Activated partial thromboplastin time kit

Abstract

The invention relates to the technical field of medicine detection, and provides an activated partial thromboplastin time kit which comprises R1 and R2, the R1 comprises the following components in percentage by mass: 0.08%-0.12% of rabbit cephalin, 0.02 per mill to 0.050 per mill of an activating agent, 0.08%-0.15% of a preservative, 0.08%-0.12% of a buffering agent and the balance of a solvent; and R2 is a calcium chloride solution. And the variety of activated partial thromboplastin time kits is expanded.

Description

Technical Field

[0001] The present application belongs to the field of medical detection technology, and in particular relates to an activated partial thromboplastin time kit. Background Art

[0002] The activated partial thromboplastin time (APTT) measurement is an important indicator of coagulation function detection and also an important indicator of preoperative examination.

[0003] The industry standard for determining the activated partial thromboplastin time is to add APTT reagent to the plasma to be tested and 2+ With the participation of the test plasma, fibrinogen is converted into insoluble fibrin, thereby coagulation occurs. The time required for coagulation is the activated partial thromboplastin time (APTT) of the test plasma.

[0004] Among the APTT reagents currently used, most use minerals as activators, such as kaolin, diatomaceous earth, etc. Therefore, it can be seen that the types of APTT reagents need to be expanded urgently. Summary of the invention

[0005] The purpose of the present application is to provide an activated partial thromboplastin time kit, aiming to solve the problem of a single type of APTT reagent.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] The present application provides an activated partial thromboplastin time kit, comprising R1 and R2, wherein R1 comprises the following components in mass percentage: 0.08% to 0.12% rabbit cephalin, 0.02‰ to 0.050‰ activator, 0.08% to 0.15% preservative, 0.08% to 0.12% buffer, and the remainder is solvent; and R2 is a calcium chloride solution.

[0008] The beneficial effects of the present invention are:

[0009] The present invention uses rabbit cephalin as a substitute for platelets and adds it to plasma. 2+ With the participation of, the intrinsic coagulation system is activated, factors XII and XI are activated, fibrinogen is converted into insoluble fibrin, and the time required for coagulation is measured, which is the activated partial thromboplastin time (APTT) of the plasma to be tested, expanding the types of activated partial thromboplastin time kits. DETAILED DESCRIPTION

[0010] The purpose of the present application is to provide an activated partial thromboplastin time kit, aiming to solve the problem that the main components of the current activated partial thromboplastin time kit are too single and have poor substitutability.

[0011] The first aspect of an embodiment of the present application provides an activated partial thromboplastin time kit, comprising R1 and R2, wherein R1 comprises the following components in mass percentage: 0.08% to 0.12% rabbit cephalin, 0.02‰ to 0.050‰ activator, 0.08% to 0.15% preservative, 0.08% to 0.12% buffer, and the remainder is solvent; R2 is a calcium chloride solution.

[0012] The detection principle of the activated partial thromboplastin time kit disclosed in the present invention is: add APTT reagent to the plasma to be tested, preheat it uniformly at 37°C for a certain period of time, 2+ With the participation of, the intrinsic coagulation system is activated, factors XII and XI are activated, fibrinogen is converted into insoluble fibrin, and the time required for coagulation is measured, which is the activated partial thromboplastin time (APTT) of the plasma to be tested.

[0013] As a preferred method, an activated partial thromboplastin time kit includes R1 and R2, R1 includes the following components in mass percentage: 0.1% rabbit cephalin, 0.035‰ activator, 0.1% stabilizer, 0.1% buffer, and the balance is solvent; R2 is calcium chloride solution.

[0014] In the present invention, the activator may be ellagic acid, a salt of ellagic acid or a metal chelate of ellagic acid, preferably ellagic acid, which mainly activates factors XII and XI, thereby converting fibrinogen into insoluble fibrin.

[0015] In the present invention, the preservative is used to prolong the storage time of the activated partial thromboplastin time kit, mainly to prevent the activator from denaturing and losing the activation effect. The preservative can be a common preservative reagent in the art such as ProClin-300.

[0016] In the present invention, the buffer is mainly used to adjust the pH value of the entire reagent, which is conducive to the activator to fully exert its effect. The buffer can be a common buffer reagent in the art such as 4-hydroxyethylpiperazineethanesulfonic acid.

[0017] In the present invention, the solvent may be pure water, plasma or other common reagents in the art.

[0018] In the present invention, partial thromboplastin refers to phospholipids derived from rabbit brain or bovine brain.

