Whole blood quality control product from Macaca thibetana for hematology analyzer and its preparation method

Abstract

The present invention discloses a Tibetan macaque-derived whole blood quality control product for a blood cell analyzer and its preparation method, which relates to the technical field of biological product reagents. It includes: white blood cells: (4-6)×10 9 / L; red blood cells: (1.5-2.5)×10 12 / L; platelets: (1.5-2.5)×10 12 / L; balanced solution: pH 7.2-7.4, osmotic pressure 280-320 mOsm. The present invention uses the peripheral blood of Tibetan macaques as raw materials to prepare a whole blood quality control product with good stability, good uniformity, meeting the requirements of clinical quality control products, and similar to human blood samples. In addition, the quality control product preparation method provided by the present invention is simple and easy to implement, can meet the requirements for the quality control of blood cell analyzers, and has great market promotion and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biological product reagents, and in particular to a Tibetan macaque-derived whole blood quality control product for a blood cell analyzer and a preparation method thereof. Background Art

[0002] As an indispensable diagnostic instrument for medical and health institutions and testing institutions, blood cell analyzers play an important role in the diagnosis, treatment and prognosis of clinical diseases. Therefore, it is very important to do a good job in the indoor quality control of blood cell analyzers to ensure the accuracy of test results. Good indoor quality control is inseparable from quality control products that are similar in nature to the specimens to be tested and have stable quality.

[0003] At present, domestic medical institutions, third-party inspection institutions and other cell analyzer quality control products mainly rely on imports. However, due to various factors such as the need for refrigerated transportation of whole blood quality control products and the need for control of blood product imports, it is not convenient to purchase blood cell analyzer quality control products in China, and their prices are also relatively expensive. In China, blood cell analyzer quality control products are prepared by partially or completely replacing human blood components with pig blood, poultry blood, hoofed animal blood, and particle simulants, but the clinical effect is unsatisfactory. The reason is that the whole blood quality control products prepared with the above-mentioned animal blood as the source are quite different from the clinical human blood sample species, there is a matrix effect, and the cell morphology and structure are different from human blood cells. There are still deficiencies in stability and uniformity, and it is difficult to achieve ideal quality control effects, and clinical quality control requirements cannot be fully met. Therefore, the development of indoor quality control products for the quality control of blood cell analyzers to replace imported products has become an urgent task for laboratory medicine.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a Tibetan macaque-derived whole blood quality control product for a blood cell analyzer and a preparation method thereof to solve the above-mentioned technical problems.

[0006] The present invention is achieved in that:

[0007] In the first aspect, the present invention provides a Tibetan macaque-derived whole blood quality control product for a blood cell analyzer, which comprises: white blood cells: (4-6)×10 9 / L; red blood cells: (1.5-2.5)×10 12 / L; Platelets: (1.5-2.5)×10 12 / L; Balanced solution: pH 7.2-7.4, osmotic pressure 280-320 mOsm; Balanced solution accounts for 50-70% of the volume of whole blood quality control product;

[0008] Among them, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.12 - 0.14 mol / L, mannitol at 0.4 - 0.6 mol / L, glycerol at 0.1 - 0.3%, glucose at 0.4 - 0.6 mol / L, a biological preservative at 0.05 - 0.15% and water.

[0009] In a preferred embodiment of the application of the present invention, the whole blood quality control product includes: white blood cells: 5×10 9 / L; red blood cells: 2×10 12 / L; platelets: 2×10 12 / L.

[0010] In a preferred embodiment of the application of the present invention, the balanced solution accounts for 60% of the volume of the whole blood quality control product.

[0011] In a preferred embodiment of the application of the present invention, the pH of the balanced solution is 7.26 - 7.3;

[0012] In an alternative embodiment, the osmotic pressure of the balanced solution is 304 - 320 mOsm.

[0013] In a preferred embodiment of the application of the present invention, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.129 - 0.14 mol / L, mannitol at 0.5 - 0.6 mol / L, glycerol at 0.2 - 0.3%, glucose at 0.5 - 0.6 mol / L, a biological preservative at 0.1 - 0.15% and water;

[0014] In an alternative embodiment, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.129 mol / L, mannitol at 0.5 mol / L, glycerol at 0.2%, glucose at 0.5 mol / L, a biological preservative at 0.1% and water.

[0015] In a preferred embodiment of the application of the present invention, the biological preservative is selected from any one of ProClin150, ProClin200 and ProClin300.

