• The calibration curve may be extended beyond the

lowest and highest point.

 *The calibration curve is valid only for the same lot of

Fibroquant thrombin reagent.

Test Procedure for Sample

1. Prepare a 1:10 dilution of plasma specimen with

Owrens buffer solution.

2. To a 10 × 75 mm test tube at 37°C add 0.2 mL of 1:10

dilution of plasma sample to be tested.

3. Incubate at 37°C for one minute.

4. To the test tube add 0.1 mL of Fibroquant thrombin

reagent (prewarmed at 37°C for one minute) and

start the stopwatch simultaneously.

5. Stop the stopwatch at the first appearance of the

fibrin web, as the gel clot begins to form and record

the time in seconds.

6. Repeat steps 1-5 for a duplicate test.

7. If at the sample dilution of 1:10 the observed clotting

time is usually between 8 and 25 seconds, the

fibrinogen content is normal (Fibrinogen content

between 150 and 400 mg/dL). Assay results can be

read off directly from the graph paper provided with

the Fibroquant kit for the fibrinogen concentration.

8. If the fibrinogen content is high the clotting time

will be less than 8 seconds. In such cases repeat the

test at 1:20 dilution of the sample or 1:30 dilution

of the sample. The results read off the graph will be

multiplied by a factor 2 or 3 for the respective dilution.

Clinical Hematology: Bleeding Disorders 301

9. Conversely, if fibrinogen content is low, the clotting

time will be over 25 seconds. Repeat the assay at 1:5

dilution, or if necessary at 1:2 dilution. In this case the

results read off the graph will be divided by a factor of

2 or 5 for the respective dilutions.

This procedure can also be performed on an automated/

semiautomated mechanical/optical instrument but the

equipment manufacturer’s methodology should be strictly

adhered to.

Remarks

1. Significant levels of heparin and elevated levels of

fibrinogen degradation products (FDP) in the patient

plasma can cause falsely low fibrinogen results.

2. Insufficient prewarming of plasma and reagent or

contaminated glassware may cause erroneous results.

3. EDTA should not be used as an anticoagulant.

4. Use reagents of the same lot for performing the test.

5. Do not interchange reagents from different lots.

FIBRINOLYTIC ACTIVITY

The three methods presented below are measures of

fibrinolytic activity in general and are influenced by

many factors. These serve as screening procedures, but

the specific contribution of the various factors must be

determined by other means. The lack of suitable standards

makes quantitative measurements unavailable for most

diagnostic laboratories.

Euglobulin Lysis Time

Principle

Euglobulin fraction of plasma contains fibrinogen and all

the plasminogen activator and plasminogen of plasma but

only traces of the anti-plasmins. The lysis of a fibrin clot

formed by the addition of thrombin is a measure of the

fibrinolytic activity.

Requirements

1. Equipment for collection of blood

2. Centrifuge

3. Topical thrombin

4. Serological pipettes

5. Carbon dioxide. A tank of CO2 fitted with a valve to

allow control of the rate of flow.

Method

1. Blood is collected in the usual manner and mixed

immediately with 0.11 M sodium citrate in a ratio of 1

part citrate solution to 9 parts blood.

2. Plasma is obtained by centrifugation.

3. 0.4 mL plasma is placed in a test tube and 7.6 mL

distilled water is added.

4. CO2 is bubbled into the solution through a capillary

tube for 30 seconds.

5. The precipitate which forms is collected by centrifugation at about 3,000 rpm for 15 minutes.

6. The precipitate is dissolved in 1 mL M/15 phosphate

buffer, pH 7.2.

7. To the euglobin in phosphate buffer, 0.1 mL thrombin

(topical thrombin diluted to 100 units per mL with

saline) is added. The solution is mixed.

8. Clotting should be rapid. After clotting has occurred,

the tube is placed in water bath (37°C) and observed

for lysis of clot, which is the end-point.

Result

In normal plasma, a period of 2 to 4 hours is required

for euglobin clot lysis to occur (the technique should be

standardized in each laboratory).