Device and method for tissue staining quality control

Abstract

The present invention provides a quality control device for histopathology staining comprising a cellulose film containing less than two plasticizers.

Description

[Technical field]

[0001] The present invention relates to a quality control device for histopathology staining. [Background technology]

[0002] Histopathology relies on the use of a variety of complex staining processes to allow visualization of cells and tissues within a sample. However, there are known degrees of variability in such staining processes depending on the dyes and process conditions used. For example, dye time, dye concentration, and tissue thickness can all affect the resulting stained image.

[0003] Digital imaging in histopathology is becoming increasingly important in the medical community, but digitizing these images adds additional complexities due to variations in the imaging process, such as changes in light intensity or color response of the camera used. These variations risk compromising the confidence that can be given to digital pathology.

[0004] If the differences that occur during the staining and imaging process due to variations in staining and imaging conditions can be quantified, the resulting images can be corrected to compensate for the differences. This ensures that the resulting digitally processed images contain the smallest possible degree of differences, thereby calibrating the images and improving their quality.

[0005] There is therefore a need for a means to quantify the differences that occur during the pathological staining process and the subsequent imaging process. This can be achieved using a quality control device such as that discussed in GB2522231. This document discloses a dye evaluation target that comprises a substrate formed from an optically transparent material that resembles biological tissue. The substrate is stained under similar conditions as the tissue sample, providing a calibration device for comparing the resulting images.

[0006] Similarly, WO2013 / 186530 discloses an imaging calibration device in which dye is applied to areas of a surface, which may include individual tissue blocks. These stained areas can be used to calibrate the resulting images.

[0007] Any such quality control device must absorb the dye with low variability so that it can provide a reliable comparison to quantify differences in the staining and imaging process. This is discussed in GB2524227, which discloses an imaging reference device having a first region of a first substrate and a second region of a second substrate, where the regions are stained with different tissue dyes and then overlaid. This creates an improved reference device that can closely match the spectral characteristics of the tissue sample. However, this is a more complicated system and requires that a separate device be made for each staining protocol. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] GB2522231 [Patent Document 2] WO2013 / 186530 [Patent Document 3] GB2524227 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a need for a quality control device for histopathology staining that exhibits low variability in dye uptake yet is simple and easy to use. [Means for solving the problem]

[0010] It has been discovered that cellulose films provide excellent dye uptake when used as a quality control device for histopathology staining and imaging. The cellulose films can be stained simultaneously and under the same conditions as the tissue samples of interest, and stained proportionally to the tissue samples, using a variety of dyes, including hematoxylin and eosin (H&E) or diaminobenzidine (DAB) and horseradish peroxide (HRP).

[0011] Thus, the cellulose film provides an internal color control, allowing characterization of color changes during sectioning, staining, and imaging. This allows independent measurements of the illumination, imaging device, and individual dyes applied to the slide, thereby allowing variations arising from the staining and imaging steps to be quantified and corrected, as shown in Figure 1.

[0012] Quantitative evaluation of the tissue staining process and any subsequent imaging steps is then possible, which can reduce inter-observer variability and allow for digitization of the staining results. This quantitative evaluation allows the digitally processed images to be corrected to compensate for any variability, thereby improving the quality of the collected images.

[0013] According to a first aspect of the present invention there is provided a quality control device for histopathology staining comprising a cellulose film containing less than two (i.e. one or zero) plasticizers.

[0014] Cellulose films generally have a complex plasticizer package that includes several different plasticizers. This allows the properties of the resulting film to be adapted to the technical requirements of different applications and also has commercial implications, such as balancing high and low costs or supplies. For example, plasticizers are generally considered essential to reduce the brittleness of conventional cellulose films.

[0015] It was surprisingly discovered that cellulose films with a single plasticizer or no plasticizers (i.e., with less than two different plasticizers) exhibited reduced variability in dye uptake, thereby yielding a more consistent product. Without wishing to be bound by theory, it is believed that cellulose films with multiple plasticizers have an increased source of variability in dyeing. Predictability of dye uptake is important in quality control devices to provide reproducible comparative measurements.

