Method for in situ antigen retrieval and imaging of biological samples

Abstract

To provide a method for in situ temperature-induced antigen retrieval of a sample.SOLUTION: The present invention relates to a device and method for in situ temperature-induced antigen retrieval of a sample, where all steps are performed under pressure above atmospheric pressure on a sample fixed on a sample support. The sample can further undergo staining and imaging on the same sample support optionally by cycle multiplexing, which allows imaging of different molecular targets through multi-molecular readout on the same sample in a rapid, sensitive and reliable way.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the field of biological sample preparation, and more particularly to antigen retrieval allowing for in situ imaging of samples. [Background technology]

[0002] Immunohistochemistry (IHC) is a technique that involves the use of specific probe molecules, such as antibodies, to detect the presence of specific biomarkers (e.g., antigens) that may be expressed by cells in a tissue sample. IHC is widely used in both clinical and research settings to diagnose certain diseases, such as certain types of cancer, or to investigate correlations between disease prognosis and the expression of novel biomarkers. The primary application of IHC is in the diagnosis of cancer, but other applications include the detection of infectious agents such as viruses (McMahon et al., 1996, Histochem J., 28(3):157-64) and aiding in the diagnosis of other diseases, such as Alzheimer's disease (Dandrea et al., 2001, Biotech Histochem., 76(2):97-106).

[0003] Antigen retrieval (AR) is a pretreatment technique for IHC used to enhance the antigenicity of various types of tissue samples, primarily formalin-fixed paraffin embedded (FFPE) tissue sections. Since its initial introduction, it has been rapidly adopted as the industry gold standard for a wide range of tissue types and target markers (Shi et al., 1991, J Histochem Cytochem. 39:741-748; U.S. Patent No. 5,244,787).

[0004] Although the exact mechanism behind the efficacy of antigen retrieval treatment remains unclear, there is consensus regarding its effectiveness in reversing the formation of formalin-induced peptide bonds and activating target epitopes. There are two main methods used to enhance epitope responsiveness: enzyme-induced antigen retrieval (EIAR) and heat-induced antigen retrieval (HIAR). Because enzymatic substances are catalytic in nature, EIAR is more likely to disrupt tissue morphology and render target protein sites nonfunctional (Werner et al., 1996, Histochemistry and Cell Biology, 105:4 253-260). Therefore, HIAR is the more widely used method of the two.

[0005] There are various devices and methodologies for performing HIAR. The traditional method is incubation of the sample in a heat bath containing a basic buffer. In this method, the general trend is that a longer incubation time is required to obtain the same antigenicity for lower AR temperatures. For example, overnight incubation at 60°C or 1 hour incubation at 95°C have both been reported to be successfully used for pre-IHC AR (Yamashita, 2007, Prog Histochem Cytochem., 41(3):141-2007). Microwave treatment is another common method in which the sample undergoes either constant power microwave treatment or cycles of heating and resting (U.S. Pat. No. 5,244,787). It has also been used to improve antigenicity in immunofluorescence (Long and C. Buggs, 2008, J Mol Histol. 39(1): 1-4) and special staining applications (Temel et al., 2005, Biotech Histochem. 80(3-4): 123-32).

[0006] It is also possible to combine HIAR and EIAR methods. Key et al. disclosed an AR buffer containing a thermostable enzyme for use in microwave treatment of FFPE samples. For these methods, where the AR temperature is increased to 100°C, successful AR was shown for various breast cancer markers (WO 2009 / 110936). Another alternative is pressure-assisted HIAR, which allows the temperature of the AR medium to be raised above its atmospheric boiling temperature. Angros et al. disclosed an automated device for performing AR in a high-pressure environment for this purpose (US Pat. No. 7,951,612).

[0007] HIAR can be performed in various environments or in various solutions modified to improve AR performance. For example, Gourevitch disclosed a novel solution that promotes sample integrity during AR (US Patent Application Publication No. 2004 / 0029184), and Namimatsu disclosed the use of an AR solution containing CCA and NaOH to improve AR performance (US Patent Application Publication No. 2002 / 0182653). Eriksen disclosed an AR solution to which various non-ionic surfactants were added to reverse specific cross-links between amine groups present in target epitopes (US Patent No. 9,506,928).

[0008] Numerous AR solution formulations are known in the art that increase the boiling point of the solution at atmospheric pressure so that AR can be performed at high temperatures. Key et al. disclosed AR solutions with various additives and viscosities with increased boiling points up to 135°C, in addition to a device for automatic sample temperature control (US 2006 / 0134793, WO 2009 / 085574). Christensen et al. disclosed a horizontal AR device and method that achieved AR temperatures of 120°C to 130°C using improved buffers (US 2010 / 0136612). Kram et al. disclosed AR solutions containing organic salts or ionic liquids that reach boiling points above 200°C (WO 2006 / 066039).

[0009] It is also known in the art to apply different buffer solutions successively to improve AR efficiency.For example, Gerdes et al. disclose a method of treating samples with AR buffer solutions of different pH values ​​for the detection of multiple antigens (US Patent Application Publication No. 2012 / 009666).

[0010] Another approach to reduce sample pretreatment time before immunostaining is to provide a buffer that can be used in both deparaffinization and antigen retrieval of FFPE samples. Aghassi et al. disclosed a buffer containing various detergents that was used to simultaneously perform deparaffinization and AR (U.S. Patent No. 6,649,368).

[0011] The above methods do not adequately address the issues of long sample processing times and low efficiency, especially since antigen retrieval is performed as part of the sample preparation prior to the staining process.

[0012] Thus, there is a need for new techniques, equipment, and tools for performing tissue processing methods that require reduced processing times, as reduced processing times lead to increased throughput in pathology workflows and more efficient use of limited clinical laboratory resources. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Pat. No. 5,244,787 [Patent Document 2] International Publication No. 2009 / 110936 [Patent Document 3] U.S. Patent No. 7,951,612 [Patent Document 4] US Patent Application Publication No. 2004 / 0029184 [Patent Document 5] US Patent Application Publication No. 2002 / 0182653 [Patent Document 6] U.S. Pat. No. 9,506,928 [Patent Document 7] US Patent Application Publication No. 2006 / 0134793 [Patent Document 8] International Publication No. 2009 / 085574 [Patent Document 9] US Patent Application Publication No. 2010 / 0136612 [Patent Document 10] International Publication No. 2006 / 066039 [Patent Document 11] US Patent Application Publication No. 2012 / 009666 [Patent Document 12] U.S. Patent No. 6,649,368 [Non-patent literature]

[0014] [Non-Patent Document 1] McMahon et al., 1996, Histochem J., 28(3):157-64 [Non-Patent Document 2] Dandrea et al., 2001, Biotech Histochem., 76(2):97-106 [Non-Patent Document 3] Shi et al., 1991, J Histochem Cytochem., 39:741-748 [Non-Patent Document 4] Werner et al., 1996, Histochemistry and Cell Biology, 105:4, 253-260 [Non-Patent Document 5] Yamashita, 2007, Prog Histochem Cytochem., 41(3):141~2007 [Non-Patent Document 6] Long and C. Buggs, 2008, J Mol Histol. 39(1): 1-4. [Non-Patent Document 7] Temel et al., 2005, Biotech Histochem., 80(3-4):123-32 [Non-Patent Document 8] MA Hayat, "Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy", 2002, Springer Science+Business Media New York [Non-Patent Document 9] Modern Pathology, 2011, 24, 613~623, doi:10.1038 / modpathol.2010.228 [Non-Patent Document 10] Dabbs, Diagnostic Immunohistochemistry: theranostic and diagnostic applications, 4th edition, 2014, ISBN 978-1-4557-4461-9 Summary of the Invention [Problem to be solved by the invention]

[0015] The object of the present invention is to provide a method for in situ temperature-induced antigen retrieval of a sample, in which all steps are performed under pressure higher than atmospheric pressure on a sample fixed on a sample support, allowing antigen retrieval prior to staining and imaging of said sample on the same sample support.

[0016] It would be advantageous to provide a method for in situ temperature-induced antigen retrieval of samples under pressures higher than atmospheric that allows for a fully automated and rapid high-temperature-induced denaturing effect and cold-induced refolding temperature to be achieved, allowing rapid and reproducible temperature-induced unmasking of target epitopes.

[0017] It would be advantageous to provide a method of in situ temperature-induced antigen retrieval of tissue or cell samples fixed on a sample support that maintains sample integrity and allows for subsequent staining and imaging of said samples on the same sample support with reduced processing times due to the avoidance of subjecting the sample to multiple processing steps for antigen retrieval prior to staining, as well as mounting the sample on different sample supports for antigen retrieval and staining.

