Sample Pretreatment and Preservation Method for Flow Cytometry Detection of Cytokines in Rheumatism Patients

Abstract

The present invention provides a method for sample pretreatment and preservation for cytokine flow detection of rheumatism patients, and a diagnostic kit for determining the type of autoimmune disease and diagnosing autoimmune diseases (such as rheumatism). Specifically, the method and kit provided by the present invention have clinical significance for improving the stability and accuracy of cytokine detection results based on flow cytometry in rheumatism patients. In addition, the method established according to the particularity of cytokine expression in the bodies of rheumatism patients has accurate detection results, good repeatability, and is easy to be clinically popularized and applied.

Description

Technical Field

[0001] The present invention relates to the field of medical tests, and particularly to a method for sample pretreatment and preservation for flow cytometry detection of cytokines in rheumatism patients, and a kit for diagnosing rheumatism. Background Art

[0002] Cytokines are a class of bioactive polypeptides or glycoproteins produced by various immune cells and non-immune cells. Commonly referred to cytokines include lymphokines, monokines and cytokines produced by other cells. There is a wide variety of cytokines, and their biological activities are also complex and diverse. There are more and more research reports on their roles in maintaining the normal state of the body and the occurrence of different diseases. With the rapid development of related disciplines such as molecular biology, various detection methods for cytokines have been constructed one after another.

[0003] Flow cytometry (FCM) technology is to apply a flow cytometer, combined with monoclonal antibody technology and immunofluorescence staining technology, to measure and analyze multiple parameters of single cells in a rapidly flowing liquid, with a fast detection speed and high precision. The method for quantitatively detecting multiple cytokines by a flow cytometer can qualitatively and quantitatively detect multiple indicators in a sample at the same time. It has been widely used in China. However, due to the reagents of each manufacturer, especially in the field of special rheumatism patients, there is no corresponding standardized operation process established. In order to make the experimental results more truly reflect the situation in the patient's body, it is crucial to develop a set of standardized operation processes for cytokine detection by flow cytometry.

[0004] The pathogenesis of autoimmune diseases is very complex, but the activation of autoreactive T cells and B cells is an inevitable link in the pathogenesis of autoimmune diseases. Therefore, many scholars divide autoimmune diseases into three categories: T cell-mediated, autoantibody-mediated, and jointly mediated autoimmune diseases. Autoantibodies, an important product after B cell activation, have been used as markers for diagnosing autoimmune diseases, while there are currently no specific indicators for routinely detecting autoreactive T cells, which makes it difficult to accurately determine whether an autoimmune disease is a T cell-mediated autoimmune disease or a jointly mediated autoimmune disease.

[0005] The detection of cytokines produced by T cells reflects to a certain extent the activation degree of T cells. The combined detection of multiple cytokines can reflect the activation of different subsets of helper T cells (Th). Due to the uncertainty of T cell activity in rheumatism patients, these cytokines secreted into serum or plasma not only have low content, but are also not easy to detect.

[0006] Therefore, it is crucial to establish a detection method that can accurately detect autoimmune diseases mediated by autoreactive T cells. Summary of the Invention

[0007] The object of the present invention is to provide a method for sample pretreatment and preservation for cytokine flow detection in patients with rheumatism.

[0008] In the first aspect of the present invention, a diagnostic kit is provided, and the kit includes:

[0009] (a) A blood collection tube for blood collection, and the blood collection tube is a blood collection tube with a separation gel and containing a coagulant;

[0010] (b) Detection reagents for detecting cytokines, and the cytokines are selected from the group consisting of: IL-8, IL-17A, TNF-β, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17F, IL-22, TNF-α, IFN-γ, or a combination thereof;

[0011] The kit is used to determine whether an autoimmune disease is an autoimmune disease mediated by T cells, or whether it is an autoimmune disease jointly mediated by T cells and autoantibodies.

[0012] In another preferred example, the cytokines at least include IL-8, IL-17A, TNF-β, or a combination thereof.

[0013] In another preferred example, the kit further includes a label or an instruction manual, and the label or the instruction manual indicates that: the kit is used to determine whether an autoimmune disease is an autoimmune disease mediated by T cells, or whether it is an autoimmune disease jointly mediated by T cells and autoantibodies.

[0014] In the second aspect of the present invention, a method for sample pretreatment before detection is provided, and the method includes the following steps:

[0015] (a) Provide a blood collection tube with a separation gel;

[0016] (b) Use the blood collection tube with a separation gel to collect a blood sample;

[0017] (c) Centrifuge the blood sample in the blood collection tube to obtain a separated serum sample;

[0018] (d) Detect the separated serum sample.

