Absolute counting tube containing various absolute counting microspheres and application thereof
By using two independent absolute counting microspheres in a flow cytometer for cell counting and averaging the results, the bias problem of single microsphere counting was solved, resulting in more stable and accurate cell counting results while reducing production costs.
Patent Information
- Application Number
- CN202511118104.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-11-14
AI Technical Summary
In existing technologies, when using single microspheres for cell counting, especially in samples with a very small number of target cells, the detection results are prone to deviation, affecting the accuracy of the results.
Two different absolute counting microspheres were used to completely distinguish them in flow cytometry and detected independently on different fluorescence channels. The average of the results from the two microspheres was taken as the final counting result.
It improves the stability and accuracy of test results, reduces deviations, simplifies operation procedures, and lowers production costs.
Smart Images
Figure CN120948329A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell detection and counting analysis technology, and in particular to an absolute counting tube containing multiple absolute counting microspheres and its applications. Background Technology
[0002] The microsphere method is the earliest method used in flow cytometry for absolute counting and is now widely used and accepted. Absolute counting microspheres can be used to calculate the absolute number of cells in a complex, mixed cell population (such as a blood sample).
[0003] Absolute counting microspheres are microspheres of known quantity or concentration that emit bright fluorescence over a wide range of excitation and emission wavelengths. In the preparation of the target cell suspension sample, the sample is first added directly to an absolute counting tube containing a known absolute number of fluorescent reference microspheres, or the sample and antibody for labeling the target cells are first added to a flow cytometer. After antibody labeling, an equal volume and a known absolute number of fluorescent reference microspheres are added to the target cell suspension sample. The cell suspension and fluorescent reference microspheres are then mixed and analyzed by flow cytometry, while the counting results of the target cells and fluorescent reference microspheres are recorded simultaneously. Because there is a certain proportional relationship between the two, the absolute count value of the target cells can be calculated from the counting results and absolute number of the fluorescent reference microspheres. The calculation formula is:
[0004]
[0005] Different cells exhibit abnormal changes in certain diseases such as immunodeficiency diseases, allergic diseases, viral infections, and malignant tumors. Detecting the absolute number of different cell populations is of great significance for controlling the occurrence and development of these diseases, understanding the disease mechanisms, and guiding clinical treatment.
[0006] Current conventional absolute counting methods use only one type of microsphere, which can lead to biased results, especially in samples with very few target cells, causing significant deviations and affecting the accuracy of the detection results. This invention provides a method for simultaneously counting the same sample using multiple microspheres, with the results collectively used in statistical analysis. Compared to using a single microsphere, this method results in smaller deviations, higher accuracy, and more stable results. Summary of the Invention
[0007] The purpose of this invention is to provide an absolute counting tube containing multiple absolute counting microspheres and its application. It uses two different absolute counting microspheres to count the same sample simultaneously. The two types of microspheres can be completely distinguished in flow cytometry detection, and they are completely independent and do not overlap in different fluorescence channels, so as not to interfere with each other. All detection results can be obtained simultaneously through one test of a sample, and the average of the two results is taken as the final absolute counting result.
[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0009] This invention provides a method for preparing an absolute counting tube containing multiple absolute counting microspheres, comprising the following steps:
[0010] (1) Mix disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100 and phosphate buffer to obtain a dispersion buffer;
[0011] (2) Mix absolute count microspheres I and absolute count microspheres II with dispersion buffer to obtain microsphere solution I and microsphere solution II respectively. Mix microsphere solution I and microsphere solution II in equal proportions to obtain microsphere mixed solution;
[0012] (3) Dispense the microsphere mixture into a flow cytometer, ensuring that the microsphere mixture is completely added to the bottom of the flow cytometer during dispensing, and then perform vacuum drying to obtain the absolute counting tube containing multiple absolute counting microspheres.
[0013] Preferably, in step (1), the mass-to-volume ratio of disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100, and phosphate buffer is 0.2–1.0 g: 0.2–1.0 g: 0.2–1.0 mL: 80–120 mL;
[0014] The concentration of the phosphate buffer solution is 0.005–0.015 mol / L.
[0015] Preferably, in step (2), the concentration of microsphere solution I is 1800-2200 particles / μL, and the concentration of microsphere solution II is 1800-2200 particles / μL; the two microsphere solutions have the same concentration and are mixed in equal proportions.
