Urine formed element analysis multi-item quality control liquid and preparation method thereof

Abstract

A urine formed element analysis multi-item quality control solution is composed of a buffer, red blood cells, white blood cells, epithelial cells, casts, crystals and bacilli. In 1L of the quality control solution, the molar percentage of the buffer is 10-20 mmol / L, the number of the red blood cells is 1×10 6 ~2×10 10 , the number of the white blood cells is 1×10 6 ~1×10 10 , the number of the epithelial cells is 1×10 6 ~5×10 8 , the number of the casts is 1×10 6 ~1×10 8 , the number of the crystals is 1×10 6 ~2×10 8 , and the number of the bacilli is 1×10 6 ~2×10 10 . The pH value of the buffer is controlled at 6.0-9.0. The multi-item quality control solution uses animal-derived raw materials to simulate human cell morphology through process treatment, and can be stored for 24 months in unopened and sealed conditions at 2-8℃ in the dark. After opening, it can be stored for 60 days in a refrigerator at 2-8℃.

Description

Technical Field

[0001] This invention relates to the field of urine testing equipment technology, and in particular to a multi-component quality control solution for urine formation analysis and its preparation method. Background Technology

[0002] Urine sediment analysis, due to its ease of operation, non-invasiveness, and low cost, is hailed as the most economical "kidney biopsy" and is a key means of early screening for kidney disease. However, the analysis of sedimentary elements in urine must be performed by professionals using external instruments, reagents, and methods. Therefore, before conducting sedimentary element analysis, it is essential to verify the reliability of the testing system, including instruments, reagents, methods, and personnel, to ensure accurate and consistent results. The medium used in verifying the reliability of the testing system is called a quality control product. Quality control products, also known as quality control substances, are standard substances with known properties and stable composition, used to verify the reliability of the testing system and ensure accurate and consistent results.

[0003] Specifically, firstly, quality control products are needed to verify the performance of the instrument, such as confirming the accuracy and counting precision of the urine formed element analyzer in identifying components such as red blood cells, white blood cells, epithelial cells, casts, crystals, and bacilli. Secondly, quality control products are needed to monitor reagents and consumables, such as checking the stability of consumables like staining solutions, sheath fluid, and reaction buffers to avoid misidentification of components due to consumable issues, such as confusing white blood cells with epithelial cells. Thirdly, quality control products are also used to standardize operating procedures, such as verifying the standardization of sample processing steps like centrifugation speed and smear thickness, reducing errors caused by human operation. Furthermore, quality control products are used to ensure the reliability of clinical diagnosis, such as avoiding misdiagnosis due to distorted test results, such as missing pathological casts or misdiagnosis. Finally, quality control products are used to support result verification; for example, when quality control results are abnormal, it prompts the need to verify samples from patients in the same period, reducing medical risks.

[0004] However, existing quality control products suffer from three major drawbacks. First, their coverage is incomplete. Market-available quality control products often have single-component profiles, primarily focusing on red blood cells and white blood cells. Comprehensive quality control products targeting key pathological components such as epithelial cells, casts, crystals, and bacilli are extremely scarce. Second, standardized traceability remains a challenge. Manual identification of formed elements is highly subjective, and the morphology and optical characteristics of simulated components differ from those in real urine. Furthermore, simulated components are prone to dissolution and degradation during storage, making manual microscopic examination impossible. This lack of standardized traceability between instruments, quality control products, and human intervention leads to poor comparability of results across laboratories. Third, there is still a gap between stability and morphological accuracy. Existing products typically have a stability period of 6 months, but clinical requirements for the morphological integrity of formed elements (red blood cells, casts, etc.) are extremely high. A negative example is the potential for morphological distortion (shrunken red blood cells or broken white blood cell nuclei) caused by human-derived red blood cells and white blood cells. Furthermore, differences in recognition rates between instruments from different brands can easily lead to result bias, and the toxicity risks of preservatives such as sodium azide used in traditional quality control products also increase operational hazards. Therefore, the field of urine analysis urgently needs a quality control product that can solve the above problems. Summary of the Invention

[0005] With the widespread adoption of fully automated urine analysis workstations, laboratories urgently need composite quality control solutions capable of simultaneously covering multiple formed elements, maintaining long-term stability, and compatible with instruments from multiple brands. These products not only need to meet the morphological and fidelity requirements of manual microscopic examination, but also require optical signal stability adapted to the detection logic of automated equipment, while completely avoiding biosafety risks. In view of this, the present invention provides a multi-component quality control solution for urine formed element analysis and its preparation method to meet the aforementioned needs.

