Method and device for determining the layer thickness of histological sectional specimens and determining the concentration of biological substances in histological specimens

Abstract

The invention relates to a method for determining a layer thickness of a sectional specimen in medical histology. In a further aspect, the invention relates to a method for determining the concentration of a target substance in a tissue contained in the sectional specimen, in particular for the diagnostic determination of the concentration in medical sectional specimens. In a further aspect, the invention relates to a device for measuring the layer thickness of a sectional specimen and optionally for determining the concentration of a target substance in the sectional specimen.

Description

[0001] Method and device for determining the layer thickness of histological sections and for determining the concentration of biological substances in histological sections

[0002] DESCRIPTION

[0003] The invention relates to the fields of medicine, diagnostics, histology (or section preparations of cell blocks), staining and evaluation of tissue sections, and the concentration measurement of target substances in tissue sections. The invention particularly relates to the field of medical histology and the diagnostic concentration measurement of target substances in tissue sections.

[0004] The invention relates to a method for determining a layer thickness of a section preparation, comprising (a) measuring values ​​generated by scattering and / or absorption by the section preparation, (b) providing a model of the layer thickness of the section preparation based on calibration values, and (c) determining the layer thickness of the section preparation by using the measured values ​​and the modeling according to (b).The invention preferably relates to a method for determining a layer thickness of a section preparation, comprising (a) the measurement of values ​​which were generated by scattering and / or absorption of a light beam through the section preparation with one or more light sources of different wavelengths, and (b) the provision of a model of the layer thickness of the section preparation based on calibration values ​​which were generated by measuring the scattering and absorption of a light beam through one or more calibration preparations with one or more light sources of different wavelengths, and (c) the determination of the layer thickness of the section preparation by using the measured values ​​according to (a) and a model according to (b).

[0005] In a further aspect, the invention relates to a device for measuring the layer thickness of a section preparation and optionally for determining the concentration of a target substance in the section preparation, comprising (a) one or more light sources of different wavelengths, (b) at least one sensor for measuring the scattering and / or absorption of a light beam, (c) a device for positioning the section preparation during the measurement, and (d) a computing unit for modeling the layer thickness of a section preparation or for using the modeling in determining the layer thickness of a section preparation.

[0006] In a further aspect, the invention relates to a slide for use in the method or device, comprising a calibration preparation containing a target substance in two or more spatially separated concentrations, to which the histological section preparation can additionally be applied. BACKGROUND AND STATE OF THE ART

[0007] Histopathology involves the examination of tissue sections, such as biopsies, using special staining techniques and is an important diagnostic tool in medicine. It is used, for example, to assess malignancy and early diagnosis of tumors, to detect metabolic diseases, parasitic bacterial, and inflammatory diseases, and thus to guide decisions regarding treatment selection.

[0008] Since its establishment approximately 150 years ago, the method of histopathology has involved obtaining and preparing sections of human or animal tissue, and then using dyes to visualize the tissue structures or components for microscopic examination. The process of obtaining, preparing, and staining the sections is referred to as histology, and the resulting sections are known as histological specimens.

[0009] Four steps that are common to all procedures of classical histology and histopathology are (i) preparation including fixation and embedding or freezing, (ii) sectioning, (iii) staining and (iv) microscopic assessment of the sample.

[0010] (i) Various types of histological specimens are known, each requiring different preparation techniques, such as paraffin-embedded specimens or frozen section specimens. The choice of histological specimen and the associated preparation steps depend on the properties of the tissue, the structures to be examined within the tissue, their stability during dissection and preparation, as well as the clinical question and urgency. Paraffin-embedded specimens are generally used, whereby the tissue to be examined is fixed in formalin and embedded in paraffin.

[0011] (ii) In the next step, the embedded or frozen tissue is usually sectioned manually on a microtome. The section thickness is mechanically adjusted to a thickness between 1 and 9 μm, with 1 to 5 μm being particularly common. The section curls considerably during sectioning and is further influenced by the tissue it contains. The section is then stretched on a water surface and from there transferred to a microscope slide for further processing and examination. The precision of these devices varies greatly and is also subject to significant fluctuations due to manual operation. The fluctuations in the actual section thickness of the sectioned histological specimen can be more than 200%. In addition, the section thickness set on the microtome often differs considerably from the actual section thickness of the specimen. This was investigated, for example, in a study by Masuda et al., 1998.In the study, cross-sections of numerous paraffin sections prepared using routine methods with a layer thickness of 3 μm and greater were examined using a confocal laser scanning microscope. The thinner sections, in particular, showed a significant upward deviation from the microtome's preset setting. The layer thickness has a significant influence on the intensity of the staining, as thicker sections often exhibit more intense staining, or the staining process itself can be influenced by the layer thickness. A significant deviation from the preset layer thickness is therefore particularly problematic for subsequent staining, the assessment of the staining, and the resulting histopathologist's findings.

[0012] (iii) The sectioned specimen, mounted on a slide, is then stained with various dyes. Staining of these sections is necessary for assessment by the histopathologist, as the unstained components of the cells and tissue are difficult to distinguish under the light microscope. The goal of staining is therefore to create a visual distinction between the tissue components. The standard procedure is double staining with hematoxylin and eosin, which stains the basic components of the cell (nucleus and cytoplasm). For special structures, such as connective tissue fibers, additional special stains (e.g., Goldner stain) are used. Another fundamental advance was the introduction of immunohistochemistry. In this method, antibodies bind specifically to selected target proteins under investigation.These specifically bound antibodies can be visualized on the microscope using a detection system that binds to the antibodies, dyes in the visible wavelength range or fluorescent dyes.

[0013] (iv) The subsequent evaluation of the stained histological section is subject to the experience of the assessing histopathologist, who performs a subjective assessment of the sections. The histopathologist typically assesses the morphological appearance of the specimen, the presence, location, and staining of specific structures, and the intensity of staining. The assessment of staining intensity is usually qualitatively or semi-quantitatively by estimating and classifying it as "strong," "moderate," "weak," or "negative," as well as the distribution of stained structures in the section.

[0014] To date, a finer grading using more detailed scores has only been performed for individual diseases and associated target structures and specific proteins. This is then done according to a defined evaluation system, in which various individual criteria are often combined to produce a score. Examples include the immunoreactive score 0-12 for estrogen receptors and progesterone receptors in breast cancer, scores for Her2neu, the proliferation-associated antigen Ki 67, the protein c-Myc, PD-L1, and others. More precise quantification of these factors is so important because they are used to determine a prognosis group and also therapeutic decisions. Therefore, there is a need for precise and objective quantification, especially for such target structures and substances. However, these scores are also based on the subjective assessment and experience of the histopathologist.A quantitative measurement of a substance concentration in a histological specimen is not yet possible in everyday clinical practice, but is generally of great interest in order to enable more precise assessment and diagnostics as a basis for, for example, treatment decisions. To date, the quantitative determination of substance concentrations has required the use of clinical chemistry methods in clinical settings. In contrast to histology, clinical chemistry offers the possibility of quantifying target substances in biological material using highly precise methods. It predominantly involves analyzing liquid samples (blood, blood serum, and other body fluids). For specific protein determination, an immunoassay is usually carried out. The protein is bound to an antibody specific for this protein. This antibody can be coupled to an enzyme (ELISA), which produces color from a substrate.The color density can be measured photometrically, thus assigning a concentration of the bound protein. However, this does not allow for localization of the substance in the tissue; only the determination of an average value of the concentration of the target substance in the biological sample.

[0015] While the steps of embedding and staining can now be largely automated, especially in clinical settings, and are therefore subject to minor variations, the above points make it clear that the steps of sectioning the sample on the microtome and the assessment of the sample by the histopathologist are not yet automated and standardized, making a precise and quantitative assessment of histological sections difficult.

[0016] Various approaches have been investigated to enable a more objective assessment of histological specimens. Various approaches to standardization have already been undertaken, particularly with regard to the assessment of histological specimens, images taken from histological specimens, and the determination of target substances in histological specimens.

[0017] In recent years, it has become possible to scan histological sections by combining special microscope optics with an image sensor, creating a high-resolution image of the entire specimen from a multitude of individual images. This so-called slide scan can be used instead of viewing the section under a microscope and is gradually finding its way into routine histopathology. However, even the slide scan has so far been evaluated subjectively by the histopathologist through the assignment of scores. The evaluation of the scan using image analysis and image measurement algorithms, including those using artificial intelligence (AI), is under development and already partially in use, although the focus remains on evaluating only relative color differences.

[0018] Nguyen et al., 2013 discloses the determination of a relative staining intensity by measuring the optical density and then subtracting the intensity of the stained area from the maximum intensity.

[0019] Choudhury et al., 2009, discloses the use of an ATM (Average Threshold Measure) score to automatically set thresholds for positive staining in immunohistochemistry. This also demonstrated good repeatability and comparability, but does not allow for an absolute measurement of protein concentration. Jensen et al., 2017, discloses an immunohistochemistry method for the determination of Her2neu protein based on an amplification system that allows the detection of a protein in fixed and paraffin-embedded tissue by counting spots in an image analysis. However, this method cannot measure individual components of the cell (particularly cytoplasm and nucleus). The result is also presented as a histoscore. The so-called histoscore is based on the staining density and the percentage of stained cells and consists of four categories: negative (0), weak (1+), moderate (2+), and strong (3+).

[0020] Quantitative methods for determining an absolute substrate concentration in a histological preparation are disclosed in US 2004 / 0253649 A1 and US 5610022.

[0021] US 2004 / 0253649 A1 discloses a method for quantifying cellular proteins in tissue samples using a cell imaging densitometer in conjunction with immunohistological staining and a reference standard that is stained in the same way as the actual tissue sample. The method enables the quantification of a target protein in terms of the absolute amount of the protein or the number of protein molecules per cell / volume / fraction in a tissue sample that expresses the target protein.

[0022] US 5610022 also discloses an internal control that can be used for quantifying substance concentrations in tissue samples using immunohistochemistry. The control is treated under the same conditions as the sample, including immunostaining of the tissue sample. The immunoreactivity of the control and the sample is compared before and after processing.

[0023] The aforementioned methods, particularly those described in US 2004 / 0253649 A1 and US 5610022, enable a more precise, semi-quantitative or quantitative determination of substance concentrations in histological specimens. However, the large fluctuations in slice thickness during manual sectioning on a microtome, with fluctuations of up to 200% in the actual slice thickness, are not taken into account, but they have a significant impact on the color intensity of the staining, which forms the basis for the substance concentration determination in the respective methods. The fluctuations in the actual slice thickness thus significantly impair the accuracy and precision of the respective methods.

[0024] EP 1200799 B1 discloses a method and an apparatus for determining the thickness of thin films, which is based on ellipsometric measurement of the rotation of polarized light reflected by the thin film.

[0025] US 2013 / 0314712 A1 also discloses a device and a method for determining the optical properties of a thin film, in which the thin film is simultaneously irradiated with light of different wavelengths and interference effects are measured. Reflection and transmission coefficients are then calculated and compared with calibration values. Based on this, the thickness and concentration of analytes in the thin film can be determined in an image of the thin film by a grayscale analysis of the pixels of an image of the thin film and comparison with calibration or reference values. However, a concrete method for determining the concentration or a targeted analysis of selected areas of the thin film is not disclosed. Matenaers et al.(PLoS ONE 13 (2), 2018, p. 1-21; e0192879) discloses the determination of the layer thickness of a histological specimen using a reflectometer (measurement of spectral reflectance as a function of wavelength) as an alternative to the orthogonal reembedding technique (ORE) commonly used in histology. However, the determination of layer thickness using reflection is only described for hard sections such as synthetic resin, and not for paraffin sections. However, paraffin sections represent the majority of sections prepared in histology. At the same time, in contrast to synthetic resin, for example, paraffin sections exhibit low homogeneity, which can sometimes severely impair the determination of layer thickness using reflection. The applicability of this method to paraffin sections is therefore questionable.

