Stabilization of Tissue Specimens for Pathological Examination and Biomedical Research

Abstract

Human tissue specimens are critical reagents in the diagnosis of disease and biomedical research. Tissues experience rapid degradation immediately after ligation from their blood supply. A variety of processing techniques are employed to prevent the degradation of tissue samples, principally chemical fixation and thermal processing. The success of processing techniques is measured by the preservation of tissue morphology, as well as the critical biomarkers. In preservation of tissue specimens, formaldehyde is the most widely used fixative that maintains tissue morphology. However, the cross-links resulting from chemical interactions between formaldehyde and biomolecules in the specimens introduce difficulties in detection and extraction of antigens for analysis. Alternative processing methods, such as chemical fixation (e.g., alcohol-based) or thermal processing (e.g., freezing) help avoid the loss of antigenicity due to cross-linking, but introduce morphological artifacts. In this article, we review methods of processing of fresh tissue samples, as well as the effects of these procedures on morphology and antigenicity of the preserved tissues as assessed by histology, immunohistochemistry, proteomics, and genomics.

Introduction

Tissue biopsies and biospecimens collected during surgery are valuable resources for diagnostic and research purposes. These samples facilitate diagnosis, selection of treatment, and monitoring of response. As soon as a tissue is disconnected from its blood supply, it experiences hypoxic conditions. The enzymatic alteration of proteins and nucleic acids ensues, leading to loss of antigens.^1–3 Chemical or thermal processing of tissue is used to arrest degradation. Each method of preservation has its advantages and limitations. This article will review the parameters that influence the quality of critical biomarkers after preservation by thermal and chemical processing.

Tissue Ischemia

Ischemia is the state in which tissue has experienced hypoxia due to lost or restricted blood supply. The hypoxic conditions and enzymatic alteration of proteins and nucleic acids cause the loss of antigens.^1–3 Moreover, this process is selective, affecting certain molecules more than others.^4,6 In particular, nucleic acids and signaling molecules exhibit significant degradation in tissues subjected to hypoxia.^5–9 Chung et al.¹ simulated warm ischemia in the laboratory using rat kidneys and found a decrease in the rRNA quality (assayed as a ratio of 18S and 28S bands by electropherogram) and RNA yield within 4 h at 25°C or 37°C, compared to 4°C. However, the use of RNAlater, a stabilizing storage buffer, maintained the RNA quality for up to 24 h at 25°C.¹

Huang et al.⁶ investigated the effect of warm ischemia on gene expression by using cDNA microarrays. They demonstrated that, although the standard assay of mRNA stability based on the 18S and 28S bands showed that the mRNA was stable, the microarray technique revealed significant alteration in gene expression profiles within 20 min after excision of human colon cancer specimens. In similar studies, Spruessel et al.⁹ investigated the effect of ischemia on gene and protein expression in normal and malignant colon tissues. They found that the expression of 10%–15% of the genes differed significantly from baseline within 15 min of tissue resection. Also, analysis of protein profiles by mass spectrometry (SELDI-TOF) showed that 30% of the peaks in protein profiles changed more than 2-fold within 30 min, with most of the changes occurring within the first 15 min. In contrast, investigation of gene expression patterns in radical prostatectomy specimens by Dash et al.⁸ yielded very little overall variation after 5 h of ischemia. However, among the genes associated with prostate cancer, EGR1 showed almost a 2-fold increase in expression, while hepsin exhibited a relatively steady expression throughout the 5 h of ischemia. These results suggest that tissue ischemia is a significant factor in detection and interpretation of molecular biomarkers.

Ischemic time can also influence other methods of biomarker detection, including immunohistochemistry. Khoury et al.¹⁰ investigated the effects of delay in fixation on the expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2. Immunohistochemical (IHC) staining of breast cancer tissues for ER and PR showed a decrease in staining when tissue fixation had been delayed for 1–2 h, with a significant loss of expression after a 4 to 8 h delay. HER2 staining assayed by fluorescence in situ hybridization (FISH) showed a decrease in fluorescence with increasing delay in fixation, starting within 1 h, which prompted the authors to recommend fixation of breast cancer biopsies within 1 h of resection to avoid degradation of these markers and hence avoid inappropriate therapeutic decisions. Yildiz-Aktas et al. also noted reduced staining of ER, PR, and HER2 markers in breast carcinoma samples.¹¹ Pinhel and colleagues¹² compared the expression of breast cancer biomarkers in resected tumor specimens that were processed immediately or after known periods of delay in a clinical setting. The markers Ki-67, ER, PR, and HER2 did not vary significantly among the core-cut biopsies taken immediately after tumor resection and after 20 to 80 min delays in fixation.

