A composite calcium agent and its preparation method and application

Abstract

The present invention provides a composite calcium agent, its preparation method and application. The active ingredients are composed of CaCl2, Tris-HCl and potassium sorbate. Among them, the pH of Tris-HCl is 6.8-7.5, and the mass ratio of each component is CaCl2:Tris-HCl:potassium sorbate = 2.5-5.0:1:1, which is prepared by the way of component mixing. The composite calcium agent is applied to blood coagulation detection, which can make the test results more stable, reduce experimental variance and stabilize calcium ions. At the same time, the appearance of the composite calcium agent also enriches the blood coagulation detection method and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of blood coagulation detection, and in particular to a composite calcium agent and a preparation method and application thereof. Background Art

[0002] Blood coagulation activity is an important item for obtaining an index, which is used to screen for deficiency of exogenous coagulation factors or to monitor abnormal liver function and oral anticoagulant therapy. At present, coagulation function test assessment mainly includes prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), D-dimer detection (D-dimer). When PT determination and APTT determination are used for detection, protein and calcium ions of coagulated blood are generally mixed as coagulation reaction triggers in plasma, and the time (freezing point) from the beginning of mixing to the completion of coagulation is determined, and the lag time is estimated by comparing the measured time with the standard plasma result.

[0003] This PT method for determining the blood coagulation activity of the intrinsic / extrinsic blood coagulation pathway has been standardized and widely used internationally. For example, the current method, magnetic bead method, and optical method detection are methods in which the sample (specimen) is introduced together with the activator, and the blood coagulation time is measured by the increase in resistance, viscosity, and turbidity.

[0004] However, these tests mainly reflect the lack of certain factors in the intrinsic and exogenous coagulation pathways or the inhibition of their activity, which manifests as a prolonged coagulation time, and cannot reflect the abnormal coagulation function caused by the metabolic imbalance of pathological procoagulants. They are mainly used to diagnose and differentiate hemorrhagic diseases and monitor bleeding risks. However, since such tests are insensitive to the enhanced activity of coagulation factors, the phenomenon of shortened coagulation time mostly has no clear clinical significance, lacks correlation with pathological hypercoagulable states, and cannot effectively evaluate various clinical thrombosis risks. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a stable and multi-purpose composite calcium agent.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned composite calcium agent.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned composite calcium agent in blood coagulation related detection.

[0008] The technical solution adopted by the present invention is:

[0009] A compound calcium preparation, the active ingredient of which is CaCl 2 , Tris-HCl and potassium sorbate, wherein the pH of Tris-HCl is 6.8-7.5, and the mass ratio of each component is CaCl2 : Tris-HCl: potassium sorbate = 2.5-5.0: 1: 1.

[0010] Preferably, the mass ratio of each component of the above-mentioned composite calcium agent is CaCl 2 : Tris-HCl (PH 6.8-7.5): potassium sorbate = 2.55-3:1:1.

[0011] The preparation method of the above-mentioned composite calcium agent comprises the following specific steps:

[0012] (1) Weigh each component and set aside;

[0013] (2) Mix the components according to the mass ratio of CaCl 2 : Tris-HCl (PH 6.8-7.5): potassium sorbate: = 2.5-5.0: 1: 1, add water to make up to volume, mix and obtain.

[0014] Preferably, in the preparation method of the above-mentioned composite calcium agent, in step (2), CaCl 2 : Tris-HCl (PH 6.8-7.5): potassium sorbate = 2.55-3:1:1.

[0015] The above-mentioned composite calcium agent is used as a blood coagulation detection reagent.

[0016] Preferably, the above-mentioned composite calcium agent is used to measure the time of change of blood viscosity after adding the composite calcium agent to whole blood in vitro by vibration, rotation and crystal resonance viscosity method.

[0017] Preferably, the above-mentioned composite calcium agent is used to measure the time when the fibrin formation of ex vivo whole blood changes significantly after adding the composite calcium agent through a viscosity change detection method.

[0018] Preferably, the application of the above-mentioned composite calcium agent comprises the following specific steps:

[0019] a. Add anticoagulant to the isolated venous blood and let it stand for later use;

[0020] b. For the test of the spare blood in step a, first turn the blood vessel upside down and mix it, take part of the whole blood and slowly add it to the reaction cup, add the complex calcium agent, mix it at 37°C, and then test it through the coagulation function tester;

[0021] c. Take the remaining whole blood in step b and centrifuge it in a high-speed centrifuge tube to prepare platelet-free plasma (ppp);

[0022] d. Take 0.1-0.5 ml of platelet-free plasma after centrifugation in step c, add calcium complex, mix at 37°C, and test on the machine to read the GCT value (overall clotting time) of PPP;

[0023] e. Continue centrifugation of the remaining whole blood sample, take out 0.1-0.5 ml of platelet-rich plasma, add calcium complex, mix at 37°C and test on the machine.