[0026] Overall, all these methods for measuring section thickness are based either on surface measurement alone or on reflection and transmission. None of the methods investigates or considers the specific properties of paraffin layers, whose structure is more complex than, for example, the structure of hard sections such as synthetic resin. This complex structure can cause multiple optical effects to overlap, significantly complicating and impairing the determination of layer thickness using conventional optical methods.

[0027] US 2006 / 0154234 A1 describes standardization and quantification methods for histology, immunohistochemistry, and cytology. A reference standard for cytology and histology is disclosed, which contains an embedding medium and a compact particle, such as a cell or a non-biological particle with cell-like dimensions. The cell or particle contains a known amount of a substance (e.g., a protein or nucleic acid) that is to be quantitatively determined in the actual sample. However, the actual section thickness of the histological specimens is not examined when determining the substance concentration. Furthermore, individual images of the section are used for the concentration determination, which may exhibit inhomogeneity in the exposure and thus impair the accuracy of the concentration determination.Furthermore, as explained above, only defined particles are measured, not the entire or any desired area of ​​the section. To enable accurate and precise determination of substance concentrations in combination with the localization of the substance in histological specimens, and thus a sound basis for diagnosis and treatment decisions based on these specimens, there is a need to further develop and automate the histological procedure. This further development is particularly necessary with regard to those steps of the procedure that are subject to strong, including local, fluctuations, such as the actual layer thickness of the specimens to be examined. SUMMARY OF THE INVENTION

[0028] The object of the present invention was to eliminate the disadvantages of the prior art and to provide a method that enables the accurate and precise determination of substance concentrations and the localization of substances in section preparations. One object of the present invention was to provide a method that determines the actual layer thickness of a section preparation and a calibration preparation. A further object of the present invention was to provide a correction factor for the actual local or spatially varying layer thickness of the section preparation, which can be used for the accurate determination of a substance concentration in the section preparation. Furthermore, one object of the present invention was to provide a calibration preparation that contains the target substance to be determined and can be used for quantifying the substance concentration in the section preparation.Additionally, the present invention aimed to provide a device for determining the thickness of a section. Furthermore, the present invention aimed to provide a microscope slide that can be used in the method and device according to the invention and on which both the section and the calibration slide can be applied and stained simultaneously.

[0029] The object of the invention is achieved by the features of the independent claims. Advantageous embodiments of the invention are described in the dependent claims.

[0030] In one aspect, the invention relates to a method for determining a layer thickness of a section preparation, comprising (a) measuring one or more values ​​generated by scattering and / or absorption of a light beam through the section preparation with one or more light sources of different wavelengths, (b) providing a model of the layer thickness of the section preparation based on calibration values ​​generated by measuring the scattering and / or absorption of a light beam through one or more calibration preparations with one or more light sources of different wavelengths, and (c) determining the layer thickness of the section preparation by using the measured values ​​according to (a) and a model according to (b).

[0031] A major advantage of the method according to the invention is that it enables simple and precise determination of the actual slice thickness of a section. Sections are typically prepared by cutting on a microtome. The actual slice thickness can deviate considerably from the set slice thickness, particularly at low slice thicknesses. The slice thickness can have a significant impact on the subsequent staining and assessment by the histopathologist or the quantification of target substances in the section. Common methods for determining the slice thickness of a section involve complex and time-consuming microscopic procedures such as the use of a laser scanning microscope.It is therefore a particular advantage of the method according to the invention that the actual slice thickness of a slice can be determined quickly and without the use of special microscopes or special knowledge on the part of the user by measuring the scattering and / or absorption of a slice. The provision of a model of the slice thickness through the use of calibration preparations and calibration values ​​based thereon enables broad application of the method, since the actual slice thickness of each slice present can be directly determined based on the modeling and the measured values. Furthermore, in contrast to microscopic methods, the method enables high-throughput determination of the actual slice thickness of slices, making it particularly advantageous for use in everyday clinical practice where a large quantity of slices is generated.

[0032] Measuring scattering and / or absorption proves particularly advantageous for the method according to the invention, since the measured scattering and / or absorption values ​​exhibit a high sensitivity with respect to the layer thickness. By measuring scattering and / or absorption, in contrast to other optical effects such as partial transmission and, in particular, reflection, it is possible to precisely and accurately determine the actual layer thickness of the section specimen over a broad wavelength range, thus increasing the precision and accuracy of the assessment of section specimens and the determination of the concentration of target substances in these specimens.Measurement using scattering and / or absorption particularly enables the measurement of the layer thickness of sections with an irregular structure and surface, such as paraffin sections, which represent a large proportion of the sections used in histology. For example, measuring the transmittance alone is not suitable for determining the layer thickness of such sections with an irregular structure, as several optical effects overlap to varying degrees, so that no continuous relationship between layer thickness and transmittance can be established.

[0033] In a further embodiment, the method is characterized in that the light source(s) have a wavelength of 200 to 2000 nm, preferably 300 to 1800 nm, particularly preferably 400 to 1700 nm. In a further preferred embodiment, the light source(s) may have a wavelength of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 nm or other wavelengths in this range which achieve the desired properties of the present invention.

[0034] In a preferred embodiment, the method is characterized in that the modeling of the layer thickness comprises (a) providing the calibration values ​​determined by measuring the scattering and / or absorption of a light beam by one or more calibration preparations with one or more light sources of different wavelengths, wherein the calibration preparations have a known layer thickness of 0.1 to 10 pm, preferably a known layer thickness of 1 to 7 pm, (b) determining one or more calibration functions, wherein the calibration function comprises the correlation of the layer thickness of the one or more calibration preparations with the calibration values, (c) comparing the measured values ​​of a section preparation with the one or more calibration functions according to (b), preferably taking into account an inaccuracy of the measurement of the values ​​of 5%, (d) outputting a layer thickness of the section preparation,which corresponds to the mean of one or more values ​​which, according to (c), agree with one or more of the calibration functions. In a further preferred embodiment, the light source(s) may have a wavelength of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 nm or other wavelengths in this range that achieve the desired properties of the present invention.

[0035] By providing a model of the actual slice thickness of a section, a simple and broad application of the method according to the invention is possible. In contrast to microscopic methods for determining the actual slice thickness, the method can be applied without special knowledge and with easy-to-operate devices, such as the device according to the invention, and enables a precise and accurate determination of the actual slice thickness and its spatial distribution. Furthermore, the use of modeling in the method according to the invention enables an automated and high-throughput determination of slice thickness, which can be used, particularly in everyday clinical practice, even by personnel without special knowledge.

[0036] Advantageously, a correction factor can be determined for several spatially or locally separated areas of the section preparation by means of the calibration function, which enables locally different corrections and, for example, the creation of a height profile of the section preparation as explained in detail below.

[0037] In a further embodiment, the method is characterized in that the section preparation has a layer thickness of 0.1 to 10 pm, preferably 1 to 7 pm. In a further embodiment, the section preparation can have a layer thickness of 0.1 pm, 0.2 pm, 0.3 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 1.5 pm, 2 pm, 2.5 pm, 3 pm, 3.5 pm, 4 pm, 4.5 pm, 5 pm, 5.5 pm, 6 pm, 6.5 pm, 7 pm, 7.5 pm, 8 pm, 8.5 pm, 9 pm, 9.5 pm, 10 pm or another thickness in this range which achieves the desired properties of the present invention.

[0038] The method of the present invention can advantageously be used for the entire range of slice thicknesses of sections commonly used in histology.

[0039] Surprisingly, the method according to the invention exhibits high sensitivity across the entire range and enables accurate and precise determination of the actual slice thickness. Surprisingly, the method also enables accurate determination of the slice thickness in the slice thickness range of 1 to 2 μm, which is common in histology and for which a particularly high deviation from the slice thickness set on the microtome was observed. Thus, the method according to the invention can be widely used, particularly in histology, and thus enables the accurate assessment and quantification of target substances and, based on this, diagnostics and therapeutic decisions.

[0040] In a further embodiment, the method is characterized in that the section preparation contains tissue.

[0041] In a further embodiment, the method additionally comprises determining the concentration of a target substance in the tissue contained in a section preparation.

[0042] In a further embodiment, the method is characterized in that the determination of the concentration of the target substance comprises: (a) providing a section preparation and a calibration preparation, wherein the calibration preparation comprises a predetermined concentration of the target substance, (b) determining the layer thickness of the section preparation and the calibration preparation, (c) determining a correction factor by using the layer thickness according to (b), (d) simultaneously staining the section preparation and the calibration preparation, preferably on the surface of a slide, (e) measuring the color intensity of the tissue contained in the section preparation and the target substance contained in the calibration preparation, and (f) determining the concentration of the target substance in the tissue by using the color intensity according to (e) and the correction factor according to (c).In a further embodiment, the tissue-free part of the section preparation and / or the calibration preparation containing a dye that absorbs light in a wavelength range of 200 to 2000 nm is covered or separated before staining.

[0043] In a further embodiment, the determination of the layer thickness is carried out only after the simultaneous staining of the section preparation. In a further embodiment, the determination of the locally varying layer thickness is carried out only after the simultaneous staining of the section preparation.

[0044] In a further embodiment, the method is characterized in that the determination of the concentration of the target substance comprises: (a) providing a section preparation and a calibration preparation, wherein the calibration preparation comprises a predetermined concentration of the target substance, and the section preparation and the calibration preparation comprise one or more regions containing a dye that absorbs light in a wavelength range of 200 to 2000 nm, (b) covering or separating the regions of the section preparation and the calibration preparation that contain the dye, (c) simultaneously staining the section preparation and the calibration preparation, preferably on the surface of a slide, (d) determining the layer thickness of the section preparation and the calibration preparation by a method according to one of the preceding claims, (e) determining a correction factor by using the layer thickness according to (d),(f) measuring the color intensity of the tissue contained in the section preparation and the target substance contained in the calibration preparation, and (g) determining the concentration of the target substance in the tissue by using the color intensity according to (f) and the correction factor according to (e).

[0045] The method according to the present invention can advantageously be used to absolutely quantify a target substance in a section preparation. The use of a calibration preparation, which contains a known concentration of the target substance and is stained and measured simultaneously with the section preparation in the method according to the invention, enables the determination of the absolute concentration of the target substance. In combination with the determination of the actual layer thickness of the section preparation and the calibration preparation, which may vary locally, a correction factor (for example, a correction factor area based on a height profile, also called a correction area) is provided, which significantly increases the accuracy and correctness of the concentration determination.In the methods used to date for the absolute concentration determination of a target substance in histological specimens, the actual layer thickness of the sections is not determined, and no correction factor is calculated based on this. However, since the layer thickness of sections such as histological specimens can vary considerably, for example due to the sectioning method (e.g., using a microtome) or the embedding material used (e.g.,Since the concentration (due to local differences in layer thickness, as occurs with paraffin) can thus differ considerably within the section preparation or between the section and the calibration preparation and at the same time has a significant influence on the intensity of the color and thus the determination of the concentration of a target substance, the method according to the invention achieves a significantly increased accuracy and precision in the absolute concentration determination compared to the prior art by determining a correction factor for the layer thickness. A correction of these local or regional differences in layer thickness, as occurs with paraffin, and their consideration in the concentration determination, for example by creating a height profile as described below, has not yet been disclosed in the prior art.

