Application of plasma cell-like dendritic cell as marker in children sepsis prognosis product

Abstract

The invention relates to the technical field of medical immunology, in particular to application of plasma cell-like dendritic cells as markers in children sepsis prognosis products. The present invention detects the pDC level in the peripheral blood of a patient based on flow cytometry and assesses the prognosis of the patient based on its expression level. The pDC expression level is in significant positive correlation with children sepsis scores (Phoenix and pSOFA scores), and is in positive correlation with the death rate of children suffering from sepsis. When the expression of the pDC is higher than 0.96%, 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, has high specificity and sensitivity, helps doctors to adjust treatment schemes in time and optimize 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 prognosis evaluation of plasmacytoid dendritic cells (pDCs) for childhood sepsis and an application method thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which mainly leads to multiple organ dysfunction. Especially in pediatric patients, sepsis has a high morbidity and mortality rate (Singer M, et al. The ThirdInternational Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Jama 2016;315;Karakike E, et al. The early change of SOFA score as aprognostic marker of 28-day sepsis mortality: analysis through a derivationand a validation cohort. Critical Care 2019;23;Rudd KE, et al. Global,regional, and national sepsis incidence and mortality, 1990-2017: analysisfor 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 predict the prognosis of children and provide personalized treatment remains a major challenge in clinical treatment due to the rapid progression of sepsis and complex clinical manifestations.

[0003] The current prognostic assessment of sepsis mainly relies on clinical scoring systems, such as pSOFA (sepsis-related organ failure assessment) and Phoenix sepsis score. However, these systems are mainly based on the patient's organ dysfunction to help clinicians assess the severity of the disease. However, these methods often lack a timely reflection of the state of the immune system, especially the immune imbalance of sepsis patients in the early stages. This makes it difficult for doctors to identify patients with severe immune impairment in the early stages of the disease, thus missing the opportunity for timely intervention.

[0004] Plasmacytoid dendritic cells (pDCs) are an important cell type in the human immune system, responsible for initiating antiviral immune responses and mediating immunoregulatory functions in viral infections and systemic inflammatory responses. pDCs play an important immunoregulatory role in sepsis. The dynamic changes in pDC levels can reflect the immune status of sepsis patients and their ability to clear pathogens. The dynamic changes in pDCs are closely related to the progression of the patient's disease. When pDC levels are abnormally elevated, it often indicates that the patient is facing a serious immune imbalance and may lead to an aggravation of the immunosuppressive response, which is closely related to the mortality rate of sepsis patients.

[0005] Existing methods for evaluating the prognosis of sepsis mainly rely on the patient's organ function scoring system and conventional inflammatory markers, but these methods often cannot effectively reflect the state of the patient's immune system, especially the early immunosuppression phenomenon. Immune imbalance in sepsis patients is one of the main causes of death, but conventional clinical markers such as C-reactive protein and procalcitonin have low sensitivity in assessing the state of immunosuppression. In addition, although the role of type I interferon in regulating immune response has been widely studied, its dynamic relationship with pDC and its role in the prognosis of sepsis have not been fully explored.

[0006] Therefore, there is an urgent need to develop a detection method based on immunoregulatory cells that can accurately assess the immune status and disease progression of pediatric sepsis patients, thereby helping doctors make more accurate prognostic judgments in the early stages of the disease. Summary of the invention

[0007] In view of this, the present invention provides a method for evaluating the prognosis of childhood sepsis based on pDC (plasmacytoid dendritic cells). The method uses flow cytometry to detect the level of pDC in the patient's peripheral blood, and determines its optimal critical value of 0.96% based on the ROC curve. When the patient's pDC level is higher than this value, it indicates that the patient faces a higher risk of death. The innovation of this method lies in its high specificity and sensitivity, which 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 plasmacytoid dendritic cells as markers in the preparation of a child sepsis prognosis product.

[0010] In some specific embodiments of the present invention, the above-mentioned application of the prognosis determination rule includes: when the ratio of plasmacytoid dendritic cells to total lymphocytes is higher than 0.96, it indicates that the patient has a higher risk of death.

