凝血功能相關檢驗

凝血功能相關檢驗 Hsu Chia Chun 97.08.26 Hemostasis • Primary Hemostasis: Describes the interaction between platelets and the vessel wall. • Mechanism: – Blood vessel wall is injured. – Circulating blood is exposed to the subendothelium. – Platelets adhere to the injured area. • Von Willebrand's Factor – Mediator of platelet adhesion to subendothelial collagen. – Binds to subendothelial collagen and platelet receptors, initiating adhesion. • Platelets adhere to the underlying tissues and undergo aggregation, forming the temporary primary hemostatic plug which is short-lived (seconds to minutes). platelet activation and aggregation • Platelets contain a large number of procoagulants and clotting factors providing an optimum environment for activating the coagulation cascade (secondary hemostasis). • Secondary Hemostasis: Describes the formation of fibrin through the activation of the coagulation cascade. • Traditionally referred to as the coagulation cascade. – Intrinsic pathways (APTT) – Extrinsic pathways (PT) – Common pathways • Involves the clotting factors: – Clotting factors are produced by the hepatocytes of the liver. – Some factors are Vitamin K dependent: require Vitamin K to bind to clotting sites. • II • VII • IX • X • Fibrinolysis: Refers to the lysis of a clot or thrombus through the activation of plasminogen into plasmin. -The role of the fibrinolytic system is to destroy or lyse fibrin clots or thrombi. • Plasminogen (proenzyme) is activated into plasmin by factor XIIa or other tissue activators. • Plasmin – Biodegrades fibrinogen and fibrin into fibrin degradation products (FDP's) which can be measured. • The fibrinolytic system has built-in inhibitory mechanisms to control fibrinolysis. • Natural Anticoagulants: Oppose the effects of the components of the coagulation cascade. • Maintain the balance between clotting and bleeding. • Anticoagulants: – Antithrombin III – Heparin – Protein C • Vitamin K dependent • Inhibits Factors V and VIII – Protein S • Vitamin K dependent • Inhibits Factors V and VIII Screening Tests 1. Platelet count & morphology 2. Bleeding Time 3. Clotting Time 4. Prothrombin Time 5. Activated Partial Thromboplastin Time 6. Fbg (fibrinogen) 7. D-dimer The Sysmex® CA-1500 Analyzer (CA-1500全自動血液凝固分析儀) performs clotting, chromogenic and immunologic tests, including an automated D-Dimer assay. The system offers mid-size laboratories Multiple Dilution Analysis for detection of inhibitors during factor assays, accurate sample identification with barcode reading at the time of aspiration and cap piercing to enhance productivity. The The SysmexSysmex®® CACA--540 Analyzer 540 Analyzer (CA(CA--540540全自動血液凝固分析儀全自動血液凝固分析儀)) features automated clotting, chromogenic and immunologic detection capabilities. These onboard methodologies allow even the smallest labs to offer specialty testing including heparin anti-Xa and quantitative D-Dimer. 分析項目 • PT (prothrombin time) • APTT (activated partial thromboplastin time) • Fbg (fibrinogen) • D-dimer CA-1500測定原理 <1> 凝固法 ( PT /APTT /Fbg)：以散射光偵測檢體在未加入試劑與加入試劑後 散射光強度的變化。並計算檢體加入試劑後散色光50 %時的秒數為測定值。 ‧ 0 % ：檢體預溫後加入試劑時的散色光強度。 ‧ 50 %：檢體加入試劑後50 %凝固的秒數；取50%凝固秒數為測定值，50%凝固 測定的秒數與手工拉絲法的結果較為接近；100%：完全凝固所需時間。 百分比終點反應法 <2>免疫比濁法Immunoturbidity (D-dimer )：檢體和試劑中的單株抗體形成免 疫複合物反應，經由575 nm下測透射光改變量得知待測檢體的量。 免疫混濁法分析原理 (1) PT ((1) PT (prothrombinprothrombin time)time) 測定原理: ‧ Thromboplastin–CaCl2與測試血漿於 37℃下混合後； 纖維蛋白凝塊形成的 時間就是PT秒數，可測定外在和共同路徑和凝血因子7、10、5、2、1等因子 是否正常。 ‧ 凝固法：以散射光偵測檢體在未加入試劑與加入試劑的散射光強度的變化， 並藉由百分比終點反應法測定凝固時間。 zz For defects of deficits in Factors II, V, VII, and X and severe For defects of deficits in Factors II, V, VII, and X and severe dysdys-- or or hypofibrinogenemiahypofibrinogenemia; includes monitoring of long term anticoagulant therapy ; includes monitoring of long term anticoagulant therapy affecting these factors, such as affecting these factors, such as coumadincoumadin. . zz A PT value A PT value higher than normalhigher than normal means your blood is taking longer than usual means your blood is taking longer than usual to form a clot. This prolonged PT may happen if: to form a clot. This prolonged PT may happen if: taking taking warfarinwarfarin. . have liver disease. have liver disease. need more vitamin K. need more vitamin K. have an inherited blood disorder. have an inherited blood disorder. have had a lot of heavy bleeding recently.have had a lot of heavy bleeding recently. 