[0016] Preferably, the vacuum degree of vacuum drying in step (3) is <10 Pa;
[0017] The vacuum drying temperature is 20–35°C;
[0018] The vacuum drying time is 12 to 24 hours.
[0019] The present invention also provides a method for preparing an absolute counting tube containing multiple absolute counting microspheres.
[0020] This invention also provides an application of an absolute counting tube containing multiple absolute counting microspheres in detecting the absolute number of cell subpopulations.
[0021] The present invention has the following beneficial effects:
[0022] 1. This invention employs a method of counting multiple microspheres simultaneously, generating multiple results for a single sample, with the final result being the average. This method yields more stable, less biased, and more accurate results compared to the traditional method of counting only one type of microsphere.
[0023] 2. This invention simultaneously prepares corresponding absolute counting tubes containing multiple microspheres, avoiding the tediousness of repeated experiments and the waste of samples and reagents when using different counting microspheres, making the operation simpler. Compared with traditional absolute counting tubes with a single microsphere, the same operating steps can yield more stable and accurate results.
[0024] 3. The absolute counting tube in this invention does not require freeze-drying technology, but is produced using vacuum drying. Compared with traditional freeze-drying, vacuum drying only requires determining the temperature, vacuum degree and drying time, eliminating the need to explore the conditions for product pre-freezing, sublimation and desorption, which greatly shortens the product production cycle. Moreover, the cost of vacuum drying equipment is lower than that of freeze drying, which has a greater economic advantage. Attached Figure Description
[0025] Figure 1 This is a clustering diagram of two types of microspheres detected by flow cytometry using an absolute counter tube in Example 1.
[0026] Figure 2 This is a clustering diagram of two types of microspheres in the FITC and PE channels for absolute counting tube flow cytometry analysis in Example 1.
[0027] Figure 3 This is a diagram of CD3 cell populations stained with the T lymphocyte subset detection reagent (CD45 / CD3 / CD4 / CD8 flow cytometry antibody) of Example 1.
[0028] Figure 4 This is a diagram of CD3+CD4+ cell clusters stained with the T lymphocyte subset detection reagent (CD45 / CD3 / CD4 / CD8 flow cytometry antibody) of Example 1.
[0029] Figure 5 This is a diagram of CD3+CD8+ cell clusters stained with the T lymphocyte subset detection reagent (CD45 / CD3 / CD4 / CD8 flow cytometry antibody) of Example 1. Detailed Implementation
[0030] This invention provides a method for preparing an absolute counting tube containing multiple absolute counting microspheres, comprising the following steps:
[0031] (1) Mix disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100 and phosphate buffer to obtain a dispersion buffer;
[0032] (2) Mix absolute count microspheres I and absolute count microspheres II with dispersion buffer to obtain microsphere solution I and microsphere solution II respectively. Mix microsphere solution I and microsphere solution II in equal proportions to obtain microsphere mixed solution;
[0033] (3) Dispense the microsphere mixture into a flow cytometer, ensuring that the microsphere mixture is completely added to the bottom of the flow cytometer during dispensing, and then perform vacuum drying to obtain the absolute counting tube containing multiple absolute counting microspheres.
[0034] In this invention, the preferred mass-to-volume ratio of disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100, and phosphate buffer in step (1) is 0.2–1.0 g: 0.2–1.0 g: 0.2–1.0 mL: 80–120 mL, more preferably 0.3–0.8 g: 0.3–0.8 g: 0.3–0.8 mL: 85–115 mL, and even more preferably 0.4–0.6 g: 0.4–0.6 g: 0.4–0.6 mL: 90–110 mL.
[0035] In this invention, the concentration of the phosphate buffer solution is preferably 0.005 to 0.015 mol / L, more preferably 0.007 to 0.013 mol / L, and even more preferably 0.008 to 0.012 mol / L.
[0036] In this invention, the mixing process requires thorough stirring to dissolve the contents, followed by filtration to obtain a dispersion buffer.
[0037] In this invention, sodium dodecyl sulfate and Triton X-100 are surfactants that can prevent the aggregation of microspheres; disodium ethylenediaminetetraacetate, as an antioxidant, can make the reagent more stable.
[0038] In this invention, the concentration of microsphere solution I in step (2) is preferably 1800-2200 particles / μL, more preferably 1850-2150 particles / μL, and even more preferably 1900-2100 particles / μL.