Jones et al.¹³ measured variations in the levels of Src tyrosine kinase markers, due to delays in processing, in breast and bladder cancer specimens. Delays in processing in breast cancer biopsy specimens for 60 min after ligation led to statistically significant increases in the levels of phosphopaxillin, while the levels of total paxillin decreased in the same time period. In contrast, the same markers did not vary significantly in bladder cancer specimens under similar processing conditions. The stability of phosphoproteins was examined by Espina et al.¹⁴ who quantified 53 phosphoprotein biomarkers in 6 different tissue types over a period up to 2 h past tissue excision. Fluctuations were recorded in the expression levels of different phosphoproteins in different tissue types and storing tissues at 4°C was ineffective at reducing changes in phosphoprotein levels. Phosphatase and kinase inhibitors in the fixative or storage solution stabilized the phosphoproteins.¹⁵ The authors recommended tissue harvesting and processing within 20 min post-excision in order to preserve phosphoproteins.

Thermal Processing of Frozen Sections

Another strategy to reduce tissue degradation involves quick freezing. This technique is commonly used when nucleic acids or proteins are to be extracted.^{4,6,14,16–20} Sample fixation by freezing is a rapid process (when compared to chemical fixation)²¹ and therefore is useful for interoperative diagnostics.^22–24 Frozen tissue biospecimens are also commonly analyzed using immunohistochemistry, proteomics, and genomics.^4,7,18–20 The most commonly used freezing technique involves immersion of the sample in an ultra-cold bath of quenching fluid such as liquid nitrogen or isopentane.^{6,17,19,26–35} Briefly, a bath of isopentane in a metal container is cooled using a liquid nitrogen bath to the stage where the isopentane starts to solidify around the edges, resulting in a temperature of about −150°C. A tissue sample is dissected into small pieces and transferred to standard cryovials and frozen by immersing the sealed cryovial into the isopentane bath.²⁹ Alternatively, liquid nitrogen quenching (−196°C) or an isopentane bath cooled using dry ice (−80°C) may be employed, depending upon the tissue type, specimen size, and availability of resources. These protocols for biospecimen freezing, termed “snap freezing” or “flash freezing”, are recommended by the National Cancer Institute (NCI),²⁹ European Organization for Research and Treatment of Cancer,¹⁹ and International Society for Biological and Environmental Repositories (ISBER).³⁵

Another method of processing tissue samples involves first embedding the tissue into Tissue Freezing Medium (TFM) or Optimal Cutting Temperature (OCT) compound,^20,29 then freezing the tissue. NCI and ISBER recommend placing the tissue sample into a plastic cryomold, which is then filled with the liquid embedding medium and the entire system is frozen (e.g., in the vapor phase of liquid nitrogen).^29,35 For small specimens, Loken and Demetrick²⁰ proposed using a type ‘00’ VCap capsule that is partially filled with OCT. By this technique, a small piece of tissue is placed in the capsule and the rest of the capsule is filled with more OCT, and then immediately frozen by immersion in liquid nitrogen. The capsules can be stored in cryovials and the specimen can be harvested by slicing the capsule as needed while maintaining the remaining sample in the cryovial for subsequent use.

The main disadvantage of allowing liquid nitrogen to contact tissues directly (i.e., with or without a cryovial) as a quenching fluid, is that when a biospecimen at room temperature is introduced into the liquid, a vapor film forms around the tissue which reduces the heat transfer efficiency drastically.^36–38 Moline and Glenner²¹ showed that coating tissues with fine powders (e.g., confectioners' sugar, talcum powder, flour, or starch) can achieve very fast cooling rates. The coated samples were frozen at least twice as fast as uncoated tissues frozen with liquid nitrogen or isopentane. Damage to tissue morphology was dependent upon the size of the coating granule; the best and most consistent morphology preservation was achieved when tissues were coated with talcum powder.

