Application of a flow cytometry reagent in a product for classifying bleeding risk levels in tumor diseases

Abstract

This invention provides an application of flow cytometry reagents in a product for classifying bleeding risk levels in tumor diseases, belonging to the field of biomedical testing. This invention also provides a non-disease diagnostic method, comprising the following steps: S1, adding flow cytometry reagents to peripheral blood from healthy individuals for testing, collecting and analyzing the ratio of the fluorescence intensity of the positive and negative groups of biomarkers (S / N), and establishing a reference interval; S2, collecting and processing peripheral blood samples to be tested, and then testing the samples using the same flow cytometry reagents and instrument parameters as in S1, collecting and analyzing the S / N results; S3, comparing the S / N results from S2 with the reference interval from S1 to assess bleeding risk. Compared with traditional platelet counting methods, this method has advantages such as speed, high specificity, high sensitivity, and low sample volume requirements.

Description

Technical Field

[0001] This invention belongs to the field of biomedical testing, specifically relating to the application of a flow cytometry reagent in a product for classifying bleeding risk levels in tumor diseases. Background Technology

[0002] Drug-related bleeding is a common adverse reaction to anti-tumor drugs. Bleeding not only prolongs hospital stays and increases medical costs, but can also lead to patient death. It can also reduce treatment dosage intensity, delay treatment, or even force treatment cessation, thus affecting the efficacy of anti-tumor drugs and negatively impacting long-term survival. After radiotherapy and chemotherapy, bone marrow is suppressed to varying degrees in cancer patients.

[0003] Currently, peripheral blood platelet count is the primary method for assessing bleeding risk. Platelets are irregular blood cells produced by mature megakaryocytes, possessing physiological characteristics such as adhesion, release, and aggregation, and are mainly involved in the body's physiological hemostasis. Platelet count is one of the important indicators for screening hemostasis and coagulation disorders in the human body. Elevated or decreased platelet counts, in addition to individual physiological fluctuations, are closely related to various bleeding and thrombotic diseases. Pathologically decreased platelet counts are associated with platelet production disorders, platelet destruction, and increased bleeding risk. While there is a close link between platelet dysfunction and bleeding disorders, there is currently no risk assessment method for tumor drug-related bleeding.

[0004] Flow cytometry is a detection method that performs quantitative analysis of single cells or other biological particles at the functional level. It can analyze the physical and chemical characteristics of a large number of cells or biological particles in a short time at high speed. It has been used in various fields of clinical medical testing and scientific research, such as the detection of immune cell subsets, platelet activation status, hematological detection such as lymphoma, and prognostic monitoring after organ transplantation.

[0005] Platelet membrane glycoproteins (GPs) are specific glycoprotein components found within the platelet membrane, on its surface, and in plasma. More than ten have been identified, including GPⅠb-IX-V, GPⅠa / Ⅱa, GPⅥ, GPⅡb / Ⅲa, and P-selectin. They play crucial roles in initial hemostasis, platelet adhesion to the extracellular matrix, and subsequent platelet aggregation, acting as important mediators of platelet function. GPⅡb and GPⅢa (CD41 and CD61) are the main glycoproteins distributed on the platelet membrane surface. Analyzing the expression of CD41 and CD61 using flow cytometry with fluorescein-labeled CD41 or CD61 antibodies has clinical significance in platelet counting, bleeding risk assessment, and diagnosis of platelet dysfunction. In addition to CD41 and CD61, CD9, CD36, CD107a, CD107b, CD42c, CD42d, CD51, and CD63 can be used to assess platelet function.

[0006] Compared with traditional platelet counting methods, flow cytometry can simultaneously detect the expression levels of multiple platelet membrane glycoproteins, comprehensively reflecting the functional status of platelets. It can complete the detection of a large number of cells in a short time, making it suitable for rapid clinical diagnosis. Through the establishment of a bleeding risk assessment model, combined with clinical data, it can more accurately assess the bleeding risk of cancer patients, avoiding the limitations of relying solely on platelet counts.