[0019] As a possible implementation, the activated partial thromboplastin time kit includes R1 and R2. In 1L of R1: the activator is ellagic acid, with a content of 0.035g, the partial thromboplastin is rabbit phospholipids, with a content of 1.0g, the preservative is ProClin-300, with a content of 1ml, the buffer is 4-hydroxyethylpiperazineethanesulfonic acid, with a content of 1g, and the balance is pure water; in R2: the concentration of the calcium chloride solution is 25mM.

[0020] The range of activated partial thromboplastin time kits is expanded by using ellagic acid as an activator to activate factors XII and XI, instead of minerals as activators.

[0021] The following describes the invention in conjunction with specific embodiments.

[0022] Example 1

[0023] This embodiment discloses an activated partial thromboplastin time kit, including R1 and R2, 1L R1 includes the following components by mass or volume: 0.035g ellagic acid, 1.0g rabbit cephalin, 1mL ProClin-300, 1g 4-hydroxyethylpiperazineethanesulfonic acid, and the balance is pure water, wherein the pure water is deionized water. R2 is a calcium chloride solution with a concentration of 25mM.

[0024] This example discloses a method for preparing an activated partial thromboplastin time kit. Ellagic acid, rabbit cephalin, ProClin-300 and 4-hydroxyethylpiperazineethanesulfonic acid are sequentially dissolved in pure water and stirred evenly to obtain R1.

[0025] When using the above reagents, add R2 to the plasma and stir evenly, then add R1 to the system and stir evenly, and record the coagulation time.

[0026] Example 2

[0027] This embodiment discloses an activated partial thromboplastin time kit, including R1 and R2, 1L R1 includes the following components by mass or volume: 0.02g of ellagic acid, 1.2g of rabbit cephalin, 0.9mL of ProClin-300, 1.2g of 4-hydroxyethylpiperazineethanesulfonic acid, and the balance is pure water, wherein the pure water is deionized water. R2 is a calcium chloride solution with a concentration of 25mM.

[0028] This example discloses a method for preparing an activated partial thromboplastin time kit. Ellagic acid, rabbit cephalin, ProClin-300 and 4-hydroxyethylpiperazineethanesulfonic acid are sequentially dissolved in pure water and stirred evenly to obtain R1.

[0029] When using the above reagents, add R2 to the plasma and stir evenly, then add R1 to the system and stir evenly, and record the coagulation time.

[0030] Example 3

[0031] This embodiment discloses an activated partial thromboplastin time kit, including R1 and R2, 1L R1 includes the following components by mass or volume: 0.05g ellagic acid, 0.8g rabbit cephalin, 1.5mL ProClin-300, 0.8g 4-hydroxyethylpiperazineethanesulfonic acid, and the balance is pure water, wherein the pure water is deionized water. R2 is a 25mM calcium chloride solution.

[0032] This example discloses a method for preparing an activated partial thromboplastin time kit. Ellagic acid, rabbit cephalin, ProClin-300 and 4-hydroxyethylpiperazineethanesulfonic acid are sequentially dissolved in pure water and stirred evenly to obtain R1.

[0033] When using the above reagents, add R2 to the plasma and stir evenly, then add R1 to the system and stir evenly, and record the coagulation time.

[0034] 1. The kit prepared in Example 1 was used to measure the normal plasma samples. The results are shown in Table 1.

[0035] Table 1 Mean evaluation data record table of samples tested by the kit

[0036]

[0037]

[0038] Results: From the above data analysis, there were no outliers, and the APTT results of 30 normal human plasma samples were no more than 35s, which met the experimental technical requirements.

[0039] 2. Precision

[0040] Precision APTT quality control product test original data record

[0041] Table 2

[0042]

[0043]

[0044] 3. Precision verification

[0045] Using one measurement system, two normal and abnormal plasma samples were tested, and each sample was repeated 10 times.

[0046] Table 3 Precision plasma sample verification data record

[0047] Number of detections Normal plasma sample (unit: s) 1 27.4 2 28.2 3 26.9 4 26.0 5 27.3 6 27.3 7 28.1 8 26.9 9 27.8 10 26.4 M 27.23 SD 0.71 M±2SD 【25.82，28.64】 CV 2.59％

[0048] Table 4

[0049]

[0050]

[0051] The test results of the kit for detecting normal / abnormal plasma samples showed that the precision was no higher than 5%, which met the requirements of the research and development design.

[0052] 4. Analytical specificity

[0053] 4.1 Analysis of interfering substances

[0054] The reagent detects plasma samples with added interfering substances and plasma samples without added interfering substances. When the deviation between the test results with added interfering substances and the test results without added interfering substances exceeds ±10%, it is considered that the concentration of the interfering substances at this time will affect the performance of the detection reagent.

