Preparation method of fully soluble animal plasma
Through anticoagulant treatment and graded centrifugation technology, the problems of hemolysis and insoluble particle residue in animal plasma preparation are solved, and the preparation of high-quality fully soluble animal plasma is achieved to meet clinical testing needs.
Patent Information
- Application Number
- CN202511060050.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-10-21
AI Technical Summary
In the existing technology, the animal plasma preparation process has problems of hemolysis and residual insoluble particles, which affects the quality and stability of the plasma and cannot meet the clinical detection requirements of high specificity and high sensitivity.
Whole blood is treated with anticoagulants, combined with temperature-controlled standing, graded centrifugation and siphon transfer technology. A 3.2%~3.8% sodium citrate aqueous solution or a composite anticoagulant is used for full-process temperature control, including cold water bath, incubator transportation and graded centrifugation to separate red blood cells, white blood cells and insoluble particles.
It significantly reduces the incidence of hemolysis, maintains the stability of plasma components, and improves plasma purity and quality. It is suitable for laboratory and transportation applications and is operationally controllable and practical.
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Figure CN120820385A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a method for preparing fully soluble animal plasma. Background Art
[0002] In the development of quality control products for in vitro diagnostic reagents and the production of clinical simulation samples, animal plasma (including porcine, bovine, and equine species) has become an important alternative resource for alleviating the imbalance between supply and demand of clinical samples due to its relatively wide availability and similar composition to human plasma. Currently, mainstream methods for preparing animal plasma include centrifugation, filtration, precipitation, chemical separation, and membrane separation. Taking into account key factors such as production cost and the retention of plasma bioactivity, centrifugation is the most widely used method in industry practice. However, existing centrifugation preparation technologies still face numerous technical bottlenecks that need to be addressed. First, the lack of systematic temperature monitoring and control mechanisms during animal whole blood processing. Prolonged exposure of blood samples to suboptimal temperatures can easily lead to denaturation or inactivation of bioactive components in plasma (such as coagulation factors and enzymes), severely compromising the reliability and stability of plasma as a quality control or simulated sample. Second, the lack of standardized operating procedures and specialized transfer equipment during plasma transfer leads to improper control of mechanical stress during sample transfer, frequently causing hemolysis. Hemolysis not only alters the physicochemical properties of plasma, but also releases substances such as hemoglobin, which can interfere with the accuracy of subsequent test results, impacting the quality of quality control and simulated samples. Third, centrifugation often relies on a single, crude centrifugation step. This simple, crude centrifugation method fails to effectively remove insoluble particulate impurities from plasma, leaving a large number of platelets, white blood cells, and other mononuclear cells in the plasma.
[0003] It is worth noting that to meet the clinical testing requirements for highly specific and sensitive test samples, animal plasma is often used to prepare cryoprecipitates to obtain high-value plasma. However, the components produced by the aforementioned hemolysis phenomenon and the residual insoluble particles can severely hinder the formation and growth of ice crystals during the cryoprecipitate preparation process, reducing the dissolution rate and ultimate yield of the cryoprecipitate. This makes the goal of preparing fully soluble animal plasma difficult to achieve and makes it impossible to provide high-quality plasma samples for clinical diagnosis and scientific research. Summary of the Invention
[0004] The present invention aims to provide a method for preparing fully soluble animal plasma to solve the hemolysis problem and the problem of residual insoluble particles in the animal plasma preparation process in the prior art.
[0005] To achieve the above-mentioned object, the present invention adopts the following technical scheme: a method for preparing fully soluble animal plasma, which comprises treating the plasma with an anticoagulant, and then performing standing, transfer and centrifugation under temperature control throughout the entire process. The anticoagulant is a 3.2% to 3.8% sodium citrate aqueous solution or a composite anticoagulant containing sodium citrate and / or citric acid, and the volume ratio of the anticoagulant to the animal whole blood is 1:8 to 1:10. Preferably, as an improvement, the composite anticoagulant contains 3.0% to 3.6% sodium citrate, 0.11% to 0.14% citric acid and 1% to 2% glucose, or the composite anticoagulant contains 3.0% to 3.6% sodium citrate and 1% to 2% glucose.
