Methods, Reagents and Instrumentation for Preparing Impregnated Tissue Samples Suitable for Histopathological and Molecular Studies

• US 20080274496A1
• Filed: 07/31/2006
• Published: 11/06/2008
• Est. Priority Date: 07/29/2005
• Status: Active Grant

• Current Assignee: HistoGeneX N.V.
• Original Assignee: HistoGeneX N.V.
• Inventors: Kockx, Marc, Duymelinck, Carla
• Granted Patent: US 8,703,440 B2
• Time in Patent Office: Days
• US Class Current: 435/40.52
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/049 Valves integrated in closure
• CPC Class Codes: B01L 2300/0609 Holders integrated in container to position an object
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/508 rigid containers not provided for above
• CPC Class Codes: B01L 3/523 with means for closing or opening
• CPC Class Codes: B01L 3/563 Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• 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 2001/315 Basket-type carriers for tissues
• CPC Class Codes: G01N 2035/00782 reprogrammmable code
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A process and kit for the production of paraffin sections of biological tissue, especially for molecular pathology studies, is described. In the process, the tissue sample is simultaneously fixed, dehydrated and cleared in a first step, subsequently dehydrated and cleared in a second step and infiltrated with an inert specimen matrix in a third step. The specimen can then be further embedded in a casting supporting matrix according to the standard procedures followed by any local pathology or research laboratory. The method is uncomplicated, requires little hands-on time, is reproducible and is standardized thereby limiting cross-centre pre-analytical variance. A bio-indicator system for measuring the degree of crosslinking is also disclosed.

Claims

1. A method for preparing a tissue sample for morphological and/or immunohistochemical analysis, and molecular analysis comprising the steps of:

1) fixing, dehydrating and initially clearing the sample simultaneously using a fixation-dehydration-clearing, FDC, solution, that comprises

a cross-linker,

an excess of aliphatic or aromatic organic solvent, and

an ether

2) dehydrating and finally clearing the sample using a dehydrating-clearing solution, and

3) infiltrating the sample with an inert specimen matrix, ISM.

2. The method according to claim 1, wherein said cross-linker comprises an aldehyde, preferably at a concentration between 0.2% and 10% (v/v).

3. The method according to claim 1, wherein said organic solvent is methanol.

4. The method according to claim 1, wherein said ether is at a concentration between 5% and 25% (v/v).

5. The method according to claim 1, wherein said ether is one that can bind between 0.5 to 7 g water/100 g ether at 20 deg C., and can dissolve between 10 to 60 g paraffin/100 g ether at 20 deg C.,

6. The method according to claim 1, wherein said ether is a monoether, a straight or branched ether, an alkyl ether, an alkenyl ether, a C₂ to C₆ ether, a cyclic ether or a C₃ to C₅ cyclic ether.

7. The method according to claim 1, wherein said ether is any of diethyl ether, methyl tertiary butyl ether, dihydrofuran or tetrahydrofuran.

8. The method according to claim 1, wherein said FDC solution further comprises a pH modifying substance.

9. The method according to claim 8, wherein said pH modifying substance is a weak acid.

10. The method according to claim 8, wherein said pH modifying substance is at a concentration between 0.1% and 10% (v/v).

11. The method according to claim 8, wherein said pH modifying substance is acetic acid.

12. The method according to claim 1, wherein said FDC solution further comprises formaldehyde, methanol and optionally acetic acid.

13. The method according to claim 12, wherein said FDC solution comprises 0.2 to 10% formaldehyde, 30 to 90% methanol, 5 to 25% diethylether and 0 to 10% acetic acid (v/v).

14. The method according claim 12, wherein the FDC solution consists of 10% formaldehyde, 65% methanol, 20% diethylether and 5% acetic acid (v/v).

15. The method according to claim 1, wherein said dehydration-clearing solution can bind between 0.5 to 7 g water/100 g solution, and can dissolve between 10 to 60 g paraffin/100 g solution.

16. The method according to claim 1, wherein the dehydration-clearing solution comprises an ether.

17. The method according to claim 16, wherein said ether is any which can bind between 0.5 to 7 g water/100 g ether at 20 deg C., and can dissolve between 10 to 60 g paraffin/100 g ether at 20 deg C.

18. The method according to claim 16, wherein said ether is a monoether, a straight or branched ether, alkyl, an alkenyl ether, a C₂ to C₆ ether, a cyclic ether or a C₃ to C₅ cyclic ether.

19. The method according to claim 1, wherein said dehydration-clearing solution comprises one or more of diethylether, methyl tertiary butyl ether, hydrofuran, tetrahydrofuran, dioxane or dimethoxypropane.

20. The method according to claim 1, wherein said ISM is low melting point paraffin.

21. The method according to claim 20, wherein said paraffin infiltrates the sample at a temperature between 45 and 56 deg C.

22. The method according to claim 1, wherein step 1) further comprises indicating the degree of fixation by the use of a crosslinking indicator.

31. A tissue sample obtainable by the method according to claim 1.

33. Data obtainable by using a paraffin-embedded sample that has been produced according to the method of claim 1.

34. Data according to claim 33, which is one or more of micrograph 2D or 3D (virtual) images of sections, morphological analysis data, nucleic acid concentration and integrity data and data from downstream nucleic acid analyses.

23. A kit comprising:

a) an FDC solution comprising;

a cross-linker,

an excess of aliphatic or aromatic organic solvent, and

an ether

24. The kit according to claim 23, further comprising

b) a dehydrating-clearing solution.

25. The kit according to claim 23, further comprising

c) an inert specimen matrix, ISM, for infiltrating the tissue samples.

26. The kit according to claim 23, wherein a), b) and c) are each in separate containers for separate and/or sequential application to the sample.

27. The kit according to claim 23, wherein said cross-linker comprises an aldehyde, said organic solvent is methanol, and said ether is at a concentration between 5% and 25% (v/v).

28. The kit according to claim 23, wherein said dehydrating-clearing solution can bind between 0.5 to 7 g water/100 g solution, and can dissolve between 10 to 60 g paraffin/100 g solution.

29. The kit according to claim 23, wherein said ISM is low-melting paraffin suitable for impregnating the tissue at a temperature between 45 and 56 deg C.

30. The kit according to claim 23, further comprising a crosslinking indicator system to indicate the degree of fixation.

32. (canceled)