 g / L of osmotic pressure regulator, 0.3-0.6 g / L of surfactant, and 0.5-3.0 g / L of organic acid and / or organic acid salt; Alternatively, the hemolytic agent includes 0.5-1.2 g / L osmotic pressure regulator, 0.3-0.6 g / L surfactant, 0.5-3.0 g / L organic acid and / or organic acid salt, and also includes at least one of 5-15 mmol / L buffer, 0.05-1.0 g / L chelating agent, and 0.05-0.5 g / L preservative.

10. The use of the hemolytic agent according to claim 8 in blood sample detection, characterized in that: The osmotic pressure regulator is selected from at least one of sodium chloride, glucose or mannitol; And / or, the surfactant is selected from at least one of a quaternary ammonium salt cationic surfactant or a betaine zwitterionic surfactant; and / or, the organic acid and / or organic acid salt is selected from at least one of citric acid, malic acid, sodium salicylate, maleic acid, tartaric acid, phthalic acid or succinic acid; And / or, the buffer is selected from at least one of a phosphate buffer, a citrate buffer, a 2-morpholineethanesulfonic acid buffer, a glycine-hydrochloric acid buffer, a phthalic acid-hydrochloric acid buffer or an acetic acid-sodium acetate buffer; And / or, the chelating agent is selected from at least one of disodium ethylenediaminetetraacetate or dipotassium ethylenediaminetetraacetate; And / or, the preservative is selected from at least one of the Proclin series, sodium azide, potassium hydrogen phthalate or isothiazolinone.

11. Use of the hemolytic agent according to any one of claims 8 to 10 in blood sample detection, characterized in that: The pH of the hemolytic agent is 2.5-4.

0.

12. A blood sample detection method, characterized in that: The following steps are involved: (1) mixing the blood sample to be tested with a first hemolytic agent to dissolve the red blood cells in the sample to be tested, and treating the white blood cells so that they can be stained with a first fluorescent dye; (2) mixing the reaction solution of step (1) with a first fluorescent dye to stain the leukocytes to obtain a first test solution, and performing a test on the first test solution to obtain the four-category classification and / or counting results of the leukocytes of the test blood sample; (3) mixing part or all of the first test solution with the second hemolytic agent to dissolve the nucleated red blood cells and the white blood cells other than the basophils to a semi-naked nucleus state, and quenching the fluorescence of the first fluorescent dye and / or weakening the fluorescence intensity; (4) Mixing the reaction solution of step (3) with a second fluorescent dye to differentially stain the nucleic acid substance in the nucleated red blood cells to obtain a second test solution, and testing the second test solution to obtain the classification and / or counting results of the nucleated red blood cells and basophils in the test blood sample.

13. The method according to claim 12, characterized in that Step (4) is to add a second fluorescent dye under acidic conditions.

14. A blood sample analyzer, characterized in that: include: A sampling device, used for drawing a blood sample to be tested; A test solution preparation device, comprising a reaction pool and a reagent supply unit, wherein the reaction pool is used to receive part / all of the blood sample to be tested sucked by the sampling device, and the reagent supply unit is used to provide a first hemolytic agent and a first fluorescent dye to the reaction pool and mix and react with the blood sample to be tested to form a first test solution, and provide a second hemolytic agent and a second fluorescent dye to the reaction pool and mix and react with the first test solution to form a second test solution; An optical detection device having a detection area for detecting a first liquid to be tested and generating a first signal, and detecting a second liquid to be tested and generating a second signal; The processor obtains four classification and / or counting results of white blood cells of the blood sample to be tested based on the first signal, and obtains classification and / or counting results of nucleated red blood cells and basophils of the blood sample to be tested based on the second signal.

15. The analyzer according to claim 14, characterized in that The first liquid to be tested and the second liquid to be tested are both generated in the same reaction pool; Alternatively, the first liquid to be tested and the second liquid to be tested are generated in different reaction pools.

16. The analyzer according to claim 14 or 15, characterized in that The detecting of the first liquid to be tested and generating the first signal and the detecting of the second liquid to be tested and generating the second signal are both performed in the same detection zone.

Images