Cell preserving fluid, red blood cell simulant and preparation method and application of red blood cell simulant

Abstract

The invention relates to the field of cell preservation solutions, and provides a cell preservation solution, an erythrocyte simulant and a preparation method and application of the erythrocyte simulant. The cell preservation solution comprises a polyphenol compound, a buffer system, a metal chelating agent, a nutrient substance, a metabolism regulator, mannitol, poloxamer and an optional preservative. The preparation method of the red blood cell simulant comprises the following steps: taking mammal venous blood and purifying to obtain red blood cells; fixing the obtained red blood cells by using a fixing agent; and dispersing the red blood cells obtained by immobilization treatment in the cell preserving fluid provided by the invention. The cell preserving fluid is beneficial for improving the stability of red blood cells and enabling the red blood cells to maintain good uniformity; the red blood cells in the red blood cell simulant have good bottle opening stability, thermal stability and long-term stability, the variable coefficient of the red blood cells is low, and the product batch-to-batch repeatability is good.

Description

Technical Field

[0001] The invention belongs to the technical field of blood cell analysis, and in particular relates to a cell preservation solution and a red blood cell simulant, and a preparation method and application thereof. Background Art

[0002] The number and morphology-related parameters of red blood cells are key indicators for routine blood screening. Modern blood cell analyzers play an important role in clinical testing, especially in red blood cell analysis, with the following advantages: ① Blood cell analyzers use a variety of technologies (such as flow cytometry, electrical impedance method, etc.), which can quickly provide high-precision and accurate red blood cell parameters; ② Comprehensive red blood cell morphology analysis: Advanced blood cell analyzers have image analysis functions, which help to identify pathological morphological changes of red blood cells while automatically analyzing and classifying red blood cell morphology. In blood research and applications, red blood cell stability is a core concern. Taking the quality control material / calibration material for blood cell analyzers as an example, the red blood cell particle component accounts for about 70% or more, and its stability greatly affects the overall shelf life and performance of the product; containing stable red blood cell particle clusters can not only effectively ensure the accuracy and reliability of blood analysis, but also expand the market potential of the product.

[0003] The red blood cell simulants obtained through conventional preparation processes such as hemoglobin oxidation and fixation can achieve the effect of instrument identification of red blood cell related parameters (such as RBC, HGB, HCT, etc.), but there are still problems in this application field, including the following: (1) Insufficient uniformity, increasing the risk of hemolysis: Studies have shown that the deterioration of uniformity is related to the fragility of the cell membrane structure, and poor uniformity will lead to different tolerance of different cells to osmotic pressure, thereby increasing the risk of hemolysis. In order to standardize cell morphology, inducing spheroidization by adding reagents is an effective means. However, it is difficult to control the balance between osmotic pressure and cell membrane tension. Moreover, during long-term storage, partial spheroidization cannot completely solve the problem of deterioration of uniformity. (2) Insufficient stability of mean corpuscular size (MCV): In order to match the instrument reagents, it has become a consensus in the field that the fixation strength of red blood cells should not be too high. As the research deepened, we found that in a low-temperature storage environment, the fixed red blood cell particles will have storage damage, such as the accumulation of endogenous reactive oxygen species, lipid peroxidation, and accumulation of metabolites; the performance characteristics on the blood cell analyzer are: poor MCV stability, large coefficient of variation (CV) and significant trend changes, and there is a risk of loss of control. (3) Insufficient stability of red blood cell scatter plot: In order to improve the accuracy of analysis, special reagents will pre-spheroidize the red blood cell particles. If the stability of the red blood cell membrane is good, the graph displayed by the detection channel has a good aggregation degree and presents an elliptical shape; if the stability of the red blood cell membrane is insufficient, the graph displayed by the detection channel presents a vertical spindle shape, with relatively concentrated particles at the head and a "tailing phenomenon" with a high degree of dispersion at the bottom. (4) Batch difference: Due to individual differences in the source of raw materials and complex human factors in the process, batch differences may be uncontrollable.

[0004] Therefore, obtaining red blood cell analogs with good uniformity, good stability and small batch-to-batch differences has certain application value in the field of blood cell analysis; solving the existing problems in this application field (raw material uniformity and cell preservation system stability) is a direction worth exploring. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a cell preservation fluid and a red blood cell simulant and a preparation method and application thereof. The cell preservation fluid of the present invention is beneficial to improving the stability of red blood cells and maintaining good uniformity of red blood cells; the red blood cells in the red blood cell simulant of the present invention have good opening stability, thermal stability and long-term stability, and the coefficient of variation of the red blood cells is low, and the product batch-to-batch repeatability is good.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a cell preservation solution, which comprises a polyphenol compound, a buffer system, a metal chelator, nutrients, a metabolic regulator, mannitol, poloxamer and an optional preservative.

[0008] In some embodiments of the present invention, the concentration of the polyphenolic compound in the cell preservation solution is 50 to 300 mg / L, preferably 100 to 200 mg / L. For example, the concentration of the polyphenolic compound is 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg / L.