[0016] In a preferred embodiment of the application of the present invention, the volume ratio of the fixing solution to the blood cells is 1:(4 - 6).

[0017] Secondly, the present invention also provides a preparation method of a Macaca thibetana - derived whole blood quality control product, which includes the following steps:

[0018] Mix the separated white blood cells, red blood cells and platelets from normal Macaca thibetana with the fixing solution according to a volume ratio of 1:(4 - 6) respectively. After fixation, adjust the concentration so that the white blood cell concentration is 5×1010 / L, the platelet concentration is 2×10 13 / L, and the red blood cell concentration is 1×10 13 / L;

[0019] Then, white blood cells, platelets, red blood cells and balanced salt solution are mixed according to a volume ratio of 1:1:2:6.

[0020] The fixing solution includes: 2-3% (v / v) formaldehyde and 0.4-0.6% (v / v) glutaraldehyde.

[0021] In a preferred embodiment of the application of the present invention, the isolated white blood cells, red blood cells and platelets from the normal Tibetan macaque are respectively mixed with the fixing solution according to a volume ratio of 1:5;

[0022] In an alternative embodiment, the fixation is low-temperature fixation;

[0023] In an alternative embodiment, the fixation is carried out at 0-8°C.

[0024] In a preferred embodiment of the application of the present invention, during the fixation process, suspension is carried out every 8-10 minutes.

[0025] In a preferred embodiment of the application of the present invention, the fixation time is 20-60 minutes; in an alternative embodiment, the fixation time is 40 minutes;

[0026] In an alternative embodiment, after fixation, centrifugation is further included, and the supernatant is discarded;

[0027] In an alternative embodiment, the rotation speed of centrifugation is 1800-2200 rpm, centrifugation is carried out at 2-8°C for 20 minutes, after discarding the supernatant, resuspension is carried out with PBS, and then centrifugation is carried out at 2800-3200 rpm for 5 minutes, and the supernatant is discarded.

[0028] In a third aspect, the present invention also provides a kit, which includes the above-mentioned whole blood quality control product from Tibetan macaque or the whole blood quality control product from Tibetan macaque prepared by the above-mentioned preparation method.

[0029] The present invention has the following beneficial effects:

[0030] The present invention uses the peripheral blood of Tibetan macaque as the raw material to prepare a whole blood quality control product with good stability, good uniformity, meeting the requirements of clinical quality control products and being similar to human blood samples. The function of sodium citrate in the quality control product is to adjust the pH value of the solution within a stable range, maintain the osmotic pressure balance, prevent the aggregation of each component of the cells and the lysis of red blood cells; mannitol and glycerol play a role in maintaining the integrity of the cell membrane and preventing hemolysis; glucose provides the energy required for the metabolism of red blood cells; the biological preservative plays a role in preventing the contamination of pathogenic bacteria.

[0031] Compared with the existing human whole blood quality control products, the Tibetan macaque whole blood quality control product adopted by the present invention has convenient and feasible raw material sources, low costs, a simple and easy quality control product preparation method, can meet the requirements for the quality control of hematology analyzers, and has great market promotion and application value.

[0032] The whole blood quality control product provided by the present invention has very good stability. After being stored for 90 days, each index is stable in the long term and is very suitable for the application of hematology analyzers.

[0033] In addition, the present invention also provides the application of the Tibetan macaque whole blood quality control product in a reagent kit. Detailed Description of the Invention

[0034] Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, and not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0035] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0036] In a first aspect, the present invention provides a Tibetan macaque-derived whole blood quality control product for a hematology analyzer, which comprises: white blood cells: (4 - 6)×10 9 / L; red blood cells: (1.5 - 2.5)×10 12 / L; platelets: (1.5 - 2.5)×10 12 / L; balanced solution: pH 7.2 - 7.4, osmotic pressure 280 - 320 mOsm; and the balanced solution accounts for 50 - 70% of the volume of the whole blood quality control product;

[0037] Among them, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.12 - 0.14 mol / L, mannitol at 0.4 - 0.6 mol / L, glycerol at 0.1 - 0.3%, glucose at 0.4 - 0.6 mol / L, a biological preservative at 0.05 - 0.15%, and water.

