Cell stabilizer and application thereof

Abstract

The cell stabilizer is prepared from the following components in concentration: 3 to 10 percent (w / v) of a cell separating agent, 3 to 8 mu M of a caspase inhibitor, 0.33 to 0.40 mM of mimic peptide, 3.35 to 7.25 mM of alpha-D-glucose, 0.50 to 1.50 mM of N-acetyl-L-cysteine, 2 to 15 mM of NaCl, 0.02 to 90 mu g / ml of a platelet inhibitor and 20 to 40 mM of anhydrous sodium acetate. According to the cell stabilizer provided by the invention, the preservation time of a whole blood sample after being in vitro can be prolonged, so that the detection duration of the collected sample of the detection kit can be prolonged, and the stability of an ATP detection value of the detection kit can be ensured.

Description

Technical Field

[0001] The present invention relates to the field of blood storage, and in particular to a cell stabilizer and application thereof. Background Art

[0002] Peripheral blood is the tissue most frequently approached by biomedicine. The detection items of its biochemical indicators are complex and diverse. It is often used as one of the bases for clinical medical diagnosis. It can not only objectively reflect the health status of the human body and diagnose the functions of the main systems of the body, but also has important significance for clinical treatment effects and prognosis evaluation. For example, the separation of blood-derived cells in hematology, blood transfusion, immunology, regenerative medicine and oncology has extensive clinical and scientific importance. Therefore, the accuracy of peripheral blood biochemical test results is closely related to the quality of blood samples. However, after blood samples are collected, they are often not tested in time due to the influence of conditions. They need to be stored and transported for a long time, and whole blood (WB) will deteriorate rapidly after leaving the body. For example, nutrient loss, oxidative stress, changes in osmolarity and pH, and accumulation of toxic metabolic byproducts begin to occur rapidly; within a few hours, neutrophils are activated, undergo sudden oxidation, necrosis and apoptosis; the transportation of blood samples produces uncontrolled oscillations that induce hemolysis and platelet activation. These damages not only affect the viability and function of target cells in the sample, but also greatly affect the widely used enrichment techniques.

[0003] After blood leaves the body's circulation, it may suffer from "storage damage" due to the limitations of storage and maintenance conditions, thus affecting various blood functional indicators. With the extension of blood storage period, changes in red blood cell survival conditions, the generation of metabolic waste, and factors such as shortened lifespan and increased damage of some stored red blood cells, the metabolism and function of red blood cells in stored blood will change to varying degrees.

[0004] Blood preservation fluid is one of the main factors affecting blood storage. In the prior art, in order to preserve the blood for transfusion for a certain period of time, appropriate anticoagulants are added to the blood preservation fluid and deterioration during the storage period is prevented as much as possible.

[0005] Lymphocytes in human peripheral blood are almost all in the GI phase (G0 phase), and generally do not continue to divide. If mitogens and antigenic substances such as concanavalin A (ConA), lipopolysaccharide (LPS), interleukin 2 (IL-2), and phytohemagglutinin (pHA) are added to the preservation solution, lymphocytes will be stimulated by the inducer to transform into lymphoblasts and then enter mitosis. After a short period of cell culture, some cells in the metaphase of mitosis can be obtained.

[0006] Based on the above principle, existing in vitro detection kits, such as in vitro qualitative detection of adenosine triphosphate (ATP) in human whole blood CD4 and T lymphocytes after stimulation and activation, and CD4 cell ATP release test by luciferase method, are used to monitor the body's immune status, that is, when the immune system function is abnormal, the antibody level or T and B cell function in the body will be abnormally reduced or increased, and the number and status of CD4 and T lymphocytes will usually change significantly. In the body's immune activity, ATP is needed as an energy substance to provide energy for the entire process. Therefore, by measuring the concentration of adenosine triphosphate (ATP) in CD4 and T lymphocytes after pHA activation stimulation, the body's immune status can be reflected.

