Freeze-dried cells for quality control, preparation method, use and quality control kit

Abstract

This application discloses frozen stem cells for quality control, their preparation method and uses, and a quality control kit, including two core technical routes: (1) fixation + lyophilization protectant to maintain cell morphology integrity; (2) pre-staining followed by lyophilization, followed by reconstitution for direct imaging without staining. The frozen stem cells of this invention can be stored and transported at room temperature. After reconstitution, the cells exhibit stable morphology, no hemolysis, and no aggregation, making them suitable for quality control, instrument calibration, and / or algorithm verification in blood analyzers, cell morphology analyzers, and AI image recognition equipment. This invention is simple to operate, highly stable, and widely applicable, solving problems such as cold chain dependence, short shelf life, cumbersome staining, and easy morphological damage associated with traditional quality control products.

Description

Technical Field

[0001] This invention belongs to the field of medical device quality inspection technology, specifically relating to frozen stem cells for quality inspection of microscopic testing instruments, their preparation method, quality control kit, and applications. Background Technology

[0002] Currently, there is a lack of stable, convenient, and room-temperature-storable quality control products for cell morphology microscopy analyzers, blood analyzers, and AI image recognition equipment used in clinical and laboratory settings.

[0003] Traditional liquid quality control products require low-temperature storage, have a short shelf life, and are susceptible to microbial contamination; ordinary frozen stem cells are not stained or adequately fixed and protected, and their morphology is severely damaged after reconstitution, which cannot meet the requirements of high-precision imaging.

[0004] Definition of terms: Microscopic sample: A sample into which a detection chip can be injected to obtain a magnified microscopic image.

[0005] Quality control: In this application, quality control refers to the calibration, standardization, and functional verification of microscopic testing instruments; it is applicable to the quality control, instrument calibration, standardization, functional verification, and / or algorithm verification of formed elements such as blood, urine, and fecal suspensions, cell morphology analyzers, and AI image recognition equipment. Summary of the Invention

[0006] The purpose of this invention is to provide a frozen stem cell quality control product that can be stored at room temperature, reconstituted and used immediately, with intact cell morphology and stable staining, suitable for quality inspection, calibration and / or verification of various microscopic testing instruments.

[0007] This invention solves the above-mentioned defects through a dual technical solution: a fixation + protective agent stabilization route and a pre-dyeing freeze-drying route.

[0008] To address the problems of traditional liquid quality control products, the applicant proposes a frozen stem cell preparation for quality inspection in microscopic testing instruments. This frozen stem cell preparation is obtained by treating cell-containing liquid with the addition of a fixative and a lyophilization protectant, followed by individual fractionation and freeze-drying. The fixative is used to preserve the original morphology and structure of the cells, while the lyophilization protectant inhibits cell deformation, hemolysis, or aggregation during freeze-drying and reconstitution. The frozen stem cell preparation is a single-fraction freeze-dried formulation, which, after reconstitution, is used for microscopic image acquisition and / or instrument quality inspection. The lyophilization protectant can also be referred to as an excipient.

[0009] Frozen stem cells are frozen stem cells that have been treated with fixatives and lyophilization protectants. They are resistant to storage. Due to the effect of the fixative, after rehydration, the cells retain their morphology and can produce microscopic images that are the same as or similar to those of fresh blood. This significantly reduces the cost of calibration, verification, and functional validation in the entire industry chain during the instrument production and use process.

[0010] In the quality inspection, calibration and / or validation of the instrument, it is very difficult to test the color reproduction of stained cells. The applicant proposed that the frozen stem cells be stained during or after rehydration and then used for microscopic image acquisition and / or instrument quality inspection.

[0011] Reconstituted cells have properties close to those of fresh cells and can be stained. By staining reconstituted cells, the performance indicators of the instrument, such as capturing, acquiring, and / or analyzing cell colors, can be verified.

[0012] Staining cells requires certain skills from the operators, and the staining process has specific requirements for environmental temperature and staining time. In order to reduce the complexity of the operation for on-site personnel and the training requirements for their staining skills, the applicant proposes that the cell-containing liquid used to prepare the frozen stem cells be stained.

[0013] The applicant proposes that the preparation process of the frozen stem cells is as follows: first, the liquid sample containing cells is stained, then a freeze-drying protectant is added, and the cells are individually packaged according to the set specifications, and finally, they are freeze-dried. The cells have been stained before freeze-drying, forming a stable stained morphology; the finished product is a single-component freeze-dried preparation, which can be used for microscopic image acquisition and / or instrument quality inspection after reconstitution.

[0014] By staining during the frozen stem cell production process, pre-stained frozen stem cells are obtained. This eliminates the need for on-site personnel to perform staining operations, significantly reducing staining training workload and minimizing on-site operation time and errors caused by operator mistakes. While cells not treated with fixatives have a relatively shorter storage life, the reduced difficulty of on-site operation makes them commercially viable.

[0015] The applicant discovered that some components in the liquid before the lyophilization protectant treatment could affect the integrity of the cells after reconstitution. The applicant proposed that the cells be cleaned before the lyophilization protectant treatment, and then the lyophilization protectant be added for further treatment.

[0016] The cleaning process removes other extracellular components, improving product quality and allowing the cells to retain their complete morphology after reconstitution.

[0017] The applicant proposes that the staining uses one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green.

[0018] Extensive experimental verification has shown that pre-treatment with the above-mentioned staining agents can produce good staining results after reconstitution.

[0019] The applicant states that the staining process is used to stain one or more of the following: cell nucleus, cytoplasm, granules, intracellular structures, and RNA / DNA.

[0020] Extensive experimental verification has shown that pre-staining can meet the staining requirements of the above-mentioned internal cell structures, and good staining results can be achieved after reconstitution.

[0021] The applicant stated that the fixative is one or more of formaldehyde, paraformaldehyde, and glutaraldehyde.

[0022] Through numerous experiments, the applicant discovered that frozen stem cells, after being treated with a fixative, can be preserved for a long time and exhibit a good morphology after rehydration.

[0023] Through extensive experimentation, the applicant discovered that if excess fixative is not washed away after fixation treatment, it will affect cell rehydration and unfolding after a period of storage. In order to extend the storage time, the cells need to be washed away before freeze-drying to remove excess fixative.

[0024] The applicant states that the freeze-drying protectant includes one or more of trehalose, sucrose, and hydroxyethyl starch. The freeze-drying protectant is used to maintain cell morphology, size, and structural integrity.

[0025] Through extensive experiments, the applicant found that the above-mentioned components can effectively maintain the morphology of cells during the freeze-drying process, and after a certain period of storage, they can exhibit a good morphology after reconstitution.

[0026] The applicant states that the cells are one or more of blood cells, body fluid cells, and tissue cells.

[0027] After extensive experimentation, the applicant discovered that all of the above-mentioned cells could be prepared as frozen stem cell quality control products.

[0028] The applicant states that the cells are derived from one or more of the following: human, cow, monkey, dog, cat, turtle, rabbit, and parrot.

[0029] The applicant stated that the cells were derived from bovine blood.

[0030] Through extensive experimentation, the applicant discovered that both human and animal cells can be prepared as frozen stem cell quality control products. Considering the ease of collection for commercial applications and the volume requirements for mass production, using bovine blood cells is a relatively better option, as bovine blood cells can be collected from living individuals, are available in large quantities, and can meet the needs of periodic batch collection.

[0031] The applicant stated that the cells were derived from vertebrates or invertebrates.

[0032] For certain special testing, research, and teaching applications, this solution can also meet the cell quality inspection needs of different species.

[0033] The applicant proposed to repackage the product into single-use portions or multiple-use portions according to the specified quantities.

[0034] This product is a biological product. Once opened, it readily absorbs moisture from the air and undergoes changes. It is packaged in pre-defined portions and used immediately after opening to ensure the quality requirements of experiments or tests are met.

[0035] Meanwhile, this type of instrument is a high-tech device with stringent requirements for the quality control products. Using single-use samples can effectively ensure the stability of the quality control products.

[0036] For tests that need to be repeated two or more times, two or more portions of lyophilized powder can be packaged together and used up within a short period of time after opening.

[0037] The applicant proposed that the resolution was performed using pure water or a resolution solution.

[0038] Using pure water for reconstitution reduces the difficulty of operation for on-site personnel. The operation procedure is designed to be simple and easy to understand, making it easy for on-site personnel to master quickly.