[0006] A multi-component quality control solution for urine formation analysis comprises buffer solution, red blood cells, white blood cells, epithelial cells, casts, crystals, and bacilli. In 1 L of this quality control solution, the molar percentage of the buffer solution is 10–20 mmol / L, and the number of red blood cells is 1 × 10⁻⁶. 6 ~2×10 10 The number of white blood cells is 1 × 10⁶. 6 ~1×10 10 The number of epithelial cells is 1 × 10⁶. 6 ~5×10 8 The number of the tubular types is 1 × 10. 6 ~1×10 8 The number of crystals is 1 × 10⁻⁶. 6 ~2×10 8 The number of the bacteria is 1 × 10⁶. 6 ~2×10 10 The pH value of the buffer solution is controlled between 6.0 and 9.0.

[0007] Furthermore, both the red blood cells and white blood cells are derived from human or animal cells.

[0008] Further, the buffer solution is one of sodium dihydrogen phosphate-disodium hydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-morpholine ethanesulfonic acid, and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane).

[0009] Furthermore, the red blood cells are derived from animal blood, and the processing technology for obtaining the red blood cells includes the following steps: STEP101: Treat the blood of quarantined animals with an anticoagulant; STEP102: Wash animal blood at least 3 times by centrifugation with cell washing buffer at a volume of 1 to 10 times the blood volume. Centrifugation parameters: 1000 rpm to 3000 rpm, 3 to 5 min. After centrifugation, discard the supernatant to obtain primary erythrocytes. STEP103: Mix the primary erythrocytes with a composite aldehyde solution consisting of 1% to 5% (by mass) of formaldehyde-glutaraldehyde and cell washing solution of the same volume as the discarded supernatant for cross-linking for 1 to 5 days. STEP104: After cross-linking, the cells are washed three times by centrifugation with cell washing buffer. The precipitate is mixed with glycerol-containing PBS preservation solution and stored in a sealed container at 2℃~8℃ to obtain the red blood cells.

[0010] Furthermore, the leukocytes are derived from animal blood, and the processing technology for the leukocytes includes the following steps: STEP201: Treat the blood of quarantine-qualified animals with an anticoagulant; STEP202: Add 3% to 5% gelatin solution by weight in equal proportions, mix well, and let stand to separate into layers; STEP203: After centrifuging the supernatant, repeat the lysis with 1 to 10 times the volume of cell lysis buffer, centrifuge 3 times, with centrifugation parameters of 2000 rpm to 5000 rpm for 5 min. After centrifugation, discard the supernatant to obtain the primary leukocyte pellet. STEP204: Mix the obtained leukocyte precipitate with a composite aldehyde solution consisting of 1% to 5% formaldehyde-glutaraldehyde (by weight) and cell washing solution of the same volume as the discarded supernatant, and crosslink for 3 to 7 days. STEP205: After cross-linking, wash three times with cell washing buffer by centrifugation. The precipitate is then precipitated with glycerol-PBS buffer and stored in a sealed container at 2℃-8℃ for 24 months of sterile and stable preservation.

[0011] Furthermore, the method for preparing the high-purity crystal includes the following steps: STEP301: Provide a raw material for preparing the crystals, wherein the raw material may be cystine; STEP302: Dissolve the raw material solid in a mixed solution of concentrated hydrochloric acid and purified water to obtain the stock solution; STEP303: While the stock solution is being stirred, add a 1% to 10% sodium hydroxide solution by weight to the reaction system. During the addition, monitor the pH value in real time with a pH meter to keep it between 1.5 and 3.0. The addition of the sodium hydroxide solution should be precisely stopped at the moment when the cloud point first appears. STEP304: Stir continuously for 1-3 minutes to generate a stable crystalline suspension; STEP305: Purification by gradient sieving: The crystallized suspension is filtered and precipitated using a 600-mesh filter; STEP306: After washing the precipitate with acetone eluent to remove impurities, filter it through a 300-mesh filter to obtain high-purity crystals.

[0012] Furthermore, the cell washing solution is a phosphate buffer or a tris(hydroxymethyl)aminomethane buffer, and the pH of the cell washing solution is 7.4–8.0.

[0013] Furthermore, the formaldehyde-glutaraldehyde composite aldehyde solution is obtained by mixing formaldehyde and glutaraldehyde in a weight ratio of 1:1 to 3:1.

[0014] Furthermore, the volume ratio of glycerol to phosphate buffer in the glycerol-containing preservation solution is 1:4.

[0015] Furthermore, the anticoagulant can be one of potassium oxalate, sodium fluoride, heparin, sodium diethylaminetetraacetate, or sodium citrate.

[0016] Compared with existing technologies, the urine formed element analysis multiple quality control solution of the present invention has the following advantages: Using animal-derived raw materials and processing techniques to simulate human cell morphology, the resulting biconcave disc-shaped red blood cells, white blood cells with lobed nuclear structures, flat, upright, and columnar epithelial cells, cylindrical tubular cells, rod-shaped bacteria, and hexagonal cystine crystals are morphologically very similar, with a similarity to human cells greater than or equal to 98%.

