Bleeding Time, Clotting Time,

Prothrombin Time/PTT

CHAMBERLAIN I. AGTUCA, JR., MD

Department of Pathology
Vicente Sotto Memorial Medical Center
Platelets (Thrombocytes)

Function interact with injured vascular wall

structures, plasma proteins, other circulating
blood cells
a. Adhesion: adhere to basement membrane of
injured blood vessel
b. Release action: release of alpha & dense
granules, & lysosomes
c. Aggregation: formation of secondary, irreversible
platelet aggregation
 Coagulation:
Starts with activation of Thromboplastin, from
injured tissue
Fibrinolytic System:
Endothelial cells
Heparin
Antithrombin
Evaluation of Coagulation Factor Disorders

Medical History:
1. Symptoms
2. Onset of Bleeding
3. Location of Bleeding
4. Family History
5. Drugs & Medications
6. Other Systemic diseases
Physical Exam:
Laboratory Screening Tests

1. Prothrombin Time (Protime/PT)

2. Partial Thromboplastin Time (PTT)
3. Platelet count

*If abnormal add:

1. Bleeding Time
2. Clotting Time
I. Prothrombin time /Protime/PT

Screen for deficiencies in: Extrinsic Pathway

FI, FII, V, VII, X
Test of choice for monitoring anticoagulant
therapy by Vit K antagonists (II, VII, IX, X)

Principle: Thromboplastin + Platelet-poor

plasma & Ca++ react w/ FVII to activate FX ---
convert Prothrombin to Thrombin
 Specimen source:
Citrated platelet-poor plasma (PPP)
Reference Range: Values differ
10 12 seconds or 12 14 seconds
Reporting:
1. Patient time in sec w/ Ref range
2. Patient time in sec w/ control time
3. Prothrombin ratio (PT divided by mean of Ref
range multiplied by 100)
4. Percent Activity (outdated?)
5. International Normalized Ratio (INR):
- Widely used nowadays
- Comparison of Patient value with the normal value =
result is normalized to an international reference
preparation (IRP) of thromboplastin
- Values usually provided by manufacturers of
reagents
 Interpretation of results:
Sensitivity: detect deficiencies of <40% - 50% of normal
Prolonged PT indicates abnormality in:
Common pathway
Extrinsic pathway
Abnormality may be acquired or hereditary
Factor inhibitors
II. Partial Thromboplastin Time (PTT)

Test of choice for factor deficiencies of the

Intrinsic & Common Pathway
Test of choice for monitoring Heparin therapy
PTT Reagent: 2 components
1. Platelet substitute (phospholipid) prepared from
brain & plant phospholipids
2. Activator - Kaolin, Celite, micronized silica, or
ellagic acid
 Specimen source:
Citrated platelet-poor plasma (PPP)
Reagents:
Phospholipid w/ Activators (PTT reagent)
0.025 M CaCl2
Procedure: PPP (0.1mL) + 0.1mL PTT reagent,
incubate at 37C for 3-5min, + 0.1mL warm CaCl2.
 Reporting:
Reported in seconds, to the nearest tenth, along
with reference range.
Reference range: vary according to reagent,
method, instrument used
Lower limit of 20 sec to an upper limit of 45 sec
 Interpretation:
Prolonged PTT in the absence of Heparin use
indicate:
Factor deficiency
Acquired circulating anticoagulant (e.g. Lupus inhibitor)
Antibody to a specific Factor (e.g. anti-FVIII)
Prolonged PTT if factor measured is <40% to 50%
of normal
 Sources of Error:
1. Sample collection & preparation
a. Improper collection & specimen processing
b. Incorrect anticoagulant-to-plasma ratio
c. Hemolysis
d. Platelets in plasma sample
e. Unexpected heparin contamination
2. Reagent preparation
a. Improper storage
b. Water impurities
c. Incorrect dilution

3. Instrumentation
a. Failing light source
b. Fluctuations in temperature
c. Loss of calibration of tubing
d. Contamination
III. Platelet Count

Formed in the BM from Megakaryocytes

Difficult to count:
a. Small & difficult to discern
b. Attached to surfaces/particles in diluting fluid
c. Disintegrate easily
d. Form clumps with other platelets
 Specimen:
Capillary blood from finger puncture
Values are lower because of clumping at puncture site
Venous blood
EDTA (Lavender tube) as anticoagulant of choice
 Method of Determination:
1. Manual Count
a. Unopette system
b. Hemacytometer
2. Automated Method
a. Optical methods
b. Impedance methods
Calculation: Manual Platelet Count

Ave # of Plt in 4 squares X 1mm2 x 100 X 106 = Platelets X 109/L

0.1 mm

Platelet Reference Range: 150 450 x 109/L

 Clinical Significance:
Thrombocytopenia:
Platelet values lower than normal

Thrombocytosis:
Platelet values higher than normal
TESTS FOR PLATELET FUNCTION

1. Platelet Closure time

2. Platelet Aggregation Studies
3. Automated Platelet Function Analysis
Platelet closure time:
Assess platelet-related primary hemostasis; greater
accuracy & reliability than Bleeding Time (BT).
Instrument and test cartridge system which simulates the
process of platelet adhesion and aggregation following a
vascular injury.
Rapid evaluation of platelet function on samples of
anticoagulated whole blood.
The time required to obtain full occlusion of the aperture
is reported as the closure time (CT) in seconds.
Sensitive to platelet adherence and aggregation
abnormalities ; allows discrimination of aspirin-like defects
and intrinsic platelet disorder.
Platelet Aggregation Studies:

Measure response of platelet during hemostatic

process
Change in shape
Increase in surface adhesiveness
Platelet plug formation

Use of Aggregating Agent

Measurement: Turbidimetry Method
CLOTTING TIME

Time required for a blood sample to coagulate/clot in

vitro under standard conditions.
Most common method: Capillary Tube Method.
Affected by calcium ion levels and many diseases.
Normal value: 5 to 8 minutes.
Time taken for blood to clot reflects time required for
the generation of thrombin
BLEEDING TIME

Principle: BT is the time it takes for a standard wound

to stop bleeding
Comprehensive test of platelet action in vivo & is
sensitive to the following abnormalities:
1. Platelet numbers & function
2. Plasma VIII:vWF deficiencies
3. Vessel wall composition
Standardization of Wound size is a problem
Employs BP cuff inflated at 40 mmHg
 Reference range:
2 to 9 minutes
Considerations for the Test:
Volar skin must be dry
Adequate incision/proper depth
Site should not touch filter paper
Aspirin& aspirin-containing meds must be discontinued 1
week prior to testing
NSAIDS must be discontinued 24 hrs prior to testing