[0016] One of the additional challenges of using cellulose film as a quality control reference material is to hold it in place on the slide surface throughout the process of slide preparation so that it remains intact, flat, and suitable for imaging. This is because the dimensions of cellulose film change in different hydration states, meaning that cellulose films can undergo a variety of dimensional changes that are strong enough to cause detachment from the slide or another surface to which the film is attached.

[0017] It was surprisingly found that a simpler plasticizer package (i.e., containing one plasticizer) or the absence of a plasticizer helped to reduce lifting of the cellulose film from the surface to which it was attached, such as a slide. Without wishing to be bound by theory, it is believed that the plasticizer leaches out of the cellulose film and into the adhesive between the film and the surface, thereby destroying its integrity and allowing liquid ingress behind the film. This leaching may further increase image variability that occurs during the staining and imaging process.

[0018] It has also been found that increased brittleness of the resulting cellulose film was not an issue in the quality control devices of the present invention due to the low stress profile of such devices, especially when attached to a surface or provided with a protective sheet.

[0019] The variability of dye uptake increased with the number of different plasticizers present in the cellulose films. The most predictable dye uptake was seen in cellulose films without any plasticizers.

[0020] Plasticizers are well known for use in cellulose films and any conventional plasticizer may be used in the cellulose films of the present invention. Suitable plasticizers include glycerin, polyethylene glycol, monopropylene glycol, triethylene glycol and urea.

[0021] The cellulose film may contain one or more reference items. Reference items are particles that are visible in the film after the staining process, thereby allowing size comparisons to be made to items found in the tissue sample and allowing easier focusing of the image. Reference items may also be used to visually represent the tissue itself to aid the pathologist in viewing the sample.

[0022] The reference item is preferably of known size, orientation and / or concentration within the cellulose film. The reference item is preferably embedded within the cellulose film. The reference item may be embedded in the film during its manufacture.

[0023] The reference item may include inert particles, such as PMMA or silica particles. The inert particles may be of known particle size. The inert particles may be of varying particle size having a known range of sizes. The inert particles may be of known concentration within the cellulose film.

[0024] The reference item may include cellulose fibers. The cellulose fibers may be of known fiber length and / or diameter. The cellulose fibers may be of known orientation and / or known concentration within the cellulose film.

[0025] The reference item may include air bubbles. The air bubbles may be of a controlled and / or known size. The air bubbles may be of a known concentration within the cellulose film.

[0026] The reference item may include a protein. The protein may be one found in a tissue sample of the subject, e.g., her-2 and estrogen receptor proteins. The concentration of the protein in the cellulose film may correspond to the concentration of the protein expected in a tissue sample of the subject. The protein may be of known size and / or known concentration in the cellulose film.

[0027] The cellulose film may contain a combination of different reference items that may be used to replicate one or more characteristics of a tissue sample of interest.

[0028] Thus, the reference items may have similar properties, e.g., size and / or staining, as the particles of interest in the tissue sample. Different reference particles having different values ​​of a particular property, e.g., size or staining, may be included on the film to provide a fast reference when imaging.

[0029] The reference items may have a random distribution within the film, and thus the pattern of the reference items may be used to generate a unique "digital fingerprint" of each quality control device, allowing traceability of the device throughout its history.

[0030] The cellulose film may contain chitosan. The chitosan may be a fungal chitosan. This may reduce the dye uptake variability. The staining variability decreases with increasing chitosan levels in the film. The film may contain chitosan at 1 to 10%, preferably 3 to 10% and most preferably 5 to 10%.

[0031] The cellulose film may contain dopants that aid in dye uptake in the cellulose film. Different tissues have different degrees of dye uptake, especially for dyes such as eosin and hematoxylin. These dopants may be included in amounts such that the dye uptake in the cellulose film matches that of the tissue sample of interest. Thus, the cellulose film may be matched to the tissue sample of interest.

