Kit for prognosis judgment of sepsis and application of kit

Abstract

The invention provides a kit for prognosis judgment of sepsis and application of the kit. The kit comprises a CD45 / CD14 / CD16 / HLA-DR / VSIG4 / IL1R2 antibody which is used for multicolor flow cytometry detection and is provided with a fluorescent label. A mononuclear cell is expressed and circled according to CD45 / CD14 / CD16, and a VSIG4 + HLA-DRlowCD14 + mononuclear cell (a cell proportion P1) and an IL1R2 + HLA-DRlowCD14 + mononuclear cell (a cell proportion P2) are expressed and circled according to HLA-DR / VSIG4 / IL1R2 in the mononuclear cell. When P1gt; 20% or P2gt; if the content is 35%, the sepsis patient is poor in prognosis; when P1lt; 20% and P2lt; if the percentage is 35%, the sepsis patient has good prognosis. The kit is high in prognosis sensitivity and specificity for distinguishing different sepsis patients, and high-risk patients can be rapidly distinguished.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a reagent kit for predicting the prognosis of sepsis and its application. Background Technology

[0002] Sepsis is a major infectious disease that seriously threatens human health. Given its high morbidity and mortality rates, the World Health Organization has identified it as a global health priority.

[0003] Accurate prognosis assessment for sepsis patients is crucial for clinical treatment decisions and resource allocation. However, a rapid, accurate, and highly specific prognostic method for sepsis remains lacking in clinical practice. Existing prognostic methods largely rely on a comprehensive analysis combining clinical symptoms (such as level of consciousness), physical signs (such as blood pressure and respiratory rate), and laboratory indicators (such as complete blood count and liver and kidney function tests). For example, the Sequential Organ Failure Assessment (SOFA) score, which comprehensively assesses the degree of organ dysfunction based on clinical and laboratory indicators, is currently the most commonly used method for assessing sepsis prognosis, but it has many limitations, including insufficient sensitivity and specificity, difficulty in data acquisition, and inapplicability to emergency patients.

[0004] In summary, developing a rapid and accurate prognostic method for sepsis based on biomarkers is of significant clinical importance. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for predicting the prognosis of sepsis and its application. This kit, combined with multicolor flow cytometry, can rapidly and accurately predict the prognosis of sepsis patients.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, a kit for prognostic assessment of sepsis is provided, the kit comprising: fluorescently labeled CD45 antibody, CD14 antibody, CD16 antibody, HLA-DR antibody, VSIG4 antibody and IL1R2 antibody for multicolor flow cytometry detection.

[0007] Furthermore, the fluorescently labeled antibodies can be distinguished in multicolor flow cytometry.

[0008] In one specific implementation, the CD45 antibody is labeled with V500, the CD14 antibody is labeled with BV421, the CD16 antibody is labeled with PE-Cy7, the HLA-DR antibody is labeled with BV605, the VSIG4 antibody is labeled with PE, and the IL1R2 antibody is labeled with APC.

[0009] In a second aspect of the invention, a method for predicting the prognosis of sepsis is provided, the method comprising: Add the fluorescently labeled antibody from the kit to peripheral blood; CD14 was detected by multicolor flow cytometry. + VSIG4 in monocytes + HLA-DR low Cells and IL1R2 + HLA-DR low Cell percentage.

[0010] Furthermore, the detection of CD14 by multicolor flow cytometry... + VSIG4 in monocytes + HLA-DR low Cells and IL1R2 + HLA-DR low Cell percentage, specifically including: Flow cytometry was used for analysis. Nucleated cell phyla were identified by the forward scattering angle (FSC) and side scattering angle (SSC). Monocyte phyla were initially identified within the nucleated cell phyla based on CD45 and SSC. CD14 was then identified within the initial monocyte phyla based on CD14 and CD16. + Monocytes (the combined CD14 and CD16 gating helps exclude interference from lymphocytes and neutrophils), in CD14 + In the monocyte phylum, VSIG4 is circled based on its correlation with HLA-DR. + HLA-DR low Cells, simultaneously in CD14 + In the monocyte phylum, IL1R2 is circled based on IL1R2 and HLA-DR. + HLA-DR low cell.