[0046] The method according to the invention thus makes it possible for the first time to perform a precise, absolute quantitative measurement of the concentration of a target substance with simultaneous localization of the target substance in a histological section. Substances, particularly proteins, for which quantitative measurement methods are otherwise available can be objectively quantified by creating a calibration curve using the calibration preparation in comparison with the section preparation. Thus, the method according to the invention combines the high quantitative accuracy of clinical chemistry with the high spatial resolution of histology. The method according to the invention enables localized and precise measurements of concentrations of a target substance in individual cells and even in individual cell components (cell membrane, cytoplasm, nucleus, parts of the cytoplasm or nucleus). This opens up new possibilities, particularly in medical diagnostics and research.By using the method according to the invention, threshold values ​​for diagnostic decisions / therapy decisions can be established and, in research, a high-resolution and absolute quantitative analysis of pathological processes is also enabled.

[0047] The present method can be particularly relevant for diseases with a heterogeneous appearance, such as prostate cancer, in which the prostate-specific antigen (PSA) is produced. PSA can be detected immunohistochemically in the carcinoma and in the blood (serum) using clinical chemistry methods. Elevated blood levels indicate an increased probability of the presence of a carcinoma or metastasis, but can also be elevated in inflammation. Furthermore, expression in an existing tumor varies from individual to individual, is heterogeneous, and currently cannot be precisely quantified. By using the method according to the invention, PSA quantification and localization in the tumor tissue can be achieved, and based on this, individual cutoff values ​​can be determined for each patient. This can replace the cutoff currently statistically determined for all patients.This could significantly improve and individualize prognosis and treatment decisions for this tumor. Similarly, the method can also be applied to other tumor diseases and markers, such as the protein S100, which is expressed in malignant melanomas.

[0048] In a further embodiment, the method is characterized in that the calibration preparation contains the target substance in two or more concentrations which are spatially separated from each other.

[0049] In a further embodiment, the method is characterized in that the layer thickness of the section preparation and the calibration preparation is determined in tissue-free areas of the section preparation and the calibration preparation.

[0050] In a further embodiment, the method is characterized in that the values ​​generated by scattering and / or absorption of a light beam by the section preparation, the calibration preparation, and / or calibration preparation are measured at one or more positions in the tissue-free regions of the preparation, preferably at two or more positions, particularly preferably at two or four positions. In a further embodiment, the method is characterized in that the values ​​generated by scattering and / or absorption of a light beam by the section preparation, the calibration preparation, and / or calibration preparation are measured at two or more positions in the tissue-free regions of the preparation, which are arranged symmetrically around the tissue contained in the section preparation, preferably at opposite tissue-free regions.In a preferred embodiment, the method is characterized in that an average value is calculated from the values ​​measured at two or more positions, which is used to determine the slice thickness of the section. In a further embodiment, a two-dimensional function (correction surface) is determined. Such a correction surface takes into account any local or regional differences in slice thickness that may be present in the section and can thus lead to locally or regionally different correction factors.

[0051] In a further embodiment, the method is characterized in that the tissue-free areas of the section preparation, the one or more calibration preparations and / or the calibration preparation comprise one or more areas which contain a dye which absorbs light in a wavelength range from 200 to 2000 nm, for example 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1050 nm, 1100 nm, 1150 nm, 1200 nm, 1250 nm, 1300 nm, 1350 nm, 1400 nm, 1450 nm, 1500 nm, 1550 nm, 1600 nm, 1650 nm, 1700 nm, 1750 nm, 1800 nm, 1850 nm, 1900 nm, 1950 nm or 2000 nm, preferably in a range of 300 to 800 nm,

[0052] In a further embodiment, the method is characterized in that the section preparation, the one or more calibration preparations and the calibration preparation are a paraffin section.

[0053] In a further embodiment, the method is characterized in that the section preparation and / or the calibration preparation are obtained by embedding tissue and / or one or more calibration cones containing prepared tissue of a known concentration in a paraffin block, which is subsequently sectioned. In a further embodiment, the method is characterized in that the section preparation is obtained by embedding tissue in a paraffin block (sample block), which is subsequently sectioned. In a further embodiment, the method is characterized in that the calibration preparation is obtained by embedding one or more calibration cones containing prepared tissue of a known concentration in a paraffin block (calibration block), which is subsequently sectioned.In a further embodiment, the method is characterized in that the calibration preparation is obtained by injecting a solution containing a defined concentration of the target protein into one or more pre-prepared calibration cones, which are subsequently embedded in paraffin and sectioned. In a further embodiment, after injecting a solution containing a defined concentration of the target protein, the calibration cone is stored in formalin to solidify the protein. In a further embodiment, the target protein is a protein isolated from a tissue and / or a recombinantly produced protein.

[0054] In a further embodiment, the method is characterized in that one or more dyes that absorb light in a wavelength range of 200 to 2000 nm are introduced into the tissue-free area of ​​the sample block and / or the calibration block, and the sample block and / or the calibration block are subsequently sectioned to obtain a sectioned preparation and / or a calibration preparation. In a further embodiment, the method is characterized in that the dye is introduced into the sample block and / or calibration block by inserting one or more punches (calibration punches). In a further embodiment, the method is characterized in that the dye is introduced into a separate paraffin block, from which one or more samples are taken by punching and introduced into the sample block and / or the calibration block, preferably by punching.

[0055] In a further embodiment, the method is characterized in that the layer thickness of a section is determined by measuring values ​​obtained by absorption of a light beam by the section in the one or more regions in which a dye has been introduced. In a further embodiment, the method is characterized in that the layer thickness of a section is determined by measuring values ​​obtained by absorption of a light beam by the section in the one or more regions in which the calibration punch has been introduced.

[0056] In a further embodiment, the method is characterized in that the layer thickness of a section preparation is determined by measuring values ​​obtained by absorbing a light beam through the section preparation in the two or more regions in which a dye has been introduced. In a further embodiment, the method is characterized in that a profile of the layer thickness (height profile) of the paraffin section is created based on the determination of the layer thickness of a section preparation by measuring values ​​obtained by absorbing a light beam through the section preparation in the two or more regions in which a dye has been introduced.

[0057] In one embodiment, the creation of the height profile comprises a two-dimensional fitting function. In another embodiment, the two-dimensional fitting function comprises layer thicknesses or equivalent layer thicknesses (support points) determined at two or more different positions on the section specimen. In another embodiment, the creation of the height profile additionally comprises interpolation from the support points determined by the two-dimensional fitting function to the entire section specimen.

[0058] In a further embodiment, a correction factor is determined based on the height profile for each area of ​​the section preparation and / or the calibration preparation (control area), and the concentration of a target substance in an area of ​​the section preparation is determined using the correction factor for this area. In a further embodiment, the concentration of a target substance in an area of ​​the section preparation is determined using a correction factor for this area, wherein the correction factor is a constant correction value for the entire section preparation or a spatially resolved correction factor (correction area created using a height profile).

[0059] In a further embodiment, the method is characterized in that a digital scan of the section preparation, the calibration preparation, and / or the calibration preparation is created, which is used to measure the values ​​and / or the color intensity, wherein the measurement of the values ​​and / or the color intensity comprises the determination of a brightness value. Creating a digital scan in the method according to the invention enables a standardized and automated creation of calibration curves based on calibration preparations and / or calibration preparations, as well as evaluation of the section preparation and determination of the concentration of a target substance. Simply photographing a section, calibration preparation, or calibration preparation leads to considerable fluctuations in image quality due to variable magnifications, vignetting of the image field, and different lamp brightness, color temperature, and exposure time.By contrast, creating a digital scan produces an image with consistent brightness and white balance. Calibration allows consistent results even for different sections. The resulting image can then be standardized and automatically evaluated. It can be used to determine the slice thickness of a section using the correction factor or multiple correction factors (correction area) determined from the height profile for the actual slice thickness, and then used to determine the concentration. Standard photo editing programs familiar to those skilled in the art can also be used for the analysis.

[0060] In contrast to the method disclosed in US 2013 / 0314712 A1, in which only the entire section or specific regions is measured in the apparatus, but not the scanned image, a digital scan of the section specimen according to the present invention enables pixel-precise resolution of the section specimen. A digitally scanned image, which is composed of numerous individual images, ensures a uniform image brightness / base signal intensity across the entire specimen, since optically generated images always exhibit a more or less pronounced edge darkening.

[0061] In a further embodiment, a mathematical program is used to determine the correction area. In a further embodiment, a false-color representation with a color key is created using the mathematical program, wherein the local concentrations of the target substance are encoded in the color key. Advantageously, such a mathematical program thus provides information about the local concentrations of the target substance in a section preparation.

[0062] In a further embodiment, the method is characterized in that, based on the measured color intensity of the target substance present in the calibration preparation, a calibration curve for the concentration of the target substance is created, which is preferably determined from two or more concentrations of the target substance.

[0063] By providing two or more concentrations of the target substance, it is possible to create a calibration curve that can be used to determine the concentration accurately and precisely. The spatial separation of the concentrations in the calibration preparation prevents overlaps and mixing of the concentrations, which can lead to incorrect concentration determinations. The spatial separation and provision of different concentrations can be achieved very easily using known techniques.

[0064] In a further embodiment, (a) the preparation or tissue containing the target substance is prepared according to methods known in the art, preferably by mechanical comminution and / or dissolution of the tissue in a lysis buffer, (b) the concentration of the target substance is determined, preferably by the methods of clinical chemistry, (c) the prepared preparation is diluted so that it contains a specific concentration of the target substance, and (d) the prepared preparation or tissue according to (c) is bound in a gel in the form of a calibration cone (cell block). In a further embodiment, one or more of the calibration cones are spatially separated from one another in a paraffin block (calibration block). In a further embodiment, this calibration block can be used in the method according to the invention for the production of a calibration preparation, preferably by producing a section of the calibration block.

[0065] The calibration blocks according to the present invention can advantageously be produced using standardized procedures for any target substance, such as proteins, and provided to the user of the method. This allows a high level of worldwide standardization of the method according to the invention to be achieved.

[0066] In a preferred embodiment, the method is characterized in that the target substance is a substance within the tissue contained in a section preparation, preferably a protein, and the target substance is localized in the tissue contained in the section preparation using the calibration preparation and the calibration curve, and the concentration of the target substance is determined.

[0067] In a further aspect, the invention relates to a device for measuring the layer thickness of a section preparation and optionally for determining the concentration of a target substance in the section preparation, comprising (a) one or more light sources of different wavelengths in the range from 200 to 2000 nm, preferably 300 to 1800 nm, particularly preferably 400 to 1700 nm, (b) at least one sensor for measuring the scattering and / or absorption of a light beam, (c) a device for positioning the section preparation during the measurement, and (d) a computing unit for modeling the layer thickness of a section preparation or for using the modeling in determining the layer thickness of a section preparation.In a further preferred embodiment, the one or more light sources may have a wavelength of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 nm or other wavelengths in this range which achieve the desired properties of the present invention.

