CN110741302B - Process recording slide for immunohistochemical staining - Google Patents
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- CN110741302B CN110741302B CN201880039196.1A CN201880039196A CN110741302B CN 110741302 B CN110741302 B CN 110741302B CN 201880039196 A CN201880039196 A CN 201880039196A CN 110741302 B CN110741302 B CN 110741302B
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Abstract
An apparatus and method for determining the effect of paraffin removal, antigen retrieval and the use of primary and secondary staining reagents in an assay procedure, particularly a multi-step Immunohistochemistry (IHC) assay. The device includes an adhesive coated microscope slide containing a plurality of compounds spotted in a 2D or 3D configuration and sealed under a paraffin coating. Subsequently, tissue sections or loose cells are added to the same slide and all undergo IHC processing steps applied from tissue capture to coverslip. The compounds were reacted with primary or secondary IHC staining reagents to record the processing history of the coexisting tissue sections or loose cells.
Description
Cross Reference to Related Applications
The present application claims priority of U.S. provisional application No. 62/520,319 entitled "process recording slide for immunohistochemical staining" filed on day 6, 15 of 2017, U.S. provisional application No. US62/539,281 entitled "process recording slide for immunohistochemical staining" filed on day 31 of 2017, U.S. provisional application No. 62/520,169 entitled "barrier coating for microscope slide protein deposits" filed on day 6, 15 of 2017, U.S. provisional application No. 62/520,178 entitled "immunohistochemical imaging baseline reference" filed on day 6, 15 of 2017, U.S. provisional application No. US 62/520,187 entitled "immunohistochemical antigen imaging scale extrapolation" filed on day 6, 15 of 2017, the entire contents of each of which are incorporated herein by reference.
Technical Field
The present invention relates to a novel process recording slide. The invention particularly relates to a process recording slide and a staining method for immunohistochemistry. More particularly, the present invention discloses a process recording slide that provides control targets for coexisting patient samples to undergo a staining process together. After staining, stained controls immediately showed possible errors, i.e. deviations from the baseline of the known target, if any. The above-described process recording slides provide efficient quality control with high accuracy and precision at a cost-effective price and an easily acceptable threshold.
Background
All immunohistochemical methods, as well as other immunochemical methods, are multi-step procedures involving a series of reagent exchanges, incubations and washes. Most of these procedures require trained personnel and results may vary widely from laboratory to laboratory. Automated systems have been developed that employ cost savings, consistent slide preparation, and reduce procedural human error.
There are some key points to consider for both automated and manual methods. Care must be taken to avoid sample loss on the slide. Thorough washing of the sample during reagent application is very necessary, especially to remove unbound antibody, as residues can be enlarged. Excess liquid must be removed to avoid carryover of previous reagents and/or unnecessary dilution of subsequent reagents, yet never allow the sample to dry. Sufficient antibody reagent must be used to completely cover the area of the slide where the sample may be present, but waste must be kept to a minimum.
In addition, many of the reagents used in immunohistochemical and immunochemical methods, such as enzyme solutions and peroxidase developers, have limited stability at operating temperatures, even at room temperature. This requires frequent preparation of the reagents. Furthermore, non-specific antibody binding leading to erroneous results remains a problem.
Methods and reagents that improve results and minimize reagent preparation would facilitate both manual and automated immunohistochemical methods. Many improvements can be readily applied to relevant immunochemical methods such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays and in situ hybridization.
Reference may be made to "Use of cut cells as a control for quantitative immunological analysis of estrogenic receptor in the branched cancer. the quick method", which discloses that variations in tissue fixation, processing and staining are the main cause of poor reproducibility in Estrogen Receptor (ER) immunohistochemical assays. Frozen agar suspension particles of MCF-7 cells with known ER content were added to each of 55 samples of Invasive Breast Cancer (IBC) as controls. Image analysis determined the percentage of MCF-7 cells and IBC positive area (positive nuclei/total nuclei analyzed) and the percentage of positive staining (sum of optical density of the positive nuclear region divided by the sum of optical density of all nuclei studied). The average ER value in MCF-7 cells was 150fmol/mg as determined by dextran-coated activated carbon assay. Image analysis of 55 MCF-7 cells showed an average positive area of 70.81. The positive staining range for IBC cases was 0 to 98.5. The conversion factor was used to convert the positive area of clinical samples to femtomolar equivalents by using MCF-7 cells of known ER content and positive area, 55 cases of IBC with positive area ranging from 0 to 1790 (mean, 187). An ER control containing a known femtomolar amount provides an internal standard for quality control and ER quantification.
Reference may be made to CN102435728, which discloses a method for preparing a positive reference for quality control in immunohistochemical processes. The method comprises the following steps: the polypeptide or protein which can react with the antibody specifically and has different concentrations is adsorbed on the glass slide in advance, or the polypeptide or protein with different concentrations is placed on the glass slide in advance, the polypeptide or protein and the pathological tissue section are simultaneously subjected to the conventional immunohistochemical step, and the color development result of the polypeptide or protein is used as a positive control of the immunohistochemical process. The invention adopts a method of setting positive control protein or polypeptide on a glass slide to realize positive control and quality control standard, and the method is an important supplement of the existing quality assurance program and is a new method for quality control of immunohistochemical test.
The above invention reports the problems listed below:
a. since the binding of the peptide fragments to the dextran polymer depends on the viscosity of the mixed solution, the temperature and size of the precipitated polymer particles will vary depending on bath concentration, reaction temperature and NaOH injection reproducibility, and the excess peptide washed from the dextran will not be consistent with the target density.
b. Since the peptide concentration available on the polymer particles is unknown, the constructed targets of known reactivity (staining density) are limited. The result is only a yes/no primary antibody detector.
c. Although dextran can support protein capture of secondary IgG targets, only yes/no results. Thus, a baseline detection scale supporting digital imaging cannot be established.
d. During antigen retrieval, the target will leak proteins/peptides onto the remaining slides and tissue sections. The target is placed above the tissue section and therefore there is background and tissue contamination from the target during processing.
Reference may be made to Horizon Diagnostics, which made control slides similar to CN102435728, but the construction of the target is very different. Targets are prepared from DNA-modified tissue culture cell lines to naturally place the desired antigenic peptide with the cells. These cell lines can be replicated as needed and are referred to as "renewable resources". Cells were fixed in formalin and paraffinized into tissue blocks in loose cell paste form. In use, the targets may comprise non-reactive cells to produce a mixture of positive and negative reactive targets in the same region. To generate a target array, each set of cell elements can be formed into cylindrical cores aligned with other cores, and the entire array cut into individual sections for application to a slide.
The above invention reports the problems listed below:
a. controls were limited to only a yes/no result simply because there was too much unknown of the reactive target site density. Simply represent: by changing the cross-sectional section of the cells, the staining intensity results will change. Since the cell mass must pass through the antigen retrieval process, whatever antigen is present will be affected empirically, resulting in unknown variables.
b. Upon cleavage of a portion, the formation of a mixture of known cells (antigen versus blank) is statistically ineffective because the electrostatic charge on the cells can differ, causing them to separate and clump together. Thus, the ratio of cross-section to cross-section through the cell mass is variable, which makes the generation of a ratio of antigen density unlikely.
c. Cell lines do not perform consistently due to the limited replicative life span of cell propagation. There is no guarantee that the antigen density of the new cell line is the same as the previous cell line.
d. Since the tissue block, the cutting block, and the section are manually constructed and used for the slide, it is not cost-effective.
Reference may be made to US2016/0274006a1, which discloses a method and apparatus for use as a control and calibrator for performing assays on cells and tissues mounted on microscope slides. The device includes a quality control moiety, such as a peptide epitope, attached to a particulate object, such as a transparent spherical bead and the bead is preferably approximately the size of the cell. The quality control portion is designed to exhibit similar behavior in the assay as the analyte, thereby producing a positive assay reaction. During the step of staining with the novel liquid matrix, the beads are stored on the microscope slide, and the matrix solidifies upon drying and causes the beads to adhere to the microscope slide.
This control and calibration solution, while interesting, is impractical in practical use because the stability of the target to the substrate is weak and target data is difficult to extract due to target material on sparsely distributed coated beads. When a single bead is imaged, the staining color changes from the top center of the bead to the edge of the bead, so it is difficult to know at which point on the bead surface the image data is correct.
Reference may be made to US7271008B2, which discloses a device and a method for determining the quality of a reagent during an assay, in particular a multi-step immunohistochemical assay. In particular, the device comprises a substrate to which a plurality of compounds are attached, wherein each compound reacts with a reagent used in the assay.
The immunostaining disclosed in the above-mentioned patent document is intended to serve as a quality control slide to evaluate the performance of the secondary staining kit, rather than to support tissue sections that are subjected to immunohistochemical processing. The slide substrate is an aminosilane that does not support covalent bonds that can participate in the antigen retrieval process for any target. In addition, alkaline phosphatase targets are degraded by exposure to antigen retrieval temperatures.
Disclosure of Invention
In general, in one aspect, the invention provides a process recording slide for immunohistochemical staining.
In another aspect of the invention, a device and method are provided for determining the effect of paraffin removal, antigen retrieval, and the use of primary and secondary staining reagents during an assay, particularly a multi-step Immunohistochemistry (IHC) assay.
In another aspect, the invention provides a device comprising an adhesive-coated microscope slide containing a plurality of compounds spotted in 2D or 3D structures and sealed under a paraffin coating.
In another aspect, the invention provides a process recording slide in which tissue sections or loose cells are subsequently added to the same slide and all undergo an IHC processing step applied from tissue capture to coverslip.
In another aspect, the invention provides a process recording slide in which a compound is reacted with a primary or secondary IHC staining reagent to record the processing history of coexisting tissue sections or loose cells.
In another aspect, the invention provides a process recording slide in which the primary target consists of bound antigen on a carrier protein.
In another aspect, the invention creates a process record slide in which the primary target consists of bound antigen on a carrier protein that is mixed with other carrier proteins bound with different antigens to form targets with multiple capture capabilities. Only one can be used at a time, but this method amplifies the number of primary targets, beyond the actual number of primary targets on the slide.
In another aspect, the invention creates a process record slide in which the primary target consists of a bound antigen of a carrier protein, which is mixed with other non-reactive proteins to create a gradient density array of all the same antigens.
In another aspect, the invention provides a process recording slide in which two secondary target arrays are used, one of which is a mouse and the other of which is a rabbit.
In another aspect, the invention provides a process recording slide in which a secondary array is applied to a substrate in a 2D gradient density array from a concentration of 10% to 100% along with a 2D/3D 100% concentration target.
In particular, a process recording slide is provided in accordance with the present invention in which the concentration of each antigen on a coexisting tissue section is objectively measured by extrapolation of a secondary array and, if possible, establishment of a scale or ruler aided by a primary array. IHC staining undergoes permanent locking with coexisting tissue sections or loose cells to support quality control of IHC procedures and objective measurement of antigen density.
