Glyoxal/zinc fixative

• US 20060088813A1
• Filed: 10/27/2004
• Published: 04/27/2006
• Est. Priority Date: 10/27/2004
• Status: Active Grant

• Current Assignee: Medical Chemical Corporation
• Original Assignee: Medical Chemical Corporation
• Inventors: Rocha, Andrew J.
• Granted Patent: US 7,368,132 B2
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

This invention provides compositions and methods for fixing a biological sample, particularly fecal samples for diagnosis of parasitic infection. The fixative composition of the present invention comprises glyoxal (pyruvate aldehyde) and zinc sulfate and permits staining of biological samples without use of toxic compounds, such as formaldehyde and mercury-containing compounds. The fixative is compatible with many diagnostic assays, including trichrome stains, hematoxlin, ELISA, fluorescent assays, and lateral flow assays.

Claims

1. A mercury-free, formaldehyde-free fixative composition for preserving a biological specimen, comprising:

a. reagent alcohol;

b. polyvinyl alcohol;

c. zinc sulfate;

d. glyoxal;

e. buffer; and

f. water.

2. The fixative composition of claim 1, comprising

a. from about 0 milliliters per liter to about 300 milliliters per liter of reagent alcohol;

b. from about 10 grams per liter to about 100 grams per liter of polyvinyl alcohol;

c. from about 10 grams per liter to about 150 grams per liter of zinc sulfate;

d. from about 1 gram per liter to about 50 grams per liter of glyoxal;

e. from about 0.01M per liter to about 1.0 M per liter of buffer; and

f. water.

3. The fixative composition of claim 2, comprising:

a. between about 50 milliliters and about 250 milliliters of reagent alcohol;

b. between about 25 grams per liter and about 75 grams per liter of polyvinyl alcohol;

c. from about 50 grams per liter to about 100 grams per liter of zinc sulfate;

d. from about 5 grams per liter to about 25 grams per liter of glyoxal;

e. from about 0.05 M per liter to about 0.5 M per liter of buffer; and

f. water.

4. The fixative composition of claim 3, comprising about 225 milliliters per liter of reagent alcohol.

5. The fixative composition of claim 3, comprising about 40 grams per liter of polyvinyl alcohol.

6. The fixative composition of claim 3, comprising about 75 grams per liter of zinc sulfate.

7. The fixative composition of claim 3, comprising about 12 grams per liter of glyoxal.

8. The fixative composition of claim 3, comprising about 0.1 M per liter of buffer.

9. The fixative composition of claim 1, wherein the reagent alcohol is selected from the group consisting of low molecular weight alcohols.

10. The fixative composition of claim 1, wherein the buffer has a pKa of about 6.

11. The fixative composition of claim 1, wherein the buffer is an acetate buffer.

12. The fixative composition of claim 1, wherein the pH is from about 3.0 to about 6.0.

13. The fixative composition of claim 12, wherein the pH is between about 3.5 and about 5.0.

14. The fixative composition of claim 13 wherein the pH is about 4.3.

15. A fixative composition for preserving biological specimens, comprising:

a. from about 200 milliliters per liter to about 250 milliliters per liter of reagent alcohol;

b. from about 25 grams per liter to about 75 grams per liter of polyvinyl alcohol;

c. from about 50 grams per liter to about 100 grams per liter of zinc sulfate;

d. from about 5 grams per liter to about 25 grams per liter of glyoxal;

e. from about 0.05 M per liter to about 0.5 M per liter of buffer; and

f. water, wherein the pH of the fixative composition is from about 3.5 to about 5.0

16. The fixative composition of claim 15, wherein the pH is about 4.3.

17. A fixative composition for preserving biological specimens, comprising:

a. about 225 milliliters per liter of reagent alcohol;

b. about 40 grams per liter of polyvinyl alcohol;

c. about 75 grams per liter of zinc sulfate;

d. about 12 grams per liter of glyoxal;

e. about 0.10 M per liter of buffer; and

f. water.

18. The fixative of claim 17, wherein the pH is from about 3.5 to about 5.0.

19. The fixative of claim 18, wherein the pH is about 4.3.

20. A method for preserving fecal specimens, said method comprising the step of:

contacting a fecal specimen with an amount of mercury-free, formaldehyde-free fixative composition effective for preserving the fecal specimen;

wherein

said fixative composition comprises from about 200 milliliters per liter to about 250 milliliters per liter of reagent alcohol;

from about 20 grams per liter to about 60 grams per liter of polyvinyl alcohol;

from about 50 grams per liter to about 100 grams per liter of zinc sulfate;

from about 0.01 M per liter to about 0.5 M per liter of buffer; and

water.

21. The method of claim 20, wherein the fixative composition comprises about 225 milliliters per liter of reagent alcohol;

about 40 grams per liter of polyvinyl alcohol;

about 75 grams per liter of zinc sulfate;

about 12 grams per liter of glyoxal;

about 0.1 M per liter of buffer; and

water.

22. A method for preparing a mercury-free, formaldehyde-free fixative composition for preserving biological specimens, said method comprising the steps of:

a. adding buffer salts and zinc sulfate to water to form a zinc sulfate solution;

b. adding polyvinyl alcohol to water to form a polyvinyl alcohol solution;

c. heating the polyvinyl alcohol solution to between about 60°

C. to about 100°

C.;

d. maintaining the temperature of the polyvinyl alcohol solution above about 90°

C. for between about fifteen minutes and about sixty minutes;

e. mixing the polyvinyl alcohol solution and the zinc sulfate solution together; and

f. adding reagent alcohol.

23. The method of claim 22, wherein the polyvinyl alcohol solution is heated to about 90°

C.

24. The method of claim 22, wherein the temperature of the polyvinyl alcohol solution is maintained above about 90°

C. for about thirty minutes before mixing the polyvinyl alcohol solution and the zinc sulfate solution together.

25. The method of claim 22, further comprising the step of filtering the fixative composition.