[0006] Preferably, as an improvement, the centrifugal treatment process adopts a graded centrifugation method, including low-speed centrifugation and high-speed centrifugation performed sequentially.
[0007] Preferably, as an improvement, the conditions for low-speed centrifugation are a centrifugation temperature of 2-8°C, a rotation speed of 500-1100g, and a centrifugation time of 5-20min.
[0008] Preferably, as an improvement, the conditions for high-speed centrifugation are a centrifugation temperature of 2-8°C, a rotation speed of 7000-15000g, and a centrifugation time of 5-20 min.
[0009] Preferably, as an improvement, a method for preparing fully soluble animal plasma comprises the following steps: S1. Preparation: Take whole blood from an animal, mix it in a volume ratio of 1:8 to 1:10 between an anticoagulant and the animal's whole blood, and immediately mix it to prepare anticoagulated whole blood; the anticoagulant is a 3.2% to 3.8% sodium citrate aqueous solution or a composite anticoagulant containing sodium citrate and / or citric acid, the composite anticoagulant containing 3.0% to 3.6% sodium citrate, 0.11% to 0.14% citric acid, and 1% to 2% glucose, or the composite anticoagulant containing 3.0% to 3.6% sodium citrate and 1% to 2% glucose; S2, cold water bath: take anticoagulated whole blood and seal it in a cold water bath; S3. Transportation: transport the anticoagulated whole blood in an incubator at 2-8°C; S4, let it stand; S5, siphon transfer: siphon transfer the supernatant; S6. Centrifugation: Using a graded centrifugation method, including sequential low-speed centrifugation and high-speed centrifugation, the conditions for low-speed centrifugation are centrifugation temperature 2-8°C, speed 500-1100g, and centrifugation time 5-20 min; the conditions for high-speed centrifugation are centrifugation temperature 2-8°C, speed 7000-15000g, and centrifugation time 5-20 min, and the high-speed centrifugation supernatant is collected; S7. Preparation: Take the supernatant from high-speed centrifugation and add at least one of Proclin 300, potassium sorbate, sodium diacetate, and benzalkonium bromide to obtain the product.
[0010] Preferably, as an improvement, in step S2, the temperature of the cold water bath is 2-25°C, the cold water at least covers 2 / 3 of the container, and the cold water bath time is 0.5-2 h.
[0011] Preferably, as an improvement, in step S4, the standing time is 0.5 to 2 h.
[0012] Preferably, as an improvement, in step S5, a silicone tube with an inner diameter of 2.4 to 3.1 mm is used for transfer.
[0013] Preferably, as an improvement, in step S7, Proclin 300 is added to a final concentration of 0.02-0.1%, potassium sorbate is added to a final concentration of 0.1-1%, sodium diacetate is added to a final concentration of 0.1-0.25%, and benzalkonium bromide is added to a final concentration of 0.1%-0.5%.
[0014] The principles and advantages of this solution are: in actual application, in this technical solution, the inventors have comprehensively optimized the preparation method and key control points of animal whole blood in response to the problems of hemolysis and residual insoluble particles in the preparation of animal whole blood in the prior art: blood cells are prone to hemolysis due to mechanical impact during long-term storage, transportation, and transfer after being removed from the body, and the blood loses its nutrient circulation supply after being removed from the body, and is prone to activity reduction or loss. In the prior art, it is generally believed that only removing red blood cells and fully retaining the quality of plasma components is the best and most stable. However, through long-term research, the inventors have found that monocytes, platelets, and insoluble particles in whole blood are often prone to aggregation, reducing the quality and stability of plasma. This discovery and viewpoint have overcome the prejudice of the prior art.
