Use of kynurenine in the preparation of a diagnostic kit for diabetic cardiovascular complications

Abstract

The application relates to the field of biological diagnosis, and discloses the use of kynurenine in the preparation of a diabetes cardiovascular complication diagnosis kit. The diabetes is type 2 diabetes; and the cardiovascular complication includes stable coronary heart disease and acute myocardial infarction. The application discloses for the first time that kynurenine can be used as a diagnosis marker for type 2 diabetes combined with cardiovascular complications such as stable coronary heart disease and acute myocardial infarction, fills the blank of early specific diagnosis indexes for diabetes cardiovascular complications, provides a brand-new technical scheme for a diabetes and complication prevention system, and has important clinical transformation significance and a wide market application prospect.

Description

Technical Field

[0001] This application relates to the field of biological diagnostics, and more specifically, to the use of kynurenine in the preparation of diagnostic kits for cardiovascular complications of diabetes. Background Technology

[0002] In recent years, the prevalence of diabetes has continued to rise, and it is estimated that the total number of people with diabetes worldwide will exceed 1.3 billion by 2050, placing a heavy burden on healthcare systems and socioeconomic systems around the world. Cardiovascular disease (CVD), such as stable coronary artery disease (CAD) and acute myocardial infarction (MI), is the most common and serious complication of diabetes and the leading cause of death among diabetic patients.

[0003] Kynurenine (Kyn) is a major metabolite of the essential amino acid tryptophan (Trp). Under pathological conditions such as chronic inflammation, endothelial dysfunction, or local ischemia, pro-inflammatory cytokines (especially interferon-γ) significantly upregulate the expression of indoleamine 2,3-dioxygenase (IDO) in immune cells and endothelial cells, driving over 95% of Trp degradation along the Kyn pathway. Extensive basic and epidemiological literature confirms that abnormally elevated Kyn levels in peripheral blood and an increased Kyn / Trp ratio are reliable surrogate markers of systemic immune activation and high oxidative stress.

[0004] In cardiovascular pathophysiology, overactivation of the Kyn pathway is closely associated with macrophage infiltration, smooth muscle cell proliferation, and the risk of plaque rupture. Multiple prospective cohort studies have shown that in patients with coronary artery disease, acute coronary syndrome, and heart failure, high levels of circulating Kyn and its metabolites can significantly and independently predict the risk of future major adverse cardiovascular events (MACE) and all-cause mortality, independent of traditional cardiovascular risk factors (such as lipid abnormalities and hypertension). Furthermore, and more importantly, our previous studies confirmed that Kyn levels are significantly elevated in platelets in patients with type 2 diabetes mellitus (T2DM), significantly promoting platelet activity and thrombus formation. Therefore, Kyn is not only a key molecule in elucidating the inflammatory pathogenesis of cardiovascular disease but also a highly promising independent early warning biomarker. Introducing Kyn as a core target into cardiovascular disease risk assessment systems and developing related efficient detection methods and predictive models has significant clinical translational value and broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a diagnostic product for early screening of cardiovascular diseases in diabetic patients, thereby reducing the mortality rate of CVD in diabetic patients.

[0006] To achieve the above-mentioned objectives, this application provides the following technical solutions:

[0007] In one aspect, this application provides the use of kynurenine in the preparation of diagnostic kits for cardiovascular complications of diabetes.

[0008] Furthermore, the diabetes is type 2 diabetes.

[0009] Furthermore, the cardiovascular complications include stable coronary artery disease and acute myocardial infarction.

[0010] Secondly, this application provides a diagnostic kit for cardiovascular complications of diabetes, including a reagent for detecting kynurenine concentration.

[0011] Furthermore, the test sample for the kit is peripheral blood, plasma, or platelets.

