A cell preservation solution and red blood cell simulant and preparation method and application thereof

Abstract

The present invention relates to the field of cell preservation fluid, and provides a cell preservation fluid and a red blood cell simulant, and a preparation method and application thereof. The cell preservation fluid comprises a polyphenol compound, a buffer system, a metal chelator, a nutrient, a metabolic regulator, mannitol, a poloxamer, and an optional preservative. The preparation method of the red blood cell simulant comprises the following steps: taking venous blood from a mammal and purifying it to obtain red blood cells; then fixing the obtained red blood cells with a fixative; and then dispersing the red blood cells obtained by the fixation in the cell preservation fluid of the present invention. 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 reproducibility is good.

Description

Technical Field

[0001] The present 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, as well as a preparation method and application thereof. Background Art

[0002] The number and morphology of red blood cells are key indicators in routine blood screening. Modern hematology analyzers play an important role in clinical testing, especially in red blood cell analysis, with the following advantages: ① Hematology analyzers use a variety of technologies (such as flow cytometry and electrical impedance spectroscopy) to quickly provide high-precision and accurate red blood cell parameters; ② Comprehensive red blood cell morphology analysis: Advanced hematology analyzers have image analysis capabilities that help identify pathological morphological changes in 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 quality control materials / calibration materials used in hematology analyzers as an example, red blood cell particle components account for approximately 70% or more, and their 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 in this application field, there are still problems 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 tolerances 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 research deepened, we found that in a low-temperature storage environment, fixed red blood cell clusters would suffer from storage damage, such as the accumulation of endogenous reactive oxygen species, lipid peroxidation, and accumulation of metabolites. The performance characteristics of the hematology analyzer were as follows: poor MCV stability, large coefficient of variation (CV), and significant trend changes, which posed a risk of loss of control. (3) Insufficient stability of the red blood cell scatter plot: In order to improve the accuracy of the analysis, the special reagents will pre-spheroidize the red blood cell clusters. If the red blood cell membrane is stable, the graph displayed by the detection channel will have a good aggregation degree and appear elliptical. If the red blood cell membrane is unstable, the graph displayed by the detection channel will appear vertically spindle-shaped, with relatively concentrated particles at the head and a high degree of dispersion at the bottom, which is a "tailing phenomenon." (4) Batch-to-batch variation: Due to individual differences in the source of raw materials and the complexity of human factors in the process, batch-to-batch variation 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 their preparation method and application. 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 reproducibility 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 comprising a polyphenol compound, a buffer system, a metal chelating agent, 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 50-300 mg / L, preferably 100-200 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, 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, 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 (bisimidazolidinyl urea 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 a polyphenol compound, a buffer system, a metal chelator, nutrients, a metabolic regulator, mannitol, poloxamer and an optional preservative in a solvent; preferably, controlling the pH value of the prepared solution to 7±0.5 and the osmotic pressure to 200-300 mOsm / kg.

[0026] In the 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 cell mimics; preferably, the preparation of cell mimics refers to a process of preparing cell mimics based on the hypotonic method, and / or, the use includes using the cell preservative solution to preserve cells during the preparation of cell mimics.

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

[0029] (1) Take mammalian venous blood and purify it to obtain red blood cells;

[0030] (2) fixing the red blood cells obtained in step (1) with a fixative;

[0031] (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.

[0032] In some embodiments of the present invention, the fixative comprises the following components: formaldehyde, glutaraldehyde, and optionally a protein denaturant.

[0033] 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%.

[0034] In some embodiments of the present invention, the protein denaturant comprises guanidine thiocyanate; more preferably, the concentration of guanidine thiocyanate in the fixative 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, or 2.5 g / L.

[0035] In some embodiments of the present invention, the pH value of the fixative is 7±0.5.

[0036] In some embodiments of the present invention, the osmotic pressure of the fixative is 250-350 mOsm / kg.

[0037] 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.

[0038] 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 of the concentrated red blood cell concentrate is controlled to be (8.0±0.5) x10 12 / L.

[0039] In some embodiments of the present invention, 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).

[0040] In some embodiments of the present invention, the fixation treatment time in step (2) is 15 to 20 hours.

[0041] In some embodiments of the invention, the purifying comprises filtering out leukocytes from the mammalian venous blood.

