Application of CD39 + regulatory T cell as marker in children sepsis prognosis evaluation product

Abstract

The invention relates to the technical field of medical immunology, in particular to application of CD39 + regulatory T cells as markers in children sepsis prognosis evaluation products. The CD39 + Tregs level in peripheral blood of a patient is detected based on flow cytometry, and prognosis of the patient is dynamically evaluated based on change of expression of the CD39 + Tregs level. The CD39 + Tregs expression level is in significant negative correlation with children sepsis scores (Phoenix and pSOFA scores), and is in negative correlation with the death rate of children suffering from sepsis. And when the expression of CD39 + Tregs is lower than 12.45%, the patient is prompted to have a relatively high death risk. The method provided by the invention can provide accurate prognosis evaluation in early screening of sepsis in children, and has high specificity and sensitivity. The method can also be used for dynamic monitoring of patients, helps doctors to adjust treatment schemes in time, optimizes personalized treatment, and has good practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of medical immunology, and in particular to a method for evaluating the prognosis of childhood sepsis using CD39+ regulatory T cells and an application method thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which often leads to multi-organ dysfunction. Especially in children, sepsis has a high morbidity and mortality rate (Singer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Jama 2016; 315; Karakike E, et al. The early change of SOFA score as a prognostic marker of 28-day sepsis mortality: analysis through a derivation and a validation cohort. Critical Care 2019; 23; Rudd KE, et al. Global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease Study. Lancet 2020; 395: 200-211). Although modern medicine has made significant progress in the treatment of sepsis, how to accurately and effectively predict the prognosis of patients remains a core challenge in clinical treatment.

[0003] At present, the prognosis assessment of sepsis mainly relies on clinical scoring systems (such as pSOFA score and Phoenix sepsis score), but these systems are mainly based on the patient's organ dysfunction and cannot reflect the individual's immune response and immune imbalance. Therefore, finding new and specific immune biomarkers to assess the prognosis of sepsis patients is the key to improving the effectiveness of clinical intervention.

[0004] The immune system plays a vital role in the progression of sepsis, especially regulatory T cells (Tregs), which act as the "brakes" of the immune system and are crucial to the control of inflammatory responses and the maintenance of immune homeostasis. In particular, regulatory T cells expressing CD39 (CD39+ Tregs) play a key role in inhibiting excessive inflammation and maintaining immune balance through the ATP / adenosine metabolic pathway mediated by their exoenzyme CD39. Studies have found that the level of CD39+ Tregs is closely related to the immune status and disease progression of patients with sepsis.

[0005] Existing methods for evaluating the prognosis of sepsis mostly rely on clinical indicators, such as the patient's hemodynamic status and the severity of organ dysfunction, but these indicators usually only appear when the condition has worsened and cannot be used for early prediction. In addition, although conventional biomarkers (such as C-reactive protein and procalcitonin) can reflect the inflammatory state in the body, they have low specificity and are difficult to accurately evaluate an individual's immune response and prognosis.

[0006] Therefore, there is an urgent need for a biomarker that can combine the patient's immune status to assess the prognosis of patients with sepsis. CD39+ Tregs is a functional cell population directly related to immune imbalance and has the potential to become a highly specific prognostic marker. Summary of the invention

[0007] In view of this, the present invention provides a method for evaluating the prognosis of childhood sepsis using CD39+ regulatory T cells and its application. The method uses flow cytometry to detect the level of CD39+ Tregs in the patient's peripheral blood, and determines its optimal critical value of 12.45% based on the ROC curve. When the patient's CD39+ Tregs level is lower than this value, it indicates that the patient faces a higher risk of death. The method has good specificity and high sensitivity, and can provide a scientific basis for early risk screening and personalized treatment of patients with sepsis.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides the use of CD39+ regulatory T cells as a marker in the preparation of a child sepsis prognosis product.

