Fibrinogen determination kit

Abstract

The invention relates to a fibrinogen determination kit, and relates to the technical field of medical examination. The fibrinogen determination kit comprises a thrombin reagent, a quality control product and a calibration product, the thrombin reagent comprises thrombin, trisodium citrate, glycine, anhydrous sodium sulfate, a preservative, mannitol and a buffer solution; the quality control material comprises sodium citrate anticoagulant human plasma, and the calibration material comprises normal human plasma, mannitol, cane sugar and a preservative. The test results of various indexes of the analysis performance meet the design requirements, the clinical application requirements can be met, and the good clinical detection performance is achieved.

Description

Technical Field

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

[0002] Coagulation is an important part of the body's hemostasis function, preventing excessive blood loss due to injury. Coagulation function testing is a necessary examination item before clinical surgery. It can understand the patient's coagulation function abnormality in advance and effectively avoid unexpected situations due to bleeding before and after surgery. Currently, there are four common coagulation tests, namely fibrinogen (FIB), thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT).

[0003] Fibrinogen is a glycoprotein with coagulation function synthesized by the liver and is a precursor of fibrin. Fibrinogen, also known as coagulation factor I, is an important coagulation factor in the coagulation process in the body. Under the action of thrombin, FIB is converted from a soluble protein to an insoluble protein polymer to form a fibrin clot, and the blood will coagulate. The level of FIB affects various thrombotic diseases. High FIB content is prone to diseases such as hypertension, diabetes, and atherosclerosis, while low FIB content is prone to fibrinolysis, DIC, cirrhosis and other diseases. Fibrinogen is an important risk factor for various thrombotic diseases. Therefore, the detection of fibrinogen has very important clinical diagnostic significance.

[0004] When testing fibrinogen, problems such as reduced analytical sensitivity may occur due to the influence of antibody titer and various factors in the sample. Currently, the detection kits on the market are affected by low antibody titer and the ratio of the reagent system, which can easily lead to low reagent analysis sensitivity and false negatives. Summary of the invention

[0005] The present application provides a fibrinogen assay kit to solve the current problems.

[0006] The present application provides a fibrinogen assay kit, comprising a thrombin reagent, a quality control product and a calibrator; the thrombin reagent comprises thrombin, trisodium citrate, glycine, anhydrous sodium sulfate, a preservative, mannitol and a buffer; the quality control product comprises sodium citrate anticoagulated human plasma, mannitol and glycine, and the calibrator comprises normal human plasma, mannitol, sucrose 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] Reaction principle: Based on the principle that fibrinogen (FIB) reacts with thrombin to eventually form fibrin, a standard curve is prepared using standard plasma as the calibration sample, and thrombin is used to measure the plasma coagulation time. The obtained coagulation time is negatively correlated with the fibrinogen concentration in the plasma, thereby obtaining the fibrinogen content.

[0009] Trisodium citrate and anhydrous sodium sulfate can adjust the pH value, enhance stability, adjust the repeatability of the measurement, and adjust the absorbance of the measurement; mannitol and preservatives can increase the stability of thrombin and extend the shelf life of the kit; the combination of various raw materials can improve the analytical sensitivity of the reagent. The fibrinogen assay kit of the present application has a detection limit (0.45 g / L), a linear range ([0.50, 8.0] g / L), a reportable range (0.45 g / L to 16 g / L), accuracy (relative deviation <10%), precision (intra-batch precision (CV): coefficient of variation (CV) of low-value quality control products <8%, high-value quality control products (CV) <8%; inter-batch precision (CV) is not higher than 15%), bottle-to-bottle difference <8%, interfering substances (hemoglobin is not higher than 500 mg / dL, fat emulsion is not higher than 400 mg / dL, bilirubin is not higher than 20 mg / dL, heparin sodium is not higher than 1.2 U / dL), and the test results of various indicators of analytical performance have all met the design requirements 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 marketed, and the test results are equivalent, and the kit has good clinical detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a schematic diagram of the correlation between the 210124 batches of freeze-dried test kits and clinical sample assignments in Experimental Example 7 of this application;

[0011] Figure 2 This is a schematic diagram of the correlation between the 210124 batches of liquid-type test kits and clinical sample assignments in Experimental Example 7 of this application;

