血栓与止血检测的临床应用进展

1,血栓与止血检测的临床应用进展,上海交通大学医学院附属瑞金医院王学锋,2,一、初期止血的特殊检测,3,凝血机制 Factor VIII and vWF,4,出血时间延长,血小板计数减少,血小板计数正常,血小板减少,血小板功能缺陷,某些凝血因子缺乏,血小板增多,血管性血友病,低（无）纤维蛋白原血症,血小板计数升高,原发性,继发性,原发性,继发性,遗传性,遗传性,遗传性,获得性,获得性,获得性,初期止血缺陷的诊断,筛选试验特殊检测,5,筛选试验,血小板计数出血时间（测定器法）一期止血功能初筛仪,6,In Vivo Haemostasis,PFA100.avi,capillary 200m,aperture150m,epinephrine or ADP,platelet,vWF,erythrocyte,FLOW,- 40 mbar,collagen,membrane,PFA-100 Test Principle,lumen,fibrinogen,platelet,collagen fibrils,erythrocyte,endothelial cell,vWF,7,Revolutionizing the Clinical Routine,PFA-100 General Clinical Use,8,Sensitivity to Aspirin,A total of 120 samples were tested in duplicate between2 and 30 hours after aspirin ingestion (325 mg)Platelet dysfunction detected in 95%Aspirin indicated in 72.5%,5,87,27,Col/ADP,Col/ADP,Col/Epi,Col/Epi,CT 170 sec.,CT 114 sec.,1,Evaluate ASA-Induced Platelet Dysfunctions,PFA-100 Clinical Application,9,Sensitivity in vWD Type 1,Comparison of Bleeding Time (BT) and Closure Time (CT) in Type 1 vWD,BT (min),20,5,10,15,0,Epinephrine,ADP,CT (s),250,200,150,100,50,10,Sensitivity in vWD Type 2,Comparison of Bleeding Time (BT) and Closure Time (CT) in Type 2 variants,BT (min),20,5,10,15,0,Epinephrine,ADP,CT (s),250,200,150,100,50,Type 2A,Type 2B,Type 2N,PFA-100 Clinical Application,11,Sensitivity in acquired vWD,Comparison of Bleeding Time (BT) and Closure Time (CT) in acquired vWD,Epinephrine,ADP,CT (s),250,200,150,100,50,BT (min),20,5,10,15,0,12,Sensitivity to Hereditary Platelet Defects,0,50,100,150,200,250,300,300,250,200,150,100,50,0,Closure Time for Col/ADP (sec),Closure Time for Col/Epi (sec),Normals,Glanzmann,Bernard S.,Giant Pl.S.,Storage P.D.,Data scatter of all study groups :,13,Data scatter of all study groups :,0,50,100,150,200,250,300,300,250,200,150,100,50,0,Closure Time for Col/Epi (sec),Closure Time for Col/ADP (sec),Normals,vWD Type 1,vWD Type 2,vWD Type 3,Identify von-Willebrand-Disease,14,特殊检测,血小板聚集功能检测血小板膜特异糖蛋白检测von Willebrand Factor 相关检测,15,VWF与生理性止血,16,血管性血友病分型,1型, VWF 量部分缺乏 (75%),2型, VWF 质异常 (25%),3型, VWF 完全缺乏 (5%), 2A型,血浆中缺乏大中分子量VWF多聚物 2B型,血浆中缺乏大分子量VWF多聚物，与血小板GPIb的 结合能力增强 2M型,血浆中VWF多聚物分布正常，血小板黏附能力下降 2N型,血浆中VWF多聚物分布正常，与因子VIII结合能力 明显下降,90%以上漏诊,17,血管性血友病实验室诊断,出血筛查 PLT ; PTT; PT; Fibrinogen or TT,VWD初步诊断 VWF:Ag; VWF:Rco; FVIII,确诊和分型 VWF:Rco /VWF:Ag比值 多聚物检测 胶原结合试验 RIPA FVIII 结合试验 DNA 测序,费时,经验,设备,18,快速诊断von Willebrand Factor Antigen von Willebrand Factor