US 6,994,984 B2 · Hematological assay

Abstract

A hematological assay is described in which the blood coagulation potential of a body fluid is assessed by reacting a sample of the body fluid with an amount of an activator reagent comprising: (a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof, and (b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof or an enzyme activating endogenous factor V, (c) (optionally) phospholipids. The reagent may be dry (e.g. lyophilised) or in an aqueous solution preferably buffered to a pH from 6 to 10 (preferably 7 to 8), if desired incubating, if necessary inducing coagulation by the addition of one or more coagulation accelerants such as calcium chloride, and establishing a value indicative of the coagulation potential, e.g. by measuring the time to clotting on an optical coagulometer or through use of a chromogenic substrate. It is preferred to use at (b) factor V activator from purified Russell's Viper venom (RVV-V). An activator reagent is also described containing the components mentioned above preferably in one or more buffer solutions or in dried, e.g. lyophilised form.

Claims29

Dependent claims

A method of determining the blood coagulation potential of a body fluid selected from whole blood, citrated or equivalently stabilized blood, and plasma, comprising:

(i) contacting an activator reagent with a sample of the body fluid, wherein the reagent reacts with the body fluid, said reagent comprising:
(a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof;
and
(b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof, or an enzyme which activates endogenous factor V;
(ii) if necessary, inducing coagulation by adding one or more coagulation accelerants;
and
(iii) establishing a value indicative of the coagulation potential,
wherein the establishment of the value indicative of the coagulation potential comprises measuring one of the following:
(a) the time between the contacting of the activation reagent with or without the coagulation accelerant to said sample and the onset of clotting;
(b) the time between the addition of said reagent with or without the coagulation accelerant to said sample and the detection of free thrombin activity;
(c) the thrombin activity;
and
(d) the factor Xa activity,
and wherein the time between the addition of said reagent with or without the coagulation accelerant to said sample and the onset of clotting is measured using an optical coagulometer.

11 more elements

A method of determining the blood coagulation potential of a body fluid selected from whole blood, citrated or equivalently stabilized blood, and plasma, comprising:

(i) contacting an activator reagent with a sample of the body fluid, wherein the reagent reacts with the body fluid, said reagent comprising:
(a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof;
and
(b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof, or an enzyme which activates endogenous factor V;
(ii) if necessary, inducing coagulation by adding one or more coagulation accelerants;
and
(iii) establishing a value indicative of the coagulation potential,
and further comprising incubating the sample reacted with the activator reagent;
and then adding to the reaction mixture the accelerator and optionally an analytical accessory agent, wherein the method further comprises:
(i) preparing an activator reagent comprising:
(a) a predetermined amount from 0.01 to 10 nkat/ml of factor Xa;
(b) a predetermined amount from 0.05 to 10 U/ml of RVV-V;
and
(c) optionally a predetermined amount from 1 to 200 μg/ml of phospholipids,
in an aqueous buffer solution containing from 10 to 200 mM Tris/HCl, from 0.6 to 1.2% w/v NaCl, and from 0.01 to 1.0% w/v albumin;
(ii) mixing a predetermined amount from 1 to 100 μl of the sample with a predetermined amount from 1 to 100 μl of the activator reagent, wherein the sample is citrated or equivalently stabilized platelet-poor plasma;
(iii) incubating the mixture;
(iv) adding a predetermined amount from 10 to 100 μl of from 2 to 200 mM CaCl2;
(v) optionally adding an analytical accessory agent providing a detectable value;
and
(vi) establishing the value indicative of the coagulation potential.

