CN115702637B - Protective agent for gastrin 17 blood sample - Google Patents
Protective agent for gastrin 17 blood sample Download PDFInfo
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- CN115702637B CN115702637B CN202110929189.2A CN202110929189A CN115702637B CN 115702637 B CN115702637 B CN 115702637B CN 202110929189 A CN202110929189 A CN 202110929189A CN 115702637 B CN115702637 B CN 115702637B
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a protective agent for a gastrin 17 blood sample. The protective agent comprises a stabilizer, a preservative and water; the stabilizer is 2-bis (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or 1, 3-bis [ (trimethylol) methylamino ] propane; the concentration of the stabilizer in the protective agent is 0.5-1mol/L. The protective agent can effectively avoid the degradation of the gastrin 17 in the blood sample, and greatly prolongs the preservation time of the sample, thereby ensuring the accuracy of the detection result.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a protective agent for a gastrin 17 blood sample.
Background
The method is an effective way for changing the severe gastric cancer diagnosis and treatment situation by pushing early gastric cancer screening in natural population and further performing an endoscopic screening strategy in high-risk population. Therefore, the expert consensus of the early gastric cancer screening process in China (draft) (2017, shanghai) proposes a novel gastric cancer screening scoring system, in which 5 variables of age, sex, helicobacter pylori (Hp) antibody, pepsinogen (PG) and gastrin 17 (G-17) are given different scores (weights) for comprehensively evaluating the risk of gastric cancer of a target population. Different measures are taken according to the scoring, such as an endoscope investigation strategy is adopted for people with high risk of gastric cancer occurrence, so that early gastric cancer diagnosis rate is improved; and a proper follow-up strategy is adopted for the crowd with relatively low risk, so that the aim of saving medical resources is fulfilled.
Gastrin 17 is an important marker in scoring systems, but clinical use is greatly plagued by the instability of the gastrin 17 sample. Liu Zhimin, and the like, shows that compared with the immediate detection results, the detection results of serum separation in 2h, 4h, 8h and 24h all have obvious descending trend, and the descending percentages are respectively: 17.03%, 32.53%, 52.06%, 64.52%, the isolated serum fraction showed a tendency to decrease under room temperature and 4 ℃ storage conditions, no significant change (P > 0.05) was seen in the results over 4 days of-20 ℃, -70 ℃ storage and stabilizer addition (Liu Zhimin, zhou Haiqing, li Jing. Effect of different separation times and storage methods on gastrin-17 assay [ J ]. Experimental and assay medicine, 2017,035 (005): 717-718.). Studies in wheat, et al, showed that gastrin 17 began to degrade after ex vivo, at a rate of approximately parabolic. The storage time at room temperature 25℃and 37℃was only 4 hours and 2.5 hours (wheat, tang Yi, king reinforcement, etc. the effect of serum sample storage temperature and time on the results of gastrin-17 detection [ J ]. J. Practical hospital clinical journal, 2018,015 (002): 26-29.).
Gastrin 17 is a small molecule polypeptide consisting of 17 amino acids, and gastrin 17 begins to degrade after blood is isolated, if a sample cannot be timely detected, a detection result is inaccurate, and the accuracy of a novel gastric cancer scoring system is affected, so that the risk of misdiagnosis is increased.
In order to minimize the extent of degradation of gastrin 17 after ex vivo, the specification of the gastrin 17 assay kit from Biohit corporation prescribes that no more than 30 minutes should be allowed to stand at room temperature (20-25 ℃) after serum collection, if no more than 60 minutes should be allowed to condense in a crushed ice bath. The detection should be done after serum coagulation or after plasma separation by centrifugation within 3 hours. The new industry company specifications require that the blood sample should be coagulated for no more than 30 minutes at room temperature (20-25 ℃) and if coagulated in a crushed ice bath no more than 60 minutes. Placing the coagulated blood sample in a refrigerated centrifuge for centrifugation, and immediately detecting the obtained serum; if temporarily undetectable, it is preferable to aliquote the blood sample and freeze it to-20℃or below. The centrifugation and detection are required to be completed within 3 hours after the blood is isolated, the conditions are too harsh, and the centrifugation and detection cannot be realized in the actual operation process of many small and medium hospitals. The customer is required to use a refrigerated centrifuge to centrifuge, so that the degradation of the gastrin 17 in the centrifuging process can be reduced, and the problem of the degradation of the gastrin 17 before and after centrifugation cannot be solved.
