  The prothrombin time is therefore the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added.  The PT measures functional activity of the extrinsic and common pathways.  The PT evaluates patients suspected of having an inherited or acquired deficiency in these pathways. Protime /Prothrombin(PT)

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  Calcium ions and Thromboplastin from brain tissue (Rabbit).  Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels.  Thromboplastins were originally tissue extracts obtained from different species and different organs containing tissue factor and phospholipid. Because of the potential hazard of viral and other infections from handling human brain, it should no longer be used as a source of thromboplastin. The majority of animal thromboplastins now in use are extracts of rabbit brain or lung. PT Reagent

  The introduction of recombinant thromboplastins has resulted in a move away from rabbit brain thromboplastin. They are manufactured using recombinant human tissue factor produced in Escherichia coli and synthetic phospholipids, which do not contain any other clotting factors such as prothrombin, factor VII, and factor X.  Therefore they are highly sensitive to factor deficiencies and oral anticoagulant–treated patient plasma samples and have an International Sensitivity Index (ISI) close to 1. PT Reagent

 PT Reagent Calibration  Reagents are calibrated against standard PT reagent established by the WHO.  ISI = International Sensitivity Index.  ISI is assigned by the manufacturer for each lot of reagent using reference material from WHO.  The lower the ISI the more sensitive the Reagent 1.ISI of 1.8 to 2.4 = Low sensitivity 2.ISI of 1.4 to 1.8 = Average sensitivity 3.ISI 1.0 to 1.4 = High Sensitivity

  Principle:  The procedure uses a tissue thromboplastin reagent with CaCl (Calcium Chloride) to provide a one step procedure for evaluating plasma clotting.  This test was devised on the assumption that when an optimal amount of calcium and an excess of thromboplastin are added to decalcified plasma, the rate of coagulation depends on the concentration of prothrombin in the plasma. Principle:

  The normal values for the prothrombin time range from 10.0 to 13.0 seconds.  An elevated prothrombin time may indicate the presence of 1.Vitamin K deficiency, 2.DIC, 3.liver disease, 4.Presence of FSP’s Principle:

 5.a deficiency in one or more of the following factors: 1)Factor I (Fibrinogen) 2)Factor II (Prothrombin) 3)Factor V (Proaccelerin, Labile Factor) 4)Factor VII (Proconvertin, Stable Factor) 5)Factor X (Stuart-Prower Factor) 6)Factor XIII (Fibrin Stabilizing Factor) 6.In addition, inhibitors can cause prolonged PT’s. Principle:

  Used to monitor oral anticoagulant therapy (Warfarin / Coumadin).  When a patient who is not taking anti-coagulant drugs has signs or symptoms of a bleeding disorder  When a patient is to undergo an invasive medical procedure, such as surgery, to ensure normal clotting ability. When is it ordered?

  Reconstitute tissue thromboplastin according to instructions. Label the thromboplastin with the time, date and initials. The thromboplastin reagent is stabile for 7 days after reconstitution. Allow to sit minutes, then invert gently several times. Mix well prior to pipetting any of this reagent at any step in this procedure.  Pipet 1-2 mls, using a plastic pipet, of the tissue thromboplastin-calcium chloride reagent ( PT reagent) into a glass test tube and place in a 37 water bath incubator. Procedure

  Pipet 100 µL or.1 mL of normal control, Patient PPP into each of the test tubes.  Allow at least one (1) minute for the control to reach 37°C.  Pipet 200 µL or.2 mL of PT reagent into the tube containing the control. Start the stop watch simultaneously.  Mix the tube and leave in the water bath for a minimum of 7-8 seconds. Then remove, wipe the exterior, tilt back and forth gently until a visible clot is formed. As the clot forms, the mixture will gelatinize and may turn cloudy. Procedure

  Stop the stop watch immediately when the clot begins to form and record the time in seconds. Carry out 1 significant figure passed the decimal point. For example, if your result is seconds, report as 12.2 seconds.  Repeat the procedure for the second run of normal control. Record the time. Procedure

  If the results from run 1 and run 2 are within + 1 second from each other, average the two results and report with appropriate units. For manual PT, results should match within 1.0 second (if result is less than 20 seconds). Results over 20 seconds should match within 2.0 seconds.  If results are not within required limits, a third run should be performed and average the two that match within acceptable limits. Be sure and cross out any values you are not using for the final calculation. Include measurement unit of seconds on report sheet. Procedure

  The Mean Normal Plasma is used to evaluate routine result.  The INR is not used to evaluate ROUTINE PT results.  The International Normalization Ratio (INR) value is only used on patients who are on oral anticoagulant therapy such as Coumadin. Because these patients must be continuously monitored and their dosage adjusted accordingly, it is very important to standardize the reported PT results. Standardization ensures uniformity and safety when a traveling patient is treated in different geographical areas. Interpretation of Result

 1.Associated with specimen a.Inappropropriate ratio of anticoagulant to blood b.Failure to correct citrate volume if hematocrit > 55% c.Clotted, hemolyzed or lipemic samples d.Lack of PPP e.Delay in testing or processing f.Inappropriate storage Sources of Error

 1.Associated with storage a.Incorrect preparation of reagents b.Failure to properly store reagents c.Use of reagents beyond reconstituted stability time or expiration date d.Contaminated reagents Sources of Error

 1. Associated with procedure a.Incorrect temperature b.Incorrect incubation times c.Incorrect volumes of sample, reagents or both Sources of Error