[0042] In some embodiments of the present invention, the step (1) further comprises performing hypotonic treatment on the purified red blood cells using a hypotonic cell membrane protective agent.

[0043] 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 and can enhance the stability and elasticity of the red blood cell membrane and prevent the rupture and dissolution of the red blood cell membrane.

[0044] 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, or 8 g / L.

[0045] 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.

[0046] 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.

[0047] In some embodiments of the present invention, the hypotonic treatment time is 30 to 90 minutes, preferably 45 to 75 minutes.

[0048] In some embodiments of the present invention, the solvent of the cell membrane protective agent is water, preferably pure water.

[0049] 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 red blood cells.

[0050] 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 residual cell membrane protective agents and separate cell debris generated by the aforementioned operation steps; further preferably, the centrifugal speed is 2000 to 3000 rpm, and the centrifugation time is 5 to 15 min.

[0051] In some embodiments of the present invention, step (1) further comprises screening the mean corpuscular size of mammalian venous blood using a fully automatic blood cell analyzer before purification.

[0052] In some embodiments of the present invention, the screening method allows EDTA-anticoagulated venous blood to meet a range of 50 to 105 fL (femtoliters). For example, when the mammalian venous blood is from a human, the EDTA-anticoagulated venous blood meets a range of 90 to 105 fL; when the mammalian venous blood is from a pig, the EDTA-anticoagulated venous blood meets a range of 58 to 68 fL; and when the mammalian venous blood is from a rabbit, the EDTA-anticoagulated venous blood meets a range of 65 to 75 fL.

[0053] In some embodiments of the present invention, the step (1) also includes 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 steps ① and 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 steps ① and 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) only includes step ② or performs steps ② and ① in sequence.

[0054] 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.

[0055] In a sixth aspect, the present invention provides a red blood cell mimic prepared by the preparation method described in the fifth aspect.

[0056] In a seventh aspect, the present invention provides use of the red blood cell mimic described in the fourth aspect or the sixth aspect in the preparation of quality control materials and / or calibration materials for blood cell analysis.

[0057] 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.

[0058] In some embodiments of the present invention, the quality control material further comprises leukocyte-simulating particles and / or platelet-simulating 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.

[0059] In some embodiments of the present invention, the concentration of red blood cell simulating particles in the quality control material is 800-1250 / μL, the concentration of white blood cell simulating particles is 184-280 / μL, and the concentration of platelet simulating particles is 50-500 / μL.

[0060] The beneficial effects of the present invention are as follows:

[0061] The present invention proposes a cell preservation solution, which includes an antioxidant selected from polyphenolic compounds. The polyphenolic compounds can improve the antioxidant capacity of red blood cells, overcome the problems of reduced cell deformability and reduced brittleness 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 helps to reduce the shear stress of the cells. The combination of the two can effectively maintain the deformability of the cells and thus improve the stability of the cells. Furthermore, the cell preservation solution of the present invention also includes a buffer system, a metal chelator, nutrients, a metabolic regulator and an optional preservative, and these components are conducive to ensuring the viability of the cells and further improving the stability of the cells. The preparation method of the cell preservation solution of the present invention is simple and is convenient for industrial production of cell preservation solution.

[0062] The present invention also provides a red blood cell mimetic dispersed or preserved in the cell preservation fluid of the present invention and a method for preparing the same. Dispersing the red blood cells in the cell preservation fluid of the present invention can impart high stability to the red blood cell mimetic. Furthermore, the method for preparing the red blood cell mimetic of the present invention is simple to operate, and the resulting red blood cell mimetic exhibits not only high stability but also high homogeneity and good batch-to-batch reproducibility. The red blood cell mimetic of the present invention has promising application prospects in the preparation of quality control materials and / or calibration materials for blood cell analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Indicates the components and dosage of cell preservation solutions 1 to 20, as well as the pH value and osmotic pressure of each cell preservation solution.

[0064] Figure 2Indicates the corresponding relationship between quality control substances 1-22, red blood cell simulants 1-22 and cell preservation solutions 1-20.

[0065] Figure 3 The results of open-bottle stability analysis and 37°C heat-accelerated stability analysis of red blood cell mimetics 1 to 22 are shown.