To solve the problem of degradation of gastrin 17, biohit recommends that a certain proportion of sample stabilizer be added to serum or plasma obtained after centrifugation, and the storage time of gastrin 17 can be prolonged from 3 hours to 72 hours after the stabilizer is added. It is known from the prior patent to Biohit (CN 1950398A-peptide stabilization) and related literature that the component of the gastrin 17 stabilizer provided is dimethyl sulfoxide (DMSO), but DMSO can only be added to serum or plasma after centrifugation. Liu Zhimin et al showed that the inability of DMSO to be added to whole blood resulted in severe hemolysis and affected the assay results. And can only be preserved at room temperature after adding the stabilizing agent, and can not be preserved at the temperature of 2-8 ℃ or below-20 ℃ because white flocculent precipitate can be precipitated (Liu Zhimin, zhou Haiqing, li Jing. Influence of different separation times and preservation methods on gastrin-17 determination [ J ]. Experimental and inspection medicine, 2017,035 (005): 717-718). In addition, DMSO has certain toxicity, permeability to human skin, eye irritation and certain safety hidden trouble for users.
At present, the conventional on-market gastrin 17 detection reagent cannot well solve the problem of degradation of gastrin 17 after in vitro, and even though a DMSO sample stabilizer is provided by Biohit company, the practical application effect is not ideal. There is a great need in clinic for a better gastrin 17 sample stabilizer which solves the problem of degradation of gastrin 17, thereby ensuring the accuracy of the detection result of gastrin 17.
Disclosure of Invention
In view of this, the present invention provides a protectant for a blood sample of gastrin 17. The protective agent can effectively avoid the degradation of the gastrin 17 in the blood sample, and greatly prolongs the preservation time of the sample, thereby ensuring the accuracy of the detection result.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a protective agent for a gastrin 17 blood sample, which comprises a stabilizer, a preservative and water; the stabilizer is 2-bis (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or 1, 3-bis [ (trimethylol) methylamino ] propane; the concentration of the stabilizer is 0.5-1mol/L.
The protective agent provided by the invention takes 2-di (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or 1, 3-bis [ (trimethylol) methylamino ] propane as the stabilizer, so that the degradation of the gastrin 17 can be effectively avoided, the preservation time of a blood sample of the gastrin 17 can be greatly prolonged, and the accuracy of a detection result can be ensured.
In some embodiments, the stabilizer is present at a concentration of 0.5mol/L or 1mol/L.
In some embodiments, the preservative comprises 0.2 to 1% by volume of the protective agent.
In some embodiments, the preservative comprises Proclin300 and/or MIT. In some embodiments, the preservative consists of Proclin300 and MIT; the Proclin300 has a volume percentage of 0.2% and the MIT has a volume percentage of 0.2%.
In the present invention, the blood sample is plasma, serum or whole blood.
The invention also provides a preservation method of the gastrin blood sample, and the protective agent disclosed by the invention is used for preserving the gastrin 17 blood sample.
The invention also provides a blood collection tube, which comprises a tube body, a rubber plug, an outer cap and the protective agent. In some embodiments, the tube body is a test tube, the blood collection tube comprises a test tube 1, a sealing rubber plug 2 and a plastic outer cap 3 are arranged at the opening of the test tube 1, and a protective agent 4 (shown in fig. 1) of a blood sample of the gastrin 17 is pre-filled in the test tube 1, so that the degradation of the gastrin 17 can be effectively prevented, and the preservation time of serum or blood plasma samples can be prolonged. The form of the protecting agent 4 of the present invention is not particularly limited, and may be a liquid form, or may be a lyophilized cake obtained by lyophilization treatment using a vacuum freeze dryer, and a uniform powder obtained by treating the lyophilized cake using a homogenizer. The solid component of the stabilizer with the weight of 0.1-1% (w/v) of the blood volume of the blood collection tube can be directly weighed to prepare the blood collection tube, but the operation is more complicated, the required stabilizer addition amount is smaller, and the operation is inconvenient.