[0039] Specialized reconstitution solutions can be prepared by adding staining agents or calibration particles to the reconstitution solution, enabling more functions. For example, adding calibration particles can calibrate the accuracy of the magnification of the microscope system. Alternatively, adding irregular particles can verify the AI ​​software's ability to identify and / or resist interference from non-standard objects.

[0040] The osmotic pressure of the liquid and reconstituted solution before freeze-drying should be maintained between 280 mosm / L and 310 mosm / L, and the pH value should be between 7.2 and 7.5.

[0041] The applicant stated that the microscopic testing instruments are a cell morphology analyzer, a blood, urine, or fecal suspension formed element analyzer, and an AI image recognition instrument.

[0042] The aforementioned instruments all require AI-based cell recognition capabilities and demand a very high degree of cell restoration. Through extensive and long-term experiments by the applicant, frozen stem cells, or stained frozen stem cells reconstituted, have been developed to meet the quality testing requirements of these devices, significantly reducing the costs of quality testing, calibration, and functional testing. This also simplifies the traditional sample acquisition process.

[0043] The applicant proposed that the evaluation should include one or more of the following: image clarity, color reproduction, recognition accuracy, classification accuracy, cell size, cell volume, and repeatability of multiple measurements.

[0044] The applicant's extensive experiments and repeated verifications have shown that frozen stem cells or stained frozen stem cells after rehydration can support the above assessment.

[0045] The applicant proposed that the evaluation content could be used to determine whether the instrument's optics, image acquisition, or analysis software was functioning properly.

[0046] The applicant's extensive experiments and repeated verifications have shown that frozen stem cells or stained frozen stem cells after rehydration can support the above-mentioned evaluation work.

[0047] Through repeated testing and verification, the applicant has demonstrated that the lyophilized powder can be stored and transported at room temperature. After being stored and transported at room temperature, the product, protected by a preservative, can support the aforementioned evaluation work upon reconstitution.

[0048] After repeated experiments and verifications, the applicant determined that the shelf life of the freeze-dried powder is ≥18 months.

[0049] The lyophilized powder treated with fixative and preservative agents still supports the above assessment after 18 months of storage.

[0050] The freeze-dried powder treated with the preservative has a shorter shelf life, and within a shorter period of time, it can support the above evaluation.

[0051] After repeated experiments and verifications, the applicant determined that the cells in question are one or more of the following: red blood cells, white blood cells, platelets, and reticulocytes.

[0052] In blood testing, some specialized tests require different cell types to verify the instrument's functionality. After verification, reconstituted red blood cells, white blood cells, platelets, and reticulocytes all passed the instrument's tests.

[0053] After repeated experiments and verifications, the applicant determined that the white blood cells include one or more of neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0054] In blood testing, specific types of white blood cells are required to verify the testing function and / or performance. Neutrophils, lymphocytes, monocytes, eosinophils, and basophils can all be detected by these instruments after reconstitution.

[0055] After repeated experiments and verifications, the applicant has obtained RNA structure staining for the reticulocytes used in the quality control of the reticulocyte detection instrument.

[0056] In blood testing, reticulocytes, due to their small size and diverse morphology, are important indicators. Many instruments cannot effectively perform these tests. Through extensive experimentation, reticulocytes were extracted from large quantities of animal blood. After long-term, repeated experiments, a freeze-dried powder of reticulocytes was produced. Following transportation and storage experiments, this powder met the quality control requirements of reticulocyte testing instruments. This represents a successful technological breakthrough that was previously difficult to achieve.

[0057] The applicant proposes that the specified dosage be in individually packaged small portions for immediate use and disposal. After repeated research into usage scenarios, small-portion packaging ensures the safety requirements of the biological products used in the product, while also meeting the precision requirements of high-tech equipment. The factory's high-precision equipment configuration enables accurate proportioning of these small-portion packages, meeting the precision requirements of scientific instruments.

[0058] After repeated experiments and verifications, the applicant found that there was no cell aggregation or hemolysis after reconstitution.

[0059] The applicant has verified through extensive transportation and storage experiments that the product, after reconstitution within its 18-month shelf life, shows no cell aggregation or hemolysis and can be used normally.

[0060] If a product exceeds its shelf life or is improperly stored, and cell sedimentation, cell aggregation, or hemolysis occurs after reconstitution, it must not be used in the quality control process of the instrument.

[0061] The applicant proposes a method for preparing frozen stem cells for quality control of microscopic testing instruments, comprising: Step A20: Add fixative to the cell-containing fluid for fixation treatment; Step A30: Add a freeze-drying protectant to the cells. The freeze-drying protectant is used to prevent cell morphology from being damaged during the freeze-drying process. Step A40: The liquid containing cells and lyophilization protectant is dispensed into individual portions; the dispensed samples are then freeze-dried to obtain individual lyophilized powders. When used, the freeze-dried powder is reconstituted with water to form a test sample for microscopic image capture.

[0062] After treatment with a fixative, a freeze-drying protectant is added before freeze-drying. Precise portions are then packaged and freeze-dried directly to obtain frozen stem cells. This ensures that the cells retain their morphology after rehydration and meets the cell quantity and / or cell concentration requirements for quality inspection.

[0063] The applicant proposes that, before or after step A20, step A10 includes adding a staining agent to the cell-containing fluid for staining treatment; or, in step A20, the staining agent is mixed into the fixative, and staining is completed simultaneously with fixation.

[0064] Cells treated with staining can meet the color calibration requirements in quality inspection.

[0065] The applicant proposes to stain the cells by adding a staining agent to a cell-containing fluid, wherein the staining agent includes a fixative.

[0066] Adding a fixative to the dyeing agent can effectively reduce production steps and control product quality.

[0067] The applicant proposed that in step A30, the cell-containing liquid should first be washed to remove residual fixative or staining agent, and then the cells should be added to a liquid containing a lyophilization protectant.

[0068] By cleaning to remove residual fixatives and other unknown components, it is ensured that no unknown components remain after reconstitution, while also eliminating the impact of fixatives on subsequent processes.

[0069] After repeated experiments and verifications, the applicant determined that the staining method uses one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green.

[0070] After repeated experiments and verifications, the applicant found that the fixative includes one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; the fixation temperature is 2℃~8℃, and the fixation time is 30min~24h.

[0071] After repeated experiments and verifications, the applicant has determined that step A09 may be included before step A20: cells in liquid containing cells are obtained by removing plasma from whole blood containing EDTA or heparin anticoagulant; After repeated experiments and verifications, the applicant determined that before step A40, the following steps are included: the assignment of target values, i.e., the calibration of target values; the calibrated target values ​​include CBC baseline parameter calibration target values, white blood cell five-part differential calibration target values, and reticulocyte parameter calibration target values; the CBC baseline parameter calibration target values ​​include: WBC baseline parameter calibration target values, RBC baseline parameter calibration target values, MCV baseline parameter calibration target values, MCH baseline parameter calibration target values, HGB baseline parameter calibration target values, HCT baseline parameter calibration target values, MCHC baseline parameter calibration target values, and PLT baseline parameter calibration target values, totaling 8 items; the white blood cell five-part differential calibration target values ​​include: neutrophil calibration target values, lymphocyte calibration target values, monocyte calibration target values, eosinophil calibration target values, and basophil calibration target values; the reticulocyte parameter calibration target values ​​include: Ret% calibration target values, Ret# calibration target values, and IRF calibration target values.

[0072] Through repeated experiments and verifications, the applicant has determined that the freeze-drying protectant includes one or more of trehalose, sucrose, and hydroxyethyl starch. The final concentration of the freeze-drying protectant is 0.5%–10%. The cell-containing fluid is an isotonic buffer system.

[0073] After repeated experiments and verifications, the applicant determined that the volume of each individual portion is 200μL to 1000μL.

[0074] The applicant proposed that the repackaging be done in single-use aseptic packaging; and that the repackaged products be temporarily stored at low temperature and protected from light. The applicant stated that freeze drying includes pre-freezing, sublimation drying, or desorption drying; The applicant proposed a pre-freezing temperature of -35℃ to -50℃; After repeated experiments and verifications, the applicant determined that the final moisture content of the finished product is ≤3%.