[0019] In another preferred example, the time from step (b) to step (c) does not exceed 4 hours, preferably 1 hour.

[0020] In another preferred example, before step (d), it may further include immediately storing the separated serum sample described in step (c) at -20°C to -80°C, preferably -20°C, and performing step (d) within 24 hours after storage.

[0021] In another preferred example, in step (c), the rotational speed of the centrifugation is 4000 rpm, the centrifugation time is 5 min, and the centrifugation temperature is 20°C ± 2°C.

[0022] In another preferred example, the sample is a serum sample separated from a blood sample.

[0023] In another preferred example, the sample is derived from a human or non-human mammal.

[0024] In another preferred example, the sample is derived from a human.

[0025] In another preferred example, the detection is the detection of cytokines in the serum sample.

[0026] In another preferred example, the detection is performed using a flow cytometer.

[0027] In another preferred example, the purpose of the detection is to determine whether the autoimmune disease is a T cell-mediated autoimmune disease, or whether it is an autoimmune disease jointly mediated by T cells and autoantibodies.

[0028] In another preferred example, the cytokines include IL-8, IL-17A, TNF-β, or a combination thereof.

[0029] In another preferred example, the cytokines are selected from the group consisting of: IL-8, IL-17A, TNF-β, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17F, IL-22, TNF-α, IFN-γ, or a combination thereof.

[0030] In another preferred example, the blood collection tube is a vacuum blood collection tube.

[0031] In another preferred example, the blood collection tube further contains a coagulant for promoting blood coagulation.

[0032] In another preferred example, the blood sample is derived from a human or non-human mammal.

[0033] In another preferred example, the blood sample is derived from a human.

[0034] In another preferred example, the blood sample is derived from a patient with an autoimmune disease, and the autoimmune disease is a T cell-mediated autoimmune disease or an autoimmune disease jointly mediated by T cells and autoantibodies.

[0035] In another preferred example, the blood sample is derived from a patient with rheumatism.

[0036] In another preferred example, the method comprises the following steps:

[0037] (a) Providing a blood collection tube with a separating gel;

[0038] (b) Collecting a blood sample using the blood collection tube with the separating gel;

[0039] (c) Centrifuging the blood sample in the blood collection tube within 1 hour after collection to obtain a separated serum sample; and

[0040] (d) Detecting the separated serum sample.

[0041] In another preferred example, the method comprises the following steps:

[0042] (a) Providing a blood collection tube with a separating gel;

[0043] (b) Collecting a blood sample using the blood collection tube with the separating gel;

[0044] (c) Centrifuging the blood sample in the blood collection tube within 1 hour after collection to obtain a separated serum sample;

[0045] (d) Immediately storing the separated serum sample obtained in step (c) at -20 °C to obtain a serum sample stored at -20 °C; and

[0046] (e) Detecting the serum sample stored at -20 °C within 24 hours after storage.

[0047] In a third aspect of the present invention, there is provided a method for storing a serum sample, the method comprising storing the serum sample separated from a blood collection tube with a separating gel at -20 °C to -80 °C, preferably -20 °C, within 24 hours, wherein the sample is used to determine whether an autoimmune disease is a T cell-mediated autoimmune disease or an autoimmune disease jointly mediated by T cells and autoantibodies by detecting cytokines therein using a flow cytometer.

[0048] In another preferred example, the serum sample is derived from a patient with an autoimmune disease, and the autoimmune disease is a T cell-mediated autoimmune disease or an autoimmune disease jointly mediated by T cells and autoantibodies.

[0049] In another preferred example, the serum sample is from a rheumatism patient.

[0050] In the fourth aspect of the present invention, there is provided a use of a diagnostic kit as described in the first aspect of the present invention, wherein the kit is used to determine whether an autoimmune disease is a T cell-mediated autoimmune disease or an autoimmune disease jointly mediated by T cells and autoantibodies.

[0051] In another preferred example, the kit is used to determine whether a subject has rheumatism.

[0052] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the effects of four blood collection tubes on cytokine detection by flow cytometry in samples from 100 normal individuals. Among them, the detection results are expressed as mean ± SD, and the gray horizontal line is the cut-off value for each cytokine.

