Patent Application
20170336303
The present application relates to fixation methods for preserving tissue samples.
Proper medical diagnosis and patient safety require properly fixing prior to staining. The most common method of fixation for clinical diagnostic purposes is to immerse the tissue sample in 10% neutral buffered formalin (NBF) at room temperature. Unfortunately, many downstream analytical methods are highly sensitive to the amount of time spent in NBF. For example, if a tissues that have been exposed to formalin for a substantially extended period of time often do not work well for subsequent histochemical processes. The widely expressed cancer marker protein p53, for example, gradually loses all of its reactivity toward monoclonal antibody PAb1801 when fixed in formaldehyde for between 6 and 24 hours. Silvestrini et al., 87 J. Nat. Cancer Inst. 1020 (1995). Similarly, the diagnostically important epithelial cell marker protein keratin gradually becomes unable to bind with a monoclonal anti-keratin antibody if the tissue is fixed in formaldehyde for up to 24 hours. Battifora & Kopinski, 34 J. Histochem. Cytochem. 1095-1100 (1986). Other antibodies are sensitive to fixation time in room temperature NBF, including, for example, lymphocyte antigens, vimentin, desmin, neurofilaments, cytokeratins, S100 protein, prostate specific antigen, thyroglobulin, and carcinoembryonic antigen. Leong & Gilham, 4 Pathology 266-268 (1989). Similarly, nucleic acid analyses are often sensitive to fixation time. See Srinivasan, Am J Pathol., vol. 161, issue 6, p. 1961-71 (2002); O'Leary et al., 26 Histochem. J. 337-346 (1994); Greer et al., 95 Am. J. Clin. Pathol. 117-124 (1991); F. Karisen et al., 71 Lab. Invest. 604-611 (1994). Others have shown that post-translational modifications to some proteins, such as phosphorylation, are sensitive to extended room temperature NBF exposure. See Mueller et al., PLoS One, Vol. 6 (8): e23780 (2011).
Thus, under current clinical practice, it is important to control the tissue fixation time to achieve a compromise between the preservation of tissue morphology and the loss of antigenicity. For example, ASCO guidelines suggest fixation of tissues for at least 6 hours but no more than 72 hours if the sample is to be assayed for HER2 expression immunohistochemically. However, it often is not practical to minimize the extent of exposure to NBF. For example, tissue sample collected toward the end of the week may often be stored at room temperature in fixative over a weekend before they can be further processed. In other cases, the tissue sample may be collected at one site and then transported to a second site for further processing, which can add to processing times. In each of these cases, it is not uncommon for the amount of time in room temperature NBF. Indeed, Leong and Gilham report that the bulk of a typical surgical resection is often retained in NBF for future resampling, which may occur after 3 or more days. Similarly, autopsy specimens are usually fixed for between 3 and 14 days, depending on convenience of the technician. As a result, the quality of fixation for tissue samples is inconsistent, which can lead to variable results in downstream analytical methods and even missed diagnoses.
Some methods have been developed to address these problems.
For example, it is known to use fast freezing methods in order to halt the action of modification enzymes. See Lawson et. al. Cryobiology, vol. 62, issue 2, 115-22 (2011). Although fast freezing may initially slow down the action of such enzymes, it does not completely inhibit their action upon thawing of the sample and thus does not always ameliorate loss of labile biomarkers. Additionally, fast freezing methods are not commonly used in commercial histology laboratories, and thus would require adoption of completely different reagents and systems.
U.S. Pat. No. 8,460,859 B2 discloses the use of a three-part special fixative to achieve the stabilization of phosphoproteins. The fixative comprises a preservation component, a stabilizer component and a permeability enhancing component. In order to obtain long term preservation, the patent requires that the tissue sample be frozen. However, these methods are more complicated than can practically be applied on a commercial scale.
Others have tried to mitigate the effect of endogenous degradation pathways by fixing the tissues in the presence of exogenous protease and nuclease inhibitors to prevent loss of potential analytes during fixation. See WO 2011-130280 A1 and WO 2008-073187 A2. However, direct inhibition of naturally occurring pathways in the tissue can affect the end results. For example, WO 2008-073187 A2 teaches that treatment of tissues with phosphatase inhibitors can cause “highly abnormal upward accumulation of abnormal levels of phosphoproteins.” These methods thus do not yield reliable results. Moreover, the amounts of inhibitors necessary to adequately block enzyme activity makes the methods cost-prohibitive to implement on a wide scale.
The present inventors are not aware of any existing methods to sufficiently mitigate negative effects of extended exposure of tissue samples to fixative solutions without resorting to special reagents or complicated processing steps.
The present invention is directed to improved methods for preserving biomarkers when a tissue sample is subjected to aldehyde fixation. The aldehyde-based fixative solution and tissue sample are typically in contact with each other at the first temperature range for a period of time effective to allow the aldehyde-based fixative solution to diffuse throughout substantially the entire cross section of the tissue sample without significant diffusion inhibiting cross-linking occurring for up to 14 days. After exposure to fixative at the first temperature or temperature range the tissue sample is exposed to a second higher temperature for a second period of time sufficient to induce cross-linking. The methods enable post-fixation processing of tissue samples to be delayed up to 14 days and perhaps longer while maintaining excellent preservation of tissue morphology, antibody reactivity, and labile biomarkers.
