Reagent related to occurrence and development of sepsis-related coagulopathy and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a reagent related to generation and development of sepsis-related blood coagulation diseases and application of the reagent. When the CD10-containing reagent is used for detecting the sepsis-related coagulopathy, the detection comprises the following steps: S1, collecting peripheral blood of a sepsis patient, and storing the peripheral blood for later use; s2, carrying out grouping on the neutrophile granulocytes by adopting CD45, CD15 and CD66b, and detecting the immunophenotypes of the neutrophile granulocytes by adopting CD10; adding an Fc blocking antibody into the collected peripheral blood according to the ratio of 1: 100; preparing an antibody premixed solution, and adding the antibody premixed solution for dyeing after the closing is finished; s3, diluting the erythrocyte lysate to a working concentration; the working solution is added into the dyed peripheral blood for red cracking, and after red cracking is finished, a PBS solution is added; performing centrifugal treatment to obtain single-cell precipitate and supernate; s4, removing the supernate, and repeating the step S3; adding a PBS (Phosphate Buffer Solution) for resuspending to obtain a colorless clear transparent single-cell suspension, carrying out flow-type on-machine detection on the single-cell suspension, and calculating the proportion of mature neutrophils to immature neutrophils.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a reagent related to the occurrence and development of sepsis-related coagulopathy and an application thereof. Background Art

[0002] Sepsis is a heterogeneous acute syndrome caused by infection and characterized by organ dysfunction. It poses a serious threat to human health, with a hospital mortality rate of up to 20%-30%. With the development of critical care medical diagnosis and treatment technology, the overall mortality rate of sepsis has decreased compared with before, but the management of its complications is still a major challenge for clinicians. Among them, coagulation dysfunction is the most common complication of sepsis, namely sepsis-related coagulopathy, which manifests as dysfunction of the coagulation system. Further development may lead to disseminated intravascular coagulation (DIC) and multiple organ dysfunction syndrome (MODS). Therefore, early and efficient diagnosis of sepsis-related coagulopathy is the key to standardized treatment and improved prognosis.

[0003] During infection, pathogenic microorganisms enter the body, and circulating neutrophils quickly infiltrate into the infection focus to eliminate pathogenic microorganisms. In this process, a large number of neutrophils are consumed, so bone marrow hematopoietic stem cells quickly differentiate into mature neutrophils and replenish into the circulation. When the consumption of neutrophils exceeds the normal replenishment capacity of the bone marrow, it will cause immature granulocytes to be mobilized into the circulation in advance. This phenomenon is called acute granulocytosis. The appearance of immature granulocytes in the circulation means the occurrence of systemic inflammatory response and is related to the development of sepsis and poor prognosis. Therefore, as early as when sepsis was defined as systemic inflammatory response syndrome (SIRS), the observation of rod-shaped or rod-shaped immature neutrophils in blood smears was used as one of the diagnostic criteria. Studies in recent years have shown that neutrophils play an important role in sepsis-related coagulopathy, especially the formation of immune microthrombi. CD10 is a neutrophil enzyme that participates in a variety of biological processes, including peptide degradation and regulation of cell surface receptors. In recent years, studies have shown that CD10 can effectively characterize the level of immature neutrophils. Studies have shown that the expression level of neutrophil CD10 in patients with sepsis is significantly reduced, but whether it can be used as an early diagnostic marker for sepsis-related coagulopathy remains to be clinically verified. In the field of diagnosis of sepsis-related coagulopathy, there are currently no similar products. The closest technology is the patent applied for by Zhongnan Hospital of Wuhan University, "Sepsis coagulation-related prognostic marker genes and their application in the preparation of sepsis prognosis prediction and diagnosis products", with patent number CN202310856437.4 and publication (announcement) number CN116987778A. The patent discloses sepsis coagulation-related prognostic marker genes composed of WFDC1, FGFBP2, CX3CR1, GIMAP4, ELANE and DEFA4, and establishes a prognostic scoring system based on these genes (sepsis prognosis risk prediction model). However, the patent mainly studies the predictive efficacy of related genes for the prognosis of sepsis-related coagulopathy, and cannot be used for early diagnosis of the occurrence of sepsis-related coagulopathy.