In particular, the invention includes the following embodiments:
1. a process recording slide for immunohistochemical staining comprising:
optionally, a label on top of the slide label area that identifies the type of slide and a code that identifies the antigen supported by the primary target;
optionally, a lot number printed under a label on the slide;
space for processing by IHC and subsequent examination of tissue slices;
an IHC target located below the tissue section;
imaging reference points located on both sides of the protein array; and
optionally, a glass microscope coating.
2. The process recording slide according to embodiment 1, wherein the combination of targets can be secondary, secondary and antigen repair monitors alone, or secondary and antigen repair monitors and primary antigens.
3. The process recording slide according to embodiment 1, wherein the tissue section may be applied to the space selected from the tissue sections of any biological origin.
4. The process recording slide according to embodiment 1, wherein the secondary IHC 2D target consists of 4% formalin and (a) mouse and donkey proteins to form a gradient density series from 100-10% mouse concentration, and (b) rabbit and donkey proteins to form a mixture of gradient density series from 100-10% rabbit concentration.
5. The process recording slide according to embodiment 1, wherein secondary IHC 3D 100% target is formed from a mixture of polysaccharide as backbone and 100% of example 4a (a) mouse protein and (b) rabbit protein.
6. The process recording slide according to embodiment 1, wherein the enlarged antigen repair monitor target is a 100% mouse and rabbit protein 50:50 mixture, deposited in two structures: (a)2D fixed with 2% formalin; (b)
3D was fixed with polysaccharide and then 6% formalin.
7. The process recording slide according to embodiment 1, wherein the primary IHC 2D target is an antigenic peptide covalently linked to a carrier protein, such as Keyhole Limpet Hemocyanin (KLH), which can be mixed with neutral KLH protein and 4% formalin to prepare a gradient density series of 3 to 5-fold dilutions, or with different antigen-linked KLH proteins to form multiple antigenic targets.
8. The process recording slide according to embodiment 1, wherein the imaging reference point is a black and white imaging reference.
9. The process recording slide according to embodiment 1, wherein the glass microscope slide is coated with a bioadhesive which is covalently attached to the glass and which is conformal to reactive end groups selected from the group consisting of amine groups, amide groups, carboxyl groups and hydroxyl groups and which is slightly hydrophilic, such as the seemer feishel SuperFrost Plus # GL 4951P.
10. The process recording slide according to embodiment 1, wherein the target co-existence targets the tissue section.
11. A method of immunohistochemical staining of a process recording slide according to embodiment 1, comprising the steps of:
a. removing paraffin from the paraffin-embedded tissue sections and covering the PRS target with a paraffin barrier coating;
b. removing the formaldehyde fixation by an antigen retrieval buffer to expose antigenic sites of the tissue section;
c. applying one or two primary binding antibodies to bind to any matching antigenic sites found in the tissue section or primary antigenic target site;
d. applying a staining reagent to the exposed antigenic sites obtained in step (b) to produce a visible color indicative of the presence of the target antigen;
e. optionally, multiple amplification steps to obtain a sufficient density of staining reagents;
f. hematoxylin was used to provide a contrasting color (blue) to make the physical form visible.
g. The slide was covered with a coverslip and finally prepared for testing.
12. The method according to embodiment 11, wherein the paraffin removal in step (a) is carried out by heating the paraffin at a temperature ranging between 65-75 ℃ for 3-10 minutes to obtain a semi-liquid state, followed by serial liquefaction with organic solvents until rehydration in a buffer solution.
13. The method according to embodiments 11 and 12, wherein the organic solvent is selected from the group consisting of an aliphatic solvent starting series of solvents such as xylene or mixed xylenes, absolute ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and salt-based buffer solutions, the exposure time of each solution being designated 3 minutes.
14. The method of embodiment 11, wherein formaldehyde fixation can be removed by an antigen retrieval process, such as: heat-induced epitope removal (HIER) process or antigen retrieval process with multiple water exchanges by longer warm distilled water.
15. The method according to embodiment 11, wherein the secondary staining reagent used in step (d) may be selected from the group consisting of an enzyme-labeled secondary, an enzyme-labeled tertiary antibody reactive with the enzyme-labeled secondary antibody, an APAAP immune complex reactive with the secondary antibody, an enzyme-labeled (strept) avidin reactive with the biotinylated secondary antibody, an avidin-/streptavidin-biotin-enzyme complex reactive with the biotin-labeled secondary antibody, a streptavidin-enzyme complex on the biotinylated secondary antibody in the primary antibody, and a polymer containing the secondary antibody and an enzyme site bound to the primary antibody.
16. The method according to embodiment 11, wherein the antigen retrieval buffer used may be selected from the range of pH 6-9.
17. The method according to embodiment 11, wherein the primary antibody in step (c) is selected from the group consisting of antibodies wherein the host protein is a mouse or rabbit, including the common examples: ER, PR, Her2, Ki 67.
18. The method of embodiment 11, wherein the chromogen can be selected from the group consisting of 3,3' -Diaminobenzidine (DAB), amino-9-ethylcarbazole (AEC), 3' -diaminobenzidine + nickel enhancer, fast red, 3',5,5' -Tetramethylbenzidine (TMB), Yellow-preserving (Stay Yellow), 5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium (BCIP/NBT), 5-bromo-4-chloro-3-indole-phosphate/tetranitro blue tetrazolium (BCIP/TNBT), naphthol AS-MX phosphate + fast blue BB, naphthol AS-MX phosphate + fast red TR, naphthol AS-MX phosphate + neofuchsin, Green-preserving (Stay Green), and Nitro Blue Tetrazolium (NBT).
19. The method of embodiment 11, wherein the method is cost effective, repeatable, robust, and facilitates identifying IHC processing steps that lead to misanalysis.
20. The method of embodiment 11, wherein said method is used as a quantitative standard for process control of antigen concentration on coexisting tissue sections or loose cells.
Drawings
FIG. 1 shows the types of primary and secondary targets that may be utilized in accordance with an embodiment of the present invention.
Fig. 2A shows the basic slide fabricated. The slide has the smallest ID by lot number and has a blank area in the colored label area. There are two thick colored long axis stripes that extend beyond the area of the target spot. These bars are recently added to address how the label printer dispenses slides from the bottom of the stack. These strips ensure that the slides stacked on top do not damage the slide coating and more importantly the paraffin mask coating and the target points under the paraffin.
Fig. 2B shows the base slide as it has printed the additional data in the label area: date, 2D barcode and tissue section that has been captured. Note that the tissue sections are more like paraffin. The tissue is substantially transparent and therefore the paraffin color is dominant. The "patient tissue" and "control target" text were not printed on the slide, but rather the reader was instructed here what the paraffin covered area was.
Figure 2C shows the basic slide as it appears after IHC treatment. Both the target and the tissue section are visible and ready for interpretation.
Figure 3 is a close-up view of the effects of AR damage.
Fig. 4 shows the effect on the image when the illumination level is too dark (optimally-5%), best state (+0) and too bright (e.g. +10 or + 15%).
Figure 5 shows how the paraffin barrier coating ensures the sealing of the edge of its deposit.
Detailed Description
The present invention may be understood more readily by reference to the following detailed description of the invention taken in conjunction with the accompanying drawings, which form a part hereof. It is to be understood that this invention is not limited to the particular devices, methods, conditions or parameters described and/or illustrated herein, and that the terminology used herein is for the purpose of illustration only and is not intended to be limiting of the claimed invention. Also, as used in the specification, including the appended claims, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value, unless specifically indicated otherwise. When such a range is expressed in another embodiment, the range can be expressed from "about" or "approximately" another particular value. In addition, it should be understood that the dimensions and material properties described herein are by way of example and not by way of limitation, unless otherwise specified, and are for the purpose of better understanding an example embodiment for suitable use, and that variations other than the recited values may be within the scope of the invention depending on the particular application.
The invention is not limited in its application to the details of construction and the arrangement of the components set forth. In or shown in the following description or drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein for the purpose of description should not be regarded as limiting. The use of "including," "comprising," "having," "consisting of …," "involving," and variations thereof, as well as other items.
Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings and the description to refer to the same or like parts.
Definition of
As used herein, the term "slide," also referred to as a "microscope slide," refers to a thin, flat sheet (typically made of glass, and therefore sometimes referred to as a "glass slide") typically 75 x 26mm (3 x 1 inch) thick about 1mm thick for holding an object under examination under a microscope.
The slide in the present invention is also referred to as a "Procedure Record Slide (PRS)", which may be interchangeably referred to as a PRS-IHC slide.
The term "detection zone" as used herein refers to the space in a slide where a sample, such as tissue and loose cells of any biological origin, is placed for subsequent immunohistochemical or immunochemical detection.
The term "control zone" as used herein refers to a space that accommodates targets of known reactivity behavior for assessing antigen repair status, primary antibody agent efficacy, and secondary agent efficacy, including one or more selected from primary and secondary target arrays, imaging references, and antigen repair monitors.
It should be noted that the "detection zone" and "control zone" have clear label boundaries on the slide; it is preferable to classify them only according to their functions.
As used herein, the term "primary target" refers to a target to which a primary antibody used in an IHC assay can bind. Likewise, it may refer to any unspecified antigenic peptide fragment that can be recognized by an antibody. The type of antigenic peptide fragment can be determined by the primary antibody used in the IHC procedure and then combined with the carrier protein to obtain the desired primary target array. Alternatively, the primary targets are prepared in advance with a common antigenic peptide fragment for later use.
The term "antigenic peptide fragment" as used herein refers to a full length or portion of an antigenic protein that has the same or nearly the same antigenic specificity as the antigenic protein, and a halogen.
As used herein, the term "secondary target" refers to a target to which a secondary antibody used in an IHC procedure binds. Typically, secondary antibodies bind to primary antibodies in IHC, and thus, secondary targets typically comprise IgG of different origins, such as mouse and rabbit.
The term "host protein" refers to proteins (particularly IgG) of the same origin as the primary antibody, such as mouse, rat, rabbit and goat proteins (IgG).
The term "pseudoprotein" refers to a protein that is non-reactive with secondary antibodies and is used in admixture with host proteins to obtain a gradient dilution. Preferably the pseudoprotein is donkey protein (IgG) or horse protein (IgG).
As used herein, the term "loading point" is also interchangeably referred to as "point" and refers to an entity formed by immobilizing a desired peptide or protein on a slide. The "dots" may have any shape such as, but not limited to, circles, ovals, squares, diamonds, and the like.
