CN113295496A - Sample pretreatment and preservation method for flow detection of cytokines of rheumatism patient - Google Patents
Sample pretreatment and preservation method for flow detection of cytokines of rheumatism patient Download PDFInfo
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Abstract
The invention provides a sample pretreatment and preservation method for flow detection of a cytokine of a rheumatism patient, and a diagnostic kit for judging the type of an autoimmune disease and diagnosing the autoimmune disease (such as rheumatism). Specifically, the method and the kit provided by the invention have clinical significance for improving the stability and accuracy of the cytokine detection result of the rheumatism patient based on the flow cytometry. In addition, the method established according to the specificity of the expression of the cytokines in the body of the rheumatism patient has accurate detection result and good repeatability, and is easy to popularize and apply clinically.
Description
Technical Field
The invention relates to the field of medical examination, in particular to a sample pretreatment and preservation method for flow detection of cytokines of a rheumatism patient and a kit for diagnosing rheumatism.
Background
Cytokines (cytokines) are a class of biologically active polypeptides or glycoproteins produced by a variety of immune and non-immune cells. Cytokines are known to include lymphokines, monocytes, and other cytokines produced by cells. Cytokines are in a wide variety of varieties, and their biological activities are complex and diverse, and there are also increasing reports on research on their roles in maintenance of normal states of the body and development of different diseases. With the rapid development of related disciplines such as molecular biology, detection means for various cytokines have been constructed one after another.
The Flow Cytometry (FCM) technology is a flow cytometer, which combines the monoclonal antibody technology and the immunofluorescence staining technology to measure and analyze various parameters of single cells in a fast flowing liquid, and has fast detection speed and high accuracy. The method for quantitatively detecting the multiple cytokines by using the flow cytometer can qualitatively and quantitatively detect a plurality of indexes in a sample at the same time. Has been widely applied in China. However, due to the reagents of the respective manufacturers, in particular in the field of special rheumatic patients, there is no established corresponding standardized procedure. In order to enable the experimental result to reflect the internal condition of the patient more truly, it is crucial to the flow-type cytokine detection to establish a set of standardized operation procedures.
The pathogenesis of the autoimmune disease is complex, but the activation of autoreactive T cells and B cells is an inevitable link in the pathogenesis of the autoimmune disease. Thus, many scholars classify autoimmune diseases into three major classes: t cell mediated, autoantibody mediated, and combination mediated autoimmune diseases. The autoantibody, an important product after B cell activation, has been used as a marker for diagnosing autoimmune diseases, but no specific index for conventionally detecting autoreactive T cells exists at present, so that it is difficult to accurately judge whether the autoimmune diseases are T cell mediated autoimmune diseases or combined mediated autoimmune diseases.
The detection of cytokines produced by T cells reflects to some extent the degree of activation of T cells. The combined detection of multiple cytokines may reflect the activation of different subsets of helper T cells (Th). Due to the uncertainty of T cell activity in rheumatic patients, these cytokines secreted into serum or plasma are not only low in content, but also not easily detectable.
Therefore, it is important to establish a detection method capable of accurately detecting autoimmune diseases mediated by autoreactive T cells.
Disclosure of Invention
The invention aims to provide a sample pretreatment and preservation method for flow detection of a cytokine of a rheumatic patient.
In a first aspect of the invention, there is provided a diagnostic kit comprising:
(a) the blood collection tube is used for collecting blood, and the blood collection tube is provided with a separation gel and contains a coagulant;
(b) a detection reagent for detecting a cytokine selected from the group consisting of: IL-8, IL-17A, TNF-beta, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17F, IL-22, TNF-alpha, IFN-gamma, or a combination thereof;
the kit is used for judging whether the autoimmune disease is T cell mediated autoimmune disease or whether the autoimmune disease is T cell and autoantibody combined mediated autoimmune disease.
In another preferred embodiment, the cytokines include at least IL-8, IL-17A, TNF-beta, or a combination thereof.
In another preferred embodiment, the kit further comprises a label or instructions that states: the kit is used for judging whether the autoimmune disease is T cell mediated autoimmune disease or whether the autoimmune disease is T cell and autoantibody combined mediated autoimmune disease.
