WO2013179279A2

WO2013179279A2 - Method and device for analyzing biological sample

Info

Publication number: WO2013179279A2
Application number: PCT/IL2013/050433
Authority: WO
Inventors: Yaron Front; Chanan Sluszny; Limor SHIPOSH
Original assignee: Applied Spectral Imaging Ltd.
Priority date: 2012-05-28
Filing date: 2013-05-21
Publication date: 2013-12-05
Also published as: WO2013179279A3

Abstract

A device for analyzing a biological sample is disclosed. The device comprises, a substrate having thereon a sample region for carrying the sample and a reference region; and a reference substance being immobilized on the reference region, and being bindable to at least one label that is specific to at least one component of the sample.

Description

METHOD AND DEVICE FOR ANALYZING BIOLOGICAL SAMPLE

RELATED APPLICATION

This application claims the benefit of priority of U.S. Provisional Patent

Application No. 61/652,250 filed on May 28, 2012, the contents of which are incorporated herein by reference in their entirety

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to the analysis of substances and, more particularly, but not exclusively, to method and device for analyzing biological samples, e.g., by means of labeling.

The field of pathology involves the examination of tissue specimens to determine if the tissue is normal or diseased. The tissue specimens can be individual cells in a smear, body fluid or cell block (cytology specimens) or cell aggregates that form a structure with a specific function (histology specimens). Typically, pathology determines the structural and functional changes in cells, tissues and organs which cause or are caused by disease.

Histology serves as an invaluable tool in pathology since it deals with microanatomy of tissues and their cellular structure. Pathology typically involves the examination of histological sections of suspected tissues. A specimen is processed and applied to a microscope slide and then stained to make the normally transparent cells brilliantly colored for easier observation and to distinguish the various cellular elements, which have differing affinities for the various stains such as Hematoxylin and Eosin, Fuchsin, Giemza, and the like. Different colors are thus associated with different tissue components. However, it is recognized that these stains are not always accurate because their appearance depends on many factors including solution preparation, environmental factors (temperature, etc.), co-existence of other stains, affinity of the stained element and the like.

In immunohistochemistry (IHC), spectrally marked antibodies are applied to the specimen to detect specific protein manifestations within the tissue thereby to obtain higher level of resolution compared to histology staining and information regarding their functionality. Over the past three decades, IHC, also known as immunocytochemistry (ICC) when applied to cells, has become an indispensable tool in diagnostic pathology and has virtually revolutionized the practice of surgical pathology. The terms immunohistochemistry and immunocytochemistry are used herein interchangeably.

Panels of monoclonal antibodies can be used in the differential diagnosis of undifferentiated neoplasms (e.g. , to distinguish lymphomas, carcinomas, and sarcomas); to reveal markers specific for certain tumor types; to diagnose and phenotype malignant lymphomas; and to demonstrate the presence of viral antigens, oncoproteins, hormone receptors, and proliferation-associated nuclear proteins. Not only do such markers have diagnostic significance, but there is a growing body of evidence that some tumor markers have prognostic significance, as has been most extensively demonstrated, for example, in breast cancer. These marker studies can be performed by IHC on a variety of specimen types, including cytological preparations, paraffin-embedded tissue and frozen sections. Most clinical assays employ a single antibody for each slide but by using chromogens of different colors, it is possible to observe/demonstrate two or more markers simultaneously. The specific (or primary) antibody may be labeled directly, or a second antibody carrying the label can be used to specifically bind to the first antibody.

For most applications of IHC, qualitative assessment of the staining patterns is sufficient, because it is the overall staining pattern of the tissue and the cells of interest that imparts the diagnostic information. However, there are certain markers for which image analysis can play an important role. These include detection of hormone receptors, tumor proliferation markers, oncoproteins, tumor suppressor gene products, chemotherapy resistance factors, tumor angiogenesis, lymphocyte subset analysis, etc.

The general goal of quantitative IHC (QIHC) is to provide objective measurements of immuno staining reactions. These measurements may be useful in data collection for experimental purposes, but the greatest potential of such measurements lies in exploring their use as aids for determining diagnosis, prognosis, or therapy of human disease, especially cancer. Aside from the mechanics of making such measurements, which will be discussed, a number of issues are related to the peculiarities of IHC which potentially affect how the measurements should be made.

The ability to measure reactions in tissue sections or cytological preparations depends on the choice of staining and imaging methods. In IHC, for example, this is addressed by carefully titering the antibodies (primary and secondary) to maximize the specific staining and minimize the background staining. The tissues are visualized using histological stains (also known as counterstains) having a different color than the chromogen, so that the staining reaction can be visualized. For example, immunoperoxidase reactions, which yield a brown reaction product, are typically combined with methyl green or toluidine blue histological stains (counterstains) to provide visual contrast.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a device for analyzing biological sample. The device comprises, a substrate having thereon a sample region for carrying the sample and a reference region; and a reference substance being immobilized on the reference region, and being bindable to at least one label that is specific to at least one component of the sample.

According to some embodiments of the invention the substrate and the reference substance is packed in a sealed encapsulation devoid of the sample.

According to some embodiments of the invention the substrate is selected from the group consisting of a microscope slide, a fluid chamber or channel of a microfluidic device and titer plate and in general any biological-sample mount.

According to an aspect of some embodiments of the present invention there is provided a device for use in an analysis of a biological sample using at least one label being specific to at least one component of the sample. The device comprises: a sticker assembly having a substrate layer; a reference substance being immobilized on a first side of the substrate layer and being bindable to the at least one label; and an adhesive layer at least partially coating a second side of the substrate layer.

According to an aspect of some embodiments of the present invention there is provided a kit for analyzing biological sample. The kit comprises the device as delineated above and optionally as further detailed hereinunder.

According to an aspect of some embodiments of the present invention there is provided a method of analyzing a biological sample. The method comprises, placing the biological sample on the sample region of a device; contacting the device with the at least one label; and analyzing a signal effected by at least one label, wherein the device is as delineated above and optionally as further detailed hereinunder.

According to an aspect of some embodiments of the present invention there is provided a method of analyzing a biological sample. The method comprises, placing the biological sample on a sample region of a substrate; attaching a sticker assembly to a reference region of the substrate; contacting the substrate with the at least one label; and analyzing a signal effected by the at least one label, wherein the sticker assembly is as delineated above and optionally as further detailed hereinunder.

According to some embodiments of the invention the method comprises capturing the signal using an imaging system, prior to the analysis.

According to some embodiments of the invention the analysis of the signal is by a data processor configured for performing the analysis.

According to some embodiments of the invention the analysis comprises piecewise histogram normalization of signal received from the sample region based on signal received from the reference region.

According to some embodiments of the invention the signal comprises a plurality of color channels and wherein the analysis is executed separately for each of at least two of the color channels.

According to some embodiments of the invention the method comprises receiving a characteristic signal profile from a source other than the substrate, wherein the analysis is based in part on the characteristic signal profile.

According to an aspect of some embodiments of the present invention there is provided a method of fabricating a device for analyzing a biological sample. The method comprises, immobilizing on a substrate a reference substance being bindable to at least one label that is specific to at least one component of the sample, the substrate having thereon a sample region for carrying the sample and a reference region, and the reference substance being immobilized on the reference region; and packing the substrate but not the sample in a sealed disposable encapsulation.

According to an aspect of some embodiments of the present invention there is provided a method of fabricating a device for analyzing a biological sample. The method comprises, immobilizing a reference substance on a first side of substrate layer having, on a second side thereof, an adhesive layer at least partially coating the second side, wherein the reference substance is bindable to at least one label that is specific to at least one component of the sample.

According to some embodiments of the invention the reference substance is chemically attached to the reference region.

According to some embodiments of the invention the reference substance is imprinted on the reference region.

According to some embodiments of the invention the reference substance is embedded in a structure immobilized on the reference region.

According to some embodiments of the invention the structure is a porous structure.

According to some embodiments of the invention the structure is a non-porous structure.

According to some embodiments of the invention the structure is a membrane structure.

According to some embodiments of the invention the structure is a powder.

According to some embodiments of the invention the structure comprises gel. According to some embodiments of the invention the structure is an artificial tissue.

According to some embodiments of the invention the reference substance is a reference tissue identified as having at least one pathology.

According to some embodiments of the invention the reference tissue comprises at least two regions having different histological scores for the pathology.

