Method for separating panda peripheral blood cells

Abstract

The invention relates to the technical field of cell engineering, and particularly provides a method for separating peripheral blood cells of pandas, which comprises the following steps: step 1, collecting a fresh anticoagulant blood sample of the pandas, and mixing with a Tycode solution buffered by HEPES to obtain a mixed solution; step 2, carrying out first centrifugal operation on the mixed solution to obtain middle leukocytes, lower-layer erythrocytes and upper-layer platelet-rich plasma; and step 3, performing secondary centrifugation operation on the platelet-rich plasma to obtain lower-layer platelets and upper-layer platelet-deficient plasma. According to the method disclosed by the invention, the integrity of the peripheral blood cells in the separation operation can be protected, the determination of other physiological and biochemical parameters of blood cannot be influenced, the separated platelets are high in purity, and the platelet recovery efficiency can reach 95% or above.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a method for separating peripheral blood cells of giant pandas. Background Art

[0002] The giant panda (Ailuropoda melanoleuca) is a rare and endangered protected animal unique to my country, and has extraordinary conservation and research significance. In recent years, rapid assessment of the health status of giant pandas and timely adjustment of diagnosis, treatment, care and feeding management plans based on the assessment results have increasingly become an inevitable requirement for the formulation of scientific protection plans for giant pandas. Blood physiological and biochemical parameters, blood cell status and analysis results can, to a certain extent, reflect the metabolism and physiological function status of giant pandas in real time. Therefore, the detection of blood physiological and biochemical parameters and the separation of various blood cells can provide important scientific basis for clinical veterinarians to quickly judge the health status of animals, formulate diagnosis and treatment plans, and cooperate with animal management personnel to adjust feeding management plans.

[0003] In other animals, the operation procedures and detection techniques for analyzing whole blood, serum and plasma are relatively mature. With the help of these analytical methods, many reports have been made on the physiological and biochemical parameters of giant panda blood. Unlike the limited information provided by whole blood, serum and plasma, analyzing the state of animal peripheral blood cells can provide researchers with a large amount of real-time changes in the immune system. Peripheral blood leukocytes are an important component of the animal immune system and are of great significance for the evaluation of animal immune function and disease diagnosis. Platelets can not only interact with leukocytes and the coagulation system to mediate thrombotic inflammation, but also change the inflammatory environment by releasing various factors and extracellular vesicles. Red blood cells are not only oxygen carriers, but also cooperate with immune cells in inflammatory states to accelerate the immune process. However, due to the lack of research on the separation technology of giant panda peripheral blood cells, there are currently almost no research reports on the dynamic changes of giant panda peripheral blood cells and immune system function. In other animals, the method of separating peripheral blood cells mainly adopts the relatively low-cost density gradient centrifugation method. However, this separation method has high operational requirements for technicians and requires the purchase of expensive inert gradient medium solutions. The cell composition ratio, blood components, blood viscosity and other physiological characteristics of peripheral blood of different animals are different, so the centrifugal conditions and operation steps required for separating blood cells may need to be optimized and tested for giant pandas. At present, there is no research specifically on the separation method of peripheral blood cells of giant pandas. Therefore, finding a rapid separation method suitable for giant panda peripheral blood cells is of great significance to assist veterinarians in quickly judging the function of the giant panda's immune system, adjusting and formulating diagnosis and treatment plans. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problems that the current density gradient centrifugation method has high operating requirements for technicians, needs to purchase inert gradient medium solutions, and lacks technical parameters for the operation of giant panda peripheral blood cells. A method for separating giant panda peripheral blood cells is provided. The method can not only efficiently separate red blood cells, immune cells and platelets, but also use whole blood samples before the separation operation and plasma samples during the separation operation to measure blood physiological and biochemical parameters.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows: The present invention provides a method for separating peripheral blood cells of giant pandas, comprising the following steps: Step 1, collecting a fresh anticoagulated blood sample from a giant panda and mixing it with a HEPES-buffered Tyrode solution to obtain a mixed solution; Step 2, subjecting the mixed solution to a first centrifugation operation to obtain intermediate white blood cells, lower red blood cells and upper platelet-rich plasma; Step 3, subjecting the platelet-rich plasma to a second centrifugation operation to obtain a lower layer of platelets and an upper layer of platelet-poor plasma.

[0006] Preferably, in step 1, the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:7-15.

[0007] Further preferably, in step 1, the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:9.5-11.5.