[0075] The applicant proposes a quality control kit for microscopic testing instruments, comprising a single-packaged pre-stained frozen stem cell powder; the frozen stem cell powder is prepared by freeze-drying pre-stained cells in a solution containing a lyophilization protectant; a reconstitution solution is added during use to form a test sample for microscopic image capture; the test sample is used to evaluate whether the instrument's imaging, identification, classification, and counting functions are qualified.

[0076] By repackaging the individual kits, the impact on the individual packages during transportation can be avoided.

[0077] The applicant proposed that the kit includes K1 single-packaged frozen stem cell powder and K2 single-packaged frozen stem cell powder. After reconstitution with the same volume V of water, the cell concentration of the K1 test sample is higher than that of the K2 test sample, or the K1 concentration is an integer multiple of the K2 concentration.

[0078] By packaging with different cell contents, testing needs for different concentrations can be met.

[0079] By configuring samples with concentration differences that are integer multiples, the measurement accuracy of the instrument can be verified, and its ability to accurately identify such integer multiple concentration differences can be tested.

[0080] The applicant stated that the kit includes frozen stem cell powder for a five-part differential hematology test.

[0081] The applicant stated that the kit includes frozen stem cell powder for reticulocyte detection.

[0082] The applicant stated that the reagent kit should be accompanied by a standard image library, judgment rules, and quality control range instructions.

[0083] The applicant argues that the aforementioned frozen stem cells are the cell source corresponding to the frozen stem cell powder.

[0084] To reduce the difficulty for on-site personnel in obtaining reconstitution solutions, the applicant proposed including individually packaged reconstitution solutions. These individually packaged solutions would allow on-site personnel to accurately prepare the liquid at the desired cell concentration.

[0085] The applicant stated that the reconstitution solution is pure water. Even ordinary pure water is included with the reagent kit, significantly reducing the operational steps and difficulty for on-site staff.

[0086] The applicant stated that the kit includes single-volume encapsulated reconstituted solutions of varying volumes, with the larger volume being an integer multiple of the smaller volume.

[0087] By using single-volume encapsulated reconstitution solutions of different volumes, on-site personnel can accurately prepare liquids with different cell concentrations.

[0088] The applicant states that the kit is suitable for model validation and / or calibration of AI cell morphology analyzers; The applicant stated that the reconstitution solution volume is marked on the packaging. By marking the volume on the reconstitution solution packaging, on-site personnel can accurately determine the required concentration of the cell suspension to be prepared.

[0089] The applicant proposes a use of pre-stained frozen stem cells in the quality inspection of microscopic testing instruments, which involves reconstituted pre-stained, individually packaged, and freeze-dried cell powder with water. Microscopic images of the reconstituted samples were captured to obtain microscopic images of the stained cells. The instrument's qualification is determined based on the morphology, structure, color, or clarity of the stained cells in the image. The cells were stained before freeze-drying and did not need to be stained again after reconstitution.

[0090] By using pre-stained frozen stem cells reconstituted with water, the difficulty of sample acquisition for quality control of microscopic testing instruments is significantly reduced, eliminating the need for on-site staining and greatly improving sample quality. This also enables accurate calibration of on-site equipment.

[0091] The applicant proposed that it could be used for routine quality control of blood, feces, or urine sediment analyzers.

[0092] By rehydrating pre-stained frozen stem cells with water, routine quality control of formed element analyzers can be performed at low cost. Routine quality control or instrument calibration can be performed on a weekly, monthly, or quarterly basis, thereby improving the instrument's testing accuracy.

[0093] The applicant proposed quality control for white blood cell differential and reticulocyte parameters.

[0094] The applicant proposes algorithm verification and / or consistency assessment for AI image recognition devices.

[0095] The applicant proposed that it be used for taking microscopic images to determine whether the instrument's imaging, identification, classification, or counting functions are qualified.

[0096] The applicant states that the instrument is used for the detection of biological samples from animals or plants.

[0097] The applicant states that the instrument is used for the detection of human biological samples.

[0098] Beneficial effects: Can be stored and transported at room temperature, with a shelf life of ≥18 months; cells remain morphologically intact, do not hemolyze, and do not aggregate; pre-stained solutions are ready to use after reconstitution, eliminating the need for on-site staining; suitable for AI microscopy instruments, blood analyzers, and morphology analyzers; individually packaged, disposable, and free from cross-contamination. Attached Figure Description

[0099] Figure 1 This is part of the report output after analyzing a urine sample under a 40x magnifying microscope; Figure 2 This is part of the report output after a blood sample under a 40x magnifying microscope is analyzed by the equipment. Figure 3 This is part of the report output after analyzing a fecal sample under a 40x magnifying microscope; Figure 4 This describes the preparation process of frozen stem cells in Example 1; Figure 5 This is a schematic diagram of the rehydration of frozen stem cells in Example 2; Figure 6 This describes the preparation process of frozen stem cells in Example 3; Figure 7 This describes the preparation process of frozen stem cells in Example 4; Figure 8 This describes the preparation process of frozen stem cells in Example 5; Figure 9 This describes the preparation process of frozen stem cells in Example 6; Figure 10 This is a list of component formulations and effect descriptions for multiple embodiments and comparative examples; Figure 11 These are magnified microscopic images of the quality inspection sample after reconstitution in Example 11; Figure 12 This is a magnified microscopic image of the quality inspection sample after reconstitution in Example 13; Figure 13 This is a magnified microscopic image of the quality inspection sample after reconstitution in Example 14; Figure 14 This is a magnified microscopic image of the quality inspection sample of Comparative Example 1 after reconstitution; Figure 15 This is a magnified microscopic image of the quality inspection sample of Comparative Example 2 after reconstitution; Figures 11 to 15 The magnified micrograph is 40x. Detailed Implementation

[0100] The contents of this application will be further described in detail below with reference to the accompanying drawings. It should be noted that the following description is of preferred embodiments of the present invention and does not constitute any limitation on the present invention. The description of the preferred embodiments of the present invention is merely an explanation of the general principles of the invention. The use of terms such as "first," "second," and "A," "B," etc., in the present invention is for ease of explanation only and does not represent a temporal or spatial order.

[0101] Figure 1 This is part of the report output after the urine sample under a 40x magnifying microscope is analyzed by the equipment. Figure 2 This is part of the report output after a blood sample under a 40x magnifying microscope is analyzed by the equipment. Figure 3 This is part of the report generated after analyzing a fecal sample under a 40x magnifying microscope. With the development of science and technology, formed element detection instruments have gradually become essential high-tech testing equipment in pet hospitals.

[0102] These devices not only have the ability to identify cells, crystals, and food residues in samples, but also measure the volumetric content of tiny objects such as cells in a sample, making them high-tech metrology equipment. Their functionality and / or measurement accuracy need to be tested during the production process, factory quality inspection, and operation.

[0103] These types of devices require cell identification and / or volume concentration measurement. Cells can be used to verify and / or calibrate the device's function and / or detection accuracy. However, standard samples are lacking for calibration and functional verification of such devices. The applicant proposed using cells for functional verification and quality control. However, cell collection, standard solution preparation, and staining procedures require specialized knowledge and training from operators, making them difficult to perform. Different animals and individuals have different cell morphologies and sizes, making accurate calibration challenging. More importantly, while newly collected cells meet the morphological requirements for instrument quality control, they maintain their morphology for a very short time, making practical use difficult.

[0104] Existing liquid cell samples or frozen stem cell samples in the industry cannot be used as standard quality control products because it is difficult to restore the original morphology of the cells.

[0105] Pet hospitals and hospitals serving humans have a high demand for standard cells in their equipment, with specific requirements regarding cell morphology, size, and type. Traditional products and methods are insufficient in terms of both performance and price.

[0106] These types of devices often require staining to clearly visualize the interior of cells, such as substances within the cell nucleus. The equipment has strict requirements for stain recognition and precise measurement, thus necessitating standardized stained cells for calibration and / or functional testing. The staining process for cells demands a high level of expertise, which is difficult for ordinary equipment maintenance personnel to master.

[0107] Figure 4 Example 1 describes the preparation process of frozen stem cells.

[0108] To address the problems of traditional liquid quality control products, such as Figure 4A frozen stem cell for quality control of a microscopic testing instrument is disclosed. The frozen stem cells are prepared by treating a liquid containing cells with a fixative and a lyophilization protectant, then individually dispensing them into single portions and freeze-drying them. The fixative is used to lock in the original morphological structure of the cells, and the lyophilization protectant is used to inhibit cell deformation, hemolysis, or aggregation during freeze-drying and reconstitution. The frozen stem cells are a single-portion freeze-dried preparation, which is used for microscopic image acquisition and / or instrument quality control after reconstitution.