[0007] However, the whole blood sample is a sample that has been out of the body for more than 30 hours. After stimulation, the test value drops significantly, affecting the accuracy of the test results. The existing invention CN 106796219 A (A method for stabilizing whole blood samples) discloses a whole blood sample stabilizer and method. After being placed for 30 hours or longer, the preservation stability effect is still good for the non-stimulated group of samples in the test. However, it has a greater impact on the stimulation value, and the ATP test value of the stimulated group blood sample is extremely unstable.

[0008] Therefore, it is necessary to study a cell stabilizer. After the blood sample preserved by the stabilizer is stimulated after being out of body for more than 30 hours, its ATP detection value can remain stable, thereby extending the storage time of the whole blood sample after being out of body and ensuring the test results of the in vitro detection kit. Summary of the invention

[0009] The object of the present invention is to overcome at least one deficiency of the prior art and provide a cell stabilizer and application thereof.

[0010] The technical solution adopted by the present invention is: In a first aspect, the present invention provides a cell stabilizer comprising the following components: a cell separation agent, a caspase inhibitor, a platelet inhibitor, a mimetic peptide, α-D-glucose, acetyl-L-cysteine, NaCl, and anhydrous sodium acetate.

[0011] In some examples, the concentrations of the components in the cell stabilizer are: cell separation agent 3-10% (w / v), caspase inhibitor 3-8 uM, peptide mimetic 0.33-0.40 mM, α-D-glucose 3.35-7.25 mM, N-acetyl-L-cysteine ​​0.50-1.50 mM, NaCl 2-15 mM, platelet inhibitor 0.02-90 ug / ml, anhydrous sodium acetate 20-40 mM.

[0012] In some examples, the cell separation agent is selected from Percoll reagent or Ficoll 70 reagent.

[0013] In some examples, the platelet inhibitor is selected from clopidogrel, ticlopidine, dipyridamole, ifetroban, abciximab, tirofiban, epGIFibatide, or anagrelide.

[0014] In some examples, the caspase inhibitor is selected from at least one of (3S) 5 (2,6-difluorophenoxy) 3[[(2S) 3 methyl 2 (quinoline 2-carbonylamino)butyryl]amino]4-oxopentanoic acid, methyl (3S) 5 fluoro 3[[(2S) 2 [[(2S) 3 methyl 2 (phenylmethoxycarbonylamino)butyryl]amino]propionyl]amino]4-oxopentanoate, (S) methyl 5 (2,6-difluorophenoxy) 3 ((S) 3 methyl 2 (quinoline 2-carboxamido)butyryl) 4-oxopentanoate or 5-fluoro 3 [(2-methylpropane 2-yl)oxycarbonylamino]4-oxopentanoic acid methyl ester.

[0015] In some examples, the concentrations of the components in the cell stabilizer are: Percoll reagent 3-4% (w / v), caspase inhibitor 4-6 uM, mimetic peptide 0.35-0.39 mM, α-D-glucose 5.35-7.00 mM, N-acetyl-L-cysteine ​​0.60-1.00 mM, NaCl 3-10 mM, clopidogrel 0.5-20 ug / ml, and anhydrous sodium acetate 20-40 mM.

[0016] In a second aspect, the cell stabilizer provided in the first aspect of the present invention is used in the storage of whole blood.

[0017] In a third aspect, the present invention provides a whole blood preservation method, comprising adding the cell stabilizer described in the first aspect to the collected fresh blood.

[0018] In some examples, the cell stabilizer is added within 1 hour after fresh blood is collected or is added to the blood collection tube before blood collection.

[0019] In some examples, the blood is stored at a temperature of 0 to 37° C.; and / or the storage time does not exceed 78 h.