[0008] Preferably, the anticoagulant used in the fresh anticoagulated blood sample of the giant panda is at least one of the following: EDTA, heparin, sodium citrate, sodium citrate.

[0009] Preferably, in step 2, the control conditions for the first centrifugation are: temperature 15-25°C, 80-100g centrifugation for 10-30min.

[0010] Further preferably, in step 2, the control conditions of the first centrifugation are: temperature 15-25°C, 85-95g centrifugation for 15-25min.

[0011] Preferably, in step 3, the control conditions of the second centrifugation are: temperature 15-25° C., 350-600 g centrifugation for 5-20 min.

[0012] Further preferably, in step 3, the control conditions of the second centrifugation are: temperature 15-25° C., 400-500 g centrifugation for 10-15 min.

[0013] In addition, the method of the present invention is currently only applicable to giant panda species and is not applicable to other species.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (I) The present invention uses HEPES-buffered Tyrode solution to dilute giant panda EDTA anticoagulated blood and giant panda platelet-rich plasma, and optimizes the volume ratio of HEPES-buffered Tyrode solution to giant panda EDTA anticoagulated blood or platelet-rich plasma, so that the integrity of peripheral blood cells is protected during the separation operation. It is expected to solve the problem that the current density gradient centrifugation method has high operating requirements for technicians and requires the purchase of expensive inert gradient medium solutions.

[0015] (ii) The present invention optimizes the centrifugal conditions and operating steps required for separating blood cells from the peripheral blood of giant pandas.

[0016] (III) The platelets separated by the present invention have high purity and the platelet recovery efficiency can reach over 95%. Under ideal conditions, the platelet recovery rate can even reach over 99%.

[0017] (IV) The operation of separating peripheral blood cells in the present invention will not affect the determination of other blood physiological and biochemical parameters. The giant panda anticoagulated blood before the blood cell separation operation, the platelet-rich plasma and platelet-poor plasma during the operation can be used for the determination of blood parameters such as blood routine, hormones, liver function, tumor markers, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a microscope observation diagram of the giant panda blood cell morphology in Example 1 of the present invention.

[0019] Figure 2 This is the cell and plasma stratification after the centrifugation step is completed after the giant panda EDTA anticoagulated blood and HEPES buffered Tyrode solution are mixed in Example 1 of the present invention.

[0020] Figure 3 This is the situation of the middle white blood cell layer and the lower red blood cell layer in Example 1 of the present invention.

[0021] Figure 4 This is the cell stratification after the first centrifugation in Comparative Example 1 of the present invention.

[0022] Figure 5 This is the cell stratification after the first centrifugation in Comparative Example 2 of the present invention.

[0023] Figure 6 This is the cell stratification after the first centrifugation and the second centrifugation in Comparative Example 3 of the present invention.

[0024] Figure 7This is the cell stratification after the first centrifugation and the second centrifugation in Comparative Example 4 of the present invention.

[0025] Figure 8 This is the cell stratification after the first centrifugation and the second centrifugation in Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0026] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0028] Embodiment 1: This embodiment provides a method for separating peripheral blood cells of giant pandas. The separation method comprises the following steps: (1) Collect fresh EDTA anticoagulated blood sample from a female giant panda and observe the blood cell morphology under a microscope. Figure 1 For blood samples with normal blood cell status, routine blood tests are first performed to obtain whole blood parameter results including platelet count (PLT1) and hematocrit (HCT).

[0029] (2) Add 10 mL of HEPES-buffered Tyrode solution to 1 mL of EDTA-anticoagulated blood sample. Invert the tube to mix thoroughly.

[0030] (3) Take the mixture from (2) and centrifuge it at 90g for 20 minutes at room temperature (15-25°C). After centrifugation, observe the cell stratification ( Figure 2 ).

[0031] (4) Separately collect the middle white blood cell layer and the lower red blood cell layer ( Figure 3 ), transferred into two centrifuge tubes for storage.

[0032] (5) Perform plasma parameter analysis on the platelet-rich plasma in (3) to obtain the platelet count (PLT2) result of the platelet-rich plasma.

[0033] (6) Take the platelet-rich plasma from (3) and centrifuge it at 450 g for 10 min at room temperature (15-25°C).

[0034] (7) Separately collect the lower layer of platelets and the upper layer of platelet-poor plasma and transfer them into two centrifuge tubes.

[0035] (8) Perform plasma parameter analysis on the platelet-poor plasma in (7). Measure blood parameters such as hormones, liver function, and tumor markers.