[0012] Figure 3 This is a schematic diagram of the correlation between the 210325 batches of freeze-dried test kits and clinical sample assignments in Experimental Example 7 of this application;

[0013] Figure 4 This is a schematic diagram of the correlation between the 210325 batches of liquid-type test kits and clinical sample assignments in Experimental Example 7 of this application;

[0014] Figure 5 This is a schematic diagram of the correlation between the 210523 batches of freeze-dried test kit and clinical sample assignment in Experimental Example 7 of this application;

[0015] Figure 6 This is a schematic diagram of the correlation between the 210523 batches of liquid-type test kits and clinical sample assignments in Experimental Example 7 of this application. DETAILED DESCRIPTION

[0016] 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.

[0017] 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.

[0018] The present application provides a fibrinogen assay kit, comprising a thrombin reagent, a quality control product and a calibrator; the thrombin reagent comprises thrombin, trisodium citrate, glycine, anhydrous sodium sulfate, a preservative, mannitol and a buffer; the quality control product comprises sodium citrate anticoagulated human plasma, and the calibrator comprises normal human plasma, mannitol, sucrose and a preservative.

[0019] Preferably, the thrombin concentration is 40000-60000 IU / L, and the buffer concentration is 40-60 mM.

[0020] Preferably, the concentration of trisodium citrate is 2-8 g / L, the concentration of glycine is 80-120 g / L, the concentration of anhydrous sodium sulfate is 2-8 g / L, the concentration of preservative is 4-9 ml / L, and the concentration of mannitol is 20-30 g / L.

[0021] Preferably, the buffer is an imidazole buffer.

[0022] Preferably, the concentration of sodium citrate anticoagulant in human plasma is ≥ 90%.

[0023] Preferably, the calibrator comprises 900-1100 mL of normal human plasma, 15-25 g of mannitol, 25-35 g of sucrose and 2-8 mL of a preservative.

[0024] Preferably, the preservative is one or more of Pc-300, sodium azide, sodium thimerosal, potassium sorbate and sodium benzoate.

[0025] Preferably, the thrombin reagent is in a lyophilized or liquid form.

[0026] Preferably, the preparation method of the freeze-dried thrombin reagent is: mixing and dissolving the various raw materials and then freeze-drying them.

[0027] Preferably, the preparation method of the liquid thrombin reagent is: mixing and dissolving the various raw materials.

[0028] 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.

[0029] Example 1

[0030] This embodiment provides a fibrinogen assay kit, which uses the following raw materials:

[0031] Thrombin reagent: trisodium citrate 5g, glycine 100g, anhydrous sodium sulfate 5g, Pc-300 5mL, mannitol 25g, the concentration of the buffer is 50mM, and the concentration of thrombin is 50000IU / L;

[0032] Calibrators: 1000 mL normal human plasma, 20 g mannitol, 30 g sucrose and 5 mL Pc-300;

[0033] The preparation method of the above-mentioned freeze-dried thrombin reagent comprises the following steps:

[0034] Prepare 1 L of 50 mM imidazole buffer, add the following into 700 mL of 50 mM imidazole buffer respectively: trisodium citrate, glycine, anhydrous sodium sulfate, Pc-300, mannitol, 50000 IU of thrombin, after dissolution, make up to 1 L with imidazole buffer, and freeze-dry to obtain the product.

[0035] Example 2

[0036] This embodiment provides a fibrinogen assay kit, which uses the following raw materials:

[0037] Thrombin reagent: 2 g trisodium citrate, 80 g glycine, 2 g anhydrous sodium sulfate, 4 mL Pc-300, 20 g mannitol, the concentration of the buffer is 40 mM, and the concentration of thrombin is 40000 IU / L;

[0038] Calibrators: 900 mL normal human plasma, 15 g mannitol, 25 g sucrose and 2 mL Pc-300;

[0039] The preparation method of this embodiment is the same as that of embodiment 1.