[0012] In summary, this application has the following beneficial effects: This application reveals for the first time that kynurenine can serve as a diagnostic biomarker for cardiovascular complications in type 2 diabetes mellitus, including stable coronary artery disease and acute myocardial infarction, filling a gap in early specific diagnostic indicators for diabetic cardiovascular complications. The provided diagnostic kit, by detecting kynurenine concentrations in peripheral blood, plasma, or platelets, enables rapid and accurate assessment of the risk of cardiovascular complications in diabetic patients. The kit is easy to use and samples are readily available, facilitating large-scale clinical application. Compared to traditional cardiovascular risk assessment methods, this kit can detect potential inflammatory pathological signals earlier, significantly improving diagnostic sensitivity and specificity. It helps clinicians identify high-risk individuals and implement interventions in a timely manner, effectively reducing the mortality rate of diabetic cardiovascular complications. This provides a novel technical solution for the prevention and treatment of diabetes and its complications, possessing significant clinical translational value and broad market application prospects. Attached Figure Description

[0013] Figure 1(AB) Untargeted metabolomics analysis was performed on platelets from WT and db / db mice (n=5). Principal component analysis (PCA) model (A) and orthogonal partial least squares discriminant analysis (OPLS-DA) model (B) showed significant differences in metabolite composition between the two groups. (C) Alignment test results of the OPLS-DA models for the two groups of mice. The R²Y and Q² values ​​indicate satisfactory predictability and interpretability of the models, respectively. (D) Hierarchical analysis clustering heatmap of differentially expressed metabolites between the two groups. Alignment IDs were used to label some metabolite names in the figure due to their excessive length. POS6672 represents O1-(2-Hydroxy-[6]chinolyl)-beta-D-glucopyranuronsaeure|O1-(2-hydroxy-[6]quinolyl)-beta-D-glucopyranuronic acid; POS6834 represents 1-methyl-N-(2-(nicotinamido)ethyl)-1H-indole-3-carboxamide; POS9544 represents 2-((R)-4-((3R,5S,7S,8R,9S,10 S,12R,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido) ethane-1-sulfonic acid; NEG4210 represents O1-(4-Hydroxy-[3]chinolyl)-beta-D-glucopyranuronsaeure|O1-(4-hydroxy-[3]quinolyl)-beta-D-glucopyranuronic acid; NEG5473 represents (Z)-7-[(1R,2R,3R,5S)-2-(1,2-Dihydroxy-3-oxooctyl)-3,5-dihydroxycyclopentyl]hept-5-enoic acid; POS3504 represents (1xi,3xi)-1,2,3,4-Tetrahydro-1-methyl-beta-carboline-3-carboxylic acid; NEG3287 represents O1-(3,5-dimethyl-phenyl)-beta-D-glucopyranuronic acid|O1-(3,5-Dimethyl-phenyl)-beta-D-glucopyranuronsaeure.(E) Log2 (FC) values ​​and p-values ​​of the top 10 metabolites with the greatest difference. (F) β-d-glucuronic acid had no effect on thrombin- and collagen-induced human platelet aggregation (n=5). This figure presents representative curves of platelet aggregation function and corresponding statistical analysis results. Statistical analysis was performed using independent samples t-tests. Data are described as mean ± standard error, and ns indicates that the difference did not reach statistical significance.

[0014] Figure 2 (A) ELISA experiments showed that the intraplatelet Kyn level in patients with type 2 diabetes mellitus (T2DM) was significantly higher than that in healthy individuals (n=80). (B) A Pearson correlation model was used to analyze the correlation between Kyn level and PRP aggregation rate. The results showed that the intraplatelet Kyn level in patients with T2DM was positively correlated with PRP platelet aggregation induced by 4 μM ADP or 0.5 μg / mL collagen (n=80). (A) Statistical analysis used independent samples t-tests, and data are described as mean ± standard error. P<0.001.

[0015] Figure 3 (A) Platelet aggregation experiments showed that regardless of the dosage or strength of collagen, thrombin, or ADP used as agonists, aggregation and ATP release were significantly amplified by Kyn in a positive correlation (n=5). This figure presents representative curves of platelet aggregation and secretion function, along with corresponding statistical analysis results. One-way ANOVA and Dunnett's multiple comparisons were used, and data are described as mean ± standard error. P<0.05, P<0.01, P<0.001.

[0016] Figure 4 (AB) Flow cytometry was used to assess CD62P expression (reflecting P-selectin release) and PAC-1 binding (indicating integrin activation). Results showed that after Kyn treatment, thrombin (0.05 U / mL)-stimulated human platelets significantly increased both P-selectin release (A) and integrin αIIbβ3 activation (B) levels (n=5). (AB) shows representative peak plots and statistical graphs from flow cytometry, all presented using one-way ANOVA and Sidak multiple comparisons. Data are described as mean ± standard error. P<0.001.