Glass slide
Typically, Immunohistochemical (IHC) staining is used to assess the presence of specific anti-home sites in patient tissue sections. Subjective interpretation was performed for staining density on tissue sections to determine the level of diagnosis of abnormal or cancerous conditions. In general, it is assumed that IHC treatment always works correctly and that tissue sections will be marked with visible chromogen markers to identify abnormal or cancerous conditions, if any. However, failure of antigen repair or staining reagents does not leave any features that identify artifacts. Thus, there is a great chance that a laboratory technician or pathologist will not be able to make an effective diagnostic decision. In other words, the physical morphology may not be sufficient to reveal an abnormal condition, but if the antigenic sites are not labeled, the slides only provide what is seen on hematoxylin and eosin (H & E) slides.
In one embodiment of the present invention, a novel adhesive-coated slide is disclosed, which may be interchangeably referred to as a "process recording slide" (hereinafter, an adhesive-coated slide may be referred to as a process recording slide having the same scope and meaning). The above "procedure record slide-immunohistochemistry" (PRS-IHC) slide incorporates targets of known reaction behavior for assessment of antigen repair status, primary antibody reagent efficacy and secondary antibody efficacy. In another embodiment of the invention, the primary antigen target site is but not limited to deparaffinization, antigen retrieval process, primary antibody performance, secondary amplification of precipitated chromogens and the cumulative results of coverslipping. In another embodiment of the invention, the secondary target site is, but is not limited to, the cumulative result of the primary antigen target minus the performance of the primary antibody reagent.
In another embodiment of the invention, the stained secondary target group sites provide a baseline at which the antigen density of the primary site can be objectively determined. Notably, each of the secondary protein arrays can also be printed as 3D targets using polysaccharides as 3D scaffolds. Furthermore, the ratio of chromogen precipitation between 2D and 3D targets at the same concentration determines a scaling factor that can be applied to the primary antigen array, enabling objective measurement of antigen concentration on the 3D material. By identifying the 3D antigen density as the degree of chromogen precipitation, a scale or scale can be applied to the co-existing tissue sections to objectively quantify the presence of antigen in the tissue sections. The presence of secondary and primary reactive targets plays a crucial role in identifying staining reagent viability and exposure through antigen retrieval processes. It is emphasized that any of the above-described steps or defects in the reagents can be easily identified in the PRS target and therefore are likely to signal a false diagnosis.
Reference may be made to fig. 1, which shows possible targets employed during an immunohistochemical assay.
Reference may be made to fig. 2, which shows the structure of an adhesive coated slide or process recording slide, where the IHC target is located below the tissue section to reduce the possibility of protein release from the target material, which may be swept onto the tissue section and captured. The first row of targets is the mouse gradient density array, in the middle is the rabbit gradient density array. The bottom row can support twelve targets, which can be primary antigens or mixtures or combinations of primary antigens. The black and white imaging reference point is located to the left of the secondary protein array. To the right of the staining target are 3D mouse and rabbit targets. The secondary targets and the rest of all primary targets are in 2D configuration. In another embodiment of the invention, a useful glass microscope slide adhesive coating was found on the semer fiyer SuperFrost slide GL 4951P. In another embodiment, the optimal adhesive coated slide has the property of covalently binding to glass and presents to the biological material, without limitation, two or more (-ROH, -R (C ═ O) OH, -RNH3、-R(C＝O)NH2and-RNH2) End groups and adjustable surface wetting properties during manufacture.
In one embodiment of the invention, the "process record slide-immunohistochemistry" (PRS-IHC) slide described above may incorporate targets of known reaction behavior to assess antigen repair status, primary antibody reagent efficacy, and secondary reagent efficacy. The primary antigen target site is the cumulative result of deparaffinization, antigen retrieval process, primary antibody performance, secondary amplification to precipitate chromogen, and coverslipping. The secondary target site is the cumulative result of the primary antigen target minus the performance of the primary antibody reagent.
In another embodiment of the invention, the PRS-IHC slide described above binds a bio-based target in a gradient density array comprising black and white imaging reference targets in order to conform to the structure of the adhesive coated slide or process recording slide described above. Since the target is of biological origin, a thin paraffin film can be applied to prevent oxidation and microbial attack. The parafilm can be removed in the same IHC treatment step as the embedding paraffin of the tissue section. It is mainly emphasized that PRS-IHC underwent the same tissue capture procedure to cover the slide to record the IHC procedure and remain permanently in the tissue section. The second opinion and remote diagnosis become feasible when the processing experience is known, recorded and available.
In one embodiment of the invention, the adhesive coated slide or process recording slide helps to keep the control and patient material co-existing so that they are not replaced and lost as with the Laboratory Information System (LIS). Therefore, the control must complete all the experience by capturing from the biological material to the cover slip.
In another embodiment of the invention, the adhesive coated slide or process recording slide is repeatable, stable over time, supports one or more antigens, each in a gradient density array, is stable to current processing of IHC slides, and is cost effective. The process record slides provide quantitative standards for process controls and objective measurements of antigen concentration on coexisting tissue sections or loose cells, with mouse and rabbit proteins used as gradient density arrays.
In one embodiment of the invention, the steps of the IHC staining process may be described to understand immunohistochemical staining performed in an adhesive coated slide or process record slide. The fixed tissue sections are embedded in paraffin, which must first be heated to a semi-liquid state, then liquefied by xylene (or mixed xylenes), then gradually washed with diluted ethanol and finally removed with buffer to expose the cellular structure of the sections. Next, formaldehyde must be removed to expose the antigenic sites. Most commonly, the fixation is removed by a heat-induced epitope repair (HIER) process or a longer warm water antigen repair process. The HIER process breaks the schiff base bond between formaldehyde and tissue by applying heat (89 ℃ optimum and not more than 95 ℃) when the tissue is exposed to a buffer (pH 6-10, depending on the tissue type). At this point, the antigenic site is exposed and a staining reagent can be used to produce a visible color indicative of the presence of the target antigen. The operating temperature of the water-based antigen retrieval process is about 10 c higher than the embedding paraffin melting temperature, about 60-65 c). The soap and many successive washing solutions slowly dissolve and remove the paraffin. Operator or processing defects in wax removal and fixation repairs can clog the staining process and produce false negative results.
Once the antigenic sites are separated from the formaldehyde fixation, one or two primary binding antibody reagents are applied. These will bind to any matching antigen sites found in tissue sections or PRS primary antigen target sites. The primary antibody is conjugated to a mouse or rabbit protein and then acted upon by a secondary staining reagent.
In another embodiment of the present invention, in order to obtain a sufficient density of the staining reagent for human visual inspection, a multi-step amplification process may be performed. From single-step amplification to three-step amplification, there are various secondary detection kits. All reach the same final state of chromogen precipitation. Typically, three commonly used sets of secondary staining reagents are used, along with one of several counterstains: horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase and nuclear counterstain. The chromogen colors that may be available for precipitation may be selected from, but are not limited to, those in the following list:
horseradish peroxidase (HRP)
Alkaline Phosphatase (AP)
Nuclear staining
Hematoxylin (most commonly) > > blue
DAB is well known and widely used in the united states. AECs are used in many other regions of the world. The reason why AEC is rejected by people using DAB is that the red saturation is too low compared to the reddish-brown color of DAB. Experiments show that the original DAB can obviously age in a short time, so that the color saturation is obviously reduced within 4 hours. Newer versions of DAB contain stabilizers, which extend the stability of DAB from hours to days. DAB also has a tendency to be washed away in subsequent buffer wash cycles, on the other hand AEC can remain stable for weeks to months.
Regulatory standards throughout the world seek or persist to recognize that once such techniques are viable and available, validated controls can be used to examine reagents, methods and instruments for treating tissue sections and loose cells. For hematology and clinical chemistry, such regulatory controls have long existed to validate results and ensure quality. The results of the control tests are plotted in the form of a Levey-Jennings diagram (Westgard et al, 1981). Westgard J, Barry P, Hunt M, Groth T (1981) "A Multi-rule Shewhart Chart for quality control in clinical chemistry". Clin Chem27: 493-.
In another embodiment of the invention, the control used above must not be significantly affected by the pretreatment step: paraffin removal and antigen retrieval. The results measure the efficacy of IHC staining reagents and develop a ratio or scale for determination of antigen concentration on tissue sections. It is important to understand the identification of certain steps or reagent failures, which helps to prevent misdiagnosis.
In another embodiment of the present invention, all of the above 2D secondary staining targets are fixed with formaldehyde. Secondary targets were as follows:
The 2D secondary staining targets combined protein gradient density arrays, one of which was mouse and donkey, and the other was rabbit and donkey. The gradient ratio follows a known profile curve between 10% and 100% density. As donkey does not support non-specific staining, sometimes donkey is used more often than regular cattle in the second staining kit. ABC secondary staining kit goat antibody (mouse or rabbit) was used as the first step reagent and the second staining reagent (containing anti-goat). The goat is too close in species to the cow, supporting the capture of the secondary staining reagent of the second step. Donkeys or other equine animals avoid such accidental reactions.
In another embodiment of the invention, the chromogens for the different secondary stains and precipitates vary greatly in color density between type and manufacturer. To illustrate the changes, 2D gradient density arrays formed a relationship between chromogen density and mouse and rabbit protein concentration. While the second order gradient array mixture can produce absolute concentration ratios, it does not account for the physical structure of the slide coating. All IHC slides that showed hydrophilic behavior and tissue retention by HIER treatment had a porosity factor. For tissue sections, this has little effect, except that the slide coating is to conform to the physical irregularities caused by the slicing blades on the surface of the section and the dispersion of reagents on the surface of the slide. However, for proteins, the porosity and wettability variables affect how much deposition is needed to fill the voids, and how much active area is on the surface. The porosity is variable between slides, but is substantially uniform across any slide. Primary dilutions of individual proteins were assessed by absorbance at 280 nm. The data was then used to formulate the deposited protein array target mix to ensure consistent performance between different IgG batches. All secondary targets were fixed with formaldehyde.
In another embodiment of the invention, the 2D primary antigen staining target is coupled to the carrier protein by way of cysteine residues and sulfo-SMCC cross-linking using a peptide chain of the desired antigen. The currently bound carrier protein is mixed with the pseudoprotein to adjust the concentration. All primary antigen targets were fixed with formaldehyde. Primary targets can be generated in two forms:
a. gradient density pairs, where the maximum density exceeds the ability of the antibody to bind, the second is 50% concentration. Each target pair comprises a single reactive antigen.
b. A series of antigen targets, each target comprising up to ten different antigens, all antigens having the greatest density. The mixing of antigen types in each target is such that only one antigen will react during use.
The antigen consists of a peptide chain with cysteine residues and is covalently bound to a carrier protein that has been previously activated by sulfo-SMCC. Keyhole Limpet Hemocyanin (KLH) was used because it is unreactive with any human antibody and is known to support a range of sites for sulfo-SMCC. Other similar proteins with equivalent properties may be used.