[0007] Flow cytometry results in large volumes of data, requiring specialized data analysis methods and models, and demanding high interpretation capabilities. Developing simpler and lower-cost flow cytometry methods will lower the threshold for clinical application. Flow cytometry is expected to be more widely used in assessing bleeding risk in tumor diseases, providing clinicians with more accurate treatment decision-making support, and improving patients' treatment outcomes and quality of life. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides an application of a flow cytometry reagent in a product for classifying bleeding risk levels in tumor diseases.

[0009] On one hand, the present invention provides a method for predicting the bleeding risk level of tumor diseases based on flow cytometry reagents, characterized by comprising the following steps:

[0010] S1. After collecting and processing peripheral blood samples from normal individuals, flow cytometry reagents were added for detection. The ratio of the fluorescence intensity of the positive group to that of the negative group of the biomarker (S / N) was collected and analyzed to establish a reference range for the S / N value of platelet biomarkers in normal individuals.

[0011] S2. After collecting and processing the peripheral blood samples to be tested, the samples were tested using the same flow cytometry reagents as in S1. The same instrument parameters as in S1 were used to test the samples, and the ratio of the fluorescence intensity of the positive group to the fluorescence intensity of the negative group of the biomarker (S / N) was collected.

[0012] S3. Compare the S / N result of S2 with the reference interval of S1 to assess the bleeding risk. The judgment criteria are as follows: if the S / N of S2 is less than the reference interval of S1, it is judged as high risk; if the S / N of S2 is within the reference interval of S1, it is judged as low risk; if the S / N of S2 is greater than the reference interval of S1, it is judged as medium risk.

[0013] The flow cytometry reagent includes a monoclonal antibody conjugated with a platelet marker and fluorescein.

[0014] The method described is a non-disease diagnostic method.

[0015] Specifically, the platelet markers include at least one of CD41, CD61, CD9, CD36, CD107a, CD107b, CD42c, CD42d, CD51, or CD63.

[0016] Specifically, the fluorescein can be excited by light of at least one wavelength selected from 405 nm, 488 nm, and 633 nm.

[0017] More specifically, the fluorescein includes at least one of FITC, PE, PE-Cy7, PerCP, APC, and APC-Cy7.

[0018] In some specific embodiments of the present invention, the platelet marker is CD61, the fluorescein is APC-Cy7, and the risk assessment criteria are as follows: a S / N range below 104 is considered high risk; a S / N range of 104-250 is considered low risk; and an S / N range above 250 is considered medium risk.

[0019] Specifically, the volume ratio of the peripheral blood sample to the flow cytometry reagent is 1:1.

[0020] Specifically, in steps S1 and S2, the processing steps of the peripheral blood sample include:

[0021] (1) Prepare one branch pipe;

[0022] (2) Add 3-8 μL of flow cytometry reagent to the bottom of the test tube;

[0023] (3) Add the same volume of anticoagulated blood as in step (2) to the bottom of all flow cytometry tubes;

[0024] (4) Add 150-250 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0025] (5) Gently shake for 1-2 seconds to mix, and incubate at room temperature in the dark for 15-30 minutes;

[0026] (6) Add 1-2 mL of 1× platelet incubation solution to all flow cytometer tubes, shake to mix, store at 2-8℃, and analyze and detect using flow cytometer within 4 hours.

[0027] More specifically, in step (1), the amount of peripheral blood used is 4-6 μL.

[0028] More specifically, in step (2), the platelet incubation solution is phosphate buffer, and the amount used is 180-220 μL.

[0029] On the other hand, the present invention provides the application of the above-mentioned flow cytometry reagent in a product for classifying bleeding risk levels in tumor diseases.

[0030] Specifically, the product also includes other reagents required for flow cytometry detection, including at least one of NaN3 solution, PBS, EDTA, or phosphate buffer.

[0031] Specifically, the products include reagent kits, chips, or risk classification systems.

[0032] Specifically, the steps for using the bleeding risk classification product for tumor diseases include: collecting and analyzing the ratio (S / N) of the positive fluorescence intensity to the negative fluorescence intensity of platelet markers in the sample to be tested, and assessing the risk.

