CN115711997A - Kit for detecting lupus anticoagulant based on SCT method - Google Patents
Kit for detecting lupus anticoagulant based on SCT method Download PDFInfo
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Abstract
The application discloses a kit for detecting lupus anticoagulant based on an SCT method, which comprises a screening reagent, a confirmation reagent and a calcium chloride aqueous solution, wherein the screening reagent or the confirmation reagent consists of silicon dioxide, phospholipid, a buffering agent, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent, a preservative and water; the phospholipid content in the screening reagent is lower than the phospholipid content in the confirmation reagent; the calcium chloride aqueous solution contains heparin neutralizer. The kit has the advantages that the heparin neutralizer is added into the calcium chloride aqueous solution, the kit is different from the existing system, the kit has long stable period and high positive detectable rate, can efficiently resist the interference of heparin/low-molecular heparin and oral anticoagulant drugs, and can be used for screening and confirming detection of systemic lupus erythematosus and anti-phospholipid syndrome diseases.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a kit for detecting lupus anticoagulant based on an SCT method.
Background
Lupus Anticoagulant (LA) is a heterogeneous immunoglobulin that can be produced naturally in vivo or by autoimmunity, and is an autoantibody to negatively charged phospholipids, mainly by binding to beta 2 Glycoprotein I (. Beta.) and its use 2 －glycoproteinⅠ，β 2 GP I), human Prothrombin (PT) and other negatively charged phospholipid complexes interfere with the phospholipid-dependent coagulation process, resulting in prolonged clotting times. The lupus anticoagulant substance is used for diagnosing, evaluating and prognosing autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS)Important indexes of the interruption.
Indications for lupus anticoagulant detection include an unexpected extension of the clotting screening test (APTT, PT, TT) time to evaluate patients with arteriovenous thrombosis or morbid pregnancy. Depending on the coagulation pathway, there are three pathways for LA detection: exogenous pathway, endogenous pathway, common pathway. The detection method of the endogenous pathway comprises APTT, SCT, KCT and CSCT; the exogenous pathway is dPT; the common approaches are dRVVT, ECT, textarin. In order to improve the detection accuracy and the detection rate of LA, two different approaches are usually adopted. The detection of LA comprises a screening test, a mixing test and a confirmation test, and the basic test principle is as follows: (1) extension of phospholipid-dependent coagulation assays; (2) Confirm that the prolongation of clotting time is not caused by a deficiency of one or more clotting factors; (3) confirming that the circulating inhibitor has phospholipid dependence. The LA screening reagent is a reagent containing phospholipid at a low concentration, the confirming reagent is a reagent containing phospholipid at a high concentration, and the presence of LA in the sample to be tested is confirmed by obtaining the ratio of the coagulation time of each reagent. The screening reagent and the confirmation reagent are used in the screening test stage and the confirmation test stage, respectively. The mixing test was performed by mixing healthy normal plasma with the test plasma at a ratio (1:1 or 1:4) to see if the extended screening time was corrected. If the blood plasma to be tested lacks one or more coagulation factors, the prolonged coagulation time after mixing is corrected; on the other hand, if an anticoagulant such as LA is present in the blood plasma to be measured, it cannot be corrected.
Most of the kits available on the market for detecting LA are classified into two major categories, dvrvvt and SCT, according to the activation pathway and the sensitivity of reagents. Compared with the dRVVT method, the SCT method has higher sensitivity, can greatly improve the detection accuracy and reduce the false negative result. Furthermore, the drvtv method is mostly a freeze-dried preparation in order to maintain the long-term stability of the reagent, and the freeze-dried powder preparation needs a reconstitution operation when in use, and the reconstitution operation is affected by reconstitution water quality, reconstitution operation process and the like, so that false positive or false negative of the detection result is easily caused. Therefore, the SCT method has wide prospect in clinical application.
According to the CLSI H60-A guideline, the LA monitoring should be carried out after patients stop taking anticoagulant drugs, however, some patients need to take the anticoagulant drugs orally or receive heparin/low-molecular-weight heparin anticoagulant therapy frequently due to the high coagulation state of the patients, the interference of the anticoagulant drugs often exists in a sample to be detected in the LA detection process, and in order to resist the interference of the heparin/low-molecular-weight heparin and the oral anticoagulant drugs, heparin neutralizing agents are added in the existing screening reagents and confirmation reagents. Although the interference of heparin/low molecular heparin and oral anticoagulant can be effectively resisted by adding heparin neutralizer into the screening reagent and the confirmation reagent, the applicant finds that the SCT liquid reagent added with heparin neutralizer has the problem of instability mainly manifested in the phenomena of easy turbidity and generation of precipitate by adding heparin neutralizer into the reagent during related experiments, which seriously influences the popularization and use of the SCT reagent.
