CN108226539B - Activated partial thromboplastin time detection reagent and detection method - Google Patents
Activated partial thromboplastin time detection reagent and detection method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a reagent and a method for detecting activated partial thromboplastin time. The detection reagent consists of an activator, phospholipid, a divalent metal ion salt, a buffering agent, a surfactant, an antioxidant and a stabilizer, can accurately and quickly detect APTT, and has good stability. The stability is still kept for 15 days at 37 ℃, and the detection effect is not influenced. Compared with a control group without adding a surfactant, the detection reagent disclosed by the invention can realize accurate detection on samples with low fibrinogen content, reduces the detection limit, improves the sensitivity, shows good precision on detection of various samples, and has a cv value of less than 3%.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a reagent and a method for detecting activated partial thromboplastin time.
Background
Activated Partial Thromboplastin Time (APTT) is a screening test for examining intrinsic coagulation factors and can be used to confirm the deficiency of congenital or acquired coagulation factors VIII, IX, XI or the presence of their corresponding inhibitors. Also, APTT can be used to confirm the absence of blood coagulation factor XII, prokallikrein, and high molecular weight prokallikrein. Since the high sensitivity of APTT and the action pathway of heparin are mainly intrinsic coagulation pathways, APTT becomes the first indicator for monitoring common heparin.
The Activated Partial Thromboplastin Time (APTT) detection reagent mainly comprises an activator, phospholipid and calcium ions, wherein the activator is mixed with blood plasma and then incubated at 37 ℃ for a period of time, then the calcium ions are added to activate an endogenous coagulation system, and the time for forming fibrin clots is measured.
At present, the activators for APTT determination mainly comprise kaolin, diatomite and the like, and natural mineral materials, but the reagents usually exist in a suspension state, the uniformity of the reagents is difficult to guarantee, and if precipitation occurs, the accuracy of the test is directly influenced. Most of APTT reagents in China at present are in a freeze-dried powder preparation form, the reagents need to be redissolved before use, operation is troublesome, and errors of measurement results are easily caused by inaccurate redissolved liquid volume and the like. Ellagic acid is a common activator, and is widely used in preparation of reagents for measuring activated partial thromboplastin time, compared with activators such as kaolin, diatomite and the like, ellagic acid has good solubility in water and is easily prepared into a liquid reagent, but ellagic acid is a polyphenol compound and is easily oxidized, so that the coagulation time is easily prolonged. And many commercially available reagents are prone to inaccurate reading of clotting time due to too low a reaction intensity when testing samples with very low fibrinogen content. Therefore, how to improve the stability of the APTT detection reagent still remains an urgent problem to be solved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a reagent and a method for detecting activated partial thromboplastin time, wherein the reagent provided by the present invention has good stability, can realize accurate, rapid and sensitive detection of APTT, and has good precision of detection result.
The detection reagent for the activated partial thromboplastin time consists of an activator, phospholipid, a divalent metal ion salt, a buffering agent, a surfactant, an antioxidant and a stabilizer;
the activator is ellagic acid;
the phospholipid is cephalin;
the metal ion in the divalent metal ion salt is Cu2+、Ni2+、Mn2+、Mg2+、Zn2+One or more of the above;
the buffer is any one of trihydroxy aminomethane buffer (Tris-HCl), piperazine-1, 4-diethylsulfonic acid buffer (Pipes), 4-hydroxyethyl piperazine ethanesulfonic acid buffer (Hepes) and 3- ((N-morpholine) propanesulfonic acid buffer (Mops);
the surfactant is one or more of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Triton (TX-100) and Tween series;
the antioxidant is one or more of phenol, 4-hydroxyphenol, Butylated Hydroxyanisole (BHA), Propyl Gallate (PG), chlorogenic acid, and ascorbic acid (VC);
the stabilizer consists of glycine, mannitol, BSA and sodium azide.
In the embodiment of the invention, the concentration of the ellagic acid in the detection reagent is 0.01 g/L-0.05 g/L.
In some embodiments, the concentration of ellagic acid in the detection reagent is 0.03 g/L;
in the embodiment of the invention, the cephalin is rabbit cephalin or bovine cephalin; the concentration of the phospholipid in the detection reagent is 0.05 g/L-0.5 g/L.
In some embodiments, the cephalin is rabbit cephalin; the concentration of phospholipid in the detection reagent is 0.2 g/L.
in the embodiment of the invention, the divalent metal ion salt is chloride, sulfate or nitrate, and the concentration of the divalent metal ion salt in the detection reagent is 0.5 × 10-4mol/L～2.0×10-4mol/L。
in some embodiments, the divalent metal ion salt is copper sulfate and the concentration in the detection reagent is 1.56 × 10- 4mol/L。
In the embodiment of the invention, the buffer in the detection reagent is 4-hydroxyethyl piperazine ethanesulfonic acid buffer.
