JP7369120B2 - Microscope slide with integrated black and white targets - Google Patents

Description

[Cross reference to related applications]
This application claims priority to U.S. Provisional Patent Application No. 62/520,178, filed June 15, 2017, the disclosure of which is incorporated herein by reference in its entirety. form part of the statement.

The present invention relates to microscope slides with integrated targets. The invention particularly relates to a microscope slide with integrated black and white targets. The microscope slide with integrated black and white targets disclosed in the present invention allows for the simultaneous use of process control target arrays and co-located tissues to achieve similar results in digital imaging in a variety of systems. , the irradiation level can be set.

In conventional methods, imaging systems are calibrated to standard white and black targets by adjusting the intensity and contrast of the illuminating light source. Both targets need to be non-reflective in surface finish. As a baseline white reference, freshly pressed barium sulfate tablets, such as those established for use with MacBeth PCM II, are available. A black reference is typically provided by a void that prevents reflected light from returning to the imaging device.

This white target is some type of metal oxide that can stain upon exposure to eosin if the oxide surface is not protected. A black target containing carbon compounds can be printed, but it will not be completely black because there will be some first surface reflection. However, it is sufficiently black for any staining that may occur with biomaterials.

Many imaging systems incorporate a white reference target and a black void reference target that are used to set the illumination level prior to the introduction of the imaged object. The dynamic range of the slide, even at maximum range, is less than 50% of the ideal value.

The lack of consistent results from slide to slide over time and different staining experiences is a significant disadvantage in imaging baseline processing of microscope slides.

The invention therefore aims to integrate a black reference target and a white reference target into the slide itself. As a result, the imaging reference becomes part of the slide, ensuring similar results between different imaging systems.

In general, in one aspect of the present invention, a microscope slide with integrated black and white targets is disclosed that allows for the use of process control target arrays and co-located targets in digital imaging in various systems. It is ensured that the irradiation level can be set so that tissue sections can be used simultaneously to achieve similar results.

In another aspect of the invention, it is disclosed that the white target is not completely white, but represents the whitest object on the slide.

In yet another aspect of the invention, it is disclosed that the black target sets the dynamic range of the camera.

In yet another aspect of the invention, it is disclosed that co-located targets are incorporated into microscope slides to provide objective values by which staining specific targets and tissue sections themselves can be evaluated.

a. Integrated black and white targets coexist with tissue sections or free 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 against exposure to antigen retrieval activities, buffers, heat, time, 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 target/white target is exposed to a staining reagent capable of reacting with the illustrated end groups. Thus, the terminal groups such as -CH ₃ or -C=O must be essentially inert to avoid staining the target. In most cases, staining occurs with specific staining reagents designed to specifically bind to the exemplified end groups. For example, eosin Y binds to metal oxides.

Other aspects of the invention are disclosed in the accompanying drawings and the description below.

FIG. 2 is an image of an H&E slide containing a black imaging target and a white imaging target collinear with a chemical target reactive to hematoxylin and eosin Y stain. The black target is on the right side and the white target is on the right side and above. Three images are shown for the same slide. The left side is an image of the slide before application of tissue sections, the center is an image with three tissue sections applied, and the right side is an image of the slide after staining. Areas shown in gray indicate captured tissue sections fixed in paraffin shield or formaldehyde on the target.

The invention may be more readily understood by reference to the following detailed description of the invention, which forms a part of this disclosure. The invention is not limited to the particular apparatus, methods, conditions, or parameters described and/or shown herein; the terminology used herein is by way of example only. It should be understood that no limitations on the claimed invention are intended. Also, as used in the specification, including the appended claims, references to the singular ('a', 'an', and 'the') include the plural and refer to specific numerical values. shall include at least that specific numerical value, unless the content clearly indicates otherwise. As expressed herein in other embodiments, ranges may be expressed as "about" or "approximately" from another particular value. In addition, unless otherwise indicated, dimensions and material properties referred to herein are illustrative rather than limiting, and are intended to provide a better understanding of preferred practical sample embodiments. It should be understood that variations outside these values may also be included within the scope of the invention depending on the particular application.