18 more elements

A method of determining the blood coagulation potential of a body fluid selected from whole blood, citrated or equivalently stabilized blood, and plasma, comprising:

(i) contacting an activator reagent with a sample of the body fluid, wherein the reagent reacts with the body fluid, said reagent comprising:
(a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof;
and
(b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof, or an enzyme which activates endogenous factor V;
(ii) if necessary, inducing coagulation by adding one or more coagulation accelerants;
and
(iii) establishing a value indicative of the coagulation potential,
and further comprising:
(i) preparing an activator reagent comprising:
(a) a predetermined amount from 0.01 to 10 nkat/ml of factor Xa;
(b) a predetermined amount from 0.05 to 10 U/ml of RVV-V;
and
(c) optionally a predetermined amount from 1 to 200 μg/ml of phospholipids,
in an aqueous buffer solution containing from 10 to 200 mM Tris/HCl, from 0.6 to 1.2% w/v NaCl, and from 0.01 to 1.0% w/v albumin;
(ii) mixing a predetermined amount from 1 to 100 μl of the sample with a predetermined amount from 1 to 100 μl of the activator reagent, wherein the sample is citrated or equivalently stabilized platelet-poor plasma;
(iii) adding a predetermined amount from 10 to 100 μl of from 2 to 200 mM CaCl2;
(iv) optionally adding an analytical accessory agent providing a detectable value;
and
(v) establishing the value indicative of the coagulation potential.

16 more elements

A method of determining a blood coagulation component or treatment additive in a sample containing a predetermined amount of human body fluid, comprising comparing the coagulation potential of the sample with that of a comparable normal human body fluid and/or that of a comparable human body fluid which either lacks the component or additive, or contains a known amount of the component or additive in excess, wherein coagulation potential is determined by a method comprising:

(i) contacting an activator reagent with a sample of the body fluid, wherein the reagent reacts with the body fluid, said reagent comprising:
(a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof;
and
(b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof, or an enzyme which activates endogenous factor V;
(ii) if necessary, inducing coagulation by adding one or more coagulation accelerants;
and
(iii) establishing a value indicative of the coagulation potential,
said method further comprising:
(i) preparing an activator reagent comprising:
(a) a predetermined amount from 0.01 to 10 nkat/ml of factor Xa;
(b) a predetermined amount from 0.05 to 10 U/ml of RVV-V;
and
(c) optionally a predetermined amount from 1 to 200 μg/ml of phospholipids,
in an aqueous buffer solution containing from 10 to 200 mM Tris/HCl, from 0.6 to 1.2% w/v NaCl, and from 0.01 to 1.0% w/v albumin;
(ii) mixing a predetermined amount from 1 to 100 μl of the sample with a predetermined amount from 1 to 100 μl of the activator reagent, wherein the sample is citrated or equivalently stabilized platelet-poor plasma;
(iii) incubating the mixture;
(iv) adding a predetermined amount from 10 to 100 μl of from 2 to 200 mM CaCl2;
(v) optionally adding an analytical accessory agent providing a detectable value;
and
(vi) establishing the value indicative of the coagulation potential.

17 more elements

(i) contacting an activator reagent with a sample of the body fluid, wherein the reagent reacts with the body fluid, said reagent comprising:
(a) a predetermined amount of factor Xa or a hematologically equivalent mutant thereof;
and
(b) a predetermined amount of factor Va, a hematologically equivalent mutant thereof, or an enzyme which activates endogenous factor V;
(ii) if necessary, inducing coagulation by adding one or more coagulation accelerants;
and
(iii) establishing a value indicative of the coagulation potential,
and further comprising:
(i) preparing an activator reagent comprising:
(a) a predetermined amount from 0.01 to 10 nkat/ml of factor Xa;
(b) a predetermined amount from 0.05 to 10 U/ml of RVV-V;
and
(c) optionally a predetermined amount from 1 to 200 μg/ml of phospholipids,
in an aqueous buffer solution containing from 10 to 200 mM Tris/HCl, from 0.6 to 1.2% w/v NaCl, and from 0.01 to 1.0% w/v albumin;
(ii) mixing a predetermined amount from 1 to 100 μl of the sample with a predetermined amount from 1 to 100 μl of the activator reagent, wherein the sample is citrated or equivalently stabilized platelet-poor plasma;
(iii) adding a predetermined amount from 10 to 100 μl of from 2 to 200 mM CaCl2;
(iv) optionally adding an analytical accessory agent providing a detectable value;
and
(v) establishing the value indicative of the coagulation potential.

16 more elements

Dependencies are inferred from the claim text.

Hematological assay

Life of this patent

Hematological assay

Life of this patent

Abstract

Claims29