[0024] Preferably, in the application of the above-mentioned composite calcium agent, the anticoagulant used in combination with the composite calcium agent (the anticoagulant in the above-mentioned step a) is 3.8% sodium citrate / sodium citrate, and the ratio of the anticoagulant to venous blood is 1:9.

[0025] The beneficial effects of the present invention are:

[0026] The above-mentioned composite calcium agent overcomes the defect of variable quality of previous calcium reagents and can be stored stably. When applied to blood coagulation detection, it can make the test results more stable, reduce experimental variance, and stabilize calcium ions; at the same time, the emergence of composite calcium agents has also enriched the blood coagulation detection method. The composite calcium agent cannot activate the coagulation system in plasma without procoagulants, but can induce the activation of the coagulation system in whole blood or plasma containing procoagulants, forming fibrin, and causing the apparent changes in physical properties such as whole blood or plasma viscosity and conductivity. This change can sensitively reflect the procoagulant strength of procoagulants in the blood, as well as the activation ability and degree of the coagulation system; the time taken from the activation of thrombin to the formation of fibrin (plasma coagulation) can reflect the procoagulant activity of related substances in human blood. The length of the coagulation time is related to the fragments and extracellular microvesicles produced by cells and organ tissues under pathological conditions such as acute damage to tissue cells and their procoagulant function, and then reflects the hypercoagulable state and severity of the human body under pathological conditions. This method has good repeatability, high sensitivity, and simple operation, and has important clinical application value. The specific advantages of this method are:

[0027] 1. The coagulation function test method using compound calcium is different from the current clinical routine coagulation function test method. It does not require the addition of other procoagulants. Only the addition of compound calcium can induce coagulation substances to produce an activation effect on the coagulation system, so it can more objectively reflect the true state of blood coagulation function.

[0028] 2. It can sensitively reflect the accumulation of procoagulant substances in the early stage of coagulation disorder (the content of procoagulant substances exceeds the body's metabolic capacity and the compensatory capacity of the coagulation system), and can be used for the early diagnosis of coagulation disorders in various clinical diseases such as trauma, pathological pregnancy, autoimmune diseases, tumors, etc., making up for the shortcomings of previous clinical routine coagulation function detection methods in the detection of hypercoagulable state;

[0029] 3. This method is easy to operate and is less affected by interference factors during the detection process. With the help of small instruments, early, real-time and rapid coagulation function monitoring can be achieved at the bedside, providing strong support for the early diagnosis and treatment of clinical coagulation disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 For different types of CaCl 2 Relative GCT time of solutions in whole blood coagulation assay.

[0031] Figure 2 For different blood volumes in CaCl 2 Graph of GCT changes in solution.

[0032] Figure 3 Add different concentrations of CaCl to whole blood 2 Graph showing the relationship between solution and GCT time.

[0033] Figure 4 Adding different concentrations of CaCl to PRP 2 Graph showing the relationship between solution and GCT time.

[0034] Figure 5 Adding different concentrations of CaCl to PPP 2 Graph showing the relationship between solution and GCT time.

[0035] Figure 6 CaCl in GCT experiment 2 The whole blood coagulation time is tested after the reagent has been left at room temperature for more than 24 hours.

[0036] Figure 7 Different proportions of CaCl in the complex calcium reagent 2 Effects of reagents on whole blood GCT.

[0037] Figure 8 These are whole blood GCT detection graphs of the same blood sample at different time points.

[0038] Fig. 9 This is a comparison chart of GCT detection using whole blood, PPP, and PRP in different populations.

[0039] Fig.10 This is a comparison chart of APTT coagulation time of different calcium agents.

[0040] Fig.11 This is a comparison chart of parameters of different calcium agents in thrombus elasticity experiments. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0042] Example 1

[0043] Choice of calcium chloride

[0044] The whole blood GCT (General Clotting Time, GCT, overall coagulation time) is used to detect the coagulation process without adding other procoagulants. Only the blood is observed in the CaCl 2 Blood viscosity changes under the action of CaCl 2 The stability of reagents is particularly important for producing accurate and reproducible experimental results.

[0045] CaCl 2 Reagent production and storage require special conditions, CaCl 2 After dissolving in water, the solution is neutral, but as time goes by, Ca 2+ Can react with CO in the air 2 Interaction to form CaCO 3 , while when CaCl 2 After dissolving in water, it forms Ca(OH) 2 and HCl to reduce the pH value of the solution; and it is very easy to replace the metal ions produced in water or in its production and purification, making CaCl 2 The concentration and pH value of the solution change greatly in a short time. Therefore, it is necessary to purify CaCl with different purification methods and different purities. 2 Reagents for comparative analysis.