[0017] Figure 5 (A) Kyn incubation enhanced the spreading area of ​​human platelets at all time points (n=5). (B) Kyn incubation accelerated the retraction rate of thrombin (10 U / mL)-induced human platelets at all time points, manifested as a reduction in thrombus area (n=5). (AB) presents representative figures and statistical graphs for each experiment, all using two-way ANOVA and Sidak multiple comparisons. Data are described as mean ± standard error. P<0.05, P<0.01, P<0.001.

[0018] Figure 6 (A) Kyn significantly enhanced collagen, thrombin, or ADP-induced platelet aggregation and collagen or thrombin-induced ATP release in WT mice (n=5). (B) Kyn incubation enhanced thrombin-induced P-selectin release from WT mouse platelets (n=5). (C) Kyn incubation enhanced thrombin-induced integrin αIIbβ3 activation in WT mouse platelets (n=5). (D) Kyn incubation increased the platelet spreading area at all time points in WT mice (n=5). (E) Kyn incubation accelerated the platelet retraction rate at all time points in WT mice induced by thrombin (n=5). Statistical analysis in (A) used independent samples t-test; (BC) used one-way ANOVA and Sidak multiple comparisons; (DE) used two-way ANOVA and Sidak multiple comparisons. Data are described as mean ± standard error. P<0.01, P<0.001. Detailed Implementation

[0019] The present invention will be further illustrated below with specific examples. It should be noted that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise stated, all scientific terms have the same meaning as those well known to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may be applied to the present invention. The experimental methods and materials described herein are for illustrative purposes only.

[0020] Example 1: Kyn was significantly elevated in platelets in db / db (diabetic model) mice and T2DM patients, and significantly activated platelet activity and thrombosis in both humans and mice. db / db mice were purchased from Cyagen Suzhou Co., Ltd. Mice were divided into WT mice and db / db mice, and patients were divided into healthy individuals and T2DM patients. Non-targeted metabolomics assays were provided by Baipu Co., Ltd. The simplified procedure is as follows: Platelets from each group of mice, after being uniformly concentrated, were added to 5× platelet IP lysis buffer and placed on ice for 10 minutes to allow for complete platelet lysis. The lysis products were rapidly stored in liquid nitrogen and then subjected to non-targeted metabolomics analysis.

[0021] The main steps are as follows: After extracting metabolites from platelet samples using a pre-cooled organic solvent, the precipitate was removed by centrifugation, and the supernatant was collected. The supernatant was then concentrated, dried, and reconstituted with methanol-water solution for detection. Metabolite separation was performed using an ultra-high performance liquid chromatography (UHPLC) system, and data were acquired by a high-resolution mass spectrometer in both positive and negative ion electrospray ionization (PSI) modes. The obtained raw data underwent peak extraction, retention time correction, and peak alignment. Combined with precise mass numbers and secondary mass spectrometry information, metabolite annotation was performed in public and self-built databases. Subsequently, the data were normalized and standardized, and follow-up statistical analysis was conducted to screen for differentially expressed metabolites.

[0022] To investigate changes in platelet metabolism in diabetic patients, peripheral blood was collected from db / db and WT mice in this study. Platelets were isolated, and non-targeted metabolomics analysis was performed on platelet lysates. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) revealed significantly different clustering patterns between the two groups. Figure 1 (AB), the OPLS-DA model exhibits good predictability and interpretability, with an R² of 0.983 and a Q² of 0.74 ( Figure 1 C). Further hierarchical cluster analysis revealed differences in multiple metabolites in platelets between the two groups (P<0.05, VIP>1). Figure 1 D), of which 10 metabolites showed significant differences, including 5 upregulated metabolites and 5 downregulated metabolites ( Figure 1 E). Notably, the three most abundant differentially metabolites in db / db mouse platelets were two isomers of β-D-glucopyranuronic acid and Kyn. The two isomers of β-D-glucopyranuronic acid had no direct effect on thrombin- and collagen-induced platelet aggregation in human platelets. Figure 1 Therefore, the effects of these two metabolites on platelet function were not further investigated (F). In contrast, Kyn levels were significantly elevated in db / db mouse platelets, and its potential role was further analyzed in subsequent studies.

