Simplified tissue processing

• US 20050090017A1
• Filed: 10/25/2004
• Published: 04/28/2005
• Est. Priority Date: 10/24/2003
• Status: Active Grant

• Current Assignee: Miami University
• Original Assignee: Miami University
• Inventors: Morales, Azorides R.
• Granted Patent: US 7,470,401 B2
• Time in Patent Office: Days
• 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 system for tissue processing of one or more tissue specimens, wherein the system uses only two different chemical compositions, comprising:

(a) a first module comprising (i) a first reaction chamber which is heated and (ii) a non-aqueous admixture comprised of fixing, dehydrating, and clearing functions, wherein the non-aqueous admixture is optionally contained within the first reaction chamber;

(b) a second module comprising (i) a second reaction chamber which is heated and also connected to a vacuum pump and (ii) a wax solution, wherein the wax solution is optionally contained within the second reaction chamber; and

(c) optionally a conveyance which at least transfers the tissue specimen from the first module to the second module.

2. The tissue processing system of claim 1, wherein the first reaction chamber has an interior geometry comprised of a whispering gallery and the first module further comprises (iii) a closure adapted to isolate the first reaction chamber, (iv) thermal insulation surrounding the first reaction chamber, and (v) a source of microwave radiation which is transmitted into the whispering gallery.

3. The tissue processing system of claim 2, wherein the tissue specimen is substantially hardened by the non-aqueous admixture, the microwave radiation, or both.

4. The tissue processing system of claim 1, wherein the first module further comprises an agitator within the first reaction chamber to promote chemical exchange between the non-aqueous admixture and the tissue specimen.

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

C. and/or below about 75°

C.

6. The tissue processing system 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.

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

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

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

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

11. The tissue processing system 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.

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

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

14. The tissue processing system 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.

15. The tissue processing system 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.

16. The tissue processor system of claim 1, wherein the first module further comprises a holding chamber connected to the first reaction chamber, the non-aqueous admixture is transferred to the first reaction chamber after the tissue specimen is loaded into the first module and transferred to the holding chamber before the tissue specimen is conveyed to the second module, and the non-aqueous admixture is optionally heated while in the holding chamber.

17. The tissue processing system of claim 1 configured such that the tissue specimen is substantially hardened after being in the first module for about 15 minutes or less.

18. The tissue processing system of claim 1 configured such that the tissue specimen is substantially hardened after being in the first module for about 30 minutes or less.

19. The tissue processing system of claim 1 configured such that the tissue specimen is substantially hardened after being in the first module for about 45 minutes or less.

20. The tissue processor system of claim 1, wherein the second module further comprises (iii) a closure adapted to isolate the second reaction chamber, (iv) thermal insulation surrounding the second reaction chamber, and (v) a heater to maintain wax in molten form in the second reaction chamber.

21. The tissue processor system of claim 1, wherein pressure within the second reaction chamber is above about 100 torr and/or below about 760 torr.

22. The tissue processor system of claim 1, wherein pressure within the second reaction chamber is cyclically varied by the vacuum pump.

23. The tissue processor system of claim 1, wherein the wax solution is above about 55°

C. and/or below about 85°

C.

24. The tissue processor system of claim 1, wherein the wax solution has been degassed and dehydrated.

25. The tissue processor system of claim 1, wherein the wax solution is made from paraffin which is solid below about 30°

C.

26. The tissue processor system of claim 1, wherein the second module further comprises a heated chamber connected to the second reaction chamber, solid wax is optionally melted in the heated chamber, and the wax solution is transferred from the heated chamber and to the second reaction chamber before the tissue specimen is conveyed to the second module.

27. The tissue processor system of claim 1 configured such that the tissue specimen is substantially impregnated after being in the second module for about 15 minutes or less.

28. The tissue processor system of claim 1 configured such that the tissue specimen is substantially impregnated after being in the second module for about 30 minutes or less.

29. The tissue processor system of claim 1 configured such that the tissue specimen is substantially impregnated after being in the second module for about 45 minutes or less.

30. The tissue processor system of claim 1, wherein the conveyance comprises a track connecting the first module and the second module.

31. The tissue processor system of claim 1, wherein the conveyance comprises an armature connecting the first module and the second module.