[0066] Figure 4 The results of open-bottle stability analysis, 37°C accelerated thermal stability analysis, and long-term stability analysis of quality control substances 1 to 22 are shown.

[0067] Figures 5 to 26 The order 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. 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

[0068] The following examples further illustrate the technology of the present invention. These examples are for illustration and example only and are not intended to limit the scope of the present invention in any form.

[0069] Components of cell preservation solution 1 to 20 and preparation method thereof

[0070] The components and dosage of each component in each 1L cell preservation solution are as follows: Figure 1 As shown, the solvent was purified water.

[0071] 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, a 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; 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 with sodium hydroxide; and finally obtaining a cell preservation solution having a pH value and an osmotic pressure as follows: Figure 1 shown.

[0072] 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.

[0073] Erythrocyte Simulant Example 1

[0074] A method for preparing a red blood cell mimic, comprising:

[0075] (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 meet the RBC value of (8.0±0.5)x10 12 / L to obtain red blood cell concentrate.

[0076] (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, mix well, and let it stand at room temperature for 1 hour.

[0077] (3) The red blood cells treated in step (2) were washed twice by centrifugation with 100 mmol / L phosphate buffer solution at a centrifugal speed of 2700 rpm for 10 min, and the supernatant was discarded.

[0078] (4) Formaldehyde 3.5 mL, glutaraldehyde 2 mL, and guanidine isothiocyanate 1.5 g are added to the cell preservation solution 1 to prepare a fixative having a pH of 7.33 and an osmotic pressure of 290 mOsm / kg, with each liter of fixative containing 3.5 mL of formaldehyde, 2 mL of glutaraldehyde, and 1.5 g of guanidine isothiocyanate. The erythrocytes treated in step (3) are added to the fixative and allowed to stand at room temperature for 16 h.

[0079] (5) The red blood cells fixed in step (4) were washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant was discarded; then washed twice with cell preservation solution 1, and then the red blood cells were dispersed and suspended in cell preservation solution 1 and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 1.

[0080] Erythrocyte Simulant Example 2

[0081] 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:

[0082] 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, and the red blood cell concentrate obtained in step (1) is mixed with the treatment solution in a volume ratio of 1:2, and after mixing, the mixture is placed at room temperature for 1 hour.

[0083] 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 minutes, 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 to obtain red blood cell simulant 2.

[0084] Erythrocyte Simulant Example 3

[0085] 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:

[0086] 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, and the red blood cell concentrate obtained in step (1) is mixed with the treatment solution in a volume ratio of 1:2, and after mixing, the mixture is placed at room temperature for 1 hour.

[0087] 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 minutes, 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 to obtain red blood cell simulant 3.

[0088] Erythrocyte Simulant Example 4

[0089] 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 to obtain red blood cell simulant 4.

[0090] Erythrocyte Simulant Example 5

[0091] 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 5, and then the red blood cells are dispersed and suspended in cell preservation solution 5, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 5.

[0092] Erythrocyte Simulant Example 6

[0093] 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 6, and then the red blood cells are dispersed and suspended in cell preservation solution 6, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 6.

[0094] Erythrocyte Simulant Example 7

[0095] 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 7, and then the red blood cells are dispersed and suspended with cell preservation solution 7, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 7.

[0096] Erythrocyte Simulant Example 8

[0097] 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 to obtain red blood cell simulant 8.

[0098] Erythrocyte Simulant Example 9

[0099] 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 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 to obtain red blood cell simulant 9.

[0100] Erythrocyte Simulant Example 10

[0101] 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 to obtain red blood cell simulant 10.

[0102] Erythrocyte Simulant Example 11

[0103] 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:

[0104] 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 then 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.

[0105] 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 minutes, 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.

[0106] Erythrocyte Simulant Example 12

[0107] 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 with cell preservation solution 12, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 12.

[0108] Erythrocyte Simulant Example 13

[0109] 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 in cell preservation solution 13, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 13.

[0110] Erythrocyte Simulant Example 14

[0111] 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 with cell preservation solution 14, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 14.

[0112] Erythrocyte Simulant Example 15

[0113] 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 in cell preservation solution 15, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 15.

[0114] Erythrocyte Simulant Example 16

[0115] 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 with cell preservation solution 16, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 16.

