THROMBOSIS RESEARCH bol. 11. pp. 1207-1208. % Pereamon Pr5.s Ltd. 19’8. Primer! inGreat Bnr31n.
LETTER
'!‘I) THE
EDlTORS-IN-CHTEF
A RAPID SLIDE TEST FOR THE DETERMINATION PLASMA FIBRINOGEN USING RISTOCETIN
OF
L.N.McF. Hargreaves and P.C. Hayes, Department of Pathology, University of Dundae, Scotland, U.K.
(Received
in revised form by A.L. CoQley)
10.3.1978: Accepted
1j.G. 1978.
ISTRODUCTION
Methods for the assay of fibrinogen ability
by thrombin
depend upon:
or salt fractionation,
heat precipitability;
antiganicity;
but have disadvantages such as complexity,
coagul-
volume of
plasma or time required (1). Simple, quick methods of fibrinogen determination are therefore worthexploring. We describe here a method based on the observation by Howard and Firkin (2) that fibrinogen is precipitated by the antibiotic ristocetin. The slide test assay was carried out as follows: One drop of plasma was placed on a microscope slide. To this, one drop of ristocetin (6 mg/ml) was added, mixed, and precipitation was timed with a stopwatch, by observation under 3 x magnification. A standard curve for fibrinogen precipitation was prepared by plotting reciprocal precipitation times versus standard pooled plasma dilutions. The standard dilutions used were l/5, l/10, l/20 and 1140; test plasmas were diluted to l/15. The ammonium sulphate turbidity method, an established assay for fibrinogen
measurement,
was used as described by Coles and Roman (3) for comparison. Fig. 1 shows the curves obtained
when fibrinogen
in dilutions
of the standard pooled plasma
was estimated using the slide test assay method and the ammonium sulphate turbidity technique. These graphs were used as the standard curves for the measurement of fibrinogen in all the experiments described.
,.I’
,,i.” _.I’ ,,,’ ,_.I ..-* _.I’
) 0%
31,) ,,:” ‘.U
c.2,...,,.*b” ‘,
,.I’ ,,.’
Fig.
,.,’ ,.A” ,,,’ ,,.’
0%
,.-‘,
1. Change in l/precipitation
time
(slide
test assay) and optical density ((NH4)2S04 precipitation method) when plotted against plasma dilutions.
. . ..2.*,1..9* ” 1207
1208
R=\PID FTBRISOGEX
When fibrinogen
method.
vol.l2.so.~
levels in eight normals were estimated by the two above methods, the carrel.
etion coefficient was 0.85. Fig. 2 demonstrates the correlation arthritis
DETER!4IYATION
obtained
when fibrinogen
levels in a series of rheumatoid
patients were measured using the slide test assay and the ammonium The correlation
coefficient
sulphate turbidity
was 0.86, and in the group of 25 patients, the range of fibrin-
ogen values was 1.6 - 6.4 g/litre.
Correlation Fig. 2 levels in rheumatoid
between fibrinogen arthritis patients as
measured by the slide test and (NH4)2S04 turbidity method.
No precipitation occurred when ristocetin (6 mg/ml) was added to an undiluted serum sample containing 80 fig/ml fibrin degradation products (FDP). This value would be equivalent to a level of 1200 pg/ml FDP in our test, as the samples are diluted 15 fold before testing. The effect of late FDP was therefore likely to be negligible although early FDP and fibrin monomer may interfere. This assay is carried out on a microscope slide, requiring very small amounts of reagents, and is quick, simple and inexpensive. The use of the assay therefore is suggested for cases where a rapid fibrinogen estimation is required, especially when only small amounts of plasma are available. REFERENCES 1.
2. 3.
In Thrombosis and Bleeding Disorders, edited by HUSEBY, R.M., BANG, N.U. Fibrinogen. Academic Press, London and New York, N.U. Bang, F.K. Belier, E. Deutsch, E.F. Mammen. pp 222-247,197l. HOWARD, M.A., FIRKIN, B.G. Ristocetin - anew tool in the investigation of plateletaggregation. Thromb. Diath. Haemorrh., 26,362,1971. COLES, M., ROMAN, W. A rapid method for estimating fibrinogen in plasma. J.Clin.Path., 10,282,1957.
