Method for deparaffinizing formalin-fixed paraffin-embedded tissue

Abstract

A method for deparaffinizing formalin-fixed paraffin-embedded tissue (FFPE) in a centrifugal microfluidic biochip 10 with a fluidic system including a plurality of chambers 20, the method comprising the steps of: a. placing the formalin-fixed paraffin-embedded tissue (FFPE) in a first chamber 20 of the fluidic system of the centrifugal microfluidic biochip 10; b. introducing a fluid 30 into the first chamber 20; c. melting the paraffin 40 by adjusting the temperature of the first chamber 20 to a temperature above the melting temperature of the paraffin 40; d. separating the molten paraffin 40 by rotating the centrifugal microfluidic biochip 10 around a rotation axis 50; e. solidifying the separated liquid paraffin 40 by adjusting at least a sub-region of the first chamber 20 to a temperature below the melting temperature of the paraffin 40; and f. discharging a liquid phase containing the tissue from which the paraffin 40 has been removed from the first chamber 20.

Description

[Technical Field]

[0001] The present invention relates to a process for deparaffinizing formalin-fixed, paraffin-embedded tissue (FFPE). [Background technology]

[0002] Fixing tissue in formalin followed by embedding in paraffin is one of the most common methods for preserving and stabilizing biological tissues used for histological examination. Tumor tissues in particular are mostly available as formalin-fixed, paraffin-embedded material in the context of pathological and oncological examinations, where they are also used in the diagnosis of inflammatory or autoimmune diseases.

[0003] Although the use of formalin-fixed, paraffin-embedded tissue has proven advantageous in histopathology, this type of tissue preservation poses problems for molecular analysis, especially genetic analysis, especially because the chemical modifications caused by fixation with formalin must first be reversed. For this reason, various extraction and restoration methods have been developed to make formalin-fixed, paraffin-embedded tissue (FFPE) usable for molecular diagnostic procedures even years after biopsy.

[0004] For example, Patent Document 1 describes a process for deparaffinizing formalin-fixed, paraffin-embedded tissue (FFPE) in which the paraffin is transferred to a microemulsion and washed away from the tissue, while Patent Document 2 describes a process for removing paraffin from tissue by heating the paraffin and converting it into a lipophilic phase consisting of silicone and wax.

[0005] Furthermore, US Pat. Nos. 5,999,049 and 5,999,049 disclose methods for centrifugation-based processing of biochemical samples in which paraffin is used.

[0006] These known methods require relatively high manual effort or complex equipment setup before the paraffin-free tissue is available for further molecular analysis, and in either case, the known methods are largely unsuitable for automated processing of samples as part of molecular analysis, including preparative steps such as deparaffinization. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent No. 1825246 [Patent Document 2] European Patent No. 2780453 [Patent Document 3] U.S. Patent Application Publication No. 2009 / 0246782 [Patent Document 4] Korean Patent No. 101796110 Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to create a method for the molecular analysis of formalin-fixed, paraffin-embedded tissue (FFPE) that is largely automated and can be performed with only minimal manual effort and small-scale equipment setup. [Means for solving the problem]

[0009] According to the invention, this object is solved by a method having the features of claim 1. The dependent claims disclose advantageous embodiments of the invention.

[0010] The basic idea of ​​the present invention is to integrate the process of deparaffinizing formalin-fixed, paraffin-embedded tissue (FFPE) into a centrifugal microfluidic biochip, in particular a centrifugal microfluidic biodisc, so that steps known per se can be subsequently carried out in a manner known per se to analyze the tissue with respect to at least one molecular characteristic. The analysis can for example concern the presence, absence, or increased or decreased presence of a genetic characteristic or a characteristic at the protein or gene expression level.

[0011] The lab-on-a-disk system (centrifugal microfluidic biochip, centrifugal microfluidic biodisk, or other centrifugal microfluidic test cartridge) designed according to the present invention, which combines the preparation and analysis of formalin-fixed, paraffin-embedded tissue (FFPE), automates labor-intensive and error-prone routine laboratory tasks and enables various workflows from sample preparation to data analysis to be performed in a single step. In particular, the proposed system allows for substantially faster analysis of tissue samples and corresponding treatments.

[0012] To enable sensitive diagnosis of molecular markers, especially in FFPE tissue, it is necessary to first separate the paraffin from the sample, particularly from the tissue block, to allow subsequent lysis of cellular components and access to DNA and / or RNA, without the need for additional wax / silicone mixtures, complex lysis reagents with additional components for paraffin liquefaction, or ultrasonic units in the processing equipment, as proposed in the prior art.