[0018] In one form of another implementation, it is advantageous to provide a method for in situ temperature-induced antigen retrieval of a tissue or cell sample fixed on a sample support and subject said sample to subsequent staining and imaging on the same sample support, where the temperature of the sample after the temperature-induced antigen retrieval step is cooled to a temperature higher than room temperature and the subsequent staining and imaging steps are performed at a temperature higher than room temperature, reducing processing time and increasing efficiency of staining.

[0019] In another form of implementation, it would be advantageous to provide a method for in situ temperature-induced antigen retrieval of tissue or cell samples fixed on a sample support, where said samples undergo a subsequent fully controllable flow of imaging probes directly at the surface of the sample in a specific sequence to perform a complete cycle of sample labeling and imaging on the same sample support as well as to repeat such cycles in a high-throughput manner facilitating immunostaining of various markers on the same sample and increasing the number of possible sample labeling cycles without compromising the reproducibility and / or sensitivity of the measured imaging signal.

[0020] The object of the present invention is achieved by providing a method as claimed in claim 1. [Means for solving the problem]

[0021] According to a first aspect of the present invention, disclosed herein is a method for in situ temperature induced antigen retrieval of a biological sample fixed on a sample support, comprising: a) providing said biological sample immobilized on said sample support; b) providing a microfluidic device comprising a microfluidic chamber, at least one fluid inlet at one end of the microfluidic chamber and at least one fluid outlet at an other end of the microfluidic chamber, configured to direct a fluid supplied under pressure from a fluid supply system through the microfluidic chamber, wherein at least one wall of the microfluidic chamber is formed by a sample support and is attached to a seal in a fluid-tight and removably manner by a clamping mechanism to a first wall of the microfluidic chamber, and wherein the volume of the microfluidic chamber is between 2.5 μl and 200 μl; c) mounting said microfluidic chamber and said sample support together in a fluid-tight manner, with the sample facing the inside of the microfluidic chamber; d) performing a temperature-controlled antigen retrieval step at a pressure higher than atmospheric pressure, said retrieval step comprising: - pressure P in the microfluidic chamber above atmospheric pressure chamber filling the microfluidic chamber with an epitope unmasking solution via a fluid inlet at - Set the microfluidic chamber to the incubation temperature set point (T AR ) and incubation duration t i maintaining the temperature of the microfluidic chamber at said incubation temperature set point during said incubation; - Cooling duration t c cooling the microfluidic chamber to a cooling temperature below the incubation temperature set point and above room temperature during said incubation period; and the incubation temperature set point T AR is included between about 60 °C and 200 °C, and the pressure P in the microfluidic chamber chamberis greater than 1.5 bar and the incubation duration t i is between 0.5 and 30 minutes, and The method includes:

[0022] In one advantageous embodiment, the cooling duration t c can be comprised between 1 and 30 minutes.

[0023] In one advantageous embodiment, the incubation temperature set point T during the temperature-controlled antigen retrieval step S1 AR may be comprised between 110°C and 200°C.

[0024] In one advantageous embodiment, the pressure P in the microfluidic chamber during the temperature-controlled antigen retrieval step S1 chamber may be comprised between about 2.5 and 5 bar.

[0025] In one advantageous embodiment, the incubation duration t i may be comprised between 2 and 20 minutes, preferably between 2 and 15 minutes.

[0026] In one advantageous embodiment, the method of the invention comprises the steps of: e) sequentially injecting a plurality of reagents including at least one imaging probe into the microfluidic chamber via the fluid inlet at a flow rate ranging between about 1 μl / s and about 100 μl / s; f) imaging signals emitted by components of the sample reacted with said at least one imaging probe; and g) repeating steps (e) and (f) with different imaging probes.

[0027] In one advantageous embodiment, the step of sequentially injecting a plurality of reagents comprises: - an elution step in which an elution buffer is injected to remove any unwanted material that may remain on the sample; - an optional non-specific binding blocking step in which a blocking buffer is injected; - a sample labelling step in which an imaging probe is injected; Including, Each of these steps is preceded by an optional wash step in which a wash buffer is injected; The at least one imaging probe results from the injection of a series of specific antibodies as labeling probes and chromogenic or fluorescent detection molecules that target the molecular entity to be analyzed within the sample.

[0028] In one advantageous embodiment, each step in a sequence of multiple reagents to be injected includes two flow rate steps for each reagent, namely: - a first flow rate step in which the reagent is injected at an initial flow rate in the range between about 1 μl / s and about 100 μl / s; - a second flow rate step in which the same reagent is injected at a lower flow rate (typically about 0.001 to about 1.0 μl / s) to ensure incubation of the reagent with the sample before injecting the next reagent in the sequence; Includes.

[0029] In one advantageous embodiment, the first flow step lasts between 1 and 120 seconds.

[0030] In one advantageous embodiment, the second flow step of the reagent lasts from about 1 minute to about 30 minutes.

[0031] In one advantageous embodiment, the sample labeling step may include the step of injecting at least one labeled probe for in situ hybridization with any DNA / RNA material in the sample, such as a labeled RNA or DNA probe.

[0032] In an advantageous embodiment, the sample labelling step may further comprise the step of applying temperature cycling within the microfluidic chamber in order to hybridise DNA / RNA material within the sample with the RNA or DNA probes.

[0033] In an advantageous embodiment, the imaging step may be performed by fluorescence microscopy or bright field microscopy.

[0034] In an advantageous embodiment, the elution step can be performed with temperature cycling to ensure removal of any unwanted in situ hybridized probes or markers that may remain on the sample before repeating the method with another sample labeling step.

[0035] Another object of the present invention is achieved by providing an apparatus as claimed in claim 7.

[0036] According to a second aspect of the invention, disclosed herein is a biological sample processing apparatus suitable for use in a method according to any of the preceding claims, comprising a support mechanism, a thermal unit, a microfluidic device, a pressurized fluid supply system and a control system, the microfluidic device comprising a microfluidic chamber having a volume comprised between 2.5 μl and 200 μl, at least one fluid inlet at one end of said microfluidic chamber and at least one fluid outlet at an other end of said microfluidic chamber, the microfluidic device being configured to direct a fluid through the microfluidic chamber to react with a sample inside said microfluidic chamber, at least one wall of the microfluidic chamber being formed by a sample support, and a pressure P in the microfluidic chamber greater than 1.5 bar, preferably greater than 2 bar, in particular greater than 2.5 bar, more preferably greater than 3 bar. chamberThe seal is attached in a fluid-tight and removable manner to a first wall of the microfluidic chamber by a clamping mechanism configured to compress the seal between the sample support and the microfluidic device to a sufficient extent to accommodate the sample.

[0037] In one advantageous embodiment, the microfluidic device may be in the form of a substantially planar substrate of optically transparent material having therein a microfluidic inlet channel network connected to a fluid inlet orifice and a microfluidic outlet channel network connected to a fluid outlet orifice, both of which open onto a first side of the substrate corresponding to a chamber boundary side of the substrate.

[0038] In an advantageous embodiment, the height of the chamber between the first surface and the sample support is less than 100 μm configured to ensure advective transport of reagents along a biological tissue sample disposed on the sample support.

[0039] In one advantageous embodiment, the support mechanism includes a base structure and the clamping mechanism described above, the clamping mechanism including a clamping plate with a window that allows the biological sample to be viewed under a microscope through the transparent substrate.

[0040] In one advantageous embodiment, the microfluidic device may be placed on top of the sample support with the chamber side and seal of the microfluidic device facing the sample support and facing downwards over the biological sample.

[0041] In one advantageous embodiment, the fluid supply system under pressure comprises a fluid supply channel and a fluid outlet channel attached to a base structure, each including a seal at its orifice end facing a movable clamping plate of the clamping mechanism, the seals being compressed against a chamber-facing side of a substrate of the microfluidic device at locations around the fluid inlet and outlet orifices of the microfluidic device, respectively, said seals under compression being configured to support a pressure of at least 1.5 bar, preferably greater than 2 bar, in particular greater than 2.5 bar, more preferably greater than 3 bar.

[0042] In an advantageous embodiment, the thermal unit is attached to the base structure below the position of the sample support, the thermal unit comprising a heating unit that conducts heat to the sample support and is optionally attached to a heat transfer unit that is located immediately below the sample support.

[0043] In one advantageous embodiment, the heating unit comprises a Peltier element or a number of stacked Peltier elements.

