Umbilical cord blood unfreezing reference specimen cell counting and flow cytometry dilution method

Abstract

The invention relates to the technical field of biomedical detection, and discloses an umbilical cord blood unfreezing reference specimen cell counting and flow cytometry dilution method which comprises the following steps: S1, preparing an unfreezing solution which is composed of HES, AB plasma and PBS, and placing the unfreezing solution at room temperature for later use; s2, taking the unfrozen umbilical cord blood unfreezing reference specimen, adding an unfreezing solution into the umbilical cord blood, firstly gently and uniformly mixing, then standing at room temperature, then continuing to add the unfreezing solution, gently and uniformly mixing again, then standing at room temperature, and stabilizing; s3, taking the diluted umbilical cord blood, and carrying out cell counting by adopting a counting instrument; and S4, taking the diluted umbilical cord blood again, and dyeing. The unfreezing solution composed of HES, AB plasma and PBS synergistically regulates osmotic pressure, provides cell nutrition, stabilizes the pH value, reduces the damage of the cryoprotectant to cord blood cells, maintains the cell activity, and improves the problem of result deviation caused by direct detection of cell damage in the prior art.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and more specifically, it relates to a method for cell counting and flow cytometry detection of diluted thawed umbilical cord blood reference specimens. Background Technology

[0002] Umbilical cord blood thawing reference specimens are samples prepared by standardized cryopreservation of umbilical cord blood and subsequent thawing according to standardized procedures. Their core value lies in providing a benchmark sample for the detection of hematopoietic stem and progenitor cell-related indicators in umbilical cord blood. The CD34+ hematopoietic stem and progenitor cells abundant in umbilical cord blood are key targets for assessing umbilical cord blood hematopoietic function, guiding hematopoietic stem cell transplantation, and treating hematological diseases. Therefore, accurate detection of indicators such as cell count, CD34+ cell proportion, and cell activity in this specimen is an important prerequisite for ensuring the scientific nature of clinical decision-making and the reliability of test results.

[0003] Currently, for the testing of thawed umbilical cord blood reference specimens, existing technologies involve directly sampling after thawing for cell counting and flow cytometry. Some protocols use simple dilution with physiological saline before testing. However, in practice, because umbilical cord blood cells undergo drastic environmental changes during freezing and thawing, the cell membrane integrity and physiological activity are already fragile. Existing technologies, such as direct testing or single-component dilution, cannot provide cells with synergistic protection in terms of osmotic pressure regulation, nutrient supply, and pH stability. This leads to cells being prone to swelling and rupture due to sudden changes in osmotic pressure, or decreased activity due to lack of nutrient support. Ultimately, this results in significant deviations in cell counting results and insufficient accuracy in CD34+ cell ratio detection, failing to meet the precision requirements of clinical and experimental testing. Summary of the Invention

[0004] To address the issues of significant deviations in cell counting results and insufficient accuracy in CD34+ cell proportion detection in existing technologies, which fail to meet the precision requirements of clinical and experimental settings, this application provides a method for cell counting and flow cytometry analysis of thawed umbilical cord blood reference specimens.

[0005] This application provides a method for cell counting and flow cytometry analysis of thawed umbilical cord blood reference specimens, employing the following technical solution: The thawing of umbilical cord blood followed the same dilution method as the specimen cell counting and flow cytometry analysis, including the following steps: S1. Prepare a thawing solution composed of HES, AB plasma, and PBS. Place the thawing solution at room temperature for later use. S2. Take a thawed reference sample of umbilical cord blood, add the thawing solution to the cord blood, gently mix, and let it stand at room temperature. Then, continue adding the thawing solution, gently mix again, and let it stabilize at room temperature. S3. Take the diluted cord blood and perform cell counting using a cell counter. S4. Collect diluted umbilical cord blood again, stain it, add ammonium chloride hemolysin after staining, and then perform flow cytometry analysis.