[0038] The genomes of non - human primate experimental animals have nearly 98% homology with humans, and their physiology, hematology, immunology, etc. are highly consistent with humans. They are the preferred animals for current life medicine research. The Tibetan macaque is a unique non - human primate in China and is the largest species in the genus Macaca in China. The previous research results of the inventor show that its genetic relationship is closer to humans than that of macaques, its hematological index range is basically the same as that of humans, and its cell stability is better.

[0039] Therefore, choosing whole blood from Tibetan macaques to prepare the corresponding whole - blood quality control product has the advantages of convenient and feasible raw material source and low cost compared with human - sourced whole blood.

[0040] The function of sodium citrate in the quality control product is to adjust the solution pH value within a stable range, maintain osmotic pressure balance, prevent the aggregation of each component of cells and the lysis of red blood cells; mannitol and glycerol play the role of maintaining the integrity of cell membranes and preventing hemolysis; glucose provides the energy required for red blood cell metabolism; the biological preservative plays the role of preventing pathogen contamination. Compared with other buffer solutions, HEPES has better buffering capacity as a buffer solution. The quality control product contains sodium citrate, glucose, biological preservative, mannitol and glycerol, and has higher stability.

[0041] After long - term and extensive screening, the inventor found that when the white blood cells, red blood cells, platelets, balanced solution, and fixing solution in the quality control product are set within the above - mentioned ranges, it can simulate the normal human blood state and ensure the long - term stable preservation of cells. The whole - blood quality control product provided by the present invention has the advantages of good stability and good uniformity, and meets the requirements of clinical quality control products.

[0042] The balanced solution accounts for 60% of the volume of the whole - blood quality control product to simulate the 60% ratio of plasma in whole blood from Tibetan macaques.

[0043] In a preferred embodiment of the application of the present invention, the whole - blood quality control product includes: white blood cells: 5×10 9 / L; red blood cells: 2×10 12 / L; platelets: 2×10 12 / L.

[0044] Among them, the balanced solution includes: 2×HEPES with a pH of 7.2, 7.3 or 7.4, sodium citrate at 0.12, 0.13 or 0.14 mol / L, mannitol at 0.4, 0.5 or 0.6 mol / L, glycerol at 0.1, 0.2 or 0.3%, glucose at 0.4, 0.5 or 0.6 mol / L, a biopreservative at 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15% and water.

[0045] In a preferred embodiment of the application of the present invention, the pH of the balanced solution is 7.26 - 7.3.

[0046] In an alternative embodiment, the osmotic pressure of the balanced solution is 304 - 320 mOsm. For example, the osmotic pressure is 304, 305, 310, 315, 318 or 320 mOsm. Under this osmosis, the balanced solution can maintain the cells in a suitable pressure solution, thereby maintaining the integrity and stability of the cell morphology.

[0047] In a preferred embodiment of the application of the present invention, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.129 - 0.14 mol / L, mannitol at 0.5 - 0.6 mol / L, glycerol at 0.2 - 0.3%, glucose at 0.5 - 0.6 mol / L, a biopreservative at 0.1 - 0.15% and water.

[0048] In an alternative embodiment, the balanced solution includes: 2×HEPES with a pH of 7.2 - 7.4, sodium citrate at 0.129 mol / L, mannitol at 0.5 mol / L, glycerol at 0.2%, glucose at 0.5 mol / L, a biopreservative at 0.1% and water.

[0049] In a preferred embodiment of the application of the present invention, the biopreservative is selected from any one of ProClin150, ProClin200 and ProClin300. The biopreservative plays a role in preventing pathogen contamination.

[0050] In a preferred embodiment of the application of the present invention, the volume ratio of the fixing solution to the blood cells is 1:(4 - 6).

[0051] In a second aspect, the present invention also provides a method for preparing a quality control product of whole blood from Macaca thibetana, which includes the following steps:

[0052] The separated white blood cells, red blood cells and platelets from normal Macaca thibetana are respectively mixed with the fixing solution according to a volume ratio of 1:(4 - 6). After fixation, the concentration is adjusted so that the white blood cell concentration is 5×10 10 / L, the platelet concentration is 2×10 13 / L, the red blood cell concentration is 1×10 13 / L;

[0053] Then mix white blood cells, platelets, red blood cells and balanced salt solution according to a volume ratio of 1:1:2:6.

[0054] The volume ratio of blood cells to fixative is 1:4, 1:5 or 1:6, or any volume ratio point value between the above point values.

[0055] Formaldehyde and glutaraldehyde in the fixative play a role in separately fixing and preserving white blood cells, red blood cells and platelets. Among them, glutaraldehyde can connect with the amino groups and peptide bonds of protein molecules to form crosslinks and maintain the activity of enzymes and proteins.