Activity,vWD诊断、分型VWD1三者至少有1项减低 VWF Activity/VWFAg；F VIII/VWF Ag比值近1VWD2A、2B、2MVWF Activity/VWFAg；比值小于0.7VWD2N&血友病A F VIII/VWF Ag比值小于0.7VWD3VWF Ag极低或者检测不到血栓前状态VWF变化血管内皮损伤VWF Ag增高,+F:C,19,不同类型 vWD的特异性试验,* The cut off of 0.7 is suggested in table in Veyradier et al., Int. J. Clin. Lab. Res., 1998.,20,检测结果,21,二、二期止血的特殊检测,22,凝血因子VII缺陷症（遗传性、获得性）,外源途径缺陷,共同途径缺陷,凝血因子I、II、V、X缺陷症 （遗传性、获得性）,因子XIII定性实验阳性,内源途径缺陷,有出血症状,无出血症状,凝血因子VIII、IX、XI缺陷症 （遗传性、获得性）,凝血因子XII、PK、HMWK缺陷症 （遗传性、获得性）,凝血因子XIIII缺陷症（遗传性、获得性）,出血病的诊断,23,凝血酶生成潜力测定,24,all clinical states influencing thrombin are reflected by the ETP,Thrombin Generation,Thrombin (Factor IIa) the central enzyme of coagulation,25,凝血酶是血液凝固的核心酶,增加出血风险的任何机制都减少凝血酶的产生增加血栓风险的任何机制都增加凝血酶的产生凝血酶的增加，有利止血，但增加血栓倾向,实时的凝血酶测量是一个很好的评估止血功能的工具,26,H. C. Hemker et al., Thromb Haemost 1993,ETP: Thrombin Generation Assay,0,30,60,90,0,5,10,15,20,25,Time (min),Thrombin activity nM,ETP - area of the thrombin generation curve,Lag-phase Clotting time,a measure for concentrationand time of action,27,Evaluation of ETP (1),1. Chromogenic substrate is split by thrombin OD measured,Continuous assessment of thrombin formation and inhibition using a chromogenic substrate,2. 1st derivative thrombin generation curve,3. Area = ETP,28,快速、准确、特异性高，为临床提供更多、更直接的实验数据,凝血酶直接测定法（CAT法),29,特殊检测,凝血因子II、V、VII、X凝血因子VIII、IX、XI、XII凝血因子XIII、Fg,30,临床意义,遗传性出血病的诊断、鉴别诊断遗传性出血病预后判断遗传性出血病疗效监测特殊价值,31,出血病围手术期疗效监测,（1）达到止血的要求；（2）节省血浆制品用量1/2-1/3（3）减少经济负担；（4）减少抗体产生和输血传染病,32,肝病并发DIC的实验室诊断标准（1）plt50109/L或进行性，或以下指标有二项以上异常：-TG、PF4、TXB2、P-选择素；（2）Fg1.0g/L或进行性；（3）F：C5s,APTT 10S；（5）3P（+），FDP60mg/L或D-D；（6）凝血因子标志物：TAT、FPA、sFMC、F1+2,必备,33,三、血栓形成的检测,34,Dr. Rudolph Virchow1821-1902,AbnormalBlood Flow,AbnormalVessel Wall,AbnormalBlood,The Hypercoagulable State,Thrombosis Multigenic + Environmental Factors,35,Types of Thrombosis,Arterial: platelet-based (white)thrombusPlatelet-VWF interactions criticalAssociated with end-stage atherosclerosis,Venous: Fibrin-based (red) thrombus Coagulation factors critical Venous stasis,36,筛选试验,PFA-100测试凝血酶生成潜力测定,37,血小板功能测定PFA-100,platelet-based (white)thrombus Platelet-VWF interactions critical,38,39,40,凝血酶生成潜力测定,Fibrin-based (red) thrombus Coagulation factors critical Venous stasis,41,TIA,42,PPP (0.5pM Innovin/4M PPL)TM/APC,不服用口服避孕药,服用口服避孕药,ETP升高 (没有观察到凝血酶原时间PT),TM: 血栓调节蛋白,与凝血酶共同作用的蛋白C激活因子APC: 活化蛋白CInnovin: 一种促凝血酶原激酶，可将凝血酶原酶转变为凝血酶PPL: 促凝血磷脂,当口服避孕药时产生APC抗性,43,分类检测,44,内皮细胞的抗血栓功能,45,内皮细胞的促血栓功能,46,vWF的相关检测与应用,表3 vWF的相关检测,注：凝血酶致敏蛋白-1，血管性血友病因子裂解蛋白酶,47,细胞微粒的活性检测,48,细胞微粒的活性 促凝血活性 ：PS外露，形成一个阴离子磷脂酶表面 促血栓活性：形成内皮下胶原,血小板微粒（PMP）-纤维蛋白-GPb/a复合物；来自单核细胞的单核细胞微粒（MMP）可通过表达P-选择素糖蛋白配体-1（PSGL-1）与血栓中血小板P-选择素结合，参与血栓形成。 