[0010] In some specific embodiments of the present invention, the prognosis determination rule used above includes: relative to total regulatory T cells, the expression level of CD39+ regulatory T cells is below 12.45%, indicating that the patient has a higher risk of death.

[0011] In some specific embodiments of the present invention, the CD39+ regulatory T cells used above are CD39+ regulatory T cells derived from peripheral blood.

[0012] The present invention also provides the use of antibodies in preparing a product for the prognosis of childhood sepsis, wherein the antibodies include anti-CD39 antibodies.

[0013] In some specific embodiments of the present invention, the antibodies used above also include anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies and anti-CD127 antibodies.

[0014] In some specific embodiments of the present invention, the above application is based on the antibody to obtain the proportion of CD39+ regulatory T cells in the total regulatory T cells in the sample to be tested by flow cytometry to obtain the prognostic result;

[0015] The prognostic result determination rules include: relative to the total regulatory T cells, the expression level of CD39+ regulatory T cells is below 12.45%, indicating that the patient has a higher risk of death.

[0016] In some specific embodiments of the present invention, the sample to be tested in the above application is a peripheral blood sample.

[0017] The present invention also provides a kit for the prognosis of childhood sepsis, using CD39+ regulatory T cells as a marker.

[0018] In some specific embodiments of the present invention, the above-mentioned kit uses specific antibodies to capture regulatory T cells to obtain the expression level of CD39+ regulatory T cells, and then performs prognosis based on the expression level of CD39+ regulatory T cells;

[0019] The prognostic determination rules include: relative to total regulatory T cells, the expression level of CD39+ regulatory T cells is below 12.45%, indicating that the patient has a higher risk of death;

[0020] The specific antibodies include anti-CD39 antibodies, anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies and anti-CD127 antibodies.

[0021] In some specific embodiments of the present invention, the above-mentioned kit comprises a specific antibody combination for detecting CD39+ regulatory T cells in the patient's peripheral blood, which is detected in combination with flow cytometry and used to evaluate the patient's prognosis.

[0022] In some specific embodiments of the present invention, the above kit further comprises reagents for sample processing, buffers, and fluorescent dyes for antibody labeling, so as to accurately detect the expression level of CD39+ Tregs in flow cytometry.

[0023] In some specific embodiments of the present invention, the above-mentioned kit includes anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies and anti-CD127 antibodies to identify and measure the patient's CD39+ regulatory T cell subpopulation.

[0024] In some specific embodiments of the present invention, the above-mentioned kit is suitable for early risk assessment of sepsis patients, and is particularly suitable for early screening and prognosis judgment of sepsis in children.

[0025] The present invention also provides a device for the prognosis of childhood sepsis, which uses CD39+ regulatory T cells as a marker and is coated with specific antibodies for capturing CD39+ regulatory T cells.

[0026] The present invention also provides an immune monitoring system for risk assessment of sepsis prognosis in children, based on the prognosis of CD39+ regulatory T cell expression levels;

[0027] The CD39+ regulatory T cell expression level includes the ratio of CD39+ regulatory T cells to total regulatory T cells;

[0028] The prognosis determination rules include: relative to total regulatory T cells, the expression level of CD39+ regulatory T cells is below 12.45%, indicating that the patient has a higher risk of death.

[0029] In some specific embodiments of the present invention, the immune monitoring system detects the expression level of CD39+ Tregs in the patient's peripheral blood, determines the patient's prognosis in combination with a specific threshold, and helps doctors adjust treatment plans by dynamically monitoring immune changes during disease progression.

[0030] In some specific embodiments of the present invention, the above-mentioned immune monitoring system comprises:

[0031] A sample collection device for collecting peripheral blood samples;

[0032] Flow cytometry module, used to detect the percentage of CD39+ Tregs in total Tregs;

[0033] A data analysis module is used to analyze the patient's prognostic risk according to the percentage.