[0020] The beneficial effects of the present invention are: The cell stabilizer provided by the present invention can prolong the storage time of whole blood samples after ex vivo storage, thereby prolonging the detection time of the collected samples of the detection kit, and ensuring the stability of the ATP detection value of the detection kit. In addition, the cell stabilizer can also preserve bone marrow, peripheral blood, tissues, etc. for a long time, and ensure the stability and accuracy of the results of various cell-related tests such as immunophenotyping and lymphocyte subpopulation detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the change in the proportion of granulocytes, monocytes and lymphocytes in long-term stored samples. DETAILED DESCRIPTION

[0022] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0023] In the examples and comparative examples of the present invention, the mimetic peptide is selected from EPO mimetic peptide, and the AIMdex kit (produced by Guangzhou Red Biotechnology Co., Ltd.) is used for detection. The detection steps of the AIMdex kit include: S1. Blood sample stimulation: a) Invert the heparin anticoagulated whole blood 8 times to mix, take out 250 μL of anticoagulated whole blood and 750 μL of sample diluent with a 1 mL pipette, add them to a sterile 1.5 mL centrifuge tube, cover the centrifuge tube, and mix them 8 times by inverting; b) Using a U-shaped cell culture plate, each whole blood sample to be tested uses 2 wells, one of which is a non-stimulation well and the other is a stimulation well. Two replicates are set for each whole blood sample; c) Add 25 μL of sample diluent to each non-stimulation well to serve as a non-stimulation control well for the test sample; d) Add 25 μL of pre-diluted stimulant to each stimulation well, which will serve as the stimulation well for the test sample; e) Add 100 μL of the diluted sample to each well; f) Cover the U-shaped cell culture plate with a lid and shake it on a microplate shaker for 2 minutes at 400-600 rpm.

[0024] S2. Incubate at 37°C for 15-17 hours: The 96-well U-shaped cell culture plate was incubated in a 37°C incubator for 15 to 17 hours.

[0025] S3. Cell sorting: a) Take out the U-shaped cell culture plate from the 37°C incubator and add CD4 magnetic beads; b) Cover the U-shaped cell culture plate with a lid, shake on a microplate shaker, and incubate at room temperature for 20 minutes; c) Place the U-shaped cell culture plate on a magnetic rack to adsorb magnetic beads for 2 to 3 minutes to separate CD4 cells; d) Aspirate the whole blood after CD4 cells are separated to avoid taking away the magnetic beads; e) Wash CD4 cells.

[0026] S4. ATP detection: a) Remove the magnetic stand, add 80 μL of lysis buffer to each well, gently pipette 3 to 5 times until the magnetic beads are evenly dispersed, react for 3-5 minutes, and place on the magnetic stand for adsorption for 3 minutes; b) Take out the reaction microplate strips required for this experiment, and the remaining ones should be sealed in time; c) Add lysate supernatant and calibrator to the detection wells, 30 uL / well; d) Add 70 μL of reaction working solution to each of the sample wells and calibration wells, and gently pipette 2 to 3 times; e) The reaction microplate was left to react at room temperature and away from light for 5 minutes; f) Fluorescence measurement is performed using a chemiluminescent immunoassay analyzer, wherein a zero-concentration calibrator dilution is set as a blank control well.

[0027] Example 1 A cell stabilizer was prepared, wherein the control group was a blank control, and the content of anhydrous sodium acetate in the cell stabilizer in the experimental groups 1-1, 1-2, 1-3, 1-4, 1-5, and 1-6 was shown in Table 1, and the other compositions were as follows: Percoll 3% (w / v), caspase inhibitor 5 uM, EPO mimetic peptide 0.38 mM, α-D-glucose 6.75 mM, N-acetyl-L-cysteine ​​(NALC) 0.77 mM, NaCl 8.5 mM, platelet inhibitor PI (clopidogrel) 5 ug / ml.

[0028] Percoll is used as a cell separation agent in whole blood to reduce the squeezing of upper layer cells on lower layer cells or between cells when whole blood is stored for a long time; EPO mimetic peptides act directly on EPO receptors to promote erythropoiesis; α-D-glucose, a nutrient necessary for red blood cell metabolism, can prolong the storage time of red blood cells and prevent hemolysis; it can also slow down the disappearance of organic phosphorus in cells and prevent red blood cell storage damage; Acetyl-L-cysteine ​​NALC; mucolytic agent, inhibits the stimulation of viruses by reactive oxygen intermediates; Platelet inhibitors inhibit platelet adhesion and aggregation.