[0046] There are 25 kinds of CaCl on the official website of Sigma-Aldrich 2 Reagents have different purification processes, material purity, and hydrate content. Due to the high requirements of the experiment, 5 kinds of CaCl with a purity of more than 97% were selected. 2 The experiment was conducted and contained its high-purity hydrate CaCl 2 6H 2 O, respectively: 1, 0.1 M CaCl 2 Solution (Sigma-Aldrich 21059, analytical standard for ion-selective electrodes); 2. CaCl 2 6H 2 O solid crystal (Sigma-Aldrich 21108, BioUltra, ≥99.0%); 3. CaCl 2 Powder (Sigma-Aldrich 746495, anhydrous, free-flowing, Redi-Dri TM ,≥97%)4、CaCl 2 Powder (Sigma-Aldrich 449709, AnhydroBeads TM,-10mesh,≥99.9%trace metals basis)5, 20mM CaCl 2 Solution (Wolfen APTT coagulation fixation reagent)

[0047] Experimental steps: 1) Mix 5 kinds of CaCl 2 1) Prepare the reagent to a concentration of 1M; 2) Dilute with deionized water to a 100mM reaction solution; 3) Draw 3ml to 20ml of blood, add it to a sodium citrate anticoagulant at a concentration of 9:1, slowly invert and mix 5 times, and let it stand at room temperature for 30 minutes; 4) Slowly invert and mix the whole blood 3 times before loading, take 200ul of fresh whole blood, add it to the reaction cup, and then add 170ul of different types of 20mM CaCl 2 Solution (21108, 21059, 746495, 449709, Werfen APTT coagulation reagent); 5) after thorough stirring and mixing, the GCT coagulation time was measured.

[0048] Experimental results: Since the GCT coagulation time is different when blood is drawn from different people, the measured data is only stable within 2 to 3 hours after blood drawing. Therefore, compared with CaCl 2 When processing reagent data, select relative time, that is, the GCT time of a single test is divided by the average value of multiple parallel tests ( Figure 1 ).

[0049] The results showed that in multiple comparative tests, CaCl 2 (746495) solution stability is better, its stability is similar to that of Ca 2+ The purity of Redi-Dri TM This manufacturing process is related to Redi-Dri TM Make solid powdered CaCl 2 Always keep dry and not easily exposed to H in the air 2 O reacts to form hydrates and becomes denatured.

[0050] Example 2

[0051] Establishing GCT testing system

[0052] 1. Determination of blood volume for GCT test

[0053] Experimental steps: 1) Use deionized water to dissolve CaCl 2(746495) The reagent was prepared to a concentration of 1M; 2) Deionized water was diluted to 70mM and 100mM reaction solutions; 3) 20ml of blood was drawn and placed in a sodium citrate anticoagulant at a concentration of 9:1, slowly inverted and mixed 5 times, and allowed to stand at room temperature for 30min; 4) Before loading the whole blood, slowly inverted and mixed 3 times, 200ul, 250ul, 300ul, and 350ul of fresh whole blood were taken and added to the reaction cups, and then 70mM, 100mM CaCl at different concentrations were added to each reaction cup in turn. 2 30ul of solution was used to measure the GCT coagulation time (repeated 7 times, n=7), see Table 1.

[0054] Table 1

[0055] Health 200ul 250ul 300ul 350ul <![CDATA[70mM CaCl 2 Group Mean]]> 348 341.29 351.29 356.86 Mean value of 100mM CaCl2 group 411.71 352.86 348.57 340.14 Mean Difference -63.43 -11.57 2.71 16.71 CV value of 70mM CaCl2 group 7.3％ 6.7％ 6.1％ 7.7％ CV value of 100mM CaCl2 group 3.0％ 4.8％ 6.6％ 6.3％

[0056] Experimental conclusion: 1) 200ul, 250ul, 350ul whole blood in 70mM and 100mM CaCl 2 The average value of GCT in the solution varies greatly; 300ul whole blood in 70mM and 100mM CaCl 2 The average GCT value in the solution changes slightly ( Figure 2 ). Explain the relationship between blood volume and CaCl 2 There is a nonlinear dose-effect relationship between the solution concentration reaction and the GCT blood coagulation time, so the choice of blood volume is related to the calcium ion concentration in the blood and the added calcium ion concentration. Since the coagulation function detector used in this experiment is the coagulation function analyzer of Century Yikang (Tianjin) Medical Technology Development Co., Ltd., the optimal detection dosage is 300-350ul, so the test results of 200ul and 250ul whole blood have a large variability, which may be related to the calcium ion concentration and the relevant parameters of the detection instrument. Therefore, we will explore the best CaCl in the 350ul whole blood system. 2 Solution concentration.

