CN102980993A - Platelet aggregation function detection kit and detection method - Google Patents
Platelet aggregation function detection kit and detection method Download PDFInfo
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- CN102980993A CN102980993A CN2012104401399A CN201210440139A CN102980993A CN 102980993 A CN102980993 A CN 102980993A CN 2012104401399 A CN2012104401399 A CN 2012104401399A CN 201210440139 A CN201210440139 A CN 201210440139A CN 102980993 A CN102980993 A CN 102980993A
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Abstract
The present invention relates to a platelet aggregation function detection kit, and a method using the kit to detect platelet aggregation function. The detection kit mainly includes the following detection reagents: a sodium citrate whole blood activator, a fibrin activator and a platelet activator. The fibrin activator comprises: recombinant batroxobin and activated clotting factor. By using the kit of the invention to detect, blood sample processing requirements are low, repeat testing variability is small, accuracy and stability are high, use and storage are convenient, detection cost is low, and promotion is easy.
Description
Technical field
The present invention relates to the platelets analysis field, particularly, relate to the platelet aggregation detection kit, and utilize described kit to detect the method for platelet aggregation.
Background technology
Blood platelet stops blooding, keeps vascular wall integrality and some pathologic process, as playing an important role in the processes such as thrombosis, atherosclerotic, unstable angina, metastases and inflammatory reaction in physiological.Studies show that in a large number hematoblastic activation and gathering are the initiating agents that thrombus in vivo forms, and also are one of compositions of most critical in the thrombosis.Clinical common critical acute disease such as disposition sudden death, myocardial infarction, cerebral apoplexy, pulmonary infarction etc. all are thrombotic diseases.Therefore, platelet function assay has great significance to diagnosis and the treatment of early detection thrombus risk and blood platelet relevant disease.
Clinical research is the result also confirm, use the antiplatelet drugs such as aspirin, Ticlopidine, clopidogrel, platelet glycoprotein IIb/IIIa receptor antagonist (GPI) and can significantly reduce atherosclerotic thrombus risk, so Antiplatelet therapy is used widely, and obtained good effect in cardiovascular and cerebrovascular diseases one-level, secondary prevention.
But because the clinical setting complexity of cardiovascular and cerebrovascular disease is various, standard Antiplatelet therapy scheme also is not suitable for all patients.Even part patient has accepted the treatment of routine dose antiplatelet drug, Cardioversion still occurs, namely the patient is not good to the antiplatelet therapeutic response, clinical being referred to as " antiplatelet drug opposing " phenomenon.In the clinical treatment, the incidence of antiplatelet drug opposing is about 10% ~ 50%, and consequence is comparatively serious, and the 3-5 that the risk of this type of patient's main cardiovascular and cerebrovascular ischemic event (dead, myocardial infarction or palsy) is about non-opposing patient doubly.Therefore, EARLY RECOGNITION and deal carefully with the prognosis that antiplatelet drug opposing can improve Patients with Cardiovascular/Cerebrovascular Diseases to a great extent.The domestic and foreign literature report, between platelet function assay and the clinical Cardioversion good correlativity is arranged, detect by platelet aggregation, can know that patient is to the reactivity of Antiplatelet therapy, prediction patient's thrombus risk, thereby can take targetedly the individuation Antiplatelet therapy, reduce the Cardioversion complication.
At present the method that can quantize to detect platelet function commonly used mainly contains the optics turbidimetry clinically, and pore closure Time Method, thrombocytometry, blood platelet are induced and assembled turbidimetry and thrombelastogram method, are described below:
1, optics turbidimetry for Determination platelet aggregation rate (LTA) is the most classical platelet function assay method, and the goldstandard that the Chang Zuowei diagnosis research is used is fine with the correlativity of clinical events.Concrete grammar: at first separate and be rich in blood platelet blood plasma, add the polymerization that promotes behind the activator between blood platelet and the blood platelet, with the platelet poor plasma penetrability of working sample in contrast, if platelet aggregation, then penetrability reduces again.The characteristics of LTA method are to adopt the specificity activator, thereby detect the effect of different antiplatelet drugs, as the platelet aggregation rate that adopts arachidonic acid-induction can reflect body to the reactivity of aspirin for treatment, adopts ADP then mainly to reflect reactivity to clopidogrel (or other Thienopyridines medicines) treatment as derivant.Its major defect comprises: the blood sample processing requirements is finished at short notice, duplicate detection variation is large, blood sampling volume is relatively large, need to separate that to be rich in blood platelet blood plasma thereby sample preparation time long etc.