[0009] In some embodiments of the present invention, the concentration of the metal chelator in the cell preservation solution is 2 to 3 g / L, for example, the concentration of the metal chelator is 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 g / L.

[0010] In some embodiments of the present invention, the concentration of the nutrient in the cell preservation solution is 5 to 10 g / L, for example, the concentration of the nutrient is 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 g / L.

[0011] In some embodiments of the present invention, the concentration of mannitol in the cell preservation solution is 2 to 10 g / L. For example, the concentration of mannitol is 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L.

[0012] In some embodiments of the present invention, the concentration of poloxamer in the cell preservation solution is 100-200 μL / L. For example, the concentration of poloxamer is 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μL / L.

[0013] In some embodiments of the present invention, the concentration of the metabolic regulator in the cell preservation solution is 0.5 to 2 g / L. For example, the concentration of the metabolic regulator is 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g / L.

[0014] In some embodiments of the present invention, the concentration of the preservative in the cell preservation solution is 0 to 0.5 g / L. For example, the concentration of the preservative is 0, 0.1, 0.2, 0.3, 0.4 or 0.5 g / L.

[0015] In some embodiments of the present invention, the pH value of the cell preservation solution is 7±0.5.

[0016] In some embodiments of the present invention, the osmotic pressure of the cell preservation solution is 200-300 mOsm / kg.

[0017] In some embodiments of the present invention, the buffer system includes dipotassium hydrogen phosphate and disodium hydrogen phosphate. Preferably, the concentration of dipotassium hydrogen phosphate in the cell preservation solution is 1-2 g / L, and the concentration of disodium hydrogen phosphate is 10-20 g / L.

[0018] In some embodiments of the present invention, the nutrients include one or more of glucose, trehalose, glycine, and glucose zinc.

[0019] In some embodiments of the invention, the metabolic regulator comprises folic acid and / or inositol.

[0020] In some embodiments of the present invention, the metal chelator comprises EDTA-2Na.

[0021] In some embodiments of the present invention, the preservative includes one or more of GML-2 antibacterial agent (bisimidazole uron antibacterial agent), PC950 antibacterial agent, and PC300 antibacterial agent, preferably GML-2 antibacterial agent.

[0022] In some embodiments of the present invention, the solvent of the cell preservation solution is purified water.

[0023] In some embodiments of the present invention, the polyphenol compound is one or more of resveratrol, piceatannol, stilbene glycoside, and catechin.

[0024] In some embodiments of the present invention, the poloxamer is selected from poloxamer 407 and / or poloxamer 188.

[0025] In a second aspect, the present invention provides a method for preparing the cell preservation solution described in the first aspect, comprising the following steps: dispersing polyphenol compounds, a buffer system, a metal chelator, nutrients, a metabolic regulator, mannitol, poloxamer and an optional preservative in a solvent; preferably, the pH value of the prepared solution is controlled to be 7±0.5 and the osmotic pressure is controlled to be 200-300mOsm / kg.

[0026] In a third aspect, the use of the cell preservative solution described in the first aspect or the cell preservative solution prepared by the preparation method described in the second aspect in the preparation of a cell mimic; preferably, the preparation of a cell mimic refers to a process of preparing a cell mimic based on a hypotonic method, and / or, the use includes using the cell preservative solution to preserve cells during the preparation of a cell mimic.

[0027] In a fourth aspect, the present invention provides a red blood cell simulant, comprising the cell preservative fluid described in the first aspect or the cell preservative fluid prepared by the preparation method described in the second aspect, and red blood cells dispersed or preserved in the cell preservative fluid described in the first aspect or the cell preservative fluid prepared by the preparation method described in the second aspect.

[0028] In a fifth aspect, the present invention provides a method for preparing a red blood cell mimetic, comprising: (1) Take mammalian venous blood and purify it to obtain red blood cells; (2) fixing the red blood cells obtained in step (1) with a fixative; (3) Dispersing the red blood cells obtained by the fixation treatment in step (2) in the cell preservation solution described in the first aspect or the cell preservation solution prepared by the preparation method described in the second aspect.

[0029] In some embodiments of the present invention, the fixative comprises the following components: formaldehyde, glutaraldehyde and optionally a protein denaturant.

[0030] In some embodiments of the present invention, in the fixative, the volume concentration of the formaldehyde is 0.1% to 0.5%, and the volume concentration of the glutaraldehyde is 0.1% to 0.5%.

[0031] In some embodiments of the present invention, the protein denaturant includes guanidine thiocyanate; more preferably, in the fixative, the concentration of guanidine thiocyanate is 0.5 to 2.5 g / L. For example, the concentration of guanidine thiocyanate is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 g / L.

[0032] In some embodiments of the present invention, the pH value of the fixative is 7±0.5.

[0033] In some embodiments of the present invention, the osmotic pressure of the fixative is 250-350 mOsm / kg.

[0034] In some embodiments of the present invention, the solvent of the fixative is the cell preservation solution described in the first aspect or the cell preservation solution prepared by the preparation method described in the second aspect.