[0040] Example 3

[0041] This embodiment provides a fibrinogen assay kit, which uses the following raw materials:

[0042] Thrombin reagent: trisodium citrate 8g, glycine 120g, anhydrous sodium sulfate g, Pc-300 9mL, mannitol 30g, the concentration of the buffer is 60mM, and the concentration of thrombin is 60000IU / L;

[0043] Calibrators: 1100 mL normal human plasma, 25 g mannitol, 35 g sucrose and 8 mL Pc-300;

[0044] The preparation method of the above thrombin reagent comprises the following steps:

[0045] The preparation method of this embodiment is the same as that of embodiment 1.

[0046] It should be noted that Test Examples 1-8 were all tested using the fibrinogen assay kit produced in Example 1.

[0047] Test Example 1

[0048] The performance of the kit was evaluated, and the reagent specifications and batch numbers are as follows:

[0049] Table 1 Reagent specifications and batch numbers

[0050] Dosage form R1 Thrombin Reagent R2 Imidazole Buffer R3 reconstitution solution Batch number (three batches) liquid 5×2mL 2×50mL / 210124、210325、210523 liquid 5×4mL 1×100mL / 210126、210327、210525 liquid 5×5mL 1×100mL / 210128、210329、210527 liquid 10×8mL 2×200mL / 210130、210401、210529 Dry powder 5×2mL 2×50mL 1×50mL 210124、210325、210523 Dry powder 5×4mL 1×100mL 2×50mL 210126、210327、210525 Dry powder 5×5mL 1×100mL 2×50mL 210128、210329、210527 Dry powder 10×8mL 2×200mL 2×50mL 210130、210401、210529

[0051] Appearance test method: Visual inspection with corrected vision under natural light. All three batches of kits should meet the experimental requirements. The results are as follows:

[0052] Table 2 Appearance record of three batches of freeze-dried test kits

[0053]

[0054] Table 3 Appearance record of three batches of liquid test kits

[0055]

[0056]

[0057] The results showed that the appearance of both liquid reagents and dry powder reagents met the specified requirements.

[0058] Test Example 2

[0059] 1) Linear range

[0060] Dilute the high-concentration sample close to the upper limit of the FIB test range with buffer into at least 5 samples of different concentrations within the linear range (high value close to the upper limit of the linear range, low value close to the lower limit of the linear range), and measure each concentration of sample 3 times. Record the measurement results of each sample, and calculate the average of the 3 measurement values ​​of each sample. Calculate the linear correlation coefficient r, which should not be less than 0.9900. The results should meet the technical requirements.

[0061] Table 4 Record sheet of reagent measurement

[0062]

[0063]

[0064] The results showed that within the range of [0.50, 8.00] g / L, the linear correlation coefficient r between the average value of the result and the theoretical concentration value was greater than 0.9900, and different specifications of dry powder and liquid reagents met the linear requirements and were verified to be qualified.

[0065] The deviation of the measurement results exceeding ±10% is considered an outlier. The deviation of the test results of three batches of reagents with different specifications and dosage forms within the range of [0.50, 8.00] g / L does not exceed ±10%, so there is no outlier.

[0066] The regression coefficients r of three batches of freeze-dried and liquid reagents of different specifications were calculated in the range of [0.50, 8.00] g / L, and they were all greater than 0.9900, meeting the design requirements.

[0067] The results of polynomial regression analysis of the results of three batches of freeze-dried and liquid reagents showed that within the range of [0.50, 8.00] g / L, the coefficients of the cubic equation, quadratic equation b2 and cubic equation b3 had no significant effect on linearity, and the coefficient values ​​were all ≤10-2, so the data set was linear.

[0068] The test results show that the linearity of the kit produced in three consecutive batches meets the design requirements. The correlation coefficient r of FIB in the range of [0.50, 8.00] g / L is not less than 0.9900 and there is no outlier.

[0069] Based on the above evaluation results and relevant information of marketed products, it was determined that the linear range of the fibrinogen (FIB) detection kit (coagulation method) produced by Chuangning Biotechnology (Beijing) Co., Ltd. was [0.50, 8.00] g / L.

[0070] 2) Limit of quantification

[0071] Selection of the upper limit of the reportable range: The maximum dilution factor when the reduction concentration deviates from the theoretical concentration (≤10.0%) is selected as the maximum dilution factor recommended by the method, and the product of the upper limit of the linear range and the maximum dilution factor is the upper limit of the reportable range of the method.