Disclosure of Invention
Aiming at the defect of instability of the existing SCT method liquid reagent, the applicant aims to provide a liquid SCT method kit which has long stability period and high positive detection rate, can efficiently resist the interference of heparin/low molecular heparin and oral anticoagulant, and can be used for screening, confirming and detecting systemic lupus erythematosus and phospholipid syndrome resistant diseases.
In the process of carrying out experiments by using the SCT method, the applicant unexpectedly discovers that the heparin neutralizing agent influences the long-term stability of the SCT liquid reagent, and in order to overcome the defect, a large number of single-factor experiments related to kit components are carried out, and finally, the components influencing the long-term stability of the reagent are silicon dioxide and phospholipid, and the mixture of the heparin neutralizing agent and the silicon dioxide or the phospholipid in a liquid state can generate turbidity and sedimentation because the heparin neutralizing agent is a cationic polymer with adsorbability, and the silicon dioxide and the phospholipid are both anionic mixtures, so that after the heparin neutralizing agent is mixed with the silicon dioxide and the phospholipid, anionic polymerization reaction is easy to occur, and further turbidity and sedimentation occur. In order to prolong the stability of the SCT liquid reagent, the applicant does not add heparin neutralizer into the screening reagent and the confirmation reagent, and simultaneously adds the heparin neutralizer into a separately packaged calcium chloride solution so as to enable the heparin neutralizer to have stronger anti-interference capability, and separates the heparin neutralizer from the screening reagent or the confirmation reagent, thereby prolonging the stability of the SCT liquid reagent and maintaining the anti-interference capability.
In order to achieve the technical purpose of the present application, the present application is specifically realized by the following technical solutions:
the application provides a kit for detecting lupus anticoagulant based on an SCT method, which comprises a screening reagent, a confirmation reagent and a calcium chloride aqueous solution;
the screening reagent or the confirmation reagent consists of silicon dioxide, phospholipid, a buffering agent, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent, a preservative and water; the phospholipid content in the screening reagent is lower than the phospholipid content in the confirmation reagent; the calcium chloride aqueous solution contains heparin neutralizer.
According to the kit, the heparin neutralizer is added into the calcium chloride aqueous solution, and during storage, the phenomenon that the heparin neutralizer is directly mixed with phospholipid or silicon dioxide to generate anion and cation polymerization reaction to generate turbidity and settlement is avoided, so that the stability of a screening reagent and a confirmation reagent in the kit is ensured. When the kit is used, a sample to be detected is mixed with phospholipid and silicon dioxide, then calcium ions are added, and the silicon dioxide can activate blood coagulation factors in the sample under the participation of the calcium ions, so that a blood coagulation process is excited, and the time required by sample coagulation is observed. The screening reagent is used for detecting a blood coagulation experiment containing a small amount of phospholipid, the confirming reagent is used for detecting a blood coagulation experiment containing excessive phospholipid, and when lupus anticoagulant exists, the screening blood coagulation time is prolonged, the blood coagulation time is confirmed to be normal, and the ratio is increased.
As an embodiment of the present application, in order to ensure the homogeneity of the calcium chloride aqueous solution, and the accuracy of the detection reagent, the calcium chloride aqueous solution is composed of the following components by weight: 3 per mill to 5 per mill of calcium chloride dihydrate, 1 percent to 3 percent of heparin neutralizer, 0.2 per mill to 1 per mill of preservative, and the balance of water.
In the scheme, the test value of the quality control blood plasma of the kit can fall within the quality control range, and the negative/positive quality control test values are close to the target value, so that the kit shows extremely high accuracy.
Preferably, in order to further improve the detection precision and stability of the kit, the calcium chloride aqueous solution consists of the following components in percentage by weight: 3.6 per mill of calcium chloride (containing two crystal waters), 2.5 per mill of heparin neutralizer, 0.8 per mill of preservative, and water for the balance.
As one embodiment of the application, the screening reagent is composed of the following components in parts by weight: 0.5-1.5 per mill of silicon dioxide, 0.08-0.2 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative, and the balance of water.
Preferably, the screening reagent consists of the following components in percentage by weight: 1 per mill of silicon dioxide, 0.1 per mill of phospholipid, 1.5 per mill of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative, and the balance of water.
As one embodiment of the present application, the confirmation reagent consists of the following components in weight content: 0.5-1.5 per mill of silicon dioxide, 0.1-8 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative and the balance of water.
Preferably, the confirmation reagent consists of the following components in parts by weight: 1 per mill of silicon dioxide, 6 per mill of phospholipid, 1.5 percent of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative and the balance of water.