[0011] In some specific embodiments of the present invention, the plasmacytoid dendritic cells used in the above application are plasmacytoid dendritic cells derived from peripheral blood.

[0012] The present invention also provides the use of antibodies in the preparation of a child sepsis prognosis product, wherein the antibodies include anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

[0013] In some specific embodiments of the present invention, the above application is based on the antibody to obtain the proportion of plasmacytoid dendritic cells in total lymphocytes in the sample to be tested by flow cytometry to obtain the prognostic result;

[0014] The judgment rule of the prognosis result includes: when the proportion is higher than 0.96, it indicates that the patient has a higher risk of death.

[0015] In some specific embodiments of the present invention, the sample to be tested in the above application is a peripheral blood sample.

[0016] The present invention also provides a kit for the prognosis of childhood sepsis, using plasmacytoid dendritic cells as a marker.

[0017] In some specific embodiments of the present invention, the above-mentioned kit uses specific antibodies to capture cells in a peripheral blood sample, obtains the proportion of plasmacytoid dendritic cells in total lymphocytes, and then makes a prognosis based on the proportion;

[0018] The prognosis determination rule includes: when the proportion is higher than 0.96, it indicates that the patient has a higher risk of death;

[0019] The specific antibodies include anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

[0020] In some specific embodiments of the present invention, the above diagnostic reagent comprises a specific antibody combination for detecting plasmacytoid dendritic cells (pDC) in the patient's peripheral blood, combined with flow cytometry for detection, and is used to evaluate the patient's prognosis.

[0021] In some specific embodiments of the present invention, the diagnostic kit further comprises reagents for sample processing, buffers, and fluorescent dyes for antibody labeling, so as to accurately detect the expression level of pDC in flow cytometry.

[0022] In some specific embodiments of the present invention, the above diagnostic kit includes anti-CD3 antibodies, anti-CD19 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies to identify and measure pDC subpopulations of patients.

[0023] In some specific embodiments of the present invention, the above-mentioned diagnostic kit is suitable for early risk assessment of sepsis patients, and is particularly suitable for early screening and prognosis judgment of sepsis in children.

[0024] The present invention also provides a device for the prognosis of childhood sepsis, which uses plasmacytoid dendritic cells as markers and is coated with specific antibodies for capturing plasmacytoid dendritic cells.

[0025] In some specific embodiments of the present invention, the specific antibodies in the above device include anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

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

[0027] The prognosis determination rule includes: when the expression level of the plasmacytoid dendritic cells is higher than 0.96, it indicates that the patient has a higher risk of death;

[0028] The plasmacytoid dendritic cell expression level is the ratio of plasmacytoid dendritic cells to total lymphocytes.

[0029] In some specific embodiments of the present invention, the immune monitoring system detects the expression level of pDC 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 detection module, used to detect the percentage of pDC in total lymphocytes;

[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 pDC 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 plasmacytoid dendritic cells (pDC) in the patient's peripheral blood. The prognosis of children with sepsis is evaluated by analyzing the expression level of pDC cells. When the pDC expression level is higher than 0.96%, it indicates that the patient has a higher risk of death.

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

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

[0039] S2, the percentage of pDC in total lymphocytes was 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 pDC in the above detection method is 0.96%. When the pDC expression in the patient's peripheral blood is higher 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 above detection method also includes dynamic monitoring of the patient at different time points, specifically 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] By monitoring the level of pDC, the present invention can detect high-risk patients in the early stage of sepsis and help doctors make timely treatment decisions. pDC is an important cell for immune regulation in sepsis patients, and its level is closely related to the severity of sepsis. Compared with traditional inflammatory indicators (such as C-reactive protein and procalcitonin), the detection of pDC has higher specificity and sensitivity.

[0047] The present invention proposes to dynamically monitor pDC 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.