[0109] Frozen stem cells are stem cell products treated with fixatives and lyophilization protectants, and they have storage resistance. Thanks to the effect of fixatives, their cell morphology remains intact after rehydration, and microscopic images similar to or the same as those of fresh blood can be obtained. This can significantly reduce the working costs of calibration, verification, and functional verification in the entire industrial chain of instrument production and use.

[0110] like Figures 1 to 3 In the quality inspection, calibration and / or validation of instruments, it is very difficult to test the color reproduction performance of stained cells. Traditional methods generally do not use staining for biological products such as cells, or it is difficult to meet the detection requirements of the equipment to perform accurate staining.

[0111] The applicant proposed that the frozen stem cells be stained during or after rehydration before being used for microscopic image acquisition and / or instrument quality inspection.

[0112] like Figure 5 Example 2: Schematic diagram of frozen stem cell rehydration.

[0113] By adding water and staining agents to frozen stem cells, the cells can be stained upon reconstitution.

[0114] The reconstituted cells have properties close to those of fresh cells and can be stained. By staining them, the performance indicators of the instrument in cell color imaging, acquisition and / or analysis can be verified.

[0115] Cell staining operations require certain skills from operators, and the staining process also has strict standards for environmental temperature, staining time, and other conditions. In order to reduce the complexity of on-site operations and the cost of staining skills training, we need to address these issues.

[0116] If staining is completed during the cell-containing liquid stage of frozen stem cell preparation, the operational difficulty for on-site testing personnel can be greatly reduced.

[0117] like Figure 6 Example 3 describes the preparation process of frozen stem cells.

[0118] When fixing cells, fixatives and staining agents are added to complete cell fixation; liquid samples with cells are first stained, then lyophilization protectant is added, and after being individually packaged according to the set specifications, they are freeze-dried to obtain the finished product; the cells have been stained before freeze-drying to form a stable stained morphology.

[0119] During the production process, frozen stem cells are stained to obtain stained frozen stem cells. On-site personnel do not need to perform staining operations, which greatly reduces the workload of staining training for personnel and significantly reduces on-site operation time and various errors caused by operational mistakes.

[0120] like Figure 7 Example 4 describes the preparation process of frozen stem cells.

[0121] Cells that have not been treated with fixatives have a relatively short shelf life, but they can reduce the difficulty of on-site operations and have commercial application value.

[0122] like Figure 8 Example 5 describes the preparation process of frozen stem cells.

[0123] The applicant discovered that, prior to lyophilization treatment, some components in the liquid could affect the intact morphology of cells after reconstitution, such as... Figure 8 After cleaning, other extracellular components are removed, which improves the quality of the product and allows the complete morphology of the cells to be well displayed after reconstitution.

[0124] like Figure 9 Example 6 describes the preparation process of frozen stem cells.

[0125] After treatment with the fixative, the cells are washed, and then a freeze-drying protectant is added for further treatment.

[0126] The staining agent is one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green.

[0127] Extensive experimental verification has shown that when staining is performed in advance, the above-mentioned staining agents can all exhibit good staining effects after reconstitution.

[0128] The staining process is used to stain one or more of the following: cell nucleus, cytoplasm, granules, intracellular structures, and RNA / DNA.

[0129] Extensive experimental verification has shown that pre-staining can meet the above-mentioned staining requirements for the inside of cells, and good staining results can be achieved after reconstitution.

[0130] The fixative is one or a combination of formaldehyde, paraformaldehyde, and glutaraldehyde.

[0131] Through numerous experiments, the applicant discovered that frozen stem cells can be preserved for a long time after treatment with fixatives, and can also exhibit a good morphology after rehydration.

[0132] like Figure 9After extensive experimentation, the applicant discovered that if the excess fixative is not washed away after fixation treatment, it will affect cell rehydration and unfolding after a period of storage. In order to extend the storage time, the cells need to be washed away before freeze-drying to remove the excess fixative.

[0133] Lyophilization protectants include one or more of trehalose, sucrose, and hydroxyethyl starch. Lyophilization protectants are used to maintain cell morphology, size, and structural integrity.

[0134] Through extensive experiments, the applicant found that the above-mentioned components can effectively maintain the morphology of cells during freeze-drying, and after a certain period of storage, they can exhibit good morphology upon reconstitution.

[0135] The cells can be one or more of the following: blood cells, body fluid cells, and tissue cells.

[0136] Through extensive experimental verification, the applicant discovered that all of the above-mentioned cells can be prepared as frozen stem cell quality control products.

[0137] The cells are derived from one or more of the following: human, cow, monkey, dog, cat, turtle, rabbit, and parrot.

[0138] The cells are derived from bovine blood cells. Through extensive experimentation, the applicant discovered that both human and animal cells can be prepared into frozen stem cell quality control products. Considering the ease of collection for commercial applications and the volume requirements for mass production, using bovine blood cells is a relatively better option because bovine blood cells can be collected from living individuals, are available in large quantities, and can meet the needs of periodic batch collection.

[0139] The cells can be derived from vertebrates or invertebrates, and this product can provide support for the quality inspection needs of different species of cells in special testing, scientific research and teaching scenarios.

[0140] This product can be dispensed into single-use portions or multiple-use portions according to the set dosage. As a biological product, it readily absorbs moisture from the air and changes after opening. By dispensing it into the set dosage and using it immediately after opening, the quality requirements of experiments or tests can be guaranteed.

[0141] These instruments are high-tech equipment with stringent quality control requirements. Using single-use packaging can effectively ensure the stability of the quality control products. For some tests that require repeated experiments two or more times, two or more portions of lyophilized powder can be packaged together, opened together, and used up within a short period of time.

[0142] Reconstitution can be performed using pure water or a reconstitution solution. Using pure water reduces the difficulty of operation for on-site personnel, and the procedure is simple and easy to understand, allowing for quick mastery by on-site staff. Specialized reconstitution solutions can be prepared by adding staining agents or calibration particles to the reconstitution solution, enabling more functions. For example, calibration particles can be used to calibrate the magnification accuracy of the microscope system. Alternatively, irregular particles can be added to verify the AI ​​software's ability to identify and / or resist interference from non-standard objects.

[0143] The microscopic testing instruments include a cell morphology analyzer, a blood, urine, or fecal suspension formed element analyzer, and an AI image recognition instrument.

[0144] The aforementioned instruments all require AI-based cell recognition capabilities and demand a very high degree of cell restoration. Through extensive and long-term experiments by the applicant, frozen stem cells, or stained frozen stem cells reconstituted, have been developed to meet the quality testing requirements of these devices, significantly reducing the costs of quality testing, calibration, and functional testing. This also simplifies the traditional sample acquisition process.

[0145] The evaluation criteria include one or more aspects such as image clarity, color reproduction, identification accuracy, classification accuracy, cell size, cell volume, and the reproducibility of multiple measurements. Extensive experiments and repeated validations have shown that frozen stem cells or stained frozen stem cells rehydrated after staining can support the above evaluation criteria.

[0146] The applicant stated that the evaluation was intended to determine whether the instrument's optics, image acquisition, or analysis software was functioning correctly. Extensive experimentation and repeated verification have shown that frozen stem cells, or thawed stained frozen stem cells, can support this evaluation.

[0147] Through repeated testing and verification, the applicant has demonstrated that the lyophilized powder can be stored and transported at room temperature. The product, after being stored and transported at room temperature and protected by a preservative, can support the aforementioned evaluation work upon reconstitution.

[0148] Through repeated testing and verification, the applicant has determined that the shelf life of the lyophilized powder is ≥18 months. Lyophilized powder treated with fixation and preservatives, after 18 months of storage, can be used to support the above assessment. Lyophilized powder treated with preservatives has a shorter shelf life, and within a shorter timeframe, it can support the above assessment.

[0149] Through repeated experiments and verifications, the applicant has determined that the cells in question are one or more of the following: red blood cells, white blood cells, platelets, and reticulocytes. In blood testing, some specialized tests require different cell types to verify the instrument's functionality. Verification has shown that red blood cells, white blood cells, platelets, and reticulocytes, after rehydration, can all pass the instrument's detection.

