WO2019104438A1

WO2019104438A1 - Coated glass slide for enhanced thin tissue section adhesion

Info

Publication number: WO2019104438A1
Application number: PCT/CA2018/051527
Authority: WO
Inventors: Martin DUFRESNE; Pierre Chaurand; Dominique TRUDEL; Jean-François MASSON
Original assignee: Valorisation-Recherche, Limited Partnership; Val-Chum, Limited Partnership
Priority date: 2017-12-01
Filing date: 2018-11-30
Publication date: 2019-06-06

Abstract

It is provided a substrate such as a microscopic slide coated with a layer of metal, such as chrome, titanium or aluminum, particularly a layer of less than 5 nm, or between 3 nm and 1 nm for adhering a sample, such as a tissue sample.

Description

COATED GLASS SLIDE FOR ENHANCED THIN TISSUE

SECTION ADHESION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application Serial Number 62/593292 filed on December 1 , 2018, the content of which is herewith incorporated in its entirety

TECHNICAL FIELD

[0002] It is provided a chrome coated slides for tissue analysis.

BACKGROUND

[0003] It has been known for quite some time that certain types of formalin-fixed, paraffin embedded (FFPE) tissues tend to detach from glass slides when undergoing antigen retrieval procedure prior to immunohistochemistry (IHC). Due to this, various types of “glue” and surface modified glass slides have been developed to enhance adhesion of these tissues during antigen retrieval. As of today, glue type approaches (such as chrome alum or polylysine) and commercially available high adhesion slides (Superfrost™ Plus) still have limited adhesiveness with certain types of tissues. The same type of adhesion issues are also observed during antigen retrieval on imaging mass spectrometry (I MS) dedicated indium thin oxide (ITO)-coated slides. ITO slides are quite expensive and the ITO coating is of no use for routine IHC analysis.

[0004] When happening in a medical set-up, tissue detachment from glass slides have a major impact on the evaluation of histological samples, impairing our capacities to obtain an accurate diagnosis. Impact will range from a delay in specimen evaluation due to repeated histopathological procedures, to impossibility to obtain a diagnosis. For patients, this means delay in treatment and/or repeated procedures to obtain more tissue.

[0005] When a tissue cannot be analyzed on a slide, either more expensive methods have to be used to achieve tissue characterization (such as microsatellite instability analysis), or a second specimen needs to be obtained. This is in all case associated with a delay of the diagnostic process, eventually delaying treatment. Repeated procedures to obtain new tissue are associated with side effects and significant cost. [0006] There is thus a need to turn a very poor adhesion surface like ITO-coated glass slides and plain glass slides to a high adhesion one while maintaining optical transparency for the analysis of thin tissue sections by various biochemical methods like imaging mass spectrometry or histopathology.

SUM MARY

[0007] It is provided a substrate coated a with a layer of metal for adhering a sample.

[0008] It is further provided a method of preparing a substrate coated with a layer of metal for adhering a sample comprising the steps of depositing a metal layer on the substrate, wherein the metal layer providing the adhesion for the sample.

[0009] It is also provided a slide coated with a layer of metal for adhering a sample.

[0010] In an embodiment, the metal is chrome, titanium or aluminum.

[0011] In another embodiment, the metal is chrome.

[0012] In an embodiment, the substrate is a slide.

[0013] In another embodiment, the substrate is a microscopic slide.

[0014] In a further embodiment, the substrate is made of glass.

[0015] In another embodiment, the substrate is an indium thin oxide (ITO) coated slide.

[0016] In a further embodiment, the slide is an aminosilane slide, aplastic slide or a metal slide.

[0017] In an embodiment, the layer of metal is of less than 1 nm.

[0018] In another embodiment, the layer of mtal is of 3 nm or 1 nm.

[0019] In an embodiment, the sample is a tissue section or cells.

[0020] In another embodiment, the sample is frozen, formalin fixed, or formalin fixed and paraffin embedded. [0021] In an embodiment, the metal layer is sputtered or evaporated on said substrate.

[0022] In another embodiment, the method described herein further comprises the step of mounting the sample on the substrate coated with the layer of metal.

[0023] In an embodiment, the sample is mounted by cryosectioning followed by thaw mounting or floating the sample on water and drying.

[0024] In a further embodiment, the method described herein further comprises the step of modifying the mounted sample by matrix deposition, antigen retrieval or staining.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Reference will now be made to the accompanying drawings.

[0026] Fig. 1 illustrates a workflow for the production of chrome surfaces from any type of (transparent) substrates to sample deposition and analysis according to one embodiment.