促血管生成和调节血管舒张活性：PMP促进人脐静脉内皮细胞的增生、迁移和管状形成；也可促进癌肿者血管增生和远程转移。微粒可下调血管松弛因子的活性而上调紧张性因子的活性,49,冠心病（CAD）:Bernal-Mizrochi等（2003）对38例心肌梗死（MI）、26例不稳定型心绞痛（UA）、20例稳定型心绞痛（SA）和42例正常人，CD31（+）的EMP依次是MIUASA,内皮细胞微粒（EMP）的应用,CAD患者中内皮细胞损伤,50,内皮细胞微粒（EMP）的应用,高血压:未经治疗的高血压患者EMP明显升高，升高幅度与血压升高呈正相关，其中CD31（+）的EMP提示微血管内皮细胞的调亡，严重而持久的CD31(+)升高提示弥漫性内皮细胞调亡 促进血栓形成,51,血小板微粒（PMP）的应用:,PTCA后不给予abciximab组外周动脉血中PMP水平较PTCA前升高；给予abciximab组PMP水平则在PTCA前后无明显变化冠脉搭桥术后，患者外周血液中PMP表达组织因子（TF）的水平升高水平高低反映血栓形成的活动度,52,凝血启动因子的检测与应用,53,以细胞为基础的止血概念,Hoffman M et al. Thromb Haemost 2001;85:958-65,起始阶段,放大阶段,凝血酶的爆发,54,临床应用 TF:A / TF:Ag增高、F:C / F:Ag增高或TFPI:A / TFPI:Ag减低是血栓形成的标志物之一常见于静脉血栓（DVT）、脓毒血症/败血症、肾病综合征、肾功能衰竭；系统性炎症反应综合征、急性呼吸窘迫综合征、弥散性血管内凝血（DIC）和恶性肿瘤等,55,纤溶启动因子的检测与应用,56,图5 纤溶启动因子的作用及调节,注：sct（tct）-PA：单链（双链）组织型纤溶酶原激活物；scu(tcu)-PA：单链（双链）尿激酶型纤溶酶原激活物；PAI1、2：纤溶酶原激活抑制物1、2；TAFI：凝血酶激活的纤溶抑制物；2-AT：2-抗纤溶酶；HMWK：高分子量激肽原,内皮细胞,内皮细胞,sct-PA,scu-PA,tct-PA,tcu-PA,PAI1,PAI2,纤溶酶原,纤溶酶,降解纤维蛋白（原）,激肽释放酶（K）,激肽释放酶原（PK）,F,表面接触,HMWK,Fa,2-AP,57,图6 抗凝启动因子的抗凝作用和调节,TAFI,胰蛋白酶、纤溶酶,TAFIa,低浓度,TM+凝血酶,高浓度,蛋白C,活化PC(APC)EPCR,PS,蛋白C抑制物（PCI）,Fva，Fa，PAI-1,58,随着年龄增长，内皮细胞的纤溶活性有减低倾向，导致老年人处于血栓前状态动脉粥样硬化患者血管内皮细胞的t-PA/u-PA的活性明显减低或消失，而纤溶抑制物（PAI、TAFI）明显增强，导致血栓形成,应用,59,抗凝启动因子的检测与应用,60,XIa,61,thrombomodulin,Protein C Anticoagulant Pathway,62,XII XIIa,XI XIa,IX IXa,X Xa,TF / VIIa,II IIa,Fibrinogen Fibrin,VIIIa+Ca+Pl,Va+Ca+Pl,63,抗凝因子活性/抗原含量减低，意味着凝血活性增强，可导致血栓形成常见于遗传性抗凝因子缺陷症和获得性血栓病，如静脉血栓、动脉血栓和微血栓等TAFI水平增高，可导致纤溶活性减低，也可引起血栓性疾病,应用,64,纤维蛋白原的相关检测和应用,65,参与血液凝固形成凝血块与血小板GPIIb/IIIa结合介导血小板聚集参与动脉粥样硬化斑块的形成及恶性肿瘤的转移参与创伤的修复和创面的愈合影响血液流变学,功能,Fg（AB）2,sFM （AB）2,Fb,凝血酶,2个A肽和2个B肽,XIIIa,66,应用,在血栓病的诊断和研究中，不仅要测定Fg含量，而且要测定Fg的功能异常纤维蛋白原血症患者55无症状，20为血栓形成，25为出血异常,67,一般疾病不能解释的出血或血栓形成,TT,正常,延长,缩短,爬虫酶时间,纤维蛋白原活性/抗原比例,排除应用右旋糖苷或羟乙基淀粉的情况,怀疑遗传性异常纤维蛋白原血症，进行家系分析，或者纤维蛋白原蛋白和基因分析,肝功能试验,降低,升高,进行家系分析，排除遗传性异常纤维蛋白原血症,怀疑遗传性异常纤维蛋白原血症，进行家系分析及纤维蛋白原蛋白和基因分析,怀疑获得性异常纤维蛋白原血症，在肝功能正常后，重复检测TT、爬虫酶时间、纤维蛋白原活性/抗原比例,正常,正常,正常,延长,图8 异常纤维蛋白原血症的实验室诊断步骤,68,异常纤维蛋白原检测,表10 异常纤维蛋白原检测与意义,69,易栓症的检测,70,检测对象的选择,50岁前曾有过反复静脉血栓栓塞症，有血栓形成阳性家族史者 新生儿发生内脏血栓、暴发性紫斑、皮肤出血性坏死时 服用华法林抗凝治疗时发生血栓形成者 复发性流产三次或以上妇女应考虑抗磷脂抗体血栓形成综合征 围产期或口服避孕药、雌性素替代疗法后发生血栓形成者,71,检测项目,1、初筛试验：全血细胞数，血涂片，血小板，APTT，PT2、易栓症试验：（APCR，FV Leiden，PC，PS，AT，狼疮抗凝物，抗心磷脂抗体） 血浆蛋白（活性及含量） 