Thrombin time detection kit

Abstract

The invention relates to a thrombin time detection kit, and relates to the technical field of medical examination. The thrombin time detection kit comprises thrombin, mannitol, trisodium citrate, anhydrous sodium sulfate and a preservative. The measured values of normal plasma samples are not more than 20s, and the intra-batch precision (CV): the coefficient of variation (CV) of a normal quality control product and an abnormal quality control product is not more than 5%; the inter-batch precision (CV) is not higher than 10%; and the inter-bottle difference (CV) of the dry powder reagent is not higher than 8%. According to the detection kit, the content of the interfering substance hemoglobin is not higher than 800mg / dL, the content of the fat emulsion is not higher than 400mg / dL, and the content of the bilirubin is not higher than 20mg / dL, so that the detection kit can meet the requirements of clinical application and has relatively good clinical detection performance.

Description

Technical Field

[0001] The present application relates to the field of medical testing technology, and in particular to a thrombin time detection kit. Background Art

[0002] There are two substances in the human blood that maintain the health of the human body, one is the coagulation substance, and the other is the anticoagulant substance. The dynamic balance between the two is the key to maintaining blood flow and preventing blood loss in the normal body. Coagulation is the process of blood changing from a sol state to a gel state. When the body lacks anticoagulants or fibrinolysis is abnormal, it will lead to the formation of thrombus. With the acceleration of the aging process of the population in today's society, various thrombotic diseases are increasingly becoming factors that cause disease, death and deteriorate the quality of human life, especially cardiovascular and cerebrovascular thrombotic diseases. Thrombotic diseases have become an important cause of global total mortality. Therefore, it is very necessary to examine thrombosis and coagulation clinically, and it is of great significance to develop a reliable kit for detecting coagulation.

[0003] Thrombin is a biologically active protein that plays an important role in the anticoagulation and procoagulation cascade reactions of the human body. The thrombin time (TT) is a method for quickly detecting the conversion of fibrinogen into fibrin on the common coagulation pathway. It is a simple test for detecting the function of the coagulation, anticoagulation and fibrinogenolysis systems.

[0004] At present, the determination of TT has all been made of semi-automatic or automated instruments. The detection principle is that the plasma to be measured adds an appropriate amount of thrombin solution to convert fibrinogen into insoluble fibrin, and the plasma coagulation required time is the plasma thrombin time (TT) to be measured. Due to the difference in the quality of the reagents used to measure TT, the results of the same patient measured in different hospitals vary greatly, causing the inconsistency of the test results, affecting the correct and timely diagnosis of the disease. Therefore, the quality of the TT reagent has become the key to obtaining accurate results and diagnosis. Therefore, it is undoubtedly necessary to develop a thrombin time (TT) determination kit that is effective, reliable, stable, and easy to use. Summary of the invention

[0005] The present application provides a thrombin time detection kit to solve the current problems.

[0006] The present application provides a thrombin time detection kit, comprising thrombin, mannitol, trisodium citrate, anhydrous sodium sulfate and a preservative.

[0007] The above technical solution provided by the embodiment of the present application has at least the following advantages compared with the prior art:

[0008] Trisodium citrate and anhydrous sodium sulfate can adjust pH value, enhance stability, adjust repeatability of determination, and adjust the size of absorbance. Mannitol and preservatives can increase the stability of thrombin and extend the shelf life of the kit. The normal plasma sample measurement value of the thrombin time test kit is no more than 20s, and the intra-batch precision (CV): the coefficient of variation (CV) of normal quality control products and abnormal quality control products is no more than 5%; the inter-batch precision (CV) is no more than 10%; the bottle difference (CV) of dry powder reagents is no more than 8%. Interfering substances, hemoglobin is no more than 800mg / dL, fat emulsion is no more than 400mg / dL, bilirubin is no more than 20mg / dL, and the test results of various indicators have met the requirements of the test design and can meet the needs of clinical application. In addition, the test results of the kit have good consistency with the test results of similar kits that have been on the market, and the test results are equivalent, and it has good clinical detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a schematic diagram of the correlation between the first batch of lyophilized test kit clinical samples in Experimental Example 4 of this application and the similar reagents produced by Metron in Germany;

