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Different tissue fixatives have various effects on tissue histology and on the molecular integrity. This report will explore the various fixatives used in diagnostic and research laboratories today and highlight which are best suited for different purposes and the compromises which must be made. Molecular studies involves analysing tissues at the molecular level, the goal is to decipher the building blocks of normal and diseased structures. Every human disease has some basis in genetic makeup, medical genetics is no longer constrained to the study of birth defects and inherited disorders. It is already being incorporated into the diagnostic laboratory and defective or mutated genes are frequently being identified in aetiological studies in all branches of medicine. It is the future goal of medical researchers to understand the genetics behind all diseases and thus design specific new treatments. Pharmacogenomics, is an emerging field involving the development of tailored drug therapies based on the individuals genetic code.
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It is possible to direct therapeutic agents to specific products expressed by diseased cells without affecting normal tissues.
There are many applications for molecular studies in research.
The human genome project (ref) http://www.genome.gov/10001772 and the HapMap Project http://genome.gov/10005336 http://hapmap.ncbi.nlm.nih.gov/abouthapmap.html (a comprehensive map of human genetic variation) are large scale studies with worldwide collaboration which primarily utilised molecular studies. These studies generated vast amounts of data about human DNA which has provided scientists and clinicians with powerful tools to study the genetic causes in complex disorders such as cancer, diabetes and cardiovascular disease. The Cancer Genome Atlas is a current endeavour which aims to develop and test strategies for a comprehensive exploration of the universe of genetic factors involved in cancer using genome analysis technology, such as large-scale genome sequencing , identifying DNA mutations, copy number variation and methylation status alterations. http://cancergenome.nih.gov/publications/edu_materials/genomics.asp The cancer genome anatomy project sought to determine the gene expression profiles of normal, precancerous and cancer cells, leading eventually to improved detection, diagnosis and treatment. Strausberg
The cancer genome characterization initiative http://cgap.nci.nih.gov/ incorporates multiple genomic characterization methods including exome and transcriptome analysis using second generation sequencing. The entire cancer research community is engaged in generating, characterizing and analysing the data. This integrated approach to exploring the cancer genome is providing valuable information that can be used to develop diagnostic and prognostic biomarkers and targeted therapies. Ultimately the aim is to provide the foundation information needed to develop a personalised approach to the prevention, diagnosis and treatment of cancers. http://cancergenome.nih.gov/objects/pdfs/TCGA_Portal_factsheet_FINAL_040909-508.pdf
Cancer is ultimately a genetic disease and therefore the majority of current cancer research strives to understand the role of oncogenes and tumour suppressor genes in carcinogenesis. The study of these abnormalities has been boosted by molecular techniques permitting molecular analysis such as the ability to detect mutations using the polymerase chain reaction and DNA sequencing, and comparative genomic hybridisation on genomic microarrays to detect gene amplifications and deletions on a genome wide basis. These techniques are expensive and produce poor results in formalin fixed paraffin wax embedded (FFPE) specimens, and thus require alternative fixation methods. Therefore they have not been widely employed in the routine diagnostic histopathology laboratory to date. Molecular histology in the study of solid tumours'R J Campbell, M Pignatelli J Clin Pathol: Mol Pathol 2002;55:80-82
Molecular pathology, the study of diseases at a molecular level has become increasingly important and prevalent since the creation of the human genome project and the development of technologies that enable the collection of gene and protein expression profiles. the evaluation of the molecular processes in disease demands that fixatives permit the recovery of macromolecules such as DNA, mRNA and proteins without significant biochemical modifications from fixed and embedded tissues. Molecular profiles derived from tissue specimens can give information about the disease process itself such as pathogenesis and in locating diagnostic, prognostic and therapeutic molecular targets for clinical use. Therefore tissues collected for diagnosis reasons in clinical practise are extremely important for medical research, especially molecular studies which is why maintaining the integrity of the tissues in its native state is of paramount importance. Laboratory diagnosis of disease is also progressing and advancing beyond histological analysis to the molecular level, and therefore improved tissue preservation is essential.