[0044] In an advantageous embodiment, the thermal unit further comprises a cooling unit, for example in the form of a cooling block comprising a labyrinth of fluid flow channels through which a cooling fluid flows, providing active cooling of the sample support.

[0045] In one embodiment, the sample processing device further comprises one or more temperature sensors integrated into the base structure and / or clamping plate configured to measure the temperature at the sample support or the microfluidic device and / or at the heating unit and / or the heat transfer unit, the one or more temperature sensors forming part of a temperature control system configured to control the operation of the thermal unit. [Brief description of the drawings]

[0046] [Figure 1a] 1 shows a biological sample processing device 2 according to one embodiment of the present invention, the device being shown in a closed position ready for use. [Figure 1b] FIG. 1b is a perspective view of the device of FIG. 1a showing the clamping mechanism in an open position with the microfluidic network device removed. [Figure 1c] FIG. 1c is a cross-sectional view taken along line 1c-1c in FIG. [Figure 1d] FIG. 1d is a cross-sectional view taken along line 1d-1d in FIG. [Figure 1e] FIG. 1b is a cross-sectional view taken along line 1e-1e in FIG. [Figure 1f] FIG. 1f is a cross-sectional view taken along line 1f-1f in FIG. [Figure 1g] FIG. 1b is an enlarged view of a portion of the cross-sectional view of FIG. [Figure 2a] FIG. 1 shows the process sequence used in the in situ antigen retrieval, staining and imaging step cycle of the method of the present invention, omitting the optional wash buffer steps between each major flow step (S1 to S4). [Figure 2b] 1 shows the temperature control protocol used under step S1 for AR. [Diagram 3] Fluorescence images in the DAPI (A) and PR (B) channels and combined (C) obtained with immunostaining performed after in situ antigen retrieval as described in Example 2 are shown. [Figure 4a] FIG. 1 shows various temperature control protocols used in the temperature controlled AR processing step of the method of the invention as described in Example 3 (heating from room temperature to 105° C. in about 1 min, incubating at this temperature for about 10 min, cooling to 85° C. in 3° C. increments over about 7 min, and cooling to room temperature). [Figure 4b] 1 shows various temperature control protocols used in the temperature-controlled AR treatment step of the method of the present invention as described in Example 3 (heating from room temperature to 98°C in about 1 minute, incubating at this temperature for about 10 minutes, cooling to 85°C in 3°C increments over about 4 minutes, and cooling to room temperature). [Diagram 5]Sequential images are shown obtained during the colocalization staining of Example 4 for various biomarkers on breast cancer tissue sections that underwent either (1) a standard temperature-induced antigen retrieval procedure prior to immunostaining, or (2) an in situ antigen retrieval and elution step according to the method of the present invention. The sequence of images A to D corresponds to the acquisition order indicated in the protocol. [Figure 6a] 1 is a schematic cross-sectional view of a microfluidic device and sample support of a bioprocessing device according to one embodiment of the present invention. [Figure 6b] FIG. 6b is a perspective view of a cross-section of the microfluidic device taken through line VIb-VIb in FIG. 6a, showing a microfluidic channel network for inlet and outlet of fluids into the microfluidic chamber. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The expression "epitope unmasking solution" refers to any solution suitable for heat-induced epitope retrieval and known to those skilled in the art. Typical epitope unmasking solutions are those described by MA Hayat in "Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy", 2002, Springer Science+Business Media, New York.

[0048] Referring now to the drawings, and in particular initially to Figures 1a-1g and 6a-6b, a biological sample processing device 2 according to one embodiment of the present invention is shown. The biological sample processing device 2 is configured for performing in situ temperature-induced antigen retrieval of a biological sample, and may further be configured for performing in situ imaging of the sample by cycle multiplexing. The biological sample processing device 2 is configured to inject reagents into the microfluidic chamber 40 at a pressure higher than atmospheric pressure, in order to heat the reagents injected into the chamber 40 at a temperature equal to or higher than the boiling temperature of the reagents at atmospheric pressure. In an advantageous embodiment, the device is configured such that the pressure in the chamber is in the range of 1.5 to 5 bar.

[0049] In one embodiment of the present invention, the sample processing device 2 includes a support mechanism 4, a thermal unit 6, a microfluidic device 8, a pressurized fluid supply system 10, and a control system (not shown).

[0050] The control system includes a microprocessor that receives temperature and pressure inputs from the temperature and pressure sensors of the device 2 for automatic or user-based control of the thermal unit and pressurized fluid supply system to control the temperature and fluid pressure parameters of the device, in particular the fluids and reagents injected into the microfluidic chamber 40.

[0051] The sample processing device 2 houses therein a biological sample support 12. The biological sample support may for example be in the form of a glass slide for visualization under a microscope, as commonly known per se for the microscopic examination of tissue samples and other biological samples.

[0052] The microfluidic device 8 is attached to the sample support 12 in a closed position, as best seen in Figure 6a, forming a microfluidic chamber 40 therebetween. The microfluidic device includes a seal 42 configured to completely enclose the chamber 40, such that when the sample support 12 is secured to the microfluidic device 8 under a constant clamping force F, the seal 42 is compressed between the sample support 12 and the microfluidic device 8 to a degree sufficient to withstand a pressure of at least greater than 1.5 bar, and in some embodiments, greater than 5 bar. Reagents injected into the chamber 40 can thus be provided under pressures ranging from 1.5 to 5 bar or more during the antigen retrieval process, to allow the reagents to be heated above their boiling point at atmospheric pressure.

[0053] The microfluidic device 8 is in one embodiment in the form of a substantially planar substrate 9 of optically transparent material. The substrate includes therein a microfluidic inlet channel network 36 connected to a fluid inlet orifice 32, and a microfluidic outlet channel network 38 connected to a fluid outlet orifice 34. Both the fluid inlet orifice 32 and the fluid outlet orifice 34 open onto a first side 33 of the plate corresponding to a chamber boundary side of the substrate 9.

[0054] The microfluidic inlet channel network 36 feeds into a plurality of orifices 37 extending along the inlet edge of the chamber 40 through which reagents flow into the chamber. The microfluidic outlet channel network 38 feeds into a plurality of orifices 39 extending along the outlet edge of the chamber 40 through which reagents flow out of the chamber. The microfluidic inlet and outlet channel networks 36, 38 are formed in the substrate and configured to interconnect the single inlet 32 ​​to the plurality of chamber inlet orifices 37 and the single outlet to the plurality of chamber outlet orifices 39, respectively.

[0055] According to a particular embodiment, the microfluidic chamber 40 is configured for advective transport of fluids inside said microfluidic chamber 40. The height of the chamber between the first surface 33 and the sample support 12 is preferably less than 100 μm in order to allow and ensure advective transport of reagents along the biological tissue sample 1 placed on the sample support. Advective transport provides an optimal exchange between the reagents and the tissue sample. The low chamber height also ensures a minimum volume in the chamber 40 that allows the chamber 40 to be heated very quickly and, if necessary, cooled quickly or at a desired controlled cooling rate, thus reducing the time for antigen retrieval at high temperatures.

[0056] The seal 42 may be in the form of a gasket seal partially embedded in corresponding grooves in the substrate 9 surrounding the chamber 40 , the chamber inlet orifice 37 , and the chamber exit orifice 39 .

[0057] The support mechanism 4 includes a base structure 14 and a clamping mechanism 16, which includes a clamping plate 18 actuated by an actuating lever mechanism 22. As shown in FIG. 1d and FIG. 1f, the clamping plate 18 is in an up and open position. The sample support 12 can be placed on the base 14 with the biological sample 1 facing upwards. The microfluidic device 8 can be placed on top of the sample support 12 with the chamber side 33 and the seal 42 facing the sample support 12 and facing downwards over the biological sample. The clamping plate 18 can be provided with a window 20 to allow the biological sample 1 to be viewed through the transparent substrate 9 under a microscope. A spacer plate 19, for example in a transparent polymer or glass, can be provided at the bottom of the clamping plate to act as an interface between the clamping plate 18 and the microfluidic device 12.

[0058] The pressurized fluid supply system 10 includes a fluid supply channel 44 and a fluid outlet channel 46, both of which include a seal 48 at their orifice ends facing the clamping plate 18. The seal 48 may be in the form of, for example, a small gasket or O-ring that fits partially into a corresponding groove in the base 14. When the device is closed, the seals are compressed against the chamber-facing side 33 of the substrate 9 at locations around the fluid inlet orifice 32, the fluid outlet orifice 34 of the microfluidic device 8, respectively. Thus, when the microfluidic device is placed on top of the sample support 12, the seal 42 of the microfluidic device is compressed against the sample support 12, and the seal 48 of the fluid supply system is compressed between the substrate 9 and the base 14. This compression and sealing arrangement is configured to support a pressure of at least 1.5 bar, and preferably in the range of 2 to 5 bar or more.