Tissue from the nervous system such as the brain is delicate and particularly susceptible to processing artifacts, requiring more extensive processing. For example, after dissection, brain tissue blocks may be fixed chemically for 12–24 h, followed by sucrose filtration.³⁹ To reduce tissue shrinkage resulting from the osmotic effects of exposure to sucrose, these tissue blocks are transferred to 20% sucrose in phosphate buffer for 2–3 days, followed by infiltration with 30% sucrose in phosphate buffer for another 2–3 days. These blocks can then be frozen by placing them into metal trays covered by powdered dry ice.^39,40 Though infiltration of 30% sucrose is often recommended in the freezing of brain tissue, Rosene et al.⁴¹ noted that this procedure might not provide optimum protection against freezing artifacts, especially in large blocks of brain tissue. They suggested infiltrating large brain tissue blocks (larger than 60 cc) with 10% glycerol plus 2% dimethylsulfoxide (DMSO) in fixative or buffer for 1–3 days, followed by 3–5 days of immersion in 20% glycerol plus 2% DMSO. The tissue blocks were then encased into albumin-gelatin blocks and frozen by putting the blocks onto a metal plate surrounded by dry ice pellets or by using an isopentane bath cooled using alcohol-dry ice slush. This method yielded tissue blocks with fewer freezing artifacts. Additionally they reported that the use of cryoprotectants facilitated sectioning on the cryostat.⁴¹

Chemical Processing

Chemical fixation is an alternative method of preventing degradation of tissue samples. Chemically fixed specimens are subsequently dehydrated and embedded in paraffin wax, which facilitates long-term storage as well as thin sectioning of the preserved specimen for analysis. The fixatives used in histotechnology are broadly categorized into two types, cross-linking fixatives and coagulating fixatives.

Buffered formalin or formaldehyde is the most common cross-linking fixative. Formalin, which is a solution of formaldehyde in water, mainly consists of monomeric formaldehyde or methylene hydrate. The fixation and cross-linking process requires several hours, if not days, to be completed. The cross-links and conformational changes induced by fixation may result in the masking of epitopes and hinder detection of antigens by antibodies.^43–45 The cross-linking process is reversible in most cases and antigens can be retrieved by breaking the bonds through the application of heat or chemical reactions.^34,43,45 Heat-induced antigen retrieval reverses most of the cross-linking artifacts and has become a standard procedure for tissue processing in diagnostic pathology.^{34,43,45–47}

The most common coagulating fixatives are alcohol- or acetone-based solutions. They function by removal and replacement of water in the tissue and precipitating or coagulating the proteins, which results in preservation of tissue structure.^42–44 Furthermore, the displacement of water from the vicinity of the proteins causes a reduction in the repulsive forces acting on hydrophobic moieties. This then causes a destabilization of hydrogen bonds in the hydrophilic areas of the protein, leading to the loss of tertiary structure. The irreversible disruption of tertiary structure or denaturation makes the proteins insoluble and also causes a loss of function.^42,43

The chemical fixation process is preferred in diagnostic pathology.^5,34,44 Formalin-fixed paraffin-embedded (FFPE) specimens exhibit superior tissue morphology and are most suitable for microscopic analysis and immunohistochemistry. However, FFPE samples have disadvantages: it is difficult to extract high quality DNA and RNA from FFPE samples, there are health hazards related to the use of formaldehyde, and the fixation process is quite slow.^48–50 Several reports have demonstrated that alcohol-based fixatives achieve comparable immunohistochemical staining and morphology and achieve superior preservation of nucleic acids compared with formaldehyde fixation.^48–52 Zinc-based fixatives, which contain salts of zinc in buffered solution, are non-cross-linking or noncoagulative. They show excellent preservation of morphology, superior antigenicity and immunohistochemical staining, as well as good quality RNA and DNA yield from fixed specimens.^53,54