基因分析,72,检测项目,静脉血栓主要与AT、PC、PS缺陷和FVLeiden有关，仅20%PS缺陷症的年青人可能发生动脉血栓高同型半胱氨酸血症及抗磷脂抗体血栓形成综合征中则可有动脉血栓形成和静脉血栓形成,73,先作遗传表型测定，后作基因分析，有些疾病目前尚缺乏有意义的遗传表型指标，则可以直接进行基因分析，如凝血酶原G20210A在检测遗传表型中，应先检测蛋白分子的活性，随后作蛋白抗原水平测定,检测项目,74,遗传性易栓症实验诊断方法,75,PC系统可能,AT,FV Leiden凝固法缺FV血浆,PC凝固法,游离型PS凝固法,阳性,纯合子或杂合子,发色法免疫法,总PS免疫法,阳性,正常,PC缺陷症,PC缺乏,基因分析,阳性,正常,PS缺陷症,游离型PS免疫法,正常,阳性,PS缺乏,C4b结合蛋白增高,阳性,正常,AT缺陷症,FXII凝固法,PC、PSFV Leiden结果正常,狼疮抗凝物APTT+dRTT磷脂依赖性研究,FVIII凝固法,阳性,阳性,阳性,可能为获得性缺乏时,76,四、抗凝治疗的监测,77,Warfarin Mechanism of Action,78,Warfarin: Indications,Prophylaxis and/or treatment of:Venous thrombosis and its extensionPulmonary embolismThromboembolic complications associated with AF and cardiac valve replacementPost MI, to reduce the risk of death, recurrent MI, and thromboembolic events such as stroke or systemic embolizationPrevention and treatment of cardiac embolism,79,Warfarin: Major Adverse EffectHemorrhage,Factors that may influence bleeding risk:Intensity of anticoagulationConcomitant clinical disordersConcomitant use of other medicationsQuality of management,80,Mean Warfarin Daily Dose (mg)Patient Age80Gurwitz, et al, 19926.45.14.23.6ND(n=530 patients total study)James, et al, 19926.15.34.33.93.5(n=2,305 patients total study),Increasing age has been associated with anincreased response to the effects of warfarin,Gurwitz JH, et al. Ann Int Med 1992; 116(11): 901-904.James AH, et al. J Clin Path 1992; 45: 704-706.,Warfarin Dosing in Elderly Patients,81,(,),Patients PT in SecondsMean Normal PT in Seconds,INR =,ISI,INR = International Normalized Ratio ISI = International Sensitivity Index,INR Equation,82,* Effective below 2.5,Relationship Between INR and Efficacy/Safety,Low-intensity treatment:Efficacy rapidly diminishes below INR 2.0*No efficacy below INR 1.5High-intensity treatment:Safety compromised above INR 4,83,Hylek, et al, studied the risk of intracranial hemorrhage in outpatients treated with warfarin. They determined that an intensity of anticoagulation expressed as a prothrombin time ratio (PTR) above 2.0 (roughly corresponding to an INR of 3.7 to 4.3) resulted in an increase in the risk of bleeding.,Adapted from: Hylek EM, Singer DE, Ann Int Med 1994;120:897-902,Risk of Intracranial Hemorrhage in Outpatients,84,Hylek EM, et al. NEJM 1996;335:540-546.,INR below 2.0 results in a higher risk of stroke,Lowest Effective Intensity for Warfarin Therapy for Stroke Prevention in Atrial