[0010] Figure 2 A schematic diagram of the correlation between the second batch of lyophilized test kit clinical samples in Experimental Example 4 of this application and the similar reagents produced by Metron in Germany;

[0011] Figure 3 A schematic diagram of the correlation between the third batch of clinical samples of the freeze-dried test kit in Experimental Example 4 of this application and the similar reagents produced by Metron in Germany;

[0012] Figure 4 This is a schematic diagram of the correlation between the first batch of clinical samples of liquid test kits in Experimental Example 4 of this application and the similar reagents produced by Metron in Germany;

[0013] Figure 5 This is a schematic diagram of the correlation between the second batch of clinical samples of liquid test kits in Experimental Example 4 of this application and the similar reagents produced by Metron in Germany;

[0014] Figure 6 This is a schematic diagram of the correlation between the third batch of clinical samples of liquid-type test kits in Experimental Example 4 of this application and the similar reagents produced by Mectronic of Germany. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0016] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0017] The present application provides a thrombin time detection kit, comprising thrombin, mannitol, trisodium citrate, anhydrous sodium sulfate and a preservative.

[0018] Preferably, the concentration of thrombin is 7000-9000 IU / L, the concentration of mannitol is 10-40 g / L, the concentration of trisodium citrate is 2-3 g / L, the concentration of anhydrous sodium sulfate is 4-6.5 g / L, and the concentration of preservative is 0.5-1.5 ml / L.

[0019] Preferably, the preservative is one or more of Pc-300, sodium azide, sodium thimerosal, potassium sorbate and sodium benzoate.

[0020] Preferably, the thrombin time detection kit is in a lyophilized or liquid form.

[0021] Preferably, the preparation method of the freeze-dried thrombin time detection kit is: dissolving and mixing the raw materials and then freeze-drying them.

[0022] Preferably, the preparation method of the liquid thrombin time detection kit is: dissolving and mixing the raw materials.

[0023] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0024] Example 1

[0025] This embodiment provides a thrombin time detection kit, which uses the following raw materials:

[0026] Mannitol 20g, trisodium citrate 2.5g, anhydrous sodium sulfate 5g, preservative 1mL, thrombin concentration is 8000IU / L; wherein the preservative is Pc-300;

[0027] The preparation method of the above-mentioned thrombin time detection kit comprises the following steps:

[0028] The raw materials are added into purified water to dissolve and mix, and then freeze-dried to obtain the product.

[0029] Example 2

[0030] This embodiment provides a thrombin time detection kit, which uses the following raw materials:

[0031] Mannitol 10g, trisodium citrate 2g, anhydrous sodium sulfate 4g, preservative 0.5g, thrombin concentration is 7000IU / L; wherein the preservative is sodium azide;

[0032] The preparation method of the above-mentioned thrombin time detection kit comprises the following steps:

[0033] Add all raw materials into purified water and dissolve and mix them.

[0034] Example 3

[0035] This embodiment provides a thrombin time detection kit, which uses the following raw materials:

[0036] Mannitol 40g, trisodium citrate 3g, anhydrous sodium sulfate 6.5g, preservative 1.5g, thrombin concentration is 9000IU / L; wherein the preservative is potassium sorbate;

[0037] The preparation method of the above-mentioned thrombin time detection kit comprises the following steps:

[0038] The raw materials are added into purified water to dissolve and mix, and then freeze-dried to obtain the product.

[0039] Test Example 1

[0040] Normal plasma sample values:

[0041] Test instrument: Chuangning Biotechnology (Beijing) Co., Ltd., fully automatic coagulation analyzer, model: ZK-6000, serial number: ZK60A100221M.

[0042] 30 human plasma samples with normal thrombin time (TT) were selected and tested using the kits produced in Examples 1-3. Example 1 was the first batch, Example 2 was the second batch, and Example 3 was the third batch. Each sample was tested once. The results are shown in the table below. According to the experimental results of the kit for testing normal plasma samples, values ​​exceeding 20s were considered outliers:

[0043] Table 1 Data record of the measured values ​​of normal plasma samples detected by three batches of freeze-dried test kits

[0044]

[0045] Results: From the above data analysis, there were no outliers, and the TT results of 30 normal human plasma samples were no more than 20s, which met the experimental technical requirements.