[0048] The present invention is suitable for early screening and prognostic evaluation of sepsis patients, especially for children with sepsis. By detecting pDC, 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 pDC levels of the present invention can identify high-risk sepsis patients at an early stage by detecting the proportion of pDCs 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 pDCs in children with sepsis;

[0053] Figure 3 shows the correlation analysis between the pDC level and Phoenix score and pSOFA score in children with sepsis;

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

[0055] Figure 5 The relationship between pDC and death outcome in children with sepsis was shown;

[0056] FIG. 6 shows that pDCs can be dynamically monitored over a 72-hour period. DETAILED DESCRIPTION

[0057] The present invention discloses a method for evaluating the prognosis of sepsis in children based on pDC. 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 pDCs, which determines and evaluates the prognosis risk of patients by detecting the pDC level in the peripheral blood of patients.

[0059] By dynamically monitoring the changing trend of pDC, doctors can understand the patient's immune status in real time and adjust the treatment strategy in time. 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.

[0060] The specific technical solutions are as follows:

[0061] 1. Collect peripheral blood samples from children with sepsis for immune cell analysis;

[0062] 2. Flow cytometry was used to detect the percentage of pDC in peripheral blood, and its level was closely related to the immune status of sepsis;

[0063] 3. Prognostic risk assessment: When the pDC level is higher than 0.96%, it indicates that the patient has a higher risk of death; combined with the receiver operating characteristic (ROC) curve, 0.96% is determined as the optimal critical value to evaluate the patient's prognosis;

[0064] 4. Dynamically monitor the level of pDC 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;

[0065] 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.

[0066] 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.

[0067] In some embodiments of the present invention, the anti-CD3 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD14 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD123 antibody and anti-HLA-DR antibody are subjected to flow cytometry analysis, CD3 - CD19 - CD20 - CD14 - CD16 - CD56 - CD123 + HLA-DR + Representing pDC cells, the proportion of pDC in peripheral blood was determined. The expression of pDC was detected by antibody staining combined with flow cytometry.

[0068] In some embodiments of the present invention, the relationship between pDC level and sepsis prognosis is evaluated using ROC curve. According to the analysis results, when the pDC level is higher than 0.96%, it is determined that the patient faces a higher risk of death.

[0069] In some embodiments of the present invention, blood samples are collected from patients at different time points (24 hours, 48 ​​hours, 72 hours) to perform dynamic monitoring of pDCs. By monitoring the changes in pDCs during the course of the disease, the strength of the patient's immune response is evaluated, and clinical intervention strategies are guided.

[0070] This invention focuses on the method of evaluating the prognosis of pediatric sepsis patients by the level of plasmacytoid dendritic cells (pDC). By detecting the proportion of pDC in peripheral blood, high-risk sepsis patients can be identified early, and the changes in the patient's immune status can be tracked in real time through dynamic monitoring.

[0071] The inventors of the present application used flow cytometry to detect 99 children with sepsis, divided them into two subgroups: Non-remote group and Remote group, and survival group and Non-survival group, and followed up for 28 days to evaluate the mortality rate. Flow cytometry analyzed the percentage of pDC cells, and found that the pDC percentage in the Remote group was significantly higher than that in the Non-remote group, and in the death group was significantly higher than that in the survival group. The expression of pDC was significantly positively correlated with the sepsis-related score. When the percentage of pDC cells in human peripheral blood lymphocytes is higher than 0.96%, children with sepsis may be at risk of death. There was no difference in pDC between the death group and the inventory group within 72 hours. The present invention provides a method for evaluating the prognosis of patients with sepsis by detecting pDC levels.

[0072] 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 release of the new Phoenix sepsis criteria for pediatric sepsis and septic shock by the Society of Critical Care Medicine (SCCM) Pediatric Sepsis Definition Working Group in 2024, this application only analyzed 99 children out of 102 who met the new Phoenix sepsis criteria in 2024. According to the 2024 criteria, the children were divided into Remote and Non-remote groups based on whether they had organ dysfunction away from the primary infection site. According to the in-hospital outcomes of the children, they were divided into Survival and Non-survival groups. At the same 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 according to the blood collection time. 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.

[0073] 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.

[0074] 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 6cells. Centrifuge, remove the supernatant, add anti-CD3 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD14 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD123 antibody and anti-HLA-DR antibody for flow cytometry staining, and incubate at room temperature for 30 min. Use flow cytometer for cell detection, and use flow cytometric analysis software (such as FlowJo) to analyze the data. Determine the proportion of pDC to total lymphocytes (obtained by FSC-A and SSA-A gates of flow cytometer) and record the percentage of pDC.