[0150] Through repeated experiments and verifications, the applicant has confirmed that the white blood cells include one or more of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In blood testing, specific types of white blood cells are required to verify the detection function and / or performance. Neutrophils, lymphocytes, monocytes, eosinophils, and basophils, after rehydration, can all pass the detection of the aforementioned instrument.

[0151] After repeated experiments and verifications, the applicant has obtained RNA structure staining for the reticulocytes used in the quality control of the reticulocyte detection instrument.

[0152] In blood testing, reticulocytes, due to their small size and diverse morphology, are crucial indicators. Many instruments cannot effectively perform these tests. Through extensive experimentation, reticulocytes were extracted from large quantities of animal blood. After long-term, repeated experiments, a freeze-dried reticulocyte powder was produced. Following transportation and storage experiments, this powder met the quality control requirements of reticulocyte testing instruments. This transformed what was previously impossible into a reality.

[0153] The applicant proposes that the specified dosage be in individually packaged, disposable portions. After repeated research into usage scenarios, the use of small-portion packaging ensures the safety requirements of the biological products used in the product, while also meeting the precision requirements of high-tech equipment. The factory is equipped with high-precision equipment to accurately control the dosage of the small-portion packaging, thus meeting the precision requirements of scientific instruments.

[0154] After repeated experiments and verifications, the applicant confirmed that the product did not exhibit cell aggregation or hemolysis after reconstitution.

[0155] The applicant has conducted extensive transportation and storage experiments to verify that the product, after reconstitution within its 18-month shelf life, exhibits no cell aggregation or hemolysis and can be used normally.

[0156] If the product exceeds its shelf life or is improperly stored, and cell sedimentation, cell aggregation, or hemolysis occurs after reconstitution, it cannot be used for instrument quality control.

[0157] The applicant proposes a method for preparing frozen stem cells for quality inspection of a microscopic testing instrument, comprising: step A20: adding a fixative to a cell-containing liquid for fixation; step A30: adding a lyophilization protectant to the cells to prevent cell morphology from being damaged during lyophilization; step A40: dispensing the liquid containing cells and lyophilization protectant into individual portions; lyophilizing the dispensed samples to obtain individually dispensed lyophilized powder; and reconstituted the lyophilized powder with water before use to form a test sample for microscopic image capture.

[0158] After treatment with a fixative, a freeze-drying protectant is added before freeze-drying and the cells are individually aliquoted according to accurate proportions. The resulting frozen stem cells are then freeze-dried directly. This ensures that the cells retain their morphology after rehydration and meets the cell quantity and concentration requirements for quality inspection.

[0159] Before or after step A20, including step A10: adding a staining agent to the cell-containing liquid for staining treatment; or, in step A20, mixing the staining agent into the fixative, completing the staining simultaneously with fixation. The stained cells meet the color calibration requirements of the quality inspection process. Adding a staining agent to the cell-containing liquid, where the staining agent includes a fixative, effectively reduces production steps and controls product quality. In step A30, the cell-containing liquid is first washed to remove residual fixative or staining agent, and then the cells are added to a liquid containing a lyophilization protectant. Washing removes residual fixative, staining agent, and other unknown components, ensuring no impurities remain after reconstitution and eliminating the adverse effects of the fixative on subsequent operations.

[0160] The staining uses one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green.

[0161] After repeated experiments and verifications, the applicant found that the fixative includes one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; the fixation temperature is 2℃~8℃, and the fixation time is 30min~24h.

[0162] Through repeated experiments and verifications, the applicant has determined that the freeze-drying protectant includes one or more of trehalose, sucrose, and hydroxyethyl starch. The final concentration of the freeze-drying protectant is 0.5%–10%. The cell-containing fluid is an isotonic buffer system.

[0163] After repeated experiments and verifications, the applicant determined that the individual single-part packaging volume is 200μL to 1000μL. Single-part aseptic packaging is employed; after packaging, the product is temporarily stored at low temperature in the dark; lyophilization includes pre-freezing, sublimation drying, or desorption drying; the applicant proposed a pre-freezing temperature of -35℃ to -50℃; and after repeated experiments and verifications, the final moisture content of the finished product is ≤3%.

[0164] The applicant proposes a quality control kit for microscopic testing instruments, comprising a single-packaged pre-stained frozen stem cell powder; the frozen stem cell powder is prepared by freeze-drying pre-stained cells in a solution containing a lyophilization protectant; a reconstitution solution is added during use to form a test sample for microscopic image acquisition; the test sample is used to evaluate whether the instrument's imaging, identification, classification, and counting functions are qualified.

[0165] By repackaging the individual kits, the impact on the individual packages during transportation can be prevented.

[0166] The kit includes K1 single-packaged frozen stem cell powder and K2 single-packaged frozen stem cell powder. After reconstitution with the same volume V of water, the cell concentration of the K1 test sample is greater than that of the K2 test sample, or the K1 concentration is an integer multiple of the K2 concentration.

[0167] By packaging products with different concentrations, testing needs for varying concentrations can be met. Packaging products with concentration differences in multiples of each other allows us to assess the instrument's metrological accuracy and determine whether the instrument can detect concentration differences in multiples of each other.

[0168] The kit includes frozen stem cell powder for a five-part differential hematology test. The kit also includes frozen stem cell powder for a reticulocyte count. The kit includes a standard image library, judgment rules, and quality control range instructions. The frozen stem cell powder mentioned above is the same as the frozen stem cells described earlier.

[0169] To reduce the difficulty for field personnel in obtaining reconstitution solutions, the kit includes individually packaged reconstitution solutions. These individually packaged solutions allow field personnel to accurately prepare the solution to the desired cell concentration.

[0170] The reconstitution solution is pure water. Pure water is supplied with the kit, significantly simplifying the operation steps for on-site personnel and reducing operational difficulty. The kit includes individually packaged reconstitution solutions of different volumes, with larger volumes being integer multiples of smaller volumes. Providing individually packaged reconstitution solutions of different volumes allows on-site personnel to accurately prepare liquids with different cell concentrations. The kit is suitable for model validation and calibration of AI cell morphology analyzers. The volume of the reconstitution solution is marked on the package. The volume marking on the reconstitution solution packaging helps on-site personnel accurately calculate the concentration of the cell suspension to be prepared.

[0171] The applicant proposes the use of pre-stained frozen stem cells in the quality inspection of a microscopic testing instrument. The method involves reconstituted pre-stained, individually packaged, and freeze-dried cell powder with water; capturing microscopic images of the reconstituted samples to obtain microscopic images of the stained cells; and determining the instrument's qualification based on the morphology, structure, color, or clarity of the stained cells in the images. The cells are stained before freeze-drying and do not require further staining after reconstitution.

[0172] By using pre-stained frozen stem cells reconstituted with water, the difficulty of sample acquisition for quality control of microscopic testing instruments is significantly reduced, eliminating the need for on-site staining and greatly improving sample quality. This also enables accurate calibration of on-site equipment.

[0173] The applicant proposed that this method can be used for routine quality control of blood, feces, or urine sediment analyzers. By rehydrating pre-stained frozen stem cells with water, routine quality control of sediment analyzers can be performed at low cost. This allows for weekly, monthly, or quarterly quality control or instrument calibration, improving the instrument's testing accuracy.

[0174] The applicant proposes the following: Quality control for white blood cell five-part differential and reticulocyte parameters; algorithm verification and consistency evaluation for AI image recognition equipment; and microscopic image capture to determine the quality of the instrument's imaging, recognition, classification, or counting functions. The instrument can be used for the detection of animal or plant biological samples. Plant cells, after discrete processing, can also be prepared into frozen stem cells for instrument calibration. When the instrument is used for the detection of human biological samples, the need for calibration, routine calibration, and functional testing also applies to instruments detecting ex vivo human samples; this invention is equally applicable.

[0175] This invention belongs to the field of in vitro diagnostic medical device technology, specifically relating to a pre-stained lyophilized blood cell quality control product and its preparation method, which is suitable for quality control of blood cell analysis, covering basic parameters of complete blood cell count (CBC), white blood cell differential count, and reticulocyte (Ret) parameters.

[0176] Hematology analysis is one of the most basic and commonly used tests in clinical laboratory and veterinary medicine, covering routine parameters such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), and platelet count (PLT), as well as extended parameters such as five-part differential white blood cell count and reticulocyte count (Ret). To ensure the accuracy and reliability of test results, hematology analyzers require routine quality control using quality control materials.

