A method for assessing the degree of blood contamination in a sample

Abstract

The present invention provides a method for evaluating the degree of blood contamination in a sample, comprising: extracting a peptide mixture including a target peptide from a sample and a fresh blood sample respectively, detecting the content of the target peptide after mixing, obtaining the relationship between the concentration of the target peptide and the blood content in the sample according to the detection result, and evaluating the degree of blood contamination in the sample according to the relationship, wherein the target peptide is TVAAPSVFIFPPSDEQLK. By using a specific peptide as a key reference for the method of evaluating the proportion of blood in an FNA tissue sample, an accurate evaluation of the blood content of the sample can be completed with a sample injection volume as low as 1 μg, thereby assisting doctors and testing institutions in evaluating the degree of blood contamination in FNA samples.

Description

Technical Field

[0001] The invention belongs to the technical field of sample detection, and in particular relates to a method for evaluating the degree of blood contamination in a sample. Background Art

[0002] In recent years, the incidence of thyroid cancer has gradually increased. The cure rate of early thyroid cancer with standardized treatment has reached more than 90%, so early screening for thyroid cancer is particularly important. A clear diagnosis is the prerequisite for tumor treatment, and thyroid fine needle aspiration (FNA) is the preferred method for distinguishing benign and malignant thyroid nodules. The FNA sample obtained by this method is to puncture the thyroid nodule with a fine needle and extract the pathological tissue of the thyroid nodule through a syringe. The collection method is often accompanied by the production of blood, and blood contamination will affect the subsequent test results of the FNA sample.

[0003] At present, there are few methods on the market to evaluate the proportion of blood content or tissue sample in FNA samples. The degree of blood contamination of FNA samples is mainly graded by experienced pathologists, and the main grading method is: 0 = blood clots, 1 = red blood cell contamination, 2 = no blood, and it can also be classified into high, medium and low according to the degree of blood contamination (blood contamination ratio <25% - low, blood contamination ratio 25-50% - medium, blood contamination ratio >25% - high). The existing technology basically relies on pathologists to evaluate the blood volume of FNA samples, and the accuracy of FNA sample quality assessment is also related to whether the pathologist is experienced. FNA samples are usually deblooded before pretreatment to reduce the impact of blood contamination, but the judgment of the adequacy of blood deblooding mainly depends on the subjectivity of the experimenter, and there is no unified standard. Protein spectrum detection cannot evaluate the proportion of FNA samples or the proportion of blood content from the perspective of polypeptide production. If the proportion of blood in clinical samples is large, false positive results will be caused due to the small amount of clinical samples input, so a method for evaluating the proportion of blood content or tissue proportion is urgently needed.

[0004] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the invention

[0005] Technical issues

[0006] In order to solve the above problems, the purpose of the present invention is to provide a method for detecting the blood content in FNA samples based on the LC-MS / MS method. By using specific peptide segments as the key reference for the method of evaluating the blood proportion in FNA tissue samples, an accurate evaluation of the blood content of the sample can be completed with a sample injection volume as low as 1 μg, thereby assisting doctors and testing institutions in evaluating the degree of blood contamination of FNA samples.

[0007] Technical Solution

[0008] In order to achieve the above-mentioned purpose, the inventors of the present application conducted in-depth research and found that a peptide segment (TVAAPSVFIFPPSDEQLK, IGKC protein - a partial amino acid sequence of a high-abundance antibody protein in the blood, which can be abbreviated as TVAA, TVAA peptide) has a very high correlation with the blood content, and can be used as a key reference for the method of evaluating the blood ratio in FNA tissue samples, thereby determining the amount of tissue input in FNA samples and establishing correlation research on clinical samples.

[0009] Scheme 1, a method for evaluating the degree of blood contamination in a sample, extracting a peptide mixture including a target peptide from a sample and a fresh blood sample respectively, detecting the content of the target peptide after mixing, obtaining the relationship between the concentration of the target peptide in the sample and the blood content based on the test results, and evaluating the degree of blood contamination in the sample based on the relationship, wherein the target peptide is TVAAPSVFIFPPSDEQLK.