LETTER
'!‘I) THE
EDlTORS-IN-CHTEF
A RAPID SLIDE TEST FOR THE DETERMINATION PLASMA FIBRINOGEN USING RISTOCETIN
OF
L.N.McF. Hargreaves and P.C. Hayes, Department of Pathology, University of Dundae, Scotland, U.K.
(Received
in revised form by A.L. CoQley)
10.3.1978: Accepted
1j.G. 1978.
ISTRODUCTION
Methods for the assay of fibrinogen ability
by thrombin
depend upon:
or salt fractionation,
heat precipitability;
antiganicity;
but have disadvantages such as complexity,
coagul-
volume of
plasma or time required (1). Simple, quick methods of fibrinogen determination are therefore worthexploring. We describe here a method based on the observation by Howard and Firkin (2) that fibrinogen is precipitated by the antibiotic ristocetin. The slide test assay was carried out as follows: One drop of plasma was placed on a microscope slide. To this, one drop of ristocetin (6 mg/ml) was added, mixed, and precipitation was timed with a stopwatch, by observation under 3 x magnification. A standard curve for fibrinogen precipitation was prepared by plotting reciprocal precipitation times versus standard pooled plasma dilutions. The standard dilutions used were l/5, l/10, l/20 and 1140; test plasmas were diluted to l/15. The ammonium sulphate turbidity method, an established assay for fibrinogen
measurement,
was used as described by Coles and Roman (3) for comparison. Fig. 1 shows the curves obtained
when fibrinogen
in dilutions
of the standard pooled plasma
was estimated using the slide test assay method and the ammonium sulphate turbidity technique. These graphs were used as the standard curves for the measurement of fibrinogen in all the experiments described.
,.I’
,,i.” _.I’ ,,,’ ,_.I ..-* _.I’
) 0%
31,) ,,:” ‘.U
c.2,...,,.*b” ‘,
,.I’ ,,.’
Fig.
,.,’ ,.A” ,,,’ ,,.’
0%
,.-‘,
1. Change in l/precipitation
time
(slide
test assay) and optical density ((NH4)2S04 precipitation method) when plotted against plasma dilutions.
. . ..2.*,1..9* ” 1207
1208
R=\PID FTBRISOGEX
When fibrinogen
method.
vol.l2.so.~
levels in eight normals were estimated by the two above methods, the carrel.
etion coefficient was 0.85. Fig. 2 demonstrates the correlation arthritis
DETER!4IYATION
obtained
when fibrinogen
levels in a series of rheumatoid
patients were measured using the slide test assay and the ammonium The correlation
coefficient
sulphate turbidity
was 0.86, and in the group of 25 patients, the range of fibrin-
ogen values was 1.6 - 6.4 g/litre.
Correlation Fig. 2 levels in rheumatoid
between fibrinogen arthritis patients as
measured by the slide test and (NH4)2S04 turbidity method.
No precipitation occurred when ristocetin (6 mg/ml) was added to an undiluted serum sample containing 80 fig/ml fibrin degradation products (FDP). This value would be equivalent to a level of 1200 pg/ml FDP in our test, as the samples are diluted 15 fold before testing. The effect of late FDP was therefore likely to be negligible although early FDP and fibrin monomer may interfere. This assay is carried out on a microscope slide, requiring very small amounts of reagents, and is quick, simple and inexpensive. The use of the assay therefore is suggested for cases where a rapid fibrinogen estimation is required, especially when only small amounts of plasma are available. REFERENCES 1.
2. 3.
In Thrombosis and Bleeding Disorders, edited by HUSEBY, R.M., BANG, N.U. Fibrinogen. Academic Press, London and New York, N.U. Bang, F.K. Belier, E. Deutsch, E.F. Mammen. pp 222-247,197l. HOWARD, M.A., FIRKIN, B.G. Ristocetin - anew tool in the investigation of plateletaggregation. Thromb. Diath. Haemorrh., 26,362,1971. COLES, M., ROMAN, W. A rapid method for estimating fibrinogen in plasma. J.Clin.Path., 10,282,1957.
