1. Tests to measure fibrin clot
General Approach in Investigation of Haemostasis
Lecture 9:
Tests to measure fibrin clot

2. Tests to Measure Fibrin formation
Thrombin Time TT
Reptilase Time
Fibrinogen Activity assay

3. 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 and direct thrombin inhibitors such as hirudin and FDPs.
Hirudin is a naturally occurring peptide in the salivary glands of medicinal leeches (such as Hirudo medicinalis) that has a blood anticoagulant property.
Fibrin degradation product (FDPs), also known as fibrin split products, are components of the blood produced by clot degeneration. These are produced by the action of plasmin on deposited fibrin. The most notable subtype of fibrin degradation products is D-dimer. The levels of these FDPs rises after any thrombotic event.

4. 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 = / µg
National Institutes of Health

5. Interpretation Normal value 14 to 18 sec.
The TT is prolonged in patients with
hypofibrinogenemia (usually less than 100 mg/dl.)
dysfibrinogenemia
And in the presence of anticoagulants such as heparin and hirudin, or FDPs.

6. 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 generate the fibrin clot.

7. 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

8. 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.

9. May show some prolongation
Reptilase Time
Thrombin Time
Normal ↑
Presence of unfractionated heparin
May show some prolongation
Presence of LMWH
Presence of direct thrombin inhibitors
Warfarin
Decreased/absent fibrinogen, Dysfibrinogenaemia
DIC, Liver disease
Heparin-like anticoagulants
Paraproteinaemias,
Thrombolytic therapy,
Neonate,
Amyloid
Hyperfibrinogenaemia
Hypoalbuminaemia
FDPs: These interfere with fibrin polymerisation and can at high concentration lead to a prolonged thrombin time
Paraprotiens: May interfere with fibrin polymerisation leading to a prolonged thrombin time
Amyloidosis: Prolongation of the Thrombin time and the Reptilase time has been observed in patients with amyloidosis due to the inhibition of the conversion of fibrinogen to fibrin.

10. 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
Nephelometry is performed by measuring the turbidity in a water sample by passing light through the sample being measured. In nephelometry the measurement is made by measuring the light passed through a sample at an angle. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually about 70 or 75 degrees).
Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. The amount of light scatter is measured and compared to the amount of scatter from known mixtures. The amount of the unknown is determined from a standard curve.

11. 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 Clauss method measures the rate of fibrinogen fibrin conversion in the presence of excess thrombin, when diluted plasma is clotted with excess thrombin, the fibrinogen levels is inversely propotional to the clotting time, a calibration curve is prepared from a fibrinogen reference and plotted on log-log paper,

12. 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, versus the clotting time on the 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.
***The patient specimen is diluted 1: 10. This dilution minimizes the effects of heparin or antithrombic proteins (FDPs, and para­proteins).
Heparin levels below 0.6 U/mL and FDP lev­els below 100 µg/ dL do not affect the results of the fibrinogen assay of diluted PPP if the fibrinogen level is 150 mg/dL or greater.

13. 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.

15. 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)

16. Procedure
Collect blood by clean venipuncture technique according to recommended procedures previously described.
Process and store plasma samples following recommended guidelines.
Reconstitute the thrombin reagent according to the manufacturer's directions.
This assay is commonly performed on a coagulation analyzer.
The reagent is reconstituted according to manufacturer instructions and used immediately or aliquotted and frozen. If thrombin is to be frozen, it should be prepared in a stock solution of 1000 NIH units/mL and frozen at -70oC until it is ready for use. Once thawed, thrombin is stable for only a few hours and cannot be refrozen for later use.

17. Fibrinogen standard (mL)
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.
Calibrator = 317mg/dl
Dilution
Buffer (mL)
Fibrinogen standard (mL)
Tube
63.4
1:5
0.8
0.2 1
31.7
1:10
0.4
0.4 of tube 1 (mixed) 2
15.85
1:20
0.4 of tube 2 (mixed) 3
7.925
1:40
0.4 of tube 3 (mixed) 4

18. Perform determinations on each dilution of the fibrinogen standard as follows:
Incubate 0.1 mL of fibrinogen standard dilution at 37°C for at least 2 minutes but no more than 5 minutes.
Add 0.05 mL of thrombin reagent.
Measure the clotting time. performed in duplicate, and average the results.

19. Sample Assay**
Prepares a 1: 10 dilution of each patient PPP and control with Imidazole buffer.
Incubate 0.1 ml of the patient dilution at 37°C for at least 2 minutes but no more than 5 minutes.
Add 0.05 mL of thrombin reagent.
Measure the clotting time. run in duplicate.
Limitations **
Care must be taken that the thrombin reagent is pure and has not degenerated. Exposure to sunlight or oxidation will result in rapid breakdown of thrombin. The working dilution lasts only 1 hour at 2-8°C, and should remain cold until just before testing. Specimens with clots, hemolysis, icterus, or lipemia are unacceptable.

20. 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.

21. 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.

22. Clinical Significance:
There are several causes for a deficiency of fibrinogen.
Severe hemorrhaging may result in any case.
congenital deficiencies may be due to **
Afibrinogenemia (a lack of fibrinogen)
a dysfibrinogenemia (abnormal fibrinogen)
Acquired deficiencies may be due to
liver disease
disseminated intravascular coagulation (DIC)
fibrinolysis

23. 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.

24. NOTE:
For fibrinogen values out of the linearity range ( 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).