Quantitative Fibrinogen

1. Quantitative Fibrinogen Mr. Mohammed A. Jaber

2. 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. Fibrinogen I thrombin IIa> Fibrin clot Ia

3. 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. 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.

4. 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.

5. 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.

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

8. Reference range: 200-400 mg/dL Clinical Significance: There are several causes for a deficiency of fibrinogen. Severe hemorrhaging may result in any case. Afibrinogenemia (a lack of fibrinogen) or a dysfibrinogenemia (abnormal fibrinogen) may be congenital. Acquired deficiencies may be due to liver disease, disseminated intravascular coagulation (DIC), or during a therapeutic plasma pheresis procedure. Elevated fibrinogen levels may be found in pregnancy, following surgery, or in patients in a hypercoagulable state such as with thrombosis.

9. Procedure: • Allow thrombin to warm to room temperature, at least 15 minutes. • Prepare normal control by making a 1:10 dilution. Place 100 µL of control to 900 µL of Owren’s Buffer into a labeled plastic tube. Mix gently. Store refrigerated until ready to use. • Pipette 200 µl diluted control into the bottom of 3 labeled test tubes and place in the heat block for 3 minutes.

10. Procedure • After incubation, add 100 µL of warmed thrombin reagent to the first test tube, and start the timer. • Observe the mixture for clot formation. Stop timer once clot forms. Record results in seconds. • Repeat steps 2-5 for the abnormal control and the patient sample. The fibrinogen results should be within +0.5 seconds of each other. • Take an average of the two best matching results, then read out fibrinogen concentration using the chart provided. It is this value that is reported out as the final result.

11. NOTE: For fibrinogen values out of the linearity range (46-904 mg/dL for this fibrinogen standard curve) a 1:10 dilution of the plasma will not work and a different dilution must be used.

12. NOTE For extremely high fibrinogen levels (>904 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).

13. 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).