[0034] In some specific embodiments of the present invention, the data analysis module of the immune monitoring system also analyzes the patient's prognostic risk in combination with the ROC curve and the clinical scoring system.

[0035] In some specific embodiments of the present invention, the immune monitoring system can be used to dynamically monitor the CD39+ Tregs level of patients and perform real-time prognostic evaluation at different stages of treatment of childhood sepsis.

[0036] The present invention also provides a detection method for evaluating the prognosis risk of sepsis in children, which is based on the detection of CD39+ regulatory T cells (Tregs) in the patient's peripheral blood. The prognosis of children with sepsis is evaluated by analyzing the expression level of CD39+Tregs cells. When the CD39+Tregs expression level is below 12.45%, it indicates that the patient has a higher risk of death.

[0037] In some specific embodiments of the present invention, the above detection method is flow cytometry, comprising:

[0038] S1. Collect peripheral blood samples from patients;

[0039] S2, the percentage of CD39+ Tregs in Tregs obtained by flow cytometry;

[0040] S3. Determine the patient's immune status and prognosis based on the test results.

[0041] In some specific embodiments of the present invention, S3 of the above detection method further comprises judging the patient's prognosis in combination with ROC curve analysis.

[0042] In some specific embodiments of the present invention, the optimal critical value of the CD39+ Tregs in the above detection method is 12.45%. When the expression of CD39+ Tregs in the patient's peripheral blood is lower than this value, it indicates that the patient is at a higher risk of death.

[0043] In some specific embodiments of the present invention, the detection of the above detection method also includes dynamic monitoring of the patient at different time points, including collecting samples for testing within 24 hours, 48 ​​hours and 72 hours during the course of the disease.

[0044] In some specific embodiments of the present invention, the above detection method can be further combined with other clinical scoring systems (such as pSOFA and Phoenix score) to improve the accuracy of sepsis prognosis assessment.

[0045] In some specific embodiments of the present invention, the above detection method is applicable to children with sepsis, and can be used for the prognosis assessment of sepsis patients of other age groups.

[0046] This invention focuses on exploring the method of evaluating the prognosis of pediatric sepsis patients by the level of CD39+ regulatory T cells (Tregs). By detecting the proportion of CD39+ Tregs in peripheral blood, it is possible to identify high-risk sepsis patients early and track the changes in the patient's immune status in real time through dynamic monitoring. Dynamic monitoring can detect high-risk patients in the early stages of sepsis and help doctors make timely treatment decisions. CD39+ Tregs are important cells for immune regulation in sepsis patients, and their levels are closely related to the severity of sepsis. Compared with traditional inflammatory indicators (such as C-reactive protein and procalcitonin), the detection of CD39+ Tregs has higher specificity and sensitivity.

[0047] By dynamically monitoring the changing trend of CD39+ Tregs, the present invention enables doctors to understand the patient's immune status in real time and adjust the treatment strategy in a timely manner. The present invention can also be combined with existing clinical scoring systems (such as Phoenix and pSOFA scores) to further improve the accuracy of prognostic assessment.

[0048] The present invention is suitable for early screening and prognostic evaluation of sepsis patients, especially for children with sepsis. By detecting CD39+ Tregs, high-risk patients can be identified in time and sepsis-related mortality can be reduced. In addition, this method can also be combined with other prognostic evaluation tools to provide a reference for individualized treatment. With the development of detection technology, this method is expected to be widely used in clinical practice and become a standard tool for sepsis diagnosis and management.