2, pore closure Time Method: the PFA-100 system as far back as nineteen ninety-five by propositions such as Kundu, the platelet function of anticoagulated whole blood is quantitatively detected.Hemostasis environment in the PFA-100 parody during injury of blood vessel, this system is controlled by microprocessor, uses disposal reaction cup, in one deck biological membrane is arranged, the surface is with collagen, and contains ADP or adrenaline.When the whole blood with the sodium citrate anti-freezing pumps out from the aperture of film, platelet adhesion reaction further activates in collagen and by ADP or adrenaline, form platelet thrombus aperture is blocked, instrument records the time of aperture total blockage automatically, and the required time is called " off-period ".Collagen and ADP are used for distinguishing congenital and posteriority dysfunction of platelet, and it is unusual that collagen and adrenaline all are fit to the Normal Human Platelets that detection aspirin induces.PFA-100 is simple to operate, and the result is accurate, is used for clinically the screening of vWD and blood platelet disorders, and the evaluation of elementary hemostasis in the monitoring of Anti-platelet therapy and the surgical procedures.Yet PFA-100 is low for the sensitivity of the diagnosis of fibrinogen deficiency disorders.
3, thrombocytometry: Plateletworks is researched and developed by U.S. Helena company.Its mechanism is similar to the platelet count instrument.Whole blood is inserted respectively in the test tube that is mixed with EDTA anti-freezing (basis contrast) or platelet activating agent (collagen, arachidonic acid, ADP), carry out respectively platelet count after 5 minutes, platelet count in the test tube that more different activators are processed and the basis contrast test tube can be calculated platelet aggregation rate (PAG).Platelet count after the original platelet count of PAG=(-gathering)/original platelet count * 100%.The advantage of Plateletworks is to adopt whole blood sample, and is easy and simple to handle, quick, but shortcoming is can't monitor aspirin to the inhibition of platelet function, and not good with the correlativity of clinical Cardioversion.
4, blood platelet induces gathering turbidimetry: VerifyNow by U.S. Accumetrics company research and development, is used for platelet function assay through drugs approved by FDA in 2006.Its principle is the globule that is mixed with in advance platelet activating agent and fibrin primordial covering in vitro.After adding behind the whole blood sample, blood platelet is subjected to the activator effect and activates, and the fibrinogen of its surperficial IIb/IIIa receptor complex and little bead surface is crosslinked, makes platelet aggregation combine in the globule surface, and in vitro light transmission strengthens.VerifyNow belongs to the other checkout equipment of bed, can be respectively with arachidonic acid, ADP and thrombin receptor activating peptide (TRAP) as activator, detect specifically blood platelet to the reactivity of aspirin, P2Y12 retarding agent and GPI.The advantage of VerifyNow technology is to adopt whole blood as sample, and whole testing process only needs 3 minutes, and is consuming time the shortest in present all platelet function assay methods.Up to now, the bibliographical information that pieces of writing existing up to a hundred are formally delivered adopts VerifyNow to detect platelet aggregation, but the method only is to detect the initial period of platelet aggregation, and testing cost is higher, is difficult in a short time promote.
5, TEG (TEG) method: German Helmut Hartert professor develops corresponding instrument in the forties in last century, its principle is in-vitro simulated slow venous blood flow, measure thrombotic time and intensity with sensor, and go out the blood clotting intensity curve by computer drawing.The probe that one seal wire links to each other is inserted in the whole blood sample, the container that whole blood sample is housed rotates between the suitable inverse time alternately with certain angle, behind the thrombosis, be bonded in detecting probe surface, and rotate therewith, clot strength is larger, then the probe motion amplitude is larger, by the filament that links to each other with probe the motion amplitude of probe is recorded, can be judged speed and the intensity of blood clotting, and then judge the size of thrombus risk.The advantage of TEG is to adopt whole blood test, except detecting platelet function, also can understand thromboclastic situation, all can estimate thrombus and bleeding risk.