[0023] Example 2: Kyn levels are closely related to platelet hyperreactivity. The ELISA method (using a human-canine kynurenine kit, purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.) was used to detect the kynurenine content in platelets of patients with type 2 diabetes mellitus (T2DM). The correlation between kynurenine levels and platelet aggregation rate was analyzed by detecting the ADP-induced PRP aggregation reaction.

[0024] Among them, the diabetic patients were hospitalized patients from Zhongshan Hospital affiliated to Fudan University, and informed consent was obtained from all participants. Ultimately, 80 diabetic patients were included in this study.

[0025] The results showed that, compared with healthy controls, the level of Kyn in platelets was significantly increased in diabetic patients. Figure 2 A), and was positively correlated with PRP platelet aggregation rate induced by 4 μM ADP (r=0.56, P<0.001) and 0.5 μg / mL collagen (r=0.45, P<0.01). Figure 2 B). This suggests that the intraplatelet Kyn level in diabetic patients is closely related to platelet hyperresponsiveness.

[0026] Example 3: Kyn enhances human platelet aggregation To investigate the specific effects of Kyn on platelet function, platelet aggregation response was first detected using light transmission chromatography, considered the gold standard for studying platelet function. Platelets were incubated for 5 minutes with different concentrations of Kyn (1, 2, and 5 μM) from healthy volunteers, simulating Kyn levels under physiological to pathological conditions. Platelets were then activated using various agonists.

[0027] The specific process is as follows: Platelet aggregation and ATP release detection experiment 1) Turn on the platelet aggregation analyzer in advance, stabilize the temperature at 37℃, and set the detection channel and paper feed speed, etc.

[0028] 2) Pipette 500 μL of TB solution into a new aggregation tube as a blank control; simultaneously, pipette 300 μL of RPs that have been allowed to stand for 15 minutes into another aggregation tube. Similar to the PRP aggregation experiment, if drug treatment is required, place the aggregation tube into the detection well, quickly add the corresponding drug, stir magnetically at 1200 rpm for 30 seconds, remove, and incubate at room temperature for 5 minutes; if no treatment is required, directly place the aggregation tube into the detection well for subsequent detection.

[0029] 3) If it is necessary to simultaneously detect adenosine triphosphate (ATP) release, the fluorescence detection channel of the focusing instrument must be selected in advance, and luciferase pre-incubation must be performed. If the experiment involves drug treatment, luciferase and the drug can be added to the platelet suspension simultaneously; if no drug treatment is performed, luciferase is added directly to the sample and incubated for about 5 minutes under light-protected conditions.

[0030] 4) After drug and / or luciferase incubation, start the detection program on the computer and begin recording the aggregation curve. First, perform baseline calibration to ensure the curve starts at zero. Once the curve stabilizes, quickly add the corresponding platelet agonist to the sample and record for approximately 5 minutes before ending the experiment. Avoid introducing air bubbles into the aggregation tube throughout the process to prevent affecting the results. When detecting ATP release, maintain a light-protected environment and close the instrument's dark door.

[0031] The results showed that, compared with the control group, Kyn gradually enhanced collagen, thrombin, and ADP-induced human platelet aggregation with increasing concentration, while also promoting the release of ATP stimulated by collagen and thrombin. Figure 3 A).

[0032] Example 4: Following vascular injury, platelets are activated by various agonists, which initiate their respective intracellular signaling pathways. Although these agonists exhibit receptor-specific pathways, they ultimately converge on a common downstream effector process—activation of integrin αIIbβ3 ligand binding and secretion of granule contents—to further amplify platelet activation. This process is termed "inside-out signal transduction." Therefore, flow cytometry was used to detect the expression level of P-selectin on the surface of human platelets (measured as CD62P expression) and the degree of αIIbβ3 activation (assessed by its binding to PAC-1).

