A blood cell counting detection method
Patent Information
- Application Number
- CN202611102663.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-23
- Publication Date
- 2026-10-27
AI Technical Summary
[0004]本发明提供了一种血液细胞计数检测方法，以解决现有方法无法实现一个视野内白细胞和血小板的检测，难以在同一计数板上一次性完成白细胞、红细胞和血小板三大类血细胞的精确计数，操作繁琐，检测效率低下，从而实现在一个视野内实现WBC、PLT的检测，步骤简单，效率高，且保证红细胞溶解的同时不影响WBC的形态且不影响PLT的数量的问题，最终可实现在同一计数板上一次性完成白细胞、红细胞和血小板三大类血细胞的精确计数
1.本发明提供的一种血液细胞计数检测方法，包括如下步骤：对待测全血样本进行第一次处理，获得第一溶液；取部分第一溶液进行第二次处理，获得第二溶液；取部分第一溶液，加入固定剂处理液和溶血剂处理液，获得第三溶液；将第二溶液注入计数板第一检测腔；将第三溶液注入计数板第二检测腔；分别获取第一检测腔和第二检测腔对应的图像；根据第一检测腔的图像确定待测样本中红细胞计数；根据第二检测腔的图像确定待测样本中白细胞的计数和血小板的计数。在上述方法中，本发明通过对稀释的样本先用固定剂处理液固定，后进行溶血剂处理，固定+溶血联合实现了先对白细胞和血小板固定保护、再选择性溶血红细胞，保证红细胞溶解的同时不影响白细胞的形态且不影响血小板的数量，将固定+溶血处理的样本注入计数板第二检测腔，将未经固定+溶血处理的样本注入计数板的第一检测腔，从而在细胞计数时，根据第一检测腔的图像实现红细胞计数，根据第二检测腔的图像实现在一个视野内血小板和白细胞的同时计数检测，使血小板和白细胞的计数在同一个检测窗中完成，且在图像识别中显著提升白细胞与血小板的轮廓清晰度和计数准确性，避免红细胞残留干扰，操作简单，极大的节省时间，效率高，最终可实现在同一计数板上的二个检测窗即可一次性完成白细胞、红细胞和血小板三大类血细胞的精确计数。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of blood cell counting detection, and more specifically to a method for blood cell counting detection. Background Technology
[0002] Traditional blood routine tests are represented by microscopic examination. This method can not only directly identify abnormal cell morphology, but is also unaffected by sample abnormalities such as hyperlipidemia, hemolysis, and cold agglutination. It can also correct false results from automated instruments, such as false thrombocytopenia caused by platelet aggregation. Furthermore, this method only requires a regular microscope, making it suitable for the needs of primary healthcare institutions. As the gold standard for blood cell morphology analysis, manual microscopic examination has irreplaceable value in clinical diagnosis.
[0003] However, due to limitations in the methodology itself and differences in cell concentration, this method cannot accurately count the three major types of blood cells—white blood cells (WBC), red blood cells (RBC), and platelets (PLT)—on the same counting plate at once. The operation process is cumbersome and the detection efficiency is low. Summary of the Invention
[0004] This invention provides a blood cell counting method to address the limitations of existing methods, which cannot detect white blood cells and platelets within a single field of view. These methods are difficult to perform accurate counting of the three major blood cell types (white blood cells, red blood cells, and platelets) simultaneously on a single counting plate, resulting in cumbersome operations and low detection efficiency. This invention enables the detection of WBCs and PLTs within a single field of view. The procedure is simple, efficient, and ensures that red blood cell lysis does not affect the morphology of WBCs or the number of PLTs. Ultimately, it allows for accurate counting of the three major blood cell types (white blood cells, red blood cells, and platelets) simultaneously on a single counting plate.
[0005] In a first aspect, the present invention provides a method for detecting blood cell counts, comprising the following steps: The whole blood sample to be tested is processed for the first time to obtain the first solution; A portion of the first solution is subjected to a second treatment to obtain the second solution; Take a portion of the first solution and add fixative treatment solution and hemolytic agent treatment solution to obtain the third solution; The second solution is injected into the first detection chamber of the counting plate; The third solution is injected into the second detection chamber of the counting plate; The images corresponding to the first detection cavity and the second detection cavity are acquired respectively; The red blood cell count in the sample to be tested is determined based on the image from the first detection chamber. The white blood cell count and platelet count in the sample to be tested are determined based on the image from the second detection chamber.