[0057] 2. GCT detection of CaCl in whole blood 2 Determination of solution calcium concentration

[0058] Experimental steps: 1) Use deionized water to dissolve CaCl 2 (746495) reagent was prepared into 1M concentration; 2) deionized water was diluted into 18, 28, 35, 42, 46, 70, 150, 180, 210, 268, 326, 373 mM CaCl 2 3) Draw 20ml of blood and put it into sodium citrate anticoagulant at a concentration of 9:1, slowly invert and mix 5 times, and let it stand at room temperature for 30min; 4) Slowly invert and mix the whole blood 3 times before loading, take 350ul of fresh whole blood, add it to the reaction cup, and then add different concentrations of CaCl 230ul of solution was used to measure the GCT coagulation time (repeated measurement 7 times, n=7).

[0059] Experimental conclusion: GCT time was not detected when 18, 28, and 373 mM solutions were added to whole blood, but GCT time was not detected when 35 and 326 mM CaCl was added. 2 GCT times exceeding 700 seconds were detected in the solution. 2 The GCT time after solution was gradually shortened, and the standard deviation was gradually reduced. When 150, 180, 210, 268, and 326 mM CaCl was added, the 2 After solution, GCT time gradually prolonged ( Figure 3 ). When the calcium ion concentration is low, the whole blood coagulation time shortens as the calcium ion concentration increases, and then enters a plateau period where the change in calcium ion concentration and the whole blood coagulation time is not obvious. Then, as the calcium ion concentration increases, the whole blood coagulation time prolongs. The point with the smallest variability is 70-150mM, and it is more stable to select this point for detection.

[0060] 3. GCT detection of CaCl in platelet-rich plasma (PRP) 2 Determination of solution concentration

[0061] Experimental steps: 1) Use deionized water to dissolve CaCl 2 (746495) reagent was prepared to 1M concentration; 2) deionized water was diluted to 42, 56, 70, 100, 150, 180, 210, 268, 326, 373, 420, 500 mM CaCl 2 3) Draw 20ml of blood and put it into sodium citrate anticoagulant at a concentration of 9:1, and slowly invert and mix it 5 times; 4) Put it into a centrifuge at room temperature with slow speed increase and slow speed decrease, centrifuge at 200g for 15 minutes, take the supernatant, and transfer it to a new EP tube for testing. Slowly invert and mix PRP 3 times before putting it on the machine, take 350ul PRP, add it to the reaction cup, and then add different concentrations of CaCl 2 30ul of the solution was added and the GC time was measured (repeat the measurement 5 times, n=5).

[0062] Experimental conclusion: PRP did not detect GCT time when added to 42 and 500 mM solution, but when added to 56 and 420 mM CaCl 2 The GCT time was more than 600 seconds in the solution. 2 The GCT time after solution was gradually shortened, and the standard deviation was gradually reduced. When 150, 180, 210, 268, 326, 373, and 420 mM CaCl was added, the 2 After solution, GCT time gradually prolonged ( Figure 4 ). When the calcium ion concentration is low, the PRP coagulation time shortens as the calcium ion concentration increases, then enters a plateau period where the change in calcium ion concentration and the change in whole blood coagulation time are not obvious, and then the whole blood coagulation time prolongs as the calcium ion concentration increases. The point with the smallest variability is 150mM CaCl 2 , it is more stable to select this point for detection.

[0063] 4. Determination of CaCl2 concentration in platelet-free plasma (PPP) by GCT

[0064] Experimental steps: 1) Use deionized water to dissolve CaCl 2 (746495) reagent was prepared into 1M; 2) deionized water was diluted into 56, 70, 85, 100, 150, 180, 210, 268, 326, 373, 420, 500 mM CaCl 2 Solution; 3) Draw 20ml of blood and put it into sodium citrate anticoagulant at a concentration of 9:1, and slowly invert and mix 5 times; 4) Put it into a centrifuge with slow rise and slow fall at room temperature, centrifuge at 200g for 15 minutes, take the supernatant, and transfer it to a new EP tube for testing; 5) Centrifuge the supernatant for another 12000g, take the supernatant again, and transfer it to a new EP tube. Slowly invert and mix PPP 3 times before putting it on the machine, take 350ul PPP, add it to the reaction cup, and then add different concentrations of CaCl 2 30ul of the solution was added and the GC time was measured (repeated 4 times, n=4).