[0049] In summary, the sepsis prognosis assessment method based on the CD39+ regulatory T cells (Tregs) level of the present invention can identify high-risk sepsis patients at an early stage by detecting the proportion of CD39+ Tregs in peripheral blood, and track the changes in the patient's immune status in real time through dynamic monitoring. The method is simple to operate, has high specificity and sensitivity, can provide a reliable prognosis assessment method for clinical practice, and provide a scientific basis for the formulation of personalized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0051] Figure 1 Indicate the inclusion criteria and grouping of children;

[0052] Figure 2 shows the percentage of CD39+ Tregs in children with sepsis;

[0053] Figure 3 shows the correlation analysis between the levels of CD39+ Tregs and Phoenix scores and pSOFA scores in children with sepsis;

[0054] Figure 4 The ROC curve is shown to analyze the predictive efficacy of CD39+ Tregs on in-hospital mortality in patients with sepsis;

[0055] Figure 5 The results show the relationship between CD39+ Tregs and the mortality outcome in children with sepsis;

[0056] FIG. 6 shows that CD39+ Tregs can maintain stable predictive efficacy within 72 hours. DETAILED DESCRIPTION

[0057] The present invention discloses a method for evaluating the prognosis of childhood sepsis using CD39+ regulatory T cells and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] The present invention proposes a method for evaluating the prognosis of childhood sepsis based on CD39+ Tregs, which determines and evaluates the prognosis risk of patients by detecting the level of CD39+ Tregs in the patient's peripheral blood. CD39+ Tregs can also be dynamically monitored at multiple time points, which can reflect the changes in the patient's immune status in real time, help predict the course of the disease and adjust the treatment plan.

[0059] The method for evaluating the prognosis of childhood sepsis based on CD39+ Tregs levels proposed in the present invention can screen high-risk patients at an early stage and provide a basis for personalized treatment. It has the advantages of high specificity, early detection and dynamic monitoring, and has a wide range of clinical application potential, especially suitable for early screening and prognosis evaluation of childhood sepsis patients. The specific technical scheme is as follows:

[0060] 1. Collect peripheral blood samples from children with sepsis for immune cell analysis;

[0061] 2. Flow cytometry was used to detect the percentage of CD39+ Tregs in peripheral blood. CD39+ Tregs refers to a subpopulation of regulatory T cells that express CD39 molecules, and its level is closely related to the immune status of sepsis;

[0062] 3. Prognostic risk assessment: When the CD39+ Tregs level is lower than 12.45%, it indicates that the patient has a higher risk of death; combined with the receiver operating characteristic (ROC) curve, 12.45% is determined as the optimal critical value to evaluate the patient's prognosis;

[0063] 4. Dynamically monitor the level of CD39+ Tregs at different time points (24 hours, 48 ​​hours, 72 hours), and conduct continuous prognostic evaluation in combination with the patient's clinical manifestations and scoring system. Dynamic detection can reflect changes in the patient's immune status and facilitate real-time adjustment of treatment plans;

[0064] 5. The method of the present invention can be combined with other clinical scoring systems (such as pSOFA and Phoenix score) to further improve the accuracy of prognosis prediction. By combining immune cell detection with organ function scoring, a more comprehensive risk assessment can be provided.

[0065] In some embodiments of the present invention, patients diagnosed with pediatric sepsis are recruited as research subjects, and peripheral blood samples are collected from the patients within 6 hours of admission.

[0066] In some embodiments of the present invention, the anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD25 antibody and anti-CD127 antibody are subjected to flow cytometry analysis, CD45 + CD3 + CD4 + CD25 + CD127 - CD39 + Represents CD39+ Tregs cells, and the proportion of CD39+ Tregs in peripheral blood was determined. The expression of CD39 in Tregs was detected by antibody staining combined with flow cytometry.

[0067] In some embodiments of the present invention, the ROC curve is used to evaluate the relationship between the level of CD39+ Tregs and the prognosis of sepsis. According to the analysis results, when the level of CD39+ Tregs is lower than 12.45%, it is determined that the patient faces a higher risk of death.

[0068] In some embodiments of the present invention, blood samples are collected from patients at different time points (24 hours, 48 ​​hours, and 72 hours) to dynamically monitor CD39+ Tregs. By monitoring their changes during the course of the disease, the strength of the patient's immune response is evaluated and clinical intervention strategies are guided.

