System for measuring neutrophil extracellular traps and assessing thrombus risk

Abstract

A system for measuring neutrophil extracellular traps (NETs) and assessing the thrombus risk, a use of screening for a drug for promoting or inhibiting NETs, and a method for rapidly measuring NETs with a non-diagnostic therapy purpose. By means of microscopic observation of a peripheral blood sample, the NETs and the neutrophil lobulated granulocytes are identified on the basis of the morphological characteristics of cells, and the severity of thrombus formation and even the risk of death are assessed by means of quantitative analysis of the ratio of the NETs. The method is semi-automatic or automatic, the operation steps are few, the consumed time is short, and a quantitative result can be quickly obtained within 10 min. The method has high stability, the NETs in each stage can be accurately identified, and the condition of a patient can be assessed more accurately.

Description

A system for detecting neutrophil extracellular traps and assessing thrombosis risk Technical Field

[0001] The present invention relates to a system for detecting neutrophil extracellular traps and evaluating thrombosis risk, and belongs to the field of clinical medicine. Background Art

[0002] The new coronavirus infection caused by SARS-CoV-2 is a major public health event affecting the entire world. Viral infection can cause death or severe cascade reactions, such as cytokine storm and thrombosis.

[0003] Neutrophil extracellular traps (NETs) are special structures formed after neutrophil necrosis or apoptosis. Studies have shown that NETs play an important role in thrombosis in patients infected with the new coronavirus. Not only that, NETs are also associated with a variety of diseases. In infectious diseases, NETs help capture and kill microorganisms; in autoimmune diseases, excessive activation of NETs may lead to inflammation; NETs are associated with thrombotic diseases and increase the risk of diseases such as venous thromboembolism and atherosclerosis. In studies of cardiovascular diseases, NETs are closely related to the pathogenesis of diseases such as coronary artery disease and myocardial infarction.

[0004] However, in current literature, NETs are difficult to observe directly, and the main methods for NET detection include enzyme-linked immunosorbent assay (ELISA), immunofluorescence, fluorescence spectroscopy, flow cytometry, and electron microscopy. ELISA is sensitive, rapid, and low-cost, and can be automated, but its reproducibility is poor. Detection of plasma DNA content requires centrifugation pretreatment, which can easily cause other NET components to be mixed in the precipitate and interfere with detection. Cell immunofluorescence staining, based on antibodies against NET components combined with intercalating DNA dyes, has gradually become the preferred method for qualitative and semi-quantitative detection of NETs.

[0005] However, the antibodies used in immunofluorescence mainly target peroxidases and histones, making them difficult to distinguish from other substances. Fluorescence spectroscopy allows for high-throughput and rapid detection, but not all circulating DNA (cf-DNA) is derived from NETs. Flow cytometry, a method that can rapidly and reliably analyze thousands of cells per sample and eliminates interobserver bias, has the primary advantage of not requiring neutrophil purification. However, its limitation lies in its focus on neutrophils currently undergoing the neutrophil cell death program (NETosis), potentially overlooking cells that have already lysed or are in the late stages of NETosis and potentially missing citrullinated histone (cit-H3)-negative NETs. Electron microscopy, due to its visual properties, has gradually become an important tool for detecting NETs. However, when SEM is used to detect NETs in exudates, the morphological differences between NETs and fibrin in blood are not obvious, necessitating verification of the results using immunofluorescence microscopy. These NET detection methods are cumbersome and fail to meet the clinical requirements for rapidity and simplicity.

[0006] Therefore, developing a method that can rapidly detect NETs in vitro and assess patients' disease risk is of great clinical value. Summary of the Invention

[0007] To address the shortcomings of existing methods, the present invention aims to provide a system for detecting neutrophil extracellular traps and assessing thrombosis risk, achieving the effect of rapid in vitro detection and assessment of a patient's thrombosis risk. The present invention provides a system for detecting neutrophil extracellular traps and assessing thrombosis risk, comprising a detection device and an assessment device, wherein the detection device is used to process a whole blood sample, prepare a blood smear, and obtain an optical microscopic image of the blood smear; wherein the assessment device is used to count the number of neutrophils in the microscopic image and assess the risk of thrombosis in the sample based on the ratio of neutrophil extracellular traps in the sample.