The invention is not limited in its application to the details of construction and arrangement of components set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways. Further, the terms and terminology used herein are used for purposes of explanation and should not be construed as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving” and variations thereof includes additional items. .

Hereinafter, preferred embodiments of the present invention will be described in detail.

Historically, the results of reference control slides have been completely subjective since they do not include black and white reference targets. These targets are inconsistent in density and have unique reactivity to a single staining reagent. Reference control slides are, at best, sausage-like structures containing portions containing known antigenic sites, and since the antigen concentration is not known, the stained slides do not allow any reagents to contain the antigen. It can be seen that it is working at a level sufficient to indicate its existence. In the case of IHC-treated slides, objective measurements are not possible because the results are obtained as a cumulative product of antigen retrieval, primary staining reagent, and secondary staining reagent. Therefore, the reference control slide merely provides some reassurance to the user that all reagents are working.

Digital imaging of microscope slides containing stained biological material is evolving to pre-screen and potentially complete diagnostic determination of stained material. Typically, in an imaging system, it is necessary to adjust the illumination light level so that the digital image is not compressed at either the white limit or the black limit. Traditional solutions place a black target and a white target where the markings are supposed to be located. This assumes that the white and black targets represent the extreme values that the slide can represent. However, in doing so, blacks are compressed on a digital scale so that they are much blacker and whites much whiter than can be achieved by staining tissue sections.

In one preferred embodiment of the invention, a microscope slide is disclosed that incorporates a standard control reference target co-located with tissue sections or free cell deposits. Reference targets and tissue sections record the processing steps between tissue capture and coverslipping of stained slides.

In other embodiments, co-existing black and white reference targets are present in the target array of the microscope slide. The black and white reference targets were exposed to reagents and treated in the same manner as the other target and tissue sections.

In other embodiments, a black reference target and a white reference target can vary the illumination level of the imaging system to increase the operating range. Depending on the type of biomaterial, the processing process can vary significantly.

In other embodiments, the various processing routes consist of immunohistochemical staining (IHC), hematoxylin and eosin (H&E) plus special staining, urine specimen H&E plus special staining, and three solutions containing five dyes. It can be classified as, but is not limited to, Papanicolaou stain (PAP) specimens.

Generally, pure black deposited targets are difficult to fabricate because by definition they must absorb all light. The traditional solution is simply to provide a void that bends the optical path so that reflections cannot return. A black target is somewhat reflective at best, but since there is nothing else that black on the slide, that is usually sufficient.

In other embodiments, both targets, the black target and the white target, are printed paint deposits or printed ink deposits that are unreactive to either reagent used to process the slide. A white target has a completely white color under ideal conditions. However, the usefulness of dyed biomaterials that are halfway between black and white is extremely low. Thus, the white color may deviate from perfect white by 5% to 10%, but still be a useful value. The main requirement is that the white target consists of a metal oxide or metal sulfate composition that is stable over time unless exposed to sunlight. Aluminum oxide and titanium oxide meet that requirement very well.

In other embodiments, preferred solutions to the problems described above are disclosed. Both the black and white targets are based on anhydrous epoxy paint bases catalyzed by direct UV light exposure at nominal 365 nm. The anhydride catalyst consists of methyltetrahydrophthalic anhydride and diphenyliodonium hexafluoroarsenate. In addition to being initiated by UV, the anhydride requires thermal addition to function to catalyze and crosslink the epoxy. Although preferred UV-initiated anhydrides and their allies are listed, a search of anhydride manufacturers will reveal other possible solutions. Such paints/inks can be constructed as desired, but are often commercially available components optimized for the printing method used. Paint/ink manufacturing has to deal with the difficulty of achieving good wetting between pigment particles and epoxy binder. In anhydrous paints (also called inks when the viscosity is low), the anhydride is often mixed with an epoxy to provide a pot life of several months. Reducing viscosity is a known knowledge associated with the paint industry, and formulations vary depending on the surface to which the epoxy mixture is applied and the printing method used. Although heat-driven anhydride-epoxy paints/inks are commonly used, the heat required to initiate the reaction can reduce the amount of biomaterials (proteins, peptides, and chemical targets) may be damaged.