U.S. Chimoio Copco Company develops the scheme (number of patent application: CN 200480012116.1) that the monitoring blood platelet suppresses on the basis of TEG method.Under the environment that sodium citrate, heparin, hirudin exist, adopt reptilase (batroxabin) to replace human thrombin, make fibrinogen change fibrin into, and add the activator such as ADP and activate blood platelet, measure maximum blood clot intensity.The activation blood coagulation XIII factor only is mentioned in a kind of alternatives of specific embodiment as a kind of activator, and nonessential material.The shortcoming that this technology exists is: accuracy and stability are lower.
Summary of the invention
One of purpose of the present invention is for the deficiencies in the prior art, and the platelet aggregation detection kit is provided, and can be used for assessing the clinically result for the treatment of of various antiplatelet drugs.
Another object of the present invention provides the method for utilizing described kit to detect platelet aggregation.
Platelet aggregation detection kit of the present invention mainly comprises following detection reagent: sodium citrate whole blood activator, fibrin activator and platelet activating agent.
Described fibrin activator contains: recombinant batroxobin (RecombinantBatroxobin) and activated clotting factor (Activated Coagulation Factor).
Described recombinant batroxobin effect is to change fibrinogen into fibrin, and it prepares in the yeast body by the mode of genetic recombination.
Wherein, described activated clotting factor contains activated clotting factor V at least, VIII, X, a kind of among the XIIIa.Their modes by genetic recombination prepare or extract from human plasma, and Activation In Vitro also carries out purifying.
Described sodium citrate whole blood activator contains: porcelain earth and lime chloride (CaCl
2), be used for activating the sodium citrate anticoagulated whole blood.
Described platelet activating agent is: arachidonic acid (AA) or its sodium salt, sylvite, adenosine diphosphate (ADP) (ADP) or its sodium salt, sylvite, thrombin receptor activating peptide (TRAP), adrenaline or collagen etc.
Wherein, described sodium citrate whole blood activator fully is dissolved in the sodium citrate anticoagulated whole blood after, wherein the final concentration of porcelain earth in this whole blood is 1 ~ 10mg/L; CaCl
2The final concentration of solution in this whole blood is 2 ~ 40mmol/L, preferred 2 ~ 15mmol/L.
Wherein, described fibrin activator fully is dissolved in the anticoagulant heparin whole blood after, the final concentration of recombinant batroxobin in this whole blood is 2 ~ 15 μ g/mL, preferred 10 ~ 15 μ g/mL; The final concentration of activated clotting factor in this whole blood is 8 ~ 50 μ g/mL, preferred 8 ~ 10 μ g/mL.
Described fibrin activator also contains: enzyme stabilizers, plastotype agent and antiseptic.
Wherein, described enzyme stabilizers is: bovine serum albumin(BSA) (BSA).Its final concentration in being dissolved in the anticoagulant heparin whole blood is 0.2% ~ 4.6%(w/v), preferred 0.3% ~ 0.6%.
Wherein, described plastotype agent is: lactose is or/and sucrose.Their final concentrations in being dissolved in the anticoagulant heparin whole blood are 0.5% ~ 2.2%(w/v), and preferred 0.5% ~ 0.8%.
Wherein, described antiseptic is sodium azide.Its final concentration in the anticoagulant heparin whole blood is 0.05% ~ 0.20%(w/v), preferred 0.05% ~ 0.07%.
Wherein, the final concentration that described arachidonic acid or its sodium salt, sylvite are dissolved in the anticoagulant heparin whole blood is 0.5 ~ 1.0mmol/L, and the final concentration that adenosine diphosphate (ADP) or its sodium salt, sylvite are dissolved in the anticoagulant heparin whole blood is 5.0 ~ 7.5 μ mol/L.The final concentration that thrombin receptor activating peptide, adrenaline, collagen are dissolved in the anticoagulant heparin whole blood is 0.3 ~ 1.8mg/L.
The detection kit of platelet aggregation of the present invention, also comprise: the mensuration cup that described sodium citrate whole blood activator is housed separately, the mensuration cup of fibrin activator is housed separately, the mensuration cup of fibrin activator and platelet activating agent is housed, and the packing box of separating and merge these mensuration cups of packing.
Above-mentioned detection reagent is single glass of single part testing package, for difference in functionality and the purposes of distinguishing each reagent, adopt measure cup with the different colours packing to show difference.
Kit of the present invention, each detects the equal freeze-drying of reagent and packs in measuring the cup cup at the end.
