Prothrombin Time (PT) Test

Clot Based Screening Tests

• Designed to assess coagulation as a laboratory procedure
• Originated from the Lee-White Method for Coagulation test
• Standard Tests include PT, PTT, and THROMBIN TIME
• The principle is based on the relationship between the time it takes for clot formation and the function of the coagulation system

Prothrombin Time

• The principle measures the clotting time of plasma after tissue factor (thromboplastin) and calcium are added
• A test of the extrinsic and common clotting pathways
• It is frequently used to monitor oral anticoagulant therapies
• Sodium Citrate is the preferred anticoagulant
• Testing should occur within 2 hours after blood collection

Prothrombin Time Reagents

• Thromboplastin reagent stored at 2-8 C
  • Tissue source: Lyophilized extract of rabbit brain
• Calcium chloride

Prothrombin Time Reagent Preparation

• Reconstitution requires 2mL of distilled water, agitate gently
• After reconstitution, the reagent remains stable for:
  • 7 days when stored at 2-8 C
  • 24 hrs at 15-25C
  • 24 hrs at 37C
• Storage between 2-8 C when not in use is recommended
• Normal Value: 10-14 seconds

Prothrombin Time Procedure

• Prewarm thromboplastin reagent at 37C
• Pipette into pre-warmed test tube
• Incubate at 3-5 minutes at 37C
• Add 0.2 mL of thromboplastin reagent
• Start timer with addition of reagent

Prothrombin Time Manual Procedure

• Check for clot formation by tilting the test tube, submerge it back in the water bath
• Record the time it takes for clot formation
• Use result to calculate the prothrombin time

Prothrombin Time Semi/Fully Automated

• Wait for timed results from the machine
• Clot formation will be displayed by the machine's time result when ready
• Normal Value= 12.6-14.6 secs

Prothrombin Time Calculating Results

• INR (International Normalized Ratio) normalizes results of prothrombin time from different labs
  • Independent of patient population, reagent, and machine used
• Useful in Coumadin Monitoring

Prothrombin Time INR formula

• INR = (PTtest/PTnormal)^ISI
  • INR= international normalized ratio
  • PTtest = tested prothrombin time
  • PTnormal = normal prothrombin time
  • ISI = international sensitivity index

International Sensitivity Index

• The International Sensitivity Index is provided by the manufacturer
  • Seen in package insert
  • The manufacturer's Tissue Factor is compared to the International Reference TF

Review question

• Formulas include patient PT and the INR
• Sample patient values are
  • Patient sample 1: 12.65
  • Patient sample 2: 12.90
  • PT normal: 12.6
  • ISI: 1.4

Materials for Prothrombin Time Test

• Two-way needle and Citrated evacuated tube
• Centrifuge
• Pipettes: 0.1 mL serological pipette and 0.2 mL serological
• Two Wassermann tubes
• 37C water bath
• Thromboplastin reagent
• Tissue paper and a timer
• Venous blood is collected in a citrate tube, then centrifuged at 1,500 RPM for 5 min
• Separate the plasma by placing it into a clean dry tube

Hemostat Prothrombin Time

• PT-SI (Hemostat thromboplastin SI) is a high sensitivity reagent to perform the one-stage prothrombin time (PT)
• Prolongation of Pt indicates either ACQUIRED OR CONGENITAL DISORDER that affect coagulation factors I, II, V, VII, and X
• It is a means to monitor patients on oral anticoagulant therapy due to reduced activity if vitamin K-dependent clotting factors
• Assays coagulation factors in both extrinsic and common pathways of coagulation

Hemostat Prothrombin Time Procedure

• Perform samples and control in duplicates
• Pre-warm the Hemostat Thromboplastin-SI reagent at 37C
• Pipette 0.1mL of plasma/control into a prearmed Test tube
• Incubate at 3-5 minutes at 37 C
• Add PT-SI reagent
• Start timer with addition of reagent
• Record the time required for clot formation
• Normal value = 10-14 seconds