In the scheme of screening the reagent and confirming the reagent, the stability of the screening reagent can be improved on the premise of not influencing the sensitivity of a reaction system by using the protective agent and the heavy metal ion chelating agent. Wherein the protective agent can inhibit nonspecific adsorption and aggregation in the reagent solution, and reduce precipitation. The heavy metal ion chelating agent can chelate the heavy metal ions with positive charges in the reagent, and reduce the polymerization reaction of the heavy metal ions in the solution and the anions and cations of the effective components. The combined synergistic effect of the protective agent and the heavy metal ion chelating agent greatly improves the appearance characteristic and the functional stability of the reagent.
As an embodiment of the present application, the heparin neutralization agent is selected from one of polyacrylamide, coagulated polyamine, or protamine.
As an embodiment of the present application, the phospholipid is selected from one of rabbit cephalin, lecithin, pig cephalin, cow cephalin, soybean phospholipid, peanut phospholipid or synthetic phospholipid.
As an embodiment of the present application, the buffer is selected from one of Tris buffer, hepes buffer, PBS buffer, MES buffer, imidazole buffer, MOPS buffer, or citrate buffer.
As one embodiment of the present application, the screening reagent and the confirmation reagent are adjusted to pH 7.4 to 7.6 by the buffer.
As an embodiment of the application, the protective agent is glycine, the glycine is nonpolar amino acid, and the glycine does not have polymerization reaction with anion mixture (silicon dioxide and phospholipid), so that the efficacy of the effective components is ensured, and the precipitation is avoided.
As an embodiment of the present application, the ionic strength regulator is selected from NaCl, KCl or MgCl 2 One of them.
As an embodiment of the present application, the heavy metal ion chelating agent is aluminum chloride.
As an embodiment of the present application, the preservative is selected from one of sodium azide, sodium benzoate, sodium thiomersalate, PC950, potassium sorbate, gentamicin, sodium lactate, or nitrite.
The beneficial effect of this application does:
the application provides a kit for detecting lupus anticoagulant based on an SCT method, and by adding a heparin neutralizing agent into a calcium chloride aqueous solution, turbidity and sedimentation caused by anion and cation polymerization reaction of the heparin neutralizing agent, phospholipid and silicon dioxide are avoided, so that the storage stability of the kit is improved. Meanwhile, a protective agent and a heavy metal ion chelating agent are added into the screening reagent and the confirmation reagent, and the stability of the screening reagent and the confirmation reagent is further improved through the synergistic effect of the protective agent and the heavy metal ion chelating agent. Finally, the kit has excellent stability, and the stability of the expiration date, the stability of the bottle opening and the stability of the machine are all superior to those of similar product kits.
In addition, a reagent system of adding a heparin neutralizer into a calcium chloride aqueous solution is established, so that the anti-interference performance of the kit is ensured, tests prove that the kit can achieve the concentration of resisting common heparin of 1.2U/mL, the concentration of resisting low molecular heparin of 2.0U/mL, the concentration of resisting rivaroxaban of 160ng/mL and the concentration of resisting dabigatran of 140ng/mL, and the anti-interference performance is superior to that of similar kits.
Therefore, the method provides a reliable detection method for clinical diagnosis and treatment of systemic lupus erythematosus and antiphospholipid syndrome diseases, can realize import substitution, and is favorable for further popularization and use in the market.
Drawings
FIG. 1 is a graphical representation of the expiration date, decap, and on-machine stability of the inventive reagents relative to commercially available homogeneous reagents;
FIG. 2 is a schematic diagram of the correlation curve between the reagent of the present invention and the commercially available similar reagents for resisting normal heparin, low molecular heparin, rivaroxaban and dabigatran interference.
Detailed Description
The technical solutions of the present application will be described clearly and completely with reference to specific embodiments of the present application, and it should be apparent that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making creative efforts belong to the protection scope of the present application.
The application provides a kit for detecting lupus anticoagulant based on an SCT method, and the kit comprises a screening reagent, a confirmation reagent and a calcium chloride aqueous solution. In order to overcome the problem of poor stability of the existing SCT reagent, a heparin neutralizer is not added in a screening reagent and confirmation reagent system of the kit, so that the heparin neutralizer is prevented from carrying out anion and cation polymerization reaction with silicon dioxide and phospholipid, and the screening reagent and the confirmation reagent can be stably stored for a long time. Meanwhile, in order to enable the kit to have strong anti-interference capability, the applicant adds a heparin neutralizer into a calcium chloride aqueous solution to form a new system for detecting lupus anticoagulant based on the SCT method.
In a specific embodiment of the present application, the screening reagent is composed of silicon dioxide, phospholipids, a buffer, a protective agent, a heavy metal ion chelating agent and water, after the sample to be tested and the screening reagent are mixed, a calcium chloride aqueous solution is then added, the phospholipids and the silicon dioxide in the screening reagent can activate the intrinsic coagulation factor of the sample to be tested, and then the coagulation process is triggered in the presence of calcium ions, so as to detect the coagulation time of the sample to be tested.