[0054] Figure 2 Shows the effects of four blood collection tubes on cytokine detection in samples from normal individuals at different temperatures and storage times; among them, purple: EDTA anticoagulated plasma tube, green: heparin anticoagulated plasma tube, red: ordinary serum tube without anticoagulant (containing clot promoter), yellow: serum tube with separator gel (containing clot promoter).

[0055] Figure 3 Shows the detection results of cytokines in samples from normal individuals by an ordinary serum tube without anticoagulant (containing clot promoter) and a serum tube with separator gel at different temperatures and storage times. * in the table indicates a statistically significant difference compared with the detection results on the 0th day (i.e., 4h).

[0056] Figure 4 Shows the effects of four blood collection tubes on cytokine detection by flow cytometry in samples from a rheumatism population. Among them, the detection results are expressed as mean ± SD, * indicates a statistically significant difference in the detection values between the ordinary serum tube without anticoagulant and the two plasma tubes, and # indicates a statistically significant difference in the detection values between the serum tube with separator gel and the two plasma tubes.

[0057] Figure 5Shows the effects of serum collection tubes at different temperatures and storage times on the detection of cytokines in samples from the rheumatic population. Among them, red: ordinary serum tubes without anticoagulant (containing clot activators), yellow: serum tubes with separator gel (containing clot activators); * indicates that there is a statistical difference in the detection values between the ordinary serum tubes without anticoagulant and the two plasma tubes, and # indicates that there is a statistical difference in the detection values between the serum tubes with separator gel and the two plasma tubes.

[0058] Figure 6 Shows the detection results of cytokines in samples from the rheumatic population by ordinary serum tubes without anticoagulant (containing clot activators) and serum tubes with separator gel at different temperatures and storage times. * in the table indicates a statistical difference compared with the detection results on the 0th day (i.e., 4h). Detailed implementation mode

[0059] Through extensive and in-depth research, a large number of screenings and tests, the present inventor has unexpectedly developed for the first time a diagnostic kit for determining the types of autoimmune diseases and diagnosing autoimmune diseases (especially rheumatism), and also provided a method for sample pretreatment and a storage method for flow cytometry detection of cytokines in patients with rheumatism. The kit of the present invention mainly includes a blood collection tube with separator gel and containing clot activators, and a detection reagent for detecting cytokines, and the cytokines mainly include: IL-8, IL-17A, TNF-β, or a combination thereof. Experiments have proved that by using a blood collection tube with separator gel and containing clot activators to collect blood samples from patients with rheumatism, separated serum samples are obtained by centrifugation within 1 hour, and the serum samples are immediately subjected to flow cytometry detection; or the serum samples obtained by the above centrifugation are stored at -20 °C and subjected to flow cytometry detection within 24 hours, and accurate and reliable cytokine detection results can be obtained. On this basis, the present invention has been completed.

[0060] Terms

[0061] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used in this article have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used in this article are only intended to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will only be limited by the appended claims.

[0062] The term "sample" or "specimen" used in this article refers to a material specifically associated with a subject, from which specific information related to the subject can be determined, calculated or inferred. The sample can be wholly or partly composed of biological materials from the subject.

[0063] Rheumatism

[0064] Rheumatism is a group of autoimmune diseases mainly involving joints, bones, muscles, blood vessels and related soft tissues or connective tissues. The main pathogenesis of rheumatism is the occurrence of immune responses: the body is directly stimulated by exogenous or endogenous antigenic substances or through the presentation of macrophages, which activates corresponding T cells. Some T cells produce a large number of various pro-inflammatory cytokines, causing varying degrees of damage or destruction to various tissues and organs; some T cells then activate B cells, producing a large number of antibodies, which directly or combine with antigens to form immune complexes, causing damage or destruction to tissues or organs. Therefore, the onset of rheumatism is closely related to the activation of T cells and the production of cytokines. It is a type of autoimmune disease mediated by autoreactive T cells or jointly mediated by T cells and B cells.

[0065] IL-8, IL-17F and TNF-β are all monokines produced by monocytes / macrophages. They are hormone-like molecules that can regulate the maturation and differentiation of T and B lymphocytes. They play important roles in inflammatory responses and immune regulation, and TNFα plays a central role in the cytokine network of rheumatism. TNF-β is produced in the early stage of immune responses and is an endogenous pyrogen that can cause the body to have a fever reaction. The levels of TNF-β, IL-17F and IL-8 expressed in the bodies of patients with rheumatism are closely related to the degree of disease activity and other immune indicators, further proving their important roles in the pathogenesis of rheumatic immune diseases.