[0068] In a further embodiment, the device is characterized in that the device comprises a shaping structure into which one or more light sources and one or more sensors can be introduced, preferably in opposing positions. In a further embodiment, the device is characterized in that a slide, on which a section preparation and / or a calibration preparation is applied, can be introduced between the light source and the sensor, and the light beam emanating from the light source is guided through the slide, and the scattering and / or absorption of the light beam by the sensor can be measured. In a further embodiment, the light source is a light-emitting diode or a laser diode. In a further embodiment, the sensor is a photodiode and / or a scanner.In a further embodiment, the device is characterized in that the device comprises one or more apertures that limit the light beam emanating from the light source to one or more specific positions on the slide on which a section specimen and / or a calibration specimen or a calibration specimen are applied. In a further embodiment, the device comprises a camera that can record the transillumination position of the slide through the light source, limited by the one or more apertures.In a further embodiment, the camera can be used to create an image of the section preparation, for example for image evaluation and / or for determining further properties of the section preparation, for example for determining the homogeneity of the preparation in the form of measured values ​​which can be used to determine the layer thickness or a concentration of a target substance in a section preparation.

[0069] In a further aspect, the invention relates to a microscope slide for use in the method and / or with the device, comprising a calibration preparation containing a target substance in two or more spatially separated concentrations, onto which the section preparation can additionally be applied. In a further embodiment, the microscope slide comprises a coverable and / or detachable region. In a further embodiment, the tissue-free part of the calibration preparation is applied in the detachable region, said part containing a dye that absorbs light in a wavelength range of 200 to 2000 nm, preferably in the form of a calibration punch. In a further embodiment, the tissue-free part of the section preparation is applied in the detachable region, said part containing a dye that absorbs light in a wavelength range of 200 to 2000 nm, preferably in the form of a calibration punch.

[0070] By providing a slide onto which the section and calibration specimen can be applied, simultaneous sample processing is enabled. Simultaneous sample processing allows the method according to the invention to be carried out within a short time, as the sample preparation steps only need to be performed once and not separately for the section and calibration specimen. Furthermore, the method according to the invention is further standardized by simultaneous sample preparation, as potential fluctuations that could arise from separate sample processing are eliminated. Providing a slide for the method according to the invention further facilitates applicability, for example, in a clinical setting, as only the section needs to be applied and the standardized steps of the method need to be carried out.By covering or separating the calibration punches, decomposition of the calibration punches can be advantageously prevented, making it possible to determine the layer thickness after staining the specimens in a single step, for example, by scanning the specimens, which can then be used to determine the layer thickness, the calibration curve, and the concentration of the target substance. If the calibration punches are not covered, the layer thickness must be determined before staining, as the calibration punches decompose during staining, making subsequent determination of the layer thickness impossible.

[0071] The above-mentioned features in relation to the method for determining the layer thickness of a section preparation have structural and functional effects on the other aspects of the invention which comprise the method for determining the concentration of a target substance in the tissue contained in a section preparation, the device for measuring the layer thickness of a section preparation and the slide for use in the method according to the invention, so that the method for determining the layer thickness, the method for determining the concentration of a target substance, the device for measuring the layer thickness and the slide can each be described by the features of the other aspects or derived from these.

[0072] DETAILED SUMMARY OF THE INVENTION

[0073] All cited patent and non-patent literature documents are hereby incorporated by reference in their entirety. Each term described below is to be understood in its general meaning as understood by one of ordinary skill in the art. The definitions are provided below by way of example to clarify how each term is applied in the context of the invention and may be used in preferred embodiments of the invention.

[0074] Histology and histopathology involve the microscopic examination of biological material, such as tissue samples, for morphology. In histopathology, the sample examined under the microscope is usually biological material in the form of a tissue sample taken during surgery or a medical examination, also called a biopsy. A biopsy is a procedure for removing and obtaining a small amount of tissue from a person or patient. The different types of biopsy include core biopsy, needle biopsy, endoscopic biopsy, excisional biopsy, and perioperative biopsy. Larger samples consist of organs or parts of organs (“resected specimens”) and require the pathologist to additionally remove representative material prior to further examination. These procedures are familiar to those skilled in the art.Typical biopsy samples include tissue from the colon, kidney, pancreas, cervix, lung, breast, prostate, or soft tissue.

[0075] For histological or pathohistological examination, the removed specimen is then (i) fixed, (ii) cleared / embedded, (iii) sectioned, and (iv) stained. Alternatively, frozen sections can be prepared using a cryostat if rapid results are required or if fixation would compromise the target structures.

[0076] Specimen fixation is performed to prevent autolysis and decay, and to preserve existing structures in the tissue during subsequent steps. Tissue specimens can be fixed using physical or chemical methods. Physical methods include heat and microwave treatment, as well as cryopreservation. Chemical fixation is usually performed by immersing the specimen in a fixative. In addition, there are vapor fixatives used for some specialized histochemical procedures, such as the vapor fixation of freeze-dried tissues using paraformaldehyde and osmium tetroxide. Chemical fixatives include alcohols, aldehydes such as formalin, neutral buffered formalin (4–10% formaldehyde in isotonic buffered saline), paraformaldehyde, glutaraldehyde, and acrolein; oxidizing agents such as potassium permanganate, osmium tetroxide, and chromic acid; and tannins.Biological tissue samples are often transferred to a fixative, usually 10% neutral buffered formalin, for 24 to 48 hours immediately after collection.

[0077] For subsequent dehydration, also called "dehydration," the sample is immersed in increasing concentrations of alcohol to remove water and formalin. Dehydration is followed by clarification with an organic solvent such as xylene to remove the alcohol and enable infiltration and thus embedding in paraffin wax. During embedding, the sample is infiltrated with the embedding medium—usually paraffin. The embedding medium forms a support matrix and subsequently allows the sample to be cut into very thin sections that can be examined microscopically.

[0078] A section, also called a "slice preparation," is usually created using a microtome. A microtome is a cutting device with which very thin sections with a layer thickness in the range of 0.1 to 100 μm can be obtained. The term "slice thickness" describes the diameter or height of the section, which can be uniform (constant height across the surface) or variable (spatially variable). A microtome usually consists of a knife block, a specimen holder, and a feed mechanism. During sectioning, the knife is pressed through the specimen, producing a thin section, which is then pushed out of the device by the feed mechanism to allow for the next section. Various types of microtomes are known, such as sliding microtomes, rotary microtomes, vibratomes, saw microtomes, and laser microtomes.A specialist can select a suitable device for preparing section preparations from these devices. For histological examinations, section preparations are prepared with a layer thickness of 1 to 10 μm, preferably 2 to 7 μm. The prepared sections are collected in a water basin and stretched out on the surface of the water by surface tension. They can then be transferred to a slide for further sample preparation.

[0079] The term "paraffin section" refers to a section obtained by cutting a paraffin block. A histological specimen containing tissue may be embedded in this paraffin block. The areas of the paraffin block where no tissue is embedded are referred to as "tissue-free areas." Embedding in a paraffin block serves to support the tissue structure, allows for the preparation of very thin tissue sections, and allows for the mounting of these sections on a slide for subsequent examination, such as microscopic examination. The term "histological section" encompasses a section containing tissue obtained, for example, from a biopsy.

[0080] Paraffin is a mixture of saturated acyclic hydrocarbons with the general formula C nH2n+2, where n is between 18 and 32. Paraffin is fluid, oily, or waxy, and inert to many chemicals. Paraffin is used in histology as an embedding medium for histological specimens. At a wavelength of 300 nm or higher, paraffin is highly translucent (Li et al. 2015). In the very short wavelength range (near 200 nm and below), paraffin becomes opaque, even at very thin layers.

[0081] The term "extinction" describes the reduction of radiation, such as a light beam (also called a "light source"), as it passes through a medium, such as a paraffin section. The extinction coefficient characterizes the strength of the interaction between the radiation and the medium. It is a material constant that depends on the wavelength of the incident radiation. Extinction is the overall effect of attenuation and is divided into the components of reflection, absorption, scattering, and diffraction. The attenuation of intensity along the path through the medium is proportional to the absorption coefficient of the material being irradiated and its thickness. This results in the Beer-Lambert law.

[0082] The Lambert-Beer law describes the attenuation of radiation intensity relative to its initial intensity when passing through a medium containing an absorbing substance, depending on the concentration of the absorbing substance and the layer thickness of the medium. Absorbing substances can be, for example, paraffin, natural pigments contained in histological specimens such as melanin, or dyes used to stain histological specimens. Absorbing substances can also be dyes or mixtures of dyes that are introduced into the sample block, calibration block, or calibration block in the form of a calibration punch. These dyes preferably absorb in a wavelength range from 200 to 2000 nm, particularly preferably in the range from 300 to 800 nm.Dyes that absorb in this range include, for example, anthraquinones (e.g., alizarin and carmine), azo dyes (e.g., aniline yellow and Congo red), dioxazine dyes (e.g., Sirius light blue and carbazole violet), indigoid dyes (e.g., indigo and indirubin), formazan dyes (e.g., Reactive Blue 235), methine dyes (e.g., ß-carotene, lycopene and Basic Red 12), nitro and nitroso dyes (e.g., Acid Orange 3 and Acid Green 1).

[0083] There are several variations of the notation of the Lambert-Beer law. One of them is: where I ois the intensity of the incident light beam, ^ is the intensity of the transmitted light, (A) is the wavelength-dependent absorption coefficient or extinction coefficient, c is the molar concentration of the absorbing substance, and d is the thickness of the medium through which the light passes. The Lambert-Beer law can be applied for any wavelength A to calculate the specific absorption of light of that wavelength with intensity / 0(A), which leads to the intensity of the transmitted light ^(A).

[0084] The term “reflection” describes the reflection of radiation when it hits the interface between two different media. Reflection is therefore a phenomenon that occurs on surfaces and is not dependent on the layer thickness of a section. The term “absorption” describes the absorption of radiation by a medium and is reciprocal to transmission, which describes the relative optical transmittance of a medium. Absorption depends on the wavelength of the radiation and the medium. The term “scattering”, also called “light scattering”, describes the fragmentation of a light beam by irregularities in a permeable medium, such as atoms, molecules, bacteria or droplets in an emulsion or in fog. Depending on the type, shape and size of the scattering object, the scattered light intensity is distributed differently in the surrounding space. This attenuates the radiation just passing through a medium.This attenuation of radiation is measured in an extinction measurement of a medium, along with the effects of absorption, reflection, and diffraction. According to the present invention, the effects of reflection and diffraction when passing through a sectioned specimen are negligible, and the extinction measurement is essentially based on the measurement of scattering and / or absorption. Devices for measuring extinction include, for example, turbidimeters, nephelometers, and spectrometers such as UV / VIS spectrometers or infrared spectrometers, and are known to those skilled in the art. In the context of transmitted light measurements in liquids, the term turbidimetry is also used by those skilled in the art, which is distinguished from nephelometry (scattered light measurement).In the present invention, the principles of turbidimetry are preferably used to determine the layer thickness of a section preparation, while the principles of angle-dependent resolved nephelometry can preferably be used for further indications of the spatial distribution of concentrations (for example, in the thickness direction of the section).

[0085] The term "light source" encompasses a physical system that emits electromagnetic radiation from the X-ray to the infrared range, usually within a limited spectral region. Common light sources that can be used, for example, for extinction measurements such as scattering and / or absorption measurements include, without limitation, continuum emitters such as halogen lamps and xenon lamps, line emitters such as cadmium and mercury lamps, light-emitting diodes (LEDs), fluorescent tubes, and lasers.

