General Approach in

Investigation of Haemostasis

:Lecture 9

Tests to measure
fibrin clot
Tests to Measure Fibrin
formation
Thrombin Time TT
Reptilase Time
Fibrinogen Activity
assay
Thrombin Time (TT)
Also Called Thrombin Clotting Time
(TCT)

The thrombin time (TT) is the time required

for thrombin to convert fibrinogen to an
insoluble fibrin clot.
It does not measure defects in the intrinsic or
extrinsic pathways.
The test is affected by
Abnormal levels of fibrinogen (usually less than
100 mg/dl.) and dysfibrinogenemia
The presence of antithrombins such as heparin
Principle of
TT
Commercially
prepared bovine
thrombin reagent
at 2 NIH units/mL
cleaves
fibrinopeptides A
and B from
plasma
fibrinogen to
form a detectable
polymer

1 WHO unit = 0.56 NIH unit

1 NIH unit = 0.324 +/- 0.073
g
Interpretation
Normal value 14 to 18 sec.
The TT is prolonged in patients with
1. hypofibrinogenemia (usually less than 100
mg/dl.)
2. dysfibrinogenemia
3. And in the presence of anticoagulants such
as heparin and hirudin, or FDPs.
Reptilase (Atroxin)Time

The Reptilase time is a modification of the

thrombin time in which the purified enzyme
Reptilase is used to replace thrombin.
It is a thrombin-like enzyme, isolated from the
venom of Bothrops atrox, that catalyzes the
conversion of fibrinogen to fibrin in a manner
similar to thrombin.
Reptilase cleaves fibrinogen releasing
fibrinopeptide A (FpA) generating fibrin. In
contrast thrombin cleaves both fibrinopeptide A
and fibrinopeptide B from fibrinogen to
Interpretation
Normal values are approximately 15 to 20
seconds
All the congenital dysfibrinogenemias have an
infinite reptilase time.
The reptilase time is also infinitely prolonged
in cases of congenital afibrinogenemia.
In states of hypofibrinogenemia, the reptliase
time may be variable, depending on the levels
of fibrinogen present.
The reptilase time is moderately prolonged in
the presence of FDPs and is unaffected by
heparin
Comment
In the presence of heparin, thrombin is
inhibited through the interaction of
antithrombin (AT-III). However, heparin
does not interfere with the ability of
reptilase to cleave fibrinopeptide A from
fibrinogen
Ancrod a similar enzyme from Agkistrodon
rhodostoma can also be used to replace
thrombin in the thrombin clotting time test.
 Thrombin Reptilase Time
Time
Presence of unfractionated
heparin
Normal
Presence of LMWH May show
some Normal
prolongation
Presence of direct
thrombin inhibitors
Normal
Warfarin Normal Normal
Decreased/absent
fibrinogen,
Dysfibrinogenaemia
DIC, Liver disease
Heparin-like Normal
anticoagulants
Paraproteinaemias,
Thrombolytic therapy,
Neonate,

Amyloid
Fibrinogen
Fibrinogen concentration can be
measured in 3 ways. Fibrinogen
concentration is usually reported in
milligrams per deciliter (mg/dl).
Heat precipitation:
Clotting method - thrombin clot time:
Immunologic assays: Fibrinogen
antigen levels may be assayed by
means of radial immunodiffusion
(RID) or nephelometry
Fibrinogen activity test
(Rolf Greiner // Biochemica)

Principle:
Fibrinogen assays are quantitative
techniques to measure the amount of
functional fibrinogen present in the
plasma.
The assay is based on the Clauss assay,
which is the reference method.
This fibrinogen assay measures the time
required for thrombin to convert
fibrinogen to fibrin.
 The procedure is a determination based on
fibrinogen activity, but results are converted to
concentration (mg/dL) by comparison with
control plasma results.
In the fibrinogen procedure, thrombin is added to
various dilutions of known concentrations of
fibrinogen to produce a thrombin-clotting time in
seconds.
The clotting times are then plotted on a graph,
with the known concentrations on the x-axis,
x-axis
versus the clotting time on the y-axis.
y-axis
The clotting times are performed using controls
and the patient sample at a 1:10 dilution.
An excess amount of thrombin reagent is added
and the time it takes for the specimen to clot is
recorded in seconds.
 This time is then converted to mg/dL of
fibrinogen by comparing these results to
results obtained on a fibrinogen standard
curve.
Patient results may be read directly off of
the standard curve graph, or off of a data
chart prepared from the graph that already
converts time in seconds to mg/dL.
The time it took for the specimen to clot is
inversely proportional to the fibrinogen
concentration in mg/dL.
For instance, a prolonged fibrinogen
clotting time means the fibrinogen level
(mg/dL) is low.
Reagents and Equipment