[0029] Table 1. Components of the cell stabilizer formulation in Example 1 After the preparation, 4 freshly collected whole blood samples from healthy people were used. The original blood was tested using the AIMdex kit within 1 hour of collection. The results of the stimulated group and the non-stimulated group are shown in Table 2.

[0030] Table 2 Within 1 hour after the original blood samples were obtained, the control group, experimental groups 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, a total of 7 groups of stabilizers were added and stored at room temperature (24±1°C). Stimulated and non-stimulated blood sample tests were performed 54h, 72h and 78h after the addition of cell stabilizers. The test results of whole blood sample storage time and whether it was stimulated were compared. The results are shown in Tables 3 to 5.

[0031] Table 3 Comparison of ATP detection deviations of whole blood in the stimulated group and the non-stimulated group after storage for 54 h in Example 1 Table 4 Comparison of ATP detection deviations of whole blood in the stimulated group and the non-stimulated group after storage for 72 hours in Example 1 Table 5 Comparison of ATP detection deviations of whole blood in the stimulated group and the non-stimulated group after storage for 78 h in Example 1 From the data in Tables 2 to 5, we can see that: 1) In the control group (without any stabilizer), the whole blood had expired after being stored for 54 hours or 72 / 78 hours, and could not be tested using the AIMdex kit. The detection deviation within 1 hour from fresh blood was more than 60% in both the stimulated and non-stimulated groups.

[0032] 2) The amount of anhydrous sodium acetate added in experimental groups 1-1 and 1-2 was 60mM and 50mM, respectively. After the whole blood of these two groups of stabilizers was stored for 54h and 72 / 78h, the detection deviation of the non-stimulated group was less than 20%, and a deviation of 21.2% occurred at 78 hours, which was a small deviation, indicating that the cell stabilizers of these two groups can effectively preserve the whole blood, while the detection deviations of the stimulated groups exceeded 20%, indicating that although the stabilizer did not have a large deviation effect on the non-stimulated group, the stimulated group had a large deviation and failed to achieve the effect of stable preservation of whole blood.

[0033] 3) In experimental groups 1-3, 1-4 and 1-5, the addition amount of anhydrous sodium acetate was 40mM, 30mM and 20mM respectively, and ATP detection was performed at storage time of 54h and 72 / 78h. The detection deviation was kept within 20%, which shows that the cell stabilizer within this concentration range can effectively and stably preserve whole blood for a long time, thereby extending the time from sample collection to detection of the detection kit and ensuring the stability of the ATP detection value of the detection kit.

[0034] 4) Experimental groups 1-6 (zero amount of anhydrous sodium acetate added) were also unable to preserve whole blood. After 54 hours of storage, the whole blood was tested by the AIMdex test kit and showed a deviation of more than 50% (compared with the test within 1 hour of fresh blood), indicating that the whole blood had expired.

[0035] Comparative Example 1 A cell stabilizer was prepared, wherein the control group was a blank control, the content of disodium hydrogen phosphate in the cell stabilizer in comparative examples 1-1, 1-2, 1-3, 1-4, 1-5, and 1-6 was shown in Table 6, and the other compositions were as follows: Percoll 3% (w / v), caspase inhibitor 5 uM, EPO mimetic peptide 0.38 mM, α-D-glucose 6.75 mM, N-acetyl-L-cysteine ​​(NALC) 0.77 mM, NaCl 8.5 mM, platelet inhibitor PI (clopidogrel) 5 ug / ml.

[0036] Table 6, Comparative Example 1 Cell Stabilizer Formula Components The effects of different cell stabilizers on ATP detection after different whole blood storage times were detected according to the method described in Example 1. The test results are shown in Tables 7 to 9.