The protective agent is added into the screening reagent, so that nonspecific adsorption and aggregation in a reagent solution can be inhibited, the reaction is slowed down, chemical balance is kept, surface tension is reduced, photothermal decomposition or oxidative decomposition and the like are prevented, and the stability of the solution is improved, so that the performance of a screening reagent system is stable for a long time, and the detection accuracy is improved. The heavy metal ion chelating agent can chelate heavy metal ions in a reagent system, reduce the polymerization reaction of the heavy metal ions in an aqueous solution and anions and cations of active ingredients, reduce the denaturation of proteins such as enzyme and the like caused by the heavy metal ions, and improve the stability of the reagent system.
Preferably, the phospholipid may be one of rabbit cephalin, lecithin, pig cephalin, bovine cephalin, soybean phospholipid, peanut phospholipid or synthetic phospholipid. More preferably, in this example, the phospholipid is rabbit cephalin, which applicants have found to help enhance the stability and shelf life of the screening agent.
Preferably, the protective agent is glycine, the glycine is nonpolar amino acid, and the glycine does not generate polymerization reaction with an anion mixture (silicon dioxide and phospholipid), so that the efficiency of effective components is ensured, and the precipitation is avoided. Compared with other protective agents, such as polyethylene glycol 2000, span-80, BSA, HSA, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin or mannitol and the like, the composition has good stability.
Preferably, aluminum chloride is selected as the heavy metal ion chelating agent, and the aluminum chloride is added into the screening reagent and the confirmation reagent to chelate the heavy metal ions in the reagent, so that the polymerization reaction of the heavy metal ions contained in the reagent, silicon dioxide and phospholipid is reduced, and the stability of the screening reagent and the confirmation reagent is further improved. And the aluminum chloride is selected as the chelating agent, so that the chelating agent can be prevented from chelating or complexing the ionic strength regulator in the solution, the activity of the reagent is not influenced, and the detection accuracy is improved.
In this example, the confirmation reagent system is the same as the screening reagent except that the phospholipid content of the screening reagent is lower than the phospholipid content of the confirmation reagent.
Preferably, the weight percentage content of the phospholipid in the screening reagent is 0.08 to 0.2 per mill, and the weight percentage content of the phospholipid in the confirmation reagent is 0.1 to 8 per mill. More preferably, the weight percentage content of the phospholipid in the screening reagent is 0.1%, and the weight percentage content of the phospholipid in the confirmation reagent is 6%. Through the setting of the phospholipid content of the screening reagent and the confirmation reagent, the detection rate of positive samples is greatly improved, the detection result of false negative is reduced, and the method is favorable for clinical popularization and application.
In this embodiment, the calcium chloride aqueous solution contains a heparin neutralizer, the concentration of the heparin neutralizer is 1% to 3%, a detection system in which the heparin neutralizer is added to the calcium chloride aqueous solution is established, and at this concentration, the kit of the present application has good performance of resisting interference of heparin/low molecular heparin, resisting interference of rivaroxaban, and resisting interference of dabigatran, and exhibits excellent anti-interference capability.
Preferably, the heparin neutralization agent may be one of polyacrylamide, coagulated polyamine, or protamine.
In this embodiment, a buffer, an ionic strength adjuster, and a preservative are further added to the screening reagent and the confirmation reagent. The calcium chloride aqueous solution is added with a preservative.
Wherein the buffer can be one of Tris buffer, hepes buffer, PBS buffer, MES buffer, imidazole buffer, MOPS buffer or citric acid buffer. In this example, the buffer is imidazole buffer, and the pH of the screening reagent or the confirmation reagent is adjusted to 7.4 to 7.6 using imidazole buffer, so that the reagent system maintains good appearance characteristics and performance stability characteristics.
The ionic strength regulator may be NaCl, KCl or MgCl 2 One of them. In this embodiment, the ionic strength modifier is sodium chloride, and through the addition of the ionic strength modifier, the ionic strength of the solution is kept high, and the ionic strength modifier is used in cooperation with an imidazole buffer solution to help control the pH value, so that interfering ions can be masked to a certain extent, and meanwhile, the ionic strength modifier is used for stabilizing the ionic activity coefficient of the solution and improving the detection accuracy.
The preservative can be one of sodium azide, sodium benzoate, thimerosal, PC950, potassium sorbate, gentamicin, sodium lactate or nitrite. In this embodiment, the antiseptic is PC950, realizes the anticorrosive effect of reagent system through the addition of antiseptic, prevents to use along with uncapping many times of reagent, causes the pollution of microorganism to the reagent, is favorable to prolonging the storage of kit and has certain effect to the stability of reagent simultaneously.