[0072] Based on the test results of the high limit samples, the reportable range results are shown in the following table:

[0073] Table 5: Reportable range of results for lyophilized and liquid kits

[0074]

[0075] Summary of the maximum dilution ratio and reportable range experimental results:

[0076] The fibrinogen (FIB) test kit (coagulation method) can report a lower limit of FIB of 0.45 g / L;

[0077] The dilution factor of FIB in the fibrinogen (FIB) test kit (coagulation method) that meets the detection accuracy is 2 times dilution, so the reportable upper limit of FIB is 16.0g / L.

[0078] Test Example 3

[0079] 1) Intra-batch and inter-batch precision

[0080] The intra-batch and inter-batch precision of three consecutive batches of fibrinogen (FIB) detection kits in different dosage forms and specifications were evaluated using FIB normal value quality control products.

[0081] Normal value quality control batch number: 210202; abnormal value quality control batch number: 210202.

[0082] According to the CLSI EP05-A3 method for precision evaluation, the same operator or a group of operators were used on the same instrument, using lyophilized and liquid reagents of different specifications in 3 batches, and the FIB normal value quality control samples and abnormal value quality control samples were tested 10 times. At the end of the evaluation, there were 10 pairs of low value quality control samples and high value quality control samples in each batch, that is, 10 test results for low value quality control products and high value quality control products.

[0083] The obtained results should ensure that the valid data is within the range of M±2S, and the intra-batch precision is calculated from the measurement results of the normal value and abnormal value quality control products. The inter-batch precision is calculated from the data of all normal value quality control products in each batch.

[0084] Table 6 Intra-batch and inter-batch precision records

[0085]

[0086] The results show that all data are within the range of M±2S, the data are valid, the intra-batch precision CV values ​​of each batch, dosage form and specification are no more than 8%, the inter-batch precision is no more than 15%, and the performance analysis data meet the requirements.

[0087] 2) Inter-operator precision and inter-day precision

[0088] Table 7 Precision between different operators and daily precision records

[0089]

[0090]

[0091]

[0092] The results showed that the inter-day precision of three batches of different specifications of lyophilized and liquid fibrinogen (FIB) detection kits and the inter-day precision verification test results of different operators showed that the CV of normal value quality control plasma and abnormal value quality control plasma were no more than 8%, which met the requirements of the research and development design.

[0093] 3) Bottle-to-bottle variation of freeze-dried reagents

[0094] Use normal quality control plasma and abnormal quality control plasma to test 10 bottles of the same batch of reagents once each, and calculate the average of the 10 measured values and standard deviation (s 1 ). Repeat the test 10 times with normal quality control plasma on one bottle of the reagent to be tested, and calculate the mean of the results. and standard deviation (s 2 ).

[0095] Table 8 Bottle Difference Record Table of Freeze-dried Reagents

[0096]

[0097]

[0098] The results showed that the CV of the two-level quality control results of the three batches of kits were all <8%, which met the requirements of the research and development design.

[0099] Test Example 4

[0100] Three batches of reagents were used to determine the national standard substance of fibrinogen according to the conventional test method. Each sample was measured three times and the average value X was taken. The relative deviation between the average value and the national standard substance was calculated and discussed.

[0101] Table 9 Accuracy record of the first batch of freeze-dried reagents

[0102]

[0103] Table 10 Accuracy record of the first batch of liquid reagents

[0104]

[0105] Table 11 Accuracy record of the second batch of freeze-dried reagents

[0106]

[0107]

[0108] Table 12 Accuracy record of the second batch of liquid reagents

[0109]

[0110] Table 13 Accuracy record of the third batch of freeze-dried reagents

[0111]

[0112] Table 14 Accuracy record of the third batch of liquid reagents

[0113]

[0114] Result analysis: It can be seen from the data that the test result deviations of the three national standard substances with different concentrations are all less than 10%, which is in line with the experimental expectations.

[0115] Test Example 5

[0116] 1. Analysis of interfering substances

[0117] 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.