[0033] In another aspect, the present invention provides a bleeding risk classification system for tumor diseases, characterized in that the system includes a detection device, a calculation device, and an output device; the detection device includes a sample injector, a sample processor, and a detector;

[0034] The sampler is used to collect samples from the subject;

[0035] The sample processor is used to mix the collected peripheral blood samples with flow cytometry detection reagents;

[0036] The detector includes a flow cytometer;

[0037] The computing device is used to collect and analyze the ratio (S / N) of positive fluorescence intensity to negative fluorescence intensity of markers detected by flow cytometry, and to make the following judgments: if the S / N of the test sample is less than the reference range for S / N in normal individuals, it is judged as high risk; if the S / N of the test sample is within the reference range for normal individuals, it is judged as low risk; if the S / N of the test sample is greater than the reference range for normal individuals, it is judged as medium risk.

[0038] Specifically, the subjects included healthy individuals whose routine blood tests showed normal results and whose platelet counts were between 100 and 300 × 10⁻⁶. 9 Peripheral blood samples were used to establish a reference range for the S / N values ​​of platelet markers in normal individuals.

[0039] Specifically, the output device is used to output the judgment result and judgment basis of the computing device.

[0040] Specifically, the tumor diseases include acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, multiple myeloma, breast cancer, cervical cancer, endometrial cancer, esophageal cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, thyroid cancer, nasopharyngeal carcinoma, oral cancer, lung cancer, bladder cancer, gastric cancer, colorectal cancer, squamous cell carcinoma, melanoma, basal cell carcinoma, choriocarcinoma, piloblastic leukemia, salivary gland cancer, sarcoma, connective tissue cancer, peritoneal cancer, eye cancer, or testicular cancer.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] Compared with traditional platelet counting methods, this method has advantages such as speed, high specificity, high sensitivity, and low sample volume required. Attached Figure Description

[0043] Figure 1 Scatter plot of CD61-APC-Cy7 / SSC.

[0044] Figure 2 Scatter plot of CD61-APC-Cy7 / PE.

[0045] Figure 3 This is a scatter plot of CD41-PerCP / SSC.

[0046] Figure 4 This is a scatter plot of CD41-PerCP / PE.

[0047] Figure 5 This is a scatter plot of CD42a-FITC / SSC.

[0048] Figure 6 Scatter plot of CD42a-FITC / PE.

[0049] Figure 7 This is a scatter plot of CD42b-APC / SSC.

[0050] Figure 8 Scatter plot of CD42b-APC / PE.

[0051] Figure 9 Scatter plot of CD61-APC-Cy7 / SSC.

[0052] Figure 10 Scatter plot of CD61-APC-Cy7 / PE. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0054] Example 1

[0055] The samples were peripheral blood samples from healthy individuals, and the CD61-APC-Cy7 reagent was used for testing on a flow cytometer. The CD61-APC-Cy7 reagent contains phosphate buffer, 1% BSA, APC-Cy7-labeled CD-61, and 0.1% sodium azide.

[0056] Sample preparation:

[0057] 1. Prepare one flow meter;

[0058] 2. Add 5 μL of CD61-APC-Cy7 reagent to the bottom of the test tube;

[0059] 3. Add 5 μL of anticoagulated (EDTA) blood to the bottom of all flow cytometry tubes (gently invert to mix before use) to avoid the sample touching the tube wall;

[0060] 4. Add 200 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0061] 5. Gently vortex for 2 seconds to mix, then incubate at room temperature in the dark for 15 minutes.

[0062] 6. Add 2 mL of 1× platelet incubation solution to all flow cytometry tubes, vortex to mix, store at 2-8℃, and analyze using a flow cytometer within 4 hours.

[0063] Data Analysis:

[0064] 1. First, construct a CD61-APC-Cy7 / SSC scatter plot and circle the platelet-positive clusters with high CD61-APC-Cy7 expression and low SSC expression. For example... Figure 1 As shown.

[0065] 2. Construct a CD61-APC-Cy7 / PE scatter plot and circle the platelet-negative groups with low expression of CD61-APC-Cy7 and low expression of PE. For example... Figure 2 As shown.

