Determination of fibrinogen

A total knowledge about the Determination of fibrinogen

1. Determination of fibrinogen

2. Introduction Fibrinogen (Fib), a blood coagulating protein with its highest content in plasma, is one of the acute phase proteins, synthesized by liver cells and megakaryocytes. Fib is closely associated with consumption coagulopathy, liver diseases, nephropathy syndrome, cardiovascular diseases, diabetes and malignant tumors , etc . High-levels of Fib is one of the dangerous factors contributing to coronary heart diseases . Previous reports often focused on hemorrhagic diseases, thromboembolic diseases and recurrent spontaneous abortions caused by genetic variation of Fib . More attention needs to be paid to Fib determination in the clinical field.

3. Principle There are three types of Fib determination method physical-chemical methods (thermal/salt precipitation method, etc .) congealable protein methods immunology methods

4. More recently, with the popularity of automatic coagulometers , the application of paramagnetic particle methods has gradually increased. Although the thermal/salt precipitation method possesses the largest application scope, specificity is not ideal because the results are easily influenced by other proteins in the specimen. The congealable protein method reflects the blood agglutination function of Fib, but the method is comparatively complex and time-wasting. Therefore, this method is only applicable to establish reference scheme. The immunology methods, including the immune turbidimetry method, single diffusion method and ELISA, adopt polyclonal antibodies against Fib to react. However, because of common antigen determinants among fibrin monomer, fibrin degradation product (FDPS) and abnormal Fib, the specificity is not ideal. The results using an automatic instrument, especially for the paramagnetic particle method, is precise, rapid and well-reproducible, but the instrument is expensive and the determination cost is high .

5. (1) The schematic structure of the equipment. (2) The schematic process of the measurement. Structure Of detection system

6. A : Before the reaction, Fib is free in the detection pool on the piezoelectric quartz crystal oscillator surface, and the viscosity/density remains stable. B : Fib reaction reagent (thrombin) is added, and the reaction starts. C : After the agglutination reaction, fib gradually changes into fibrous protein monomer and complex, and the density and viscosity increase gradually and grid structure is formed; the mass load on crystal surface also increases; the common role of mass effect and non mass effect result in frequency changes. D and E: Near the agglutination reaction terminal, the blood clots shrink; the fibrous protein complex adhered to crystal surface leaves the surface; mass load decreases; the crystal frequency increases for a short period and then decreases with the deposit of blood clots, forming terminal peak.

7. Fibrinogen activity test 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.

8. 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.

9. 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.

10. 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)

11. 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.

12. 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.

13. 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.

14. 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

15. 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.

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