[0013] Therefore, according to the present invention, there is provided a method for deparaffinizing formalin-fixed, paraffin-embedded tissue in a centrifugal microfluidic biochip with a fluidic system including multiple chambers, the method comprising: a. Placing formalin-fixed, paraffin-embedded tissue (FFPE) in a first chamber of a fluid system of a centrifugal microfluidic biochip; b. introducing a fluid into the first chamber; c. melting the paraffin by adjusting the temperature of the first chamber to a temperature above the melting temperature of the paraffin; d. Separating the molten paraffin by rotating the centrifugal microfluidic biochip around a rotation axis; e. adjusting the temperature of at least a portion of the first chamber to a temperature below the melting temperature of the paraffin, thereby allowing the separated liquid paraffin to solidify; f. Discharging the deparaffinized tissue-containing liquid phase from the first chamber; A method is provided, comprising:

[0014] The centrifugal microfluidic biochip is preferably designed as a centrifugal microfluidic biodisc, i.e., a disk, with the axis of rotation representing the center of the centrifugal microfluidic biodisc. In either case, the centrifugal microfluidic biochip is designed substantially as a cylinder with a bottom, side, and top. The axis of rotation of the centrifugal microfluidic biochip particularly extends throughout the height of the centrifugal microfluidic biochip. In particular, the centrifugal microfluidic biochip is designed to include a first chamber configured to hold formalin-fixed, paraffin-embedded tissue (FFPE).

[0015] The fluid used is preferably an aqueous solution, particularly in the form of a buffer solution. Most preferably, the fluid is a lysis buffer (adapted to the type of tissue) suitable for lysing tissue to make the molecules contained in the tissue available for molecular biological analysis. This can be DNA or RNA analysis, or protein analysis. The lysis buffer used can contain buffer salts (e.g., Tris-HCl) and ionic salts (e.g., NaCl) to adjust the pH value and osmolality of the lysate, as well as detergents (e.g., Triton X-100, SDS, etc.). The method according to the present invention is most preferably carried out using a lysis buffer preferably containing the enzyme proteinase K, which is used to degrade proteins and release nucleic acids in the cell lysate.

[0016] In either case, the fluid has a density different from that of the paraffin, and the paraffin melted by the heat input can separate due to the density difference. In particular, because the fluid is denser than the liquid paraffin, the melted paraffin floats on top of the fluid, which surrounds and potentially dissolves the tissue.

[0017] It does not matter whether the fluid is already present in the first chamber before the formalin-fixed, paraffin-embedded tissue (FFPE) is introduced into the first chamber, or whether the fluid is introduced into the first chamber together with the formalin-fixed, paraffin-embedded tissue (FFPE), or whether the fluid is introduced into the first chamber after the formalin-fixed, paraffin-embedded tissue (FFPE) is introduced into the first chamber. In other words, the order of steps a and b is not critical, so step b can also be performed before step a. The only critical factor is that a liquid phase is provided that allows the paraffin that embeds the tissue to be separated.

[0018] A sample in the form of formalin-fixed, paraffin-embedded tissue (FFPE) is preferably heated in a lysis buffer / proteinase K mixture in the first chamber, thereby melting the paraffin. Upon rotation of the biochip, the liquefied paraffin floats on top of the fluid, or most preferably the lysis buffer / proteinase K mixture used, due to the density difference between the lysis buffer and the paraffin, inside the pivot point. Enzymatic digestion and decrosslinking by proteinase K and lysis buffer occur preferentially in the liquid phase, within which the deparaffinized tissue fragments / particles reside. This, in particular, allows the transfer of RNA into the liquid phase.

[0019] The temperature of the first chamber in steps c. and e. is preferably controlled by introducing the temperature into the centrifugal microfluidic biochip through the bottom surface of the centrifugal microfluidic biochip, and therefore through the first chamber wall arranged transversely to the rotation axis. Particularly preferably, the temperature is applied by a heating device provided in a rotating device that rotates the centrifugal microfluidic biochip of an appropriate analytical device that accommodates the centrifugal microfluidic biochip.

[0020] The first chamber is thermostated in step c. to a temperature above 49°C, and thus above the melting point of paraffin, for a first predetermined time. In particular, the first chamber is thermostated to a temperature between 55°C and 65°C.

[0021] It is further preferred that step c. comprises adjusting the temperature of the first chamber to a temperature of 70°C to 90°C for a second predetermined time period, which is generated to decrosslink molecules contained in the tissue.