The use of peptide chains presents potential problems because they are only short fragments of the antigen. If the antibodies are not configured to match, they will not be detected even if they bind correctly to the antigen on the tissue section or the loose cells. Thus, for certain antibodies, the primary target must support up to ten different antigen segments. Most laboratories use between 75 and 100 different antigens. Many laboratories choose to use a primary antibody and a secondary stain bundled together with the stain they use. For those antibodies that are intact antibody fragments, the matching antigen is very specific. Since the staining manufacturers have developed their own antibodies in large quantities, PRS supports each staining reagent kit by using option B above, thereby better serving the market.
However, in developing new antibodies, it is important that multiple antigen preparations be tested to achieve optimal detection conditions. Option a above may solve this problem better because it is unclear what the optimal antibody concentration and sensitivity are.
In another embodiment of the present invention, the 3D target converts the 2D target results into the necessity of measurements applicable to tissue sections. The height of the tissue sections and cells were between 4 and 10 microns. The antigenic site can be anywhere on or within the cellular structure. Those antigenic sites on the vertical structure can have a considerable depth where the chromogen can be precipitated by the enzyme located at the top of the section. Thus, more chromogens can be precipitated than in 2D protein deposits. This is why 2D protein and polypeptide based targets are never as dark as loose cells and tissue sections.
The aging effect of the DAB reagent causes considerable changes in the staining results in a short period of time. However, the PRS-IHC solution was able to correct the shift reduction in chromogen precipitation only because the 3D antigen concentration ratio was not related to the change in DAB performance. Since the protein and antigen target arrays are composed of known concentrations, the staining results remain the same relative relationship even when compressed. Thus, when the observed intensity (darkness) is diminished, the ratio will continue to provide the same measure of antigen density on the tissue section or the loose cells.
Imaging reference target
In addition to the 2D and 3D secondary protein target arrays, black and white imaging reference targets were printed.
Digital imaging of microscope slides containing stained biological material is being developed to perform pre-screening and possibly comprehensive diagnostic determinations of the stained material. Typically, the imaging system must adjust the illumination light level so that the digital image is not in a compressed state at either the white or black borders. The conventional solution is to place a black and white target at the desired location of the tag. The basic assumption is that white and black targets represent the limits that a slide can assume. However, in doing so, there is a compression in the numerical scale, as black is much darker and white is much whiter than the staining of tissue sections.
In a preferred embodiment of the invention, a microscope slide is disclosed that contains control and reference target standards in coexistence with patient tissue sections or loose cell deposits. The reference target and tissue section record the processing history between tissue capture and the stained slide covered with the coverslip.
In another embodiment of the invention, there are co-existing black and white reference targets between the microscope slide target arrays. Black and white reference targets undergo the same reagent exposure and treatment as other targets and tissue sections.
In another embodiment of the invention, both reference targets, i.e. black and white targets, are printed paint deposits that are not reactive with any reagents used to treat the slides. Ideally the white target would be perfectly white. However, almost unstained biomaterials have more than half the effective performance of changing from black to white. Thus, white can differ from perfect white by 5-10%, and still have useful value. In a preferred embodiment, the white color is a metal oxide or sulfate composition that is stable over time when not exposed to sunlight. In a more preferred embodiment, the white color is aluminum and titanium oxide.
In another embodiment, both black and white targets are based on anhydride-based epoxy binders, catalyzed by direct exposure to the specified 365nm uv light. The anhydride catalyst consists of methyl tetrahydrophthalic anhydride and diphenyl iodonium hexafluoroarsenate. In addition to uv initiation, the anhydride requires heat to catalyze the crosslinking of the epoxy resin. The preferred uv-initiated anhydrides and their partners are listed, but other solutions can be found when searching for anhydride production companies. Although such coatings/inks can be configured as desired, the purchased components are typically optimized according to the printing method used. The manufacture of coatings/inks must address the difficulty of achieving good wetting between the pigment particles and the epoxy binder. Anhydride-based coatings (also referred to as inks when the viscosity is low) typically mix together the anhydride and the epoxy resin because the pot life can be as long as several months. Viscosity reduction is common knowledge of persons involved in the printing industry and the formulation will vary depending on the surface of the epoxy mixture and the printing method used. While thermally initiated anhydride-epoxy coatings/inks are commonly used, the heat required to initiate the reaction can damage biological materials (proteins, peptides, and chemical targets) that may co-exist with the coating/ink.
In another embodiment, the anhydride catalyst eliminates unreacted amine found with the amino silane based catalyst that would otherwise support non-specific staining. The free amine end groups can and will capture biological materials and certain specific stains. In particular, this solves the problem of unwanted staining of white targets by slide treatment reagents, in particular staining reagents. As a free amine end group on the surface of the coating/ink, it can capture both the primary antibody and the secondary staining reagent and be stained. Thus, the value of having an intact white target on the slide is undermined.
In another embodiment, black pigments use carbon dust less than 2 microns in diameter, while white pigments use aluminum, titanium oxide, or barium sulfate beads; barium sulfate is preferably used for white color.
In another embodiment, the preferred epoxy ink/paint formulation avoids surfactants altogether to prevent the ink/paint from reacting to the staining and reagents that these slides may undergo.
In another embodiment, the printing of the target may be accomplished by a stamp or syringe. In another embodiment, a syringe is preferred because it supports better control of the characteristic dimensions of target deposition.
In another embodiment, the white target consists of a metal oxide or sulfate pigment in an anhydride catalyzed epoxy resin. In another embodiment, the black target is comprised of a carbon pigment in an ultraviolet light initiated anhydride catalyzed epoxy. In another embodiment, the anhydride catalyst is initiated by ultraviolet light by direct ultraviolet light exposure. The main advantage of using uv-initiated anhydride catalysts is that the heat required to initiate the anhydride-epoxy reaction exceeds that which biomaterials (proteins, peptides and chemical targets) can withstand without damage. More importantly, any free amine that may react with the staining reagent is eliminated.
Shielding coating
Paraffin is generally a white or colorless, soft solid derived from petroleum, coal or oil shale, and is composed of a mixture of hydrocarbon molecules containing twenty to forty carbon atoms. It is a solid at room temperature and begins to melt above about 37 ℃ (99 ° F); its boiling point is >370 ℃ (698 ° F). Common applications for paraffin wax include lubrication, electrical insulation, and candles; the dyed paraffin can be made into crayons. It is different from kerosene and other petroleum products sometimes referred to as paraffins.
In pathology laboratories, paraffin is used to impregnate tissue prior to sectioning of thin tissue samples. Water is removed from the tissue by increasing the alcohol concentration (75% to absolute concentration) and the tissue is cleared in an organic solvent, such as xylene, or an aliphatic substitute, such as mixed xylenols. The tissue was then placed in paraffin for several hours, then placed in a mold with wax to cool and solidify, and then cut into pieces on a microtome.
Embedding tissue sections in paraffin is a routine procedure for long-term preservation of tissue sections. However, paraffin has not been reported to be applied as a thin coating to selected areas of a microscope slide. In the present invention, the target protein deposited on the microscope slide, glass or plastic provides a rich food source for the bacterial or fungal antagonist. In addition, antigenic sites (e.g., epitopes) of the protein are readily oxidized, thereby effectively neutralizing the ability of the detection antibody to bind to the protein. Many of the subsequent reactive binding sites are hydroxyl groups, which can be destroyed by reaction with acids and bases in the air. Typically, slides containing protein deposits are stored at temperatures below those that support microbial growth. However, such constraints limit the effective use of the deposits. In addition, the slide on which the protein is deposited is packaged in a vacuum-sealed container to prevent oxidative damage. The open-air retention period for unprotected protein-deposited slides is between 2 and 5 days, depending on the ambient temperature and the level of contaminants in the air.
Paraffin is inherently referred to as containing antifungal and antibacterial agents that prevent oxidation of the antigenic site and airborne acid/base degradation of the exposed site. The paraffin barrier coating changes the useful life of the biomaterial from 3-5 days to 1-2 years, thereby providing a useful product life to the end user.
Removal of the embedded paraffin is also a routine procedure in order to expose the tissue sections to subsequent Immunohistochemical (IHC) staining. The same or similar paraffin formulation is used to protect other deposited materials on the same microscope slide, ensuring that no additional slide treatment is required before IHC staining is initiated.
In one embodiment of the present invention, paraffin is mixed with a solvent to change the state of the material from solid to liquid at room temperature. The blend uses laplace X-tra (paramlast X-tra) or a product comparable to xylene or an aliphatic solvent, such as mixed xylenes, to reduce viscosity and slow cure speed after deposition.
In another embodiment, the solvent may be selected from, but is not limited to, toluene, paint thinner, turpentine or a 50:50 mixture of acetone and kerosene. Paraplast X-tra specifically incorporates the phenolic antioxidant butylated hydroxytoluene to reduce oxidative degradation of proteins, peptides and inorganic targets.
In another embodiment, the paraffin wax is melted to a liquid at a temperature not exceeding 75 ℃ above the melting temperature of the paraffin wax, and then the aliphatic solvent is slowly added until a saturation point is observed (solids formation). The mixture was cooled to 45 ℃ and more fatty compound was added slowly until completely clear.
In another embodiment, the paraffin coating described above is applied to a biological material, which may include but is not limited to proteins, peptides, binding proteins, protein-coated beads, peptide-coated beads, or binding-coated beads, and a specific stain-reactive end group that uniquely captures a specific staining material that reacts with the applied antibody and secondary staining reagent, and the specific stain-reactive deposit is first applied to an adhesive that is coated on a microscope slide.
In another embodiment, a paraffin layer is selectively applied to the target on the slide. In another embodiment, the paraffin layer may be deposited on a microscope slide including, but not limited to: spray coating, ink jet deposition, transfer printing (such as pad printing), screen printing, and vapor deposition. In a preferred embodiment, the paraffin is a thin layer, preferably no more than 5 microns. In another preferred embodiment, the paraffin wax has a melting temperature of less than 60 ℃, preferably less than 56 ℃, and dissolves when exposed to xylene or mixed xylene (aliphatic substitute) solvents. In another preferred embodiment, the paraffin wax has an ambient temperature hardness similar to that of the embedded paraffin wax.
In another embodiment, the tissue mass embedding the paraffin material may include, but is not limited to, tiheupupupip (tissue prep) and tiheupip 2(tissue prep2) of siemer fly (Thermo Fisher), melting point 56 ℃, laplacian (Paraplast) and laplacian (Paraplast plus) of laika (Leica), melting point 56 ℃, laplacian X-tra (Paraplast X-tra) of laika, melting point 50-54 ℃.
In another embodiment, each is a mixture of purified paraffin wax, synthetic polymer, and other materials to determine melt temperature, hardness, and viscosity. The inherent properties of paraffin wax do not support the growth of microorganisms.