In a second aspect of the present invention, a sample pretreatment method is provided, which includes the following steps:
(a) providing a blood collection tube with a separation gel;
(b) collecting a blood sample by using the blood collection tube with the separation gel;
(c) centrifuging the blood sample in the blood collection tube, thereby obtaining an isolated serum sample;
(d) and detecting the separated serum sample.
In another preferred embodiment, the time from step (b) to step (c) does not exceed 4 hours, preferably 1 hour.
In another preferred embodiment, the method further comprises storing the separated serum sample in step (c) at-20 ℃ to-80 ℃, preferably-20 ℃ immediately before step (d), and performing step (d) within 24 hours after storage.
In another preferred example, in step (c), the rotation speed of the centrifugation is 4000rpm, the centrifugation time is 5min, and the centrifugation temperature is 20 ℃. + -. 2 ℃.
In another preferred embodiment, the sample is a serum sample isolated from a blood sample.
In another preferred embodiment, the sample is derived from a human or non-human mammal.
In another preferred example, the sample is derived from a human.
In another preferred embodiment, the assay is an assay for a cytokine in a serum sample.
In another preferred embodiment, the detection is detection using a flow cytometer.
In another preferred embodiment, the purpose of the assay is to determine whether the autoimmune disease is a T cell mediated autoimmune disease, or whether it is an autoimmune disease mediated by a combination of T cells and autoantibodies.
In another preferred embodiment, the cytokine comprises IL-8, IL-17A, TNF-beta, or a combination thereof.
In another preferred embodiment, the cytokine is selected from the group consisting of: IL-8, IL-17A, TNF-beta, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17F, IL-22, TNF-alpha, IFN-gamma, or a combination thereof.
In another preferred example, the blood collection tube is a vacuum blood collection tube.
In another preferred example, the blood collection tube further comprises a coagulant for promoting blood coagulation.
In another preferred embodiment, the blood sample is derived from a human or non-human mammal.
In another preferred example, the blood sample is derived from a human.
In another preferred embodiment, the blood sample is derived from a patient suffering from an autoimmune disease, which is a T cell mediated autoimmune disease, or an autoimmune disease mediated by a combination of T cells and autoantibodies.
In another preferred embodiment, the blood sample is derived from a patient suffering from rheumatism.
In another preferred example, the method comprises the steps of:
(a) providing a blood collection tube with a separation gel;
(b) collecting a blood sample by using the blood collection tube with the separation gel;
(c) centrifuging the blood sample in the blood collection tube within 1 hour after collection, thereby obtaining an isolated serum sample; and
(d) and detecting the separated serum sample.
In another preferred example, the method comprises the steps of:
(a) providing a blood collection tube with a separation gel;
(b) collecting a blood sample by using the blood collection tube with the separation gel;
(c) centrifuging the blood sample in the blood collection tube within 1 hour after collection, thereby obtaining an isolated serum sample;
(d) immediately storing the isolated serum sample of step (c) at-20 ℃, thereby obtaining a-20 ℃ stored serum sample; and
(e) the serum samples stored at-20 ℃ were tested within 24 hours after storage.
In a third aspect of the present invention, there is provided a method for preserving a serum sample, which comprises preserving a serum sample isolated from a blood collection tube with a separation gel at-20 ℃ to-80 ℃, preferably-20 ℃ for less than 24 hours, wherein the serum sample is used for determining whether the autoimmune disease is a T cell-mediated autoimmune disease by detecting cytokines in the serum sample by a flow cytometer, or whether the autoimmune disease is a T cell-mediated autoimmune disease in combination with an autoantibody.
In another preferred embodiment, the serum sample is derived from a patient suffering from an autoimmune disease that is a T cell mediated autoimmune disease, or an autoimmune disease mediated by a combination of T cells and autoantibodies.
In another preferred embodiment, the serum sample is from a patient suffering from rheumatism.