[0116] Erythrocyte Simulant Example 17

[0117] 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 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 to obtain red blood cell simulant 17.

[0118] Erythrocyte Simulant Example 18

[0119] 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 with cell preservation solution 18, and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 18.

[0120] Erythrocyte Simulant Example 19

[0121] A method for preparing a red blood cell mimic, comprising:

[0122] (1) After human EDTA anticoagulated venous blood was placed in a low temperature environment of 2-8°C for 3 days, the mean corpuscular volume (MCV) of the human EDTA anticoagulated venous blood was screened using an automatic blood cell analyzer to ensure that the mean corpuscular volume (MCV) of the human EDTA anticoagulated venous blood met the range of 90-105 fL.

[0123] (2) The human EDTA-anticoagulated venous blood obtained in step (1) was treated and the upper plasma was discarded. The blood was washed once with 100 mmol / L phosphate buffer and centrifuged at 2700 rpm for 10 min. The supernatant was discarded and the leukocytes were removed by a leukocyte filter. The red blood cell concentration was adjusted to a value of (8.0 ± 0.5) x 10 12 / L to obtain red blood cell concentrate.

[0124] (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, mix well, and let it stand at room temperature for 1 hour.

[0125] (4) The red blood cells treated in step (3) were washed twice by centrifugation with 100 mmol / L phosphate buffer solution at a centrifugal speed of 2700 rpm for 10 min, and the supernatant was discarded.

[0126] (5) Formaldehyde 3.5 mL, glutaraldehyde 2 mL, and guanidine isothiocyanate 1.5 g are added to the cell preservation solution 1 to prepare a fixative having a pH of 7.33 and an osmotic pressure of 290 mOsm / kg, with each liter of fixative containing 3.5 mL of formaldehyde, 2 mL of glutaraldehyde, and 1.5 g of guanidine isothiocyanate. The erythrocytes treated in step (4) are added to the fixative and allowed to stand at room temperature for 16 h.

[0127] (6) The red blood cells fixed in step (5) were washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant was discarded; then washed twice with cell preservation solution 19, and then the red blood cells were dispersed and suspended using cell preservation solution 19 and stored at a low temperature of 2 to 8°C to obtain red blood cell simulant 19.

[0128] Erythrocyte Simulant Example 20

[0129] A method for preparing a red blood cell simulant, wherein steps (1) to (5) of the preparation method are the same as those of Example 19 of the red blood cell simulant, 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 minutes, 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.

[0130] Erythrocyte Simulant Example 21

[0131] A method for preparing a red blood cell simulant, wherein steps (1) to (5) of the preparation method are the same as those of Example 19 of the red blood cell simulant, 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 minutes, and the supernatant is discarded; then washed twice with cell preservation solution 3, and then the red blood cells are dispersed and suspended in cell preservation solution 3, and stored at a low temperature of 2 to 8°C, thereby obtaining red blood cell simulant 21.

[0132] Comparative Example 1 of Red Blood Cell Simulants

[0133] A method for preparing a red blood cell mimic, comprising:

[0134] (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 meet the RBC value of (8.0±0.5)x10 12 / L to obtain red blood cell concentrate.

[0135] (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 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.

[0136] (3) The erythrocytes fixed in step (2) were washed once with 100 mmol / L phosphate buffer, centrifuged at 2700 rpm for 10 min, and the supernatant was discarded; the erythrocytes were then washed twice with cell preservation solution 20, and then the erythrocytes were dispersed and suspended in cell preservation solution 20 and stored at a low temperature of 2 to 8°C to obtain erythrocyte simulant 22.

[0137] Application Examples

[0138] 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 once with 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 commercially available from MacBio Inc. using pig blood as raw material once with cell preservation fluid, centrifuging at 2700rpm for 5 minutes and discarding the supernatant; the platelet simulation particles are obtained by washing the platelet simulation commercially available from MacBio Inc. using pig blood as raw material once with 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 material and according to Figure 2 Prepare quality control substances 1 to 22 according to the corresponding relationships in .