[0033] The specific process is as follows: Flow cytometry was used to detect platelet P-selectin release and integrin αIIbβ3 activation. 1) After obtaining platelet precipitate according to the above method, add TB to resuspend and dilute the platelet concentration to 10×109 / L, and let it stand in a 37℃ oven for 15 minutes.

[0034] 2) Divide the platelets into a resting group and an activated group. Aspirate 100 μL of RPs into a 1.5 mL EP tube for each group. If drug treatment is required, add the corresponding drug or control solution according to the experimental design, mix gently, and incubate for 5 minutes.

[0035] 3) Add 0.05 U / mL thrombin to the activated platelets to induce platelet activation, mix gently and incubate for 5 minutes.

[0036] 4) After 5 minutes, add 1 μL of CD41 antibody to each EP tube to label platelets. At the same time, add the corresponding fluorescently labeled antibody according to the purpose of detection: 1 μL of PE (APC for mouse samples) antibody labeled with P-selectin, or 1 μL of PAC-1 (JoN / A for mice) antibody labeled with integrin αIIbβ3. Mix gently and incubate in a 37°C oven in the dark for 30 minutes.

[0037] 5) After incubation, 200 μL of TB solution was added to each EP tube to terminate the reaction, and 75 μL of 4% paraformaldehyde was added for fixation. The samples were then analyzed by flow cytometry, and the data were processed using FlowJo software.

[0038] The results showed that, compared with the control group, after treatment with 5 μM Kyn, the expression level of P-selectin and the activation degree of integrin αIIbβ3 on the surface of human platelets were significantly increased under stimulation with 0.05 U / mL thrombin. Figure 4 AB).

[0039] Once activated, integrin αIIbβ3 binds to its ligand fibrinogen, thereby stabilizing platelet adhesion and initiating an "outside-inside signaling" cascade. This process induces a series of platelet functional alterations, including platelet spreading (an early outside-inside signaling event) and clot retraction (a late outside-inside signaling event), ultimately leading to stable platelet thrombi. Therefore, the effects of Kyn on human platelet outside-inside signaling-related functional events were further evaluated.

[0040] Experimental group: platelets incubated with Kyn; control group: platelets incubated with physiological saline as the control solvent.

[0041] Platelet spreading test 1) Take out a new clean glass slide and draw a square of appropriate size on the back with a marker. On the front, use an immunohistochemistry pen to trace a frame along the square on the back. Add 150 μL of fibrinogen solution (final concentration 100 μg / mL) into the frame, ensuring it evenly covers the surface. Then, incubate the slide at 4°C for 12 hours for coating.

[0042] 2) After coating, remove the fibrinogen from the surface of the slide and wash the slide three times with phosphate-buffered saline (PBS) to remove any residue. To further eliminate non-specific binding sites, inject 150 μL of 5% bovine serum albumin (BSA) into the frame for blocking.

[0043] 3) After blocking at room temperature for 1 hour, discard the blocking solution and wash three times with PBS. Adjust the concentration to 20 × 10⁻⁶ by taking 150 μL of the solution and allowing it to stand. 9 / L of RPs were added to a glass slide and incubated at a constant temperature of 37°C for different times (20, 40, 60 minutes).

[0044] 4) After incubation, remove the RPs and wash three times with sample-grade PBS. Then add approximately 150 μL of fixation and membrane-breaking solution to each frame and incubate at room temperature for 20 minutes.

[0045] 5) After 20 minutes, discard the fixative, rinse 3 times with PBS, and add 100 μL of phalloidin diluted with 3% BSA solution to the box. Incubate at room temperature in the dark for 60 minutes.

[0046] 6) After staining, discard the staining solution and wash three times with PBS. Add 10 μL of anti-fluorescence quencher to the center of the sample area, then cover with a coverslip to complete the mounting.

[0047] 7) After mounting, observe the platelet spreading morphology and acquire images using a fluorescence microscope. Import the images into ImageJ software to quantitatively measure the platelet spreading area and perform statistical analysis.

[0048] The results showed that, compared with the control group, after treatment with 5 μM Kyn, platelets exhibited more significant expansion behavior at all set observation points, and their spreading area showed a much larger expansion trend compared with the control group. Figure 5 A); meanwhile, in the clot retraction experiment, the clot volume observed at each time point was significantly shrunken compared to the control group, indicating that the retraction process was significantly accelerated ( Figure 5 B).

