Application of plasmacytoid dendritic cells as a marker in pediatric sepsis prognostic products

Abstract

The present application relates to the technical field of medical immunology, and particularly relates to application of plasma cell-like dendritic cells as a marker in a prognosis product for children sepsis. The present application detects the pDC level in the peripheral blood of a patient based on flow cytometry, and evaluates the prognosis of the patient based on the expression level. The pDC expression level is significantly positively correlated with the children sepsis score (Phoenix and pSOFA score), and is positively correlated with the mortality rate of children with sepsis. When the expression of pDC is higher than 0.96%, it indicates that the patient has a higher risk of death. The method of the present application can provide accurate prognosis evaluation in the early screening of children sepsis, has high specificity and sensitivity, helps doctors to timely adjust the treatment scheme, optimizes personalized treatment, and has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical immunology, in particular to a method for evaluating and applying plasmacytoid dendritic cells (pDC) in prognosis of sepsis in children. BACKGROUND

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, mainly leading to multiple organ dysfunction, especially in pediatric patients, sepsis has a high morbidity and mortality (Singer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Jama 2016; 315: Karkarke 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). Despite the significant progress in the treatment of sepsis in modern medicine, due to the rapid progression of sepsis and complex clinical manifestations, how to accurately predict the prognosis of children and provide personalized treatment is still a major challenge in clinical treatment.

[0003] Current prognosis 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 organ dysfunction of patients, helping clinicians to assess the severity of the disease. However, these methods often lack timely reflection of the immune system status, especially the immune imbalance status of sepsis patients in the early stage. This makes it difficult for doctors to identify patients with severely impaired immune function in the early stage of the disease, thus missing the opportunity for timely intervention.

[0004] Plasmacytoid dendritic cells (pDC) 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. pDC plays an important immunoregulatory role in sepsis, and the dynamic changes in pDC levels can reflect the immune status of sepsis patients and their ability to clear pathogens, and the dynamic changes of pDC are closely related to the progression of the disease. When the level of pDC is abnormally high, it often indicates that the patient is facing a serious immune imbalance, and may lead to the exacerbation of immunosuppressive response, which is closely related to the mortality of sepsis patients.

[0005] The existing sepsis prognosis evaluation methods mainly rely on the organ function scoring system and conventional inflammatory markers of patients, but these methods often cannot effectively reflect the state of the immune system of patients, especially the early immune suppression 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 evaluating the immune suppression state. In addition, although the role of type I interferon in regulating immune response has been widely studied, the dynamic relationship between pDC and type I interferon and its role in sepsis prognosis have not been fully explored.

[0006] Therefore, it is urgent to develop a detection method based on immune regulatory cells to accurately evaluate the immune status and disease progression of children with sepsis, so as to help doctors make more accurate prognosis in the early stage of the disease. SUMMARY

[0007] Therefore, the present application provides a pDC (plasmacytoid dendritic cell) based prognosis evaluation method for children with sepsis, which detects the level of pDC in the peripheral blood of patients using flow cytometry, and determines the optimal critical value of 0.96% based on the ROC curve. When the level of pDC in patients is higher than the value, it indicates that the patient faces a higher risk of death. The innovation of this method is its high specificity and sensitivity, which can provide scientific basis for early risk screening and individualized treatment of sepsis patients.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0009] The present application provides the use of plasmacytoid dendritic cells as a marker in the preparation of a prognosis product for children with sepsis.

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

[0011] In some embodiments of the present application, the pDCs used in the above-mentioned applications are peripheral blood-derived pDCs.

[0012] The present application also provides the use of antibodies in the manufacture of a product for the prognosis of sepsis in children, 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 embodiments of the present application, the above-mentioned use of the antibodies obtains the proportion of pDCs in total lymphocytes in the sample to be tested by flow cytometry, and obtains the prognosis result.

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

[0015] In some embodiments of the present application, the above-mentioned use of the sample to be tested is a peripheral blood sample.

[0016] The present application also provides a kit for the prognosis of sepsis in children, wherein pDCs are used as a marker.

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

[0018] The determination rule of the prognosis includes that 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 embodiments of the present application, the above-mentioned diagnostic reagent contains a combination of specific antibodies for detecting pDCs in the peripheral blood of a patient, and flow cytometry is used for detection and for evaluating the prognosis of the patient.

[0021] In some embodiments of the present application, the above-mentioned diagnostic reagent further includes reagents, buffers for sample processing, and fluorescent dyes for antibody labeling, so as to accurately detect the expression level of pDCs in flow cytometry.