[0035] In some embodiments of the present invention, the step (1) further comprises concentrating the purified mammalian venous blood; preferably, the RBC (red blood cell count) value in the concentrated red blood cell concentrate is controlled to be (8.0±0.5) x10 12 / L.

[0036] In some embodiments of the present invention, the volume of the fixative during the fixation process in step (2) is 10 to 20 times that of the red blood cells obtained in step (1).

[0037] In some embodiments of the present invention, the fixation treatment time in step (2) is 15 to 20 hours.

[0038] In some embodiments of the invention, the purifying comprises filtering out leukocytes from the venous blood of the mammal.

[0039] In some embodiments of the present invention, the step (1) further comprises subjecting the purified red blood cells to hypotonic treatment using a hypotonic cell membrane protective agent.

[0040] In some embodiments of the present invention, the cell membrane protectant includes a choline reagent; preferably, the choline reagent includes citicoline and / or choline chloride, preferably citicoline. Citicoline is one of the main components of the red blood cell membrane, which can enhance the stability and elasticity of the red blood cell membrane and prevent the rupture and dissolution of the red blood cell membrane.

[0041] In some embodiments of the present invention, the concentration of the choline agent in the cell membrane protective agent is 2 to 8 g / L. For example, the concentration of the choline agent is 2, 3, 4, 5, 6, 7, 8 g / L.

[0042] In some embodiments of the present invention, the pH value of the cell membrane protective agent is 6.5±0.5; preferably, the pH value of the cell membrane protective agent is adjusted by sodium chloride.

[0043] In some embodiments of the present invention, the osmotic pressure of the cell membrane protective agent is 200-300 mOsm / kg; preferably, the osmotic pressure of the cell membrane protective agent is adjusted by sodium chloride.

[0044] In some embodiments of the present invention, the hypotonic treatment time is 30 to 90 minutes, preferably 45 to 75 minutes.

[0045] In some embodiments of the present invention, the solvent of the cell membrane protective agent is water, preferably pure water.

[0046] In some embodiments of the present invention, the volume of the cell membrane protective agent during the hypotonic treatment is 1 to 3 times that of the red blood cells.

[0047] In some embodiments of the present invention, the preparation method further includes washing the red blood cells with a phosphate buffer after the hypotonic treatment; preferably, the concentration of the phosphate buffer is 50 to 200 mmol / L; more preferably, the preparation method further includes centrifuging the red blood cells after washing with the phosphate buffer and discarding the supernatant to remove the residual cell membrane protective agent and separate the cell debris generated by the aforementioned operation steps; further preferably, the centrifugal speed is 2000 to 3000 rpm, and the centrifugal time is 5 to 15 min.

[0048] In some embodiments of the present invention, step (1) further comprises screening the mean red blood cell size of mammalian venous blood using a fully automatic blood cell analyzer before purification.

[0049] In some embodiments of the present invention, the screening allows EDTA anticoagulated venous blood to meet 50 to 105 fL (femtoliters). For example, when the mammalian venous blood comes from humans, the EDTA anticoagulated venous blood meets 90 to 105 fL; when the mammalian venous blood comes from pigs, the EDTA anticoagulated venous blood meets 58 to 68 fL; when the mammalian venous blood comes from rabbits, the EDTA anticoagulated venous blood meets 65 to 75 fL.

[0050] In some embodiments of the present invention, the step (1) includes both step ①: using a hypotonic cell membrane protective agent to perform hypotonic treatment on the purified red blood cells and step ②: using a fully automatic blood cell analyzer to screen the mean red blood cell size of the mammalian venous blood before purification, or only includes any one of step ① and step ②. When both step ① and step ② are included, the order of the two steps can be arbitrary. Preferably, when the mammalian venous blood in step (1) is fresh blood, the step (1) includes both step ① and step ② or only includes step ①; when the mammalian venous blood in step (1) is blood that has been isolated for more than 7 days, the step (1) includes only step ② or performs step ② and step ① in sequence.

[0051] In some embodiments of the present invention, the step (2) further comprises washing the red blood cells with a phosphate buffer after the fixation treatment; preferably, the concentration of the phosphate buffer is 50 to 200 mmol / L.

[0052] In a sixth aspect, the present invention provides a red blood cell mimic prepared by the preparation method described in the fifth aspect.

[0053] In a seventh aspect, the present invention provides use of the red blood cell analogue described in the fourth aspect or the sixth aspect in the preparation of quality control materials and / or calibration materials for blood cell analysis.

[0054] In an eighth aspect, the present invention provides a quality control material for blood cell analysis, wherein the quality control material comprises the red blood cell simulant described in the fourth aspect or the sixth aspect.

[0055] In some embodiments of the present invention, the quality control material further comprises leukocyte simulation particles and / or platelet simulation particles, the cell preservation fluid described in the first aspect, or the cell preservation fluid prepared by the preparation method described in the second aspect.

[0056] In some embodiments of the present invention, the red blood cell simulation particles in the quality control material are 800-1250 / μL, the white blood cell simulation particles are 184-280 / μL, and the platelet simulation particles are 50-500 / μL.