[0056] The fixative includes: 2-3% (v / v) formaldehyde and 0.4-0.6% (v / v) glutaraldehyde. For example, the fixative includes: 2% (v / v) formaldehyde and 0.4% (v / v) glutaraldehyde, 3% (v / v) formaldehyde and 0.4% (v / v) glutaraldehyde, 3% (v / v) formaldehyde and 0.5% (v / v) glutaraldehyde or 2% (v / v) formaldehyde and 0.6% (v / v) glutaraldehyde.

[0057] In a preferred embodiment of the application of the present invention, the isolated white blood cells, red blood cells and platelets from normal Tibetan macaques are respectively mixed with the fixative according to a volume ratio of 1:5. At the above mixing ratio, there is a better effect of fixing white blood cells, red blood cells and platelets. Less fixative will result in insufficient cell fixation, and more fixative will result in higher economic costs.

[0058] In an alternative embodiment, the fixation is low-temperature fixation; to prevent cell inactivation.

[0059] In an alternative embodiment, the fixation is carried out at 0-8°C.

[0060] In a preferred embodiment of the application of the present invention, during the fixation process, suspension is carried out every 8-10 minutes. Such a setting is to make the cells more thoroughly fixed by the fixative.

[0061] In a preferred embodiment of the application of the present invention, the fixation time is 20-60 minutes; in an alternative embodiment, the fixation time is 40 minutes. The fixation time is, for example, selected from 20 minutes, 25 minutes - 40 minutes, 30 - 60 minutes or 40 minutes - 60 minutes. If the fixation time is too short, the cells will not be sufficiently fixed, affecting the stability and uniformity of the quality control product, and the cells are prone to breakage during the subsequent processing. If the fixation time is too long, it will affect the lysis of red blood cells during the subsequent detection of the quality control product (there is a red blood cell lysis step in the detection process of the hematology analyzer).

[0062] In an optional embodiment, after fixation, centrifugation is further included to discard the supernatant;

[0063] In an optional embodiment, the centrifuge speed is 1800-2200 rpm, centrifuged at 2-8°C for 20 min, the supernatant is discarded, and PBS is added for resuspending, and the centrifuge is performed at 2800-3200 rpm for 5 min, and the supernatant is discarded.

[0064] In an optional embodiment, the centrifuge is set to rise steeply and fall slowly, so as to avoid cell resuspension and cell fragmentation and inactivation as much as possible.

[0065] In a third aspect, the present invention further provides a kit, which includes the above-mentioned Tibetan macaque-derived whole blood quality control product or the Tibetan macaque-derived whole blood quality control product prepared by the above-mentioned preparation method.

[0066] In other embodiments, the above-mentioned kit further includes a diluent, instructions, etc.

[0067] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0068] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0069] Example 1

[0070] This example provides a Tibetan macaque-derived whole blood quality control product, which includes: white blood cells: 5×10 9 / L; red blood cells: 2×10 12 / L; Platelets: 2×10 12 / L; Balanced solution: pH 7.26, osmotic pressure 304mOsm; Fixative: The volume ratio of fixative to blood cells is 1:5;

[0071] The balance solution includes: 2×HEPES with a pH of 7.2-7.4, 0.129 mol / L sodium citrate, 0.5 mol / L mannitol, 0.2% glycerol, 0.5 mol / L glucose, 0.1% ProClin300 and deionized water;

[0072] The fixative solution consisted of 2.5% (v / v) formaldehyde and 0.5% (v / v) glutaraldehyde.

[0073] The human peripheral blood lymphocyte separation solution was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number P8610. Its main components are polysucrose and meglumine diatrizoate, with a density of 1.077. The 2×HEPES was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number H1080. Its main component is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, with a pH of 7.2 - 7.4.

[0074] Preparation of required reagents and consumables: All reagents used in the present invention are of chemical analytical purity. The equilibration solution was prepared according to the above formula (except ProClin300), autoclaved at 121 °C for 15 min, and after being placed at room temperature, the broad-spectrum bacteriostatic agent ProClin300 was added according to the formula ratio and stored at 2 - 8 °C for later use.

[0075] The 5 ml silanized glass vials were autoclaved at 121 °C for 15 min and stored at room temperature for later use.