Test tubes
Commercial fibrinogen determination kit:
Thrombin, 100 National Institutes of Health
(NIH) units/mL, bovine lyophilized
(reconstitute with 2 ml Distilled water)
Fibrinogen standard
Imidazole buffer,
Control (with a known fibrinogen
concentration)
Procedure
1. Collect blood by clean venipuncture
technique according to recommended
procedures previously described.
2. Process and store plasma samples
following recommended guidelines.
3. Reconstitute the thrombin reagent
according to the manufacturer's directions.
4. This assay is commonly performed on a
coagulation analyzer.
Preparation of Calibration
Curve
The calibration curve is prepared from the reference
standard .
Make dilutions of the fibrinogen standard with Imidazole
buffer as follows: 1:5, 1:10, 1:20 and 1:40. Make all
transfers from the first test tube.
Fibrinogen Buffer Calibrator
Tube standard (mL) Dilution = 250
(mL) mg/dl
1 0.2 0.8 1:5 50
0.4 of tube
2 0.4 1:10 25
1 (mixed)
0.4 of tube
3 0.4 1:20 12.5
2 (mixed)
0.4 of tube
4 0.4 1:40 6.25
3 (mixed)
Perform determinations on each
dilution of the fibrinogen
standard as follows:
A. Incubate 0.1 mL of fibrinogen standard
dilution at 37C for at least 2 minutes but
no more than 5 minutes.
B. Add 0.05 mL of thrombin reagent.
C. Measure the clotting time. performed in
duplicate, and average the results.
Sample Assay**
1. Prepares a 1: 10 dilution of each patient
PPP and control with Imidazole buffer.
2. Incubate 0.1 ml of the patient dilution at
37C for at least 2 minutes but no more
than 5 minutes.
3. Add 0.05 mL of thrombin reagent.
4. Measure the clotting time. run in duplicate.
Interpretation
Reference range: 200-400 mg/dL
Prolonged clotting times may indicate either
A low fibrinogen concentration
The presence of inhibitors such as heparin or
circulating FDPs.
Some manufacturers include a heparin neutralizer
in the fibrinogen reagent that will negate any
interference by therapeutic levels of heparin.
 The effect of heparin may also be
excluded by
treatment of the sample with a heparin-
digesting enzyme
performing the reptilase time, because
reptilase is unaffected by heparin.
Clinical Significance:
There are several causes for a deficiency of
fibrinogen.
Severe hemorrhaging may result in any case.
congenital deficiencies may be due to **
1. Afibrinogenemia (a lack of fibrinogen)
2. a dysfibrinogenemia (abnormal fibrinogen)
Acquired deficiencies may be due to
1. liver disease
2. disseminated intravascular coagulation (DIC)
3. fibrinolysis
High fibrinogen levels are seen
During pregnancy
In women taking oral contraceptives.
In patients in a hypercoagulable state
such as with thrombosis.
Fibrinogen is considered an acute-phase
reactant, and, therefore, high levels may
be seen in states of acute infection,
neoplasms, collagen disorders, nephrosis,
and hepatitis along with other conditions
causing physical stress.
NOTE:
For fibrinogen values out of the linearity range
(46-700 mg/dL for this fibrinogen standard
curve) a 1:10 dilution of the plasma will not
work and a different dilution must be used.
For extremely high fibrinogen levels (>700 mg/dL) a
1:20 dilution of the plasma is used for the procedure.
However, due to the change in dilution, the result read
off of the fibrinogen data table must be multiplied by a
factor of 2 (since our 1:20 dilution is 2 times the 1:10
dilution originally meant for the data table).
For extremely low fibrinogen levels (<46 mg/dL) a 1:5
dilution of the plasma is used for the procedure. The
result read off of the data table must then be divided by
a factor of 2 (since our 1:5 dilution is half of the 1:10
dilution originally meant for the data table).