Blood chyle degree detection reference product and preparation method thereof

Abstract

The invention discloses a blood chyle degree detection reference and a preparation method thereof. The reference comprises the following components: triglyceride, cholesteryl ester, free cholesterol, phosphatidylcholine, bovine serum albumin, a buffer solution and an auxiliary agent. The blood chyle degree detection reference product provided by the invention can be stably stored, is low in cost, can be used for instrument calibration, can be used for judging and grading clinical plasma chyle degree and the like, and can unify detection standards and eliminate artificial interference; based on the blood chyle degree detection reference product provided by the invention, detection can be realized by utilizing image gray calculation, and compared with manual visual inspection, the detection accuracy is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of blood detection, and particularly relates to a reference product for detecting the chylomicron degree of blood and a preparation method thereof. Background Art

[0002] Chylous blood has become one of the main factors affecting the rejection of blood in blood banks, causing a waste rate of 3% - 10%; the causes of chylous blood include bad living habits and lipid metabolism diseases, etc.; its characteristic is that it contains lipoprotein particles > 200nm; clinical transfusion of chylous blood will cause transfusion adverse reactions such as allergy, fever, fat embolism, etc., even bronchial asthma and asphyxia death, and will also increase the probability of arteriosclerosis, coronary heart disease, hypertension, gallstones and diabetes in people with high blood pressure, high blood sugar and high blood fat.

[0003] Currently, most clinical blood is donated by unpaid blood donors. As the proportion of chylous blood in the blood of unpaid blood donors gradually increases, it is crucial to quickly and accurately judge different degrees of chylous blood before blood donation and collect it appropriately. In actual blood collection, the chylomicron degree of blood is usually determined by naked-eye observation, and the judgment is often based on experience. Each person has a different scale, with a large degree of subjectivity. Often due to the error in the judgment of the chylomicron degree of plasma by the judge, chylous blood is collected, resulting in blood rejection. At the same time, chylomicron particles (CM) in conventional chylous blood will interfere with optical-based biochemical tests, causing result deviations. Therefore, correctly judging the chylomicron degree of plasma can effectively save blood resources and ensure safe blood transfusion.

[0004] The currently adopted traditional chylous blood detection methods mainly include:

[0005] 1. Physiological saline dilution method

[0006] In Appendix A of the health industry standard "WST 550-2017 Guidelines for Quality Monitoring of Whole Blood and Blood Components", a method for detecting the chylomicron degree of blood based on manual visual judgment is proposed. The chylous blood is diluted with physiological saline, and the turbidity is visually compared with normal plasma. (Clinically, the current method for judging the chylomicron degree of lipemic blood by dilution method is: the chylous blood is diluted in a multiple ratio, that is, if it is diluted 2 times and reaches the clarity of normal blood, it is mild lipemic blood; if it is diluted 3 - 5 times, it is moderate lipemic blood; if it needs to be diluted about 5 times, it is severe lipemic blood.

[0007] 2. Turbidimetry card

[0008] In Appendix C of the group standard of the Chinese Blood Transfusion Association "Guidelines for Appearance Inspection of Whole Blood and Blood Components", a turbidimetry card (black circle diagram) is prepared, and the chylomicron degree of plasma is manually visually judged by observing the plasma perspective effect.

[0009] 3. Spectrophotometry

[0010] Judging by detecting the OD value of the solution, there is a defect that the detection result is inaccurate.

[0011] Currently, there is a lack of chylous blood detection reference products or quality control products on the market. Traditional detection methods are based on diluting severe chylous blood with physiological saline and judging by visual inspection, and the standards cannot be unified. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a reference product for detecting the chylous degree of blood and its preparation method in view of the above deficiencies in the prior art.

[0013] To solve the above technical problem, the technical solution adopted by the present invention is: a reference product for detecting the chylous degree of blood, which is characterized by comprising the following components: triglyceride, cholesterol ester, free cholesterol, phosphatidylcholine, bovine serum albumin, buffer solution and auxiliary agent.

[0014] Preferably, the auxiliary agent is at least one of a surfactant or a dispersant.

[0015] Preferably, the auxiliary agent is at least one of sucrose, polyethylene glycol, and polyvinylpyrrolidone.

[0016] Preferably, the buffer solution is at least one of PBS buffer solution, physiological saline, tris buffer solution, and borate buffer solution.

