PROTHROMBIN TIME

Prothrombin time is the plasma clotting time obtained when excessive

thromboplastin and optimum calcium are added to citrated plasma under
standardized conditions. PT is an essential test of the extrinsic pathway of
clotting and of the common pathway. It detects deficiencies in factors I, II V,
VII, and X. The test is least sensitive to factor II and is insensitive to factor IX
and to other factors involved in the intrinsic thromboplastin generation.

Prothrombin time is frequently used to monitor oral anticoagulant therapy.

Factors II, V, VII and X are inhibited by oral anticoagulant.

Sodium citrate is the preferred anticoagulant. The test should be performed

within two hours after blood collection.

Preparation of specimen for PT:

1.

Collect venous blood in a citrated tube.

2.
3.

Centrifuge at 1500 rpm for 5 minutes.

4.
5.

Separate the plasma by placing it into a clean dry tube.

6.

Hemostat Prothrombin Time

Principle:
The one-stage PT measures the clotting time of plasma after adding a source
of tissue factor and calcium. The recalcification of plasma in the presence of
tissue factor generates activated factor X, with the consequent formation of
thrombin and a fibrin clot.

Test procedure:

1.

Performs samples and controls in duplicate.

 2.
3.

Prewarm the hemostat thromboplastin reagent at 37C

4.
5.

Pipette 0.1 ML of plasma or control in a prewarmed tube.

6.
7.

Incubate at 3-5 minutes at 37C

8.
9.

Add the reagent.

10.
11.

Start the timer with the addition of reagent.

12.
13.

Record the time required for clot formation.

14.

Normal Value : 10-14 seconds

QUESTIONS:

1.

When is prothrombin time test being requested?

2.
3.

What does prothrombin time indicate?

4.
 5.

What is the importance of incubation at 37 C the reagent and the

specimen or control?

6.
7.

Give at least three conditions wherein prothrombin time is prolonged.

8.
9.

How is protime reported?

10.
11.

Why is protime employed in monitoring patients who is on warfarin

therapy?

12.
13.

What does INR means?

14.
15.

What happens when PT INR is high? If low?

16.
17.

What will happen if the blood sample was processed for PT after 3
hours of collection?

18.
HEMATOLOGY II
LABORATORY ACTIVITY FOR APTT

Activated partial thromboplastin time is a routine test for screening

coagulation disorders in the intrinsic pathway by measuring factors VIII, IX, XI, XII
and Prekallikrein or Fletcher Factor but not platelets and Factor XIII. APTT is also
used to detect the presence of circulating anticoagulants or inhibitors, and to monitor
heparin therapy.
Sodium citrate is the preferred anticoagulant. The test should be performed within
two hours after blood collection.

Preparation of specimen for APTT

1.

Collect venous blood in a citrated tube.

2.
3.

Centrifuge at 1500 rpm for 5 minutes.

4.
5.

Separate the plasma by placing it into a clean dry tube.

6.

HEMOSTAT ACTIVATED PARTIAL THROMBOPLASTIN TIME

Determination of Activated Partial Thromboplastin Time Utilizing Ellagic Acid

Activator

The activated partial thromboplastin time (aPTT) is a simple and versatile test which
is sensitive to deficiencies of all plasma clotting factors except Factor VII. However, it
is principally used to detect deficiencies in stage 1 of the coagulation mechanism,
namely Factors VIII, IX, XI and Prekaliikrein (or Fletcher Factor).

Test Principle

The HemoStat aPTT - EL test is performed by adding aPTT reagent containing a

plasma activator and phospholipids to the test specimen; the phospholipids serve as
a substitute for platelets. This mixture is incubated for three minutes at 37 degrees
Celsius for optimum activation. The incubated mixture is then recalcified with a
calcium chloride solution and clot formation is timed.

The aPTT-EL reagent can also be used to perform quantitative factor assays.

Procedure:
 1.

Perform samples and controls in duplicates.

2.
3.

Prewarm the HemoStat CaCl2 (0.02 M) to 37 C.

4.
5.

Pipette into 0.1 mL of plasma/control prewarmed test tube.

6.
7.

Incubate for 1-2 minutes at 37 degree celsius.

8.
9.

Add 0.1 mL aPTT-EL reagent.

10.
11.

Incubate for 3 minutes at 37 degree Celsius.

12.
13.

Add 0.1 ml of prewarmed CaCl2.

14.
15.

Start timer with addition of CaCl2.

16.
17.

Record time required for clot formation.

18.

Calculate the mean time of duplicate aPTT determinations for each test plasma and
report to the nearest 0.1 seconds.

Normal reference range : 23.4-36.2 seconds

QUESTIONS:

1.