[0027] Fig. 2 illustrates the adhesion between Superfrost™ (A) and chrome coated (B) glass slides test using FFPE endometrium tissue sections. Following antigen retrieval and the IHC procedure, 3 out of 4 sections of endometrium detached from the Superforst™ Plus slides (A) while no section detachment was observed from the chrome-coated glass slides (B).

DETAILED DESCRIPTION

[0028] In accordance with the present description, there is provided a substrate such as a microscopic slide coated with a layer of less than 3 nm of a metal for adhering a sample, such as a tissue sample.

[0029] In an embodiment, it is provided a metal coated plain glass slides for tissue analysis. The metal encompassed herein is chrome, titanium and/or aluminum.

[0030] The metal coated plain glass slides described herein were produced with two different coating thicknesses. The slide received thin tissue section of endometrium tissues, which showed problematic adhesion on commercially available high adhesion slides. After exposition to both the antigen retrieval procedures and immunohistochemistry (IHC) process (3 different IHC stains were tested), it was found as disclosed herein that the exemplified chrome coated glass slides described herein outperformed the SuperFrost™ Plus slides with regards to tissue adhesion based on the amount of the tissue sections remaining attach to the surface, without interfering with analysis.

[0031] Other types of tissues for both IHC and standard color (hematoxylin and eosin) staining were tested and it was demonstrated that chrome coated glass slides as described herein provided improved adhesion.

[0032] The chrome coated glass slides described herein provide significant increase in tissue adhesion which increase the survivability of the sample through most biochemical processes and this without any change to existing protocols.

[0033] Commonly accepted mechanism for increasing tissue adhesion to a surface is through manipulation of the electrostatic interactions that tissue sections have with surfaces. Since tissue sections carry a small negative charge (RCOC and R_aP0₄ R_b), the obvious strategy is installing a positive charge on the slide surface to increase section adhesion. The most widely used chemical modification to increase tissue adhesion is aminosilane (RNH₃ ⁺) modification. The formation of an aminosilane surface over glass greatly increases the net positive charge of the surface along with increasing its hydrophilic properties resulting in greater tissue surface interactions and thus better adhesion.

[0034] On the other hand, the generated metal surface, such a chrome, titanium or aluminum surface, doesn’t bring a positive charge (chrome and chrome oxide for example are neutral) to the system and also decreases the surface hydrophilic properties especially compared to plain glass. While a metal surface as encompassed herein does exactly the opposite of what would be expected from a modern day high adhesion surface modifier, it still increases tissue adhesion to the surface as described herein. Chrome, Titanium and Aluminum for example have very high affinity for oxygen, which is the reason why it binds so well to glass (Si0₂) and other oxide substrates such a silicon oxide, forming a thin film when in contact with oxygen. This same high affinity for oxygen in inorganic substrates explain the adhesive properties with tissue sections since oxygen is one of the prevailing atoms in biological samples (carbohydrate, phospholipids, organic acids, etc.) along with carbon and hydrogen. Most notably, the exterior and interior part of all cell is composed primarily of phospholipids which all carry a R_aP0₄ R_b functional site which can interact with the chrome layer.

[0035] It is thus provided metal coated plain glass slides with a chrome thickness bellow 1 nm since it provides better light transmission. (<1 nm to 3 nm). In an embodiment, other substrates than glass such as for example aminosilane slides, plastic slides and/or metal slides, are also encompassed.

[0036] It is thus provided metal coated glass slides. In an embodiment, the glass slides can be indium thin oxide (ITO) coated slides (commercially available). A nanometer scale (< 3 nm) of a chrome layer for example is deposited over commercially available glass substrates for pathology or imaging mass spectrometry (IMS) analyses, which drastically increases tissue adhesion to these substrates.

[0037] Both pathology and IMS rely on the deposition of a thin tissue section (3-20 pm thick) on transparent glass slides or an ITO-coated glass slide. These tissue sections can either originate from fresh frozen or formalin fix paraffin embedded (FFPE) tissue samples. In all cases, these thin tissue sections must remain attached to the substrate during the entire sample preparation and analysis. In pathology, it has been known for quite some time that certain types of FFPE tissues tend to detach from the glass slides when undergoing antigen retrieval procedure prior to immunohistochemistry (IHC). Due to this, various types of“glue” and surface modified glass slides have been developed to enhance adhesion of these tissues during antigen retrieval. As of today, glue type approaches (e.g. chrome alum or polylysin) and commercially available high adhesion slides (Superfrost™ Plus) still have limited adhesiveness with certain types of tissues. The same type of adhesion issues are also observed during antigen retrieval on IMS dedicated ITO-coated slides.