[0017] Preferably, the auxiliary agent is PEG20000 and the buffer solution is tris buffer solution.

[0018] Preferably, the reference product for detecting the chylous degree of blood comprises the following components: triglyceride 4 - 17 g / L, cholesterol ester 0.5 - 3 g / L, free cholesterol 0.05 - 0.2 g / L, phosphatidylcholine 4.5 - 18.2 g / L, bovine serum albumin 12.5 - 50 g / L, PEG20000 6 - 24 g / L, and tris buffer solution 10 - 40 mmol / L.

[0019] Among them, triglyceride is the main body of neutral lipid core and simulates the lipid core of chylomicrons; cholesterol ester is the nuclear structure stabilizer and inhibits lipid crystallization; free cholesterol regulates the fluidity of the interfacial membrane and synergistically reduces the membrane phase transition temperature with phospholipids; phosphatidylcholine is the membrane construction unit and is used to form the hydrophilic-hydrophobic interface; bovine serum albumin (BSA) is the stabilizer and simulates the colloidal protection of apolipoprotein B-48; PG20000 is the intermediate stabilizer and prevents particle aggregation through steric hindrance.

[0020] Preferably, the blood chylomicron level detection reference product comprises the following components: triglyceride 8.5 g / L, cholesteryl ester 1.5 g / L, free cholesterol 0.1 g / L, phosphatidylcholine 9.1 g / L, bovine serum albumin 25 g / L, PEG20000 12 g / L, and tris buffer 20 mmol / L.

[0021] The present invention also provides a method for preparing the above-mentioned blood chylomicron level detection reference product, comprising the following steps:

[0022] S1. Prepare the lipid phase:

[0023] Dissolve triglyceride, cholesteryl ester, free cholesterol, and phosphatidylcholine in a mixed solvent, rotate and evaporate to form a uniform lipid film, and vacuum dry to remove the residual mixed solvent to obtain the lipid phase; wherein, the mixed solvent is obtained by mixing chloroform and methanol.

[0024] S2. Prepare the aqueous phase: Add bovine serum albumin and an auxiliary agent to a buffer solution in proportion, and stir until completely dissolved to obtain the aqueous phase.

[0025] S3. Emulsification: Add the preheated aqueous phase to the lipid film, and perform ultrasonic emulsification to obtain the blood chylomicron level detection reference product.

[0026] During use, aspirate the chylomicrons suspended above the liquid in the blood chylomicron level detection reference product for detection.

[0027] Preferably, the method for preparing the blood chylomicron level detection reference product comprises the following steps:

[0028] S1. Prepare the lipid phase:

[0029] Dissolve triglyceride, cholesteryl ester, free cholesterol, and phosphatidylcholine in a mixed solvent, rotate and evaporate at 30 - 50 °C and 75 - 300 rpm to form a uniform lipid film, and vacuum dry for 6 - 24 h to remove the residual mixed solvent to obtain the lipid phase;

[0030] wherein, the mixed solvent is obtained by mixing chloroform and methanol at a volume ratio of 3:1.

[0031] S2. Prepare the aqueous phase: Add bovine serum albumin and an auxiliary agent to a 10 - 40 mM buffer solution in proportion, and stir until completely dissolved to obtain the aqueous phase.

[0032] S3. Emulsification: Add 25 - 100 mL of the aqueous phase preheated to 40 - 60 °C to the lipid film, perform ultrasonic emulsification for 5 - 30 min, and repeat 2 - 5 times to obtain the blood chylomicron level detection reference product.

[0033] During use, aspirate the chylomicrons suspended above the liquid in the blood chylomicron level detection reference product for detection.

[0034] Preferably, the method for preparing the reference product for detecting the degree of blood chylomicron includes the following steps:

[0035] S1. Prepare the lipid phase:

[0036] Dissolve triglyceride, cholesteryl ester, free cholesterol, and phosphatidylcholine in a mixed solvent, and rotary evaporate at 40 °C and 150 rpm to form a uniform lipid film. Vacuum dry for 12 h to remove the residual mixed solvent to obtain the lipid phase. Among them, the mixed solvent is obtained by mixing chloroform and methanol in a volume ratio of 3:1;

[0037] S2. Prepare the aqueous phase: In a 20 mM buffer solution, add bovine serum albumin and an auxiliary agent in proportion and stir until completely dissolved to obtain the aqueous phase;

[0038] S3. Emulsification: Add 50 mL of the aqueous phase preheated to 50 °C to the lipid film, and perform ultrasonic emulsification for 15 min, repeating 3 times to obtain the reference product for detecting the degree of blood chylomicron. Among them, the ultrasonic emulsification parameters are: 20 kHz, 60% amplitude, pulse mode: 5 s on / 5 s off;

[0039] During use, aspirate the chylomicrons suspended above the liquid in the reference product for detecting the degree of blood chylomicron for detection.