Prothrombin Time: Evaluation and Clinical Significance

• The prothrombin time checks for deficiencies in the extrinsic and common pathways
• It is a diagnostic tool for coagulopathy and prolonged PT
  • Conditions tested for include Disseminated DIC, Liver Disease, Vitamin K deficiency, Single Factor deficiency (Factor X, VII / V), and Profound prothrombin deficiency and fibrinogen deficiency
• Also used for Coumadin monitoring and Pre-operative Workup

Partial Thromboplastin Time

• The test is performed by adding aPTT reagent of a plasma activator silica, kaoilin, ellagic acid or celite and phospholipids
  • The phospholipids serve as a substitute for platelets
• This mixture is incubated 3 min at 37 C for optimum activation
• The recalified incubated mixture is timed with a calcium chloride solution for clot formation

Activated Partial Thromboplastin Reagents

• APTT Reagent
  • Chloroform extract of rabbit brain
  • Ellagic acid
• CaCl2 to recalcify

Activated Partial Thromboplastin Time Procedure

• Prewarm aPTT reagent and CaCl2 at 37°C
• Pipette into pre-warmed test tube
  • 0.1 ml of Plasma/Control
• Incubate for 1-2 minutes at 37°C
• Add 0.1 ml of aPTT reagent
• Incubate at 3 minutes at 37oC
• Add 0.1 ml of CaCl2
• Start timer with addition of CaCl2

Activated Partial Thromboplastin Time Manual

• Check clot formation by tilting the test tube and submerging it back again to water bath
• Record the time required for clot formation

Activated Partial Thromboplastin Time Semi/Fully Automated

• Wait for timed results from the machine
• Normal Values= 25- 35 secs

Activated Partial Thromboplastin Time: Evaluation and Clinical Significance

• Used for suspected hemorrhagic disorders or recurrent thrombosis and prolonged aPTT
  • Including DIC, Liver Disease, Hemophilia A/B → factor VIII deficient, Rosenthal Syndrome → factor XI deficient, and Single Factor deficiencies (Factor VIII, IX, XI)
• Looks for The presence of specific inhibitors (Prolonged aPTT) like:
  • Anti-Factor VIII and IX, Lupus Anticoagulant and Interfering substances like Fibrin degradation products (FDP) or paraproteins- seen in myeloma
• Used in Heparin Therapy Monitoring

PT and Aptt Interfering Factors

• Specimen and solution variations can interfere
• UFH therapy uses use reagent known to be insensitive to UFH or one that includes a UFH neutralizer such as polybrene
• Lupus anticoagulant has invalid results, requiring use of use alternative methods
• Incorrect calibration requires analytical error checking and repeat testing
• PT, Prothrombin time; PTT, partial thromboplastin time; UFH, unfractionated heparin

Thrombin Time

• Used to measure the availability of functional fibrinogen
• Thrombin cleaves fibrinogen (fibrinopeptides A and B) to form a fibrin monomer
• These fibrin monomers aggregate to form insoluble fibrin polymers, recognized as the end product in thrombin clotting assays
• Clot formation is timed with low potency thrombin added to undiluted plasma

Materials for Thrombin Time:

• Two-way needle
• Citrated evacuated tube
• Centrifuge
• Pipettes: 0.5 mL serological and 0.6 mL serological
• Tissue paper, two Wassermann tubes, 37C water bath
• aPTT reagent, Calcium chloride, and a timer

Preparation of Thrombin Time specimen

• Collect venous blood in a citrated tube
• Centrifuge at 1,500 rpm for 5 mins
• Separate the plasma by placing it into a clean dry tube

Thrombin Time Reagents

• Thrombin time reagent
  • Bovine thrombin time reagent
  • Lyophilisate for 1 mL

Thrombin Time Procedure

• Pipette into pre-warmed test tube: Plasma/Control 0.2 mL
• Incubate at 3 minutes at 37°C
• Add 0.1 mL Thrombin Time reagent
• Start timer with the addition of reagent

Thrombin Time Manual

• Check clot formation by tilting the test tube, then submerge it back in the water bath
• Record time required for clot formation

Thrombin Time Semi/Fully Automated

• Wait until Time results are released by machine and report results to the nearest tenth of a second
• N.V.= <21 secs