The functional units of the components of the screening reagent and the confirmation reagent in this embodiment are simplified, and each functional unit only comprises a single component, namely, a buffering agent, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent and a preservative. The other similar reagents comprise a plurality of functional units of components such as a buffering agent, an antifreezing agent, a stabilizing agent, a protective agent, a blood coagulation factor protective agent, a surfactant, an ion regulator, an antioxidant, a preservative, a lysozyme and the like, and each functional unit comprises a plurality of components. The kit simplifies functional units and corresponding components, simplifies the preparation process and reduces the production cost.
In this embodiment, preferably, the screening reagent is composed of the following components by weight: 0.5-1.5 per mill of silicon dioxide, 0.08-0.2 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative, and the balance of water. More preferably, the screening reagent consists of the following components in percentage by weight: 1 per mill of silicon dioxide, 0.1 per mill of phospholipid, 1.5 per mill of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative, and the balance of water. Most preferably, the screening reagent consists of the following components in weight percentage: 1 per mill of silicon dioxide, 0.1 per mill of phospholipid, 1.5 per mill of imidazole, 10 percent of glycine, 1.2 per mill of sodium chloride, 1.8 per mill of aluminum chloride, 0.8 per mill of PC950, 7.4 to 7.6 of pH and the balance of water.
Preferably, the confirmation reagent consists of the following components in weight content: 0.5-1.5 per mill of silicon dioxide, 0.1-8 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative and the balance of water. More preferably, the confirmation reagent consists of the following components in parts by weight: 1 per mill of silicon dioxide, 6 per mill of phospholipid, 1.5 percent of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative and the balance of water. Most preferably, the confirmation reagent consists of the following components in weight content: 1 per mill of silicon dioxide, 0.6 per mill of phospholipid, 1.5 per mill of imidazole, 10 per mill of glycine, 1.2 per mill of sodium chloride, 1.8 per mill of aluminum chloride, 0.8 per mill of PC950, 7.4 to 7.6 of pH and the balance of water.
Preferably, the calcium chloride aqueous solution consists of the following components in percentage by weight: 3 per mill to 5 per mill of calcium chloride (containing water of recrystallization), 1 percent to 3 percent of heparin neutralizer, 0.2 per mill to 1 per mill of preservative, and water for the balance. More preferably, the calcium chloride aqueous solution consists of the following components in percentage by weight: 3.6 per mill of calcium chloride (containing water of recrystallization), 2.5 per mill of heparin neutralizer, 0.8 per mill of preservative, and the balance of water. Most preferably, the calcium chloride aqueous solution consists of the following components in percentage by weight: 3.6 per mill of calcium chloride (containing water of recrystallization), 2.5 percent of heparin neutralizer, 0.8 per mill of PC950 and the balance of water.
As another specific embodiment of the present application, a method for detecting lupus anticoagulant using the above-mentioned kit is provided, in which the volume ratio of the sample to be detected (the mass volume concentration of the component to be detected is 90%) to the activating preparation prepared from the reagent (the mass volume concentration of the reagent provided by the present invention is 4.98%) is 1:1, and the method includes the following steps:
(1) preparing a sample to be tested: centrifuging the sample for 10 minutes at 1500g, transferring the supernatant, repeating the centrifugation for 10 minutes at 1500g, and reserving the supernatant to be tested;
(2) preparation of normal human pooled plasma (NPP): 40 samples of plasma from normal persons (male and female halves, age 17-70 years) were collected at 1500 g.times.10 min each, centrifuged 2 times, the supernatant was collected, and the 40 centrifuged plasma samples were mixed.
(3) Mixing the sample to be detected with the SCT screening reagent according to the volume ratio of 1:1, pre-heating at 37 ℃ for 180S, mixing with an equal volume of calcium chloride solution, and detecting the screening time (S) of the sample to be detected t )。
(4) Mixing the sample to be detected with the SCT confirmation reagent according to the volume ratio of 1:1, pre-heating at 37 ℃ for 180s, mixing with an equal volume of calcium chloride solution, and detecting the confirmation time (C) of the sample to be detected t )。
(5) Mixing the NPP and the SCT screening reagent according to the volume ratio of 1:1, pre-heating at 37 ℃ for 180S, mixing with an equal volume of calcium chloride solution, and detecting the NPP screening time (S) nt )。
(6) Mixing the NPP and the SCT confirmation reagent according to the volume ratio of 1:1, pre-heating at 37 ℃ for 180s, mixing with an equal volume of calcium chloride solution, and detecting the NPP confirmation time (C) nt )。
(7) And (3) calculating:
screening Ratio (SR) = screening time (S) of sample to be tested t ) /NPP screening time (S) nt )；
Confirmation Ratio (CR) = confirmation time (C) of sample to be measured t ) /NPP acknowledgement time (C) nt )；
Normalized Ratio (NR) = Screening Ratio (SR)/Confirmation Ratio (CR).
The technical scheme and effects of the present application are further described below with reference to specific kits and experiments.