[0008] The first object of the present invention is to provide a system for assessing the risk of thrombosis based on detecting neutrophil extracellular traps, comprising a detection device and an evaluation device, wherein the detection device is used to process whole blood samples, prepare blood smears, and obtain optical microscopic images of blood smears; the evaluation device is used to count the number of neutrophils and neutrophil extracellular traps in the microscopic image, and evaluate the risk of thrombosis in the sample based on the ratio of neutrophil extracellular traps to neutrophils in the sample.

[0009] In one embodiment, the detection device includes the following modules:

[0010] The sample collection module places the whole blood sample in an anticoagulation tube for anticoagulation and mixes the whole blood; and

[0011] a smear preparation and staining module, which prepares a blood smear from whole blood and performs Liu staining on the blood smear; and

[0012] The image acquisition module obtains optical microscopic images of the morphology of white blood cells in blood samples.

[0013] In one embodiment, the evaluation device comprises the following modules:

[0014] An information processing module, which counts the number of neutrophil rod-shaped granulocytes Ns, the number of neutrophil segmented granulocytes Nn, and the number of neutrophil extracellular trapping nets Nnets in the white blood cells in the microscopic image according to the cell morphology; and

[0015] The data processing module calculates the neutrophil extracellular trap ratio R. The calculation formula of the ratio R is as follows:

[0016] and,

[0017] Result judgment module, compares R with the safety threshold R θ ; R is less than R θ , the risk of fatal thrombosis is considered low; R is greater than R θ The risk of fatal thrombosis is considered to be greater;

[0018] Among them, neutrophils with a rod-shaped cell morphology are counted as neutrophil band cells; neutrophils with a segmented nucleus are counted as neutrophil segmented nucleus granulocytes; and cells with one of the following morphologies are counted as neutrophil extracellular traps:

[0019] (1) The lobed nucleus is swollen, the cell body is enlarged, the structure is blurred, and the edges are unclear;

[0020] (2) The swelling of the lobed nucleus intensifies, accompanied by nuclear dissolution and nuclear fragmentation, the nuclear chromatin structure is unclear, the color is light, and it begins to erupt, and the nuclear outline is blurred;

[0021] (3) The nuclear membrane breaks down, the nucleus dissociates, and fibrous chromosome material is released into the cytoplasm, and the neutrophil structure is destroyed;

[0022] (4) The nuclear membrane breaks down, the nucleus dissociates, and the cell ejects to form a chromosome network structure;

[0023] (5) The cell membrane ruptures, releasing particles and forming a chromosome network structure.

[0024] In the present invention, the inventors propose a method for detecting NETs using blood smears based on the cell morphological characteristics of NETs during their formation. This method allows for rapid, accurate, and simple identification of NETs. In the prior art, NETs in peripheral blood preparations are often treated as smear cells, which are generally believed to be formed by the rupture of lymphocytes in the peripheral blood during the blood smear preparation process.

[0025] The method of the present invention is based on the detection of NETs and the statistical analysis of the NETs / segmented neutrophil ratio to evaluate the fatal risk of thrombosis caused by NETs. It is not only used to evaluate the risk of fatal thrombosis in patients infected with the new coronavirus, but is also applicable to the risk evaluation of thrombosis caused by other infections that increase NETs.

[0026] In one embodiment, the whole blood sample in the sample collection module comes from a patient infected with the new coronavirus, a patient with cardiovascular disease, a patient with autoimmune disease, or a patient with cancer.

[0027] In one embodiment, after the anticoagulation treatment, 18.0%-23.0% W / V bovine serum albumin is further added, mixed and allowed to stand.

[0028] In one embodiment, bovine serum albumin and anticoagulated whole blood are mixed at a volume ratio of 0.8-1.2:4-6 and allowed to stand for 5-10 minutes.

[0029] In one embodiment, the staining steps in the slide preparation and staining module are:

[0030] S1, add 1-2 mL of Liu A Solution;

[0031] S2. Add Liu B Solution dropwise onto Liu A Solution to fully mix the two solutions. The amount of Liu B Solution added should be 1.5 to 3 times that of Liu A Solution. Dye for 20 to 40 seconds.

[0032] S3. Wash with water, dry, and examine under a microscope.

