Prothrombin Time (PT) Manual Test Procedure

Principle

     The addition of pre-warmed (37°C) platelet-poor plasma to thromboplastin-calcium reagent activates the coagulation cascade at Factor VII. The time required for clot formation is recorded. Clot formation may be detected by optical or electromechanical methods using manual, semi-automated, or automated devices.

Reagents and Equipment

1. Thromboplastin-calcium reagent
2. Fibrometer system
   a. Fibrometer
   b. Thermal prep block, 37 + 1°C
   c. Automatic pipet
3. Coagulation cups, non-wettable surface
   
4. Fibro-tips

Quality Control

     Quality control materials (normal and abnormal) with established control limits should be run. NCCLS recommends controls be tested at the beginning of each testing day, followed by testing during each subsequent shift or with each batch of assays.

Specimen

     Whole blood anticoagulated with 3.2% sodium citrate is the specimen of choice. The specimen should be processed to obtain platelet-poor plasma.

Procedure (Fibrometer Testing)

1. Pre-warm a sufficient quantity of thromboplastin-calcium reagent to 37°C for the number of tests to be performed.
   
2. Pipet 0.1 mL patient sample or control sample into labeled coagulation cup using the automatic pipet.
   
3. Allow sample to pre-warm to 37°C for 1-2 minutes. No longer than five minutes.
   
4. Pipet 0.2 mL pre-warmed thromboplastin-calcium reagent into coagulation cup containing patient or control sample using the automatic pipet (switch in the "on" position). When the thromboplastin-calcium reagent is dispensed the timer will automatically start. Alternatively, the timer may be started by touching the timer plate. Reagent should be forcibly added to ensure mixing of reagent and sample.
   
5. The timer will stop when clot formation occurs.
   
6. Record time taken for clot formation.
   
7. Each sample (patient or control) should be run in duplicate.

Results
     The average of the duplicate prothrombin times is recorded to the nearest half -second. To report results in INR, use the following calculation:

INR = (patient's PT/mean PT of the normal range)^ISI

Reference Interval

     Each laboratory should establish its own reference interval following a recommended procedure. The reference interval should be re-established with changes in instrumentation, reagent lot number, or at least once a year.

Comments

1. Precision between duplicate measurements is said to be acceptable if the difference between duplicates is 10% or less of the mean of the duplicate results.
   
2. If the patient's hematocrit exceeds 55%, NCCLS recommends adjusting the amount of anticoagulant used in the collection tube to prevent over-anticoagulation of the specimen. This correction formula is:
   
   C = (1.85 x 10^-3) X (100 - Hct) X V
   
   Where: C = volume of sodium citrate, V = volume of whole blood drawn, Hct = patient's hematocrit.
   
   
3. Significant variation in PT results is observed among different commercial thromboplastin-calcium reagents due to the source of thromboplastin. To normalize these variations, each thromboplastin-calcium reagent has an established international sensitivity index (ISI) that is determined by comparison to the gold standard thromboplastin used by the World Health Organization. The reagent's ISI is used in the calculation of the international normalized ratio (INR). Failure to use the ISI established for a particular lot number of thromboplastin-calcium reagent will result in errors in the INR result. Since the INR is used to monitor patients on oral anticoagulant therapy, an error could lead to misinterpretation of the patient's anticoagulation status.
   
4. The prothrombin time is prolonged in individuals with inherited single factor deficiencies of factors X, VII, V, II, I; individuals with liver disease; individuals with vitamin K deficiency; individuals with disseminated intravascular coagulation; and individuals taking oral anticoagulants.
   
   NOTE:
   
   Potential sources of error:-
   
   
   1. Associated with specimen:- Inappropriate ratio of anticoagulant to blood.
      Failure to correct citrate volume if hematocrit >55% .
      Clotted, hemolyzed, icteric, or lipemic specimen.
      Delay in processing or testing.
      Inappropriate storage.
      
   2. Associated with reagents:- Incorrect preparation of reagents.
      Failure to properly store reagents.
      Use of reagents beyond reconstituted stability time.
      Use of reagents beyond expiration date.
      Contaminated reagents.
   3. Associated with procedure:- Incorrect temperature.
      Incorrect incubation times.
      Incorrect volumes of sample, reagents, or both.
      Improperly functioning instrument.