[0015] Based on this, this technical solution utilizes an anticoagulant to treat animal whole blood: sodium citrate (3.2%-3.8% aqueous solution), a weak acid-strong base salt, is readily soluble in water and binds to calcium ions in the blood to form a difficult-to-dissociate soluble complex (calcium citrate). Calcium ions are essential for both intrinsic and extrinsic coagulation cascades, participating in coagulation signaling and regulating enzyme activity. After binding to calcium ions, sodium citrate reduces the concentration of free calcium ions in the blood, blocking the coagulation process and achieving anticoagulation. Furthermore, this technical solution develops a composite anticoagulant. The combined use of sodium citrate and citric acid preserves the coagulation activity of whole blood and maintains its pH balance after ex vivo treatment. Glucose provides nutrients to blood cells, preventing rupture caused by lack of circulation after ex vivo treatment.
[0016] Maintaining enzyme activity using a low-temperature environment: Conventional whole blood is stored and transported at room temperature. High temperatures in summer and inside train compartments can easily cause enzyme activity in the animal's plasma to decrease or even disappear. This new technology utilizes a cold water bath for storing whole blood. Low temperatures (2°C to 8°C) slow molecular motion, reduce the frequency of enzyme-substrate collisions, and inhibit the rate of enzymatic reactions. This also maintains the stability of the enzyme's spatial structure, preventing denaturation and inactivation due to high temperatures. Transport is controlled in an insulated box, and the centrifugation process is performed at a low temperature of 2-8°C to prevent enzyme activity in the plasma from decreasing or disappearing.
[0017] In response to the problem of hemolysis, the present invention conducts a reverse analysis of the causes of hemolysis and finds that improper temperature control and treatment under high temperature conditions can easily damage red blood cells; the existing technology uses standing and then mixing again, which increases the risk of red blood cell rupture; the dumping transfer does not result in obvious separation of red blood cells and supernatant, and the back-and-forth transfer can easily further damage the red blood cells. Based on this, the present invention uses a cold water bath to quickly cool down the whole blood of the animal after collection, to avoid the continuous damage of high temperature to the animal plasma, accelerate the contraction of red blood cells, and facilitate subsequent preparation. Standing is combined with siphon transfer, so that blood cells naturally sink due to density differences, achieving preliminary separation of plasma and blood cells and avoiding external mechanical damage; siphon transfer uses the pressure difference generated by the difference in liquid level height to only absorb the upper plasma without contacting the blood cell layer, thereby reducing red blood cell rupture and improving the final plasma quality.
[0018] To address the issue of insoluble particles, the inventors first analyzed the density differences among blood components: red blood cells (high density) > white blood cells / platelets (intermediate density) > plasma (low density). The present invention utilizes fractionated centrifugation, with low-speed centrifugation primarily removing red blood cells and some insoluble particles, and high-speed centrifugation removing the remaining insoluble particles. This fractionated centrifugation effectively separates plasma from insoluble particles such as red blood cells, white blood cells, monocytes, and platelets, yielding fully soluble animal plasma.
[0019] In summary, the beneficial effects of this technical solution are: 1. This technical solution solves the problem of hemolysis: rapid cooling in a cold water bath prevents high-temperature damage to red blood cells, while low-temperature transportation and centrifugation reduce the risk of membrane damage. Static standing and siphon transfer replace traditional mixing and pouring operations, reducing mechanical damage and significantly reducing the incidence of hemolysis.
[0020] 2. This technical solution can maintain the stability of plasma components: low-temperature control throughout the process (cold water bath, incubator, low-temperature centrifugation) inhibits enzyme activity attenuation, maintains the activity of enzymes and functional proteins in plasma, avoids the release of lysosomal enzymes caused by hemolysis, and prevents the degradation of plasma components.
[0021] 3. This technical solution can improve plasma purity: graded centrifugation accurately separates components of different densities, low-speed centrifugation removes red blood cells and large particles, and high-speed centrifugation removes fine impurities, resulting in plasma purity ≥99%. Siphon transfer prevents the contamination of blood cells and further improves the clarity of the supernatant.
[0022] 4. This technical solution is operationally controllable and practical: Equipment such as cold water baths and incubators are low-cost, making it suitable for both laboratory and transportation applications. The fractional centrifugation parameters are well-defined and highly reproducible, facilitating large-scale production.