[0036] This example not only obtains the red blood cells, white blood cells and platelets in the peripheral blood of giant pandas, but also measures the blood physiological and biochemical parameters before and during the separation of peripheral blood cells. The original measurement results of the blood physiological and biochemical parameters are shown in Table 1.

[0037] Table 1. Physiological and biochemical parameters of giant panda whole blood and plasma

[0038] The platelet count (PLT1), hematocrit (HCT) in whole blood, and the platelet count (PLT2) and white blood cell count (WBC) in platelet-rich plasma in Table 1 are imported into the formula to calculate the platelet recovery efficiency. The calculation formula is as follows: Platelet recovery rate = PLT2×(1- HCT) / PLT1×100%, Platelet purity = PLT2 / (PLT2+WBC) × 100%; Among them, the number of platelets (PLT1) in whole blood is 5.64×1011 / L, the hematocrit (HCT) is 25.7%, the number of platelets (PLT2) in platelet-rich plasma is 7.57×1011 / L, and the number of white blood cells (WBC) is 0.17×109 / L. The platelet recovery rate of the giant panda in Example 1 is calculated to be 99.73%, and the platelet purity is 99.98%. It can be seen that the method of the present invention is used to separate platelets with a high recovery rate and purity. The platelet recovery rate and platelet purity results calculated above are compared with the results obtained by the existing centrifugation method. The comparison results are shown in Table 2. Among them, the method provided by Chinese patent CN201710075710.4 can make the platelet recovery rate of mammals reach 83-89%, and the platelet purity reaches 96-99%. The platelet recovery rate calculated in Example 2 is higher than that in Chinese patent CN201710075710.4, and the platelet recovery rate can reach 99.73%, which is a significant improvement in the recovery rate; the purity value is not weaker than that in Chinese patent CN201710075710.4, and can reach 99.98%, and the leukocyte removal efficiency is extremely high.

[0039] Table 2. Statistical results of mammalian platelet recovery and purity obtained by different methods

[0040] The present invention further provides some parameter experiments done in the early stage of experimental exploration, which are as follows: Comparative Example 1: This comparative example provides a method for separating peripheral blood cells of giant pandas. The difference between this separation method and Example 1 is that the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:20. After the first centrifugation is completed, the cell stratification is observed ( Figure 4 ). The results showed that a large number of cells were precipitated at the bottom of the centrifuge tube, but the platelet-rich plasma layer was red. This indicates that the volume ratio of the anticoagulated blood sample to the HEPES-buffered Tyrode solution is not appropriate, resulting in moderate hemolysis in the sample. Hemolysis is the phenomenon of blood cells rupturing and releasing contents and cell fragments into the plasma. Hemolyzed samples will not only directly affect the red blood cell count, but the ruptured cell fragments will also interfere with platelet detection, resulting in distortion of platelet recovery rate and purity calculation. Therefore, when using the volume ratio of this comparative example, the values ​​of platelet count and platelet purity will deviate from the truth, and the determination of physiological and biochemical parameters of platelet-rich plasma may be affected.

[0041] Comparative Example 2: This comparative example provides a method for separating peripheral blood cells of giant pandas. The difference between this separation method and Example 1 is that the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:5. After the first centrifugation is completed, the cell stratification is observed ( Figure 5 ). The results showed that a large number of cells were precipitated at the bottom of the centrifuge tube, but the platelet-rich plasma layer was red. This indicated that the volume ratio of the anticoagulated blood sample to the HEPES-buffered Tyrode solution was not appropriate, resulting in mild hemolysis in the sample. Therefore, when the volume ratio of this comparative example is used, the values ​​of platelet count and platelet purity will deviate from the true value, and the determination of physiological and biochemical parameters of platelet-rich plasma may be affected.