[0006] By adopting the above technical solution, a thawing solution composed of HES, AB plasma and PBS is prepared in S1 and the thawing solution is kept at room temperature for later use. HES adjusts the osmotic pressure of the thawing solution, AB plasma provides nutrients for cells to be processed in the next step, and PBS maintains the pH value of the thawing solution. The three work together to reduce the damage of residual cryoprotectants to cells and maintain cell morphology and activity. In S2, a thawed reference specimen of umbilical cord blood is taken, thawed, and added to it. After gently mixing, it is placed at room temperature to stand. Then, thawed solution is added again, and the mixture is gently mixed again and placed at room temperature to stabilize. The thawed solution is added in stages and allowed to stand for a period of time to allow the cells to gradually adapt to the thawed solution environment. This avoids sudden changes in local osmotic pressure and excessively high concentration of thawed solution caused by adding the solution all at once, reduces cell aggregation, ensures cell morphology integrity, and improves sample homogeneity. Diluted umbilical cord blood was collected in S3, and cell counting was performed using a cell counter. In S4, diluted umbilical cord blood is taken again for staining. After staining, ammonium chloride hemolysin is added, followed by flow cytometry. Ammonium chloride hemolysin lyses the red blood cells in the sample, avoiding interference from red blood cells in cell signal recognition during flow cytometry. This allows the counter to obtain accurate cell-related detection results from both counting and flow cytometry, solving the problems of counting deviation and abnormal CD34+ cell ratio that easily occur in direct detection in existing technologies.

[0007] Preferably, in step S1, the amount of HES used is 0.8-1.2 ml, the amount of AB plasma used is 0.4-0.6 ml, and the amount of PBS used is 8.3-8.7 ml.

[0008] By adopting the above technical solution, this invention addresses the technical problem that after thawing umbilical cord blood reference specimens, cells are in a fragile state due to the freezing and thawing process, and are prone to decreased activity and deviations in test results due to residual cryoprotectants. A thawing solution is prepared using HES, AB plasma, and PBS in specific dosage ranges. Specifically, HES, through its dosage and ratio with other components, adjusts the osmotic pressure of the thawing solution to match the intracellular environment, preventing cell swelling or rupture due to osmotic pressure imbalance. AB plasma, added in the same amount, provides nutrients to the umbilical cord blood cells for subsequent processing, maintaining their normal physiological state. PBS, in the same amount, stabilizes the pH of the thawing solution, preventing pH fluctuations from causing cellular metabolic disorders. The synergistic effect of these three components reduces damage to umbilical cord blood cells from residual cryoprotectants, maintains cell activity and morphological integrity, and provides a suitable sample for subsequent dilution and testing steps, reducing the result deviations caused by cell damage in existing direct detection methods.

[0009] Preferably, in step S2, the volume of the thawed umbilical cord blood is 90-110 μL.

[0010] By adopting the above technical solution, thawed umbilical cord blood within a certain volume range is collected. After thawing, it can form a suitable dilution system with the thawing solution added in subsequent stages, ensuring that sufficient sample can be obtained after dilution for cell counting in step S3 and flow cytometry detection in step S4. At the same time, it avoids the risk of cell aggregation due to insufficient space during dilution caused by excessively large sampling volume, or insufficient sample volume due to insufficient sample volume to complete effective detection. This ensures the integrity of cell morphology and sample homogeneity during dilution, provides a stable sample basis for subsequent detection steps, and reduces detection errors caused by sampling volume issues.

[0011] Preferably, in step S2, the volume of thawing solution added to the umbilical cord blood is 90-110 μL.

[0012] By adopting the above technical solution, it is possible to avoid the technical problem that improper initial addition of thawing solution can easily lead to swelling, rupture, or aggregation of fragile umbilical cord blood cells after thawing due to sudden changes in osmotic pressure, affecting subsequent dilution operations and cell status. Thawing solution allows cells to initially come into contact with a system composed of HES, AB plasma, and PBS. The osmotic pressure regulated by HES forms a smooth transition with the intracellular environment, the nutrients provided by AB plasma initially maintain the physiological state of the cells, and the stable pH of PBS avoids environmental fluctuations. Combined with subsequent gentle mixing and standing operations, the cells gradually adapt to the new environment, avoiding drastic changes in osmotic pressure caused by excessive initial addition of solution, thereby reducing cell damage and aggregation, and ensuring the integrity of cell morphology.

[0013] Preferably, in step S2, when the mixture is gently mixed and then left to stand at room temperature, the standing time is 4-6 minutes.