It is a common practice in both the clinical as well as research settings to store precut, unstained tissue sections on slides for a variety of reasons, including later use as positive controls, retrospective analysis, specimen conservation by limiting the sectioning sessions, ease of storage of slides, and ability to maintain FFPE blocks at a central repository while keeping slides in different locations.^55–57 However, there is strong evidence that the immunoreactivity of antigens in these precut tissue sections decreases significantly with storage time.^55–62 ER, PR, Her-2, and p53 showed moderate to significant decreases in antigenicity with storage times of 3 to 6 months.^55,57,59 Cytokeratins, smooth muscle actin, PS-100, CD45, CD20, and CD30 exhibited no significant changes in expression over 3 to 10 years, and a moderate increase in immunostaining of vimentin was also observed.⁵⁸ The mechanisms involved in loss of antigenicity in stored sections are not fully understood. Low temperature (4°C) storage does not prevent the loss of antigenicity.^55,59 Exposure to air resulting in oxidation was thought to be one of the important mechanisms affecting antigenicity in stored slides.^60,62 Yet the addition of paraffin layers or storage in a nitrogen environment did not offer sufficient protection to precut tissue sections, while the use of wax-soluble antioxidants diminished the protective effects of the paraffin coating.⁶⁰ Recent studies in which precut tissue sections were stored in a vacuum chamber with desiccant showed that endogenous or exogenous water in the tissues protected the antigens and thereby significantly affected their immunoreactivity.⁶¹

Analysis Using Preserved Tissue Sections

Histology

Any method of preserving tissue biospecimens (chemical or thermal) will result in processing artifacts. Trained pathologists are accustomed to seeing artifacts in the processed tissue sections and can make accurate diagnostic decisions despite the presence of alterations in morphology of the tissue. Using intra-operative frozen sections, trained pathologists can make diagnoses with an accuracy level of 94%–99%.^22–24 Although the tissue is expected to shrink due to fixation and subsequent processing due to dehydration^54,63–65 and may have other fixation artifacts, FFPE sections are preferred by most pathologists mainly because of their familiarity with FFPE morphological patterns.⁶⁴ Examination of histomorphology of the tissue is critical to the making of diagnostic decisions by pathologists. For example, in prostatic carcinoma the epithelial lining of lumens is distorted, (specifically a loss of basal cells or alterations of lumen appearance and spacing is observed); whereas high-grade prostate intraepithelial neoplasia (HGPIN) is characterized by an increase in nuclear size or loss of nuclear content.^30,66

Chemical processing

Several investigators have studied the effects of fixatives and embedding media on the morphology of preserved specimens. Hoetelmans and colleagues⁶⁷ used several microscopic techniques to assess the efficacy of acetone, methanol, paraformaldehyde, and glutaraldehyde in preserving the structure and morphology in cultured MCF-7 cells. They demonstrated that fixation with acetone and methanol resulted into disruption of cellular structures, plasma membranes, and nuclear integrity. Acetone and methanol fixation resulted in suboptimal preservation of subcellular organelles compared to aldehyde-based fixatives. Though ethanol-fixed paraffin-embedded tissue sections showed comparable or superior morphology when compared to formalin-fixed or snap frozen samples, the level of nuclear details was not on par with a formalin-zinc fixative.⁴⁹ UMFIX, a fixative cocktail containing methanol and propylene glycol, adequately preserved tissue architecture, cellular and nuclear morphology, and tinctorial reaction compared to formalin-fixed tissue.⁶⁸ Cox et al.⁶³ did not find any significant differences in the morphology of rat liver samples preserved using various fixatives (e.g., formaldehyde, ethanol, methacarn, as well as UMFIX). However, modified methacarn fixative resulted in the best histomorphological features among the fixatives tested. A significant benefit of using zinc-based fixatives was exhibited for a variety of tissue types when histology was compared to formalin-fixation and snap freezing.^53,54 Wester and colleagues⁵⁴ also showed that exposure to zinc-based fixatives prior to snap freezing yielded crisper, more well-preserved tissue morphology. Gillespie et al.⁴⁹ also reported that ethanol fixation or formalin fixation of prostate cancer specimens yielded superior structural morphology and staining quality, though it did not affect the clinical diagnosis.