According to some embodiments of the invention the device comprises a chemical, biological and/or physical array being immobilized on the substrate and configured for providing signals indicative of each of a plurality of labels or label concentrations contacting the chemical, biological and/or array.

According to some embodiments of the invention the device comprises electronic circuitry being immobilized on the substrate and configured for generating electric or magnetic signals indicative of each of a plurality of labels or label concentrations contacting the electronic circuitry.

According to some embodiments of the invention the electronic circuitry is configured for recording the electric or magnetic signals. According to some embodiments of the invention a concentration of the reference substance varies across the reference region.

According to some embodiments of the invention the concentration varies continuously.

According to some embodiments of the invention the concentration varies discretely.

According to some embodiments of the invention the reference region comprises a plurality of wells each containing a different reference substance or a different concentration of reference substance.

According to some embodiments of the invention the reference region and the sample region engage different planes.

According to some embodiments of the invention the substrate is thinner at the reference region than at the sample region.

According to some embodiments of the invention the reference substance is hydrophilic.

According to some embodiments of the invention the reference substance is hydrophobic.

According to some embodiments of the invention the reference substance comprises a naturally occurring compound.

According to some embodiments of the invention the reference substance comprises a synthetic compound.

According to some embodiments of the invention the reference substance comprises an organic compound.

According to some embodiments of the invention the reference substance comprises an inorganic compound.

According to some embodiments of the invention the reference substance comprises a biological cell or tissue.

According to some embodiments of the invention the reference substance comprises at least one molecule selected from the group consisting of a polymer, a protein, a protein L, a polysaccharide, an antibody, an antigen, a peptide, a dipeptide, a polypeptide, and an aptamer. According to some embodiments of the invention the label comprises at least one substance selected from the group consisting of a direct immunohistochemical stain, a secondary immunohistochemical stain, a histological stain, a immunofluorescence stain, a DNA ploidy stain, a nucleic acid sequence specific probe, a dye, an enzyme, a nanoparticle and any combination thereof.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

Implementation of the method and/or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.

For example, hardware for performing selected tasks according to embodiments of the invention could be implemented as a chip or a circuit. As software, selected tasks according to embodiments of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In an exemplary embodiment of the invention, one or more tasks according to exemplary embodiments of method and/or system as described herein are performed by a data processor, such as a computing platform for executing a plurality of instructions. Optionally, the data processor includes a volatile memory for storing instructions and/or data and/or a non- volatile storage, for example, a magnetic hard-disk and/or removable media, for storing instructions and/or data. Optionally, a network connection is provided as well. A display and/or a user input device such as a keyboard or mouse are optionally provided as well. BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A and IB are schematic illustrations of a top view (FIG. 1A) and a side view (FIG. IB) of a device suitable for analyzing a biological sample, according to some embodiments of the present invention;

FIG. 1C is a schematic illustration of a sticker_assembly, according to some embodiments of the present invention;

FIG. 2 is a schematic illustration of the device in embodiments of the invention in which the substrate of the device is sealed in an encapsulation 22;

FIG. 3 is a schematic illustration of the device in embodiments of the invention in which the substrate of the device comprises a plurality of wells;

FIGs. 4A-B are flowchart diagrams describing a method suitable for analyzing a biological sample, according to some embodiments of the present invention;

FIG. 5 is a flowchart diagram describing a method suitable for fabricating a device for analyzing a biological sample, according to some embodiments of the present invention;

FIGs. 6A-C show variations in immunohistochemistry staining, obtained during experiments performed according to some embodiments of the present invention;

FIG. 7 is a graph showing the optical density (OD units) as a function of the concentration of a substance (ppm), as obtained during experiments performed according to some embodiments of the present invention; and

FIGs. 8A-D are images showing variations in HER2/Neu staining of breast ductal carcinoma, obtained during experiments performed according to some embodiments of the present invention. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the drawings and/or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

It was found by the present inventors that there is a large amount of variability in the results obtained when conventional analysis techniques are applied to a biological sample. This is particularly the case for analysis that involves labeling e.g., IHC assays. For example, the analysis of IHC-stained samples by pathologists in a conventional pathology laboratory involves solving a number of problems, including variation of stain intensity or variation of optical densities of stained cell objects and calibration of the staining procedure. The present inventors found that there are problems that are associated with the analysis of biological samples even when substantially the same technique is applied in two separate assays. When, for example, two histology slides are subjected to the same staining protocol, color-intensity variations oftentimes exist between slides. This variability is undesired because it adds a level of complexity that may result in a difference in the final assessment of the sample by different pathologists. The present inventors found a device, kit and method that at least partially overcome the problems associated with the variability in the results obtained from analysis of a biological sample.

Reference is now made to FIGs. 1A and IB which are schematic illustrations of a top view (FIG. 1A) and a side view (FIG. IB) of device 10 suitable for analyzing a biological sample 12, according to some embodiments of the present invention. Typically, device 10 is designed and configured for determining the presence, absence or level of at least one component in sample 12. For example, device 10 can be designed and configured for determining the presence, absence or level of at least one component selected from the group consisting of an antigen, a DNA, an RNA, a protein, an enzyme, a cell organelle, a ribosome, a virus and the like.

Device 10 comprises a substrate 14 having thereon a sample region 16 and a reference region 18. Substrate 14 can be, for example, a slide such as, but not limited to, a microscope slide, or any other substrate suitable for analyzing biological samples, particularly, but not necessarily, substrates suitable for histology optionally and preferably IHC. In some embodiments of the present invention substrate 14 is made of glass.

Sample region 16 serves for carrying sample 12, and reference region 18, serves for carrying reference substance 20. The locations and relative coordinates of regions 16 and 18 are optionally and preferably addressable by a machine, including, without limitation, mechanical addressing e.g., by a robotic arm, optical addressing e.g., by a scanning light beam, and/or computational addressing, e.g., by means of processing an image of substrate 14.

In some embodiments of the present invention substrate 14 is sealed in an encapsulation 22, optionally and preferably a disposable encapsulation. In various exemplary embodiments of the invention the encapsulation encapsulate the substrate and the reference substance but not the sample. This embodiment is illustrated in FIG. 2.

Reference substance 20 is preferably immobilized on reference region 18. For example, in some embodiments reference substance 20 is chemically attached to reference region 18, and in some embodiments reference substance 20 is imprinted to reference region 18. Other immobilization techniques are not excluded from the scope of the present invention. Substance 20 can be immobilized either directly or it can be embedded in a structure that is immobilized on region 18, e.g., by chemical attachment, physical attachment or imprint. Representative of structures suitable for the present embodiments include, without limitation, a porous structure (e.g., a structure having an average pore size of from about 0.05 microns to about 100 microns), a non-porous structure, a membrane structure, a scaffold structure, a gel, a sticker, an artificial tissue and a natural tissue. Also contemplated are embodiments in which substance 20 is embedded in a powder wherein the powder is immobilized on region 18. Any combination of the above structures can be employed. For example, a sticker can be provided with any other of the structures. FIG. 1C is a schematic illustration of a sticker assembly 30, according to some embodiments of the present invention. Sticker assembly 30 comprises a substrate layer 32 having a first side 34 and a second side 36. Reference substance 20 or the structure in which it is embedded is immobilized on first side 34. The immobilization can be by chemical attachment, physical attachment or imprint. Second side 36 is coated, at least partially, by an adhesive layer 38, selected to allow adhering layer 32 to a receiving surface, such as, but not limited to, a surface of substrate 14.

Substrate layer 32 is optionally and preferably flexible. Substrate layer 32 can be made of any material suitable on which the reference substance can be immobilized directly or indirectly. Representative examples include, without limitation, a polyester film, a vinyl film, a polyvinylchloride film, a polyethylene film, a polycarbonate film, a polyolefin film, a Polyvinylidene fluoride (PVDF) film, a nitrocellulose film, or any film having a combination of one or more of the above materials, a paper, a synthetic paper, a coated paper (e.g., a silicon-coated paper) and the like.

Sticker assembly 30 optionally and preferably comprises a backing sheet 39 attached to second side 36. Backing sheet 39 is preferably provided with a surface selected so as to allow, in a per se known manner, the separation of backing sheet 39 from adhesive layer 38 which remains connected to the second side 36 of layer 32. For example, the surface of sheet 39 that contacts adhesive layer 38 can be siliconized, or otherwise processed such that the adhesive forces between layer 38 and sheet 39 are weaker than the adhesive forces between layer 38 and layer 32.