[0066] Cytokine

[0067] Cytokines (CK) are low-molecular-weight soluble proteins produced by a variety of cells induced by immunogens, mitogens or other stimulants, and have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, and repair of damaged tissues. Cytokines can be classified into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. Cytokines are mainly produced by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and some non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.).

[0068] The sample pretreatment and preservation method provided by the present invention is for the flow cytometry detection of cytokines in serum samples. The cytokines mainly include IL-8, IL-17A, TNF-β, or a combination thereof. In another preferred example, the cytokines include (but are not limited to): IL-8, IL-17F, TNF-β, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A, IL-22, TNF-α, IFN-γ, etc.

[0069] Blood collection tube of the present invention

[0070] The method for pre - treatment of sample detection of the present invention defines the blood collection tube used in the process of collecting blood samples. The blood collection tube is a blood collection tube with separation gel. In a preferred embodiment of the present invention, the blood collection tube is an inert separation gel clot - activator tube, and an inert separation gel and a clot - activator are added into the blood collection tube. The clot - activator can quickly activate the blood coagulation mechanism and accelerate the blood coagulation process. The sample type obtained is serum. After collecting the blood sample, invert and mix it evenly 5 - 8 times, stand upright for 20 - 30 min, and the supernatant above the separation gel after centrifugation is the separated serum.

[0071] Both the clot - activator and the separation gel described in the present invention are selected from the products currently on the market. It can also be referred to, for example, as described in Patent CN1046036C.

[0072] Separation gel is an important raw material used in vacuum blood collection tubes. Its function is that the specific gravity of the separation gel is between serum and blood cells. After the blood is centrifuged by rotation and precipitated, the separation gel separates the serum and blood cells. The separation gel has a high viscosity and is not easy to be encapsulated at room temperature. According to the blood collection purpose of the vacuum blood collection tube and national standards, the weight of blood separation gel added to each blood collection tube is 1g ± 10%. Serum separation gel, as an isolation medium in blood collection tubes for serum biochemical or plasma biochemical tests, can completely separate serum (or plasma) from red blood cells, obtain high - quality serum (or plasma), and effectively ensure the accuracy of blood tests. After blood collection, after the blood is coagulated or anticoagulated, serum (or plasma) and red blood cells can be separated by centrifugation. Since the specific gravity of serum separation gel is between serum (or plasma) and red blood cells, it moves and turns to between the two components during centrifugation. After centrifugation stops, it separates and blocks serum (or plasma) from red blood cells, playing a role of separation and isolation.

[0073] The method of the present invention

[0074] The present invention provides a method for pre - treatment of sample detection for detecting cytokines in serum samples by flow cytometry. The method includes the steps:

[0075] (a) Provide a blood collection tube with separation gel;

[0076] (b) Use the blood collection tube with separation gel to collect a blood sample;

[0077] (c) Centrifuge the blood sample in the blood collection tube to obtain a separated serum sample; and

[0078] (d) Detect the separated serum sample.

[0079] Among them, the time taken from step (b) to step (c) does not exceed 4 hours, preferably 1 hour, and a separated serum sample is obtained by centrifuging at 4000 rpm for 5 minutes under the condition of 20°C ± 2°C.

[0080] After obtaining the separated serum sample, the sample can be immediately detected by flow cytometry; when detection cannot be carried out immediately, the separated serum sample can be stored at -20°C to -80°C, preferably -20°C, and detected by flow cytometry within 24 hours after storage.

[0081] Therefore, the present invention also provides a method for storing a sample for flow cytometry detection of cytokines, the method comprising storing a serum sample separated from a blood collection tube with a separator gel at -20°C to -80°C, preferably -20°C, within 24 hours.

[0082] The main advantages of the present invention are as follows:

[0083] (1) Flow cytometry is currently the mainstream method for detecting multiple cytokines, with simple operation and no need for specific instrument equipment, and can be carried out in most clinical laboratories and research laboratories.

[0084] (2) Due to the differences in the physicochemical properties of each cytokine, the present invention has formulated appropriate sample storage conditions for detecting multiple cytokines by the same detection method.

[0085] (3) The present invention has formulated a standardized pre-treatment protocol for detection samples for the method of detecting multiple cytokines by flow cytometry, which is convenient for correctly detecting the expression levels of multiple cytokines, especially IL-8, IL-17F, and TNF-β.

[0086] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0087] The materials, reagents, instruments, etc. used in the embodiments can be obtained from commercial sources without special instructions.