Kit provided by the invention by detecting hematoblastic aggregation capability, is assessed the clinical drug effect of various antiplatelet drugs, is to realize by the inhibition (inhibiting rate) that detects antiplatelet drug.
Be that the present invention also provides the application of described kit in assessment antiplatelet drug clinical efficacy.
Wherein, described antiplatelet drug is specially aspirin (Aspirin), Thienopyridines medicine, Abciximab (Abciximab), GKWEWGGPK nonapeptide, phentolamine (Phentolamine) or Propranolol (Propranolol) etc.
Further, described Thienopyridines medicine is clopidogrel (Clopidogrel), Ticlopidine (Ticlopidine), prasugrel (Prasugrel) or ADZ6140 (Ticagrelor).
Kit utilization provided by the invention is measured the method for blood viscosity and is carried out the detection of platelet aggregation, by adding excessive platelet activating agent, blood platelet corresponding membrane receptor channel activated (the corresponding different platelet membrane receptor channels of different activator are such as the arachidonic acid corresponding A
2The membrane receptor passage activates, and aspirin suppresses this passage specially), the clot intensity of (being generally thrombin activation) under the contrast blood platelet full activation state can draw the inhibition (usually being expressed as a percentage) of all kinds of antiplatelet drugs.
Concrete principle is as follows: under the anticoagulant heparin environment, fibrin ferment and other coagulation factor activities are all suppressed, by the recombinant batroxobin activation fiber proteinogen in the fibrin activator, then under the activated clotting factor effect, make fibrin monomer be cross-linked into solid reticulate texture.In this process, blood platelet is not activated, and the clot intensity of mensuration only has fibrinous contribution.
When platelet activating agent adds fashionablely, the corresponding membrane receptor channel is activated, and the clot intensity of measuring this moment just contains the contribution of fibrin and blood platelet.The value that both subtract each other namely is the independent contribution of blood platelet.When using some corresponding antiplatelet drug, (can suppress A such as aspirin
2The activation of receptor channel, clopidogrel or other Thienopyridines medicines can suppress the activation of adp receptor passage), blood platelet just descends for the contribution of clot intensity.The amplitude of weighing its decline is the inhibiting rate of medicine.Usually, the benchmark of the contribution of blood platelet in clot intensity that so that blood platelet is activated fully, records as suppressing without medicine.In practical operation, this benchmark is finished test by sodium citrate whole blood activator.The computing formula of inhibiting rate is as follows:
MA wherein
xFor the anticoagulant heparin whole blood adds the clot intensity that records behind fibrin activator of the present invention and the platelet activating agent, MA
FineFor the anticoagulant heparin whole blood only adds the clot intensity that the fibrin activator records, both subtract each other and are under the antiplatelet drug effect contribution margin of blood platelet in clot intensity; MA
AlwaysFor the sodium citrate anticoagulated whole blood adds the clot intensity that sodium citrate whole blood activator records, deduct MA
FineBe without medicine and suppress the lower contribution margin of blood platelet in clot intensity.Generally speaking, MA
Fine, MA
x, MA
AlwaysCurve opening amplitude change from small to big successively.
The method of utilizing described kit to detect platelet aggregation provided by the invention may further comprise the steps:
(1) gathers sodium citrate anticoagulated whole blood and anticoagulant heparin whole blood sample;
(2) the sodium citrate anticoagulated whole blood is injected the mensuration cup that contains sodium citrate whole blood activator, measure clot intensity MA
Always
(3) the anticoagulant heparin whole blood is injected the mensuration cup that contains the fibrin activator, measure clot intensity MA
Fine
(4) the anticoagulant heparin whole blood is injected the mensuration cup that contains fibrin activator and platelet activating agent, measure clot intensity MA
x
(5) calculate inhibiting rate according to above-mentioned formula.
The present invention compared with prior art has following advantage:
1, adopts gene recombination technology, utilize recombinant batroxobin to substitute reptilase, under the synergy of activated clotting factor, obviously strengthened the specificity of activation fiber proteinogen, greatly promote duplicate detection variability, accuracy and the stability of reagent;
2, adopt single glass of single part testing package, need not pre-treatment step, directly adding blood sample to be measured gets final product in the cup toward measuring accordingly, has has effectively reduced or remitted the manual operation error, and repeatability obviously strengthens, and makes things convenient for the user, reduced time;
3, not limited by service time behind reagent Kaifeng, namely continue service time and exceed 8 hours also unaffected, the convenient storage and transportation.