[0014] By employing the above-mentioned technical solution, improper settling time can easily lead to umbilical cord blood cells being unable to fully adapt to the initial thawing solution environment after thawing. This can result in damage or aggregation due to an unstable transition in osmotic pressure, affecting subsequent dilution operations and cell status. The solution involves gently mixing the umbilical cord blood and thawing solution first, then allowing the mixture to stand at room temperature. This settling process allows cells sufficient time to gradually balance intracellular and extracellular osmotic pressure in the mixture of HES, AB plasma, and PBS. HES continuously regulates the system's osmotic pressure, AB plasma provides nutrients to maintain cell physiological activity, and PBS stabilizes the system's pH. Through the synergistic effect of these three factors, cells can slowly adapt to the new environment, avoiding osmotic pressure shock damage caused by insufficient settling time or abnormal cell metabolism caused by excessive settling time. This reduces cell aggregation and ensures cell morphology integrity.

[0015] Preferably, the volume of thawing fluid added after step S2 is 580-620 μL.

[0016] By adopting the above technical solution, thawing solution is added to the mixture of umbilical cord blood and thawing solution after it has been left to stand. Together with the thawing solution added initially and the initial umbilical cord blood, it forms a complete dilution system. At the same time, the total amount of diluted sample can meet the sampling requirements of S3 cell counting and S4 flow cytometry detection, control the cell concentration within an appropriate range, reduce aggregation, ensure cell morphology integrity and sample homogeneity, and reduce deviations for subsequent detection.

[0017] Preferably, in step S2, when the mixture is gently mixed again and then allowed to stabilize at room temperature, the stabilization time is 28-32 minutes.

[0018] By adopting the above technical solution, after gently mixing again, the diluted system is placed at room temperature to stabilize. During the stabilization process, the thawing solution composed of HES, AB plasma and PBS continues to play a role. HES maintains the stability of the system's osmotic pressure, ensuring complete balance of intracellular and extracellular osmotic pressure. AB plasma continuously provides nutrients to the cells to maintain physiological activity. PBS keeps the pH of the system within a suitable range, avoiding pH fluctuations from affecting cell metabolism. At the same time, this stabilization time can prevent damage or aggregation of cells due to insufficient stabilization time or decreased cell viability due to excessive stabilization time, thereby ensuring cell morphology integrity and sample homogeneity.

[0019] Preferably, in step S3, the sample volume of diluted umbilical cord blood is 190-210 μL, and the counting is performed using the pre-dilution mode of the counter. The sample volume of diluted umbilical cord blood is then taken again to be 480-520 μL.

[0020] By adopting the above technical solution, diluted umbilical cord blood is used for cell counting, meeting the sample volume required for the counter and avoiding fluctuations in counting results due to insufficient sample volume. Simultaneously, a specific volume of diluted umbilical cord blood is used again for subsequent flow cytometry analysis, ensuring sufficient cell quantity for signal analysis. The counter uses a pre-dilution mode during counting. Since manual dilution has already been performed using thawing fluid, the pre-dilution mode allows the counter to calculate the original cell concentration based on the manual dilution factor, avoiding secondary dilution caused by the instrument's dilution function after startup, thus reducing counting errors. Combined with the cell viability and morphological integrity maintained by the thawing fluid, accurate cell counting results are ensured, providing a foundation for subsequent flow cytometry analysis and reducing the counting inaccuracies found in existing technologies.

[0021] Preferably, in step S4, the staining uses 7-AAD / CD45 / CD34 staining solution, and the staining time is 18-22 minutes.

[0022] By employing the above-mentioned technical solution, diluted umbilical cord blood is stained with 7-AAD / CD45 / CD34 staining solution, with a specific staining time controlled. 7-AAD binds to the nucleic acids of dead cells, CD45 specifically labels nucleated cells, and CD34 targets and binds to CD34+ cells. The three work synergistically to distinguish cell viability and cell type. The staining time ensures sufficient binding of the staining solution to the corresponding target sites, avoiding insufficient binding and weak fluorescence signals due to too short a staining time, which would prevent accurate cell type identification, or excessive non-specific binding due to too long a staining time, which would increase interference with the detection signal. Simultaneously, it accurately identifies CD34+ cells, providing a clear basis for cell signal differentiation in subsequent flow cytometry detection and reducing the problem of abnormally low CD34+ cell proportions in existing technologies.

