Process for processing organic specimens

• US 6,042,874 A
• Filed: 11/23/1998
• Issued: 03/28/2000
• Est. Priority Date: 08/02/1996
• Status: Expired due to Fees

• Current Assignee: Milestone S.R.L. (Embracer Group AB)
• Original Assignee: Milestone S.R.L. (Embracer Group AB)
• Inventors: Visinoni, Francesco, Lautenschlager, Werner
• Primary Examiner(s): DUDASH, DIANA L
• Application Number:
  US09/051,031
• Time in Patent Office: 491 Days
• Field of Search: 427/4, 427/2, 427/11, 427/336, 427/595, 427/296, 427/553
• US Class Current: 427/2.11
• 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

Abstract

This invention relates to the processing of organic tissues to prepare them for further investigation such as by light microscopy, electron microscopy, microwave staining, immunostaining, decalcification, hybridisation etc.

The present invention discloses a process for processing organic tissue, which comprises the steps of fixing an organic sample to be processed, dehydrating and clearing the sample and subsequently infiltrating and embedding the sample in a supporting medium to prepare it for further examination wherein the steps of dehydrating and clearing are carried out simultaneously by applying a mixture of a dehydration agent and an essentially lipophilic agent simultaneously to the sample.

Claims

1. A process for processing tissue comprising the steps of

(i) fixing an organic sample to be processed,

(ii) dehydrating the sample with a dehydration agent,

(iii) clearing the sample with an essentially lipohilic agent, and

(iv) impregnating the sample,

wherein said steps (ii) and (iii) are carried out simultaneously by applying a mixture of said dehydration agent and said essentially lipophilic agent simultaneously to the sample.

2. The process according to claim 1, wherein said sample comprises organic tissue.

3. The process according to claim 2, wherein said organic tissue is from a group comprising blood, nerve tissue and organ tissue.

4. The process according to claim 1, wherein the process further comprises between said steps (ii/iii) and (iv) a step (v) of drying the sample at reduced pressure.

5. The process according to claim 2, wherein the process further comprises between said steps (ii/iii) and (iv) a step (v) of drying the sample at reduced pressure.

6. The process according to claim 2, wherein the dehydration agent is selected from the group consisting of C_1-8 -alcohols, C_1-8 -ethers, ketones and phenols and anilines, tetrahydrofuran, 2,2-dimethoxypropane and 2,2-diethoxypropane and creosote or a mixture thereof.

7. The process according to claim 2, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol and t-butanol or a mixture thereof.

8. The process according to claim 2, wherein the ether is selected from the group consisting of glycolethers, 2-ethoxyethanol, dioxane and polyethylene glycol or a mixture thereof.

9. The process according to claim 2, wherein the ketone is acetone.

10. The process according to claim 2, wherein the essentially lipophilic agent is selected from the group consisting of aliphatic, aromatic and chlorinated hydrocarbons, alkanes, esters and terpenes or a mixture thereof.

11. The process according to claim 2, wherein the essentially lipophilic agent is selected from the group consisting of n-olein toluene, xylene, benzene, petroleum, paraffin, carbon disulfide, chloroform, carbon tetrachloride, trichloro, n-butylacetate, amylacetate, methyl benzoate, methyl salicylate, cedarwood oil, clove oil, inhibisol, limonene and terpineol or a mixture thereof.

12. The process according to claim 2, wherein the dehydration agent comprises ethanol and isopropanol.

13. The process according to claim 2, wherein the dehydration agent comprises ethanol and isopropanol and the essentially lipophilic agent comprises an alkane.

14. The process according to claim 12, wherein the ethanol, the isopropanol and the essentially lipophilic agent are used in equal amounts.

15. The process according to claim 1, wherein the impregnation step (iv) comprises embedding the sample in a medium selected from the group consisting of agar, gelatine, polyvinylalcohol, polyetherglycols, polyethylene glycol monostearate, diethylene glycol distearate, ester wax, polyester wax, nitrocellulose, paraffin wax or a mixture thereof.

16. The process according to claim 2, wherein the impregnation step (iv) comprises embedding the sample in a medium selected from the group consisting of agar, gelatine, polyvinylalcohol, polyetherglycols, polyethylene glycol monostearate, diethylene glycol distearate, ester wax, polyester wax, nitrocellulose, paraffin wax or a mixture thereof.

17. The process according to claim 1, wherein said steps (ii) and (iii) are carried out under pressure in the range of from 0.11 to 1.0 MPa.

18. The process according to claim 2, wherein said steps (ii) and (iii) are carried out under pressure in the range of from 0.11 to 1.0 MPa.

19. The process according to claim 1, wherein said steps (ii) and (iii) are carried out at a temperature in the range of from 70 to 90°

C.

20. The process according to claim 2, wherein said steps (ii) and (iii) are carried out at a temperature in the range of from 70 to 90°

C.

21. The process according to claim 17, wherein said steps (ii) and (iii) are carried out at a temperature in the range of from 70 to 90°

C.

22. The process according to claim 18, wherein said steps (ii) and (iii) are carried out at a temperature in the range of from 70 to 90°

C.

23. The process according to claim 4, wherein said step (v) is carried out at a pressure in the range of from 0.01 to 0.05 MPa.

24. The process according to claim 23, wherein said step (v) is carried out at a temperature in the range of from 30 to 60°

C.

25. The process according to claim 4, wherein said step (v) is carried out at a temperature in the range of from 30 to 60°

C.

26. The process according to claim 1, wherein the process is carried out in a microwave heating device.

27. The process according to claim 2, wherein the process is carried out in a microwave heating device.

28. The process according to claim 17, wherein the process is carried out in a microwave heating device.

29. The process according to claim 18 wherein the process is carried out in a microwave heating device.

30. The process according to claim 19, wherein the process is carried out in a microwave heating device.

31. The process according to claim 20, wherein the process is carried out in a microwave heating device.

32. The process according to claim 21, wherein the process is carried out in a microwave heating device.

33. The process according to claim 22, wherein the process is carried out in a microwave heating device.

34. The process according to claim 23, wherein the process is carried out in a microwave heating device.

35. The process according to claim 24, wherein the process is carried out in a microwave heating device.

36. The process according to claim 25, wherein the process is carried out in a microwave heating device.

37. The process according to claim 1, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

38. The process according to claim 4, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

39. The process according to claim 26, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

40. The process according to claim 27, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

41. The process according to claim 28, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

42. The process according to claim 29, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

43. The process according to claim 30, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

44. The process according to claim 31, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

45. The process according to claim 32, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

46. The process according to claim 33, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

47. The process according to claim 34, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

48. The process according to claim 35, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.

49. The process according to claim 36, wherein said step (iv) is carried out under alternant pressure and vacuum conditions.