[0038] As seen in Fig. 1 , a substrate 1 (e.g. glass slide modified or not by other means) with a dimension for example of 25 mm by 75 mm (corresponding to a standard microscope format), but not limited to, is provided. A nanoscale (< 3 nm) metal layer by metal sputtering, metal evaporation or any other means of deposition is deposited 2 on the substrate 1 , producing the now modified substrate or slide 3 with a metal layer with increased adhesion properties for the analysis of biological samples such as thin tissue sections. Afterwards, a biological sample 4 is deposited on the metal coated slide 3. In an embodiment, the sample 4 can be either fresh frozen, formalin fixed, formalin fixed and paraffin embedded or even cells from fine needle aspirates to name a few. The sample can be mounted on the slide using different techniques such as cryosectioning followed by thaw mounting (fresh frozen tissue sections) of by floating the section on water and drying (FFPE tissue sections). The slide 3 with sample 4 attached to the metal surface 5 is ready to be used in a biochemical workflow. The sample attached to the metal surface 5 can further be modified 6 through various biochemical approaches such as matrix deposition for imaging MALDI, antigen retrieval towards IHC or staining for optical observation. The sample is now ready for analysis 7.

[0039] A high adhesion microscopic slide compatible with both pathology and IMS is thus provided. This new high adhesion plate consists in a nanometer scale (< 3 nm) chrome layer, which can either be sputtered or evaporated (other metal deposition approach will also work) on any type of substrate such as glass and ITO-coated slides for the analysis of thin tissue sections. Chrome nanolayers was tested and is proposed herein for tissue adhesion even though chrome nanolayers lower surface hydrophilicity and do not incorporate net positive charges to the substrate.

[0040] As proposed herein, a metal such as chrome, titanium or aluminum is successfully used for adhering thin tissue sections, which are composed of a wide variety of organic compounds while retaining transparency of the substrate. A known low adhesion FFPE tissue section was initially tested, more particularly from an endometrium tissue sample, with chrome-coated slides compared and adhesion was compared to a commercially available high adhesion surface modified glass slides (Superfrost™ Plus). A standard IHC approach was carried out for which one of the crucial steps is antigen retrieval. Fig. 2 shows the raw results of this test where 3 out of 4 tissue sections peeled off the Superfrost™ Plus slides while all 5 sections deposited on either a =1 nm or =3 nm chrome-coated slides have remained attached to the surface with minimal alteration enabling the histological analysis of the sample..

[0041] Accordingly, it is provided a method for replacing widely used adhesion methods such as glue type approaches (e.g. Chrome Alum glue) or surface modified approaches (e.g. amine modified glass surfaces for increasing hydrophilicity such as Superfrost™ Plus, or polylysine coating).

[0042] Typical surface modification methods did not provide the necessary adhesion enhancement needed for samples. These methods rely on chemical modification of the surface to make it more polar and/or positively charged. The increase in adhesion comes from an enhancement in the interaction with the now positively charged surface and the negatively charged tissue section.

[0043] The metal coated slides as proposed herein increase adhesion properties and provide the ability to analyze tissue types with very few loss (< 10% tissue loss) which can detach from commercially available surfaces (in some cases > 80% tissue loss) or increase sample turn over by reducing sample preparation time due to shortening of drying step of the sections on the slides and reduction of sample loss.

[0044] The reduction of sample preparation time as proposed herein is thus an advantage for histological diagnostic compared to currently used fixing or attaching techniques where drying time of the sections on the slides can be as long as a several days for reluctant samples with no guaranties of remaining attached to the surfaces during further sample processing.

[0045] The most common commercial approach is the use of chemically modified glass by aminosilane to introduce a net positive charge to the surface which increases adhesion of biological samples (thin tissue sections) by increasing the electrostatic interactions. This happens because tissue sections (most biological samples) are slightly negatively charged and thus using a positively charge surface will increase attraction and adhesion. Other methods are also used like glue type approach (chrome alum glue) or by polymerizing amino compounds such as lysine over the surface to increase its positive charge.

[0046] Using these methods, tissue still detach from the glass slides, and consequently the standard drying step (once the tissue is deposited on the slide, it is dried for a few hours before actual tissue processing occurs) is gradually increased up to one week to favor adhesion, without warranty of success.

[0047] This new high tissue adhesion slide described herein can be used for example by any histopathology laboratory (research or service) which currently uses positively charged slides such as the SuperFrost™ Plus, or any other aminosilane modified plates. Pathology labs are found in numerous institutions such as hospitals for patient diagnostic establishment, universities for research purposes and even biotechnology (pharmaceutical) companies which work with animals.

