Use of cd39+ regulatory t cells as a marker in a product for the assessment of prognosis in pediatric sepsis

Abstract

This invention relates to the field of medical immunology, and particularly to the application of CD39+ regulatory T cells as a biomarker in products for prognostic assessment of childhood sepsis. This invention is based on flow cytometry detection of CD39+ T cells in peripheral blood of patients. + Treg levels, and dynamically assess patient prognosis based on changes in their expression. CD39 + Treg expression levels were significantly negatively correlated with childhood sepsis scores (Phoenix and pSOFA scores) and negatively correlated with mortality rates in children with sepsis. When CD39... + When Treg expression is below 12.45%, it indicates a higher risk of death in the patient. The method of this invention can provide accurate prognostic assessment in the early screening of childhood sepsis, with high specificity and sensitivity. This method can also be used for dynamic monitoring of patients, helping doctors to adjust treatment plans in a timely manner and optimize personalized treatment, demonstrating significant practical application value.

Description

Technical Field

[0001] This invention relates to the field of medical immunology, and in particular to a method for prognostic assessment and application of CD39+ regulatory T cells in children with sepsis. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, often leading to multiple 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, accurately and effectively predicting the prognosis of patients remains a core challenge in clinical treatment.

[0003] Currently, prognostic assessment of sepsis mainly relies on clinical scoring systems (such as the pSOFA score and the Phoenix sepsis score). However, these systems are primarily based on organ dysfunction and cannot reflect individual immune responses and immune imbalances. Therefore, identifying new, highly specific immune biomarkers to assess the prognosis of sepsis patients is crucial for improving the effectiveness of clinical interventions.

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

[0005] Existing methods for assessing sepsis prognosis largely rely on clinical indicators, such as the patient's hemodynamic status and the severity of organ dysfunction. However, these indicators typically only become apparent when the condition has already worsened, making early prediction impossible. Furthermore, while conventional biomarkers (such as C-reactive protein and procalcitonin) can reflect the body's inflammatory state, their low specificity makes it difficult to accurately assess an individual's immune response and prognosis.

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

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

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of CD39+ regulatory T cells as biomarkers in the preparation of prognostic products for childhood sepsis.

[0010] In some specific embodiments of the present invention, the prognostic criteria for the above-mentioned application include: a CD39+ regulatory T cell expression level of less than 12.45% relative to total regulatory T cells indicates 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 the preparation of prognostic products for childhood sepsis, said antibodies including anti-CD39 antibodies.

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

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

[0015] The prognostic criteria include: a CD39+ regulatory T cell expression level of less than 12.45% relative to total regulatory T cells indicates a higher risk of death for the patient.

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

[0017] This invention also provides a kit for the prognosis of sepsis in children, using CD39+ regulatory T cells as a marker.

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

[0019] The prognostic criteria include: a CD39+ regulatory T cell expression level of less than 12.45% relative to total regulatory T cells indicates a higher risk of death for the patient;

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

[0021] In some specific embodiments of the present invention, the kit contains a combination of specific antibodies for detecting CD39+ regulatory T cells in the peripheral blood of patients, which are then detected by flow cytometry and used to assess the prognosis of patients.

[0022] In some specific embodiments of the present invention, the kit further includes reagents for sample processing, buffer solutions, 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 kit includes antibodies against CD45, CD3, CD4, CD8, CD25, and CD127 to identify and measure the CD39+ regulatory T cell subsets of a patient.

[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 of sepsis in children.

[0025] The present invention also provides a device for the prognosis of sepsis in children, which uses CD39+ regulatory T cells as a marker and is coated with a specific antibody that captures CD39+ regulatory T cells.

[0026] This invention also provides an immune monitoring system for assessing the prognostic risk of childhood sepsis, based on the prognostic level of CD39+ regulatory T cell expression;

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

[0028] The prognostic criteria include: a CD39+ regulatory T cell expression level of less than 12.45% relative to total regulatory T cells indicates a higher risk of death for the patient.