[0086] The term "calibration" encompasses the measurement of a specimen or sample with a known property using a procedure or measurement method in order to use this procedure to determine this property in a specimen with an unknown property. The property to be determined can, for example, include the concentration of a specific substance in the specimen or the layer thickness of the specimen. The property to be determined can also include, for example, constants in the mathematical model for evaluating the measurement data. The specimen with a known property used for calibration is referred to as the "calibration specimen" or "calibration specimen," and the values ​​measured using this calibration specimen or calibration specimen are referred to as "calibration values."Using these calibration values, the properties such as the concentration of a specific substance can be determined and calculated from the values ​​obtained by the method for a preparation with unknown properties.

[0087] The term "calibration curve," also called "standard curve," encompasses a mathematical relationship or correlation between several calibration values ​​determined, for example, by measuring several known concentrations of a sample or measuring the scattering and / or absorption of different samples with known path lengths. This calibration curve can be used to determine the properties of a sample with an unknown property using the method used to create the calibration curve. The calibration curve typically includes measured values ​​within the range expected for the sample or sample with unknown properties.

[0088] For the purposes of the present invention, the term "modeling" encompasses the mathematical representation (calculation) of the properties of a preparation with an unknown property based on one or more calibration curves for this property. This involves providing the calibration values, creating one or more calibration curves, comparing the measured values ​​for a preparation with unknown properties with the calibration curves, and calculating a corresponding value for the unknown property of this preparation based on the calibration curves. Furthermore, "modeling" can include the application of additional mathematical formulas that describe physical relationships, such as the Beer-Lambert law.

[0089] Based on modeling, the present invention can calculate a correction factor for a sample. A correction factor is a quantity introduced into a calculation that is necessary to compensate for the influence of a specific quantity or effect that would otherwise distort or influence the calculation. In the present invention, a correction factor is calculated based on the actual layer thickness of a section preparation. This layer thickness correction factor is used in the calculation of the concentration of a target substance in the preparation to compensate for the influence of the layer thickness on the color intensity of the staining on which the concentration calculation is based.

[0090] The term "histological staining" refers to the use of histochemical dyes to treat tissue sections (preferably paraffin-embedded or frozen). Histological staining may also include one or more additional intermediate steps, such as the addition of an enzyme. Immunohistochemistry is a special form of staining in which the target protein is labeled with an antibody in an intermediate step; a second antibody binds to the first and is coupled, for example, with a fluorescent dye or an enzyme, which secondarily generates the color from a specific substrate. Commercial antibodies are available for a variety of proteins, which a person skilled in the art can select accordingly. In the context of the present invention, the terms "histological staining," "histochemical staining," and "immunohistochemical staining" are used synonymously.Sections are typically stained for histological examination to highlight structural features of the tissue and increase tissue contrast between structural features. Specific target structures and target substances can be stained by carefully selecting the appropriate staining agent. Target substances relevant for histology include, without limitation, proteins such as cell receptors (e.g., estrogen receptors, progesterone receptors, Her2neu, and c-Myc) and other intracellular and / or extracellular proteins (e.g., Ki67 and PSA) or other components such as nucleic acids, mucous substances, and lipids. Typical histochemical stains include, for example, staining for cytochrome c oxidase, ATPases, and NADH.

[0091] Common histological stains include, without limitation, staining with hematoxylin and eosin (HE stain), azocarmine and aniline blue (Azan stain), resorcinol, fuchsin and orcein (Elastica stain), azure, eosin and methylene blue (Giemsa stain), iron hematoxylin, picric acid and acid fuchsin (van Gieson stain), iron hematoxylin and light green (Masson-Goldner trichrome stain), iron hematoxylin (Weigert stain), iron hematoxylin (Heidenhain, EH stain), silver ions (Golgi stain), copper phthalocyanine (Luxol Fast Blue stain), cresyl violet, toluidine blue or thionine (Nissl stain), hematoxylin according to Harris, Orange G and polychrome solution (Papanicolaou stain), periodic acid (PAS stain), Sirius Red (picro-Sirius Red stain) and silver (Wartin-Starry stain).Other dyes that may be used alone or in combination with other chemicals for histological staining include, without limitation, alizarin, Victoria Blue B, Acid Red 88, eosin, acid fuchsin, Congo Red, safranin, basic fuchsin, crystal violet, methylene blue, hematoxylin, nuclear fast red, ethyl green, DRAQ5™, DRAQ7™, Nuclear Yellow (Hoechst S769121), Nuclear Green DCS1, Hoechst, DAPI, propidium iodide, Acid Fast Bacteria stain, Alcian Blue (pH 1.0), Alcian Blue (pH 2.5), alizarin red, colloidal iron, hydroxystilbamidine, Prussian Blue, Oil Red O, safranin O stain, Sudan Black B and toluidine blue.

[0092] The term "color intensity," also called "signal intensity," refers to the strength of the measurable output signal that reflects the concentration of the dye bound to any region of a tissue section, e.g., a cytoplasmic region of a cell, a nuclear region of a cell, an interstitial region, or a voided region. Color intensity can be determined, for example, using commonly used image processing programs as a brightness value or tone value, which is the brightness value of an image pixel in a color channel.

[0093] The term "calibration preparation" or "comparison preparation" refers to a section preparation that contains a known concentration of a target substance and can be used to create a calibration curve or calibration curve, which, for example, correlates a measured color intensity with the known concentration of the target substance. According to the present invention, calibration preparations can be prepared from tissue, cells, or substances that have been proven to contain the target substance. The concentration of the target substance can be determined, for example, using methods commonly used in clinical chemistry, such as ELISA or mass spectrometry. Based on the known substance concentration, different concentrations within the appropriate concentration range can be produced by dilution. Clinical chemistry normally uses only liquid samples.According to the present invention, the liquefied tissue is solidified and used as a reference preparation (calibration preparation). The calibration preparations are produced in the form of cell blocks, also called "calibration cones," in which the tissue, cells, or substances are bound in a gel at a defined concentration. One or more of these cell blocks of varying concentrations can then be embedded in a matrix such as paraffin in a so-called calibration block. By embedding them in a matrix, the cell blocks are spatially separated in the calibration block at different concentrations. A calibration block can also be produced by injecting a target protein in the form of a solution containing a defined concentration of the target protein into one or more calibration cones. The target protein can be a protein isolated from a tissue and / or a recombinantly produced protein.The protein is solidified by subsequently storing the calibration cone in formalin and embedding it in paraffin. A calibration block can be cut using a microtome and used as a calibration preparation in the present invention. One or more calibration punches containing absorbing substances such as dyes or mixtures of dyes can be inserted into a calibration block. A calibration preparation produced from such a calibration block can thus contain calibration punches containing absorbing substances such as dyes or mixtures of dyes.

[0094] The term “calibration preparation” refers to a section preparation which has a known slice thickness, contains no tissue, and can be used to create a calibration curve which, for example, correlates values ​​measured by scattering and / or absorption with the known slice thickness. A calibration preparation preferably has a known slice thickness of 0.1 to 10 μm. A calibration preparation can be produced from a calibration block which is sectioned using a microtome. In preferred embodiments, the calibration block is a paraffin block which contains no tissue. One or more calibration punches which contain absorbing substances such as dyes or mixtures of dyes in a carrier material can be introduced into the calibration block.A calibration preparation made from such a calibration block can thus contain calibration punches containing absorbing substances such as dyes or mixtures of dyes. Such a calibration punch can contain a carrier material that dissolves during staining and / or fixation of the histological section and / or contain material that does not dissolve during staining and / or fixation of the histological section.

[0095] A microscope slide is an object on which objects and specimens, such as sections, can be mounted and viewed microscopically. Materials used for microscope slides include soda-lime glass, quartz glass, or plastics such as polystyrene. Preferably, the microscope slides of the present invention are made of quartz glass.

[0096] The term "sensor," also referred to as "detector," encompasses a device that can qualitatively and / or quantitatively detect certain physical or chemical properties (e.g., the brightness, extinction, absorption, scattering of a light beam) and / or the material properties of its environment, or as a measurand. These quantities are detected by means of physical, chemical, or biological effects and converted into a further processable signal, such as an electrical signal or an image. According to the present invention, a sensor can be a light sensor, such as a photodiode, a camera, or a scanner.

[0097] The term "digital scan" encompasses the creation of an image of the section, calibration, or calibration specimen using a scanner. The image has the same brightness and white balance across all sections and is captured using a standardized procedure for all specimens. The resulting digital scan can be used for analyzing the brightness or tonal values, for example, using a photo editing program familiar to those skilled in the art, or for automated evaluation.

[0098] In some embodiments, the device according to the invention comprises a computing unit for modeling or using the modeling. The computing unit is preferably a conventional computer device with a processor, an input device such as a keyboard or mouse, memory such as a hard disk and volatile or non-volatile memory, and computer code (software). The computer may also comprise a programmable circuit board, a microcontroller, or other device for receiving and processing data signals received from local controllers, programmable manufacturing equipment, programmable material handling equipment, and robot manipulators. The device may comprise a computing device preloaded with the required computer code or software that performs the modeling steps according to the invention.In certain embodiments, multiple clients, such as desktop, laptop, or tablet computers, can be connected to a server, allowing, for example, multiple users to use the modeling according to the invention simultaneously. The computer system can also be networked with other computers via a local area network (LAN) or via an internet connection. The system can also include a backup system that keeps a copy of the data obtained according to the invention.

[0099] The "correction area" is a mathematical area function used to correct the height profile (locally varying heights in the paraffin and embedded tissue). In this context, correction means achieving a uniform layer thickness (or equivalent layer thickness) at every location in the section (both in the calibration punches and in the areas of the specimen containing tissue). This enables the subsequent conversion of the locally determined color values ​​(e.g., in a digital scan of the section) into concentration values ​​of the target substance.

[0100] The components of the computing device and computer code may be conventional, although the computing device and computer code will be individually configured for each particular implementation. The computer code may run on any architecture, such as personal microcomputers, minicomputers, microcontrollers, or mainframe systems. Example operating systems include Apple Mac OS X and iOS, Microsoft Windows and UNIX / Linux, SPARC-, POWER-, and Itanium-based systems, and the z / architecture. The computer code for carrying out the invention may be written in any programming language or model-based development environment, such as, but not limited to, C / C++, C#, Objective-C, Java, Basic / VisualBasic, MATLAB, Simulink, StateFlow, Lab View, or assembler.The computer code may include subprograms written in a proprietary computer language specific to the manufacturer of a circuit board, controller, or other computer hardware component used in connection with the invention.

[0101] In certain embodiments, the computing unit may contain amplification electronics for the output signal of a photodiode in the form of a transimpedance amplifier, which is implemented as an integrated circuit on a circuit board. In further embodiments, the measurement signal may be read and processed using a microcontroller (e.g., Arduino). In further embodiments, the measurement signal may be conditioned in a microcontroller and then further processed in a connected computing device. In certain embodiments of the invention, a human supervisor may be present to monitor the modeling and correct any errors or malfunctions.However, in preferred embodiments of the automated or semi-automated execution of the method, the supervisor is not substantially involved in the method beyond preparing and attaching a slide to the microscopic equipment and therefore does not need to routinely intervene in the computer-implemented aspects of the invention.