[0046] Table 2 Data record of the measured values ​​of normal plasma samples detected by three batches of liquid test kits

[0047]

[0048] Results: From the above data analysis, there are no outliers, and the TT results of 30 normal human plasma samples are no more than 20s, which meets the experimental requirements.

[0049] Test Example 2

[0050] Precision testing:

[0051] According to the method for precision evaluation of CLSI EP05-A3, the same operator or a group of operators were used on the same instrument to test the kits produced in Examples 1-3. Example 1 was the first batch, Example 2 was the second batch, and Example 3 was the third batch. The TT normal quality control samples and abnormal quality control samples were tested 10 times respectively. At the end of the evaluation, there were 10 pairs of normal quality control samples and abnormal quality control samples in each batch, that is, 10 test results for normal quality control samples and abnormal quality control samples. The intra-batch precision was calculated from the measurement results of the positive / abnormal quality control products. The inter-batch precision was calculated from the data of all positive / abnormal quality control products in 3 batches.

[0052] Two different operators tested each sample 5 times a day for 5 consecutive days according to each batch of reagent kits to evaluate the inter-operator and inter-day precision: three consecutive batches of reagent kits were used to test samples at two concentration levels on the same instrument for 5 consecutive days. At the end of the evaluation, each operator had a total of 25 test results per sample per batch to calculate the inter-operator and inter-day precision.

[0053] 1) Intra-batch precision

[0054] Table 3 Three batches of freeze-dried reagents batch precision TT normal quality control product test data record table

[0055]

[0056] Table 4 Data record of the test of quality control products with abnormal precision TT within three batches of freeze-dried reagents

[0057]

[0058] Table 5 Test data record table of intra-batch precision TT of three batches of liquid reagents and normal quality control products

[0059]

[0060] Table 6 Data record of abnormal quality control product test for intra-batch precision TT of three batches of liquid reagents

[0061]

[0062] First, outliers are judged based on (M) ± 2SD. If the measured value of a single sample exceeds the mean (M) ± 2SD, it is considered an outlier. From the analysis of Table 3-6, it can be judged that there are no outliers.

[0063] Using the results in Table 3-6, the intra-batch precision CV was calculated, and the results are as follows:

[0064] Table 7 Results of intra-batch precision tests for three batches of freeze-dried reagents

[0065]

[0066]

[0067] Table 8 Results of intra-batch precision tests for three batches of liquid reagents

[0068] Precision Sample Concentration First batch Second batch The third batch TT normal quality control 0.43％ 0.39％ 0.51％ TT abnormal quality control product 0.32％ 0.87％ 1.18％

[0069] Results: The intra-batch precision CV of three batches of freeze-dried reagents and liquid reagents was less than 5%, which met the experimental design requirements.

[0070] 2)Batch-to-batch precision

[0071] Table 9 Data record of inter-batch precision TT of three batches of freeze-dried reagents for normal quality control products

[0072]

[0073] Table 10 Data record of abnormal quality control products of three batches of freeze-dried reagents with different batches of precision TT

[0074]

[0075] Table 11 Data record of inter-batch precision TT of three batches of liquid reagents for normal quality control products

[0076]

[0077] Table 12 Data record of abnormal quality control products of three batches of liquid reagent batch precision TT

[0078]

[0079] Results: The precision verification of three batches of freeze-dried and liquid TT assay kits. The test results of normal and abnormal quality control products showed that the intra-batch precision (CV) was no higher than 5%.

[0080] Using the results in Tables 9-12, the batch precision CV was calculated and the results are as follows:

[0081] Table 13 Results of inter-batch precision tests of three batches of freeze-dried reagents

[0082] Precision Sample Concentration Inter-batch precision CV TT normal quality control 1.22％ TT abnormal quality control product 1.44％

[0083] Table 14 Results of inter-batch precision tests of three batches of liquid reagents

[0084] Precision Sample Concentration Inter-batch precision CV TT normal quality control 0.48％ TT abnormal quality control product 2.30％

[0085] Results: Inter-batch precision (CV): The coefficient of variation (CV) of normal quality control products and abnormal quality control products was no higher than 10%, which met the requirements of the research and development design.