Give some conditions in which APTT is prolonged.

2.
3.

What does activated partial thromboplastin indicate?

4.
5.

Why is APTT employed in monitoring a patient who is on heparin therapy?

6.
7.

What are the other tests that might be requested along with APTT?

8.
9.

What factors might affect the result of APTT?

10.
11.

Give at least one method on APTT determination and state the reagents and
procedures used.

12.
LABORATORY TESTS FOR SECONDARY HEMOSTASIS

1.COAGULATION OR CLOTTING TIME

- measures period to clot after it has been removed from the body
- detects coagulation factor deficiency
2. PROTHROMBIN TIME – PROTIME-PT
- evaluates the generation of thrombin and the formation of fibrin via
the extrinsic and common pathway.
- detects extrinsic and common pathway factors deficiency
- thromboplastin reagent is used
- thromboplastin is a mixture of tissue factor, phospholipid and
calcium ions
- the time required for a fibrin clot to form
3. ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)
- measures the time required to generate thrombin and fibrin
polymers via the intrinsic and common pathway.
-calcium ions and phospholipids are added to blood plasma
- reflects the activity of prekallikrein, HMWK, factors XII, XI, VIII,V,II
AND I
4. THROMBIN TIME – measures the rate of thrombin induced cleavage
of fibrinogen to fibrin monomers and subsequent polymerization to
form insoluble fibrin clot
- detects fibrinogen deficiency
5. REPTILASE TIME
- reptilase – enzyme found in the venom of Bothrops atrox snake
which is thrombin like in nature
- unaffected by heparin therapy
- detect fibrinogen deficiency
6. UREA SOLUBILITY TEST / DUCKERT’S TEST
- detects Factor XIII deficiency
- reagent used : 5M urea

FACTOR PT APTT
DEFICIENCY
I prolonged prolonged

II prolonged prolonged

V prolonged prolonged

VII prolonged normal

VIII normal prolonged

IX NORMAL PROLONGED

X PROLONGED PROLONGED

XI NORMAL PROLONGED

XII NORMAL PROLONGED

QUESTIONS:

1.

How does thrombin time differs from

reptilase time?
 2.

3.

What are the coagulation factors that

are both prolonged in PT and APTT in
case of deficiencies? Please explain
why.
4.

DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION

SCREENING TESTS

Abnormal APTT only – detects the deficiency of

Factors VIII, IX, XI, XII
Abnormal PT only – detects deficiency in
Factor VII
Abnormal PT & APTT – detects deficiency in
Factors X, V, II, I
- DIC, liver disease and Vit. K deficiency

Coagulation laboratory tests

Mixing studies
Definition / general
•Mixing studies are typically used to investigate abnormal
clotting time results
•Mixing studies help distinguish clotting time prolongation
due to a coagulation factor deficiency or an inhibitor
(specific or nonspecific)
•Mixing study may direct further coagulation testing but it is
not by itself diagnostic

Essential features
•Mixing studies help distinguish clotting time
prolongation due to a coagulation factor deficiency or
an inhibitor, e.g. lupus anticoagulant
•To perform a mixing study, mix patient plasma and
normal pooled plasma and measure the clotting time
that was initially prolonged
•If the clotting time corrects, this suggests a factor
deficiency; if the clotting time does not correct, this
suggests presence of a circulating inhibitor
•Mixing study may direct further coagulation testing but
it is not by itself diagnostic, e.g. may aid in selecting
factor assays.

Differential diagnosis of prolonged PT or aPTT incorporating mixing study results

(correction versus noncorrection)

Clotting time Mixing study Key differential diagnoses

prolonged result

Prothrombin time (PT) Corrects •Factor deficiency (VII, X, V, II)

•Warfarin
•Vitamin K deficiency
•Liver disease

Does not correct •Specific factor inhibitor (VII, X, V, II)

•Direct Xa inhibitor

Activated partial Corrects •Factor deficiency (VIII, IX, XI, contact factors)
thromboplastin time •Rarely, von Willebrand disease (if VIII is low
(aPTT) enough to prolong aPTT)

Does not correct •Specific factor inhibitor (VIII, IX, XI, contact
factors)
•Heparin
•Direct thrombin inhibitor
•Lupus anticoagulant
PT and aPTT Corrects •Common pathway factor deficiency (X, V, II)
•Warfarin (high doses) or superwarfarin
•Vitamin K deficiency
•Disseminated intravascular coagulation
•Liver disease

Does not correct •Common pathway factor inhibitors (X, V, II)

•Direct thrombin inhibitors
•Direct Xa inhibitors
•Lupus anticoagulant (rarely)
•Heparin (very high doses)