[0006] In some alternative implementations, the first treatment includes: mixing the whole blood sample to be tested with a diluent for a first-order dilution.
[0007] In some optional embodiments, in the first dilution, the volume ratio of the whole blood sample to be tested and the diluent is 1:5 to 1:10, such as any one of the values of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range between two values. In a preferred embodiment, the volume ratio is 1:6.
[0008] In some alternative embodiments, the second process includes: mixing the first solution and the diluent to perform a second dilution; In some optional embodiments, in the second dilution, the volume ratio of the first solution to the diluent is 1:40 to 1:120, such that the volume ratio can be any one of 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120 or any range between two values.
[0009] In some alternative embodiments, the diluent comprises components at the following concentrations: The concentration of NaCl is 1-8 g / L, the concentration of EDTA is 0.05-0.3 g / L, the concentration of preservative is 0.5-1 g / L, the concentration of buffer solution is 10-100 mM, and the pH is adjusted to 7.45-7.55.
[0010] In some alternative embodiments, the preservative is selected from at least one of PC300, PC950, sodium azide, and thimerosal; In some alternative implementations, the buffer solution is selected from at least one of PB, Tris, MOPS, and HEPES.
[0011] In some optional embodiments, the preparation of the third solution includes: taking a portion of the first solution, first adding a fixative treatment solution for fixation, and then adding a hemolytic agent treatment solution for hemolysis after the fixation is completed.
[0012] In some optional embodiments, the volume ratio of the first solution to the fixative treatment solution is 2:1 to 3:1.
[0013] In some optional embodiments, the fixation conditions are: 37°C, fixation time 30s~90s, and then the hemolytic agent is added.
[0014] In some optional embodiments, the volume ratio of the mixture of the first solution and the fixative treatment solution to the hemolytic agent treatment solution is 3:1 to 5:1.
[0015] In some alternative embodiments, the hemolysis conditions are: 37°C, and homogenization.
[0016] In some alternative embodiments, the fixative treatment solution is a solution containing a fixative, which is a substance with cell fixation properties.
[0017] In some alternative embodiments, the fixative is selected from any one of glutaraldehyde, formaldehyde, paraformaldehyde, methanol, and ethanol.
[0018] In some optional embodiments, the concentration of the fixative in the fixative treatment solution is 1-8 g / L, such that the concentration of the fixative can be any one of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or a range between any two values.
[0019] In some alternative embodiments, the fixative treatment solution also contains at least one of a preservative and a pH adjuster.
[0020] In some alternative embodiments, the preservative is selected from any one of PC300, PC950, sodium azide, and thimerosal.
[0021] In some alternative embodiments, the pH adjuster is selected from any one of KH2PO4, sodium citrate, glycine, and potassium hydrogen phthalate.
[0022] In some optional embodiments, the concentration of the preservative in the fixative treatment solution is 0.5-1 g / L; and / or, the concentration of the pH adjuster is 5-20 mM, adjusting the pH to 2.0-4.0.
[0023] In some alternative embodiments, the hemolytic agent treatment solution is a solution containing a hemolytic agent, which is a substance with cell hemolytic activity.
[0024] In some alternative embodiments, the hemolytic agent is selected from any one of sodium dodecyl sulfate, saponin, Triton 100, and DTAC; In some optional embodiments, the concentration of the hemolytic agent in the hemolytic treatment solution is 2.5-8 g / L, such that the concentration of the hemolytic agent can be any one of 2.5 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, or 8 g / L, or a range between any two values.
[0025] In some alternative embodiments, the hemolytic agent treatment solution also contains at least one of a dye, a preservative, and a pH adjuster.
[0026] In some alternative embodiments, the dye is selected from any one of neomethylene blue, methylene blue, crystal violet, and Nile blue; In some alternative embodiments, the preservative is selected from any one of PC300, PC950, sodium azide, and thimerosal; In some alternative embodiments, the pH adjuster is selected from any one of PB, Tris, MOPS, and HEPES; In some optional embodiments, the dye concentration in the hemolytic agent treatment solution is 0.2-0.6 g / L; and / or, the preservative concentration is 0.5-1 g / L; and / or, the pH adjuster concentration is 50-100 mM, adjusting the pH to 6.0-7.5.