[0042] In addition, in the present invention, if the first centrifugal speed is higher than 200g, cell stratification will occur in the whole blood, but some red blood cells and white blood cells will rupture and be released into the platelet-rich plasma in the upper layer, resulting in hemolysis. The blood physiological parameter values ​​of the hemolyzed sample will be abnormal. When the second centrifugal speed is lower than 300g, the platelets cannot be fully enriched, which will cause the number of recovered platelets to be seriously low, thereby reducing the platelet recovery efficiency. In order to solve this problem, the present invention controls the first centrifugal speed at 80~100g for 10~30min, preferably at 85~95g for 15~25min, and at the same time controls the second centrifugal speed at 350~500g for 5~20min, preferably at 400~500g for 10-15min, which greatly improves the separation effect of the three cells and the credibility of the blood physiological and biochemical parameters. The following are some failed cases in the experimental exploration process: Comparative Example 3: This comparative example provides a method for separating peripheral blood cells of giant pandas. The difference between this separation method and Example 1 is that the first centrifugation speed and the second centrifugation speed are both 220g. After the first and second centrifugation are completed, the cell stratification and plasma state ( Figure 6 ). The results show that, compared with Example 1, the color of the platelet-poor plasma after the second centrifugation in this comparative example is reddish. This indicates that the first centrifugation speed is too high, resulting in a small amount of blood cell breakage and mild hemolysis in the sample; the second centrifugation speed is too low, which is not conducive to the precipitation of platelets and blood cell fragments in the plasma. Therefore, when using the centrifugation speed of this comparative example, the values ​​of platelet count and platelet purity will deviate from the true value, and the determination of physiological and biochemical parameters of platelet-rich plasma and platelet-poor plasma may be affected.

[0043] Comparative Example 4: This comparative example provides a method for separating peripheral blood cells of giant pandas. The difference between this separation method and Example 1 is that the first centrifugation speed is 50g, and the second centrifugation speed is 220g. After the first and second centrifugation are completed, the cell stratification and plasma state ( Figure 7 ). The results show that, compared with Example 1, the color of the platelet-rich plasma after the first centrifugation in this comparative example is reddish, and the color of the platelet-poor plasma after the second centrifugation is still reddish, and a large amount of red substances are precipitated in the platelet layer. This shows that the first centrifugation speed is too low, and the red blood cells and white blood cells cannot be fully precipitated; although the second centrifugation speed is also too low, the number of blood cells suspended in the platelet-rich plasma is relatively large, resulting in the red blood cells and other blood cells that were not fully precipitated before gradually enriched in the platelet layer during the second centrifugation. Therefore, when using the centrifugation speed of this comparative example, the values ​​of platelet count and platelet purity will seriously deviate from the truth, and the determination of physiological and biochemical parameters of platelet-rich plasma and platelet-poor plasma may be affected.

[0044] Comparative Example 5: This comparative example provides a method for separating peripheral blood cells of giant pandas. The difference between this separation method and Example 1 is that the first centrifugation speed is 220g, and the second centrifugation speed is 800g. After the first and second centrifugation are completed, the cell stratification and plasma state ( Figure 8 ). The results show that, compared with Example 1, a large amount of red material is precipitated in the platelet layer after the second centrifugation in this comparative example. This indicates that the two centrifugation speed settings are not appropriate, which ultimately leads to the enrichment of blood cells or cell debris in the platelet layer. Therefore, when the centrifugation speed of this comparative example is used, the values ​​of platelet count and platelet purity will seriously deviate from the true value, and the determination of physiological and biochemical parameters of platelet-rich plasma and platelet-poor plasma may be affected.

[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for isolating peripheral blood cells of giant pandas, characterized in that: The steps include: Step 1, collecting a fresh anticoagulated blood sample from a giant panda and mixing it with a HEPES-buffered Tyrode solution to obtain a mixed solution; Step 2, subjecting the mixed solution to a first centrifugation operation to obtain intermediate white blood cells, lower red blood cells and upper platelet-rich plasma; Step 3, subjecting the platelet-rich plasma to a second centrifugation operation to obtain a lower layer of platelets and an upper layer of platelet-poor plasma.

2. The method according to claim 1, characterized in that In the step 1, the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:7-15.

3. The method according to claim 1, characterized in that In the step 1, the volume ratio of the fresh anticoagulated blood sample of the giant panda to the HEPES-buffered Tyrode solution is 1:9.5-11.

5.

4. The method according to any one of claims 1 to 3, characterized in that The anticoagulant used in the fresh anticoagulated blood sample of the giant panda is at least one of the following: EDTA, heparin, sodium citrate, and sodium citrate.

5. The method according to claim 4, characterized in that In step 2, the control conditions for the first centrifugation are: temperature 15-25° C., 80-100 g centrifugation for 10-30 min.

6. The method according to claim 5, characterized in that In step 2, the control conditions of the first centrifugation are: temperature 15-25°C, 85-95g centrifugation for 15-25min.

7. The method according to claim 6, characterized in that In step 3, the control conditions of the second centrifugation are: temperature 15-25° C., 350-600 g centrifugation 5-20 min.

8. The method according to claim 7, characterized in that In step 3, the control conditions for the second centrifugation are: temperature 15-25° C., 400-500 g centrifugation for 10-15 min.

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