Anticoagulant for high-precision animal blood cell analyzer and application thereof in white blood cell classification

Abstract

The application belongs to the technical field of biological medicine, and relates to an anticoagulant and application thereof in white blood cell classification for high-precision animal blood cell analyzers. The anticoagulant comprises two parts of buffer and anticoagulant matrix components and active components. In the anticoagulant, the buffer and anticoagulant matrix component formula comprises 1.5-2 g of citrate, 0.3-0.5 g of citric acid and 0.15-0.20 g of EDTA-K2 per 100 mL of the anticoagulant, and the active component comprises 0.08-0.10 g of low-molecular enzymatic hydrolysis carboxymethyl chitosan, 1.0-1.2 g of hydroxypropyl-beta-cyclodextrin, 10-15 mg of dipyridamole and 8-10 mg of aspirin. The anticoagulant effectively inhibits platelet activation, prevents platelet aggregation and white blood cell morphological deformation, accurately identifies in white blood cell five classification, and has a classification accuracy as high as 98%, and is suitable for veterinary clinical hematology examination and animal disease monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an anticoagulant, specifically an anticoagulant for high-precision animal blood cell analyzers and its application in white blood cell classification. Background Technology

[0002] Anticoagulants are chemical substances that maintain the liquid state of blood by interfering with the coagulation cascade or binding to key coagulation factors (such as calcium ions), and their performance directly affects the accuracy of blood cell analysis. Existing anticoagulants have significant limitations: while traditional EDTA inhibits coagulation, it easily induces changes in platelet morphology or non-specific activation (such as increased CD62p expression), interfering with leukocyte classification; furthermore, anticoagulants are prone to degradation during storage, leading to inaccurate test results. To address these problems, this invention provides an anticoagulant that inhibits platelet activation, providing a high-precision and highly stable basis for animal blood cell analysis, and has significant application value, especially in the differentiation of leukocyte subsets. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide an anticoagulant for a high-precision animal blood cell analyzer and its application in white blood cell classification.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] Firstly, this invention provides an anticoagulant for a high-precision animal blood cell analyzer. The anticoagulant comprises two parts: a buffer and anticoagulant matrix component and an active ingredient. Specifically, per 100 mL of the anticoagulant, the buffer and anticoagulant matrix component comprises 1.5-2 g of citrate, 0.3-0.5 g of citric acid, and 0.15-0.20 g of EDTA-K2. The active ingredient comprises 0.08-0.10 g of low-molecular-weight enzymatically hydrolyzed carboxymethyl chitosan, 1.0-1.2 g of hydroxypropyl-β-cyclodextrin, 10-15 mg of dipyridamole, and 8-10 mg of aspirin.

[0006] Preferably, the citrate is sodium citrate.

[0007] Secondly, this invention provides a method for preparing an anticoagulant, the specific steps of which are as follows:

[0008] S1. Preparation of low molecular weight enzymatically hydrolyzed carboxymethyl chitosan: Carboxymethyl chitosan powder was dissolved in citric acid solution to prepare a 3% (w / v) carboxymethyl chitosan solution; then the pH was adjusted to 6.2, and papain was added at 55℃ for 4-5 h of enzymatic hydrolysis; after inactivating the enzyme by heating at 92℃ for 12 min, the solution was cooled to 40℃, the pH was adjusted to 5.5, chitosanase was added, and enzymatic hydrolysis was continued at 50℃ for 6-7 h; finally, the enzyme was inactivated by boiling water bath for 15 min, and the precipitate was removed by centrifugation at 8000-9000 rpm for 20 min. The ultrafiltration permeate with a molecular weight of less than 2 kDa was placed in a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed at 4℃ with 0.1 M NH4HCO3 dialysate and distilled water in sequence (until the external conductivity ≤10 μS / cm). The contents of the dialysis bag were freeze-dried and pulverized to finally obtain low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder.