[0055] 4.1.1 Hemoglobin

[0056] Samples containing different concentrations of hemoglobin were tested, with the APTT time of about 31s for mixed plasma samples, and the hemoglobin content was 50, 100, 200, and 400 mg / dL, respectively. The test results are as follows:

[0057] Table 5 Detection results of hemoglobin interference samples

[0058]

[0059] Result analysis: Hemoglobin content has negative interference on APTT detection. The APTT time of normal plasma samples is about 31s. When the relative deviation of the test results of the interference sample exceeds ±10%, the hemoglobin concentration will affect the performance of the test reagent. Therefore, when the hemoglobin is not higher than 400mg / dL, there is no interference with the test results.

[0060] 4.1.2 Fat emulsion

[0061] Samples containing fat emulsions of different concentrations were tested, among which the APTT time was about 31s for fresh mixed plasma samples, and the fat emulsion contents were 50, 100, 200, and 400 mg / dL, respectively. The test results were as follows:

[0062] Table 6 Test results of fat emulsion interference samples

[0063]

[0064] Result analysis: The fat emulsion content has a positive interference on the APTT test. The APTT time of normal plasma samples is about 31s. When the relative deviation of the test results of the interference sample exceeds ±10%, the fat emulsion concentration will affect the performance of the test reagent. Therefore, when the fat emulsion is not higher than 400mg / dL, it will not interfere with the test results.

[0065] 4.1.3 Bilirubin

[0066] Samples containing different concentrations of bilirubin were tested, including a mixed plasma sample with an APTT time of about 32 seconds, and the bilirubin content was 5, 10, and 20 mg / dL, respectively. The test results are as follows:

[0067] Table 7 Test results of bilirubin interference samples

[0068]

[0069] Result analysis: Bilirubin content has positive interference on APTT detection. The APTT time of normal plasma samples is about 32s. When the relative deviation of the test results of the interference sample exceeds ±10%, the bilirubin concentration will affect the performance of the test reagent. Therefore, when bilirubin is not higher than 20mg / dL, there is no interference with the test results.

[0070] 5. Stability

[0071] 5.1 Accelerated Destabilization

[0072] Three batches of finished test kits at the near-expiration date and medium-expiration date were stored at 37±1℃, and inspected according to the assessment indicators every three or four days (3d, 7d and 10d). The operations were strictly in accordance with the product instructions and compared with the 0d test results (control) to examine the accelerated destruction stability of the product.

[0073] Table 8 Accelerated failure stability test results

[0074]

[0075] Table 9 Accelerated failure stability repeatability test results

[0076]

[0077]

[0078] The results showed that after the near-expiration and mid-expiration test kits were stored at 37℃±1℃ for 10 days, the stability of the reagents met the technical requirements and clinical application needs.

[0079] 5.2 Stability during shelf life

[0080] The test kits were stored at 2°C to 8°C, and tested according to the assessment indicators at different times (0, 6, 12, and 18 months (M)). The operation was strictly in accordance with the product instructions, and the test results were compared with the 0d test results (control) to examine the shelf life stability of the product.

[0081] Table 10 Results of shelf life stability test

[0082]

[0083] Table 11 APTT normal plasma measurement values

[0084]

[0085]

[0086] Table 12 Results of shelf life stability repeatability test

[0087]

[0088] The results show that after the test kit is stored at 2℃~8℃ for 18 months, all evaluation indicators meet the technical requirements. Therefore, the reagent is stable for 18 months, and the shelf life analysis of the medium-term test kit is also within the technical range. The stability experiment is still under observation.

[0089] 5.3 Transport stability

[0090] The main focus is on the effects of physical extrusion and temperature during transportation on the reagents. Therefore, it is designed that in hot summer weather, the 191011 liquid test kit will be placed in a cold chain transport box (the reagents are concentrated in the middle of the foam box, and the foam box is placed in the turnover box) and sent from Beijing to Chengdu and then returned to Beijing (this test kit was sent from Beijing on 2022-7-22, transported to Chengdu, and returned to Beijing on 2022-7-28); in severe winter weather, the test kit will be placed in a transport turnover box (cold chain transport foam box) and sent from Beijing to Chengdu and then returned to Beijing (this test kit was sent from Beijing on 2021-12-17, transported to Chengdu, and returned to Beijing on 2021-12-23). ​​The returned 191011 liquid test kit will be inspected according to the assessment indicators, strictly operated in accordance with the product instructions, and compared with the 0d test results.

