CN112067575A - Method for detecting fibrinolysis function of blood - Google Patents
Method for detecting fibrinolysis function of blood Download PDFInfo
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- CN112067575A CN112067575A CN202010941978.3A CN202010941978A CN112067575A CN 112067575 A CN112067575 A CN 112067575A CN 202010941978 A CN202010941978 A CN 202010941978A CN 112067575 A CN112067575 A CN 112067575A
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- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 120
- 238000002835 absorbance Methods 0.000 claims abstract description 70
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
Abstract
The invention relates to a method for detecting fibrinolysis function of blood, which comprises the following steps: a, preparing an experimental group reagent and a control group reagent; b, detecting absorbance; and c, calculating the fibrinolysis capacity value and the fibrinolysis time value. The invention can directly detect the fibrinolysis function of blood; the invention has relatively simple operation; the fibrinolysis function of blood can be quantitatively evaluated, namely the fibrinolysis capacity and the fibrinolysis time are reflected; traditional fibrinolytic markers such as FDP, D dimer and the like have high non-specificity and are influenced by inflammation, bleeding, thrombus load and the like, and the invention can reflect real fibrinolytic capacity and has high specificity. In addition, the invention is not influenced by factors such as blood chyle, jaundice, hemolysis and the like, and has strong anti-interference capability.
Description
Technical Field
The invention relates to the technical field of blood fibrinolysis function detection, in particular to a blood fibrinolysis function detection method.
Background
Fibrin clots are formed after blood coagulation and require a fibrinolytic system to dissolve it for revascularization. The fibrinolysis function is weakened, and thrombosis can be caused, such as venous thrombosis caused by congenital fibrinolysis defect and venous thrombosis caused by hypofibrinolysis after orthopedics operation. Excessive fibrinolysis may cause bleeding, such as post-traumatic bleeding, post-urinary surgery bleeding, severe obstetric bleeding, and the like. Patients with tumors can exhibit either inhibition or hyperfibrinolysis, and clinically can experience hemorrhage or thrombosis.
The blood system is maintained in balance by the coagulation system, the anticoagulation system and the fibrinolysis system. The patient will neither bleed nor form a thrombus. At present, the fibrinolytic system detection is that a detected marker indirectly reflects the fibrinolytic state and cannot represent the fibrinolytic activity. The existing fibrinolysis products on the market at present comprise a thromboelastogram Ly30, fibrin degradation products FDP and D dimer, a fibrinolysis marker tissue-type plasminogen activator-inhibitor compound tPAIC and a plasmin-antiplasmin compound PIC. They all indirectly detect fibrinolytic activating molecules to represent the degree of fibrinolytic activation, have high non-specificity, are influenced by inflammation, bleeding, thrombus load and the like, and are not the detection of fibrinolytic function.
The detection of the existing fibrinolysis marker cannot represent the real-time fibrinolysis functional level. Since the concentration of the fibrinolytic marker depends on the substrate concentration, the enzyme concentration, and the metabolic effects of liver and kidney functions, real-time fibrinolytic enzyme activity cannot be reflected.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for detecting the fibrinolysis function of blood.
In order to achieve the purpose, the invention adopts the following technical scheme: the method for detecting the fibrinolysis function of blood comprises the following steps:
a preparing experimental group reagent and control group reagent
The experimental group reagent comprises plasma to be detected, actin reagent, thrombin and tissue plasminogen activator reagent (tPA);
the contrast group reagent comprises plasma to be detected, actin reagent, thrombin and distilled water;
b absorbance detection
Respectively mixing the experimental group reagent and the control group reagent before absorbance detection, and detecting the absorbance change of the experimental group reagent and the control group reagent at the wavelength of 980nm by using a light density scanner every 10 seconds; automatically drawing the time-varying curves of the absorbance values of the blood plasma in the experimental group reagent and the control group reagent by using a densitometer; subtracting the absorbance value of the control group reagent from the absorbance value of the experimental group reagent to obtain absorbance difference values at different time points, then tracing a curve of the absorbance difference values along with the change of time, and calculating a first derivative of the curve;
c determining parameters
(1) Fibrinolytic capacity (LP), which reflects the integrated fibrinolytic capacity of the sample, i.e. the area between two zeros under the first derivative curve, LP ═ Log10 (area × 100);
(2) fibrinolysis time (LT) (min), representing the maximum rate of clot degradation by plasmin, defined as the time difference between the extreme point of the first derivative and the first zero point under the first derivative curve;
from the values of LP and LT, the fibrinolytic function of blood can be quantitatively evaluated.