[0066] 3. The ratio (S / N) of the fluorescence intensity of CD61 positive groups to that of CD61 negative groups was analyzed. The results showed that the fluorescence intensity of CD61 positive groups was 7660, the fluorescence intensity of CD61 negative groups was 62, and the S / N value was 123.

[0067] Example 2

[0068] The samples were derived from peripheral blood samples from healthy individuals and were tested using the CD41-PerCP reagent on a flow cytometer. The CD41-PerCP reagent contains phosphate-buffered saline, 1% BSA, PerCP-labeled CD-41, and 0.1% Proclin 300.

[0069] Sample preparation:

[0070] 1. Prepare one flow meter;

[0071] 2. Add 8 μL of the CD41-PerCP reagent for test 1 to the bottom of the test tube;

[0072] 3. Add 8 μL of anticoagulated blood to the bottom of all flow cytometry tubes (invert and mix before use) to avoid the sample touching the tube wall;

[0073] 4. Add 250 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0074] 5. Gently vortex for 2 seconds to mix, then incubate at room temperature in the dark for 20 minutes.

[0075] 6. Add 2 mL of 1× platelet incubation solution to all flow cytometry tubes, vortex to mix, store at 2-8℃, and analyze using a flow cytometer within 4 hours.

[0076] Data Analysis:

[0077] 1. First, construct a CD41-PerCP / SSC scatter plot and circle the platelet-positive clusters with high CD41-PerCP expression and low SSC expression. For example... Figure 3 As shown.

[0078] 2. Construct a CD41-PerCP / PE scatter plot and circle the platelet-negative groups with low CD41-PerCP expression and low PE expression. For example... Figure 4 As shown.

[0079] 3. The ratio (S / N) of the fluorescence intensity of CD41 positive groups to that of CD41 negative groups was analyzed. The results showed that the fluorescence intensity of CD41 positive groups was 6656, the fluorescence intensity of CD41 negative groups was 43, and the S / N value was 155.

[0080] Example 3

[0081] The samples were derived from peripheral blood samples from healthy individuals and were tested using the CD42a-FITC reagent on a flow cytometer. The CD42a-FITC reagent contains phosphate buffer, 1% BSA, FITC-labeled CD-42a, and 0.1% Proclin 300.

[0082] Sample preparation:

[0083] 1. Prepare one flow meter;

[0084] 2. Add 3 μL of the CD42a-FITC reagent for test 1 to the bottom of the test tube;

[0085] 3. Add 3 μL of anticoagulated blood to the bottom of all flow cytometry tubes (invert and mix before use) to avoid the sample touching the tube wall;

[0086] 4. Add 150 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0087] 5. Gently vortex for 1 second to mix, then incubate at room temperature in the dark for 15 minutes.

[0088] 6. Add 1 mL of 1× platelet incubation solution to all flow cytometry tubes, vortex to mix, store at 2-8℃, and analyze using a flow cytometer within 4 hours.

[0089] Data Analysis:

[0090] 1. First, construct a CD42a-FITC / SSC scatter plot and circle the platelet-positive clusters with high CD42a-FITC expression and low SSC expression. For example... Figure 5 As shown.

[0091] 2. Construct a CD42a-FITC / PE scatter plot and circle the platelet-negative groups with low CD42a-FITC and low PE expression. For example... Figure 6 As shown.

[0092] The fluorescence intensity ratio (S / N) of the CD42a positive group to the CD42a negative group was analyzed. The results showed that the fluorescence intensity of the CD42a positive group was 7325, the fluorescence intensity of the CD42a negative group was 65, and the S / N value was 113.

[0093] Example 4

[0094] The samples were peripheral blood samples from healthy individuals, and the CD42b-APC reagent was used for testing on a flow cytometer. The CD42b-APC reagent contains phosphate buffer, 1% BSA, APC-labeled CD-42b, and 0.1% sodium azide.