[0057] The beneficial effects of the present invention are as follows: The present invention proposes a cell preservation solution, which includes an antioxidant selected from polyphenolic compounds, and the polyphenolic compounds can improve the antioxidant capacity of red blood cells, overcome the problems of reduced cell deformability and reduced fragility caused by processes such as oxidative stress of cells under low-temperature storage, and thus improve the stability of cells. In addition, the cell preservation solution of the present invention also includes mannitol and poloxamer, wherein mannitol is conducive to maintaining the osmotic pressure balance inside and outside the cell membrane, and poloxamer is conducive to reducing the shear stress of cells. The combination of the two can effectively maintain the deformability of cells and thus improve the stability of cells. Furthermore, the cell preservation solution of the present invention also includes a buffer system, a metal chelator, a nutrient, a metabolic regulator and an optional preservative, and these components are conducive to ensuring the viability of cells and further improving the stability of cells. The preparation method of the cell preservation solution of the present invention is simple, which is convenient for the industrial production of cell preservation solution.

[0058] The present invention also proposes a red blood cell simulant dispersed or stored in the cell preservation solution of the present invention and a preparation method thereof. The red blood cells are dispersed in the cell preservation solution of the present invention, so that the red blood cell simulant has high stability; in addition, the preparation method of the red blood cell simulant of the present invention is simple to operate, and the obtained red blood cell simulant has not only high stability, but also high uniformity, and the product batch reproducibility is good. The red blood cell simulant of the present invention has good application prospects in the preparation of quality control materials and / or calibration materials for blood cell analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Indicates the components and dosages of cell preservation solutions 1 to 20, as well as the pH value and osmotic pressure of each cell preservation solution.

[0060] Figure 2 It indicates the corresponding relationship between the quality control substances 1-22, the red blood cell simulants 1-22 and the cell preservation solutions 1-20.

[0061] Figure 3 The results of open-bottle stability analysis and 37°C thermal accelerated stability analysis of red blood cell mimetics 1 to 22 are shown.

[0062] Figure 4 The results of open bottle stability analysis, 37°C thermal accelerated stability analysis, and long-term stability analysis of quality control substances 1 to 22 are shown.

[0063] Figures 5 to 26 The sequence is red blood cell simulant 1 and quality control substance 1, red blood cell simulant 2 and quality control substance 2, red blood cell simulant 3 and quality control substance 3, red blood cell simulant 4 and quality control substance 4, red blood cell simulant 5 and quality control substance 5, red blood cell simulant 6 and quality control substance 6, red blood cell simulant 7 and quality control substance 7, red blood cell simulant 8 and quality control substance 8, red blood cell simulant 9 and quality control substance 9, red blood cell simulant 10 and quality control substance 10, red blood cell simulant 11 and quality control substance 11, red blood cell simulant 12 and quality control substance 13. The analysis results of control substance 12, red blood cell simulant 13 and quality control substance 13, red blood cell simulant 14 and quality control substance 14, red blood cell simulant 15 and quality control substance 15, red blood cell simulant 16 and quality control substance 16, red blood cell simulant 17 and quality control substance 17, red blood cell simulant 18 and quality control substance 18, red blood cell simulant 19 and quality control substance 19, red blood cell simulant 20 and quality control substance 20, red blood cell simulant 21 and quality control substance 21, red blood cell simulant 22 and quality control substance 22. DETAILED DESCRIPTION

[0064] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0065] Components of cell preservation solution 1-20 and preparation method thereof The components and dosage of each component in each 1L of cell preservation solution are as follows: Figure 1 As shown, the solvent was purified water.

[0066] The preparation method of the cell preservation solution comprises: dispersing a polyphenol compound, a buffer system, a metal chelating agent, a nutrient, a metabolic regulator, mannitol, poloxamer and an optional preservative in purified water to obtain a cell preservation solution; detecting the pH value and osmotic pressure of the cell preservation solution, and if the pH value of the cell preservation solution is not within the range of 7±0.5, adjusting the pH value of the cell preservation solution to within the range of 7±0.5 using sodium hydroxide, and finally obtaining a pH value and osmotic pressure of the cell preservation solution such as Figure 1 shown.

[0067] The obtained cell preservation solutions 1 to 20 will be used in the following examples and comparative examples to prepare red blood cell simulants and quality control materials.

[0068] Erythrocyte Simulant Example 1 A method for preparing a red blood cell mimic, the method comprising: (1) Take human EDTA anticoagulated venous blood and discard the upper plasma. Wash once with 100mmol / L phosphate buffer, centrifuge at 2700rpm for 10min, discard the supernatant, remove white blood cells through a leukocyte filter, and then adjust the red blood cell concentration to make the RBC value meet (8.0±0.5)x10 12 / L to obtain red blood cell concentrate.

[0069] (2) Add 4 g of citicoline and 7.5 g of sodium chloride to 1 L of pure water to prepare a treatment solution with a pH of 6.35 and an osmotic pressure of 247 mOsm / kg. Mix the red blood cell concentrate obtained in step (1) with the treatment solution in a volume ratio of 1:2. After mixing, place the mixture at room temperature for 1 hour.