In another embodiment, specific staining reagents may include, but are not limited to: almonblue (Alcian Blue), aniline Blue-light-Fast Orange G Solution (Analine Blue-Orange G Solution), azocarmine staining (Azan Stain), Bielschowski silver staining (Bielschowsky silver Stain), Brownian-gram staining (Brow & Benn-Gramm Stain), Tar Violet (Cresyl Violet), Diaminobenzidine (DAB), Totanama pine (Fontana Masson), Goden-Swaiter immersion silver staining (Gordon and Sweet's silver staining), Gosset hexammine staining (Grocett's Methanamine silver staining), Holcoc Bilirubin staining (Hall's Bilrubin Stain), Jones hexammine silver staining (Jones Metaplexine staining), Fast Blue (Luckhol Blue), Luoshu-light Orange G Solution (Analine Blue-Orange G Solution), Blue Azalcaine Red staining (Azain staining), Bielshol-gram silver staining (Gordon and Sweet's silver staining), Gordon-Muller Blue staining (Luakura Blue staining), Melalex-red staining (Melalex Blue staining), Melalex Blue (Luakura Blue-red staining), Melalex-red staining (Melalex Blue staining), Melalex Blue staining (Melalex Blue), Melalex Blue (Melalex Blue), Melalex Blue (Melalex Blue), Melalex Blue (Melalex Blue), Melalex Blue (Melalex Blue), Melalex Blue (Melalex Blue), Melalex Blue (Melalex Blue), Melalex, light-Fast orange G (orange G), Nuclear Fast Red (Nuclear Red), Centrol periodate Schiff's with amylase Digestion (PAS with Diastase digest), Centrol periodate Schiff (PAS) (Periodic Acid Schiff (PAS)), Phosphotungstic Acid (Phosphotungstic Acid), hematoxylin (Haematoxylin), Sirius Red (Picro Sirius Red), Acidified Toluidine Blue (Toluidine Blue Acid), One-Step Golomicro's stain (Trichrome-Gomoris One-Step), Masson Trichrome stain (Trichrome-Masson's), Victoria Blue (Victoria Blue), calcium salt stain (Von Kossa), Wegener's stain (Weigert's stain), Wegener's stain (Weigerton's stain), Wegnel Red (Weigerton's stain (Weigerton's-Zehnder Method).
In another embodiment, the selection of targets may be, but is not limited to, pigmented deposits, such as black and white, but may include any pigment color.
In another embodiment, the microscope slide on which the paraffin coating described above may be applied may be selected from, but not limited to, glass, plastic, or any polymeric material. In another embodiment, the paraffin wax may be purified and anhydrous.
In another embodiment, the resulting microscope slide can be post-heated to melt and/or blend the paraffin particles into the bulk surface coating, thereby sealing the deposit and the surface of the slide surrounding the deposit.
In another embodiment, the resulting microscope slide is heated after the paraffin wax is deposited to force the solvent out of the paraffin wax, thereby ensuring its return to a hardened state. This must start from the paraffin side of the slide, preferably using infrared light. Melting the paraffin from top to bottom ensures that the solvent can rise and evaporate from the paraffin without hindrance. The results are shown in fig. 5, which shows how the melted paraffin ensures a good seal of the edge of its deposit.
Antigen retrieval monitor
In another embodiment of the invention, the antigen retrieval monitor is performed by an antigen retrieval (hereinafter AR) process, which varies greatly from slide to slide and stain to stain, depending on the process used and its embodiment. AR is an open-loop process because direct measurements of AR buffer and buffer temperatures are actually unknown and only estimated. The AR process assumes that the temperature of the AR buffer is always the same since the heater is told to be used at the desired temperature. The AR procedure also assumes that the AR buffer used is the correct mixture using the correct reagent components. These two assumptions will result in the inability to perform AR without providing any tangible feedback to the laboratory.
In another embodiment of the invention, the PRS binds two AR targets: ARM3D and ARM2D plus a 2D secondary array. There are three AR states: under-recovery, normal and over-recovery.
An insufficient recovery condition is a result of the AR temperature being too low, the exposure time being insufficient, or not running at all. The ARM2D target is, for example, a 50:50 mixed mouse and rabbit protein (or proteins from other species, not limited to mouse and rabbit proteins) at 100% concentration with the lowest formaldehyde fixation rate. If the target stains, the AR treatment fails. Preferably, the protein is IgG.
Conditions > excessive recovery are caused by too high an AR temperature, too much exposure time or an AR buffer pH of more than 9.5 or less than 5.5. ARM3D target was a 50:50 mixed mouse and rabbit protein (or proteins from other species, not limited to mouse and rabbit proteins) at 100% concentration deposited in 3D scaffolds that had been over-immobilized with formaldehyde. If the target is stained, this indicates that the AR process is excessive and the slide is not used for diagnostic evaluation.
Normal recovery occurs when 10% to no more than 30% of the targets on the mouse and rabbit gradient density arrays show no visible staining. The extent of AR damage can then be assessed by the amount of low concentration secondary target that is not stained. This lesion can also be seen in tissue sections.
Antigen imaging ratio extrapolation
It is well known that primary antibodies consist of processed host serum obtained by inoculating a host animal (e.g., mouse or rabbit) with the desired antigenic fragment. The host then produces serum proteins in which the antigenic site now contains antibody responses to the antigen antagonist. When the antibody is subsequently contacted with a protein comprising the target antigen, the antigen and antibody are bound together. The result is that antibodies from the host species (mouse or rabbit) can react freely with the secondary staining kit.
The primary and secondary targets have well-defined and regular (such as circular) deposition areas onto which a known dispensed volume of target material is applied. Since the protein deposits contain cross-linking coupling agents, they do not sink into the pores in the slide coating beyond the depth of the protein. When proteins are even loosely cross-linked together, their effective size inhibits the ability to sink into the pores of the adhesive coating. The porosity is not much greater than for a pair of proteins cross-linked together. Thus, the protein is largely left as a much thicker coating. Crosslinking does not occur completely until the baking step after the protein spots are deposited on the slide. It is important to know that the protein deposit is not absorbed by the coating because when absorbed into the coating it cannot then react with the staining reagent because there is not enough space at all available to accommodate the amplification chemicals. From an imaging perspective, some proteins are lost during antigen retrieval, but new proteins are exposed. Thus, the target spots appear as a monolayer of protein, since only those on top of the sediment can react with the staining.
Thus, knowing the atomic mass of the protein, the amount of protein per protein type in the deposit, the area of the target, and the active surface protein density of the target can be calculated.
The applied concentration, dispensed volume and surface area on the slide exposed to the primary antibody reagent are known. It is reasonable to assume that most of the suspended antibody will fall off and be captured by the acceptor antigenic site during the exposure time of the reagent. Only those that directly land on the antigenic site will be captured, the remainder will be washed away by a buffer wash step. Thus, the deposited antibody concentration can be determined under appropriate conditions, for example, when the concentration is greater than a cutoff value of 25% above and less than a saturation value of 25%, where the cutoff value is defined as insufficient target site density to capture the applied protein concentration; saturation values are defined as the concentration at which all applied proteins cannot be captured.
Knowing the primary dilution ratio, the correct primary target density target can be selected and the primary concentration can be verified.
In one embodiment of the invention, each secondary and primary target is a mixture of [ (mouse or rabbit) + (donkey + crosslinker + fungal inhibitor) ] or [ (KLH with antigen a or KLH with antigen B) + (unbound KLH + crosslinker + fungal inhibitor) ]. The total protein volume for each spot is the same, but since the atomic mass may differ between the proteins that make up a particular target, the mixing ratio must be fine-tuned.
Mouse IgG 155kDa
Rabbit IgG 150kDa
Donkey IgG 160kDa
KLH subunits conjugated to an antigenic peptide chain, where the subunits are KLH1 and KLH2 ═ 350 and 390 kDa.
In another embodiment of the invention, the 2D secondary target gradient is 1 to 1000: a stepwise dilution increment of 1, preferably, follows a-20 log (dilution) curve, with the dilution increment being-3 dBd steps. The term-20 log (dilution) ═ dBd all refer to describing the dilution in a semilogarithmic fashion to linearize the data so that the modifying term can be easily applied. The term (dilution) refers to dilution X, wherein X is [1..1000], equals 1:1 to 1000: 1. the term dBd is defined as the degree of dilution or decibels of dilution intensity. The modified terms include: antigen repair damage, enzyme gain, primary antibody reagent dilution. A single 2D/3D target was used to measure the dye density gain between the 2D substrate and the 3D particle. Increments can be applied to the rest of the 2D array to produce a color density ratio that closely matches the 3D representation seen in or on the tissue section.
The secondary 100% 2D/3D and 2D targets confirmed that the two deposits were matched in 2D staining density. This demonstrates that the 3D particle component does not consume enough 100% of the protein material to cause movement of the 2D component.
The secondary stain has an enzymatic gain function of between 1 and 20 times that of the stain, which is its structural function. Thus, as the gain is increased, lower concentrations of secondary target will become saturated, and when the gain is decreased to 1, only higher concentrations of secondary target will be visibly stained.
Since the size difference between the secondary and primary target proteins is considerable, the protein concentration density is established by the primary protein.
In the case of an average primary antibody atomic mass of 150kDa, the individual antibodies had a weight of 150kDa (1.6605X 10)12) Equivalent to 249X 10-12ng weight. If we choose to have a single area of the slide as the only exposed portion, we can know the amount of primary reagent applied. Thus, the inside dimension is 20.3mm2Closed capillary gap 0.14mm high, volume 57.2. mu.L. A ratio of target regions of 1mm in diameter that will yield 2.832nl of applied primary antibody reagent.
The primary antibody reagent was diluted from its concentrate to an intermediate dilution of 10 ug/ml. The intermediate dilutions were then run from 1:1 dilution to 1000:1, coating on a glass slide. This resulted in a dilution ratio from 1:1 to 25.1: 1 on a1 square micron area, 31.5 to 7.08 antibodies were deposited.
To ensure 100% capture capacity, the primary target should have a safety factor of 100 to 1000-fold. Selecting a 1000-fold option, the primary target would need to contain 4 × 106And (c) antigenic sites. Although the KHL subunit is larger than the applied antibody, this increase is not sufficient to increase the number of captured antibodies beyond 1: 1. the average atomic mass of each KLH subunit was 370kDa, corresponding to 614.4X 10-12ng weight.
The volume of a protein molecule can be estimated very simply and reliably from the molecular weight of the protein and the average specific volume of the protein fraction. (volume/molecular weight) the average of the soluble globular protein partial specific volumes determined experimentally was 0.73cm3(ii) in terms of/g. This value varies from protein to protein, but the range is narrow. The reaction formula is reduced to (1.212X 10)3×MW)nm3Protein volume of (c). Thus, the individual volume for the KLH subunit is 448.44nm3. If the protein is modeled as a sphere, the diameter of the sphere will become 0.132 x MW1/3In nm. For the KLH subunit, 9.436 nm.
For a target diameter of 1mm, the monolayer of KLH subunits required 11.237 × 1027A protein. For 4X 106Of proteinsEffective target density, minimum dilution ratio becomes 1: 2.8X 1021. In fact, any approach to 1: a dilution of 1000 is feasible because the evaluation of the primary antibody depends mainly on the concentration of its active protein. Thus, the target density is limited only by its low concentration lower limit.