[0095] Sample preparation:

[0096] 1. Prepare one flow meter;

[0097] 2. Add 5 μL of the CD42b-APC reagent for test 1 to the bottom of the test tube;

[0098] 3. Add 5 μL of anticoagulated blood to the bottom of all flow cytometry tubes (invert and mix before use) to avoid the sample touching the tube wall;

[0099] 4. Add 200 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0100] 5. Gently vortex for 1 second to mix, then incubate at room temperature in the dark for 15 minutes.

[0101] 6. Add 1 mL of 1× platelet incubation solution to all flow cytometry tubes, vortex to mix, store at 2-8℃, and analyze using a flow cytometer within 4 hours.

[0102] Data Analysis:

[0103] 1. First, construct a CD42b-APC / SSC scatter plot and circle the platelet-positive clusters with high CD42b-APC expression and low SSC expression. For example... Figure 7 As shown.

[0104] 2. Construct a CD42b-APC / PE scatter plot and circle the platelet-negative groups with low CD42b-APC expression and low PE expression. For example... Figure 8 As shown.

[0105] 3. The ratio (S / N) of the fluorescence intensity of CD42b positive groups to that of CD42b negative groups was analyzed. The results showed that the fluorescence intensity of CD42b positive groups was 6582, the fluorescence intensity of CD42b negative groups was 58, and the S / N value was 113.

[0106] Example 5

[0107] The samples were peripheral blood samples from healthy individuals and tumor patients with low platelet counts. The CD61-APC-Cy7 reagent was used for testing on a flow cytometer.

[0108] The inclusion criteria for cancer patients were those with low platelet counts (platelet count below 100 × 10⁶). 9 (pieces / L).

[0109] Sample preparation:

[0110] 1. Prepare the flow cytometer tubes required for the test;

[0111] 2. Add 5 μL of the CD61-APC-Cy7 reagent for test 1 to the bottom of the test tube;

[0112] 3. Add 5 μL of anticoagulated blood to the bottom of all flow cytometry tubes (invert and mix before use) to avoid the sample touching the tube wall;

[0113] 4. Add 200 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes;

[0114] 5. Gently vortex for 2 seconds to mix, then incubate at room temperature in the dark for 15 minutes.

[0115] 6. Add 2 mL of 1× platelet incubation solution to all flow cytometry tubes, vortex to mix, store at 2-8℃, and analyze using a flow cytometer within 4 hours.

[0116] Data Analysis:

[0117] 1. First, construct a CD61-APC-Cy7 / SSC scatter plot and circle the platelet-positive clusters with high CD61-APC-Cy7 expression and low SSC expression. For example... Figure 9 As shown.

[0118] 2. Construct a CD61-APC-Cy7 / PE scatter plot and circle the platelet-negative groups with low expression of CD61-APC-Cy7 and low expression of PE. For example... Figure 10 As shown.

[0119] 3. The ratio of fluorescence intensity of CD61 positive group to fluorescence intensity of CD61 negative group (S / N) was analyzed. The S / N results are shown in Tables 1-2.

[0120] Table 1. CD61 S / N Results in Normal Human Samples

[0121]

[0122] Table 2. CD61 S / N results in tumor patient samples

[0123]

[0124] The experimental results in Tables 1 and 2 show that the CD61 S / N ratio of normal human samples was generally high, while the CD61 S / N ratio of tumor patient samples with low platelet counts was generally low.

[0125] 4. Determine the reference interval

[0126] Based on the "Guiding Principles for Registration Review of Reference Range for In Vitro Diagnostic Reagents", 150 normal human samples were measured. By analyzing the ratio of fluorescence intensity of CD61 positive group to fluorescence intensity of CD61 negative group (S / N), the reference range of CD61 S / N was determined to be 104-250.

[0127] Example 6

[0128] Peripheral blood samples from 200 cancer patients were collected; these patients were not duplicated in Example 5. The same CD61-APC-Cy7 reagent and flow cytometer as in Example 5 were used for testing, and the results were recorded to obtain the S / N ratio. These 200 patients were then followed up for 90 days to observe and analyze the occurrence of endpoint events. Finally, the correlation between the S / N ratio and bleeding events was analyzed.