[0069] This application uses flow cytometry to detect 99 children with sepsis, and divides them into two subgroups: Non-remote group and Remote group, and survival group and Non-survival group, and follows up for 28 days to evaluate the mortality rate. Flow cytometry analysis of the percentage of CD39+ Tregs cells found that the percentage of CD39+ Tregs in the Remote group was significantly lower than that in the Non-remote group, and in the death group was significantly lower than that in the survival group. The expression of CD39+ Tregs was significantly negatively correlated with sepsis-related scores. When the percentage of CD39+ Tregs cells in human peripheral blood lymphocytes is lower than 12.45%, children with sepsis may be at risk of death. CD39+ Tregs can maintain stable predictive efficacy within 72 hours.

[0070] A specific embodiment is a method for evaluating the prognosis of a patient with sepsis by detecting the level of CD39+ regulatory T cells (Tregs), specifically:

[0071] Study population: A total of 102 children with sepsis who were admitted to the PICU of the Children's Hospital Affiliated to Chongqing Medical University and met the inclusion and exclusion criteria between July 2022 and April 2024 were included. Inclusion criteria were: (1) diagnosis in accordance with the definition of "severe sepsis" or "septic shock" in the 2005 International Pediatric Sepsis Consensus Conference; (2) age older than 28 days and younger than 18 years; (3) the guardian of the child signed the informed consent form. Exclusion criteria were: (1) patients with autoimmune diseases, hematological malignancies, and immunodeficiency diseases; (2) children who had received immunotherapy (hormone shock, IVIG, immunosuppressants, monoclonal antibodies, etc.) or blood purification therapy (hemodiafiltration, perfusion, plasma exchange) before enrollment. In view of the fact that the pediatric sepsis definition working group of the Society of Critical Care Medicine (SCCM) released the new Phoenix sepsis standard for pediatric sepsis and septic shock in 2024, this example only analyzed 99 children who met the new Phoenix sepsis standard in 2024 out of 102 cases. According to whether the children had organ dysfunction far away from the primary infection site according to the 2024 standard, they were divided into Remote group and Non-remote group. According to the in-hospital outcomes of the children, they were divided into Survival group and Non-survival group. At the same time, according to the blood collection time, some immune indicators of children with different in-hospital outcomes in the early stage (<3 days before the course of the disease) (survival (e) group / nonsurvival (e) group) and children with different in-hospital outcomes in the late stage (>3 days before the course of the disease) (survival (l) group / nonsurvival (l) group) were analyzed. The basic information (sex, age), vital signs, test results (blood routine, biochemical, etiology, inflammatory indicators), Phoenix sepsis score, treatment plan, in-hospital outcome and 28-day survival of all children were recorded. Peripheral blood was obtained within 6 hours of enrollment. Blood samples were additionally obtained from some patients 24 hours, 48 ​​hours and 72 hours after enrollment.

[0072] Sample collection: Blood samples were collected from the enrolled children using ethylenediaminetetraacetic acid (EDTA) anticoagulation tubes, and peripheral blood mononuclear cells (PBMCs) were immediately separated by density gradient centrifugation using human lymphocyte separation medium for flow cytometry.

[0073] Flow cytometry: Add 1640 medium containing 2% fetal bovine serum to freshly extracted peripheral blood mononuclear cells (PBMCs) and mix well. Add 100 μL of the cell mixture to the flow cytometry tube so that each tube contains 10 6The cells were centrifuged, the supernatant was removed, and anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD25 antibody and anti-CD127 antibody were added for flow cytometry staining, and incubated at room temperature for 30 min. Cells were detected using a flow cytometer.

[0074] Analyze the data using flow cytometry analysis software (such as FlowJo). + CD3 + CD4 + CD25 + CD127 - CD39 + ) accounted for the total Tregs (CD45 + CD3 + CD4 + CD25 + CD127 - ) and recorded the percentage of CD39+ Tregs.