The concentration of the Hepes buffer was 20 mM.
In the embodiment of the invention, the concentration of the surfactant in the detection reagent is 0.5 g/L-3 g/L.
In the embodiment of the invention, the polymerization degree of the polyethylene glycol is 2000-12000; the average molecular weight of the polyvinylpyrrolidone is 8000-100000.
In some embodiments, the surfactant is polyvinylpyrrolidone (PVP), specifically PVP-K30. The concentration of polyvinylpyrrolidone in the detection reagent is 1 g/L.
The test of the invention shows that the surfactant can improve the reaction strength, so that the sample with low fibrinogen content can be accurately detected.
in the embodiment of the invention, the antioxidant consists of butylated hydroxyanisole and phenol, and the concentration of the antioxidant in the detection reagent is 0.5 × 10-4mol/L～1.5×10-4mol/L。
In some embodiments, the detection reagentthe concentration of butylated hydroxyanisole is 2.5 × 10-4mol/L, the concentration of phenol is 1.0 × 10-4mol/L。
In some embodiments, the concentration of glycine is 1g/L, the concentration of mannitol is 6g/L, the concentration of BSA is 5g/L, and the concentration of sodium azide is 0.5 g/L.
The pH value of the detection reagent provided by the invention is 7.4.
In some embodiments, the detection reagent provided by the invention consists of the following components:
20mmol/LHepes buffer solution and ellagic acid 0.03g/L, rabbit brain cephalin 0.2g/L, copper sulfate 25mg/L, PVP-K301g/L, butyl hydroxy anisol 2.5 × 10-4mol/L, phenol 1.0 × 10-4mol/L, 10g/L glycine, 6g/L mannitol, 5g/LBSA, 0.5g/L sodium azide, and the pH value is 7.4.
The preparation method of the detection reagent comprises the following steps:
dissolving ellagic acid in 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, and sequentially adding phenol, BHA and copper sulfate;
stirring for 10min, and adding cephalin; continuously stirring for 60min, and adding glycine, mannitol, BSA and sodium azide;
and adjusting the pH value to 7.4, fixing the volume, and continuously stirring for 2h to prepare the detection reagent.
The invention also provides a detection kit for the activated partial thromboplastin time, which comprises the detection reagent and 25mmol/L calcium chloride aqueous solution.
The invention also provides a method for detecting the time of activated partial thromboplastin, which adopts the detection reagent and 25mmol/L calcium chloride aqueous solution to detect the serum to be detected by adopting a coagulation method.
Specifically, the detection is carried out by a sysmex CS-1600 full-automatic coagulation analyzer.
The invention provides a detection reagent for activating partial thromboplastin time and a detection method, the detection reagent consists of an activator, phospholipid, a divalent metal ion salt, a buffering agent, a surfactant, an antioxidant and a stabilizer, and the reagent can accurately and quickly detect APTT and has good stability. The stability is still kept for 15 days at 37 ℃, and the detection effect is not influenced. Compared with a control group without adding a surfactant, the detection reagent disclosed by the invention can realize accurate detection on samples with low fibrinogen content, reduces the detection limit, improves the sensitivity, shows good precision on detection of various samples, and has a cv value of less than 3%.
Detailed Description
The invention provides a reagent and a method for detecting the time of activated partial thromboplastin, and a person skilled in the art can use the contents to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials and instruments adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1:
30mg of ellagic acid is weighed and dissolved in 980mL2 mMpH7.4 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, stirred and dissolved, 25mg of copper sulfate is added, stirred for 10min, fully mixed, 200mg of cephalin is added, stirred for 60min, 10g of glycine, 6g of mannitol, 5g of BSA and 0.5g of sodium azide are added. The pH was adjusted to 7.4 and the volume was 1L. Stirring for 2h to obtain the liquid Activated Partial Thromboplastin Time (APTT) measuring reagent.
Example 2:
weighing 30mg ellagic acid, dissolving in 980mL of 2 mL of 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with pH7.4, stirring to dissolve, adding 1.0 × 10-4M phenol and 2.5X10-4MBHA, adding copper sulfate 25mg, stirring for 10min, mixing, adding cephalin 200mg, stirring for 60min, adding glycine 10g, mannitol 6g, and BSA 5gAnd 0.5g of sodium azide. The pH was adjusted to 7.4 and the volume was 1L. Stirring for 2h to obtain the liquid Activated Partial Thromboplastin Time (APTT) measuring reagent.