[0102] REFERENCES

[0103] Masuda, Tomoyuki, et al. "How thick are the paraffin-embedded tissue sections routinely prepared in laboratory? A morphometric study using a confocal laser scanning microscope." Pathology international 48.3 (1998): 179-183.

[0104] Nguyen, David. "Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity." (2013).

[0105] Choudhury, Kingshuk Roy, et al. "A robust automated measure of average antibody staining in immunohistochemistry images." Journal of Histochemistry & Cytochemistry 58.2 (2010): 95-107.

[0106] Jensen, Kristian, et al. "A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2." Modern Pathology 30.2 (2017): 180-193.

[0107] Li, Dong, et al. „Optical properties of a liquid paraffin-filled double glazing unit“ Energy and Buildings 108 (2015) 381-386.

[0108] Matenaers et al., Practicable methods for histological thickness measurement in quantitative stereological analyses, PLoS ONE 13 (2), 2018, S- 1-21 ; e0192879.

[0109] FIGUREN

[0110] Die Erfindung soll im Folgenden anhand der Abbildungen und Beispiele näher erläutert werden.

[0111] The figures and examples do not limit the scope of the invention, but represent preferred embodiments of the aspects of the invention which serve for illustration purposes.

[0112] Fig.1 : Tissue with natural tissue pigment (melanin / iron) sectioned using a microtome at different layer thicknesses (A) 1 pm, (B) 3 pm, (C) 5 pm and (D) 7 pm.

[0113] Fig. 2: Transmittance curve of liquid paraffin measured at different layer thicknesses, depending on the wavelength (according to Li et al. 2015) (state of the art).

[0114] Fig. 3: Measurement signal (in mV) after passage of UV-C (A = 260 nm) through tissue-free paraffin sections as a function of the section thickness in pm (p) (average of several measurements), O=blank value (glass only).

[0115] Fig. 4: Measurement of the scattering of a red laser (650 nm) through tissue-free paraffin sections as central brightness in the projection photo as a function of the section thickness in pm (p), left blank value (layer thickness 0, glass only), far left black value (darkest section of the photo without laser) as average values ​​from several measurements (n > 6).

[0116] Fig. 5: (A) to (E) Comparison of the measurement curves (scattering) at different wavelengths, plotted against the section thicknesses 1, 3, 5 and 7 pm (p) (blank value = 0) of a tissue-free paraffin section: (A) infrared (direct measurement photo), (B) red (central brightness in the projection photo), (C) blue (central brightness in the projection photo), (D) violet (central brightness in the projection photo) and (E) UV-C transmittance (sensor, in mV). (With the exception of the measurement in the UV-C range, the maximum is at 3 pm. When comparing 1 pm and 5 pm, a shift of the maximum towards 1 pm in the infrared range and towards 5 pm in the violet wavelength range can be seen, illustrated by a straight line. The angle of inclination of the straight line is plotted against the wavelength in (F) and shows a clear monotonic relationship.

[0117] Fig. 6: Gradients with associated fitting functions. TW denotes tonal values ​​TW te [0; 255] and U the measured voltage.

[0118] Fig. 7: Example measurements for a thickness of 5 pm. Plausible thicknesses detected by the algorithm in the respective curve are highlighted (in pm). The cross indicates the range of agreement between all curves. The average of these values ​​is determined as the output value.

[0119] Fig. 8: Examples of color intensity measurements. Detail photos of a section scan with histogram. Immunohistochemistry for Melan A (red color substrate in the positive image), shown here is the inverted image, where the brightness value is determined in the corresponding green color channel. Section thickness A: 7 μm, B: approximately 3 μm. The thicker the section thickness, the higher the color intensity.

[0120] Fig. 9: (A) Calibration cones containing liquefied brain tissue, which was diluted to a standardized standard, resolidified in gel, and embedded in paraffin. For each block, the concentration of the target protein (S100) was determined in parallel by clinical chemistry. (B) Reassembly of these calibration cones at different concentrations C1 to C3 in a new common calibration block (schematic).

[0121] Fig. 10: Schematic of a microscope slide 1 which can be used for measuring layer thickness and determining the concentration of a target substance in a section preparation 3. In the control area 2, a section of a calibration block (calibration preparation) is applied, which contains calibration cones (A-D), each of which contains a specific concentration of the determining target substance. The section preparation 3, which comprises a tissue sample 4, is applied in the middle area of ​​the microscope slide. Calibration punches 7 can be inserted into the section preparation 3 and the calibration preparation. Information for identifying, for example, the sample and the coloration can be applied in the data field 6. The microscope slide is inserted into the device according to the invention. The layer thickness is then measured in the dark gray marked areas and / or in the areas of the calibration punches 7.In a preferred embodiment, a measurement is carried out at four measuring positions 5 in the area of ​​the section specimen.

[0122] Fig. 11: (A) Calibration curve, created from the tone values ​​determined for the calibration blocks, which allows a clear assignment of each tone value to a concentration (the concentrations were determined in parallel in the same dilution series in clinical chemistry). x-axis: dilution series (10x10' x (B) Reverse immunohistochemistry image for SWO of the patient sample on the same section. Using the calibration curve and individual calibration, each area of ​​the image can be assigned a concentration of S100. (C) shows the measured values ​​determined for two adjacent tumor cells of a malignant melanoma (metastasis). At least one image pixel is required for the measurement, but ideally several.

[0123] Fig.12: Section of a calibration block (calibration preparation) containing three differently concentrated calibration cones of brain tissue, stained with an immunohistochemistry for S 100 (brown color reaction).

[0124] Fig. 13: Correlation of the tonal value measured in a digital scan of the calibration preparation (inverted image, blue color channel) with the S100 protein concentration contained in the calibration cones.

[0125] Fig.14: The mean values ​​of the values ​​(scattering and absorption) measured at the four measuring positions (“North”, “East”, “South” and “West”) show constantly decreasing measured values ​​(y-axis, in % of the interpolated blank value) depending on the wavelength (infrared, red, blue and violet) and the layer thickness in pm (x-axis).

[0126] Fig. 15: Section series (1-7 pm) of a paraffin block containing a sphere of colored wax with a known color concentration (e.g., red), from which the calibration dies are also obtained. It is already visible to the naked eye that the color density increases with section thickness.

[0127] Fig. 16: Dependence of the color intensity (brightness value, also referred to as tone value) of the wax spheres described in Fig. 15 on the layer thickness of the section series in pm (p) (1-7 pm, inverted image, green color channel). Fig. 17: Unstained paraffin sections of varying thicknesses (top (calibration preparation) from left to right: 7, 5, 3, 1 pm; bottom (section preparation) 1, 3, 5, 7 pm) with the calibration punches included.

[0128] Fig.18: Standard curves (mean values ​​of different measurements) created from the test section series (1, 3, 5 and 7 pm) for diluted (right) and undiluted (left) wax.

[0129] Fig. 19: Staining of the test section series using immunohistochemistry for S 100. Tumor tissue is shown at the top, and calibration blocks are shown at the bottom (the microtome section thickness is marked on the slide).

[0130] Fig.20: Calibration curves: On the left, the calibration of the calibration specimen, on the right, the calibration of the section specimen (tumor sample) after correction of the section thickness difference between the calibration specimen and the section specimen.

[0131] Fig. 21: Is an isometric view of a preferred embodiment of the device according to the present invention. The device comprises a shaping structure 8 (housing) into which a light source 9 (for example a light-emitting diode) and a sensor 11 (for example a photodiode) can be inserted in opposite positions. The specimen slide 1, on which a section specimen and / or a calibration specimen is applied, can be inserted between the light source 9 and the sensor 11 in such a way that the light beam emanating from the light source 9 is guided through the specimen slide 1 and the scattering and / or absorption of the light beam by the sensor 11 can be measured.

[0132] Fig. 22: Is a plan view of a preferred embodiment of the device according to the present invention. The device comprises one or more apertures 12 (position of the apertures shown, apertures themselves not shown in the figure), which can be introduced into the shaping structure 8 in order to limit a light beam emanating from the light source 9 (not shown in the present figure, shown in Figure 23) to one or more specific positions on the specimen slide 1, on which a section specimen and / or a calibration specimen or a calibration specimen are applied, and thus to measure the scattering and / or absorption of the light beam by the section specimen, calibration specimen or calibration specimen.

[0133] Fig. 23: Is a front view of a preferred embodiment of the device according to the present invention. The device also comprises a camera 13 (position of the camera shown, camera itself not shown), which can record the transillumination position of the slide 1 by the light source 9 and is used to check the positioning of the one or more apertures 12 between the light source 9 and the slide 1 and the sensor 11 and the slide 1, thus enabling a measurement of the scattering and / or absorption of the light beam by the section preparation, calibration preparation, or calibration preparation applied to the slide 1, at one or more specific positions.Furthermore, the camera can be used to create an image of the section specimen or to determine other properties of the section specimen, for example, to determine the homogeneity of the specimen in the form of measured values, which can be used to determine the layer thickness or the concentration of a target substance in a section specimen. The light source 9 and / or the sensor 11 can be inserted into the shaping structure 8 of the device on an insert 10 and can thus be advantageously replaced by removing the insert and inserting an insert containing a different light source (for example, an LED of a different wavelength), thus enabling, for example, a measurement at a different wavelength.

[0134] Fig. 24: Is a side view of a preferred embodiment of the device according to the present invention. As described in Figure 23, the light source 9 and / or the sensor 11 can be inserted and exchanged on an insert 10 in the shaping structure 8 of the device. This enables, for example, the measurement of the scattering and / or absorption of a light beam by the section specimen, calibration specimen, or calibration specimen applied to the slide 1 at different wavelengths by exchanging the light source 9 for one of a different wavelength.

[0135] Fig. 25: Images from the Laserscan microscope. (A) Photomicrograph of the edge of a section with contour lines drawn from the 3D scan. (B) The measured contour line, drawn along the arrow from the upper right to the lower left edge. A slice thickness of 7.56 pm was determined.

[0136] Fig. 26: Summary of the measured values ​​of all height profiles of the paraffin sections from the laser coherence tomography measurement, (A) individual values, (B) mean values. A strong scatter (A) of the measured section thicknesses is evident, as is a large upward deviation of the mean values ​​(B) compared to the target section thickness (microtome setting), especially for thin sections.

[0137] Fig. 27: Similarity of the scans. (A) After a "rigid" image registration (translation & rotation) to compare the images, they were superimposed using rigid-body transformations (translation, rotation, no stretching). Subsequently, 10 x 8 measurement windows (squares) were defined. (B) The color measurement values ​​(from 0-250) are plotted for each color channel and each window (1-80). It can be seen that the error bars / standard deviations in each window are very small.

[0138] Fig. 28: (A) Original scan with calibration punches (circles) and protein test substance (area, dark gray). (B) Determination of the mean color value (ignoring the white surrounding area) with the calibration punch at the top center.

[0139] Fig. 29: (A) Correction area (in color values) created by the calibration punches at the respective positions in the section. (B) False-color image of the correction area. Fig. 30: (A) Corrected image within the correction area (dashed line). (B) Highly exaggerated image to illustrate the unevenness and differences in layer thickness within the section.

[0140] Fig. 31: Tissue preparation (brain tissue) with Histogel, poured, sectioned, and paraffinized. (A) Immunohistochemistry for S 100: High homogeneity of the material without any discernible systematic concentration gradients; small irregularities due to air bubbles and presumed section artifacts. (B) Immunohistochemistry for NeuN, which labels nerve cells punctually; here, too, a generally very homogeneous distribution.