[0010] Preferably, the extracting of the peptide mixture including the target peptides is specifically to extract the peptides in the sample and the fresh blood sample using a pre-treatment method for preparing peptides.

[0011] Preferably, the relationship between the target peptide concentration and the blood content is a linear correlation equation.

[0012] Preferably, the LC-MS / MS method is used to detect the content of the target peptide segment.

[0013] Preferably, the evaluation of the degree of blood contamination in the sample includes qualitative and quantitative detection of the blood content in the sample.

[0014] Scheme 2, use of a reagent for detecting the peptide TVAAPSVFIFPPSDEQLK in the preparation of a kit for assessing the degree of blood contamination in a sample.

[0015] Scheme 3, a kit for evaluating the degree of blood contamination in a sample, including reagents for detecting the peptide TVAAPSVFIFPPSDEQLK.

[0016] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0017] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0018] Beneficial Effects

[0019] According to the present invention, a pretreatment method for preparing polypeptides is used to extract peptides from intraoperative tissue samples and fresh blood samples, and then the two polypeptides are mixed in different proportions. The LC-MS / MS method is then used to detect the content of the target peptide TVAAPSVFIFPPSDEQLK in the mixed peptide solution. A linear correlation equation between the target peptide concentration in the sample and the blood content is obtained based on the test results. This method only requires a sample injection volume as low as 1 μg to evaluate the quantitative blood content, thereby assisting doctors and testing agencies in evaluating the degree of blood contamination of FNA samples, providing a judgment on the hemolysis of FNA samples before testing the FNA samples, and facilitating subsequent processing by doctors and testing agencies.

[0020] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It represents the sample detection flow chart;

[0023] Figure 2 Represents a standard curve graph;

[0024] Figure 3 Represents the correlation function between the concentration of FNA samples and the blood content. DETAILED DESCRIPTION

[0025] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0027] Unless otherwise specified, all percentages, parts, ratios, etc. are by weight; otherwise specified, including but not limited to, "%", "wt%", "mass%" means weight percentage, "mol%" means molar percentage, and "vol%" means volume percentage.

[0028] When a quantity, concentration or other numerical value or parameter is given as a range, a preferred range or a series of upper preferred values ​​and lower preferred values, it should be understood that it specifically discloses all ranges formed by any pair of numerical values ​​of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether the range is disclosed separately. For example, when describing a range of "1 to 5 (1-5)", the described range should be understood to include a range of "1 to 4 (1-4)", "1 to 3 (1-3)", "1 to 2 (1-2)", "1 to 2 (1-2) and 4 to 5 (4-5)", "1 to 3 (1-3) and 5", etc. Unless otherwise stated, where a numerical range is described herein, the range includes the range end values ​​and all integers and fractions within the range.

[0029] Unless specifically stated, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are still described herein.

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0031] In order to facilitate understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention are first explained or defined:

[0032] FNA: Fine Needle Aspiration, fine needle aspiration biopsy;

[0033] LC-MS / MS: high performance liquid chromatography tandem mass spectrometry;

[0034] ABB: ammonium bicarbonate;

[0035] RapiGest: A mild MS-compatible protein denaturant that helps solubilize and unfold proteins, making them easier to cleave without denaturing or inhibiting proteolytic enzymes like conventional denaturants do;

[0036] TCEP: It is a very effective thiol reducing agent and is widely used as a quantitative reducing agent for disulfide bonds in protein chemistry and proteomics research.

[0037] IAA: iodoacetamide;

[0038] EtOH: ethanol;

[0039] TFA: trifluoroacetic acid.

[0040] Summary of the Invention

[0041] The present invention aims to establish a method for detecting the degree of blood contamination in FNA samples based on the LC-MS / MS method. The inventors found in relevant experimental studies that a target peptide (TVAAPSVFIFPPSDEQLK) has a high correlation with the blood content, which can be used as a method for evaluating the blood ratio in FNA tissue samples, thereby determining the amount of tissue input in FNA samples and establishing a correlation study of clinical samples. Therefore, it should be clear and undoubted that the content involved in and claimed for protection in the present invention is only the situation where the sample is contaminated by blood, and there is no content related to the health of the sample host, and therefore does not involve any diagnostic method or treatment method for any disease.