Examples 1 to 6
Adding phospholipid, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent and a preservative into the silicon dioxide solution, uniformly stirring, adding a buffering agent to regulate the pH value to 7.4-7.6, and then fixing the volume to obtain the screening reagent or the confirmation reagent. Weighing calcium chloride dihydrate, heparin neutralizer and preservative, mixing and stirring in water to a constant volume to obtain the calcium chloride aqueous solution. And respectively packaging the quantitative screening reagent, the quantitative confirmation reagent and the calcium chloride aqueous solution separately, and assembling to obtain the kit.
TABLE 1 composition of screening reagent, confirmation reagent and aqueous calcium chloride solution in the kit
After the reagent systems of the examples 1 to 6 with different concentration ratios are kept still for 72 hours, the corresponding standardized ratios are detected by quality control plasma respectively, the reagents of each example are detected for three times respectively, and then the average value is taken to compare the NR conditions of the reagent systems of the different examples. The Negative quality Control selected in the experiment is HemosIL LA Positive Control, the target value and the range are 0.98 (less than 1.16), the Positive quality Control selected is HemosIL LA Positive Control, and the target value and the range are 3.20 (more than or equal to 1.16). The relevant experimental data are shown in table 2 below.
TABLE 2 detection of NR in quality control blood samples by kits for different concentrations in examples 1-6
As can be seen from tables 1 and 2, the reagent systems of examples 1 to 6 all showed a uniform solution after standing for a period of time, indicating that the reagent systems of the reagent kits had excellent stability. The detection of the quality control plasma shows that the quality control test values of the kit systems of the embodiments 1 to 6 are all in the quality control range, and the negative/positive quality control test values are close to the target value, which shows that the kit has better accuracy and also indirectly proves that the kit has good stability. The reagent ratio of example 1 is the best embodiment of the present application, as the deviation of the test value and the target value of the reagent system of example 1 is minimum.
Example 7 influence of the addition of glycine and aluminum chloride at different concentrations on the stability of the reagent system is based on the reagent system of example 1, experimental groups to which glycine and/or aluminum chloride are not added and glycine and/or aluminum chloride at different concentrations are added are respectively arranged, each experimental group is provided with three parallel control groups, after standing for 72h, quality control plasma is used for detecting corresponding standardized ratios of the reagent system at different glycine and/or aluminum chloride concentration ratios, an average value is obtained, and the NR condition of the reagent under different glycine and/or aluminum chloride concentration conditions is compared. The Negative quality Control selected in the experiment is HemosIL LA Positive Control, the target value and the range are 0.98 (less than 1.16), the Positive quality Control selected is HemosIL LA Positive Control, and the target value and the range are 3.20 (more than or equal to 1.16). The experimental result data are shown in table 3 below.
TABLE 3 Effect of the addition of different concentrations of Glycine and aluminum chloride on the stability of the reagent System
As can be seen from the data in Table 3, the reagent of the experimental group 1 has different degrees of precipitation after 72 hours because glycine and aluminum chloride are not added, and when the reagent is used for detecting quality control plasma, the test value exceeds the range of the quality control plasma, thus the accuracy of the detection reagent is seriously influenced. And after the experimental groups 2 and 3 added with 8-12% of glycine and 1.5-7.5% of aluminum chloride are stood for 72 hours, the appearance is still good, the quality control plasma is detected, the test value falls within the quality control range, and the negative/positive quality control test values are close to the target value. The experiment group 4 only adds amino acid, but not adds aluminum chloride, the appearance is slightly turbid after standing for 72h, which shows that the addition of aluminum chloride is helpful for improving the stability of the reagent, because the aluminum chloride can chelate a small amount of heavy metal ions with positive charges in the reagent, and because of the strong chelating effect of the aluminum chloride, the polymerization reaction of heavy metal cations with silicon dioxide and phospholipid is reduced, thereby realizing the purpose of stabilizing the reagent. The test of the quality control plasma in the experimental group 4 shows that the deviation from the target value is smaller than that in the example 1 and larger than that in the examples 2 and 3, and the stabilizing effect of the aluminum chloride on the reagent system is indirectly proved.
Example 8 Effect of heparin neutralizers on screening and confirming reagent stability
Based on the reagent system in example 1, experimental groups respectively adding heparin neutralizers with different concentrations into a screening reagent, a confirmation reagent and a calcium chloride solution are arranged, each experimental group is provided with three parallel control groups, after standing for 72 hours, quality control plasma is used for detecting corresponding standardized ratios of the reagent system under different glycine and/or aluminum chloride concentration ratios, an average value is taken, and the NR conditions of the reagent under different glycine and/or aluminum chloride concentration conditions are compared. The Negative quality Control selected in the experiment is HemosIL LA Positive Control, the target value and the range are 0.98 (less than 1.16), the Positive quality Control selected is HemosIL LA Positive Control, and the target value and the range are 3.20 (more than or equal to 1.16). The experimental result data are shown in table 4 below.