[0009] S2. Preparation of buffer and anticoagulant matrix components: Dissolve 1.5-2g of sodium citrate and 0.3-0.5g of citric acid in 60mL of sterile water, adjust the pH to 6.8, add 0.5-0.8g of EDTA-K2 and stir at 50℃ until completely dissolved;

[0010] S3. Preparation of anticoagulant: Take 5-7 mL of anhydrous ethanol, add 10-15 mg of dipyridamole and 8-10 mg of aspirin, mix well to obtain a mixture. Slowly add this mixture, 1.0-1.2 g of hydroxypropyl-β-cyclodextrin and 0.08-0.10 g of low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder to the buffer and anticoagulant matrix solution prepared in step S2. Make up the volume to 100 mL with sterile water, stir well, adjust the pH of the solution to 7.0±0.2, and filter sterilize using a 0.22 μm sterile filter membrane to obtain the anticoagulant.

[0011] Preferably, the citric acid solution in step S1 of the present invention has a mass-volume percentage of 2%.

[0012] Preferably, the pH of the citric acid solution in step S1 of the present invention is 4.5-5.0.

[0013] Preferably, in step S1 of the present invention, the papain substrate concentration ratio is 1:45 (w / w), and the activity is 3500 U / g.

[0014] Preferably, in step S1 of the present invention, the chitosanase substrate concentration ratio is 1:80 (w / w), and the activity is 900 U / g.

[0015] Preferably, the reagent used to adjust the pH to 6.8 in step S2 of the present invention is NaOH.

[0016] Preferably, the reagent used to adjust the pH to 7.0±0.2 in step S3 of the present invention is phosphate buffer.

[0017] The present invention has the following beneficial effects:

[0018] The anticoagulant in this invention has the effect of inhibiting platelet activation.

[0019] 2. This anticoagulant can stably preserve whole blood samples from animals such as dogs and cattle for 48 hours, effectively preventing platelet aggregation and white blood cell morphology deformation. It accurately identifies white blood cells in the five differential counts (neutrophils / lymphocytes / monocytes / eosinophils / basophils) with a classification accuracy rate of up to 98%. It is suitable for veterinary clinical hematology testing and animal disease monitoring. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a comparison chart showing the real-time accuracy of the high-precision animal blood cell analyzer in Examples 1-3 and Comparative Examples 3-5 for the classification of white blood cells into five categories (neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

[0022] Figure 2 This is a comparison chart showing the 24-hour accuracy of high-precision animal blood cell analyzers in Examples 1-3 and Comparative Examples 3-5 for the classification of white blood cells into five categories (neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

[0023] Figure 3 This is a comparison chart of the 48-hour accuracy of high-precision animal blood cell analyzers in Examples 1-3 and Comparative Examples 3-5 for the classification of white blood cells into five categories (neutrophils, lymphocytes, monocytes, eosinophils, and basophils). Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0025] Low molecular weight enzymatically hydrolyzed carboxymethyl chitosan was purchased from Shanxi Aisen Biotechnology Co., Ltd.; EDTA-K2, sodium citrate, and citric acid were purchased from Shanghai Ebi Chemical Reagent Co., Ltd.; dipyridamole was purchased from Guangdong Huanan Pharmaceutical Group Co., Ltd.; aspirin was purchased from Shandong Kangdi Pharmaceutical Co., Ltd.; and hydroxypropyl-β-cyclodextrin was purchased from Xi'an Dongfeng Biotechnology Co., Ltd.

[0026] Example 1

[0027] This embodiment provides an anticoagulant for a high-precision animal blood cell analyzer, and the specific steps are as follows:

[0028] S1. Preparation of low-molecular-weight enzymatic hydrolysis of carboxymethyl chitosan: Carboxymethyl chitosan powder was dissolved in 2% (w / v) citric acid solution (pH 4.5) to prepare a 3% (w / v) carboxymethyl chitosan solution. The pH was then adjusted to 6.2, and papain was added at 55°C with a substrate concentration ratio of 1:45 (w / w). Enzymatic hydrolysis was carried out for 4 hours. The enzyme was inactivated by heating at 92°C for 12 minutes, then cooled to 40°C, and the pH was adjusted to 5.5. Chitosanase with an activity of 900 U / g (substrate concentration ratio 1:80, w / w) was added, and enzymatic hydrolysis continued at 50°C for 6 hours. Finally, the enzyme was inactivated by boiling in a water bath for 15 minutes, and the precipitate was removed by centrifugation at 8000 rpm for 20 minutes. The ultrafiltration permeate with a molecular weight less than 2 kDa was placed in a dialysis bag with a molecular weight cutoff of 500 Da and subjected to 0.1 M... The NH4HCO3 dialysate and distilled water were dialyzed (until the external fluid conductivity was ≤10μS / cm). The contents of the dialysis bag were freeze-dried and pulverized to finally obtain low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder.

