INR = (patient PT/mean normal PT) ISI.

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1 The Relationship of the International Normalized Ratio () to the Prothrombin Time (PT) By: William DePond MD, President and Chief Medical Officer MEDLAB In 1983, it was determined that patients receiving long-term anticoagulant therapy may be subject to unnecessary risks of bleeding or thromboembolism because of variability in the commercial thromboplastins used to determine prothrombin time (PT) and consequent uncertainty about the actual intensity of anticoagulation. The accuracy of the PT was noted to be system-dependent showing variations in results due to marked variability in the response of commercial thromboplastin reagents to clotting factors (II, VII, X). This led to the introduction of the International Normalized Ratio (), a method developed to normalize the clotting time value by correcting for differences in reagent responsiveness. Thus, laboratory monitoring of oral anticoagulant therapy could be standardized. Each lot of thromboplastin is tested against an international standard and the relationship is expressed as the International Sensitivity Index (ISI). The uses the ISI to equate all thromboplastins to the reference thromboplastin through the following equation: = (patient PT/mean normal PT) ISI. This logarithmic relationship is easily seen in the table below comparing various ISI reagents and the variation of seconds compared to the. A patient with an of 2.0 (remember the s will be equivalent) will show a PT of 26.1 seconds using a sensitive ISI reagent (1.0) versus 16.0 seconds an insensitive ISI reagent (2.4). PT seconds vs. ISI ISI=1.0 ISI=1.6 ISI=2.0 ISI= Reagents with lower ISI values are more responsive to the effects of Warfarin therapy and thus have longer PTs. When a sample from a patient stabilized on Warfarin therapy is tested using two thromboplastin reagents with two different ISIs, the PT (seconds) will be higher with the more sensitive (lower ISI) thromboplastin. Conversely, the PT (seconds) will be lower with the less sensitive (higher ISI) reagent. Nevertheless, the values will be equivalent. The College of American Pathologists (CAP) Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy published a recommendation that laboratories use thromboplastins with an ISI between 0.9 and 1.7.

2 The use of sensitive thromboplastins is supported by how variations (or errors) in ISI values influence the. Variations in ISI can occur due to inherent imprecision in the manufacturer-assigned ISI, changes during transportation or local instrument effect. A thromboplastin reagent with a lower ISI value results in a wider range of PT ratios to obtain a therapeutic. This enhances patient safety. Despite the system, significant inter-laboratory variation and inaccuracies persist in laboratories which can be can reduce by using a sensitive thromboplastin with an instrument-specific ISI value, determining the geometric mean normal PT for each lot of thromboplastin and ensuring the calculation uses the appropriate ISI for reagent lot. If a laboratory uses the PT (seconds) to trigger a critical value call to a clinician, when a change of thromboplastin reagents is made to one with a lower ISI, the laboratory will need to adjust the PT critical value. If a laboratory uses the for triggering a critical value call, then a change in thromboplastin reagent would have little impact to critical value reporting. The normal range for the for a healthy person is Recommendations for therapeutic levels of anticoagulation based on can be found in "Antithrombotic and Thrombolytic Therapy, 8th Ed: ACCP Guidelines", published in the June 2008 (Supplement) issue of Chest. An of 2.0 to 3.0 is recommended for most indications. The guidelines are helpful but do replace clinical judgment in monitoring patients, as patients vary in their response to oral anticoagulation. Such variations in patient response may be due to intrinsic factors such as genetic factors or extrinsic factors such as medications, disease processes and/or diet. The tables below show some of these factors and their impact on a patient s response to anticoagulation therapy.

3 Cytochrome P450 2C9 and VKORC1 Mutation Analysis Clinical Significance: Warfarin (coumadin) therapy is associated with significant complications because of its narrow therapeutic index and large interpatient dosage variation necessary to achieve an optimal therapeutic response. This variation is due to both genetic and environmental factors. A promoter variant (-1639 G>A) of the Vitamin K epoxide complex subunit 1 (VKORC1) accounts for 25%-44% of this variability and variants of the cytochrome P450 enzyme 2C9 (CYP2C9) account for 10%-15% of this variability. Identification of these Warfarin sensitive variants of the VKORC1 and the CYP2C9 genes may allow a more individualized therapy and reduced risk of bleeding complications. CYP2C9*2 or CYP2C9*3, vitamin K epoxide reductase (VKORC1) Metabolize coumarins slowly Metabolize coumarins slowly Twice as likely to have a laboratory or clinical adverse event Homozygous VKORC1 promoter polymorphism 1639 G>A (aka VKOR 3673, haplotype A, or haplotype*2) Reduce Warfarin dosage Reduce Warfarin dosage compared to genotype GG patients Condition Prothrombin time Partial thromboplastin time Bleeding time Platelet count Vitamin K deficiency or prolonged prolonged unaffected unaffected Warfarin Disseminated intravascular prolonged prolonged prolonged decreased coagulation Von Willebrand disease unaffected prolonged prolonged unaffected Hemophilia unaffected prolonged unaffected unaffected Aspirin unaffected unaffected prolonged unaffected Thrombocytopenia unaffected unaffected prolonged decreased Early Liver failure prolonged unaffected unaffected unaffected End-stage Liver failure prolonged prolonged prolonged decreased Uremia unaffected unaffected prolonged unaffected Congenital afibrinogenemia prolonged prolonged prolonged unaffected Factor V deficiency prolonged prolonged unaffected unaffected Factor X deficiency as seen prolonged prolonged unaffected unaffected in amyloid purpura Glanzmann's thrombasthenia unaffected unaffected prolonged unaffected Bernard-Soulier syndrome unaffected unaffected prolonged decreased