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

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

[0031] Sample collection device for collecting peripheral blood samples;

[0032] The flow cytometry detection module is used to detect the percentage of CD39+ Tregs in the total Tregs;

[0033] The data analysis module is used to analyze the patient's prognostic risk based on the percentage.

[0034] In some specific embodiments of the present invention, the data analysis module of the above-mentioned immune monitoring system also combines ROC curves and clinical scoring systems to analyze the patient's prognostic risk.

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

[0036] The present invention also provides a detection method for prognostic risk assessment of childhood sepsis, which is based on the detection of CD39+ regulatory T cells (Tregs) in the peripheral blood of patients. By analyzing the expression level of CD39+ Tregs cells, the prognosis of children with sepsis is assessed. When the expression level of CD39+ Tregs is below 12.45%, it indicates that the patient has a high risk of death.

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

[0038] S1. Collect peripheral blood samples from the patient;

[0039] S2. The percentage of CD39+ Tregs among all 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 includes combining ROC curve analysis to determine the patient's prognosis.

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

[0043] In some specific embodiments of the present invention, the detection method described above further includes dynamic monitoring of the patient at different time points, including collecting samples for detection at 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 scores) to improve the accuracy of sepsis prognosis assessment.

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

[0046] This invention focuses on exploring a method for assessing the prognosis of children with sepsis by detecting the level of CD39+ regulatory T cells (Tregs). By detecting the proportion of CD39+ Tregs in peripheral blood, high-risk sepsis patients can be identified early, and dynamic monitoring can track changes in the patient's immune status in real time. Dynamic monitoring can identify high-risk patients in the early stages of sepsis, helping 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 markers (such as C-reactive protein and procalcitonin), the detection of CD39+ Tregs has higher specificity and sensitivity.

[0047] This invention allows physicians to monitor the changing trends of CD39+ Tregs in real time, enabling them to understand the patient's immune status and adjust treatment strategies accordingly. Furthermore, this invention can be integrated with existing clinical scoring systems (such as the Phoenix and pSOFA scores) to further improve the accuracy of prognostic assessment.

[0048] This invention is applicable to early screening and prognostic assessment of sepsis patients, especially children with sepsis. By detecting CD39+ Tregs, high-risk patients can be identified promptly, reducing sepsis-related mortality. Furthermore, this method can be combined with other prognostic assessment 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, becoming a standard tool for the diagnosis and management of sepsis.

[0049] In summary, this invention provides a sepsis prognostic assessment method based on CD39+ regulatory T cells (Tregs). By detecting the proportion of CD39+ Tregs in peripheral blood, it can identify high-risk sepsis patients at an early stage and track changes in the patient's immune status in real time through dynamic monitoring. This method is simple to operate, has high specificity and sensitivity, and can provide a reliable prognostic assessment tool for clinicians, as well as a scientific basis for the development of personalized treatment plans. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 Show 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 CD39+ Tregs levels and Phoenix score and pSOFA score in children with sepsis.

[0054] Figure 4 ROC curve analysis was performed to assess the predictive efficacy of CD39+ Tregs for in-hospital mortality in sepsis patients.

[0055] Figure 5 This study demonstrates the relationship between CD39+ Tregs and mortality outcomes in children with sepsis.

[0056] Figure 6 shows that CD39+ Tregs can maintain stable predictive performance over 72 hours. Detailed Implementation

[0057] This invention discloses a method for prognostic assessment and application of CD39+ regulatory T cells in children with sepsis. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] This invention proposes a prognostic assessment method for childhood sepsis based on CD39+ Tregs. This method assesses the patient's prognostic risk by detecting the level of CD39+ Tregs in the patient's peripheral blood. It also allows for dynamic monitoring of CD39+ Tregs at multiple time points, reflecting changes in the patient's immune status in real time, which helps predict disease progression and adjust treatment plans.