Reference substance 20, or the structure in which it is embedded is typically designed to provide detectable signal output, based on the type of measurement. For example, for fluorescence assays, substance 20 or the structure in which it is embedded can be black. For transmittance assays, substance 20 or the structure in which it is embedded is configured to allow transmission of light of the respective wavelength therethrough, for example, substance 20 or the structure in which it is embedded can be made of a material which is transparent to the applied light, or it can be made from a material which is generally non-transparent to the applied light, but sufficiently thin so as not to completely block the light.

In embodiments in which reference substance 20 is attached to substrate 14 by means of sticker assembly 30, the layer 32 of sticker assembly 30 can be selected so as not to interfere with the signal provided by substance 20. Thus, for example, when sticker assembly 30 is designed for fluorescence assays, layer 32 can be dark (e.g. , black), and when sticker assembly 30 is designed for transmittance assays, layer 32 can be transparent.

The thickness of reference 20 or the structure in which it is embedded can be from about 1 micron to about 1 mm. The thickness of sample 12 depends on the analysis that is employed. For histological or IHC analysis, the thickness of sample 12 is typically about 5 microns.

In some embodiments, reference region 18 and sample region 16 engage different planes. For example, substrate 14 can be thinner at reference region 18 than at sample region 16. This embodiment is particularly useful when substance 20 is thicker than sample 12. When substance 20 is immobilized on a plane lower than the plane of region 16, the levels of the top surfaces of substance 20 and sample 12 are approximately equal, so that during the analysis both substance 20 and sample 12 are exposed to substantially the same conditions.

In various exemplary embodiments of the invention substance 20 is selected such that it is generally analogous to sample 12 in terms of the analysis assay. Typically, substance 20 is capable of binding to at least one label that is specific to the component of sample 12 which the assay is designed to detect. In some embodiments of the present invention substance 20 comprises the same component of sample 12 to which the label is specific.

As used herein, "label" refers to any molecule that has a sufficiently high binding affinity to at least one component of a biological sample.

As used herein, "binding affinity" refers to the apparent association constant or K_a, which is the reciprocal of the dissociation constant ¾ at equilibrium between the label and the respective component, and which describes the likelihood of the label and sample component to be in the bound state.

As used herein, "sufficiently high binding affinity" refers to a binding affinity corresponding to a dissociation constant K_d of less than 10 μΜ or less than 1 μΜ or less than 0.1 μΜ or less than 10 nM or less 1 nM or less than 0.1 nM.

Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in PBS (phosphate buffered saline) at pH 7.2 at 30 °C. These techniques can be used to measure the concentration of bound and free binding label as a function of label concentration. The concentration of bound label B is related to the concentration of free label F and the concentration of binding sites for the label on the component by the following equation: B=N-F/((1/K_a)+F), where N is the number of binding sites per one molecule of the component.

Representative example of labels suitable for the present embodiments include, without limitation, a stain (e.g. , a direct immunohistochemical stain, a secondary immunohistochemical stain, a histological stain, a immunofluorescence stain, a DNA ploidy stain, etc.), a nucleic acid sequence specific probe (for example aptamer), a dye, an enzyme, a nanoparticle (e.g. , a quantum dot, a metal nanoparticle, a metal oxide nanoparticle, a transition metal complex nanoparticle) a protein L, a peptide and any combination thereof.

As used herein, the term "stain" or "stains" refers to colorants, either fluorescent, luminescent and/or chromogenic and further to reagents or matter used for effecting coloration.

As used herein, the term "immunohistochemical stain" refers to colorants, reactions and associated reagents in which a primary antibody which binds a cytological or receptor (e.g. protein receptor) marker is used to directly or indirectly (via "sandwich" reagents and/or an enzymatic reaction) stain the biological sample examined. Immunohistochemical stains are in many cases referred to in the scientific literature as immunostains, immunocytostains, immunohistopathological stains, etc.

As used herein, the term "histological stain" refers to any colorant, reaction and/or associated reagents used to stain cells and tissues in association with cell components such as types of proteins (acidic, basic), DNA, RNA, lipids, cytoplasm components, nuclear components, membrane components, etc. Histological stains are in many cases referred to as counterstains, cytological stains, histopathological stains, etc.

As used herein, the term "DNA ploidy stain" refers to stains which stoichiometrically bind to chromosome components, such as, but not limited to, DNA or histones. When an antibody is involved, such as anti-histone antibody, such stains are also known as DNA immunoploidy stains. Lists of known stains are provided in U.S. Pat. application No. 6,007,996, filed July 27, 1998, the contents of which are hereby incorporated by reference.

As used herein, the phrase "nucleic acid sequence specific probe" refers to polynucleotides labeled with a label moiety which is either directly or indirectly detectable, which polynucleotides being capable of base-pairing with matching nucleic acid sequences present in the biological sample.

Reference substance 20 is optionally, but not necessarily, hydrophilic, so as to allow embedding a water-based solution, such as salt or buffer. Alternatively, reference substance 20 can be hydrophobic. Substance 20 can comprise a naturally occurring compound a synthetic compound, an organic compound and/or an inorganic compound. In some embodiments of the present invention substance 20 comprises a biological cell. For example, substance 20 can be a reference tissue that is identified as having at least one pathology (e.g. , cancer). Additional examples for substances suitable for use as reference substance include, but are not limited to, at least one molecule selected from the group consisting of a polymer, a protein, a protein L, a polysaccharide, an antibody, an antigen, a peptide, a dipeptide, a polypeptide, and an aptamer.

One or more of the properties of substance 20 may vary across region 18, so as to form over region 18 sub-regions, wherein in each sub-region substance 20 is characterized by a different property. For example, in some embodiments, the concentration of substance 20 varies, so that in each sub-region of region 18, there is a different, more preferably unique, amount of binding between substance 20 and the label(s). In embodiments in which substance 20 comprises reference tissue, the reference tissue can be treated (for example, chemically) to form two or more regions having different histological scores for the pathology.

The variation of the property of substance 20 across region 18 can be continuous or discrete, and can have any dependence on the location across region 18. In some embodiments of the present invention the property or properties of substance 20 vary to form a monotonic gradient along one direction. For example, substance 20 can be immobilized along a strip within region 18 wherein the property or properties (for example, concentration) of substance 20 varies monotonically along the stripe.

In use, the encapsulation 22 (in embodiments in which device 10 includes an encapsulation) is removed. When substance 20 is immobilized on sticker assembly 30, sheet 39 (in embodiments in which assembly 30 includes a backing sheet) is separated from the assembly and layer 32 is attached to a substrate, e.g., substrate 14.

The biological sample is then placed on the substrate (e.g. , on sample region 16). The substrate is contacted with one or more labels, for example, according to a histological or immunohistochemical protocol. The labeling process affects both reference substance 20 and sample 12, resulting in a change in the color, color intensity, morphology, opaqueness or other properties of substance 20, and sample 12.

The signal generated by the label(s) is then analyzed. The signal is typically an optical signal in the form of emitted, reflected or transmitted light having a central wavelength that is characteristic to the bound label.

Prior to analysis the signal is optionally and preferably captured, for example, using an imaging system. In these embodiments, the analysis is based on the image of the reference substrate.

In various exemplary embodiments of the invention the signal generated by the label that binds to sample 12 is normalized or calibrated using the signal generated by the label that binds to reference substance 20. This is optionally and preferably done using normalization or calibration data that describe the relation between a concentration or amount of substance 20 and a signal generated by the label(s) responsively to substance 20. Such data can be prepared in advance and be supplied together with device 10 or with sticker assembly 30, for example, as an analysis kit.

In embodiments in which one or more of the properties of substance 20 varies across region 18, the signal received from sample 12 can optionally and preferably be compared to the signal received from each of the sub-regions of region 18 to increase the resolution of the normalization and/or calibration. In embodiments in which substance 20 comprises reference tissue which includes two or more regions having different histological scores for the pathology, the signal received from the sample can be compared to each of the regions of the reference tissue to allow accurate scoring of the pathology.