[0027] The technical solution of this invention has the following advantages: 1. The present invention provides a blood cell counting detection method, comprising the following steps: performing a first processing on a whole blood sample to be tested to obtain a first solution; taking a portion of the first solution for a second processing to obtain a second solution; taking a portion of the first solution and adding a fixative treatment solution and a hemolysin treatment solution to obtain a third solution; injecting the second solution into the first detection chamber of a counting chamber; injecting the third solution into the second detection chamber of a counting chamber; acquiring images corresponding to the first and second detection chambers respectively; determining the red blood cell count in the sample to be tested based on the image of the first detection chamber; and determining the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber. In the above method, the present invention first fixes the diluted sample with a fixative solution, and then treats it with a hemolytic agent. The combination of fixation and hemolysis achieves the first fixation and protection of white blood cells and platelets, and then selective hemolysis of red blood cells. This ensures that the morphology of white blood cells and the number of platelets are not affected while the red blood cells are dissolved. The fixed and hemolyzed sample is injected into the second detection chamber of the counting plate, and the untreated sample is injected into the first detection chamber of the counting plate. Thus, during cell counting, red blood cells are counted based on the image of the first detection chamber, and platelets and white blood cells are counted and detected simultaneously in one field of view based on the image of the second detection chamber. This allows the counting of platelets and white blood cells to be completed in the same detection window, and significantly improves the clarity of the outline of white blood cells and platelets and the accuracy of counting in image recognition. It avoids interference from residual red blood cells, is simple to operate, saves a lot of time, and is highly efficient. Ultimately, it can achieve accurate counting of the three major blood cell types—white blood cells, red blood cells, and platelets—in one go using only two detection windows on the same counting plate. Detailed Implementation
[0028] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.
[0029] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0030] HEPES, or 4-hydroxyethylpiperazine ethanesulfonic acid, is a Chinese name for this substance. MOPS stands for 3-morpholinopropanesulfonic acid. The Chinese name for Tris is tris(hydroxymethyl)aminomethane; PB stands for Phosphate Buffer. EDTA's Chinese name is ethylenediaminetetraacetic acid; The Chinese name for DTAC is dodecyltrimethylammonium chloride.
[0031] Example 1 The diluent, hemolytic agent, and fixative are formulated as shown in Table 1 below: Table 1. Formula
[0032] According to the formula in Table 1 above, weigh the materials and dissolve them in deionized water to prepare the solution. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolytic agent treatment solution and fixative treatment solution respectively.
[0033] This embodiment provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 150 μL of diluent for first-order dilution to obtain the first solution.
[0034] (2) Take 15 μL of the first solution and 600 μL of the diluent and mix them for a second dilution to obtain the second solution.
[0035] (3) Take 53 μL of the first solution and 20 μL of the fixative treatment solution and mix them for fixation. The fixation conditions are 37℃ and the fixation time is 90s. After the fixation is completed, add 20 μL of the hemolysin treatment solution and mix them evenly at 37℃ to obtain the third solution.
[0036] (4) Inject 20 μL of the second solution into the first detection chamber of the counting plate and 20 μL of the third solution into the second detection chamber of the counting plate. Adjust the microscope to focus and take pictures to obtain the images corresponding to the first and second detection chambers respectively. Determine the red blood cell count in the sample to be tested based on the image of the first detection chamber. Determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0037] Example 2 The diluent, hemolytic agent, and fixative are formulated as shown in Table 2 below: Table 2, Formula
[0038] According to the formula in Table 2 above, weigh the materials and dissolve them in deionized water for preparation. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolysin treatment solution and fixative treatment solution respectively.
[0039] This embodiment provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 150 μL of diluent for first-order dilution to obtain the first solution.
[0040] (2) Take 15 μL of the first solution and 600 μL of the diluent and mix them for a second dilution to obtain the second solution.
[0041] (3) Take 53 μL of the first solution and 20 μL of the fixative treatment solution and mix them for fixation. The fixation conditions are 37℃ and the fixation time is 90s. After the fixation is completed, add 20 μL of the hemolysin treatment solution and mix them evenly at 37℃ to obtain the third solution.