[0059] The prognostic assessment method for childhood sepsis based on CD39+ Treg levels proposed in this invention can screen high-risk patients at an early stage, providing a basis for personalized treatment. It has advantages such as high specificity, early detection, and dynamic monitoring, and has broad clinical application potential, especially suitable for early screening and prognostic assessment of childhood sepsis patients. The specific technical solution 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 refer to the regulatory T cell subset that expresses CD39 molecules, and their level is closely related to the immune status of sepsis.

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

[0063] 4. Dynamically monitor CD39+ Treg levels at different time points (24 hours, 48 ​​hours, and 72 hours), and combine this with the patient's clinical manifestations and scoring system for continuous prognostic assessment. Dynamic monitoring can reflect changes in the patient's immune status, facilitating real-time adjustments to the treatment plan;

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

[0065] In some embodiments of the present invention, patients diagnosed with childhood 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 analyzed by flow cytometry. + CD3 + CD4 + CD25 + CD127 - CD39 + The proportion of CD39+ Tregs in peripheral blood was measured, representing CD39+ Tregs cells. The expression of CD39 in Tregs was detected by antibody staining combined with flow cytometry.

[0067] In some embodiments of the present invention, ROC curves are used to assess the relationship between CD39+ Treg levels and sepsis prognosis. Based on the analysis results, when CD39+ Treg levels are below 12.45%, the patient is considered to face 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) for dynamic monitoring of CD39+ Tregs. By monitoring changes in these Tregs during the course of the disease, the strength of the patient's immune response is assessed, and clinical intervention strategies are guided.

[0069] This application used flow cytometry to examine 99 children with sepsis, dividing them into a Non-remote group and a Remote group, and a survival group and a Non-survival group, with a 28-day follow-up to assess mortality. Flow cytometry analysis of the percentage of CD39+ Treg cells revealed that the percentage of CD39+ Treg cells was significantly lower in the Remote group than in the Non-remote group, and significantly lower in the death group than in the survival group. CD39+ Treg expression was significantly negatively correlated with sepsis-related scores. Children with sepsis may have a risk of death when the percentage of CD39+ Treg cells in human peripheral blood lymphocytes is below 12.45%. CD39+ Tregs maintained stable predictive efficacy over 72 hours.

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

[0071] Study population: 102 children with sepsis who were admitted to the PICU of Children's Hospital of Chongqing Medical University between July 2022 and April 2024 and met the inclusion and exclusion criteria. Inclusion criteria were: (1) diagnosis meeting the definition of "severe sepsis" or "septic shock" in the 2005 International Pediatric Sepsis Consensus Conference; (2) age greater than 28 days and less than 18 years; (3) informed consent signed by the child's guardian. Exclusion criteria were: (1) patients with autoimmune diseases, hematologic malignancies, or immunodeficiency diseases; (2) children who had received immunotherapy (hormone pulse, IVIG, immunosuppressants, monoclonal antibodies, etc.) or blood purification therapy (hemodialysis filtration, perfusion, plasma exchange) before enrollment. Given that the Society of Critical Care Medicine (SCCM) Pediatric Sepsis Definition Working Group released the new Phoenix sepsis criteria for childhood sepsis and septic shock in 2024, this embodiment only analyzed 99 children out of 102 who met the new 2024 Phoenix sepsis criteria. Children were divided into Remote and Non-remote groups based on whether they had organ dysfunction far from the primary infection site according to the 2024 criteria. They were also divided into Survival and Non-survival groups based on their in-hospital outcomes. Furthermore, some immune indicators were analyzed based on blood collection time for children with different in-hospital outcomes in the early stage (<3 days before the onset of illness) (survival(e) group / nonsurvival(e) group) and in the late stage (>3 days before the onset of illness) (survival(l) group / nonsurvival(l) group). Record all basic information (gender, age), vital signs, laboratory results (complete blood count, biochemistry, etiology, inflammatory markers), Phoenix sepsis score, treatment plan, in-hospital outcome, and 28-day survival for all children. Peripheral blood samples were collected from children within 6 hours of enrollment. Additional blood samples were collected from some patients at 24, 48, and 72 hours after enrollment.