[0023] Preferably, when adding ammonium chloride hemolysin in step S4, the amount of ammonium chloride hemolysin added is 0.8-1.2 ml, and the reaction time is 9-11 min.

[0024] By adopting the above technical solution, ammonium chloride hemolysin can specifically act on red blood cells, destroy the red blood cell membrane and cause them to lyse, thereby removing the interference of fluorescence signals from CD34+ cells and nucleated cells in red blood cell flow cytometry, ensuring that the hemolysin concentration is sufficient to cover the red blood cells in the sample, and ensuring that the reaction time guarantees sufficient lysis of red blood cells, while avoiding damage to nucleated cells, including CD34+ cells, caused by ammonium chloride due to excessive reaction time.

[0025] In summary, this application has the following beneficial effects: 1. This application uses a thawing solution composed of HES, AB plasma and PBS. The three components work synergistically to regulate osmotic pressure, provide cell nutrition, stabilize pH value, reduce damage to umbilical cord blood cells by cryoprotectants, maintain cell viability, and improve the problem of result deviation caused by cell damage in the direct detection of existing technologies.

[0026] 2. This application adopts a dilution step of adding thawing solution in stages and allowing the cells to gradually adapt to the environment, avoiding the osmotic pressure shock and excessively high local concentration caused by adding solution all at once, reducing cell aggregation, ensuring the integrity of cell morphology, and improving sample homogeneity and detection repeatability.

[0027] 3. The sample processing and detection process of this application is compatible. The pre-dilution mode counting can avoid the instrument's secondary dilution error. Hemolysin is used to remove red blood cell interference before flow cytometry to ensure accurate detection of key indicators and improve the problem of abnormally low CD34+ cell ratio and inaccurate counting in the existing technology. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method in this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0030] Example 1 This application provides a method for cell counting and flow cytometry analysis of thawed umbilical cord blood reference specimens, including the following steps: S1. Prepare the thawing solution, which consists of HES, AB plasma and PBS. Place the thawing solution at room temperature for later use. The dosage of HES was 0.8 ml, the dosage of AB plasma was 0.4 ml, and the dosage of PBS was 8.3 ml.

[0031] S2. Take a thawed reference specimen of umbilical cord blood, add thawing solution to the umbilical cord blood, mix gently first and then let it stand at room temperature. Then add more thawing solution, mix gently again and let it stabilize at room temperature. The procedure involved taking 90 μL of thawed cord blood, adding 90 μL of thawing solution to the cord blood initially, gently mixing the solution, and allowing it to stand at room temperature for 4 minutes. After standing, adding another 580 μL of thawing solution, gently mixing the solution again, and allowing it to stabilize at room temperature for 28 minutes.

[0032] S3. Collect diluted umbilical cord blood and perform cell counting using a cell counter. The sample volume of diluted umbilical cord blood was 190 μL, and the counting was performed using the pre-dilution mode of the counter.

[0033] S4. Collect diluted umbilical cord blood again, stain it, add ammonium chloride hemolysin after staining, and then perform flow cytometry analysis. The sample volume of the diluted umbilical cord blood was 480 μL; 7-AAD / CD45 / CD34 staining solution was used for staining, and the staining time was 18 min; 0.8 ml of ammonium chloride hemolysin was added, and the reaction time of ammonium chloride hemolysin was 9 min.

[0034] Example 2 This application provides a method for cell counting and flow cytometry analysis of thawed umbilical cord blood reference specimens, including the following steps: S1. Prepare the thawing solution, which consists of HES, AB plasma and PBS. Place the thawing solution at room temperature for later use. The dosage of HES was 1.0 ml, the dosage of AB plasma was 0.5 ml, and the dosage of PBS was 8.5 ml.

[0035] S2. Take a thawed reference specimen of umbilical cord blood, add thawing solution to the umbilical cord blood, mix gently first and then let it stand at room temperature. Then add more thawing solution, mix gently again and let it stabilize at room temperature. The procedure involved taking 100 μL of thawed cord blood, adding 100 μL of thawing solution to the cord blood, gently mixing it, and then allowing it to stand at room temperature for 5 minutes. After standing, another 600 μL of thawing solution was added, and the mixture was gently mixed again and allowed to stabilize at room temperature for 30 minutes.