[0033] In one embodiment, the information processing module counts the number of neutrophil bands Ns, the number of neutrophil segmented nuclei Nn, and the number of neutrophil extracellular traps Nnets per 100 white blood cells.

[0034] In one embodiment, the safety threshold R in the result determination module θ It is derived from the actual medical statistical model, and the method for constructing the actual medical statistical model is as follows:

[0035] (1) Outliers were excluded according to the interquartile range (IQR) method, that is, observations falling outside the inner column were considered outliers;

[0036] (2) Determine the probability distribution satisfied by each data set using the Kolmogorov-Smirnov test; transform each skewed distribution data set into a normal distribution using the lognormal method;

[0037] (3) Calculate the confidence interval CI for each group at a 95% confidence level.

[0038] Calculate the confidence interval CI according to modern medical statistical formula

[0039] Among them: Y=log(X), Y~N(μ,σ^2), Sample mean, S X 、S Y : The difference between each sample, n: sample size, t n : Quantile of t distribution, Y LB 、Y UB are the upper and lower bounds of the confidence interval after logarithmic transformation; X LB 、X UB are the upper and lower bounds of the original sample confidence interval;

[0040] Calculate the confidence interval CI of each group in a case according to the formula i (a i , b i ), where a and b represent X LB 、X UB , namely CI i (a i , b i )=CI i (X LB , X UB ), i represents the severity group of the case;

[0041] Set up control group CI0 (a0, b0); mild group CI1 (a1, b1), moderate group CI2 (a2, b2); severe group CI3 (a3, b3); critical group CI4 (a4, b4); death group CI5 (a5, b5);

[0042] (4) Comparing the confidence intervals of each group, those less than or equal to b2 are considered to be the safe reference range, i.e., R0(0, b2); those greater than b2 and less than or equal to a5 are considered to be the low-risk area, i.e., R1(b2, a5); those greater than a5 are considered to be the high-risk area, i.e., R3(a5, ∞), i.e., R θ is (b2, a5).

[0043] In one embodiment, the safety threshold R θ It is 32.5% to 50.4%.

[0044] The second object of the present invention is to provide an application of the above system in screening drugs that promote or inhibit NETs.

[0045] A third object of the present invention is to provide a method for rapid detection of neutrophil extracellular traps for non-diagnostic and therapeutic purposes, comprising the following steps:

[0046] S01. placing the collected whole blood sample in an anticoagulant tube for anticoagulation treatment, and mixing the whole blood; and,

[0047] S02, preparing a blood smear from the whole blood, and performing Liu staining on the blood smear; and,

[0048] S03, reading the stained blood smear to obtain an optical microscopic image of the leukocyte morphology of the blood sample; and,

[0049] S04. Cells with any of the following morphologies are counted as neutrophil extracellular traps:

[0050] (1) The lobed nucleus is swollen, the cell body is enlarged, the structure is blurred, and the edges are unclear;

[0051] (2) The swelling of the lobed nucleus intensifies, accompanied by nuclear dissolution and nuclear fragmentation, the nuclear chromatin structure is unclear, the color is light, and it begins to erupt, and the nuclear outline is blurred;

[0052] (3) The nuclear membrane breaks down, the nucleus dissociates, and fibrous chromosome material is released into the cytoplasm, and the neutrophil structure is destroyed;

[0053] (4) The nuclear membrane breaks down, the nucleus dissociates, and the cell ejects to form a chromosome network structure;

[0054] (5) The cell membrane ruptures, releasing particles and forming a chromosome network structure.

[0055] In one embodiment, the whole blood sample in the sample collection module comes from a patient infected with the new coronavirus, a patient with cardiovascular disease, a patient with autoimmune disease, or a patient with cancer.

[0056] In one embodiment, after the anticoagulation treatment, 18.0%-23.0% W / V bovine serum albumin is further added, mixed and allowed to stand.

[0057] In one embodiment, bovine serum albumin and anticoagulated whole blood are mixed at a volume ratio of 0.8-1.2:4-6 and allowed to stand for 5-10 minutes.

[0058] In one embodiment, the staining steps in the slide preparation and staining module are:

[0059] S1, add 1-2 mL of Liu A Solution;

[0060] S2. Add Liu B Solution dropwise onto Liu A Solution to fully mix the two solutions. The amount of Liu B Solution added should be 1.5 to 3 times that of Liu A Solution. Dye for 20 to 40 seconds.

