HEMATOLOGY II – Laboratory
M1.5 EXPLAINNNG THE PRINCIPLES & PROCEDURES OF
LABORATORY DAGNOSTIC TESTS EVALUATING FIBRINOLYTIC
SYSTEM
PROTHROMBIN TIME (PT)
❖ It is the plasma clotting time obtained when excessive
thromboplastin and optimum calcium are added to citrated
plasma under standardized conditions.
❖ Anticoagulant of the tube: sodium citrate (blue)
Anticoagulant to blood ratio: 1:9
❖ Oral anticoagulant therapy: warfarin
❖ It is essentially a test of the extrinsic pathway of clotting and
the common pathway.
❖ It detects deficiencies in factors I, II, V, VII, and X.
❖ It is least sensitive to factor II and insensitive to factor IX and
to other factors involved in the intrinsic thromboplastin
generation.
❖ It is frequently used to monitor oral anticoagulant therapy.
(Factors II, IX, VII, and IX are inhibited by oral
anticoagulants)
❖ Vitamin K dependent coagulation factor: 9,10,7,2
❖ It should be performed within two (2) hours after blood
collection.
❖ Prolonged PT is seen in Vitamin K deficiency, certain liver
diseases, specific coagulation deficiencies, disseminated
intravascular coagulation (DIC), some dysproteinemias, and
the presence of circulating anticoagulants and
fibrin/fibrinogen split products.
❖ Factor IV: Ionized calcium ; Factor III: Tissue cells
❖ Sodium citrate is the preferred anticoagulant.
Materials:
• Two-way needle • Wassermann tube (2)
• Citrated evacuated tube • 37 degrees Celsius water bath
• Centrifuge • Thromboplastin reagent
• 0.1 mL serological pipette • Tissue paper
• 0.2 mL serological pipette • Timer
Preparation of Specimen for PT:
1. Collect venous blood in a citrated tube.
2. Centrifuge at 1,500 rpm for five minutes.
3. Separate the plasma by placing it into a clean dry tube.
HEMOSTAT PROTHROMBIN TIME
Intended Use
Hemostat Thromboplastin SI (PT-SI) is a high-sensitivity
reagent that is used to perform the one-stage prothrombin time
(PT). Prolongation of PT indicates either acquired or congenital
disorders that affect coagulation factors I, II, V, VII, and X. The PT
has been widely accepted as the means to monitor patients on oral
anticoagulant therapy due to the reduction in the activity of
vitamin K-dependent clotting factors (II, VII, IX, X, Protein C, and
Protein S). HemoStat Thromboplastin SI can be used to assay
coagulation factors in the extrinsic and common pathways of
coagulation.
Specimen of choice: citrated poor plasma
Principle
The one-stage PT measures the clotting time of plasma
after adding a source of tissue factor (thromboplastin) and
calcium. The recalcification of plasma in the presence of tissue
factor generates activated factor X, with the consequent
formation of thrombin and ultimately a fibrin clot.
Reagent
Thromboplastin reagent (stored @ 2-8 degrees Celsius)
*Lyophilized extract of rabbit brain
*Calcium chloride
Reagent Preparation:
• Reconstitute with exactly 2 mL distilled water. Agitate gently
until the solution is complete.
• After reconstitution, the reagent is stable for seven days when
stored at 2-8 degrees Celsius, 24 hours @ 15-25 degrees Celsius
and 24 hours @ 37 degrees Celsius. Storing the reagent between
2-8 degrees Celsius when it is not in use is recommended.
Test Procedure:
1. Perform samples and controls in duplicates.
2. Prewarm the HemoStat Thromboplastin-SI reagent at 37
degrees Celsius.
3. Pipette 0.1 ml of plasma/control into a prewarmed test tube.
4. Incubate at 3-5 minutes at 37 degrees Celsius.
5. Add PT-SI reagent.
6. Start timer with the addition of reagent.
7. Record the time required for clot formation.
Normal Value: 10-14 seconds
ACTIVATED PARTIAL THROMBOPLASTIN TIME
❖ It is a routine test for screening coagulation disorders in the
intrinsic pathway by measuring factors VIII, IX, XI, XII, and
Prekallikrein (PK) or Fletcher factor but not platelets and
factor XIII.
❖ It is also used to detect the presence of circulating
anticoagulants or inhibitors, and to monitor heparin
therapy.
❖ It should be performed within two (2) hours after blood
collection.
❖ Sodium citrate is the preferred anticoagulant.
Materials:
• Two-way needle • Wassermann tube (2)
• Citrated evacuated tube • 37 degrees Celsius water bath
• Centrifuge • aPPT reagent
• 0.3 mL serological pipette • Tissue paper
• 0.4 mL serological pipette • Timer
• Calcium Chloride
Preparation of Specimen for PT:
1. Collect venous blood in a citrated tube.
2. Centrifuge at 1,500 rpm for five minutes.
3. Separate the plasma by placing it into a clean dry tube.
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
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 HEMATOLOGY II – Laboratory

mechanism, namely factors VIII, IX, XI, and Prekallikrein (or

Fletcher factor).

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 platelet
factor 3. 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.
Plasma Activators: Cauline, ellagic acid, celite
Contents and Reagent Preparation:
➢ HemoStat aPTT-EL
• Chloroform extract of rabbit brain
• Ellagic acid
➢ CaCl2
• Calcium chloride (0.02 mol/L)

Test Procedure:
1. Perform samples and controls in duplicates.
2. Prewarm the HemoStat CaCl2 (0.02 M) to 37 degrees Celsius.
3. Pipette into 0.1 mL of plasma/control prewarmed test tube.
4. Incubate for 1-2 minutes at 37 degrees Celsius.
5. Add 0.1 mL aPTT-EL reagent.
6. Incubate for 3 minutes at 37 degrees Celsius.
7. Add 0.1 ml of prewarmed CaCl2
8. Start timer with addition of CaCl2
9. Record time required for clot formation.

*Calculate the mean time of duplicate aPTT determinations for

each test plasma and report to the nearest 0.1 seconds.

Normal Reference Range: 24 – 35 seconds

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