[0004] The current diagnosis of sepsis-related coagulopathy still lacks specificity. The existing diagnostic criteria are a composite diagnosis of sepsis combined with clinical and laboratory indicators such as coagulation function and platelets. At present, the conventional coagulation indicators commonly used in clinical practice include prothrombin time (PT) and activated partial thromboplastin time (APTT). Although they are helpful in the diagnosis of sepsis-related coagulopathy to a certain extent, they are mainly used to comprehensively assess the risk of bleeding, and the anticoagulant therapy based on this has failed to improve the prognosis of patients. Their sensitivity and specificity are not enough to meet clinical needs. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a reagent related to the occurrence and development of sepsis-related coagulopathy and its application.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A use of a reagent containing CD10 in the preparation of a product for diagnosing sepsis-related coagulopathy.

[0008] As a preferred embodiment of the present invention, the reagent containing CD10 specifically comprises the following components: Anti-humanCD45-APC-CY7, Anti-human CD10-PE, Anti-human CD15-Alexa fluor 488 and Anti-humanCD66b-PE / Dazzle 594; the volume ratio of Anti-human CD45-APC-CY7, Anti-human CD10-PE, Anti-humanCD15-Alexa fluor 488 and Anti-human CD66b-PE / Dazzle 594 is 5:2:2:2.

[0009] Furthermore, when a reagent containing CD10 is used to detect sepsis-related coagulopathy, the detection includes the following steps:

[0010] S1. Collect peripheral blood from patients with sepsis and store it for future use;

[0011] S2. Neutrophils were grouped using CD45, CD15, and CD66b, and then the neutrophil immune phenotype was detected using CD10. Specifically, Fc blocking antibody was added to the collected peripheral blood at a ratio of 1:100, and the mixture was shaken and incubated at room temperature for 15 minutes. After the blocking was completed, staining was performed directly, and the antibody premix was prepared.

[0012] S3, diluting the red blood cell lysis solution into a working solution; adding the working solution to the peripheral blood after staining and performing red cell lysis in an ice bath away from light, after the red cell lysis is completed, adding an equal volume of pre-cooled PBS solution to terminate the red cell lysis reaction; and then performing centrifugation to obtain a single cell precipitate and a supernatant;

[0013] S4, remove the supernatant, add the working solution again, and repeat the red lysis step in step S3; then add PBS solution to resuspend to obtain a colorless, clear and transparent single-cell suspension, and perform flow cytometry on the colorless, clear and transparent single-cell suspension to calculate the ratio of mature neutrophils to immature neutrophils.

[0014] Furthermore, in step S1, the peripheral blood collection volume is 3 ml and the storage temperature is 4°C.

[0015] Furthermore, the antibody premix solution is prepared by adding 5 μl Anti-human CD45-APC-CY7, 2 μl Anti-human CD10-PE, 2 μl Anti-human CD15-Alexa fluor488 and 2 μl Anti-humanCD66b-PE / Dazzle 594 into a test tube, gently shaking to mix, mixing at a low speed and incubating in the dark for 45 minutes to prepare the antibody premix solution.

[0016] Furthermore, the volume ratio of red blood cell lysate to pure water in the working solution is 1:10; when the volume of peripheral blood in step S2 is 100ul, the amount of the working solution is 2ml.

[0017] Furthermore, the centrifugal treatment conditions in step S3 are a rotation speed of 300G and a centrifugal time of 10 minutes.

[0018] Furthermore, the instrument used for flow cytometry in step S3 is Cytek Northern Lights TM Model full spectrum flow cytometer; the specific steps of flow cytometry detection are as follows:

[0019] Step 1. In the FSC / SSC scatter plot, set a gate to encircle all cells including granulocytes, lymphocytes and monocytes;

[0020] Step 2: In the SSC-A / SSC-H dot plot, set a gate to identify single cells

[0021] Step 3. In the APC-cy7 / SSC scatter plot, the cells circled in step 2 are divided into APC-cy7+ and APC-cy7- cells. APC-cy7+ are all immune cells that express positive CD45, and APC-cy7- are non-immune cells that express negative CD45.

[0022] Step 4: Circle the Alexa fluor 488+ / PE / Dazzle 594+ neutrophils in the Alexa fluor 488 / PE / Dazzle 594 scatter plot;

[0023] Step 5. In the PE / Count scatter plot, analyze the PE+ / PE- cells of the neutrophils and calculate the ratio of mature neutrophils to immature neutrophils.

[0024] A product for diagnosing sepsis-related coagulopathy, wherein the product is a reagent containing CD10.

[0025] Flow cytometry (FCM) is a detection method for quantitative analysis and sorting of single cells or other biological particles at the functional level. It can analyze tens of thousands of cells at high speed and measure multiple parameters from one cell at the same time.

[0026] Beneficial effects of the present invention:

[0027] 1. The present invention only requires 300ul of extremely small amount of whole blood sample, which has little impact on patients.