[0076] PBS (1X, pH 7.2) was pretreated at low temperature before use. The centrifuge tubes and pipette tips are sterile disposable consumables.

[0077] Its preparation method is as follows:

[0078] (1) Prepare the equilibration solution and the fixing solution respectively according to the above formula.

[0079] (2) Collection of peripheral blood from Macaca thibetana: Select adult male healthy Macaca thibetana aged 8 - 12 years old and weighing 15 - 20 kg, which are negative for monkeypox virus, monkey B virus, simian T-cell tropic virus, simian retrovirus type D, simian immunodeficiency virus, and Mycobacterium tuberculosis in monkeys. Fast the animals for 8 h before blood collection without restricting water intake. Use Zoletil (tiletamine hydrochloride, zolazepam hydrochloride) at 0.5 ml / kg for intramuscular anesthesia. After the animal's blink reflex disappears, shave the hair on the hind limb, disinfect with iodophor, and collect peripheral whole blood from the saphenous vein of the hind limb with an EDTA anticoagulant bag. The blood collection volume per single animal does not exceed 10% of the total body blood volume. The fresh blood is stored at low temperature, and the subsequent processing interval does not exceed 2 h.

[0080] (3) Separation and fixation of each component of blood cells:

[0081] (3.1) Platelet enrichment: Transfer the fresh whole blood to a 50 ml sterile centrifuge tube. Set the centrifuge to rise steeply and descend slowly, centrifuge at 1000 rpm at 2 - 8 °C for 5 min. Carefully aspirate the supernatant and transfer it to a new sterile centrifuge tube. Set the centrifuge as before, centrifuge at 2000 rpm at 2 - 8 °C for 10 min. Discard two-thirds of the upper supernatant, and the bottom is the enriched platelets. Gently resuspend and mix with PBS to depolymerize, and place at low temperature for later use;

[0082] (3.2)Leukocyte enrichment: Add PBS equal in volume to the supernatant to the precipitate after the first centrifugation in (3.1), gently resuspend, and slowly add it to the human peripheral blood lymphocyte separation medium equal in volume to the whole blood. Set the centrifuge to rise steeply and descend slowly, centrifuge at 1500 rpm at 2 - 8 °C for 20 min. Carefully aspirate the middle leukocyte layer, add PBS equal in volume, set the centrifuge as before, centrifuge at 3000 rpm at 2 - 8 °C for 5 min, discard the supernatant, add PBS equal in volume to the aspirate to resuspend, set the centrifuge as before, centrifuge at 3000 rpm at 2 - 8 °C for 5 min, discard the supernatant to obtain leukocytes, gently resuspend and mix well with PBS to depolymerize, and place at low temperature for standby;

[0083] (3.3)Erythrocyte enrichment: After taking the leukocyte layer in (3.2), discard the supernatant, and the bottom precipitate is erythrocytes. Add PBS equal in volume to the precipitate, gently suspend, set the centrifuge to rise steeply and descend slowly, centrifuge at 3000 rpm at 2 - 8 °C for 5 min, discard the supernatant, add PBS equal in volume to gently resuspend, set the centrifuge as before, centrifuge at 3000 rpm at 2 - 8 °C for 5 min, discard the supernatant to obtain erythrocytes, gently resuspend and mix well with PBS to depolymerize, and place at low temperature for standby;

[0084] (3.4)Fixation and quantification of each component of blood cells: Fix the platelets, leukocytes, and erythrocytes enriched in the above (3.1), (3.2), and (3.3) with the fixative at a ratio of 1:5 at low temperature, gently suspend once every 10 min, and fix for 20 - 60 min, preferably 40 min. After the fixation ends, set the centrifuge to rise steeply and descend slowly, centrifuge at 2000 rpm at 2 - 8 °C for 20 min, carefully discard the supernatant, add PBS equal in volume to gently resuspend, set the centrifuge as before, centrifuge at 3000 rpm for 5 min, discard the supernatant to obtain the fixed cells. Add the fixative to gently resuspend, and adjust the concentration of cell components through a cell counter. Adjust the leukocyte concentration to 5×10 10 / L, the platelet concentration to 2×10 13 / L, and the erythrocyte concentration to 1×10 13 / L.