[0070] (3) The red blood cells treated in step (2) were washed twice by centrifugation with 100 mmol / L phosphate buffer solution at 2700 rpm for 10 min, and the supernatant was discarded.

[0071] (4) Formaldehyde, glutaraldehyde and guanidine thiocyanate are added to the cell preservation solution 1, with each liter of the fixative containing 3.5 mL of formaldehyde, 2 mL of glutaraldehyde and 1.5 g of guanidine thiocyanate, to prepare a fixative with a pH value of 7.33 and an osmotic pressure of 290 mOsm / kg; the red blood cells treated in step (3) are added to the fixative and allowed to stand at room temperature for 16 h.

[0072] (5) The red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 1, and then the red blood cells are dispersed and suspended with cell preservation solution 1, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 1.

[0073] Erythrocyte Simulant Example 2 A method for preparing a red blood cell simulant, wherein steps (1), (3) and (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that: Step (2) is as follows: (2) 8 g of citicoline and 7 g of sodium chloride are added to 1 L of pure water to prepare a treatment solution having a pH value of 6.43 and an osmotic pressure of 244 mOsm / kg; the red blood cell concentrate obtained in step (1) is mixed with the treatment solution in a volume ratio of 1:2; after mixing, the mixture is placed at room temperature for 1 hour.

[0074] Step (5) is as follows: (5) the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 2, and then the red blood cells are dispersed and suspended with cell preservation solution 2, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 2.

[0075] Erythrocyte Simulant Example 3 A method for preparing a red blood cell simulant, wherein steps (1), (3) and (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that: Step (2) is as follows: (2) 4 g of citicoline and 8 g of sodium chloride are added to 1 L of pure water to prepare a treatment solution having a pH value of 6.35 and an osmotic pressure of 261 mOsm / kg; the red blood cell concentrate obtained in step (1) is mixed with the treatment solution in a volume ratio of 1:2; after mixing, the mixture is placed at room temperature for 1 hour.

[0076] Step (5) is as follows: (5) the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 3, and then the red blood cells are dispersed and suspended with cell preservation solution 3, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 3.

[0077] Erythrocyte Simulant Example 4 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 4, and then the red blood cells are dispersed and suspended with cell preservation solution 4, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 4.

[0078] Erythrocyte Simulant Example 5 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 5, and then the red blood cells are dispersed and suspended using cell preservation solution 5, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 5.

[0079] Erythrocyte Simulant Example 6 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of red blood cell simulant Example 1; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 6, and then the red blood cells are dispersed and suspended using cell preservation solution 6, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 6.

[0080] Erythrocyte Simulant Example 7 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 7, and then the red blood cells are dispersed and suspended using cell preservation solution 7, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 7.

[0081] Erythrocyte Simulant Example 8 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 8, and then the red blood cells are dispersed and suspended with cell preservation solution 8, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 8.

[0082] Erythrocyte Simulant Example 9 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 9, and then the red blood cells are dispersed and suspended with cell preservation solution 9, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 9.

[0083] Erythrocyte Simulant Example 10 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 10, and then the red blood cells are dispersed and suspended with cell preservation solution 10, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 10.

[0084] Erythrocyte Simulant Example 11 A method for preparing a red blood cell simulant, wherein steps (1), (3) and (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that: Step (2) is as follows: (2) 5 g of citicoline and 7.5 g of sodium chloride are added to 1 L of pure water, and the pH value is adjusted to 6.5±0.5 with sodium hydroxide to prepare a treatment solution with an osmotic pressure of 247 mOsm / kg. The red blood cell concentrate obtained in step (1) is mixed with the treatment solution in a volume ratio of 1:3, and after mixing, the mixture is placed at room temperature for 1 hour.

[0085] Step (5) is as follows: (5) the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 11, and then the red blood cells are dispersed and suspended with cell preservation solution 11, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 11.

[0086] Erythrocyte Simulant Example 12 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 12, and then the red blood cells are dispersed and suspended using cell preservation solution 12, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 12.

[0087] Erythrocyte Simulant Example 13 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 13, and then the red blood cells are dispersed and suspended using cell preservation solution 13, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 13.

[0088] Erythrocyte Simulant Example 14 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 14, and then the red blood cells are dispersed and suspended using cell preservation solution 14, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 14.

[0089] Erythrocyte Simulant Example 15 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 15, and then the red blood cells are dispersed and suspended using cell preservation solution 15, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 15.

[0090] Erythrocyte Simulant Example 16 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 16, and then the red blood cells are dispersed and suspended using cell preservation solution 16, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 16.

[0091] Erythrocyte Simulant Example 17 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of red blood cell simulant Example 1; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 17, and then the red blood cells are dispersed and suspended using cell preservation solution 17, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 17.

[0092] Erythrocyte Simulant Example 18 A method for preparing a red blood cell simulant, wherein steps (1) to (4) of the preparation method are the same as those of Example 1 of the red blood cell simulant; the difference is that step (5) is as follows: the red blood cells fixed in step (4) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 18, and then the red blood cells are dispersed and suspended using cell preservation solution 18, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 18.

