PT, APTT, TT

Prothrombin Time
• The prothrombin time: Is the time required for the plasma to clot
after an optimal concentration of thromboplastin and calcium have
been added.

• Measures the function of the Extrinsic Pathway(as well as Common

pathway).

• Sensitive to Factors I, II, V, VII, X.

• The PT evaluates patients suspected of having an inherited or

acquired deficiency in these pathways.
 History
• The Prothrombin time was discovered by Dr
Armand Quick and colleagues in 1935. It aided
in the identification of the anticoagulants
dicumarol and warfarin, and was used
subsequently as a measure of activity for
warfarin when used therapeutically.
 Normal Range
• The Reference range for Prothrombin time is
usually around 11-16 seconds( With Rabbit
thromboplastins); For recombinant human
thromboplastin it is somewhat shorter(10-12s)
 When is it ordered?

• Used to monitor oral anticoagulant therapy (Warfarin /

Coumadin).

• When a patient who is not taking anti-coagulant drugs

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.
An elevated Prothrombin time may indicate the
presence of:
Vitamin K deficiency
(Vitamin K is needed to make prothrombin and other clotting factors)
DIC
liver disease
 a deficiency in one or more of the following factors:
 I, II, V, VII, X.
 Anticoagulant (warfarin)
 Blood Sample Anticoagulation
• For the prothrombin time test and other
coagulation assay the appropriate
anticoagulant is 3·2% Tri-sodium citrate (in the
ratio of 9 volume of blood to 1 volume of
anticoagulant).
• If the tube is underfilled or overfilled with
blood, the standardized dilution of 1 part
anticoagulant to 9 parts whole blood is no
longer valid and may give erroneus reult.
 EQUIPMENTS
• WATERBATH AT 37°C
• TEST TUBE (75x10mm)
• STOPWATCH
• MICROPIPETTE
 REAGENTS
• PLATELET POOR PLASMA(PPP)
• NORMAL CONTROL PLASMA
• PT REAGENT (NEOPLASTINE Cl Plus)
- Reagent Preparation : Reagent2 (calcium) were
added to Reagent1(lyophilized thromboplastin)
and allowed the reconstituted agent to stand at
room temperature (18-25°C) for 30 minutes.Then,
swirled the reagent 1 vial gently to obtain a
homogenous suspension.
 PROCEDURE
• 0.2ml reagent was taken in test tube in
waterbath at 37°C and then added 0.1ml
platelet poor plasma and started the
stopwatch immediately. As soon as the clot
was formed in the test tube stop the
watch.Record the clotting time.
• Test was also done for control plasma.
 International normalized ratio(INR)

• INR stands for a way of standardizing the

results of prothrombin time tests, no matter
the testing method .
• The result (in seconds) for a prothrombin time
performed on a normal individual will vary
according to the type of analytical system
employed. This is due to the variations between
different batches of manufacturer's tissue factor
used in the reagent to perform the test. The INR
was devised to standardize the results.
 HISTORY
• The INR was introduced in the early 1980s, by Dr.
Jack Hirsh and colleagues at McMaster University
School of Medicine, when it turned out that there
was a large degree of variation between the
various prothrombin time assays, a discrepancy
mainly due to problems with the purity of the
thromboplastin (tissue factor) concentrate.The
INR became widely accepted worldwide,
especially after endorsement by the World Health
Organization.
 International Sensitivity Index(ISI)
• Each manufacturer assigns an ISI value for any
tissue factor they manufacture. The ISI value
indicates how a particular batch of tissue
factor compares to an international reference
tissue factor. The ISI is usually between 1.0
and 2.0. The INR is the ratio of a patient's
prothrombin time to a normal (control)
sample, raised to the power of the ISI value
for the analytical system used
INTERNATIONAL NORMALISED RATIO (INR)

INR = [PTpt] ISI

[PTRef]
PTpt – prothrombin time of patient
PTRef –prothrombin time of normal(control) sample
ISI – International Sensitivity Index
 NORMAL RANGE
• INR in absence of anticoagulation therapy is 0.8-1.2.
The target range for INR in anticoagulant use (e.g.
warfarin) is 2 to 3. In some particular situations, such
as for those with a mechanical heart valve, or
bridging warfarin with a low-molecular weight
heparin (such as enoxaparin) perioperatively cases, if
more intense anticoagulation is thought to be
required, the target range may be 2.5-3.5. An INR of
1.0 means that the patient PT is normal.A high INR
level such as INR=5 indicates that there is a high
chance of bleeding, whereas if the INR=0.5 then
there is a high chance of having a clot.
 USE
• Monitoring oral anticoagulant therapy
(eg. Warfarin);
• note that heparin will not prolong INR
(heparinase is included within the INR
reagent)
• For heparin therapy we monitor aPTT
 Activated Partial Thromboplastin
Time(APTT)
• The Activated Partial Thromboplastin Time: Is the
time required for the plasma to form fibrin clot after an
optimal concentration of phospholipid and calcium have
been added.

• Measures the function of the Intrinsic Pathway(as well as

Common pathway).

• Sensitive to Factors XII,XI,IX,VIII, I, II, V, , X.

