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General Approach of Haemostasis

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General Approach of Haemostasis Lecture 4: Coagulation Assay (PT) Recombinant Thromboplastin: producing a sensitive human thromboplastin reagent from a non ... – PowerPoint PPT presentation

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Title: General Approach of Haemostasis


1
General Approach of Haemostasis
  • Lecture 4
  • Coagulation Assay (PT)

2
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).

3
  • 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.

4
6
5
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

6
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.

7
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8
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.

9
  • 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.

10
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)

11
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

12
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

13
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.

14
  • 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.

15
  • 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.

16
  • Results are expressed as the mean of the
    duplicate reading in
  • Seconds
  • Prothrombin ratio
  • International Normalized Ratio (INR)

17
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)
18
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19
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.

20
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.

21
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.

22
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.

23
Sources of Error
  • Associated with specimen (Preanalytical)
  • Inappropropriate ratio of anticoagulant to blood
  • Failure to correct citrate volume if hematocrit gt
    55
  • Clotted, hemolyzed or lipemic samples
  • Lack of PPP
  • Delay in testing or processing
  • Inappropriate storage

24
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

25
Thank you
http//site.iugaza.edu.ps/wael/courses/practical-h
aemostasis-and-thrombosis http//site.iugaza.edu
.ps/ialaswad/courses/practical-hemostasis-thrombos
is/lectures/
  • Next Lecture

26
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