Thermal processing

When compared to FFPE tissue specimens, frozen tissue sections exhibit a variety of morphological artifacts. The most prominent artifact seen in frozen sections is due to nucleation and growth of ice crystals in the samples during freezing, subsequent storage, and thawing.^17,41,69,70 Steu et al.¹⁷ demonstrated that tumor samples frozen using carbon dioxide gas showed significant ice crystal artifacts compared to tissue frozen using isopentane or liquid nitrogen. However, the complex and irregular morphological patterns seen in carcinomas of lung, endometrium, and kidney were preserved in tissue samples frozen using an isopentane bath pre-cooled to −80°C. Falconeiri et al.⁷⁰ noted that snap-frozen specimens exhibit technical artifacts due to the crushing of tissues during processing, nuclear smudges caused by tissue tears, ridges, or folds, and ice crystal artifacts that may preclude diagnosis or make the microscopic examination difficult. They commented that the frozen section technique may be used with confidence in cases where tumor sizes are large, but it is problematic to diagnose smaller cancers or assign Gleason scores for prostatic cancers. Scott et al.³⁰ also noted prominent artifacts in frozen sections from prostatectomy biopsies. They observed that in frozen sections, the nuclear enlargement and size variations in cancerous cells appeared to be accentuated. While histologic patterns that are usually seen in FFPE samples were maintained in frozen sections, nuclear chromatin textural features were lost and heterochromatin was homogeneous. Despite these artifacts, they were able to make accurate diagnoses and the frozen tissues could be used in immunohistochemistry and gene expression studies.

Immunohistochemistry

Immunohistochemistry (IHC) is used for the detection and localization of specific biomarkers within tissue sections.⁷¹ IHC is extremely sensitive to tissue fixation and processing protocols. Conventional fixation techniques inherently alter the biomarkers through cross-linking, dehydration, precipitation, and coagulation; each of these affects IHC analysis. For example, hepatocellular carcinoma is characterized by the expression of CD34 in a unique sinusoidal pattern with collagen around tumor nests.⁷² Also, in breast cancer patients, IHC staining for ER and PR is the determinant for anti-hormonal therapy, while IHC staining for the HER2/neu receptor determines the treatment with trastuzumab.^73–75

Chemical processing

Formaldehyde fixation protocols result in masking of the epitopes due to cross-linking of the proteins and hinder detection of these antigens with specific antibodies.^43–45 This necessitates the use of antigen retrieval techniques, which reverse the cross-links and facilitate antigen detection.^{34,43,45–47} By using individual peptide spots as well as cellular and tissue controls, Sompuram et al.⁴⁶ demonstrated that IHC techniques are unable to detect antigens in samples after formalin fixation, which can be reversed by applying heat to the samples. Sample processing protocols and antibodies have been developed for formalin-fixed samples⁴⁹ and as a result IHC staining can be achieved in most tissue types for a variety of antigens.^73–77

Several alternative fixatives have been evaluated for their ability to preserve antigenicity as assessed by IHC. By analyzing the expression of epidermal growth factor receptor in various cancerous tissues, Atkins et al.⁷⁵ noted that the best IHC staining was observed in samples fixed using 4% unbuffered formalin, acetic formalin alcohol, and a proprietary fixative called Pen-Fix. In contrast, tissues fixed with 4% neutrally buffered formalin or Bouin's fixative solution displayed slightly weaker staining. Cerio et al.⁷⁸ reported that the immunoreactivity of the antigens in preserved cutaneous tissue samples decreased rapidly when fixation lasted more than 4 h in acetone, ethanol, or formalin, whereas fixation with ammonium sulfate medium for 7 days did not affect the immunoreactivity. When compared to formalin fixation, tissue samples fixed in acetone,^52,78 ethanol,^49,52,78 modified methanol-Carnoy,⁵² and methanol-based UMFIX⁶⁸ demonstrated adequate preservation of antigenicity. Furthermore, the zinc-based fixatives resulted in excellent antigen preservation and showed superior IHC staining results in absence of any antigen retrieval processes compared to the FFPE sections.^53,54

Thermal processing

Antibodies that recognize native epitopes frequently require the use of frozen tissue sections for IHC.³⁰ Shi et al.⁷³ noted that frozen sections are considered “gold standards” for the evaluation of new markers or new reagents. They compared acetone or ethanol-fixed frozen sections to formaldehyde-fixed frozen sections, and showed that the latter technique with antigen retrieval yielded the best IHC detection for many antibodies. In another study, Scott et al.³⁰ used frozen sections from prostatectomy specimens for the investigation of integrin expression. Three antibodies specific for α3, α6, and β4 integrins, each detected their antigen in the frozen tissue sections. However, the integrins were not detected in FFPE sections, even after antigen retrieval. Steu et al.,¹⁷ reported that Ki-67 was detected in various frozen tumor tissues and was not affected by the processing methods they tested.