TABLE 4 Effect of heparin neutralization agent on screening and validation of reagent stability
As can be seen from the data in Table 4, when heparin neutralization agents were added to the screening reagents and confirmation reagents, anion and cation polymerization occurred to generate precipitates, and the test values of the quality-controlled plasma were not within the quality-controlled range. After (1% -3%) heparin neutralizing agent is added into calcium chloride solution, the appearances of screening reagent and confirmation reagent are all kept in uniform state, which shows that heparin neutralizing agent is the main reason for producing precipitate of screening reagent and confirmation reagent, because heparin neutralizing agent is cationic polymer with adsorptivity, silicon dioxide and phospholipid are anionic mixture, if the above three substances are mixed in screening reagent and confirmation reagent, anion and cation polymerization reaction is easy to occur, and further turbidity and precipitation appear.
Meanwhile, the experiment groups 6 and 7 add different contents of heparin neutralizer into the calcium chloride solution, which proves that the quality control detection results of the kit are kept within the quality control range within 1-3% of the weight content, and the negative/positive quality control test values are close to the target values.
Example 9 stability testing of the kits of the present application
According to the actual situation of clinical detection, the reagent and the commercial kit in the example 1 are tested for the stability of 29 days accelerated at 37 ℃, the stability of 130 days refrigerated at 2-8 ℃ after bottle opening and the stability of 15 days at 16 ℃ after bottle opening, the test method is to utilize Negative quality Control plasma to detect the blood coagulation time, the Negative quality Control selected in the experiment is HemosIL LA Negative Control, each detection is repeated for three times to obtain an average value, and then the stability performance of the reagent in the example 1 and the stability performance of the same type of reagents in the market are compared. The enterprise standard requires that the deviation of the test value of the reagent in the monitoring period is not more than +/-2 s, and the reagent can be judged to be qualified.
1) The stability condition of the effective period of the reagent is simulated by 37 ℃ accelerated test
TABLE 5 test values of reagents at 37 deg.C/29 days
As is clear from Table 5, the reagent kit of the present application maintained the difference between the blood coagulation time on days 1 to 23 and the blood coagulation time on day one at 37 ℃ within 1s, and the maximum difference within 29 days was 1.6s; whereas commercial screening reagents deviated by more than 1s on day three, 15.6 on day 23 and 32.7s maximum within day 29. The combination of A in FIG. 1 proves that the stability of the kit of the application at 37 ℃ is far better than that of the commercial kit.
2) Simulating the cold storage stability of 2-8 ℃ after the bottle is opened
TABLE 6 reagent test values at 2-8 deg.C/130 days after decapping
As can be seen from Table 6, the reagent in the kit of the present application was stored at 2-8 ℃ after decapping, and the deviation between the blood coagulation time during day 130 and the blood coagulation time on day one was kept within 1s, with the maximum deviation being 0.7s; the deviation of the commercial screening reagent at day 30 was more than 1s, and the maximum deviation was 6.4s within 130 days. The kit disclosed by the invention is proved to be far better than the commercial kit in stability at the temperature of 2-8 ℃ by combining B in figure 1.
3) Simulating the stability of the reagent at 16 ℃ in the machine after opening the bottle
TABLE 7 test values of reagent at 16 deg.C/15 days after decapping
As can be seen from Table 7, the reagent kit of the present invention was unpacked and the difference between the blood coagulation time on day 15 and the blood coagulation time on day one was kept within 1s at 16 ℃ with the maximum difference being 0.6s; the deviation of the commercial screening reagent on day 7 was more than 1s, and the maximum deviation was 6.6s within 15 days. The combination of C in figure 1 proves that the stability of the kit of the application under the on-machine condition at 16 ℃ is far better than that of the kit sold on the market.
In conclusion, the detection results of the reagent meet the enterprise standard requirements (the detection values do not exceed 2.0s before and after the detection), the reagent is comprehensively judged to be qualified, and the deviation of the detection values of the reagent, namely the reagent is far smaller than that of the reagent sold in the market, wherein the stability of the reagent at 37 ℃ for 29 days is accelerated, the stability of the reagent at 2-8 ℃ for 130 days after the bottle is opened, and the stability of the reagent at 16 ℃ after the bottle is opened for 15 days.
Example 10 comparison of detection rates of positive samples of the kit and the commercially available kit according to the present application, the reagent of example 1 and the commercially available similar reagent were used for comparative analysis of lupus anticoagulant positive clinical samples, and response of the corresponding reagent system to lupus anticoagulant was evaluated. The experiment is divided into a commercially available reagent group and a reagent group in example 1, 30 lupus anticoagulant positive clinical samples are subjected to related detection, each sample is subjected to repeated detection twice, and the mean value of each group of detection is recorded.
