DIRECT FIBRINOGEN ASSAY

DOSAGE DIRECT DE LA FIBRINOGENE

English Abstract

2068221 9108460 PCTABS00005
An endpoint assay for measuring fibrinogen utilizing strong
thrombin and weak plasma. In a preferred embodiment the assay is based
on the direct conversion of a normalized signal from a sensor to
fibrinogen concentration.

Claims

WO 91/08460 PCT/US90/06988
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WHAT IS CLAIMED IS:

1. A method for optically measuring a concentration of
fibrinogen in a blood plasma sample, said method compris-
ing:
providing a sample of plasma containing fibrinogen in
a container;
adding thrombin to the sample;
mixing the thrombin with the sample to form a reaction
mixture;
measuring an initial optical transmittance for the
reaction mixture;
allowing the thrombin and fibrinogen to react with
each other in the reaction mixture;
measuring a final optical transmittance for the
reaction mixture;
comparing the final transmittance measurement to the
initial transmittance measurement to compute a delta value;
and
determining the concentration of fibrinogen based on
the delta value.


2. The method of claim 1, wherein the thrombin is at
a concentration of about 100 NIH unit and the plasma
sample is diluted in about a 1:10 ratio with sodium
barbital.


3. The method of claim 1, wherein the delta value is
computed according to the equation:

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where
D is a normalized value of delta;
Ti is the initial optical transmittance of the sample;

WO 91/08460 PCT/US90/06988

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Tf is the final optical transmittance of the sample;
So is an offset which is dependent on the device used
to perform said method; and
K is a predetermined constant.
4. The method of claim 1, wherein said step of
determining fibrinogen concentration further comprises:
deriving a correlation equation which allows
fibrinogen concentration of a sample to be determined from
a value R, where R is computed from the equation

Image

where
D is the delta value of the sample plasma; and
Dc is a delta value of a calibrator plasma; and
determining the fibrinogen concentration of the plasma
sample based on the derived correlation equation.

5. The method of claim 4, wherein the correlation
equation is derived by:
measuring a delta value for a plurality of standard
plasmas of different known concentrations;
measuring a delta value for a calibrator plasma of
known concentration;
and plotting Rs versus the log(Cs/Cc) for the
plurality of standard plasmas where

Image

where
Rs is the R value for a standard plasma;
Ds is a measured delta value for the standard
plasma;
Dc is a measured delta value for a calibrator
plasma;

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Cs is the concentration of fibrinogen in the
standard plasma; and
Cc is the concentration of fibrinogen in the
calibrator plasma.

Description

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DIRECT FI~RINOGEN ASSAY


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.BACKGROUND OF THE INVENTION
The invention relates to a methold for determining the
,concentration o~ fibrinogen in blood ,plasma using thrombin
as a reagent.
Prior methods of using thrombin to measure fibrinogen
concentration, include the Clauss met~hod which is based on
measuring the time it takes for a pla,sma-thrombin reaction ,.
to occur (clotting time) and the ACL3 ~ibrinogen assay. : .
10~ The Clauss method is described in Manual of Hemostasis and
Thrombosis, ed. 3, by Arthur R. Thompson and haurence A.
Harker, Appendix A, p. 179 (1983) and~in Gerrinnunqs
: hv~iologische ~chnell Methode ur B~stimmun~ des ~,.
EL~irgge~ by A- Clauss, Acta Haematol, 17:237 (1957).
:, 15 The ACL3 method is describe~ in Method for the Determina~
: `tio~ of Functional ~Çlottable) Fibr:inoqen bv ~he_New Family ,.: :
e5_~5l.95~9~ 5~ by E. Rossi, P. Mondonico, A. Lomabar~
di,~L. Preda, Thrombosis ~esaarch 52;~453-469~(1988). ~ ,
~These methods rely on th~ measurement of a relevant::
~0 paraméter such as clotting time or changes in;optical
transmission and on multiple dilutions of a calibrator
plasma to compensiate conditions o~ the instrument and
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reagent at a given time. Using calibrator plasmas (i.e.
plasma having known fibrinogen concentrations) "standard
lines" or "calibration curves" must be constructed
repeatedly whenever conditions warr~nt. In the determiina-
tion of ~ibrinogen concentration of an unknown sample, therelevant quantity, such as clotting time, is measured and
the concentration is then "read" from the standard curve.
This process can involve considerable calculation, and is
often tedious and time consuming.
Another deficiency o~ these prior methods is that the
relevant quantity b~ing measured is often instrument depen-
dent, as well as reaction dependent. For example, if the
instrument used to measure the relevant parameter employs
an electro-optical system in which scattered or transmiitted
light is detected, the value obtained from the measurement
will depend on the signal level measured by the optical
sensor, which in turn depends on the amount of light
incident on the reaction vessel as well as the electronic
gains used in association with the optical sensor. The
values of these quantities do not remain constant in time,
nor do they remain constant from channel to channel or
instrument to instrumient.