[0036] S3. Collect diluted umbilical cord blood and perform cell counting using a cell counter. The sample volume of diluted umbilical cord blood was 200 μL, and the counting was performed using the pre-dilution mode of the counter.

[0037] S4. Collect diluted umbilical cord blood again, stain it, add ammonium chloride hemolysin after staining, and then perform flow cytometry analysis. The sample volume of the diluted umbilical cord blood was 500 μL; 7-AAD / CD45 / CD34 staining solution was used for staining, and the staining time was 20 min; 1.0 ml of ammonium chloride hemolysin was added, and the reaction time of ammonium chloride hemolysin was 10 min.

[0038] Example 3 This application provides a method for cell counting and flow cytometry analysis of thawed umbilical cord blood reference specimens, including the following steps: S1. Prepare the thawing solution, which consists of HES, AB plasma and PBS. Place the thawing solution at room temperature for later use. The amount of HES used is 1.2 ml, the amount of AB plasma is 0.6 ml and the amount of PBS is 8.7 ml.

[0039] S2. Take a thawed reference specimen of umbilical cord blood, add thawing solution to the umbilical cord blood, mix gently first and then let it stand at room temperature. Then add more thawing solution, mix gently again and let it stabilize at room temperature. The procedure involved taking 110 μL of thawed cord blood, adding 110 μL of thawing solution to the cord blood, gently mixing it, and then allowing it to stand at room temperature for 6 minutes. After standing, another 620 μL of thawing solution was added, and the mixture was gently mixed again and allowed to stabilize at room temperature for 32 minutes.

[0040] S3. Collect diluted umbilical cord blood and perform cell counting using a cell counter. The sample volume of diluted umbilical cord blood was 210 μL, and the counting was performed using the pre-dilution mode of the counter.

[0041] S4. Collect diluted umbilical cord blood again, stain it, add ammonium chloride hemolysin after staining, and then perform flow cytometry analysis. The sample volume of the diluted umbilical cord blood was 520 μL; 7-AAD / CD45 / CD34 staining solution was used for staining, and the staining time was 22 min; 1.2 ml of ammonium chloride hemolysin was added, and the reaction time of ammonium chloride hemolysin was 11 min.

[0042] Comparative Example 1 The difference from Example 2 is that in step S1, when preparing the thawing solution, HES is missing and it consists only of AB plasma and PBS.

[0043] Comparative Example 2 The difference from Example 2 is that in step S1, when preparing the thawing solution, AB plasma is missing, and it consists only of HES and PBS.

[0044] Comparative Example 3 The difference from Example 2 is that in step S1, when preparing the thawing solution, an equal volume of physiological saline is used instead of PBS.

[0045] Comparative Example 4 The difference from Example 2 is that step S2 is missing the step of gently mixing first and then letting it stand at room temperature.

[0046] Comparative Example 5 The difference from Example 2 is that in step S2, the thawing solution is not added in stages, but is added to the thawed umbilical cord blood all at once.

[0047] Comparative Example 6 The difference from Example 2 is that the counting in step S3 is not done in the pre-dilution mode of the counter, but in the post-dilution mode of the counter.

[0048] Comparative Example 7 The difference from Example 2 is that the step of adding ammonium chloride hemolysin is omitted in step S4, and flow cytometry detection is performed directly.

[0049] Comparative Example 8 The difference from Example 2 is that steps S1 and S2 are completely omitted.

[0050] Performance Experiment (1) Cell count recovery rate detection experiment Umbilical cord blood thaw reference specimens from the same batch and the same donor were selected and thawed in a constant temperature water bath until completely melted to ensure that all samples to be tested were in the same initial state. A fully automated cell counter, model XX-C100, was used. Before the experiment, the instrument was calibrated using the calibration solution provided with the instrument. For Examples 1-3, Comparative Examples 1-5, and 7-8, the pre-dilution mode was selected, with the injection volume set to 50 μL / sample, the detection channel set to the white blood cell detection channel, and the data acquisition time set to 10 s / sample. For Comparative Example 6, the post-dilution mode was selected, and the instrument automatically set the internal dilution factor to 10 times. All other parameters were the same as in the pre-dilution mode. Aspirate the diluted samples from each technical solution using a pipette with 1% accuracy and inject it into the sample cell of the counter to start the detection. Each sample is tested in parallel 3 times. After each test, the sample cell is rinsed 3 times with PBS to avoid cross-contamination. Based on the cell concentration of the diluted sample displayed by the counter, the cell count recovery rate (%) was calculated according to the formula: (total dilution factor of the cell concentration after dilution) / original cell concentration before freezing 100%. The average of three parallel test results was taken as the final data.

