Red blood cell function screening method for preventing femoral head necrosis
By designing a red blood cell function screening method, and using dilution and counting technology to automatically calculate the ratio of surviving cells, the problem of high cost of detecting cytoskeleton protein gene mutations and cumbersome operation in the prior art is solved, efficient and simple cell distinction is achieved, and detection efficiency is improved.
Patent Information
- Application Number
- CN202411748471.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art detects mutations in the cytoskeleton protein gene, which is costly and cumbersome, making it difficult to efficiently distinguish cells carrying mutated genes from normal cells, affecting the detection efficiency.
A red blood cell function screening method was designed, and red blood cells were isolated by EDTA anticoagulation by adding phosphate buffer, diluting and counting, and the ratio of surviving cells was automatically calculated using plate reading method and counting formula to distinguish mutated cells from normal cells.
It achieves simple and efficient distinction between mutant cells and normal cells, improves detection efficiency, reduces costs, and is suitable for high-throughput experimental needs.
Smart Images
Figure CN119804272A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical detection technology, and in particular to a method for screening red blood cell function for preventing femoral head necrosis. Background Art
[0002] In current medical research, cytoskeletal protein gene mutation is considered one of the most critical risk factors for femoral head necrosis. This gene mutation will destroy the stability of red blood cells in a hypotonic environment, greatly increasing the possibility of their rupture. However, there are many sites of cytoskeletal protein gene mutations. If the whole genome is sequenced directly, the cost is very high, which brings huge challenges to related detection work in terms of manpower, material resources and financial resources.
[0003] In this context, the international calibration standard proposes a more reasonable preliminary screening method, which is to first conduct a red blood cell lysis (hemolysis) experiment. This experiment is of great significance for the preliminary screening of gene mutations related to femoral head necrosis. It can simulate a hypotonic environment, observe the state of red blood cells in this environment, and then confirm whether the red blood cells are prone to rupture. If the red blood cells show a higher tendency to rupture in the experiment, it can be preliminarily inferred that the individual may carry a gene mutation related to femoral head necrosis.
[0004] In traditional testing methods, a series of complex operations are required to distinguish cells carrying mutant genes from normal cells. These operations include diluting the sample multiple times and then carefully counting the cells. The entire process is not only time-consuming, but also cumbersome, and requires a high level of patience and technical skills from the experimenter, which seriously affects the efficiency of the test. Summary of the invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for screening red blood cell function for preventing femoral head necrosis, so as to achieve a simpler and more efficient distinction between mutant cells and normal cells.
[0006] In order to achieve the above purpose, a red blood cell function screening method for preventing femoral head necrosis is designed, comprising: S1. Add 1X PBS to EDTA anticoagulated blood and centrifuge at room temperature to separate the red blood cells; S2. After centrifugation, the supernatant is discarded, and only the red blood cell pellet is retained. 1X PBS with a volume of 20 times the original blood volume is added to these red blood cells to resuspend them for diluting the red blood cells; S3. Take 10 μl of the resuspended red blood cells and add it to a test tube containing 1.1 ml of 1X PBS. Mix well to further dilute the red blood cell sample. S4. After thorough mixing, add 10 uL of red blood cell sample to several first wells; S5. At the same time, add 0.9 ml of deionized water to another test tube, and then take 10 uL of the sample and add it to the second well with the same number as the first well; S6. After the sample has been allowed to rest for a set period of time, the device is used to count the cells; S7. The device obtains the average number of cells in the first well and the average number of cells in the second well; S8. The device is also provided with a calculation module, and the calculation module formula is as follows: residual red blood cell percentage = [average cell number of the second well / (average cell number of the first well*1.1 / 2.0)]*100%.
[0007] Preferably, 0.9 mL of pure water is added to the 1.1 mL of red blood cells to induce hypotonicity and distinguish mutant cells from normal cells.
[0008] Preferably, the first hole and the second hole are both thin-bottomed holes with a hole depth of 0.1 mm.