In other embodiments, the anhydride catalyst removes unreacted amines found in amino-silane-based catalysts that support non-specific staining. Free amine end groups can and do trap both biological materials and certain stains. In particular, anhydride catalysts address the problem of undesirable staining of white targets by slide processing reagents, especially staining reagents. As free amine end groups on the surface of the paint/ink, both the primary antibody and the secondary staining reagent can be captured and thereby stained. This negates the value of having an integrated white target on the slide.

In other embodiments, the black pigment is carbon dust less than 2 microns in diameter, while the white is aluminum oxide beads or titanium oxide beads. Titanium oxide is the preferred metal oxide for this application.

In other embodiments, it is known to adjust viscosity and drying time with various short chain alcohols, long chain alcohols, and surfactants when making epoxy ink/paint formulations. The choice of surfactant allows the ink/paint to remain reactive to the range of stains and reagents that these slides can tolerate. Preferred formulations are those that avoid surfactants altogether.

In other embodiments, printing of the target can be done by stamp pad or syringe, but a syringe is the preferred method as it supports adjustment to size, which is a better characteristic of target deposits.

In other embodiments, the white target described above is comprised of a metal oxide pigment within an anhydride catalyzed epoxy. In other embodiments, the black target described above consists of a carbon pigment in the range of UV-initiated anhydride-catalyzed epoxies. In other embodiments, the anhydride catalyst is UV initiated by direct UV light exposure. A major advantage of using UV-initiated anhydride catalysts is that the heat required to initiate the anhydride-epoxy reaction does not damage biomaterials (proteins, peptides, and chemical targets). It is to surpass what one can endure without. More importantly, any free amines that can react with the staining reagents are removed.

Example 1
An image of an H&E slide containing aligned targets is shown in Figure 1. These targets are bounded at the edges by black pigment targets and white pigment targets. Pigment targets are printed away from specific dye targets. Black targets/white targets are printed using a modified dispensing pump. The dispensing pump applies a spit and suck cycle to the epoxy mixture coming down a hollow tube held onto the slide surface. When the print head is raised following the ejection/aspiration cycle, the deposits remain as dots. The remainder of the chemical target is printed at another station, and the slide is passed through a UV exposure tunnel to prime the anhydride catalyst and cure the epoxy.