[0141] Fig. 32: Example of a calibration cone (tissue preparation thickened in a defined dilution, HE staining). The calibration cone shows a high homogeneity of the contained tissue.

[0142] Fig. 33: Tissue (unfixed brain tissue) after passing twice through an ultrafine sieve (400 mesh). Macroscopically (A) and microscopically (B) (HE staining, section after embedding), individual fragments are no longer distinguishable; the cell nuclei (dark dots) and fibers (everything else in between) appear almost homogeneously distributed.

[0143] Fig. 34: Measured concentrations of S 100 (Clinical Chemistry) in serial dilutions of three different batches prepared immediately one after the other from the same sample material. The variance of the results is very small.

[0144] EXAMPLES

[0145] Paraffin section

[0146] Formalin-fixed and paraffin-embedded tissue is sectioned manually on a microtome. The section thickness is mechanically adjusted to a thickness between 1 μm and 9 μm, with 1 to 5 μm being the most common. During sectioning, the section typically curls significantly. This effect is further amplified by the tissue contained within. After sectioning, the section is stretched on a water surface and then transferred to a microscope slide.

[0147] The actual slice thickness usually deviates significantly from the set slice thickness and simultaneously influences the color intensity. This effect is based on the increasing optical density of the section with increased slice thickness and the Lambert-Beer law. The influence of slice thickness on color intensity can be observed even in unstained specimens based on the natural coloration of the tissue (Figure 1: natural pigments hemosiderin and melanin, Figure 15: histologically stained section).

[0148] Accuracy of the layer thickness set on the microtome

[0149] First, a standardized series of sections was created from a test block consisting solely of paraffin (without tissue) using the test device (Leica RM2125 RTS microtome): four sections each with the section thicknesses set on the device: 0.5 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm. For each of these sections, an ultra-precise height profile was created at the edge and in the center, along with a 3D scan using a laser scanning microscope (Keyence VK-X1050 coherence tomograph). This allowed the height of the section to be measured at the edge and in the center with an accuracy of approximately 0.01 pm. It was found that the height of this profile showed a significant deviation, sometimes by several times the set thickness, particularly at thin section thicknesses (see Figures 25 and 26). This suggests that complex folding of the paraffin layer is present.

[0150] Determination of layer thickness by scattering and / or absorption

[0151] Comparison example - Transmission in the UV-C range

[0152] Transmittance describes the relative permeability of a medium (here, paraffin) in percent and is reciprocal to absorption (the absorption of radiation when passing through a medium). This varies depending on the wavelength of the electromagnetic radiation and is described by transmission curves. Figure 2 shows the transmission curves for paraffin (according to Li et al. 2015).

[0153] It turns out that paraffin is highly transparent, largely independent of layer thickness, starting at a wavelength of 300 nm. In the very short wavelength range (near 200 nm and below), paraffin becomes opaque at all measured layer thicknesses. In the borderline range between 200 and 300 nm, however, there is a strong sensitivity of the measurement (i.e., variability of the measurement result with changing input parameters) depending on the layer thickness.

[0154] For measurement in this wavelength range, a circuit with a UV-C-sensitive photodiode was selected. A commercially available UV-C fluorescent tube (OSRAM Puritec HNS 5W) was used as the light source, whose emission spectrum exhibits a strong peak at 254 nm. Slides containing paraffin sections with layer thicknesses set on the microtome of 1, 3, 5, and 7 pm, initially without a tissue sample, were placed between the light source and the sensor in the beam path. A section of the slide without paraffin was measured as a blank value.

[0155] For measurements in the UV-C range, high-purity quartz glass (synthetic fused silica) was required. Standard microscope slides can be used for all other wavelengths.

[0156] The results of the measurements are shown in Figure 3 as mean values ​​of the absorbance with UV-C as radiation source in mV of the sensor (sensor type: UVC-T10GM-LA, UV sensor module, 220-280 nm).

[0157] Contrary to expectations, there was no continuous decrease in the measurement signal with increasing layer thickness, but rather a curve. The maximum of the curve lies at an average layer thickness of 5 pm and exceeds the blank value. This may be due to a possible fluorescence effect, which is common with UV radiation. In this fluorescence effect, radiation emission is caused by an originally shorter wavelength of a radiation source. This makes the emission of the sample appear stronger than would be expected based on the intensity of the actual radiation source (known effects with signal colors, whitening agents or so-called black light) and can therefore exceed a blank value. Furthermore, scattering effects and reflections in the measuring chamber can be important. The key hypothesis based on these considerations was that at low layer thicknesses, a second effect plays a role in addition to absorption.Scattering was investigated as the most likely second effect after absorption.

[0158] Invention Example - Scattering and Absorption

[0159] Scattering describes the fragmentation of a light beam (deflection of photons) due to irregularities in a fundamentally transparent medium (e.g., fog). A paraffin section appears cloudy and only vaguely transparent to the naked eye. The cloudiness of a paraffin section can be explained, for example, by the formation of unevenly distributed solidification zones during the cooling of the liquid paraffin and the resulting transitions at which scattering occurs. Scattering in an optical medium leads to a reduction in image contrast. Bright light points are therefore attenuated disproportionately. Initially, a laser light source seemed particularly suitable for the measurement. This was not directed directly at a sensor, but rather at a projection surface (matte photo cardboard). This had the advantage of also capturing surrounding scattering patterns.The projection was captured in a standardized manner using a digital camera (same exposure data for each photo, no automatic exposure or sensitivity adjustment). The brightness or attenuation compared to the blank value (slide without paraffin section) was recorded as the tonal value of the photo in the corresponding color channel (red, 0-255). Based on this, the device described below was developed, which performs a direct measurement using photodiodes.

[0160] The method demonstrated very good repeatability with multiple measurements in different sections of paraffin sections and highly significant differences between different layer thicknesses. It was assumed that the scattering increases uniformly with layer thickness. However, analogous to the UV-C measurement, a curve with a maximum at an average layer thickness of 3 pm was observed (Figure 4). The conclusion from this was that the scattering is particularly high at low layer thicknesses, thereby reducing the transmittance more sharply at low layer thicknesses and causing the curve shape with a maximum. Micrographs and laser microscopy contributed to the clarification: These indeed show stronger folding of the section at low section thicknesses (not shown). All effects together are taken into account in the modeling.

[0161] A further observation was that the maximum of the curve occurs at different layer thicknesses depending on the wavelength of the light source. While the maximum for the measurement in the UV-C range is at a layer thickness of 5 pm, the maximum for red laser light (650 nm) was at a layer thickness of 3 pm. Further measurements were subsequently performed with radiation sources of different wavelengths. Lasers with blue light (A = approx. 450 nm) and violet light (A = 405 nm) and an infrared source (A = approx. 980 nm) were used.

[0162] Differences in the maxima of the curves are particularly evident between 1 pm and 5 pm layer thicknesses (Figure 5). The 1 pm section yields significantly higher values ​​in the infrared range than the 5 pm section; the opposite is true for a violet light source (405 nm). To visualize the effect, a straight line was drawn between the measured values ​​of the 1 pm and 5 pm section thicknesses, its inclination angle was recorded, and plotted against the wavelength of the radiation source. A clear and regular effect is evident here, which is also visible for small differences—for example, between blue and violet light. Because the maxima of the curves are far enough apart, a clear retrograde assignment to the section thickness can be made with sufficient accuracy both by measuring at one wavelength and by combining measurements at different wavelengths. Measuring at multiple wavelengths increases the accuracy.

[0163] Paraffin sections containing tissue often exhibit irregular structures. These irregularities can be visualized in scans of unstained sections or under a laser microscope. Accuracy can be improved by performing a "multi-position measurement" of a section at various positions within the section. For this purpose, scattering and absorption were measured at various wavelengths (infrared to violet) at four defined positions ("north," "east," "south," and "west"; see Figure 10) in the paraffin section containing unstained tumor tissue. As previously observed, transmittance decreased with increasing section thickness. The mean values ​​of the four measurement positions around the tissue showed a clear dependence on wavelength and section thickness (Figure 14).It should be noted that the curve of paraffin sections with tissue contained shows a different course than without tissue contained (no maximum), which must be taken into account during calibration.

[0164] Inventive Example - Absorption

[0165] The accuracy can also be increased by introducing so-called "wax punches", also called "calibration punches" (areas containing a dye that absorbs in the wavelength range from 200 to 2000 nm). First, spheres of colored wax (here red) with a known color concentration were poured into a new paraffin block. A series of sections (1 - 7 pm) was taken from such a block. It can be seen that the color density and thus absorption increases with section thickness (Figure 15). The sections were scanned and the color density was determined as a tonal value (0-255). This shows a clear linear dependence of the values ​​on the section thickness (Figure 16). By introducing one or more calibration punches into a section preparation, the layer thickness can be determined by measuring the absorption in these areas.Using several calibration punches made of colored wax in different areas of the section, a height profile of the section can also be created. This information can be used to determine a correction factor for each section, and the staining scan can subsequently be corrected accordingly for each area (Figures 28 to 30).

[0166] Script for modeling layer thickness from calibration values

[0167] To evaluate the layer thickness from the obtained measured values ​​(calibration values) (scattering and / or absorption), an evaluation script was developed in the Matlab software. This script performs a fitting of each individual curve for different light wavelengths, designated IR (980 nm), LR (650 nm), LB (450 nm), LL (405 nm), and UV (260 nm), using a smoothing spline function (a piecewise interpolation function that includes each support point). The curves are shown in Figure 6. This script initially applies to paraffin sections without any tissue.

[0168] The software uses the scatter values ​​measured for a paraffin section in one or more of the five curves to determine the actual layer thickness. To do this, the respective scatter value (separately for IR, LR, LB, LL and UV) is subjected to the known measurement uncertainty (e.g. ±5%) as the measured value range. The values ​​of the curves that lie within the corresponding range are then marked in the curve (Figure 7). These are the possible layer thicknesses for which the measured value can be measured within the measurement accuracy and which the script calculates as plausible layer thicknesses for the paraffin section. The accuracy of the determination can be increased by using several curves measured at different wavelengths.

[0169] When using multiple gradients, a range of possible layer thicknesses results from the matching layer thicknesses (marked by the cross in Figure 7). The layer thickness of the paraffin section is determined by calculating the mean and standard deviation of these possible layer thicknesses.

[0170] In the following, "artificial measurement values" were generated for various thicknesses, for which the quality of the evaluation was compared. For this purpose, a test layer thickness ("real layer thickness") was defined. This corresponds to the measurement of the layer thickness of a section to be analyzed using the script. Based on this, "example measurement values" were generated, i.e., the expected measurement values ​​or sensor outputs for the given layer thickness according to the known curves. To account for random error in the measurement, these values ​​were subjected to a random "measurement error" of 5% (a pseudo-random number generated using the Mersenne Twister Generator and a seed of 1).

[0171] The analysis over a slice thickness range of 1-7 pm results in a high degree of accuracy for the script. Deviations occur in the range of 2-3.5 pm. If the section thickness set on the microtome ("nominal section thickness") is known, deviations in this range are significantly reduced.

[0172] Measuring color intensity

[0173] The first step involved creating a digital scan of the sections. The scanner produces an image that is equally bright and has the same white balance across all sections. Calibration ensures consistent results even for different sections. To measure color intensity, the tonal value of the nearest color channel (red, green, or blue) is determined in an image processing program (possibly in reverse).