Embodiments of the method comprise applying a first aldehyde-based fixative solution at a first temperature to a tissue sample, followed by applying a second aldehyde-based fixative solution to the tissue sample. In some embodiments of the present invention, a first temperature range is from at least 0° C. to about 10° C. In at least one embodiment the temperature can be in the range from about 2° C. to about 8° C., while in another embodiment can be in the range from about 4° C., plus or minus 3° C. Embodiments of the invention may have a time range during which the tissue sample is exposed to the aldehyde-based fixative solution at the first temperature of from about 72 hours up to about 14 days or more.
The second aldehyde-based fixative solution may be different from the first aldehyde-based fixative solution. For example, the solutions can be at different concentrations, or the second aldehyde-based fixative solution may comprise an aldehyde different from the first aldehyde. The aldehyde typically is a lower alkyl aldehyde, such as formaldehyde, glyoxal, glutaraldehyde, or combinations thereof.
One disclosed exemplary embodiment of the present invention comprises immersing a tissue sample into a formalin solution at a temperature of from equal to or greater than 0° C. up to 7° C. for a first period of from greater than 72 hours up to about 14 days. The tissue sample is then immersed into a formalin solution at a second temperature greater than about 20° C. up to at least 45° C. for a second time period of from about 1 hour to about 4 hours. The formalin solution generally is 10%-30% NBF. These processing steps typically are followed by a series of alcohol washes, further followed by a clearing solution wash, such as a xylene wash, of from greater than 0 minutes up to at least about 30 minutes, or to about 1, about 2, about 3, or about 4 hours. Wax is then applied to the tissue sample to form a wax impregnated block.
Without being bound by a theory of operation, it currently is believed that at reduced temperature, very little cross-linking occurs but fixative solution does penetrate into substantially the whole tissue section. Additionally, it may be that metabolic or enzymatic processes are dramatically reduced. Once diffused, the temperature is rapidly raised, where cross-linking kinetics are greatly increased. In addition, since fixative solution has substantially diffused into the sample, more even morphologic and antigen preservation are observed. This protocol differs from the prior art by separating the fixation process into a first process step that permits diffusion of fixative solution into a tissue sample but minimizes cross-linking, and a second process step that increases the rate of cross-linking, during the time periods typically used for fixing a tissue sample in disclosed working embodiments.
In typical embodiments, the methods preserve post-translation modification signals of proteins in the tissue sample significantly, for example, by preserving at least 30%, 50%, 70%, or 90% post-translation modification signals for up to 14 days. The tissue fixation methods of the present invention can significantly halt the enzyme activities destroying the post-translation modification signals, such as halting the enzyme activities of phosphatase.
In another typical embodiment, the methods preserve signals of proteins in the tissue sample significantly, for example, by preserving at least 30%, 50%, 70%, or 90% post-translation modification signals. The tissue fixation methods of the present invention can significantly halt the enzyme activities degrading proteins, such as halting the enzyme activities of protease for up to 14 days.
In one exemplary embodiment, formaldehyde fixed-paraffin embedded (FFPE) tissue samples are used. The present method offers several advantages over existing attempts to preserve modification states from FFPE tissue. The method uses a standard formalin solution that is in wide use in histology practice. The cold step can be carried out in a simple manner consisting of cold formalin for up to 14 days followed by heated formalin. The present invention for the first time in the art accomplishes long term preservation of modification states in FFPE tissue.
In summary, the present method offers at least three improvements over existing methods in the art. First, by allowing formalin to penetrate into the tissue section in a cold environment can significantly reduce enzyme activities for up to 14 days. Second, by increasing the cross-linking kinetics by quickly raising the tissue sample temperature, the cellular constituents and biomarkers are “locked” into place more rapidly than what would be observed at room temperature. This combination makes this technique superior over existing methods and for the first time allows modification states to be preserved in FFPE tissues. Third, this represents a general method believed to be applicable to a wide variety of modification states and enzymes. While other methods target a specific set of modification enzymes, this method rapidly disables all modification enzymes and therefore preserve the general cellular status much better than gold standard room temperature procedures. Since the invention is not limited to a specific set of biomolecules or biomolecules containing specific post-translations modifications, it is believed that this method represents a general method for preservation of any biomolecule or modification state. Thus, this invention can preserve with high quality quantities of biomolecules and biomolecules containing specific post-translations modifications.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee.
In order to facilitate review of the various examples of this disclosure, the following explanations of abbreviations and specific terms are provided:
H&E: Hematoxylin and eosin staining.
FFPE tissue: Formalin-fixed, paraffin-embedded tissue.
ISH: In situ hybridization.
NBF: neutral buffered formalin.
Affinity histochemistry: A histochemical method in which the analyte-binding entity is an agent other than an antibody, antibody fragment, or nucleic acid probe.
Aldehyde-based fixative: Any composition suitable for fixation of a tissue sample in which at least one of the agents primarily responsible for tissue fixation is an aldehyde.
Analyte: An entity (such as a molecule, group of molecules, macromolecule, subcellular structure, or cell) that is to be specifically detected in a sample.