In a fourth aspect of the invention, there is provided use of a diagnostic kit according to the first aspect of the invention for determining whether an autoimmune disease is a T cell mediated autoimmune disease or whether it is an autoimmune disease mediated by a combination of T cells and autoantibodies.
In another preferred embodiment, the kit is used for judging whether a subject has rheumatism.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows the effect of four blood collection tubes on flow-based cytokine detection in samples from 100 normal populations. Wherein, the detection result is expressed by mean + -SD, and the gray horizontal line is cut-off value of each cytokine.
FIG. 2 shows the effect of four blood collection tubes at different temperatures and storage times on cytokine detection in samples from normal populations; wherein, purple: EDTA anticoagulant plasma tube, green: heparin anticoagulant tube, red: normal serum tube without anticoagulant (with coagulant), yellow: with a separation gel serum tube (containing coagulant).
FIG. 3 shows the results of measurements of cytokines in samples from normal persons at different temperatures and storage times for normal serum tubes without anticoagulant (with coagulant) and with separator gel. The table indicates the statistical difference compared to the test results on day 0 (i.e. 4 h).
Figure 4 shows the effect of four blood collection tubes on flow-based detection of cytokines in samples from rheumatic populations. Wherein, the detection result is expressed by the average number +/-SD, which indicates that the detection values of the common serum tube without anticoagulant and 2 plasma tubes have statistical difference, and # indicates that the detection values of the separation serum tube and 2 plasma tubes have statistical difference.
Figure 5 shows the effect of serum collection tubes at different temperatures and storage times on cytokine detection in samples from rheumatic populations. Wherein, red: normal serum tube without anticoagulant (with coagulant), yellow: with a separation gel serum tube (containing coagulant); indicates that the common serum tube without anticoagulant has statistical difference compared with the detection values of 2 plasma tubes, and indicates that the separation gel serum tube has statistical difference compared with the detection values of 2 plasma tubes.
FIG. 6 shows the results of measurements of cytokines in samples from a rheumatic human at different temperatures and storage times for a common serum tube without anticoagulant (with coagulant) and with separator gel. The table indicates the statistical difference compared to the test results on day 0 (i.e. 4 h).
Detailed Description
The inventor of the invention has conducted extensive and intensive research, and through a large number of screening and testing, has unexpectedly developed a diagnostic kit for judging the type of autoimmune disease and diagnosing autoimmune disease (especially rheumatism) for the first time, and also provides a sample pretreatment method and a preservation method for the flow detection of cytokines of a rheumatism patient. The kit of the invention mainly comprises a blood collection tube with a separation gel and a coagulant, and a detection reagent for detecting the cell factor, wherein the cell factor mainly comprises: IL-8, IL-17A, TNF-beta, or a combination thereof. Experiments prove that a blood sample of a patient with rheumatism is collected by a blood collection tube with separation gel and a coagulant, the separated serum sample is obtained by centrifugation within 1 hour, and the serum sample is immediately subjected to flow cytometry; or the serum sample obtained by centrifugation is stored at the temperature of minus 20 ℃ and is detected by a flow cytometer within 24 hours, so that an accurate and reliable cytokine detection result can be obtained. On the basis of this, the present invention has been completed.
Term(s) for
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other 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 belongs. Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject.
Rheumatism
Rheumatism is a group of autoimmune diseases that mainly invade joints, bones, muscles, blood vessels and related soft tissues or connective tissues. The pathogenesis of the disease is mainly the occurrence of immune response: the stimulation of exogenous or endogenous antigen substances by the organism directly or through macrophage presentation enables corresponding T cells to be activated, and partial T cells generate a large amount of various inflammatory cytokines to cause damage or destruction of various tissues and organs to different degrees; some of the T cells reactivate B cells, produce large amounts of antibodies, form immune complexes, either directly or in combination with antigens, and cause tissue or organ damage or destruction. Thus, rheumatic onset is closely related to T cell activation and cytokine production, and is a type of autoimmune disease mediated by autoreactive T cells, or a combination of T cells and B cells.