Proteomics

Advances in techniques for protein extraction, separation (e.g., electrophoresis and chromatography), and quantitation (e.g., mass spectrometry) have led to increasing interest and application of proteomics in diagnostic pathology.⁷⁹ Traditionally, fresh or frozen tissues are used commonly for proteomic analysis due to minimal alterations in the native state of the proteins that is an inherent characteristic of the chemical fixation methods.^4,79–82 Proteomic analysis is utilized largely in the investigation of disease pathogenesis and identification of important diagnostic, predictive, and prognostic biomarkers of disease progression.⁸⁰ Detection of phosphoproteins has become essential for the development of individualized cancer therapy.¹⁴ In the clinical setting, proteomic techniques enable the differential diagnosis of tumors that appear histologically identical.⁸³

Chemical processing

One of the goals of chemical fixation is to preserve the proteome in the tissue sample. However, it has been reported that soluble proteins are lost when alcohol-based fixatives are used, while cross-linking fixatives like paraformaldehyde preserve the localization of these soluble proteins.⁸¹ Soluble proteins move with the water and are consequently lost to the aqueous fixative solutions, as well as to the dehydrating agents such as ethanol during tissue processing, even in presence of aldehyde-based fixatives.⁸⁴ Although stabilization of tissue specimens with formalin fixation achieves superior structural preservation, the inter-molecular and intra-molecular cross-links that are formed as a result of the fixation process actually hinder the effective extraction of the proteins of interest and subsequent analysis.^80–82,85 O'Leary et al.⁴⁷ showed that fixation with formalin led to the aggregation of proteins and the formation of heterogeneous protein-formaldehyde adducts. By SDS-PAGE, they observed spreading of the characteristic protein spots and the presence of multiple uncharacteristic bands. The antigen retrieval process successfully reversed the covalent methylene bridge cross-links and restored immunoreactivity. However, they also showed that formalin-fixation followed by ethanol dehydration resulted in the loss of protein tertiary structure in cross-linked proteins, which essentially protected the cross-links from reversing during heat-induced antigen retrieval. Recently, it has been demonstrated that using a buffer solution containing SDS improved the efficacy of the heat-induced antigen retrieval techniques, allowing the extraction of full length, reactive proteins from FFPE tissue sections.⁸⁰ FFPE tissue blocks can be used for proteomic analysis even after prolonged storage.⁷⁹ Coagulative fixatives such as ethanol^49,82 and methanol-based UMFIX⁶⁸ are compatible with proteomic analysis, resulting in a well-preserved protein profile that is superior to the formalin-fixed samples in the absence of any antigen retrieval.

Thermal processing

Fresh frozen tissues are preferred for proteomic analysis since their proteome is typically well preserved. The proteomic profile of frozen tissue sections has been analyzed by a variety of assays and demonstrates only minimal alteration.^{4,17,49,68,82,86} Steu et al.¹⁷ showed that protocols involving isopentane freezing and OCT embedding did not negatively influence the preservation of key cancer protein biomarkers such as Ki-67 when compared to snap-frozen specimens. Also, when compared to ethanol-fixed specimens, 25%–50% more proteins were extracted from frozen sections; the proteins from frozen sections appeared as sharp bands by 2D SDS-PAGE as opposed to the smeared appearance of protein bands extracted from ethanol-fixed samples.^49,82

One of the advantages of using frozen sections for proteomic analysis is that phosphoproteins and proteins with other post-translational modifications (PTMs), which are otherwise difficult to preserve using standard fixation techniques, are found in higher abundance in frozen samples. PTMs are critical components of signaling pathways and influence biochemical interactions and cellular responses to various conditions. Many of the PTMs are dynamic and they are sensitive to tissue processing techniques. Hence, the ability to capture and measure the PTMs provides an important tool for investigating cellular regulatory mechanisms, as well as irregularities and abnormalities that are characteristic of a disease and have the potential to assist in personalized molecular therapies.^14,87 Recent reports indicate that snap-freezing specimens soon after resection can capture the PTM states of several biomarkers which would otherwise be lost or further modified.^13,14,87 Furthermore, Ahmed et al.⁸⁷ showed that heat stabilization of resected brain tissue to 95°C and the presence of a stabilizer buffer immediately after resection improved the preservation of specific phosphoproteins.