[0065] Experimental conclusion: No GCT time was detected when PPP was added to 56 mM solution, but it was detected when 70 and 420 mM CaCl 2 In the solution with 70, 70, 85, 100, 150 CaCl 2 The GCT time after solution was gradually shortened, and the standard deviation was gradually reduced. When 180, 210, 268, 326, 373, and 420 mM CaCl was added, the 2 After solution, GCT time gradually prolonged ( Figure 5 ). When the calcium ion concentration is low, the PPP coagulation time shortens as the calcium ion concentration increases, then enters a plateau period where the change in calcium ion concentration and the change in whole blood coagulation time are not obvious, and then the whole blood coagulation time prolongs as the calcium ion concentration increases. The point with the smallest variability is 150 mM CaCl 2 .

[0066] The experiment found that the CaCl in the GCT time plateau of 350ul whole blood 2The solution concentration is 70-150mM, and the CaCl in the GCT plateau of 350ulPRP 2 The solution concentration is 150-180 mM, 350 ul PPP of CaCl in the GCT plateau 2 The solution concentration is 150mM. Therefore, the CaCl 2 The solution concentration was 100 mM, which had the least variability, and 150 mM was used for PPP and PRP detection.

[0067] Example 3

[0068] CaCl 2 Improved solution stability

[0069] During the GCT experiment, CaCl 2 If the reagent is left at room temperature for more than 24 hours, the clotting time will change significantly when performing the GCT test. Figure 6 ), at this time, the CaCl 2 The pH value of the reagent is 6.3. Therefore, it is necessary to increase the CaCl 2 Stability of reagents.

[0070] CaCl 2 The pH value of the solution is neutral in theory, but it is actually acidic. The reason is that the calcium ions in calcium chloride can combine with the hydroxide ions ionized by water molecules to form calcium hydroxide. Calcium hydroxide is slightly soluble in water, so as time goes by, the hydrogen ions in the solution are more than the hydroxide ions, which is weakly acidic. Saturated CaCl 2 When exposed to air, the pH value can drop to 5 quickly. 2 The Ca ion content in the solution will also decrease over time. Therefore, it is necessary to add Na ions or K ions to the solution to competitively bind to hydroxide ions to form soluble sodium hydroxide or potassium hydroxide. Normal blood also contains a certain amount of Na ions or K ions, so it is necessary to stabilize CaCl without affecting the coagulation reaction. 2 At the same time, the pH value of normal blood is 7.35-7.45, which is weakly alkaline, and a buffer solution must be added to maintain the pH value of the solution. Therefore, alkaline Tris-HCl (pH 6.8-7.4), 0.9% NaCl saline, and potassium sorbate are added to CaCl 2 Solution of choice.

[0071] 1. Preliminary exploration of CaCl 2 Determination of the applicable range of solution pH value

[0072] Experimental steps: 1) Use deionized water to dissolve CaCl 2(746495) reagent was prepared into 1M concentration; 2) deionized water was diluted into 100mM reaction solution; 3) 20ml blood was drawn and placed into sodium citrate anticoagulant at a concentration of 9:1, slowly inverted and mixed 5 times, and allowed to stand at room temperature for 30min; 4) 1M Tris-HCl (PH 6.8-PH 7.4, Solebol) with different pH values ​​was selected and mixed in different proportions, and its pH value was tested (Table 2); 5) CaCl was added according to the mass ratio 2 :Tris-HCl=3:1 ratio, prepare the reaction solution, and test the CaCl with different pH values 2 Solution (Table 2); 5) Slowly invert the whole blood 3 times before loading, take 350ul of fresh whole blood, add it to the reaction cup, and then add 100mM CaCl to each reaction cup in turn. 2 30ul of solution was used to measure the GCT coagulation time (repeated 6 times, n=6). The coagulation function analyzer (Century Yikang (Tianjin) Medical Technology Development Co., Ltd.) was used for measurement.

[0073] Table 2 Effect of different pH values ​​on GCT coagulation detection function

[0074]

[0075] The results showed that 1M Tris-HCl with pH values ​​of 6.8, 7.0, and 7.4 was mixed in proportion to obtain a 1M Tris-HCl solution with a pH of 6.75-7.3, and then CaCl was added. 2 The pH value of the solution increased after adding Tris-HCl (pH 7.4). The buffer with only CaCl 2 The GCT test value of the solution is also closest to the pH value of blood, 7.35-7.45.