In a word, use kit of the present invention to detect, the blood sample processing requirements is low, and the duplicate detection variability is little, and Stability and veracity is high, conveniently uses and stores, and testing cost is low, is easy to promote.
Description of drawings
Fig. 1 is clot intensity MA
Fine, MA
x, MA
AlwaysTotal synoptic diagram.
Fig. 2 shows that the aspirin that detects according to embodiment 1 is to blood platelet A
2The inhibiting rate of membrane receptor passage is 69.3%.
Fig. 3 shows that the clopidogrel that detects according to embodiment 2 is 33.1% to the inhibiting rate of platelet ADP membrane receptor passage.
Fig. 4-1 is to the test result of Fig. 4-3 for detecting according to the U.S. Chimoio Copco Company kit in the Comparative Examples 1, wherein MA
FineBe respectively 20.7mm, 16.2mm and 13.2mm; The test result that Fig. 4-4 detects for the kit by the embodiment of the invention 1 to Fig. 4-6, wherein MA
FineBe respectively 17.2mm, 19.5mm and 17.2mm.
Fig. 5-1 is the stacking diagram according to two groups of contrast tests of the kit of Comparative Examples 2 U.S. Chimoio Copco Company, MA
FineBe 12.2mm, MA
xBe 31.7mm; Fig. 5-2 is the stacking diagram by two groups of contrast tests of the embodiment of the invention 1 used kit, MA
FineBe 14.7mm, MA
xBe 44.3mm.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Used concrete reagent is this area common agents that can buy from the market among the present invention, and the preparation method of kit also is the ordinary skill in the art.The thrombelastogram instrument can be bought in market and obtain.
Activated clotting factor XIIIa is available from U.S. Haematologic Technologies company.
Embodiment 1:
One, the preparation of AA activated pathway platelet aggregation detection kit
Sodium citrate whole blood activator: wherein porcelain earth is formulated as 0.12g/L with physiological saline, every glass of packing 10 μ L; CaCl
2Be formulated as 0.2mol/L with physiological saline, every glass of packing 20 μ L.The equal freeze-drying of above component was preserved in the colourless mensuration cup cup end.
The fibrin activator: wherein recombinant batroxobin is formulated as 1mg/mL with pure water, every glass of packing 5 μ L; Activated clotting factor is selected XIIIa, is formulated as 1mg/mL with pure water, every glass of packing 3.5 μ L; Enzyme stabilizers is selected bovine serum albumin(BSA) (BSA), is formulated as 20%(w/v with pure water), every glass of packing 10 μ L; Lactose is selected in the plastotype agent, is formulated as 20%(w/v with pure water), every glass of packing 10 μ L; Sodium azide is formulated as 3%(w/v with pure water), every glass of packing 8 μ L.The equal freeze-drying of above component is measured the cup cup end in blueness and is preserved.
Platelet activating agent is sodium arachidonate, is formulated as 20mmol/L with physiological saline, and every glass of packing 10 μ L measure the cup cup end with the freeze-drying of fibrin activator in purple and preserve.
The separation of said determination cup and merging are packaged in the packing box.
Two, detect aspirin to A with above-mentioned detection kit
2The inhibiting rate of membrane receptor passage
Detecting step:
(1) the same patient's of collection Aspirin sodium citrate anticoagulated whole blood and anticoagulant heparin whole blood sample;
(2) with the above-mentioned colourless clot intensity MA that cup is measured the sodium citrate anticoagulated whole blood that measures
Always
(3) with the above-mentioned blue clot intensity MA that cup is measured the anticoagulant heparin whole blood that measures
Fine
(4) measure the clot intensity MA that cup is measured the anticoagulant heparin whole blood with above-mentioned purple
x
(5) calculate medicine to hematoblastic inhibiting rate according to following formula:
Three, adopt the detecting step of above-mentioned detection kit as follows:
1) software interface setting: be chosen in three passage input patient's (Aspirin) names, test name.In the combobox of test-types, select respectively " CK-Citratedkaolin ", " A-Activated ", " AA-Activated+AA ".
2) dress cup: on three passages of thrombelastogram instrument, load successively above-mentioned colourless mensuration cup, blue mensuration cup and purple and measure cup.