[0022] In some embodiments of the present application, the diagnostic kit comprises an antibody against CD3, an antibody against CD19, an antibody against CD14, an antibody against CD16, an antibody against CD123, and an antibody against HLA-DR, so as to identify and determine the pDC subpopulation of the patient.

[0023] In some embodiments of the present application, the diagnostic kit is suitable for early risk assessment of patients with sepsis, and is particularly suitable for early screening and prognosis of pediatric sepsis.

[0024] The present application also provides a device for prognosis of pediatric sepsis, taking plasmacytoid dendritic cells as a marker, and coated with specific antibodies for capturing plasmacytoid dendritic cells.

[0025] In some embodiments of the present application, the specific antibodies of the device comprise an antibody against CD3, an antibody against CD19, an antibody against CD20, an antibody against CD14, an antibody against CD16, an antibody against CD56, an antibody against CD123, and an antibody against HLA-DR.

[0026] The present application also provides an immunological monitoring system for risk assessment of prognosis of pediatric sepsis, based on the expression level of plasmacytoid dendritic cells;

[0027] The determination rule of the prognosis comprises: 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 expression level of the plasmacytoid dendritic cells is the proportion of plasmacytoid dendritic cells in total lymphocytes.

[0029] In some embodiments of the present application, the immunological monitoring system detects the expression level of pDC in the peripheral blood of the patient, combines with a specific threshold to determine the prognosis of the patient, and dynamically monitors the immune changes in the progression of the disease, so as to help doctors adjust the treatment plan.

[0030] In some embodiments of the present application, the immunological monitoring system comprises:

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

[0032] A flow cytometry detection module for detecting the percentage of pDC in total lymphocytes;

[0033] A data analysis module for analyzing the prognosis risk of the patient according to the percentage.

[0034] In some embodiments of the present application, the data analysis module of the immunological monitoring system further analyzes the prognosis risk of the patient in combination with a ROC curve and a clinical scoring system.

[0035] In some embodiments of the present application, the immune monitoring system described above can be used to dynamically monitor the pDC level of the patient, and to perform real-time prognosis evaluation at different stages of the treatment of pediatric sepsis.

[0036] The present application also provides a detection method for the prognosis risk evaluation of pediatric sepsis, which is based on the detection of plasmacytoid dendritic cells (pDC) in the peripheral blood of the patient, and evaluates the prognosis of the pediatric sepsis patient by analyzing the expression level of the pDC cells, wherein when the expression level of the pDC is higher than 0.96%, it indicates that the patient has a higher risk of death.

[0037] In some embodiments of the present application, the method for detecting pDC in the detection method described above is flow cytometry, which comprises:

[0038] S1, collecting a peripheral blood sample of the patient;

[0039] S2, obtaining the percentage of pDC in total lymphocytes by flow cytometry;

[0040] S3, determining the immune status of the patient according to the detection result, and judging the prognosis of the patient.

[0041] In some embodiments of the present application, the S3 of the detection method described above further comprises ROC curve analysis to judge the prognosis of the patient.

[0042] In some embodiments of the present application, the optimal critical value of the pDC in the detection method described above is 0.96%, and when the expression of pDC in the peripheral blood of the patient is higher than this value, it indicates that the patient has a higher risk of death.

[0043] In some embodiments of the present application, the detection method described above further comprises dynamic monitoring of the patient at different time points, specifically collecting samples within 24 hours, 48 hours and 72 hours in the course of the disease for detection.

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

[0045] In some embodiments of the present application, the detection method described above is suitable for pediatric sepsis patients, and can be used for the prognosis evaluation of sepsis patients of other age groups.

[0046] The present application can find high-risk patients in the early stage of sepsis by monitoring the level of pDC, helping doctors make timely treatment decisions. As an important cell for immune regulation in sepsis patients, the level of pDC is closely related to the severity of sepsis. Compared with traditional inflammatory markers (such as C-reactive protein and procalcitonin), the detection of pDC has higher specificity and sensitivity.

[0047] The present application proposes dynamic monitoring of pDC at multiple time points, which can reflect the changes in the immune status of patients in real time, helping to predict the development of the disease and adjust the treatment plan.

[0048] The present application is suitable for early screening and prognosis evaluation of sepsis patients, especially for children with sepsis. Through the detection of pDC, high-risk patients can be identified in time, and the mortality rate of sepsis can be reduced. In addition, this method can also be combined with other prognosis 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 the diagnosis and management of sepsis.