[0075] CD39+ Tregs level assessment: The test results show the percentage of CD39+ Tregs. When the CD39+ Tregs expression level is lower than 12.45%, it indicates that the patient has a higher risk of death. This critical value is based on the receiver operating characteristic (ROC) curve analysis and has good sensitivity and specificity.

[0076] Dynamic monitoring: In order to improve the monitoring effect of patient prognosis, it is recommended to collect and test blood samples at multiple time points during the course of sepsis, usually testing CD39+ Tregs levels again 24 hours, 48 ​​hours and 72 hours after admission. By dynamically monitoring the changing trend of CD39+ Tregs, the recovery or deterioration of the patient's immune function can be evaluated, and the progression of the disease can be judged. Patients with particularly low CD39+ Tregs levels or those that continue to decline during the course of the disease indicate a higher risk of death.

[0077] Combination with clinical scoring system: The method of the present invention can be used in combination with existing clinical scoring systems for sepsis (such as pSOFA and Phoenix score). By comparing the detection results of CD39+ Tregs with the clinical score, the accuracy of prognosis assessment can be improved. If the patient's CD39+ Tregs level is lower than 12.45% and the clinical score shows that the condition is serious, it can be more certain that the patient is at high risk of death, so that more active treatment measures can be taken.

[0078] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0079] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0080] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0081] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0082] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0083] In the flow cytometric analysis involved in the present application, the CD39+ Tregs ratio=CD39+ Tregs cell number / Tregs cell number×100%.

[0084] Abbreviations involved in this application: WBC, white blood cell; ANC, absolute neutrophil count; ALC, absolute lymphocyte count; CRP, C-reactive protein; PCT, procalcitonin; ALT, alanine transaminase; APTT, activated partial thromboplastin time; INR, international normalized ratio; OFI, organ failure index; PSS, phoenix sepsisscore; pSOFA, pediatric sequential failure organ failure assessment; D, day; CNS, centralnervous system; UTI, urinary tract infection; GCC, glucocorticoid; CRRT, continuous renal replacement therapy; IVIG, intravenous immunoglobulin.

[0085] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0086] The present invention will be further described below in conjunction with the embodiments.

[0087] Example 1: Detection of CD39+Tregs levels in children with sepsis

[0088] Samples to be tested: 99 children with sepsis were included in the study according to the inclusion and exclusion criteria ( Figure 1 ), clinical characteristics are shown in Table 1. Among them, the primary infection sites of children with sepsis in the Non-remote group (28 cases, 28.3%), Remote group (71 cases, 71.7%); Survival group (87 cases, 87.9%), and death group (12 cases, 12.1%) were mainly respiratory tract (67 cases, 67.7%), followed by digestive tract (20 cases, 20.2%) and central nervous system (9 cases, 9.1%). The main diagnosis of children was severe pneumonia (63 cases, 63.6%). All deaths in this application were in-hospital deaths, and half of the children died within 3 days after admission (6 cases, 50.0%).

[0089] Table 1: Clinical characteristics of the survival group and the death group

[0090]

[0091]

[0092]

[0093]

[0094] Data are presented as n (number, percentage), mean ± SD, or median (first quartile, third quartile).

[0095] Materials: Fluorescently labeled FITC anti-CD45 antibody, anti-CD3 (APC-Cy7) antibody, anti-CD4 (BV650) antibody, anti-CD8 (BV510) antibody, anti-CD25 (BV421) antibody, anti-CD127 (APC-R700) antibody and CD39 (PE-CF594) antibody were purchased from BD Pharmingen; human lymphocyte separation medium (Cedarlane) was purchased from Beijing Dakoway Biotechnology Co., Ltd.

[0096] Experimental methods:

[0097] 1 mL of anticoagulated peripheral blood sample was taken for peripheral blood mononuclear cell separation, and the separated PBMCs were subjected to flow cytometry detection.