Specimen
•Platelet poor plasma in 3.2% sodium citrate
Principles
•To perform a mixing study, mix patient plasma and
normal pooled plasma (NPP) and measure the clotting
time that was initially prolonged
•In factor deficiency: NPP adds sufficient clotting factors
to overcome the deficiency and correct the clotting time
•If an inhibitor is present, it typically inhibits the clotting
factors in patient plasma and NPP so the clotting time
remains prolonged
•Inhibitors can be specific inhibitors (antibodies directed
against a specific coagulation factor), nonspecific
inhibitors (e.g. lupus anticoagulant) or medications such
as heparin, direct thrombin inhibitors or direct Xa
inhibitors
•Mixing study results aid in selection of further
coagulation testing, such as assays for specific factor
deficiencies or inhibitors.
 QUESTIONS

1.

What is mixing studies?

2.
3.

Give at least two advantages and disadvantages.

4.

FIBRINOLYSIS
- physiological process that removes insoluble fibrin deposits by
enzymatic digestion of the stabilized fibrin polymers.
- as healing occurs, clots themselves are dissolved by plasmin

PLASMIN – digest fibrin and fibrinogen by hydrolysis to produce

progressively smaller fragments
- a slow acting process gradually dissolves away the clot as
tissue repair takes place

PLASMIN - particulate matter being phagocytized by

mononuclear phagocytic cells

- active enzyme of fibrinolysis

- responsible for fibrin degradation
- its precursor is the PLASMINOGEN

PLASMINOGEN – normally in the plasma

- synthesized in the liver
- its activation to plasmin is the result of proteolytic enzymes,
known as plasminogen activators
2 types of plasminogen activators:
1.Endogenous – e.g. tissue type plasminogen activator, urokinase
2.Exogenous – e.g.streptokinase, acyl- plasminogen streptokinase
activator complex
 ASSAYS FOR FIBRIN FORMATION
1.Fibrinogen levels –useful in detecting deficiencies of fibrinogen
quantitated by the following
●Precipitation or denaturation methods
●Fibrin clot density method
●Coagulable protein assays
●Immunological assays

2. Thrombin time – determines the rate of thrombin-induced

cleavage of fibrinogen to fibrin monomers
3. Latex D –Dimer Assay –
D-dimers are evidence of intravascular fibrin formation

For D-dimers to be formed, three major hemostatic stages must

be functioning
oCoagulation to form a clot
oCovalent cross linking of fibrin by activated factor XIII
oFibrinolysis to dissolve a fibrin clot into smaller fragments
4.Determination of Lysis Time
oEuglobulin Lysis Test
- euglobulin : proteins that precipitate when plasma is diluted
with water and acidified
5. Determination of Lysis Products
oProtamine Sulfate Dilution Test
oEthanol Gelation Test

ANTICOAGULANT THERAPY
1.Heparin – intravenous anticoagulant
- acts as anticoagulant by accelerating the binding of
antithrombin to target enzymes
- action: inhibits thrombin
- neutralized by : Protamine Sulfate
 2. Warfarin/Coumadin/Coumarin – oral anticoagulant
- Action : Vitamin K antagonist that interfere with the normal
synthesis of Factors II, VII, IX, X as well as Protein C and Protein
S
3. Low Molecular Weight Heparin – less capable than the
standard heparin to activate resting platelets

QUESTIONS

1.

What is fibrinolysis? What is its importance

in hemostasis and coagulation?
2.
3.

What is the function of an oral

anticoagulant?

4.

COAGULATION PROCEDURES
SPECIMEN QUALITY
●Minimum tissue trauma
●Avoidance of hemolysis
ANTICOAGULANTS
●Sodium citrate
●3.2% buffered sodium citrate – standard coagulant for
coagulation studies
●One part of anticoagulant to nine parts of whole blood
●Coagulation tests are sensitive to excess of citrate
 Effect of pH
●Changes in the pH of samples prolong clotting time
●Samples should remain in unopened tubes if testing cannot be
done immediately
Effect of temperature
●V and VIII deteriorate at room temperature
●VII and XI tend to be prematurely activated at 4C

SPECIMEN HANDLING
●Once sample is in vitro, changes begin to occur and labile factors
such as factor VIII begin to deteriorate
●PT test should be done within 24 hours and APTT should be
completed within 4 hours
●Samples should be held for a maximum of 2 hours at 4C

●If testing is not conducted, plasma should be separated from the

cells by centrifugation to produce platelet poor plasma
●Specimen can be stored at -20C for up to 2 months and -70C for
6 months

SOURCES OF ERROR
●Collection
●Temperature
●pH
●Reagent concentration

QUESTIONS:

1.

How does pH affect the clotting time?

2.