[0129] The inclusion criteria for cancer patients were those with low platelet counts (platelet count below 100 × 10⁶). 9 (pieces / L).

[0130] Endpoint events include:

[0131] • Bleeding events:

[0132] Major bleeding: meets the criteria of the International Society for Thrombosis and Haemostasis (ISTH).

[0133] Clinically relevant non-major bleeding: bleeding events requiring medical intervention.

[0134] • Non-bleeding events:

[0135] No major bleeding or clinically relevant non-major bleeding events occurred at the end of the 90-day follow-up period.

[0136] The preparation of the test samples and the data analysis methods are the same as in Example 5.

[0137] The statistical S / N results are shown in Table 3:

[0138] Table 3. CD61 S / N results from 200 cancer patients.

[0139]

[0140] The experimental results in Table 3 show a significant correlation between the CD61 S / N ratio and the occurrence of bleeding events in 200 cancer patients during follow-up. The results indicate that a CD61 S / N ratio less than 104 carries the highest risk of bleeding, while a CD61 S / N ratio greater than 250 carries a relatively high risk. This demonstrates that the CD61 S / N ratio can be used to assess the bleeding risk level in cancer patients. Preliminary findings suggest that an S / N ratio below the reference range indicates a high incidence of bleeding; an S / N ratio above the reference range indicates a moderate incidence of bleeding; and an S / N ratio within the reference range indicates a low incidence of bleeding.

[0141] Example 7

[0142] Peripheral blood samples from 100 cancer patients (no duplicates of those in Examples 5 or 6) were collected. The same CD61-APC-Cy7 reagent and flow cytometer as in Example 5 were used for testing, and the results were recorded and the S / N ratio was obtained. These 100 patients were then followed up for 90 days to observe and analyze the occurrence of endpoint events. The correlation between the S / N ratio and bleeding events was verified.

[0143] Endpoint events include:

[0144] • Bleeding events:

[0145] Major bleeding: meets the criteria of the International Society for Thrombosis and Haemostasis (ISTH).

[0146] Clinically relevant non-major bleeding: bleeding events requiring medical intervention.

[0147] • Non-bleeding events:

[0148] No major bleeding or clinically relevant non-major bleeding events occurred at the end of the 90-day follow-up period.

[0149] The preparation of the test samples and the data analysis methods are the same as in Example 5.

[0150] The statistical S / N results are shown in Table 4:

[0151] Table 4. CD61 S / N results from 100 cancer patients

[0152]

[0153] The experimental results in Table 4 show that the CD61 S / N ratio in 100 cancer patients was significantly correlated with the occurrence of bleeding events during follow-up. A CD61 S / N ratio less than 104 indicated the highest risk of bleeding, while a CD61 S / N ratio greater than 250 indicated a relatively high risk of bleeding. This further demonstrates that the CD61 S / N ratio can be used to assess the bleeding risk level in cancer patients.

[0154] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for predicting the bleeding risk level of tumor diseases based on flow cytometry reagents, characterized in that, Includes the following steps: S1. After collecting and processing peripheral blood samples from normal individuals, flow cytometry reagents were added for detection. The ratio of the fluorescence intensity of the positive group to that of the negative group of the biomarker (S / N) was collected and analyzed to establish a reference range for the S / N value of platelet biomarkers in normal individuals. S2. After collecting and processing the peripheral blood samples to be tested, the samples were tested using the same flow cytometry reagents as in S1. The same instrument parameters as in S1 were used to test the samples, and the ratio of the fluorescence intensity of the positive group to the fluorescence intensity of the negative group of the biomarker (S / N) was collected. S3. Compare the S / N result of S2 with the reference interval of S1 to assess the bleeding risk. The judgment criteria are: if the S / N of S2 is less than the reference interval of S1, it is judged as high risk; if the S / N of S2 is within the reference interval of S1, it is judged as low risk. If the S / N of S2 is greater than the reference range of S1, it is judged as medium risk; The flow cytometry reagent includes a monoclonal antibody conjugated with a platelet marker and fluorescein. The positive cluster refers to the platelet cluster in the sample that highly expresses a monoclonal antibody conjugated with a platelet marker. The negative group refers to the platelet population in the sample that shows low expression of monoclonal antibodies conjugated with platelet markers; The platelet markers include at least one of CD41, CD61, CD9, CD36, CD107a, CD107b, CD42c, CD42d, CD51, or CD63; The fluorescein includes at least one of FITC, PE, PE-Cy7, PerCP, APC, and APC-Cy7; The method described is a non-disease diagnostic method.