[0011] Analysis of VSIG4 + HLA-DR low and IL1R2 + HLA-DR low Cells in CD14 + The percentage of mononuclear cells was used as the measurement result: If VSIG4 + HLA-DR low CD14 + Monocyte percentage >20% or IL1R2 + HLA-DR low CD14 + A monocyte percentage >35% indicates a poor prognosis for the patient; If VSIG4 +HLA-DR low CD14 + Monocyte percentage <20% and IL1R2 + HLA-DR low CD14 + A monocyte percentage of <35% indicates a good prognosis for the patient; In a third aspect of the invention, the use of the kit for sepsis prognosis assessment in the preparation of products for assessing sepsis prognosis is provided.

[0012] In a fourth aspect of the invention, a sepsis prognostic biomarker detection system is provided, comprising: Antibody combo module: contains fluorescently labeled antibodies against CD45, CD14, CD16, HLA-DR, VSIG4, and IL1R2; Flow cytometry module: Equipped with a multi-color laser light source and optical filter array for acquiring fluorescence signals; The data analysis module is programmed to perform the following operations: delineate nucleated cell populations based on forward scattering (FSC) and side scattering (SSC); preliminarily delineate monocyte populations within the nucleated cell populations based on CD45 / SSC; and delineate CD14+ cells within the preliminarily delineated monocyte populations based on CD14 / CD16. + Monocyte population (CD14 and CD16 combined gating helps exclude interference from lymphocytes and neutrophils); in CD14 + VSIG4 was circled in the monocyte population based on its correlation with HLA-DR. + HLA-DR low Cells, simultaneously in CD14 + In the monocyte phylum, IL1R2 is circled based on IL1R2 and HLA-DR. + HLA-DR low Cells; Calculating VSIG4 + HLA-DR low Cell percentages of P1 and IL1R2 + HLA-DR low Cell percentage P2; Output module: Displays the values ​​of P1 and P2 and a scatter plot.

[0013] Furthermore, the output module also includes: When P1>20% or P2>35%, output a "poor prognosis" signal; When P1 < 20% and P2 < 35%, output a "good prognosis" signal.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. This invention provides a kit for predicting the prognosis of sepsis, which detects VSIG4 in the peripheral blood of sepsis patients. + HLA-DR low CD14 + Monocytes and IL1R2 + HLA-DR low CD14 + The proportion of monocytes can be used to quickly determine the prognosis of sepsis patients. The operation is simple and can quickly identify high-risk patients.

[0015] 2. The kit provided by this invention for predicting the prognosis of sepsis has high specificity and sensitivity: using VSIG4 alone... + HLA-DR low CD14 + Monocyte ratio and IL1R2 + HLA-DR low CD14 + The sensitivity of monocyte proportion in differentiating sepsis prognosis is only 53.85% and 46.15%, respectively. However, when these two indicators are used together to distinguish between poor and good prognosis in sepsis patients, the sensitivity reaches 92.3% and the specificity reaches 90%. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A represents the detection of VSIG4 by flow cytometry in an embodiment of the present invention. + HLA-DR low CD14 + Monocytes and IL1R2 + HLA-DR low CD14 + A diagram illustrating the gate strategy for the proportion of monocytes; Figure 1 B is a flow cytometry graph representing representative healthy controls, patients with good prognosis (survival), and patients with poor prognosis (death).

[0018] Figure 2 VSIG4 included 13 patients with poor sepsis prognosis (death) and 30 patients with good sepsis prognosis (survival). + HLA-DR low CD14 + and IL1R2 + HLA-DRlow CD14 + Results of monocyte proportion.

[0019] Figure 3 For using VSIG4 alone + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + ROC curve analysis results of monocyte ratio distinguishing between dead and surviving patients.

[0020] Figure 4 To unite VSIG4 + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + Venn diagram showing the ratio of monocytes in distinguishing between dead and surviving patients.