• The APTT evaluates patients suspected of having an

inherited or acquired deficiency in these pathways.
 History

• The aPTT was first described in 1953 by

researchers at the University of North Carolina
at Chapel Hill
 NORMAL RANGE
• The reference values for the aPTT test, using
manual methods, are 26-40 seconds.
 PRINCIPLE
• It measures the clotting time by recalcification
of plasma in the presence of an optimal
concentration of cephalin (platelet substitute)
and a factor activator(kaolin) but without
added thromboplastin and indicate overall
efficiency of the intrinsic clotting system.
 EQUIPMENT
• Waterbath at 37°C
• TestTube(75x10mm)
• Stopwatch
 REAGENTS
• Platelet poor Plasma(PPP)
• Normal control Plasma
• APTT Reagent(C.K.Prest)
Reagent preparation – reagent2 (cephalin) were
added to Reagent (buffered suspension of kaolin
5mg/ml) and allowed the reconstituted reagent
to stand at room temprature (18-25°C) for
30minutes.Then swirled the Reagent 1 vial gently
to obtain homogenous suspension.
• Calcium chloride 0.025M
 PROCEDURE
• 0.1ml PPP was taken in a test tube and 0.1ml APTT
reagent was added then incubated in waterbath at
37°C for 30 minutes.Then added 0.1ml CaCl2 0.025M
and started the stopwatch immediately. Mix the tube
once, immediately after adding the calcium reagent.
Allow the tube to remain in the water bath,
approximately 20 seconds, mixing occasionally.
• After 20 seconds, remove the tube from the water
bath. Wipe off the outside of the tube. Gently tilt the
tube back and forth until a visible clot forms.As soon as
clot is formed stop the watch immediately. Clotting
time was recorded.Test was also done for control
plasma.
 Procedure

Add 100
µL CaCl2
100 µL APTT reagent Incubation at 37° C

100 µL plasma 3 min.

Time to clot
formation
 Clinical Significance:
• The aPTT is prolonged with deficiencies XII, XI, IX,
VIII,(intrinsic pathway) and X, V, II, I or XIII
(common pathway).
• Disseminated Intrravascular Coagulation
• Liver Disease
• Massive transfusion with plasma depleted red
blood cells
• A circulating Anticoagulant (inhibitor)
 Clinical Significance:
• In addition to screening for coagulation defects,
the APTT test is used to monitor heparin therapy.
• In general the aPTT of a patient on heparin therapy
should be 1½ to 2½ times normal.
 Clinical Significance:
• When the partial thromboplastin time is used in
combination with the prothrombin time, most
procoagulant disorders can be classified.
1. Plasma with a long prothrombin time and a normal partial
thromboplastin time is deficient in factor VII.
2. If both the prothrombin time and the partial
thromboplastin time are long, the plasma is deficient in X,
V, II, I or XIII.
3. If the prothrombin time is normal but the partial
thromboplastin time is long, the Plasma is deficient in
factor XII, XI, IX, or VIII.
 THROMBIN TIME
• The Thrombin Time (TT), is a blood test that measures
the time it takes for a plasma sample to clot after an
optimal concentration of thrombin has been added.
• It is a measure of conversion of Fibrinogen to Fibrin,
which is prolonged by Afibrinogemia,Abnormal
Fibrinogen, or the presence of inhibitory substances
,e.g. Fibrin degradation products,or Heparin.
• Reptilase/Ancrod, a thrombin like enzyme unaffected
by the presence of Heparin, may be used in place of
Thrombin.
 EQUIPMENTS
• Waterbath at 37°C
• Test Tube(75x10mm)
• StopWatch
 REAGENTS
• Platelet poor plasma(PPP)
• Normal control plasma
• Thrombin solution( ̴7-8NIH U/ml)
• Bufferd normal saline
 PROCEDURE
• 0.1 ml PPP was taken in test tube and added
0.1ml bufferd saline incubated for 4 minutes
at 37°C, then added 0.1ml thrombin and
started the stopwatch and recorded the time
recquired for clot formation.
 INTERPRETATION
• A patient’s TT should be within 2 sec of
control (i.e.15-19seconds). Time of 20 sec and
longer are definetely abnormal.
 Prolonged TT
• Dysfibrinogenemia,either inherited or acquired,in
liver disease or in neonates.
• Afibrinogenemia
• Hypofibrinogenemia as found in DIC and more
rarely,in conginital defect or deficiency.
• In the presence of Heparin, which interfares with
the Thrombin-Fibrinogen reaction.
• Raised concentration of FDP, as encounterd in DIC
or liver disease.
• Hypoalbuminaemia
 Sources of Error

Associated with specimen

a. Inappropropriate ratio of anticoagulant to blood
b. Failure to correct citrate volume if hematocrit > 55%
c. Clotted, hemolyzed or lipemic samples
d. Lack of PPP
e. Delay in testing or processing
f. Inappropriate storage
 Sources of Error

Associated with Reagent

a. Incorrect preparation of reagents
b. Failure to properly store reagents
c. Use of reagents beyond reconstituted stability
time or expiration date
d. Contaminated reagents
 Sources of Error

Associated with procedure

a. Incorrect temperature
b. Incorrect incubation times
c. Incorrect volumes of sample, reagents or both
 REFERENCES
• DACIE AND LEWIS – PRACTICAL
HAEMATOLOGY