In step a, the volume ratio of the plasma to the actin reagent is 1: 1.
in the step a, an experimental group reagent and a control group reagent are respectively contained in two reagent holes of a 96-hole transparent plate, the reagent hole containing the experimental group reagent is called an experimental hole, the reagent hole containing the control group reagent is called a control hole, the optimal detection volume of liquid in the reagent holes is 200ul, so the total volume of blood plasma and the 4 reagents is 200ul, and the detection volume of 200ul in the experimental hole comprises: plasma 70ul, actin reagent 70ul, thrombin 20ul, tPA20ul, control wells with the same volume of distilled water in place of tPA, 200ul assay volume comprising in total: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of distilled water and 20ul of calcium ions.
In step a, the concentration of thrombin is 0.5IU/ml, the concentration of tissue plasminogen activator is 600ng/ml, and the concentration of calcium ion solution is 0.025 mol/L.
In step a, the actin reagent is a mixture of cephalin and ellagic acid, and is used for providing a reaction interface.
In step b, when the plasma is the frozen platelet-poor plasma, the plasma is required to be bathed at 37 ℃ for 5 min.
In step b, the experiment was carried out at 37 ℃ for 2 hours with the change in absorbance being continuously detected.
In step b, the optical density scanner was an InfiniteM200_ pro optical density scanner manufactured by teiken, switzerland.
In the step b, a plurality of experimental holes and a plurality of comparison holes are arranged, the optical density scanner averages the absorbance values of the experimental holes, a curve of the plasma absorbance values in the experimental holes along with the change of time is drawn by using the average value, the optical density scanner averages the absorbance values of the comparison holes, and a curve of the plasma absorbance values in the comparison holes along with the change of time is drawn by using the average value.
The invention has the beneficial effects that: the invention can directly detect the fibrinolysis function of blood; the invention has relatively simple operation; the fibrinolysis function of blood can be quantitatively evaluated, namely the fibrinolysis capacity and the fibrinolysis time are reflected; the invention can reflect the real fibrinolytic capacity and has high specificity. In addition, the invention is not influenced by factors such as blood chyle, jaundice, hemolysis and the like, and has strong anti-interference capability.
Drawings
FIG. 1 is a graph of absorbance values as a function of reagent for 600ng/mltPA in combination with different concentrations of thrombin;
FIG. 2 is a graph of absorbance values as a function of reagent for combinations of 0.5IU thrombin with different concentrations of tPA;
FIG. 3 is a graph of fibrinolytic function;
The following detailed description will be made in conjunction with embodiments of the present invention with reference to the accompanying drawings.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
as shown in fig. 1 to 3, the method for detecting fibrinolytic function of blood comprises the following steps:
a preparing experimental group reagent and control group reagent
The experimental group reagent comprises plasma to be detected, actin reagent, thrombin and tissue plasminogen activator reagent (tPA);
the contrast group reagent comprises plasma to be detected, actin reagent, thrombin and distilled water;
b absorbance detection
Respectively mixing the experimental group reagent and the control group reagent before absorbance detection, and detecting the absorbance change of the experimental group reagent and the control group reagent at the wavelength of 980nm by using a light density scanner every 10 seconds; automatically drawing the time-varying curves of the absorbance values of the blood plasma in the experimental group reagent and the control group reagent by using a densitometer; subtracting the absorbance value of the control group reagent from the absorbance value of the experimental group reagent to obtain absorbance difference values at different time points, then tracing a curve of the absorbance difference values along with the change of time, and calculating a first derivative of the curve;
c determining parameters
(1) Fibrinolytic capacity (LP), which reflects the integrated fibrinolytic capacity of the sample, i.e. the area between two zeros under the first derivative curve, LP ═ Log10 (area × 100);
(2) fibrinolysis time (LT) (min), representing the maximum rate of clot degradation by plasmin, defined as the time difference between the extreme point of the first derivative and the first zero point under the first derivative curve;
from the values of LP and LT, the fibrinolytic function of blood can be quantitatively evaluated.