[0049] Example 5: Kyn enhances platelet activation and related functional responses in mice In the above embodiments, it has been determined that Kyn can enhance human platelet activation induced by various agonists. Next, to further verify whether Kyn has a similar effect on mouse platelets, peripheral blood of WT mice was collected, centrifuged and suspended to obtain RPs, and co-incubated with Kyn (5 μM) for 5 minutes, and the above series of platelet functional experiments were repeated.

[0050] The specific steps are as follows: Isolation of mouse platelets and preparation of suspended platelets 1) This study selected 8-12 week old WT or db / db mice as experimental subjects. Mice were first anesthetized by intraperitoneal injection of 1% pentobarbital solution. The pentobarbital dosage was selected within the range of 1-1.5 mL based on the mouse's body weight. Within 5 minutes after administration, the reflexes were assessed by gently touching the mouse's toes with forceps. If no obvious avoidance response was observed, the anesthesia was considered sufficient, and subsequent experimental procedures could proceed.

[0051] 2) Place the mouse in a supine position with its limbs fixed to the operating table. Disinfect the mouse's chest and abdomen with 75% ethanol. Cut the skin and subcutaneous fascia along the midline of the abdomen and pull them to both sides to fully expose the abdomen. Then, locate the abdominal aorta by blunt dissection of the abdominal tissue. After locating the abdominal aorta, insert a 1mL syringe thoroughly moistened with 3.8% sodium citrate anticoagulant and slowly pull the syringe. If aspiration is difficult, slightly adjust the needle direction. If blood still cannot be obtained, change the blood sampling site and avoid forced aspiration.

[0052] 3) If the amount of blood drawn is small, it is difficult to obtain platelet precipitation by centrifugation. Therefore, blood from mice of the same strain can be mixed to ensure that there is 3 mL of blood in the centrifuge tube. For the blood sample to be processed, before the first centrifugation, 2 mL of physiological saline for dilution should be added, mixed well, and centrifuged at 270 g at 37°C for about 3 minutes.

[0053] 4) After the first centrifugation, aspirate the upper PRP layer for a second centrifugation. Since the mouse blood volume is small, the remaining blood sample can be centrifuged again (the operation is the same as the first centrifugation, but the speed is adjusted to 300g, and the resulting PRP is combined with the PRP obtained in the first centrifugation).

[0054] 5) For the second centrifugation, increase the speed to 450g and the time to the plasma volume, keeping other conditions unchanged. After centrifugation, discard the upper PPP layer, add an appropriate amount of TB pre-warmed at 37°C to the platelet precipitate in the centrifuge tube, and gently agitate to ensure uniform resuspending of the platelets.

[0055] 6) Finally, aspirate 60 μL of RPs into an automated hematology analyzer for platelet counting. Based on the count, supplement with TB to bring the platelet concentration to 300 × 10⁻⁶. 9 / L, and finally place the RPs in a 37°C oven for 15 minutes to stand before use in subsequent experiments.

[0056] The results showed that, compared with the control group, the Kyn treatment group significantly enhanced collagen, thrombin, and ADP-induced platelet aggregation in mice, as well as collagen and thrombin-induced ATP release from dense platelet granules in mice. Figure 6 A); simultaneously, Kyn also promoted thrombin (0.05 U / mL)-induced P-selectin levels on mouse platelet surface ( Figure 6 B), integrin activation ( Figure 6 C), and the spread of platelets ( Figure 6 D) and the clot retraction process ( Figure 6 E). The above results indicate that Kyn can also enhance the activation of mouse platelets and their related functional responses.

[0057] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Use of kynurenine in the preparation of diagnostic kits for cardiovascular complications of diabetes.

2. The use according to claim 1, characterized in that, The diabetes mentioned is type 2 diabetes.

3. The use according to claim 1, characterized in that, The cardiovascular complications include stable coronary artery disease and acute myocardial infarction.

4. A diagnostic kit for cardiovascular complications of diabetes, characterized in that, This includes reagents for detecting kynurenine concentration.

5. The reagent kit according to claim 4, characterized in that, The test samples for the kit are peripheral blood, plasma, or platelets.

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