Analyte-binding entity: Any compound or composition that is capable of specifically binding to an analyte. Examples of analyte-binding entities include: antibodies and antibody fragments (including single chain antibodies), which bind to target antigens; t-cell receptors (including single chain receptors), which bind to MHC:antigen complexes; MHC: peptide multimers (which bind to specific T-cell receptors); aptamers, which bind to specific nucleic acid or peptide targets; zinc fingers, which bind to specific nucleic acids, peptides, and other molecules; receptor complexes (including single chain receptors and chimeric receptors), which bind to receptor ligands; receptor ligands, which bind to receptor complexes; nucleic acid probes, which hybridize to specific nucleic acids; and engineered specific binding structures, including ADNECTINs (scaffold based on 10th FN3 fibronectin; Bristol-Myers-Squibb Co.), AFFIBODYs (scaffold based on Z domain of protein A from S. aureus; Affibody AB, Solna, Sweden), AVIMERs (scaffold based on domain A/LDL receptor; Amgen, Thousand Oaks, Calif.), dAbs (scaffold based on VH or VL antibody domain; GlaxoSmithKline PLC, Cambridge, UK), DARPins (scaffold based on Ankyrin repeat proteins; Molecular Partners AG, Zürich, CH), ANTICALINs (scaffold based on lipocalins; Pieris AG, Freising, Del.), NANOBODYs (scaffold based on VHH (camelid Ig); Ablynx N/V, Ghent, BE), TRANS-BODYs (scaffold based on Transferrin; Pfizer Inc., New York, N.Y.), SMIPs (Emergent Biosolutions, Inc., Rockville, Md.), and TETRANECTINs (scaffold based on C-type lectin domain (CTLD), tetranectin; Borean Pharma A/S, Aarhus, DK). Descriptions engineered specific binding structures are reviewed by Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the content of which is incorporated by reference in its entirety.
Antibody: The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
Antibody fragment: A molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
Anti-phospho-antibody: An antibody or antibody fragment that binds to a phosphorylated protein or amino acid residue, but not to a non-phosphorylated version of the same protein or amino acid residue. Examples of anti-phospho antibodies include:
Fixation preserves a cellular sample for subsequent examination. Chemical fixation involves immersing the sample in a volume of chemical fixative. The fixative diffuses through the tissue sample and preserves structures (both chemically and structurally) as close to that of living cells as possible. Cross-linking fixatives, typically aldehydes, create covalent chemical bonds between endogenous biological molecules, such as proteins and nucleic acids, present in the sample. Formaldehyde is the most commonly used fixative in histology. Formaldehyde may be used in various concentrations for fixation, but it primarily is used as 10% neutral buffered formalin (NBF), which is about 3.7% formaldehyde in an aqueous phosphate buffered saline solution. Paraformaldehyde is a polymerized form of formaldehyde, which depolymerizes to provide formalin when heated. Glutaraldehyde operates in similar manner as formaldehyde, but is a larger molecule having a slower rate of diffusion across membranes. Glutaraldehyde fixation provides a more rigid or tightly linked fixed product, causes rapid and irreversible changes, provides good overall cytoplasmic and nuclear detail, but is not ideal for immunohistochemistry staining. Some fixation protocols use a combination of formaldehyde and glutaraldehyde. Glyoxal and acrolein are less commonly used aldehydes. Many other aldehyde-based fixatives are also known.
It is well known that tissue fixation kinetics can be increased by raising the temperature of the fixative. However, placing a tissue sample directly into a heated fixative can cause the outside of the tissue to cross-link well before formalin penetrated to the center of the tissue, which in turn retards or even prevents further diffusion of the fixative into the tissue. As a result, biomolecules in the center of the tissue are heated without any significant cross-linking, rendering these molecules more susceptible to degradation and damage. It is also well-known that extended exposure of samples to fixative solutions can compromise the integrity of the sample and lead to loss of certain biomarkers, particularly labile biomarkers.
It was previously demonstrated that the degree of degradation and damage could be reduced by first pre-soaking the tissue samples in cold fixative to allow the fixative to diffuse throughout the sample, followed by a higher temperature treatment to spur cross-linking. See US 2012-0214195 A1. We have unexpectedly found that the cold pre-soaking step can be extended for as long as 14 days without significant loss of tissue.
In principle, the present methods may be used with any cellular sample type that can be fixed with aldehyde-based fixatives, including tissue samples and cytology samples.
In one embodiment, the sample is a tissue sample. Typically, tissue samples for immersion fixation are limited in size to ensure that fixative diffusion occurs quickly enough and adequately enough to preserve tissue morphology. Thus, certain tissue samples, such as tumor resections and whole organs, must be dissected before fixation to ensure adequate diffusion of the fixative. This is particularly true when the tissue contains analytes of interest that are subject to degradation by residual enzyme activity in the tissue. The present methods, however, increase diffusion speed and thus enable fixation of thicker-than-normal tissue samples. In an embodiment, the tissue may be as large as a tumor resection or a whole organ. In another embodiment, the tissue sample is a tissue biopsy, such as a core needle biopsy.