[0028] 2. The present invention does not require intervention or pre-labeling of neutrophils and has simple steps, little interference or impact on cells, and no problem of marker leakage.

[0029] 3. CD10 of the present invention, as a characteristic marker of mature neutrophils, can effectively distinguish mature and immature neutrophils in peripheral blood, and has a good degree of differentiation for sepsis-related coagulopathy. Figure 2 , so the detection range of the present invention is wide.

[0030] 4. The present invention can evaluate the coagulation function status of sepsis patients by identifying the ratio of mature to immature neutrophils. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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 will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 This is the flow gate scheme of the present invention; Note: P1 gate: blood sample after erythrocyte lysis; P2 gate: de-adhesion; WBC gate: all CD45 + Leukocytes; Total Neu gate: all CD66b + CD15 + Neutrophils; CD10 - Imm Neu:CD10 - Immature neutrophil ratio; CD10 + Imm Neu:CD10 + The proportion of mature neutrophils.

[0033] Figure 2 CD10 + Results of mature neutrophil detection; Note: Negative control: healthy volunteers without CD10 staining; Positive control: healthy volunteers with CD10 antibody added; Test tube: sepsis patients with CD10 antibody added. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] To achieve the above object, the present invention adopts the following technical solutions:

[0036] S1: Collect blood samples from clinical patients, disinfect the patient's peripheral superficial vein with iodine, use a 22G disposable venous blood collection needle to puncture the blood vessel, and then draw 3 ml of peripheral blood from the patient using a vacuum EDTA anticoagulation blood collection tube. The specimens are stored at 4°C and completed flow cytometry detection within 6 hours.

[0037] S2: Flow cytometry staining: CD45, CD15, and CD66b were used to group neutrophils, and then CD10 was used to detect the neutrophil immune phenotype.

[0038] 1) Set up 3 tubes: the first tube is a blank control tube, the second tube is a fluorescence minus one control (Fluorescence MinusOne, FMO) tube, and the third tube is a test tube.

[0039] 2) Fc antibody blocking: Take 100ul of whole blood from three tubes respectively, add 1ul of Fc blocking antibody at a ratio of 1:100, shake well, and incubate at room temperature for 15 minutes;

[0040] 3) Multicolor flow cytometry staining: After blocking, the sample is stained directly, and the following antibody premix is ​​prepared:

[0041] ① The negative control tube does not contain antibodies.

[0042] ②FMO tube:

[0043]

[0044]

[0045] ③Test tube:

[0046] Mix gently by shaking, then incubate at low speed for 45 minutes in the dark.

[0047] S3: Red blood cell lysis:

[0048] 1) Dilute 10X BD erythrocyte lysis buffer with pure water at a ratio of 1:10 to make a working solution. After staining, add 2 mL of working solution to each sample tube and place in an ice bath away from light for 10 minutes. After the lysis is completed, add an equal volume of pre-cooled PBS to terminate the lysis reaction. Then centrifuge slowly at 300g for 10 minutes.

[0049] 2) After centrifugation, discard the supernatant, add 2 ml of erythrocyte lysis working solution again, repeat the above erythrocyte lysis steps, then add 200 ul 1× PBS to resuspend to obtain a colorless, clear and transparent single cell suspension, ready for flow cytometry detection.

[0050] S4: Streaming on-machine testing.

[0051] Use: Cytek Northern Lights TM Model full spectrum flow cytometer to establish flow cytometry analysis template.

[0052] Step 1. In the FSC / SSC scatter plot, set a gate to encircle all cells including granulocytes, lymphocytes and monocytes;

[0053] Step 2: In the SSC-A / SSC-H dot plot, set a gate to identify single cells

[0054] Step 3. In the APC-cy7 / SSC scatter plot, the cells circled in Step 2 are divided into APC-cy7+ and APC-cy7- cells. APC-cy7+ are all immune cells that express positive CD45, and APC-cy7- are non-immune cells that express negative CD45.

[0055] Step 4: Circle the Alexa fluor 488+ / PE / Dazzle 594+ neutrophils in the Alexa fluor 488 / PE / Dazzle 594 scatter plot;

[0056] Step 5. In the PE / Count scatter plot, analyze the PE+ / PE- cells of the neutrophils in Step 4 and calculate the ratio of mature neutrophils to immature neutrophils.

[0057] 7) The scatter plots obtained by flow cytometry analysis are generated by any national standard flow cytometry analysis software.