[0061] S3. Wash with water, dry, and examine under a microscope. Beneficial effects

[0062] Compared with the prior art methods, the present invention has the following beneficial effects:

[0063] (1) The method of the present invention can detect NETs quickly, fully automatically and / or semi-automatically within 10 minutes with an accuracy rate of up to 95%. Compared with existing immunohistochemistry methods (such as immunofluorescence, 24 hours, 90%) and immunological techniques (such as ELISA, 90 minutes, 56%), the method can also observe intuitive NETs images using an automatic blood analyzer, overcoming the problems of cumbersome operation steps, easy interference, high cost and consumables in the existing technology.

[0064] (2) The present invention proposes a NETs / neutrophil segmentation ratio indicator, which provides a theoretical basis and method for the diagnosis, prognosis and treatment of NETs-related clinical diseases.

[0065] (3) The method of the present invention is very important in establishing a rapid detection of NETs or screening for drugs that promote or inhibit NETs or targeted therapeutic drugs, and is also of significant significance in the field of clinical medical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0067] Figure 1 is a schematic diagram of NETs staining;

[0068] Figure 2 is a schematic diagram of NETs entrapping blood cells: A shows NETs entrapping red blood cells; B shows NETs entrapping abnormal red blood cells; C shows NETs entrapping platelets;

[0069] Figure 3 shows the results of NETs ratio in patients with sudden thrombosis. A and B are the distribution diagrams (quartiles) of FIB (g / L) and D-dimer (ug / L) in peripheral blood samples of patients infected with the new coronavirus; C and D are the distribution diagrams of neutrophil and NETs counts in peripheral blood of patients, which were collected from samples infected with severe acute respiratory syndrome coronavirus 2 at a specific time; E shows the difference between the groups (25–75%) by the defined index (NETs / Segmented Neutrophil (%)); F shows the specific density of each group along with the percentage (NETs / Segmented Neutrophil), highlighting the difference in symptoms of patients infected with the new coronavirus. The dotted line indicates the threshold value of the neutrophil extracellular trap ratio (R) that divides the reference interval in the log-normal analysis. The valid data range is the mean ± 95% confidence interval. Among them, *p < 0.05, ***p < 0.01, ***p < 0.001, non-parametric multivariate analysis of variance with Dunn's post hoc test for multiple comparisons;

[0070] Figure 4 is a statistical chart of the patients' nets ratio. DETAILED DESCRIPTION

[0071] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the following will provide a clear and complete description of the technical solutions in the embodiments of the present invention. It is apparent that the described embodiments are only a portion of the embodiments of the present invention, rather than all of them. The following embodiments are not intended to limit the present invention, but are merely intended to illustrate the present invention.

[0072] Example 1: Method and system for assessing the risk of thrombosis-related death in patients infected with the novel coronavirus

[0073] 1. Detect neutrophil extracellular traps. The specific steps are as follows:

[0074] (1) 450 patients were recruited (38 negative patients and 412 positive patients). According to the Diagnosis and Treatment Plan for Novel Coronavirus Infection (Trial Version 10), the positive patients were clinically classified as mild (73 patients), moderate (181 patients), severe (58 patients), critical (46 patients), and deaths (54 patients, the measured values ​​were within four days before the patient's death);

[0075] (2) Collect peripheral blood (2 mL) from the patient and place it in an EDTA-K2 anticoagulant tube, and mix the whole blood;

[0076] (3) 20% bovine serum albumin (BSA, w / v) and EDTA-K2 anticoagulated whole blood were mixed at a ratio of 1:5 (v / v) and allowed to stand for 5 min;

[0077] (4) A blood smear was prepared from EDTA-K2 anticoagulated whole blood mixed with BSA, and the blood smear was stained with Liu's stain using an automatic slide machine;

[0078] (4.1) Liu staining:

[0079] S1. Add Liu A Solution (about 1-2 mL);

[0080] S2. Add Liu B Solution dropwise to Liu A Solution (approximately twice the amount of Solution A), mix the two solutions thoroughly, and stain for 30 seconds.

[0081] S3. Wash with water, dry, and examine under a microscope.