[0039] In this invention, the concentration of the microsphere solution II is preferably 1800-2200 particles / μL, more preferably 1850-2150 particles / μL, and even more preferably 1900-2100 particles / μL.
[0040] In this invention, the vacuum degree of vacuum drying in step (3) is preferably <10 Pa.
[0041] In this invention, the vacuum drying temperature is preferably 20-35°C, more preferably 22-34°C, and even more preferably 25-32°C.
[0042] In this invention, the vacuum drying time is preferably 12-24 hours, more preferably 14-22 hours, and even more preferably 16-20 hours.
[0043] In this invention, during the dispensing process in step (3), ensure that the microsphere solution is completely added to the bottom of the flow cytometer and not added to the tube wall. After vacuum drying, remove the flow cytometer, put on the matching cap, and store it at room temperature away from light to obtain an absolute counting tube containing two types of absolute counting microspheres. The absolute counting microspheres after vacuum drying are firmly attached to the bottom of the flow cytometer and are not easy to fall off.
[0044] The present invention also provides a method for preparing an absolute counting tube containing multiple absolute counting microspheres.
[0045] This invention also provides an application of an absolute counting tube containing multiple absolute counting microspheres in detecting the absolute number of cell subpopulations.
[0046] In this invention, taking T lymphocyte subsets as an example, the method for detecting the absolute number of cell subsets includes the following steps:
[0047] (1.1) Draw a blood sample and add it into the absolute counting tube containing multiple absolute counting microspheres, avoiding blood from coming into contact with the upper part of the tube wall;
[0048] (1.2) Add the T lymphocyte subset detection reagent to the tube in step (1.1), mix well, and react in the dark;
[0049] (1.3) Add the hemolysin used for flow cytometer to the tube in step (1.2), mix well and react in the dark. After the reaction is complete, perform the instrument detection.
[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0051] Example 1
[0052] Mix 0.5 g of disodium ethylenediaminetetraacetate, 0.5 g of sodium dodecyl sulfate, 0.5 mL of Triton X-100, and 100 mL of 0.01 mol / L phosphate buffer. After thorough stirring and dissolution, filter the solution through a 0.22 μm pore membrane and store at 2–8 °C to obtain a dispersion buffer. Take absolute count microspheres I and II, and dilute them separately with the above dispersion buffer to a concentration of 2000 particles / μL. Then mix them in equal proportions and thoroughly to obtain a microsphere mixed solution.
[0053] The microsphere mixture was thoroughly shaken and mixed. 50 μL / pipette was then dispensed into 12*75 mm flow cytometry tubes, ensuring the solution reached the bottom and not the tube walls. Vacuum dryer setup: The machine was run to 30°C. The flow cytometry tubes containing the microsphere solution were placed inside. A vacuum was created (vacuum degree < 10 Pa). After 20 hours of vacuum drying, the pressure inside the dryer was restored to atmospheric pressure. The flow cytometry tubes were removed, capped, and stored at room temperature away from light. This yielded absolute counting tubes containing two types of absolute counting microspheres. The vacuum-dried absolute counting microspheres adhered firmly to the bottom of the flow cytometry tube and were not easily detached.
[0054] The absolute counting tube obtained in this embodiment is used to detect the absolute number of T lymphocyte subsets in a blood sample. The specific detection steps are as follows:
[0055] Using reverse pipetting, 50 μL of well-mixed blood sample was aspirated and added to the absolute counting tube prepared in this embodiment, avoiding contact between the blood and the upper part of the tube wall. Then, T lymphocyte subset detection reagent (CD45 / CD3 / CD4 / CD8 flow cytometry antibody) was added to the tube, and the mixture was vortexed for 3 seconds. The mixture was then incubated at 25°C in the dark for 30 minutes. After the reaction, 450 μL of hemolysin for flow cytometry was added to the tube, and the mixture was vortexed for 15 seconds. The mixture was then incubated at room temperature in the dark for 10 minutes. After the reaction, the sample was analyzed by the flow cytometer.
[0056] Results Analysis and Data Statistics
[0057] In this embodiment, the flow cytometer detected the following... Figure 1 or Figure 2 The flow cytometry diagram shown has microspheres I and II circled in the gates (the number of microspheres acquired in each gate is A and B, respectively). It can be seen that the two types of microspheres are clearly clustered in each fluorescence channel and do not interfere with each other.