[0029] S2. Preparation of buffer and anticoagulant matrix components: Dissolve 1.5g sodium citrate and 0.3g citric acid in 60mL sterile water, adjust the pH to 6.8 with NaOH, add 0.15g EDTA-K2 and stir at 50℃ until completely dissolved;

[0030] S3. Preparation of anticoagulant: Take 5 mL of anhydrous ethanol, add 10 mg of dipyridamole and 8 mg of aspirin, mix well to obtain a mixture. Add this mixture, 1.0 g of hydroxypropyl-β-cyclodextrin and 0.08 g of low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder to the buffer and anticoagulant matrix solution prepared in step S2 in sequence and slowly add to the volume with sterile water to 100 mL. After stirring evenly, adjust the pH of the solution to 6.8 with phosphate buffer. After filtration and sterilization using a 0.22 μm sterile filter membrane, the anticoagulant is obtained.

[0031] Example 2

[0032] This embodiment provides an anticoagulant for a high-precision animal blood cell analyzer, and the specific steps are as follows:

[0033] S1. Preparation of low-molecular-weight enzymatic hydrolysis of carboxymethyl chitosan: Carboxymethyl chitosan powder was dissolved in 2% (w / v) citric acid solution (pH 4.7) to prepare a 3% (w / v) carboxymethyl chitosan solution. The pH was then adjusted to 6.2, and papain was added at 55°C with a substrate concentration ratio of 1:45 (w / w). Enzymatic hydrolysis was carried out for 4-5 hours. The enzyme was inactivated by heating at 92°C for 12 minutes, then cooled to 40°C. The pH was adjusted to 5.5, and chitosanase with an activity of 900 U / g (substrate concentration ratio 1:80, w / w) was added. Enzymatic hydrolysis continued at 50°C for 6.5 hours. Finally, the enzyme was inactivated by boiling in a water bath for 15 minutes, and the precipitate was removed by centrifugation at 8500 rpm for 20 minutes. The ultrafiltration permeate with a molecular weight less than 2 kDa was placed in a dialysis bag with a molecular weight cutoff of 500 Da and subjected to 0.1 M... The NH4HCO3 dialysate and distilled water were dialyzed (until the external fluid conductivity was ≤10μS / cm). The contents of the dialysis bag were freeze-dried and pulverized to finally obtain low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder.

[0034] S2. Preparation of buffer and anticoagulant matrix components: Dissolve 1.7g sodium citrate and 0.4g citric acid in 60mL sterile water, adjust the pH to 6.8 with NaOH, add 0.17g EDTA-K2 and stir at 50℃ until completely dissolved;

[0035] S3. Preparation of anticoagulant: Take 6 mL of anhydrous ethanol, add 12 mg of dipyridamole and 9 mg of aspirin, mix well to obtain a mixture. Add this mixture, 1.1 g of hydroxypropyl-β-cyclodextrin and 0.09 g of low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder to the buffer and anticoagulant matrix solution prepared in step S2 in sequence and slowly add to the solution. Make up to 100 mL with sterile water, stir well, adjust the pH of the solution to 7.0 with phosphate buffer, and filter sterilize using a 0.22 μm sterile filter membrane to obtain the anticoagulant.

[0036] Example 3

[0037] This embodiment provides an anticoagulant for a high-precision animal blood cell analyzer. The specific steps are as follows:

[0038] S1. Preparation of low-molecular-weight enzymatic hydrolysis of carboxymethyl chitosan: Carboxymethyl chitosan powder was dissolved in 2% (w / v) citric acid solution (pH 5.0) to prepare a 3% (w / v) carboxymethyl chitosan solution. The pH was then adjusted to 6.2, and papain was added at 55°C with a substrate concentration ratio of 1:45 (w / w). Enzymatic hydrolysis was carried out for 5 hours. The enzyme was inactivated by heating at 92°C for 12 minutes, then cooled to 40°C, and the pH was adjusted to 5.5. Chitosanase with an activity of 900 U / g (substrate concentration ratio 1:80, w / w) was added, and enzymatic hydrolysis continued at 50°C for 7 hours. Finally, the enzyme was inactivated by boiling in a water bath for 15 minutes, and the precipitate was removed by centrifugation at 9000 rpm for 20 minutes. The ultrafiltration permeate with a molecular weight less than 2 kDa was placed in a dialysis bag with a molecular weight cutoff of 500 Da and subjected to 0.1 M... The NH4HCO3 dialysate and distilled water were dialyzed (until the external fluid conductivity was ≤10μS / cm). The contents of the dialysis bag were freeze-dried and pulverized to finally obtain low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder.

