Methods of preparing tissue samples

• US 4,839,194 A
• Filed: 07/05/1985
• Issued: 06/13/1989
• Est. Priority Date: 07/05/1985
• Status: Expired due to Fees

• Current Assignee: BONE Diagnostic Center
• Original Assignee: BONE Diagnostic Center
• Inventors: Malluche, Hartmut H., Malluche, Gisela
• Primary Examiner(s): Hill, Jr., Robert J.
• Application Number:
  US06/751,960
• Time in Patent Office: 1,439 Days
• Field of Search: 435/3, 436/174, 436/164, 427/45.1, 427/4, 427/2, 427/54.1, 427/322, 427/443, 424/3, 264/109, 264/239
• US Class Current: 427/2.11
• CPC Class Codes: A01N 1/00 Preservation of bodies of humans or animals, or parts thereof
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 1/44 Sample treatment involving radiation, e.g. heat
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method is described for preparing tissue samples for use in diagnostic evaluation involving the rapid fixation, dehydration, and embedding of a tissue sample as well as rapid mounting of plastic sections to glass slides, dissolution of plastic and optimal staining of tissure components. The rapid and controllable fixation and the dehydration of the tissue sample utilizes the denaturing effects of microwaves on the proteins in combination with a heat sink. Alternatively, the dehydration of the tissue sample utilizes the principles of freeze drying. The tissue sample can be embedded using conventional paraffin embedding or plastic embedding techniques. Rapid penetration of plastic into the tissue is facilitated by microwaves, adhesion of plastic sections to glass slides is promoted by microwaves, as is the fast and optimal staining of tissue components. The method of this invention provides a very rapid technique for processing of tissue for diagnostic and analytic evaluation.

Claims

1. A method of fixation of a tissue specimen comprising placing a tissue specimen in a liquid fixation medium at a temperature below about 10°

C., said liquid fixation medium being present in an amount sufficient to provide a heat sink to allow the non-thermal effects of microwave radiation to act on said tissue specimen, and exposing said specimen in said fixation medium to microwave radiation for a time sufficient to fix said tissue specimen while controlling the temperature of said medium so as not to exceed about 40°

C.

2. The method according to claim 1 wherein said liquid fixation medium is ethanol at a temperature between about 0°

C. and -40°

C.

3. A method of dehydrating a tissue specimen comprising placing a tissue specimen in an alcoholic solution, said alcoholic solution being present in an amount sufficient to provide a heat sink to allow the non-thermal effects of microwave radiation to act on said tissue specimen, and exposing said specimen in said alcoholic solution to microwave radiation for a time sufficient to fix said tissue specimen while controlling the temperature of said medium so as not to exceed about 40°

C.

4. A method of embedding a tissue specimen in plastic comprising

(1) placing a tissue specimen in a solution of plastic monomer and alcohol;

(2) thereafter, placing said tissue specimen in a solution of plastic monomer and exposing said specimen to microwave radiation;

(3) repeating step (2) twice so that the plastic monomer penetrates into said tissue specimen; and

(4) hardening said plastic monomer which has penetrated into said tissue specimen.

5. A method of removing plastic from a tissue specimen which is embedded in plastic and adhered to a slide comprising

(1) placing a slide having a tissue specimen adhered thereto in a container filled with an organic solvent;

(2) exposing said slide while in said organic solvent to microwave radiation;

(3) repeating steps (1) and (2), each time transferring said slide to a container filled with a fresh organic solvent solution; and

(4) transferring said slide to a fresh organic solvent solution and allowing said slide to cool.

6. The method according to claim 5 wherein steps (1) and (2) are repeated twice and said organic solvent is selected from the group consisting of 2-methoxy-ethyl-acetate and methyl glycol acetate.

7. A method of staining a tissue specimen comprising placing a tissue specimen in a container filled with a 100% alcoholic solution at room temperature with said tissue specimen remaining in said container for about 2-3 minutes, removing said tissue specimen from said alcoholic solution, placing said tissue specimen in a stain and exposing said tissue specimen to microwave radiation.

8. A method of preparing a tissue sample for use in diagnostic evaluation comprising -

(1) placing a tissue specimen in a liquid fixation medium at a temperature below about 10°

C., said liquid fixation medium being present in an amount sufficient to provide a heat sink to allow the non-thermal effects of microwave radiation to act on said tissue specimen, and exposing said specimen to microwave radiation present in an amount sufficient to provide a heat for a time sufficient to fix said tissue specimen while controlling the temperature of said medium so as not to exceed about 40°

C.;

(2) dehydrating said tissue specimen of step (1); and

(3) embedding said tissue specimen of step (2) in an embedding material.

9. The method according to claim 8 wherein said liquid fixation medium is ethanol at a temperature between about 0°

C. and -40°

C.

10. The method according to claim 8 wherein step (2) comprises placing said tissue specimen in an alcoholic solution and exposing said specimen in said solution to microwave radiation.

11. The method according to claim 8 wherein step (2) comprises immersing said tissue specimen in ethanol at -5°

C. followed by freeze-drying said tissue specimen for 0.25 to 3 hours.

12. The method according to claim 8 wherein said tissue specimen is embedded in plastic.

13. The method according to claim 8 wherein said tissue specimen is embedded in paraffin.

14. The method according to claim 8 wherein the embedding step comprises

(1) placing the tissue specimen in a container filled with a mixture of ethanol and methyl-methacrylate monomer and continuously stirring the mixture for about 30-45 minutes;

(2) transferring said tissue specimen to another container filled with fresh methyl-methacrylate monomer and exposing said tissue specimen to microwave radiation for about 5 minutes;

(3) repeating step (2) twice so that the methyl-methacrylate monomer penetrates into said tissue specimen; and

(4) hardening said monomer which has penetrated into said tissue specimen by polymerization.

15. The method according to claim 14 wherein the step of polymerization comprises

(1) placing said tissue specimen in a polymerization solution and keeping said tissue specimen in a refrigerator for a period of about 2-8 hours;

and

(2) transferring said tissue specimen into a container filled with about 10 to 12 ml of polymerization solution, and keeping said container in a warm-water bath at about 48°

C. to 50°

C. until hardening is complete.