CN101587063A - Method for detecting hemostatic capability - Google Patents
Method for detecting hemostatic capability Download PDFInfo
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- CN101587063A CN101587063A CNA2009100229965A CN200910022996A CN101587063A CN 101587063 A CN101587063 A CN 101587063A CN A2009100229965 A CNA2009100229965 A CN A2009100229965A CN 200910022996 A CN200910022996 A CN 200910022996A CN 101587063 A CN101587063 A CN 101587063A
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Abstract
The invention discloses a method for detecting hemostatic capability. The method comprises: preparing anticoagulant blood from venous blood and anticoagulant with 0.13mol/L of sodium citrate in a volume ratio of 9:1; centrifuging an anticoagulant blood sample for 15 minutes at a speed of 3,000 r/min and sucking plasma to be detected; performing OHP determination of overall hemostatic capability, OCP determination of overall blood coagulation capability and OFP judgment of overall fibrinolytic capability; indicating the existence of blood coagulation disturbance when OCP and OHP are less than the lower limit of normal reference values; indicating the existence of hypercoagulable state when the OCP and the OHP are greater than the upper limit of the normal reference values; and indicating the reduction of fibrinolytic function when OFP value is less than 41.61 percent. The method has the advantages of good stability and repeatability, easy operation and low cost, and is worthy of popularization.
Description
Technical field
The present invention relates to the experimental technique that a kind of hemostasis and thrombus detect, particularly a kind of detection method that is used for the blood plasma hemostatic capability.
Background technology
For a long time, people attempt to set up a kind of test that can reflect overall hemostatic capability always in effort, are used to estimate body hemostasis balance.In fact, as far back as nineteen ninety-five, Hemker etc. have just set up so-called interior source thrombase ability, and (endogenous thrombin potential, ETP) assay method are used to estimate body blood coagulation balance.This method is to activate blood plasma with low concentration TF and mixture of phospholipids, measures fibrin ferment with artificial chromophoric substrate and generates, and generates figure (Thrombogram) measuring and calculating ETP according to fibrin ferment.ETP increases and sees state and/or hypercoagulative state case before the thrombus, comprises that antithrombase lacks, the factor height expresses that (fraction A prothrombin 20210C), PROTEIN C/Protein S lacks, factor V Leiden sudden change and oral contraceptive individuality; The ETP attenuating sees various blood coagulation disorders diseases.The ETP method adopts TF and mixture of phospholipids, can activate coagulation process comprehensively, can reflect preceding state of some thrombus of body and blood coagulation disorders, but this method is generated as the monitoring terminal point with fibrin ferment, form the coagulation function in above stage so can only reflect fibrin ferment, phase (being transformed into fibrin as fibrinogen) in the time of can not reflecting blood coagulation whole does not comprise the fibrinolytic process yet, thereby can't say that this method can reflect overall hemostatic function.He in 1999 etc. have reported a kind of OHP assay method.This method is in the presence of plasminogen activator tPA, with a small amount of thrombin activation blood plasma, directly observes fibrin form and degradation process.Though this method can observe blood coagulation and fibrinolytic process, can reflect blood coagulation and fibrinolytic system function substantially, but this method with fibrin ferment as the coagulation activation thing, directly make fibrinogen be transformed into fibrin, ignored factor VII and other all hemostatic compositions impetus of reaction pair blood coagulation each other.In fact, the very difficult blood coagulation situation that reflects truly or all sidedly of this method.
Summary of the invention
In order to overcome above-mentioned prior art deficiency, the invention provides a kind of hemostatic capability detection method, this method can reflect the overall hemostatic capability of body comprehensively, not only can be used as the detection means of hypercoagulative state, also can be used as the screening means of blood coagulation disorders.The foundation of this method will have great importance to early diagnosis, early prevention and the treatment of thrombotic disease.Simultaneously, this method has stability and repeated preferably, and easy and simple to handle, with low cost, is worth generally promoting.
The method of detection hemostatic capability of the present invention has following steps:
(1) gathers venous blood, prepare anticoagulation in venous blood and anti-coagulants (0.13mol/L arrests rafter acid sodium) 9: 1 ratios of volume ratio.
(2) with the centrifugal blood sample of 3000r/min 15 minutes, it is standby to draw blood plasma.