[0086] 3) Bottle-to-bottle precision

[0087] Use 10 boxes of freeze-dried test kits to test 2 normal and abnormal quality control samples, and test the quality control sample once with each box of reagent; select another box from the above 10 boxes of freeze-dried reagents and repeat the test of the quality control sample 10 times.

[0088] Table 15 Data record of precision verification between bottles of three batches of freeze-dried reagents

[0089]

[0090]

[0091] Using the data in Table 15, the bottle-to-bottle precision CV was calculated, and the results are as follows:

[0092] Table 16 Calculation results of precision between bottles of three batches of freeze-dried test kits

[0093] Precision Sample Concentration First batch Second batch The third batch Normal quality control 4.45％ 3.71％ 2.87％ Abnormal quality control products 1.81％ 1.58％ 0.00％

[0094] Results: The results of testing normal / abnormal quality control products of three batches of freeze-dried reagents showed that the coefficient of variation (CV) of the bottle difference of freeze-dried reagents was no more than 8%, which met the requirements of the research and development design.

[0095] Test Example 3

[0096] Analytical Specificity:

[0097] The kits produced in Examples 1-3 were tested, Example 1 was the first batch, Example 2 was the second batch, and Example 3 was the third batch. The reagents tested 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 exceeded ±10%, it was considered that the concentration of the interfering substances at this time would affect the performance of the test reagent. Interfering substances hemoglobin, fat emulsion, and bilirubin were analyzed.

[0098] 1) Hemoglobin

[0099] Samples containing different concentrations of hemoglobin were tested, among which the normal mixed sample plasma with a TT value of about 16s, and the hemoglobin content was 0, 400, 600, and 800 mg / dL respectively. The test results are as follows:

[0100] Table 17 Test results of hemoglobin interference samples of three batches of freeze-dried test kits

[0101]

[0102]

[0103] Table 18 Test results of hemoglobin interference samples of three batches of liquid test kits

[0104]

[0105] Results: Hemoglobin content interferes with TT detection. The TT value of normal plasma samples is about 16s. 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 hemoglobin is not higher than 800mg / dL, the relative deviation of the test result is less than 10%, and there is no interference with the test result.

[0106] 2) Fat emulsion

[0107] Samples containing different concentrations of fat emulsion were tested, where the TT value was a 16s mixed plasma sample with fat emulsion contents of 0, 100, 200, and 400 mg / dL, respectively. The test results are as follows:

[0108] Table 19 Test results of three batches of freeze-dried test kit fat emulsion interference samples

[0109]

[0110] Table 20 Test results of three batches of liquid type test kit fat emulsion interference samples

[0111]

[0112] Results: The fat emulsion content has a positive interference on TT detection. The TT time of normal plasma samples is about 16s. When the relative deviation of the test results of the interference sample exceeds ±10%, the fat emulsion concentration will affect the performance of the detection reagent. Therefore, when the fat emulsion is not higher than 400mg / dL, the relative deviation of the test result is less than 5%, and there is no interference with the test result.

[0113] 3) Bilirubin

[0114] Samples containing different concentrations of bilirubin were tested, where the TT value was a 16s mixed plasma sample with bilirubin contents of 0, 5, 10, and 20 mg / dL, respectively. The test results are as follows:

[0115] Table 21 Test results of bilirubin interference samples from three batches of freeze-dried kits

[0116]

[0117] Table 22 Test results of bilirubin interference samples from three batches of liquid kits

[0118]

[0119] Results: Bilirubin content has positive interference on TT detection. The TT time of normal plasma samples is about 16s. 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 the bilirubin is not higher than 20mg / dL, the relative deviation of the test result is less than 5%, and there is no interference with the test result.

[0120] Test Example 4

[0121] Comparative test of the kit of Example 1 of the present application with similar kits on the market:

[0122] Three batches of freeze-dried and liquid test kits were used to test clinical samples assigned with TT reagents produced by Metron, Germany, and the results were statistically analyzed. If k∈[0.80, 1.20] and r>0.975, it is considered to have good consistency with the same product on the market. The test results are as follows:

[0123] Table 23 Comparison results between three batches of freeze-dried reagents and marketed products

[0124]

[0125]

[0126] According to Table 23, Appendix Figure 1-3 Analysis shows that the three batches of freeze-dried reagents had a correlation coefficient r∈[0.9777, 0.9830]≥0.975 with similar reagents produced by Metronic in Germany in the test of 40 clinical samples. The test results were highly positively correlated, and the kit had good clinical detection performance.