[0021] Figure 5 To unite VSIG4 + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + Survival curves distinguishing between dead and surviving patients by the proportion of monocytes. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] Explanation of relevant terms in this invention: 1. VSIG4 + HLA-DRˡᵒʷ cells Cell type: CD14 + A functional subset of monocytes; Phenotypic characteristics: HLA-DR: Human leukocyte antigen-DR (HLA-DR) expression level below a threshold (the threshold is set based on HLA-DR expression in healthy individuals, where HLA-DR is expressed at CD14). + Monocyte expression is typically above 90%. VSIG4 + :V-set and immunoglobulin domain-containing 4 (VSIG4) positive expression.

[0025] 2. IL1R2 + HLA-DR cells Cell type: CD14 + Another functional subset of monocytes; Phenotypic characteristics: HLA-DRˡᵒʷ: Same as the above definition; IL1R2 + Interleukin-1 receptor 2 (IL1R2) positive expression.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0027] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0028] V500-labeled CD45 antibody (clone number 2D1; catalog number 662912), BV421-labeled CD14 antibody (clone number MφP9; catalog number 563743), PE-Cy7-labeled CD16 antibody (clone number 3G8; catalog number 302016), BV605-labeled HLA-DR antibody (clone number L243; catalog number 307640), PE-labeled VSIG4 antibody (clone number 6H8; catalog number sc-53977), and APC-labeled IL1R2 antibody (clone number 34141; catalog number FAB663A).

[0029] The overall concept of this invention is as follows:

[0030] According to a typical embodiment of the present invention, a kit for predicting the prognosis of sepsis is provided, the kit comprising: fluorescently labeled CD45 antibody, CD14 antibody, CD16 antibody, HLA-DR antibody, VSIG4 antibody and IL1R2 antibody for multicolor flow cytometry detection.

[0031] Specifically, it is used for flow cytometry detection of CD14. +VSIG4 in monocytes + HLA-DR low Cells and IL1R2 + HLA-DR low Antibodies of varying cell percentages can be packaged separately into kits, such as kits containing fluorescently labeled antibodies for CD45, CD14, CD16, HLA-DR, VSIG4, and IL1R2.

[0032] In some preferred embodiments, antibodies against CD45, CD14, CD16, HLA-DR, VSIG4, and IL1R2 are labeled with V500, BV421, PE-Cy7, BV605, PE, and APC fluorescent markers, respectively. In some other embodiments, other fluorescent molecules may be attached to the antibodies, with the aim of enabling differentiation during multicolor flow cytometry.

[0033] According to another typical embodiment of the present invention, a method for predicting the prognosis of sepsis is provided, comprising: Add the fluorescently labeled antibody from the kit to peripheral blood; CD14 was detected by multicolor flow cytometry. + VSIG4 in monocytes + HLA-DR low Cells and IL1R2 + HLA-DR low Cell percentage.

[0034] As one specific implementation method, the method involves detecting CD14 using multicolor flow cytometry. + VSIG4 in monocytes + HLA-DR low Cells and IL1R2 + HLA-DR low Cell percentage includes: Flow cytometry was used for analysis. Nucleated cell phyla were identified by the forward scattering angle (FSC) and side scattering angle (SSC). Monocyte phyla were initially identified within the nucleated cell phyla based on CD45 and SSC. CD14 was then identified within the initial monocyte phyla based on CD14 and CD16. + Monocytes (the combined CD14 and CD16 gating helps exclude interference from lymphocytes and neutrophils), in CD14 + In the monocyte phylum, VSIG4 is circled based on its correlation with HLA-DR. + HLA-DR low Cells, simultaneously in CD14 + In the monocyte phylum, IL1R2 is circled based on IL1R2 and HLA-DR. +HLA-DR low cell.