[0118] 1) Hemoglobin

[0119] Dissolve hemoglobin in purified water to obtain a high hemoglobin solution (500 mg / dL). Use purified water as a blank control solution. Use the high hemoglobin solution and blank samples to mix with fresh mixed plasma to obtain 4 gradients (62.5, 125, 250, 500 mg / dL) of hemoglobin mixed plasma and blank control plasma. Use reagents to detect the fibrinogen concentration in the sample, and the test results are as follows:

[0120] Table 15 Hemoglobin interference test record table for reagents

[0121]

[0122]

[0123]

[0124] Result analysis: Hemoglobin content has a linear positive interference on FIB detection. When the relative deviation of the test result exceeds ±10%, the hemoglobin concentration will affect the performance of the test reagent. When the hemoglobin reaches 500mg / dL, the test result has changed significantly, and the relative deviation is less than 10%. To ensure the correctness of the test result, it is confirmed that the hemoglobin does not exceed 500mg / dL and has no interference with the test result.

[0125] 2) Fat emulsion

[0126] The fat emulsion solution (4.0 g / dL) was mixed with fresh mixed plasma to obtain 4 gradient (50, 100, 200, 400, 800 mg / dL) fat emulsion plasma samples, and purified water was used as the blank control solution to obtain blank control samples. The fibrinogen concentration in the sample was detected using the reagent. The test results are as follows:

[0127] Table 16 Record of fat emulsion interference with reagents

[0128]

[0129]

[0130] Results analysis: The fat emulsion content has a linear positive interference on FIB detection. When the fat emulsion concentration is 800 mg / dL, the relative deviation of the test results exceeds 5%, reaching more than 8%, which significantly affects the test results; when the fat emulsion is not higher than 400 mg / dL, the relative deviation of the test results is less than 5%, which has no significant effect on the test results.

[0131] 3) Bilirubin

[0132] Dissolve bilirubin in purified water to obtain a bilirubin solution (70 mg / dL). Use purified water as a blank control solution. Mix the bilirubin solution with fresh plasma to prepare samples of different concentrations. The bilirubin contents are 2.5, 5, 10, 20, and 40 mg / dL, respectively. The test results are as follows:

[0133] Table 17 Bilirubin interference detection record table for reagents

[0134]

[0135]

[0136] Result analysis: Bilirubin content has a linear negative interference on FIB detection. When the bilirubin content is 40mg / dL, the relative deviation of the test result is close to or exceeds ±10%, which significantly affects the performance of the detection reagent. When the bilirubin content is 20mg / dL, the relative deviation of the test result is less than 5%, which has no interference on the test result.

[0137] 4) Heparin Sodium

[0138] The heparin sodium and fresh mixed plasma samples containing 12U / dL were diluted to obtain 4 gradient (0.1, 0.3, 0.6, 1.2, 2.4U / dL) heparin sodium plasma samples and a blank control sample mixed with purified water and fresh plasma. The fibrinogen concentration in the sample was detected using the reagent, and the results were as follows:

[0139] Table 18 Heparin sodium interference test record table for reagents

[0140]

[0141]

[0142]

[0143] Result analysis: Heparin sodium content has a linear negative interference on FIB detection. When the heparin sodium content is 2.4U / ml, the relative deviation of the test result is close to or exceeds ±10%, which significantly affects the performance of the detection reagent, while there is no interference with the test result when the heparin sodium content is 1.2U / ml.

[0144] Test Example 6

[0145] Calibrator analysis performance:

[0146] 1. Appearance

[0147] Table 19 Calibrator Appearance Observation Record

[0148]

[0149] The results showed that the appearance of the dry powder reagent and the reconstituted reagent met the specified requirements.

[0150] 2. Loading quantity

[0151] Table 20 Calibrator filling quantity record

[0152]

[0153] The results showed that the reagent loadings after reconstitution exceeded the labeled amounts.

[0154] 3. Accuracy

[0155] Table 21 Calibrator Accuracy Record

[0156]

[0157] The results showed that the relative deviation between the measured value and the labeled value of the calibration material met the requirements.

[0158] 4. Uniformity within and between bottles

[0159] Table 22 Record of uniformity within and between calibrants

[0160]

[0161]

[0162] The results showed that the uniformity within and between calibrants bottles met the specified requirements.

