Method of glucose stabilization and analysis in dried blood spot samples

• US 5,204,267 A
• Filed: 12/17/1991
• Issued: 04/20/1993
• Est. Priority Date: 12/17/1991
• Status: Expired due to Term

• Current Assignee: Osborn Laboratories Incorporated
• Original Assignee: Osborn Laboratories Incorporated
• Inventors: Pflatzgraff, Linda R., Van Hercke, Melinda L., Sangha, Jangbir S.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): BHAT, NINA NMN
• Application Number:
  US07/812,066
• Time in Patent Office: 490 Days
• Field of Search: 436/14, 436/18, 436/66, 436/95, 436/176, 436/178, 436/811, 436/826
• US Class Current: 436/14
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/144444 Glucose
• CPC Class Codes: Y10T 436/2525 Stabilizing or preserving
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A method of stabilizing the glucose content of a blood sample applied to a sorbent for drying is provided. A solution of sodium fluoride or other glycolysis inhibitor is applied to a sorbent. A blood sample for glucose determination is then applied to the sorbent whereupon glycolysis is immediately inhibited. The blood sample may then be dried for shipping without loss of glucose content during the drying period.

Claims

1. A method of preparing a blood sample and determining the glucose concentration thereof, said method comprising the steps of:

contacting a sorbent with a solution of a means for inhibiting glycolysis;

evaporating said solution to present a sorbent having said means for inhibiting glycolysis thereon;

applying a fluid blood sample to said sorbent;

drying said blood sample on said sorbent to present a dried blood sample;

dissolving a glucose-containing portion of said dried blood sample in a solvent such that a blood protein portion of said dried blood sample substantially remains as a solid;

separating said solvent from said solid;

reacting said glucose-containing solvent with glucose oxidase enzyme and peroxidase enzyme in the presence of a peroxidase enzyme substrate molecule to produce an oxidized substrate molecule; and

measuring the absorbance of said oxidized substrate molecule to proportionally determine the glucose concentration in the original blood spot.

2. The method as claimed in claim 1, wherein said means for inhibiting glycolysis is an earth metal fluoride.

3. The method as claimed in claim 1, wherein said means for inhibiting glycolysis is an organic phosphate.

4. The method as claimed in claim 1, wherein said means for inhibiting glycolysis is selected from the group consisting of sodium fluoride, potassium fluoride, potassium oxalate, sodium oxalate, citrate, adenosine diphosphate, adenosine triphosphate and sodium iodoacetate.

5. The method as claimed in claim 1, wherein said sorbent is selected from the group consisting of cellulose fiber, glass, fritted glass, cotton fiber, paper and diatomaceous earth.

6. The method as claimed in claim 1, wherein said solvent is an acid selected from the group consisting of sulfosalicylic acid, trichloroacetic acid, phosphotungstic acid, perchloric acid and benzoic acid.

7. The method as claimed in claim 1, wherein said solvent is an alcohol.

8. The method as claimed in claim 7, wherein said alcohol is selected from the group consisting of methyl alcohol, ethyl alcohol, and butyl alcohol.

9. A method of preparing a blood sample and determining the glucose concentration thereof, said method comprising the steps of:

contacting a sorbent with a solution of a means for inhibiting glycolysis;

evaporating said solution to present a sorbent having said means for inhibiting glycolysis thereon;

applying a fluid blood sample to said sorbent;

drying said blood sample on said sorbent to present a dried blood sample;

dissolving a glucose portion of said dried blood sample in a solvent such that a blood protein portion of said dried blood sample substantially remains as a solid;

separating the glucose-containing solvent from said solid;

combining said glucose-containing solvent with o-toluidine and acetic acid to form a colored complex of glucose and o-toluidine; and

determining the absorbance of said glucose and o-toluidine complex whereby to proportionally determine the glucose concentration in the original blood spot.

10. The method as claimed in claim 9, wherein said means for inhibiting glycolysis is an earth metal fluoride.

11. The method as claimed in claim 9, wherein said means for inhibiting glycolysis is an organic phosphate.

12. The method as claimed in claim 9, wherein said means for inhibiting glycolysis is selected from the group consisting of sodium fluoride, potassium fluoride, potassium oxalate, sodium oxalate, citrate, adenosine diphosphate, adenosine triphosphate and sodium iodoacetate.

13. The method as claimed in claim 9, wherein said sorbent is selected from the group consisting of cellulose fiber, glass, fritted glass, cotton fiber, paper and diatomaceous earth.

14. The method as claimed in claim 9, wherein said solvent is an acid selected from the group consisting of sulfosalicylic acid, trichloroacetic acid, phosphotungstic acid, perchloric acid and benzoic acid.

15. The method as claimed in claim 9, wherein said solvent is an alcohol.

16. The method as claimed in claim 15, wherein said alcohol is selected from the group consisting of methyl alcohol, ethyl alcohol, and butyl alcohol.

17. A method of preparing a blood sample and determining the glucose concentration thereof, said method comprising the steps of:

contacting a sorbent with a solution of a means for inhibiting glycolysis;

evaporating said solution to present a sorbent having said means for inhibiting glycolysis thereon;

applying a fluid blood sample to said sorbent;

drying said blood sample on said sorbent to present a dried blood sample;

dissolving a glucose-containing portion of said dried blood sample in a solvent such that a blood protein portion of said dried blood sample substantially remains as a solid;

separating said solvent from said solid;

converting the glucose in said solvent by introducing a glucose oxidase enzyme portion to produce a hydrogen peroxide portion;

combining said hydrogen peroxide portion with a peroxidase enzyme substrate molecule;

introducing a peroxidase enzyme portion to said hydrogen peroxide portion;

oxidizing said substrate molecule with said peroxidase enzyme portion and said hydrogen peroxide portion to produce an oxidized substrate molecule; and

measuring the absorbance of the oxidized substrate molecule whereby to proportionally determine the glucose concentration in the original blood spot.

18. The method as claimed in claim 17, wherein said means for inhibiting glycolysis is an earth metal fluoride.

19. The method as claimed in claim 17, wherein said means for inhibiting glycolysis is an organic phosphate.

20. The method as claimed in claim 17, wherein said means for inhibiting glycolysis is selected from the group consisting of sodium fluoride, potassium fluoride, potassium oxalate, sodium oxalate, citrate, adenosine diphosphate, adenosine triphosphate and sodium iodoacetate.

21. The method as claimed in claim 17, wherein said solvent is an acid selected from the group consisting of sulfosalicylic acid, trichloroacetic acid, phosphotungstic acid, perchloric acid and benzoic acid.

22. The method as claimed in claim 17, wherein said solvent is an alcohol.