Novel means and method for diagnostic quantitation of serum or plasma bilirubin

Claim 1

A method for the determination of total bilirubin in blood serum or plasma, which comprises: (a) providing a first set of three and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain: (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in each of said reservoirs in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsules within said reservoirs thereby bringing said sodium nitrite into admixture with the acid reagent; (c) adding equally measured proportions of blood serum or plasma to one reservoir of each of said sets; (d) adding equally measured proportions of a standard bilirubin solution to a second reservoir of each of said sets; (e) adding equally measured proportions of a control serum to each of a third reservoir in each of said sets; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about ten minutes; (g) observing the degree of color developed in each of said reservoirs within about 30 minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine total bilirubin in the mixture of step (c) above.

Claim 2

A method for the determination of total bilirubin in blood serum or plasma, which comprises: (a) providing a first set of three and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain: (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in each of said reservoirs in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsules within said reservoirs thereby bringing said sodium nitrite into admixture with the acid reagent; (c) adding equally measured proportions of blood serum or plasma to one reservoir of each of said sets; (d) adding equally measured proportions of a standard bilirubin solution to a second reservoir of each of said sets; (e) adding equally measured proportions of a control serum to each of a third reservoir in each of said sets; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about ten minutes; (g) observing the degree of color developed in each of said reservoirs within about 30 minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine total bilirubin in the mixture of step (c) above.

Claim 4

A method for the determination of direct bilirubin in blood serum or plasma, which comprises; (a) providing a first set of one and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain; (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in said reservoir in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsules within said reservoirs, thereby bringing said sodium nitrite into admixture with the acid reagent in said reservoirs; (c) adding equally measured proportions of blood serum or plasma to one reservoir of each of said sets; (d) adding a measured proportion of a standard bilirubin solution to a second reservoir in said second set; (e) adding a measured proportion of a control serum to the remaining reservoir of said second set; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about 10 minutes; (g) observing the degree of color developed in each of said reservoirs within about 30 minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine direct bilirubin in the mixtures of step (c) above.

Claim 5

A method for the determination of direct bilirubin in blood serum or plasma, which comprises; (a) providing a first set of one and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain; (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in said reservoir in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsules within said reservoirs, thereby bringing said sodium nitrite into admixture with the acid reagent in said reservoirs; (c) adding equally measured proportions of blood serum or plasma to one reservoir of each of said sets; (d) adding a measured proportion of a standard bilirubin solution to a second reservoir in said second set; (e) adding a measured proportion of a control serum to the remaining reservoir of said second set; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about 10 minutes; (g) observing the degree of color developed in each of said reservoirs within about 30 minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine direct bilirubin in the mixtures of step (c) above.

Claim 7

A method for the determination of total and direct bilirubin in blood serum or plasma, which comprises; (a) providing a first set of four and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain: (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in each of said reservoirs in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and one of said reservoirs of said first set, each capsule enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsule within said reservoirs thereby bringing said sodium nitrite into admixture with the acid reagent; (c) adding equally measured proportions of blood serum or plasma to two reservoirs of said first set and one reservoir of said second set; (d) adding equally measured proportions of a standard bilirubin solution to a third reservoir in said first set and to a second reservoir in said second set; (e) adding equally measured proportions of a control serum to each of the remaining reservoirs in said first and second sets; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about 10 minutes; (g) observing the degree of color developed in each of said reservoirs within about thirty minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine total and direct bilirubin in the mixtures of step (c) above.

Claim 8

A method for the determination of total and direct bilirubin in blood serum or plasma, which comprises; (a) providing a first set of four and a second set of three separate reagent reservoirs, each of which comprises a flexible walled, closed container having a thin puncturable zone for access to the contents of said reservoirs and which contain: (i) a first acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids disposed in each of said reservoirs in said first set; (ii) a second acid reagent consisting essentially of an aqueous solution of sulfanilic and hydrochloric acids and dimethyl sulfoxide disposed in each of said reservoirs in said second set; (iii) a frangible capsule disposed within each of said reservoirs of said second set and one of said reservoirs of said first set, each capsule enclosing under hermetic conditions a proportion of sodium nitrite; (b) breaking each of said capsule within said reservoirs thereby bringing said sodium nitrite into admixture with the acid reagent; (c) adding equally measured proportions of blood serum or plasma to two reservoirs of said first set and one reservoir of said second set; (d) adding equally measured proportions of a standard bilirubin solution to a third reservoir in said first set and to a second reservoir in said second set; (e) adding equally measured proportions of a control serum to each of the remaining reservoirs in said first and second sets; (f) simultaneously incubating the mixtures obtained in steps (c) - (e) above at room temperature for about 10 minutes; (g) observing the degree of color developed in each of said reservoirs within about thirty minutes of said incubating; and (h) comparing the degree of color developed in each of said reservoirs to determine total and direct bilirubin in the mixtures of step (c) above.