[0023] 5. After testing, the animal plasma prepared by this technical solution has no hemolysis and no insoluble particles. The intra-batch CV: R, K, Angle, and MA intra-batch coefficient of variation is less than 8%; the inter-batch relative range: R, K, Angle, and MA inter-batch relative range is less than 10%. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a process flow chart for fully soluble animal plasma according to an embodiment of the present invention.
[0025] Figure 2 This is the sedimentation curve of animal whole blood at different temperatures in the embodiment of the present invention.
[0026] Figure 3 This is the coagulation time curve (enzyme activity) of animal whole blood at different temperatures in the embodiment of the present invention. DETAILED DESCRIPTION
[0027] The following is further described in detail through specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.
[0028] Program Overview: like Figure 1 As shown, a method for preparing fully soluble animal plasma comprises the following steps: S1. Preparation: This includes the preparation of anticoagulants and the collection of whole blood from animals. The anticoagulant is a 3.2% to 3.8% sodium citrate aqueous solution or a compound anticoagulant containing sodium citrate and / or citric acid (the compound anticoagulant contains 3.0% to 3.6% sodium citrate, 0.11% to 0.14% citric acid and 1% to 2% glucose, or the compound anticoagulant contains 3.0% to 3.6% sodium citrate and 1% to 2% glucose). ), wherein the preparation method of 3.2%~3.8% sodium citrate aqueous solution is as follows: take a container, weigh 32.0~38.0g of sodium citrate and put it into the container, then add purified water to dissolve it completely and make up to 1000mL, and mix it well as an anticoagulant; during the animal whole blood collection process, take freshly collected animal whole blood, mix it according to the volume ratio of anticoagulant to animal whole blood of 1:8~1:10, and immediately mix it well as anticoagulant whole blood.
[0029] S2. Cold water bath: Take anticoagulated whole blood, seal it, and place it in cold water (2-25°C), with the cold water covering at least 2 / 3 of the container, and bathe in cold water for 0.5-2 hours.
[0030] S3. Transportation: Place the anticoagulated whole blood in an incubator at 2-8°C and complete transportation within 2 hours.
[0031] S4. Let the anticoagulated whole blood stand for 0.5 to 2 hours.
[0032] S5. Siphon transfer: Use a silicone tube with an inner diameter of 2.4~3.1mm to siphon transfer the supernatant.
[0033] S6. Centrifugation: Take the siphon supernatant, weigh and balance it, centrifuge it at low speed at low temperature (2~8℃, 500~1100g, 5~20min), and collect the supernatant of low-speed centrifugation; then take the supernatant of low-speed centrifugation, weigh and balance it, centrifuge it at high speed at low temperature (2~8℃, 7000~15000g, 5~20min), and collect the supernatant of high-speed centrifugation.
[0034] S7. Preparation: Take the supernatant from high-speed centrifugation, add Proclin 300 to a final concentration of 0.02-0.1%, and mix well.
[0035] Example 1 Study on the concentration of a single sodium citrate aqueous solution anticoagulant To determine the appropriate anticoagulant concentration, this study investigated the anticoagulant effects of different concentrations of anticoagulants on fresh animal whole blood. Sodium citrate was used as the anticoagulant, and different concentrations of sodium citrate anticoagulant (0%, 1%, 2%, 3.2%, 3.4%, 3.6%, 3.8%, 4.5%, and 5%) were prepared. Freshly collected animal whole blood was mixed at a volume ratio of anticoagulant:animal whole blood of 1:9 to obtain animal anticoagulant whole blood. The anticoagulant whole blood was observed at 0, 0.5, 2, 4, 8, 16, and 24 hours for the presence of clots. The serum was also observed for hemolysis after centrifugation. The results are shown in Tables 1 and 2. The results showed that the control group and the 1% sodium citrate group had blood clots after 0.5 h, the 2% sodium citrate group had blood clots after 2 h, while the other groups had no blood clots; the 1% sodium citrate group, the 2% sodium citrate group, the 4.5% sodium citrate group, and the 5% sodium citrate group began to show obvious hemolysis after the whole blood was placed for 8 h, 16 h, 24 h, and 16 h, respectively, while the other groups had no hemolysis, indicating that 3.2%~3.8% sodium citrate can not only have a good anticoagulant effect on animal whole blood, but also prevent hemolysis to a certain extent.