[0085] (4)Maintenance and aliquoting of fixed cells

[0086] Add the cell components fixed and adjusted in cell concentration above to the equilibration solution, and the addition volume ratio is leukocytes:platelets:erythrocytes:equilibration solution = 1:1:2:6. After suspending and mixing evenly, aliquot into sterile siliconized glass vials, 2 ml per vial, to obtain whole blood quality control products, and store at 2 - 8 °C. Among them, the leukocyte concentration of the quality control product is 5×10 9 / L, the platelet concentration is 2×10 12 / L, and the erythrocyte concentration is 2×10 12 / L.

[0087] The following experiments were carried out in accordance with reference standards such as CNAS-CL02:2012 "Accreditation Criteria for the Quality and Competence of Medical Laboratories", CNAS-GL003:2018 "Guidelines for the Evaluation of the Homogeneity and Stability of Proficiency Testing Samples", and ISO Guide 35 "Certification of Reference Materials / Reference Samples - General Principles and Statistical Principles".

[0088] Experimental Example 1

[0089] In this experimental example, a sterility spot-check experiment was conducted.

[0090] Three quality control products prepared in Example 1 above were randomly selected. Streaking on blood agar plates was performed with an inoculation needle in a sterile laminar flow hood and incubated upside down in a 37°C incubator for 3 days. Whether colonies appeared was observed every day. The results showed that no colonies appeared, indicating that the whole blood quality control product prepared according to the preparation process described in the present invention was free of pathogenic bacteria contamination.

[0091] Experimental Example 2

[0092] Experiment for evaluating the homogeneity of whole blood quality control products

[0093] Twelve whole blood quality control products prepared in Example 1 above were randomly selected for homogeneity testing. The instrument used was an automated hematology analyzer (Mindray BC-7500). Before sample testing, the machine was calibrated with the original quality control product, and the original reagents and standard methods provided by the instrument were used. After the samples were left at room temperature, they were gently mixed and then loaded onto the machine. Each sample was randomly detected in parallel three times. The detection parameters were: red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), platelet count (PLT), and white blood cell count (WBC). Statistical analysis results are shown in Table 1.

[0094] Table 1 Homogeneity analysis of whole blood quality control products of Macaca thibetana

[0095]

[0096] The results showed that the CV values of each index were lower than the standard requirements, and the measured values of the three indexes of RBC, WBC, and PLT were basically consistent with the counting results of the counter. The coefficient of variation of each index was close to the precision of the instrument. The P values of the detection data of the five indexes analyzed by one-way ANOVA were all greater than 0.05, indicating no statistical significance, and the F value was less than F crit, indicating that the homogeneity of the quality control product was good.

[0097] Experimental Example 3

[0098] In this experimental example, the stability evaluation after opening the bottle was carried out.

[0099] Randomly select 3 whole blood quality control products prepared above to carry out the stability of the quality control products within 7 days of repeated bottle opening (1 day, 3 days, 5 days, 7 days). The instrument is an automatic blood cell analyzer (Mindray BC-7500). Before sample detection, the machine uses the original quality control product for quality control, and the instrument is equipped with original reagents and standard methods. After the test samples are at room temperature, gently mix them evenly and load them onto the machine. Each sample is randomly detected in parallel three times. The detection parameters are: red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), platelet count (PLT), white blood cell count (WBC), and statistical analysis is carried out. The results are shown in Table 2.

[0100] Table 2 Analysis of the stability of the whole blood quality control product of Macaca thibetana after bottle opening

[0101]

[0102] Note: A represents the percentage deviation analysis of the test results at each time point compared with the test results on the first day.

[0103] The percentage deviation analysis shows that the overall change range of RBC, HCT, HGB, WBC, and PLT is within the acceptable range. The simple linear regression analysis and variance analysis show that the significant differences between the test results on the 3rd, 5th, and 7th days and those on the first day are all greater than 0.05, without statistical significance, indicating that within 7 days of preparation, repeated bottle opening and detection, the indicators are all within the stable range and do not show a linear relationship with the detection time.

[0104] Experimental Example 4

[0105] This experimental example conducts a long-term stability evaluation.

[0106] Randomly select 1 whole blood quality control product prepared above to carry out the stability of the quality control product within 90 days (1 day, 30 days, 60 days, 90 days). The instrument is an automatic blood cell analyzer (Mindray BC-7500). Before sample detection, the machine uses the original quality control product for quality control, and the instrument is equipped with original reagents and standard methods. After the test samples are at room temperature, gently mix them evenly and load them onto the machine. Each sample is randomly detected in parallel three times. The detection parameters are: red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), platelet count (PLT), white blood cell count (WBC), and statistical analysis is carried out. The results are shown in Table 3 and Table 4.