[0032] Both keratin and gelatin were found to increase dye uptake in cellulose films. Chitosan also increases dye uptake in cellulose films. Thus, cellulose films can contain one or more of chitosan, keratin, and gelatin to tailor the dye uptake of the cellulose film to reflect the staining of the tissue sample of interest.

[0033] The cellulose film may be 10 to 50 μm thick. The cellulose film may be 20 to 30 μm thick. This thickness helps reduce changes in film dimensions due to different hydration states, which can reduce lifting of the film from surfaces such as slides, thereby reducing dye uptake variability.

[0034] The degree of variation in the cellulose film thickness is preferably low, as this helps to further reduce variability in the dye uptake in the cellulose film. The thickness variation may be less than 3% of its target value, preferably less than 2%.

[0035] The quality control device may include pre-colored red, blue, and green regions, which can improve accuracy when quantifying variations resulting from the staining and imaging process by providing color standards in the device.

[0036] The cellulose film may have an adhesive on at least one side thereof.The cellulose film may have an adhesive on both sides thereof.

[0037] The cellulose film can be attached to a protective sheet. The cellulose film can be laminated to a protective sheet. This can reduce dimensional changes in the film due to different hydration states, thereby reducing dye uptake variability. The protective sheet can also protect the cellulose film from damage during use as a quality control device.

[0038] The protective sheet may include a polymer. The protective sheet may be a film.

[0039] The quality control device may further include an adhesive on the opposite side of the cellulose film to the protective sheet. This allows the quality control device to be attached to and stained on the same surface as the tissue, thereby ensuring that the tissue and the quality control device are subject to the same staining conditions. The surface may be a microscope slide.

[0040] The cellulose film may comprise a coating on at least one side thereof. The cellulose film may also include a primer layer between the cellulose film and the coating. The cellulose film may be free of a coating and / or primer layer. If present, the coating is preferably on the side of the film adjacent to the surface, opposite the protective sheet (if present), so that the dye is not prevented from reaching the cellulose film. An adhesive may be applied over the coating to adhere the cellulose film to the surface.

[0041] The coating may be a barrier coating. This may prevent plasticizers from leaching into the adhesive and thereby destroying its integrity and reducing the adhesion between the film and the surface. The coating may also increase the adhesion between the adhesive and the cellulose film itself. The coating may include ethylene acrylic acid, polyvinylidene chloride, acrylic or any other barrier coating material.

[0042] According to a second aspect of the present invention there is provided a surface comprising a quality control device as described above attached thereto. The surface may be the surface of a microscope slide.

[0043] The quality control device does not have to cover the entire surface. A tissue sample can then be positioned on the surface and stained simultaneously with the quality control device, thereby ensuring that the tissue sample and the quality control device are subjected to the same staining conditions.

[0044] According to a third aspect of the present invention there is provided the use of a cellulose film containing less than two plasticizers in a quality control device for histopathological staining.

[0045] As previously mentioned, the presence of one or zero plasticizer reduces dye uptake variability in cellulose films, thereby producing a more consistent quality control device for histopathology staining.

[0046] According to a fourth aspect of the present invention there is provided the use of a quality control device as discussed herein for correcting image variations in a pathological staining and imaging process.

[0047] This is possible due to the predictable and linear dye uptake of the cellulose films of the present invention, which produces reliable color control that can be used to quantify variations arising from the staining and imaging processes, thereby allowing for the correction of such variations in the resulting images.

[0048] According to a fifth aspect of the present invention there is provided a method of correcting image variations resulting from staining and imaging a tissue sample comprising: a. staining said quality control device and tissue sample under the same conditions; b. imaging the quality control device; c. Quantifying staining and imaging variability in quality control device images; and d. Imaging the tissue sample; and e. Correcting tissue sample images using quantification of staining and imaging variations in quality control device images.

[0049] The quality control device and the tissue sample can be stained simultaneously, at the same time and under the same staining conditions. Preferably, the quality control device and the tissue sample are mounted on the same surface, e.g., a microscope slide, and stained together, as this ensures that they are subjected to the same staining conditions.