[0082] (5) The stained blood smears were read using a fully automatic blood cell morphology analyzer to obtain optical microscopic images of the leukocyte morphology of the blood samples. Some typical cell morphology results are shown in Figures 1 and 2.

[0083] If the cells to be tested are neutrophils with segmented nuclei or rod-shaped neutrophils, the counting results are obtained respectively; if the cells to be tested show neutrophils with swollen segmented nuclei, enlarged cell bodies, blurred structures, and unclear edges (as shown in Figure 1a), or neutrophils with aggravated swollen segmented nuclei, accompanied by nuclear dissolution and nuclear fragmentation, unclear nuclear chromatin structure, light staining, blurred nuclear outlines, and the beginning of eruption (as shown in Figure 1b), or neutrophils in the process of NETs eruption, with broken nuclear membranes, nuclear dissociation, release of fibrous chromosome material into the cytoplasm, and destruction of neutrophil structure (as shown in Figure 1c), or neutrophils releasing reticular structures, ejecting the formed chromosome reticular structure and entrapping red blood cells (as shown in Figure 1d), or neutrophils with ruptured cell membranes, releasing granules, and forming reticular structures (as shown in Figure 1e), they are counted as neutrophil extracellular traps.

[0084] 2. The method for assessing the risk of fatal thrombosis caused by neutrophil extracellular traps is as follows:

[0085] (1) The number of neutrophils with segmented nuclei, Nn, and the number of neutrophil extracellular traps, Nnets, after observing every 100 white blood cells;

[0086] (2) Calculate the neutrophil extracellular trap ratio (R); the calculation formula of the ratio R is as follows:

[0087] (3) Data processing and analysis, deriving the standard index R based on the actual medical statistical model θ , R θ is the safety threshold. By comparing R θand R, predict the risk of critical illness or even death of the patient. If the patient's R is higher than R θ If the risk is lower than R θ are considered to have lower risk;

[0088] Among them, R θ It is calculated based on the test data of 450 recruited patients according to the following method:

[0089] (3.1) Outliers were excluded according to the interquartile range method (IQR), that is, observations falling outside the inner column were considered outliers;

[0090] (3.2) The Kolmogorov-Smirnov test is used to determine the probability distribution that each group of data may satisfy. In this embodiment, each group satisfies the lognormal distribution.

[0091] (3.3) Calculate the confidence interval CI for each group at a 95% confidence level. Calculate the confidence interval CI according to the modern medical statistical formula:

[0092] Among them: Y=log(X), Y~N(μ,σ^2), Sample mean, S X 、S Y : The difference between each sample, n: sample size, t n : Quantile of t distribution, Y LB 、Y UB are the upper and lower bounds of the confidence interval after logarithmic transformation; X LB 、X UB are the upper and lower bounds of the original sample confidence interval;

[0093] Calculate the confidence interval CI of each group in a case according to the formula i (a i , b i ), where a and b represent X LB 、X UB , namely CI i (a i , b i )=CI i (X LB , X UB ), i represents the severity group of the case;

[0094] Set up control group CI0 (a0, b0); mild group CI1 (a1, b1), moderate group CI2 (a2, b2); severe group CI3 (a3, b3); critical group CI4 (a4, b4); death group CI5 (a5, b5);

[0095] (3.4) Comparing the confidence intervals of each group, those less than or equal to b2 are considered to be the safe reference range, i.e., R0(0, b2); those greater than b2 and less than or equal to a5 are considered to be the low-risk area, i.e., R1(b2, a5); those greater than a5 are considered to be the high-risk area, i.e., R3(a5, ∞), i.e., R θ is (b2, a5).

[0096] By counting the number of neutrophils and NETs in the peripheral blood of samples of acute respiratory syndrome coronavirus 2 with specific severity, the results are shown in E in Figure 3. The ratio of the number of NETs to the number of neutrophils was calculated. After calculation, b2 was 32.5% and a5 was 50.4%, which means the safety threshold R was obtained. θ The reference range was 32.5% to 50.4%. The specific density of samples in different severity groups is shown along with the percentage of the NETs / Segmented Neutrophil ratio, as shown in Figure 3F. The dotted line indicates the threshold of the neutrophil extracellular trap ratio (R) that divides the reference interval in lognormal analysis. The valid data range is the mean ± 95% confidence interval.