[0048] More particularly, the high tissue adhesion slide described herein can be used in the field of imaging mass spectrometry where tissue sections are also analyzed. Any other fields of research which uses tissue samples or other biological samples such as cell cultures or even single cell analysis could take advantage of this new high adhesion slide.

[0049] Accordingly, the high tissue adhesion slide described herein is intended to replace the use of commercial glues for tissue adhesion to surfaces like the chrome alum glue, and/or replace poly-lysine adhesive.

[0050] In an embodiment, the metal coated slide encompassed herein can be manufactured alongside plain ITO-coated slides for MALDI imaging analysis of poorly adhesive samples. In the case of IMS, there are very little alternatives to ITO-coated slides aside from glues and poly-lysine when a tissue is non-adhesive.

[0051] Accordingly, the metal coated slides described herein provide a mean for high adhesion of tissue sections or other types of biological samples compared to commercially available high adhesion slides. The slides described herein have an infinite shelf life, good light transmission in the visible spectrum, do not require any modification of current biochemical/histological procedures, and are inexpensive

[0052] While the description has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations, including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A substrate coated with a layer of metal for adhering a sample.

2. The substrate of claim 1 , wherein the substrate is a slide.

3. The substrate of claim 1 or 2, wherein said substrate is a microscopic slide.

4. The substrate of any one of claims 1-3, wherein said metal is chrome, titanium or aluminum.

5. The substrate of any one of claims 1-3, wherein said metal is chrome.

6. The substrate of any one of claims 1-5, wherein said substrate is made of glass.

7. The substrate of any one of claims 1-5, wherein said substrate is an indium thin oxide (ITO) coated slide.

8. The substrate of claim 2, wherein the slide is an aminosilane slide, aplastic slide or a metal slide.

9. The substrate of any one of claims 1-8, wherein the layer of metal is of less than 5 nm.

10. The substrate of any one of claims 1-8, wherein the layer of chrome is of less than 3 nm.

11. The substrate of any one of claims 1-8, wherein the layer of chrome is of 3 nm or 1 nm.

12. The substrate of any one of claims 1-11 , wherein the sample is a tissue or cells.

13. The substrate of any one of claims 1-12, wherein the sample is frozen, formalin fixed, or formalin fixed and paraffin embedded. - I Q -

14. A method of preparing a substrate coated with a layer of metal for adhering a sample comprising the steps of depositing a metal layer on the substrate, wherein the metal layer providing the adhesion for the sample.

15. The method of claim 14, wherein the metal layer is sputtered or evaporated on said substrate.

16. The method of claim 14 or 15, wherein said metal is chrome, titanium or aluminum.

17. The metal of claim 14 or 15, wherein said metal is chrome.

18. The method of any one of claims 14-17, wherein the layer of metal is of less than 5 nm.

19. The method of any one of claims 14-17, wherein the layer of metal is of less than 3 nm.

20. The method of any one of claims 14-17, wherein the layer of metal is of 3 nm or 1 nm.

21. The method of any one of claims 14-20, further comprising the step of mounting the sample on the substrate coated with the layer of metal.

22. The method of claim 21 , wherein the sample is mounted by cryosectioning followed by thaw mounting or floating the sample on water and drying.

23. The method of claim 21 or 22, further comprising the step of modifying the mounted sample by staining, antigen retrieval, enzymatic digestion, matrix deposition, in-situ hybridization or immunochemistry.

24. The method of any one of claims 14-23, wherein the substrate is a slide.

25. The method of any one of claims 14-24, wherein said substrate is a microscopic slide.

26. The method of any one of claims 14-25, wherein said substrate is made of glass.

27. The method of any one of claims 14-25, wherein said substrate is an indium thin oxide (ITO) coated slide.

28. The method of claim 24, wherein the slide is an aminosilane slide, aplastic slide or a metal slide.

29. The method of any one of claims 14-28, wherein the sample is a tissue or cells.

30. The method of any one of claims 14-29, wherein the sample is frozen, formalin fixed, or formalin fixed and paraffin embedded.

31. A slide coated with a layer of metal for adhering a sample.

32. The slide of claim 31 , wherein said slide is a microscopic slide.

33. The slide of claim 31 or 32, wherein said metal is chrome, titanium or aluminum.

34. The slide any one of claims 31-33, wherein said metal is chrome.

35. The slide of any one of claims 31-34, wherein said slide is made of glass.

36. The slide of any one of claims 31-34, wherein said slide is an indium thin oxide

(ITO) coated slide.

37. The slide of any one of claims 31-34, wherein the slide is an aminosilane slide, aplastic slide or a metal slide.