[0036] Table 1
[0037] Table 2
[0038] Example 2 Study on the concentration of composite anticoagulant To determine the appropriate concentration of a composite anticoagulant, this study investigated the anticoagulant effects of different concentrations of anticoagulants on fresh animal whole blood. Sodium citrate-citric acid-dextrose was selected as the composite anticoagulant. Different concentrations of sodium citrate (3.0%, 3.2%, and 3.6%), citric acid (0.11%, 0.12%, and 0.14%), and glucose (1%, 1.5%, and 2%) were prepared. Freshly collected animal whole blood was mixed at a volume ratio of anticoagulant to animal whole blood (1:9). The anticoagulated whole blood was then observed at 0, 0.5, 2, 4, 8, 16, 24, 36, and 48 hours for the presence of clots and for hemolysis of the serum after centrifugation. The results are shown in Tables 3 and 4. The results showed that blood clots appeared in the 3.2% sodium citrate group and the 3.8% sodium citrate group and plasma hemolysis occurred after 36 hours of storage, while no blood clots appeared in the other groups. At the same time, the plasma was light yellow and no hemolysis occurred. This shows that the composite anticoagulant composed of 3.0%-3.6% sodium citrate, 0.11%-0.14% citric acid, and 1%-2% glucose can achieve a good anticoagulant effect and prevent hemolysis for a longer period of time (48 hours).
[0039] Table 3
[0040] Table 4
[0041] Example 3 Study on the mixing ratio of anticoagulants and animal whole blood To determine the optimal mixing ratio of anticoagulant to animal whole blood, this study investigated the anticoagulant effects of different mixing ratios of anticoagulant to animal whole blood. 3.2% and 3.8% sodium citrate were prepared, respectively, and freshly collected animal whole blood was mixed at different anticoagulant:animal whole blood volume ratios (1:2, 1:4, 1:8, 1:9, 1:10, and 1:11). The anticoagulant whole blood was observed at 0, 0.5, 2, 4, 8, 16, and 24 hours for the presence of clots.
[0042] The control group used fresh porcine whole blood collected directly without the addition of any anticoagulant. The results are shown in Table 5. The results show that blood clots formed in the control group after 0.5 hours. Mixing 3.2% sodium citrate with animal whole blood at 1:2, 1:4, 1:8, 1:9, 1:10, and 1:11, and mixing 3.8% sodium citrate with animal whole blood at 1:2, 1:4, 1:8, 1:9, and 1:10, no blood clots formed within 0-24 hours. This indicates that 3.2% to 3.8% sodium citrate mixed with animal whole blood at 1:8 to 1:10 ratios all have a good anticoagulant effect.
[0043] Table 5
[0044] Example 4 Study on the Effect of Temperature on Animal Whole Blood To determine the extent of temperature effects on animal whole blood, this study investigated the hemolysis, sedimentation, and enzyme activity of animal whole blood at different temperatures. 3.8% sodium citrate was prepared. Freshly collected animal whole blood was mixed at a volume ratio of anticoagulant to whole blood (1:9) to create animal anticoagulated whole blood. The blood was aliquoted at 1 mL per tube and stored at different temperatures (2°C, 4°C, 8°C, 20°C, 25°C, 30°C, 40°C, and 45°C). Hemolysis, sedimentation, and coagulation time were measured or observed at 0, 1, 2, 4, 8, 16, and 24 hours. Hemolysis was observed by centrifuging the anticoagulated whole blood at 4°C at 1000g for 5 minutes. The supernatant color was visually inspected: light yellow indicated no hemolysis, while light orange to red indicated hemolysis. Sedimentation was measured by measuring the supernatant height with a ruler. Coagulation time was measured using a thrombelastograph.