[0051] (2) Detection experiment of CD34+ cell proportion (CD34+%) A flow cytometer, model XX-F400, was used. Before the experiment, the 488nm laser excitation channel and fluorescence signal detection channel were calibrated using a fluorescence microsphere calibrator. The 7-AAD signal corresponds to the 620nm channel, the CD45 signal corresponds to the 525nm channel, and the CD34 signal corresponds to the 670nm channel. The number of cells to be acquired was set to 10,000 nucleated cells to ensure that the statistical sample size met the accuracy requirements. Place the flow cytometer tube into the sample stage, start the instrument, and acquire the fluorescence signal. Each sample is tested in parallel three times. After each test, flush the sample inlet tube with sheath fluid to avoid antibody residue. Using the flow cytometer software, first use the CD45 fluorescence signal to delineate the total nucleated cell population and exclude interference from anucleated cells. Then, within this cell population, use the 7-AAD fluorescence signal to exclude dead cells. Finally, calculate the percentage of CD34-positive cells in the CD45-positive + 7-AAD-negative cell population, which is the CD34+%, and take the average of the three parallel test results.

[0052] (3) Nucleated cell viability detection experiment The flow cytometry parameters are set the same as those for CD34+% detection, focusing only on CD45 and 7-AAD signals. There is no need to enable the CD34 signal channel. Place the flow cytometer tube into the instrument and start the detection. Use the CD45 signal to delineate the total nucleated cell population and count the percentage of 7-AAD-negative cells in the population. Each sample is tested in parallel three times, and the tubing is cleaned after each test. The calculation formula is: Nucleated cell viability (%) = (Number of CD45-positive + 7-AAD-negative cells / Total number of CD45-positive cells) × 100%.

[0053] (4) CD34+ cell viability assay The flow cytometry parameters are completely consistent with those for CD34+% detection, ensuring that the laser excitation intensity and fluorescence channel sensitivity are the same. The instrument was started to acquire signals. Each sample was tested in parallel three times. The tubing was cleaned after each test to avoid cross-contamination. The total nucleated cell population was first delineated by CD45 signal using software. Then, the CD34+ cell subpopulation was delineated by CD34 signal within the total nucleated cell population. Finally, the percentage of 7-AAD signal-negative cells in the CD34+ cell subpopulation was counted, which is the live CD34+ cells. The calculation formula is: CD34+ cell activity (%) = (number of CD34 positive + 7-AAD negative cells / total number of CD34 positive cells) × 100%. The average of the three parallel results was taken.

[0054] Table 1 Performance Experiment Data Group Cell count recovery rate (%) CD34+ cell percentage (CD34+%) Nucleated cell viability (%) CD34+ cell viability (%) Example 1 96.851.23 0.510.03 80.121.56 91.950.87 Example 2 98.110.95 0.550.02 82.591.13 92.770.64 Example 3 97.521.08 0.530.04 81.361.32 92.310.79 Comparative Example 1 90.231.87 0.420.05 72.452.01 89.861.21 Comparative Example 2 89.761.92 0.400.06 71.882.15 89.231.34 Comparative Example 3 88.512.14 0.390.05 70.352.32 88.971.46 Comparative Example 4 92.671.65 0.450.04 75.621.89 90.151.08 Comparative Example 5 91.341.78 0.430.05 74.282.03 89.721.15 Comparative Example 6 85.382.46 0.370.06 78.951.67 91.030.92 Comparative Example 7 97.851.02 0.290.07 Unable to detect accurately Unable to detect accurately Comparative Example 8 89.712.25 0.340.05 68.052.58 91.730.85 Experimental conclusion: 1. Based on the results of the performance testing experiments in Examples 1-3 and Comparative Example 1, it can be seen that Comparative Example 1, due to the absence of HES in the preparation of the thawing solution, had significantly lower cell count recovery rate, CD34+ cell ratio, and nucleated cell activity compared to Examples 1-3. As an osmotic pressure regulator, HES can maintain the osmotic pressure balance of the environment in which umbilical cord blood cells are located after thawing, preventing cells from swelling or rupturing due to sudden changes in osmotic pressure. At the same time, it can stabilize cell morphology and reduce aggregation. It can form a synergistic effect with AB plasma and PBS in terms of osmotic pressure regulation, nutrient supply, and buffering stability. When HES is missing, this synergistic relationship is disrupted, cells are easily damaged, leading to decreased activity, reduced CD34+ cell recognition accuracy, and worse counting accuracy. Ultimately, Comparative Example 1 is inferior to Examples 1-3 in terms of cell count and key flow cytometry indicators.