[0022] When the temperature control in steps c. and e. is performed by temperature input through a first chamber wall arranged transverse to the rotation axis, it is further provided that step e. includes forming a temperature gradient extending through the height of the centrifugal microfluidic biochip, wherein the temperature of the first chamber wall through which the temperature input is performed exceeds the melting point of paraffin, and the temperature of a second chamber wall (e.g., the upper surface of the centrifugal microfluidic biochip) opposite the first chamber wall (e.g., the bottom surface of the centrifugal microfluidic biochip) is below the melting point of paraffin. This can be particularly preferably achieved by step e. of thermoregulating the first chamber wall to a temperature above the melting point of paraffin, particularly a temperature of 65°C, in which the centrifugal microfluidic biochip is rotated around the rotation axis at a frequency higher than in step d. to cool the second chamber wall below the melting point of paraffin.

[0023] However, in principle, the temperature gradient can be formed in any direction, and in this case the geometry of the first chamber must be designed in such a way that the temperature input can be controlled so that the temperature on the first chamber wall corresponds to or exceeds the melting point of the paraffin, and the temperature on the second chamber wall opposite the first chamber wall is below the melting point of the paraffin so that the paraffin can solidify on the second chamber wall.

[0024] Separation of the paraffin by partial solidification is preferably achieved by simultaneously thermostating and rotating the chamber above the melting point of the paraffin, which is sufficient to create a temperature gradient in the liquid paraffin column (the chamber is cooler than the melting point of the paraffin on the upper side of the biochip facing the heating device due to air turbulence and therefore heat dissipation), causing the top of the column to harden while the bottom remains liquid.

[0025] In particular, steps e and f are intended to be performed at least partially simultaneously. It can also be provided that further processing steps of the tissue are performed in the first chamber before the tissue is expelled from the first chamber together with the liquid phase. The tissue is preferably lysed and suspended in the liquid phase, but can also be expelled together with the liquid phase without lysis. Thus, deparaffinization, lysis, and decrosslinking of the tissue can be performed separately in terms of both time and space.

[0026] Finally, since deparaffinization is to be understood as a preparatory step for the molecular analysis of tissue, a method for the molecular analysis of formalin-fixed, paraffin-embedded tissue is also expressly claimed, which comprises a method of deparaffinization according to the invention and a subsequent step g. of analyzing the liquid phase containing the dissolved tissue for at least one molecular property in a manner known per se.

[0027] For RNA purification of samples available as formalin-fixed paraffin-embedded tissue (FFPE), the following steps are exemplified: 1. The sample is heated in a lysis buffer in the first chamber of a centrifugal microfluidic biochip, which melts the paraffin. The paraffin then floats on top of the lysis buffer as a hydrophobic phase. Enzymatic digestion and decrosslinking occur in the liquid (hydrophilic) phase, allowing the RNA to migrate into the liquid (hydrophilic) phase. 2. Separation by partial solidification is achieved by heating the chamber to a temperature above the melting point of paraffin and simultaneously rotating it at high speed, which creates a temperature gradient in the liquid paraffin column (the chamber is cooler than the melting point of paraffin at the top due to air turbulence and therefore heat dissipation), causing the top of the column to harden while the bottom remains liquid. 3.The paraffin then separates due to the solidification of the paraffin. 4. The lysate is drained and mixed with binding buffer and magnetic particles in another chamber. 5. The magnetic particles are transferred into a third chamber where they are washed. 6. Finally, the magnetic particles are transferred into a fourth chamber where the RNA is eluted, making the RNA extracted from the formalin-fixed paraffin-embedded tissue (FFPE) available for further processing as known per se. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows the state of an FFPE sample being processed in a centrifugal microfluidic biochip during the sequential steps of the method according to the invention in the example shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] The invention will be explained in more detail below on the basis of particularly preferred exemplary embodiments shown in the accompanying drawings.

[0030] 1 shows in particular the state of an FFPE sample being processed in a centrifugal microfluidic biochip during the sequential steps of the method according to the present invention in the illustrated embodiment. Specifically, FIG. 1A shows a schematic cross-sectional view of a centrifugal microfluidic biochip 10 arranged in an analytical device 100. The centrifugal microfluidic biochip 10 is designed in a manner known per se and includes a fluid system, which in the illustrated embodiment shows a first chamber 20 into which formalin-fixed, paraffin-embedded FFPE tissue has been introduced and which is surrounded by a fluid 30 stored in the centrifugal microfluidic biochip 10 and configured as a lysis buffer. The buffer 30 can be provided before the formalin-fixed, paraffin-embedded FFPE tissue is introduced into the chamber 20, or can be added manually from the outside afterwards, or can be introduced into the first chamber 20 from a further chamber in the centrifugal microfluidic biochip 10 containing a fluid.