[0042] (4) Inject 20 μL of the second solution into the first detection chamber of the counting plate and 20 μL of the third solution into the second detection chamber of the counting plate. Adjust the microscope to focus and take pictures to obtain the images corresponding to the first and second detection chambers respectively. Determine the red blood cell count in the sample to be tested based on the image of the first detection chamber. Determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0043] Example 3 The diluent, hemolytic agent, and fixative are formulated as shown in Table 3 below: Table 3, Formula
[0044] According to the formula in Table 3 above, weigh the materials and dissolve them in deionized water for preparation. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolysin treatment solution and fixative treatment solution respectively.
[0045] This embodiment provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 150 μL of diluent for first-order dilution to obtain the first solution.
[0046] (2) Take 15 μL of the first solution and 600 μL of the diluent and mix them for a second dilution to obtain the second solution.
[0047] (3) Take 53 μL of the first solution and 20 μL of the fixative treatment solution and mix them for fixation. The fixation conditions are 37℃ and the fixation time is 90s. After the fixation is completed, add 20 μL of the hemolysin treatment solution and mix them evenly at 37℃ to obtain the third solution.
[0048] (4) Inject 20 μL of the second solution into the first detection chamber of the counting plate and 20 μL of the third solution into the second detection chamber of the counting plate. Adjust the microscope to focus and take pictures to obtain the images corresponding to the first and second detection chambers respectively. Determine the red blood cell count in the sample to be tested based on the image of the first detection chamber. Determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0049] Example 4 The diluent, hemolytic agent, and fixative are formulated as shown in Table 4 below: Table 4, Formula
[0050] According to the formula in Table 4 above, weigh the materials and dissolve them in deionized water for preparation. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolytic agent treatment solution and fixative treatment solution respectively.
[0051] This embodiment provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 250 μL of diluent for first-order dilution to obtain the first solution.
[0052] (2) Take 15 μL of the first solution and 900 μL of the diluent and mix them for a second dilution to obtain the second solution.
[0053] (3) Take 60 μL of the first solution and 30 μL of the fixative treatment solution and mix them for fixation. The fixation conditions are 37℃ and the fixation time is 30s. After the fixation is completed, add 30 μL of the hemolysin treatment solution and mix them evenly at 37℃ to obtain the third solution.
[0054] (4) Inject 20 μL of the second solution into the first detection chamber of the counting plate and 20 μL of the third solution into the second detection chamber of the counting plate. Adjust the microscope to focus and take pictures to obtain the images corresponding to the first and second detection chambers respectively. Determine the red blood cell count in the sample to be tested based on the image of the first detection chamber. Determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0055] Example 5 The diluent, hemolytic agent, and fixative are formulated as shown in Table 5 below: Table 5. Formula
[0056] According to the formula in Table 5 above, weigh the materials and dissolve them in deionized water for preparation. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolysin treatment solution and fixative treatment solution respectively.
[0057] This embodiment provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 125 μL of diluent for first-order dilution to obtain the first solution.
[0058] (2) Take 15 μL of the first solution and 1800 μL of the diluent and mix them for a second dilution to obtain the second solution.
[0059] (3) Take 60 μL of the first solution and 20 μL of the fixative treatment solution and mix them for fixation. The fixation conditions are 37℃ and the fixation time is 30s. After the fixation is completed, add 16 μL of the hemolytic agent treatment solution and mix them evenly at 37℃ to obtain the third solution.
[0060] (4) Inject the second solution into the first detection chamber of the counting plate, inject the third solution into the second detection chamber of the counting plate, adjust the microscope to focus and take pictures, and obtain the images corresponding to the first detection chamber and the second detection chamber respectively; determine the red blood cell count in the sample to be tested based on the image of the first detection chamber; determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0061] Comparative Example 1 The diluent, hemolytic agent, and fixative are formulated as shown in Table 6 below: Table 6. Formula
[0062] According to the formula in Table 6 above, weigh the materials and dissolve them in deionized water for preparation. Adjust the pH value to the target pH by adding HCl or NaOH to obtain the diluent, hemolysin treatment solution and fixative treatment solution respectively.
[0063] This comparative example provides a method for detecting blood cell counts, including the following steps: (1) Mix 25 μL of the whole blood sample to be tested with 125 μL of diluent for first-order dilution to obtain the first solution.
[0064] (2) Mix 20 μL of the first solution and 150 μL of the diluent for a second dilution to obtain the second solution.