[0076] 2. CaCl 2 The optimal mass ratio of potassium sorbate and Tris-HCl added to the solution

[0077] Experimental steps: 1) Prepare 1M CaCl with deionized water 2 (746495), potassium sorbate, Tris-HCl pH 7.4 (Solabo); 2) according to the mass ratio of CaCl 2 :Tris-HCl:potassium sorbate=1:1:1;2:1:1;2.55:1:1;3:1:1;4:1:1;5:1:1;6:1:1;7:1:1;8:1:1 to make 100mM CaCl 2 Reagents and 100 mM CaCl without Tris-HCl or potassium sorbate 23) Draw 20ml of blood and put it into sodium citrate anticoagulant at a concentration of 9:1, slowly invert and mix 5 times, and let it stand at room temperature for 30 minutes; 4) Slowly invert and mix the whole blood 3 times before loading, take 350ul of fresh whole blood, add it to the reaction cup, and then add 100mM of the above different ratios of CaCl in each reaction cup in turn. 2 Reagent 30ul, measure GCT coagulation time ( Figure 7 ), (repeated 6 times n=6). Coagulation function analyzer (Century Yikang (Tianjin) Medical Technology Development Co., Ltd.) was used for determination.

[0078] The results showed that when the concentration of calcium ions in the solution was 100 mM and the mass ratio of CaCl2:Tris-HCl:potassium sorbate was 1:1:1, GCT did not show a value. When the test cup was taken out for observation, the blood was in a semi-coagulated state. The reason was unknown. As the mass of Tris-HCl and potassium sorbate in the reagent increased, there was no trend of prolonged coagulation time with increasing concentration. 2 The detection mean values ​​of Tris-HCl: potassium sorbate at mass ratios of 2.55:1:1 and 5:1:1 were similar to those of CaCl 2 The mean values ​​detected by the solutions are closer, and the group with the smallest variability is 3:1:1. Therefore, the acceptable component ratios are 2.55:1:1, 3:1:1, and 5:1:1.

[0079] 3. At the same time, by extending the detection time, check CaCl 2 Whether the reagent can obtain a relatively stable GCT coagulation time as the detection time increases.

[0080] Use CaCl 2 The mass ratio of Tris-HCl: potassium sorbate is 2.55:1:1

[0081] The experimental procedure was the same as the previous experiment, using the same blood sample for testing at different time points ( Figure 8 ). The change of GCT value over time after adding stabilizer is compared with that of CaCl without adding stabilizer. 2 Solution, the measured values ​​are stable, and no large extreme values ​​appear.

[0082] Example 4

[0083] Detection of GCT-whole blood, PPP-GCT and PRP-GCT in normal people, patients with pregnancy-induced hypertension and cancer patients.

[0084] Experimental steps: According to CaCl 2 : Tris-HCl: potassium sorbate mass ratio is 2.55:1:1 to prepare 100mM CaCl 2The reagents were placed at room temperature and re-prepared every 4 days. Over a period of several months, 35 normal subjects, 35 patients with gestational hypertension, and 35 patients with different types of cancer were tested.

[0085] Blood samples from 35 people aged 25-50 from health check-up centers were collected. Through questionnaire surveys and previous physical examination data, healthy people who took anticoagulants and had no chronic diseases such as heart disease and diabetes were excluded. Blood samples from 35 patients with gestational hypertension and 35 cancer patients admitted for screening were included. The test was performed according to the experimental steps established above:

[0086] 1. Routinely puncture the vein to draw human venous blood (note: avoid excessive squeezing, repeated punctures and other injuries when drawing blood to avoid false activation of the coagulation system);

[0087] 2. Obtain anticoagulated whole blood with a ratio of 3.8% sodium citrate to whole blood of 1:9 (or use a clinically commercialized citrate anticoagulation tube to draw 2 ml of venous blood);

[0088] 3. Add 200ul whole blood and 170ul complex calcium reagent (CaCl 2 : Tris-HCl: potassium sorbate = 2.55:1:1), and the measurement was performed at 37°C;

[0089] 4. Use a blood viscosity or conductivity meter to continuously test the blood viscosity or conductivity value at multiple points (not less than once per second), and use computer software to perform continuous and repeated calculations. When the average viscosity or conductivity value tested within a unit time (2 or 3 seconds) is significantly higher than the average of all previous points (P<0.05), or the blood viscosity value within a unit time (2 or 3 seconds) exceeds the average of all previous viscosity points by 4 centipoise units, record the total time (unit: seconds) after the sample test begins as the measured value.

[0090] The results showed that: GCT coagulation detection method and composite CaCl 2 The reagent can reflect the coagulation status of different populations ( Fig. 9 ).

[0091] Example 5

[0092] CaCl 2 Application of the reagent in other related coagulation detection methods

[0093] 1. Composite CaCl 2 Application of the reagent in activated partial thromboplastin time (APTT).

[0094] The activated partial thromboplastin time test method and principle of HemosIL SynthAsil (Warfin, USA) is as follows: the plasma sample is incubated with an appropriate amount of phospholipids, and the negatively charged contact activator and the buffer work together to activate the endogenous coagulation pathway. After incubation at 37 degrees for a certain period of time, calcium is added to trigger the coagulation process and the time required for blood coagulation is measured. The CaCl used in the experiment 2 The reagents were labeled as 0.020 mol / L.