The signal from substance 20 can, according to some embodiments of the present invention be used for performing at least one of the following operations: asses the quality and other parameters of the staining process and the quality of stains; determine slide preparation quality; provide a measure of the variability of the procedure with respect to previously used preparations; provide the user with information showing how the sample may have been affected based on the specific staining procedure; modify colors and intensities of the measured sample to mimic the expected appearance as if it would have been stained using a different procedure; compensate for variations caused by different staining methods, chemicals, reagents and protocols; and compensate for different protocols provided by different manufacturers or different ingredients used during sample preparation.

Before providing a further detailed description of the device, kit and method, as delineated hereinabove and in accordance with some embodiments of the present invention, attention will be given to the advantages and potential applications offered thereby.

The device, kit and method of present embodiments can be used in many staining techniques. The Feulgen staining technique, for example, may be used to stain DNA in cell objects with dyes, for example, with thionin, Azure A, Azure C, pararosa- nilin and methylene blue. Proteins may be stained with congo red, eosin, an eosin/hematoxylin combination, or fast green. Enzymes may be made visible with diaminobenzidine or 3- amino-9 ethylcarbazole or alkaline phosphatase in combination with a dye substrate; cell organelles may be stained with methylene blue; and ribosomes with methylene blue and mi- tochrondia with giemsa stain. Dyes containing thionin groups can be used to determine RNA distribution in cells.

In IHC, some of these stains are used in combination with monoclonal antibodies, e.g. , monoclonal antibodies detect estrogen or progesterone receptors. Antigen analysis may include the steps of binding of monoclonal antibodies to the sample. Later, the monoclonal antibody may be conjugated with an enzyme stain. Also, the monoclonal antibody may be conjugated with a fluorescent material or stain. Then the fluorescent stain may be excited at a wavelength to induce the fluorescence and then this may be observed at another wavelength at which fluorescent emission occurs.

Many monoclonal antibodies directed against cell cycle-related nuclear antigens are commercially available and may be used for immunohistochemical staining of tissues and cells.

A commonly employed antibody is Anti-Ki-67, which stains a proliferation- related nuclear antigen in human cells of all lineages. This antibody has the useful property of staining nuclei of cells in Gl, S, G2, and M phases of the cell cycle, but not the nuclei of resting (GO phase) cells. Several studies have demonstrated that the proportion of tumor cell nuclei stained by Anti-Ki-67 correlates with tumor grade and other prognostic features for a variety of tumor types, including breast carcinoma, lymphoma, meningioma, glial and astrocytic brain tumors, malignant melanoma, and sarcoma. In non-Hodgkin's lymphoma, Ki-67 staining is associated with working classification grade. Increasing proliferative fraction as measured by Ki-67 staining is associated with a worse prognosis. In breast cancer, Ki-67 staining has been correlated with nuclear grade and lymph node status and several studies have shown the prognostic significance of Ki-67 staining in this type of cancer. The monoclonal antibody MIB 1 appears to stain the same antigen as stained by Ki-67. This antibody can be used on paraffin sections. The antibody Ki-Sl appears to have similar staining characteristics as Ki-67 and can also be used on paraffin sections. The relative fraction of tumor cell nuclei positive for Ki-Sl staining appears to have prognostic significance in breast cancer.

Other proliferation- associated nuclear markers may also be used according to some embodiments of the present invention. PCNA/cyclin is a 36 kDa nuclear protein present in proliferating cells, and is an accessory protein to DNA polymerase-delta. Two fractions of PCNA/cyclin exist in cell nuclei, the fraction that is insoluble in nonionic detergent being more restricted to the S phase of the cell cycle. Immunohistochemical staining for this protein can be applied according to some embodiments of the present invention in frozen and paraffin-embedded tissues fixed in alcohol or formalin, thus permitting study of archival tissues. Detection of PCNA in paraffin embedded tissue can be improved using a microwave technique. Nuclei stained for PCNA/cyclin exhibit varying degrees of positivity, and studies have indicated that the more intensely stained nuclei correspond to S-phase cells. Thus, the present embodiments can be used for quantitation of antigen expression.

Antibodies to alpha DNA polymerase can be applied to tissue sections of normal and malignant tissue. P105 is a nuclear antigen expressed starting at the G0/G1 phase transition, with increasing expression through M phase.

The commercial availability of monoclonal antibodies to estrogen receptor (ER) and progesterone receptor (PR) has made possible the visual detection of hormone receptors using standard immunohistochemical methods. It is recognized that there is a high correlation between immunohistochemical determination of ER/PR and standard biochemical methods. Some features of interest that can be measured according to some embodiments of the present invention are the percent tumor cell nuclei stained for ER/PR, as well as the intensity of the staining.

The device, kit and method of the present embodiments can be used in quantitative IHC (QIHC) and/or semi-QIHC.

As used herein, QIHC refers to an automated method of scanning and scoring samples that have undergone IHC to identify and quantify the presence of a specified biomarker such as an antigen or other protein.

The score given to the sample in QIHC may be a numerical representation of the intensity of the immunohistochemical staining of the sample, and may represent the amount of target biomarkers present in the sample. For example, Optical Density (OD) is a numerical score that represents intensity of staining.

As used herein, semi-QIHC refers to scoring of immunohistochemical results by the human eye, where a trained operator ranks results numerically (e.g., as 1+, 2+, or 3+).

It is recognized that QIHC score correlates with biochemical determination of

ER.

Some embodiments of the present invention relate to the analysis of cell surface and cytoplasmic antigens, particularly, but not necessarily, the products of oncogenes. For example, it is recognized that both amplification and overexpression of HER-2/neu oncogene in breast and ovarian cancer correlate with a worsened prognosis. The intensity of immuno staining is known to correlate well with other measures of gene expression, and the level of expression in ovarian cancer correlates with survival.

The device, kit and method of the present embodiments can be used in a QIHC assay, for example, for analysis of oncogene expression. Nuclear oncoproteins can be measured using image analysis in a manner similar to that described for hormone receptors and Ki-67 antigen, and expressed either as percent positive nuclear area or as a value that combines staining intensity with positivity.

Another example of a cellular protein is the product of the multiple drug resistance (mdr) gene, P-glycoprotein, a cell surface-associated ATPase that appears to be responsible for one mechanism of non-specific chemotherapy resistance. Expression of P-glycoprotein is known to correlates with relapse in multiple myeloma, and use of image analysis can aid in measuring this protein as detected by IHC. Other markers of therapy resistance, include, without limitation, glutathione transferase and topoisomerase II.

Following are a few examples of sets of labels having prognostic and/or therapeutic significance in various cancers.

For breast cancer, the label can include at least one of the anti-ER, anti-PR, anti- Her2/neu, anti-p53, anti-Ki-67 and anti-CD31 immunohistochemical marker stains, a DNA ploidy stain and a H&E counterstain. In some embodiments of the present invention device 10 is prepared for the analysis of a sample for the presence or grade of breast cancer, and reference substance 20 is selected to have sufficiently high affinity to at least one of these labels. For example, reference substance 20 can include at least one of ER, PR, Her2/neu, p53, Ki-67 and CD31.

For ovarian and/or endometrial cancer, the label can include at least one of the anti-Her2/neu, anti-p53, anti-Ki-67 and anti-CD31 immunohistochemical stains, a DNA ploidy stain and a H&E counterstain. In some embodiments of the present invention device 10 is prepared for the analysis of sample for the presence or grade of ovarian and/or endometrial cancer, and reference substance 20 is selected to have sufficiently high affinity to at least one of these labels. For example, reference substance 20 include at least one of Her2/neu, p53, Ki-67 and CD31.

For prostrate and/or bladder cancer, the label can include at least one of the anti- Ki-67, anti-p53, anti-CD31 and anti-retinoblastoma protein (Rb) marker stains, a DNA ploidy stain and a H&E counterstain. In some embodiments of the present invention device 10 is prepared for the analysis of sample for the presence or grade of prostrate and/or bladder cancer, and reference substance 20 is selected to have sufficiently high affinity to at least one of these labels. For example, reference substance 20 include at least one of Ki-67, p53, CD31 and Rb.

For colorectal cancer, the label can include at least one of the anti-Ki-67, anti- p53, anti-CD31 and anti-p21 (ras oncoprotein) marker stains, a DNA ploidy stain and a H&E counterstain. In some embodiments of the present invention device 10 is prepared for the analysis of sample for the presence or grade of colorectal cancer, and reference substance 20 is selected to have sufficiently high affinity to at least one of these labels.

