WO2018228574A1

WO2018228574A1 - Microscope slide with integrated black and white target

Info

Publication number: WO2018228574A1
Application number: PCT/CN2018/091685
Authority: WO
Inventors: Frederick Knute Husher; Jee Jong Shum
Original assignee: Sunstone Scientific Ltd
Current assignee: Sunstone Scientific Ltd
Priority date: 2017-06-15
Filing date: 2018-06-15
Publication date: 2018-12-20
Anticipated expiration: 2019-12-15
Also published as: EP3638995A4; JP2020523646A; EP3638995A1; JP7369120B2; KR20200040746A; CN110770553A

Abstract

Provided is a microscope slide with integrated targets, which comprises a black reference target and a white reference target co-resident on the microscope slide. The microscope slide with integrated black and white target enables the digital imaging on different systems to be able to set the illumination level do that the process control target arrays and co-resident tissue can be used together and yield similar results.

Description

MICROSCOPE SLIDE WITH INTEGRATED BLACK AND WHITE TARGET

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priorities of US provisional Application number 62/520178 filed on June 15, 2017, the disclosures of which are incorporated herein by reference in its entirety.

FIELD

The present invention relates to a microscope slide with integrated targets. The present invention particularly relates to the microscope slide with integrated black and white targets. The microscope slide as disclosed in the present invention with integrated black and white target enables the digital imaging on different systems to be able to set the illumination level do that the process control target arrays and co-resident tissue can be used together and yield similar results.

BACKGROUND

In the classic fashion an imaging system is calibrated against standard white and black targets by adjusting the illumination source intensity and contrast. Both are to be non-reflective in surface finish. The accepted baseline white reference is a freshly pressed barium sulfate tablet, as established for use with the MacBeth PCM II. Black is generally provided by a void that prevents reflected light from returning to the imaging device.

The white target will be some sort of metal oxide which will stain with exposure to eosin unless the oxide surface is protected. While a black target could be printed containing a carbon compound it will have some amount of 1st surface reflection and thus not be a perfect black. However, it will be black enough relative to any staining that could occur with the biomaterials.

Many imaging systems incorporate white and black void reference targets which are used to set the illumination level prior to the introduction of the object to be imaged. While providing the widest range, the dynamic range on the slide is less than 50%of ideal.

The inconsistency for producing the same results between slides with passage of time and differing stain experience is the major disadvantage of processing imaging baseline on the microscope slide.

It is therefore the object of the present invention to integrate the black and white reference targets on the slide itself. As a result, the imaging reference is a part of the slide and can ensure close results between different imaging systems.

SUMMARY

Generally, in one aspect of the present invention discloses a microscope slide with integrated black and white targets ensures that digital imaging on different systems will be able to set the illumination level so that the process control target arrays and co-resident tissue section can be used together and yield similar results.

In another aspect, the present invention discloses that the white targets will not be perfect it will represent the whitest object on the slide.

In yet another aspect, the present invention discloses the black targets sets the dynamic range for the camera.

In still another aspect, the present invention discloses that the co-resident targets are included among the microscope slide to provide objective values by which the stain specific targets and the tissue section itself can be evaluated against.

Other aspects of the present invention are disclosed in the appended drawings and the following description.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 illustrates a slide for H&E usage that contains black and white imaging targets on the same line as the chemical targets reactive to Hematoxylin and Eosin Y stains. The black target is at the right and the white target is on the right. Three images are shown for the same slide: left is the slide before the application of tissue sections, the center has three tissue sections applied, and the right is the slide after being stained. The grayed out zones represent a paraffin shield over the targets or captured formaldehyde fixed tissue sections.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to the following detailed description of the invention, which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, conditions or parameters described and/or shown herein and that the terminology used herein is for the example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms ‘a’ , ‘an’ , and ‘the’ include the plural, and references to a particular numerical value includes at least that particular value unless the content clearly directs otherwise. Ranges may be expressed herein as from ‘about’ or ‘approximately’ another particular value, when such a range is expressed another embodiment. Also, it will be understood that unless otherwise indicated, dimensions and material characteristics stated herein are by way of example rather than limitation, and are for better understanding of sample embodiment of suitable utility, and variations outside of the stated values may also be within the scope of the invention depending upon the particular application.

This invention is not limited in application to the details of construction and the arrangement of components set forth. In the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein for the purpose of description and should not be regarded as limiting. The use of “including” , “comprising” , “having” , “containing” , “involving” , and variations thereof as well as additional items.

Reference will now be made in details to the preferred embodiments of the present invention.