[0017] By designing a microenvironment buffer solution, which is free of surfactants and preservatives, it ensures that five types of components, namely red blood cells, white blood cells, epithelial cells, casts, and crystals, do not aggregate, dissolve, or deform in the suspension, and that they are well distributed uniformly under a microscope with a uniformity CV of less than or equal to 5%.

[0018] The raw materials used are animal by-products, which reduces costs by more than 80% compared to human-derived materials; It matches the detection thresholds of mainstream instruments such as Maxside CM-4500, Sysmex UF-5000, and Dirui FUS-2000, specifically for red blood cell diameters of 6–9 μm and white blood cell diameters of 10–14 μm, avoiding misjudgments by impedance / light scattering instruments due to size deviations, with a detection consistency CV value of less than or equal to 3%. Unopened and sealed at 2-8℃ away from light, it can be stored for 24 months. Once opened, it can be stored for 60 days when refrigerated at 2-8℃. Attached Figure Description

[0019] Figure 1 The image shows the morphological observation of red blood cells in Example 2 of a urine formed element analysis multiple quality control solution provided by the present invention.

[0020] Figure 2 This is an observation image obtained from the morphology of white blood cells in Example 2.

[0021] Figure 3 This is an observation image obtained from the morphology of epithelial cells in Example 2.

[0022] Figure 4 This is an observation image obtained from the tubular morphology of Example 2.

[0023] Figure 5 This is an observation diagram obtained from the crystal morphology of Example 2.

[0024] Figure 6 This is an observation image obtained from the morphology of the bacillus in Example 2. Detailed Implementation

[0025] The following provides a more detailed description of specific embodiments of the present invention. It should be understood that the description of the embodiments of the present invention herein is not intended to limit the scope of protection of the present invention.

[0026] This invention provides a multi-component quality control solution for urine formed elements analysis, which consists of buffer solution, red blood cells, white blood cells, epithelial cells, casts, crystals, and bacilli. In 1 L of the quality control solution, the molar percentage of the buffer solution is 10–20 mmol / L, and the number of red blood cells is 1 × 10⁻⁶. 6 ~2×10 10 The number of white blood cells is 1 × 10⁶. 6 ~1×10 10 The number of epithelial cells is 1 × 10⁶. 6 ~5×10 8 The number of the tubular types is 1 × 10. 6 ~1×10 8 The number of crystals is 1 × 10⁻⁶. 6 ~2×108 The number of the bacteria is 1 × 10⁶. 6 ~2×10 10 There are several requirements for different media particles used in urine analysis, such as red blood cells, white blood cells, epithelial cells, casts, crystals, and bacilli, when mixed into the buffer solution. This is because the physicochemical properties of the system required for the preparation or subsequent preservation of each media particle, such as pH or osmotic pressure, are different. Therefore, it is necessary to provide a suitable system that is compatible with the physicochemical characteristics required by each media particle, or to modify the preparation or preservation process of certain media particles. For example, the pH requirements for the preservation system of epithelial cells and the preparation and preservation system of crystals are different.

[0027] The buffer solution is one of the following: sodium dihydrogen phosphate-disodium hydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution, 2-morpholine ethanesulfonic acid (MES) solution, or bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) (Bistris) solution. The pH of the buffer solution must be controlled between 7.0 and 9.0. A pH greater than 9.0 will cause crystals to become rounded, losing their characteristic features and leading to misinterpretation of results. A pH less than 7.0 will cause morphological changes or instability in epithelial cells or casts, resulting in cell breakdown and ultimately misinterpretation of results and decreased stability. Preferably, the buffer solution is a HEPES buffer with a pH of 7.2.

[0028] The red blood cells and white blood cells are derived from human or animal cells, such as the blood of humans, pigs, chickens, rabbits, etc. Preferably, the cell source is pig.

[0029] The processing steps for obtaining red blood cells from animal blood include the following: STEP101: Treat the blood of quarantined animals with an anticoagulant; STEP102: Centrifuge the animal blood three times with cell washing buffer at a volume of 1 to 10 times the blood volume. Centrifugation parameters: 1000 rpm to 3000 rpm, 3 to 5 min. After centrifugation, discard the supernatant and record the volume of the discarded supernatant to obtain primary erythrocytes. STEP 103: Mix the primary erythrocytes with a composite aldehyde solution of formaldehyde-glutaraldehyde and cell washing solution at a mass percentage of 1% to 5% (equal to the volume of the discarded supernatant) for cross-linking for 1 to 5 days. STEP 104: Centrifuge and wash at least 3 times with cell washing buffer. After precipitation, mix with glycerol-containing preservation solution and seal and refrigerate at 2℃~8℃ to obtain the red blood cells.