[0045] Table 6
[0046] Table 7
[0047] Table 8
[0048] The results are shown in Tables 6, 7, and 8. Figure 2 、 Figure 3 As shown, the test results showed that: when the temperature was 2℃, 4℃, 8℃, 20℃, 25℃, and 30℃, there was no hemolysis in the whole blood of animals from 0h to 24h (Table 6), and the height of the supernatant increased with time. Before 8h, the lower the temperature, the greater the supernatant height. After 8h, there was no significant difference in the supernatant height between the temperatures, and the coagulation time basically did not change significantly within 24h; while at 40℃ and 45℃, obvious hemolysis occurred after 1h, and the supernatant height increased with time, but the overall stratification was not obvious. The supernatant height was significantly less than 2℃~30℃, and the coagulation time showed a significant difference from 0h from 1h, and was significantly prolonged. The comprehensive analysis of hemolysis, sedimentation and coagulation time shows that low temperature can effectively prevent hemolysis, promote blood cell sedimentation, and maintain good enzyme activity, while high temperature is prone to hemolysis, slows cell sedimentation, and significantly reduces enzyme activity.
[0049] Example 5: Study on the inner diameter screening of siphon silicone tubes To determine the optimal inner diameter for siphoning silicone tubing, this study investigated the siphoning performance of silicone tubing with different inner diameters. Siphoning of static, anticoagulated whole blood from animals was performed using silicone tubing with inner diameters of 0.8 mm, 1.6 mm, 2.4 mm, 3.1 mm, and 4.8 mm. The results showed that the 0.8 mm and 1.6 mm silicone tubings had slow siphoning speeds, while the 4.8 mm silicone tubing, while fast, readily aspirated red blood cells, making it suitable for siphoning large supernatant volumes. The 2.4 mm and 3.1 mm silicone tubings had moderate siphoning speeds, siphoning fluid without noticeable red blood cells, making them suitable for both large and small supernatant siphoning.
[0050] Example 6 Low-speed centrifugation study To obtain optimal low-speed centrifugation conditions, this study investigated the effects of different centrifugal forces and times. Siphon supernatant was centrifuged at 4°C for 5 minutes at various centrifugal forces (100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, and 1100g). The separation between the supernatant and the precipitate was observed. Siphon supernatant was centrifuged at 4°C for 5 minutes at 500g and different times (5 minutes, 10 minutes, 15 minutes, and 20 minutes). The results showed that when centrifuged at 100g, 200g, 300g, and 400g for 5 minutes, the supernatant and precipitate were not completely separated, and the precipitate easily mixed with the supernatant during separation. When centrifuged at 500g, 600g, 700g, 800g, 900g, 1000g, and 1100g for 5 minutes, the supernatant and precipitate were clearly separated and easily separated, and the two were not easily mixed. When the centrifugal force was 500g, the supernatant and precipitate were quickly and effectively separated for 5 minutes, 10 minutes, 15 minutes, and 20 minutes. Overall, low-speed centrifugation with a centrifugal force of 500g to 1100g and a centrifugation time of 5 minutes to 20 minutes can effectively remove red blood cells.
[0051] Example 7 High-speed centrifugation study To identify optimal high-speed centrifugation conditions, this study investigated the effects of different centrifugal forces and times. Supernatants from low-speed centrifugations were centrifuged for 1 minute at 4°C at various centrifugal forces (7000g, 8000g, 9000g, 10000g, 11000g, 12000g, 13000g, 14000g, and 15000g). The supernatants from high-speed centrifugations were then centrifuged again at 15000g for 5 minutes and observed for the presence of a precipitate. The presence of a precipitate indicated the presence of insoluble particles, while the absence of a precipitate indicated minimal or no insoluble particles. The results showed that no significant precipitate was observed after centrifugation for 1 minute at 11000g, 12000g, 13000g, 14000g, and 15000g. The supernatant from the low-speed centrifugation was centrifuged at 7000g for 1, 2.5, 5, 10, 15, and 20 minutes at 4°C. The supernatant from the high-speed centrifugation was then centrifuged again at 15000g for 5 minutes to observe for the presence of precipitation. The results showed that no significant precipitation was observed in the supernatant after centrifugation at 7000g for 5, 10, 15, and 20 minutes. This suggests that high-speed centrifugation with a centrifugal force between 7000g and 15000g and a centrifugation time between 5 and 20 minutes can effectively remove insoluble particles.