Example 3:
weighing 30mg ellagic acid, dissolving in 980mL of 2 mL of 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with pH7.4, stirring to dissolve, adding 1.0 × 10-4M phenol and 2.5X10-4MBHA, then adding 25mg copper sulfate, stirring for 10min, mixing well, adding 200mg cephalin, stirring for 60min, adding 1.0g polyvinylpyrrolidone (PVP-K30), 10g glycine, 6g mannitol, 5g BSA and 0.5g sodium azide. The pH was adjusted to 7.4 and the volume was 1L. Stirring for 2h to obtain the liquid Activated Partial Thromboplastin Time (APTT) measuring reagent.
Comparative example 1
Weighing 30mg ellagic acid, dissolving in 980mL of 2 mL of 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with pH7.4, stirring to dissolve, adding 1.0 × 10-4M phenol and 2.5X10-4MBHA, then adding 25mg copper sulfate, stirring for 10min, mixing well, adding 200mg cephalin, stirring for 60min, adding 4.0g polyvinylpyrrolidone (PVP-K30), 10g glycine, 6g mannitol, 5g BSA and 0.5g sodium azide. The pH was adjusted to 7.4 and the volume was 1L. Stirring for 2h to obtain the liquid Activated Partial Thromboplastin Time (APTT) measuring reagent.
Identification of detection Effect
1. Reagent stability
The reagents of examples 1 to 3 and comparative example 1 were combined with 25mM calcium chloride, and quality Control plasma (normal quality Control plasma Control N: 24.2-32.8s, abnormal quality Control plasma Control P: 51.7-85.7s) purchased from siemens was tested using a sysmex CS-1600 full-automatic coagulometer. Specifically, the APTT reagent prepared in example 3 was stored at 37 ℃ and sampled every 1 day, and the stability of the reagent was examined for 15 days, and the results are shown in Table 1.
TABLE 1 partial thromboplastin activation time(s)
From the table, it can be seen that after a certain amount of antioxidant is added into the reagent, the stability of the reagent is obviously improved, the reagent in example 1 is obviously changed after being placed at 37 ℃ for 5 days, and the result is still stable after the reagents in examples 2-3 are measured for 15 days.
2. Sensitivity of the probe
APTT assay reagents prepared in examples 2 and 3, in combination with 25mM calcium chloride, were tested on quality Control plasma (normal quality Control plasma Control N: 24.2-32.8s, abnormal quality Control plasma Control P: 51.7-85.7s) purchased from siemens using a sysmex CS-1600 full-automatic coagulometer. Specifically, the APTT reagents prepared in example 2 and example 3 were tested on two quality control plasma samples, each sample was tested 6 times, and the change of the reaction intensity (dH) was examined, and the results are shown in Table 2.
TABLE 2 reaction Strength (dH)
As can be seen from the above table, the reaction intensity after adding a certain amount of polyvinylpyrrolidone into the APTT reagent is about twice higher than that without adding the APTT reagent, which undoubtedly greatly improves the test sensitivity for the optical test. For further validation, we performed APTT tests on sysmex CS-1600 with samples with very low fibrinogen content, and controls with commercially available APTT reagents (from siemens) associated with the instrument, with the results shown in Table 3:
TABLE 3 results of sample detection with very low fibrinogen content
As can be seen from the above table, when a sample with extremely low fibrinogen content is tested, the reagent with PVP (example 3) can successfully measure the activated partial thromboplastin time, and has good correlation with the Siemens reagent, while the reagent without PVP (example 2) can not obtain the coagulation time due to too low reaction strength (i.e. test error), and even if slight agglutination is detected to obtain the coagulation time, the risk of inaccurate value is high. Increasing the content of PVP can increase the reaction strength, but when the concentration exceeds 3g/L, the reaction strength is increased less and less obviously, and after adding excessive surfactant, the coagulation time of a sample is obviously shortened, so that the test result is inaccurate.
3. Precision degree
Two samples (clinical sample 1 and clinical sample 2) were extracted from the samples with extremely low fibrinogen content, and the quality Control plasma (normal quality Control plasma Control N: 24.2-32.8s, abnormal quality Control plasma Control P: 51.7-85.7s) purchased from siemens was subjected to precision measurement using the reagents of example 3, 10 times for each sample, and the measurement results are shown in Table 4.