For example, reference substance 20 includes at least one of Ki-67, p53, CD31 and p21.

Referring now again to the drawings, device 10 and/or sticker assembly 30 can optionally and preferably comprise a chemical, biological or physical array and/or electronic circuitry, collectively shown at 24. When array and/or electronic circuitry 24 is immobilized on substrate 14, it is optionally and preferably immobilized (e.g. , attached to or formed on) region 18 of substrate 14.

When a chemical, biological or physical array is employed, it is preferably configured for providing signals indicative of each of a plurality of labels or label concentrations contacting the array. The signal is typically, but not necessarily, an optical signal in the form of light emitted, reflected or absorbed from the array. For example, each element of the array can change color responsively to chemical interaction between the respective element and the respective label. When the assay includes the use of a plurality of labels, the changes in the property of each element (e.g. , color,) can serve for recording the type of labels that are used in the assay.

The array is typically embodied as a plurality of array elements arranged over a two-dimensional arrangement of spatial locations. In a chemical array, two separate array elements can be different compositions or the same composition but with different concentrations. In a biological array, two separate array elements can be different biological material or the same biological material but with a different pathology. In a physical array two separate array elements can be materials of different size or structure

(e.g. , powder of different particle size).

When electronic circuitry is employed, it is preferably configured for generating electric or magnetic signals indicative of each of a plurality of labels or label concentrations contacting the electronic circuitry. For example, the electronic circuitry can include an array of electrochemical sensors each configured for generating a signal responsively to an interaction of the sensor with a different label or label concentration.

The electronic circuitry can, in some embodiments of the present invention, be configured for recording the electric or magnetic signals.

In some embodiments of the present invention reference region 18 comprises a plurality of wells 26 each containing a different reference substance 20 or a different concentration of reference substance 20. A cross- sectional view of this embodiment is illustrated in FIG. 3. Wells 26 can by microwells. The advantage of having wells 26 is that it effects a discrete variation in the property or type substance 20 across region 18.

Reference is now made to FIG. 4A which is a flowchart diagram of a method suitable for analyzing a biological sample. The method begins at 40 and continues to 41 at which the biological sample is placed on sample region 16 of device 10. In embodiments in which substrate 14 is sealed, the encapsulation is removed prior to the placement of the sample. The method continues to 42 at which device 10 is contacted with at least one label, according to a biological labeling protocol, including, without limitation, histology, histopathology, IHC, and the like. The method optionally and preferably continues to 43 at which the signal generated by the label(s) is captured, for example, using an imaging system.

The present embodiments contemplate any imaging technique. In some embodiments of the present invention, a spectral imaging system is employed. A representative example of a spectral imaging system suitable for the present embodiments is disclosed in U.S. Patent No. 5,539,517, the contents of which are hereby incorporated by reference. This system utilizes the information available from the collected incident light of the image to substantially decrease the required frame time and/or to substantially increase the signal to noise ratio, as compared to slit- or filter- type imaging spectrometer, and does not involve line scanning. In this system the light is passed through an interferometer which outputs modulated light corresponding to a predetermined set of linear combinations of the spectral intensity of the light emitted from each pixel. The light from the interferometer is focused on a detector array, and the optical path difference (OPD) generated in the interferometer is scanned for all pixels.

The method continues to 44 at which the signal received by the label is analyzed.

The analysis can be done by an expert, such as, but not limited to, as a trained pathologist that can inspect the substrate or its image, or it can be done by a data processor configured for performing analysis. For example, the data processor can be supplemented by a computer software product comprising a computer-readable medium in which program instructions are stored, which instructions, when read by the data processor, cause the data processor to receive an image of the substrate and perform the analysis. When the analysis is executed by the data processor, the term "image" is to be understood as values (grey-levels or intensities) at picture elements, treated collectively, as an array. Thus, the term "image" as used herein includes a collection of picture- elements, and does not necessarily correspond to a physical image, although the imagery data certainly do correspond to a physical image.

When the analysis is executed by the data processor, the analysis typically, but not necessarily, comprises piecewise histogram normalization of signal received from sample region 16 based on signal received from reference region 18. When one or more of the properties of substance 20 varies across region 18, the piecewise histogram normalization is optionally and preferably also performed with respect to each of the sub-regions of reference region 18.

The normalization can be according to calibration data that describes the relation between a concentration of substance 20 and the signal affected by the label(s) responsively to substance 20. For example, the calibration data can be a calibration curve, describing the signal (expressed, for example, as an optical density measure) as a function of the concentration. The calibration data can be contained in a medium that is provided together with device 10, for example, as a kit.

The medium containing the calibration data can, in some embodiments of the present invention, be a computer readable medium, such as, but not limited to, a flash memory device, an optical disk (CD ROM) or the like. The calibration data can also be provided in the form of downloadable data, wherein the kit packaging includes device 10 (optionally and preferably encapsulated by encapsulation 22) and a code or a barcode or any other type of permission for downloading the calibration data from the internet. The code can be in the form of a sticker attached to the packaging of the kit or to encapsulation 22. The calibration data can alternatively or additionally be provided as a printed material contained in the kit packaging. A representative example of a calibration curve prepared according to some embodiments of the present invention is provided in the Examples section that follows.

The normalization is optionally and preferably with respect to the intensity of the picture-elements (e.g., pixels, groups of pixels) of the obtained image. In various exemplary embodiments of the invention the intensity of each color channel (e.g., a red channel, a green channel and a blue channel) is normalized separately. The color channels can be provided separately to the data processor or the data processor can decompose the image according to the wavelength prior to the normalization. For example, it is oftentimes desired to perform the analysis separately for each of the colors that the labels produce, rather than for generic colors (e.g. , red, green and blue). In these embodiments the data processor can use the colors produced by the labels as the basis according to which the decomposing is performed.

In some embodiments of the present invention the method receives a characteristic signal profile from an external source, wherein the analysis is at least partially based on the received profile. The external source can be any source other than the labels themselves. For example, the external source can be a computer readable medium, or provided in the form of downloadable data, as further detailed hereinabove.

In some embodiments of the present invention the external source is a database including history data pertaining to the signal profiles, e.g. , the shapes of the histograms, obtained in the past during assays performed in a particular laboratory or a particular group of laboratories. The database can include an entry regarding the obtained signal profiles for each of a plurality of laboratories or a plurality of groups of laboratories. When such database is employed the characteristic signal can be the result of a supervised or unsupervised machine learning procedure (for example, a neural network algorithm) which determines the expected signal profile based on the history of signal profiles for each laboratory or for each group of laboratories. The characteristic signal can be used in the analysis of signals received from the labels. For example, if the histogram is expected to have a particular shape (for example, a single peak histogram, a double peak histogram, a Gaussian histogram, a Lorentzian histogram), the normalization can attempt to reshape the signal from sample region 16 to that particular shape, using the calibration data.

Also contemplated are embodiments in which a calibration dataset is prepared in advance for a particular characteristic signal profile. In these embodiments, it is not necessary for the method to receive the characteristic profile separately from the calibration data, since the calibration data already ensures that the normalization is based, at least in part, on the characteristic profile. In various exemplary embodiments of the invention a plurality of calibration datasets are prepared in advance, one calibration dataset for each characteristic signal profile. When the kit of the present embodiments is distributed to a receiving entity (e.g. , a user, a laboratory) for which the characteristic signal profile is known, the kit includes the calibration dataset that has been prepared based on this characteristic signal profile.

A representative example of a protocol that can be employed for making a record of a characteristic signal profile, which is not to be considered as limiting, is as follows. A staining protocol with a specific label (e.g. , estrogen receptor), is applied to substrate 14, including the immobilized reference substance 20 and/or chemical array and/or electronic circuitry 24, but without sample 12. A signal (e.g. , an optical signal) is then collected from reference substance 20 and/or chemical array and/or electronic circuitry 24, and a record of this signal is stored in a medium, such as, but not limited to, a computer readable medium, wherein the record is associated with the employed staining protocol and label, thereby making the record specific to that staining protocol and that label. Optionally and preferably the record is also associated with the laboratory at which the staining protocol is employed thereby making the record specific to that laboratory. The medium storing the record can then be used for the calibration. A representative example of such a record is shown in FIG. 7 of the Examples section that follows.