In step a, the volume ratio of the plasma to the actin reagent is 1: 1.
in the step a, an experimental group reagent and a control group reagent are respectively contained in two reagent holes of a 96-hole transparent plate, the reagent hole containing the experimental group reagent is called an experimental hole, the reagent hole containing the control group reagent is called a control hole, the optimal detection volume of liquid in the reagent holes is 200ul, so the total volume of blood plasma and the 4 reagents is 200ul, and the detection volume of 200ul in the experimental hole comprises: plasma 70ul, actin reagent 70ul, thrombin 20ul, tPA20ul, control wells with the same volume of distilled water in place of tPA, 200ul assay volume comprising in total: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of distilled water and 20ul of calcium ions.
In step a, the concentration of thrombin is 0.5IU/ml, the concentration of tissue plasminogen activator is 600ng/ml, and the concentration of calcium ion solution is 0.025 mol/L.
In step a, the actin reagent is a mixture of cephalin and ellagic acid, and is used for providing a reaction interface.
In step b, when the plasma is the frozen platelet-poor plasma, the plasma is required to be bathed at 37 ℃ for 5 min.
In step b, the experiment was carried out at 37 ℃ for 2 hours with the change in absorbance being continuously detected.
In step b, the optical density scanner was an InfiniteM200_ pro optical density scanner manufactured by teiken, switzerland.
In the step b, a plurality of experimental holes and a plurality of comparison holes are arranged, the optical density scanner averages the absorbance values of the experimental holes, a curve of the plasma absorbance values in the experimental holes along with the change of time is drawn by using the average value, the optical density scanner averages the absorbance values of the comparison holes, and a curve of the plasma absorbance values in the comparison holes along with the change of time is drawn by using the average value.
Example 1
The invention is carried out in a 96-hole transparent plate, the optimal detection volume of liquid in a reagent hole is 200ul, so the total volume of blood plasma and the 4 reagents is 200 ul. The ratio of plasma to actin reagent volume in FLPA experiments was 1: 1. each sample assay consists of a test well and a control well. The test wells contained 200ul of test volumes in total: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of tPA and 20ul of calcium ions; tPA was replaced by the same volume of distilled water in the control wells, and a 200ul assay volume included: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of distilled water and 20ul of calcium ions.
Before starting the reading, the mixture was shaken gently for 5 seconds to mix well. Subsequently, changes in absorbance in the experimental and control wells at a wavelength of 980nm were detected every 10 seconds using an InfiniteM200_ pro optical density scanner; the experiment was performed at 37 ℃ and the change in absorbance in the wells was continuously examined for 2 hours. The frozen platelet-poor plasma used in the experiment needs to be bathed at 37 ℃ for 5min, and each sample is detected by adopting double complex holes (namely, an optical density scanner averages the absorbance values of two experimental holes, a curve of the plasma absorbance values in the experimental holes along with time is drawn by using the average value, an optical density scanner averages the absorbance values of two comparison holes, a curve of the plasma absorbance values in the comparison holes along with time is drawn by using the average value), and LP and LT are calculated by taking the average values, wherein the LP and LT are collectively called as FLPA parameters.