[0009] Compared with the prior art, the present invention has the following advantages: 1. The plate reading method can quickly process a large number of samples, which is suitable for high-throughput experimental needs, making the experimental process more convenient and greatly improving experimental efficiency.
[0010] 2. By designing duplicate wells, the accuracy of experimental data can be ensured, which can effectively reduce the systematic errors in the experiment; 3. Designing thin-bottomed wells helps reduce the movement and stacking of cells during the culture process, helps observe cell morphology, avoids errors in cell technology, and improves the accuracy of the technology; 4. Using a simple visible light cell counter and counting method, the plate is automatically read for cell counting, and the ratio of surviving cells (% surviving red blood cells) is automatically calculated according to the formula. Normal cells are about 100%, while mutant cells are generally less than 60%, making data processing faster and less costly. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 , are diagrams of the experimental area and counter results of the present invention.
[0012] In the figure: 1.1 mL of 1000X diluted red blood cells; 2. 0.9 mL of pure water. DETAILED DESCRIPTION
[0013] In order to make the purpose and principle of the present invention more clear, it is further described below in conjunction with the accompanying drawings and specific embodiments.
[0014] This embodiment provides a method for screening red blood cell function for preventing femoral head necrosis. Figure 1 , the experimental conditions and experimental steps include: 1. Add 1.0 mL of 1X phosphate buffered saline (PBS) (Gibco, C14190500CP) to the EDTA anticoagulated blood and centrifuge at 500 g for 5 minutes at room temperature.
[0015] 2. Discard the supernatant and add 1X PBS 20 times the original blood volume to resuspend the red blood cell pellet.
[0016] 3. Place 10 uL of the final red blood cell dilution in a tube containing 1.1 mL of 1X PBS.
[0017] 4. After thorough mixing, add 10 uL of red blood cell sample to well 1 of the control group (three replicate wells).
[0018] 5. Add 0.9 mL of deionized water to the test tube and add 10 uL of red blood cell sample to well 2 of the experimental group (3 replicate wells).
[0019] After 6.2 minutes of waiting time, use a specific device to count the cells. The "percentage of residual red blood cells" can reflect the anti-hypotonicity of red blood cells. The calculation formula is: Percentage of residual red blood cells = [average number of cells in well 2 / (average number of cells in well 1 * 1.1 / 2.0)] * 100%. If the "percentage of residual red blood cells" is greater than 100%, it will be recorded as "100%". Normal people are generally greater than 90%, while patients are less than 90%. The lower the risk, the greater the risk.
[0020] The experimental results are shown in Table 1: Table 1 The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes according to the technical solutions and novel concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for screening red blood cell function for preventing femoral head necrosis, characterized in that include S1. Add 1X PBS to EDTA anticoagulated blood and centrifuge at room temperature to separate the red blood cell sample; S2. After centrifugation, the supernatant is discarded, and only the red blood cell pellet is retained. 1X PBS with a volume of 20 times the original blood volume is added to these red blood cells to resuspend them for diluting the red blood cells; S3. Take 10 μl of the resuspended red blood cells and add it to a test tube containing 1.1 ml of 1X PBS. Mix well to further dilute the red blood cell sample. S4. After thorough mixing, add 10 uL of red blood cell sample to several first wells; S5. At the same time, add 0.9 ml of deionized water to another test tube, and then take 10 uL of the sample and add it to the second well with the same number as the first well; S6. After the sample has been allowed to rest for a set period of time, the device is used to count the cells; S7. The device obtains the average number of cells in the first well and the average number of cells in the second well; S8. The device is also provided with a calculation module, and the calculation module formula is as follows: residual red blood cell percentage = [average cell number of the second well / (average cell number of the first well*1.1 / 2.0)]*100%.
2. A method for screening red blood cell function for preventing femoral head necrosis according to claim 1, characterized in that: The 1.1 mL of red blood cells were added with 0.9 mL of pure water to induce hypotonicity and distinguish mutant cells from normal cells.
3. A red blood cell function screening method for preventing femoral head necrosis as described in claim 1, wherein the first hole and the second hole are both thin-bottomed holes with a hole depth of 0.1 mm.