[0030] In STEP101, the anticoagulant is a blood anticoagulant, which is existing technology and can be potassium oxalate, sodium fluoride, heparin, sodium diethylaminetetraacetate, sodium citrate, etc. This anticoagulant is used to prevent the freshly obtained blood from clotting and becoming unusable for subsequent operations.

[0031] In STEP 102, the purpose of using cell washing buffer at 1 to 10 times the volume of blood is to dilute the animal blood. After dilution, washing is performed to remove various inorganic salts, lipids, proteins, and other impurities from the blood. After washing, centrifugation is performed to remove white blood cells and enrich red blood cells, facilitating aldehyde formation in STEP 103.

[0032] In STEP103, the formaldehyde-glutaraldehyde composite aldehyde solution is obtained by mixing formaldehyde and glutaraldehyde in a weight ratio of 1:1 to 3:1. The purpose of using the composite aldehyde solution to aldehyde-aldehyde the red blood cells is to solidify the red blood cells, thereby allowing the morphology of the red blood cells and the cytoplasm within them to be maintained and preserved for a long period of time.

[0033] In STEP104, the cell washing solution can be phosphate buffer or tris(hydroxymethyl)aminomethane buffer, and the pH of the cell washing solution is 7.4–8.0. The PBS preservation solution is prepared by disodium hydrogen phosphate (Na₂HPO₄), potassium dihydrogen phosphate (KH₂PO₄), sodium chloride (NaCl), etc., in a specific ratio, and the pH value is usually maintained at 7.2–7.4, with an osmotic pressure similar to that of human body fluids. In the glycerol-containing PBS preservation solution, the volume ratio of glycerol to phosphate buffer is 1:4. The purpose of using the red blood cell washing solution to perform a second washing of aldehyde-treated red blood cells is to remove the aldehyde solution from the aldehyde treatment step, as the aldehyde solution itself is toxic. The pH of the red blood cell washing solution must be controlled between 7.4 and 8.0, because a pH value less than 7.4 will cause red blood cells to shrink and deform, changing their shape, which will affect instrument recognition after solidification. When the pH value is greater than 8.0, it will damage the red blood cell membrane, causing red blood cells to swell and rupture, resulting in hemolysis.

[0034] Red blood cells processed using the above steps can be sterile and stably preserved for 24 months.

[0035] The processing steps for obtaining white blood cells from animal blood include the following: STEP201: Treat the blood of quarantine-qualified animals with an anticoagulant; STEP202: Add 3% to 5% gelatin solution by weight in equal proportions, mix well, and let stand to separate into layers to obtain leukocyte precipitate; STEP203: After centrifuging the supernatant, repeat the lysis with cell lysis buffer at 1 to 10 times the volume of the leukocyte precipitate, and centrifuge at least 3 times. The centrifugation parameters are 2000 rpm to 5000 rpm and 5 min. After centrifugation, discard the supernatant and record the volume of the discarded supernatant to obtain the primary leukocyte precipitate. STEP204: Mix the obtained leukocyte precipitate with the discarded supernatant at a weight percentage of 1% to 5% of formaldehyde-glutaraldehyde and cell washing solution, and crosslink for 3 to 7 days. STEP205: The cross-linked leukocyte precipitate is then centrifuged and washed at least 3 times with cell washing solution. The leukocytes are then mixed with a glycerol-containing preservation solution and sealed and refrigerated at 2℃-8℃ to achieve sterile and stable preservation for 24 months.

[0036] In STEP201, the anticoagulant can be potassium oxalate, sodium fluoride, heparin, sodium diethylaminetetraacetate, sodium citrate, etc.

[0037] In STEP 202, the purpose of using the gelatin solution is to initially separate red and white blood cells in the blood, facilitating their individual collection. Because the gelatin molecules in the solution carry a positive charge, they neutralize the negative charge on the surface of red blood cells, promoting red blood cell aggregation and significantly increasing their sedimentation rate, thus separating them from white blood cells. Simultaneously, because white blood cells are fewer in number and have a lower charge density, this effect is less pronounced. The concentration of the gelatin solution must be between 3% and 5%, as higher concentrations of gelatin would greatly increase blood viscosity, potentially counteracting the accelerating aggregation effect.

[0038] In STEP203, the sediment volume refers to the volume of supernatant that remains after the supernatant is poured out, forming a suspension that covers the sediment. 1-10 times is a multiple of this suspension volume.

[0039] In STEP203, the cell lysis buffer is a mixture of 0.1%–0.5% by weight of ethyl phenyl polyethylene glycol and 0.1%–0.5% by weight of sodium dodecyl sulfate. The purpose of using this cell lysis buffer on the initially separated red and white blood cells is to lyse and remove red blood cells while retaining white blood cells. This is because the above-mentioned separation is preliminary, and a small number of red blood cells still remain in the white blood cell supernatant, requiring lysis and purification of the white blood cells.

