The document discusses prothrombin time (PT) and activated partial thromboplastin time (APTT) tests, which are essential for assessing blood coagulation. It details the procedures, reagents, and interpretation of results alongside their clinical significance in diagnosing bleeding disorders and monitoring anticoagulant therapy. Additionally, it emphasizes the importance of consistency and calibration in laboratory testing to ensure accurate results.

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PT & APTT
MITHUN VENUGOPAL
HAEMATOLOGY & TRANSFUSION MEDICINE

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PROTHROMBIN TIME
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.
The prothrombin time (PT) is a global coagulation assay. A
prolongation of the PT clotting time evaluates the integrity
of the extrinsic and final common coagulation pathway.

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METHODS
1) Quicks method
2) Electromechanical: Impedance steel ball. A steel ball
rotates in magnetic field until formation of fibrin clot.
3) Optical methods
 Turbidimetric method
 Nephelometric/ light scattering

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PROTHROMBIN TIME
QUICK’S (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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REAGENTS & REQUIREMENTS
Thromboplastin-CaCl2
Control plasma
Test tubes (12mm x 75mm)
Stopwatch
Centrifuge
Micropipette
3.2% sodium citrate anticoagulated blood sample
Water bath 37o
C

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Specimen collection and
storage
Specimen collected by venepuncture using vacutainer and
mix the sample. Centrifuge at 2500 rpm -3000 rpm for 15
minutes.
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.

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SAMPLE REJECTION CRITERIA
 Haemolysed sample
Clotted sample
Inadequate sample

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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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METHOD

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PROCEDURE
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 and add 0.1ml control
plasma.
Incubate at 37o
C for 1 min.

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Add 0.2 ml of prewarmed thromboplastin reagent into the
tube.
Start the stop watch.
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.
PROCEDURE

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PROCEDURE
SL NO REAGENT TUBE-I TUBE-2 CONTROL
1 PATEINT PLASMA 0.1 ML 0.1 ML ---
2 CONTROL PLASMA --- --- 0.1 ML
INCUBATE AT 37o
C 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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INTERPRETATION

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NORMAL VALUE
11-14 Seconds
Critical values for urgent clinical notification: > 60seconds

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INR-International Normalized
Ratio
Introduced in1983 by WHO.
PT results of different labs using different thromboplastin
reagent may lead to different result and misinterpretation.
Therefore all thromboplastin reagents distributed are
calibrated against WHO reference preparation.
The calibration number is called international sensitivity
index (ISI).
INR = [PT Patient ÷ MNPT]ISI

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INR
The INR has no units (it is a ratio) and is determined to one decimal
place.
The first step of the INR calculation is to “normalize” the PT by
comparing it to the mean normal prothrombin time (MNPT), the
geometric mean of the prothrombin times of the healthy adult
population.
In the second step, this ratio is raised to a power designated the ISI, or
international sensitivity index. The ISI is a function of the thromboplastin
reagent.
Two groups of data are used to derive the ISI (i) normal healthy
individuals and (ii) patients stabilized on warfarin. Paired PT data are
obtained from multiple samples using both the working thromboplastin
reagent and the international thromboplastin standard.

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USES OF INR
Allows PT results to be compared among labs across
world.
Used to monitor patients taking oral anticoagulant and it is
not used for initial evaluation of hemostatic system.
DISADVANTAGE:
Incorrectly calculated INR values.
Incorrect ISI value assigned to thromboplastin reagent.

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CLINICAL SIGNIFICANCE
DIC
Liver diseases
Vitamin K deficiency
Oral anticoagulant therapy
FV, FVII, F X deficiency

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ACTIVATEDPARTIAL
THROBOPLASTINTIME(APTT)
The activated partial thromboplastin time (APTT) is a
global coagulation assay.
A prolongation of the APTT clotting time indicates an
abnormality of the intrinsic and final common coagulation
pathway.

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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 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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REQUIREMENTS
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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PROCEDURE
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.
0.1 ml of APTT reagent is added to the 1 first. Incubate at
37o
C , stop watch is started as soon as the reagent is added.
Incubate the tube labeled test “2” one minute after test 1.
At the end of 3 minutes add 0.1 ml of pre-warmed CaCl2 &
start the stop watch.

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PROCEDURE
Mix the tube gently in the water bath for 20 seconds.
Take out & observe for clot.
Take the average of the 2 readings.

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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 37o
C 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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NORMAL RANGE
Normal range : 25 – 35 seconds

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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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THANK YOU