[0095] Experimental steps: According to CaCl 2 : The mass ratio of Tris-HCl: potassium sorbate is 2.55:1:1 to prepare 20mM composite CaCl 2 The reagent was left to stand at room temperature for 1 hour before use. Two identical blood samples were prepared according to the APTT test procedure. Phospholipids were added to the blood samples for incubation. Then, a negatively charged contact activator and a buffer were added. After incubation at 37 degrees for a certain period of time, CaCl in APTT was added to the same treated blood samples. 2 Reagents and self-prepared 20 mM complex CaCl 2 After testing 43 clinical samples, it was found that the self-prepared 20mM complex CaCl 2 CaCl 2 The test results of the reagents were basically consistent ( Fig.10 ).

[0096] 2. Composite CaCl 2 Application of reagents in thromboelastography.

[0097] Thromblelastograph (Haemoneticse, USA) is a thromboelastogram test (viscosity measurement method) used to detect the coagulation time (R), coagulation rate (Angle), clot formation time (K), clot strength (MA) and myofascin index (CI) of human whole blood in vitro, and is used to evaluate the function of human coagulation and fibrinolytic system. Add CaCl to the sample measuring cup. 2 The blood begins to coagulate and fibrinolysis occurs after the addition of a coagulant. By measuring the shear stress between the cup body and the cup cover using a thromboelastogram instrument, a curve of blood clot strength changing over time can be drawn. The CaCl used in this experiment 2 The reagents are marked as 0.20 mol / L and are prepared in normal saline.

[0098] Experimental steps: According to CaCl 2 : The mass ratio of Tris-HCl: potassium sorbate is 2.55:1:1 to prepare 200mM composite CaCl 2The reagent was allowed to stand at room temperature for 1 hour before use. The blood samples were prepared according to the thromboelastometry test steps and kaolin was added to the blood samples for incubation. 2 Reagents and self-prepared 20 mM CaCl 2 The reagents were added to blood samples for parallel testing. After testing 8 clinical samples, it was found that the self-prepared 200mM complex CaCl 2 The reagent was the same as the CaCl 2 The test results of the reagents were basically consistent ( Fig.11 ).

[0099] In summary, the composite calcium reagent of the present invention has made many explorations in the selection of calcium ions, the establishment of an experimental blood volume detection system, the determination of the solution pH value, and the determination of the proportion of the added composite calcium reagent components, and finally found a composite calcium reagent that meets the needs of GCT detection. The composite calcium reagent is not only suitable for GCT detection, but also for the APTT experiment of HemosIL Synth Asil (U.S. Wolfen Company) and the thromboelastograph experiment of Thrombelastograph (U.S. Haemoneticse Company).

[0100] Example 6

[0101] A compound calcium preparation, the active ingredient of which is CaCl 2 , Tris-HCl and potassium sorbate, wherein the pH of Tris-HCl is 6.8-7.5, and the mass ratio of each component is CaCl 2 : Tris-HCl:potassium sorbate=2.55:1:1.

[0102] The preparation method of the above-mentioned composite calcium agent comprises the following specific steps:

[0103] (1) Weigh each component and set aside;

[0104] (2) Mix the components according to the mass ratio of CaCl 2 : Tris-HCl: potassium sorbate: = 2.55:1:1, add water to make up to 1.5 ml, mix and you have it.

[0105] Example 7

[0106] A compound calcium preparation, the active ingredient of which is CaCl 2 , Tris-HCl and potassium sorbate, wherein the pH of Tris-HCl is 6.8-7.5, and the mass ratio of each component is CaCl 2 : Tris-HCl:potassium sorbate=3.0:1:1.

[0107] The preparation method of the above-mentioned composite calcium agent comprises the following specific steps:

[0108] (1) Weigh each component and set aside;

[0109] (2) Mix the components according to the mass ratio of CaCl 2 : Tris-HCl: potassium sorbate: =3.0:1:1, add water to make up to 1.5 ml, mix and obtain.

[0110] Example 8

[0111] A compound calcium preparation, the active ingredient of which is CaCl 2 , Tris-HCl and potassium sorbate, wherein the pH of Tris-HCl is 6.8-7.5, and the mass ratio of each component is CaCl 2 : Tris-HCl:potassium sorbate=5.0:1:1.

[0112] The preparation method of the above-mentioned composite calcium agent comprises the following specific steps:

[0113] (1) Weigh each component and set aside;

[0114] (2) Mix the components according to the mass ratio of CaCl 2 : Tris-HCl: potassium sorbate: =5.0:1:1, add water to make up to 1.5ml, mix and obtain.

