High quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method

• US 6,207,408 B1
• Filed: 08/19/1998
• Issued: 03/27/2001
• Est. Priority Date: 08/20/1997
• Status: Expired due to Term

• Current Assignee: Miami University, University of Miami
• Original Assignee: University of Miami
• Inventors: Essenfeld, Ervin, Essenfeld, Harold, Morales, Azorides
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Moran, Marjorie A.
• Application Number:
  US09/136,292
• Time in Patent Office: 951 Days
• Field of Search: 435/40.5, 435/40.51, 435/40.52, 435/173, 436/60, 219/751
• US Class Current: 435/40.5
• CPC Class Codes: B01L 9/50 Clamping means, tongs in general F16B2/06
• CPC Class Codes: G01N 1/06 providing a thin slice, e.g. microtome
• 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 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 1/36 Embedding or analogous mounting of samples
• CPC Class Codes: G01N 1/44 Sample treatment involving radiation, e.g. heat
• CPC Class Codes: G01N 2001/305 Fixative compositions
• CPC Class Codes: G01N 2001/315 Basket-type carriers for tissues

Abstract

A process and apparatus for rapid, continuous flow histological processing of tissues is disclosed. The steps of fixation, dehydration, clearing and impregnation are performed in less than one hour; this allows a pathologist to evaluate samples shortly after receipt, perhaps while the patient is still in the operating room. Rapid and continuous processing is accomplished by decreasing the thickness of tissue sections, use of non-aqueous solutions composed of admixtures of solutions, solution exchange at elevated temperature and with agitation, and impregnation under vacuum pressure. The patient in surgery is thus provided with point-of-care surgical pathology.

Claims

1. A method of processing a tissue specimen for histology comprising:

(a) slicing a solid tissue to provide at least one tissue specimen to a thickness of up to 2 mm,

(b) fixing the tissue specimen,

(c) dehydrating the tissue specimen,

(d) removing fat from the tissue specimen,

(e) impregnating the tissue specimen with a mixture of waxes having different melting points to provide a block comprised of the tissue specimen and wax, and

(f) sectioning the block to provide a tissue section which is only then placed on a slide;

wherein said tissue specimen is processed in steps (b)-(d) by a series of all non-aqueous solutions, at least one of which is a first solution comprising fixative and dehydrating agent;

wherein fixation, dehydration, and fat removal occur with agitation of said non-aqueous solutions and heating by microwave energy;

wherein impregnation is conducted under less than atmospheric pressure; and

whereby said tissue specimen is fixed, dehydrated, defatted, and impregnated in about 90 minutes or less.

2. A method of continuous tissue processing for histology comprising:

(a) providing a series of tissue specimens up to 1.5 mm in thickness,

(b) fixing at least one of said tissue specimens,

(c) dehydrating at least one of said tissue specimens,

(d) removing fat from at least one of said tissue specimens, and

(e) impregnating at least one of said tissue specimens under less than atmospheric pressure with a mixture of waxes having different melting points, wherein steps (b)-(e) are completed in about 90 minutes or less;

wherein all solutions for processing said tissue specimens are non-aqueous and said series of tissue specimens is processed by initiating step (b) for a later-processed tissue specimen before step (e) for an earlier-processed tissue specimen has been completed, and

wherein steps (b), (c), and (d) occur with agitation and heating by microwave energy.

3. A method of continuous tissue processing for histology comprising:

(a) slicing a solid tissue to provide a tissue specimen of up to 2 mm in thickness,

(b) fixing and dehydrating said tissue specimen by incubation in a series of all non-aqueous solutions with agitation and heating by microwave energy,

(c) impregnating said fixed and dehydrated tissue specimen under less than atmospheric pressure, and

(d) repeating steps (b) and (c) at least once with another tissue specimen, wherein at least one repetition of steps (b) and (c) takes about 90 minutes or less;

whereby tissue specimens are continuously processed by initiating step (b) for a later-processed tissue specimen before step (c) has been completed for an earlier-processed tissue specimen.

4. A method according to claim 3, wherein said tissue specimen is sliced to a thickness between 1 mm and 2 mm before processing.

5. A method according to claim 3, wherein said tissue specimen is sliced to a thickness between 1 mm and 1.5 mm before processing.

6. A method according to claim 3 further comprising sectioning said fixed, dehydrated, and impregnated tissue specimen to provide a tissue section of between 1 micron and 50 microns.

7. The method of claim 3, wherein at least one of said series of non-aqueous solutions is a first solution comprised of fixative and dehydrating agent.