[0049] In summary, the present application provides a sepsis prognosis evaluation method based on the level of pDC. By detecting the proportion of pDC in peripheral blood, high-risk sepsis patients can be identified early, and the immune status of patients can be tracked in real time through dynamic monitoring. This method is simple to operate, has high specificity and sensitivity, and can provide reliable prognosis evaluation means for clinical practice and provide scientific basis for the development of individualized treatment plan. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description.

[0051] Figure 1 The enrollment and exclusion criteria and group grouping of children are shown;

[0052] Figure 2A 、 Figure 2B 、 Figure 2C The percentage of pDC in sepsis children is shown;

[0053] Figure 3A 、 Figure 3B 、 Figure 3C The correlation analysis of the level of pDC in sepsis children with Phoenix score and pSOFA score is shown;

[0054] Figure 4 The ROC curve analysis of the predictive performance of pDC in sepsis patients for in-hospital death is shown;

[0055] Figure 5 The relationship between pDC and the death outcome of sepsis children is shown;

[0056] Figure 6A 、 Figure 6B 、 Figure 6C The pDCs can be dynamically monitored within 72 hours. DETAILED DESCRIPTION

[0057] The application discloses a pDC-based prognosis evaluation method for children with sepsis, and those skilled in the art can refer to the content herein and appropriately improve process parameters to realize. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as being included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technical field of the application.

[0058] The application provides a pDC-based prognosis evaluation method for children with sepsis. The method can judge and evaluate the prognosis risk of a patient by detecting the pDC level in peripheral blood of the patient.

[0059] By dynamically monitoring the change trend of pDC, a doctor can master the immune state of a patient in real time, and timely adjust the treatment strategy. The application can also be combined with existing clinical scoring systems (such as Phoenix and pSOFA scores) to further improve the accuracy of prognosis evaluation.

[0060] The specific technical solutions are as follows:

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

[0062] 2. Detecting the percentage of pDC in peripheral blood by flow cytometry, and the level is closely related to the immune state of sepsis;

[0063] 3. Prognosis risk evaluation: when the pDC level is higher than 0.96%, it indicates that the patient has a higher death risk; in combination with a receiver operating characteristic (ROC) curve, 0.96% is determined as the best critical value to evaluate the prognosis of the patient;

[0064] 4. Dynamically monitoring the level of pDC at different time points (24 hours, 48 hours and 72 hours), and combining the clinical manifestations and scoring systems of the patient to continuously evaluate the prognosis; dynamic detection can reflect the change of the immune state of the patient, and facilitate real-time adjustment of the treatment scheme;

[0065] 5. The method can be combined with other clinical scoring systems (such as pSOFA and Phoenix scores) to further improve the accuracy of prognosis prediction. By combining immune cell detection with organ function scoring, more comprehensive risk evaluation is provided.

[0066] In some embodiments of the present application, patients diagnosed with pediatric sepsis are recruited for the study, and peripheral blood samples are collected within 6 hours of hospitalization.

[0067] In some embodiments of the present application, 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 is determined. Through antibody staining combined with flow cytometry detection, the expression of pDC is detected.

[0068] In some embodiments of the present application, the relationship between pDC level and prognosis of sepsis 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 application, blood samples are collected from patients at different time points (24 hours, 48 hours, 72 hours) for dynamic monitoring of pDC. By monitoring its changes in the course of the disease, the intensity of the immune response of the patient is evaluated, and the clinical intervention strategy is guided.

[0070] The present application focuses on exploring the method of evaluating the prognosis of pediatric sepsis patients by investigating 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 immune status of patients can be tracked in real time through dynamic monitoring.

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

[0072] Study population: 102 children with sepsis who met the inclusion and exclusion criteria and were admitted to the PICU of the Children's Hospital of Chongqing Medical University for treatment from July 2022 to April 2024 were included. The inclusion criteria were: (1) The diagnosis met the definition of "severe sepsis" or "septic shock" in the 2005 International Pediatric Sepsis Consensus Conference (2) Age greater than 28 days, less than 18 years old; (3) The guardian of the child signed the informed consent form. The 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 (hemodialysis filtration, perfusion, plasma replacement) before enrollment. In view of the release of the Phoenix sepsis new standard for children with sepsis and septic shock by the 2024 American Society of Critical Care Medicine (Society of Critical Care Medicine, SCCM) Pediatric Sepsis Definition Working Group, this application only analyzed 99 cases of 102 cases that met the 2024 Phoenix sepsis new standard. According to whether the children met the 2024 standard and had organ dysfunction away from the primary infection site, they were divided into Remote and Non-remote groups. According to the in-hospital outcome of the children, they were divided into Survival and Non-survival groups. At the same time, according to the blood sampling time, the partial immune indexes of early (course <3 days) and late (course >3 days) children with different in-hospital outcomes (survival(e) / nonsurvival(e) group) were analyzed. The basic information (gender, age), vital signs, test results (blood routine, biochemistry, etiology, inflammatory markers), Phoenix sepsis score, treatment plan, in-hospital outcome, and 28-day survival of all children were recorded. Within 6 hours of enrollment, peripheral blood was obtained from the children. Some patients additionally obtained blood samples at 24 hours, 48 hours, and 72 hours after enrollment.