[0058] 2.1 Calculate the absolute number of CD3+ T lymphocytes
[0059] Figure 3 The CD3 cell clustering diagram shows that CD3+ cells are prominent, and the number of cells obtained within the phylum is C. Therefore, the absolute CD3+ cell count calculated using microsphere I is C*50000 / A*50; the absolute CD3+ cell count calculated using microsphere II is C*50000 / B*5. The detection was repeated 10 times, and the calculation results are shown in Table 1.
[0060] Table 1. Absolute number of T lymphocytes and CD3+ cells
[0061] Number of tests Microsphere I Microsphere II Average values of the two types of microspheres 1 987 1021 1004 2 939 1073 1006 3 1034 1024 1029 4 1044 1045 1045 5 1026 1137 1081 6 982 1059 1020 7 869 1050 960 8 965 1035 1000 9 1069 1070 1069 10 977 1247 1112 AV 989 1076 1033 SD 62 69 45 CV 6％ 6％ 4％
[0062] 2.2 Calculate the absolute number of T lymphocyte subsets CD3+CD4+ cells
[0063] Figure 4 The diagram shows the CD3+CD4+ cell clustering. The number of CD3+CD4+ cells within each phylum is D. Therefore, the absolute CD3+CD4+ cell count calculated using microsphere I is D*50000 / A*50; the absolute CD3+CD4+ cell count calculated using microsphere II is D*50000 / B*5. The detection was repeated 10 times, and the results are shown in Table 2.
[0064] Table 2. Absolute number of T lymphocytes (CD3+CD4+ cells)
[0065] Number of tests Microsphere I Microsphere II Average values of the two types of microspheres 1 591 643 617 2 610 673 642 3 700 623 661 4 650 672 661 5 637 701 669 6 684 686 685 7 656 681 668 8 698 688 693 9 644 663 654 10 592 784 688 AV 646 681 664 SD 40 43 23 CV 6％ 6％ 3％
[0066] 2.3 Calculate the absolute number of T lymphocyte subsets CD3+CD8+ cells
[0067] Figure 5 The diagram shows the CD3+CD8+ cell clustering. The number of CD3+CD8+ cells within each phylum is E. Therefore, the absolute CD3+CD8+ cell count calculated using microsphere I is E*50000 / A*50; the absolute CD3+CD8+ cell count calculated using microsphere II is E*50000 / B*5. The detection was repeated 10 times, and the results are shown in Table 3.
[0068] Table 3. Absolute number of T lymphocytes (CD3+CD8+ cells)
[0069]
[0070]
[0071] Calculations and statistics show that the average value of the two microspheres calculated separately has a smaller CV than the result of any single microsphere, indicating a more stable result.
[0072] Example 2
[0073] Mix 0.2 g of disodium ethylenediaminetetraacetate, 0.2 g of sodium dodecyl sulfate, 0.2 mL of Triton X-100, and 80 mL of 0.005 mol / L phosphate buffer. After thorough stirring and dissolution, filter the solution through a 0.22 μm pore membrane and store at 2–8 °C to obtain a dispersion buffer. Take absolute count microspheres I and II, and dilute them separately with the above dispersion buffer to a concentration of 1800 particles / μL. Then mix them in equal proportions and thoroughly to obtain a microsphere mixed solution.
[0074] The microsphere mixture was thoroughly shaken and mixed. 50 μL / pipette was then dispensed into 12*75 mm flow cytometry tubes, ensuring the solution reached the bottom and not the tube walls. Vacuum dryer setup: The machine was run to 25°C. The flow cytometry tubes containing the microsphere solution were placed inside. A vacuum was created (vacuum degree < 10 Pa). After 15 hours of vacuum drying, the pressure inside the dryer was restored to atmospheric pressure. The flow cytometry tubes were removed, capped, and stored at room temperature away from light. This yielded absolute counting tubes containing two types of absolute counting microspheres. The vacuum-dried absolute counting microspheres adhered firmly to the bottom of the flow cytometry tube and were not easily detached.