[0127] Table 24 Comparison results between three batches of liquid test kits and marketed products

[0128]

[0129]

[0130]

[0131] According to Table 24, Figure 4-6 Analysis shows that in the test of 40 clinical samples, the three batches of liquid reagents had a correlation coefficient r∈[0.9797, 0.9851]≥0.975 with similar reagents produced by Metronic in Germany, and the test results were highly positively correlated. The kit has good clinical detection performance.

[0132] Test Example 5

[0133] Evaluate the stability of continuously produced test kits and provide a basis for determining the actual shelf life of the test kits.

[0134] Main instruments: Fully automatic blood coagulation analyzer, model: ZK-6000SN number: ZK60A100221M.

[0135] 1) Reagent stability

[0136] 1. Accelerate the destruction of stability

[0137] The test kits of near-effectiveness and medium-effectiveness were stored at 37±1℃ respectively, and tested every three or four days (3d, 7d and 10d) according to the assessment indicators. 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 accelerated destruction stability of the product.

[0138] Table 25 Results of accelerated destruction stability test of freeze-dried kit

[0139]

[0140] Table 26 Results of accelerated destruction stability repeatability test of freeze-dried kit

[0141]

[0142] Table 27 Liquid type test kit accelerated destruction stability test results

[0143]

[0144]

[0145] Table 28 Liquid type test kit accelerated destruction stability test results

[0146]

[0147] The results showed that after the near-expiration and mid-expiration test kits were stored at 37℃±1℃ for 10 days, all assessment indicators met the technical requirements, and all indicators remained unchanged on the 10th day. Therefore, they are stable when stored at 37℃ for 10 days.

[0148] 2. Stability during shelf life

[0149] The freeze-dried and liquid test kits were stored at 2°C to 8°C, respectively, 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 results were compared with the 0d test results (control) to examine the shelf life stability of the product.

[0150] Table 29 Results of the stability test of the freeze-dried kit

[0151]

[0152] Table 30 Results of the stability repeatability test of the freeze-dried kit

[0153]

[0154]

[0155] Table 31 Liquid type test kit shelf life stability test results

[0156]

[0157] Table 32 Liquid type test kit shelf life stability repeatability test results

[0158]

[0159] The results show that when 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.

[0160] 3. Transportation stability

[0161] The design is to place the freeze-dried and liquid test kits in cold-chain transport boxes in summer (the test kits are placed in the middle of the foam box, and the foam box is placed in the transport box) and then send them from Beijing to Chengdu and then return them to Beijing; in winter, the test kits are placed in cold-chain transport boxes (cold-chain transport foam boxes) and then sent from Beijing to Chengdu and then returned to Beijing. The returned freeze-dried and liquid test kits are inspected according to the assessment indicators, strictly operated in accordance with the product instructions, and compared with the 0d test results.

[0162] Table 33 Freeze-dried kit winter transportation stability test results

[0163]

[0164] Table 34 Winter transportation stability test results of liquid test kit

[0165]

[0166] Table 35 Results of summer transportation stability test of freeze-dried test kit

[0167]

[0168] Table 36 Liquid type test kit summer transportation stability test results

[0169]

[0170]

[0171] Table 37 Results of repeatability test on stability of freeze-dried test kit during winter transportation

[0172]

[0173] Table 38 Liquid type test kit winter transportation stability repeatability test results

[0174]

[0175]

[0176] Table 39 Results of repeatability test on summer transportation stability of freeze-dried test kit

[0177]

[0178] Table 40 Results of repeatability test on summer transportation stability of liquid test kit

[0179]

[0180]

[0181] The results showed that during the transportation process, all assessment indicators of the three batches of test kits met the technical requirements.