3) colourless mensuration cup detects: pipette 360 μ L sodium citrate anticoagulated whole bloods and add in the colourless mensuration cup, test cup is pushed into the top, reference test bar is allocated to test position.Choose corresponding test channel, click " beginning " or " F10 " on the keyboard on the software, begin test.
4) the blue cup of measuring detects: pipette 360 μ L anticoagulant heparin whole bloods and add blue mensuration in the cup, and mixed the suction three times, the rising glass stand moves on to test position to the top with reference test bar.Choose corresponding test channel, click " beginning " or " F10 " on the keypad on the software, begin test.
5) purple is measured the cup detection: draw 360 μ L anticoagulant heparin whole bloods and add in the purple mensuration cup, and mixed suction three times, the rising glass stand moves on to test position to the top with reference test bar.Choose corresponding test channel, click " beginning " or " F10 " on the keyboard on the software, begin test.
6) finish detection: after test is finished, click " stopping " or " Stop " at software interface, shed the disposable test cup, press the Laboratory Request discard processing.
7) interpretation of result is namely checked inhibiting rate: click " combination " on the software, click the colourless test result figure that cup, blue mensuration cup and purple are measured cup that measures again, click " finishing " button and carry out compound demonstration, can check inhibiting rate.As shown in Figure 2, the aspirin that records of present embodiment is to blood platelet A
2The inhibiting rate of membrane receptor passage is 69.3%, and drug effect is arranged.
The correspondence table of inhibiting rate and drug effect
| Inhibiting rate (AA) % | Drug effect |
| ≤50% | Inhibition is poor |
| >50% | Drug effect is arranged, suppress obviously |
Embodiment 2:
One, the preparation of ADP activated pathway platelet aggregation detection kit
Other are all with embodiment 1, and difference is that platelet activating agent is ADP, are formulated as the solution of 0.2mmol/L with physiological saline, and every glass of packing 10 μ L measure a cup cup end with the freeze-drying of fibrin activator in green and preserve.
Two, detect clopidogrel (also can be other Thienopyridines medicines) to the inhibiting rate of ADP membrane receptor passage with above-mentioned detection kit
Detecting step:
(1) the same patient's of clopidogrel sodium citrate anticoagulated whole blood and anticoagulant heparin whole blood sample are taken in collection;
(2) with the colourless clot intensity MA that measures cup mensuration sodium citrate anticoagulated whole blood
Always
(3) with the blue clot intensity MA that measures cup mensuration anticoagulant heparin whole blood
Fine
(4) with the above-mentioned green clot intensity MA that cup is measured the anticoagulant heparin whole blood that measures
x
(5) calculate medicine to hematoblastic inhibiting rate according to following formula:
Three, adopt the detecting step of above-mentioned detection kit as follows:
1) software interface setting: be chosen in three passage input patient's (taking clopidogrel) names, test name.In the combobox of test-types, select respectively " CK-Citratedkaolin ", " A-Activated ", " ADP-Activated+ADP ".
2) dress cup: on three passages of thrombelastogram instrument, load successively colourless mensuration cup, blue cup and the green cup of measuring measured.
3) colourless mensuration cup detects: pipette 360 μ L sodium citrate anticoagulated whole bloods and add in the colourless mensuration cup, test cup is pushed into the top, reference test bar is allocated to test position.Choose corresponding test channel, click " beginning " or " F10 " on the keyboard on the software, begin test.
4) the blue cup of measuring detects: pipette 360 μ L anticoagulant heparin whole bloods and add blue mensuration in the cup, and mixed the suction three times, the rising glass stand moves on to test position to the top with reference test bar.Choose corresponding test channel, click " beginning " or " F10 " on the keypad on the software, begin test.
5) the green cup of measuring detects: draw 360 μ L anticoagulant heparin whole bloods and add green mensuration in the cup, and mixed the suction three times, the rising glass stand moves on to test position to the top with reference test bar.Choose corresponding test channel, click " beginning " or " F10 " on the keyboard on the software, begin test.
6) finish detection: after test is finished, click " stopping " or " Stop " at software interface, shed the disposable test cup, press the Laboratory Request discard processing.
7) interpretation of result is namely checked inhibiting rate: click " combination " on the software, click colourless cup, blue cup and the green test result figure that measures cup of measuring of measuring again, click " finishing " button and carry out compound demonstration, can check inhibiting rate.As shown in Figure 3, the clopidogrel that present embodiment records is 33.1% to the inhibiting rate of platelet ADP membrane receptor passage, and inhibition is poor.