[0035] Analysis of VSIG4 + HLA-DR low and IL1R2 + HLA-DR low Cells in CD14 + The percentage of monocytes was used as the measurement result: If VSIG4 + HLA-DR low CD14 + Monocyte percentage >20% or IL1R2 + HLA-DR low CD14 + A monocyte percentage >35% indicates a poor prognosis for the patient; If VSIG4 + HLA-DR low CD14 + Monocyte percentage <20% and IL1R2 + HLA-DR low CD14 + A monocyte percentage of <35% indicates a good prognosis for the patient.

[0036] The aforementioned VSIG4 + HLA-DR low CD14 + Monocytes and IL1R2 + HLA-DR low CD14 + Mononuclear cell percentage: This is automatically obtained after gating on a flow cytometer, which can easily and efficiently differentiate the prognosis of sepsis patients.

[0037] The present application will now be described in detail with reference to embodiments and experimental data.

[0038] Example 1: A kit for predicting the prognosis of sepsis patients This invention provides a kit for predicting the prognosis of sepsis, comprising: V500-labeled CD45 antibody; BV421-labeled CD14 antibody; PE-Cy7 labeled CD16 antibody; BV605-labeled HLA-DR antibody; PE-labeled VSIG4 antibody; APC-labeled IL1R2 antibody.

[0039] As a specific implementation method, the antibody concentration and dosage standards are as follows: Table 1

[0040] Example 2: A method for predicting the prognosis of sepsis Forty-three hospitalized patients with evidence of infection and meeting the diagnostic criteria for septic shock were enrolled. Patients were grouped according to their survival within 28 days of testing, including 13 patients with poor prognosis (death within 28 days) and 30 patients with good prognosis (survival within 28 days). Evidence of infection was defined as positive pathogen culture and / or nucleic acid testing in clinical samples (e.g., blood, urine, and pleural effusion). The diagnostic criteria for sepsis were evidence of infection and a Sequential Organ Failure Assessment (SOFA) score ≥2. The diagnostic criteria for septic shock were patients diagnosed with sepsis who, despite adequate volume resuscitation, required vasoactive agents to maintain a mean arterial pressure (MAP) of 65 mmHg, and simultaneously had a serum lactate concentration >2 mmol / L.

[0041] I. Specimen Collection: Collect 4 mL of heparin-anticoagulated peripheral blood from patients who tested positive for the pathogen.

[0042] II. Multicolor flow cytometry detection: 100 μL of peripheral blood was added to a flow cytometry tube, followed by 5 μL each of V500-labeled CD45 antibody, BV421-labeled CD14 antibody, PE-Cy7-labeled CD16 antibody, BV605-labeled HLA-DR antibody, PE-labeled VSIG4 antibody, and APC-labeled IL1R2 antibody. The mixture was gently vortexed and incubated at room temperature in the dark for 20 minutes. 2 mL of PBS was added for washing, followed by vortexing and centrifugation, after which the supernatant was discarded. 2 mL of erythrocyte lysis buffer was added, followed by vortexing and incubation at room temperature in the dark for 5 minutes, after which the supernatant was discarded. 2 mL of PBS was added to wash the cells again, followed by vortexing and centrifugation, after which the supernatant was discarded. 300 μL of PBS was added to resuspend the cells, and the cells were then analyzed using a flow cytometer.

[0043] III. Flow Cytometry 1. Circle the nucleated cell phylum within the forward scattering angle (FSC) and side scattering angle (SSC). Within the nucleated cell phylum, preliminarily circle the monocyte phylum based on CD45 and SSC. Additionally, circle lymphocytes within both CD45 and SSC as a control within the monocyte phylum.

[0044] 2. In the preliminary mononuclear phylum, circle CD14 based on CD14 and CD16. + Mononuclear cells.

[0045] 3. In CD14 + In the monocyte phylum, VSIG4 is circled based on its correlation with HLA-DR. + HLA-DR low cell.

[0046] 4. Simultaneously in CD14 + In the monocyte phylum, IL1R2 is circled based on IL1R2 and HLA-DR. + HLA-DR low cell.