As shown in fig. 3, the densitometer automatically plots the absorbance of plasma over time in the test and control wells. And subtracting the absorbance value of the control hole from the absorbance value of the experimental hole to obtain the absorbance difference value at different time points, and then tracing the curve of the absorbance difference value changing along with time. Calculate the first derivative of the curve, calculate the FLPA parameters: fibrinolysis capacity (LP), which reflects the integrated fibrinolysis capacity of the sample, i.e. the area between point a and point B under the first derivative curve, LP ═ Log10 (area × 100); fibrinolysis time (LT) (min), representing the maximum rate of degradation of the clot by plasmin, is defined as the time difference between point C and point a under the first derivative curve.
Example 2
The method for detecting the fibrinolysis function of blood comprises the following steps:
a preparing experimental group reagent and control group reagent
The experimental group reagent comprises plasma to be detected, actin reagent, thrombin and tissue plasminogen activator reagent (tPA);
the contrast group reagent comprises plasma to be detected, actin reagent, thrombin and distilled water; the ratio of plasma to actin reagent volume is 1: 1. experiment group reagent and contrast group reagent are held respectively in two reagent holes of 96 hole transparent plates, and the reagent hole that holds experiment group reagent is called experimental hole, and the reagent hole that holds contrast group reagent is called contrast hole, and the liquid optimum detection volume in the reagent hole is 200ul, and the total volume of the above-mentioned 4 kinds of reagents of event plasma is for 200ul, and 200 ul's detection volume includes altogether in the experimental hole: plasma 70ul, actin reagent 70ul, thrombin 20ul, tPA20ul, control wells with the same volume of distilled water in place of tPA, 200ul assay volume comprising in total: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of distilled water and 20ul of calcium ions.
As shown in figure 1, under the condition of constant tPA concentration, when the concentration of thrombin is 0.5IU/ml, the curve of the change graph is smoother, and the change rate is more stable, therefore, the concentration of thrombin adopted in the experiment of the invention is 0.5 IU/ml;
as shown in FIG. 2, under the condition that the concentration of thrombin is not changed, when the concentration of tPA is 600ng/m, the curve of the change graph is smooth, and the change rate is stable, so that the concentration of tPA adopted in the experiment of the invention is 600 ng/m;
the concentration of the calcium ion solution is 0.025mol/L, and the actin reagent is a mixture of cephalin and ellagic acid and is used for providing a reaction interface. Both calcium ion solution and actin reagent can be used as blood activating surface for plasma, thrombin and tPA reaction.
b absorbance detection
Respectively mixing the experimental group reagent and the control group reagent before absorbance detection, and detecting the absorbance change of the experimental group reagent and the control group reagent at the wavelength of 980nm by using a light density scanner every 10 seconds; automatically drawing the time-varying curves of the absorbance values of the blood plasma in the experimental group reagent and the control group reagent by using a densitometer; subtracting the absorbance value of the control group reagent from the absorbance value of the experimental group reagent to obtain absorbance difference values at different time points, then tracing a curve of the absorbance difference values along with the change of time, and calculating a first derivative of the curve;
c determining parameters
(1) Fibrinolytic capacity (LP), which reflects the integrated fibrinolytic capacity of the sample, i.e. the area between two zeros under the first derivative curve, LP ═ Log10 (area × 100);
(2) fibrinolysis time (LT) (min), representing the maximum rate of clot degradation by plasmin, defined as the time difference between the extreme point of the first derivative and the first zero point under the first derivative curve;
from the values of LP and LT, the fibrinolytic function of blood can be quantitatively evaluated.
In step b, when the plasma is the frozen platelet-poor plasma, the plasma is required to be bathed at 37 ℃ for 5 min.
In step b, the experiment was carried out at 37 ℃ for 2 hours with the change in absorbance being continuously detected.
When the invention works, the invention can directly detect the fibrinolysis function of blood; the invention has relatively simple operation; the fibrinolysis function of blood can be quantitatively evaluated, namely the fibrinolysis capacity and the fibrinolysis time are reflected; traditional fibrinolytic markers such as FDP, D dimer and the like have high non-specificity and are influenced by inflammation, bleeding, thrombus load and the like, and the invention can reflect real fibrinolytic capacity and has high specificity. In addition, the required absorbance difference is obtained by subtracting the absorbance value of the control group reagent from the absorbance value of the experimental group reagent, so that the method is not influenced by factors such as blood chyle, jaundice and hemolysis, and has strong anti-interference capability.