TABLE 8 Positive detection Rate of lupus anticoagulant of example 1 with commercial congeners kit
As can be seen from Table 8, the detection rate of the reagent in example 1 on a positive sample of clinical lupus anticoagulant is 96.7%, the correlation (r) with a clinical original value can reach 0.99, and the detection value is closer to the clinical original value than the detection value of the same type of reagents sold in the market, which shows that the reagent system of the invention has good response on lupus anticoagulant and can be used for detecting clinical lupus anticoagulant.
EXAMPLE 11 interference rejection of the reagents of the present application
The experiment is divided into a commercially available reagent and a reagent group in example 1, 2 reagents are randomly selected from each group to detect Negative quality Control plasma containing interferents with different concentrations, the influence of the content of the interferents with different concentrations on the reagents is compared, wherein the Negative quality Control is HemosIL LA Negative Control, the target value and the range are 0.98 (less than 1.16), each reagent is repeatedly detected twice, the mean value of each group of detection values and the deviation of each group of mean values and unused medicine groups are respectively calculated, and the anti-interference capability of the anticoagulant is further evaluated.
TABLE 9 antijamming Capacity of example 1 with commercially available reagents against anticoagulants
As can be seen from Table 9, the reagent of example 1 had an anti-interference ability of 1.0U/mL for heparin, 2.0U/mL for low molecular weight heparin, 160ng/mL for rivaroxaban, and 140ng/mL for dabigatran. With reference to fig. 2, it can be proved that the kit of the present application has superior anti-interference ability to common heparin, low molecular heparin, rivaroxaban, and dabigatran compared to commercially available kits.
Claims (10)
1. A kit for detecting lupus anticoagulant based on an SCT method comprises a screening reagent, a confirmation reagent and a calcium chloride aqueous solution, and is characterized in that:
the screening reagent or the confirmation reagent consists of silicon dioxide, phospholipid, a buffering agent, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent, a preservative and water; the phospholipid content in the screening reagent is lower than the phospholipid content in the confirmation reagent;
the calcium chloride aqueous solution contains heparin neutralizer.
2. The kit for detecting lupus anticoagulant based on the SCT method according to claim 1, wherein: the calcium chloride aqueous solution comprises the following components in percentage by weight: 3-5 per mill of calcium chloride, 1-3 percent of heparin neutralizer, 0.2-1 per mill of preservative, and water for balancing.
3. The kit for detecting lupus anticoagulant based on the SCT method according to claim 2, wherein: the calcium chloride aqueous solution comprises the following components in percentage by weight: 3.6 per mill of calcium chloride, 2.5 per mill of heparin neutralizer, 0.8 per mill of preservative and water for balancing.
4. The kit for detecting lupus anticoagulant based on the SCT method according to claim 1, wherein: the screening reagent consists of the following components in percentage by weight: 0.5-1.5 per mill of silicon dioxide, 0.08-0.2 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative, and the balance of water.
5. The SCT method-based kit for detecting lupus anticoagulant according to claim 4, wherein: the screening reagent consists of the following components in percentage by weight: 1 per mill of silicon dioxide, 0.1 per mill of phospholipid, 1.5 per mill of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative, and the balance of water.
6. The kit for detecting lupus anticoagulant based on the SCT method according to claim 1, wherein: the confirmation reagent comprises the following components in percentage by weight: 0.5-1.5 per mill of silicon dioxide, 0.1-8 per mill of phospholipid, 0.5-3 percent of buffering agent, 8-12 percent of protective agent, 0.8-3 per mill of ionic strength regulator, 1.5-7.5 per mill of heavy metal ion chelating agent, 0.2-1 per mill of preservative and the balance of water.
7. The SCT method-based kit for detecting lupus anticoagulant according to claim 6, wherein: the confirmation reagent comprises the following components in percentage by weight: 1 per mill of silicon dioxide, 6 per mill of phospholipid, 1.5 percent of buffering agent, 10 percent of protective agent, 1.2 per mill of ionic strength regulator, 1.8 per mill of heavy metal ion chelating agent, 0.8 per mill of preservative and the balance of water.
8. The SCT-based kit for detecting lupus anticoagulant as claimed in any one of claims 1~7 wherein: the heavy metal ion chelating agent is aluminum chloride.
9. The SCT-based kit for detecting lupus anticoagulant as claimed in any one of claims 1~7 wherein: the protective agent is glycine.
10. The SCT-based kit for detecting lupus anticoagulant as claimed in any one of claims 1~7 wherein: the pH value of the screening reagent or the confirmation reagent is 7.4-7.6.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211581680.1A CN115711997A (en) | 2022-12-09 | 2022-12-09 | Kit for detecting lupus anticoagulant based on SCT method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211581680.1A CN115711997A (en) | 2022-12-09 | 2022-12-09 | Kit for detecting lupus anticoagulant based on SCT method |
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