The test tube 1 of the blood collection tube can be made of glass or plastic materials, and the sealing rubber plug can be made of butyl rubber.
The vacuum negative pressure of the test tube of the blood collection tube provided by the invention can be designed to be 3ml or 5ml, and can be other volumes, so that the detection requirement can be met.
The protective agent for the gastrin blood sample provided by the invention comprises a stabilizer and a preservative; the stabilizer is 2-di (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or 1, 3-bis [ (trimethylol) methylamino ] propane. Compared with the existing gastrin 17 sample stabilizer, the gastrin 17 protective agent provided by the invention has the following advantages:
(1) The protective agent can be added not only to serum or plasma obtained after centrifugation but also to whole blood without causing hemolysis.
(2) The addition of stabilizers to the serum or plasma obtained after centrifugation does not block the degradation of gastrin 17 from the time of blood collection to centrifugation. The protective agent can be added into whole blood, and is a precondition for preparing a vacuum blood collection tube containing a gastrin 17 stabilizer, so that a key technology for blocking the degradation of the gastrin 17 from the source is realized.
(3) The protective agent can effectively avoid degradation of gastrin 17 in blood samples, and greatly prolong the preservation time of the samples, thereby ensuring the accuracy of detection results.
Drawings
FIG. 1 shows a schematic representation of a evacuated blood collection tube containing a gastrin 17 protecting agent in accordance with the present invention.
Detailed Description
The invention provides a protective agent for a gastrin 17 blood sample. Those skilled in the art can, given the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation method of gastrin 17 protectant of the invention
1000ml of purified water is measured by a measuring cylinder, 104.62g of 2-di (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and the preservatives Proclin 3002ml and MIT 2ml are sequentially added, fully and uniformly stirred, and the mixture is packaged into 10ml bottles for later use at room temperature.
Example 2 preparation method of gastrin 17 protective agent provided by the invention
1000ml of purified water is measured by a measuring cylinder, 141.165g of 1, 3-bis [ (trimethylol) methylamino ] propane and 3002ml of preservative Proclin and 2ml of MIT are sequentially added, and the mixture is fully and uniformly stirred, split-packed into 10ml of each bottle and kept at room temperature for standby.
Example 3 preparation method of gastrin 17 protective agent provided by the invention
1000ml of purified water is measured by a measuring cylinder, 282.44g of 1, 3-bis [ (trimethylol) methylamino ] propane and 3002ml of preservative Proclin and 2ml of MIT are sequentially added, and the mixture is fully and uniformly stirred, split-packed into 10ml of each bottle and kept at room temperature for standby.
Example 4 preparation method of gastrin 17 protective agent provided by the invention
1000ml of purified water is measured by a measuring cylinder, 104.62g of 2-di (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol, 141.165g of 1, 3-bis [ (trimethylol) methylamino ] propane and the preservatives Proclin 3002ml and MIT 2ml are sequentially added, fully and uniformly stirred, split-packed into 10ml bottles and stored at room temperature for standby.
Example 5 preparation method of a blood collection tube containing a gastrin 17 protective agent according to the present invention
In a first step, a protectant was prepared as in examples 1-4.
Secondly, cleaning the inner wall of the test tube 1, and adding a protective agent 4 into the bottom of the test tube 1 in an ultra-clean bench sterile working environment, wherein the addition amount is 5-10% (w/v) of the blood volume of the blood collection tube;
and thirdly, vacuumizing according to the measurement of the blood collection tube, and then assembling the sealing rubber plug 2 and the plastic outer cap 3, wherein the structure of the finished blood collection tube is shown in figure 1.
Comparative example 1 preparation of gastrin 17 protectant
1000ml of purified water is measured by a measuring cylinder, 70.58g of 1, 3-bis [ (trimethylol) methylamino ] propane and 3002ml of preservative Proclin and 2ml of MIT are sequentially added, and the mixture is fully and uniformly stirred, split-packed into 10ml of each bottle and kept at room temperature for standby.
Comparative example 2 preparation of gastrin 17 protectant
DMSO is used as a protective agent, and the protective agent is preserved in shade at a dry place.
