Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2001-12-07
2004-01-20
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S004000, C435S975000, C435S283100
Reexamination Certificate
active
06680177
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The present invention relates to a dry chemistry format assay for measuring the low molecular weight heparin content of a whole blood sample, and a system and reagent for performing such an assay.
2. Discussion of the Background
Low molecular weight heparins (LMWHs) are a heterogeneous group of antithrombotic drugs produced from unfractionated heparin (UFH) using diverse chemical and enzymatic processes. LMWHs, like UFH, exhibit an anticoagulant effect by complexing with antithrombin (AT) to inactivate several of the coagulation enzymes preventing fibrin formation. Of these, Factor Xa and thrombin (IIa) are the most responsive to inhibition. LMWHs, introduced as antithrombotic drugs in the mid-1980s, are now established as the drug of choice for surgical thromboprophylaxis and are increasingly replacing UFH in the acute treatment of venous thromboembolic disorders. The low molecular weight heparin, enoxaparin, increasingly is used in patients with unstable angina (UA) and non-Q-wave myocardial infarction (NQMI) (J. Fareed et al, Past, present and future considerations on low molecular weight heparin differentiation: an epilogue. Semin Thromb Hemost, 25 Suppl 3:145-7 (1999), and J. Hirsh et al, Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest, January; 119(1 Suppl):64S-94S (2001)), who transition to percutaneous coronary intervention (PCI) (Lovenox (enoxaparin sodium) injection package insert, ©1998, rev. January, 2001). Although the activated partial thromboplastin time (aPTT) and activated clotting time (ACT) are the most common methods used to monitor UFH, they are relatively insensitive to LMWHs, such as enoxaparin. While chromogenic anti-Xa assays are commonly used to monitor the concentration of LMWHs, such assays provide an indirect measure of drug concentration and results are not routinely available in a cardiac catheterization laboratory setting.
The LMWHs have mean molecular weights between 4000 to 6000 daltons, and they have less ability to inactivate thrombin compared to UFH. Each LMWH is a specific mixture often demonstrating a unique anti-Xa/anti-IIa ratio and signature anticoagulant profile. The result is an anti-Xa/anti-IIa ratio of approximately 3 to 14:1 (depending on the brand of LMWH, dosage, and route of administration) compared to the 1:1 ratio observed with UFH
(Lovenox P.I.)
. The LMWH, enoxaparin, has a mean molecular weight of approximately 4,500 daltons and, given at a dose of 1.5 mg/kg subcutaneously (SC), is characterized by a higher ratio of anti-Factor Xa to anti-Factor IIa activity (mean±SD, 14.0±3.1) (based on areas under anti-Factor activity versus time curves) compared to the ratios observed for heparin (mean±SD, 1.22±0.13)
(Lovenox P.I.)
. This is an important distinction because the ability to prolong the aPTT and ACT is proportional to anti-IIa activity. Chromogenic anti-Xa assays provide estimates of enoxaparin concentration only in dilute, supplemented plasma and are not suitable for point-of-care (POC) testing.
More recently, clinical trials have confirmed the safety and efficacy of the LMWH, enoxaparin sodium (Lovenox®, Clexane®), in the management of acute coronary syndromes (ACS) (J. Fareed et al., Thromosis and Hemostasis, Supplement 3, Vol. 25, 3-4 (1999)).
Blood clotting reactions, in general, are employed as clinical assays to measure the time required for the formation of a fibrin clot. Blood clotting assays are principally used for screening, diagnosis, and for monitoring patients receiving anticoagulant therapy. There are many types of coagulation assays. These include prothrombin time (PT), partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), fibrinogen assay, thrombin clotting time (TCT), activated clotting time (ACT), etc.
Before performing conventional clotting tests, a blood sample is collected in a tube or syringe containing anticoagulant (citrate). The blood sample is centrifuged, and the plasma separated (e.g., by decantation) from the red blood cells. A measured quantity (usually 0.1 ml) of plasma is pipetted into the reaction vessel or cuvette. A measured amount of reagent is then added manually via pipette or automatically by means of other volumetric delivery systems capable of metering a known, preset quantity of reagent. Alternatively, the sample can be added to the reagent directly.
Typically, 0.2 ml of reagent is employed. The addition of the reagent initiates the reaction. Many existing blood clotting assays suffer from at least one of the following disadvantages: difficulty in performance, requirement of highly trained personnel, inaccuracy in measurement, reagent instability, large consumption of reagent, etc.
One solution to this problem was addressed in Oberhardt, U.S. Pat. No. 5,110,727, in which a dry reagent based reaction slide is provided for performing coagulation assays quickly, accurately and simply. Such tests are marketed by Pharmanetics, Inc.
The capacity of blood to clot, as well as to not clot, is dependent on a large number of enzymatic factors and cofactors. The ability of central clinical laboratories to reliably and conveniently assay for LMWH in whole blood or plasma samples can be critical in monitoring individuals in LMWH therapy. The blood coagulation system is dominated by sequential proteolytic activation reactions of inactive precursors, called zymogens. Forward clotting reactions are controlled by simultaneous activation of anticoagulant zymogens that serve to limit the extent of clot formation and initiate the fibrinolytic system to resolve the clot.
There is thus a strongly felt need for a simple, facile and accurate method for the performance of blood clotting assays, e.g., in medical applications. Such a method should be based on a minimum number of manipulations of either a sample or reagent. Ideally such a method should be easily utilized by persons without extensive clinical laboratory training and should require no sample or reagent-containing solution preparation. It should not suffer the problems associated with reagent instability and be very accurate. It should permit effective mixing of sample and reagent. It should require only a very small amount of sample. And it should be able to perform automatic treatments of the sample, e.g., it should not require centrifugation of the blood sample or any other off line cell separation process. Available clotting parameter assays likewise suffer salient disadvantages.
Since the tests currently used for LMWHs are chromogenic assays requiring isolation of derived plasma from whole blood samples and significant processing time for performing the assay, an assay is needed that can quantitatively measure LMWHs quickly and easily, using whole blood and be performed at the bedside, in order to provide rapid determinations of LMWH therapeutic levels.
Additionally, conventional chromogenic assays measure actual levels of LMWH in the plasma sample, but do not reflect the actual clotting dynamics of the patient's blood. Since the clotting dynamics can depend or be confounded by a variety of factors, a test is needed that will correlate the clotting time with the amount of LMWH in the sample, and will also detect other possible problems in the clotting dynamics that are independent of the LMWH.
Clotting parameter assays are referred to herein as function and structure-based assays in the broad realm of coagulation diagnostics which do not utilize clot formation or clot lysis processes to generate end points. Most of these assays utilize chromogenic synthetic substrates to quantify molecular markers or specific factors or components associated with coagulation. These are typically functional reaction based assays as opposed to most immunoassays which could detect the same molecules but utilize structure recognition and may therefore still identify inhibited components or defective components, neither of which may be functional.
SUMMARY OF THE INVENTION
Cardiovascular Diagnostics Inc.
Leary Louise N.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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