[0039] S2. Preparation of buffer and anticoagulant matrix components: Dissolve 2g of sodium citrate and 0.5g of citric acid in 60mL of sterile water, adjust the pH to 6.8 with NaOH, add 0.20g of EDTA-K2 and stir at 50℃ until completely dissolved;

[0040] S3. Preparation of anticoagulant: Take 7 mL of anhydrous ethanol, add 15 mg of dipyridamole and 10 mg of aspirin, mix well to obtain a mixture. Slowly add this mixture, 1.2 g of hydroxypropyl-β-cyclodextrin and 0.10 g of low molecular weight enzymatically hydrolyzed carboxymethyl chitosan powder to the buffer and anticoagulant matrix solution prepared in step S2, and make up to 100 mL with sterile water. After stirring evenly, adjust the pH of the solution to 7.2 with phosphate buffer, and filter sterilize using a 0.22 μm sterile filter membrane to obtain the anticoagulant.

[0041] Comparative Example 1: Aspirin was not added in step S3, and all other steps were the same as in Example 3.

[0042] Comparative Example 2: Dipyridamole was not added in step S3, and all other steps were the same as in Example 3.

[0043] Comparative Example 3: Hydroxypropyl-β-cyclodextrin was not added in step S3, and all other steps were the same as in Example 3.

[0044] Comparative Example 4: In step S3, low molecular weight enzymatic hydrolysis of carboxymethyl chitosan was not added, and all other steps were the same as in Example 3.

[0045] Comparative Example 5: In step S1, carboxymethyl chitosan is not enzymatically hydrolyzed, that is, in step S3, low molecular weight enzymatic hydrolysis of carboxymethyl chitosan is not added and the carboxymethyl chitosan is directly ultrafiltered before use. All other steps are the same as in Example 3.

[0046] Experiment 1 Study on the effects of anticoagulants on platelet activation in rats

[0047] Animals: SD rats, SPF grade, weighing 250±50g, half male and half female, purchased from Speford (Beijing) Biotechnology Co., Ltd., 10 rats per cage were acclimatized in a constant temperature and oxygen supply environment of 25±2℃ for one week before the start of the experiment.

[0048] Grouping: 60 rats were randomly divided into Example 1-3 groups, Comparative Example 1-2 groups and standard anticoagulant control group (using vacuum blood collection tubes with EDTA anticoagulant, with a routine blood collection volume of 5 mL purchased from Shandong Huabo Medical Instrument Co., Ltd.), with 10 rats in each group.

[0049] Flow cytometry assay: After grouping, 50 μl samples were taken from the anticoagulated blood of all rats to detect the natural platelet activation level. An equal volume of the sample was added to ADP solution at a final concentration of 10 μmol / L to induce activation. Subsequently, 1.0 μL of CD62pPE / Cy7 (purchased from Shanghai Yuduo Biotechnology Co., Ltd.) and CD61PE antibody (purchased from Shanghai Kemin Biotechnology Co., Ltd.) were added for double labeling (CD61 ​​labeled all platelets, CD62p labeled activated platelets). After incubation at 4℃ in the dark for 25 min, the samples were fixed for 10 min with 0.5 ml of PBS buffer containing 4% paraformaldehyde. 50 μl of the fixed sample was mixed with 1 ml of diluent, and the platelet activation level after ADP stimulation was analyzed by flow cytometry.

[0050] Data analysis: Platelet populations were defined using a two-parameter scatter plot based on CD61PE and side-scattered light (SSC). The number of CD61 and CD62p positive cells in 5000 platelets was counted. The activation rate was calculated as the percentage of CD62p positive cells to CD61 positive cells. The experimental results are shown in Table 1.