[0091] Table 13. Transport stability test results (winter)

[0092]

[0093] Table 14 Transport stability test results (summer)

[0094]

[0095] The results show that during the transportation of the test kit in winter and summer, all assessment indicators meet the technical requirements.

[0096] 5.4 Reagent freeze-thaw stability

[0097] The test kit was stored at 2℃-8℃. After opening the bottle, the reagent was placed in -20℃ for freeze-thaw three times. The results of the three freeze-thaw tests were compared with the results of zero freeze-thaw tests (control) to examine the freeze-thaw stability of the product reagents.

[0098] Table 15. Freeze-thaw stability test results

[0099]

[0100] The results showed that the relative deviation of the test results after three repeated freeze-thaw cycles of the reagent was ≤15%. It is recommended that the reagent should be frozen and thawed no more than three times.

[0101] 5.5 Reagent stability after opening

[0102] After opening the test kit, test the normal quality control products and abnormal quality control products three times each. The average of the test results is taken as the initial value (Day 0). Thereafter, test once every 5 days for 15 consecutive days. Strictly follow the product instructions for use and compare with the 0-day test result (control) to examine the stability of the product when stored at 2°C to 8°C after opening the bottle.

[0103] After the reagents in Table 16 were opened, normal quality control products and abnormal quality control products were tested on days 0, 5, 10, and 15 (d), and the results were analyzed for deviations. The results are as follows:

[0104] Table 16. Open bottle stability test results

[0105]

[0106]

[0107] The results show that 15 days after the reagent was opened, the relative deviation between the normal / abnormal quality control results and the 0-day test results was ≤10%, and the test results on the 15th day did not change significantly. Therefore, the reagent is stable within 15 days after opening the bottle, which meets the experimental requirements.

[0108] 5.6 Reagent stability in machine

[0109] After opening the bottle, place the finished test kit in the reagent compartment of the coagulometer, and inspect it according to the assessment indicators at different times (0, 5, and 7 days). Operate strictly in accordance with the product instructions and compare it with the 0-day test result (control) to examine the product's on-machine stability.

[0110] Table 17 On-machine stability test results

[0111]

[0112] The results show that the reagents are placed on the instrument at 2℃-8℃ for 0, 5, and 7 days for quality control, and the reagents can be stable on the instrument for 7 days.

[0113] 6. Applicable sample stability

[0114] 6.1 Sample storage stability at 2℃~8℃

[0115] The samples were placed at 2℃~8℃ for 0, 6, 12, and 24 hours (h) and tested, and the mean of the test results was analyzed for deviation. The results are as follows:

[0116] Table 18. Mean results of stability test of plasma samples at 2℃~8℃

[0117]

[0118] Table 19 Plasma sample stability test results at 2℃~8℃

[0119] deviation% 0h 6h 12h 24h Low value - 0.87％ 4.81％ 6.41％ High value - 1.48％ 4.60％ 6.34％

[0120] It can be seen from Table 19 that when the plasma samples were stored at 2°C to 8°C for 24 hours, the relative deviation of the test results was ≤15%. Therefore, the samples were stable when stored at 2°C to 8°C for 24 hours.

[0121] 6.2 Storage stability at room temperature (18℃~26℃)

[0122] The samples were placed at 18℃~26℃ and tested after 0, 6, 8, and 12 hours (h). The mean of the test results was analyzed for deviation. The results are as follows:

[0123] Table 20. Mean results of stability test of plasma samples at 18℃~26℃

[0124]

[0125] Table 21. Batch stability test results of plasma samples at 18℃~26℃

[0126] deviation% 0h 6h 8h 12h Low value - 0.32％ 5.35％ 8.88％ High value - 1.40％ 4.40％ 8.73％

[0127] It can be seen from Table 21 that when the plasma samples were stored at 18℃~26℃ for 8h, the relative deviation of the test results was ≤15%. Therefore, there was no obvious change when the samples were stored at 18℃~26℃ for 12h, and it is recommended to test within 8 hours.

[0128] 6.3 Sample storage stability below -20℃

[0129] The samples were placed below -20°C for 1, 2, 3, and 4 weeks (W) and then tested. The mean values ​​of the test results were analyzed for deviations. The results are as follows:

[0130] Table 22 Mean results of stability test of plasma samples below -20℃

[0131]

[0132] Table 23. Results of stability test of plasma samples below -20°C

[0133] deviation% Comparison 1W 2W 3W 4W Low value - -0.10％ 1.60％ 9.39％ 9.74％ High value - 0.74％ 2.68％ 9.19％ 9.79％

[0134] It can be seen from Table 23 that the plasma samples were stored at -20°C for 4 weeks, and the relative deviation of the test results was ≤15%. Therefore, the samples can be stored at -20°C for 4 weeks. There will be obvious changes starting from the third week, and it is recommended to test within 2 weeks.