[0040] The beneficial effects of the present invention are:

[0041] The present invention provides a reference product for detecting the degree of blood chylomicron and a preparation method thereof. The reference product for detecting the degree of blood chylomicron provided by the present invention can be stably stored, has low cost, can be used for instrument calibration and for judging and grading the chylomicron degree of clinical plasma, etc., and can unify the detection standard and eliminate manual interference; based on the reference product for detecting the degree of blood chylomicron provided by the present invention, detection can be realized by using image gray-scale calculation, which greatly improves the detection accuracy compared with manual naked-eye observation. Description of the Drawings

[0042] Figure 1 It is the detection result of the particle size and distribution of chylomicron sample 1;

[0043] Figure 2 It is the detection result of the particle size and distribution of chylomicron sample 2;

[0044] Figure 3 It is the microscopic staining detection result of chylomicrons under the microscope;

[0045] Figure 4 It is the result of detecting the chylomicrons prepared in Example 1 after gradient concentration dilution by using a detector with a chylomicron reference product;

[0046] Figure 5The correlation curve between the chylomicron degree and the gray value of the chylomicrons prepared in Example 1. Detailed implementation manners

[0047] The following examples are used to further elaborate the present invention in detail, so that those skilled in the art can implement it according to the description in the specification.

[0048] It should be understood that the terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0049] Unless otherwise specified, the test methods used in the following examples are all conventional methods. Unless otherwise specified, the materials and reagents used in the following examples can all be obtained through commercial channels. For those not specifying specific conditions in the following examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] A reference product for detecting the chylomicron degree of blood, comprising the following components: triglyceride 8.5 g / L, cholesterol ester 1.5 g / L, free cholesterol 0.1 g / L, phosphatidylcholine 9.1 g / L, bovine serum albumin 25 g / L, PEG20000 12 g / L, and tris buffer 20 mmol / L.

[0052] The preparation method of the reference product for detecting the chylomicron degree of blood includes the following steps:

[0053] S1. Prepare the lipid phase:

[0054] Dissolve triglyceride, cholesterol ester, free cholesterol, and phosphatidylcholine in a mixed solvent, and rotary evaporate at 40 °C and 150 rpm to form a uniform lipid film. Vacuum dry for 12 h to remove the residual mixed solvent to obtain the lipid phase; wherein, the mixed solvent is obtained by mixing chloroform and methanol in a volume ratio of 3:1.

[0055] S2. Prepare the aqueous phase: In a 20 mM buffer solution, add bovine serum albumin and an auxiliary agent in proportion and stir until completely dissolved to obtain the aqueous phase.

[0056] S3. Emulsification: Add 50 mL of the aqueous phase preheated to 50 °C to the lipid film, and perform ultrasonic emulsification for 15 min, repeating 3 times to obtain a suspension of the reference product for detecting the chylomicron degree of blood, wherein the ultrasonic emulsification parameters are: 20 kHz, 60% amplitude, pulse mode: 5 s on / 5 s off.

[0057] Carefully aspirate the chylomicrons suspended above the liquid in the blood chyle degree detection reference product, detect the particle size distribution of the chylomicrons by a Malvern laser particle size analyzer, and use microscopic staining for under-microscope detection.

[0058] 1. Detection of the particle size and distribution of the prepared chyle reference product by a Malvern laser particle size analyzer

[0059] Method:

[0060] After placing the chylomicron sample and the buffer at room temperature for 30 minutes of equilibration, dilute the chyle reference product suspension 50-fold with 20 mM Tris-HCl, pH 7.4, and ensure that the light intensity is within the detection range of the instrument to avoid multiple scattering caused by excessive concentration. After gentle vortex mixing, let it stand for 5 minutes and then perform the detection on the instrument.