[0040] In STEP204, the formaldehyde-glutaraldehyde composite aldehyde solution is obtained by mixing formaldehyde and glutaraldehyde in a weight ratio of 1:1 to 3:1. The purpose of using the composite aldehyde solution to aldehyde-aldehyde the leukocytes is to solidify the leukocytes, thereby allowing the morphology of the leukocytes and the cytoplasm within them to be maintained and preserved for a long period of time.

[0041] In STEP 205, the purpose of the secondary washing with "cell washing solution" is to remove the aldehyde solution from the aldehyde treatment step, which is toxic. Red blood cells treated in the above steps can be sterile and stably preserved for 24 months.

[0042] Epithelial cells, tubular cells, and bacilli can be directly processed using semi-finished products prepared by Changchun Maikeside Medical Technology Co., Ltd.

[0043] The crystals can be of various types, including cystine crystals, tyrosine crystals, calcium phosphate crystals, cholesterol crystals, and uric acid crystals. The crystallization process includes the following steps: STEP301: Provide a raw material for preparing the crystals, wherein the raw material may be cystine; STEP302: Dissolve the raw material in a mixed solution of concentrated hydrochloric acid and purified water to obtain the stock solution; STEP302: While the stock solution is being stirred, add a sodium hydroxide solution with a mass percentage of 1% to 10% dropwise to the reaction system. During the dropwise addition, monitor the pH value in real time with a pH meter to keep it between 1.5 and 3.0. The dropwise addition of the sodium hydroxide solution is precisely terminated at the moment when the cloud point first appears. STEP303: Stir continuously for 1-3 minutes to generate a stable crystalline suspension; STEP304: Purification by gradient sieving: The crystallized suspension is filtered and precipitated using a 600-mesh filter; STEP305: After washing the precipitate with acetone eluent to remove impurities, filter it through a 300-mesh filter to obtain high-purity crystals.

[0044] In STEP302, the concentration of the concentrated hydrochloric acid is specifically 12 mol / L.

[0045] In STEP303, the sodium hydroxide solution undergoes a neutralization reaction with concentrated hydrochloric acid to maintain the pH value between 1.5 and 3.0. During the dropwise addition, a cloud point will appear, which is the beginning of crystal nucleation. After stopping the dropwise addition, allowing it to grow naturally will result in stable, non-aggregated crystals. If the dropwise addition continues, a large number of crystals will aggregate and become easily broken in subsequent operations.

[0046] The entire crystallization process involves steps such as acid dissolution, alkali titration (crystallization), sieve filtration, elution, and secondary sieving. Acid dissolution aims to dissolve the amorphous cystine powder or crystals into a transparent solution for subsequent acid-base neutralization titration. Alkali titration aims to consume the hydrochloric acid used to dissolve cystine, altering the solution pH and thus reducing the solubility of cystine, facilitating crystallization. Sieve filtration aims to remove extremely small crystals while retaining the target crystals. Elution aims to remove residual moisture and impurities; secondary sieving aims to selectively retain crystals of the target particle size.

[0047] The advantages of using the urine formed element analysis multiple quality control solution of the present invention are as follows: Using animal-derived raw materials and processing techniques to simulate human cell morphology, the resulting biconcave disc-shaped red blood cells, white blood cells with lobed nuclear structures, flat, upright, and columnar epithelial cells, cylindrical tubular cells, rod-shaped bacteria, and hexagonal cystine crystals are morphologically very similar, with a similarity to human cells greater than or equal to 98%.

[0048] By designing a microenvironment buffer solution, which is free of surfactants and preservatives, it ensures that five types of components, namely red blood cells, white blood cells, epithelial cells, casts, and crystals, do not aggregate, dissolve, or deform in the suspension, and that they are well distributed uniformly under a microscope with a uniformity CV of less than or equal to 5%.

[0049] The raw materials used are animal by-products, which reduces costs by more than 80% compared to human-derived materials; It matches the detection thresholds of mainstream instruments such as Maxside CM-4500, Sysmex UF-5000, and Dirui FUS-2000, specifically for red blood cell diameters of 6–9 μm and white blood cell diameters of 10–14 μm, avoiding misjudgments by impedance / light scattering instruments due to size deviations, with a detection consistency CV value of less than or equal to 3%. Unopened and sealed at 2-8℃ away from light, it can be stored for 24 months. Once opened, it can be stored for 60 days when refrigerated at 2-8℃. Example

[0050] The processing procedure for swine-derived red blood cells is as follows: Blood from quarantine-qualified animals is treated with an anticoagulant. Five times the blood volume is then washed three times with 0.01M, pH 7.4 PBS solution by centrifugation at 2000 rpm for 5 minutes. The cell pellet is cross-linked for 3 days with an equal volume of 4% formaldehyde-glutaraldehyde composite aldehyde solution (formaldehyde to glutaraldehyde ratio 1:1) and cell washing buffer. The pellet is then washed three times with the cell washing buffer. The pellet is then mixed with a glycerol-PBS buffer (20% + 80%) preservation solution and stored under sealed conditions at 2℃-8℃ for 24 months of aseptic stability. The cell washing buffer is 0.01M, pH 7.4 PBS.