2. The method according to claim 1, characterized in that, In steps S1 and S2, the volume ratio of the peripheral blood sample to the flow cytometry reagent is 1:

1.

3. The method according to claim 1, characterized in that, In steps S1 and S2, the processing steps for the peripheral blood sample include: (1) Prepare one branch pipe; (2) Add 3-8 μL of flow cytometry reagent to the bottom of the test tube; (3) Add the same volume of anticoagulated blood as in step (2) to the bottom of all flow cytometry tubes; (4) Add 150-250 μL of 1× platelet incubation solution to the bottom of all flow cytometry tubes; (5) Gently shake for 1-2 seconds to mix, and incubate at room temperature in the dark for 15-30 minutes; (6) Add 1-2 mL of 1× platelet incubation solution to all flow cytometer tubes, shake to mix, store at 2-8℃, and analyze and detect using flow cytometer within 4 hours.

4. The method according to claim 3, characterized in that, In step (1), the amount of peripheral blood used is 4-6 μL; the platelet incubation solution is phosphate buffer, and the amount used is 180-220 μL.

5. The method according to claim 1, characterized in that, The platelet marker-coupled fluorescein combination includes at least one of CD61-APC-Cy7, CD41-PerCP, CD42a-FITC, CD42b-APC, or CD61-APC-Cy7.

6. The method according to claim 5, characterized in that, The platelet marker conjugated with fluorescein is CD61-APC-Cy7, and the reference range in step S1 is 104-250.

7. The application of the method according to any one of claims 1-6 in the preparation of a bleeding risk classification product for tumor diseases, said product comprising a bleeding risk classification system.

8. A bleeding risk classification system for tumor diseases, characterized in that, The system includes a detection device, a computing device, and an output device; the detection device includes a sample injector, a sample processor, and a detector. The sampler is used to collect samples from the subject; The sample processor is used to mix the collected peripheral blood samples with flow cytometry detection reagents; The detector includes a flow cytometer; The computing device is used to collect and analyze the ratio (S / N) of positive fluorescence intensity to negative fluorescence intensity of markers detected by flow cytometry, and to make the following judgments: if the S / N of the test sample is less than the reference range for S / N in normal individuals, it is judged as high risk; if the S / N of the test sample is within the reference range for normal individuals, it is judged as low risk; if the S / N of the test sample is greater than the reference range for normal individuals, it is judged as medium risk. The flow cytometry assay reagent includes a monoclonal antibody conjugated with a platelet marker and fluorescein. The positive fluorescence intensity refers to the fluorescence intensity of platelet populations highly expressed by a monoclonal antibody conjugated with a platelet marker in the sample. The negative fluorescence intensity refers to the fluorescence intensity of platelet populations with low expression of monoclonal antibodies conjugated with platelet markers in the sample. The platelet markers include at least one of CD41, CD61, CD9, CD36, CD107a, CD107b, CD42c, CD42d, CD51, or CD63; The fluorescein includes at least one of FITC, PE, PE-Cy7, PerCP, APC, and APC-Cy7.

9. The bleeding risk classification system for tumor diseases according to claim 8, characterized in that, The tumor diseases mentioned include acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, diffuse large B-cell lymphoma, multiple myeloma, breast cancer, cervical cancer, endometrial cancer, esophageal cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, thyroid cancer, nasopharyngeal carcinoma, oral cancer, lung cancer, bladder cancer, gastric cancer, colorectal cancer, melanoma, basal cell carcinoma, choriocarcinoma, hairy cell leukemia, salivary gland cancer, peritoneal cancer, or testicular cancer.

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