[0065] (3) Take 20 μL of the second solution and 150 μL of the diluent and mix them to perform a third dilution to obtain the third solution. (4) Take a certain amount of the first solution 20 μL and mix it with 30 μL of leukocyte hemolytic agent to perform hemolysis and staining, and obtain the fourth solution.
[0066] (5) Take a certain amount of the first solution (20 μL) and mix it with 200 μL of platelet hemolytic agent to perform hemolysis, and obtain the fifth solution; (6) Inject 20 μL of the third solution into the first detection chamber of the counting plate, inject 20 μL of the fourth solution into the second detection chamber of the counting plate, and inject 20 μL of the fifth solution into the third detection chamber of the counting plate. Adjust the microscope to focus and take pictures to obtain the images corresponding to the first detection chamber, the second detection chamber, and the third detection chamber respectively. Determine the red blood cell count in the sample to be tested based on the image of the first detection chamber. Determine the white blood cell count and platelet count in the sample to be tested based on the image of the second detection chamber.
[0067] Experimental Example 1 The processing times of Example 1 and Comparative Example 1 are compared, and the results are shown in Table 7 below: Table 7
[0068] As can be seen from the comparison in the table above, the method of Embodiment 1 of this application saves 214 seconds of total time compared with Comparative Example 1, which greatly improves efficiency.
[0069] Experiment Example 2 Repeatability This experiment examined the reproducibility of Examples 1-5 and Comparative Example 1. The method was as follows: Four whole blood samples were obtained and tested according to Examples 1-5 and Comparative Example 1. Each sample was tested 10 times, and the coefficient of variation (CV) for each example was calculated using the following formula: ; σ: Standard Deviation, representing the magnitude of absolute fluctuation in the data.
[0070] μ: Mean, representing the average level of the data.
[0071] The results are shown in Table 8 below: Table 8. Repeatability
[0072] As shown in Table 8 above, compared with Comparative Example 1, the repeatability CV of WBC in Examples 1-5 of this application is <6%, and the repeatability CV of PLT is <10%, indicating that the repeatability of this application meets the requirements.
[0073] Experimental Example 3: Linear This experiment investigated the linearity of Examples 1-5 and Comparative Example 1. The method was as follows: one sample was prepared at each of the upper and lower limits of the linear range. Six linearity verification samples were prepared according to a certain dilution ratio. The theoretical concentrations of WBC in the samples were 0.05 × 10⁻⁶. 9 / L, 20.40×10 9 / L, 40.3×10 9 / L, 60.2×10 9 / L, 80.1×10 9 / L, 100×10 9 / L, the theoretical concentration of PLT in the sample is 20×10 9 / L、216×10 9 / L、412×10 9 / L、608×10 9 / L, 804×10 9 / L, 1000×10 9 For each linear validation sample, the test was performed three times, and the mean of the test results was calculated. A linear regression was then performed with dilution on the X-axis and the mean of the measured results on the Y-axis to obtain the correlation coefficient r. The relative deviation between the measured results and the theoretical values was also calculated. The results are shown in Table 9 below. Table 9. Linear
[0074] As shown in Table 9 above, the linearity of Examples 1-5 of this application is greater than that of Comparative Example 1, all satisfying the linearity requirement of r>0.99 within the linear range, indicating that this application can meet the linearity requirements of blood routine tests.
[0075] Experiment Example 4 Stability This experiment investigated the stability of Examples 1-5 and Comparative Example 1. The method was as follows: the reagent was placed at 55℃ for 15 days, and the deviation of the test results of the reagent stored at 2-8℃ was compared with that of the reagent stored at 2-8℃ ((test mean - reference value) / reference value × 100%, where the reference value is generally the set value of the reference system (a commercially available and recognized good product)). The results are shown in Tables 10-11 below: Table 10. Results of WBC in each group
[0076] Table 11. Results of PLT in each group
[0077] As can be seen from Tables 10-11 above, compared with Comparative Example 1, and compared with 2-8℃, Examples 1-5 of this application all meet the stability requirements of WBC deviation within ±15% and PLT deviation within ±20%.