[0088] Method for detecting cytokines by flow cytometry

[0089] Use the cytokine detection kit for flow cytometry produced by Tianjin Kuangbo Company. The specific operation steps are as follows:

[0090] (1) Add 20 μL of 1* capture microsphere mixture to each test tube (vortex for 45 seconds before adding the microspheres).

[0091] (2) Take standard quality control tubes numbered 1 - 8 and add 20 μL of serially diluted standards respectively; add 20 μL of experimental diluent to tube No. 9 as the background tube; add 20 μL of the sample to be tested to each sample tube.

[0092] (3) Vortex all test tubes for 10 - 20 seconds to mix well.

[0093] (4) Add 20 μL of 1* detection antibody mixture to each test tube, vortex to mix, and incubate with shaking in the dark at room temperature (23 - 27°C) for 2 hours (shaking frequency: 500 revolutions per minute).

[0094] (5) Add 20 μL of SA-PE to each test tube and incubate with shaking in the dark at room temperature for 30 minutes.

[0095] (6) Add 300 μL of 1* buffer to each test tube, vortex for 10 - 20 seconds, centrifuge at 500g for 5 minutes, discard the supernatant, and gently invert the test tube mouth on the absorbent paper to dry the residual liquid at the tube mouth.

[0096] (7) Repeat step (6).

[0097] (8) Add 150 - 300 μL of 1* buffer to each test tube and detect using a flow cytometer.

[0098] Example 1

[0099] Effect of blood collection tubes on cytokine detection results of samples from normal population

[0100] For the healthy control group, blood was collected using EDTA anticoagulant tubes, heparin anticoagulant tubes, ordinary serum tubes without anticoagulant, and serum separator tubes with separator gel respectively. After centrifuging at 4000 rpm for 5 minutes within 1 hour, the separated plasma / serum was detected within 4 hours. The number of samples from healthy people in this example was 100 cases.

[0101] The results are as Figure 1 shown in Table 1.

[0102] Table 1 Detection values of cytokines based on flow cytometry in samples from 100 normal population using four types of blood collection tubes

[0103]

[0104] The results showed that the detection values of the 4 types of blood collection tubes did not exceed the upper limit of the reference range set by the manufacturer ( Figure 1 ).

[0105] Example 2

[0106] Effect of storage temperature and storage time of samples on cytokine detection results of samples from normal population

[0107] In this example, the effects of storage temperature and storage time of samples after sampling on the detection of cytokines by flow cytometry were tested. In the healthy control group, blood was collected using EDTA anticoagulant tubes, heparin anticoagulant tubes, ordinary serum tubes without anticoagulant (containing clot promoters), and serum separator tubes with separating gel (containing clot promoters), respectively.

[0108] For the blood samples collected using EDTA anticoagulant tubes and heparin anticoagulant tubes, centrifuge at 4000 rpm for 5 minutes to obtain plasma samples.

[0109] For the blood samples collected using ordinary serum tubes without anticoagulant (containing clot promoters) and serum separator tubes with separating gel (containing clot promoters), centrifuge at 4000 rpm for 5 minutes to obtain serum samples.

[0110] The prepared plasma samples and serum samples were respectively stored at 4°C, -20°C, and -80°C, and the cytokines in the samples in the 4 types of blood collection tubes were detected by flow cytometry on the 0th, 1st, 3rd, 5th, and 7th days of storage.

[0111] The results are as Figure 2 , and Figure 3 shown in the table.

[0112] In the healthy control group, for most cytokines, the detection values in the 4 types of blood collection tubes stored at 4°C, -20°C, and -80°C respectively did not exceed the manufacturer's set range; however, during the storage of IL-8 at 4°C and -20°C until the 7th day, except for the serum tubes with separating gel which remained within the normal range, the detection values of the other three types of tubes exceeded the upper limit of normal people, especially in the samples of EDTA anticoagulant tubes and heparin anticoagulant tubes.

[0113] In addition, the detection value of TNF-β exceeded the normal upper limit during the storage in EDTA anticoagulant tubes.

[0114] Therefore, it can be concluded that for serum or plasma test samples from normal people (healthy controls), there is no obvious difference in the samples of the 4 types of tubes at 3 temperatures and within a storage time of up to 1 week, and the serum tubes with separating gel perform the best in all sample detections.