IL-8, IL-17F and TNF-beta are mononuclear factors produced by monocytes/macrophages, hormone-like molecules capable of regulating the mature differentiation of T, B lymphocytes, which play an important role in inflammatory responses, immune regulation, whereas TNF alpha plays a central role in the cytokine network of rheumatism. TNF-beta is produced early in the immune response and is an endogenous pyrogen that can cause the body to respond with a fever. The expression level of TNF-beta, IL-17F and IL-8 in the body of a patient with rheumatism is closely related to the disease activity degree and other immune indexes, and further proves that the TNF-beta, the IL-17F and the IL-8 have important functions in the pathogenesis of the rheumatism immune disease.
Cytokine
Cytokines (CK) are low molecular weight soluble proteins that are produced by various cells induced by immunogens, mitogens or other stimulants and have a variety of functions such as modulating innate and adaptive immunity, hematopoiesis, cell growth and damaged tissue repair. Cytokines can be classified as interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, and the like. Cytokines are produced primarily by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.).
The sample detection pretreatment and storage method provided by the invention aims at the flow cytometry detection of the cytokine in the serum sample. The cytokine comprises primarily IL-8, IL-17A, TNF-beta, or a combination thereof. In another preferred embodiment, the cytokines include (but are not limited to): IL-8, IL-17F, TNF-beta, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A, IL-22, TNF-alpha, IFN-gamma, etc.
Blood collection tube of the present invention
The sample detection pretreatment method of the present invention defines a blood collection tube used in a blood sample collection process, and the blood collection tube is a blood collection tube with a separation gel. In a preferred embodiment of the present invention, the blood collection tube is an inert separation gel accelerating tube, and an inert separation gel and a coagulant are added into the blood collection tube. The coagulant can rapidly activate the coagulation mechanism and accelerate the coagulation process, and the sample type is serum. And after the blood sample is collected, reversing and uniformly mixing for 5-8 times, standing for 20-30min vertically, and centrifuging to obtain supernatant above the separation gel, namely the separated serum.
The coagulant and the separating gel of the invention are both products which circulate in the existing market. See also, for example, patent CN 1046036C.
The separating gel is an important raw material applied to the vacuum blood collecting tube, and has the functions that the specific gravity of the separating gel is between serum and corpuscles, and the serum and the corpuscles are separated by the separating gel after the blood is precipitated by rotating and centrifuging. The separating gel has high viscosity, is not easy to package at normal temperature, and the weight of the blood separating gel added into each blood collection tube is 1g +/-10% according to the blood collection application and national standard of the vacuum blood collection tube. The serum separation gel is used as an isolation medium in a blood collection tube for serum biochemical or plasma biochemical test, can completely separate serum (or plasma) from red blood cells to obtain high-quality serum (or plasma), and effectively ensures the accuracy of blood test. After blood collection, blood is subjected to procoagulant or anticoagulation, and serum (or plasma) and red blood cells can be separated by centrifugation. Because the specific gravity of the serum separation gel is between that of serum (or plasma) and red blood cells, the gel moves and reverses to be between two components during centrifugation. After the centrifugation is stopped, the serum (or the plasma) is separated from the red blood cells, and the separation and the blockage are realized.
Method of the invention
The invention provides a sample detection pretreatment method for carrying out flow cytometry detection on cytokines in a serum sample. The method comprises the following steps:
(a) providing a blood collection tube with a separation gel;
(b) collecting a blood sample by using the blood collection tube with the separation gel;
(c) centrifuging the blood sample in the blood collection tube, thereby obtaining an isolated serum sample; and
(d) and detecting the separated serum sample.
Wherein the time from step (b) to step (c) does not exceed 4 hours, preferably 1 hour, and the separated serum sample is obtained by centrifugation at 4000rpm at 20 ℃. + -. 2 ℃ for 5 min.
After obtaining the separated serum sample, the sample may be subjected to flow cytometry; where detection cannot be performed immediately, the separated serum sample may be stored at-20 ℃ to-80 ℃, preferably-20 ℃, and flow cytometric detection may be performed within 24 hours after storage.
Therefore, the invention also provides a method for preserving a cytokine flow assay sample, which comprises preserving a serum sample separated from a blood collection tube with a separation gel at-20 ℃ to-80 ℃, preferably-20 ℃ for less than 24 hours.