[0059] The clamping mechanism 16 has a hinge linkage 17 with a lever arm effect which allows a high clamping force, for example in the range of 30 to 500 Newtons, to be applied on the substrate 9 of the microfluidic network device 8. The articulation mechanisms used in the various techniques capable of providing such an effect are well known per se and need not be described in more detail. Within the scope of the present invention, a variety of clamping means may be used which are configured to generate the required force to secure the microfluidic device relative to the base and sample support.

[0060] The fluid inlets and outlets 44, 46 may be connected to tubing connected to the underside of the base structure 14 and connected to a fluid supply and waste system (not shown), as best shown in Figure 1g. The fluid supply and waste system includes a pump system connected to a supply of required reagents, the pump system configured to deliver reagents to the biological sample processing device 2 at a pressure greater than 1.5 bar, preferably greater than 2-5 bar or greater.

[0061] The thermal unit 6 is attached to the base structure 14 below the location of the sample support 12. The thermal unit includes a heating unit 26 which may advantageously include a Peltier element or a number of Peltier elements, for example a pair of stacked Peltier elements. However, the heating unit 26 may in variants include other heater devices, such as an induction coil or a resistive electric heater. The heating unit 26 may optionally be attached to a heat transfer unit 24 which conducts heat to the sample support 12 and is arranged directly below the sample support 12, as shown in the illustrated embodiment. The heating unit 26 or the heat transfer unit 24 may be in direct contact with the sample support 12 or separated therefrom by a small gap. In the illustrated embodiment, the Peltier element 26 is attached to the heat transfer unit 24 which forms an essentially conductive block, for example made of metal, which conducts heat to the sample support 12. A passage 25 or a number of passages may be provided in the heat transfer unit 24 between the heating unit 26 and the sample support 12 for heat transfer by conduction and radiation. The passage 25 may alternatively or additionally serve to mount therein a temperature sensor of a temperature control system for controlling the operation of the thermal unit 6, and in particular the heating unit 26.

[0062] The thermal unit 6 may optionally further comprise a cooling unit 28, for example in the form of a cooling block 54 including a labyrinth of fluid flow channels 56 through which a cooling fluid flows, to provide active cooling of the sample support 12 at the end of the antigen retrieval step at elevated temperature. However, depending on the variant, active cooling may not be required and passive cooling may be performed, for example, by omission of the cooling unit 28 or by air cooling of the cooling unit 28 without a cooling fluid passing through the cooling unit block. The thermal unit may be fixed to the base 14 by a fixed mounting structure or by a clamping device 50.

[0063] The clamping device may advantageously be provided with an adjustable piston mechanism 51. The piston mechanism may be mechanically or hydraulically or pneumatically actuated. In a preferred embodiment, the piston mechanism is actuated by compressed gas, e.g. compressed air, injected under the piston 51 via an inlet 53. The piston mechanism 51 serves to press the sample support 12 from below against the microfluidic device 8 to improve the clamping and fluidic sealing by the seal 42 compressed between the microfluidic device 8 and the sample support 12, as well as allowing automatic compensation of any misalignment / unflatness between the sample support 12 and the microfluidic device 8, preventing leakage under high pressure (usually above 3 bar) which may be implemented in the present invention. The piston mechanism may also serve to adjust the pressure of the heating unit 26 and the cooling unit relative to the base 14. Compressed gas actuation of the piston mechanism advantageously provides a stable and precisely controlled biasing force of the sample support 12 against the microfluidic device 8.

[0064] The biological sample processing device 2 may further comprise one or more temperature sensors integrated in the base structure 14 (e.g. in the passage 25) and / or in the clamping plate 18, configured to measure the temperature at the sample support 12 or the microfluidic device 8 and / or at the heating unit 26 and / or the heat transfer unit 24. The temperature sensors may comprise various sensors well known per se in the art of temperature measurement, for example by optical detectors, infrared detectors. In a preferred embodiment, the temperature sensor is a resistive sensor. The temperature sensor may be connected to the fluid outlet 44. The one or more temperature sensors may form part of a temperature control system configured to control, inter alia, the operation of the thermal unit during the antigen retrieval processing step.

[0065] In one embodiment, the temperature control system and the thermal unit 6 are connected to an automatic control system. The control system can include any useful combination of feedback elements known in the art, such as proportional, integral, and derivative feedback elements that control the pump and valve system for the delivery of reagents and the thermal unit 6 for heating and cooling operations as a function of specified process parameters. In one embodiment, the automatic control system can include, for example, commercially available electronic PID controllers, and the parameters of the automatic control system can be adjusted using computer software.

[0066] Referring to the drawings, and in particular to FIG. 2, a method for in situ temperature induced antigen retrieval of a sample immobilized on a sample support, according to one embodiment of the present invention, comprises: (i) providing a sample 1 fixed on a sample support 12; (ii) providing a microfluidic device 8 comprising a microfluidic chamber 40, at least one fluid inlet 32 ​​at one end of the microfluidic chamber and at least one fluid outlet 34 at the other end of the microfluidic chamber, the device being configured to direct a fluid supplied under pressure from a fluid supply system 10 through the microfluidic chamber 40 for transporting fluidic substances and reagents within said microfluidic chamber in a uniform manner, wherein at least one wall of the microfluidic chamber is formed by a sample support 12 and is attached to an other wall 33 of the microfluidic chamber 40 by a clamping mechanism 16 in a fluid-tight and removably manner against a seal 42, the volume of the microfluidic chamber being between 2.5 μL and 200 μL; (iii) mounting the microfluidic chamber and the sample support together in a fluid-tight manner, with the sample 1 facing the inside of the microfluidic chamber 40; (iv) carrying out a temperature-controlled antigen retrieval step S1 at a pressure higher than atmospheric pressure, which step comprises: - pressure P in the microfluidic chamber 40 comprised between 1.5 and 5 barchamber filling the microfluidic chamber 40 with an epitope unmasking solution via the fluid inlet 32 ​​at - Set the microfluidic chamber to the incubation temperature set point (T AR ) and incubation duration t i maintaining the temperature of the microfluidic chamber at said incubation temperature set point during said incubation; - Cooling duration t c cooling the microfluidic chamber to a cooling temperature below the incubation temperature set point and above room temperature during said incubation period; and the incubation temperature set point T AR is comprised between about 60°C and 200°C, and the incubation duration t i is comprised between about 0.5 and 30 minutes, and the cooling duration t c The steps include between about 1 and 30 minutes. It may advantageously comprise:

[0067] According to a further aspect, there is provided a method for in situ temperature induced antigen retrieval of a sample fixed on a sample support according to the invention, said fixed sample being subsequently stained, in particular immunostained and imaged, on the same sample support.

[0068] According to a further aspect, there is provided a method for in situ temperature-induced antigen retrieval of a sample immobilized on a sample support according to the invention, which method comprises, after a temperature-controlled antigen retrieval step S1: (v) sequentially injecting a plurality of reagents, including at least one imaging probe, into the microfluidic chamber via the fluid inlet at a flow rate ranging between about 1 μl / s and about 100 μl / s; (vi) imaging signals emitted by components of the sample that have reacted with the at least one imaging probe (S4); Further comprising: The step of sequentially injecting a plurality of reagents includes: - an elution step (S2, S2') during which an elution buffer is injected to remove unwanted substances, such as antibodies, that may remain on the sample from the previous cycle; - a sample labeling step (S3, including S3', S3'', etc.) in which an imaging probe is injected; Including, Each of these steps is preceded by an optional wash step in which a wash buffer is injected.