[0047] HLA-DR low Definition: HLA-DR expression levels are below a threshold; threshold: set based on HLA-DR expression in healthy individuals, where HLA-DR is present at CD14 levels. + Monocyte expression is typically above 90%. Each time, blood samples from healthy controls are simultaneously analyzed, using the crosshair gate of the flow cytometry plot of the healthy controls as a reference. Figure 1 In the B-shaped gate, the upper left and upper right quadrants are the normal HLA-DR expression areas (>90%), while the lower left and lower right quadrants are defined as HLA-DR regions. low cell.

[0048] Positive diagnosis criteria: VSIG4 + PE channel signal > 99th percentile of MFI in isotype control PE (IgG1-PE); IL1R2 + : APC channel signal > 99th percentile of MFI in isotype control APC (IgG1-APC); 5. Finally, VSIG4 + HLA-DR low CD14 + Monocytes and IL1R2 + HLA-DR low CD14 + Mononuclear cells were used as the assay result.

[0049] VSIG4 + HLA-DR low CD14 + Monocytes and IL1R2 + HLA-DR low CD14 + Single-cell flow cytometry gating strategy, such as Figure 1 As shown in Figure A. The flow cytometry scatter plot shows healthy individuals, septic shock survivors, and septic shock deceased individuals (VSIG4). + HLA-DR low CD14 +Monocytes and IL1R2 + HLA-DR low CD14 + Representative results of monocytes ( Figure 1 B).

[0050] IV. Test Results 13 patients who died from septic shock and 30 patients who survived septic shock (VSIG4) + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + The results of the monocyte proportion are as follows: Figure 2 As shown.

[0051] Depend on Figure 2 It can be known that: VSIG4 + HLA-DR low CD14 + >20% is a predictor of mortality (7 / 13 of the mortality group exceeded this threshold); IL1R2 + HLA-DR low CD14 + A mortality rate of >35% is another predictor of death (6 out of 13 deaths exceeded this threshold).

[0052] Example 3: Sensitivity and specificity analysis of two indicators used alone Using VSIG4 respectively + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + ROC curves were plotted using monocyte proportion data to analyze VSIG4. + HLA-DR low CD14 + Monocyte ratio and IL1R2 + HLA-DR low CD14 + The proportion of monocytes distinguishes the performance of patients with different prognoses, and the results are as follows: Figure 3 As shown.

[0053] Depend on Figure 3 It can be seen that using VSIG4 alone + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14+ The proportion of monocytes was poor at distinguishing between dead and surviving patients, with ROC curve areas of only 0.703 and 0.769, respectively.

[0054] When VSIG4 + HLA-DR low CD14 + A proportion greater than 20.5% is used as a threshold, and VSIG4 is used. + HLA-DR low CD14 + The sensitivity and specificity of the ratio in distinguishing between deceased and surviving patients were 53.85% and 96.67%, respectively.

[0055] When IL1R2 + HLA-DR low CD14 + A monocyte percentage greater than 34.2% was used as the cutoff value for IL-1R2. + HLA-DR low CD14 + The sensitivity and specificity of the monocyte ratio in distinguishing between dead and surviving patients were 46.15% and 93.33%, respectively.

[0056] Example 4: Performance Analysis of Two Indicators Combined Figure 4 For joint use of VSIG4 + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + Venn diagram showing the ratio of monocytes in distinguishing between dead and surviving patients.

[0057] Depend on Figure 4 It can be seen that among the 13 deceased patients, VSIG4 + HLA-DR low CD14 + Seven patients had a monocyte percentage greater than 20%, and IL1R2 + HLA-DR low CD14 + Six patients had a monocyte percentage greater than 35% and also met the VSIG4 criteria. + HLA-DR low CD14 + The proportion of monocytes is greater than 20% and IL1R2 + HLA-DR low CD14 + One patient had a monocyte percentage greater than 35%, and another had VSIG4. +HLA-DR low CD14 + The proportion of monocytes is less than or equal to 20% and IL1R2 + HLA-DR low CD14 + The proportion of monocytes was less than or equal to 35%, and the results showed that combining VSIG4... + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + The sensitivity of the monocyte ratio in distinguishing between dead and surviving patients was 92.3% (12 / 13).