The main advantages of the invention are:
(1) the flow cytometry is a mainstream method aiming at detecting a plurality of cytokines at present, is simple and convenient to operate, does not need specific instruments and equipment, and can be developed in most clinical laboratories;
(2) because the physicochemical properties of the cytokines are different, the invention aims at detecting a plurality of cytokines by the same detection method and establishes proper sample storage conditions;
(3) the invention aims at a method for detecting a plurality of cytokines by flow cytometry, and establishes a standard pretreatment scheme of a detection sample, so that the expression quantity of the plurality of cytokines can be detected correctly, especially IL-8 and IL-17F, TNF-beta.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.
Method for detecting cytokine by flow cytometry
The kit for jointly detecting the cytokines by using the flow cytometry produced by Tianjin Kuangbo company comprises the following specific operation steps:
(1) each test tube was added 20 microliters of 1 × capture microsphere mixture (microspheres added vortex 45 seconds before addition);
(2) taking a No. 1-8 standard sample tube, and respectively adding 20 microliters of standard samples for gradient dilution; adding 20 microliter of experimental diluent into a No. 9 tube to serve as a bottom tube; adding 20 microliters of samples to be detected into each sample tube;
(3) all the test tubes are vortexed for 10-20 seconds and fully and uniformly mixed;
(4) adding 20 microliters of 1-star detection antibody mixed solution into each test tube, uniformly mixing by vortex, and incubating for 2 hours (oscillation frequency is 500 rpm) at room temperature (23-27 ℃) in a dark oscillation manner;
(5) adding 20 microliters of SA-PE into each test tube, and oscillating and incubating for 30 minutes at room temperature in a dark place;
(6) adding 300 microliters of 1-buffer solution into each test tube, vortexing for 10-20 seconds, centrifuging for 5 minutes at 500g, discarding supernatant, slightly reversing the test tube orifice on absorbent paper, and sucking residual liquid at the tube orifice;
(7) repeating the step (6);
(8) 150-300 microliters of 1 × buffer was added to each test tube and detected using a flow cytometer.
Example 1
Influence of blood collection tube on cytokine detection result of normal population sample
Aiming at healthy control group population, an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, a heparin anticoagulation tube, a common serum tube without anticoagulant and a serum separation tube with separation gel are adopted to respectively collect blood, and after centrifugation is carried out for 5 minutes at 4000rpm within 1 hour, the separated plasma/serum is detected within 4 hours. The number of samples of healthy people in this example is 100.
The results are shown in FIG. 1 and Table 1.
Table 1 detection of flow-based cytokine in samples from 100 normal populations using four blood collection tubes
The results show that the detection values of the 4 blood sampling tubes do not exceed the upper limit of the reference range set by the manufacturer (figure 1).
Example 2
Effect of sample preservation temperature and preservation time on cytokine detection results of normal population samples
In this example, the effect of the storage temperature and storage time of the sampled samples on the flow assay of cytokines was tested. In healthy control group population, blood is collected by using EDTA anticoagulation tube, heparin anticoagulation tube, ordinary serum tube (containing coagulant) without anticoagulant and serum separation tube (containing coagulant) with separation gel.
For blood samples collected with EDTA and heparin anticoagulation tubes, they were centrifuged at 4000rpm for 5 minutes to obtain plasma samples.
Blood samples collected with a normal serum tube (containing a coagulant) without anticoagulant and a serum separation tube (containing a coagulant) with a separation gel were centrifuged at 4000rpm for 5 minutes to obtain serum samples.
The prepared plasma sample and serum sample are respectively stored in the environment of 4 ℃, 20 ℃ and 80 ℃ below zero, and the cytokines of the samples in the 4 blood collection tubes are respectively detected by the flow cytometry method on the 0 th, 1 th, 3 th, 5 th and 7 th days of storage.
The results are tabulated in fig. 2, as well as fig. 3.