[0051] Table 1. Comparison of CD62p expression rates in platelets of mice in different groups

[0052]

[0053] Table 1 is a comparison table of the expression rate of CD62p in platelets (spontaneous expression rate and expression rate after ADP activation) of SD rats in Examples 1-3 and Comparative Examples 1-2. This indicator can directly reflect the platelet activation level.

[0054] The spontaneous expression rate of CD62p in Examples 1-3 remained at a low level and was significantly lower than that in Comparative Examples 1-2 and the standard anticoagulant control group. This indicates that under natural conditions without external induction, the anticoagulants in Examples 1-3 can more effectively inhibit the spontaneous activation of rat platelets and reduce non-specific platelet activation.

[0055] The expression rate of CD62p after ADP activation was observed in the 1-3 groups of Examples, which remained at a stable low level, significantly lower than that of the 1-2 groups of Comparative Examples and the standard anticoagulant control group. This indicates that when dealing with ADP, a common activator, the 1-3 groups of Examples can effectively inhibit the platelet activation process and further reduce the proportion of activated platelets.

[0056] Based on the combined results of the two indicators, it can be seen that the anticoagulant in the groups of Examples 1-3 is more effective than that in the comparative groups 1-2, whether it is inhibiting spontaneous platelet activation or resisting externally induced platelet activation, thus demonstrating that it has the best regulatory effect on platelet activation in rats.

[0057] Experiment 2 Studies on the accurate effects of anticoagulants on the five differential counts of white blood cells (neutrophils / lymphocytes / monocytes / eosinophils / basophils) Analysis of rates.

[0058] Animals: Beagles were purchased from Changzhou Beile Experimental Animal Breeding Co., Ltd., weighing 8-9kg and 35-39cm in height. They were put into the experiment after 5 days of acclimatization.

[0059] Grouping: 70 beagle dogs were randomly divided into Example 1-3 groups, Comparative Examples 3-5 groups, and a standard anticoagulant control group (using vacuum blood collection tubes with EDTA anticoagulant, with a routine blood collection volume of 5 mL purchased from Shandong Huabo Medical Equipment Co., Ltd.), with 10 dogs in each group.

[0060] After grouping, venous blood was collected from the pit bulls, and each venous blood sample was divided into 10 parallel subsamples. Each subsample was then divided into two subsamples, and white blood cell classification was performed using optical microscopy and a high-precision animal hematology analyzer, respectively. This design allows for 10 replicate tests on the same original venous blood sample from the same pit bull, ensuring data accuracy. The operation procedure of the high-precision animal hematology analyzer is as follows: all samples are placed in vacuum blood collection tubes containing the anticoagulant of this invention, and after gentle and repeated inversion to mix, the white blood cells (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) are analyzed strictly according to the instrument's operating procedures. The microscopic examination procedure was performed according to clinical laboratory guidelines. Laboratory personnel conducted blinded analysis of the microscopic samples. Two blood smears of appropriate thickness were prepared for each sample. After staining with Wright's stain, the neutrophils, lymphocytes, monocytes, eosinophils, and basophils in the white blood cells were counted under an oil immersion microscope (200 white blood cells were counted at each stage). Using the microscopic examination results as the gold standard, the immediate, 24-hour, and 48-hour classification accuracy of the five-part differential high-precision animal hematology analyzer for each type of white blood cell was calculated. The experimental results are shown in Table 2. Figures 1-3 As shown.

[0061] Classification accuracy = (Number of white blood cells of this type correctly detected by the instrument / Number of white blood cells of this type detected by the microscope) × 100%

[0062] Table 2 Comparison of accuracy rates of five-part differential white blood cell count (neutrophils / lymphocytes / monocytes / eosinophils / basophils) in different groups of mice

[0063]

[0064] Table 2 shows the classification accuracy of the high-precision animal blood cell analyzer for the five differential white blood cell categories (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) in Examples 1-3 and Comparative Examples 3-5, using microscopic examination results as the gold standard. The results show that compared to the classification accuracy of each white blood cell category in Comparative Examples 3-5, Examples 1-3 demonstrated superior accuracy in classifying neutrophils, lymphocytes, monocytes, eosinophils, and basophils, achieving an accuracy of 98% or higher for all categories after 48 hours. This fully demonstrates that the anticoagulant in Examples 1-3 effectively stabilized animal whole blood samples for up to 48 hours, ensuring a white blood cell classification accuracy of up to 98%.