[0058] Of the 30 surviving patients, VSIG4 + HLA-DR low CD14 + There were 29 patients with a monocyte percentage of 20% or less, and IL1R2 + HLA-DR low CD14 + Twenty-eight patients had a monocyte percentage of 35% or less and also met the VSIG4 criteria. + HLA-DR low CD14 + The proportion of monocytes is less than or equal to 20% and IL1R2 + HLA-DR low CD14 + Among the 27 patients with a monocyte percentage of ≤35%, the results showed that combining VSIG4... + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + The specificity of the monocyte ratio in distinguishing between dead and surviving patients was 90% (27 / 30).

[0059] Figure 5 To unite VSIG4 + HLA-DR low CD14 + and IL1R2 + HLA-DR low CD14 + The monocyte ratio was used to differentiate patients with different prognoses in survival curve analysis results, by Figure 5 It can be seen that combining the two indicators can significantly distinguish between deceased and surviving patients.

[0060] Example 5: Repeatability Test 1. Test Objective Verify the consistency of the kit in repeated testing.

[0061] Test metric: VSIG4 + HLA-DRˡᵒʷ and IL1R2 + HLA-DRα cell ratio in CD14 + The coefficient of variation (CV) in monocytes.

[0062] 2. Experimental Methods Sample type: 5 peripheral blood samples with known proportions (3 with good prognosis and 2 with poor prognosis).

[0063] 3. Test conditions: Intra-batch testing: Five consecutive tests are performed on the same batch of reagents.

[0064] Inter-batch testing: 3 independent reagent batches, each tested once. 4. Results The coefficient of variation (CV) is shown in Table 2.

[0065] Table 2

[0066] High repeatability: All CV values ​​are significantly lower than the industry standard (10%), demonstrating the stability of the method.

[0067] The intra-batch CV was as low as 3.2%, indicating that the error in a single experiment was extremely small.

[0068] The highest inter-batch CV was 7.8%, indicating good consistency between different reagent batches.

[0069] Application example: Practical application of sepsis prognosis assessment kit Heparin-anticoagulated peripheral blood from patients was added to flow cytometry tubes. V500-labeled CD45 antibody, BV421-labeled CD14 antibody, PE-Cy7-labeled CD16 antibody, BV605-labeled HLA-DR antibody, PE-labeled VSIG4 antibody, and APC-labeled IL1R2 antibody were added for labeling. CD14 levels were then measured by flow cytometry. + Expression levels of HLA-DR, VSIG4, and IL1R2 on the surface of monocytes.

[0070] If VSIG4 + HLA-DR low CD14 + Monocyte percentage >20% or IL1R2 + HLA-DR low CD14 +A monocyte percentage >35% indicates a poor prognosis for the patient; If VSIG4 + HLA-DR low CD14 + Monocyte percentage <20% and IL1R2 + HLA-DR low CD14 + A monocyte percentage of <35% indicates a good prognosis for the patient.

[0071] Individual 1, a 45-year-old male, was admitted to the hospital due to "fever for one week, accompanied by cough, sputum production, and dyspnea." Physical examination revealed a temperature of 39.1℃, pulse of 115 beats / min, respiratory rate of 28 breaths / min, and blood pressure of 85 / 50 mmHg. Blood tests showed significantly elevated white blood cell and neutrophil percentages, suggesting bacterial infection. Based on the patient's clinical symptoms, signs, and preliminary laboratory results, septic shock was considered. On the third day after admission, the patient's blood culture was positive, yielding Klebsiella pneumoniae. A Sequential Organ Failure Assessment (SOFA) score was performed, resulting in a score of 4. Peripheral blood was collected on the same day as the positive blood culture and tested using this method, showing a VSIG4 result. + HLA-DR low CD14 + The proportion of monocytes was 3.5%, and IL1R2 was... + HLA-DR low CD14 + The monocyte percentage was 5.1%, which, according to the criteria presented in this invention, indicates a good prognosis for this individual. After comprehensive treatment including aggressive anti-infection therapy, fluid resuscitation, and respiratory support, the patient's body temperature gradually decreased, respiratory distress eased, and blood pressure returned to normal. Two weeks after hospitalization, the patient's condition stabilized, no other complications occurred, and the patient was discharged.