4 Antibiotics erythromycin inhibition of metabolism Avoid if possible, otherwise decrease Warfarin metronidazole inhibition of metabolism Avoid if possible, otherwise decrease Warfarin quinolones (enoxacin > ciprofloxacin > norfloxacin > nalidixic acid > ofloxacin) inhibition of metabolism; altered protein binding Avoid if possible, otherwise decrease Warfarin trimethoprim / sulfamethoxazole inhibition of metabolism; altered protein binding Avoid if possible, otherwise decrease Warfarin rifampin induction of metabolism Monitor, dose increase probably required Analgesics Anti-inflammatory s phenylbutazone inhibition of metabolism altered protein binding aspirin (high dose) direct prolongation of prothrombin time NSAIDs (except phenylbutazone) Lipid Lowering s Avoid: anti-platelet effect, increases peptic ulceration Anti-platelet effect, increases peptic ulceration. Significant risk but low dose aspirin may be justified for some clinical indications. - minor displacement interactions Cause peptic ulceration, reversible antiplatelet effect, avoid if possible but minimal pharmacokinetic interactions. clofibrate unknown Avoid cholestyramine binds Warfarin Colestipol has less effect than cholestyramine Neurological s Barbiturates induction of metabolism Potent enzyme inducer, will require a large increase in dose over several weeks carbamazepine induction of metabolism Potent enzyme inducer, will require a large increase in dose over several weeks Gastroenterological s cimetidine inhibition of metabolism Avoid: substitute another H2 antagonist Cardiovascular s amiodarone inhibition of metabolism May require decrease in dose, consider sotalol or other agent unless extrapyramidal symptoms indicate otherwise. quinidine unknown Nutritional Supplements Social s

5 vitamin K vitamin K Avoid, except when given to correct excess anticoagulation alcohol induction/inhibition of safe if intake is < 30 Gm/day metabolism Miscellaneous s disulfuram indirect potentiation Avoid heparin direct prolongation of prothrombin time Prolongation of 10-20% if activated partial thromboplastin time is in the therapeutic range antithyroid drugs altered catabolism of clotting Increased dose may be required factors sulfinpyrazone inhibition of metabolism altered protein binding anti-platelet effect Avoid Variable Enhanced Response to Warfarin Decreased Response to Warfarin Diet Malnutrition Foods high in vitamin K such as: Beef liver Pork liver Green tea Leafy green vegetables Medications and nutritional Quinidine Indomethacin Adrenal Anti-thyroid drugs Barbiturates Estrogen supplements Medical conditions corticosteroids Reduced vitamin K absorption as occurs in: Obstructive jaundice Hepatitis Cirrhosis Aluminum hydroxide Diabetes mellitus Edema Hyperlipidemia Hypothyroidism Most errors in plasma-based coagulation testing occur in the pre-analytical phase. A proper specimen is essential for accurate test results. The following table summarizes errors that can occur in specimen collection, transport, and storage.

6 Preanalytical Factor Potential Error Recommendation Blood collection tube additive Some additives will interfere with Use a collection tube containing 3.2% sodium coagulation test results. citrate. Order of draw Using a winged blood collection device for venipuncture Collecting a coagulation specimen from a vascular access device (VAD) Underfilling the blood collection tube Specimen storage time prior to prothrombin time (PT) test Specimen storage time prior to activated partial thromboplastin time (aptt) test Contamination from other additives could interfere with coagulation test results. Air that is collected into the blood specimen from the tubing dead space will alter the blood to anticoagulant ratio and may impact test results. Heparin contamination occurs if the line had been previously flushed with heparin. Ratio of blood to anticoagulant is altered, and the test result may be incorrect. Incorrect storage will alter patient test result. Incorrect storage will alter patient test result. Draw the coagulation tube (blue top) before tubes with other additives. Use a discard tube to collect a sufficient amount of blood to fill the tubing dead space before collecting the patient's specimen. If a VAD must be used, the line should first be flushed with 5 ml of saline, and then 5 ml of blood should be collected and discarded using a non-additive or coagulation tube as the discard tube. Allow the tube to completely fill so the ratio of blood to anticoagulant is 9:1. Specimen can be stored at room temperature (18-24 C) for up to 24 hours in an unopened collection tube. If stored longer than 24 hours, remove plasma and freeze at < -20 C. Must be centrifuged and tested within four hours of collection, or the plasma removed and frozen at < -20 C. Centrifuge within one hour of collection if patient is receiving unfractionated heparin.

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