Simplified tissue processing

• US 8,288,168 B2
• Filed: 11/20/2008
• Issued: 10/16/2012
• Est. Priority Date: 10/24/2003
• Status: Active Grant

• Current Assignee: University of Miami
• Original Assignee: University of Miami
• Inventors: Morales, Azorides R.
• Primary Examiner(s): Nagpaul, Jyoti
• Application Number:
  US12/274,613
• Publication Number:
  US 20090136992A1
• Time in Patent Office: 1,426 Days
• Field of Search: 422 63- 67, 422/50, 422500-501, 436/180, 436/174, 427/4, 427/296
• US Class Current: 436/174
• CPC Class Codes: B01L 2300/1866 Microwaves
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/16 Retorts
• 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 1/31 Apparatus therefor
• 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 31/22 using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

Improved systems and methods for tissue processing are described here. The chemical process and the construction of the apparatus are simplified by using only two different solutions in two separate reaction modules. They are compatible with processing of tissue specimens for genetic analysis, histology, in situ antibody binding and hybridization, archival preservation of morphology and nucleic acids, and combinations thereof.

Claims

1. A method for tissue processing of one or more tissue specimens using only two different chemical compositions and an apparatus comprising:

a microwave unit comprised of (i) a first reaction chamber with an interior geometry comprised of a whispering gallery to achieve uniform distribution of microwave energy and heating of the interior, (ii) a single non-aqueous admixture which is optionally contained within the first reaction chamber, (iii) a source of microwave energy which is transmitted into the first reaction chamber to heat the non-aqueous admixture, and (iv) an agitator within the first reaction chamber to promote chemical exchange between the non-aqueous admixture and the tissue specimen and

a vacuum unit comprised of (i) a second reaction chamber, (ii) a single wax solution which is optionally contained within the second reaction chamber, (iii) a source of radiant energy which is transmitted into the second reaction chamber to heat the wax solution, and (iv) a vacuum pump connected to the second reaction chamber;

the method comprising;

(a) initially impregnating a tissue specimen with the single non-aqueous admixture comprised of (i) fixing, (ii) dehydrating, and (iii) clearing functions which is agitated and heated; and

then

(b) substantially impregnating the tissue specimen with the single wax solution which is heated and also under less than atmospheric pressure.

2. The tissue processing method of claim 1, wherein the tissue specimen is about 3 mm or less in thickness.

3. The tissue processing method of claim 1, wherein tissue specimens are manually processed.

4. The tissue processing method of claim 1, wherein tissue specimens are mechanically processed.

5. The tissue processing method of claim 1, wherein the tissue specimen is processed without using an aldehyde.

6. The tissue processing method of claim 1, wherein the tissue specimen is processed without using a xylene.

7. The tissue processing method of claim 1, wherein the tissue specimen has not been fixed prior to processing.

8. The tissue processing method of claim 1, wherein the tissue specimen has been fixed in formaldehyde prior to processing.

9. The tissue processing method of claim 1, wherein the tissue specimen has been preserved in methanol and polyethylene glycol (PEG) prior to processing.

10. The tissue processing method of claim 1, wherein the tissue specimen is substantially hardened by the non-aqueous admixture, radiation from the microwave unit, or both.

11. The tissue processing method of claim 1, wherein the non-aqueous admixture is above about 55°

C. and/or below about 75°

C.

12. The tissue processing method of claim 1, wherein the non-aqueous admixture is comprised of fixative and dehydrant in a volume ratio greater than about 1:

6 and/or less than about 6;

1.

13. The tissue processing method of claim 1, wherein a fixative is greater than about 20% (v/v) and/or less than about 60% (v/v) of the non-aqueous admixture.

14. The tissue processing method of claim 1, wherein a dehydrant is greater than about 20% (v/v) and/or less than about 60% (v/v) of the non-aqueous admixture.

15. The tissue processing method of claim 1, wherein a clearant is greater than about 10% (v/v) and/or less than about 30% (v/v) of the non-aqueous admixture.

16. The tissue processing method of claim 1, wherein the non-aqueous admixture is further comprised of a surfactant.

17. The tissue processing method of claim 1, wherein the non-aqueous admixture is comprised of a fixative which is at least one ketone and a dehydrant which is at least one alcohol.

18. The tissue processing method of claim 1, wherein the non-aqueous admixture is comprised of a clearant which is mineral oil.

19. The tissue processing method of claim 1, wherein the non-aqueous admixture is further comprised of dimethyl sulfoxide (DMSO).

20. The tissue processing method of claim 1, wherein the non-aqueous admixture is comprised of about 15% (v/v) to about 35% (v/v) acetone, about 45% (v/v) to about 65% (v/v) isopropyl alcohol, and about 10% (v/v) to about 25% (v/v) mineral oil.

21. The tissue processing method of claim 1, wherein the non-aqueous admixture is comprised of about 40% to about 60% (v/v) of acetone, about 25% to about 35% (v/v) of isopropanol, and about 10% (v/v) to about 25% (v/v) of mineral oil.

22. The tissue processing method of claim 1, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 15 minutes or less.

23. The tissue processing method of claim 1, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 30 minutes or less.

24. The tissue processing method of claim 1, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 45 minutes or less.

25. The tissue processing method of Claim 1, wherein the tissue specimen is substantially impregnated at a pressure above about 100 torr and/or below about 760 torr.

26. The tissue processing method of claim 1, wherein the wax solution is above about 55°

C. and/or below about 85°

C.

27. The tissue processing method of claim 1, wherein the wax solution has been degassed and dehydrated.

28. The tissue processing method of claim 1, wherein the wax solution is made from paraffin which is solid below about 30°

C.

29. The tissue processing method of claim 1, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 15 minutes or less, and then cooled.

30. The tissue processing method of claim 1, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 30 minutes or less, and then cooled.

31. The tissue processing method of claim 1, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 45 minutes or less, and then cooled.

32. A method for tissue processing of one or more tissue specimens using only two different chemical compositions and an apparatus comprising:

a first module comprising (i) a first reaction chamber with an interior geometry comprised of a whispering gallery to achieve uniform distribution of microwave energy and heating of the interior, (ii) a single non-aqueous admixture which is optionally contained within the first reaction chamber, (iii) a source of microwave energy which is transmitted into the first reaction chamber to heat the non-aqueous admixture, and (iv) an agitator within the first reaction chamber to promote chemical exchange between the non-aqueous admixture and the tissue specimen and

a second module comprising (i) a second reaction chamber, (ii) a single wax solution which is optionally contained within the second reaction chamber, (iii) a source of radiant energy which is transmitted into the second reaction chamber to heat the wax solution, and (iv) a vacuum pump connected to the second reaction chamber;

the method comprising;

(a) initially impregnating a tissue specimen in the first module with the single non-aqueous admixture comprised of (i) at least one ketone, (ii) at least one alcohol, and (iii) mineral oil while being agitated and heated then

(b) substantially impregnating the tissue specimen in the second module with the single wax solution while being under vacuum and heated.

33. The tissue processing method of claim 32, wherein tissue specimens are manually processed.

34. The tissue processing method of claim 32, wherein the tissue specimen is substantially hardened by the non-aqueous admixture, radiation from the microwave unit, or both.

35. The tissue processing method of claim 32, wherein the tissue specimen is substantially impregnated at a pressure above about 100 torr and/or below about 760 torr.