The present methods and systems are especially useful in fixing clinical samples in which the presence of labile biomarkers (including post-translational modifications to proteins and labile nucleic acids) will be evaluated. In some embodiments, the sample is a clinical tissue sample.
The present methods are useful with aldehyde-based fixatives. In certain embodiments, the fixative is an aldehyde-based cross-linking fixative, such as glutaraldehyde- and/or formalin-based solutions. Examples of aldehydes frequently used for immersion fixation include:
Aldehydes are often used in combination with one another. Standard aldehyde combinations include 10% formalin+1% (w/v) Glutaraldehyde. Atypical aldehydes have been used in certain specialized fixation applications, including: fumaraldehyde, 12.5% hydroxyadipaldehyde (pH 7.5), 10% crotonaldehyde (pH 7.4), 5% pyruvic aldehyde (pH 5.5), 10% acetaldehyde (pH 7.5), 10% acrolein (pH 7.6), and 5% methacrolein (pH 7.6). Other specific examples of aldehyde-based fixative solutions used for immunohistochemistry are set forth in Table 1:
In certain embodiments, the fixative solution is selected from Table 1.
In the context of concentrations of components of the aldehyde-based fixatives, the term “about” shall be understood to encompass all concentrations outside of the recited range that do not result in a statistically significant difference in diffusion rate in the same type of tissue having the same size and shape as measured by Bauer et al., Dynamic Subnanosecond Time-of-Flight Detection for Ultra-precise Diffusion Monitoring and Optimization of Biomarker Preservation, Proceedings of SPIE, Vol. 9040, 90400B-1 (2014-Mar.-20).
Another feature of the methods and systems is that they do not need exogenous degradation inhibitors (such as phosphatase inhibitors, kinase inhibitors, protease inhibitors, or nuclease inhibitors) to substantially preserve labile biomarkers in a state that they can be detected by histochemistry. Therefore, although such degradation inhibitors may be included in the fixative solutions, they are not required. In an embodiment, the aldehyde-based fixative solutions do not contain an effective amount of exogenously added phosphatase inhibitor or kinase inhibitor. In other embodiments, the aldehyde-based fixative solutions do not contain an effective amount of phosphatase inhibitor, kinase inhibitor, protease inhibitor, or nuclease inhibitor.
Certain disclosed embodiments concern a multi-step, typically a two-step, tissue fixation process for infusing/diffusing a tissue sample using an aldehyde-based fixative solution. During a first processing step, a sample is treated with the aldehyde-based fixative solution under conditions that allow the fixative to diffuse throughout substantially the entire cross-section of the sample. This first step is conducted using a fixative composition for a first period of time, and at a first temperature, that effects substantially complete tissue infusion/diffusion. The second step is to subject the tissue sample to a fixative composition at a second, higher temperature to allow cross-linking to occur. In operation, the first and second processing steps are performed over the course of an extended time period, typically on the order of greater than two days. As shown in the Examples below, the process has been validated up to 14 days, although it likely can be extended for even longer than that.
First, an unfixed tissue sample is immersed in an aldehyde-based fixative solution at a cold temperature. The temperature of the aldehyde-based fixative solution is held at the cold temperature at least long enough to ensure that the fixative has diffused throughout the tissue sample. The minimum amount of time to allow diffusion can be determined empirically using various time and temperature combinations in cold fixatives and evaluating the resulting tissue samples looking at factors, such as preservation of tissue architecture and loss of for preservation of a target analyte by immunohistochemistry (if the analyte is a protein or phosphorylated protein, for example) or in situ hybridization (if the target analyte is a nucleic acid, such as miRNA or mRNA). Alternatively, the minimum amount of time of time to allow for diffusion can be determined by monitoring diffusion using, for example, a method as outlined in Bauer et al., Dynamic Subnanosecond Time-of-Flight Detection for Ultra-precise Diffusion Monitoring and Optimization of Biomarker Preservation, Proceedings of SPIE, Vol. 9040, 90400B-1 (2014-Mar.-20). An effective temperature range for the first step can include any temperature between the freezing point of the aldehyde-based fixative solution and below 10° C., for example, about 0° C. to about 7° C., about 2° C. to about 5° C., and about 4° C. In this context, the term “about” shall encompass temperatures that do not result in a statistically significant difference in diffusion rate in the same type of tissue having the same size and shape as measured by Bauer et al., Dynamic Subnanosecond Time-of-Flight Detection for Ultra-precise Diffusion Monitoring and Optimization of Biomarker Preservation, Proceedings of SPIE, Vol. 9040, 90400B-1 (2014-Mar.-20). Diffusion of the fixative composition into the tissue sample is continued for a time period effective for diffusion of the composition throughout substantially the entire cross section of the sample.
Once the cold fixative solution has sufficiently diffused throughout the tissue sample, it is stored for an extended period of time either in cold storage (such as a refrigerator or ice bucket) or at ambient temperature (i.e. a temperature from 18° C. to 28° C.) for a cumulative time of greater than two days. In some embodiments, the cumulative time is from greater than two days to up to two weeks or longer, such as from at least 72 hours to 14 days. “Cumulative time” in this context is the sum of the diffusion time and the following cold or ambient temperature extended storage).