The correspondence table of inhibiting rate and drug effect
| Inhibiting rate (ADP) % | Drug effect |
| ≤50% | Inhibition is poor |
| >50% | Drug effect is arranged, suppress obviously |
Embodiment 3
Detect Abciximab to the inhibiting rate of GPIIa-IIIb membrane receptor passage take TRAP as activator
Other are all with embodiment 1, and difference is that platelet activating agent is TRAP, and the activated pathway of detection is GPIIa-IIIb membrane receptor passage.The inhibiting rate that records is 48.2%, and inhibition is poor.
Embodiment 4
Detect phentolamine, Propranolol to hematoblastic inhibiting rate take adrenaline as activator
Other are all with embodiment 1, and difference is that platelet activating agent is adrenaline, and the activated pathway of detection is α
2The adrenocepter passage.The inhibiting rate that records is respectively 30.2% and 30.6%, and inhibition is poor.
Embodiment 5
Detect the GKWEWGGPK nonapeptide to hematoblastic inhibiting rate take collagen as activator
Other are all with embodiment 1, and difference is that platelet activating agent is collagen, and the activated pathway of investigation is collagen GPIa-IIb receptor channel.The inhibiting rate that records is 65.8%, and suppressing obviously has drug effect.
Comparative Examples 1
Repeated, stable in order to verify the kit that the present invention relates to, the platelet aggregation detection kit of producing with the U.S. Chimoio Copco Company of going on the market is kit in contrast, its component contains P1, P2 and P3, and wherein the P1 component mainly contains reptilase.
Get normal person's anticoagulant heparin whole blood, with Batroxobin cup (the blue cup of measuring) the duplicate detection clot intensity (MA of embodiment 1
Fine) three times, also detect clot intensity (MA with the P1 component in the contrast agents box simultaneously
Fine) three times, the statistics coefficient of variation (CV) separately.Reagent in the Batroxobin cup and the effect of P1 are similar, all be that fibrinogen is converted into fibrin, different is that P1 is not single glass of single part of testing package, pipette again 10 μ L to blood after need adding the water-soluble solution of 50 μ L, but the Batroxobin cup only need add blood single stepping (the reagent freeze-drying is bonded in measures the cup end in advance).
Clot intensity (the MA that Chimoio Copco Company's P1 reagent records
Fine) be respectively 20.7mm, 16.2mm and 13.2mm, CV are that 22.6%(sees that Fig. 4-1 is to Fig. 4-3).Clot intensity (the MA that kit Batroxobin cup of the present invention records
Fine) be respectively 17.2mm, 19.5mm and 17.2mm, CV are that 7.4%(sees that Fig. 4-4 is to Fig. 4-6).Find out obviously that from above-mentioned accompanying drawing kit of the present invention result in duplicate detection is more stable, the CV value of clot intensity is lower.
Comparative Examples 2
For confirming the superiority of recombinant batroxobin of the present invention, it is mainly contained reptilase with the reagent component P1(of U.S. Chimoio Copco Company) compare experiment.
Control group: test 360 μ L normal person anticoagulant heparin whole bloods and add the P1(of U.S. Chimoio Copco Company production by its reagent instructions operation).
Of the present invention group: the Batroxobin cup (the blue cup of measuring) that adds the embodiment of the invention 1 at 360 μ L normal person anticoagulant heparin whole bloods.
First group of contrast test: measure respectively the clot intensity of control group and of the present invention group, the result sees respectively the MA among Fig. 5-1 and Fig. 5-2
Fine, control group MA wherein
Fine=12.2mm, of the present invention group of MA
Fine=14.7mm.
Second group of contrast test: except in 360 μ L normal person anticoagulant heparin whole bloods, adding the mentioned reagent, the final concentration that also adds respectively equivalent is the adrenaline of 6.5mmol/L, also measure the clot intensity of control group and of the present invention group, the result sees respectively (the MA among Fig. 5-1 and Fig. 5-2
x) control group MA wherein
x=31.7mm, of the present invention group of MA
x=44.3mm.