[0069] According to another further aspect there is provided a method for in situ temperature induced antigen retrieval of a sample immobilised on a sample support according to the invention, comprising, after step (vi), (vii) an elution step (S0) in which an elution buffer is injected to remove undesired substances such as labeled probes (e.g. antibodies or markers) that may remain on the sample; (vi) repeating steps (iv) and (vii) with different imaging probes; Further includes:

[0070] According to one embodiment, lowering the temperature of the microfluidic chamber to a temperature below the incubation temperature set point can be achieved by turning off the heating unit 24 (natural or passive cooling) or by forced or active cooling by the cooling unit 28, for example by flowing cooling fluid through the channels 56 of the cooling block 54. The flow rate of the cooling fluid can be used to control the degree of cooling. The cooling unit 28 reduces the temperature of the microfluidic chamber below the cooling temperature set point (T) during the cooling period according to a temperature reduction profile. c ) can be controlled by setting the temperature control system, i.e., by setting the cooling time, cooling step size, and slope (active cooling). The cooling period can be controlled by setting the initial and subsequent cooling temperature setpoints (T c In other embodiments, the cooling period may be of a discrete nature, including a period of time from a first cooling temperature set point (T c)

[0071] In one embodiment, the first cooling temperature set point value (T c ) is selected from the range between 40° C. and 200° C., said cooling temperature set point value being lower than the incubation temperature set point, in particular from about 40° C. to about 60° C. For example, it may be about 50° C., 60° C., 70° C., 80° C., 90° C., 100° C., 120° C., 140° C., 160° C., 180° C. or 200° C.

[0072] In one embodiment, the internal pressure value of the microfluidic chamber (P chamber ) is comprised between about 2 and 5 bar, such as 2, 2.5, 3, 3.5, 4, 4.5 or 5 bar. In a preferred embodiment, it is between 1.5 and 3 bar, in particular between 2 and 3 bar.

[0073] In another embodiment, the incubation temperature set point value (T AR ) is comprised between about 60°C and about 200°C. For example, it can be about 60°C, 70°C, 80°C, 90°C, 95°C, 100°C, 110°C, 115°C, 120°C, 140°C, 160°C, 180°C or 200°C. In a preferred embodiment, it is between 110°C and 140°C.

[0074] In another embodiment, the duration of the incubation period (t i ) is usually between 2 and 20 minutes, particularly 2 to 5 or 5 to 10 minutes. For example, it may be about 2 minutes, 2.5 minutes, 4 minutes, 5 minutes, 10 minutes or 20 minutes. If the same desired effect can be achieved so that the overall sample processing time is kept low, t i It is advantageous to keep this as short as possible.

[0075] In another embodiment, the pH value of the epitope unmasking solution is higher than 5.5. For example, the epitope unmasking solution can have a pH of 6 or a pH of 9. Optimal AR conditions can be obtained with different buffer compositions. For example, a sodium citrate buffer with Tween 20 at a pH of 6 or a Tris-EDTA buffer with a pH of 9 can be used. In another embodiment, more acidic solutions (e.g., pH less than 5) can also be used as the epitope unmasking solution.

[0076] In one embodiment, the imaging probe is a labeled probe suitable for interacting with a specific molecular entity on the sample. For example, the imaging probe can be a labeled RNA or DNA sequence useful for in situ hybridization with an RNA or DNA sequence (complementary sequence) from the sample. In another example, the imaging probe is a labeled primary antibody (e.g., fluorescent) that directly binds the target antigen.

[0077] In another embodiment, the imaging probe results from the injection of a series of labeled probes, such as specific antibodies and chromogenic or fluorescent detection molecules, that target the molecular entities to be analyzed within the sample. In one embodiment, the imaging probe results from a labeled secondary (e.g. fluorescent) antibody that is injected after the primary antibody.

[0078] According to one particular embodiment, the flow rate of the injected reagents ranges from about 1 μl / s to about 30 μl / s, such as from about 5 μl / s to about 30 μl / s (eg, about 25 μl / s).

[0079] According to another particular embodiment, the height of the microfluidic chamber, as defined by the distance from the wall of the sample support 12 to the opposite wall 33 of the microfluidic chamber, ranges from about 10 μm to about 300 μm, and the diagonal or diameter of the microfluidic chamber ranges from about 100 μm to about 56 mm, forming a shallow and wide shape.

[0080] In another embodiment, each step in the sequence of multiple injected reagents is applied for a period of time necessary to flush the previous solution in the solution flow step sequence from the microfluidic chamber, where this flushing corresponds to a decrease in the concentration of the previous solution to 1% of the previously injected concentration.

[0081] In another embodiment, each step in the sequence of injected reagents is applied for a period of time necessary to increase the concentration of the injected solution to 99% of the intended protocol concentration in the microfluidic chamber.

[0082] In one embodiment, each step in the sequence of injected reagents lasts from about 1 second to about 120 seconds, such as from about 5 seconds to about 20 seconds (eg, about 10 seconds).

[0083] In another particular embodiment, the step of sequentially injecting a plurality of reagents comprises: - an optional blocking step (S2') during which a blocking buffer is injected; - an optional incubation step in which the previously injected blocking buffer is incubated with or without any flow conditions; - an optional washing step during which a washing buffer is injected; - a sample labeling step (S3) in which an imaging probe is injected; - an optional incubation step in which the previously injected imaging probe is incubated with or without any flow conditions; - a washing step (S3a) during which a washing buffer is injected; - an optional pre-imaging step (S4') during which an imaging buffer is injected; Including, The sample labeling step involves injecting either a labeling probe directly or a series of labeling probes leading to an imaging probe.

[0084] According to one particular embodiment, the sample labelling step S3 comprises a series of steps of injecting a labelled probe leading to an imaging probe including a first step (S3') in which a primary antibody is injected, a washing step (S3''') in which a washing buffer is injected and a further step (S3'') in which a labelled secondary antibody is injected.

[0085] In one particular embodiment, the sample labelling step S3 comprises a first step (SB3') in which a primary antibody is injected, a wash step (SB3''') in which a wash buffer is injected, a second step (SB3'') in which an enzyme-linked secondary antibody is injected, a wash step (SB3'''') in which a wash buffer is injected and a further step SB3* in which a chromogenic or fluorescent detection molecule that reacts with the enzyme linked to the secondary antibody is injected.

[0086] In another particular embodiment, the sample labelling step comprises a first step (SB3') in which a primary antibody is injected, a wash step (SC3''') in which a wash buffer is injected, a second step in which a post-primary antibody is injected, a wash step (SC3'') in which a wash buffer is injected, a third step (SC3'''') in which an enzyme-linked secondary antibody is injected, a wash step (SC3*) in which a wash buffer is injected and a further step SC3** in which a chromogenic or fluorescent detection molecule that reacts with the enzyme linked to the secondary antibody is injected.

[0087] In another particular embodiment, the sample labeling step comprises injecting at least one labeled probe for in situ hybridization with any DNA / RNA material within the sample, such as a labeled RNA or DNA probe.

[0088] In a further particular embodiment, when the sample labeling step comprises injecting at least one labeled probe for in situ hybridization with any DNA / RNA material in the sample, the method of the invention further comprises applying temperature cycles in the microfluidic chamber required for hybridization and dehybridization steps of the RNA or DNA probe (complementary sequence) with any DNA / RNA material in the sample. For example, a heater external to the microfluidic chamber or the sample support can apply such temperature cycles. In situ hybridization can be achieved, for example, as defined in Modern Pathology, 2011, 24, 613-623, doi:10.1038 / modpathol.2010.228. Imaging can then be achieved on the immobilized hybridization probe (labeled complementary sequence probe) for RNA and DNA sequence detection.

[0089] In one particular embodiment, each step in a sequence of multiple reagents to be injected includes two flow rate steps for each reagent: - a first flow rate step in which the reagent is injected at an initial flow rate in the range between about 1 μl / s and about 100 μl / s; - a second flow rate step in which the same reagent is injected at a lower flow rate (usually about 0.001 to about 1.0 μl / s) before injecting the next reagent in the sequence to ensure sufficient flow of that reagent with the sample; Includes.

[0090] In a further particular embodiment, the second flow step of the reagent lasts from about 1 minute to about 30 minutes (eg, from about 2 to about 15 minutes).

[0091] According to one particular embodiment, the duration of the second flow step depends on the volume of the microfluidic chamber 40 used and the time required for incubation of the reagent with the sample. For typical primary antibody molecules, a calculated incubation time of about 1 minute is required for a chamber height of less than 100 μm.

[0092] In one embodiment, the imaging step (vi) is carried out by confocal fluorescence microscopy.

[0093] In one embodiment, the imaging step (vi) is carried out by fluorescence microscopy.

[0094] In one embodiment, the imaging step (vi) is carried out by bright field microscopy.

[0095] According to one particular embodiment, the washing buffer is selected from phosphate buffered saline (PBS) and Tris buffered saline (TBS).

[0096] According to one particular embodiment, the elution buffer is selected from solutions with low pH (e.g. pH 2) supplemented with a detergent (TritonX). The elution buffer solution may further comprise a high ionic salt concentration (e.g. from about 0.001 M NaCl to about 1 M NaCl) or a chaotropic agent.