臨床意義: PT試驗： (1) PT為評量外在路徑及共同路徑因子活化凝固系統，包括7、10、5、2、1 等 因子是否正常，此項試驗可測出prothrombin以診斷凝血障礙疾病。 (2) PT上升於纖維蛋白原不足、膽道阻塞、循環中的抗凝劑、DIC、凝固因子缺 乏、FDP、廔管、肝臟疾病、纖維溶解活性上升、癌症、營養不良、維他命K 缺乏、毒蛇咬到、中毒休克。 (3) PT下降於動脈栓塞、深部靜脈血管栓塞、水腫、先天性coumarin無效、脊椎 傷害、心肌梗塞、肺梗塞、移植失敗。 PT (治療用)試驗： (1)經常使用於口服抗凝劑coumarin治療的監測指標。 (2) 標準治療： ‧ INR ≦2.5內視鏡步驟出血的可能性降至最低。 ‧ INR 1.6 ~ 2.5預防中風的治療範圍。 ‧ INR 2.0 ~ 3.0預防中風的治療範圍。 ‧ INR 2.0 ~ 3.0處理深部靜脈血管栓塞、預防全身性栓塞、血管人工瓣膜。 ‧ 高劑量積極治療：INR 2.5 ~ 3.5當個案最近急性心肌梗塞、心臟瓣膜、處理 全身性栓塞、血管人工瓣膜引起的栓塞、高危險手術的預防。 (2) APTT (activated partial (2) APTT (activated partial thromboplastinthromboplastin time)time) 測定原理: ‧ 以適量Activated Cephaloplastin Reagent和已離心好的血漿反應，加入適 量鈣離子驅使內在路徑活化，最後產生不溶性fibrin。 ‧ 凝固法：以散射光偵測檢體在未加入試劑與加入試劑的散射光強度的變化， 並藉由百分比終點反應法測定凝固時間點。 • Primarily for defects or deficits in Factors VIII, IX, XI, and contact activation components, but also sensitive to all other clotting factors except VII; frequently used to monitor heparin therapy. • Hereditary: * Deficiency of factor VIII, IX, XI, XII, prekallikrein, or HMWK (PT is normal) * Deficiency of fibrinogen or factor II, V, or X (PT is also prolonged) • Acquired: * Lupus anticoagulants (PT usually normal) * Heparin (PT less affected than PTT, PT may be normal) * Liver dysfunction (PT affected earlier and more than PTT) * Vitamin K deficiency (PT affected earlier and more than PTT) * Specific factor inhibitors 臨床意義: ‧APTT可以篩檢出90％血液凝固缺損的病 人，除了VII、XIII因子外，其它因子的缺 損及抑制物質的存在都可以偵測。也經常 用於評估肝素 (heparin)治療的功效。 (3) (3) FbgFbg (fibrinogen)(fibrinogen) 測定原理: ‧ Fibrinogen 是一種血漿蛋白質，經由Thrombin的切除作用後會成為 不可溶的聚合物進而形成fibrin clot。稀釋後的血漿與大量的 thrombin反應，使fibrinogen 最後成為fibrin clot。Fibrin clot 的形成與散射光強度成正比。測定波長660 nm 散射光的變化以推算 Fibrinogen秒數。再將此秒數代入fibrinogen標準曲線來換算 fibrinogen 濃度。 臨床意義: ‧ Fibrinogen上升: Fibrinogen是一種急性期蛋白，所以在發炎和組 織壞死時血中的濃度會大量上升；口服雌激素和懷孕也會有 Fibrinogen濃度上升的現象。 ‧ Fibrinogen下降: DIC。 (4) D(4) D--dimerdimer 測定原理: ‧ 使用免疫比濁法偵測D-Dimer的濃度：polystyrene particles-murine monoclonal antibody(DD5) + accelerator + 檢體裡的D-Dimer → 形成 Immuno-complexes，於575 nm下測透射光改變量來得知D-Dimer的量。 臨床意義: ‧ D-Dimer上升：Primary and secondary fibrinolysis、thrombotic problem，例如：深部靜脈血栓、肺栓塞、DIC、懷孕、癌症、手術。 干擾因素: 1. 血量和抗凝劑比例不正確。 2. 溶血(Hemolysis)：當檢體溶血時，會影響血液凝固測試，因此當檢 體有明顯溶血，應將檢體退件，重新採檢。 3. 脂血(Lipemia)：當檢體的混濁度超過儀器的偵測極限時，報告會出 現***伴隨 Turbidity Level Over＂的異常訊號。 Evaluation of Prolonged Results • Most commercial reagents will demonstrate a prolonged APTT or PT if any factor measured by the APTT or PT, respectively, is less than 30 or 40-50% of normal. • Variables affecting coagulation tests should be ruled out first. • Lupus anticoagulant or inhibitors to factors • Coagulation factor deficiency/inhibitor • Test plus control plasma - 1:1 • Repeat PT/APTT • > 50% correction – Yes - Factor deficiency – No - inhibitor Prolonged PT/APTTProlonged PT/APTT Laboratory Methods • 1:1 Mixing Study Laboratory Methods Mixing tests with PTMixing tests with PT PT of test plasma + Aged plasma adsorbed plasma Diagnosis Corrected Not corrected X Not corrected Corrected V Not corrected Partial II Mixing tests with APTTMixing tests with APTT APTT of test plasma + Aged plasma Adsorbed plasma Diagnosis No correction Corrected VIII Corrected No correction IX Corrected Corrected XI,XII 凝血功能相關檢驗 Hemostasis platelet activation and aggregation Screening Tests The Sysmex® CA-1500 Analyzer �(CA-1500全自動血液凝固分析儀) The Sysmex® CA-540 Analyzer �(CA-540全自動血液凝固分析儀) 分析項目 CA-1500測定原理 (1) PT (prothrombin time) (2) APTT (activated partial thromboplastin time) (3) Fbg (fibrinogen) (4) D-dimer Evaluation of Prolonged Results Prolonged PT/APTT Laboratory Methods Laboratory Methods