TABLE 4 partial thromboplastin activation time(s)
| Number of detections | Clinical sample 1 | Clinical sample 2 | Control N | Control P |
| 1 | 55.1 | 93.5 | 27.5 | 75.6 |
| 2 | 54.3 | 90.8 | 27.8 | 74.8 |
| 3 | 54.8 | 91.2 | 28.2 | 77.1 |
| 4 | 55.6 | 89.9 | 29.0 | 75.5 |
| 5 | 56.0 | 94.3 | 28.4 | 74.1 |
| 6 | 56.1 | 95.5 | 28.3 | 73.8 |
| 7 | 55.9 | 96.3 | 27.6 | 76.2 |
| 8 | 57.1 | 94.7 | 28.1 | 75.5 |
| 9 | 55.2 | 95.0 | 28.2 | 74.9 |
| 10 | 54.9 | 92.1 | 27.7 | 75.0 |
| Mean value of | 55.5 | 93.3 | 28.1 | 75.3 |
| Standard deviation of | 0.808 | 2.195 | 0.449 | 0.965 |
| CV | 1.5％ | 2.4％ | 1.6％ | 1.3％ |
As can be seen from the above table, no matter the reagent in example 3 is a sample with normal test reaction strength or a sample with low test reaction strength, the CV value is less than 3%, and the precision is better, and the CV values detected by using the reagents in examples 1-2 and comparative example 1 as samples with Control N are 2.2%, 1.5%, and 1.2% in sequence.
4. Accuracy of
50 clinical specimens were tested using the reagent of example 3 using a sysmex CS-1600 fully automated coagulation analyzer, and the results are shown in Table 5, using a commercially available APTT reagent in combination with the automatic coagulation analyzer.
TABLE 5 clinical specimens activated partial thromboplastin time(s)
As can be seen from the data in the table, the test result of the reagent in example 3 has good correlation with the test result of the existing reliable commercial reagent (siemens), and the deviation between the test result and the test result is small.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (4)
1. A reagent for detecting the time to activate a partial thromboplastin,
the composition consists of the following components:
20mmol/L Hepes buffer solution and ellagic acid 0.03g/L, rabbit brain cephalin 0.2g/L, copper sulfate 25mg/L, PVP-K301g/L, butyl hydroxy anisol 2.5 × 10-4mol/L, phenol 1.0 × 10-4mol/L, 10g/L glycine, 6g/L mannitol, 5g/L BSA, 0.5g/L sodium azide, and the pH value is 7.4.
2. The method for preparing a detection reagent according to claim 1, comprising:
dissolving ellagic acid in Hepes buffer solution, and then sequentially adding phenol, butyl hydroxy anisol and copper sulfate;
stirring for 10min, and adding rabbit brain cephalin; continuously stirring for 60min, and adding PVP-K30, glycine, mannitol, BSA and sodium azide;
and adjusting the pH value to 7.4, fixing the volume, and continuously stirring for 2h to prepare the detection reagent.
3. A kit for detecting activated partial thromboplastin time, comprising the detection reagent according to claim 1 and 25mmol/L aqueous calcium chloride solution.
4. A method for detecting the time required for activating a partial thromboplastin, characterized in that the detection reagent of claim 1 and a 25mmol/L aqueous solution of calcium chloride are used to detect the serum to be detected by a coagulation method.
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| CN109444438B (en) * | 2018-10-25 | 2022-03-15 | 宁波瑞源生物科技有限公司 | Stabilizer for APTT detection reagent and APTT detection reagent |
| CN109613279A (en) * | 2018-12-06 | 2019-04-12 | 太原博奥特生物技术有限公司 | A kind of novel ellagic acid compounds and activated partial thromboplastin time assay reagent is prepared using ellagic acid compounds |
| JP7444572B2 (en) * | 2019-09-24 | 2024-03-06 | シスメックス株式会社 | Reagent and reagent kit for measuring activated partial thromboplastin time, and method for suppressing precipitation of ellagic acid compound |
| CN110824154A (en) * | 2019-10-15 | 2020-02-21 | 常熟常江生物技术有限公司 | Activator for thromboelastography |
| CN113025684A (en) * | 2019-12-25 | 2021-06-25 | 深圳市帝迈生物技术有限公司 | Activated partial thromboplastin time detection reagent and kit |
| CN111638375B (en) * | 2020-06-08 | 2022-12-13 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for measuring activated partial thromboplastin time |
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| CN113848332B (en) * | 2021-09-17 | 2024-04-19 | 广州徕西姆医学诊断技术有限公司 | Thrombus elastography detection reagent and preparation method and application thereof |
| CN114113641B (en) * | 2021-10-28 | 2023-11-03 | 中科精瓒(武汉)医疗技术有限公司 | Activated blood coagulation detection reagent and preparation method thereof |
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