[0097] According to one particular embodiment, the blocking buffer is selected from sodium citrate buffer and PBS supplemented with proteins (eg bovine serum albumin or serum) and / or detergents (eg Tween).

[0098] According to one particular embodiment, the non-specific binding blocking step (S2) is optional.

[0099] According to one particular embodiment, the sample labelling step comprises a first step (S3') in which a primary antibody is injected, a washing step (S3''') in which a washing buffer is injected and a further step (S3'') in which a secondary antibody is injected.

[0100] According to a particular embodiment, the primary antibody of the present invention is a IgG antibody as described in Dabbs, Diagnostic Immunohistochemistry: theranostic and diagnostic applications, 4 th The antibody may be any suitable antibody for any immunohistochemistry and immunofluorescence assay, such as those described in the American Journal of Clinical Chemistry, vol. 13, no. 1, 2014, ISBN 978-1-4557-4461-9. For example, a suitable antibody is a mouse or rabbit anti-human immunoglobulin G or Y antibody against a clinically relevant epitope.

[0101] In another embodiment, the flow is applied in a continuous manner.

[0102] According to one particular embodiment, the method according to the invention comprises at least about 2 to 80 cycles of steps (iv) to (vii), in particular at least about 20 to 80 cycles of steps (iv) to (vii).

[0103] According to one particular embodiment, the method according to the invention comprises from 2 to about 200 cycles of steps (iv) to (vii), in particular from 2 to about 20 cycles, or from 2 to about 100 cycles.

[0104] According to one aspect, the method of the present invention allows for in situ imaging of samples with cycle multiplexing of molecular profiling on various samples, in particular biological samples including tissue sections, cell cultures, protein or nucleic acid preparations.

[0105] According to another aspect, the samples provided for analysis by the method of the invention as fixed by various types of techniques include formalin-fixed paraffin-embedded (FFPE) tissue samples, cryofixed tissue samples, cell smears, biopsy samples, and fixed cell preparations. In particular, the methods described in the invention can be applied to samples fixed by crosslinking agents, such as whole tissue samples, surgical or needle biopsies of tissue types including, but not limited to, breast, lung, tonsil, lymph node, prostate, intestine, liver or kidney. These methods can also be applied to fixed tumor samples, including biopsies from cancers, such as breast, lung, prostate, ovarian, colorectal and melanoma. These methods can also be applied to fixed samples of fluid nature, such as body fluids, such as fixed blood samples or cell smears. These methods can also be applied to samples of microbial nature, such as bacteria. Depending on the type of sample and the desired application, various types of sample preparation steps can be realized. For example, the methods described in the invention can also be applied to samples fixed by crosslinking agents that are cut into thin sections and then applied to a support, such as a microscope slide. Microscope slides suitable for use in the disclosed methods include coated or charged slides, such as polylysine-coated or gel-coated slides. The disclosed methods can also be performed with suspended samples in suspension, for example, by providing a closed chamber variant of a microfluidic device in which at least one surface is capable of immobilizing and capturing a biological target (such as a surface coated with a recognition molecule) and injecting the suspended sample into the chamber.

[0106] In this alternative, the sample immobilized on the sample support used in the method of the invention is preliminarily obtained by immobilizing a suspension sample on the surface of a sample support which has a specific affinity for the sample.

[0107] In another embodiment, the labeled probes include chemical dyes, antibodies and antibody fragments, or oligonucleotides linked to imaging probes such as in situ hybridization or amplification probes.

[0108] The above features may be combined in any suitable manner.

[0109] A notable advantage of the method of the present invention is that it removes the need to repeatedly mount and remove the sample coverslip for antigen unmasking and imaging, especially throughout each imaging cycle, which can affect sample integrity, lead to reduced reproducibility, and prevent full automation of such processes, which is also essential for reproducible labeling.

[0110] A further significant advantage of the methods of the present invention is the reduction in pre-treatment time and reagent volumes used for steps such as antigen retrieval, resulting in higher throughput with lower reagent consumption.

[0111] Apart from sample analysis, the methods according to the invention may be useful for multiplexing gene sequence detection, such as by in situ hybridization.

[0112] Other features and advantages of the invention will be apparent from the claims, detailed description, and drawings. The invention having been described, the following examples are offered by way of illustration and not by way of limitation.

[0113] (Example) Example 1 In situ antigen retrieval and immunofluorescence immunostaining with the possibility of multiplexing protocols to perform successive sample labeling cycles The method of the present invention for in situ high pressure temperature controlled antigen retrieval and immunofluorescence immunostaining of samples is carried out on formalin fixed paraffin embedded (FFPE) tissue samples in an apparatus as shown in FIG. 1 and following a protocol as shown in FIG. 2 with optional cycle multiplexing to perform successive sample labeling cycles.

[0114] a) Example of sample preparation Depending on the sample type and the desired application, different kinds of sample preparation steps can be implemented. In this example, the previously performed sample preparation steps for FFPE tissue samples are described here as a typical example.

[0115] Tissue slides of biological samples are first manually deparaffinized in Histoclear™ solution and gradually rehydrated in an EtOH series according to standard procedures. This procedure is carried out outside the microfluidic chamber of the device of the invention ("off-chip"). The sample is then ready to perform the method of the invention as shown in the exemplary device discussed above with respect to the embodiment shown in Figures 1a-1g and 6a-6b. The exemplary biological sample processing device 2 is used to perform the method of in situ antigen retrieval and immunofluorescence immunostaining ("on-chip") according to the invention, in which a sample 1 prepared, for example, under a) is fixed on a sample support 12, which is maintained relative to the microfluidic device 8 and forms a pressure-resistant fluid-tight seal around the microfluidic chamber 40 during the process as described above.

[0116] b) Example of preparing an apparatus for carrying out the method of the invention First, the microfluidic chamber 40 is filled with a solution having suitable properties as a medium for antigen retrieval (antigen retrieval solution) via a reagent delivery system of the device, which can be operated via a fluid delivery control system, which can be automated (step S1' in FIG. 2A). The pressure inside the microfluidic chamber 40 is adjusted to a pressure value above atmospheric pressure (P chamber ) (usually about 1.5-5 bar, e.g. about 2-3 bar).

[0117] Example 2 In situ high pressure temperature-induced antigen retrieval and immunofluorescence immunostaining sequence The initial in situ high pressure temperature controlled antigen retrieval step of the method of the present invention can begin after preparation of the tissue and reagents.

[0118] The temperature-controlled AR processing step (step S1″ in FIG. 2A ) is performed under high pressure (about 2 to 3 bar) via the temperature control system of the biological sample processing device 2, and the temperature in the microfluidic chamber 40 is (i) The temperature of the microfluidic chamber is controlled from room temperature to the incubation temperature set point (T AR ) during a controlled heating period; (ii) The temperature of the microfluidic chamber (T i ) is the incubation temperature set point (T AR ) an incubation period in which the (iii) a cooling period, during which the temperature of the microfluidic chamber is cooled from the incubation temperature set point to a cooling temperature set point, and the cooling of the microfluidic chamber can be either natural or active cooling, i.e., under control; and The temperature is controlled via a temperature control protocol including:

[0119] The incubation temperature set points, cooling temperature set points, heating and cooling times, temperature step sizes, and gradients depend on the type of epitope to be unmasked.

[0120] The temperature control protocol is depicted diagrammatically in FIG. 2B.

[0121] Typically, the controlled heating period (t1 to t2) lasts from about 1 to about 3 minutes, the incubation temperature set point is from about 80 to about 130° C., the incubation period (t2 to t3) lasts from about 2 to about 20 minutes, the cooling period (t3 to t4) lasts from about 1 to about 20 minutes, and the cooling temperature set point can be either room temperature for natural cooling or about 20 to about 90% lower than the incubation temperature set point. Exemplary temperature control protocols are provided under Examples 2 and 3.

[0122] An initial immunostaining / imaging sequence can be started during the cooling period or preferably immediately thereafter. The imaging reagent sequence (including washing / elution solutions, blocking solutions, labeled probe solutions, etc.) used in the method according to the invention, which is utilized to perform successive sample labeling and imaging cycles, is outlined in FIG. 2A. Such imaging reagent sequence is successively introduced into the microfluidic chamber 40 via the fluid inlet 32 ​​by the fluid supply system. When the method is used in multiplexing mode, after the first imaging step, all steps from the antigen retrieval step to the imaging step of other target markers can be repeated as detailed below.