[0042] The present invention provides a method for evaluating blood contamination in a sample. A peptide mixture including a target peptide is extracted from a sample and a fresh blood sample respectively. The content of the target peptide is detected after mixing. The relationship between the concentration of the target peptide in the sample and the blood content is obtained according to the detection result. The contamination degree of the blood in the sample is evaluated according to the relationship. The target peptide is TVAAPSVFIFPPSDEQLK.

[0043] The sample and / or the fresh blood sample only needs to be as low as 1 μg. Therefore, the quantitative detection of the blood contamination degree in the sample can be completed with extremely low impact on the total amount of FNA sample and fresh blood. On the one hand, after clarifying the blood contamination degree of the FNA sample, it is helpful for medical workers to judge whether the FNA sample can continue to be tested, avoiding false positive results caused by samples with large blood content. On the other hand, extremely low-volume FNA samples and fresh blood samples can further reduce the additional sample and blood volume brought to the patient by traditional blood contamination detection methods, so that only trace samples and blood can be collected to complete the blood contamination degree inspection and sample case inspection, reducing the pain of patients.

[0044] The extracting of the peptide mixture including the target peptides specifically involves extracting the peptides from the sample and the fresh blood sample using a pretreatment method for preparing peptides.

[0045] The pre-treatment method includes: sample lysis, reduction alkylation, magnetic bead incubation, enzymatic hydrolysis and polypeptide elution.

[0046] The sample lysis comprises: adding ABB to the sample, mixing and centrifuging, then adding RapiGest, and incubating at a constant temperature.

[0047] The sample lysis specifically includes: adding 50-100 mM ABB to the sample, mixing and briefly centrifuging, and then adding an equal volume of 0.05-0.25% RapiGest; incubation: constant temperature shaking metal bath, 500-1000 rpm, 30 min, 60°C.

[0048] The reductive alkylation comprises: adding TCEP to each reaction and incubating according to the volume of the solution during incubation, and adding IAA and reacting in the dark.

[0049] The reductive alkylation specifically includes: adding 100 mM TCEP to each reaction according to the volume of the solution during incubation, incubating for 45 to 60 minutes, 37° C., and 800 rpm. Adding 800 mM IAA to each reaction according to the volume of the solution during incubation, reacting in the dark for 45 to 60 minutes, 25° C., and 800 rpm.

[0050] The magnetic bead incubation includes: adding the washed magnetic beads to the sample, adding EtOH and gently shaking the EP tube to make the liquid uniform, leaving the EP tube still after incubation, and removing the supernatant after the magnetic beads are completely attracted to the tube wall. After removing the EP tube, add EtOH to disperse the magnetic beads, let the tube stand until the magnetic beads are completely attracted to one side of the tube wall and remove the supernatant, and after repeated washing, gently close the EP tube cap and let it stand until the residual solution on the surface of the material evaporates.

[0051] The magnetic bead incubation specifically includes: adding an appropriate amount of pure water to the magnetic beads for washing, placing them on a magnetic rack and standing for 2 minutes, then removing the supernatant, washing twice, adding an appropriate amount of pure water to resuspend the magnetic beads, and mixing. Add 5 to 10 μL of the above-washed magnetic beads to each sample; according to the volume of the original solution in the tube, add 50 to 100 μL of EtOH to each reaction (add at a close distance, try not to touch the liquid surface); gently shake the EP tube to make the liquid uniform (Note: do not blow the liquid, you can disperse the magnetic beads when adding EtOH, but do not use excessive force to flush them onto the tube wall above the liquid surface); incubation: 24℃ / 15 to 20min / 800rpm; after incubation, place the EP tube on the magnetic rack and let it stand until the magnetic beads are completely attracted to the tube wall; remove the supernatant, be careful not to touch the magnetic beads. Remove the EP tube from the magnetic rack and add an appropriate amount of 80% EtOH at a slightly faster speed to disperse the magnetic beads; place the EP tube on the magnetic rack and let it stand for 2 minutes, wait until the magnetic beads are completely attracted to one side of the tube wall, remove the supernatant, and be careful not to touch the magnetic beads; repeat the cleaning operation once, for a total of 2 times; after cleaning, gently close the EP tube cap and let it stand for 3 minutes to wait for the residual solution on the surface of the material to evaporate.