[0115] In summary, the present invention utilizes a composite calcium agent with specific components to achieve simple and rapid whole blood testing. The principle is mainly that under physiological conditions, cells, platelets and extracellular microvesicles with procoagulant substances such as phosphatidylserine (PS) on their surfaces produced by physiological metabolism exist in human blood. These components with procoagulant function can be removed through metabolic channels and do not cause an imbalance in the coagulation system (coagulation disorder).

[0116] Under pathological conditions, cells (which may be derived from damaged cells), platelets, and extracellular microvesicles (including extracellular microvesicles derived from damaged tissue cells) with procoagulant substances such as phosphatidylserine (PS) on their surfaces increase significantly, exceeding the threshold of physiological metabolism and accumulating in the blood, which can cause the blood to be in a hypercoagulable state. Once the balance of the physiological coagulation-fibrinolysis system is broken, it can cause coagulation disorders in the blood. Clinical manifestations include hypercoagulation or thrombosis of the blood, and bleeding tendency may also occur due to the consumption of a large amount of coagulation substances.

[0117] The composite calcium agent used in the present invention cannot activate the coagulation system in plasma without procoagulant components, but can induce activation of the coagulation system in whole blood or plasma containing the above-mentioned procoagulant components, forming fibrin and causing apparent changes in physical properties such as whole blood or plasma viscosity and conductivity. Such changes can sensitively reflect the metabolic state of procoagulant components in the blood and the degree of activation of the coagulation system. The apparent characteristic changes of blood viscosity or conductivity measured by special instruments objectively reflect the coagulation state in the human body, providing a basis for early clinical discovery of coagulation disorders (hypercoagulable or hypocoagulable states).

[0118] The above-mentioned composite calcium preparation is used as a blood coagulation detection reagent. During the application process, the specific cells and extracellular microvesicles produced in different diseases in the whole blood are used as coagulants (no other mediators or inducers are required to reduce in vitro interference). Based on the coagulation mechanism of microparticles under pathological conditions, in Ca 2+ The fibrin formation process is observed with the participation of . This application improves and expands the traditional coagulation function test, and provides a timely, effective and reliable means for further observation and research on the occurrence and development mechanism of various complex diseases (such as DIC, brain trauma, pregnancy-induced hypertension and other hypercoagulable diseases) and the overall picture of coagulation disorders, and also fills the gap in the monitoring of procoagulant function in traditional coagulation tests. The establishment of the application method of this composite calcium agent has improved the observation method of the entire plasma coagulation process, and achieved the purpose of comprehensively evaluating the procoagulant, coagulation and fibrinolytic functions of blood using simple technology.

[0119] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of a composite calcium agent as a blood coagulation detection reagent, characterized in that: The effective ingredients of the composite calcium preparation are composed of CaCl2, Tris-HCl and potassium sorbate, wherein the pH of Tris-HCl is 6.8-7.5, the mass ratio of each component is CaCl2:Tris-HCl:potassium sorbate=2.5-5.0:1:1, and the blood coagulation test is the global coagulation time GCT, the partial thromboplastin time APTT or the thromboelastogram test.

2. The use according to claim 1, characterized in that: The mass ratio of each component is CaCl2:Tris-HCl:potassium sorbate=2.55-3:1:

1.

3. The use according to claim 1, characterized in that: The time of significant change in blood fibrin generation after adding the complex calcium agent to whole blood in vitro is determined by the following methods: turbidimetry, current, magnetic beads, crystal resonance viscosity or electrochemical method.

4. The use according to claim 3, characterized in that: The method described enables stable detection of the time for significant changes in fibrin formation.

5. The use according to claim 3, characterized in that: The specific steps are as follows: a. Add anticoagulant to the isolated venous blood and let it stand for later use; b. For the test of the spare blood in step a, first turn the blood vessel upside down and mix it, take part of the whole blood and slowly add it to the reaction cup, add the complex calcium agent, mix it at 37°C and then test it; c. Take the remaining whole blood in step b, centrifuge in a high-speed centrifuge tube, and prepare platelet-free plasma; d. Take 0.1-0.5 ml of platelet-free plasma after centrifugation in step c, add the calcium complex, mix at 37°C and start testing, and read the GCT value of PRP; e. Continue centrifugation of the remaining whole blood sample, take 0.1-0.5 ml of platelet-rich plasma, add complex calcium, mix at 37°C, start testing, and read the GCT value of PPP.

6. The use according to claim 5, characterized in that: The anticoagulant used in combination with the complex calcium agent in step a is 3.8% sodium citrate / sodium citrate, and the ratio of the anticoagulant to venous blood is 1:9.

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