In one embodiment, the secondary target array is from 1 to 1000:1 in stepwise dilution increments. The linear slope of the dilution was dBd ═ 20log (dilution). For 1 to 1,000 listed: 1, and a semilogarithmic range of 0dB to-60 dBd. Selecting a-3 dB dilution step, the secondary target dilution becomes: -0, -3, -6, -9, -12, -15, -18, -21 dBd.
Both secondary and primary target arrays are irreversibly immobilized and degrade to a much lesser extent during AR than tissue or AR targets. Protein fragments from cleavable, but not intact, proteins are degraded. As the AR process continues on to the protein target and tissue sections, it can be seen that AR damage is due to the shift of the gradient scale mode to 100% position. On the other hand, an increase in secondary enzyme resulted in a shift of the gradient array to 10% of the positions. The gains of the enzymes were: 1.2, 4, 5, 8, 10, 15 and 20. These transitions move the secondary array to 10% target by:
1.20-fold all target movement-26 dBd
2.15 fold all target movements-23.52
3.10 times all target moves-20
4.5 times all target movements-13.98
5.4 fold all target movement-12.04
6.2 times all target movement-6.02
7.1 times only 2D 100% points near black
Typically, AR damage will cause the secondary array to move three or more spots to 100% of the positions, which is considered excessive, and the slide should be redone using a secondary staining kit with higher enzyme content or higher concentrations of antibody.
The primary antigen target color density is thus the sum of the antibody concentration multiplied by the enzyme gain of the secondary staining kit. Whereas the secondary target density is simply the enzyme gain times the secondary target protein concentration.
Depending on the digital imaging system, changes in illumination intensity can change the dynamic range of the image to either compression (dimming) or saturation (brightening). These changes will change the color scale of the antigen, while the numerical scale of the antigen density will not change. Thus, the digital scale is independent, while the color scale depends on the illumination intensity.
In one embodiment of the present invention, the secondary protein target array forms two lines: one mouse IgG and another rabbit IgG were mixed with virtual IgG serum proteins to form a five or more component gradient density series from maximum to minimum density in a-20 log (dilution) linear slope, where the dilution could vary from 1:1 to 1000:1 after the initial 1000:1 dilution.
In another embodiment, in the final processing step, those antigenic sites that are recognized are colored by precipitation of a chromogen. Thus, the mouse and rabbit target arrays reflect a-20 log (dilution) linear slope of the chromogen precipitate of the secondary staining kit.
In another embodiment, a preferred solution for the method for forming the primary antigen density ratio is to successfully compose the target mixture based by depositing it onto a binder coated slide with covalent bonds between the binder and the target material.
In another embodiment, it is inferred that the target array was successfully applied and that the primary and secondary staining reagents performed reasonably, so a fitted curve between the data sets can be readily obtained by computer algorithms. In another embodiment, the primary stain may be selected from any IHC approved antibody to the used mouse or rabbit host protein, which is also not bound to a fluorescent label or to an enzyme site (such as HRP or AP). In another embodiment, the secondary stain may be selected from, but is not limited to, secondary stains having 1-fold to 25-fold enzymatic gain that are unique between mice and rabbits, respectively, each using a different color chromogen.
In another embodiment of the present invention, it is noted that the performance results based absolutely on one slide may differ from the performance results of another slide at another time. This is due to the fact that the performance of the secondary staining kit is different from the performance of the primary bound primary antibody. However, either procedure records the performance of the slide, the antigen ratio is valid and is quite equivalent to another slide done with a different staining reagent.
In another embodiment, a primary antigen concentration ratio is applied to the coexisting tissue sections to approximate tissue sections of the detected cellular defect, e.g., cancer.
The various embodiments described herein are examples. It will be apparent to those skilled in the art that various modifications may be made and other embodiments may be used without departing from the broader scope of the invention as set forth herein. These and other variations on the exemplary embodiments are intended to be covered by the present invention.
Examples
The following examples are presented in a manner to illustrate the invention and should not be construed as limiting the scope of the invention in any way:
example 1 method for spraying paraffin wax barrier coating
Spray coating the surface with a low air flow. A low liquid to air mixture is preferred. The mixture was sprayed through a mask onto a slide to coat the PRS target. Typically requiring 1-2 passes to form a layer less than 5 microns thick without reheating to cause the wax seal to flow. The paraffin mixture reservoir and the spray head were both heated to slightly above 56 c to ensure that the paraffin was liquid and maintained liquid during the flight from the spray head to the slide. The spray coverage from the spray head is about 0.375 inches. If a single pass, reheating would be required to ensure 100% sealing.
Example 2 Screen printing method for Paraffin Shielding coating
The stainless steel wire mesh is heated by passing an electric current through the wire mesh between two parallel sides. The temperature of the screen needs to be slightly below the wax melting temperature so that the wax does not penetrate to the bottom side of the screen. Essentially, paraffin behaves more as a paste than a liquid. The PRS requires reheating to ensure 100% sealing.
Example 3 ink-jet method for paraffin barrier coating
Inkjet heads require integrated heaters within the print head to keep the paraffin in a liquid state. A post heat cycle on the slide will ensure 100% sealing.
Example 4 roll transfer printing of Paraffin barrier coating
The heated roller pulls the parafilm from the heated reservoir onto the roller. The roller transfers the parafilm to the slide in much the same way as the roller brushes the wall with a squeegee. Post heat cycles on the slides will ensure 100% sealing.
Example 5 antigen repair exposure and degradation of PRS targets
Test studies attempt to verify that changes in AR exposure will be seen in the 2D secondary and AR targets.
The expected result is a linear slope of exposure time and protein degradation. However, this is not the case. This is because the AR buffer is not applied to the slide in a pre-heated state, but must be heated to an operating temperature between 92 ℃ and 95 ℃. Therefore, neglecting the time it takes to bring the AR buffer above 89 ℃, the slope is linear. The best white balance and contrast were set using 8-bit digitization with PRS black/white targets, the slope being 1.3 lsb/min, +/-0.2 lsb. After 20 minutes of time-labeling at more than 89 ℃, 50% of the targets are under tremendous pressure and the effectiveness of the secondary target panel is compromised.
Example 6 Secondary target identity
The test study has two factors to explore:
I. point-to-point between slides within a single mixed batch of secondary protein.
Point-to-point between slides of different structures of secondary protein arrays.
Single mixed batch tests used 100% and 40% target formulation. One hundred slides were printed and all treated with Avidin-Biotin Complex (Avidin-Biotin Complex) (ABC) type mouse and rabbit secondary staining kits purchased from Scytek. Antigen retrieval was not performed because it only adds one additional variable. Both distributions are within 1.5%.
Example 7 selection of pseudoproteins
Ten different secondary dilution groups were made from two different batches of mouse, rabbit and bovine IgG proteins. Dilutions were 100%, 40% and 20% mouse and cattle and rabbit and cattle. The distribution of the 100% and 40% dilution groups was within 1.5%. The 20% dilution group showed an unexpected increase in staining density. From these data, we found an interaction between cattle and biotinylated goat anti-multivalent reagents of the ABC staining kit. This problem is solved by replacing cattle with donkeys. The test was repeated and now 20% of the groups remained within 1.5%.
Example 8 quality control use of PRS-IHC
In QC mode, as shown in fig. 3, the co-existing target provides IHC process feedback. Four rows of secondary arrays are shown, differing in the degree to which antigen retrieval is performed within the ranges of nominal, over, very over and over 5%, 10%, 30% and 40%, respectively. The antigen retrieval process attempts to reveal antigenic sites by withdrawing the schiff base bond between formaldehyde and protein. The rate at which the antigen becomes exposed depends to a large extent on the temperature of the reaction. As the temperature increases, a chance of nucleate boiling occurs. Nucleate boiling causes physical damage to both tissue and protein deposits. Ideally, the antigen retrieval activity is uniform across the slide but does not actually occur, so the resulting area has more or less antigen retrieval activity, depending on the method and environment used. Given uniform antigen retrieval activity, the following can be used to indicate that the slide can be used in diagnostic assays.
If the AR is minimal or excessive, the secondary array may not reflect the failure. However, both AR targets will signal too many fault conditions.
a. Low AR is considered to be both black in 2D/3D under the immobilized target and 2D on the immobilized target. The secondary array looks best, nor does the target have an AR moving to the left. Low AR activity can occur in IHC stainers under the following conditions:
i.AR heaters not operating or set below 80 deg.C
AR buffer having neutral pH 7, instead of 6 or 9
Too short exposure time
b. High AR is considered to be that 2D/3D under the immobilized target becomes very white and 2D on the immobilized target is less than 50% black. The secondary array will also be substantially whitened. Low AR activity can occur in IHC stainers under the following conditions:
i. the working temperature of the heater is higher than 95 DEG C
Too long exposure time
c. In both cases, errors in chromogen precipitation occur:
i. if at high concentrations, the staining intensity of the secondary target decreases, rather than at maximum darkness. The secondary array should always increase relative to the site density. If not, the chromogen precipitate will exhaust the capacity of the secondary kit. The solution is to increase the primary antibody dilution (equivalent to decreasing the antibody concentration).
The chromogen reagent has deteriorated since activation (as typically occurs in DAB). The solution is to use a new DAB mixture.
Staining may be subject to saturation or criticality depending on the concentration of primary antibody and the enzymatic gain of the secondary staining kit. Saturation refers to the density of enzyme sites exceeding the ability to precipitate colorant from the chromogen. In other words, the dyeing color is as dark as possible. When the concentration of the primary antibody and the enzymatic gain of the secondary staining kit are too low, criticality occurs, resulting in failure to see sufficient stain precipitation. These two factors result in the darkness of the secondary line shifting to saturation (100%) or critical (0%). According to fig. 3, this movement is considered as the number of targets visible. As the secondary enzyme gain increases, the 100% dot density will shift to the 0% position. Common enzyme gains are: 1.2, 4, 5, 8, 10, 15 and 20. These transitions move the secondary array to the 0% position by:
1.20-fold all target movement-26 dBd
2.15 fold all target movements-23.52
3.10 times all target moves-20
4.5 times all target movements-13.98
5.4 fold all target movement-12.04
6.2 times all target movement-6.02
7.1 times only 2D 100% points near black
If a primary target array is present, an increase in the gain of the secondary enzyme will shift the staining density to a point of lower primary concentration. This is also the case if the primary antibody concentration is increased. The antigen retrieval process will result in degradation of both primary and secondary targets to a level that reverses the critical shift. If three or more spots disappear at the end of IHC staining, the antigen retrieval time, temperature, or both of the slides can be considered too high and too much antigen is lost in the tissue, making diagnostic interpretation unimportant. Since secondary staining has been shown to be impaired, this decision is independent of the efficacy of the primary antibody. Nothing at the antibody step can overcome this level of damage.