[0072] Sample collection: Blood samples from enrolled children were collected using EDTA anticoagulant tubes. Peripheral blood mononuclear cells (PBMCs) were immediately separated by density gradient centrifugation using human lymphocyte separation medium and then subjected to flow cytometry.

[0073] Flow cytometry assay: 1640 medium containing 2% fetal bovine serum was added to freshly extracted peripheral blood mononuclear cells (PBMCs) and mixed thoroughly. 100 μL of the cell mixture was then added to flow cytometry tubes, ensuring each tube contained 10 cells. 6Cells were collected. After centrifugation and removal of the supernatant, anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-CD127 antibodies were added for flow cytometry staining. The cells were incubated at room temperature for 30 min. Cell analysis was performed using a flow cytometer.

[0074] Data were analyzed using flow cytometry software (such as FlowJo). CD39+ Tregs (CD45+ Tregs) were identified. + CD3 + CD4 + CD25 + CD127 - CD39 + ) accounts for a total of Tregs (CD45) + CD3 + CD4 + CD25 + CD127 - The percentage of CD39+ Tregs was recorded.

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

[0076] Dynamic monitoring: To improve the monitoring of patient prognosis, it is recommended to collect and test blood samples at multiple time points during the course of sepsis, typically repeating CD39+ Treg levels at 24, 48, and 72 hours after admission. By dynamically monitoring the trend of CD39+ Treg changes, the recovery or deterioration of the patient's immune function can be assessed, thereby determining disease progression. Patients with particularly low CD39+ Treg levels or those whose levels continue to decline throughout the course of the disease indicate a higher risk of death.

[0077] Integration with Clinical Scoring Systems: The method of this invention can be used in conjunction with existing sepsis clinical scoring systems such as pSOFA and Phoenix scores. By comparing the results of CD39+ Tregs testing with clinical scores, the accuracy of prognostic assessment can be improved. If a patient's CD39+ Tregs level is below 12.45% and the clinical score indicates severe illness, there is greater certainty that the patient faces a high risk of death, thus allowing for more aggressive treatment measures.

[0078] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0080] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0081] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

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

[0083] In the flow cytometry analysis involved in this 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 this invention are all commercially available products and can be purchased from the market.

[0086] The present invention will be further illustrated below with reference to the embodiments.

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

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

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

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[0094] Data are expressed as n (quantity, percentage), mean ± standard deviation, or median (first quartile, third quartile).

[0095] Materials: FITC-labeled 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 Dakowei Biotechnology Co., Ltd.

[0096] Experimental methods:

[0097] One mL of anticoagulated peripheral blood sample was used for peripheral blood mononuclear cell (PBMC) isolation, and the isolated PBMCs were analyzed by flow cytometry.

[0098] Take 100 μL containing 10 6 For each PBMC, add 1 μL each of FITC-conjugated 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). After incubating at room temperature for 30 min, add 1 mL of PBS to each cell, centrifuge at 500g for 5 min, wash once, add 200 μL of PBS to suspend the cells, and perform flow cytometry to analyze the results.

[0099] Results analysis: The percentage of CD39+Tregs in children with sepsis is shown in the table below. Figures 2A-2C . Figure 2A This embodiment illustrates the gate strategy of CD39+ Tregs. Figure 2B The results showed that CD39+ Tregs expression was significantly lower in the Remote group than in the nonromote group. Figure 2C The results showed a decrease in CD39+ Treg cells in the death group. In summary, children with sepsis who progressed or died exhibited low expression of CD39+ Treg cells.

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

[0101] The Society of Critical Care Medicine (SCCM) adopted the Phoenix criteria for the diagnosis of sepsis and septic shock in children in 2024. A Phoenix sepsis score of 2 or higher indicates potential life-threatening organ dysfunction in the respiratory, cardiovascular, coagulation, and / or neurological systems of a child with suspected or confirmed infection.