[0050] The steps of the method may be performed in a different order than outlined above, for example, imaging the tissue sample may occur before staining and imaging variations in the quality control device images are quantified.

[0051] The invention will now be more particularly described with reference to the following examples and figures. [Brief description of the drawings]

[0052] [Figure 1] 1A-1D illustrate a process for correcting images of stained tissue samples using a quality control device according to an embodiment of the present invention. [Diagram 2] FIG. 1 shows the linearity of dye uptake in zero-plasticiser cellulose film according to one embodiment of the present invention. [Diagram 3] FIG. 1 shows the linearity of dye uptake in single-plasticiser cellulose film according to one embodiment of the present invention. [Figure 4] FIG. 1 shows eosin uptake in single plasticizer cellulose films according to an embodiment of the present invention with different chitosan dosages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] [Example 1] The change in dye intensity with increasing dye time was investigated using uncoated cellulose films with one plasticizer and uncoated cellulose films with no plasticizer.

[0054] Figure 2 shows the increase in dye intensity with increasing dye time for the zero plasticizer cellulose films, while Figure 3 shows the increase in dye intensity with increasing dye time for the single plasticizer cellulose films.

[0055] R of cellulose films without plasticizers 2 The R value for the single plasticizer cellulose film is 0.9915, whereas 2 The value goes down to 0.9905. These graphs show that the addition of plasticizer causes an increase in variability in the dye uptake of the cellulose films of the invention. However, this variability is statistically acceptable for both cellulose films tested.

[0056] [Example 2] Figure 4 shows the variability of RGB values ​​in cellulose films containing a high dosage of chitosan (5% by film weight) and in cellulose films containing a low dosage of chitosan (2.5% by film weight) after staining with 2 min hematoxylin and 2 min eosin.

[0057] The film was scanned on the same AT2 scanner, and the RGB and L of the film were * a * b * The results were analyzed with respect to QuPath by measuring the values.

[0058] As shown in Figure 4, high dosage chitosan in the cellulose films reduces the variability of RGB values ​​compared to cellulose films containing low dosage chitosan. High dosage chitosan films also appeared to show preferential uptake of eosin compared to lower dosage films. Thus, increasing the level of chitosan in the films reduces the variability of the imaging process.

Claims

1. A quality control device for histopathology staining comprising a cellulose film containing less than two plasticizers.

2. The quality control device of claim 1 , wherein the cellulose film is plasticizer-free.

3. The quality control device of claim 1 , wherein the cellulose film includes one or more reference items.

4. The quality control device of claim 3 , wherein the one or more reference items comprise PMMA particles, silica particles, cellulose fibers, air bubbles and / or proteins.

5. The quality control device of claim 1 , wherein the cellulose film comprises chitosan, keratin and / or gelatin.

6. The quality control device of claim 1 , wherein the cellulose film comprises a coating on at least one side thereof.

7. 2. The quality control device of claim 1, wherein the cellulose film is 10 to 50 μm thick, preferably 20 to 30 μm thick.

8. The quality control device of claim 1 , wherein the cellulose film is attached to a protective sheet.

9. The quality control device of claim 1 further comprising an adhesive on one side thereof.

10. 10. The quality control device of claim 9, further comprising a barrier coating on the side of the cellulose film that contains the adhesive.

11. A surface having attached thereto a quality control device according to any one of claims 1 to 10.

12. Use of a cellulose film containing less than two plasticizers in a quality control device for histopathology staining.

13. Use of a quality control device according to any one of claims 1 to 10 for correcting image variations in pathological staining and imaging processes.

14. a. staining the quality control device of any one of claims 1 to 10 and a tissue sample under the same conditions; b. imaging the quality control device; c. Quantifying staining and imaging variation in said quality control device images; d. Imaging the tissue sample; e. correcting the tissue sample images using the quantification of the staining and imaging variations in the quality control device images; 23. A method for correcting image variations resulting from the steps of staining and imaging the tissue sample, comprising:

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