[0098] Take 100 μL containing 10 6 1 μL each of FITC-containing anti-CD45 antibody, anti-CD3 (APC-Cy7) antibody, anti-CD4 (BV650) antibody, anti-CD8 (BV510) antibody, anti-CD25 (BV421) antibody and anti-CD127 (Alexa Flour700) were added to each PBMCs cell. After incubation at room temperature for 30 min, 1 mL of PBS was added to each cell. The cells were centrifuged at 500 g for 5 min, washed once, and then suspended in 200 μL PBS. The cells were then placed on a flow cytometer and the results were analyzed.

[0099] Results Analysis: The percentage of CD39+Tregs in children with sepsis was shown in Figure 2A to Figure 2C . Figure 2A The gating strategy of CD39+ Tregs in this example is shown. Figure 2B The results showed that the expression of CD39+ Tregs in the Remote group was significantly lower than that in the nonromote group. Figure 2C The results showed that CD39+ Tregs cells decreased in the death group. In summary, children with sepsis who progressed or died had low expression of CD39+ Tregs cells.

[0100] Example 2: The level of CD39+ Tregs in children with sepsis is negatively correlated with Phoenix score and pSOFA score

[0101] The 2024 Phoenix criteria for the diagnosis of pediatric sepsis and septic shock by the Society of Critical Care Medicine (SCCM) stipulate that a Phoenix sepsis score of 2 or more indicates that children with suspected or confirmed infection have potentially life-threatening organ dysfunction in the respiratory, cardiovascular, coagulation, and / or neurological systems.

[0102] In the sepsis diagnostic standard Sepsis 3.0, the pSOFA score is an important indicator for diagnosing sepsis. A large amount of research data shows that for patients with infection or suspected infection in the ICU, when the pSOFA score is ≥ 2 points, they are diagnosed with sepsis. Not only that, pSOFA reflects the degree of multiple organ dysfunction in patients with multiple organ dysfunction syndrome (MODS) and is closely related to the patient's hospitalization rate.

[0103] Figure 3A to Figure 3C The following is a correlation analysis between the level of CD39+ Tregs in children with sepsis and the Phoenix score and pSOFA score. Figure 3A As shown, the correlation coefficient between the percentage change of CD39+ Tregs in children with sepsis and Phoenix sepsisscores (PSS)-4 score was r = -0.31, P < 0.002. Figure 3B The results showed that the correlation coefficient between the percentage of CD39+Tregs and PSS-8 score in children with sepsis was r = -0.31, P < 0.002. Figure 3C Correlation analysis showed that the correlation coefficient between the expression level of CD39+ Tregs in children with sepsis and their pSOFA scores was r = -0.35, P = 0.004. In summary, the level of CD39+ Tregs in children with sepsis was negatively correlated with Phoenix scores and pSOFA scores.

[0104] Example 3: ROC curve analysis found that CD39+ Tregs have a good predictive effect on in-hospital mortality in children with sepsis

[0105] The laboratory parameters and clinical data of children with sepsis were collected, and the ROC curve was used to analyze the diagnostic efficacy of CD39+ Tregs for in-hospital mortality in children with sepsis.

[0106] Figure 4 The ROC curve in this example is used to analyze the predictive efficacy of CD39+ Tregs in sepsis patients for in-hospital mortality. Figure 4 As shown in Table 2, ROC curve analysis found that CD39+ Tregs had a good predictive effect on in-hospital mortality in children with sepsis, with an area under the curve of 0.8563 (95% CI: 0.7719-0.9408, P <0.0001), and the optimal critical value was 12.45%, with a sensitivity of 91.7% and a specificity of 73.6%. From the above statistical results, it was found that the area under the curve of CD39+ Tregs for the diagnosis of childhood sepsis was >0.85, with a good diagnostic efficacy and statistical significance, indicating that CD39+ Tregs had a good predictive effect on in-hospital mortality in children with sepsis.