[0058] Case Study:

[0059] The peripheral whole blood of 28 non-sepsis-related coagulopathy groups, 38 sepsis-related coagulopathy groups and 30 healthy people was detected by the CD10 mean fluorescence intensity method of the present invention: after ROC curve statistical analysis, the area under the ROC curve was 79.79%, the cutoff value was 0.4586, the sensitivity was 0.8158, and the specificity was 0.6429. Similarly, the peripheral whole blood of 28 non-sepsis-related coagulopathy groups, 38 sepsis-related coagulopathy groups and 30 healthy people was detected by the CD10% method of the present invention: after ROC curve statistical analysis, the area under the ROC curve was 82.61%, the cutoff value was 0.5300, the sensitivity was 0.8157, and the specificity was 0.7143.

[0060] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0061] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Use of a reagent containing CD10 in the preparation of a product for diagnosing sepsis-related coagulopathy.

2. The use according to claim 1, characterized in that: The CD10-containing reagent specifically comprises the following components: Anti-human CD45-APC-CY7, Anti-human CD10-PE, Anti-human CD15-Alexa fluor 488 and Anti-human CD66b-PE / Dazzle 594; the volume ratio of Anti-human CD45-APC-CY7, Anti-human CD10-PE, Anti-human CD15-Alexa fluor 488 and Anti-human CD66b-PE / Dazzle 594 is 5:2:2:

2.

3. The use according to claim 1, characterized in that: When using a CD10-containing reagent to detect sepsis-associated coagulopathy, the test includes the following steps: S1. Collect peripheral blood from patients with sepsis and store it for future use; S2. Neutrophils were grouped using CD45, CD15, and CD66b, and then the neutrophil immune phenotype was detected using CD10. Specifically, Fc blocking antibody was added to the collected peripheral blood at a ratio of 1:100, and the mixture was shaken and incubated at room temperature for 15 minutes. Antibody premix was prepared, and after the blocking was completed, the antibody premix was added for staining. S3, diluting the red blood cell lysis solution into a working solution; adding the working solution to the peripheral blood after staining and performing red cell lysis in an ice bath away from light, after the red cell lysis is completed, adding an equal volume of pre-cooled PBS solution to terminate the red cell lysis reaction; and then performing centrifugation to obtain a single cell precipitate and a supernatant; S4. Remove the supernatant from the suspension, add the working solution again, and repeat the red lysis step in step S3; then add PBS solution to resuspend to obtain a colorless, clear and transparent single-cell suspension, and perform flow cytometry on the colorless, clear and transparent single-cell suspension to calculate the ratio of mature neutrophils to immature neutrophils.

4. The use according to claim 3, characterized in that: In step S1, the peripheral blood collection volume is 3 ml and the storage temperature is 4°C.

5. The use according to claim 3, characterized in that: The antibody premix solution is prepared by adding 5 μl Anti-human CD45-APC-CY7, 2 μl Anti-human CD10-PE, 2 μl Anti-human CD15-Alexafluor 488 and 2 μl Anti-human CD66b-PE / Dazzle 594 into a test tube and gently shaking to mix. After mixing at a low speed, the antibody premix solution can be prepared.

6. The use according to claim 3, characterized in that: The volume ratio of red blood cell lysate to pure water in the working solution is 1:10; when the volume of peripheral blood in step S2 is 100ul, the amount of the working solution is 2ml.

7. The use according to claim 3, characterized in that: The centrifugal treatment conditions in step S3 are a rotation speed of 300G and a centrifugal time of 10 minutes.

8. The use according to claim 3, characterized in that: The instrument used for flow cytometry in step S3 is Cytek Northern Lights TM Model full spectrum flow cytometer; the specific steps of flow cytometry detection are as follows: Step 1. In the FSC / SSC scatter plot, set a gate to encircle all cells including granulocytes, lymphocytes and monocytes; Step 2: In the SSC-A / SSC-H scatter plot, set a gate to circle single cells Step 3. In the APC-cy7 / SSC scatter plot, the cells circled in step 2 are divided into APC-cy7+ and APC-cy7- cells. APC-cy7+ are all immune cells that express positive CD45, and APC-cy7- are non-immune cells that express negative CD45. Step 4: Circle the Alexa fluor 488+ / PE / Dazzle 594+ neutrophils in the Alexa fluor 488 / PE / Dazzle 594 scatter plot; Step 5. In the PE / Count scatter plot, analyze the PE+ / PE- cells of the neutrophils and calculate the ratio of mature neutrophils to immature neutrophils.

9. A product for diagnosing sepsis-related coagulopathy, characterized in that: The product is a reagent containing CD10.

Images