[0075] pDC level assessment: The test results show the percentage of pDC in lymphocytes. When the pDC expression level is higher than 0.96%, 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, the present invention recommends collecting and testing blood samples at multiple time points during the course of sepsis, usually testing pDC levels again 24 hours, 48 ​​hours and 72 hours after admission. By dynamically monitoring the changing trend of pDC, the recovery or deterioration of the patient's immune function can be evaluated, and then the progression of the disease can be judged. There was no difference in pDC expression between the death group and the survival group within 72 hours.

[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 pDC with the clinical score, the accuracy of prognosis assessment can be improved. If the patient's pDC level is higher than 0.96% 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] 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.

[0084] 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.

[0085] The present invention will be further described below in conjunction with the embodiments.

[0086] Example 1: Detection of pDC levels in children with sepsis

[0087] Samples to be tested: 99 children with sepsis were included 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 invention were in-hospital deaths, and half of the children died within 3 days after admission (6 cases, 50.0%).

[0088] Table 1: Clinical characteristics of the survival group and the death group

[0089]

[0090]

[0091]

[0092]

[0093] Data are presented as n (number, percentage), mean ± SD, or median (first quartile, third quartile).

[0094] Materials: Fluorescently labeled anti-CD3 (APC-Cy7) antibodies, anti-CD19 (APC) antibodies, anti-CD14 (PE-Cy7) antibodies, anti-CD20 (APC) antibodies, anti-CD16 (BB700) antibodies, CD56 (APC-Cy7) antibodies, anti-CD123 (PE) antibodies, and anti-HLA-DR (FITC) antibodies were purchased from BD Pharmingen; human lymphocyte separation medium (Cedarlane) was purchased from Beijing Dakoway Biotechnology Co., Ltd.

[0095] Experimental methods:

[0096] 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.

[0097] Take 100 μL containing 10 61 μL each of APC-Cy7 anti-CD3 antibody, APC anti-CD19 antibody, APC anti-CD20 antibody, PE-Cy7 anti-CD14 antibody, BB700 anti-CD16 antibody, APC-Cy7 anti-CD56 antibody, PE anti-CD123 antibody and FITC anti-HLA-DR 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 suspended in 200 μL PBS. The cells were then placed on a flow cytometer and the results were analyzed.

[0098] Results analysis: The percentage of pDC in children with sepsis was Figure 2A to Figure 2C . Figure 2A Shown is the gating strategy for pDC in this example. Figure 2B The results showed that the expression of pDC in the Remote group was significantly higher than that in the nonremote group. Figure 2C The results showed that pDC cells increased in the death group. In summary, children with sepsis who progressed or died highly expressed pDC cells.

[0099] Example 2: pDC levels in children with sepsis are negatively correlated with Phoenix scores and pSOFA scores

[0100] 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.

[0101] 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.

[0102] Figure 3A to Figure 3C The following is a correlation analysis between the pDC level in children with sepsis and the Phoenix score and pSOFA score. Figure 3A As shown in the figure, the correlation coefficient between the percentage change of pDC in children with sepsis and Phoenix sepsis scores (PSS)-4 was r = 0.39, P < 0.0001. Figure 3BThe results showed that the correlation coefficient between the percentage of pDC in children with sepsis and the PSS-8 score was r = 0.37, P = 0.0002. Figure 3C Correlation analysis showed that the correlation coefficient between the expression level of pDC and the pSOFA score in children with sepsis was r = 0.38, P = 0.0001. In summary, the level of pDC in children with sepsis was significantly positively correlated with the Phoenix score and pSOFA score.

[0103] Example 3: ROC curve analysis found that pDC has a good predictive effect on in-hospital mortality in children with sepsis

[0104] The laboratory indexes and clinical data of children with sepsis were collected, and the ROC curve was used to analyze the diagnostic efficacy of pDC for in-hospital mortality in children with sepsis.