[0177] Currently, there are two main types of blood cell analysis equipment on the market: Traditional blood cell analyzers: These use principles such as electrical impedance tomography and laser scattering to detect routine hematological parameters such as WBC, RBC, HGB, and PLT.

[0178] Formed element analyzer: Based on morphological analysis or artificial intelligence image recognition technology, it performs microscopic imaging and classification analysis of formed elements (red blood cells, white blood cells, platelets, etc.) in blood. This type of equipment has higher requirements for the preservation of cell morphology in quality control samples.

[0179] Freeze-drying technology has been widely used in the field of in vitro diagnostic reagents. Its principle is to first freeze water-containing materials to below the freezing point, and then sublimate the ice directly into water vapor under vacuum conditions to obtain dried products.

[0180] According to the applicant's research, all mainstream global whole blood cell count (CBC) hematology quality control products are in liquid form, and no commercial products in lyophilized form were found.

[0181] While lyophilization technology is widely used in IVD quality control products, it has long remained a challenge in the field of CBC whole blood cell quality control products. The main reason for this is: 1. High requirements for precise preservation of blood cell volume and morphology: Among CBC parameters, the mean erythrocyte volume (MCV) and erythrocyte distribution width (RDW) of RBCs are extremely sensitive to changes in cell volume. Ice crystal formation and dehydration shrinkage during freeze-drying can cause irreversible changes in cell volume, directly affecting the accuracy of calculated parameters such as MCV, MCH, and MCHC.

[0182] 2. Multiple cell types need to be simultaneously stabilized: CBC quality control products need to cover three types of cells: red blood cells, white blood cells, and platelets. The optimal freeze-drying protection conditions for the three types may be different, so it is necessary to find a protection system with good compatibility.

[0183] 3. Stability of red blood cell hemoglobin: During the freeze-drying-reconstitution process, red blood cells may undergo hemolysis or hemoglobin leakage, leading to deviations in the target values ​​of parameters such as HGB and HCT.

[0184] 4. Platelet aggregation and activity maintenance: Platelets are extremely sensitive to changes in shear force and osmotic pressure. Irreversible aggregation can easily occur during freeze-drying, leading to distortion of PLT counts.

[0185] 5. Changes in cell scattering properties after fixation: Existing liquid quality control products typically use glutaraldehyde or formaldehyde to fix blood cells to improve stability. However, fixation may alter the scattering properties of cells (especially for laser scattering analyzers), and the freeze-drying process further increases the risk of cell membrane protein denaturation.

[0186] The aforementioned technical challenges explain why, despite possessing mature freeze-drying technology, mainstream manufacturers still use liquid methods for CBC quality control products.

[0187] In existing technologies, lyophilized whole blood is used for specific testing items (non-CBC), such as G6PD lyophilized whole blood quality control products, i.e., glucose-6-phosphate dehydrogenase activity quantitative assay kits and their lyophilized whole blood quality control products, used for G6PD enzyme activity detection. Some specific biochemical / immunological assays also have similar quality control products. There are also lyophilized platelets used in single transfusion medicine projects. However, in existing technologies, there is no known technical solution for using lyophilized whole blood or lyophilized blood cells as multi-parameter quality control products for CBC, nor is there a solution for lyophilized blood cell quality control products containing staining treatment.

[0188] The current application status of freeze-drying technology in blood cell quality control products can be summarized as follows: 1. Lyophilization technology has been widely used in IVD quality control products, but it mainly covers parameters such as clinical chemistry, immunology, and coagulation, and does not involve multiple parameters of CBC whole blood cells.

[0189] 2. In the existing technology, only freeze-dried whole blood has been used for quality control products, but it only covers the single G6PD test item.

[0190] 3. Currently, there are no commercial products worldwide that apply freeze-drying technology to CBC whole blood cell multi-parameter quality control products.

[0191] 4. Globally, there are no lyophilized blood cell quality control products containing staining treatment (especially pre-staining).

[0192] Existing blood cell quality control technology has the following defects and shortcomings: 1. Short shelf life and heavy reliance on cold chain: Liquid quality control products typically have a shelf life of only 60 to 120 days and must be stored and transported in a cold chain at 2 to 8 degrees Celsius throughout the process. This places strict requirements on the cold chain conditions for distributors and end users, resulting in high logistics costs and the risk of product failure due to chain breaks.

[0193] 2. Poor stability after opening: After opening, liquid quality control products may experience sedimentation, aggregation, metabolic changes, or morphological changes in blood cells due to increased usage and prolonged storage time. This can lead to deviations in the detection target values ​​and affect the reliability of the quality control results. Users need to strictly follow the mixing procedure daily; improper operation can easily introduce errors.

[0194] 3. Significant waste for single-person / small-batch users: For users with low daily testing volume, a bottle of quality control product may not be used up within its shelf life, resulting in reagent waste; if multiple people share a bottle, cross-operation further increases the risk of contamination and instability.

[0195] 4. Ret quality control products require back-end staining, resulting in poor consistency: All existing Ret quality control products require users to stain them themselves before using the machine. The staining results are affected by various factors such as operator skills, environmental conditions, and batches of dye solutions, resulting in poor consistency of quality control results and low comparability between different laboratories.

[0196] 5. Incomplete coverage of Ret (Reticulocyte) parameters: Most Ret quality control products only cover Ret% (Reticulocyte Percentage) and Ret# (Reticulocyte Count). There is insufficient supply of quality control products for IRF (Immature Reticulocyte Fraction) parameters, which makes it difficult to meet the clinical demand for comprehensive Ret parameter quality control.

[0197] 6. No freeze-dried CBC quality control products worldwide: Currently, all CBC whole blood cell multi-parameter quality control products worldwide, whether for human or animal use, are in liquid form. There are no freeze-dried products or patent reports, and users cannot get rid of their dependence on the cold chain.

[0198] The purpose of this invention is to provide a pre-stained lyophilized blood cell quality control product and its preparation method, in order to solve the problems of short shelf life, instability after opening, cold chain dependence, and cumbersome operation of existing liquid quality control products, as well as the poor consistency caused by the need for back-end staining of existing Ret quality control products.

[0199] Specifically, freeze-drying technology is used to process blood cells into freeze-dried products with a shelf life of up to 18 months. These products can be transported at room temperature, significantly reducing cold chain logistics costs and the risk of chain disruptions.

[0200] The product features an individual single-serving packaging design, allowing for quick and easy use and disposal, fundamentally eliminating the issue of stability upon opening traditional liquid quality control products.

[0201] The reconstitution process is simple; only a fixed amount of pure water needs to be added to restore the product to its original state. No additional reconstitution solution needs to be prepared, which lowers the barrier to entry for users.

[0202] Staining is performed simultaneously in a fixed procedure, so that after reconstitution of the quality control product, it can support the detection of multiple parameters such as five-part differential white blood cells and reticulocytes (Ret), and a single product covers more quality control needs.

[0203] The dye is added to the matrix solution before lyophilization. After lyophilization and storage, the user can reconstitute the dye and directly test it on the instrument without additional staining operations. This eliminates the consistency problem caused by back-end staining and ensures the consistency and comparability of the test results.

[0204] This invention uses whole blood as raw material. After blood cell separation, washing, fixation and secondary washing, the blood cells are suspended in a base solution containing dye, and a matrix suspension is adjusted to the target cell concentration. Freeze-drying protectants (sucrose, hydroxyethyl starch, trehalose, etc.) are added, and the product is packaged in individual vials (500 μL per person) and freeze-dried to produce single-person freeze-dried quality control products.

[0205] When using it, simply add a measured amount of pure water to reconstitute it before testing. No reconstitution solution or additional dyeing is required—the dye is already included in the base solution before freeze-drying.

[0206] This invention covers the following parameters: CBC basic parameters: WBC, RBC, MCV, MCH, HGB, HCT, MCHC, PLT (8 items); white blood cell differential: neutrophils, lymphocytes, monocytes, eosinophils, basophils (5 categories); reticulocyte parameters: Ret%, Ret#, IRF.

[0207] This invention applies the above-mentioned dyeing technology to the scenario of freeze-dried quality control products, that is, using a base solution containing dye as the matrix suspension before freeze-drying - so that the quality control products can be directly tested after reconstitution without the need for users to perform back-end dyeing operations.

[0208] Whole blood samples with added anticoagulants were collected as the starting material.