[0097] That is, patients with R lower than 32.5% are considered safe, and patients with R between 32.5% and 50.4% are considered to have a low risk; patients with R higher than 50.4% are considered to be at risk of serious illness or even death.

[0098] Based on the above method, a system for assessing thrombosis risk based on neutrophil extracellular trapping net for novel coronavirus pneumonia is constructed. The system includes: a detection device and an evaluation device. Specifically,

[0099] The detection device includes the following modules:

[0100] The sample collection module places the whole blood sample in an anticoagulation tube for anticoagulation and mixes the whole blood; and

[0101] a smear preparation and staining module, which prepares a blood smear from whole blood and performs Liu staining on the blood smear; and

[0102] The image acquisition module obtains optical microscopic images of the morphology of white blood cells in blood samples.

[0103] The information processing module counts the number of neutrophil rod-shaped granulocytes Ns, the number of neutrophil segmented granulocytes Nn, and the number of neutrophil extracellular traps Nnets in the white blood cells in the microscopic image. If the cells to be tested are neutrophil segmented granulocytes or rod-shaped neutrophils, the counting results are obtained respectively; if the cells to be tested have swollen segmented nuclei, nuclear dissolution and nuclear fragmentation, unclear nuclear chromatin structure, light coloration, blurred nuclear outline, and fiber-like chromosome material is released into the cytoplasm to form a network structure, then the cells are neutrophil extracellular traps (NETs); and,

[0104] The data processing module calculates the neutrophil extracellular trap ratio (R). The calculation formula of the ratio R is as follows:

[0105] and,

[0106] Result judgment module, compares R with the safety threshold R θ (32.5% to 50.4%); R less than 32.5 is considered safe, and R within the range of 32.5% to 50.4% is considered to have a low risk of thrombosis; R greater than R θ It is believed that the risk of thrombosis is high and there is a risk of death.

[0107] The above experimental method and evaluation system were used to verify 82 patients, including 14 negative patients and 68 positive patients. 18 positive patients died after their condition became serious, and there were no deaths among negative patients. The results are shown in Figure 4. The nets ratio of all negative patients was below 30%. After being infected with the new coronavirus, the nets ratio was below 50% (49 / 50 people) and all had a good prognosis. Among the 18 patients who died, the nets ratio showed huge fluctuations (30%-180%). The mortality rate of all patients with a nets ratio above 60% was as high as 90.1% (10 / 11 people). In this case, the accuracy rate of the above method and evaluation system was over 90%.

[0108] Comparative Example 1: Using fibrinogen (FIB) and D-dimer (D-Dimer) as clinical thrombosis test indicators for novel coronavirus pneumonia

[0109] Fibrinogen (FIB) and D-dimer (D-Dimer) are used as clinical test indicators to assess the risk of death from thrombosis. Studies examining the relationship between FIB, D-Dimer, and patient clinical symptoms revealed no statistically significant difference in the FIB index between the two groups in peripheral blood. As shown in Figure 3, A and B, these indicators indicate that FIB and D-Dimer are not effective indicators of a patient's critical illness risk.