8. The method of claim 7, wherein said tissue specimen is incubated in said first solution for at least 15 minutes.

9. The method of claim 7, wherein said first solution is comprised of alcohol and ketone.

10. The method of claim 9, wherein said first solution contains a volume ratio of alcohol to ketone between 1:

1 and 3;

1.

11. The method of claim 7, wherein said first solution is further comprised of polymers between 100 and 500 average molecular weight.

12. The method of claim 11, wherein said first solution is further comprised of a surfactant at about 1% of total volume.

13. The method of claim 3, wherein at least one of said series of non-aqueous solutions is a first solution comprised of acetone, isopropyl alcohol, and low molecular weight polyethylene glycol.

14. The method of claim 3 further comprising clearing said tissue specimen in a non-aqueous solution comprised of clearant.

15. The method of claim 14, wherein said clearant is xylene.

16. The method of claim 3, wherein tissue specimens are processed without using formalin.

17. The method of claim 3, wherein tissue specimens are processed without using xylene.

18. The method of claim 3, wherein at least one of said series of non-aqueous solutions is comprised of alcohol, ketone, and mineral oil.

19. The method of claim 3, wherein said fixed and dehydrated tissue specimen is impregnated with a mixture of waxes having different melting points.

20. The method of claim 3, wherein said fixed and dehydrated tissue specimen is impregnated with a wax solution comprised of xylene and paraffin.

21. The method of claim 3, wherein said fixed and dehydrated tissue specimen is impregnated with a wax solution comprised of mineral oil and paraffin.

22. The method of claim 3, wherein said fixed and dehydrated tissue specimen is impregnated in molten paraffin for at least 12 minutes.

23. A method of continuous tissue processing for histology comprising:

(a) slicing a solid tissue to provide tissue specimens with thickness between 1 mm and 2 mm, wherein a tissue specimen to be processed is not placed on a slide for histology until said tissue specimen has been hardened, impregnated, and sectioned;

(b) hardening said tissue specimen by incubation in a series of all non-aqueous solutions with agitation and heating by microwave energy, wherein incubation in a first non-aqueous solution comprised of ketone and alcohol is then followed by incubation in a second non-aqueous solution comprised of less concentrated ketone and more concentrated alcohol as compared to said first non-aqueous solution;

(c) impregnating said hardened tissue specimen in a wax solution under less than atmospheric pressure to provide a block comprised of said hardened and impregnated tissue specimen and wax, wherein hardening and impregnating a tissue specimen takes about 90 minutes or less; and

(d) repeating steps (b) and (c) at least once with another tissue specimen;

whereby tissue specimens are continuously processed by initiating hardening of a later-processed tissue specimen before impregnation of an earlier-processed tissue specimen has been completed.

24. A method according to claim 23 further comprising sectioning said hardened and impregnated tissue specimen to provide a tissue section of between 1 micron and 50 microns.

25. The method of claim 23 further comprising placing said tissue section on a slide for histology, and then removing wax from the slide.

26. The method of claim 23, wherein said tissue specimen is incubated in said first non-aqueous solution for at least 15 minutes.

27. The method of claim 23, wherein said first non-aqueous solution is comprised of acetone and isopropyl alcohol.

28. The method of claim 27, wherein said first non-aqueous solution contains a volume ratio of alcohol to ketone between 1:

1 and 3;

1.

29. The method of claim 23, wherein said first non-aqueous solution is further comprised of polymers between 100 and 500 average molecular weight.

30. The method of claim 29, wherein said first non-aqueous solution is further comprised of a surfactant at about 1% of total volume.

31. The method of claim 23, wherein said first non-aqueous solution is comprised of acetone, isopropyl alcohol, and low molecular weight polyethylene glycol.

32. The method of claim 23 further comprising clearing said tissue specimen in a non-aqueous solution comprised of clearant.

33. The method of claim 32, wherein said clearant is xylene.

34. The method of claim 23, wherein tissue specimens are processed without using formalin.

35. The method of claim 23, wherein tissue specimens are processed without using xylene.

36. The method of claim 23, wherein at least one of said series of non-aqueous solutions is comprised of alcohol, ketone, and mineral oil.

37. The method of claim 23, wherein said fixed and dehydrated tissue specimen is impregnated with a mixture of waxes having different melting points.

38. The method of claim 23, wherein said fixed and dehydrated tissue specimen is initially impregnated with a wax solution comprised of xylene and paraffin.

39. The method of claim 23, wherein said fixed and dehydrated tissue specimen is impregnated with a wax solution comprised of mineral oil and paraffin.

40. The method of claim 23, wherein said fixed and dehydrated tissue specimen is impregnated in molten paraffin for at least 12 minutes.