General Laboratory: Routine Coagulation Testing Online Training
Determine the factors and pathways of the coagulation cascade that are evaluated by routine coagulation tests. Describe the routine coagulation test methods and how the results are used in the diagnosis, treatment, and monitoring of patients. This clinical laboratory training qualifies for continuing education units (CEU).
Welcome to the Routine Coagulation Testing Online Training course. This course will cover the basic principles of coagulation and routine diagnostic test methods. Select Next to continue. This course was created by: Donna Castellone, MS, MT(ASCP), SH Siemens Healthcare Diagnostics Upon successful completion of this course, you will be able to: Determine which factors and pathways of the coagulation cascade are evaluated by routine coagulation tests Describe how coagulation test results are used in the diagnosis, treatment, and monitoring of patients Describe routine coagulation test methods Select Next to continue. Routine Coagulation tests: PT, APTT, Fibrinogen, and the Thrombin Time are routine assays performed in most coagulation laboratories. Provide clinicians with important information required to assess patients bleeding or thrombotic status Important to understand the principle of the test Clotting Not Clotting Pro-coagulant vs. Anti-coagulant Coagulation Cascade: Linked reactions involving a number of protein factors Each factor is given a number (I, II, III, IV, V, VI, etc.) Each factor is activated as part of the process Activated factors are noted with “a” after the number Initiated by contact activation of High Molecular Weight Kininogen (HMWK) and Prekallikrein Monitored using APTT Initiated by Trauma or injury to vessel wall and release of thromboplastin Monitored using PT Final steps to clot formation Both PT and APTT use fibrin development as end point Risk assessment: Bleeding risk assessment Thrombotic risk assessment Diagnosis and follow-up of coagulation disorders Monitoring and adjustment of therapy: Anticoagulant therapy Substitution therapy Tests can be used to evaluate: Entire coagulation cascade Specific factor in the coagulation cascade Efficiency of interactions of clotting factors to form the fibrin clot Abnormal results may indicate: acquired or congenital factor deficiency anticoagulant therapy acquired antibody Routine coagulation tests: Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT) Fibrinogen Thrombin Time (TT) Other tests used for screening: Antithrombin (AT) D-dimer Monitors: Method: PT sample: Plasma Blue top – EDTA tube + PT reagent: Tissue factor (thromboplastin) Lipids Calcium chloride Clot Result: Time to fibrin strand formation Thromboplastin reagent Challenges: great variability of reagent sensitivity wide variability of results confusion among physicians need for standardization Variables Reagent source: origin human bovine rabbit plain / combined Reporting clotting time (sec) prothrombin ratio prothrombin index percent of activity (%) instrumentation used tissue brain lung placenta . Mathematical correction for differences in PT reagents and methods International Sensitivity Index (ISI): Relates reagent sensitivity to an international standard Provided by the reagent manufacturer with each lot of reagent Reagents with a low ISI (~1.0) are similar to the WHO standard and are considered to be sensitive Reagents with a high ISI (>2.0) are less sensitive Screen for acquired or congenital factor deficiencies Extrinsic Pathway: Factor VII Common Pathway: Factors I, II, V and X Acquired factor deficiencies Liver Disease Vitamin K Preoperative screening Monitoring anticoagulation therapy Mechanism: Interferes with the synthesis of vitamin K Used for Prophylaxis or treatment of: Venous thrombosis Pulmonary embolism Thromboembolic complications of atrial fibrillation or cardiac valve replacement Used to reduce risk of death, recurrent MI, and thromboembolic events such as stroke Therapeutic Guidelines: Effective control prevents thromboembolic complications and minimizes bleeding episodes Monitored with PT/INR Recommended therapeutic range: INR = 2.0-3.0 INR = 2.5-3.5 for certain conditions Reagents: Want ISI < 1.2 Good lot-to-lot reproducibility High sensitivity for single factors, especially FVII Heparin insensitivity during induction phase of warfarin therapy Standardized INR