Method for fixing tissues and cells for analysis using oxazolidine compounds

• US 5,460,797 A
• Filed: 11/03/1994
• Issued: 10/24/1995
• Est. Priority Date: 05/08/1991
• Status: Expired due to Term

• Current Assignee: Streck Laboratories Inc.
• Original Assignee: Streck Laboratories Inc.
• Inventors: Ryan, Wayne L.
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): SAUCIER, SANDRA E
• Application Number:
  US08/333,875
• Time in Patent Office: 355 Days
• Field of Search: 424/3, 424/75, 422/40
• US Class Current: 435/40.5
• CPC Class Codes: A61K 2039/55516 Proteins; Peptides
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• 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: G01N 2001/307 non-toxic, no Hg, no formaldehyde
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

A method and composition for fixing and stabilizing tissues, cells, and cell components such that the antigenic sites are preserved for a useful period of time. The fixative employs compounds that are less toxic, non-flammable, and that stabilize the cell with minimal damage to and alteration of the cell morphology. In particular, the cell antigenic sites are left intact so that studies with monoclonal antibodies may be conducted. The invention also discloses a method for developing a positive control for test reagents and for test instrumentation.

Claims

1. In a method for the analysis of a biological material wherein said biological material is a cell or tissue preserved by a fixing or stabilizing solution, the improvement comprising:

treating said biological material with a fixative which comprises at least one oxazolidine in an amount sufficient to fix said biological material; and

analyzing said preserved biological material.

2. The method of claim 1, wherein the oxazolidine is selected from the group consisting of:

1) 24.5% by weight 5-hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo (3.3.0) octane, 17.7% by weight 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0) octane, 7.8% by weight 5-hydroxypoly methyl-1-aza-3, 7-dioxabicyclo (3.3.0) octane;

2) 50% by volume [[1 -methyl-2-(5-methyl-3-oxazolidinyl)ethoxy ]methoxy]methanol;

3) 74.7% by weight 4,4-dimethyloxazolidine, 2.5% by weight 3,4,4-trimethyloxazolidine;

4) 76.0% by weight 4,4-dimethyloxazolidine, greater than 1.0% by weight 3,4,4-trimethyloxazolidine, less than 1.5% by weight 2-amino-2-methyl-1-propanol;

5) 96% by weight 7A-ethyldihydro-1H, 3H, 5H-oxazolo (3,4-C) oxazolo, 2% by weight 4,4-dimethyl-oxazolidine and 4-ethyl-oxazolidine;

6) 76% by weight 4,4-dimethyl-1-oxa-3-azacyclopentane, greater than 1.0% by weight 3,4,4-trimethyl-1-oxa-3-azacyclopentane, greater than or equal to 1.5% by weight 2-amino-2-methyl-1-propanol;

7) 96% by weight 7A-ethyldihydro-1H,3H,5H-oxazolo (3,4-C) oxazolo, 2% by weight 4,4-dimethyl-oxazolidine and 4-ethyl-oxazolidine;

8) 96% by weight 7A-ethylodihydro-1H,3H,5H-oxazolo (3,4-C) oxazolo, 2% by weight 4,4-dimethyl-oxazolidine and 4-ethyl-oxazolidine; and

9) 55.0% by weight 5-hydroxymethyl-1-aza-3, 7-dioxabicyclo (3,3,0) octane.

3. The method of claim 1, wherein the oxazolidine is present in an amount of about 5 to about 100 grams per liter.

4. The method of claim 1, wherein the fixing or stabilizing solution further comprises water.

5. The method of claim 1, wherein the fixing or stabilizing solution further comprises alcohol.

6. The method of claim 1, wherein the fixing or stabilizing solution further comprises about 0.25 to about 1 grams per liter of an alkali metal salt of ascorbic acid.

7. The method of claim 6, wherein the salt is sodium ascorbate.

8. The method of claim 1, wherein the fixing or stabilizing solution further comprises acetic acid in an amount sufficient to improve nuclear detail.

9. The method of claim 1, wherein the fixing or stabilizing solution further comprises dimethylsulfoxide in an amount sufficient to increase the rate of penetration of the oxazolidine.

10. The method of claim 1, wherein the fixing or stabilizing solution further comprises zinc sulfate in an amount sufficient to improve staining and nuclear detail.