[0051] The processing procedure for swine-derived leukocytes is as follows: Blood from quarantine-qualified animals is treated with an anticoagulant, and gelatin solution is added at a ratio of 5% and mixed thoroughly, allowing the mixture to separate into layers. The supernatant is centrifuged, and then repeatedly lysed with 8 times the volume of 0.5% NP-40 solution, centrifuged 3 times at 3000 rpm for 5 min. The cell pellet is cross-linked for 5 days with an equal volume of 4% formaldehyde-glutaraldehyde-cell washing buffer (formaldehyde to glutaraldehyde ratio 1:1). The cell washing buffer is centrifuged and washed 3 times. The pellet is then mixed with glycerol-containing PBS preservation solution and sealed and refrigerated at 2℃-8℃ for 24 months of aseptic and stable preservation.

[0052] The cystine crystallization process is as follows: the raw material solid is dissolved in a concentrated hydrochloric acid-purified water system to obtain a stock solution. 5% sodium hydroxide solution is added dropwise to the reaction system while stirring. During this process, the pH is monitored in real time with a pH meter, and the reaction is precisely terminated at the moment when the turbidity point first appears (pH 2.5). Stirring is continued for 1 minute to generate a stable crystalline suspension. Gradient sieving purification is used: the precipitate is retained by a 600-mesh filter, impurities are removed by washing with acetone eluent, and high-purity crystals are filtered out by a 300-mesh filter.

[0053] The buffer solution is HEPES buffer with a pH of 8.0.

[0054] The urine sample obtained by the above method includes multiple quality control solutions for formed element analysis, including: buffer solution 20 mmol / L; red blood cells 1000 / μL; white blood cells 200 / μL; epithelial cells 30 / μL; casts 10 / μL; crystals 100 / μL; and bacilli 1000 / μL.

[0055] The various media obtained in Example 1 will now be tested.

[0056] After the urine formed element analysis multiple quality control solution prepared in Example 1 was placed at room temperature for 30 min, a stability test was conducted using a Maxside CM-4500 instrument.

[0057] The test results are as follows:

[0058] The test results after 24 months of storage at 2℃~8℃ are as follows:

[0059] The test results after 60 days of storage at 2℃~8℃ after opening are as follows:

[0060] The test data above shows that the attenuation was within -10% in both real-time stability and open-pack stability experiments, while the standard requires an attenuation of less than 15% and an accuracy of less than 10%, indicating good cell morphology stability.

[0061] The urine formed element analysis multiple quality control solution prepared in Example 1 at the end of the real-time stability period was placed at room temperature for 30 min. The results were then validated using different manufacturers' instruments, namely the Maxside CM-4500, Sysmex UF-5000, and Dirui FUS-2000. Manual microscopic examination was also introduced as the gold standard for comparison. Each instrument / method was tested three times, and the mean, coefficient of variation (CV), and p-value were calculated (p>0.05 indicates no statistical significance).

[0062]

[0063] The test data above show that the CV of the urine formed element analysis multiple quality control solution of the present invention is within 10% and the p value is greater than 0.05 between different manufacturers and different models, indicating that the urine formed element analysis multiple quality control solution of the present invention has strong compatibility between different manufacturers and models, cell morphology does not affect instrument / manual interpretation, and the performance is good. Example

[0064] The processing procedure for chicken-derived cells is as follows: Blood from quarantine-qualified animals is treated with an anticoagulant, and gelatin solution is added at a 5% ratio, mixed thoroughly, and allowed to separate into layers. The supernatant is centrifuged, and the cells are repeatedly lysed with 2-3 times the volume of cell lysis buffer, centrifuged three times at 5000 r / min. The cell pellet is then cross-linked for 40 minutes with an equal volume of 3% formaldehyde-glutaraldehyde composite aldehyde solution (formaldehyde and glutaraldehyde mixed in a 2:1 ratio) for centrifugation. The pellet is washed three times with washing buffer, and the precipitate is mixed with glycerol-containing PBS buffer for preservation. The pellet is then sealed and refrigerated at 2℃-8℃ for 24 months of aseptic and stable storage. The cell lysis buffer is 0.1% SDS. The washing buffer is Tris buffer, pH 8.0.

