Method for deparaffinization of 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] Fixation of tissues 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 tissues has also proven advantageous in histopathology, this type of tissue preservation creates problems for molecular analyses, especially genetic analyses, especially since 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 tissues (FFPE) usable for molecular diagnostic procedures even years after biopsy.

[0004] For example, US Patent No. 5,999,666 describes a process for deparaffinizing formalin-fixed, paraffin-embedded tissue (FFPE) in which the paraffin is transferred to a microemulsion and washed off the tissue, while US Patent No. 5,999,666 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. No. 5,399,633 and US Pat. No. 5,499,663 disclose a method 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 any case the known methods are not suitable for automated processing of samples as part of molecular analysis, including, for the most part, the preparative step of deparaffinization. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent No. 1825246 [Patent Document 2] European Patent No. 2780453 [Patent Document 3] US 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 tissues (FFPE) that is largely automated and can be performed with only a small amount of manual effort and a small 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 a process of deparaffinizing formalin-fixed paraffin-embedded tissue (FFPE) in 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 for analyzing 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-disk system (centrifugal microfluidic biochip, centrifugal microfluidic biodisk or other centrifugal microfluidic testing cartridge) designed according to the present invention, combining preparation and analysis of formalin-fixed paraffin-embedded tissue (FFPE), can automate routine labor-intensive and error-prone laboratory tasks and perform various workflows from sample preparation to data analysis in just one step. In particular, the proposed system allows substantially faster analysis of tissue samples and corresponding treatments.

[0012] To allow sensitive diagnosis of molecular markers especially in FFPE, the paraffin must first be separated from the sample, in particular from the tissue block, to allow subsequent dissolution of cellular components and accessibility of DNA and / or RNA, without the need for additional wax / silicone mixtures, complex dissolution 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, 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 a 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 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 any case, the centrifugal microfluidic biochip is substantially designed as a cylinder with a bottom, a side and a top. The axis of rotation of the centrifugal microfluidic biochip extends in particular through the height of the centrifugal microfluidic biochip. In particular, the centrifugal microfluidic biochip is designed with a first chamber configured for holding formalin-fixed paraffin-embedded tissue (FFPE).

[0015] The fluid used is preferably an aqueous solution, in particular in the form of a buffer solution. Most preferably, the fluid is a lysis buffer (adapted to the type of tissue) suitable for lysing the tissue in order 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 invention is most preferably carried out using a lysis buffer, preferably containing the enzyme proteinase K, which is used for the degradation of proteins and the release of nucleic acids in the cell lysate.

[0016] In either case, the fluid has a density different from that of the paraffin, so that 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 surrounding and potentially dissolving 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, is introduced into the first chamber together with the formalin-fixed paraffin-embedded tissue (FFPE), or is introduced into the first chamber afterwards. In other words, the order of steps a and b is not critical, so that 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] The 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, which melts the paraffin. 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, when the biochip rotates. Enzymatic digestion and decrosslinking by proteinase K and lysis buffer take place preferentially in the liquid phase, inside which the deparaffinized tissue pieces / particles are present. In particular, this 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 via the bottom surface of the centrifugal microfluidic biochip and thus via 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 for rotating the centrifugal microfluidic biochip of a suitable analysis device that accommodates the centrifugal microfluidic biochip.

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

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

[0022] If the temperature control in steps c. and e. is performed by temperature input through a first chamber wall arranged transversely to the rotation axis, it is further provided that step e. comprises the formation of a temperature gradient extending through the height of the centrifugal microfluidic biochip, the temperature of the first chamber wall through which the temperature input is made is above the melting point of paraffin, and the temperature of the second chamber wall (e.g. the top 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 thermostating the first chamber wall to a temperature above the melting point of paraffin, in particular to a temperature of 65° C., in which the centrifugal microfluidic biochip is rotated around the rotation axis at a higher frequency than in step d. in order to cool the second chamber wall below the melting temperature of paraffin.

[0023] However, in principle the temperature gradient can be formed in any direction, in which case the geometry of the first chamber must be designed in such a way that the temperature input can be controlled such 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 upper part of the column to harden while the lower part is still liquid.

[0025] In particular, it is intended that steps e and f are carried out at least partially simultaneously. It can also be provided that a further processing step of the tissue is carried out in the first chamber before the tissue is discharged from the first chamber together with the liquid phase. The tissue is preferably dissolved and suspended in the liquid phase, but can also be discharged together with the liquid phase without dissolution. Thus, deparaffinization, dissolution and decrosslinking of the tissue can be carried out separately both in terms of time and space.

[0026] Finally, since deparaffinization is to be understood as a preparatory step for the molecular analysis of tissues, a method for the molecular analysis of formalin-fixed, paraffin-embedded tissue is also explicitly claimed, which comprises a method of deparaffinization according to the invention and a subsequent step g. of analyzing the liquid phase containing the lysed 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 tissues (FFPE), the following steps are exemplified: 1. The sample is heated in a lysis buffer in the first chamber of the 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. This allows 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 is still 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 a separate 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 description of the drawings]

[0028] [Figure 1] 1 shows the state of an FFPE sample being processed in a centrifugal microfluidic biochip during the method steps according to the invention which proceed sequentially in the illustrated example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0030] Fig. 1 shows in particular the state of an FFPE sample processed in a centrifugal microfluidic biochip during the method steps according to the invention, which in the example shown proceed sequentially. In particular, Fig. 1A shows a schematic cross-sectional view of a centrifugal microfluidic biochip 10 arranged in an analysis device 100. The centrifugal microfluidic biochip 10 is designed in a manner known per se and has a fluidic system, which in the example shown shows a first chamber 20 into which a formalin-fixed paraffin-embedded tissue FFPE has been introduced, 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 tissue FFPE is introduced into the chamber 20, or can be added manually from the outside afterwards, or introduced into the first chamber 20 from a further chamber provided in the centrifugal microfluidic biochip 10, in which a fluid is at hand.

[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 fluidic 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 molten 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 lower than the melting temperature of the paraffin (40) to allow the separated liquid paraffin (40) to solidify; f. Discharging the tissue-containing liquid phase from the first chamber (20) from which the paraffin (40) has been removed; A method comprising:

2. The method of claim 1, characterized in that 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. The method according to any one of claims 1 to 3, characterized in that the fluid (30) comprises a protease.

5. The method according to claim 4, characterized in that the protease is protease K.

6. The method according to any one of claims 1 to 5, characterized in that step b. comprises introducing a fluid (30) held in the centrifugal microfluidic biochip (10) into the first chamber (20).

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

8. 8. The method according to any one of claims 1 to 7, characterized in that 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, characterized in that step c. comprises thermostating the first chamber (20) at a temperature between 55°C and 65°C.

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

11. 11. The method according to claim 1, characterized in that step e. comprises the creation of a temperature gradient, the temperature of the first chamber wall through which the temperature input is made being above the melting point of the paraffin (40) and the temperature of a second chamber wall opposite the first chamber wall being below the melting point of the paraffin (40).

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

13. A method for the molecular analysis of formalin-fixed paraffin-embedded tissue (FFPE), characterized by the method according to any one of claims 1 to 12, followed by a step g. of analyzing the liquid phase containing the tissue with respect to at least one molecular property in a manner known per se.

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