[0209] The original blood sample was centrifuged to separate blood cells and plasma components. After removing the plasma, the blood cells were washed with buffer solution to remove impurities such as plasma proteins and anticoagulants.

[0210] The fixed blood cells are washed a second time to thoroughly remove residual fixative and avoid the impact of fixative residue on subsequent freeze-drying and reconstitution processes.

[0211] The cleaned immobilized blood cells were suspended in a base solution containing dye, the cell concentration was adjusted to the target level, and lyophilization protectants (sucrose, hydroxyethyl starch, trehalose, etc.) were added to fully protect the blood cells during the lyophilization process and maintain their morphology, volume, and dispersibility after reconstitution.

[0212] Preliminary testing and target calibration were performed on various blood cell parameters (including CBC baseline parameters, white blood cell differential parameters, reticulocyte parameters, etc.) to confirm that the parameter values ​​of the quality control products were within the target range.

[0213] The adjusted quality control suspension was dispensed into individual vials, with each vial containing 500 μL, and then freeze-dried to produce freeze-dried quality control products.

[0214] The freeze-dried quality control samples underwent final quality inspection, and calibration tests were performed on multiple hematology analyzers. All parameters (basic CBC parameters + white blood cell differential + reticulocyte parameters) were finally assigned values ​​to determine the target values ​​and allowable ranges for each parameter.

[0215] Pre-stained lyophilized blood cell quality control products are prepared by lyophilizing blood cells after fixation in a base solution containing dye, and then reconstituted for direct testing without additional staining.

[0216] A combination of freeze-drying protectants (sucrose + hydroxyethyl starch + trehalose) is used to simultaneously protect the morphological and volume stability of blood cells during the freeze-drying process.

[0217] The preparation process flow (separation → cleaning → fixation → cleaning → matrix preparation (including dye) + excipients → preliminary assignment → dispensing → freeze drying → calibration assignment), and the key process parameters for each step.

[0218] The product is individually packaged for single use and freeze-dried, so it can be reconstituted and tested immediately.

[0219] The method of reconstitution with pure water resulted in good cell dispersibility after reconstitution, and the target values ​​of each parameter were within the allowable range.

[0220] The purpose of lyophilization of dye-based solutions covers basic CBC parameters, white blood cell differential, and reticulocyte parameters (Ret%, Ret#, IRF).

[0221] The dye-containing base solution allows for immediate reconstitution and testing: the dye is added to the base solution before lyophilization. After lyophilization and storage, users can directly reconstitute the dye for testing without additional staining. Compared to traditional Ret quality control products, which require post-processing staining before testing (affected by various factors such as operator skill, environmental conditions, and dye batch number), this solution eliminates variations in staining procedures, ensuring consistency of test results and comparability between laboratories.

[0222] Significantly extended shelf life and the ability to transport at room temperature: Through freeze-drying technology, the shelf life of quality control products can reach 18 months, far exceeding the 60-120 days of existing liquid quality control products (an extension of approximately 4-9 times). Freeze-dried products can be transported at room temperature, while existing liquid quality control products require a cold chain of 2-8°C throughout the entire process, significantly reducing cold chain logistics costs and the risk of chain breakage.

[0223] Individually packaged for single use, eliminating the problem of instability after opening: Each 500μL individual vial is ready to use and disposable. Existing liquid quality control products have a shelf life of only 7-30 days after opening, and parameters gradually drift over time; this invention fundamentally eliminates this problem, resulting in more stable and reliable quality control results.

[0224] The range of parameters has been significantly expanded: By using dye-based solutions after fixation, the lyophilized quality control products now support the detection of white blood cell five-part differential and reticulocyte parameters (Ret%, Ret#, IRF) in addition to the original eight CBC basic parameters (WBC, RBC, MCV, MCH, HGB, HCT, MCHC, and PLT), covering more quality control needs with a single product.

[0225] Reconstitution is simple and requires no special reagents: Only a measured amount of pure water needs to be added for reconstitution before use; no additional reconstitution solution needs to be prepared or purchased, as the dye is already included in the base solution of the lyophilized quality control sample. This further simplifies user operation compared to existing solutions.

[0226] The freeze-drying protection system effectively maintains cell morphology: The freeze-drying protection system (sucrose + hydroxyethyl starch + trehalose) of this invention is systematically designed to address the technical challenges of blood cell freeze-drying (volume retention, multi-cell synchronous stability, hemoglobin stability, and platelet anti-aggregation), ensuring intact cell morphology after reconstitution. The dye-containing base solution ensures stable staining signals after reconstitution.

[0227] Lowering the barriers to laboratory use and costs: The dyeing process is eliminated, and the dye is already included in the freeze-dried quality control product, which can be used directly by all laboratories, especially beneficial to the quality control work of primary healthcare institutions.

[0228] Preparation process overview: ① Raw material collection (EDTA or heparin anticoagulated whole blood) → ② Blood cell separation and washing, removing plasma; → ③ Fixation, treatment with fixative to lock cell morphology; → ④ Secondary washing to remove residual fixative; → ⑤ Adjusting the matrix and adding excipients, which can be a dye-containing base solution + sucrose + hydroxyethyl starch + trehalose; → ⑥ Preliminary calibration (target value calibration, including CBC + classification + Ret parameters) → ⑦ Dispensing, 500μL / vial; → ⑧ Lyophilization, vacuum freeze-drying; → ⑨ Calibration and calibration (final quality inspection, including calibration of all parameter target values); → ⑩ Finished product.

[0229] This application discloses frozen stem cells for quality control, their preparation method and uses, and a quality control kit, including two core technical routes: (1) fixation + lyophilization protectant to maintain cell morphology integrity; (2) pre-staining followed by lyophilization, followed by reconstitution for direct imaging without staining. The frozen stem cells of this invention can be stored and transported at room temperature. After reconstitution, the cells exhibit stable morphology, no hemolysis, and no aggregation, making them suitable for quality control, instrument calibration, and / or algorithm verification in blood analyzers, cell morphology analyzers, and AI image recognition equipment. This invention is simple to operate, highly stable, and widely applicable, solving problems such as cold chain dependence, short shelf life, cumbersome staining, and easy morphological damage associated with traditional quality control products.

[0230] To verify the technical effectiveness, this application has designed several embodiments, such as... Figure 10 The diagram shows the components and their reconstituted states of Examples 11 to 28. Due to the large number of examples, magnified microscopic images of some examples after reconstitution are selected, such as... Figures 11 to 13 The cells are morphologically intact and clearly stained, making them suitable for quality inspection. Figure 14 and Figure 15 The magnified microscopic images of Comparative Example 1 and Comparative Example 2 after reconstitution are shown respectively. Figure 14 In the middle stage, most cells ruptured and appeared as shadows, making them unusable for quality inspection. Figure 15 In the middle, most cells are stuck together and cannot be used for quality inspection.

[0231] Although the present invention has been described and illustrated in conjunction with preferred embodiments and several alternatives, it is not limited to the specific descriptions herein, and other alternative or equivalent components may also be used to implement the present invention.

Claims

1. A frozen stem cell for quality inspection, characterized in that, Quality control for microscopic testing instruments includes: The frozen stem cells were prepared by adding a fixative and a freeze-drying protectant to a liquid containing cells, then individually dispensing them into single portions and freeze-drying them. The fixative is used to lock in the original morphological structure of the cells, and the freeze-drying protectant is used to inhibit cell deformation, hemolysis or aggregation during freeze-drying and reconstitution. The frozen stem cells are a single-sample lyophilized preparation, which is then reconstituted for use in microscopic image acquisition and / or instrument quality inspection.

2. The frozen stem cells for quality inspection according to claim 1, characterized in that: The frozen stem cells are stained during or after rehydration and then used for microscopic image acquisition and / or instrument quality inspection.

3. The frozen stem cells for quality inspection according to claim 1, characterized in that: The cell-containing fluid used to prepare the frozen stem cells was stained.

4. A frozen stem cell for quality inspection, characterized in that, Quality control for microscopic testing instruments includes: The frozen stem cells were prepared by first staining a liquid sample containing cells, then adding a freeze-drying protectant, and then individually dispensing them according to a set procedure, followed by freeze-drying. The cells were stained before freeze-drying, forming a stable staining morphology; The frozen stem cells are a single-sample lyophilized preparation, which is then reconstituted for use in microscopic image acquisition and / or instrument quality inspection.

