Tests to Measure Fibrin formation. Mr. Mohammed A. Jaber . Tests to Measure Fibrin formation. Thrombin Time TT Reptilase Time Quantitative Fibrinogen. Tests to Measure Fibrin formation. Thrombin Time (TT) or Thrombin Clotting Time (TCT). Thrombin Clotting Time (TCT). Principle
Mr. Mohammed A. Jaber
Thrombin Time TT
Commercially prepared bovine thrombin reagent at 2 NIH units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable polymer
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.
Normal values are approximately 15 to 20 seconds.
All the congenital dysfibrinogenemias have an infinite reptilase time.
The reptilasetime is also infinitely prolonged in cases of congenital afibrinogenemia.
In states of hypofibrinogenemia, the reptliasetime 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
A comparison of both TT and reptilase time will aid in detecting the presence of thrombin inhibitors such as 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
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
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.
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.
Patient thrombin clotting time of 12.5 seconds 220 mg/dL
Note: I, II, III represent reference plasmas
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.
Preparation of Calibration Curve
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'sVeronal buffer as follows: 1:5, 1:15, and 1:40. Make all transfers from the first test tube.
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. If performed in duplicate, average the results.
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.
The clinical laboratory scientist prepares a 1: 10 dilution of each patient PPP and control with Owren's 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. Average the results if tested in duplicate.
The automated analyzer will automatically make determinations from the curve in mg/dL
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).