[0123] Elution step (step S0 or S2, Figure 2A) Each cycle of the immunostaining / imaging sequence (except the first cycle starting with S2) begins with first eluting the tissue sample by flowing an elution buffer, the composition and pH conditions of which may vary depending on the analyzed sample to remove undesired substances (e.g., labeled probes such as antibodies) that may remain on the sample. For example, a 0.1 M glycine buffer at pH 2 supplemented with 0.05% TritonX surfactant can be used as the elution buffer.

[0124] Optional Non-Specific Binding Blocking Step (Step S2', Figure 2A) A blocking buffer (e.g., sodium citrate buffer or PBS-Tween containing bovine serum albumin) is then optionally flowed through the microfluidic chamber in the sequence of the imaging reagent sequence to reduce non-specific binding of proteins in subsequent steps.

[0125] Sample labeling step (step S3 (S3'-S3'') Figure 2A) The imaging probe or a labeling probe connected to the imaging probe is then introduced into a sequence of imaging reagents flowing through the microfluidic channel. For example, a sequence of labeling probes leading to a labeling probe includes a sequence of washing the sample with a wash buffer between each step while a primary antibody and then a secondary antibody (labeling probe) are flowed and incubated. The dilution ratio of the labeling probe is determined according to an optimized protocol or vendor's instructions.

[0126] Alternatively, another example of a sample labeling step includes injecting an RNA or DNA labeled probe for in situ hybridization, where the method further includes applying an appropriate temperature cycle to ensure hybridization of the RNA or DNA material in the sample with a complementary sequence of the RNA or DNA labeled probe.

[0127] Imaging step (step S4, Figure 2A) After this cycle is completed, imaging is performed.

[0128] The entire cyclic process (steps S0 to S4) can be repeated up to about 50 times with different imaging probes.

[0129] Examples of the use of the methods of the present invention are provided herein.

[0130] - Epitope activation for progesterone receptor (PR) staining of FFPE breast tumor sections Table 1 below shows an example of sample preparation (1, 2) and of subjecting a sample to one cycle of steps S1 to S4 (3-8) of the method of the invention leading to a stained image as shown in FIG.

[0131] [Table 1]

[0132] The samples are prepared as described in Table 1, the slides are washed again in PBS and soaked for 15 minutes in PBS supplemented with 0.25% Triton-X surfactant, and then the slides are inserted into an apparatus as described herein for carrying out the methods of the invention.

[0133] In this example, the duration of the entire process of the temperature controlled AR treatment step S1 is carried out under high pressure (about 2 to 3 bar) and is kept below 10 minutes as follows. (i) controlled heating of the microfluidic chamber 40 from room temperature to the incubation temperature set point in approximately 2.5 minutes; (ii) an incubation period for incubating the sample with the AR solution at the incubation temperature set point for 2 to 5 minutes; (iii) Active cooling of the microfluidic chamber 40 from the incubation temperature set point back to room temperature within about 2.5 to 7 minutes.

[0134] After a cooling period following the high pressure temperature controlled AR processing step, immunostaining / imaging of the sample is initiated by sequentially introducing a sequence of imaging reagents into the microfluidic chamber 40 via the fluid inlets by the fluid delivery system, the imaging probes being the result of injecting a series of antibodies (primary and secondary) as labeling probes as listed in Table 1.

[0135] Primary antibody binding: applied at a flow rate of 15 μl / s for 10 seconds, then incubated for 4 minutes. (S3')

[0136] Washing: PBS is applied at a flow rate of 25 μl / s for 10 seconds. (S3''')

[0137] Secondary antibody binding: applied at a flow rate of 15 μl / s for 10 seconds, then incubated for 4 minutes. (S3'')

[0138] Washing: PBS is applied at a flow rate of 25 μl / s for 10 seconds. (S3''')

[0139] Imaging: After immunofluorescence staining, slides were counterstained, mounted with a coverslip, and imaged under a microscope (S4). Images were then visualized on Zeiss Axiovision® software and analyzed with ImageJ®. Figure 3 shows images obtained using the described sample processing and immunofluorescence staining method. The DAPI channel is shown to assist in the localization of the nuclei on the sample. Then, the PR channel and the combination of the two channels are shown separately. Using the temperature control assistance method of the present invention, sample processing time was dramatically reduced as seen in this example.

[0140] - Sequential epitope retrieval and fluorescent immunostaining of multiple markers on the same specimen Using the method of the present invention, in situ high pressure temperature controlled AR treatment steps were performed on a sample (FFPE breast section) with an area co-expressing four different markers, HER2 (human epidermal growth factor receptor 2), ER (estrogen receptor), PR (progesterone receptor) and CK (cytokeratin), with target epitopes reactivated between each step under a pressure of about 2 to 3 bar according to the method of the present invention as follows:

[0141] Sample pretreatment was performed outside the microfluidic device and deparaffinization of the samples was performed as described above for breast tumor sections. Table 2 summarizes the specific protocols of the methods used.

[0142] However, in this example, an initial temperature-controlled AR treatment step was performed prior to staining outside the microfluidic device following standard procedures in a 95°C heat bath and 40 min incubation ("off-chip") to investigate the effect of a temperature-controlled AR treatment step alone between staining cycles to reactivate epitopes.

[0143] Repetitive immunostaining of the samples was then performed on the samples after temperature-controlled AR treatment by 4 cycles of tyramide signal amplification (TSA)-based fluorescent immunostaining, which was performed in situ in the microfluidic device on the samples subjected to the method of the present invention under the conditions listed in Table 2 below. After the fourth biomarker was stained, the samples were counterstained with SlowFade Gold™ for visualization of cell nuclei.

[0144] [Table 2]

[0145] [Table 3]

[0146] [Table 4]

[0147] Images were then acquired on a confocal microscope in four separate channels corresponding to the wavelengths of the imaging probe, 350, 488, 594 and 647 nm.

[0148] It has been found that the intermediate antigen retrieval treatment method of the present invention enables the next staining cycle to be performed in a short time, thereby shortening each cycle to less than about 10 minutes, while achieving high-performance multiplexed labeling of samples.

[0149] Example 3 In situ antigen retrieval and fluorescent immunostaining of FFPE breast tumor sections for HER2 The antigen retrieval method of the present invention was used for HER2 epitope activation from FFPE breast sections fixed on standard slides according to two different protocols as follows.

[0150] The samples were pretreated as in Example 2 (deparaffinization and stepwise rehydration in EtOH). Then, an AR solution at a pH of 6 (ThermoScientific L pH 6) was filled into the microfluidic chamber and two different temperature control protocols were applied to the temperature control element as seen in Figure 4a and Figure 4b. In the first protocol, the AR solution was incubated on the sample at 105 °C for 5 to 10 min. The chamber was then gradually cooled down to 85 °C in 1 min steps of 3 °C using the temperature control system, followed by a fast active cooling step until room temperature was reached (Figure 4a). The second protocol shows another variant where the sample was incubated in the AR solution at 98 °C for 10 min, followed by a controlled 4-step cooling and a fast cooling step (Figure 4b).

[0151] Example 4 Comparison of the in situ antigen retrieval method of the present invention with standard antigen retrieval on FFPE breast tumor sections The in situ high pressure temperature controlled AR processing method of the present invention was compared to a standard AR procedure in a heat bath using FFPE breast section samples. After the AR processing step, each sample was stained for one of four different markers (HER2 (human epidermal growth factor receptor 2), ER (estrogen receptor), PR (progesterone receptor) and CK (cytokeratin), and then image quality and contrast were analyzed and compared.

[0152] The AR treatment for the control samples was carried out with an incubation time of 30 minutes in a heat bath at 95° C. Samples treated with the AR method of the present invention were subjected to the AR protocol described in Example 1.

[0153] The staining protocol was kept the same for the samples treated with the two different AR protocols: the protocol defined in Example 1 was used, with the only difference being the use of an HRP-based chromogenic detection element instead of a fluorescent secondary antibody.