Nucleic acids and genomics

Advancement in the field of genomic sequencing and transcriptional profiling stimulated the use of nucleic acid based assays for molecular profiling of diseases in clinical practice. Microarray technology facilitates analysis of the differential expression of thousands of genes simultaneously while real-time polymerase chain reaction (RT-PCR) allows rapid analysis of the transcriptional factors in the samples. These techniques allow investigators to analyze biochemical and regulatory pathways associated with disease states.^1,5,7,8,49 Furthermore, genome-wide expression analysis has paved the way to identify subsets of several diseases clinically, elucidate distinctions among highly related tumor types, and stratify patients with differential responses to therapies.⁷ The widespread use of genomic profiling depends upon the successful isolation and recovery of nucleic acids from preserved tissues; conventionally frozen tissues have been the most reliable source of nucleic acids.

Chemical processing

FFPE has been the preferred method of tissue processing for clinical applications. Therefore, it is important to understand the effects of formalin-based tissue fixation and processing on the nucleic acids within tissues. The cross-linking process that is characteristic of formaldehyde fixation is most detrimental to the nucleic acids, making it difficult to extract and isolate them. Although enzymatic digestion or heat-induced antigen retrieval helps in recovering the antigenicity of proteins, these procedures are ineffective or may cause disruptions in some noncovalent interactions in the RNA, which affects its integrity for subsequent downstream analysis.^7,88,89 Masuda et al.⁸⁸ showed that formalin fixation for 16 h at 4°C caused the addition of mono-methylol groups to almost 40% of the adenine bases from extracted RNA; this increased to 62% when samples were fixed for 7 days. However, heat treatment in Tris-EDTA buffer reversed most of the methylol additions from the oligo-RNA. RNA isolated from FFPE samples is mostly fragmented and does not extend beyond 200 to 300 bases by RT-PCR.^7,68,89 Furthermore, formalin-fixation results in artificial mutations or sequence alterations in the DNA due to cross-links as well as promotion of jumping the templates during PCR amplification.^89,90

Most of the alternative fixatives preserve the nucleic acids better than FFPE. Due to the absence of cross-linking, ethanol and methanol preserve nucleic acids quite well. Most of the conformational changes brought about by these alcohol-based fixatives can be reversed by rehydration.⁸⁹ Though the quality of the RNA extracted from ethanol-fixed paraffin-embedded tissues was reduced, as indicated by the 18S and 28S bands, the RNA was sufficient for techniques like RT-PCR and cDNA microarrays. Also, the quality of the DNA isolated from these samples was superior to formalin-fixed samples; resulting in longer fragments that could be successfully amplified using PCR.⁴⁹ However, several limitations were reported for the ethanol-fixed samples used in mRNA analysis due to increased laser microdissection time and loss of measureable transcripts.⁴⁸ Kim et al.⁹¹ reported that methacarn-fixed paraffin-embedded tissues yielded superior quality RNA with intact 18S and 28S bands; the extracted RNA could be successfully used for subsequent RT-PCR based assays. UMFIX-based,^50,68 as well as zinc-based fixation protocols,⁵⁴ have also demonstrated excellent nucleic acid recovery that is comparable to frozen specimens.

Thermal processing

Molecular profiling of clinical samples has relied on freezing as the best method to preserve proteins and nucleic acids in a way that is free from any fixation-related artifacts. Hence, frozen tissues are most commonly used for genomic analysis. Despite the issues of a lack of morphological preservation during freezing, frozen samples have been successfully used for laser capture microdissection, a technique that facilitates gene expression analysis by isolating specific regions or cells from tissues.^48,91–93 The quality of the RNA isolated from frozen specimens and the quality and yield of DNA is superior, compared to most chemically fixed samples.^{17,48–50,68,93}

Summary

Preservation of fresh tissue samples resected from the human body for diagnostic pathology is extremely important and is sensitive to processing methods. Tissues experience ischemia and intrinsic enzymatic degradation, which alter the state of the biomarkers. Quick processing of the tissue, using either chemical fixation or freezing, arrests the degradation and facilitates the use of tissues for subsequent analysis and long-term storage. Thermal processing is the favored procedure for many applications including intraoperative diagnostics, proteomics, and genomics. In contrast, chemical fixation, using cross-linking fixatives such as formalin or coagulative fixatives such as alcohols, is the preferred procedure for archiving, histology, and immunohistochemical applications. No one processing technique is perfect for all applications; rather each technique has advantages and shortcomings. Therefore, procedures should be selected to optimize the specific downstream application of the tissues.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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