(3) overall hemostatic capability (OHP) is measured: regulating the photometer wavelength is 340nm, test duration 30min is set, with damping fluid (0.1mmol/L Tris, PEG-60005-30g/L, Tween-80 10g/L, CaCl
25mmol/L, all the other are water, pH 7.35) regulate and test zero point.In cuvette, add blood plasma 100 μ l to be checked in advance, add 900 μ l reaction buffer (0.1mmol/L Tris, PEG-6000 5-30g/L, Tween-80 10g/L, CaCl subsequently
25mmol/L, tissue factor (TF) concentration is from 0-1/200000, and UK concentration is from 10U-200U/ml, and all the other are water, pH 7.35), mixing places in the photometer instrument connection then immediately, presses computing machine " Enter " key.Automatically monitoring and the record fibrin forms and the absorbance of degradation process changes of instrument, with the addition of per minute absorbance (totally 30 minutes), i.e. area in the response curve, this value is an overall hemostatic capability (OHP).
(4) overall blood coagulation ability (OCP) is measured: adopt and identical reaction system, experiment condition and the method for operating of above-mentioned OHP test, but reaction system does not contain plasminogen activator UK, the variation of the absorbance of monitoring 30min inner fibrin forming process, calculate response curve with interior total absorbance, try to achieve OCP.
(5) overall fibrinolytic ability (OFP) is judged: calculate by following formula: OFP=((OCP-OHP)/OCP) * 100%.
Judged result
Normal plasma OCP reference value is 4.84 ± 1.40, OHP is 2.37 ± 0.91, when OCP, OHP less than the normal reference value lower limit, there is blood coagulation disorders in prompting; Greater than the normal reference value upper limit, there is hypercoagulative state in prompting.The OFP value is less than 41.61%, and prompting fibrinolysis function reduces.
Description of drawings
Fig. 1 variable concentrations macrogol is to the figure that influences of OHP lag time;
Fig. 2 variable concentrations TF is to the figure that influences of OHP;
Fig. 3 variable concentrations UK is to the figure that influences of OHP;
Fig. 4 variable concentrations Factor IX is to the figure that influences of OCP;
Below in conjunction with accompanying drawing content of the present invention is described in further detail.
Embodiment
Below the present invention will be further described by specific embodiment, but the present invention is not limited only to following examples.
Embodiment 1: selecting 0.1mol/L Tris damping fluid is buffer system (pH 7.35), the coagulation activation thing that suitably filters out, an amount of lime chloride is as set accelerator, and an amount of Tween-80 adds PEG-6000 (5～30g/L) the conditioned reaction lag times of variable concentrations as stabilizing agent.
Fig. 1 shows the influence of variable concentrations macrogol to the OHP lag time, and the blood coagulation reaction is too fast or slow excessively, all can influence the susceptibility of test.According to measuring and calculating, suitable blood coagulation reaction lag time should be in 2～3 minutes scopes.Therefore, in the OHP test system, add a certain amount of macrogol-6000 (PEG-6000), in order to the lag time of regulating the blood coagulation reaction (lag time, LT) and improve the susceptibility of test.According to the The selection result to variable concentrations PEG-60000, maintenance reaction lag time is 20g/L 2～3 minutes PEG-6000 concentration.
Embodiment 2: selecting 0.1mol/L Tris damping fluid is buffer system (pH 7.35), PEG-600020g/L, Tween-80 10g/L, CaCl
25mmol/L, tissue factor (TF) concentration is measured normal plasma and Factor IX and is lacked blood plasma blood coagulation response curve from 0～1/200000.
Fig. 2 shows the influence of variable concentrations TF to OHP, and the OHP test request activates blood coagulation reaction comprehensively, but common coagulation activation thing and do not meet this requirement.In the presence of the TF of relative high concentration, the OHP value that normal control blood plasma and Factor IX lack blood plasma there is no significant difference.But, when TF concentration is reduced to 1/20000 dilutability when following, with contrast blood plasma relatively, Factor IX lacks the obviously reduction of OHP value of blood plasma.Illustrate that low concentration TF can activate inside and outside source coagulation pathway simultaneously.Therefore, select 1/20000 dilution TF as the coagulation activation thing.
Embodiment 3: selecting 0.1mol/L Tris damping fluid is buffer system (pH 7.35), PEG-600020g/L, Tween-80 10g/L, CaCl
25mmol/L, tissue factor (TF) concentration 1/20000, UK concentration is from 10U～200U/ml.
