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

• US 4,078,892 A
• Filed: 09/17/1976
• Issued: 03/14/1978
• Est. Priority Date: 06/30/1975
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Steinbrink, Charles F. Jr.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US05/724,307
• Time in Patent Office: 543 Days
• Field of Search: 23/230 B, 23/259, 252/408
• US Class Current: 436/97
• CPC Class Codes: G01N 33/728 Bilirubin; including biliverdin
• CPC Class Codes: Y10T 436/103332 Bilirubin or uric acid standard or control
• CPC Class Codes: Y10T 436/146666 Bile pigment

Abstract

Method of determining total and direct bilirubin in blood serum or plasma. A first reagent containing sulfanilic and hydrochloric acids and a second reagent containing sulfanilic acid, hydrochloric acid and dimethyl sulfoxide are disposed in a scheme of reagent reservoirs. Into some of the reservoirs is also placed sodium nitrite contained in frangible capsules. The frangible capsules are broken with the resulting formation of a diazonium reagent. Blood serum or plasma, standard bilirubin solution and control serum are added to the reservoirs according to the analytical scheme. The subsequent color developments in the reservoirs are correlated to the bilirubin contents.

Claims

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.

2. A method according to claim 1 wherein said observing is with the aid of a spectrophotometer.

3. 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.

4. A method according to claim 3 wherein said observing is with the aid of a spectrophotometer.

5. 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.