[0093] Erythrocyte Simulant Example 19 A method for preparing a red blood cell mimic, the method comprising: (1) After human EDTA anticoagulated venous blood is placed in a low-temperature environment of 2-8°C for 3 days, the mean corpuscular volume (MCV) is screened using an automatic blood cell analyzer to ensure that the human EDTA anticoagulated venous blood meets the requirement of 90-105 fL.

[0094] (2) Take the human EDTA anticoagulated venous blood obtained in step (1) and discard the upper plasma. Wash once with 100mmol / L phosphate buffer, centrifuge at 2700rpm for 10min, discard the supernatant, remove the white blood cells through a leukocyte filter, and then adjust the red blood cell concentration to make the RBC value meet (8.0±0.5)x10 12 / L to obtain red blood cell concentrate.

[0095] (3) Add 4 g of citicoline and 7.5 g of sodium chloride to 1 L of pure water to prepare a treatment solution with a pH of 6.35 and an osmotic pressure of 247 mOsm / kg. Mix the red blood cell concentrate obtained in step (2) with the treatment solution in a volume ratio of 1:2. After mixing, place the mixture at room temperature for 1 hour.

[0096] (4) The red blood cells treated in step (3) were washed twice by centrifugation with 100 mmol / L phosphate buffer solution at 2700 rpm for 10 min, and the supernatant was discarded.

[0097] (5) Formaldehyde, glutaraldehyde and guanidine thiocyanate are added to the cell preservation solution 1, with each liter of the fixative containing 3.5 mL of formaldehyde, 2 mL of glutaraldehyde and 1.5 g of guanidine thiocyanate, to prepare a fixative with a pH value of 7.33 and an osmotic pressure of 290 mOsm / kg; the red blood cells treated in step (4) are added to the fixative and allowed to stand at room temperature for 16 h.

[0098] (6) The red blood cells fixed in step (5) are washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 19, and then the red blood cells are dispersed and suspended with cell preservation solution 19 and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 19.

[0099] Erythrocyte Simulant Example 20 A method for preparing a red blood cell simulant, wherein steps (1) to (5) of the preparation method are the same as those of red blood cell simulant Example 19, except that step (6) is as follows: the red blood cells fixed in step (5) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 11, and then the red blood cells are dispersed and suspended using cell preservation solution 11, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 20.

[0100] Erythrocyte Simulant Example 21 A method for preparing a red blood cell simulant, wherein steps (1) to (5) of the preparation method are the same as those of red blood cell simulant Example 19, except that step (6) is as follows: the red blood cells fixed in step (5) are washed once with 100 mmol / L phosphate buffer, centrifuged at a centrifugal speed of 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 3, and then the red blood cells are dispersed and suspended using cell preservation solution 3, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 21.

[0101] Erythrocyte Simulant Comparative Example 1 A method for preparing a red blood cell mimic, the method comprising: (1) Take human EDTA anticoagulated venous blood and discard the upper plasma. Wash once with 100mmol / L phosphate buffer, centrifuge at 2700rpm for 10min, discard the supernatant, remove white blood cells through a leukocyte filter, and then adjust the red blood cell concentration to make the RBC value meet (8.0±0.5)x10 12 / L to obtain red blood cell concentrate.

[0102] (2) Formaldehyde, glutaraldehyde and guanidine thiocyanate are added to the cell preservation solution 1, with each liter of the fixative containing 3.5 mL of formaldehyde, 2 mL of glutaraldehyde and 1.5 g of guanidine thiocyanate, to prepare a fixative with a pH value of 7.33 and an osmotic pressure of 290 mOsm / kg; the red blood cell concentrate obtained in step (1) is added to the fixative and allowed to stand at room temperature for 16 h.

[0103] (3) The red blood cells fixed in step (2) are washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant is discarded; then washed twice with cell preservation solution 20, and then the red blood cells are dispersed and suspended with cell preservation solution 20 and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 22.

[0104] Application Examples The quality control material for blood cell analysis includes red blood cell simulation particles, white blood cell simulation particles, platelet simulation particles and cell preservation fluid; wherein the red blood cell simulation particles are obtained by washing the red blood cell simulation material once with the cell preservation fluid, centrifuging at 2700rpm for 5 minutes and discarding the supernatant; the white blood cell simulation particles are obtained by washing the white blood cell simulation material commercially available from MacBio Inc. using pig blood as the raw material once with the cell preservation fluid, centrifuging at 2700rpm for 5 minutes and discarding the supernatant; the platelet simulation particles are obtained by washing the platelet simulation material commercially available from MacBio Inc. using pig blood as the raw material once with the cell preservation fluid, centrifuging at 2700rpm for 5 minutes and discarding the supernatant; in the quality control material, the white blood cell simulation particles are 250 / μL, the red blood cell simulation particles are 1050 / μL, and the platelet simulation particles are 250 / μL. According to the composition of the above quality control materials and according to Figure 2 Prepare quality control substances 1 to 22 according to the corresponding relationships in the text.