[0061] Set the instrument detection temperature to 25 °C, preheat and calibrate the instrument. Add 1 mL of the diluted sample to a quartz cuvette, avoiding fingerprint contamination. Select the dynamic light scattering (DLS) mode to detect the particle size distribution: Measurement time: automatic mode (usually 10 - 30 seconds / time)

[0062] Data fitting algorithm: Cumulants analysis (Z-Average) and non-negative least squares (NNLS) (particle size distribution), and save the original data.

[0063] The results are as Figure 1 、 Figure 2 and shown in Table 1 below:

[0064] Table 1

[0065]

[0066] It can be seen from the detection results that the particle size of the chyle reference product prepared by the present invention is within the range of the particle size of natural chylomicrons (diameter 75 - 1200 nm). This detection method can not only evaluate the physical properties of chylomicrons, prove that the emulsification process is feasible, but also optimize the preparation process through particle size analysis (such as adjusting the phospholipid / triglyceride ratio to control the particle size).

[0067] 2. Microscopic examination of the morphology of the chylomicron reference product

[0068] Method:

[0069] Sample preparation: Take 10 μL of the suspension of the chylomicron reference product prepared in Example 1, evenly coat it on a glass slide, and let it dry naturally at room temperature. Drop 10 wt% formaldehyde solution to fix for 5 minutes to avoid lipid loss. Drop Sudan III staining solution (0.5% Sudan III dissolved in 70% ethanol), cover the sample, and let it stand for 15 minutes. Decolorize with 70% ethanol for 10 seconds to remove the excess dye. Seal the slide with glycerol gelatin, avoiding air bubbles. Perform microscopic examination and count the distribution density and morphology of the stained particles.

[0070] The results are as Figure 3 shown. It can be seen that chylomicrons are stained into orange-red particles and evenly dispersed in the field of view. Under microscopic examination, the morphology of chylomicrons is round, with uniform size, and the particle size is about 150 nm. A very small number of particles have a particle size greater than 300 nm, which is consistent with the detection results of the laser particle size analyzer. The intuitive morphology verifies the lipid composition and physical stability of chylomicrons. The particle morphology shown by this detection reflects the stability of the emulsification process, which is consistent with the staining characteristics of natural chylomicrons, supporting the effectiveness of its use as a reference product.

[0071] 3. Use a chylomicron reference product to detect the chylomicrons prepared in Example 1 with a detector (a colorimeter for measuring the concentration of a monochromatic solution, Patent ZL202022784100.1)

[0072] Method:

[0073] Dilute the chylomicrons prepared in Example 1 with gradient concentrations, add them to a cuvette, and place them in the instrument for detection to establish the matching between the chylomicron degree and the image gray-scale algorithm. [[ID=!14]]

[0074] The detection results are as Figure 4 shown. For the prepared chylomicrons, when taking pictures in the instrument, the darker the color, the greater the chylomicron degree; the numbers corresponding to the lower part of the pictures represent the chylomicron degree, and the larger the number, the greater the chylomicron degree.

[0075] The gray-scale analysis results of each picture are shown in Table 2 below:

[0076] Table 2

[0077] Sample number Pattern gray value 1 944862 2 863216 3 775160 4 703446 5 660037 6 585925 7 571605

[0078] Taking the chylomicron degree as the abscissa and the gray-scale value as the ordinate for curve fitting, a correlation curve between the chylomicron degree and the gray-scale value is obtained. As Figure 5 shown, the correlation coefficient R 2 = 0.9959, indicating that the chylomicron degree of the prepared chylomicrons shows a gradient distribution with the gray-scale value of the pictures, can simulate the serum situation of patients with natural chylomicronemia, and can be used for the judgment of the chylomicron degree.

[0079] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalent scope, the present invention is not limited to specific details.

Claims

1. A reference product for detecting the degree of blood chylomicron, characterized in that, It includes the following components: triglyceride, cholesterol ester, free cholesterol, phosphatidylcholine, bovine serum albumin, buffer solution and auxiliary agent.

2. The reference product for detecting the degree of blood chylomicron according to claim 1, characterized in that, The auxiliary agent is at least one of a surfactant or a dispersant.

3. The reference product for detecting the degree of blood chylomicron according to claim 2, characterized in that, The auxiliary agent is at least one of sucrose, polyethylene glycol, and polyvinylpyrrolidone.

4. The reference product for detecting the degree of blood chyle according to claim 3, wherein The buffer solution is at least one of PBS buffer solution, physiological saline, tris buffer solution, and borate buffer solution.