[0052] The enzymatic hydrolysis comprises: adding ABB to the system, adding trypsin to perform enzymatic digestion reaction; after the digestion is completed, centrifuging all samples, standing to attract magnetic beads, taking the supernatant and adding it to an EP tube pre-added with a reaction termination solution.

[0053] The enzymatic hydrolysis specifically includes: adding 15-30 μL 50-100 mM ABB. For protease digestion, add 1-3 μg of trypsin. The digestion reaction is carried out at 37°C, 800-1000 rpm for 12-16 hours. Add 2 μL 5% TFA to a new 1.5 mL EP tube to prepare for collecting the polypeptide solution after enzymatic digestion. After digestion, centrifuge all samples: 16000g / 24°C / 2min, place on a magnetic stand for 2min to attract magnetic beads, and take the supernatant to a 1.5 mL EP tube (2 μL of reaction termination solution has been pre-added).

[0054] The polypeptide elution comprises: adding an elution solution, slightly vortexing and then centrifuging; standing until the magnetic beads are completely attracted to the tube wall; repeating the elution to obtain the supernatant; combining the two elution supernatants with the initial enzymatic hydrolysis collection solution, centrifuging to obtain the supernatant, and spinning dry.

[0055] The polypeptide elution specifically includes: adding 40 μL of elution solution, vortexing lightly for 3 minutes, centrifuging at 16000g / 24℃ / 2min, placing on a magnetic rack and standing for 2 minutes until the magnetic beads are completely attracted to the tube wall, and taking the supernatant; adding 40 μL of elution solution again, repeating the washing operation, and taking the supernatant. The two elution supernatants are combined with the initial enzymatic hydrolysis collection solution, centrifuged at 16000g / 24℃ / 2min, taking the supernatant, and spinning dry.

[0056] The mixing is to adjust the peptide mixture extracted from the sample and the peptide mixture extracted from the fresh blood sample to the same peptide concentration and then prepare a mixed sample according to the blood content ratio of 0 to 100%.

[0057] The relationship between the target peptide concentration and the blood content is a linear correlation equation.

[0058] The LC-MS / MS method is used to detect the content of the target peptide segment.

[0059] The evaluation of the contamination degree of blood in the sample includes qualitative and quantitative detection of the blood content in the sample.

[0060] The present invention also provides the use of a reagent for detecting the peptide segment TVAAPSVFIFPPSDEQLK in preparing a kit for evaluating the degree of blood contamination in a sample.

[0061] The present invention also provides a kit for evaluating the degree of blood contamination in a sample, comprising a reagent for detecting the peptide segment TVAAPSVFIFPPSDEQLK.

[0062] The present invention is described in detail below

[0063] The method for evaluating the degree of blood contamination in a sample described in the present invention mainly includes sample pretreatment process, spin drying & re-dissolution, LC-MS / MS detection and analysis, and the establishment of a correlation method between TVAA peptide segments and blood peptide segment proportions.

[0064] like Figure 1 As shown in the figure, the sample detection process includes: sample lysis, reduction alkylation, magnetic bead incubation, enzymatic hydrolysis, peptide elution, spin drying & re-dissolution, and LC-MS / MS detection and analysis. The details are as follows.

[0065] Part 1: Pre-processing process

[0066] 1) Sample lysis:

[0067] Add 100 mM ABB to the sample, mix well and centrifuge briefly, then add an equal volume of 0.1% RapiGest; incubate: constant temperature shaking metal bath, 800 rpm, 30 min, 60°C.

[0068] 2) Reductive alkylation:

[0069] 2.1) Add 100 mM TCEP to each reaction according to the volume of the solution during incubation. Incubate for 60 min at 37°C and 800 rpm.

[0070] 2.2) Add 800 mM IAA to each reaction according to the volume of the solution during incubation. Incubate in dark for 60 min, 25°C, 800 rpm.