[0078] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for detecting blood cell counts, characterized in that, Includes the following steps: The whole blood sample to be tested is processed for the first time to obtain the first solution; A portion of the first solution is subjected to a second treatment to obtain the second solution; Take a portion of the first solution and add fixative treatment solution and hemolytic agent treatment solution to obtain the third solution; The second solution is injected into the first detection chamber of the counting plate; The third solution is injected into the second detection chamber of the counting plate; The images corresponding to the first detection cavity and the second detection cavity are acquired respectively; The red blood cell count in the sample to be tested is determined based on the image from the first detection chamber. The white blood cell count and platelet count in the sample to be tested are determined based on the image from the second detection chamber.
2. The blood cell counting detection method according to claim 1, characterized in that, The first treatment includes: mixing the whole blood sample to be tested with the diluent for first-order dilution; Optionally, the volume ratio of the whole blood sample to be tested to the diluent is 1:5 to 1:
10.
3. The blood cell counting detection method according to claim 1 or 2, characterized in that, The second process includes: mixing the first solution and the diluent to perform a second-stage dilution; Optionally, the volume ratio of the first solution to the diluent is 1:40 to 1:
120.
4. The blood cell counting detection method according to claim 2 or 3, characterized in that, The diluent comprises components of the following concentrations: The concentration of NaCl is 1-8 g / L, the concentration of EDTA is 0.05-0.3 g / L, the concentration of preservative is 0.5-1 g / L, the concentration of buffer solution is 10-100 mM, and the pH is adjusted to 7.45-7.
55. Optionally, the preservative is selected from at least one of PC300, PC950, sodium azide, and thimerosal; Optionally, the buffer solution is selected from at least one of PB, Tris, MOPS, and HEPES.
5. The blood cell counting method according to any one of claims 1-4, characterized in that, The preparation of the third solution includes: taking a portion of the first solution, first adding a fixative treatment solution for fixation, and then adding a hemolytic agent treatment solution for hemolysis after the fixation is completed.
6. The blood cell counting detection method according to claim 5, characterized in that, The volume ratio of the first solution to the fixative treatment solution is 2:1 to 3:1; And / or, the fixation conditions are: 37°C, fixation time 30s~90s, and hemolytic agent is added after fixation; And / or, the volume ratio of the mixture of the first solution and the fixative treatment solution to the hemolytic agent treatment solution is 3:1 to 5:1; And / or, the conditions for hemolysis are: 37°C, and thorough mixing.
7. The blood cell counting method according to any one of claims 1-6, characterized in that, The fixative treatment solution is a solution containing a fixative, which is a substance with cell fixation properties; Optionally, the fixative is selected from any one of glutaraldehyde, formaldehyde, paraformaldehyde, methanol, and ethanol; Optionally, the concentration of the fixative in the fixative treatment solution is 1-8 g / L.
8. The blood cell counting detection method according to claim 7, characterized in that, The fixative treatment solution also contains at least one of a preservative and a pH adjuster; Optionally, the preservative is selected from any one of PC300, PC950, sodium azide, and thimerosal; Optionally, the pH adjuster is selected from any one of KH2PO4, sodium citrate, glycine, and potassium hydrogen phthalate; Optionally, the concentration of the preservative in the fixative treatment solution is 0.5-1 g / L; and / or the concentration of the pH adjuster is 5-20 mM, adjusting the pH to 2.0-4.
0.
9. The blood cell counting method according to any one of claims 1-8, characterized in that, The hemolytic agent treatment solution is a solution containing a hemolytic agent, which is a substance with cell hemolytic activity; Optionally, the hemolytic agent is selected from any one of sodium dodecyl sulfate, saponin, Triton 100, and DTAC; Optionally, the concentration of the hemolytic agent in the hemolytic treatment solution is 2.5-8 g / L.
10. The blood cell counting detection method according to claim 9, characterized in that, The hemolytic agent treatment solution also contains at least one of dye, preservative and pH adjuster; Optionally, the dye is selected from any one of neomethylene blue, methylene blue, crystal violet, and Nile blue; Optionally, the preservative is selected from any one of PC300, PC950, sodium azide, and thimerosal; Optionally, the pH adjuster is selected from any one of PB, Tris, MOPS, and HEPES; Optionally, in the hemolytic agent treatment solution, the dye concentration is 0.2-0.6 g / L; and / or, the preservative concentration is 0.5-1 g / L; and / or, the pH adjuster concentration is 50-100 mM, adjusting the pH to 6.0-7.5.