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Method for detecting anticoagulant capacity of human prothrombin complex

A technology for human prothrombin and prothrombin is applied in the field of detecting the anticoagulation ability of human prothrombin complex, which can solve the problems of inconsistent content of anticoagulant components, difficulty in evaluating comprehensive anticoagulation ability, and inability to determine the risk of thrombosis. Easy operation and good repeatability

Inactive Publication Date: 2015-03-25
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to different processes and lack of unified standards, the contents of anticoagulant components (PC, PS, PZ, heparin, and AT) in different PCCs are inconsistent, and they are mixed with procoagulant components FII, FVII, FIX, and FX
Therefore, the detection of anticoagulant ability of PCCs products is very complicated, and it is difficult to evaluate the comprehensive anticoagulant ability, so it is impossible to determine the risk of thrombosis of PCCs products

Method used

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  • Method for detecting anticoagulant capacity of human prothrombin complex
  • Method for detecting anticoagulant capacity of human prothrombin complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The thrombin chromogenic substrate CS-01(38) was purchased from HYPEN-biomed Company, lot number: 30401-1. Embodiment 1 Detection method of the present invention

[0032] 1. Dilution of Human Prothrombin Complexes (PCCs) Preparations

[0033] Dilute the PCCs product with pure water to 0.8IU FIX / ml, and add 40μl to the microwell plate.

[0034] 2. PCCs mixed with human thrombin

[0035] Take 40 μl of human thrombin with a concentration of 4 IU / ml, add it to the above-mentioned microwell plate, shake slightly, mix well, and stand at room temperature for 1 min.

[0036] 3. Color reaction

[0037] Add 40 μl of the chromogenic substrate CS-01(38) (molecular formula: H-D-Phe-Pip-Arg-pNa·2HCl) solution at a concentration of 0.5 mg / ml to the above microwell plate, mix well, and incubate in a water bath at 22°C 4min.

[0038] 4. Absorbance measurement

[0039] The absorbance value (OD value) was measured at a wavelength of 405nm, once every 30s, and five times for a total ...

Embodiment 2

[0044] Embodiment 2 detection method of the present invention

[0045] 1. Dilution of Human Prothrombin Complexes (PCCs) Preparations

[0046] Dilute the PCCs product with pure water to 0.8-1.0 IU FIX / ml, and add 60 μl to the microwell plate.

[0047] 2. PCCs mixed with human thrombin

[0048] Take 60 μl of human thrombin with a concentration of 5 IU / ml, add it to the above-mentioned microwell plate, shake slightly, mix well, and place at room temperature for 1.5 min.

[0049] 3. Color reaction

[0050] Add 60 μl of the chromogenic substrate CS-01(38) (molecular formula: H-D-Phe-Pip-Arg-pNa·2HCl) solution at a concentration of 0.6 mg / ml to the above microwell plate, mix well, and incubate in a water bath at 25°C 5min.

[0051] 4. Absorbance measurement

[0052] The absorbance value (OD value) was measured at a wavelength of 405nm, once every 40s, and 7 times for a total of 280s.

[0053] 5. Establishment of linear equations

[0054] With the above-mentioned measured OD ...

Embodiment 3

[0057] Embodiment 3 detection method of the present invention

[0058] 1. Dilution of Human Prothrombin Complexes (PCCs) Preparations

[0059] Dilute the PCCs product with pure water to 1.3IU FIX / ml, and add 80μl to the microwell plate.

[0060] 2. PCCs mixed with human thrombin

[0061] Take 80 μl of human thrombin with a concentration of 6 IU / ml, add it to the above-mentioned microwell plate, shake slightly, mix well, and stand at room temperature for 2 minutes.

[0062] 3. Color reaction

[0063] Add 80 μl of the chromogenic substrate CS-01(38) (molecular formula: H-D-Phe-Pip-Arg-pNa 2HCl) solution with a concentration of 0.7 mg / ml to the above microwell plate, mix well, and incubate in a water bath at 28°C 6min.

[0064] 4. Absorbance measurement

[0065] The absorbance value (OD value) was measured at a wavelength of 405nm, once every 50 s, 8 times for a total of 400 s.

[0066] 5. Establishment of linear equations

[0067] With the above-mentioned measured OD valu...

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Abstract

The invention discloses a method for detecting anticoagulant capacity of a human prothrombin complex. The method comprises the following steps: (1) taking the human prothrombin complex, diluting until a blood coagulation factor IX is 0.8-1.3IU / ml, adding a human prothrombin solution with the isovolumetric concentration being 4-6IU / ml, mixing evenly, and standing at room temperature for 1-2 minutes; (2) adding a thrombin chromogenic substrate solution which is isovolumetric to the human prothrombin solution, mixing evenly, and incubating at 22-28 DEG C for 4-6 minutes, wherein the concentration of the chromogenic substrate solution is 0.5-0.7mg / ml; (3) determining a light absorption value at the wavelength of 405nm, and determining once 30-50 seconds for 5-8 times in all; and (4) drawing the time changing trend along with the light absorption value by taking the determined light absorption value as a longitudinal coordinate (y) and time (s) as a cross coordinate (x), building a linear equation y=kx+a, wherein k is slope, and calculating the value of 1 / k. The method for detecting the anticoagulant capacity of the human prothrombin complex is simple and convenient to operate and good in repeatability; the comprehensive anticoagulant ability of the PCCs product can be quantitatively detected; and the safety of the PCCs product can be evaluated.

Description

technical field [0001] The invention relates to a method for detecting anticoagulant ability of human prothrombin complex. Background technique [0002] Human prothrombin complex concentrates (PCCs) are a plasma protein product isolated from healthy human plasma, mainly containing four coagulation factors (coagulation factors, F), respectively FII, FVII, FIX, FX , these four coagulation factors have similar physical and chemical properties, and at the same time, they all need to be synthesized by vitamin K; in addition, they also contain protein C (protein C, PC), protein S (protein S, PS), protein Z (protein Z, PZ) Three anticoagulant proteins that also rely on vitamin K for synthesis. Since the product was used in the clinical prevention and treatment of hemophilia B in the 1960s, due to its complex composition, its clinical indications have continued to expand, and it can also be used for congenital or acquired FII, FVII, FX, PC, PS Deficiency, coagulation disorder caus...

Claims

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Application Information

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IPC IPC(8): G01N33/86G01N21/78
CPCG01N33/86
Inventor 曹海军田倩辛叶叶生亮李长清
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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