[0102] In the Sepsis 3.0 diagnostic criteria, the pSOFA score is an important indicator for diagnosing sepsis. Extensive research data shows that for ICU patients with infection or suspected infection, a pSOFA score ≥2 is sufficient for a diagnosis of sepsis. Furthermore, the pSOFA reflects the degree of dysfunction of multiple organs in patients with multiple organ dysfunction syndrome (MODS) and is closely related to the patient's hospitalization rate.

[0103] Figures 3A-3C This example illustrates the correlation analysis between CD39+ Treg levels and Phoenix and pSOFA scores in children with sepsis. Figure 3A As shown, the percentage change in CD39+ Tregs in children with sepsis was correlated with the Phoenix sepsisscores (PSS)-4 score, with a correlation coefficient of r = -0.31, P < 0.002. Figure 3B The results showed that the percentage of CD39+Tregs in children with sepsis was correlated with the PSS-8 score by a coefficient of r = -0.31, P < 0.002. Figure 3C Correlation analysis revealed a correlation coefficient (r = -0.35, P = 0.004) between CD39+ Treg expression levels and pSOFA scores in children with sepsis. In conclusion, CD39+ Treg levels in children with sepsis are negatively correlated with both Phoenix and pSOFA scores.

[0104] Example 3: ROC curve analysis revealed that CD39+ Tregs have good predictive efficacy for in-hospital mortality in children with sepsis.

[0105] Laboratory and clinical data of children with sepsis were collected. ROC curve analysis was used to assess the diagnostic efficacy of CD39+ Tregs for in-hospital mortality in children with sepsis.

[0106] Figure 4 This embodiment demonstrates the predictive power of ROC curve analysis on in-hospital mortality in sepsis patients using CD39+ Tregs. Figure 4 As shown in Table 2, ROC curve analysis revealed that CD39+ Tregs have good predictive efficacy for 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). The optimal cutoff value was 12.45%, at which point the sensitivity was 91.7% and the specificity was 73.6%. These statistical results indicate that for the diagnosis of sepsis in children, a CD39+ Tregs area under the curve > 0.85 indicates excellent diagnostic efficacy and is statistically significant, demonstrating that CD39+ Tregs have good predictive efficacy for 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] Further observation was conducted on CD39+ Tregs and mortality outcomes in children with sepsis, and Kaplan-Meier survival curves were constructed for children with sepsis. Figure 5 The results showed that the mortality rate was higher in the group with CD39+ Tregs ≤ 12.45% compared with the group with CD39+ Tregs > 12.45%, and the difference was statistically significant.

[0111] Example 5: CD39 + Tregs can maintain stable predictive performance for 72 hours.

[0112] To explore whether CD39+ Tregs, as a potential predictor, have stable predictive value in the short term, we analyzed the changes in CD39+ Tregs in children with sepsis at different stages and with different prognoses, and randomly monitored the level of CD39+ Tregs in 16 children with sepsis over 72 hours. Figure 6A The results showed that the percentage of CD39+Tregs was lower in the early death group of children with sepsis than in the survival group (P<0.05). Figure 6B The percentage of CD39+Tregs was lower in the late-stage death group of children with sepsis compared to the survival group (P<0.05). Figure 6C The results showed that, compared with the survival group, CD39+Tregs in the death group remained dynamically low for 72 hours after enrollment, and the difference was statistically significant.

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

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immunomonitoring system for the prognostic risk assessment of sepsis in children, characterized in that, Prognosis based on CD39+ regulatory T cell expression level; The determination rule of the prognosis comprises: if the CD39+ regulatory T cell expression level is 12.45% or less, it indicates that the patient has a higher risk of death; The CD39+ regulatory T cell expression level is the proportion of CD39+ regulatory T cells in total regulatory T cells; CD45 + CD3 + CD4 + CD25 + CD127 - CD39 + CD45 + CD3 + CD4 + CD25 + CD127 - .

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