[0065] The uric acid crystallization process is as follows: the raw material solid is dissolved in a concentrated hydrochloric acid-purified water system to obtain a stock solution, and 15% sodium hydroxide solution is added dropwise to the reaction system. During this process, the pH is monitored in real time with a pH meter, and the reaction is precisely terminated at the moment when the turbidity point first appears (pH 5.0). The mixture is stirred continuously until the pH does not change within 1 minute, and a stable crystalline suspension is generated. The mixture is purified by gradient sieving; the precipitate is retained by a 600-mesh filter membrane; the mixture is washed with acetone eluent to remove impurities; and high-purity crystals are obtained by filtration through a 300-mesh filter.

[0066] The buffer solution is a pH 7.0 MES buffer.

[0067] This urine sample contains multiple quality control solutions for formed element analysis, including: buffer solution 10 mmol / L; red blood cells 10 / μL; white blood cells 10 / μL; epithelial cells 5 / μL; casts 1 / μL; crystals 10 / μL; and bacilli 100 / μL.

[0068] The various test media obtained in Example 2 will now be tested.

[0069] After the urine sample prepared in Example 2 at the end of the real-time stability period was placed at room temperature for 30 minutes, cell morphology was observed using a Maxside CM-4500 fully automated urine analysis system. Figures 1 to 6 The morphological observations of the various media show that the five components did not aggregate, dissolve, or deform in the suspension.

[0070] Example 3 The processing procedure for rabbit-derived cells is as follows: Blood from quarantined animals is treated with an anticoagulant, and gelatin solution is added at a ratio of 4% and mixed thoroughly, allowing the cells to separate into layers. The supernatant is centrifuged, and the cells are repeatedly lysed with three times the volume of cell lysis buffer, centrifuged three times at 5000 r / min. The cell pellet is then cross-linked for 50 minutes with an equal volume of 2% formaldehyde-glutaraldehyde composite aldehyde solution (formaldehyde and glutaraldehyde mixed in a 3:1 ratio). The pellet is washed three times with washing buffer, and the precipitate is mixed with glycerol-containing PBS buffer for preservation. The pellet is then sealed and refrigerated at 2℃-8℃, achieving aseptic stability for 24 months. The cell lysis buffer consists of 0.1% NP-40 and 0.5% SDS. The washing buffer is PBS buffer, pH 8.0.

[0071] The tyrosine crystallization process is as follows: the raw material solid is dissolved in a concentrated hydrochloric acid-purified water system to obtain a stock solution, and 12% sodium hydroxide solution is added dropwise to the reaction system. During this process, the pH is monitored in real time with a pH meter, and the reaction is precisely terminated at the moment when the turbidity point first appears (pH 5.5). The mixture is stirred continuously until the pH does not change within 2 minutes, and a stable crystalline suspension is generated. The mixture is purified by gradient sieving; the precipitate is retained by a 600-mesh filter membrane; the mixture is washed with acetone eluent to remove impurities; and high-purity crystals are obtained by filtration through a 300-mesh filter.

[0072] The buffer solution is a pH 8.0 MES buffer.

[0073] This urine sample contains multiple quality control solutions for formed element analysis, including: buffer solution 15 mmol / L; red blood cells 1000 / μL; white blood cells 200 / μL; epithelial cells 30 / μL; casts 10 / μL; crystals 50 / μL; and bacilli 500 / μL.

[0074] The analytical medium obtained in Example 3 will be tested below. After placing the urine sample prepared in Example 3 at room temperature for 30 minutes, 10 bottles of the same quality control solution were randomly selected and numbered 1 to 10. Each bottle was tested 3 times in the following order: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. The test results of each bottle were recorded, and F, sr, and CV were calculated. When the statistical result F value was less than or equal to the critical F value (0.05, ν1, ν2), the homogeneity between the quality control solution bottles was considered to be good.

[0075] 7.1 Red blood cell uniformity

[0076] 7.2 White blood cell uniformity

[0077] 7.3 Epithelial cell uniformity

[0078] 7.4 Uniformity of Tube Shape

[0079] 7.4 Crystallization homogeneity

[0080] 7.4 Bacterial uniformity

[0081] All six F values ​​were less than the critical F value, indicating good homogeneity among the component bottles.

[0082] In summary, this invention, through a synergistic system of animal cells and buffer-suspension, pioneered the achievement of long-term stable coexistence of erythrocytes, leukocytes, epithelial cells, casts, crystals, and bacilli in a single solution. It overcomes the technical barriers of existing quality control solutions, such as single component, difficulty in traceability, short stability period, and poor cross-platform compatibility, filling the technical gap in multi-item quality control of urine morphology and promoting the standardization process of clinical urine analysis.