Fig. 3 shows the influence of variable concentrations UK to OHP, adds activator of plasminogen (UK) in experimental system, activates fibrinolytic system, in order to measure the fibrinolytic ability.Can be clear that from Fig. 3 when UK concentration during less than 30U/ml, the fibrin that is generated can not effectively be degraded; UK concentration is during greater than 50U/ml, though fibrin can be degraded rapidly, fibrinous formation obviously reduces.So we select UK concentration 40U/ml.
Embodiment 4: selecting 0.1mol/L Tris damping fluid is buffer system (pH 7.35), PEG-600020g/L, Tween-80 10g/L, CaCl
25mmol/L, tissue factor (TF) concentration 1/20000 is measured Factor IX and is lacked blood plasma, and Factor IX (relative activity 0,1.625,3.125,6.25,12.5,25,50,100) is observed the influence of different clotting factor to experiment, the sensitivity of verification method.
Fig. 4 shows the influence of variable concentrations Factor IX to OCP; lack the Factor IX (the normal pooled plasma that adds different amounts) that adds different amounts in the blood plasma in Factor IX, along with Factor IX content increases (relative activity 0,1.625; 3.125; 6.25,12.5,25; 50; 100), the OCP value progressively increases, and is accompanied by the shortening of reaction lag time.
Embodiment 5: get normal humanplasma 100 μ l, add 40U/ml UK (final concentration), add the damping fluid (0.1mol/L Tris, 20g/L PEG-6000,10g/L Tween-80,5mmol/LCaCl2, pH 7.35) that 900 μ l contain TF again, at 340nm test OHP.The OCP test is identical with above-mentioned experimental system, but reaction system does not contain UK.With with a collection of reagent replication with a blood plasma 10 times, recording the OCP coefficient of variation is 2.81% (n=10), OHP is 3.54% (n=10), adopt the reagent of 5 parts of different batches simultaneously, frozen dry blood plasma OCP, OHP that METHOD FOR CONTINUOUS DETERMINATION is same batch, and the calculating coefficient of variation, be respectively 4.86% and 4.38%.
Embodiment 6: detect PTS people at highest risk (patients with coronary heart disease, malignant tumor patient, normal physiological gestation middle and advanced stage pregnant woman) and blood coagulation disorders disease patient's (hemophilia) OHP, method is estimated.
The different case OHP of table 1., OCP, OFP and FIB test result
＊＊P＜0.01
＊P＜0.05
As seen from Table 1, compare with control group, malignant tumour, gestation, coronary heart disease patient's OCP and OHP value all obviously increase, and the OFP value lowers.Show that this type of patient's ubiquity hypercoagulative state and/or fibrinolytic vigor reduce.Hemophilia patient OCP and OHP value obviously reduce, and the OFP value also decreases.Illustrate that there is serious blood coagulation disorders in hemophilia.
1. the theoretical foundation set up of method
End/the blood coagulation theory according to the modern times, at activator of plasminogen urokinase (urokinse, UK) exist down, activate blood plasma with low concentration TF, 340nm monitor in real time fibrin form with degradation process in absorbance change, obtain overall hemostasis potential (OHP), overall blood coagulation potential (OCP) and overall fibrinolytic potential parameters such as (OFP) from the blood coagulation-fibrinolytic figure that obtains.Judge body hemostasis balance according to above-mentioned parameter, promptly judge thrombophilia and blood coagulation disorders.
2. practical work process
2.1 overall hemostatic capability (OHP) is measured
Regulate the photometer wavelength to 340nm, test duration 30min is set, regulate with damping fluid and test zero point.In cuvette, add blood plasma 100 μ l to be checked and an amount of UK (in order to activate the blood plasma plasminogen) in advance, add damping fluid (0.1mmol/L Tris, 20g/L PEG-6000,10g/L Tween-80,5mmol/L CaCl that 900 μ l contain tissue factor subsequently
2, pH 7.35), mixing places in the instrument connection then immediately, presses feeler switch.Instrument is monitored automatically and the record fibrin forms and the absorbance of degradation process changes, and calculates response curve inner area (according to total absorbance calculating), obtains OHP.
