Methods, reagents and instrumentation for preparing impregnated tissue samples suitable for histopathological and molecular studies

• US 8,703,440 B2
• Filed: 07/31/2006
• Issued: 04/22/2014
• Est. Priority Date: 07/29/2005
• Status: Expired due to Fees

• Current Assignee: HistoGeneX N.V.
• Original Assignee: HistoGeneX N.V.
• Inventors: Duymelinck, Carla, Kockx, Mark
• Primary Examiner(s): LEITH, PATRICIA A
• Application Number:
  US11/997,306
• Publication Number:
  US 20080274496A1
• Time in Patent Office: 2,822 Days
• Field of Search: 435/40.5
• US Class Current: 435/40.5
• 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-center pre-analytical variance. A bio-indicator system for measuring the degree of crosslinking is also disclosed.

Claims

1. A kit comprising:

a) an FDC solution comprising, v/v;

0.2 to 10% formaldehyde 40% w/v,

30 to 90% methanol, and

5 to 25% diethylether, and

0 to 10% acetic acid (v/v).

2. The kit according to claim 1, further comprising

b) a dehydrating-clearing solution.

3. The kit according to claim 1, further comprising

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

4. The kit according to claim 3, wherein said ISM is low-melting paraffin suitable for impregnating the tissue at a temperature between 45 and 56 deg C.

5. The kit according to claim 1, wherein a), b) and c) are each in separate containers for separate and/or sequential application to the sample.

6. The kit according to claim 1, further comprising a crosslinking indicator system to indicate the degree of fixation.

7. The kit according to claim 1, wherein said acetic acid is at a concentration between 0.1% and 10% (v/v).

8. The kit according to claim 1, wherein the FDC solution consists of, v/v, 10% formaldehyde 40% w/v, 65% methanol, 20% diethylether and 5% acetic acid (v/v).

9. The kit according to claim 1, for processing tissue sample containing fixation-sensitive phosphoprotein.

10. The kit according to claim 1, for processing tissue sample containing fixation-sensitive DNA and/or RNA.