[0055] 2. Combining Examples 1-3 and Comparative Example 2 with the performance test results table, it can be seen that Comparative Example 2, due to the lack of AB plasma in the preparation of the thawing solution, performed far worse than Examples 1-3 in various detection indicators. The protein components and nutrients contained in AB plasma can provide necessary nutritional support for the fragile umbilical cord blood cells after thawing, and at the same time help maintain the stability of cell surface antigens, such as CD34 antigen, ensuring that target cells can be accurately identified during flow cytometry. Together with HES and PBS, it ensures cell survival and detection accuracy. When AB plasma is missing, the lack of nutrient supply to cells leads to reduced activity, decreased stability of CD34 antigen, increased misjudgment rate of CD34+ cell proportion during flow cytometry, and the count recovery rate also decreased due to the influence of cell activity, failing to achieve the detection effect of Examples 1-3.

[0056] 3. Combining Examples 1-3 and Comparative Example 3 with the performance test results table, it can be seen that the detection performance of Comparative Example 3, which replaced PBS in the thawing solution with physiological saline, was significantly weaker than that of Examples 1-3. PBS has a stable buffering capacity and can maintain the pH value of the thawing solution within the suitable range of 7.2-7.4, providing a stable acid-base environment for cells and preventing metabolic disorders caused by pH fluctuations. It works synergistically with HES and AB plasma to maintain the normal physiological state of cells. In contrast, physiological saline has a very weak buffering capacity and cannot stabilize the pH of the thawing solution, leading to an imbalance in the cell metabolic environment, a decrease in nucleated cell activity, and interference with CD34+ cell signal recognition. Ultimately, the cell count recovery rate, CD34+ cell ratio, and nucleated cell activity of Comparative Example 3 were all lower than those of Examples 1-3.

[0057] 4. Combining Examples 1-3 and Comparative Example 4 with the performance test results table, it can be seen that Comparative Example 4, due to the absence of the static setting operation in step S2, has lower nucleated cell activity and CD34+ cell ratio than Examples 1-3. The static setting operation allows thawed umbilical cord blood cells to gradually adapt to the thawing solution environment and slowly regulate the osmotic pressure balance inside and outside the cells, avoiding damage to the cell membrane caused by sudden changes in osmotic pressure. It is a key step in the process of adding liquid in stages, static setting adaptation, and stable equilibrium dilution. When the static setting operation is missing, the cells directly face the osmotic pressure shock from the addition of thawing solution twice, resulting in damage to the cell membrane integrity and decreased activity. Some CD34+ cells cannot be accurately identified due to damage, ultimately causing the test indicators of Comparative Example 4 to be lower than those of Examples 1-3.

[0058] 5. Combining Examples 1-3 and Comparative Example 5 with the performance test results table, it can be seen that Comparative Example 5, due to its method of adding thawing solution all at once, has a much worse detection effect than Examples 1-3. Adding thawing solution in stages can avoid excessively high local thawing solution concentrations caused by a single addition, reducing cell aggregation. Combined with static incubation, it allows cells to gradually adapt to the environment, which is an important process to ensure cell morphology integrity and activity. Adding solution all at once will cause a sudden increase in local thawing solution concentration, making cells prone to aggregation and more severely affected by osmotic pressure shock. This not only leads to a decrease in the activity of nucleated cells, but also interferes with the flow cytometry detection of CD34+ cells, resulting in the CD34+ cell ratio and cell count recovery rate of Comparative Example 5 being lower than those of Examples 1-3.

