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

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.

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 = 0.324 +/- 0.073 µg

5. 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.

6. Interpretation 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. and is unaffected by heparin The reptilase time is moderately prolonged in the presence of FDPs and is unaffected by heparin

7. 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.

8. Reptilase TimeThrombin Time Normal ↑ Presence of unfractionated heparin Normal May show some prolongation Presence of LMWH Normal ↑ Presence of direct thrombin inhibitors Normal Warfarin ↑↑ Decreased/absent fibrinogen, Dysfibrinogenaemia ↑↑ DIC, Liver disease Normal ↑ Heparin-like anticoagulants ↑↑ Paraproteinaemias, Thrombolytic therapy, Neonate, Amyloid ↑↑ Hyperfibrinogenaemia Hypoalbuminaemia

9. Fibrinogen  Fibrinogen concentration can be measured in 3 ways. Fibrinogen concentration is usually reported in milligrams per deciliter (mg/dl).  Heat precipitation:(Heating at 56 o C will precipitate fibrinogen only)  Clotting method - thrombin clot time:  Immunologic assays: Fibrinogen antigen levels may be assayed by means of radial immunodiffusion (RID) or nephelometry

10. 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.

11.  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. concentrations the x-axisclotting timethe y-axis  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.

12.  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.

14. 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)

15. 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.

16. 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. Preparation of Calibration Curve Calibrator = 317 mg/dlDilution Buffer (mL) Fibrinogen standard (mL) Tube 63.4 1:50.80.21 31.7 1:100.4 0.4 of tube 1 (mixed) 2 15.85 1:200.4 0.4 of tube 2 (mixed) 3 7.925 1:400.4 0.4 of tube 3 (mixed) 4

17. A. Incubate 0.1 mL of fibrinogen standard dilution at 37°C 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. Perform determinations on each dilution of the fibrinogen standard as follows:

18. 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 37°C 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.

19. 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.

20.  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.

21. 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

22. 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.

23. 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).