[0163] Test Example 7

[0164] Comparative test with similar kits on the market:

[0165] Three batches of freeze-dried and liquid kits of different specifications were used to test clinical samples assigned by the fibrinogen content assay kit (batch number: 621041) produced by Wuhan Zhongtai Biotechnology Co., Ltd., 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:

[0166] Table 23 Comparison of the detection performance of freeze-dried and liquid test kits with similar test kits on the market

[0167]

[0168]

[0169] According to Table 23, Appendix Figure 1-6 The analysis shows that after calculation, the three batches of freeze-dried and liquid reagents of this specification were compared with the clinical samples assigned by the fibrinogen content determination kit (batch number: 621041) produced by Wuhan Zhongtai Biotechnology Co., Ltd. in the test of 40 clinical samples, and the results were statistically analyzed. The correlation coefficient k∈[0.9915, 1.0098], r≥0.975, the test results were highly positively correlated, and the kit had good clinical detection performance.

[0170] Test Example 8

[0171] Evaluate the stability of continuously produced test kits and provide a basis for determining the actual shelf life of the test kits.

[0172] Test object: Continuously produced fibrinogen test kit, specifications are as follows:

[0173] Table 24 Kit specifications

[0174]

[0175] Main instruments: Fully automatic blood coagulation analyzer, Sysmex Corporation, model: CS-5100.

[0176] 1) Reagent stability

[0177] 1. Accelerate the destruction of stability

[0178] Reagents of near-effectiveness and medium-effectiveness were stored at 37±1℃ respectively, and tested every three or four days (0d, 4d, 7d and 10d) according to the assessment indicators. The operation was strictly in accordance with the product instructions, and the results were compared with the 0d test results (control) to examine the accelerated destruction stability of the product.

[0179] Table 25 Results of accelerated destruction stability test of freeze-dried kit

[0180]

[0181]

[0182] Table 26 Liquid type test kit accelerated destruction stability test results

[0183]

[0184] The results showed that when the test kits with near-term and medium-term shelf life were stored at 37℃±1℃ for 10 days, all evaluation indicators met the technical requirements, so they were stable when stored at 37℃ for 10 days.

[0185] 2. Stability during shelf life

[0186] 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 (6, 12, 18, 21, 24, and 25 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.

[0187] Table 27 Results of the stability test of the freeze-dried kit

[0188]

[0189] Table 28 Liquid type test kit shelf life stability test results

[0190]

[0191]

[0192] The results show that after the kit is stored at 2℃~8℃ for 24 months, all the evaluation indicators meet the technical requirements, but the indicators change from the 25th month, so the reagent is stable for 24 months. The results of the shelf life stability test of the medium-term kit meet the experimental requirements.

[0193] 3. Transportation stability

[0194] 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.

[0195] Table 29 Results of winter transportation stability test of freeze-dried test kit

[0196]

[0197] Table 30 Winter transportation stability test results of liquid test kit

[0198]

[0199] Table 31 Results of summer transportation stability test of freeze-dried test kit

[0200]

[0201]

[0202] Table 32 Liquid type test kit summer transportation stability test results

[0203]

[0204] The results showed that during the transportation of the test kit, all assessment indicators met the technical requirements.

[0205] 4. Stability of reconstitution of calibrants

[0206] Three batches of test kits were taken for calibration, and the FIB calibrators (2 bottles) were tested three times using the calibrated curve. The average of the test results was taken as the initial value (0h), and the test was repeated three times at 4h, 6h and 8h, and the test was continuously tested for 8h. The operation was strictly in accordance with the product instructions, and the results were compared with the 0h test results (control) to investigate the reconstitution stability of the three batches of product calibrators.

[0207] Table 33 Results of stability test of freeze-dried reagent calibrators at 2℃~8℃ after reconstitution

[0208]

[0209]

[0210] The results show that the calibrator is qualified within 8 hours after opening the bottle. According to the actual usage of the user, the stable time after opening the bottle and re-dissolving is selected as 8 hours.