[0182] 4. Reagent freeze-thaw stability

[0183] The test kit was stored at 2℃~8℃. After opening the bottle, the reagent was placed in -20℃ for freeze-thaw for 3 times. The results of the 3 freeze-thaw tests were compared with the results of the 0 freeze-thaw tests (control). The results are as follows:

[0184] Table 41 Freeze-thaw stability test results of lyophilized kit

[0185]

[0186] Table 42 Freeze-thaw stability test results of liquid kit

[0187]

[0188] The results showed that the relative deviation of the test results after three freeze-thaw cycles was ≤5%, so the reagent is stable if the freeze-thaw cycles do not exceed three times.

[0189] 5. Reagent stability after opening the bottle

[0190] After opening the liquid and lyophilized test kits, 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). Test once every 5 days for 15 consecutive days. Strictly follow the product instructions for use and compare with the 0-day test results (control) to examine the opening / reconstitution stability of the product when stored at 2°C to 8°C after opening the bottle.

[0191] After the reagent bottle is opened, the low-value quality control product and the high-value quality control product are tested three times at 0, 5, 10, and 15 days (d), and the results are analyzed for deviations. The results are as follows:

[0192] Table 43 Freeze-dried reagent bottle stability test results

[0193]

[0194] Table 44 Liquid reagent bottle opening stability test results

[0195]

[0196] Table 45 Results of stability repeatability test of freeze-dried reagents after opening the bottle

[0197]

[0198] Table 46 Liquid reagent bottle opening stability repeatability test results

[0199]

[0200]

[0201] The results show that: 15 days after the three batches of reagents were opened, the relative deviation of the normal / abnormal quality control results and the 0-day test results was ≤10%, the repeatability was ≤5%, and the test results on the 15th day did not change significantly. Therefore, the three batches of reagents were stable within 15 days after opening the bottle, which met the experimental requirements.

[0202] 6. On-machine stability

[0203] After opening the bottle / reconstitution, the finished dry powder and liquid test kits are placed in the reagent compartment of the coagulometer, and tested according to the assessment indicators at different times (0, 5, and 7 days). The operation is strictly in accordance with the product instructions, and the test results are compared with the 0d test results (control) to examine the in-machine stability of the product.

[0204] Table 47 Results of the in-machine stability test of the freeze-dried kit

[0205]

[0206] Table 48 Liquid type test kit in-machine stability test results

[0207]

[0208]

[0209] The results show that after the dry powder reagent is reconstituted or the liquid reagent is placed on the instrument at 2℃-8℃ for 0, 5, and 7 days for quality control, there is no obvious change compared with the 4-degree reagent.

[0210] 2) Stability of applicable samples

[0211] 1. Storage stability at 2℃~8℃

[0212] Two plasma samples from the same batch of fresh samples (in the normal and abnormal ranges, respectively) were taken and stored at 2°C to 8°C. The samples were tested after 0, 18, 36, and 48 hours (h). The mean values ​​of the test results were analyzed for deviations. The results are as follows:

[0213] Table 49 Mean results of stability test of plasma samples

[0214]

[0215] Table 50 Plasma sample stability test results

[0216] deviation% 0h 18h 36h 48h Low value - 0.37％ 2.49％ 3.55％ High value - 2.08％ 4.13％ 4.72％

[0217] The results show that when the plasma samples are stored at 2℃~8℃ for 48 hours, the relative deviation of the test results is ≤15%. Therefore, the samples are stable when stored at 2℃~8℃ for 48 hours.

[0218] 2. Storage stability at 18℃~26℃

[0219] Two plasma samples from the same batch of fresh samples (in the normal and abnormal ranges, respectively) were taken and tested after being placed at 18°C ​​to 26°C for 0, 4, 6, and 8 hours (h). Deviation analysis was performed on the mean of the test results. The results are as follows:

[0220] Table 51 Mean results of plasma sample stability test

[0221]

[0222] Table 52 Plasma sample stability test results

[0223]

[0224]

[0225] The results show that when the plasma samples are stored at 18℃~26℃ for 8h, the relative deviation of the test results is ≤15%. Therefore, the samples are stable when stored at 18℃~26℃ for 8h.