[0115] Example 3

[0116] Effect of blood collection tubes on cytokine detection results of samples from patients with rheumatism

[0117] For the rheumatic population (mainly including systemic lupus erythematosus, inflammatory myopathy, Behçet's disease), blood samples were collected using EDTA anticoagulant tubes, heparin anticoagulant tubes, ordinary serum tubes without anticoagulant, and serum separator tubes with separating gel respectively. After centrifuging at 4000 rpm for 5 minutes within 1 hour, the separated plasma / serum was immediately tested. The number of samples from the rheumatic population in this example was 200 cases.

[0118] The results are as Figure 4 shown in

[0119] Table 2 Detection values of cytokines based on flow cytometry in samples from 200 cases of rheumatic population using four types of blood collection tubes

[0120]

[0121] The results show that:

[0122] For IL-8, when using heparin anticoagulant tubes and EDTA anticoagulant tubes, the detection values are higher compared to the two types of serum tubes.

[0123] For IL-17F, the detection value in the heparin anticoagulant tube is significantly higher than that in the other three types of tubes.

[0124] This result indicates that neither EDTA anticoagulant tubes nor heparin anticoagulant tubes are suitable for cytokine detection in rheumatic patients.

[0125] Example 4

[0126] Effect of sample storage temperature and storage time on cytokine detection results of samples from rheumatic population

[0127] In this example, further analysis was carried out on the effect of sample storage temperature and storage time on cytokine detection results of samples from rheumatic population when using ordinary serum tubes or serum separator tubes with separating gel.

[0128] In the rheumatic population, blood samples were collected using ordinary serum tubes (containing clot activators) without anticoagulant and serum separator tubes (containing clot activators) with separating gel respectively. After centrifuging at 4000 rpm for 5 minutes, the serum samples in the two types of blood collection tubes were respectively stored at 4°C, -20°C, and -80°C, and the cytokines in the samples in the two types of blood collection tubes were detected by flow cytometry on the 0th, 1st, 3rd, 5th, and 7th days of storage. The number of samples from the rheumatic population in this example was 200 cases.

[0129] The results are as Figure 5 and Figure 6 shown in the tables in

[0130] The results show that:

[0131] In the population of patients with rheumatism, serum samples separated from ordinary serum tubes (containing clot promoters) without anticoagulants were stored at 4°C, -20°C, and -80°C respectively. For most cytokines in the samples, the detected values on the 1st day of storage had statistical differences compared with those on the 0th day.

[0132] While serum samples separated from serum separation tubes with separating gel (containing clot promoters) were stored at 4°C, -20°C, and -80°C respectively. For most cytokines in the samples, the detected values on the 3rd day of storage had statistical differences compared with those on the 0th day. Except for IL-6 and IL-8, for most cytokines in the serum samples separated from serum separation tubes with separating gel (containing clot promoters) stored at -20°C, the detected values on the 1st day of storage had no statistical differences compared with those on the 0th day ( Figure 4 ).

[0133] Thus, it can be seen that only when using serum collection tubes with separating gel (containing clot promoters) for serum samples, the test results are closest to the immediate test when stored at -20°C for at most 1 day.

[0134] The immune system in the body of patients with rheumatism is extremely complex, so a relatively high requirement is placed on the authenticity of the detected values of cytokines. Based on the above results, blood should be collected using blood collection tubes with separating gel, centrifuged at 4000 rpm for 5 minutes within 1 hour in a timely manner and then detected, or the serum should be aliquoted after centrifugation and immediately stored at -20°C, and the test results within 24 hours are the most authentic and reliable. If the storage time exceeds 24 hours, the detected values deviate greatly.

[0135] All documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a combination of a blood collection tube and a detection reagent in the preparation of a diagnostic kit for judging rheumatism, wherein, the blood collection tube is a blood collection tube with a separation gel and containing a coagulant; and the detection reagent is a detection reagent for detecting cytokines by a flow cytometer, and the cytokines include IL-8, IL-17A and TNF-b; and, the cytokines further include IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17F, IL-22, TNF-a, IFN-γ, or a combination thereof; the diagnostic kit further comprises a label, and the label indicates: collect a blood sample using the blood collection tube with a separation gel and containing a coagulant, centrifuge the blood sample in the blood collection tube within 1 hour to obtain a separated serum sample, and then immediately perform flow cytometer detection on the separated serum sample, or immediately store the separated serum sample at -20°C and perform flow cytometer detection within 24 hours after storage.

2. The use according to claim 1, wherein, The rotation speed of the centrifugation is 4000 rpm, the centrifugation time is 5 min, and the temperature of the centrifugation is 20°C ± 2°C.

3. The use according to claim 1, wherein, The blood sample is from a rheumatism patient.

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