If the sample is stored at cold temperature, then it is subjected to a warm temperature treatment (i.e. a temperature of from 18° C. up to 55° C.) for a sufficient amount of time to permit fixation. The temperature associated with the warm temperature treatment typically is ambient or higher, such as higher than about 18° C. In an embodiment, a temperature range is from ambient up to 50° C. (such as from 20° C. to 50° C.). If the temperature is reaches around 55° C., however, the sample generally begins to degrade, which may have a deleterious effect on certain subsequent histological reactions. Therefore, temperatures significantly above 50° C. should be avoided for extended periods of time. Thus, in such an embodiment, the upper temperature and second time period should be selected so as to preserve the sample in a state that permits subsequent analyses (such as in situ hybridization, histochemical analyses and/or H&E) to proceed effectively. The optimal upper and lower time and temperature limits should be determined empirically based on the particular analysis that will be performed and the sample type being used. In particular, guardbanding of time and temperature ranges should be performed to determine acceptable time/temperature combinations that do not unacceptably compromise tissue architecture and/or analyte detection levels. In some embodiments, the warm temperature treatment is performed in the same fixative solution in which the first processing step is performed. In such an embodiment, the fixative solution may be brought to the second temperature range by active heating (for example, by using a heating element or other heat source) or passive heating (such as by moving the fixative and sample from a cold environment to a warm environment and allowing the temperature of the fixative solution to equilibrate with the environment). In other embodiments, the sample is placed in contact with a fixative solution at a second temperature range by removing the sample from the fixative solution at the first temperature range and immersing the sample in a volume of an aldehyde-based fixative solution at the second temperature range. For example, the fixative solution at the first temperature range could be disposed in a first vessel and the fixative solution at the second temperature range could be disposed in a second vessel, in which case the sample may be physically moved from the first vessel to the second vessel after the first time period has expired. Alternatively, the fixative solution at the first temperature range may be removed from a vessel and replaced with the fixative solution at the second temperature range. As yet another alternative, only a portion of the fixative solution at the first temperature range may be removed, and a hot fixative solution may be added to the remaining fixative solution, such that the resulting combination brings the temperature within the second temperature range. Many other potential arrangements can be envisioned. In any of the embodiments in this paragraph, the fixative solution at the first temperature range may be the same or different from the fixative solution at the second temperature (including differ in the concentration of aldehyde, identity of aldehyde, and/or overall composition).
If the extended storage is at ambient temperature, then additional warm temperature treatment is unnecessary before further tissue processing, although it can be done if desired.
As used herein, the phrase “further tissue processing” shall encompass any process following aldehyde fixation that is used to prepare the fixed tissue sample for storage and/or analysis. Many such processes are well-known and would be well understood by a person of ordinary skill in the art. For example, protocols for using zinc formalin, Helly's fixative and Hollande's require a water wash after fixation to remove various contaminates. Some protocols for Bouin's and B-5 suggest storing the fixed samples in 70% ethanol before processing. Additionally, some specimens may be difficult to cut on a microtome because of calcium carbonate or phosphate deposits, and thus may require decalcification. Other post-fixation tissue processing would be well-known to a person having ordinary skill in the art.
In one embodiment, post-fixation tissue processing comprises wax-embedding. In the typical example, the aldehyde-fixed tissue sample is subjected to a series of alcohol immersions to dehydrate the sample, typically using increasing alcohol concentrations ranging from about 70% to about 100%. The alcohol generally is an alkanol, particularly methanol and/or ethanol. After the last alcohol treatment step the sample is then immersed into another organic solvent, commonly referred to as a clearing solution. The clearing solution (1) removes residual alcohol, and (2) renders the sample more hydrophobic for a subsequent waxing step. The clearing solvent typically is an aromatic organic solvent, such as xylene. Wax blocks are formed by applying a wax, typically a paraffin wax, to the sample. Typically, before tissue analysis, the blocks are sliced into thin sections using a microtome. The thin sections may then be mounted on a slide and stored for later analysis and/or subjected to post-processing analysis.
In other examples, the tissue sample may be embedded in resin blocks (such as epoxy or acrylic resins) instead of wax blocks. Exemplary resins include methyl methacrylate, glycol methacrylate, araldite, and epon. Each requires specialized post-fixation processing steps, which are well known in the art.
Fixed tissue samples obtained by the processes and compositions disclosed herein can be used together with any staining systems and protocol known in the art of histochemistry, as well as affinity histochemistry, immunohistochemistry and in situ hybridization. The present invention can also be used together with various automated staining systems, including those marketed by Ventana Medical Systems, Inc. (such as the VENTANA HE600, SYMPHONY, BENCHMARK, and DISCOVERY series automated platforms), Dako (such as the COVERSTAINER, OMNIS, AUTOSTAINER, and ARTISAN series automated slide stainer), and the LEICA ST series stainers. Exemplary systems are disclosed in U.S. Pat. No. 6,352,861, U.S. Pat. No. 5,654,200, U.S. Pat. No. 6,582,962, U.S. Pat. No. 6,296,809, and U.S. Pat. No. 5,595,707, all of which are incorporated herein by reference. Additional information concerning automated systems and methods also can be found in PCT/US2009/067042, which is incorporated herein by reference.
