In this exercise, two tests will be performed to screen

for defective clotting factors.

The formation of thrombin in the plasma samples will
be inhibited by an anticoagulant.
The anticoagulant used, either citric acid or oxalic acid,
removes calcium ion (Ca2+) from the plasma.
The removal of Ca2+ has an anticoagulant effect because
calcium is a necessary cofactor in the activation of a
number of the clotting factors. This inhibition can be
easily reversed by adding calcium ion during the
clotting tests.
 Clotting time or Coagulation time
• The time required for blood to clot in a glass tube.

• The normal range for the test described below is 5 to 15 min.

but each laboratory should determine its own normal values.

 Reagent & equipment

1. water bath, 37C.
2. Glass test tube.
3. Stopwatch.
4. Plastic syringe and 20-gauge needle.

• Specimen: fresh whole blood , 4 ml .

 Procedure
1. Label 3 glass test tube with patient name and number them,
#1, #2, and #3.
2. Add 1 ml of blood in each tube. The last 1 mL of blood may
be discarded.
3. Start the stopwatch as soon as the blood is placed in tube #3.
4. Place the three test tubes in a 37°C water bath.
5. At exactly 5 min., title test tube #1 gently to a 45° angle.
Repeat this procedure every 30 seconds, until the test tube
can be completely inverted without spilling the contents
(that is, until the blood is completely clotted).
6. Record the time it took the blood in test tube
#1 to clot.
7. 30 sec. after the blood in test tube #1 is
clotted. Proceed with tube #2, and repeat the
preceding procedure, tilting the test tube
every 30 seconds, until a clot is formed.
Record the results. Repeat this procedure for
test tube #3.
 Clotting time - capillary method
Material

1. Sterile disposable pricking needle or lancet.

2. Stop watch
3. Dry glass capillary tube (narrow diameter 1 top 2
mm, minimum 10 cm long.)
4. Cotton Swab of absorbent cotton.
5. Spirit wetted, cotton swab.
6. 70 % v/v ethyl alcohol
Clotting time of whole blood
 Clotting Time - Slide Method
• The surface of the glass
tube initiates the clotting
process. This test is
sensitive to the factors
involved in the intrinsic
pathway
• The expected range for
clotting time is 4-10 min.
 Terms
• Platelet –Poor plasma (PPP)
 <10 x 10 9 /L.
• Why is PPP essential?
Contains platelet factor 4(heparin neutralizer).
Contains phospholipid (affects lupus anticoagulant
and factor assay testing).
Contains proteases (affect testing for vWF).
• Platelet-Rich plasma (PRP)
 200-300 x 10 9 /L.
 PT Reagent
• Calcium ions and Thromboplastin from brain tissue
(Rabbit).

• Thromboplastin (Tissue Factor) protein-lipid complex

found in tissues outside blood vessels.
 Principle
• The procedure uses a tissue thromboplastin reagent with CaCL
(Calcium Chloride) to provide a one step procedure for
evaluating plasma clotting.
• The normal values for the prothrombin time range from 10.0
to 13.0 seconds.
 An elevated prothrombin time may indicate the presence of
1. Vitamin K deficiency,
2. DIC,
3. liver disease,
4. Presence of FSP’s
 Performing a PT test
• Pre-warm PT reagent and sample to 37 oC
• Add 100 L sample to tube
• Add 200 L of PT reagent to tube
• Start timer
• Record time to clot in seconds
 If the results from run 1 and run 2 are within + 1 second
from each other, average the two results and report with
appropriate units.
 If results are not within required limits, a third run should
be performed and average the two that match within
acceptable limits.
• Calculate INR
 Expected PT Values
• Mean Normal Plasma = 10 to 14 seconds.
• A high sensitivity (Low ISI) PT will give a
high normal PT value (13 to 15 seconds).
• Oral anticoagulant monitoring = Target INR of
2.0 to 3.0.
• INR of greater than 5 or 5.5 = unacceptable
high risk of bleeding.
 PTT
• Reagents
• Phospholipids and a surface activator
• Calcium Chloride reagent added to start the
reaction.
• APTT reagent mimics the surface of a platelet.
 Principle
• Under carefully controlled conditions and with
properly prepared reagents, calcium, partial
thromboplastin and an activator (such as kaolin,
celite, micronized silica or ellagic acid) are added to
the plasma to be tested, which eliminates the
variability of activation by glass contect.

• The partial thromboplastin reagent stimulates

activated platelet surfaces by providing a
phospholipid platelet surface on which enzymatic
reactions can occur. which eliminates the test’s
sensitivity to platelet number and function.
 Sample
• Citrate tube; 3.2% citrate tubes
• Ratio of anticoagulant to blood should be 1:9
• After centrifugation, the Plasma contains all
the intrinsic coagulation factors except
calcium (removed during anticoagulation) and
the platelets (removed during centrifugation).
 Procedure
• Prewarm sufficient volumes of CaCl2 (0.025 M) to
37oC
• Label 2 test tube & 2 control tubes
• Add 0.1 ml (100µL) of sample or control to the
appropriate tube
• Add 0.1 ml of APTT reagent to each tube
• Incubate at 37oC for 5 minutes
• Add 0.1 ml (100µL) of prewarmed CaCl2 & start stop
watch immediately
• Record the time at which clot is detected
 Expected APTT Values

• Normal Range: 25 to 43 seconds

• Slightly Elevated: 45 to 65 seconds
• Extremely Elevated > 70 seconds
 ?What does the test result mean
• APTT prolongations are caused by either factor
deficiencies (especially of factors VIII, IX, XI, and/or
XII),
• Inhibitors (most commonly, lupus
anticoagulants{ immunoglobulin that binds to
phospholipids and proteins associated with the cell
membrane})
• Therapeutic anticoagulants such as heparin.
• DIC
• Liver disease
 Sources of error

1. 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
2. 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
3. Associated with procedure
a. Incorrect temperature
b. Incorrect incubation times
c. Incorrect volumes of sample, reagents or both
 APTT PT Defective Factor
Abnormal Abnormal V
Normal Abnormal VII
Abnormal Normal VIII
Abnormal Normal IX
Abnormal Abnormal X
Abnormal Normal XI
Abnormal Normal XII