[0037] Table 7. Comparative Example 1 Comparison of ATP detection deviations in whole blood of the stimulated group and the non-stimulated group after 54 hours of storage Table 8. Comparative Example 1: Comparison of ATP detection deviations between whole blood in the stimulated group and the non-stimulated group after storage for 72 hours Table 9. Comparative Example 1 Comparison of ATP detection deviations in whole blood of the stimulated group and the non-stimulated group after storage for 78 hours From the data in Tables 7 to 9, it can be seen that compared with Tables 3 to 5 in Example 1, when stored for 54 hours in Comparative Example 1, the detection deviation of the stimulation group was greater than 20%, and the detection deviation of the non-stimulation group was less than 20%. This shows that although the cell stabilizer in Comparative Example 1 can effectively preserve blood for 54 hours, the detection deviation of the stimulation group is large, so it is not suitable for the storage of whole blood samples in the kit. When stored for 78 hours, the detection deviation of the stimulation group and the non-stimulation group is greater than 20%.

[0038] Comparative Example 2 A cell stabilizer was prepared, wherein the control group was a blank control, the content of trisodium citrate dihydrate in the cell stabilizer in Comparative Examples 2-1, 2-2, 2-3, 2-4, 2-5, and 2-6 was shown in Table 10, and the other compositions were as follows: Percoll 3% (w / v), caspase inhibitor 5 uM, EPO mimetic peptide 0.38 mM, α-D-glucose 6.75 mM, N-acetyl-L-cysteine ​​(NALC) 0.77 mM, NaCl 8.5 mM, and platelet inhibitor PI (clopidogrel) 5 ug / ml.

[0039] Table 10, Comparative Example 2 Cell Stabilizer Formula Components The effects of different cell stabilizers on ATP detection after different whole blood storage times were detected according to the method described in Example 1. The test results are shown in Tables 11 to 13.

[0040] Table 11. Comparative Example 2: Comparison of ATP detection deviations between whole blood in the stimulation group and the non-stimulation group after 54 hours of storage Table 12. Comparative Example 2: Comparison of ATP detection deviations between whole blood in the stimulated group and the non-stimulated group after storage for 72 hours Table 13. Comparative Example 2: Comparison of ATP detection deviations between whole blood in the stimulation group and the non-stimulation group after storage for 78 hours It can be seen from the data in Tables 11 to 13 that the deviations of most groups in Comparative Example 2 are greater than 20%, and therefore the whole blood sample storage of the kit is not suitable.

[0041] Comparative Example 3 A cell stabilizer was prepared, wherein the control group was a blank control, and the contents of trisodium citrate dihydrate and sodium dihydrogen phosphate dihydrate in comparative examples 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6 in the cell stabilizer were shown in Table 14, and the other components were as follows: Percoll 3% (w / v), caspase inhibitor 5 uM, EPO mimetic peptide 0.38 mM, α-D-glucose 6.75 mM, N-acetyl-L-cysteine ​​(NALC) 0.77 mM, NaCl 8.5 mM, and platelet inhibitor PI (clopidogrel) 5 ug / ml.

[0042] Table 14, Comparative Example 3 Cell Stabilizer Formula Components The effects of different cell stabilizers on ATP detection after different whole blood storage times were detected according to the method described in Example 1. The test results are shown in Tables 15 to 17.

[0043] Table 15. Comparative Example 3: Comparison of ATP detection deviations between whole blood in the stimulation group and the non-stimulation group after 54 hours of storage Table 16. Comparative Example 3: Comparison of ATP detection deviations between whole blood in the stimulation group and the non-stimulation group after storage for 72 hours Table 17. Comparative Example 3: Comparison of ATP detection deviations between whole blood in the stimulation group and the non-stimulation group after storage for 78 hours It can be seen from the data in Tables 15 to 17 that the deviations of most groups in Comparative Example 3 are greater than 20%, and therefore the whole blood sample storage of the kit is not suitable.

[0044] Comparative Example 4 A cell stabilizer was prepared, wherein the control group was a blank control, and the specific composition and content were shown in Table 18.

[0045] The effects of different cell stabilizers on ATP detection after different whole blood storage times were detected according to the method described in Example 1. The test results are shown in Tables 19 and 20.