Evaluation of Hemostat Thrombin Time

• A simple test that screens for conditions that can interfere with the conversion of fibrinogen into fibrin
• A low-potency fibrin is added to undiluted plasma and clot formation is timed

Thrombin Time Reagent

• Bovine thrombin time reagent
• Lyophilisate for 1 mL

Hemostat Thrombin Time Procedure

• Perform samples and controls in duplicates
• Pipette 0.2 mL of plasma/control into a prewarmed test tube
• Incubate for 3mins at 37C, then add 0.1 mL of the thrombin time reagent
• Start timer with addition of reagent
• Record the time required for clot formation
• Normal Value: 8-14 seconds

Evaluation and Clinical Significance of Thrombin Time

• Cases of low, absence, or non-functional fibrinogen include conditions causing prolonged TT
  • Hypofibrinogenemia, Afibrinogenemia, or Dysfibrinogenemia
• Presence of Anti- thrombotic materials causes prolonged TT
  • Inhibiting thrombin in cleaving the fibrinogen to form a fibrin clot or Fibrin degradation products (FDP)
  • Also conditions like Paraproteins or being treated with Heparin
• Others
  • DIC, End-stage liver disease, and Malnutrition

Other Tests for Secondary Hemostatis

• These involve venom activation or solubility testing

Reptilase Time

• Batroxobin, a thrombin-like enzyme action similar to thrombin
• Isolated from the venom of Bothrops atrox
• Converts Fibrinogen to Fibrin
• Cleaves Fibrinopeptide A only
• Insensitive to Heparin and Factor XIII deficiency but is markedly prolonged in dysfibrinogenemia and presence of FDPs

Stypven or Russel Viper Venom Test

• RVV comes Daboia russelii viper
• Activates factor X
• Was previously used as an alternative to PT and for the intrinsic test
• Detects deficiencies in common pathway
  • DRVVT (Dilute RVV Time) is used to detect Lupus Anticoagulant

Duckert's Test

• Also known as urea solubility test
• Detects clinically significant deficiency of factor XIII activity
• Clots formed in normal plasma are insoluble in 5M urea
  • Fibrin clot that has not been stabilized by factor XIIIa can be dissolved by 5M urea

Clot Retraction Time

• The normal blood clot contracts and expresses serum
• The degree of clot retraction can be measured on the basis of the amount of serum expressed
• Retraction is directly parallel to platelet count

Materials for Clot Retraction:

• Castor oil
• Cotton
• Wassermann tube
• Blood lancet
• Sahli's pipette
• Timer
• 70% isopropyl alcohol

Castor oil/Hirschboeck Method

• Pour Castor oil into a Wassermann tube up to about 1 inch from the rim
• Skin puncture and discard the first drop of blood
• Aspirate blood up to the 20 cumm mark with a Sahli's pipette
• Expel the blood into the central surface of the Castor oil by touching the tip of the pipette into the oil
• Note the time
• Place the tube on a rack and observe for the formation of dimpling or nipple-like protrusion on the surface of the drop of blood
  • This indicates the end point
• Clot retraction is reported as delayed after 1 hour, standard value is 15-45 minutes

Stefanini / Test Tube Retraction Procedure

• Perform venipuncture and place 3-5 mL of blood in a chemically clean Wassermann tube
• Close the tube with a cotton plug and place in a water bath set at 37°C
• Observe the tube after 1, 2, 16, 18, and 24 hours
• Report as no retraction, partial retraction, or complete retraction

Mac Farlane Retraction Procedure

• Fill a calibrated centrifuge tube up to the 5 mL mark with freshly drawn blood.
• Immerse a glass rod by placing it into the tube with the blood column.
• Fit a cork over the projecting end of the glass rod.
• Examine the blood for evidence of coagulation at 5-10 minute interval, with the tube in a water bath set at 37°C.
• If retraction has taken place, the clot will shrink and attach to the glass rod after one hour.
• Remove the tube from the water bath
• Pull the glass rod and the clot, remove together
• Read the volume of the serum directly in the graduated scale of the tube.