Laboratory tests for secondary hemostasis
Record the coagulation time
CLOTTING TIME
Coagulation time measures the period required
for blood to clot or solidify after it has been
extracted from the body. It can indicate if the
person has an acquired defect of platelet
function.
Before operations, accompanied by bleeding
time
Methods:
1. Whole blood method ( Lee and white
method)
2. Capillary tube method ( dale and
laidlaws method)
3. Capillary slide or drop method
A. Capillary blood method
I. Slide or drop method
Procedure:
1. Performa skin puncture
2. Place a drop of blood on a clean glass
slide
3. Draw the tip of the lancet across the
drop of blood at 30-second interval
(fibrin strand)
4. Record and stop timing
II. Capillarytube: Dale and Laidlaws
Procedure:
1. Skin puncture, wipe first drop of blood
2. Fill a non heparinized capillary tube ¾
with blood
3. Start timer as soon as blood enters the
tube
4. Set a capillary tube aside in a horizontal
position for 2 minutes
5. Break off about 1cm of the capillary
tube at 30 second interval, until fibrin
thread bridges the broken ends of the
capillary tube
6.
Lee and White method
Procedure:
1. Label 3 tubes (1,2,3). Place in a 37
degrees water bath
2. Extract 3 ml venous blood, two-syringe
method.
3. Place 1 mL of blood into each tube
4. Start timer as blood is. Delivered into
tube I. Put the tubes back into water
bath
5. Gently tilt tube 3 every 30 second until
blood solidifies. Handle tube 2 in the
same way. Finally, tilt tube I until solid
clot.
6. Stop timer and record
STYPVEN TIME
• Russell’s Viper venom (Daboia russellii
snake)
• For determination of common pathway
deficiencies
• Recalcification of platelet-poor plasma
at 37 degrees celcius in presence of
Stypven (Russell viper venom) and
phospholipid
• The venom activates Factor X directly
with the presence of Factor V,
prothrombin, calcium and phospholipid,
results to fibrin clot.
• Formation of clot: normal levels
THROMBIN TIME (heparin sensitive)
Used to measure the availability of functional
fibrinogen.
• Cleaves the fibrinogen into Fibrin
peptide A and B generating fibrin to
fibrin clot.
• Evaluates the cleaving of fibrinogen to
fibrino peptide a and B
Prolonged in
- Low levels of fibrinogen cases
- Dysfibrinogenemia
- Presence of FDP/FSP
- Presence of thrombolytic agent
- Presence of heparin
Principle
Thrombin time is a simple test to screen
for conditions that can interfere with the
conversion of fibrinogen into fibrin. A low
potency thrombin is added to undiluted plasma
and clot formation is timed.
Reagents:
Bovine
Lyophilisate
Normal value: 8 to 14 seconds
Preparation of specimen
1. Collect venous blood in a citrated tube
(clotting best using ETS)
2. Centrifuge at 1500 rpm for 5 minutes
3. Separate the plasma by placing it into a
clean dry tube
Procedure
1. Perform samples and controls in
duplicates (2 sample 2 control)
2. Pipette 0.2 mL plasma/control into a
prewarmed test tube
3. Incubate for 3 minutes at 37 C
4. Add 0.1 mL of thrombin time reagent
5. Start time with addition of reagent
6. Record the time required for clot
formation
REPTILASE TIME
Reptilase- cleaves the fibrinogen peptide to
form monomers
Evaluates the cleaving of fibrinogen to fibrino
peptide A
Prolonged in:
• Fibrinogen deficiency and impaired
function
• Presence of FDP and FSP
• Presence of streptokinase
• Resistant to heparin (opposite of
thrombin time)
Reference time: 10 to 15 seconds
DUCKERT’S TEST (5 M UREA CLOT SOLUBILITY
TEST)
- For factor XIII deficiency
Principle:
Fibrin clot formed in the presence of Factor XIII
and thrombin are stable (crosslinking) for at
least 24 hours in 5M urea, whereas clots formed
in the absence of factor XIII dissolve rapidly.
Reagents:
5M urea solution
Thrombin
0.025 CaCl2
Procedure:
1. Mixed 0.2 mL patient plasma and 0.2
mL thrombin
2. Incubate at 37 C for 20 minutes. And
add 3 mL 5M urea solution for 24 hours
Interpretation
Results:
1. Normal: Clot is insoluble to urea
2. FXIII deficiency: clot is soluble