[0154] FIG. 5 shows that the contrast was increased by at least a factor for each of the stainings for these different biomarkers, and thus the image quality is much higher than stainings performed with the standard method after off-chip AR, while the process duration of the present invention was substantially reduced (approximately 5 min of high temperature incubation as opposed to 30 min). [Explanation of symbols]

[0155] 1. Sample 2. Biological sample processing device 4 Support mechanism 6 Heat Unit 8. Microfluidic Devices 9 Substrate 10 Fluid Supply System 12 Sample Support 14 Base Structure 16 Clamping mechanism 17 Hinge linkage 18 Clamp plate 19 Spacer 20 Observation Passage 22 Actuating lever mechanism 24 Heat Transfer Unit 25 Passage 26 Heating unit (Peltier element) 28 Cooling Unit 30 Cooling fluid inlet / outlet 32 Fluid inlet orifice 33 First Side 34 Fluid outlet orifice 35 Second Side 36 Microfluidic Inlet Channel Network 37 Chamber inlet orifice 38 Microfluidic Outlet Channel Network 39 Chamber Exit Orifice 40 Microfluidic Chamber 42 Seal 44 Fluid Supply Channel 46 Fluid Outlet Channels 48 Seal 50 Mounting device 51 Movable piston 53 Compressed air inlet 54 Cooling block 56 Fluid Flow Channel

Claims

1. A method for in situ temperature-induced antigen retrieval of a biological sample (1) fixed on a sample support (12), comprising: a) providing the biological sample immobilized on the sample support; b) providing a microfluidic device (8) comprising a microfluidic chamber (40), at least one fluid inlet (32) at one end of the microfluidic chamber and at least one fluid outlet (34) at an other end of the microfluidic chamber, configured to direct a fluid supplied under pressure from a pressurized fluid supply system (10) through the microfluidic chamber (40), at least one wall of the microfluidic chamber being formed by the sample support and attached in a fluid-tight and releasable manner to a seal (42) by a clamping mechanism (16) to a first wall (33) of the microfluidic chamber (40), the volume of the microfluidic chamber being between 2.5 μL and 200 μL; c) mounting the microfluidic chamber and the sample support together in a fluid-tight manner, with the biological sample (1) facing the inside of the microfluidic chamber (40); d) performing a temperature-controlled antigen retrieval step S1 at a pressure higher than atmospheric pressure, the step S1 comprising: The pressure P in the microfluidic chamber exceeds atmospheric pressure chamber filling the microfluidic chamber with an epitope unmasking solution via the fluid inlet at The microfluidic chamber is set at an incubation temperature set point (T AR ) and incubation duration t i maintaining the temperature of the microfluidic chamber at the incubation temperature set point during Cooling duration t c cooling the microfluidic chamber to a cooling temperature below the incubation temperature set point and above room temperature during the incubation period; The incubation temperature set point T AR is comprised between 60° C. and 200° C., and the pressure P chamber is greater than 1.5 bar, and the incubation duration t i is comprised between 0.5 and 30 minutes; and A method comprising: The method is carried out using a biological sample processing device (2), The biological sample processing device (2) includes a support mechanism (4), a thermal unit (6), a microfluidic device (8), a pressurized fluid supply system (10), and a control system; the microfluidic device (8) comprises a microfluidic chamber (40) having a volume comprised between 2.5 μL and 200 μL, at least one fluid inlet (32) at one end of the microfluidic chamber and at least one fluid outlet (34) at an other end of the microfluidic chamber, configured to direct a fluid through the microfluidic chamber (40) for reacting with the biological sample inside the microfluidic chamber, at least one wall of the microfluidic chamber being formed by the sample support and attached in a fluid-tight and removable manner to a first wall (33) of the microfluidic chamber (40) by a clamping mechanism (16) configured to compress a seal (42) between the sample support and the microfluidic device to a degree sufficient to accommodate a pressure Pchamber in the microfluidic chamber of greater than 1.5 bar; the thermal unit (6) is attached to a base structure (14) below the position of the sample support (12), the thermal unit comprising a heating unit (26) that conducts heat to the sample support and includes a Peltier element or a plurality of stacked Peltier elements attached to a heat transfer unit (24) located immediately below the sample support; The method, wherein the thermal unit further comprises a cooling unit (28) in the form of a cooling block (54) including fluid flow channels (56) through which a cooling fluid flows to provide active cooling of the sample support (12).

2. The cooling duration t c 2. The method according to claim 1, characterized in that: is comprised between 1 and 30 minutes.

3. The incubation temperature set point T during the temperature controlled antigen retrieval step S1 AR 3. The method according to claim 1 or 2, characterized in that the temperature is comprised between 110°C and 200°C.

4. The pressure P in the microfluidic chamber during the temperature-controlled antigen retrieval step S1 chamber 4. The method according to claim 1, wherein the pressure is between 2.5 and 5 bar.

5. The incubation duration t i 5. The method according to claim 1 , wherein the period is comprised between 2 and 20 minutes.

6. A method for in situ imaging of a sample by cycle multiplexing, comprising carrying out the method according to any one of claims 1 to 5, e) sequentially injecting any one of a plurality of reagents, including at least one imaging probe, into the microfluidic chamber via the fluid inlet at a flow rate ranging between 1 μl / s and 100 μl / s; f) imaging signals emitted by components of the sample reacted with the at least one imaging probe; g) repeating steps (e) and (f) with different imaging probes; The method of claim 1, further comprising:

7. A biological sample processing apparatus (2) for use in the method according to any one of claims 1 to 6, comprising a support mechanism (4), a thermal unit (6), a microfluidic device (8), a pressurized fluid supply system (10) and a control system, The microfluidic device (8) comprises a microfluidic chamber (40) having a volume comprised between 2.5 μL and 200 μL, at least one fluid inlet (32) at one end of the microfluidic chamber and at least one fluid outlet (34) at an other end of the microfluidic chamber, the microfluidic device being configured to direct a fluid through the microfluidic chamber (40) for reacting with the biological sample inside the microfluidic chamber, at least one wall of the microfluidic chamber being formed by the sample support, and configured to operate at a pressure P in the microfluidic chamber greater than 1.5 bar. chamber a clamping mechanism (16) configured to compress a seal (42) between the sample support and the microfluidic device sufficiently to accommodate a sample support having a first wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) configured to compress a seal (42) between the sample support and the microfluidic device sufficiently to accommodate a sample support having a first wall (33) and a second wall (33) of the microfluidic chamber (40 ... and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the microfluidic chamber (40) and a second wall (33) of the the thermal unit (6) is attached to a base structure (14) below the position of the sample support (12), the thermal unit comprising a heating unit (26) that conducts heat to the sample support and includes a Peltier element or a plurality of stacked Peltier elements attached to a heat transfer unit (24) located immediately below the sample support; 1. The biological sample processing device, characterized in that the thermal unit further comprises a cooling unit (28) in the form of a cooling block (54) including a fluid flow channel (56) through which a cooling fluid flows to provide active cooling of the sample support (12).

8. 8. The sample processing apparatus of claim 7, wherein the microfluidic device is in the form of a substantially planar substrate (9) of optically transparent material comprising therein a microfluidic inlet channel network (36) connected to an orifice of a fluid inlet (32) and a microfluidic outlet channel network (38) connected to an orifice of a fluid outlet (34), both of which open onto a first side (33) of the substrate corresponding to a chamber boundary side of the substrate.

9. 9. The sample processing device according to claim 7 or 8, characterized in that the height of the chamber between the first surface (33) and the sample support (12) is less than 100 μm configured to ensure advective transport of reagents along a biological sample (1) placed on the sample support.

10. The sample processing device according to any one of claims 7 to 9, characterized in that the support mechanism (4) comprises a base structure (14) and the clamp mechanism (16), the clamp mechanism comprising a clamp plate (18) having a window (20) allowing the biological sample to be viewed through a transparent substrate under a microscope.

11. 11. The sample processing apparatus of claim 7, wherein the microfluidic device (8) is positioned on top of the sample support (12) with the chamber side (33) and the seal (42) of the microfluidic device facing the sample support (12) and facing downwards over the biological sample (1).

12. 9. The sample processing device of claim 8, wherein the pressurized fluid supply system (10) comprises a fluid supply channel (44) and a fluid outlet channel (46) attached to a base structure, each including a seal (48) at its orifice end facing the movable clamping plate (18) of the clamping mechanism, the seals being compressed against a chamber-facing side (33) of the substrate (9) of the microfluidic device at positions around the orifices of the fluid inlet (32) and fluid outlet (34) of the microfluidic device, respectively, and the seals under compression are configured to support a pressure of at least greater than 1.5 bar.

13. The method further comprises one or more temperature sensors integrated into the base structure (14) and / or the clamping plate (18), 13. A sample processing device according to any one of claims 7 to 12, characterized in that the one or more temperature sensors form part of a temperature control system arranged to control the operation of the thermal unit.

14. 14. The sample processing device of claim 7, further comprising a clamping device including an adjustable piston mechanism (51) for pressing the sample support (12) against the microfluidic device (8) from below to improve clamping and fluid sealing by the seal (42) compressed between the microfluidic device and the sample support.

15. 15. The sample processing device of claim 14, wherein the piston mechanism is actuated by compressed gas.

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