[0110] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A system for assessing thrombotic risk based on detecting neutrophil extracellular traps, characterized in that: It comprises a detection device and an evaluation device, wherein the detection device is used to process a whole blood sample, prepare a blood smear, and obtain an optical microscopic image of the blood smear; the evaluation device is used to count the number of neutrophils and neutrophil extracellular traps in the microscopic image, and based on the ratio of neutrophil extracellular traps to neutrophils in the sample, evaluate the risk of thrombosis in the sample; The evaluation device includes the following modules: The information processing module counts the number of neutrophil rod-shaped granulocytes Ns, the number of neutrophil segmented granulocytes Nn, and the number of neutrophil extracellular trapping nets Nnets in every 100 white blood cells in the microscopic image according to the cell morphology; and, The data processing module calculates the neutrophil extracellular trap ratio R. The calculation formula of the ratio R is as follows: and, Result judgment module, compares R with the safety threshold R θ ; R is less than R θ , the risk of fatal thrombosis is considered low; R is greater than R θ The risk of fatal thrombosis is considered to be greater; Among them, neutrophils with a rod-shaped cell morphology are counted as neutrophil rod granulocytes; neutrophils with a segmented nucleus are counted as neutrophil segmented nucleus granulocytes; and cells with one of the following morphologies are counted as neutrophil extracellular traps: (1) The lobed nucleus is swollen, the cell body is enlarged, the structure is blurred, and the edges are unclear; (2) The swelling of the lobed nuclei intensifies, accompanied by nuclear dissolution and nuclear fragmentation, the nuclear chromatin structure is unclear, the color is light, and it begins to erupt, and the nuclear outline is blurred; (3) The nuclear membrane breaks down, the nucleus dissociates, and fibrous chromosome material is released into the cytoplasm, and the structure of neutrophils is destroyed; (4) The nuclear membrane breaks down, the nucleus dissociates, and the cell ejects to form a chromosome network structure; (5) The cell membrane ruptures, releasing particles and forming a chromosome network structure; The safety threshold R in the result judgment module θ It is derived from the medical actual statistical model, and the method for constructing the medical actual statistical model is as follows: (1) Collect data samples of different components and exclude outliers based on the interquartile range method; (2) Determine the probability distribution satisfied by each set of data using the Kolmogorov-Smirnov test; (3) The confidence interval of each group was calculated at the 95% confidence level: CI0 (a0, b0) for the control group; CI1 (a1, b1) for the mild group; CI2 (a2, b2) for the moderate group; CI3 (a3, b3) for the severe group; CI4 (a4, b4) for the critical group; and CI5 (a5, b5) for the death group. (4) Comparing the confidence intervals of each group, those less than or equal to b2 are considered to be the safe reference range, i.e., R0(0, b2); those greater than b2 and less than or equal to a5 are considered to be the low-risk area, i.e., R1(b2, a5); those greater than a5 are considered to be the high-risk area, i.e., R3(a5, ∞), i.e., R θ are (32.5%, 50.4%).

2. The system according to claim 1, characterized in that The detection device includes the following modules: A sample collection module, placing the whole blood sample in an anticoagulation tube for anticoagulation treatment, and mixing the whole blood; and, A smear preparation and staining module is used to prepare blood smears from whole blood and to perform Liu staining on the blood smears; and, The image acquisition module obtains optical microscopic images of the morphology of white blood cells in blood samples.

3. The system according to claim 2, characterized in that The whole blood samples in the sample collection module come from patients infected with the new coronavirus, patients with cardiovascular diseases, patients with autoimmune diseases, or patients with cancer.

4. The system according to claim 2, characterized in that 18.0%-23.0% W / V bovine serum albumin can be added to the sample collection module for processing, wherein the bovine serum albumin and anticoagulated whole blood are mixed at a volume ratio of 0.8-1.2:4-6 and left to stand for 5-10 minutes.

5. The system according to claim 2, characterized in that The steps of staining in the slide staining module are: S1, add 1-2 mL of Liu A Solution; S2. Add Liu B Solution onto Liu A Solution to mix the two solutions thoroughly. The amount of Liu B Solution added is 1.5 to 3 times that of Liu A Solution. Dye for 20 to 40 seconds. S3. Wash, dry and examine under a microscope.

6. Use of the system according to claim 1 in screening drugs that promote or inhibit NETs.

7. A method for rapid detection of neutrophil extracellular traps for non-diagnostic and therapeutic purposes, comprising the following steps: S01, placing the collected whole blood sample in an anticoagulation tube for anticoagulation treatment, and mixing the whole blood; and, S02, preparing a blood smear from the whole blood, and performing Liu staining on the blood smear; and, S03, reading the stained blood smear to obtain an optical microscopic image of the leukocyte morphology of the blood sample; and, S04. Cells with one of the following morphologies are counted as neutrophil extracellular traps: (1) The lobed nucleus is swollen, the cell body is enlarged, the structure is blurred, and the edges are unclear; (2) The swelling of the lobed nuclei intensifies, accompanied by nuclear dissolution and nuclear fragmentation, the nuclear chromatin structure is unclear, the color is light, and it begins to erupt, and the nuclear outline is blurred; (3) The nuclear membrane breaks down, the nucleus dissociates, and fibrous chromosome material is released into the cytoplasm, and the structure of neutrophils is destroyed; (4) The nuclear membrane breaks down, the nucleus dissociates, and the cell ejects to form a chromosome network structure; (5) The cell membrane ruptures, releasing particles and forming a chromosome network structure.

Images