[0059] 6. Combining Examples 1-3 and Comparative Example 6 with the performance test results table, it can be seen that Comparative Example 6, due to the use of the counter's post-dilution mode, has a significantly lower cell count recovery rate and CD34+ cell proportion than Examples 1-3. The pre-dilution mode is highly compatible with the manual fractional dilution process of this scheme, and the counter can accurately back-calculate the original cell concentration based on the preset manual dilution factor, avoiding double dilution errors. However, the post-dilution mode causes the counter to perform a second automatic dilution of the manually diluted sample, resulting in double dilution by both manual and instrument dilution. This leads to deviations in the counter's calculation of the original concentration, and the second dilution may further damage the cells. Ultimately, the cell counting accuracy and CD34+ cell identification precision of Comparative Example 6 are not as good as those of Examples 1-3.

[0060] 7. Combining Examples 1-3 and Comparative Example 7 with the performance test results table, it can be seen that Comparative Example 7, due to the absence of the addition of ammonium chloride hemolysin, had a significantly lower CD34+ cell ratio than Examples 1-3, and nucleated cell activity and CD34+ cell activity could not be accurately detected. Ammonium chloride hemolysin can specifically lyse red blood cells, removing the interference of red blood cells on the flow cytometry detection signal, ensuring that the flow cytometer can accurately identify nucleated cells and CD34+ cells. When the hemolysin operation is missing, a large number of residual red blood cells will mask the fluorescence signals of nucleated cells and CD34+ cells, leading to misjudgment of the CD34+ cell ratio, and the inability to count nucleated cells and CD34+ cell activity. The accuracy and completeness of the detection results are far inferior to those of Examples 1-3.

[0061] 8. Combining Examples 1-3 and Comparative Example 8 with the performance test results table, it can be seen that Comparative Example 8, due to the complete absence of the thawing solution preparation and dilution steps, had the worst test indicators among all groups, and was significantly lower than Examples 1-3. The dual role of the thawing solution in protection and dilution is the core to solving the defects of the prior art. HES, AB plasma, and PBS work together to protect cell viability, and fractional dilution and static incubation avoid cell damage. However, direct detection in the prior art will cause the fragile cells after thawing to be affected by the residual cryoprotectant. At the same time, the lack of osmotic pressure and environmental buffering leads to a significant decrease in the activity of nucleated cells, a low proportion of CD34+ cells, and a count recovery rate of less than 90%.

[0062] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The umbilical cord blood thawing reference specimen cell counting and flow cytometry dilution method is characterized by, Includes the following steps: S1. Prepare a thawing solution, which is composed of HES, AB plasma and PBS, and place the thawing solution at room temperature for later use. S2. Take a thawed reference specimen of umbilical cord blood, add thawing solution to the umbilical cord blood, mix gently first and then let it stand at room temperature. Then add more thawing solution, mix gently again and let it stabilize at room temperature. S3. Collect diluted umbilical cord blood and perform cell counting using a cell counter. S4. Collect diluted umbilical cord blood again, stain it, add ammonium chloride hemolysin after staining, and then perform flow cytometry analysis.

2. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S1, the amount of HES used is 0.8-1.2 ml, the amount of AB plasma used is 0.4-0.6 ml, and the amount of PBS used is 8.3-8.7 ml.

3. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S2, the volume of the thawed umbilical cord blood is 90-110 μL.

4. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S2, the volume of thawing solution added to the umbilical cord blood is 90-110 μL.

5. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S2, when the mixture is gently mixed and then left to stand at room temperature, the standing time is 4-6 minutes.

6. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, After step S2, add 580-620 μL of thawing fluid.

7. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S2, after gently mixing again and allowing to stabilize at room temperature, the stabilization time is 28-32 minutes.

8. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S3, the sample volume of diluted umbilical cord blood is 190-210 μL. When counting, the pre-dilution mode of the counter is used, and the sample volume of diluted umbilical cord blood is 480-520 μL.

9. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, In step S4, 7-AAD / CD45 / CD34 staining solution is used for staining, and the staining time is 18-22 min.

10. The method for cell counting and flow cytometry detection dilution of thawed umbilical cord blood reference specimens according to claim 1, characterized in that, When adding ammonium chloride hemolysin in step S4, the amount of ammonium chloride hemolysin added is 0.8-1.2 ml, and the reaction time is 9-11 min.

Images