SUMMARY OF THE INVENTION
There~ore, it is an object of the invention to pr~vide
a method for measuring the concentration of ~ibrinogen in a
blood sample that is more efficient and efficacious manner
than prior methods.
It is another object o~ the invention to eliminate the
effects of instrument variation and channel variation in
~easuring thè changes in optical transmission, which are
the ba~is for determining fibrinogen concentration.
It i~ also an object of the invention to employ
measured quantities in a manner that eliminates the need to
repeatedly establish a standard curve.

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The present invention provides a method for measuring
the concentration of fibrinogen in a hlood plasma sample.
According to the method of the invention, a sample of
plasma containing fibrinogen is provided in a container.
Thrombin is added to the sample and mixed with the sample
to form a reaction mixture. An initial optical transmit-
tance is measured for the reaction mixture. The thrombin
and fibrinogen are allowed to react with each other in the
reaction mixture. A final optical transmittance is
10 measured for the reaction mixture. The measurements are `
manipulated in the manner described below and concentration
of fibrinogen is determined from a previously established
standard curve.
It is an aspect of the invention that the standard
curve is constructed in such a manner that it remains
unchanged by variations in instrument, reagent or sample.
Therefore, once established, it is not necessary to
repeatedly reconstruct it.

DESC~IP~ION OF_THE PREFERREi~ ~MBOD~MENTS
The present method is preferably us~d in conjunction
with an optical monitoring system such as that disclosed in
concurrently fil~d and copending U.S. Patent Application
Serial No. 07/443,952 to Swope et al., entitled "Multichan-
nel Optical Monitoring System", assigned to the assigne~ of
the present application, the disclosure of which is
incorporated herein by reference, or in conjunction with
commercially available hemostasis instruments such a~ the
assignee's model Coag-A-Mate XC or modeI Coag-A-~ate XM. ~ ~ -
Approximate reagent/plasma concentrations that are
suitable for the method of the invention are known from the
Clauss fibrinogen method noted above. The thrombin con-
centration is preferably about 100 NI~ units (a strong
thro~bin concentration) and the plas~a sample is prPferably
diluted in a 1:10 ratio (a weak plasma concentration) with
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WOgl/0~60 P~T/US90/0~988

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Owren 's Veronal Buffer (sodium barbital). Other suitabl~
diluents ~or the plasma are described in Clauss.
In the present invention the formation of fibrinogen
is photo-optically monitored ~or total change between the
optical transmittance before the onset of the reaction and
the optical transmittance at the conclusion of the reac-
tion. According to the method, reagant is added to plasma
and, after a time which allows for co~plete sample-reagent
mixing, an initial transmittance signal (Ti) is recorded.
When the clot i5 fully formed, the final transmittance
signal ~Tf~ is processed as described below.
The relevant parameter, delta or D, is computed the
initial and final transmittance measurements by normalizing
the difference in the readings to the initial value plus
any offset using the following equation-

Ti ~ Tf
D = ~ - x R
Ti ~ So
where
D is the normalized digital value of delta;
Ti is the digital value of the transmitted light prior
to the onset of the clot;
Tf is the digital value of the transmitted light
subsequent to the formation of the clot;
S0 is the digikal offset that may have been imposed as
part o~ the instrument design; and
K is an arbitrary constant chosen for convenience.
It should be noted that in prior methods, D was
defined as the difference (Ti-Tf) only. The denominator in
the above expression represents the normalization of D to
the initial value of ths transmittance.
The next step in det~rmining the concentration of
fibrinogen of an unknown sample is to refer the above
determined value of D to the concentration by the use of a
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WO91/~60 ~ 3~, ~ PCT/U~90/069~
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standard curve. This is done by ~irst computing the ~ -
cluantity
D
R = lo~
Dc - ~-
. .
where
Dc is the previously determined delta for a
calibrator plasma of known fibri~ogen concentra-
tion.
Measurements of Dc are per~ormed relatively infrec~uently as
changes in test conditions warrant. The next step is to
use a previously determined correlation equation which -~
describes ~he relationship between R and fibrinog~n ~ ~ -
concantration to determine the fibrinogen concentration of
the sample. It has been discovered that the correlation
equation relating R and fibrinogen concentration does not
change significantly with different c1esignated reagents and
calibrator plasmas. There~ore, it can be permanently
stored as part of the computational ~;oftware and does not
require periodic recomputation.
The correlation equation is prei~erably derived as
follows: Various standard plas~as of known fibrinogen
concentration are prepared and a delt:a value Ds is deter-
mined for each standard plasma. Next, a value Rs iscalculated for each standard plasma based on the following
~` equation:
Ds
s = log(~
Dc -
. ,. .: .
where
s is the R value ~or a standard plas~a;
Ds is the m~asured delta value ~or the standard ,-
plasma; and ~
D~ is the previously determined delta ~or the ~ ~ -
~cal1brator plasma. ~ ~ ~
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WO91/0~60 PCT/US90/06988
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The correlation equation is then derived by plotting Rs
versus log(Cs/Cc) for the various standard plasmas where Cs
is the fibrinogen concentration of a standard plasma and Cc
is the fibrinogen concentration of the calibrator plasma~
It will be understood that the above description of
the present invention is susceptible to various modifica-
tions, changes and adaptations, and the same are intended
to be comprehended within the meaning and range of e-
quivalents of the appended claims.




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