[0105] Figure 4 The ROC curve in this example is used to analyze the predictive efficacy of pDC for in-hospital mortality in patients with sepsis. Figure 4 As shown in Table 2, ROC curve analysis found that pDC had good predictive efficacy for in-hospital mortality in children with sepsis, with an area under the curve of 0.7632 (95% CI: 0.5951-0.9313, P <0.005), and the optimal critical value was 0.96%, with a sensitivity of 81.8% and a specificity of 77.9%. From the above statistical results, it was found that the area under the pDC curve for the diagnosis of sepsis in children was 0.7632, with a good diagnostic efficacy and statistical significance, indicating that pDC had good predictive efficacy for in-hospital mortality in children with sepsis.

[0106] Table 2

[0107]

[0108] Example 4: Relationship between pDC and mortality outcome in children with sepsis

[0109] The relationship between pDC and the mortality 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 with the pDC expression level of 0.96% as the dividing line, the mortality rate of the group with high expression level was higher than that of the group with low expression level, and the difference was statistically significant.

[0110] Example 5: pDCs do not have stable predictive efficacy within 72 hours

[0111] To explore whether pDC has a stable predictive value as a potential predictor in the short term, the changes of pDC in children with sepsis at different stages and with different prognoses were analyzed, and the levels of pDC in 16 children with sepsis were randomly monitored within 72 hours. Fig. 6AThe results showed that the percentage of pDC in the early death group of children with sepsis was higher than that in the survival group (P<0.05). Figure 6B The results showed that there was no significant difference in the expression of pDC between the two groups in the late death group of children with sepsis. Figure 6C The results showed that there was no significant difference in pDC expression between the death group and the survival group within 72 hours.

[0112] In summary, this application provides a method for evaluating the prognosis of sepsis based on plasmacytoid dendritic cells (pDCs). The level of pDCs in peripheral blood is detected by flow cytometry, combined with dynamic monitoring and clinical scoring systems, which can effectively evaluate the prognosis of sepsis patients. 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.

[0113] 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 plasmacytoid dendritic cells as markers in the preparation of prognostic products for pediatric sepsis.

2. The use according to claim 1, characterized in that The prognosis determination rule includes: when the ratio of plasmacytoid dendritic cells to total lymphocytes is higher than 0.96, it indicates that the patient has a higher risk of death.

3. The use according to claim 1 or 2, characterized in that: The plasmacytoid dendritic cells are peripheral blood-derived plasmacytoid dendritic cells.

4. Use of antibodies in the preparation of a product for the prognosis of childhood sepsis, the antibodies comprising anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

5. The use according to claim 4, characterized in that Based on the antibody, the ratio of plasmacytoid dendritic cells to total lymphocytes in the sample to be tested is obtained by flow cytometry to obtain a prognostic result; The judgment rule of the prognosis result includes: when the proportion is higher than 0.96, it indicates that the patient has a higher risk of death.

6. A kit for the prognosis of sepsis in children, characterized in that: Plasmacytoid dendritic cells were used as markers.

7. The kit according to claim 6, characterized in that Using specific antibodies to capture cells in peripheral blood samples, the proportion of plasmacytoid dendritic cells in total lymphocytes is determined, and the prognosis is then determined based on the proportion; The prognosis determination rule includes: when the proportion is higher than 0.96, it indicates that the patient has a higher risk of death; The specific antibodies include anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

8. A device for the prognosis of sepsis in children, characterized in that: Plasmacytoid dendritic cells are used as markers and are coated with specific antibodies for capturing plasmacytoid dendritic cells.

9. The device according to claim 8, characterized in that The specific antibodies include anti-CD3 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD14 antibodies, anti-CD16 antibodies, anti-CD56 antibodies, anti-CD123 antibodies and anti-HLA-DR antibodies.

10. An immune monitoring system for risk assessment of sepsis prognosis in children, characterized in that: Prognosis based on plasmacytoid dendritic cell expression levels; The prognosis determination rule includes: when the expression level of the plasmacytoid dendritic cells is higher than 0.96, it indicates that the patient has a higher risk of death; The plasmacytoid dendritic cell expression level is the ratio of plasmacytoid dendritic cells to total lymphocytes.

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