Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2000-10-03
2002-09-10
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S004000, C435S283100
Reexamination Certificate
active
06448024
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the field of determining components of biological fluids, and more particularly relates to methods, reagents, test cartridges and devices useful for determining fibrinogen in physiological samples, preferably in undiluted whole blood and undiluted blood plasma samples.
The recognition of a potential role of fibrinogen in cardiovascular disorders has increased the need for reliable and simple fibrinogen assays. Prior art methods of determining fibrinogen include clotting-time dependent fibrinogen assays that determine the coagulation time of a diluted sample and correlate the clotting time with the fibrinogen concentration in a sample wherein the coagulation time is inversely proportional to the amount of fibrinogen. In addition, total protein assays have been performed to determine the amount of protein in a clot, which is then correlated with an initial fibrinogen concentration. The first type of technique requires dilution in order to slow down the rate of coagulation to facilitate obtaining a meaningful result, while the second type of technique requires isolation of a clot from the sample, washing the clot, and determining the protein content. In view of the desire to minimize the manipulation of biological fluids, such as blood, there is a need for improved assay methods that minimize sample manipulation. Thus, it is particularly desirable to have an assay that can utilize an undiluted sample, but which avoids the problems caused by rapid clotting.
The need for improved assay methods that minimize sample manipulation is demonstrated by U.S. Pat. No. 5,292,664, to Fickenscher, which discloses a test and reagent for determining fibrinogen in undiluted plasma samples. The method of Fickenscher involves the addition of a large excess of thrombin, or a protease with analogous activity, to ensure immediate conversion of all of the fibrinogen in the sample to fibrin monomers. In a preferred embodiment of Fickensher's method, a heparin inhibitor is added to prevent inhibition of thrombin. However, the aggregation of the fibrin monomers is slowed by the addition of a fibrin aggregation inhibitor, thus slowing the formation of clots. The clotting time at a constant concentration of a fibrin aggregation inhibitor can be correlated with the fibrin concentration, and hence lead to a determination of the fibrinogen level in the original sample. Thus, Fickensher's method of inhibiting clotting promotes a first reaction in the clotting pathway, followed by inhibition of a subsequent reaction. This method requires that the sample be combined with a fibrin aggregation inhibitor prior to the addition of excess thrombin, otherwise clotting will occur too rapidly for the method to be useful.
Further information on the physiological role of fibrinogen and prior assays is abundant and readily available and known to those of skill in the art. More background information can be obtained from: National Committee for Clinical Laboratory Standards, Procedure for the Determination of Fibrinogen in Plasma; Approved Guideline. NCCLS document H30-A. (ISBN 1-56238-221-7), NCCLS, 771 East Lancaster Avenue, Villanova, Penn. 19085 (1994); U.S. Pat. No. 5,563,041; and Canadian Patent 2,252,983. All documents mentioned herein are incorporated by reference as if reproduced in full below.
There remains a need for a one-step fibrinogen assay for whole undiluted blood, and corresponding reagents and devices. The term “one-step” refers to test sample manipulation steps, such as the number of manipulations required to determine the fibrinogen in a sample. There is also a need for clotting-time dependent fibrinogen assays in which sample clotting time is less than about three hundred seconds for samples containing a wide range of fibrinogen concentrations and in which clinically significant differences in sample fibrinogen concentration correlate to reliable and readily measurable differences in clotting time. There is also a need for fibrinogen assay reagents which are stable, cost effective, readily available, can be used in dry form and can be dissolved by the sample of interest, and that provide consistent results from sample to sample. It is also desired that sample containers and other surfaces that come into contact with samples, particularly blood samples, be disposable and provide minimum opportunity for human contact with the sample. Thus, there is also a need for a cartridge useful in an automated clotting time determination device, which fulfils all or most of these needs. There is also a need for an automated device that directly reports the amount of fibrinogen in a sample.
SUMMARY OF THE INVENTION
The present invention involves a method, reagent, test cartridge, and device for determining fibrinogen in a physiological sample, such as whole blood or undiluted blood plasma. The method, reagent, test cartridge and device make use of thrombin and a thrombin inhibitor. With the thrombin and thrombin inhibitor added to it, the clotting time of a sample is measured. Since the clotting time of a blood or blood plasma sample is inversely related to its fibrinogen concentration, the clotting time can be used to determine sample fibrinogen concentration by reference to the clotting times of fibrinogen standards that have been contacted with constant amounts of reagent active ingredients.
In a preferred embodiment of the present invention, the physiological sample is undiluted, such as an undiluted whole blood sample or an undiluted blood plasma sample. Preferably, activity of added thrombin, as well as the activity of the endogenous thrombin in blood samples, is inhibited in the assay, thus slowing the rate of conversion of fibrinogen in the sample to fibrin. In a preferred embodiment, clotting time is sufficiently slow in samples containing the range of fibrinogen concentrations of clinical interest that differences in clotting time can be readily linked to different fibrinogen levels. In a preferred embodiment, an assay reagent is utilized that comprises a predetermined amount of thrombin and a thrombin inhibitor that slows but does not stop thrombin enzymatic activity with respect to the conversion of fibrinogen to fibrin.
In a preferred embodiment, a device for determining sample fibrinogen concentration is provided, comprising a cartridge incorporating a reagent of the present invention, comprising thrombin and a thrombin inhibitor. In an embodiment, a test cartridge and reagent is provided which can be utilized in a one-step assay to determine fibrinogen in an undiluted whole blood sample.
In another embodiment, a test cartridge and reagent is utilized in a two-step assay to determine fibrinogen in undiluted blood plasma; the first step being the addition of a detectable material, such as but not limited to latex particles, as a surrogate for red blood cells that would be detected in a whole blood sample, and the second step being identical to the one-step assay for determining fibrinogen in whole blood.
In a preferred embodiment, the device of the present invention comprises a processor for converting sample clotting time to the fibrinogen concentration of the sample. The device preferably includes a display or other readout device for automatically reporting the sample fibrinogen concentration to the device operator, and optionally may also provide the clotting time of the sample. In an alternative embodiment, a reagent and method is provided which can be utilized in a one-step assay using a fibrometer to determine fibrinogen in an undiluted blood plasma sample or undiluted whole blood sample, wherein the clotting time of a sample is based on the time required, after contact of the sample with the reagent, for the fibrometer probe to detect a predetermined degree of resistance to movement through the sample. The clotting time is then used to determine the amount of fibrinogen in the sample.
Thus, one embodiment of the present invention is a device for use in determining fibrinogen in a sample suspected of containing fibrinogen, compr
Brinks Hofer Gilson & Lione
Leary Louise N.
Roche Diagnostics Corporation
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