In an embodiment, specific analytes are detected using immunohistochemistry (IHC). In the typical IHC protocol, a tissue sample is contacted first with an analyte-specific antibody under conditions sufficient to permit specific binding of the analyte-specific antibody to the analyte. In exemplary embodiments, detection of specific analytes is realized through antibodies capable of specific binding to the analyte (or antibody fragments thereof) conjugated with multiple enzymes (e.g. horse radish peroxidase (HRP), alkaline phosphatase (AP). This enzyme-antibody conjugate is referred to as an HRP or AP multimer in light of the multiplicity of enzymes conjugated to each antibody. Multimer technologies are described in U.S. Pat. No. 8,686,122, which is hereby incorporated by reference in its entirety. This type of detection chemistry technology is currently marketed by Ventana Medical Systems Inc., as ultraView Universal DAB detection kit (P/N 760-500), ultraView Universal AP Red detection kit (P/N 760-501), ultraView Red ISH DIG detection kit (P/N 760-505), and ultraView SISH DNP detection kit (P/N 760-098). In illustrative embodiments, the approach uses non-endogenous haptens (e.g. not biotin, see U.S. application Ser. No. 12/660,017 which is hereby incorporated by reference in its entirety for disclosure related to detection chemistries). In illustrative embodiments, a tyramide signal amplification may be used with this approach to further increase the sensitivity and dynamic range of the detection (See PCT/US2011/042849 which is hereby incorporated by reference in its entirety for disclosure related to detection chemistries).
Any suitable enzyme/enzyme substrate system can be used for the disclosed analysis/detection method. Working embodiments typically used alkaline phosphatase and horseradish peroxidase. If the enzyme is alkaline phosphatase, one suitable substrate is nitro blue tetrazolium chloride/(5-bromo-4-chloro-1H-indol-3-yl)dihydrogen phosphate (NBT/BCIP). If the enzyme is horseradish peroxidase, then one suitable substrate is diaminobenzidine (DAB). Numerous other enzyme-substrate combinations are known to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149, and 4,318,980. In some embodiments, the enzyme is a peroxidase, such as horseradish peroxidase or glutathione peroxidase or an oxidoreductase.
U.S. Patent Publication 2008/0102006, the entire disclosure of which is incorporated herein by reference, describes robotic fluid dispensers that are operated and controlled by microprocessors. U.S. Patent Publication 2011/0311123, the entire disclosure of which is incorporated herein by reference, describes methods and systems for automated detection of immunohistochemical (IHC) patterns. The automated detection systems disclosed in these patent applications can be used to detect analytes in the fixed tissue samples of the present invention.
In some embodiments, the fixed tissue samples are analyzed by immunohistochemistry for the presence of post-translationally modified proteins. In the typical process, the fixed tissue sample is contacted with an analyte-binding entity capable of specifically binding to the post-translationally modified protein under conditions sufficient to effect binding of the analyte-binding entity to the post-translationally modified protein; and binding of the analyte-binding entity to the post-translationally modified protein is detected. The precise conditions for effective IHC generally need to be worked on an individual basis, depending upon, for example, the precise antibody used, the type of sample used, sample size, further processing steps, et cetera. In an embodiment, the post-translational modification is one that is susceptible to loss during a standard aldehyde fixation process due to residual enzyme activity within the tissue sample. One could determine whether a given post-translational modification is susceptible to residual enzyme activity by treating a sample with an entity that leads to increased presence of the post-translational modification. The sample could then be fixed using a standard technique (such as 24 hour fixation in room temperature NBF) and a fixation process as disclosed herein and the amount of signal detectable in each of the samples can be compared. If signal is absent or significantly lower in the sample fixed according to standard techniques, then one can assume that the post-translational modification is susceptible to degradation by residual enzyme activity. Thus, in an embodiment, the post-translational modification is a post-translational modification that has a lower level of detection in a tissue fixed for 24 hours in room temperature NBF without a cold temperature pre-treatment than in a substantially identical tissue sample that has been fixed using a two-temperature fixation as described above. In an embodiment, the post-translational modification is a diagnostic or prognostic marker for a disease state of the tissue sample. In an embodiment, the post-translational modification is a predictive marker for an effect of a therapy on a disease state of the tissue. In an embodiment, the post-translational modification is a phosphorylation.
In some embodiments, the fixed tissue samples are analyzed by in situ hybridization for the presence of specific nucleic acids. In the typical process, the fixed tissue sample is contacted with a nucleic acid probe complementary to the analyte nucleic acid under conditions sufficient to effect specific hybridization of the probe to the analyte nucleic acid; and binding of the nucleic acid probe to the analyte nucleic acid is detected. The precise conditions for effective ISH generally need to be worked on an individual basis, depending upon, for example, the precise nucleic acid probe used, the type of sample used, sample size, further processing steps, et cetera. In an embodiment, the analyte nucleic acid is one that is susceptible to loss during a standard aldehyde fixation process due to residual enzyme activity within the tissue sample. One could determine whether a given nucleic acid is susceptible to residual enzyme activity by treating a sample with an entity that leads to increased presence of the nucleic acid. The sample could then be fixed using a standard technique (such as 24 hour fixation in room temperature NBF) and a fixation process as disclosed herein and the amount of signal detectable in each of the samples can be compared. If signal is absent or significantly lower in the sample fixed according to standard techniques, then one can assume that the analyte nucleic acid is susceptible to degradation by residual enzyme activity. Thus, in an embodiment, the analyte nucleic acid has a lower level of detection in a tissue fixed for 24 hours in room temperature NBF without a cold temperature pre-treatment than in a substantially identical tissue sample that has been fixed using a two-temperature fixation as described above. In an embodiment, the analyte nucleic acid is a diagnostic or prognostic marker for a disease state of the tissue sample. In an embodiment, the analyte nucleic acid is a predictive marker for an effect of a therapy on a disease state of the tissue. In an embodiment, the analyte nucleic acid is an RNA molecule, such as mRNA or miRNA.