Comparative example 3 preparation of gastrin 17 protectant
1000mL of purified water is measured, and NaH is added in sequence 2 PO 4 ·2H 2 O 0.59g、Na 2 HPO 4 ·12H 2 O5.8 g, naCl 9g, casein 10g, sodium dodecyl sulfate 20g, aprotinin 2g and ProClin300 mL, and the mixture was stirred until it was clear and transparent.
Test example 1 examination of the stabilizing Effect of the gastrin 17 protectant of the invention
Examination of serum samples isolated from ordinary blood collection tubes, addition of the samples of example 1 to serum/plasma after centrifugation, addition of the serum samples of example 1 to whole blood, and examination of sample stability at 2-8deg.C by using the gastrin 17 detection kit (magnetic particle chemiluminescence method) of Anji organisms on AutoLumoA2000 at time points of 0h, 3h, 4h, 1 day, 3 days, 5 days, and 7 days, respectively, gave the results shown in Table 1.
Table 1 2 stabilization effect (sample) of gastrin 17 protectant at-8deg.C
Example 4 was added to the gastrin 17 quality control, and the quality control stability under the preservation condition of 2-8deg.C was examined by examining the quality control stability of gastrin 17 quality control at time points of 0h, 3h, 4h, 1 day, 3 days, 5 days, and 7 days, respectively, using the gastrin 17 detection kit (magnetic particle chemiluminescence method) of Anji organism on AutoLumo A2000, and the results are shown in Table 2.
Table 2 2 stabilization effect (quality control product) of gastrin 17 protectant at-8deg.C
As can be seen from the experimental data in tables 1 and 2, the G-17 sample can be stably stored at 2-8 ℃ for at least 7 days by using the protective agent disclosed by the invention, and the reduction of the amplitude is less than 10%; the G-17 quality control product can be stably stored at 2-8deg.C for at least 7 days after redissolution, and the reduction is less than 10%.
Examination of serum samples isolated from ordinary blood collection tubes (control), addition of example 2, example 3 and comparative example 1 to serum/plasma after centrifugation, preservation time of gastrin 17 blood samples of comparative example 3, comparative example 2, were examined on an AutoLumoA2000 using gastrin 17 detection kit (magnetic particle chemiluminescence method) of animate being at time points of 0h, 3h, 4h, 1 day, 3 day, 5 day, 7 day, respectively, and examination of sample stability under preservation conditions of 2-8 ℃ was performed, and the results are shown in table 3.
Table 3 2-8 ℃ stabilization effect of different gastrin 17 protectants
As can be seen from the experimental data in Table 3, the G-17 sample can be stably stored at 2-8deg.C for at least 7 days with a reduction of less than 10% by using the protectant of the present invention; the G-17 sample can be stably stored for 3 hours at the temperature of 2-8 ℃ only by using the protective agent of the comparative example 1, and the amplitude reduction is less than 10%; the G-17 sample can be stably stored for 3 days at the temperature of 2-8 ℃ only by using the protective agent of the comparative example 2, and the amplitude reduction is less than 10%; the G-17 sample can be stably stored for 3 hours at the temperature of 2-8 ℃ only by using the protective agent of comparative example 3, and the amplitude reduction is less than 10%.
Test example 2 sample stability test of blood collection tube containing gastrin 17 protective agent according to the present invention
Blood sample treatment was performed simultaneously using a blood collection tube containing the present invention example 5 (containing 5% protectant) and a conventional common tube blood collection tube, and a total of 10 samples were examined. Samples were tested on an AutoLumoA2000 using the gastrin 17 detection kit of the ambroxol organism (magnetic particle chemiluminescence). Upside down for 8-10 times after blood drawing, placing in a 37 ℃ incubator for 30 minutes, taking out, and centrifuging for 10 minutes at 4000 rpm; centrifuging, and storing at 2-8deg.C. Samples were tested using the ambroxol bio-gastrin 17 assay kit (magnetic particle chemiluminescence) at time points of 0h, 3h, 4h, 1 day, 3 days, 5 days, 7 days, and 14 days, respectively, and the stability of the samples of the two blood collection tubes under 2-8deg.C storage conditions was examined, and the results are shown in tables 4 and 5.
