General Approach of Haemostasis

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General Approach of Haemostasis

• Lecture 4
• Coagulation Assay (PT)

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Protime /Prothrombin (PT)

• Prothrombin time is the time required for the
  plasma to clot after an excess of thromboplastin
  and an optimal concentration of calcium have been
  added.
• Although the PT was originally described as a
  specific, one-stage assay of prothrombin (FII),
  it is sensitive to a quantitative or qualitative
  abnormalities of any of the factors involved in
  the extrinsic and common pathways of the
  coagulation system (Factors II, V, VII, X, and
  Fibrinogen).

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• The PT used to determine the clotting tendency of
  blood, in the measure of warfarin dosage, liver
  damage, and vitamin K status.
• Occasionally, the test may be used to screen
  patients for any previously undetected bleeding
  problems prior to surgical procedures.

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The Test The Standard operating Procedure

• Procedure name
• Clinical significance
• Principle of method
• Specimen of choice
• Reagents and equipments
• Procedure
• Reference values
• Comments
• References Principle

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Principle

• When reagent thromboplastin--to which calcium has
  been added--is mixed with plasma (derived from
  sodium citrated whole blood), the time (in
  seconds) it takes for the formation of a clot is
  reported as the Prothrombin time (PT).
• Calcium is necessary for the correct orientation
  and binding of a number of complexes including
  tissue factor-VIIa, IXa-VIIIa, and Va-Xa.

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PT Reagent

• Thromborel S Reagent lyophilized human
  placental thromboplastin, calcium chloride,
  stabilizers
• Thromboplastin may be obtained from other sources
  like Rabbit brain or lung tissue
• Neoplastine C1 Plus - lyophilized fresh rabbit
  brain thromboplastin with a specific heparin
  inhibitor hydrated with a solvent containing
  calcium with stabilizers, polybrene, buffer and
  preservatives.
• (Tissue Factor, coagulation factor III )
  protein-lipid complex found in tissues outside
  blood vessels. It is the combination of both
  phospholipids and tissue factor, both needed in
  the activation of the extrinsic pathway.

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• Recombinant thromboplastin has been produced
  using human tissue factor in Escherichia coli.
  These synthetic phospholipids 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 anticoagulanttreated
  patient plasma samples and have an International
  Sensitivity Index (ISI) close to 1.

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SPECIMEN

• Citrated plasma 1 part of sodium citrate
  solution (0.11 mol/ L) to 9 part of venous blood,
  avoiding the formation of foam.
• Control normal plasma (Commercial, Pooled Plasma)

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Calibration of Reagent

• Each Thromboplastin Reagents must be 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
• ISI of 1.8 to 2.4 Low sensitivity
• ISI of 1.4 to 1.8 Average sensitivity
• ISI 1.0 to 1.4 High Sensitivity

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EQUIPMENTS

• 100 µL micropipettes (0.10 mL)
• 200 µL micropipettes (0.20 mL)
• Stop Watch.
• Thromborel S Reagent
• Racks
• Test tubes
• Waterbath (37ºC)
• Waste containers/ Biohazard bags

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Procedure

• 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 10-15 minutes,
  then invert gently several times.
• Mix well prior to pipetting any of this reagent
  at any step in this procedure.
• Prewarming of the reagent by pipetting 1-2 mls,
  using a plastic pipette, of the tissue PT reagent
  into a glass test tube and place in a 370 C
  water bath incubator.

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• Pipette 100 µL of normal control, Patient PPP
  into each of the test tubes.
• Allow at least one (1) minute to reach 37C.
• Pipette 200 µL 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.
• Stop the stop watch and record the result. If the
  results from run 1 and run 2 are within 10
  second from each other, average the two results
  and report with appropriate units.

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• 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.
• Carry out 1 significant figure passed the decimal
  point. For example, if your result is 12.23
  seconds, report as 12.2 seconds.
• Repeat the procedure for the samples and Record
  the time.

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• Results are expressed as the mean of the
  duplicate reading in
• Seconds
• Prothrombin ratio
• International Normalized Ratio (INR)

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Reference ranges

• PT 11.0 13.0 seconds.
• Therapeutic levels are at a P/C ratio of 2.0
  3.0
• CRITICAL VALUES
• PT critical value changed to gt 42.1 seconds
• INR changed to gt 4.5

(Refer to PCS Policy 7.01.05 Reporting
Critical Values for reporting guidelines)
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When is it ordered?

• Used to monitor oral anticoagulant therapy
  (Warfarin / Coumadin).
• When a patient who 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.

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An elevated Prothrombin time may indicate the
presence of

• Vitamin K deficiency
• DIC,
• Liver disease,
• Presence of FSPs
• A deficiency in one or more of the Concerning
  factors Factor I (Fibrinogen), Factor II
  (Prothrombin), Factor V (Proaccelerin, Labile
  Factor), Factor VII (Proconvertin, Stable Factor,
  Factor X (Stuart-Prower Factor, Factor XIII
  (Fibrin Stabilizing Factor)
• In addition, inhibitors can cause prolonged PTs.

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Interpretation of Result

• A Normal Plasma is used to evaluate routine
  result.
• The INR is not calculated to evaluate Routine PT
  results.
• For Patients who are on oral anticoagulant
  therapy such as Coumadin INR result must be
  reported.
• Standardization of Report from lab to lab, by
  using INR results.
• Patients with lupus anticoagulants are not be
  requested for PT as they have antiphospholipid.

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Interfering Factors

• Diet ingestion of excessive green, leafy
  vegetables will increase the absorption of vit-K,
  which promotes blood clots.
• Alcoholism, Prolonged PT levels
• Diarrhea and vomiting decrease PT because of
  dehydration.
• Quality of Vein puncture.
• Medication Antibiotics , Aspirin, Cimethidine.
• Prolonged Storage of plasma at 4o C.

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Sources of Error

• Associated with specimen (Preanalytical)
• Inappropropriate ratio of anticoagulant to blood
• Failure to correct citrate volume if hematocrit gt
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• Clotted, hemolyzed or lipemic samples
• Lack of PPP
• Delay in testing or processing
• Inappropriate storage

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Sources of Error

• Associated with Reagent (Analytical)
• Incorrect preparation of reagents
• Use of reagents beyond reconstituted stability
  time or expiration date
• Contaminated reagent.
• Associated with procedure (Analytical)
• Incorrect temperature
• Incorrect incubation times
• Incorrect volumes of sample, reagents or both

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