Example 9 PRS tracks illumination in proportion to its antigen Density
In terms of illumination, the observation of microscope slides by conventional microscopes is subjective. In Whole Slide Imaging (WSI), the scanner uses perfect white and black holes to establish white balance and contrast. This is not the case with manual microscopes. Fig. 4 shows the effect on the image when the illumination is too dark (optimally-5%), optimally (+0) and too bright (e.g. +10 or + 15%). When the light level is less than optimal, the dye density decreases. This may lead to a diagnosis that is one step higher than the original one with respect to the stage of cancer. When the light level is higher than optimal, the image may fade. This may lead to a diagnosis that is one step lower than the original one with respect to the stage of cancer. Antigen color density and digital scales are formed by the primary and secondary targets and can be superimposed on the WSI image. Numerical proportions are independent terms and color density is a dependent term. When applying antigen density color and digital scale to WSI, the numerical scale remains fixed as the user moves the illumination up and down. On the other hand, as the illuminance changes, the color density scale also changes. Advantageously, the user can select the best "visible" feature to move the apparent illumination up/down onto the tissue image without losing the numerical relationship of color density. This will also work as the magnification changes.
EXAMPLE 10 construction of antigen Density Scale
The antigen density scale can be established in two forms.
1. Type a is based on this assumption: primary antibodies always apply less than 10% excess antibody relative to the tissue antigenic site.
Type B uses a primary antigen gradient density array.
Type A: antigen scaleplate based on two stages only
This format uses only secondary target arrays. The information conveyed embedded in the 2D barcode includes (a) primary antibody data: host species and-dBd dilution of antibody, and (b) secondary enzyme gain.
The secondary gradient density target array consisted of known concentrations of protein between targets, decreasing at-3 dBd. The maximum concentration was selected by the minimum dilution for the primary antibody. Most users adopted the concentration specification provided by the antibody reagent manufacturer and diluted to a constant intermediate concentration of 1 ug/ml. Thus, all other dilutions were made as necessary to accommodate different tissue types. Typically, the second set of primary antibody dilutions was between 1:1 to 1000: 1.
To accommodate the gain range of the secondary enzyme, the secondary array must consist of a wider range of dilutions. Thus, at a step size of-3 dBd, the lowest dilution of the secondary array starts at 1,000: 1 or-60 dBd, which is denoted as SdBd. Then, the maximum value of the 8-point sequence becomes-0 dBd or 1: 1. the effect of antigen retrieval degrades secondary proteins as represented by ARdBd. Each of the eight points in the secondary array represents an increment of-3 dBd. The loss of antigen repair by losing both targets (no longer visible) would be +6 dBd. This means that for the 2D target, the secondary array is (-S + AR) dBd, or [ +6 to-54 dBd ]. Antibody concentration and secondary enzyme gain must now be considered. Antibody concentration was AdBd and enzyme gain was EdBd. Thus, the secondary array would be (-S + AR-E) dBd, while the tissue would be (+ AR-E + A) dBd. The next factor that must be applied is the 100% 2D to 3D difference. The staining difference between the 3D object in the 100% 2D/3D target and 100% 2D represents the secondary staining chromogen precipitation constant, which is used to give a numerical scale to the color density and is given as dbdd. The difference in color density will apply to each 2D target in the array. Thus, the 2D array had a staining color density of (+ AR-E + A + D) dBd.
If the enzyme gain is 10 times, then E-20 dBd. Then, the 2D secondary array will become: -14, -17, -20, -23, -26, -29, blank dBd. Two spots towards 0% have been completely damaged by the antigen retrieval process, so that they cannot be recovered by staining and are therefore blank. For example, if the 2D/3D color density difference is 10 times, then D ═ 20dBd, bringing the 3D secondary array to-34, -37, -40, -43, -46, -49, blank dBd. Assuming that the primary antibody reagent finds a suitable antigenic site in the primary target, 100% yield results. It is also hypothesized that even though there are more than two antigenic peptide chains per KLH protein, only one antibody per KLH protein can bind and be stained efficiently. Any additional antibodies on the same KLH protein that find a suitable antigen will be prevented from achieving secondary staining due to overlapping occupancy. Thus, each primary antigen carries one number of antigenic sites in the protein that can be detected. Since the primary target contained the same amount of protein per micron as the secondary, a primary dilution of 500ug/ml antibody stock was applied to the secondary array data to adjust the secondary color density to a numerical antigen density. Secondary targets are monitored and targets with intermediate color densities are selected. The intermediate color density is defined as the 50% point between the maximum black and maximum white. Then the point is equal to 1.5dBd which is outside the 3dBd range. This spot will then function as an anchor point from which an antigen density ruler is established. The last target range using above the midpoint will become-41.5 dBd.
The secondary protein was diluted to 10. mu.g/ml of the primary dilution. Each array was a mixture of mice or rabbits mixed with donkey IgG protein. Although the atomic weights of the proteins were different, it is assumed below that all proteins were 150kDa, and that the total number of proteins per target spot was constant and the mixing ratio was not constant. Currently, only the concentration of the reactive protein is considered. At 150kDa, the molecular weight of a single protein, MW, is 249.07X 10-12ng. The standard target point is 1mm in diameter. If the thickness of the printed precipitate is 1 μm and the precipitate concentration is 10 μ g/mL, 31.5X 10 precipitates will be6And (3) a protein. A region of 1 μm in diameter will contain 31.5 proteins. If we allow one protein to equal one antigenic site, the antigen density can be determined. The secondary array uses the same amount of protein per deposition, but as the concentration of mouse or rabbit decreases, the ratio between mouse or rabbit and donkey will also change. 100% of the targets were completely mice or rabbits and matched to the 0dBd point on the scale.
The secondary antibody will stain the tissue only when the primary antibody binds to the antigenic site on the tissue. It is not particularly dependent on the concentration of antibody applied, except that a sufficient concentration of antibody must be provided to bind to available antigenic sites. Thus, the antigen density metric on the tissue remains constant, but the values must be corrected for antigen repair damage and secondary enzyme gain. The color density must then be unified with the numerical measurement.
In the previous examples, the enzyme gain was 10-fold, and antigen retrieval has resulted in the loss of two spots from the secondary array. The enzyme gain was-20 dBd, while the loss of antigen retrieval was +6 dBd. The result was-14 dBd. After dilution will convert to:
type B: scale based on primary antigens
This format uses primary and secondary target arrays. The information conveyed embedded in the 2D barcode includes (a) primary antibody data: host species and-dBd dilution of antibody, and (b) secondary enzyme gain. Lot number data includes information about which primary target combination to use.
If there is a primary target series, it will be 3 spots, with the most concentrated spots having the same 100% concentration as the secondary array, but the spots are spaced in steps of-6 dBd. In fact, the primary and secondary arrays have the same dilution slope. The primary target becomes: -0, -6, -12dBd, and is denoted as PdBd. It is reasonable to expect that the antigen retrieval damage is nearly identical to that of the secondary array. The primary array is acted upon by the secondary stain and therefore has the same enzymatic gain function. Thus, the primary array is (-A + AR-E) dBd, with primary target density being controlled by primary antibody dilution. The only requirement is that P is always greater than a. For 10-fold enzyme gain-20 dBd and +6dbd antigen repair loss, the primary array was-20, -26, -32 dBd. Based on the impact on the secondary array, loss of antigen repair cannot act on the primary target, and is not sufficient to destroy it. Although secondary arrays are sufficient to generate a scale of antigen density, it is important to verify the correct use of the primary dilution. Thus, primary targets function in this capacity.
Claims (40)
1. A slide, comprising a detection zone and a control zone, wherein
The detection area is used for processing a tissue slice or a loose cell through Immunohistochemical (IHC) or Immunochemical (ICC) detection and/or subsequent examination; and
the control zone having a control target that reflects possible errors present in one or more intermediate steps of the immunohistochemical or immunochemical detection process and/or provides a reference for qualitatively or quantitatively determining the color density of stained tissue or cells;
applying a paraffin coating to one or more biological materials selected from the group consisting of primary targets, secondary targets, imaging references, and antigen retrieval monitor loading points on the slide; or applying the paraffin coating to an inorganic deposit, the application of the paraffin coating comprising the steps of:
(a) melting the solid paraffin at a temperature of 60-70 ℃ until the solid paraffin is in a liquid state;
(b) adding an aliphatic solvent to the product paraffin obtained in step (a) until a saturated mixture is obtained;
(c) cooling the saturated mixture obtained in step (b) at a temperature in the range of 30-33 ℃ and then by slow addition of an organic solvent until a substantially clear liquid is obtained;
(d) applying a thin film of the clarified liquid from step (c) to the biological material or the inorganic precipitate on the slide to form a barrier coating on the slide;
(e) the deposited paraffin wax mixture is exposed to infrared heating to melt and evaporate the released solvent, thereby restoring the paraffin wax mixture to its original solid state.
2. The slide of claim 1, having an adhesive coating to enable attachment of peptides, proteins, sugars, lipids, or small inorganic molecules.
3. The slide of claim 2, wherein the adhesive coating is covalently bonded to glass and presents one or more end groups selected from the group consisting of-ROH, -R (C = O) OH, -RNH3、-R(C＝O)NH2and-RNH2Group (d) of (a).
4. The slide of claim 2, wherein the adhesive coating is slightly hydrophilic.
5. The slide of claim 1 or 2, wherein the control zone comprises one or more sets of primary target arrays comprising one or more primary target load points, each said primary target load point comprising one or more primary targets that are antigenic peptide fragments immobilized on the slide and that are identical to the full length or a portion of at least one of an antigenic protein or a variant of said antigenic protein that does not alter its antigenic specificity.
6. The slide of claim 5, wherein the primary targets comprise primary antigen targets and primary antibody targets, at least one of the primary antibody targets in one of the primary target load points being identifiable by a primary antibody used for the immunohistochemical or immunochemical detection.
7. The slide of claim 1 or 2, wherein the peptide chain of the primary target is coupled to a carrier protein via a cysteine residue, and the bound carrier protein is mixed with a pseudoprotein to adjust the concentration;
the carrier protein is selected from Keyhole Limpet Hemocyanin (KLH) or other proteins which are non-reactive with secondary staining reagents, the other proteins being Ovalbumin (OVA) from chicken proteins or equine family;
the primary target is fixed by a fixing agent, and the fixing agent is paraformaldehyde or formalin.
8. The slide of claim 7, wherein the peptide chain of the primary target is coupled to the carrier protein by way of a sulfo-SMCC cross-link via a cysteine residue.
9. The slide of claim 6, wherein the primary target load points form gradient target density pairs, each of the gradient target density pairs binding a single reactive antigen; or
Each of the primary target load points is composed of two to ten different antigenic peptide fragments, all at maximum density.