[0073] Sample collection: Blood samples from enrolled children were collected using ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using human lymphocyte separation medium immediately for flow cytometry.

[0074] Flow cytometry detection: 1640 medium containing 2% fetal bovine serum was added to freshly extracted peripheral blood mononuclear cells (PBMCs) and mixed evenly, 100 μL of cell mixture was added to the flow tube, so that each tube contained 10 6Cells. Centrifuge, remove 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, incubate at room temperature for 30 min. Use flow cytometry for cell detection, and use flow cytometry analysis software (such as FlowJo) to analyze data. Determine the proportion of pDC in total lymphocytes (obtained by gating FSC-A and SSA-A of flow cytometry), and record the percentage of pDC.

[0075] pDC level evaluation: the detection result shows the percentage of pDC in lymphocytes, and when the expression level of pDC is higher than 0.96%, it indicates that the patient has a high risk of death. The critical value is obtained based on receiver operating characteristic curve (ROC) analysis, which has good sensitivity and specificity.

[0076] Dynamic monitoring: in order to improve the monitoring effect on the prognosis of patients, the present application suggests that blood samples are collected and detected at multiple time points during the course of sepsis, usually 24 hours, 48 hours and 72 hours after admission. By dynamically monitoring the change trend of pDC, the recovery or deterioration of the immune function of the patient is evaluated, and then the progress of the disease is judged. There is 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 application can be used in combination with existing sepsis clinical scoring systems (such as pSOFA and Phoenix score). By comparing the detection result of pDC with the clinical score, the accuracy of prognosis evaluation can be improved. If the pDC level of the patient is higher than 0.96% and the clinical score shows that the disease is serious, it can be more certain that the patient faces a 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 object recited after the expression and various different combinations of two or more of the recited objects, individually, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0080] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0081] The use of any and all examples, or exemplary language herein, for example, merely to better illuminate the application. The use of any such examples or exemplary language is not intended to limit the scope of the application. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0082] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only chosen to encompass the more precise values in the specification. Any numerical range or parameter is not intended to limit the scope of the application to the precise values stated. Accordingly, all ranges and numerical values are approximations.

[0083] Abbreviations used in the 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 sepsis score; pSOFA, pediatric sequential organ failure assessment; D, day; CNS, central nervous system; UTI, urinary tract infection; GCC, glucocorticoid; CRRT, continuous renal replacement therapy; IVIG, intravenous immunoglobulin.

[0084] Unless otherwise indicated, the materials, reagents, and equipment used in the application are commercially available and can be obtained from market.

[0085] The present invention will be further illustrated below with reference to 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 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 invention were in-hospital deaths, with half of the children dying within 3 days of 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 expressed as n (quantity, percentage), mean ± standard deviation, or median (first quartile, third quartile).

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

[0095] Experimental methods:

[0096] 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.

[0097] Take 100 μL containing 10 6PBMCs cells, 1 μL of APC-Cy7 anti-CD3 antibody, 1 μL of APC anti-CD19 antibody, 1 μL of APC anti-CD20 antibody, 1 μL of PE-Cy7 anti-CD14 antibody, 1 μL of BB700 anti-CD16 antibody, 1 μL of APC-Cy7 anti-CD56 antibody, 1 μL of PE anti-CD123 antibody and 1 μL of FITC anti-HLA-DR antibody were added, and incubated at room temperature for 30 min. After washing once with 1 mL of PBS at 500 g for 5 min, 200 μL of PBS was added for suspension, and the results were analyzed by flow cytometry.

[0098] Results analysis: The percentage of pDC in children with sepsis is shown in Figures 2A-2C . Figure 2A The results show the gating strategy of pDC in this example. Figure 2B The results show that the expression of pDC in the Remote group is significantly higher than that in the nonremote group. Figure 2C The results show that the pDC cells in the death group are increased. In summary, the pDC cells are highly expressed in children with sepsis and death.