5. The reference product for detecting the degree of blood chylomicron according to claim 4, wherein The auxiliary agent is PEG20000 and the buffer solution is tris buffer solution.

6. The reference product for detecting the degree of blood chyluria according to claim 1, wherein It includes the following components: 4 - 17 g / L of triglyceride, 0.5 - 3 g / L of cholesterol ester, 0.05 - 0.2 g / L of free cholesterol, 4.5 - 18.2 g / L of phosphatidylcholine, 12.5 - 50 g / L of bovine serum albumin, 6 - 24 g / L of PEG20000, and 10 - 40 mmol / L of tris buffer solution.

7. The reference product for detecting the degree of blood chyle according to claim 6, characterized in that, It includes the following components: 8.5 g / L of triglyceride, 1.5 g / L of cholesterol ester, 0.1 g / L of free cholesterol, 9.1 g / L of phosphatidylcholine, 25 g / L of bovine serum albumin, 12 g / L of PEG20000, and 20 mmol / L of tris buffer solution.

8. A method for preparing a reference product for detecting the degree of blood chylomicron as described in any one of claims 1-7, characterized in that, It includes the following steps: S1. Prepare the lipid phase: Dissolve triglyceride, cholesterol ester, free cholesterol, and phosphatidylcholine in a mixed solvent, rotate and evaporate to form a uniform lipid film, and vacuum dry to remove the residual mixed solvent to obtain the lipid phase; wherein, the mixed solvent is obtained by mixing chloroform and methanol. S2. Prepare the aqueous phase: In the buffer solution, add bovine serum albumin and the auxiliary agent in proportion and stir until completely dissolved to obtain the aqueous phase. S3. Emulsify: Add the preheated aqueous phase to the lipid film and perform ultrasonic emulsification to obtain the reference product for detecting the degree of blood chylomicron. During use, aspirate the chylomicrons suspended above the liquid in the reference product for detecting the degree of blood chylomicron for detection.

9. The method for preparing a reference product for detecting the degree of blood chylomicron according to claim 8, characterized in that, It includes the following steps: S1. Prepare the lipid phase: Dissolve triglyceride, cholesterol ester, free cholesterol, and phosphatidylcholine in a mixed solvent, rotate and evaporate at 30 - 50 °C and 75 - 300 rpm to form a uniform lipid film, and vacuum dry for 6 - 24 h to remove the residual mixed solvent to obtain the lipid phase. Among them, the mixed solvent is obtained by mixing chloroform and methanol at a volume ratio of 3:

1. S2. Prepare the aqueous phase: In 10 - 40 mM buffer solution, add bovine serum albumin and the auxiliary agent in proportion and stir until completely dissolved to obtain the aqueous phase. S3. Emulsify: Add 25 - 100 mL of the aqueous phase preheated to 40 - 60 °C to the lipid film, perform ultrasonic emulsification for 5 - 30 min, and repeat 2 - 5 times to obtain the reference product for detecting the degree of blood chylomicron. During use, aspirate the chylomicrons suspended above the liquid in the reference product for detecting the degree of blood chylomicron for detection.

10. The preparation method of the blood chylomicron degree detection reference product according to claim 9, wherein, It includes the following steps: S1. Prepare the lipid phase: Dissolve triglyceride, cholesterol ester, free cholesterol, and phosphatidylcholine in a mixed solvent, rotate and evaporate at 40 °C and 150 rpm to form a uniform lipid film, and vacuum dry for 12 h to remove the residual mixed solvent to obtain the lipid phase; wherein, the mixed solvent is obtained by mixing chloroform and methanol at a volume ratio of 3:

1. S2. Prepare the aqueous phase: In 20 mM buffer solution, add bovine serum albumin and the auxiliary agent in proportion and stir until completely dissolved to obtain the aqueous phase. S3 Emulsification: Add 50 mL of the aqueous phase preheated to 50 °C to the lipid membrane, and perform ultrasonic emulsification for 15 min, repeating 3 times to obtain a reference product for detecting the degree of blood chylomicron. Among them, the ultrasonic emulsification parameters are: 20 kHz, 60% amplitude, pulse mode: 5 s on / 5 s off; During use, aspirate the chylomicrons suspended above the liquid in the reference product for detecting the degree of blood chylomicron for detection.

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