32. The tissue processor system of claim 1 further comprising a plurality of tissue carriers which allow chemical exchange, wherein each tissue specimen is enclosed within one of the plurality of carriers and each tissue carrier is reversibly attached to the conveyance so batches of tissue specimens may be continuously processed.

33. The tissue processor system of claim 1 further comprising a loading station at which non-impregnated tissue specimens enter the tissue processor system, and are then conveyed to the first module.

34. The tissue processor system of claim 1 further comprising an unloading station to which impregnated tissue specimens are conveyed from the second module, and then optionally collected until exiting the tissue processor system.

35. A method for tissue processing of one or more tissue specimens, wherein the method uses only two different chemical compositions, comprising:

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

then

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

36. The tissue processing method of claim 35, wherein the tissue specimen is about 3 mm or less in thickness.

37. The tissue processing method of claim 35, wherein tissue specimens are manually processed.

38. The tissue processing method of claim 35, wherein tissue specimens are mechanically processed.

39. The tissue processing method of claim 35, wherein the tissue specimen is processed without using an aldehyde.

40. The tissue processing method of claim 35, wherein the tissue specimen is processed without using a xylene.

41. The tissue processing method of claim 35, wherein the tissue specimen has not been fixed prior to processing.

42. The tissue processing method of claim 35, wherein the tissue specimen has been fixed in formaldehyde prior to processing.

43. The tissue processing method of claim 35, wherein the tissue specimen has been preserved in methanol and polyethylene glycol (PEG) prior to processing.

44. The tissue processing method of claim 35, wherein the tissue specimen is processed in a microwave retort for (a).

45. The tissue processing method of claim 44, wherein the tissue specimen is substantially hardened by the non-aqueous admixture, radiation from the microwave unit, or both.

46. The tissue processing method of claim 35 further comprising agitation to promote chemical exchange between the non-aqueous admixture and the tissue specimen.

47. The tissue processing method of claim 35, wherein the non-aqueous admixture is above about 55°

C. and/or below about 75°

C.

48. The tissue processing method of claim 35, 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.

49. The tissue processing method of claim 35, wherein a fixative is greater than about 20% (v/v) and/or less than about 60% (v/v) of the non-aqueous admixture.

50. The tissue processing method of claim 35, wherein a dehydrant is greater than about 20% (v/v) and/or less than about 60% (v/v) of the non-aqueous admixture.

51. The tissue processing method of claim 35, wherein a clearant is greater than about 10% (v/v) and/or less than about 30% (v/v) of the non-aqueous admixture.

52. The tissue processing method of claim 35, wherein the non-aqueous admixture is further comprised of a surfactant.

53. The tissue processing method of claim 35, 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.

54. The tissue processing method of claim 35, wherein the non-aqueous admixture is comprised of a clearant which is mineral oil.

55. The tissue processing method of claim 35, wherein the non-aqueous admixture is further comprised of dimethyl sulfoxide (DMSO).

56. The tissue processing method of claim 35, 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.

57. The tissue processing method of claim 35, 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.

58. The tissue processing method of claim 35, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 15 minutes or less.

59. The tissue processing method of claim 35, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 30 minutes or less.

60. The tissue processing method of claim 35, wherein the tissue specimen is substantially hardened after being incubated in the non-aqueous admixture for about 45 minutes or less.

61. The tissue processing method of claim 35, wherein the tissue specimen is processed in a vacuum retort for (b).

62. The tissue processing method of claim 61, wherein the tissue specimen is substantially impregnated at a pressure above about 100 torr and/or below about 760 torr.

63. The tissue processing method of claim 35, wherein the wax solution is above about 55°

C. and/or below about 85°

C.

64. The tissue processing method of claim 35, wherein the wax solution has been degassed and dehydrated.

65. The tissue processing method of claim 35, wherein the wax solution is made from paraffin which is solid below about 30°

C.

66. The tissue processing method of claim 35, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 15 minutes or less, and then cooled.

67. The tissue processing method of claim 35, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 30 minutes or less, and then cooled.

68. The tissue processing method of claim 35, wherein the tissue specimen can be sectioned after being incubated in the wax solution for about 45 minutes or less, and then cooled.