5. The frozen stem cells for quality inspection according to any one of claims 1 to 4, characterized in that: Before treatment with the freeze-drying protectant, the cells are washed, and then the freeze-drying protectant is added for further treatment.

6. The frozen stem cells for quality inspection according to any one of claims 2 to 4, characterized in that, Includes one or more of the following features: Feature 10: The staining uses one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green; Feature 20: The staining treatment is used to stain one or more of the following: cell nucleus, cytoplasm, granules, intracellular structures, and RNA / DNA.

7. The frozen stem cells for quality inspection according to any one of claims 1 to 4, characterized in that, Includes one or more of the following features: Feature 30: The fixative includes one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; Feature 40: The freeze-drying protectant includes one or more of trehalose, sucrose, and hydroxyethyl starch; Feature 80: Lyophilization protectants are used to maintain cell morphology, size, and structural integrity; Feature 50: The cells are one or more of blood cells, body fluid cells, and tissue cells; Feature 60: The cells are derived from one or more of humans, cattle, monkeys, dogs, cats, turtles, rabbits, and parrots; Feature 61: The cells are derived from bovine blood cells; Feature 70: The cells are derived from vertebrates or invertebrates; Feature 90: It can be packaged into single-use portions or multiple-use portions according to a set amount; Feature 100: The redissolution uses pure water or a redissolution solution, the redissolution solution comprising a salt; Feature 110: The microscopic testing instrument is a cell morphology analyzer, a blood, urine or fecal suspension formed element analyzer, and an AI image recognition instrument; Feature 120: The evaluation content includes one or more of the following: image sharpness, color reproduction, recognition accuracy, classification accuracy, cell size, cell volume, and repeatability of multiple measurement results; Feature 130: The evaluation content is used to determine whether the instrument's optics, image acquisition, or analysis software is functioning properly; Feature 140: The freeze-dried powder can be stored and transported at room temperature; Feature 150: After treatment with the fixative and lyophilization protectant, a lyophilized powder is obtained with a shelf life of ≥18 months; Feature 160: The cells are one or more of red blood cells, white blood cells, platelets, and reticulocytes; Feature 170: The white blood cells include one or more of neutrophils, lymphocytes, monocytes, eosinophils, and basophils; Feature 180: The reticulocytes are stained with RNA structure for quality control of reticulocyte detection instruments; Feature 190: The specified portion size is in individually packaged small portions, ready to use and discard; Feature 200: No cell aggregation or hemolysis after reconstitution.

8. A method for preparing frozen stem cells for quality inspection, characterized in that, Frozen stem cells are used for quality control of microscopic testing instruments, including: Step A20: Add fixative to the cell-containing fluid for fixation treatment; Step A30: Add a freeze-drying protectant to the cells. The freeze-drying protectant is used to prevent cell morphology from being damaged during the freeze-drying process. Step A40: Dispense the liquid containing cells and lyophilization protectant into individual portions; freeze-dry the dispensed samples to obtain individually dispensed lyophilized powders; When used, the freeze-dried powder is reconstituted with water to form a test sample for microscopic image capture.

9. The method according to claim 8, characterized in that: Before or after step A20, including step A10: adding staining agent to the cell-containing fluid for staining treatment; Alternatively, in step A20, the dye is mixed into the fixative, and the dyeing is completed simultaneously with the fixation.

10. The method according to claim 8, characterized in that: The cells are stained by adding a staining agent to a liquid containing cells, the staining agent including a fixative.

11. The method according to claim 8, characterized in that: In step A30, the cell-containing liquid is first washed to remove residual fixative or staining agent, and then the cells are added to a liquid containing a lyophilization protectant.

12. The method according to claim 9, characterized in that: Includes one or more of the following features: Feature 110: The staining uses one or more of the following: new methylene blue, crystal violet, methylene blue, brilliant tar blue, toluidine blue, neutral red, and methyl green; Feature 120: The fixative includes one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; the fixing temperature is 2℃~30℃, and the fixing time is 30min~24h.

13. The method according to claim 10, characterized in that: Includes one or more of the following features: Feature 130: Before step A20, it includes step A09: cells in a cell-containing liquid, obtained by removing plasma from whole blood containing EDTA or heparin anticoagulant; Feature 140: Before step A40: It also includes the step of assigning values, i.e., calibrating the target value; The calibrated target values ​​include CBC baseline parameter calibration target values, white blood cell five-part differential calibration target values, and reticulocyte parameter calibration target values. The CBC basic parameter calibration target values ​​include: WBC basic parameter calibration target values, RBC basic parameter calibration target values, MCV basic parameter calibration target values, MCH basic parameter calibration target values, HGB basic parameter calibration target values, HCT basic parameter calibration target values, MCHC basic parameter calibration target values, and PLT basic parameter calibration target values, totaling 8 items; The target values ​​for the five-part differential white blood cell classification include: target values ​​for neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The target values ​​for reticulocyte parameter calibration include: Ret% target value, Ret# target value, and IRF target value.

14. The method according to claim 8, characterized in that, Includes one or more of the following features: Feature 210: The freeze-drying protectant includes one or more of trehalose, sucrose, and hydroxyethyl starch; Feature 220: The final concentration of the lyophilization protectant is 0.5% to 10%; Feature 230: The cell-containing fluid is an isotonic buffer system; Feature 240: Individually dispensed in volumes of 200 μL to 1000 μL; Feature 250: Aseptic dispensing for single-use portions; Feature 260: After repackaging, store temporarily at a low temperature and away from light; Feature 270: Freeze-drying includes pre-freezing, sublimation drying, or desorption drying; Feature 280: Pre-freezing temperature -35℃ to -50℃; Feature 290: Final moisture content of the finished product ≤3%.

15. A quality control reagent kit, characterized in that, Used for quality inspection of microscopic testing instruments, including: Single-serving pre-stained frozen stem cell powder; the frozen stem cell powder is prepared by freeze-drying pre-stained cells in a solution containing a freeze-drying protectant; When used, a reconstitution solution is added to form a detection sample for microscopic image capture; The test samples are used to evaluate whether the instrument's imaging, recognition, classification, and counting functions are up to standard.

16. The quality control kit according to claim 15, characterized in that, Includes one or more of the following features: Feature 300: The kit includes K1 single-packaged frozen stem cell powder and K2 single-packaged frozen stem cell powder. After reconstitution with the same volume V of water, the cell concentration of the K1 test sample is greater than that of the K2 test sample, or the K1 concentration is an integer multiple of the K2 concentration. Feature 310: The kit includes frozen stem cell powder for a five-part differential hematology test; Feature 320: The kit includes frozen stem cell powder for reticulocyte detection; Feature 330: The kit includes a standard image library, judgment rules, and quality control range instructions; Feature 390: The frozen stem cell powder comprises the frozen stem cells as described in any one of claims 1 to 14.

17. The quality control kit according to claim 15, characterized in that, Includes one or more of the following features: Feature 340: Includes a single-packaged reconstituted solution; Feature 350: The complex solution is pure water; Feature 360: The kit includes single-volume reconstitution solutions of varying volumes, with the volume of the larger volume reconstitution solution being an integer multiple of the volume of the smaller volume reconstitution solution; Feature 370: The kit is suitable for model validation and / or calibration of AI cell morphology analyzers; Feature 380: The volume of the reconstituted solution is marked on the package.

18. The use of pre-stained frozen stem cells, characterized in that: Used for quality inspection of microscopic testing instruments. The pre-stained, individually aliquoted, and lyophilized cell powder was reconstituted with water. Microscopic images of the reconstituted samples were captured to obtain microscopic images of the stained cells. The instrument's qualification is determined based on the morphology, structure, color, or clarity of the stained cells in the image. The cells were stained before freeze-drying and did not need to be stained again after reconstitution.

19. The use according to claim 18, characterized in that, Includes one or more of the following features: Feature 400: Used for routine quality control of blood, feces or urine formed element analyzers; Feature 410: Used for quality control of white blood cell differential and reticulocyte parameters; Feature 420: Algorithm verification and / or conformance assessment for AI image recognition devices; Feature 430: Used for capturing microscopic images to determine whether the instrument's imaging, identification, classification, or counting functions are up to standard; Feature 440: The instrument is used for the detection of biological samples from animals or plants; Feature 450: The instrument is used for the detection of human biological samples.

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