Table 4 2-8deg.C sample preservation time of the blood collection tube of the present invention
Table 5 2-8deg.C conventional common tube blood collection tube sample preservation time
As can be seen from the experimental data in tables 4 and 5, the G-17 sample can be stably stored at 2-8 ℃ for 7 days by using the blood collection tube containing the gastrin 17 sample protective agent disclosed by the invention, and the amplitude reduction is less than 10%. By using a conventional common tube blood collection tube, the G-17 sample can be stored stably for only 3 hours at 2-8 ℃ after being stored for 1 day at 2-8 ℃ and the amplitude of the G-17 sample is reduced by more than 25% after 1 day.
Test example 3 Effect of protectant on test results
The protective agent provided by the invention is used for detecting samples on an AutoLumo A2000 by using a gastrin 17 detection kit (magnetic particle chemiluminescence method) of Anji organisms, and the influence of the protective agent on the detection results of the samples is examined, and the results are shown in tables 6 and 7.
TABLE 6 influence of protectants on the detection results (example 1)
TABLE 7 influence of protectants on the detection results (examples 2 and 3)
| Sample numbering | P6 | P7 | P8 | P9 | P10 |
| Serum for common blood collection tube | 99.41 | 46.00 | 26.62 | 33.12 | 9.55 |
| Containing 5% of the serum of example 2 | 98.22 | 42.04 | 27 | 33.27 | 9.08 |
| Bias of | -1.20％ | -8.61％ | 1.43％ | 0.45％ | -4.92％ |
| Containing 5% of the serum of example 3 | 96.1 | 44.55 | 25.62 | 32.79 | 9.74 |
| Bias of | 3.44％ | 3.25％ | 3.90％ | 1.01％ | -1.95％ |
As can be seen from the experimental data in tables 6 and 7, the use of the protective agent according to the present invention has an influence deviation of 10% or less on the detection result of the sample, and the protective agent has no influence on the detection result.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A protective agent for a gastrin 17 blood sample, comprising a stabilizer, a preservative and water;
the stabilizer is 2-bis (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or 1, 3-bis [ (trimethylol) methylamino ] propane;
the concentration of the stabilizer is 0.5-1mol/L;
the preservative consists of Proclin300 and MIT; wherein, the volume percentage of Proclin300 is 0.2 percent, and the volume percentage of MIT is 0.2 percent.
2. The protectant of claim 1, wherein the blood sample is plasma, serum or whole blood.
3. A method for preserving a gastrin 17 blood sample, characterized in that the gastrin 17 blood sample is preserved by using the protective agent according to claim 1 or 2.
4. A blood collection tube comprising a tube body, a rubber plug, an outer cap and the protective agent of claim 1 or 2.
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| JPH05209857A (en) * | 1990-07-30 | 1993-08-20 | Hitachi Ltd | Ion measuring sample diluent and ion measuring method |
| CN109212189A (en) * | 2018-09-26 | 2019-01-15 | 郑州安图生物工程股份有限公司 | The kit of HER-2 protein content in a kind of quantitative detection serum, blood plasma |
| CN109307776A (en) * | 2018-11-16 | 2019-02-05 | 郑州安图生物工程股份有限公司 | A kind of gastrin 17 detection kit of improvement |
| CN110672849A (en) * | 2019-11-12 | 2020-01-10 | 郑州安图生物工程股份有限公司 | Detection kit for S100 protein |
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| CA3024996A1 (en) * | 2016-06-08 | 2017-12-14 | Cf Genome, Llc | A chemical composition to stabilize extracellular vesicles in a blood sample and method of use thereof |
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| JPH05209857A (en) * | 1990-07-30 | 1993-08-20 | Hitachi Ltd | Ion measuring sample diluent and ion measuring method |
| CN109212189A (en) * | 2018-09-26 | 2019-01-15 | 郑州安图生物工程股份有限公司 | The kit of HER-2 protein content in a kind of quantitative detection serum, blood plasma |
| CN109307776A (en) * | 2018-11-16 | 2019-02-05 | 郑州安图生物工程股份有限公司 | A kind of gastrin 17 detection kit of improvement |
| CN110672849A (en) * | 2019-11-12 | 2020-01-10 | 郑州安图生物工程股份有限公司 | Detection kit for S100 protein |
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