The following examples are provided to illustrate certain features of working embodiments of the present invention. A person of ordinary skill in the art will appreciate that the scope of the invention is not limited to the features recited in these examples.
4 mm Calu3 Xenograft tumor cores that were placed into cooled formalin at 7, 10 or 15° C., respectively, for 2, 4 or 6 hours to form a 9 panel matrix around soak temperature. After the cold soak was completed, tumors were immediately immersed into warm formalin at 45° C. for 2 hours. Samples were then processed further in a standard tissue processor set to an overnight cycle. Tissue was sliced in half and embedded cut side down to reveal the edges and middle of the tissue. Control tissues consisted of comparison pieces of the same tumors being fixed with a two-temperature protocol (2 hours 4° C.+2 hours 45° C.) and pieces of tumor fixed at RT for 24 hours. Tissues were then stained with anti-pAKT (CST #4060) at a 1:50 dilution on a Ventana DISCOVERY XT automated stainer using the OptiView DAB staining kit (Ventana Medical Systems, Inc.). Results are shown at
Calu3 Xenograft tumors were harvested and placed into the experiment with less than 10 minutes of cold ischemia time. Tumors were cored at 4 mm using a disposable biopsy device to ensure all samples were roughly the same size. To test how long samples can sit in cold formalin, pieces of Calu3 tumors (no more than 4 mm thick) were placed into 4° C. formalin for up to 14 days. After the cold soak was completed, tumors were immediately immersed into warm formalin at 45° C. for 2 hours. Samples were then processed further in a standard tissue processor set to an overnight cycle. Tissues were sliced in half and embedded cut side down to reveal the edges and middle of the tissue.
Tissues were stained with anti-pAKT (CST #4060) at a 1:50 dilution on a DISCOVERY XT automated stainer using the OptiView DAB staining kit (Ventana Medical Systems Inc.). This dilution was previously chosen based on a number of similar experiments utilizing Calu3 tumors and this same antibody. To reduce background staining from mouse tissue, staining was performed by substituting a rabbit only form of the linker in the commercial kit.
To demonstrate a real-world application of the present fixation process, a shipping study was conducted. A total of 20 Calu-3 xenograft tumors and 20 human tonsil samples were collected. Samples were staggered such that 5 Calu-3 tumors and 5 tonsil samples were shipped in a week. The shipping schedules tested are reproduced below in Table 2:
6 days
Styrofoam-insulated shipping containers were retrofit with data loggers to track the temperature of the package during shipping and frozen inserts to maintain a cold temperature.
5 Calu-3 tumors were split into 2 samples each. One half of the tumor was fixed by the 2+2 method as a positive control for controlled fixation. The other half of the tumors were placed into histology cassettes, and the cassettes were labeled and loaded into specimen containers. This procedure was repeated in the afternoon for human tonsil samples that arrive in the afternoon. Specimen containers were placed the data loggers and were placed into a Styrofoam grid which contained a top and bottom for better insulation. Once assembled, the Styrofoam block was placed into either a small or larger shipping container that has frozen inserts. After samples were shipped and received, the tissues were placed into heated formalin for an additional 2 hours, processed overnight into wax blocks and stained for a variety of IHC markers.
The temperature of the specimen containers during shipping is presented at
The setup for Shipment 2 was essentially the same as Shipment 1, except that the data loggers were placed in a refrigerator overnight to cool. Samples were harvested in an identical manner to shipment 1 and the data loggers were out of the refrigerator approximately 10 minutes. The temperature of the specimen containers during shipping is presented at
Between shipment 2 and 3, the collection procedure was modified slightly to determine if we could maintain the temperature below 7° C. for the entire collection procedure. For this shipment, data loggers were never removed from the refrigerator, only the specimen containers. For example, Calu-3 tumors were received in small batches (2-3 at a time). A corresponding number of specimen containers were placed under a chemical hood and tumors were sectioned, cassettes labeled, clipped into container lids and placed back in the refrigerator within 5 minutes. Specimen containers were placed directly into cooled data loggers and the data loggers were started. When all samples had been processed in this manner, data loggers with corresponding specimen containers were placed into foam packing and placed into a shipping box. The shipping box had been previously conditioned and waiting for the samples. As can be seen, all data loggers registered temperatures below 5.5° C. Shipment 4 was essentially identical to shipment 3.