10. The slide of claim 9, wherein binding peptide is mixed with neutral keyhole limpet hemocyanin and 4% formalin to prepare a two to ten fold dilution series of gradient target density pairs.
11. The slide of claim 1 or 2, wherein the control zone further comprises one or more sets of secondary target arrays comprising one or more secondary target load points, each of said secondary target load points being a mixture of host protein and pseudoprotein immobilized on the slide in a ratio; wherein
For different secondary target loading points in different secondary target arrays in the same secondary target array group, the host proteins are the same, and the pseudoproteins are the same or different; and
the host proteins are different and the pseudoproteins are the same or different for different sets of secondary target arrays.
12. The slide of claim 11, wherein the secondary target array forms a gradient dilution series.
13. The slide of claim 1, wherein the secondary target is fixed with a fixative that is paraformaldehyde or formalin.
14. The slide of claim 11, wherein the host protein is an animal protein that produces primary antibodies for immunohistochemical or immunochemical detection; wherein the host is selected from the group consisting of mouse, rat, rabbit and goat.
15. The slide of claim 11, wherein the pseudoprotein is donkey or horse protein.
16. The slide of claim 1 or 2, wherein the control zone further comprises one or more 3D secondary array target load points, each of the 3D secondary array target load points being formed from a mixture of a polysaccharide as a scaffold and a host protein at a concentration of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or any value in between, and loaded to the slide.
17. The slide of claim 1 or 2, wherein the control zone further comprises one or more imaging reference load points.
18. The slide of claim 17, wherein the imaging reference load points comprise at least a black reference target or a white reference target.
19. The slide of claim 18, wherein the black reference target is selected from the group consisting of carbon dust; or
The white reference target is selected from titanium oxide, aluminum sulfate or barium sulfate.
20. The slide of claim 18, wherein the black reference target and the white reference target are each based on an anhydride-based epoxy binder that is catalyzed by direct exposure to specified 365nm ultraviolet light.
21. The slide of claim 1 or 2, wherein the control zone further comprises one or more antigen repair monitor load points for detection under recovery conditions of an antigen repair process, each of the antigen repair monitor load points being a mixture of one or more host proteins.
22. The slide of claim 21, wherein the mixture of one or more host proteins is mouse and rabbit proteins as (0-100): (100: 0) mixing the mixture in the ratio.
23. The slide of claim 1 or 2, wherein the control zone further comprises one or more additional antigen repair monitor load points for detection of an excessive recovery condition of an antigen repair process, each of the antigen repair monitor load points being a mixture of one or more host proteins deposited in a 3D scaffold that has been immobilized with a fixative.
24. The slide of claim 1 or 2, wherein the paraffin coating is applied in a coating thickness of 1-5 microns.
25. The slide as claimed in claim 24, wherein the paraffin wax is selected from the group consisting of TissuePrep by selmefeier having a melting temperature of 56 ℃, TissuePrep2 by selmefeier feier having a melting temperature of 56 ℃, paramast by laika having a melting temperature of 56 ℃, paramast plus by laika having a melting temperature of 56 ℃ or paramast X-tra by laika having a melting temperature of 50-54 ℃; and
the organic solvent is selected from xylene, aliphatic xylene substitutes, toluene, paint diluents, turpentine, or a 50:50 mixture of acetone and kerosene.
26. The slide glass of claim 24, wherein the clear liquid obtained in step (c) is applied by spray coating, ink jet deposition, transfer printing, screen printing, or vapor deposition.
27. The slide of claim 1 or 2, further comprising a label identifying the slide type and a code identifying an antigen supported by the primary target; and/or lot number.
28. The slide of claim 27, wherein the label is on top of a label area of the slide, the lot number being below the label area.
29. A method of immunohistochemical staining of a slide according to any one of claims 1 to 28, comprising the steps of:
b. removing the fixative fixation to expose the tissue sections, the loose cells, or the antigenic sites of the primary antibody targets, the primary targets including the primary antigen targets and the primary antibody targets;
c1. applying one or more primary antibodies to bind to any matching antigenic sites found in the tissue section, the bulk cells, or the primary antibody target; applying one or more secondary antibodies that bind to a moiety capable of producing a color in the presence of a staining reagent to bind to a primary antibody used in the tissue section, the loose cells, a secondary antigen target, or an antigen retrieval monitor; or
c2. Applying one or more primary antibodies that bind to a moiety capable of producing a color in the presence of the staining reagent to bind to any matching antigenic sites found in the tissue section, the bulk cells, or the primary antibody target;
e. applying the staining reagent to produce a visible color indication of the presence of the target antigen;
g. quantitatively determining the color density of the stained tissue or cells based on, and/or with the aid of, the secondary antigen density gradient;
h. assessing the quality of the detection process based on the primary target, the secondary target, and the staining density of the antigen retrieval monitor.
30. The method of claim 29, wherein a paraffin coating is selectively applied to the primary and secondary targets on the slide, and the tissue section or the loose cells are also embedded in paraffin, then the following steps are performed prior to step b:
a. removing the paraffin from the paraffin-embedded tissue section or the loose cells and the paraffin coating overlaid on the primary and secondary targets.
31. The method of claim 29, further comprising after step e and before step g:
f. multiple amplification steps to achieve sufficient staining reagent density.
32. The method of claim 30, wherein the paraffin removal in step a is performed by heating the paraffin at a temperature ranging between 65-75 ℃ for 3-10 minutes to obtain a semi-liquid state, and then liquefying with an organic solvent until rehydrated in a buffer solution.
33. The method of claim 32, wherein the organic solvent is selected from xylene, mixed xylenes, absolute ethanol, 95% ethanol, 70% ethanol, or 50% ethanol.
34. The method of claim 29, wherein the destoner immobilization is performed by a heat-induced epitope retrieval (HIER) process, or by a multiple exchange warm distilled water antigen retrieval process.
35. The method of claim 29, wherein the staining reagent used is selected from the group consisting of an enzyme-labeled secondary antibody, an enzyme-labeled tertiary antibody that reacts with the enzyme-labeled secondary antibody, an APAAP immune complex that reacts with the secondary antibody, an enzyme-labeled avidin that reacts with the biotinylated secondary antibody, an enzyme-labeled streptavidin that reacts with the biotinylated secondary antibody, an avidin-/streptavidin-biotin-enzyme complex that reacts with the biotin-labeled secondary antibody, a streptavidin-enzyme complex on the biotinylated secondary antibody in the primary antibody, or a polymer containing the secondary antibody and an enzyme site that binds to the primary antibody.
36. The method of claim 29, wherein the removing the fixative fixation comprises removing the fixative fixation using an antigen retrieval buffer selected from the range of pH 6-9.
37. The method of claim 29, wherein the primary antibody in step c1 or step c2 is selected from an antibody in which the host is a mouse or a rabbit.
38. The method of claim 29, wherein the staining reagent is selected from the group consisting of 3,3' -Diaminobenzidine (DAB), amino-9-ethylcarbazole (AEC), DAB + nickel enhancer, fast red, TMB, stay yellow, BCIP/NBT, BCIP/TNBT, naphthol AS-MX phosphate + fast blue BB, naphthol AS-MX phosphate + fast red TR, naphthol AS-MX phosphate + new magenta, stay green, or NBT.
39. The method of claim 34, wherein the evaluation criteria for the antigen retrieval process are as follows:
if the antigen retrieval monitor load point used to detect the target in the restored state is stained, indicating that the antigen retrieval process has failed;
if the antigen retrieval monitor load point used to detect the over-recovery state is stained, indicating that the antigen retrieval process is excessive, the slide is not used for diagnostic evaluation;
if 10% to no more than 30% of the secondary target shows no visible staining, indicating that a normal recovery state has occurred, then the extent of antigen repair damage can be assessed by the amount of unstained low concentration secondary target.
40. A kit comprising a slide as claimed in any one of claims 1 to 28, or for performing a method as claimed in any one of claims 29 to 39.
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| PCT/CN2018/091686 WO2018228575A1 (en) | 2017-06-15 | 2018-06-15 | Process record slide for immunohistochemical staining |
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| EP4354138A1 (en) * | 2021-06-07 | 2024-04-17 | Hitachi High-Tech Corporation | Control slide glass for pathological examination and method for manufacturing same |
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| AU2003302137A1 (en) * | 2002-07-15 | 2004-06-15 | Technical Instrument San Francisco | Quality control of assays |
| JPWO2004044639A1 (en) * | 2002-10-18 | 2006-03-16 | 浜松ホトニクス株式会社 | Slide glass, cover glass and pathological diagnosis system |
| GB0304515D0 (en) * | 2003-02-27 | 2003-04-02 | Dakocytomation Denmark As | Standard |
| CN1957256B (en) * | 2004-03-24 | 2013-01-02 | 三路影像公司 | Methods and compositions for the detection of cervical disease |
| US20070141723A1 (en) * | 2005-12-16 | 2007-06-21 | Sompuram Seshi A | Immunohistochemistry staining controls |
| WO2009085576A2 (en) * | 2007-12-28 | 2009-07-09 | Spring Bioscience Corporation | Positive control cell pellets for immunohistochemistry and methods of use thereof |
| CA2797741A1 (en) * | 2010-05-07 | 2011-11-10 | F. Hoffmann-La Roche Ag | Diagnostic method for the detection of cells ex vivo |
| EP2649446B1 (en) * | 2010-12-06 | 2016-02-24 | Dako Denmark A/S | Combined histological stain |
| CN202002935U (en) * | 2011-03-21 | 2011-10-05 | 武汉大学 | Kit for detecting staphylococcus aureus infection in blood |
| CN102391655B (en) * | 2011-09-08 | 2013-04-03 | 成都理工大学 | Preparation method of octodecyl trimethyl ammonium bromide antibacterial solid paraffin |
| WO2013090567A2 (en) * | 2011-12-13 | 2013-06-20 | Lab Vision Corporation | Immunohistochemical validation devices and methods |
| US20140022631A1 (en) * | 2012-07-21 | 2014-01-23 | General Data Company, Inc. | Microscope slide for specimen tracking and verification, and method of making same |
| US10082451B2 (en) * | 2013-03-30 | 2018-09-25 | Ge Healthcare Bio-Sciences Corp. | Microscope slides with quality controls thereon |
| KR101449589B1 (en) * | 2013-10-17 | 2014-10-08 | 한국과학기술원 | Fabrication method of a stable superhydrophobic surface through the fixation of nanoparticle by using a paraffin wax |
| WO2015070075A1 (en) * | 2013-11-07 | 2015-05-14 | Medical Discovery Partners Llc | Quantitative controls and calibrators for cellular analytes |
| GB2522231B (en) * | 2014-01-17 | 2019-11-06 | Leeds Teaching Hospitals Nhs Trust | Method of forming a stain assessment target |
| CN204790174U (en) * | 2015-06-30 | 2015-11-18 | 天津市康婷生物工程有限公司 | Do not need cover glass can directly be used for slide glass of film -making |
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