Tests to Measure Fibrin formation Mr. Mohammed A. Jaber

1.Thrombin Time TT 2.Reptilase Time 3.Quantitative Fibrinogen Tests to Measure Fibrin formation

Thrombin Time (TT) or Thrombin Clotting Time (TCT) Tests to Measure Fibrin formation

 Principle  The thrombin time (TT) is the time required for thrombin to convert fibrinogen to an insoluble fibrin clot.  Fibrin formation is triggered by the addition of thrombin to the specimen and, therefore, bypasses prior steps in the coagulation cascade.  The TT does not measure defects in the intrinsic or extrinsic pathways.  The test is affected by abnormal levels of fibrinogen and dysfibrinogenemia and the presence of antithrombins such as heparin and direct thrombin inhibitors such as hirudin and FDPs. Thrombin Clotting Time (TCT)

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

14 to 18 sec.  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. Interpretation

Reptilase (Atroxin)Time Tests to Measure Fibrin formation

 Principle  Reptilase (Atroxin) 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.  Unlike thrombin, the enzyme cleaves only fibrinopeptide A from the fibrinogen molecule whereas thrombin cleaves fibrinopeptides A and B. Reptilase Time

 Normal values 15 to 20  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. and is unaffected by heparin  The reptilase time is moderately prolonged in the presence of FDPs and is unaffected by heparin Interpretation

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 Comment Test Comparison Thrombin TimeReptilase TimeDefect Infinitely prolonged Dysfibrinogenemia Infinitely prolonged Afibrinogenemia prolongedEqually prolongedHypofibrinogenemia prolongedNormalHeparin prolonged Slightly to moderately prolonged FDPs  A comparison of both TT and reptilase time will aid in detecting the presence of thrombin inhibitors such as heparin.

Quantitative Fibrinogen Tests to Measure Fibrin formation

functional fibrinogen present in the plasma.  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.  Fibrinogen I thrombin IIa > Fibrin clot Ia Principle:

 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.*** Principle

 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. Principle

 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. Principle

 For example: Patient thrombin clotting time of 12.5 seconds 220 mg/dL Note: I, II, III represent reference plasmas Principle

 Coagulation analyzer  Test tubes  Commercial fibrinogen determination kit:  Thrombin, 100 National Institutes of Health (NIH) units/mL, bovine lyophilized  Fibrinogen standard  Owren's Veronal buffer, pH 7.35  Control (with a known fibrinogen concentration) Reagents and Equipment

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. Procedure

1.The calibration curve is prepared from the reference standard by the coagulation analyzer. Alternatively, it may be prepared manually: Make dilutions of the fibrinogen standard with Owren's Veronal buffer as follows: 1:5, 1:15, and 1:40. Make all transfers from the first test tube. Procedure : 1.Preparation of Calibration Curve Dilution Buffer (mL) Fibrinogen standard (mL) Tube 1: : of tube 1 (mixed)2 1: of tube 1 (mixed)3

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. If performed in duplicate, average the results. Procedure : 2.Perform determinations on each dilution of the fibrinogen standard as follows:

 The calibration curve is plotted via the analyzer with the clotting time in seconds on the vertical (y) axis versus the concentration of fibrinogen standard dilutions on the horizontal (x) axis. Construct a linear regression line. Calibration Curve

1.The clinical laboratory scientist prepares a 1: 10 dilution of each patient PPP and control with Owren's 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. Average the results if tested in duplicate. 5.The automated analyzer will automatically make determinations from the curve in mg/dL Sample Assay**

 Reference range: mg/dL  Prolonged clotting times may indicate either 1.A low fibrinogen concentration 2.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. Interpretation

 The effect of heparin may also be excluded by 1.treatment of the sample with a heparin-digesting enzyme 2.performing the reptilase time, because reptilase is unaffected by heparin.  A comparison of clotting times using both TT and reptilase time may help to distinguish a fibrinogen deficiency from a dysfibrinogenemia. Interpretation

 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 Clinical Significance:

 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. Clinical Significance:

 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. NOTE:

 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). NOTE

 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). NOTE