Human Tonsil—Human tonsil samples were stained with Hematoxylin and Eosin to determine if there were any tissue morphology issues throughout the shipping process. Samples were compared to control tissues fixed with a 2+2 fixation protocol. All tonsil samples shipped had excellent morphology with no visible defects with any conditions tested (see upper H&E panel). Human tonsil tissues were also stained with PD-L1, FoxP3 and CD68 according to the validation data. All tissues stained identically to control tissues fixed with a 2+2 protocol with all shipping scenarios.
Calu-3—Calu-3 samples were stained with PR, Ki-67 and an antibody (CST4060) that recognizes the phosphorylated AKT protein. For total IHC protein staining (PR and Ki-67), results were indistinguishable between control samples fixed with a 2+2 protocol. Robust staining was evident regardless of the shipping conditions, even shipment 1 that had temperatures above the 7° C. zone. It appears that these two proteins are expressed to high levels in the Calu-3 cell model and are stable to slightly elevated temperatures. A different result was obtained when we stained for pAKT. Levels of this labile epitope varied depending on the shipment and temperature conditions compared to controls with a 2+2 fixation protocol. Shipment 1 had initial temperatures up to 14° C., which led to variable staining between the shipped samples and the 2+2 controls. Variable but better consistency was observed with shipment 2 which had temperatures that just peaked above 7° C. Better staining consistency was observed with shipments 3 and 4 with almost identical staining compared to the control.
Calu3 xenografts were fixed in 10% NBF under a variety of conditions as set forth in Table 3 and evaluated for morphology by H&E stain. “Hot” in table 3 denotes 45° C. for 1 hour. “Cold” indicates 4° C. Samples were scored on a +, ++, or +++ scale, where + is poor morphology and +++ is the best morphology.
Additionally, the samples were immunohistochemically stained for pAkt. Results are shown at
It has previously been demonstrated that nucleic acids (such as mRNA and miRNA) can be sensitive to standard 24 hour room temperature fixation. See, e.g., US 2012-0214195. To illustrate this, the preservation of two miRNA—miR-21 and miR-200c—was evaluated using standard 24 hour room temperature fixation and cold soak followed by 1 hour fixation at 45° C. 4 mm thick pieces of the same human tonsil organ were placed into either room temperature (21-24° C.) 10% neutral buffered formalin for 24 hours or else 2 hours in 4° C. formalin followed by 1 hour in 45° C. formalin (Cold/Hot). Tonsil samples were probed for the expression of miR-21 or miR-200c with specific DNA probe sequences to each target. After application of the probe sequence, detection of the bound probe occurred on a VENTANA DISCOVERY XT automated stainer with a silver detection kit. Cold/Hot fixation resulted in an increase in the amount of specific signal in the samples indicating a greater preservation of the miRNA species. Results are shown at
The tissue sample is immersed in an aldehyde-based fixative solution at a cold temperature (e.g., above the freezing point of the fixative solution but less than 10° C., including for example in a range of from 2 to 7° C., 2 to 5° C., or about 4° C.). The temperature of the aldehyde-based fixative solution is held at the cold temperature at least long enough to ensure that the fixative has diffused throughout the tissue sample. The minimum amount of time to allow diffusion can be determined empirically using various time and temperature combinations in cold fixatives and evaluating the resulting tissue samples for preservation of the target nucleic acid using an in situ hybridization procedure. Alternatively, the minimum amount of time of time to allow for diffusion can be determined by monitoring diffusion using, for example, a method as outlined in Bauer et al., Dynamic Subnanosecond Time-of-Flight Detection for Ultra-precise Diffusion Monitoring and Optimization of Biomarker Preservation, Proceedings of SPIE, Vol. 9040, 90400B-1 (2014-Mar.-20).
Once the cold fixative solution has sufficiently diffused throughout the tissue sample, it is stored for an extended period of time either in cold storage (such as a refrigerator or ice bucket) or at ambient temperature (i.e. a temperature from 18° C. to 28° C.) for a cumulative time of at least 72 hours. “Cumulative time” in this context is the sum of the diffusion time and the following cold or ambient temperature extended storage). If the sample is stored at cold temperature, then it is subjected to a warm temperature treatment (i.e. a temperature of from 18° C. up to 55° C.) for a sufficient amount of time to permit fixation. If the extended storage is at ambient temperature, then additional warm temperature treatment is unnecessary.
After the extended storage period, the tissue sample is subjected to post-fixation processing to prepare it for in situ hybridization to detect the target nucleic acid. The tissue sample is washed (if the fixative used requires a wash step), subjected to alcohol dehydration, a clearing solution, and then embedded in paraffin according to standard techniques. The embedded tissue is then sectioned on a microtome, mounted on a slide, and stained for a target messenger RNA (mRNA), microRNA (miRNA), or DNA molecule using an in situ hybridization technique, for example, using an automated IHC/ISH slide stainer, such as the VENTANA BENCHMARK or the VENTANA DISCOVERY automated stainer.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
Additionally, the following specific embodiments are disclosed:
This is a continuation of PCT/EP2016/051431, filed Jan. 25, 2016, and claims the benefit of U.S. Provisional Patent Application No. 62/108,248, filed on Jan. 27, 2015, the content of each of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62108248 | Jan 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2016/051431 | Jan 2016 | US |
| Child | 15660868 | US |