[0071] 3) Magnetic beads preparation:

[0072] Add appropriate amount of pure water to the magnetic beads for washing, place them on the magnetic rack and let them stand for 2 minutes, then remove the supernatant, wash twice, add appropriate amount of pure water to resuspend the magnetic beads and mix well.

[0073] 4) Magnetic bead incubation:

[0074] 4.1) Add 10 μL of the washed magnetic beads to each sample.

[0075] 4.2) Add 80 μL of EtOH to each reaction according to the volume of the original solution in the tube (add at a close distance, try not to touch the liquid surface).

[0076] 4.3) Gently shake the EP tube to make the liquid uniform (Note: Do not blow the liquid. You can disperse the magnetic beads by adding EtOH, but do not use excessive force to flush the tube wall above the liquid surface).

[0077] 4.4) Incubation: 24°C / 20 min / 800 rpm.

[0078] 4.5) After incubation, place the EP tube on a magnetic rack and let it stand until the magnetic beads are completely attracted to the tube wall.

[0079] 4.6) Remove the supernatant, being careful not to touch the magnetic beads.

[0080] 5) Cleaning:

[0081] 5.1) Remove the EP tube from the magnetic stand and add an appropriate amount of 80% EtOH at a slightly faster speed to disperse the magnetic beads.

[0082] 5.2) Place the EP tube on the magnetic rack and let it stand for 2 minutes. Wait until the magnetic beads are completely attracted to one side of the tube wall. Remove the supernatant and be careful not to touch the magnetic beads.

[0083] 5.3) Repeat the cleaning operation 1 time, a total of 2 times.

[0084] 5.4) After cleaning, gently close the EP tube cap and let it stand for 3 minutes to allow the residual solution on the surface of the material to evaporate.

[0085] 6) Enzymatic hydrolysis:

[0086] 6.1) Add 20 μL 75 mM ABB.

[0087] 6.2) Protease digestion: add 2 μg of trypsin.

[0088] 6.3) The enzyme digestion reaction was carried out at 37°C and 900 rpm for 15 h.

[0089] 6.4) Add 2 μL of 5% TFA to a new 1.5 mL EP tube to collect the peptide solution after enzymatic hydrolysis.

[0090] 6.5) After digestion, centrifuge all samples at 16000g / 24℃ / 2min, place on a magnetic stand for 2min to attract magnetic beads, and take the supernatant into a 1.5mL EP tube (pre-added with 2μL of reaction stop solution).

[0091] 7) Peptide elution:

[0092] 7.1) Elution of peptide adsorbed by materials. Add 40 μL of elution buffer, vortex lightly for 3 minutes, centrifuge at 16000g / 24℃ / 2min, place on a magnetic rack and let stand for 2 minutes until the magnetic beads are completely attracted to the tube wall, take the supernatant; add 40 μL of elution buffer again, repeat the washing operation, and take the supernatant.

[0093] 7.2) Combine the two elution supernatants with the initial enzymatic hydrolysis solution, centrifuge at 16000g / 24℃ / 2min, take the supernatant and spin dry.

[0094] Part 2: Drying and Redissolving

[0095] After the samples were completely dried by spin drying for 90 minutes, each sample was reconstituted with 40 μL of 0.1% formic acid water and the concentration was measured using an ultra-micro biochemical spectrophotometer. Finally, the samples were diluted, mixed with the internal standard and loaded onto the instrument.

[0096] Part III: LC-MS / MS Detection and Analysis

[0097] HPLC parameters

[0098] Injection volume: 20µL;

[0099] Mobile phase information: Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile.

[0100] Mass spectrometry detection parameters:

[0101] The mass spectrometry detection parameters are shown in Table 1.

[0102] Table 1. Mass spectrometry detection parameters

[0103]

[0104] Part 4: Construction of the correlation method between TVAA peptides and blood peptide proportions

[0105] Four intraoperative tissue samples without blood contamination (named FNA1, FNA2, FNA3, and FNA4) and fresh blood samples were pre-treated to obtain the corresponding peptides, which were uniformly diluted to the same concentration (about 0.05-0.1 μg / μL), and mixed samples were prepared according to the blood content of 0% to 100%, and then mass spectrometry was performed on the mixed samples.