The analysis typically includes displaying a synthesized normalized image obtained by normalizing the original image of the sample using the calibration data, such as the calibration plot shown in Fig 7. The analysis can optionally and preferably include use of other relevant data (e.g. , images of different stains) that can be coupled to the synthesized normalized image, and/or be analyzed to provide a score for the sample. The other relevant data can be presented side by side with the synthesized normalized image or they can be used to mask the synthesized normalized image.

Once the analysis is completed the method proceeds to 45 at which a report regarding the analysis is issued. The report can be transmitted to a computer readable medium in a local or remote location, displayed on a display device and/or transmitted to a printer to be provided as a printed material.

The method ends at 46.

Reference is now made to FIG. 4B which is a flowchart diagram of a method suitable for analyzing a biological sample. The method begins at 60 and continues to 61 at which the biological sample is placed on a substrate. The method continues to 62 at which a sticker assembly having a reference substance immobilized thereon (for example, sticker assembly 30) is attached to the substrate, optionally and preferably, at a location that is laterally displaced from the sample. When the sticker assembly includes a backing sheet, the backing sheet is separated from the assembly before the attachment 62. In some embodiments of the present invention 61 is executed before 62 and in some embodiments of the present invention 62 is executed before 61. The method continues to 63 at which the biological sample is analyzed. In various exemplary embodiments of the invention the biological sample is analyzed by executing at least one of operations 42-45 as further detailed hereinabove with respect the FIG. 4A. The method ends at 64.

Reference is now made to FIG. 5 which is a flowchart diagram describing a method suitable for fabricating a device for analyzing a biological sample, according to some embodiments of the present invention.

The method begins at 50 and continues to 51 at which a substrate, such as substrate 14 or substrate layer 30 as further detailed hereinabove is provided. The method continues to 52 at which a reference substance, such as substance 20 as further detailed hereinabove is immobilized on a reference region of the substrate. Any immobilization technique can be employed, including, without limitation, chemical attachment, imprinting, physical attachment, adsorption, and the like.

In various exemplary embodiments of the invention the method records the location on the substrate at which the reference substance is immobilized. When the properties of the reference substance vary over the reference region as further detailed hereinabove, the method preferably records the dependence of the properties on the location. The recorded information can be used by aforementioned method 40 for distinguishing between the signals from the reference substance and the signals received from the sample.

Method 50 can also record the shape of the reference substance. When several shapes are immobilized, the method preferably records the location and shape of each immobilized shape.

The method optionally and preferably continues to 53 at which the substrate including the reference substance, but preferably not the sample, is packed in a sealed encapsulation, optionally and preferably disposable encapsulation. As used herein the term "about" refers to ± 10 %.

The word "exemplary" is used herein to mean "serving as an example, instance or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments." Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Labels Suitable for Some Embodiments of the Present Invention

The following lists various labels which can be used to implement the method according to some embodiments of the present invention. Further included is a list of natural cell constituents having a detectable spectral signature which can be co-detected with the stains using the method according to some embodiments of the present invention.

It will be appreciated by one ordinarily skilled in the art that some staining procedures may interfere with others. Therefore the type of stains employed and their sequence of application should be well considered. These considerations can be applied by one ordinarily skilled in the art, knowing the staining procedures.

Immunohisto chemical stains for use in transmittance microscopy.

In principle, any enzyme that (i) can be conjugated to or bind indirectly to (e.g., via conjugated avidin, strepavidin, biotin, secondary antibody) a primary antibody, and (ii) uses a soluble substrate to provide an insoluble product (precipitate) could be used. Such enzymes include, for example, HRP, AP, LacZ and glucose oxidase.

Alkaline phosphatase (AP) substrates include, but are not limited to, AP-Blue substrate (blue precipitate, Zymed catalog p. 61); AP-Orange substrate (orange, precipitate, Zymed), AP-Red substrate (red, red precipitate, Zymed), 5-bromo, 4-chloro, 3-indolyphosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium/ iodonitrotetrazolium (BCIP/INT substrate, yellow- brown precipitate, Biomeda), 5-bromo, 4-chloro, 3-indolyphosphate/nitroblue tetrazolium (BCIP/NBT substrate, blue/purple), 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium/iodonitrotetrazolium (BCIP/NBT/INT, brown precipitate, DAKO, Fast Red (Red), Magenta-phos (magenta), Naphthol AS-BI- phosphate (NABP)/Fast Red TR (Red), Naphthol AS-BI-phosphate (NABP)/New Fuchsin (Red), Naphthol AS-MX-phosphate (NAMP)/New Fuchsin (Red), New Fuchsin AP substrate (red), p-Nitrophenyl phosphate (PNPP, Yellow, water soluble), VECTOR™ Black (black), VECTOR™ Blue (blue), VECTOR™ Red (red), Vega Red (raspberry red color).

Horseradish Peroxidase (HRP, sometimes abbreviated PO) substrates include, but are not limited to, 2,2' Azino-di-3-ethylbenz-thiazoline sulfonate (ABTS, green, water soluble), aminoethyl carbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC, red). Alpha-naphthol pyronin (red), 4-chloro- 1 -naphthol (4C1N, blue, blue-black), 3,3'- diaminobenzidine tetrahydrochloride (DAB, brown), ortho-dianisidine (green), o- phenylene diamine (OPD, brown, water soluble), TACS Blue (blue), TACS Red (red), 3,3',5,5' Tetramethylbenzidine (TMB, green or green/blue), TRUE BLUE™ (blue), VECTOR™ VIP (purple), VECTOR™ SG (smoky blue-gray), and Zymed Blue HRP substrate (vivid blue).

Glucose Oxidase (GO) substrates, include, but are not limited to, nitroblue tetrazolium (NBT, purple precipitate), tetranitroblue tetrazolium (TNBT, black precipitate), 2-(4-iodophenyl)-5-(4-nitorphenyl)-3-phenyltetrazolium chloride (INT, red or orange precipitate), Tetrazolium blue (blue), Nitrotetrazolium violet (violet), and 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, purple). All tetrazolium substrates require glucose as a co-substrate. The glucose gets oxidized and the tetrazolium salt gets reduced and forms an insoluble formazan which forms the color precipitate.

Beta-Galactosidase substrates, include, but are not limited to, 5-bromo-4-chloro- 3-indoyl beta-D-galactopyranoside (X-gal, blue precipitate).

The precipitates associated with each of the substrates listed have unique detectable spectral signatures (components).

Antibody which links heavy metals can be used for immuno staining using reflection contrast, bright-field or dark-field imaging, or electron microscopy. Such heavy metals include, but are not limited to, gold and silver, typically in a colloidal form.

The following references, which are incorporated herein provide additional examples. J.M Elias (1990) Immunohistopathology: A practical approach to diagnosis. ASCP Press (American Society of Clinical Pathologists), Chicago; J.F. McGinty, F.E. Bloom (1983) Double immuno staining reveals distinctions among opioid peptidergic neurons in the medial basal hypothalamus. Brain Res. 278: 145-153; and T. Jowett (1997) Tissue In situ Hybridization: Methods in Animal Development. John Wiley & Sons, Inc., New York; J Histochem Cytochem 1997 December 45(12): 1629-1641.

Histological stains for use in transmittance microscopy:

The following lists some histological stains used in transmitted light microscopy: eosin, hematoxylin, Orange G, Light Green SF, Romanowsky-Giemsa, May-Grunwald, Blue counterstain (Trevigen), ethyl green (CAS), Feulgen-naphthol yellow S, Giemsa, Methylene Blue, Methyl Green, pyronin, Naphthol-yellow, Neutral Red, Papanicolaou stain (which typically includes a mixture of Hematoxylin, Eosin Y, Light Green SF, Orange G and Bismarck Brown, Red Counterstain B (Trevigen), Red Counterstain C (Trevigen), and Sirius Red. DNA ploidy stains for use in transmittance microscopy.

The following lists some DNA ploidy stains used in transmitted light microscopy. Feulgen reagent (pararosanilin), Gallocyanin chrom-alum, Gallocyanin chrom-alum and naphthol yellow S, Methyl green-pyronin Y, Thionin-Feulgen reagent. Immunohistochemical stains for use in fluorescence microscopy.

Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5, VECTOR Red, ELF™ (Enzyme-Labeled Fluorescence), CyO, CyO.5, Cyl, Cyl.5, Cy3, Cy3.5, Cy5, Cy7, FluorX, Calcein, Calcein-AM, CRYPTOFLUOR™'S, Orange (42 kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa), BHMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexa dye family, N-[6-(7-nitrobenz-2-oxa-l, 3-diazol-4- yl)amino]caproyl] (NBD), BODIPY™, boron dipyrromethene difluoride, Oregon Green, MITOTRACKER™ Red, DiOC7(3), DilCi g, Phycoerythrin, Phycobiliproteins BPE (240 kDa) RPE (240 kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, Spectrum Aqua, Spectrum Green, Spectrum Gold, Spectrum Orange, Spectrum Red, NADH, NADPH, FAD, Infra-Red (IR) Dyes, Cyclic GDP-Ribose (cGDPR), Calcofluor White, Tyrosine and Tryptophan.

Histological stains for use in fluorescence microscopy.

4',6-diamidino-2-phenylindole (DAPI), Eosin, Fluorescein isothiocyanate

(FITC), Hoechst 33258 and Hoechst 33342 (two bisbenzimides), Propidium Iodide, Quinacrine, Fluorescein-phalloidin and Resorufin.

DNA ploidy stains for use in fluorescence microscopy.

Chromomycin A 3, DAPI, Acriflavine-Feulgen reaction, Auramine O-Feulgen reaction, Ethidium Bromide, Propidium iodide, high affinity DNA fluorophores such as

POPO, BOBO, YOYO and TOTO and others, Green Fluorescent Protein fused to DNA binding protein, such as histones, ACMA, Quinacrine and Acridine Orange.

Endogenous pigments:

Hemoglobin, myoglobin, porphyrin, hemosiderin and other ferrous pigments, lipofuscin, melanin, neuromelanin, ceroid a fluorescent oxidation product of lipid/protein, carotenoids, pyridine, flavin nucleotides.

The following lists some primary antibodies known to specifically bind their associated cytological markers and which are presently employed as components in immunohistochemical stains used for research and, in limited cases, for diagnosis of various diseases. Anti-estrogen receptor antibody (breast cancer), anti-progesterone receptor antibody (breast cancer), anti-p53 antibody (multiple cancers), anti-Her-2/neu antibody (multiple cancers), anti-EGFR antibody (epidermal growth factor, multiple cancers), anti-cathepsin D antibody (breast and other cancers), anti-Bcl-2 antibody (apoptotic cells), anti- E-cadherin antibody, anti-CA125 antibody (ovarian and other cancers), anti-CA15-3 antibody (breast cancer), anti-CA19-9 antibody (colon cancer), anti-c-erbB-2 antibody, anti-P-glycoprotein antibody (MDR, multi-drug resistance), anti-CEA antibody (carcinoembryonic antigen), anti-retinoblastoma protein (Rb) antibody, anti-ras oncoprotein (p21) antibody, anti-Lewis X (also called CD15) antibody, anti-Ki-67 antibody (cellular proliferation), anti-PCNA (multiple cancers) antibody, anti-CD3 antibody (T-cells), anti-CD4 antibody (helper T cells), anti-CD5 antibody (T cells), anti-CD7 antibody (thymocytes, immature T cells, NK killer cells), anti-CD8 antibody (suppressor T cells), anti-CD9/p24 antibody (ALL), anti-CDIO (also called CALLA) antibody (common acute lymphoblasic leukemia), anti-CDl lc antibody (Monocytes, granulocytes, AML), anti-CD13 antibody (myelomonocytic cells, AML), anti-CD14 antibody (mature monocytes, granulocytes), anti-CD15 antibody (Hodgkin's disease), anti-CD 19 antibody (B cells), anti-CD20 antibody (B cells), anti-CD22 antibody (B cells), anti-CD23 antibody (activated B cells, CLL), anti-CD30 antibody (activated T and B cells, Hodgkin's disease), anti-CD31 antibody (angiogenesis marker), anti-CD33 antibody (myeloid cells, AML), anti-CD34 antibody (endothelial stem cells, stromal tumors), anti-CD35 antibody (dendritic cells), anti-CD38 antibody (plasma cells, activated T, B, and myeloid cells), anti-CD41 antibody (platelets, megakaryocytes), anti- LCA/CD45 antibody (leukocyte common antigen), anti-CD45RO antibody (helper, inducer T cells), anti-CD45RA antibody (B cells), anti-CD39, CD100 antibody, anti- CD95/Fas antibody (apoptosis), anti-CD99 antibody (Ewings Sarcoma marker, MIC2 gene product), anti-CD106 antibody (VCAM-1; activated endothelial cells), anti- ubiquitin antibody (Alzheimer's disease), anti-CD71 (transferrin receptor) antibody, anti- c-myc (oncoprotein and a hapten) antibody, anti-cytokeratins (transferrin receptor) antibody, anti-vimentins (endothelial cells) antibody (B and T cells), anti-HPV proteins (human papillomavirus) antibody, anti-kappa light chains antibody (B cell), anti-lambda light chains antibody (B cell), anti-melanosomes (HMB45) antibody (melanoma), anti- prostate specific antigen (PSA) antibody (prostate cancer), anti-S-100 antibody (melanoma, salvary, glial cells), anti-tau antigen antibody (Alzheimer's disease), anti- fibrin antibody (epithelial cells), anti-keratins antibody, and anti-Tn-antigen antibody (colon carcinoma, adenocarcinomas, and pancreatic cancer). Immobilized Substances Suitable for Some Embodiments of the Present Invention

The following lists various substances which can be immobilized, directly or indirectly, on the substrate of the present embodiments. Diseases that correlate to the respective substance are provided in parentheses.

Estrogen receptor (breast cancer), progesterone receptor (breast cancer), p53 (multiple cancers), Her-2/neu (multiple cancers), EGFR (epidermal growth factor, multiple cancers), cathepsin D (breast and other cancers), Bcl-2 (apoptotic cells), E- cadherin , CA125 (ovarian and other cancers), CA15-3 (breast cancer), CA19-9 (colon cancer), c-erbB-2 , P-glycoprotein (MDR, multi-drug resistance), CEA (carcinoembryonic ), retinoblastoma protein (Rb) , ras oncoprotein (p21) , Lewis X (also called CD15) , Ki-67 (cellular proliferation), PCNA (multiple cancers) , CD 3 (T-cells), CD4 (helper T cells), CD5 (T cells), CD7 (thymocytes, immature T cells, NK killer cells), CD 8 (suppressor T cells), CD9/p24 (ALL), CD10 (also called CALLA) (common acute lymphoblasic leukemia), CDl lc (Monocytes, granulocytes, AML), CD13 (myelomonocytic cells, AML), CD14 (mature monocytes, granulocytes), CD15 (Hodgkin's disease), CD 19 (B cells), CD20 (B cells), CD22 (B cells), CD23 (activated B cells, CLL), CD30 (activated T and B cells, Hodgkin's disease), CD31 (angiogenesis marker), CD33 (myeloid cells, AML), CD34 (endothelial stem cells, stromal tumors), CD35 (dendritic cells), CD38 (plasma cells, activated T, B, and myeloid cells), CD41 (platelets, megakaryocytes), LCA/CD45 (leukocyte common ), CD45RO (helper, inducer T cells), CD45RA (B cells), CD39, CD100 , CD95/Fas (apoptosis), CD99 (E wings Sarcoma marker, MIC2 gene product), CD 106 (VCAM-1; activated endothelial cells), ubiquitin (Alzheimer's disease), CD71 (transferrin receptor) , c-myc (oncoprotein and a hapten) , cytokeratins (transferrin receptor) , vimentins (endothelial cells) (B and T cells), HPV proteins (human papillomavirus) , kappa light chains (B cell), lambda light chains (B cell), melanosomes (HMB45) (melanoma), prostate specific (PSA) (prostate cancer), S-100 (melanoma, salvary, glial cells), tau (Alzheimer's disease), fibrin (epithelial cells), keratins , and Tn- (colon carcinoma, adenocarcinomas, and pancreatic cancer).

Immunohistochemistry Staining Variations

FIGs. 6A-C show IHC staining variations that result from different staining procedures. The procedures employ estrogen receptor using the Dako protocol and cell cultures (see, e.g., ER/PR pharmDxTM Interpretation Manual, by Dako, 2007).