Fig. 5-1 and Fig. 5-2 is respectively the stacking diagram of two groups of contrast tests, by it seems among the figure, no matter whether adds adrenaline, substitutes reptilase with recombinant batroxobin and all can obtain larger MA value and more level and smooth curve shape.
In a word, the present invention compared with prior art improves significantly.
Although, above with a general description of the specific embodiments, confirmatory experiment, the present invention is described in detail for Comparative Examples, but on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (12)
1. a platelet aggregation detection kit mainly comprises following detection reagent: sodium citrate whole blood activator, fibrin activator and platelet activating agent; Contain in the described fibrin activator: recombinant batroxobin and activated clotting factor.
2. kit according to claim 1 is characterized in that, described activated clotting factor is selected from: activated clotting factor V, VIII, X, one or more among the XIIIa.
3. kit according to claim 1 and 2 is characterized in that, contains in the described sodium citrate whole blood activator: porcelain earth and lime chloride.
4. kit according to claim 1 and 2 is characterized in that, described platelet activating agent is: arachidonic acid or its sodium salt, sylvite, adenosine diphosphate (ADP) or its sodium salt, sylvite, thrombin receptor activating peptide, adrenaline or collagen.
5. kit according to claim 3, it is characterized in that, after described sodium citrate whole blood activator was dissolved in the sodium citrate anticoagulated whole blood, wherein the final concentration of porcelain earth in this whole blood was 1 ~ 10mg/L, and the final concentration of lime chloride in this whole blood is 2 ~ 40mmol/L.
6. kit according to claim 1 and 2, it is characterized in that, after described fibrin activator was dissolved in the anticoagulant heparin whole blood, wherein the final concentration of recombinant batroxobin in this whole blood was 2 ~ 15 μ g/mL, and the final concentration of activated clotting factor in this whole blood is 8 ~ 50 μ g/mL.
7. according to claim 1,2 or 6 described kits, it is characterized in that described fibrin activator also contains: enzyme stabilizers, plastotype agent and antiseptic.
8. kit according to claim 7 is characterized in that, described enzyme stabilizers is bovine serum albumin(BSA), and the final concentration that it is dissolved in the anticoagulant heparin whole blood is 0.2% ~ 4.6%; Described plastotype agent is lactose or/and sucrose, and the final concentration that they are dissolved in the anticoagulant heparin whole blood is 0.5% ~ 2.2%; Described antiseptic is sodium azide, and the final concentration that it is dissolved in the anticoagulant heparin whole blood is 0.05% ~ 0.20%.
9. kit according to claim 4, it is characterized in that, the final concentration that described arachidonic acid or its sodium salt, sylvite are dissolved in the anticoagulant heparin whole blood is 0.5 ~ 1.0mmol/L, the final concentration that adenosine diphosphate (ADP) or its sodium salt, sylvite are dissolved in the anticoagulant heparin whole blood is 5.0 ~ 7.5 μ mol/L, and the final concentration that thrombin receptor activating peptide, adrenaline, collagen are dissolved in the anticoagulant heparin whole blood is 0.3 ~ 1.8mg/L.
10. the described kit of any one according to claim 1 ~ 9, it is characterized in that, this kit also comprises: the mensuration cup that described sodium citrate whole blood activator is housed, the mensuration cup of fibrin activator is housed, the mensuration cup of fibrin activator and platelet activating agent is housed, and the packing box of separating and merge these mensuration cups of packing; Described mensuration cup is distinguished with different colours, and each detects the equal freeze-drying of reagent and packs in measuring the cup cup at the end.
11. the application of the described detection kit of claim 1 ~ 10 any one in assessment antiplatelet drug clinical efficacy.
12. utilize kit claimed in claim 10 to detect the method for platelet aggregation, it is characterized in that, may further comprise the steps:
(1) gathers sodium citrate anticoagulated whole blood and anticoagulant heparin whole blood, all contain antiplatelet drug in two kinds of whole bloods;
(2) the sodium citrate anticoagulated whole blood is injected the mensuration cup that contains sodium citrate whole blood activator, measure clot intensity MA
Always
(3) the anticoagulant heparin whole blood is injected the mensuration cup that contains the fibrin activator, measure clot intensity MA
Fine
(4) the anticoagulant heparin whole blood is injected the mensuration cup that contains fibrin activator and platelet activating agent, measure clot intensity MA
x
(5) calculate the inhibiting rate of antiplatelet drug according to following formula:
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