[0075] Example 3
[0076] Mix 1.0 g of disodium ethylenediaminetetraacetate, 1.0 g of sodium dodecyl sulfate, 1.0 mL of Triton X-100, and 120 mL of 0.015 mol / L phosphate buffer. After thorough stirring and dissolution, filter the solution through a 0.22 μm pore membrane and store at 2–8 °C to obtain a dispersion buffer. Take absolute count microspheres I and II, and dilute them separately with the above dispersion buffer to a concentration of 2200 particles / μL. Then mix them in equal proportions and thoroughly to obtain a microsphere mixed solution.
[0077] The microsphere mixture was thoroughly shaken and mixed. 50 μL / pipette was then dispensed into 12*75 mm flow cytometry tubes, ensuring the solution reached the bottom and not the tube walls. Vacuum dryer setup: The machine was run to 35°C. The flow cytometry tubes containing the microsphere solution were placed inside. A vacuum was created (vacuum degree <10 Pa). After 24 hours of vacuum drying, the pressure inside the dryer was restored to atmospheric pressure. The flow cytometry tubes were removed, capped, and stored at room temperature away from light. This yielded absolute counting tubes containing two types of absolute counting microspheres. The vacuum-dried absolute counting microspheres adhered firmly to the bottom of the flow cytometry tube and were not easily detached.
[0078] As can be seen from the above embodiments, the present invention provides a method for preparing an absolute counting tube containing multiple absolute counting microspheres, comprising the following steps: mixing disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100, and phosphate buffer to obtain a dispersion buffer; mixing absolute counting microspheres I and II with the dispersion buffer to obtain microsphere solution I and microsphere solution II respectively; mixing microsphere solution I and microsphere solution II in equal proportions to obtain a microsphere mixed solution; dispensing the microsphere mixed solution into flow cytometry tubes, ensuring that the microsphere mixed solution is completely added to the bottom of the flow cytometry tube during dispensing, and then vacuum drying to obtain the absolute counting tube containing multiple absolute counting microspheres. The present invention employs a method of counting multiple microspheres simultaneously, generating multiple results for a single sample, with the final result being the average value; this method yields more stable results, smaller deviations, and higher accuracy compared to the traditional method of counting only one type of microsphere. This invention simultaneously prepares corresponding absolute counters containing multiple microspheres, avoiding the cumbersome steps of adding different microspheres multiple times, making the operation simpler. Compared with traditional absolute counters containing a single microsphere, the same operating steps yield more stable and accurate results. The absolute counters in this invention do not require freeze-drying technology; instead, they are produced using vacuum drying. Compared with traditional freeze-drying, vacuum drying only requires determining the temperature, vacuum level, and drying time, eliminating the need for trial and error in pre-freezing, sublimation, and desorption, significantly shortening the product production cycle. Furthermore, the cost of vacuum drying equipment is lower than that of freeze-drying, offering greater economic advantages.
[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing an absolute counting tube containing multiple absolute counting microspheres, characterized in that, Includes the following steps: (1) Mix disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100 and phosphate buffer to obtain a dispersion buffer; (2) Mix absolute count microspheres I and absolute count microspheres II with dispersion buffer to obtain microsphere solution I and microsphere solution II respectively. Mix microsphere solution I and microsphere solution II in equal proportions to obtain microsphere mixed solution; (3) Dispense the microsphere mixture into a flow cytometer, ensuring that the microsphere mixture is completely added to the bottom of the flow cytometer during dispensing, and then perform vacuum drying to obtain the absolute counting tube containing multiple absolute counting microspheres.
2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of disodium ethylenediaminetetraacetate, sodium dodecyl sulfate, Triton X-100, and phosphate buffer is 0.2–1.0 g: 0.2–1.0 g: 0.2–1.0 mL: 80–120 mL. The concentration of the phosphate buffer solution is 0.005–0.015 mol / L.
3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of microsphere solution I is 1800-2200 particles / μL, and the concentration of microsphere solution II is 1800-2200 particles / μL; the two microsphere solutions have the same concentration and are mixed in equal proportions.
4. The preparation method according to claim 1, characterized in that, The vacuum degree of vacuum drying in step (3) is <10 Pa; The vacuum drying temperature is 20–35°C; The vacuum drying time is 12 to 24 hours.
5. An absolute counting tube containing multiple absolute counting microspheres prepared by the preparation method according to any one of claims 1 to 4.
6. The application of the absolute counting tube containing multiple absolute counting microspheres as described in claim 5 in detecting the absolute number of cell subpopulations.