[0099] Example 2: The level of pDC in children with sepsis is negatively correlated with Phoenix score and pSOFA score

[0100] Phoenix, the new standard for the diagnosis of sepsis and septic shock in children by the American Society of Critical Care Medicine (SCCM) in 2024, Phoenix sepsis score of 2 or more indicates that children with suspected or confirmed infection have potential life-threatening organ dysfunction in the respiratory, cardiovascular, coagulation and / or nervous system.

[0101] In the sepsis diagnosis standard sepsis 3.0, pSOFA score is an important indicator for the diagnosis of sepsis. A large amount of research data shows that for ICU patients with infection or suspected infection, when pSOFA score ≥ 2 points, it is diagnosed as sepsis. Not only that, pSOFA reflects the degree of multiple organ dysfunction syndrome (MODS) in multiple organ dysfunction patients, and is closely related to the hospitalization rate of patients.

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

[0103] Example 3: ROC curve analysis found that pDC had good predictive performance for in-hospital mortality in children with sepsis

[0104] Laboratory indicators and clinical data of children with sepsis were collected. ROC curve analysis was used to analyze the diagnostic performance of pDC for in-hospital mortality in children with sepsis.

[0105] Figure 4 The ROC curve analysis of pDC for predicting in-hospital mortality in children with sepsis is shown in this example. As shown in Figure 4 As shown in Table 2, ROC curve analysis found that pDC had good predictive performance 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), the best critical value was 0.96%, the sensitivity was 81.8%, and the specificity was 77.9%. From the above statistical results, the area under the curve of pDC for the diagnosis of sepsis in children was 0.7632, the diagnostic performance was good, and it was statistically significant, indicating that pDC had good predictive performance for in-hospital mortality in children with sepsis.

[0106] Table 2

[0107]

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

[0109] Further observation of the relationship between pDC and death outcome in children with sepsis, Kaplan-Meier survival curve of children with sepsis was made. Figure 5 The results showed that with pDC expression level of 0.96% as the boundary, 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: pDC does not have stable predictive performance within 72 hours

[0111] In order to explore whether pDC as a potential predictor has stable predictive value in a short period of time, 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 within 72 hours were randomly monitored. Figure 6AThe results showed that the percentage of pDCs was higher in the early death group than in the survival group (P<0.05). Figure 6B The results showed that there was no significant difference in the expression of pDCs between the two groups in the late death group. Figure 6C The results showed that there was no significant difference in the expression of pDCs between the two groups within 72 hours.

[0112] In summary, the present application provides a method for evaluating the prognosis of sepsis based on pDCs. By detecting the level of pDCs in peripheral blood through flow cytometry and combining dynamic monitoring and clinical scoring system, the prognosis of sepsis patients can be effectively evaluated. This method has high specificity and sensitivity, and is especially suitable for early risk assessment of pediatric sepsis patients, and provides a scientific basis for the development of individualized clinical treatment plans.

[0113] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Use of an agent for detecting plasmacytoid dendritic cells in the manufacture of a product for the prognosis of sepsis in children, characterized in that, The determination rule of the prognosis is that when the proportion of the plasmacytoid dendritic cells in total lymphocytes is higher than 0.96, it indicates that the patient has a higher death risk.

2. Use according to claim 1, wherein The plasmacytoid dendritic cells are peripheral blood-derived plasmacytoid dendritic cells.

3. Use of an antibody for the manufacture of a product for the prognosis of sepsis in children, characterized in that, The antibodies consist of 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; The product obtains the proportion of plasmacytoid dendritic cells in total lymphocytes in the sample to be tested by flow cytometry based on the antibodies, and obtains the prognosis result; The determination rule of the prognosis result is that when the proportion is higher than 0.96, it indicates that the patient has a higher death risk.

4. Device for the prognosis of sepsis in children, characterized in that, The proportion of plasmacytoid dendritic cells in total lymphocytes is used as a determination index, and specific antibodies for capturing plasmacytoid dendritic cells are coated; 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; The determination rule of the prognosis is that when the proportion is higher than 0.96, it indicates that the patient has a higher death risk.

5. An immunomonitoring system for the prognostic risk assessment of sepsis in children, characterized by, Prognosis based on plasmacytoid dendritic cell expression level; The determination rule of the prognosis is that when the plasmacytoid dendritic cell expression level is higher than 0.96, it indicates that the patient has a higher death risk; The plasmacytoid dendritic cell expression level is the proportion of plasmacytoid dendritic cells in total lymphocytes.

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