[0107] Table 3 Analysis of the long-term stability of the whole blood quality control product of Macaca thibetana

[0108]

[0109] Table 4 Uncertainty of long-term stability

[0110]

[0111] Note: Standard deviation of the slope of the unary linear regression equation for S(b1), u lts Uncertainty of long-term stability, u r Uncertainty of method repeatability

[0112] Within 90 days after sample preparation, the indicators are detected every 30 days. For the detection results of RBC, HCT, HGB, and WBC, as time extends, their detected values show a decreasing trend, while the detection result of PLT shows an increasing trend. The detected values of the three indicators of RBC, HCT, and PLT at 90 days do not show a linear relationship with time, indicating that these three indicators are relatively stable. Through the uncertainty calculation formula, with 90 days as the expected stable time, the uncertainty calculation results show that the uncertainty of long-term stability u of the three indicators of RBC, HCT, and PLT at 90 days lts is less than the measurement uncertainty of method repeatability u r , indicating that these three indicators are stable within 90 days.

[0113] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a whole blood quality control product from Macaca thibetana, characterized in that, It includes the following steps: The white blood cells, red blood cells and platelets isolated from the peripheral blood of healthy Tibetan macaques were respectively mixed with the fixative according to a volume ratio of 1:(4-6), fixed at low temperature, centrifuged to discard the supernatant, gently resuspended with the fixative, and the cell concentration was adjusted so that the white blood cell concentration was (4-6)×10 10 / L, the platelet concentration was (1.5-2.5)×10 13 / L, and the red blood cell concentration was (0.75-1.25)×10 13 / L; Then, white blood cells, platelets, red blood cells, and balanced salt solution are mixed at a volume ratio of 1:1:2:6 to obtain a whole blood quality control product from Macaca thibetana. Among them, the whole blood quality control product includes: white blood cells: 5×10 9 / L; red blood cells: 2×10 12 / L; platelets: 2×10 12 / L; The fixative solution includes: 2 - 3% (v / v) formaldehyde and 0.4 - 0.6% (v / v) glutaraldehyde; the fixation time is 20 - 60 min; Among them, the equilibration solution includes: 2×HEPES with a pH of 7.2 - 7.4, 0.129 - 0.14 mol / L sodium citrate, 0.5 - 0.6 mol / L mannitol, 0.2 - 0.3% glycerol, 0.5 - 0.6 mol / L glucose, 0.1 - 0.15% biopreservative and water, and the osmotic pressure of the equilibration solution is 280 - 320 mOsm.

2. The preparation method according to claim 1, characterized in that, The pH of the equilibration solution is 7.26 - 7.

3.

3. The preparation method according to claim 2, wherein, The osmotic pressure of the equilibration solution is 304 - 320 mOsm.

4. The preparation method according to claim 1, characterized in that, The equilibration solution includes: 2×HEPES with a pH of 7.2 - 7.4, 0.129 mol / L sodium citrate, 0.5 mol / L mannitol, 0.2% glycerol, 0.5 mol / L glucose, 0.1% biopreservative and water.

5. The preparation method according to claim 1, characterized in that, The biopreservative is selected from any one of ProClin150, ProClin200 and ProClin300.

6. The preparation method according to claim 5, characterized in that, The biopreservative is selected from ProClin300.

7. The preparation method according to claim 1, characterized in that, The isolated white blood cells, red blood cells and platelets from healthy Tibetan macaques are respectively mixed with the fixative solution at a volume ratio of 1:

5.

8. The preparation method according to claim 1, wherein The low-temperature fixation is carried out at 0 - 8°C.

9. The preparation method according to claim 1, characterized in that, During the fixation process, it is suspended once every 8 - 10 min.

10. The preparation method according to claim 9, characterized in that, The fixation time is 40 min.

11. The preparation method according to claim 1, characterized in that, The centrifugation speed is 1800 - 2200 rpm, centrifuged at 2 - 8°C for 20 min, the supernatant is discarded and then resuspended with PBS, and then centrifuged at 2800 - 3200 rpm for 5 min, and the supernatant is discarded.

12. A Tibetan macaque-derived whole blood quality control product for a blood cell analyzer, characterized in that, It is prepared by the preparation method according to any one of claims 1 - 11.

13. A kit, characterized in that, It includes the Tibetan macaque-derived whole blood quality control product according to claim 12.

Images