[0106] The prepared peptide mixture and standard working solution (internal standard + organic solvent that does not affect this patent, acetonitrile is selected in the present invention, and other solvents such as TCEP can also be selected) are mixed with the internal standard (the standard of the No. 40 peptide segment with a heavy label, specifically T{VAL(13C5,15N)}AAPSVFIFPPSDEQLK), and then plotted according to the above-mentioned measurement conditions with Area Ratio as the horizontal axis and fmol as the vertical axis to obtain a standard curve as shown in the figure below: Figure 2 As shown, the standard curve y=7.844×10 -3 x+1.852×10 -2 , R 2 =0.9993.

[0107] Then the concentration of the peptide mixture sample was calculated. The concentration is shown in Table 2. After removing the background, the blood concentration was plotted as the horizontal axis and the fmol amount of TVAA as the vertical axis. The linear function of the concentration of the FNA sample and the blood content was obtained as follows: y=3.435x-6.428, where x≥0.1, and the correlation r=0.939. Figure 3 shown.

[0108] Table 2. Concentration of peptide mixture samples

[0109]

[0110] Based on the above verification experiments, the linear correlation coefficient between the TVAA peptide concentration and the blood content in the sample of this example is 0.939, indicating that the two are highly correlated. The linear correlation equation between the two can be used to evaluate the blood content in FNA samples, assisting doctors and testing institutions in evaluating the degree of blood contamination in FNA samples and then judging the sample quality.

[0111] Furthermore, 216 FNA samples were selected for clinical sample testing. They were pre-treated according to the above process and tested according to the mass spectrometry detection method. The sample injection volume was 1 μg. The fmol amount of each sample was obtained according to the standard curve of the TVAA peptide segment. The formula y=3.435x-6.428 was used to obtain the blood content percentage of the clinical samples. The summary results are shown in Tables 3 and 4.

[0112] Table 3. FNA sample test results

[0113]

[0114] Table 4. Detection results of TVAA peptides in 216 clinical samples and their corresponding blood content percentages

[0115]

[0116] From the results, it can be seen that this method can evaluate the proportion of blood content and the proportion of blood in the injection volume of 1 μg, thereby determining the content of tissue samples and establishing quality control of clinical samples.

[0117] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0118] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0119] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0120] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0121] Matters not covered in the present invention are all known technologies.

Claims

1. A method for evaluating the degree of blood contamination in a sample, characterized in that: A peptide mixture including a target peptide is extracted from the sample and the fresh blood sample respectively, and the content of the target peptide is detected after mixing. The relationship between the concentration of the target peptide in the sample and the blood content is obtained based on the detection result, and the contamination degree of the blood in the sample is evaluated based on the relationship. The target peptide is TVAAPSVFIFPPSDEQLK; The sample and / or the fresh blood sample only needs to be as low as 1 μg; The mixing is to adjust the peptide mixture extracted from the sample and the peptide mixture extracted from the fresh blood sample to the same peptide concentration and then prepare a mixed sample according to the blood content ratio of 0 to 100%.

2. The method according to claim 1, characterized in that: The extracting of the peptide mixture including the target peptides specifically involves extracting the peptides from the sample and the fresh blood sample using a pre-treatment method for preparing peptides.

3. The method according to claim 2, characterized in that: The pre-treatment method includes: sample lysis, reduction alkylation, magnetic bead incubation, enzymatic hydrolysis and polypeptide elution.

4. The method according to claim 1, characterized in that: The relationship between the target peptide concentration and the blood content is a linear correlation equation.

5. The method according to claim 1, characterized in that: The LC-MS / MS method is used to detect the content of the target peptide segment.

6. The method according to claim 1, characterized in that: The evaluation of the contamination degree of blood in the sample includes qualitative and quantitative detection of the blood content in the sample.

7. Application of the reagent for detecting the peptide segment TVAAPSVFIFPPSDEQLK in the preparation of a kit for evaluating the degree of blood contamination in a sample.

Images