Epoxy plus anhydride is a commercially available premix. To ensure uniformity, the epoxy mixture is stirred and then degassed. Paints/inks are purchased with the pigments already mixed. Such paints/inks are commercially available from a number of vendors, but in all cases the formulations are proprietary to the vendor and will vary from vendor to vendor. Adjust the viscosity as necessary using the recommended diluent supplied by the vendor. The white pigment is barium sulfate, which avoids most chemical interactions with certain dyes. This is in contrast to titanium dioxide or aluminum oxide, which can react with eosin Y.
The microscope slide of this embodiment is a microscope slide that includes an integrated black reference target and a white reference target for imaging, and includes a control target arranged in a straight line. The control target includes the black reference target, the white reference target, and the stained target. The staining target is placed between the black reference target and the white reference target. The black reference target and the white reference target are arranged at both ends of the straight line. The black reference target, the white reference target, and the stained target are arranged on a plurality of separated regions.
The black reference target and the white reference target are made from a material that includes a UV-initiated anhydride-catalyzed epoxy paint or ink base.
The material of the black reference target is carbon dust.
The carbon dust has a particle size of 0.1 micron to 2 micron.
The white reference target is an oxide or sulfate of a metal selected from metals of groups 13-15 of the periodic table of elements, titanium and barium.
The white reference target is selected from titanium oxide, aluminum oxide, aluminum sulfate, barium sulfate, or mixtures thereof.
The UV-initiated anhydride catalyzed epoxy paint base is comprised of methyltetrahydrophthalic anhydride and diphenyliodonium hexafluoroarsenate.
The black reference target and the white reference target are present with tissue sections or free cells.
The black reference target and the white reference target are non-reactive with biomaterial stains.
The black reference target and the white reference target are stable to exposure to antigen retrieval activities, buffers, heat, and time.
The black reference target and the white reference target are non-reactive with hydroxyl compounds, amine compounds, amide compounds, hydrazide compounds, and aromatic benzene compounds.
The black reference target and the white reference target are printed on a microscope slide by stamp pad, syringe, or inkjet.
The staining target includes a staining reagent.
A digital imaging method using the microscope slide, wherein the stained target, tissue section or free cell is subjected to IHC staining, hematoxylin and eosin staining, urine specimen H&E plus special staining, or Papanicolaou staining (PAP) specimen, By comparing the colors of the stained target, the tissue section, or the free cells, the irradiation intensity and contrast of the stained target, the tissue section, or the free cells are determined.

Claims (14)

A microscope slide comprising an integrated black reference target and a white reference target for imaging, the slide comprising a control target arranged in a straight line, the slide comprising:
The control target has the black reference target, the white reference target and the stained target,
the staining target is located between the black reference target and the white reference target;
The black reference target and the white reference target are arranged at both ends of the straight line,
A microscope slide, wherein the black reference target, the white reference target, and the stained target are arranged on a plurality of separated areas. 2. The microscope slide of claim 1, wherein the black reference target and the white reference target are made from a material comprising a UV-initiated anhydride-catalyzed epoxy paint or ink base. A microscope slide according to claim 1, wherein the material of the black reference target is carbon dust. A microscope slide according to claim 3, wherein the carbon dust has a particle size of 0.1 micron to 2 microns. Microscope slide according to any one of claims 1 to 4, wherein the white reference target is an oxide or sulfate of a metal selected from metals of groups 13-15 of the Periodic Table of the Elements, titanium and barium. 6. A microscope slide according to claim 5, wherein the white reference target is selected from titanium oxide, aluminum oxide, aluminum sulfate, barium sulfate, or mixtures thereof. 3. The microscope slide of claim 2, wherein the UV-initiated anhydride catalyzed epoxy paint base is comprised of methyltetrahydrophthalic anhydride and diphenyliodonium hexafluoroarsenate. Microscope slide according to any one of claims 1 to 7, wherein the black reference target and the white reference target are present with tissue sections or free cells. A microscope slide according to any one of claims 1 to 7, wherein the black reference target and the white reference target are non-reactive with staining agents for biomaterials. A microscope slide according to any one of claims 1 to 9, wherein the black reference target and the white reference target are stable against exposure to antigen retrieval activities, buffers, heat, and time. 11. The black reference target and the white reference target are non-reactive towards hydroxyl compounds, amine compounds, amide compounds, hydrazide compounds and aromatic benzene compounds. Microscope slide. Microscope slide according to any one of the preceding claims, wherein the black reference target and the white reference target are printed on the microscope slide by a stamp pad, a syringe or an inkjet. A microscope slide according to any one of claims 1 to 12 , wherein the staining target comprises a staining reagent. A digital imaging method using a microscope slide according to any one of claims 1 to 13 , comprising:
Performing IHC staining, hematoxylin and eosin staining, urine specimen H&E plus special staining, or Papanicolaou staining (PAP) specimen on the stained target, tissue section or free cells,
A digital imaging method, wherein the illumination intensity and contrast of the stained target, the tissue section, or the free cells is determined by comparing the colors of the stained target, the tissue section, or the free cells.