Fibrillation,85,MeanNormal(Seconds),PTR,ISI,INR,12,12,13,11,14.5,1.3,1.5,1.6,2.2,2.6,A,B,C,D,E,Blood from a single patient,PatientsPT(Seconds),16,18,21,24,38,ThromboplastinReagent,How Different Thromboplastins Influence the PT Ratio and INR,86,MeanNormal(Seconds),PTR,ISI,INR,12,12,13,11,14.5,1.3,1.5,1.6,2.2,2.6,3.2,2.4,2.0,1.2,1.0,2.6,2.6,2.6,2.6,2.6,A,B,C,D,E,Blood from a single patient,PatientsPT(Seconds),16,18,21,24,38,Thromboplastinreagent,How Different Thromboplastins Influence the PT Ratio and INR,87,问题Loss of accuracy with high ISI thromboplastinsIncorrect ISI assignment by manufacturerIncorrect calculation of INR due to failure to use proper mean normal plasma value to derive PT ratio,解决方法Use thromboplastin reagents with low ISI values (less than 1.5)Use plasma calibrants with certified INR valuesUse “mean normal” PT derived from normal plasma samples for every new batch of thromboplastin reagent,88,IndicationINR RangeTargetProphylaxis of venous thrombosis (high-risk surgery)2.03.02.5Treatment of venous thrombosisTreatment of PEPrevention of systemic embolismTissue heart valvesAMI (to prevent systemic embolism)Valvular heart diseaseAtrial fibrillationMechanical prosthetic valves (high risk)2.53.53.0Certain patients with thrombosis and the antiphospholipid syndromeAMI (to prevent recurrent AMI)Bileaflet mechanical valve in aortic position, NSR2.03.02.5,Warfarin: Current Indications/Intensity,89,Mechanical Prosthetic Heart Valves,Patient CharacteristicsRecommendationBileaflet mechanical valve in the aortic position,Goal INR 2.5; range, 2.03.0left atrium of normal size, NSR, normal ejection fractionTilting disk valve or bileaflet mechanical valve inGoal INR 3.0; range, 2.53.5*the mitral positionBileaflet mechanical aortic valve and AFGoal INR 3.0; range, 2.53.5*Caged ball or caged disk valvesGoal INR 3.0; range, 2.53.5;and aspirin therapy (80100 mg/d)Additional risk factorsGoal INR 3.0; range, 2.53.5;and aspirin therapy (81 mg/d)Systemic embolism, despite adequate therapyGoal INR 3.0; range, 2.53.5;with oral anticoagulantsand aspirin therapy (81 mg/d)* Alternative: goal INR 2.5; range, 2.03.0; and aspirin therapy (80100 mg/d),90,PT-INR 及 TGT各参数,Patients and Methods, 23例，INR：1.512.00, 39例，INR：2.013.00, 16例，INR：3.014.26,华法林组 男53例 女25例 心瓣膜置换术后 58例 房颤 20例 口服华法林时间 442月正常对照组 男24例 女24例 正常献血员 未服用过任何药物,91,INR and paramenters of TGT in different group,注：与I组比较P0.01,与II组比较P0.01,与III组比较P0.01, 与对照组比较