[0052] Example 8 Study on the Quality of Fully Soluble Animal Plasma Appearance: Take fully soluble animal plasma, freeze and thaw, and heat at 37°C for 5 minutes. Observe the appearance of the fully soluble animal plasma. The test results show that all three batches of fully soluble animal plasma are light yellow liquids.
[0053] Intra-batch CV: Fully soluble animal plasma was taken and tested 10 times continuously using a thrombelastograph. R, K, Angle, and MA were recorded respectively. The mean (X), standard deviation (SD), and inter-batch coefficient of variation (CV) of the 10 test results were calculated according to formula (1), formula (2), and formula (3). The results showed that the coefficients of variation of R, K, Angle, and MA were all less than 8%, indicating good intra-batch precision.
[0054] Table 9
[0055] Inter-batch CV: Three batches of fully soluble animal plasma were tested as required. Each batch of fully soluble animal plasma was measured three times. The measurement results included R, K, Angle, and MA. The total mean (XT) of the nine measurement results and the mean value (Xi) of each batch of kit measurements were calculated according to formula (1). The maximum value was the maximum inter-batch mean (Xmax), and the minimum value was the minimum inter-batch mean (Xmin). The inter-batch relative range (R) was calculated according to formula (4). The results showed that the inter-batch relative range of R, K, Angle, and MA was less than 10%, indicating good inter-batch precision.
[0056] R=((Xmax-Xmin) / XT)*100% Formula (4) Table 10
[0057] Example 9 Evaluation of Insoluble Particles Direct evaluation: Take fully soluble animal plasma and, after thawing, heat at 37°C for 5 minutes. Centrifuge at 15,000g for 5 minutes at 4°C. Observe the centrifuge tube to see if there is any precipitation.
[0058] The results showed that the fully soluble animal plasma had no precipitation after centrifugation, while the control animal plasma had obvious white precipitation at the bottom after centrifugation, indicating that the animal plasma prepared by the present invention was indeed fully soluble and had no insoluble particulate matter.
[0059] Indirect evaluation: Take 2 mL each of fully soluble animal plasma and control animal plasma, store at -20℃ overnight, then place at 2℃~8℃ for cold precipitation for 12 hours, then centrifuge at 10000g at 4℃ for 5 minutes, collect the cold precipitation, add the corresponding 0.25 mL of supernatant, heat at 37℃ for 5 minutes to obtain the high-value animal plasma solution, then centrifuge at 15000g at 4℃ for 5 minutes, and observe whether there is precipitation in the centrifuge tube.
[0060] The results showed that the high-value animal plasma solution prepared from fully soluble animal plasma completely dissolved, presenting a light yellow transparent liquid with no precipitation after centrifugation. However, the high-value animal plasma solution prepared from control animal plasma could not completely dissolve, presenting a light yellow suspension with numerous insoluble particles and a significant amount of white precipitation at the bottom of the tube after centrifugation. This comparison demonstrates that fully soluble animal plasma is indeed fully soluble.
[0061] Application Example 1 Plasma stored at -20°C was thawed at room temperature and heated at 37°C for 10-30 minutes. During the process, it was found that the plasma obtained by ordinary centrifugation contained a large amount of flocculent insoluble matter after thawing, and the matter remained after heating. However, the plasma in this protocol became transparent after the ice was completely thawed and after heating, without any flocculent insoluble matter.
[0062] Application Example 2 Preparation of cryoprecipitate: The cryoprecipitate prepared from plasma obtained by ordinary centrifugation cannot be completely dissolved and contains a large amount of lumpy insoluble matter; however, the cryoprecipitate prepared in this protocol completely dissolves within 5 minutes after adding the supernatant.