[0105] Product stability analysis For the red blood cell simulations 1 to 22 prepared in the red blood cell simulation examples 1 to 21 and the red blood cell simulation comparative example 1, and the quality control materials 1 to 22 prepared in the application example, the red blood cell related numerical stability thereof was monitored using the fully automatic blood cell analyzer F880 manufactured by MacBio Inc., including RBC (red blood cell count), HGB (hemoglobin concentration), HCT (hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin content), MCHC (mean corpuscular hemoglobin concentration), RDW-SD (standard deviation of red blood cell distribution width), and RDW-CV (coefficient of variation of red blood cell distribution width); in addition, the graphs shown by the FCR channel when the red blood cell simulations 1 to 22 were analyzed using the fully automatic blood cell analyzer F880 manufactured by MacBio Inc. were recorded simultaneously. Among them, the analysis results of the red blood cell simulations were derived from the open bottle stability (15 days) and the 37°C thermal accelerated stability (5 days), and the analysis results of the quality control materials were derived from the open bottle stability (15 days), the 37°C thermal accelerated stability (5 days), and the long-term stability (90 days).

[0106] The results obtained by analyzing red blood cell simulants 1 to 22 and quality control substances 1 to 22 are as follows: Figures 3 to 26 As shown. Among them, Figure 3 Displayed are the average values ​​of RBC, HGB, HCT, MCV, MCH, MCHC, RDW-SD, and RDW-CV of red blood cell mimics 1 to 22, as well as the range of MCV. Figure 4 Displayed are the average values ​​of RBC, HGB, HCT, MCV, MCH, MCHC, RDW-SD, and RDW-CV of quality control materials 1 to 22, as well as the range of MCV. Figures 5 to 26 The left side of each figure is, from top to bottom, the MCV of the red blood cell simulant on days 1 to 15 during the open-bottle stability analysis, the MCV of the red blood cell simulant on days 1 to 5 during the 37°C thermal accelerated stability analysis, the graphs shown by the FCR channel on days 1 and 15 during the open-bottle stability analysis of the red blood cell simulant, and the graphs shown by the FCR channel on days 1 and 5 during the 37°C thermal accelerated stability analysis of the red blood cell simulant; Figures 5 to 26 On the right side of each figure, from top to bottom, are the MCVs of the quality control materials on days 1 to 15 in the open bottle stability analysis, the MCVs of the quality control materials on days 1 to 5 in the 37°C thermal accelerated stability analysis, and the MCVs of the quality control materials on days 1, 15, 30, 45, 60, 75, and 90 in the long-term stability analysis.

[0107] from Figure 3 and 4 It can be seen that the opening stability and 37°C thermal acceleration stability of red blood cell simulant 22 are the worst among all cell simulants, and the opening stability, 37°C thermal acceleration stability and long-term stability of quality control material 22 are also the worst among all quality control materials; it proves that adding polyphenolic compounds, metabolic regulators and poloxamer to cell preservation fluid can help improve the stability of red blood cells. The analysis results of red blood cell simulant 5 and quality control material 5 show that their MCV range is generally high, indicating that the lower dosage of polyphenolic compounds is not effective in reducing the batch-to-batch difference of the product. The analysis results of 37°C thermal acceleration stability and long-term stability of quality control material 15 indicate that a higher dosage of polyphenolic compounds will lead to larger batch-to-batch differences in thermal stability and long-term stability of the product.

[0108] Graphical analysis of red blood cell simulants 1, 2, 3, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21 and quality control materials 1, 2, 3, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21 showed that in the analysis of stability after bottle opening (15 days), the red blood cell graph had a high aggregation degree, and the graph on the 15th day after bottle opening did not show tailing diffusion phenomenon; in the analysis of thermal accelerated stability at 37°C (5 days), the direction of the red blood cell graph changed and there were some scattered points in the lower part, but no significant tailing diffusion phenomenon was shown. Figure 3 and Figure 4 From the data analysis, it can be seen that the hemolysis probability of the red blood cells in most of the red blood cell simulants and quality control materials of the present invention is 0% in the analysis of open bottle stability (15 days), long-term stability (90 days) and 37°C thermal accelerated stability (5 days), and the batch-to-batch repeatability is relatively good; these data indicate that these red blood cell simulants and quality control materials have a certain degree of stability.

[0109] Graphic analysis of red blood cell simulants 4, 12, 20 and quality control materials 4, 12, 20 showed that in the analysis of stability after bottle opening (15 days), the red blood cell graph had a high aggregation degree, and the graph on the 15th day after bottle opening did not show tailing and diffusion; in the analysis of thermal acceleration stability at 37°C (5 days), the direction of the red blood cell graph changed and the graph was relatively stable, with no obvious scatter points. Figure 3 and Figure 4 From the data analysis, it can be seen that the hemolysis probability of the red blood cell simulants 4, 12, 20 and the quality control substances 4, 12, 20 of the present invention is 0% in the analysis of open bottle stability (15 days), long-term stability (90 days) and 37°C thermal accelerated stability (5 days), and the batch reproducibility is good; these data indicate that the red blood cell simulants 4, 12, 20 and the quality control substances 4, 12, 20 have good stability.