The invention has been described in connection with the accompanying drawings, it is to be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover various modifications, adaptations or uses of the invention, and all such modifications and variations are within the scope of the invention.
Claims (8)
1. The method for detecting the fibrinolysis function of blood is characterized by comprising the following steps:
a preparing experimental group reagent and control group reagent
The experimental group reagent comprises plasma to be detected, actin reagent, thrombin and tissue plasminogen activator reagent;
the contrast group reagent comprises plasma to be detected, actin reagent, thrombin and distilled water;
b absorbance detection
Respectively mixing the experimental group reagent and the control group reagent before absorbance detection, and detecting the absorbance change of the experimental group reagent and the control group reagent at the wavelength of 980nm by using a light density scanner every 10 seconds; automatically drawing the time-varying curves of the absorbance values of the blood plasma in the experimental group reagent and the control group reagent by using a densitometer; subtracting the absorbance value of the control group reagent from the absorbance value of the experimental group reagent to obtain absorbance difference values at different time points, then tracing a curve of the absorbance difference values along with the change of time, and calculating a first derivative of the curve;
c determining parameters
(1) Fibrinolytic capacity, which reflects the integrated fibrinolytic capacity of the sample, i.e. the area between two zeros under the first derivative curve, LP ═ Log10 (area × 100);
(2) fibrinolysis time (min), representing the maximum rate of clot degradation by plasmin, defined as the time difference between the extreme point of the first derivative and the first zero point under the first derivative curve;
from the values of LP and LT, the fibrinolytic function of blood can be quantitatively evaluated.
2. The method for detecting fibrinolytic function of blood according to claim 1, wherein in step a, the volume ratio of plasma to actin reagent is 1: 1.
3. the method of claim 1, wherein in step a, the test reagent and the control reagent are respectively contained in two reagent wells of a 96-well transparent plate, the reagent well containing the test reagent is called an experimental well, the reagent well containing the control reagent is called a control well, the optimal detection volume of liquid in the reagent well is 200ul, so that the total volume of the plasma and the 4 reagents is 200ul, and the detection volume of 200ul in the experimental well comprises: plasma 70ul, actin reagent 70ul, thrombin 20ul, tPA20ul, control wells with the same volume of distilled water instead of tPA, 200ul of detection volume collectively comprising: 70ul of plasma, 70ul of actin reagent, 20ul of thrombin, 20ul of distilled water and 20ul of calcium ions.
4. The method of claim 2, wherein the concentration of thrombin in step a is 0.5IU/ml, the concentration of tissue plasminogen activator is 600ng/ml, and the concentration of calcium ion solution is 0.025 mol/L.
5. The method for detecting a fibrinolytic function of blood according to claim 3, wherein in the step a, the actin reagent is a mixture of cephalin and ellagic acid.
6. The method of claim 1, wherein the plasma is freeze-dried platelet-poor plasma, and the temperature of the plasma is kept at 37 ℃ for 5min in step b.
7. The method of claim 6, wherein the assay is performed at 37 ℃ in step b, and the time for continuously measuring the change in absorbance is 2 hours.
8. The method of claim 3, wherein in step b, the test wells and the control wells are provided in plurality, the optical density scanner averages the absorbance values of the test wells, and uses the averaged absorbance values to plot the time-dependent plasma absorbance values in the test wells, and the optical density scanner averages the absorbance values of the control wells and uses the averaged absorbance values to plot the time-dependent plasma absorbance values in the control wells.
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| CN111662956A (en) * | 2020-06-02 | 2020-09-15 | 上海原科实业发展有限公司 | Thromboelastography enhanced fibrinolysis system detection kit and preparation method thereof |
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