[0226] 3. Storage stability below -20℃

[0227] Two plasma samples from the same batch of fresh samples (in the normal and abnormal ranges, respectively) were taken and placed below -20°C for 1, 2, 3, and 4 weeks (W) before being reconstituted and tested. Deviation analysis was performed on the mean values ​​of the three batches of test results. The results are as follows:

[0228] Table 53 Mean results of plasma sample stability test

[0229]

[0230] Table 54 Plasma sample stability test results

[0231] deviation% Comparison 1W 2W 3W 4W Low value - -0.25％ 1.43％ 9.71％ 10.14％ High value - -1.37％ 1.24％ 9.82％ 10.65％

[0232] The results show that when plasma samples are stored at -20°C for 4 weeks, the relative deviation of the test results is ≤15%. Therefore, the samples can be stored at -20°C for 4 weeks. Obvious changes will begin to occur in the third week, and it is recommended to test within 2 weeks.

[0233] 4. Repeated freeze-thaw stability

[0234] 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 4 times, and the results were analyzed for deviation. The results are as follows:

[0235] Table 55 Mean results of stability test of plasma samples

[0236]

[0237] Table 56 Plasma sample stability test results

[0238]

[0239]

[0240] The results showed that the relative deviation of the plasma sample test results after three freeze-thaw cycles was ≤15%, which was within the industry standard; the relative deviation of the test results after four freeze-thaw cycles was >15%, which exceeded the industry standard. Therefore, the number of freeze-thaw cycles should not exceed 3.

[0241] Therefore, the thrombin time determination kit (coagulation method) of the present application 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 3 times; it is stably stored at 2°C~8°C for 15 days after opening the bottle; the dry powder reagent is stably stored at 2°C-8°C on the instrument for 7 days after reconstitution. Sample collection and processing can be maintained for at least 8 hours at 18~26°C, and can be stably stored for at least 48 hours at 2°C~8°C. It is recommended to be tested within 2 weeks when stored below -20°C, and repeated freezing and thawing should not exceed 3 times. In addition, the shelf life of the kit is 18 months when stored at 2°C~8°C.

[0242] In summary, the measured values ​​of normal plasma samples of the thrombin time detection kit provided by the present application are all no more than 20s, the intra-batch precision (CV): the coefficient of variation (CV) of normal quality control products and abnormal quality control products are all no more than 5%, the inter-batch precision (CV) is no more than 10%, the bottle-to-bottle difference (CV) of the dry powder reagent is no more than 8%, the interfering substances (hemoglobin is no more than 800 mg / dL, the fat emulsion is no more than 400 mg / dL, the bilirubin is no more than 20 mg / dL, the test results of various indicators of analytical performance have met the design requirements, can meet the needs of clinical application, and have good clinical detection performance.

[0243] 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.

[0244] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". 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 the associated objects, indicating that there may 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 "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0245] 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. A thrombin time detection kit, characterized in that: Includes thrombin, mannitol, trisodium citrate, anhydrous sodium sulfate and preservatives.

2. The thrombin time detection kit according to claim 1, characterized in that: The thrombin concentration is 7000-9000 IU / L.

3. The thrombin time detection kit according to claim 1, characterized in that: The mannitol concentration is 10-40 g / L.

4. The thrombin time detection kit according to claim 1, characterized in that: The concentration of trisodium citrate is 2-3 g / L.

5. The thrombin time detection kit according to claim 1, characterized in that: The concentration of anhydrous sodium sulfate is 4-6.5 g / L.

6. The thrombin time detection kit according to claim 1, characterized in that: The concentration of the preservative is 0.5-1.5 ml / L.

7. The thrombin time detection kit according to claim 6, characterized in that: The preservative is one or more of Pc-300, sodium azide, sodium thimerosal, potassium sorbate and sodium benzoate.

8. The thrombin time detection kit according to any one of claims 1 to 7, characterized in that: The thrombin time detection kit is a freeze-dried type or a liquid type.

9. The thrombin time detection kit according to claim 8, characterized in that: The preparation method of the freeze-dried thrombin time detection kit is as follows: various raw materials are dissolved and mixed and then freeze-dried.

10. The thrombin time detection kit according to claim 8, characterized in that: The preparation method of the liquid thrombin time detection kit is as follows: various raw materials are dissolved and mixed.

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