FIG. 6A shows inadequate staining intensity, wherein the intensity of the DAB stain (brown) in cells is below the normal range; FIG. 6B shows adequate staining intensity, wherein all relevant cells within the sample have been properly stained with DAB ; and FIG. 6C shows inadequate staining intensity, wherein the intensity of the DAB stain (brown) in cells is above the normal range.

FIGs. 8A-D show HER2/Neu staining of breast ductal carcinoma using various procedures, including PATHWAY® rmAb clone 4B5 (Ventana) and the CE IVD labelled kit Oracle™ (Leica), from serial tissue sections (see, e.g., PATHWAY HER- 2/neu, by Ventana Medical Systems, Inc, 2013). FIGs. 8A and 8C shows adequate staining, wherein most of the relevant features within the sample are properly stained; and FIGs. 8B and 8D show inadequate staining, wherein several features within the sample are not stained.

Exemplified Calibration Curve

HER2/neu protein was as a reference substance and was further used for calculating a calibration curve describing the optical density as a function of the concentration. The reference substance was embedded on a surface of a PVDF porous membrane, approximately 100 microns in thickness, and approximately 0.45 microns in pore size. The PVDF membrane was physically attached to a microscope slide using an adhesive tape.

HER2/neu protein was serially diluted to the following concentrations: 1 ppm, 2 ppm, 5 ppm 10 ppm, 20 ppm and 100 ppm. 3 microliter spots of protein solution of the various concentrations were loaded onto the membrane. This way, six protein spots representing different concentrations (or amount) of protein were embedded on the PVDF membrane, as the basis for the calibration plot. Each membrane was analyzed using the following IHC protocol: the membrane was stained with human Erb2/Her2 biotinylated antibody, followed by horseradish peroxidase and 3,3 Diaminobenzidine (DAB) according to protocols by abeam ® and R&D systems ®. Following staining, protein spots appeared as brown dots on the membrane surface. The membrane was imaged with BF microscopy using a 4X magnification, and absorbance was calculated based on transmittance measurements using an empty region as reference (10).

FIG. 7 is a graph showing the optical density (OD units) as a function of the concentration of the substance. Such graph can be used as a calibration curve according to some embodiments of the present invention for normalizing the signal received from the sample.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A device for analyzing biological sample, comprising,

a substrate having thereon a sample region for carrying the sample and a reference region; and

a reference substance being immobilized on said reference region, and being bindable to at least one label that is specific to at least one component of the sample.

2. The device according to claim 1, wherein said substrate and said reference substance is packed in a sealed encapsulation devoid of the sample.

3. The device according to any of claims 1 and 2, wherein said substrate is selected from the group consisting of a microscope slide, a fluid chamber or channel of a microfluidic device and titer plate and in general any biological- sample mount.

4. A device for use in an analysis of a biological sample using at least one label being specific to at least one component of the sample, comprising:

a sticker assembly having a substrate layer;

a reference substance being immobilized on a first side of said substrate layer and being bindable to said at least one label; and

an adhesive layer at least partially coating a second side of said substrate layer.

5. A kit for analyzing biological sample, comprising: the device according to any of claims 1-4, and a medium containing calibration data describing a relation between a concentration or amount of said reference substance and a signal effected by said at least one label responsively to said reference substance.

6. A method of analyzing a biological sample, comprising,

placing the biological sample on the sample region of the device according to any of claims 1-3;

contacting the device with said at least one label; and

analyzing a signal effected by said at least one label.

7. A method of analyzing a biological sample, comprising, placing the biological sample on a sample region of a substrate;

attaching the device according to claim 4 to a reference region of said substrate; contacting the substrate with said at least one label; and

analyzing a signal effected by said at least one label.

8. The method according to any of claims 6 and 7, further comprising capturing said signal using an imaging system, prior to said analysis.

9. The method according to any of claims 6-8, wherein said analysis of said signal is by a data processor configured for performing said analysis.

10. The method according to any of claims 6-9, wherein said analysis comprises piecewise histogram normalization of signal received from said sample region based on signal received from said reference region.

11. The method according to any of claims 6-10, wherein said signal comprises a plurality of color channels and wherein said analyzing is executed separately for each of at least two of said color channels.

12. The method according to any of claims 6-11, further comprising receiving a characteristic signal profile from a source other than said substrate, wherein said analysis is based in part on said characteristic signal profile.

13. A method of fabricating a device for analyzing a biological sample, comprising,

immobilizing on a substrate a reference substance being bindable to at least one label that is specific to at least one component of the sample, said substrate having thereon a sample region for carrying the sample and a reference region, and said reference substance being immobilized on said reference region; and

packing said substrate but not the sample in a sealed disposable encapsulation.

14. A method of fabricating a device for analyzing a biological sample, comprising, immobilizing a reference substance on a first side of substrate layer having, on a second side thereof, an adhesive layer at least partially coating said second side, wherein said reference substance is bindable to at least one label that is specific to at least one component of the sample.

15. The device, kit or method according to any of claims 1-13, wherein said reference substance is chemically attached to said reference region.

16. The device, kit or method according to any of claims 1-13, wherein said reference substance is imprinted on said reference region.

17. The device, kit or method according to any of claims 1-13, wherein said reference substance is embedded in a structure immobilized on said reference region.

18. The device, kit or method according to claim 17, wherein said structure is a porous structure.

19. The device, kit or method according to claim 17, wherein said structure is a non-porous structure.

20. The device, kit or method according to claim 17, wherein said structure is a membrane structure.

21. The device, kit or method according to claim 17, wherein said structure is a powder.

22. The device, kit or method according to claim 17, wherein said structure comprises gel.

23. The device, kit or method according to claim 17, wherein said structure is an artificial tissue.

24. The device, kit or method according to any of claims 1-18, wherein said reference substance is a reference tissue identified as having at least one pathology.

25. The device, kit or method according to claim 24, wherein said reference tissue comprises at least two regions having different histological scores for said pathology.

26. The device, kit or method according to any of claims 1-25, further comprising a chemical, biological and/or physical array being immobilized on said substrate and configured for providing signals indicative of each of a plurality of labels or label concentrations contacting said chemical, biological and/or array.

27. The device, kit or method according to any of claims 1-26, further comprising electronic circuitry being immobilized on said substrate and configured for generating electric or magnetic signals indicative of each of a plurality of labels or label concentrations contacting said electronic circuitry.

28. The device, kit or method according to claim 27, wherein said electronic circuitry is configured for recording said electric or magnetic signals.

29. The device, kit or method according to any of claims 1-28, wherein a concentration of said reference substance varies across said reference region.

30. The device, kit or method according claim 29, wherein said concentration varies continuously.

31. The device, kit or method according claim 29, wherein said concentration varies discretely.

32. The device, kit or method according to any of claims 1-28, wherein said reference region comprises a plurality of wells each containing a different reference substance or a different concentration of reference substance.

33. The device, kit or method according to any of claims 1-28, wherein said reference region and said sample region engage different planes.

34. The device, kit or method according to claim 33, wherein said substrate is thinner at said reference region than at said sample region.

35. The device, kit or method according to any of claims 1-28, wherein said reference substance is hydrophilic.

36. The device, kit or method according to any of claims 1-28, wherein said reference substance is hydrophobic.

37. The device, kit or method according to any of claims 1-35, wherein said reference substance comprises a naturally occurring compound.

38. The device, kit or method according to any of claims 1-37, wherein said reference substance comprises a synthetic compound.

39. The device, kit or method according to any of claims 1-38, wherein said reference substance comprises an organic compound.

40. The device, kit or method according to any of claims 1-39, wherein said reference substance comprises an inorganic compound.

41. The device, kit or method according to any of claims 1-40, wherein said reference substance comprises a biological cell or tissue.

42. The device, kit or method according to any of claims 1-38, wherein said reference substance comprises at least one molecule selected from the group consisting of a polymer, a protein, a protein L, a polysaccharide, an antibody, , an antigen, a peptide, a dipeptide, a polypeptide, and an aptamer.

43. The device, kit or method according to any of claims 1-42, wherein said label comprises at least one substance selected from the group consisting of a direct immunohistochemical stain, a secondary immunohistochemical stain, a histological stain, a immunofluorescence stain, a DNA ploidy stain, a nucleic acid sequence specific probe, a dye, an enzyme, a nanoparticle and any combination thereof.