[0065] The above-described embodiments are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. An anticoagulant for a high-precision animal blood corpuscle analyzer, characterized by comprising: The anticoagulant includes buffer and anticoagulant matrix components and active ingredients; wherein, in 100 mL of the anticoagulant, the buffer and anticoagulant matrix components include 1.5-2 g of citrate, 0.3-0.5 g of citric acid and 0.15-0.20 g of EDTA-K2, and the active ingredients include 0.08-0.10 g of low-molecular-weight enzymatic carboxymethyl chitosan, 1.0-1.2 g of hydroxypropyl-β-cyclodextrin, 10-15 mg of dipyridamole and 8-10 mg of aspirin; ​ The carboxymethyl chitosan is subjected to enzymatic hydrolysis by papain and chitosanase; and the citrate is sodium citrate.

2. The method for preparing an anticoagulant for a high-precision animal blood cell analyzer according to claim 1, characterized in that: The specific preparation method is as follows: S1, preparation of low-molecular-weight enzymatic carboxymethyl chitosan: carboxymethyl chitosan powder is dissolved in a citric acid solution to prepare a 3% w / v carboxymethyl chitosan solution; then, the pH is adjusted to 6.2, and papain is added at 55°C for enzymatic hydrolysis for 4-5 h; after inactivation of the enzyme by heating at 92°C for 12 min, the temperature is cooled to 40°C, the pH is adjusted to 5.5, chitosanase is added, and enzymatic hydrolysis is continued at 50°C for 6-7 h; finally, the enzyme is inactivated by boiling water bath for 15 min, the precipitate is removed by centrifugation at 8000-9000 rpm for 20 min, the supernatant is collected, and the supernatant is subjected to ultrafiltration with a molecular weight cutoff of 2 kDa; the ultrafiltration permeate with a molecular weight of less than 2 kDa is taken and loaded into a dialysis bag with a molecular weight cutoff of 500 Da, and dialysis treatment is performed with 0.1M NH4HCO3 dialysis solution and distilled water at 4°C in sequence; the contents in the dialysis bag are freeze-dried and pulverized to obtain low-molecular-weight enzymatic carboxymethyl chitosan powder; S2, preparation of buffer and anticoagulant matrix components: 1.5-2 g of sodium citrate and 0.3-0.5 g of citric acid are dissolved in 60 mL of sterile water, the pH is adjusted to 6.8, 0.15-0.20 g of EDTA-K2 is added, and stirring is performed at 50°C until complete dissolution; S3, preparation of the anticoagulant: 5-7 mL of anhydrous ethanol is taken, 10-15 mg of dipyridamole and 8-10 mg of aspirin are added, and the mixture is uniformly mixed to obtain a mixed solution; the mixed solution, 1.0-1.2 g of hydroxypropyl-β-cyclodextrin and 0.08-0.10 g of low-molecular-weight enzymatic carboxymethyl chitosan powder are slowly added to the buffer and anticoagulant matrix solution prepared in step S2, and the volume is adjusted to 100 mL with sterile water; after uniform stirring, the pH of the solution is adjusted to 7.0±0.2, and the solution is filtered and sterilized with a 0.22 μm sterile filter membrane to obtain the anticoagulant.

3. The method for preparing an anticoagulant for a high-precision animal blood cell analyzer according to claim 2, characterized in that: The mass fraction of the citric acid solution in step S1 is 2%.

4. The method for preparing an anticoagulant for a high-precision animal blood cell analyzer according to claim 2, characterized in that: The pH of the citric acid solution in step S1 is 4.5-5.

0.

5. The method for preparing an anticoagulant for a high-precision animal blood cell analyzer according to claim 2, characterized in that: The substrate concentration ratio of papain in step S1 is 1:45 w / w, and the activity is 3500 U / g.

6. The method for preparing an anticoagulant for a high-precision animal blood cell analyzer according to claim 2, characterized in that: The substrate concentration ratio of chitosanase in step S1 is 1:80 w / w, and the activity is 900 U / g.

7. Application of the anticoagulant of claim 1 to white blood cell classification in a high-precision animal blood cell analyzer.

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