[0046] Table 18, Comparative Example 4 Different Cell Stabilizer Composition Table 19. Comparative Example 4: Comparison of ATP detection deviations in whole blood from the stimulation group and the non-stimulation group after 54 hours of storage Table 20. Comparative Example 4: Comparison of ATP detection deviations in whole blood of the stimulation group and the non-stimulation group after storage for 72 hours It can be seen from the data in Tables 19 and 20 that, compared with Example 1, the components in Comparative Example 4 are not suitable for storing whole blood for use in the detection kit.

[0047] Example 2 Application of the cell stabilizer of the present invention in flow cytometry sample storage Take the cell stabilizer prepared according to Example 1, directly add 1.5 mL of normal human peripheral blood to each tube, store at 2-8°C, and conduct a long-term (June 28, 2024 to August 17, 2024, for 50 days) cell preservation test. During this period, take a tube of preserved samples every five to nine days for flow cytometry detection. The results are as follows Figure 1 shown. Figure 1 The changes in the proportions of granulocytes, monocytes, and lymphocytes in long-term stored samples are shown. The results show that for these three types of cells as a whole, they can maintain good proportion stability after long-term storage. The above experiment tested a total of 15 parallel samples, and all obtained results similar to the examples. In summary, peripheral blood samples using the cell stabilizer of the present invention can be stored for at least 50 days at 2-8°C.

[0048] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A cell stabilizer, characterized in that The composition includes the following components: cell separation agent, caspase inhibitor, platelet inhibitor, mimetic peptide, α-D-glucose, acetyl-L-cysteine, NaCl, and anhydrous sodium acetate.

2. The cell stabilizer according to claim 1, characterized in that The concentrations of the components in the cell stabilizer are: cell separation agent 3-10% (w / v), caspase inhibitor 3-8 uM, peptide simulation 0.33-0.40 mM, α-D-glucose 3.35-7.25 mM, N-acetyl-L-cysteine ​​0.50-1.50 mM, NaCl 2-15 mM, platelet inhibitor 0.02-90 ug / ml, and anhydrous sodium acetate 20-40 mM.

3. The cell stabilizer according to claim 1, characterized in that The cell separation agent is selected from Percoll reagent or Ficoll 70 reagent.

4. The cell stabilizer according to claim 1, characterized in that The platelet inhibitor is selected from clopidogrel, ticlopidine, dipyridamole, ifetroban, abciximab, tirofiban, epGIFibatide or anagrelide.

5. The cell stabilizer according to claim 1, characterized in that The caspase inhibitor is selected from at least one of (3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butyryl]amino]-4-oxopentanoic acid, methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butyryl]amino]propionyl]amino]-4-oxopentanoate, (S)-methyl 5-(2,6-difluorophenoxy)-3-((S)-3-methyl-2-(quinoline-2-carboxamido)butyryl)-4-oxopentanoate or 5-fluoro-3-[(2-methylpropane-2-yl)oxycarbonylamino]-4-oxopentanoic acid methyl ester.

6. The cell stabilizer according to any one of claims 1 to 5, characterized in that The concentrations of the components in the cell stabilizer are: Percoll reagent 3-4% (w / v), caspase inhibitor 4-6 uM, peptide mimetic 0.35-0.39 mM, α-D-glucose 5.35-7.00 mM, N-acetyl-L-cysteine ​​0.60-1.00 mM, NaCl 3-10 mM, clopidogrel 0.5-20 ug / ml, and anhydrous sodium acetate 20-40 mM.

7. Use of the cell stabilizer according to any one of claims 1 to 6 in the storage of whole blood.

8. A whole blood storage method, characterized in that: The method comprises adding the cell stabilizer according to any one of claims 1 to 6 to the collected fresh blood.

9. The whole blood storage method according to claim 8, characterized in that: The cell stabilizer is added within 1 hour after fresh blood is collected or is added to the blood collection tube before blood collection.

10. The whole blood storage method according to claim 8, characterized in that: The storage temperature of the blood is 0 to 37° C.; and / or the storage time does not exceed 78 hours.

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