[0110] Graphic analysis of red blood cell simulant 5 and quality control substance 5 showed that in the analysis of stability after opening the bottle (15 days), the red blood cell graph had a high degree of aggregation, and the graph on the 15th day after opening the bottle did not show tailing diffusion phenomenon; however, in the analysis of thermal accelerated stability at 37°C (5 days), the direction of the red blood cell graph changed, showing a vertical spindle shape, with relatively concentrated particles at the head and a "tailing phenomenon" with a high degree of diffusion at the bottom, indicating that a lower dosage of polyphenol compounds has relatively poor effect on improving graph stability.

[0111] Graphical analysis of red blood cell simulant 22 and quality control material 22 showed that in the open bottle stability analysis (15 days), scattered points appeared in the red blood cell graph on the 15th day; in the 37°C thermal accelerated stability analysis (5 days), the direction of the graph changed, showing a vertical spindle shape, with relatively concentrated particles at the head and a "tailing phenomenon" with a high degree of diffusion at the bottom.

[0112] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words.

Claims

1. A cell preservation solution, characterized in that: The cell preservation solution comprises polyphenol compounds, a buffer system, a metal chelating agent, nutrients, a metabolic regulator, mannitol, poloxamer and an optional preservative; wherein, In the cell preservation solution, the concentration of polyphenol compounds is 100-200 mg / L, the concentration of metal chelators is 2-3 g / L, the concentration of nutrients is 5-10 g / L, the concentration of mannitol is 2-10 g / L, the concentration of poloxamer is 100-200 μL / L, the concentration of metabolic regulators is 0.5-2 g / L, and the concentration of preservatives is 0-0.5 g / L.

2. The cell preservation solution according to claim 1, characterized in that The pH value of the cell preservation solution is 7±0.5; and / or The osmotic pressure of the cell preservation solution is 200-300 mOsm / kg; and / or The buffer system comprises dipotassium hydrogen phosphate and disodium hydrogen phosphate; and / or The nutrients include one or more of glucose, trehalose, glycine, and glucose zinc; and / or The metabolic regulators include folic acid and / or inositol; and / or The metal chelating agent comprises EDTA-2Na; and / or The preservative includes one or more of GML-2 antibacterial agent, PC950 antibacterial agent, and PC300 antibacterial agent; and / or The solvent of the cell preservation solution is purified water; and / or The polyphenol compound is one or more of resveratrol, piceatannol, stilbene glycoside, and catechins; and / or The poloxamer is selected from poloxamer 407 and / or poloxamer 188.

3. The method for preparing the cell preservation solution according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: dispersing polyphenol compounds, a buffer system, a metal chelating agent, nutrients, a metabolic regulator, mannitol, poloxamer and an optional preservative in a solvent.

4. A method for preparing a red blood cell mimetic, characterized in that: The preparation method comprises: (1) Take mammalian venous blood and purify it to obtain red blood cells; (2) fixing the red blood cells obtained in step (1) with a fixative; (3) Dispersing the red blood cells obtained by the fixation treatment in step (2) in the cell preservation solution described in claim 1 or 2 or the cell preservation solution prepared by the preparation method described in claim 3.

5. The preparation method according to claim 4, characterized in that: The fixing agent comprises the following components: formaldehyde, glutaraldehyde and an optional protein denaturing agent; and, In the fixative, the volume concentration of formaldehyde is 0.1% to 0.5%, and the volume concentration of glutaraldehyde is 0.1% to 0.5%; and / or The protein denaturant includes guanidine thiocyanate; in the fixative, the concentration of guanidine thiocyanate is 0.5 to 2.5 g / L; and / or The pH value of the fixative is 7±0.5; and / or The osmotic pressure of the fixative is 250-350 mOsm / kg; and / or The solvent of the fixative is the cell preservation solution according to claim 1 or 2 or the cell preservation solution prepared according to claim 3; and / or The volume of the fixative during the fixation treatment in step (2) is 10 to 20 times that of the red blood cells obtained in step (1); and / or The fixation treatment time in step (2) is 15 to 20 hours.

6. The preparation method according to claim 4 or 5, characterized in that: The purification comprises filtering out leukocytes from mammalian venous blood; and / or The step (1) further comprises subjecting the purified red blood cells to hypotonic treatment using a hypotonic cell membrane protective agent; and / or The step (1) also includes screening the mean red blood cell size of mammalian venous blood using a fully automatic blood cell analyzer before purification.

7. The preparation method according to claim 6, characterized in that: The cell membrane protective agent includes a choline agent; and / or The pH value of the cell membrane protective agent is 6.5±0.5; and / or The osmotic pressure of the cell membrane protective agent is 200-300 mOsm / kg; and / or The hypotonic treatment time is 30 to 90 minutes; and / or The solvent of the cell membrane protective agent is water.

8. An erythrocyte mimetic prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the red blood cell mimic according to claim 8 in the preparation of quality control materials and / or calibration materials for blood cell analysis.

10. A quality control substance for blood cell analysis, characterized in that: The quality control material comprises the red blood cell mimic described in claim 8.

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