[0135] 6.4 Sample stability after repeated freeze-thaw cycles

[0136] The samples were taken out from below -20℃ and restored to 18℃~26℃ for testing, and then frozen below -20℃ again. The operation was repeated 3 times, and the results were analyzed for deviation. The results are as follows:

[0137] Table 24 Mean results of repeated stability tests of plasma samples

[0138]

[0139] Table 25. Results of repeated freeze-thaw stability test of plasma samples

[0140] deviation% 0 times 1 time 2 times 3 times Low value - 1.28％ 5.54％ 9.20％ High value - 1.00％ 5.48％ 9.27％

[0141] It can be seen from Table 25 that the relative deviation of the test results of plasma samples after three repeated freeze-thaw cycles was ≤15%, and the third change was obvious. It is recommended that plasma samples be frozen and thawed no more than twice.

[0142] The results of the accelerated destruction stability test, shelf life stability, reagent freeze-thaw stability, reagent opening stability, in-machine stability, the influence of adverse conditions during transportation, and sample applicability of the activated partial thromboplastin time assay kit (coagulation method) obtained in Example 1 determined that the activated partial thromboplastin time assay kit (coagulation method) can be stably stored at 37°C ± 1°C for 10 days, including transportation stability; all indicators meet the requirements after transportation; the reagent is repeatedly frozen and thawed no more than 2 times; after opening the bottle for use, it is stably stored at 2°C to 8°C for 15 days; the reagent is stably stored at 2°C-8°C on the instrument for 7 days. Sample collection and processing can be maintained for 8 hours under 18-26°C conditions, stably stored for 24h under 2°C to 8°C storage conditions, and it is recommended to be tested within 2 weeks under storage conditions below -20°C, and it is recommended that repeated freezing and thawing should not exceed 2 times.

[0143] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0144] In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that there can be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "multiple" refers to two or more. "At least one", "the following at least one (individual)" or similar expressions refer to any combination of these items, including any combination of single (individual) or plural (individual). For example, "at least one (individual) of a, b, or c", or "at least one (individual) of a, b, and c", can all represent: a, b, c, ab (i.e. a and b), ac, bc, or abc, wherein a, b, c can be single or multiple respectively.

[0145] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. An activated partial thromboplastin time kit, characterized in that: The invention comprises R1 and R2, wherein R1 comprises the following components in mass percentage: 0.08% to 0.12% of rabbit cephalin, 0.02‰ to 0.050‰ of activator, 0.08% to 0.15% of preservative, 0.08% to 0.12% of buffer, and the balance is solvent; and R2 is calcium chloride solution.

2. The activated partial thromboplastin time kit according to claim 1, characterized in that The invention comprises R1 and R2, wherein R1 comprises the following components in mass percentage: 0.1% rabbit cephalin, 0.035‰ activator, 0.1% stabilizer, 0.1% buffer, and the balance is solvent; and R2 is calcium chloride solution.

3. The activated partial thromboplastin time kit according to claim 1 or 2, characterized in that: The activator refers to ellagic acid, a salt of ellagic acid or a metal chelate of ellagic acid.

4. The activated partial thromboplastin time kit according to claim 1 or 2, characterized in that: The preservative is ProClin-300.

5. The activated partial thromboplastin time kit according to claim 1 or 2, characterized in that: The buffer is 4-hydroxyethylpiperazineethanesulfonic acid.

6. The activated partial thromboplastin time kit according to claim 3, characterized in that: The activator is ellagic acid.

7. The activated partial thromboplastin time kit according to claim 1, characterized in that: The solvent is pure water.

8. The activated partial thromboplastin time kit according to claim 1, characterized in that: Partial thromboplastin is a phospholipid derived from rabbit brain or bovine brain.

9. The activated partial thromboplastin time kit according to claim 1, characterized in that: The concentration of the calcium chloride solution is 10-30 mM, preferably 25 mM.

10. The activated partial thromboplastin time kit according to claim 1, characterized in that: In 1L R1: the activator is ellagic acid, the content is 0.035g, the partial thromboplastin is rabbit cephalin, the content is 1.0g, the preservative is ProClin-300, the content is 1ml, the buffer is 4-hydroxyethylpiperazineethanesulfonic acid, the content is 1g, and the balance is pure water; in R2: the concentration of the calcium chloride solution is 25mM.

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