In healthy control group population, most of cytokines are respectively stored in 4 blood collection tubes at 4 ℃, 20 ℃ and 80 ℃ without exceeding the range set by the manufacturer; however, IL-8 was maintained in the normal range during storage at 4 ℃ and-20 ℃ up to day 7, except for the serum tube with the separator gel, and the values of the other three tubes were above the upper limit of normal, especially in the EDTA and heparin anticoagulation tube samples.
In addition, TNF- β was detected above the upper normal limit during storage of EDTA anticoagulant tubes.
It follows that for serum or plasma test samples from normal persons (healthy controls), the samples of 4 tubes differed insignificantly at 3 temperatures and for a storage time of up to 1 week, and the serum tube with the separation gel performed optimally in all the test samples.
Example 3
Influence of blood collection tube on cytokine detection result of rheumatism population sample
Aiming at rheumatism people (mainly including systemic lupus erythematosus, inflammatory myopathy and Behcet disease), an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, a heparin anticoagulation tube, a common serum tube without anticoagulant and a serum separation tube with separation gel are adopted for blood collection respectively, and separated plasma/serum is detected in time after centrifugation at 4000rpm within 1 hour for 5 minutes. The number of samples of the rheumatic population in this example was 200.
The results are shown in FIG. 4 and Table 2.
Table 2 detection of cytokines by flow assay in samples from 200 rheumatic patients using four blood collection tubes
The results show that:
in IL-8, the detection values were higher in both heparin anticoagulation tube and EDTA anticoagulation tube than in 2 serum tubes.
For IL-17F, the values detected in heparin anticoagulation tubes were significantly higher than those of the other 3 tubes.
The results show that neither EDTA anticoagulation tube nor heparin anticoagulation tube is suitable for detecting the cell factors of patients with rheumatism.
Example 4
Influence of sample preservation temperature and preservation time on cytokine detection result of rheumatism population sample
In this example, the effect of the preservation temperature and preservation time of the sample on the cytokine detection result of the rheumatic population sample is further analyzed when a common serum tube or a serum separation tube with separation gel is used.
In the population suffering from rheumatism, blood is respectively collected by a common serum tube (containing coagulant) without anticoagulant and a serum separation tube (containing coagulant) with separation gel, the blood is centrifuged at 4000rpm for 5 minutes, serum samples in 2 blood collection tubes are respectively stored at the environment of 4 ℃, 20 ℃ and 80 ℃ below zero after centrifugation, and the cytokines of the samples in the 2 blood collection tubes are respectively detected by a flow cytometry method on the 0 th, 1 th, 3 th, 5 th and 7 th days of storage. The number of samples of the rheumatic population in this example was 200.
The results are tabulated in fig. 5 and 6.
The results show that:
in the rheumatic population, serum samples separated from ordinary serum tubes (containing coagulant) without anticoagulant were stored at 4 ℃, -20 ℃ and-80 ℃ respectively, and the measured values of most cytokines in the samples on day 1 of storage were statistically different from those on day 0;
and storing serum samples separated from the serum separation tube (containing coagulant) with the separation gel at 4 ℃, 20 ℃ and 80 ℃ respectively, wherein the detection values of most cytokines in the samples on the 3 rd day of storage are statistically different from the detection values on the 0 th day; with the exception of IL-6 and IL-8, most cytokines in serum samples isolated from serum separator tubes with separator gel (containing coagulant) stored at-20 ℃ were not statistically different from day 0 in the values measured on day 1 of storage (FIG. 4).
It can be seen that the results of the test using the serum sample only with the separation gel serum collection tube (containing the coagulant) stored at-20 ℃ for up to 1 day are the closest to the instant test.
The immune system in the body of the patient with rheumatism is extremely complex, so that the requirement on the authenticity of the detection value of the cell factor is higher. Combining the results, a blood collection tube with separation gel is adopted for blood collection, and the blood is centrifuged for 5 minutes at 4000rpm within 1 hour in time for detection, or serum is subpackaged after centrifugation and is immediately stored at 20 ℃ below zero, and the detection result within 24 hours is the most real and reliable. The storage time is more than 24 hours, and the deviation of the detection value is large.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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