2.2 overall blood coagulation ability (OCP) is measured
Adopt and identical reaction system, experiment condition and the method for operating of above-mentioned OHP test, but reaction system does not contain plasminogen activator UK, the variation of the absorbance of monitoring 30min inner fibrin forming process is calculated response curve with interior total absorbance, tries to achieve OCP.
2.3. overall fibrinolytic ability (OFP) is judged
Calculate by following formula: OFP=((OCP-OHP)/OCP) * 100%.
Claims (1)
1, a kind of detection method of hemostatic capability is characterized in that, step is as follows:
1) by volume 9: 1 ratios of anti-coagulants of the inflexible rafter acid of venous blood and 0.13mol/L sodium prepare anticoagulation;
2) with the centrifugal anticoagulation blood sample of 3000r/min 15 minutes, it is to be checked to draw blood plasma;
3) overall hemostatic capability OHP measures:
Regulating the photometer wavelength is 340nm, test duration 30min is set, test zero point with the damping fluid adjusting, in cuvette, add blood plasma 100 μ l to be checked in advance, add 900 μ l reaction buffers subsequently, reaction buffer is 0.1mmol/L Tris, PEG-6000 5-30g/L, Tween-80 10g/L, CaCl
25mmol/L, tissue factor TF concentration is from 0-1/200000, and UK concentration is from 10U-200U/ml, and all the other are water, pH 7.35, mixing places in the photometer instrument connection then immediately, and monitoring and record fibrin form and the absorbance of degradation process changes, with the addition of per minute absorbance, totally 30 minutes, i.e. area in the response curve, this value is overall hemostatic capability OHP;
4) overall blood coagulation ability OCP measures:
Adopt and identical reaction system, experiment condition and the method for operating of above-mentioned OHP test, but reaction buffer is 0.1mmol/L Tris, PEG-6000 5-30g/L, Tween-80 10g/L, CaCl
25mmol/L, tissue factor TF concentration is from 0-1/200000, all the other are water, and pH 7.35, immediately mixing, place then in the photometer instrument connection, the variation of the absorbance of monitoring 30min inner fibrin forming process, with the addition of per minute absorbance, totally 30 minutes, be the area in the response curve, this value is overall blood coagulation ability OCP;
5) overall fibrinolytic ability OFP judges:
Calculate by following formula: OFP=((OCP-OHP)/OCP) * 100%;
Normal plasma OCP reference value is 4.84 ± 1.40, OHP is 2.37 ± 0.91, when OCP, OHP less than the normal reference value lower limit, there is blood coagulation disorders in prompting; When greater than the normal reference value upper limit, there is hypercoagulative state in prompting, when OFP value less than 41.61%, the reduction of prompting fibrinolysis function.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103604766A (en) * | 2013-11-25 | 2014-02-26 | 牡丹江友搏药业股份有限公司 | Biological quality control method of Shuxuetong injection |
| CN107300622A (en) * | 2017-08-24 | 2017-10-27 | 杨江存 | A kind of Forecasting Methodology of amniotic fluid embolism coagulation disorders |
| WO2020147252A1 (en) * | 2019-01-14 | 2020-07-23 | 浙江大学 | APPLICATION OF EXOSOME TβRII PROTEIN AS MARKER IN BREAST CANCER DETECTION KIT PREPARATION |
| CN112067575A (en) * | 2020-09-09 | 2020-12-11 | 吴俊� | Method for detecting fibrinolysis function of blood |
-
2009
- 2009-06-19 CN CNA2009100229965A patent/CN101587063A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103604766A (en) * | 2013-11-25 | 2014-02-26 | 牡丹江友搏药业股份有限公司 | Biological quality control method of Shuxuetong injection |
| CN107300622A (en) * | 2017-08-24 | 2017-10-27 | 杨江存 | A kind of Forecasting Methodology of amniotic fluid embolism coagulation disorders |
| CN107300622B (en) * | 2017-08-24 | 2020-02-18 | 杨江存 | Prediction method of amniotic fluid embolism coagulation dysfunction |
| WO2020147252A1 (en) * | 2019-01-14 | 2020-07-23 | 浙江大学 | APPLICATION OF EXOSOME TβRII PROTEIN AS MARKER IN BREAST CANCER DETECTION KIT PREPARATION |
| CN112067575A (en) * | 2020-09-09 | 2020-12-11 | 吴俊� | Method for detecting fibrinolysis function of blood |
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