[0107] Table 2

[0108]

[0109] Example 4: Relationship between CD39+ Tregs and mortality outcomes in children with sepsis

[0110] The relationship between CD39+ Tregs and the mortality outcome of children with sepsis was further observed, and the Kaplan-Meier survival curve of children with sepsis was prepared. Figure 5 The results showed that the mortality rate in the group with CD39+ Tregs≤12.45% was increased compared with that in the group with CD39+ Tregs>12.45%, and the difference was statistically significant.

[0111] Example 5: CD39 + Tregs can maintain stable predictive efficacy over 72 hours

[0112] To explore whether CD39+Tregs as a potential predictive factor has stable predictive value in the short term, the changes of CD39+ Tregs in children with sepsis at different stages and with different prognoses were analyzed, and the levels of CD39+ Tregs in 16 children with sepsis were randomly monitored within 72 hours. Fig. 6A The results showed that the percentage of CD39+Tregs in the early death group of children with sepsis was lower than that in the survival group (P<0.05). Figure 6B The results showed that the percentage of CD39+Tregs in the late death group of children with sepsis was lower than that in the survival group (P<0.05). Figure 6C The results showed that compared with the survival group, the CD39+Tregs in the death group continued to be dynamically low expressed within 72 hours after enrollment, and the difference was statistically significant.

[0113] In summary, this application provides a method for evaluating the prognosis of sepsis based on CD39+ regulatory T cells. By detecting the level of CD39+ Tregs in peripheral blood by flow cytometry, combined with dynamic monitoring and clinical scoring systems, the prognosis of sepsis patients can be effectively evaluated. This method has high specificity and sensitivity, is particularly suitable for early risk assessment of pediatric sepsis patients, and provides a scientific basis for the formulation of clinical personalized treatment plans.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of CD39+ regulatory T cells as markers in the preparation of pediatric sepsis prognosis products.

2. The use according to claim 1, characterized in that The prognosis determination rules include: if the proportion of CD39+ regulatory T cells to total regulatory T cells is below 12.45%, it indicates that the patient has a higher risk of death.

3. The use according to claim 1 or 2, characterized in that: The CD39+ regulatory T cells are CD39+ regulatory T cells derived from peripheral blood.

4. Use of antibodies in the preparation of a product for the prognosis of childhood sepsis, wherein the antibodies include anti-CD39 antibodies.

5. The use according to claim 4, characterized in that The antibodies also include anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies and anti-CD127 antibodies.

6. The use according to claim 4 or 5, characterized in that Based on the antibody, the ratio of CD39+ regulatory T cells in the total regulatory T cells in the sample to be tested is obtained by flow cytometry to obtain a prognostic result; The prognostic result determination rule includes: if the proportion is below 12.45%, it indicates that the patient has a higher risk of death.

7. A kit for the prognosis of sepsis in children, characterized in that: CD39+ regulatory T cells were used as markers.

8. The kit according to claim 7, characterized in that Using specific antibodies to capture regulatory T cells, obtaining the proportion of CD39+ regulatory T cells in total regulatory T cells, and then making a prognosis based on the proportion; The prognosis determination rules include: if the proportion is below 12.45%, it indicates that the patient has a higher risk of death; The specific antibodies include: (i), anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies and anti-CD127; and / or (ii), anti-CD39 antibody, anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD25 antibody and anti-CD127.

9. A device for the prognosis of sepsis in children, characterized in that: CD39+ regulatory T cells are used as markers and are coated with specific antibodies that capture CD39+ regulatory T cells.

10. An immune monitoring system for risk assessment of sepsis prognosis in children, characterized in that: Prognosis based on CD39+ regulatory T cell expression levels; The prognosis determination rules include: if the CD39+ regulatory T cell expression level is below 12.45%, it indicates that the patient has a higher risk of death; The CD39+ regulatory T cell expression level is the ratio of CD39+ regulatory T cells to total regulatory T cells.

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