Historically, the results of reference control slides are entirely subjective because there is no inclusion of black and white reference targets the target (s) are inconsistent in density, and unique in reactivity to a single stain reagent. The reference control slide at best has a sausage that contains portions containing known antigen sites. The antigen concentration is unknown so all that can come from the stained slide is that the reagents all appear to be working at enough of a level to signal the antigen presence. In the case of IHC processed slides no objective measure can be performed as because the result is the cumulative product of the antigen retrieval, primary stain reagents, and secondary stain reagents. Therefore, the reference control slide at best simply provides some comfort to the user that all appears to be working.

Digital imaging of microscope slides containing stained biomaterials is evolving to perform prescreening and potentially full diagnostic determination on the stained materials. In general, the imaging system must adjust the illumination light level so that the digital image is not in compression at either the white or black boundaries. The conventional solution is to have black and white targets located where the label is expected to be positioned. The underlying assumption is that the white and black targets represent the extremes that the slide can present. However, in doing so there is compression in the digital scale as the black is much blacker and the white much whiter than can realized by the staining of a tissue section.

In one preferred embodiment of the present invention, a microscope slide is disclosed that incorporates control and reference target standards which are co-resident with a tissue section or loose cell deposit. The reference targets and tissue section record the processing experience between tissue capture and cover slipping of the stained slide.

In another embodiment, amongst the microscope slide target arrays are co-resident black and white reference targets. The black and white reference targets have experienced that same exposure to reagents and processing as the other targets and tissue section.

In another embodiment, the black and white reference targets enable the imaging system to shift the illumination level so that the working range is expanded. Depending upon the biomaterial type the processing experience can change considerably.

In another embodiment, the different processing paths can be grouped as but not limited to ImmunoHistoChemical (IHC) , Hematoxylin and Eosin (H&E) plus special stains, Urine smear H&E plus special stains, and Papanicolaou stain (PAP) smear which is composed of five dyes in three solutions.

A pure black deposit target in general, is difficult to produce because, by definition, it must absorb all light. The classic solution is to simply provide a void that folds the light path in such a manner that a reflection cannot be returned. At best a black target will be somewhat reflective, but it is usually good enough as nothing else on the slide will be nearly so black.

In another embodiment, both the targets i.e. the black and white targets are printed paint or ink deposits which are non-reactive to any of the reagents used to process a slide. The white target in an ideal situation would be a perfect white. However, there is very little stained biomaterial usefulness that gets more than halfway from black to white. Thus, the white can be 5-10%away from perfect white and still be of useful value. The main requirement is that the white be of a metal oxide or sulfate composition that is stable with the passage of time when not left exposed to sunlight. Aluminum and Titanium oxides more than meet the requirements.

In another embodiment, the preferred solution to the above mentioned problem is disclosed. The black and white targets are both based on an anhydride based epoxy paint base that is catalyzed by direct UV light exposure at nominally 365nm. The anhydride catalyzer is composed of methyl tetrahydrophthalic anhydride and diphenyliodonium hexafluroroarsenate. Other than UV initiated, anhydrides require the addition of heat to function in catalyzing the epoxy to cross-link. The preferred UV initiated anhydride and its companion are listed, but there are other solutions possible that can be found when performing a search of anhydride producing companies. While such a paint/ink can be constructed as needed, it is usually a purchased component that has been optimized for the printing method being used. Fabrication of the paint/ink must address the difficulty in achieving good wetting between the pigment particles and the epoxy binder. Anhydride based paints (also called an ink when having low viscosity) often have the anhydride mixed in with the epoxy as the pot life can be many months in duration. To lower the viscosity would be known knowledge of those involved in the printing industry and the formulation varies depending on the surface the epoxy mixture is to applied onto and the printing method used. While heat triggered anhydride-epoxy paint/inks are commonly used, the heat necessary to initiate the reaction can potentially damage biomaterials (proteins, peptide, and chemical targets) that may be co-resident with the paint/ink.

In another embodiment, the anhydride catalyzer eliminates the unreacted amines found with an amino-silane based catalyzer that would otherwise support non-specific staining. Free amine end groups can and will capture both biomaterials and some of the special stains. Specifically, this addresses the issue of undesired staining of the white target by the slide processing reagents, in particular the staining reagents. As a free amine end group on the surface of the paint/ink, it can capture both the primary antibody and secondary stain reagents and become stained. Thus, defeating the value of having an integrated white target on the slide.

In another embodiment, the black pigment uses a carbon dust of less than 2 microns diameter while the white uses aluminum or titanium oxide beads. Titanium oxide is the preferred metal oxide for this application.

In another embodiment, it is well known that in formulating epoxy ink/paint formulations the viscosity and drying time is controlled by various short and long chain alcohols and surfactants. The choice of surfactants can leave the ink/paint reactive to the range of stains and reagents these slides can experience. The preferred formulation avoids surfactants altogether.