[0072] Individual 2, female, 70 years old, was admitted to the hospital due to "fever and cough with sputum for 2 weeks". The patient had a history of chronic obstructive pulmonary disease. Physical examination results: temperature 38.5℃, pulse 120 bpm, respiration 30 breaths / min, blood pressure 85 / 60 mmHg. Chest CT showed large areas of consolidation in both lower lobes with indistinct borders, suggestive of pulmonary infection. Blood routine showed significantly elevated white blood cell and neutrophil percentages, indicating bacterial infection. Based on the patient's clinical symptoms, signs, and preliminary laboratory results, septic shock was considered. On the 5th day after admission, the patient's blood culture result was positive: Acinetobacter baumannii was cultured. Sequential Organ Failure Assessment (SOFA) score was performed, and the result was 10, indicating severe multiple organ dysfunction and critical condition. Peripheral blood was collected on the same day as the positive blood culture for this individual's test using this method.

[0073] The results show VSIG4 + HLA-DR lowCD14 + The proportion of monocytes was 33.9%, and IL1R2... + HLA-DR low CD14 + A monocyte percentage of 25.1% indicates a poor prognosis according to the criteria presented in this invention. The patient developed acute respiratory distress syndrome during treatment and required mechanical ventilation after intubation. However, the patient's condition continued to deteriorate, developing multiple organ dysfunction syndrome, including acute kidney injury, liver failure, and coagulation disorders. Despite aggressive comprehensive treatment, the patient's condition did not improve significantly, and the patient ultimately died 20 days after admission.

[0074] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0075] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention is also intended to include these modifications and modifications.

Claims

1. A reagent kit for predicting the prognosis of sepsis, characterized in that, The kit includes fluorescently labeled CD45 antibody, CD14 antibody, CD16 antibody, HLA-DR antibody, VSIG4 antibody, and IL1R2 antibody for multicolor flow cytometry detection.

2. The reagent kit for predicting the prognosis of sepsis according to claim 1, characterized in that, The fluorescently labeled antibodies can be distinguished in multicolor flow cytometry.

3. The reagent kit for predicting the prognosis of sepsis according to claim 1, characterized in that, The fluorescent label can be selected from at least one of V500, BV421, PE-Cy7, BV605, PE, and APC.

4. The reagent kit for predicting the prognosis of sepsis according to claim 1, characterized in that, The CD45 antibody was labeled with V500, the CD14 antibody with BV421, the CD16 antibody with PE-Cy7, the HLA-DR antibody with BV605, the VSIG4 antibody with PE, and the IL1R2 antibody with APC.

5. The use of the kit for sepsis prognosis assessment according to any one of claims 1-4 in the preparation of products for assessing sepsis prognosis.

6. A sepsis prognostic biomarker detection system, characterized in that, include: Antibody combo module: contains fluorescently labeled antibodies against CD45, CD14, CD16, HLA-DR, VSIG4, and IL1R2; Flow cytometry module: Equipped with a multi-color laser light source and optical filter array for acquiring fluorescence signals; The data analysis module is programmed to perform the following operations: delineate nucleated cell populations based on forward scattering (FSC) and side scattering (SSC); preliminarily delineate monocyte populations within the nucleated cell populations based on CD45 / SSC; and delineate CD14+ cells within the preliminarily delineated monocyte populations based on CD14 / CD16. + Mononuclear cell population; in CD14 + VSIG4 was circled in the monocyte population based on its correlation with HLA-DR. + HLA-DR low Cells, simultaneously in CD14 + In the monocyte phylum, IL1R2 is circled based on IL1R2 and HLA-DR. + HLA-DR low cell; Calculate VSIG4 + HLA-DR low Cell percentages of P1 and IL1R2 + HLA-DR low Cell percentage P2; Output module: Displays the values ​​of P1 and P2 and a scatter plot.

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