[0031] In either case, as shown in FIG. 1B, the paraffin 40 surrounding the tissue is melted by controlling the temperature of the first chamber 20 to a temperature above the melting temperature of the paraffin 40. The temperature is applied by a heating zone 110 provided in the analysis device 100, which applies the temperature from the bottom surface of the centrifugal microfluidic biochip 10. When the centrifugal microfluidic biochip 10 is simultaneously rotated around the rotation axis 50, the density difference between the fluid 30 and the liquid paraffin 40 causes the liquid paraffin 40 floating on the fluid to separate.

[0032] If separate phases are present, the separated liquid paraffin 40 can be solidified by thermostating at least a portion of the first chamber 20 to a temperature below the melting temperature of the paraffin 40. In particular, the temperature input into the first chamber 20 is controlled by the heating zone 110 of the analysis device 100 to form a temperature gradient through the height of the centrifugal microfluidic biochip 10, whereby the liquid paraffin 40 solidifies on the cooler wall of the first chamber 20 facing the heating zone.

[0033] In Figures 1D and 1E, the meanwhile lysed tissue is discharged from the first chamber 20 and sent for further processing, in which case the volume of liquid paraffin 40 decreases and the volume of paraffin 40 solidified on the walls increases. [Explanation of symbols]

[0034] 10. Centrifugal Microfluidic Biochip 20 First Chamber 30 Fluid Buffer 40 Paraffin Liquid Paraffin 50 Rotational Axis 100 Analyzer 110 Heating Zone FFPE formalin-fixed paraffin-embedded tissue

Claims

1. A method for deparaffinizing formalin-fixed, paraffin-embedded (FFPE) tissue in a centrifugal microfluidic biochip (10) having a fluidic system including a plurality of chambers (20), comprising: a. Placing formalin-fixed, paraffin-embedded tissue (FFPE) in a first chamber (20) of a fluid system of a centrifugal microfluidic biochip (10); b. introducing a fluid (30) into the first chamber (20); c. Melting the paraffin (40) by adjusting the temperature of the first chamber (20) to a temperature above the melting temperature of the paraffin (40); d. Separating the melted paraffin (40) by rotating the centrifugal microfluidic biochip (10) around a rotation axis (50); e. adjusting the temperature of at least a portion of the first chamber (20) to a temperature below the melting temperature of the paraffin (40), thereby allowing the separated liquid paraffin (40) to solidify; and f. Discharging the tissue-containing liquid phase from the first chamber (20) from which the paraffin (40) has been removed; The method, wherein step e. includes forming a temperature gradient, wherein the temperature of a first chamber wall through which the temperature input is made is above the melting point of the paraffin (40), and the temperature of a second chamber wall opposite the first chamber wall is below the melting point of the paraffin (40).

2. 2. The method of claim 1, wherein the fluid (30) is an aqueous solution.

3. 3. The method according to claim 1 or 2, characterized in that the fluid (30) is a buffer solution.

4. 2. The method of claim 1, wherein the fluid (30) comprises a protease.

5. 5. The method of claim 4, wherein the protease is protease K.

6. 2. The method of claim 1, wherein step b. comprises introducing a fluid (30) held in the centrifugal microfluidic biochip (10) into the first chamber (20).

7. 2. The method according to claim 1, wherein the temperature regulation in steps c. and e. is performed by applying the temperature through a first chamber wall arranged transversely to the rotation axis (50).

8. 2. The method of claim 1, wherein step c. comprises thermostating the first chamber (20) at a temperature above 49°C for a first predetermined time.

9. 9. The method of claim 8, wherein step c. comprises thermostating the first chamber (20) at a temperature between 55°C and 65°C.

10. 9. The method of claim 8, wherein step c. comprises thermostating the first chamber (20) at a temperature between 70°C and 90°C for a second predetermined time to decrosslink molecules contained in the tissue.

11. 2. The method of claim 1, wherein step e. comprises thermostating the first chamber wall to a temperature above the melting point of paraffin (40), and wherein the centrifugal microfluidic biochip (10) is rotated around the rotation axis (50) at a frequency higher than in step d. to cool the second chamber wall.

12. A method for molecular analysis of formalin-fixed paraffin-embedded tissue (FFPE), characterized by the method according to claim 1 and subsequent step g. of analyzing the liquid phase containing the tissue for at least one molecular characteristic in a manner known per se.

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