[0174] Further series of 1, 3, 5, and 7 μm sections with tissue were taken, and the tissue color density was determined as a tonal value / color intensity (0-255) on the digital scan. Initially, natural pigment was used for orientation. A clearly monotonic relationship was observed, meaning a color intensity that steadily increased with section thickness. In subsequent test series, this was also performed using immunohistochemical staining (Figure 8).

[0175] The principle can be applied to any substance, provided it has its own color or can be stained. Since natural colors (e.g., the blood pigment hemoglobin) are usually too pale at the very thin layer thicknesses used, histochemical stains are generally used. Additionally, an optical kit (mounting medium) is placed between the tissue and the coverslip. This minimizes scattering by filling the irregular surface and enables a uniform, unobstructed image.

[0176] Accuracy of the specimen scan

[0177] For this purpose, a stained test specimen with various structures and some natural tissue pigments was scanned five times on the same device (panoramic desk). The scans were measured for color density in defined areas. Accuracy was high (variance was low) (Figure 27).

[0178] To establish the method, a protein was used that can be examined in histological sections and with clinical chemistry methods in both methods as standard and with high accuracy and can be reliably isolated from tissue.

[0179] Protein S100 is a low-molecular-mass (9-13 kDa) calcium-binding protein with various subtypes. The S100B subtype was detected here. This protein is expressed primarily in glial cells, but also in other tissue types such as melanocytes and melanocytic tumors. A fresh (unfixed) human cerebrum sample was used as the tissue.

[0180] For clinical chemistry, the tissue was mechanically homogenized by pureeing, largely dissolved with a lysis buffer and then strongly (10 5 -fold) to reach the usable measuring range, which is very low with this method (between 0.02 pg / L-30 pg / L) (Figure 34).

[0181] In histopathology, the process was slightly different: Since a histological specimen undergoes several rinsing steps during preparation, buffer without lysing substances was used so that tiny tissue fragments were available for the calibration preparations at the same dilution as in clinical chemistry. For this purpose, the tissue was mechanically ground into a small amount of liquid using a hand blender and mechanically bound in a gel with a defined dilution (“calibration cone”). Alternatively or additionally, the fresh tissue can be passed through a very fine sieve (100 mesh, meaning 100 meshes per inch) and mixed with Histogel to solidify. To achieve even greater homogeneity, the tissue can also be passed one or more times through a 400 mesh sieve (Fig. 33).

[0182] This calibration cone was embedded in paraffin and sectioned, revealing a homogeneous distribution of tissue within the cone (Figure 32). Several cell blocks were prepared with defined dilutions. The tissue concentration contained in these cell blocks was calculated by comparison with clinical chemistry. These cell blocks were then poured into a new paraffin block as a calibration instrument ("calibration block") (Figure 9). Furthermore, high concentrations of recombinant test protein provided by the manufacturer can be injected directly into the calibration cone as a small depot of approximately 1 mm in size.

[0183] A section was prepared from the cell block, and immunohistochemistry was performed for S100. This section was scanned, and the color concentration was determined as a tonal value of the image between 0 and 255 (blue color channel in the reversed image or brown color in the positive image). Each tonal value can be clearly assigned to a concentration of S100, which allowed a calibration curve for S100 to be created based on the calibration preparation (Figure 12, Figure 13).

[0184] Staining of the specimen using immunohistochemistry

[0185] A potential source of error in determining the concentration of a target substance in a tissue is the distribution of antibodies on the tissue section. To test this, tissue prepared as described above was poured into a mold measuring approximately 5 x 6 cm to ensure high homogeneity during solidification. After formalin fixation, this cast plate was cut into orientable strips, paraffinized, and poured into paraffin blocks. These blocks were routinely stained for the target protein (S100), as well as for spot staining (here, NeuN for the nerve cells evenly distributed in the tissue preparation). The staining showed only minimal fluctuation (Fig. 31).

[0186] Application of the complete method - scattering and absorption

[0187] For the final testing of the method, a tissue sample known to express S 100 was used: the metastasis of a malignant melanoma. The measurement slides were prepared by applying a section of the calibration block with anonymized section thickness to one half of the slide. A section of the tumor sample, also with anonymized section thickness, was applied to the other half (Figure 10). For both sections, the section thickness was determined in the tissue-free margin of the section using the above-mentioned method, and the correction factor was determined. The section was scanned, and representative photographs were taken. The color intensity (tone value, inverted image blue) was determined for each concentration on the calibration sections, and an individual calibration curve was calculated from these three values.The color intensity was also determined for the desired sections in the tumor and the concentrations of the protein S100 were directly assigned using the individual calibration curve and labeled in the photo (Figure 11 and Table 1).

[0188] Table 1 : Measured values ​​of the calibration block determined in the procedure

[0189] Application of the complete procedure - absorption

[0190] To precisely insert a wax punch ("calibration punch") into the block to be measured, which contains the tissue to be examined, a method analogous to the creation of a tissue microarray was used. Tissue cylinders were removed from a paraffin block containing the colored wax using a hollow needle and then punched into the block containing the tissue to be examined or the calibration cones. Two wax punches of different concentrations were punched into each block.

[0191] From these blocks, unstained serial sections of varying thicknesses (1, 3, 5, and 7 μm) were first cut and placed on a microscope slide (Figure 17). The unstained sections were scanned, and the color intensity (brightness values) in the areas of the calibration punches were measured using the scanned image. In parallel, the scattering of red laser light was photographed (in the area adjacent to the tissue).

[0192] By determining the color intensity of the wax punches in sections with different thicknesses of 1, 3, 5, and 7 pm, a standard curve was created for each of the two wax punch concentrations (Figure 18). Based on this, a correction factor was determined between the thickness of the calibration block and the block containing the tumor tissue.

[0193] The sections were then immunohistochemically stained (Figure 19). During this process, the calibration punches (here, wax punches) dissolved and were no longer measurable. Alternatively, a calibration punch can be used, which contains a material that does not dissolve during staining and fixation of the sections. The stained sections were then scanned, and the color intensity of the tumor tissue and the calibration cones of the calibration block were measured based on the scanned image. The measurement ranges are shown schematically in Figure 10. Based on the color intensity of the calibration cones in the calibration block and the correlation with the known concentration of the substance contained in the calibration cones (here, S 100 protein), a calibration curve was created. This calibration curve, taking into account the determined correction factor, was used to determine the concentration in the tumor tissue of the section based on the brightness value of the tumor tissue in the scanned image (Figure 20).This can be represented linearly in the core area of ​​the application. A spreadsheet has been created for this purpose, which summarizes all of the above calculations. The measured values ​​(calibration values) must be entered into the spreadsheet or a corresponding device, from which the individual calibration is calculated. The measured values ​​of the section (the area of ​​the tissue sample to be measured) are then entered, and the concentration of the target protein is calculated and output.

Claims

PATENT CLAIMS 1 . A method for determining a layer thickness of a section preparation, comprising a. measuring one or more values ​​generated by scattering and / or absorption of a light beam by the section preparation with one or more light sources of different wavelengths, b. providing a model of the layer thickness of the section preparation based on calibration values ​​generated by measuring the scattering and / or absorption of a light beam by one or more calibration preparations with one or more light sources of different wavelengths, and c. determining the layer thickness of the section preparation by using the measured values ​​according to (a) and a model according to (b).

2. The method according to claim 1, characterized in that the light source(s) have a wavelength of 200 to 2000 nm, preferably 300 to 1800 nm, particularly preferably 400 to 1700 nm.

3. The method according to one or more of the preceding claims, characterized in that the modeling of the layer thickness comprises, a. providing the calibration values ​​which were determined by measuring the scattering and / or absorption of a light beam by one or more calibration preparations with one or more light sources of different wavelengths, wherein the calibration preparations have a known layer thickness of 0.1 to 10 pm, preferably a known layer thickness of 1 to 7 pm, b. determining one or more calibration functions, wherein the calibration function comprises the correlation of the layer thickness of the one or more calibration preparations with the calibration values, c. comparing the measured values ​​of a section preparation with the one or more calibration functions according to (b), preferably taking into account an inaccuracy in the measurement of the values ​​of 5%, d.The output of a slice thickness of the section preparation which corresponds to the mean of one or more values ​​which agree with one or more of the calibration functions according to (c).

4. The method according to one or more of the preceding claims, characterized in that the section preparation has a layer thickness of 0.1 to 10 pm, preferably 1 to 7 pm.

5. The method according to one or more of the preceding claims, characterized in that the section preparation contains tissue.

6. The method according to the preceding claim, additionally comprising determining the concentration of a target substance in the tissue contained in a section preparation.

7. The method according to the preceding claim, wherein the determination of the concentration of the target substance comprises: a. providing a section preparation and a calibration preparation, wherein the calibration preparation comprises a predetermined concentration of the target substance, b. determining the layer thickness of the section preparation and the calibration preparation by a method according to one of the preceding claims, c. determining a correction factor by using the layer thickness according to (b), d. simultaneously staining the section preparation and the calibration preparation, preferably on the surface of a slide, e. measuring the color intensity of the tissue contained in the section preparation and of the target substance contained in the calibration preparation, and f. determining the concentration of the target substance in the tissue by using the color intensity according to (e) and the correction factor according to (c).

8. The method according to one or more of claims 7, wherein the calibration preparation contains the target substance in two or more concentrations which are spatially separated from each other.

9. The method according to one or more of claims 7 to 8, wherein the layer thickness of the section preparation and the calibration preparation is determined in tissue-free areas of the section preparation and the calibration preparation.

10. The method according to one or more of claims 7 to 9, characterized in that the tissue-free regions of the section preparation, the one or more calibration preparations and / or the calibration preparation comprise one or more regions which contain a dye which absorbs light in a wavelength range of 200 to 2000 nm, preferably in the range of 300 to 800 nm.

11. The method according to one or more of claims 7 to 10, characterized in that the section preparation, the one or more calibration preparations and the calibration preparation are a paraffin section.

12. The method according to any one of claims 7 to 11, wherein a digital scan of the section preparation, the calibration preparation and / or the calibration preparation is made, which is used for the measurement of the values ​​and / or the color intensity, wherein the measurement of the values ​​and / or the color intensity comprises the determination of a brightness value.

13. The method according to one or more of claims 7 to 12, wherein a calibration curve for the concentration of the target substance is created on the basis of the measured color intensity of the target substance present in the calibration preparation, which calibration curve is preferably determined from two or more concentrations of the target substance.

14. The method according to the preceding claim, wherein the target substance is a substance within the tissue contained in a section preparation, preferably a protein, and the target substance is localized in the tissue contained in the section preparation using the calibration preparation and the calibration curve and the concentration of the target substance is determined.

15. A device for measuring the layer thickness of a section preparation and optionally for determining the concentration of a target substance in the section preparation, comprising a. one light source or several light sources of different wavelengths in the range from 200 to 2000 nm, preferably 300 to 1800 nm, particularly preferably 400 to 1700 nm, b. At least one sensor for measuring the scattering and / or absorption of a light beam, c. A device for positioning the section preparation during the measurement, and d. A computing unit for modeling the layer thickness of a section preparation or for using the modeling in determining the layer thickness of a section preparation and optionally for calculating the concentration of a target substance contained in the section preparation.

16. A slide for use in a method according to any one of the preceding claims or with the device according to claim 15, comprising a calibration preparation containing a target substance in two or more concentrations which are spatially separated from one another, to which a section preparation can additionally be applied.

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