[0083] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions or improvements within the spirit of the present invention are covered within the scope of the claims of the present invention.

Claims

1. A multi-factor quality control solution for urine formation analysis, characterized in that: The urine sample multiple control solution for formed elements analysis consists of buffer solution, red blood cells, white blood cells, epithelial cells, casts, crystals, and bacilli. In 1 L of this control solution, the molar percentage of the buffer solution is 10–20 mmol / L, and the number of red blood cells is 1 × 10⁻⁶. 6 ~2×10 10 The number of white blood cells is 1 × 10⁶. 6 ~1×10 10 The number of epithelial cells is 1 × 10⁶. 6 ~5×10 8 The number of the tubular types is 1 × 10. 6 ~1×10 8 The number of crystals is 1 × 10⁻⁶. 6 ~2×10 8 The number of the bacteria is 1 × 10⁶. 6 ~2×10 10 The pH value of the buffer solution is controlled between 6.0 and 9.

0.

2. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The red blood cells and white blood cells mentioned are both derived from human or animal cells.

3. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The buffer solution is one of sodium dihydrogen phosphate-disodium hydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-morpholine ethanesulfonic acid, and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane).

4. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The red blood cells are derived from animal blood, and the processing steps for obtaining the red blood cells include the following: STEP101: Treat the blood of quarantined animals with an anticoagulant; STEP102: Wash animal blood at least 3 times by centrifugation with cell washing buffer at a volume of 1 to 10 times the blood volume. Centrifugation parameters: 1000 rpm to 3000 rpm, 3 to 5 min. After centrifugation, discard the supernatant to obtain primary erythrocytes. STEP103: Mix the primary erythrocytes with a composite aldehyde solution consisting of 1% to 5% (by mass) of formaldehyde-glutaraldehyde and cell washing solution of the same volume as the discarded supernatant for cross-linking for 1 to 5 days. STEP104: After cross-linking, the cells are washed three times by centrifugation with cell washing buffer. The precipitate is mixed with glycerol-containing PBS preservation solution and stored in a sealed container at 2℃~8℃ to obtain the red blood cells.

5. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that, The leukocytes are derived from animal blood, and the processing technology for the leukocytes includes the following steps: STEP201: Treat the blood of quarantine-qualified animals with an anticoagulant; STEP202: Add 3% to 5% gelatin solution by weight in equal proportions, mix well, and let stand to separate into layers; STEP203: After centrifuging the supernatant, repeat the lysis with 1 to 10 times the volume of cell lysis buffer, centrifuge 3 times, with centrifugation parameters of 2000 rpm to 5000 rpm for 5 min. After centrifugation, discard the supernatant to obtain the primary leukocyte pellet. STEP204: Mix the obtained leukocyte precipitate with a composite aldehyde solution consisting of 1% to 5% formaldehyde-glutaraldehyde of the same volume as the discarded supernatant and cell washing solution, and crosslink for 3 to 7 days. STEP205: After cross-linking, wash three times with cell washing buffer by centrifugation. The precipitate is then precipitated with glycerol-PBS buffer and stored in a sealed container at 2℃-8℃ for 24 months of sterile and stable preservation.

6. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The method for preparing the high-purity crystals includes the following steps: STEP301: Provide a raw material for preparing the crystals, wherein the raw material may be cystine; STEP302: Dissolve the raw material solid in a mixed solution of concentrated hydrochloric acid and purified water to obtain the stock solution; STEP303: While the stock solution is being stirred, add a 1% to 10% sodium hydroxide solution by weight to the reaction system. During the addition, monitor the pH value in real time with a pH meter to keep it between 1.5 and 3.

0. The addition of the sodium hydroxide solution should be precisely stopped at the moment when the cloud point first appears. STEP304: Stir continuously for 1-3 minutes to generate a stable crystalline suspension; STEP305: Purification by gradient sieving: The crystallized suspension is filtered and precipitated using a 600-mesh filter; STEP306: After washing the precipitate with acetone eluent to remove impurities, filter it through a 300-mesh filter to obtain high-purity crystals.

7. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The cell washing solution is a phosphate buffer or a tris(hydroxymethyl)aminomethane buffer, and the pH of the cell washing solution is 7.4–8.

0.

8. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The formaldehyde-glutaraldehyde composite aldehyde solution is obtained by mixing formaldehyde and glutaraldehyde in a weight ratio of 1:1 to 3:

1.

9. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The volume ratio of glycerol to phosphate buffer in the glycerol-containing preservation solution is 1:

4.

10. The urine formed element analysis multi-factor quality control solution as described in claim 1, characterized in that: The anticoagulant can be one of potassium oxalate, sodium fluoride, heparin, sodium diethylaminetetraacetate, or sodium citrate.

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