In another embodiment, the printing of the targets can be done by pad stamp or syringe with the syringe the preferred method as it supports better feature size control of the target deposition.

In another embodiment, the aforementioned white targets are composed of metal oxide pigments within an anhydride catalyzed epoxy. In another embodiment, the aforementioned black targets are composed of carbon pigments within a UV initiated anhydride catalyzed epoxy. In another embodiment, the anhydride catalyzer is UV initiated by direct UV light exposure. The primary advantage of using the UV initiated anhydride catalyzer is that the heat needed to initiate anhydride-epoxy reaction exceeds what the biomaterials (proteins, peptide, and chemical targets) can tolerate without damage. More importantly is the elimination of any free amines that could react with the stain reagents.

Advantages of the invention:

a. The integrated black and white targets are co-resident with a tissue section or loose cells on a microscope slide.

b. The integrated black and white targets are non-reactive to biomaterial stains.

c. The integrated black and white targets are stable through exposure to antigen retrieval activities: buffers, heat, duration and other environmental factors.

d. The integrated black and white targets are non-reactive to hydroxyl, amine, amide, hydrazide, and aromatic benzene compounds. The black/white targets will be exposed to stain reagents that can react with the end groups listed. Thus, to ensure the targets will not stain the end groups must be inherently inert, such as -CH3 or -C=O. For the most part the staining will occur by the special stains reagents that are designed to specifically bind to the end groups listed. For example: Eosin Y binding to metal oxides.

Examples

Example 1

Figure 1 illustrates an H&E usage slide that contains one line of targets, bounded on the ends by black and white pigment targets. The pigment targets are printed separately from the special stains targets. The black/white targets are printed using a modified dispensing pump. The dispensing pump applies a spit and suck cycle to the epoxy mixture fed down a hollow tube that is held to the slide surface. When the print head lifts following the spit and suck cycle the deposit remains as a dot. The balance of the chemical targets are printed at another station and the slide passed through a UV exposed tunnel, initiating the anhydride catalyzer to cure the epoxy.

The epoxy plus anhydride is purchased pre-mixed. To ensure homogeneity the epoxy mixture is stirred and then degassed. The paint/ink is purchased with the pigment already blended in. Such a paint/ink is available from a number of vendors, but in all cases the formulation is proprietary to the vendor and will likely be different from each vendor. Viscosity is adjusted as needed using the recommended thinner the vendor supplies. The white pigment is barium sulfate which avoids most chemical interactions with the special stains. This is contrast to titanium dioxide or aluminum oxide, which may react with the Eosin Y.

Claims

A microscope slide with integrated black and white reference for imaging, which comprises a black reference target and a white reference target co-resident on the microscope slide.
The microscope slide as claimed in claim 1 wherein, the black reference target and the white reference target are made of a material comprising UV initiated anhydride catalyzed epoxy paint/ink base.
The microscope slide as claimed in claim 1 wherein, the material of the black reference target is selected from carbon dust, a carbon compound, or a mixture thereof.
The microscope slide as claimed in claim 3 wherein, the carbon dust has a particle size of 0.1 to 2 micron.
The microscope slide as claimed in any one of claims 1 to 4 wherein, the white reference targets is selected from a ‘poor metals’ in the periodic chart of elements in either oxide or sulfate states.
The microscope slide as claimed in claim 5 wherein, the white reference target is selected from titanium oxide, aluminum oxide, aluminum sulfate, barium sulfate, or a mixture thereof.
The microscope slide as claimed in claim 2 wherein, the UV initiated anhydride catalyzed epoxy paint base is composed of methyl tetrahydrophthalic anhydride and diphenyliodium hexafluroroarsenate.
The microscope slide as claimed in any one of claims 1 to 7 wherein, the black reference target and the white reference target are co-resident with a tissue section or loose cells.
The microscope slide as claimed in any one of claims 1 to 7 wherein, the black reference target and the white reference target are non-reactive to biomaterial stains.
The microscope slide as claimed in any one of claims 1 to 9 wherein, the black reference target and the white reference target are stable through exposure to antigen retrieval activities, buffers, heat and duration.
The microscope slide as claimed in any one of claims 1 to 10 wherein, the black reference target and the white reference target are non-reactive to hydroxyl, amines, amide, hydrazide, and aromatic benzene compounds.
The microscope slide as claimed in any one of claims 1 to 11 wherein, the black reference target and the white reference target may be printed on the microscope slide by pad stamp, syringe, or inkjet etc.
Use of the microscope slide as claimed in any one of claims 1 to 12 for establishing illumination intensity and contrast for imaging of the same slide for IHC staining, Hematoxylin and Eosin staining, Urine Smear H&E plus special staining, and Papanicolaou stain (PAP) smear.