[0063] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.
Claims
1. A method for preparing fully soluble animal plasma, characterized in that: After treatment with an anticoagulant, the blood is allowed to stand, transferred, and centrifuged under temperature control throughout the entire process. The anticoagulant is a 3.2% to 3.8% sodium citrate aqueous solution or a composite anticoagulant containing sodium citrate and / or citric acid. The volume ratio of the anticoagulant to the animal's whole blood is 1:8 to 1:
10.
2. The method for preparing fully soluble animal plasma according to claim 1, wherein: The composite anticoagulant contains 3.0% to 3.6% sodium citrate, 0.11% to 0.14% citric acid and 1% to 2% glucose, or the composite anticoagulant contains 3.0% to 3.6% sodium citrate and 1% to 2% glucose.
3. The method for preparing fully soluble animal plasma according to claim 2, wherein: The centrifugal treatment process adopts a graded centrifugation method, including low-speed centrifugation and high-speed centrifugation performed in sequence.
4. The method for preparing fully soluble animal plasma according to claim 3, wherein: The low-speed centrifugation conditions are as follows: centrifugation temperature 2-8°C, rotation speed 500-1100g, and centrifugation time 5-20min.
5. The method for preparing fully soluble animal plasma according to claim 4, wherein: The conditions for the high-speed centrifugation are as follows: a centrifugation temperature of 2-8° C., a rotation speed of 7000-15000 g, and a centrifugation time of 5-20 min.
6. The method for preparing fully soluble animal plasma according to claim 5, characterized in that: The steps include: S1. Preparation: Take whole blood from an animal, mix it in a volume ratio of 1:8 to 1:10 between an anticoagulant and the animal's whole blood, and immediately mix it to prepare anticoagulated whole blood; the anticoagulant is a 3.2% to 3.8% sodium citrate aqueous solution or a composite anticoagulant containing sodium citrate and / or citric acid, the composite anticoagulant containing 3.0% to 3.6% sodium citrate, 0.11% to 0.14% citric acid, and 1% to 2% glucose, or the composite anticoagulant containing 3.0% to 3.6% sodium citrate and 1% to 2% glucose; S2, cold water bath: take anticoagulated whole blood and seal it in a cold water bath; S3. Transportation: transport the anticoagulated whole blood in an incubator at 2-8°C; S4, let it stand; S5, siphon transfer: siphon transfer the supernatant; S6. Centrifugation: Using a graded centrifugation method, including sequential low-speed centrifugation and high-speed centrifugation, the conditions for low-speed centrifugation are centrifugation temperature 2-8°C, speed 500-1100g, and centrifugation time 5-20 min; the conditions for high-speed centrifugation are centrifugation temperature 2-8°C, speed 7000-15000g, and centrifugation time 5-20 min, and the high-speed centrifugation supernatant is collected; S7. Preparation: Take the supernatant from high-speed centrifugation and add at least one of Proclin 300, potassium sorbate, sodium diacetate, and benzalkonium bromide to obtain the product.
7. The method for preparing fully soluble animal plasma according to claim 6, wherein: In step S2, the temperature of the cold water bath is 2-25°C, the cold water at least covers 2 / 3 of the container, and the cold water bath time is 0.5-2 h.
8. The method for preparing fully soluble animal plasma according to claim 7, wherein: In step S4, the standing time is 0.5 to 2 h.
9. The method for preparing fully soluble animal plasma according to claim 8, characterized in that: In step S5, a silicone tube with an inner diameter of 2.4-3.1 mm is used for transfer.
10. The method for preparing fully soluble animal plasma according to claim 9, characterized in that: In step S7, Proclin 300 is added to a final concentration of 0.02-0.1%, potassium sorbate is added to a final concentration of 0.1-1%, sodium diacetate is added to a final concentration of 0.1-0.25%, and benzalkonium bromide is added to a final concentration of 0.1%-0.5%.