[0211] 5. Reagent stability after opening the bottle

[0212] Three batches of test kits with near-expiry and mid-expiry periods were opened and reconstituted, and normal and abnormal quality control products were tested three times each. The average of the test results was taken as the initial value (Day 0). Tests were conducted once every 5 days before the test, and once a day on the 14th and 15th days for 15 consecutive days. The operations were strictly in accordance with the product instructions, and the results were compared with the 0-day test results (control) to examine the stability of the three batches of products after opening and reconstitution and storage at 2°C to 8°C.

[0213] After opening the bottles of three batches of reagents and re-dissolving them, the normal quality control products and abnormal quality control products were tested three times each, and the results were analyzed for deviations. The results are as follows:

[0214] Table 34 Results of stability test on opening and reconstitution of three batches of freeze-dried reagents

[0215]

[0216]

[0217] Table 35 Results of stability test on three batches of liquid reagents after opening and re-dissolving

[0218]

[0219]

[0220] The results show that: after the three batches of dry powder and liquid reagents were opened for testing 15 days, the relative deviations of the normal / abnormal quality control results and the 0-day test results were ≤10%, 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.

[0221] Therefore, the fibrinogen (FIB) detection kit of the present application can be stably stored at 37°C ± 1°C for 10 days, including transportation stability. The freeze-dried and liquid test kits are stable for 24 months under storage conditions of 2°C to 8°C. The calibrator is stably stored at 2°C to 8°C for 8 hours after opening the bottle and reconstituting. The freeze-dried and liquid test kits are stably stored at 2°C to 8°C for 15 days after opening the bottle and reconstituting.

[0222] In summary, the fibrinogen assay kit provided by the present application has a detection limit (0.45 g / L), a linear range ([0.50, 8.0] g / L), a reportable range (0.45 g / L to 16 g / L), accuracy (relative deviation <10%), precision (intra-batch precision (CV): coefficient of variation (CV) of low-value quality control products <8%, high-value quality control products (CV) <8%; inter-batch precision (CV) is not higher than 15%), bottle difference <8%, interfering substances (hemoglobin is not higher than 500 mg / dL, fat emulsion is not higher than 400 mg / dL, bilirubin is not higher than 20 mg / dL, heparin sodium is not higher than 1.2 U / dL), and the test results of various indicators of analytical performance have met the design requirements and can meet the needs of clinical application. The test results of the kit have good consistency with the test results of similar kits that have been on the market, the test results are equivalent, and the kit has good clinical detection performance.

[0223] 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.

[0224] 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.

[0225] 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 fibrinogen assay kit, characterized in that: It includes thrombin reagent, quality control product and calibration product; the thrombin reagent includes thrombin, trisodium citrate, glycine, anhydrous sodium sulfate, preservative, mannitol and buffer; the quality control product includes sodium citrate anticoagulated human plasma, and the calibration product includes normal human plasma, mannitol, sucrose and preservative.

2. The fibrinogen assay kit according to claim 1, characterized in that The thrombin concentration is 40000-60000 IU / L, and the buffer concentration is 40-60 mM.

3. The fibrinogen assay kit according to claim 1, characterized in that The concentration of trisodium citrate is 2-8 g / L, the concentration of glycine is 80-120 g / L, the concentration of anhydrous sodium sulfate is 2-8 g / L, the concentration of preservative is 4-9 ml / L, and the concentration of mannitol is 20-30 g / L.

4. The fibrinogen assay kit according to any one of claims 1 to 3, characterized in that The buffer is imidazole buffer.

5. The fibrinogen assay kit according to claim 1, characterized in that The sodium citrate anticoagulant human plasma concentration is ≥90%.

6. The fibrinogen assay kit according to claim 1, characterized in that The calibrator comprises 900-1100 mL of normal human plasma, 15-25 g of mannitol, 25-35 g of sucrose and 2-8 mL of a preservative.

7. The fibrinogen assay kit according to claim 1, characterized in that: The preservative is one or more of Pc-300, sodium azide, sodium thimerosal, potassium sorbate and sodium benzoate.

8. The fibrinogen assay kit according to any one of claims 1 to 7, characterized in that: The thrombin reagent is in a lyophilized or liquid form.

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

10. The fibrinogen assay kit according to claim 8, characterized in that: The preparation method of the liquid thrombin reagent is: mixing and dissolving various raw materials.

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