Prothrombin time and aptt

1. PT & APTT
P. SUNIL KUMAR
Haematology & Transfusion medicine
St.John’s Medical College
Bangalore
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2. Importance of PT/ Clinical Significance
• Prothrombin time (PT) is a blood test that
measures how long it takes blood to clot.
• A Prothrombin time test can be used to check
for bleeding problems.
• PT is also used to check whether medicine to
prevent blood clots is working.
• A PT test may also be called an INR test.
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3. PROTHROMBIN TIME- ( Extrinsic)
• QUICK (one stage )
method.
• Introduced by Dr. Armand
Quick in 1935.
• Time required for clotting
of citrated plasma after
addition of calcium and
tissue thromboplastin.
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4. Automated method
1) Electromechanical
 Impedance steel ball
a steel ball rotates in magnetic field until
formation of fibrin clot.
2) Optical methods-
 Turbidimetric method
 Nephelometric/ light scattering
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5. PT by Quick Onestage Method
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6. Reagents& Requirements
• Thromboplastin -CaCl2mixture reagent)
• Control plasma
• Test tubes, 12mm x 75mm
• Stopwatch
• Centrifuge
• Micropipette
• Micropipette tips
• 3.2% sodium citrate anticoagulated blood sample
• Water bath 37oC
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7. Principle :
• The calcium in whole blood is bound by
sodium citrate, thus preventing coagulation.
Tissue Thromboplastin, to which calcium has
been added, is mixed with the plasma, and
the clotting time is noted.
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12. Specimen collection and storage :
• Specimen collected by veinpuncture using
vacutainer and mix the sample .Centrifuge at
2500 rpm -3000 rpm for 15 minutes.
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13. Specimen required
• Blood anticoagulation with 3.2% sodium
citrate in the ratio of 1 part of citrate to 9
parts of blood is used .
• It is important that this ratio be maintained
accurately. An anticoagulant correction is
required when PCV is high or low.
• Formula for PCV Correction -100 -PCV for 1 ml
blood 595-PCV
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15. Rejection criteria
• Haemolysed sample.
• Clotted sample.
• Inadequate sample.
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16. Quick’ Method (Manual )
• Centrifuge the blood sample at 2500 -3000 RPM for 15
minutes. Separate the plasma from the cells as soon as
possible.
• Label 2 test tubes as test tube No.1 & 2 .
• Add 0.1 ml of patient plasma to each
• Label another test tube as control.
• Add control plasma 0.1 ml .
• Incubate at 37oC for 1 min.
• Add 0.2 ml of prewarmed thromboplastin reagent into
the tube.
• Start the stop watch.
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17. • Mix the tube and shake it in water bath for 5-6
sec, take out the test tube & observe for clot
formation against light.
• Run the duplicate test & control in the same
way.
• Take the average of the two test readings.
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18. PROCEDURE
SL NO REAGENT TEST – I TEST - II CONTROL
1 PATEINT PLASMA 0.1 ML 0.1 ML --
2 CONTROL PLASMA --- ---- 0.1 ML
INCUBATE AT 37oC FOR 60 SEC OR 1
MIN
3 CaCl2 0.025M 0.1 ML 0.1 ML 0.1 ML
START STOP WATCH
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19. Biological reference Interval:
or
Normal Range
• 11-14 Sec.
• Critical values for urgent clinical notification :
> 60sec.
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20. Inr- international normalized ratio
• Introduced in1983 by WHO
• PT results of diff labs using diff thromboplastin
reagent may lead to diff result even when plasma
warfarin conc. is same, so leads to missinterpretation
• Therefore all thromboplastin reagents distributed are
caliberated against WHO reference preparation
• The caliberation no: is called international sensitivity
index (ISI)
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21. • INR= PT patient ISI
geometric mean of
normal PT
ISI value is provided by manufacturer
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22. Uses of inr
• Allows PT results to be compared among labs
accros world
• Used to monitor patients taking oral
anticoagulant, it is not used for initial evaluation
of hemostatic system.
DISADVANTAGE-
Incorrectly calculated INR values
Incorrect ISI value assinged to thromboplastin
reagent
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23. Clinical significance/ Increased (PT)in
• DIC
• Liver diseases
• Vitamin K deficiency
• Oral anticoagulant therapy
• FV, FVII, F X deficiency
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24. ACTIVATED PARTIAL THROBOPLASTIN
TIME (APTT). Intrinsic
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25. Principle :
• The calcium in a whole blood sample is bound by sodium citrate,
thus preventing coagulation.
• The plasma, after centrifugation, contains all intrinsic coagulation
factors except calcium and platelets. In the APTT test, partial
thromboplastin (a phospholipid substitute) and an activator (to
ensure maximum activation) are added to the plasma allowing the
coagulation cascade to begin.
• During incubation, Factors XII, PK and XI are activated, building up
the levels of XIa in the reaction tube. Once CaCl2 is added, the rest
of the coagulation cascade is allowed to continue and timing of the
event is obtained. The time required for the plasma to clot is the
activated partial thromboplastin time.
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27. Type of specimen :
• 3.2 % tri sodium citrate anti-coagulated
sample, random sample.
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28. Specimen collection and storage :
• Sample collected by vein puncture in the
citrate vacutainer and mixed gently by
inverting the tube several times. Separate the
plasma by centrifuging at 2500-3000 RPM for
15 minutes. Stable for 4 hours at 4oC and 1
month at -30oC.
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29. Rejection criteria :
• Hemolysed sample.
• Clotted sample.
• Inadequate sample.
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30. Reagents required.
• Phospholipid or Partial thromboplastin
containing an activator (commercially available)
• Platelet poor plasma from the patient & control
plasma.
• CaCl2 0.025M solution freshly prepared.
• Control samples (normal and abnormal)
• Test tubes, 12mm x 75mm
• Stopwatch
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31. PROCEDURE
• 1. 0.1 ml test plasma in 2 tubes test 1 & 2 ,
add 0.1 ml of control plasma in 2 tubes
labeled control 1 & 2.
• 2. 0.1 ml of APTT reagent is added to the 1
first. Incubate at 37oC , stop watch is started
as soon as the reagent is added. Incubate the
tube labeled test “2” one minute after test 1.
• 3. At the end of 3 minutes add 0.1 ml of
prewarmed CaCl2 & start the stop watch.
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32. • 4. Mix the tube gently in the water bath for 20
seconds.
• 5. Take out & observe for clot.
• 6. Take the average of the 2 readings.
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33. PROCEDURE
SL NO REAGENT TEST - 1 TEST- II CONTROL - I CONTROL -II
1 PATIENT
PLASMA
0.1 ML 0.1 ML - -
2 CONTROL
PLASMA
- -- 0.1 ML 0.1 ML
3.
APTT REAGENT 0.1 ML 0.1 ML 0.1 ML 0.1 ML
INCUBATE AT 37oC FOR 180 SE OR 3 MIN
4 CaCl2 0.025M 0.1 ML 0.1 ML 0.1 ML 0.1 ML
START STOP
WATCH
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34. Normal range
• Normal range : 25 – 35 seconds
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35. Clinical significance
• DIC
• Haemophilia A & B
• VWD
• Liver diseases
• Massive transfusion of whole blood
• Administration of Heparin
• F IX, F XI, F XII
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37. THANKS
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