[0139] Product stability analysis

[0140] The stability of red blood cell-related parameters, including RBC count (RBC), 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), was monitored for red blood cell simulants 1-22 prepared in Examples 1-21 and Comparative Example 1, as well as quality control materials 1-22 prepared in the Application Example, using a fully automated hematology analyzer F880 manufactured by MacBio Inc. In addition, the FCR channel graphs of red blood cell simulants 1-22 analyzed using the fully automated hematology analyzer F880 manufactured by MacBio Inc. were also recorded. The analysis results for the red blood cell simulants were derived from open-vial stability (15 days) and 37°C accelerated thermal stability (5 days), while the analysis results for the quality control materials were derived from open-vial stability (15 days), 37°C accelerated thermal stability (5 days), and long-term stability (90 days).

[0141] The results of the analysis of 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 Shown 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 and the range of MCV for quality control materials 1 to 22. Figures 5 to 26 On the left side of each figure, from top to bottom, are the MCV of the red blood cell simulant on days 1 to 15 during the open-vial 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 of the red blood cell simulant on days 1 and 15 during the open-vial stability analysis as shown by the FCR channel, and the graphs of the red blood cell simulant on days 1 and 5 during the 37°C thermal accelerated stability analysis as shown by the FCR channel; 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 heat-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.

[0142] from Figure 3 and 4As can be seen, the open-vial stability and 37°C accelerated heat stability of RBC simulant 22 were the worst among all cell simulants, and the open-vial stability, 37°C accelerated heat stability, and long-term stability of control material 22 were also the worst among all quality control materials. This suggests that the addition of polyphenols, metabolic regulators, and poloxamers to cell preservation solutions can improve RBC stability. The analysis results of RBC simulant 5 and control material 5 showed generally high MCV ranges, suggesting that lower polyphenol dosages are ineffective in reducing batch-to-batch variability. The 37°C accelerated heat stability and long-term stability analysis results of control material 15 suggest that higher polyphenol dosages can lead to greater batch-to-batch variability in thermal and long-term stability.

[0143] 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 Data analysis shows that the red blood cells in most of the red blood cell simulants and quality control materials of the present invention have a hemolysis probability of 0% in the open bottle stability (15 days), long-term stability (90 days) and 37°C thermal accelerated stability (5 days) analyses, and the inter-batch reproducibility is relatively good; these data suggest that these red blood cell simulants and quality control materials have a certain degree of stability.

[0144] Graphical analysis of red blood cell simulants 4, 12, 20 and quality control materials 4, 12, 20 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 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 red blood cell simulants 4, 12, 20 and the quality control substances 4, 12, 20 of the present invention have a hemolysis probability of 0% in the open bottle stability (15 days), long-term stability (90 days) and 37°C heat-accelerated stability (5 days) analyses, 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.

[0145] Graphical analysis of red blood cell simulant 5 and quality control substance 5 showed that in the analysis of stability after bottle opening (15 days), the red blood cell graph had a high degree of aggregation, and the graph on the 15th day after bottle opening showed no 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 a relatively poor effect on improving graph stability.

[0146] 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.

[0147] It should be noted that the embodiments described above are only used to illustrate 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 red blood cell preservation solution for blood cell analysis, characterized in that: The red blood 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 red blood 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; The osmotic pressure of the red blood cell preservation solution is 200 to 300 mOsm / kg.

2. The red blood cell storage solution for blood cell analysis according to claim 1, characterized in that The pH value of the red blood cell preservation solution is 7±0.5; and / or The buffer system includes 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 includes 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 red blood cell preservation solution is purified water; and / or The polyphenol compound is one or more of resveratrol, piceatannol, stilbene glycoside, and catechin; and / or The poloxamer is selected from poloxamer 407 and / or poloxamer 188.

3. The method for preparing the red blood cell preservation solution for blood cell analysis 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 mimic, 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 red blood cell preservation solution according to claim 1 or 2 or the red blood cell preservation solution prepared by the preparation method according to claim 3; The osmotic pressure of the fixative is 250 to 350 mOsm / kg.

5. The preparation method according to claim 4, characterized in that The fixative comprises the following components: formaldehyde, glutaraldehyde and an optional protein denaturant; 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 solvent of the fixative is the red blood cell preservation solution according to claim 1 or 2 or the red blood 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 fixing 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 performing hypotonic treatment on the purified red blood cells using a hypotonic cell membrane protective agent; and / or The step (1) further comprises screening the mean red blood cell size of the 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 produced by the 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 according to claim 8.

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