reporting: Uniform reporting system improves comparability of results Increased safety and effectiveness of warfarin therapy Monitors: Method: + Add CaCl2 Clot APTT sample: Plasma Blue top – EDTA tube APTT reagent: Surface activator Phospholipid Incubate 2-3 minutes Result: Time to fibrin strand formation Preoperative screening Screen for congenital or acquired bleeding disorders: Intrinsic Pathway Factors Common Pathway: Factors I, II, V and X Hemophilia A (FVIII) and B (FIX) Coagulation factor assays One-stage APTT in combination with factor deficient plasmas Monitoring un-fractionated heparin therapy Screening for lupus anticoagulants Two types: Unfractionated (UFH) Low molecular weight (LMWH) Acts by enhancing Antithrombin activity APTT can monitor use of UFH Antithrombin III Heparin Antithrombin III Since Heparin acts immediately, it is useful for: Deep venous thrombosis Pulmonary embolism Early unstable angina Acute myocardial infarction Extracorporeal circulation during surgery or dialysis Acute and chronic disseminated intravascular coagulation (DIC) Response to dose varies between patients APTT effective way to establish correct patient specific dose Adjust dose so APTT in “therapeutic range” Therapeutic range is method / system dependent Reagents: High lot-to-lot consistency Good sensitivity to: detect factor deficiencies monitor heparin therapy detect lupus anticoagulant Heparin monitoring Unfractionated heparin monitored by APTT Therapeutic range should be specific for instrument/reagents LMWH monitored by anti-Xa assay Protein synthesized in liver Deficiency impacts both PT and APTT Measured directly specific assay Deficiencies either congenital or acquired. Elevated levels due to inflammation, infection, and pregnancy Clauss Method: Modified Thrombin Time assay Clotting time inversely related to Fibrinogen concentration Standard curve relates clotting time to mg/dl fibrinogen Method: + Fibrinogen sample: Diluted Plasma Blue top – EDTA tube Reagent: Thrombin Clot Result: Time to fibrin strand formation Compared to standard curve Bleeding screen: Screen for low fibrinogen levels (bleeding risk if < 100 mg/dL) Screen for dysfunctional fibrinogen Monitoring liver function Diagnosis and monitoring the course of disseminated intravascular coagulation (DIC) Monitoring thrombolytic therapy Method: + Clot Sample: Plasma Blue top – EDTA tube Reagent: low concentration Thrombin Result: Time to fibrin strand formation Assesses concentration and function of fibrinogen Can detect issues not picked up by fibrinogen assay Reference interval is reagent / instrument dependent Highly sensitive test for residual heparin Bleeding screen: severe fibrinogen deficiency dysfunctional fibrinogen fibrinogen degradation products (e.g. consumption coagulopathy) Determine the presence of heparin Monitoring thrombolytic therapy Both assays assess fibrinogen Fibrinogen measures quantity only Thrombin Time measures quantity and functional capability Concentration of the thrombin used in reagent differentiates assays Abnormal results can be due to: Inherited or acquired fibrinogen conditions Thrombolytic therapy- breaking down in-vivo clots Antithrombotic drugs preventing clot formation Synthesized in the liver Functions to regulate / inhibit clot formation Complex with heparin enhances activity Result is inversely proportional: increased absorbance = decreased AT concentration If AT activity is <50%, superficial or deep vein thrombosis and pulmonary embolism can occur Hereditary Deficiency: Relatively rare 85% of patients experience thrombosis by age 55 Initial thrombosis often occurs between 20-30 years and reoccurs throughout lifetime Thrombosis may occur in unusual sites Newborns: normal levels are 30-50% below adult levels Antithrombin Deficiency: Liver failure Nephrotic syndrome Severe trauma or burns Disseminated intravascular coagulation and sepsis Preeclampsia Widespread (metastatic) cancer Pregnancy Medications: heparin, oral contraceptives, leukemia therapy Antithrombin is a major inhibitor of coagulation Primary action against thrombin (IIa) and factor Xa Regulates formation of fibrin Anticoagulation by unfractionated heparin requires antithrombin Deficiency of antithrombin may lead to clot formation Antithrombin deficiency may be inherited or acquired Functional antithrombin is measured using a chromogenic assay