In diagnostic histopathology laboratories the goal in tissue fixation and processing is to maintain clear and consistent morphological features to enable accurate diagnosis. To accurately assess the microanatomy the molecular relationships amongst cells, cellular and extracellular components and local tissue chemical environment must be maintained. Any artefacts introduced must be consistent. Stained tissue sections are a compromise on the original picture of one or more features representing the living tissue. An ideal fixative has not been developed, each are selected on their ability to produce a final product, required to demonstrate a particular feature of a specific tissue (Grizzle 2001 - text book)
To enable accurate and reproducible analysis molecular techniques require ex vivo tissue samples in a condition as similar to in vivo as possible. The nature of specimen handling has a significant effect on how useful the tissue will be, to minimise degradation of biomolecules there are three major factors to be monitored: temperature, time of handling and specimen size. (Leiva) The choice of fixative also has a severe impact. Fixation stabilizes the proteins throughout the specimen and prevents putrefaction and autolysis. The goal is to harden and preserve the tissues at a specific moment in time and to prevent the loss of specific molecules. Once removed from the body tissues degrade by autolysis and microbial degradation. All fixatives are selected by compromise, the advantageous effects balanced against the less desirable effects with regards to the intended analysis of the fixed tissue. Many factors must be considered, possible molecular loss, swelling, shrinkage, variations in the quality of histochemical and immunohistochemical staining, alterations to organelle structure and finally the ability to analyse the biochemistry of the tissue accurately after fixation. (Insert ref carson 1990 or somesuch see text book)
Snap-freezing of tissue is used traditionally for molecular analysis. (leiva)It produces poor quality morphology compared to FFPE slides. It is preferred over paraffin-embedded tissues because it yields higher quality RNA and more RT-PCR product than paraffin-embedded tissues [Goldsworthy et al., 1999].
Most commonly tissues are fixed by chemical means. There are two main categories: cross-linking and coagulant fixatives. compound fixatives are a mixture of reagents e.g alcoholic formalin, which fix by addition of covalent hydroxymethyl groups and cross-links in addition to coagulation and dehydration. There are many advantages and disadvantages to be considered with each type. Therefore the fixative selected must be based upon the intended use of the tissue and molecular integrity or histology must be prioritised. Chemical fixatives have the ability to prevent destruction of the micro-architecture of the tissue by suspending metabolic activity of catabolic enzymes, causing destruction of infectious agents and hence maintaining the structural integrity. The diffusion of soluble molecules from original source is also minimised.
Cross-linking fixatives are aldehyde based such as formalin (formaldehyde), glutaraledhyde and other aldehydes. They cause extensive protein crosslinking and make the recovery of biomolecules difficult; such biomolecules are unsuitable for high throughput expression methodologies such as cDNA microarrays, SAGE and 2D-PAGE (Gillespie 2002). The crosslinks are formed within and between proteins and nucleic acids as well as between nucleic acids and proteins.
10% neutral buffered formalin (NBF) is the most common fixative used in diagnostic pathology. The reactions with macromolecules are complex and numerous. Formalin fixation and routine tissue processing methods are of limited, if any, value in preserving macromolecules (Sambrook et al, 2001). The fixative penetrates the nucleic acids-protein shell and stabilizes the structure. There are also reactions with the free amino groups of nucleotides and proteins causing modifications. Reactive groups can combine with hydrogen groups or with each other and form methylene bridges. Biomolecules recovered from FFPE tissues are of very poor quality because of formalin cross-links. FFPE is currently the standard in diagnostic pathology due to the clear morphology produced. Tissues are fixed in 10% NBF and embedded in paraffin and stained with haematoxylin and eosin. FFPE tissues are suitable for DNA analysis such as PCR, mutation analysis. However samples recovered from FFPE tissues are of lower quality and can yield less reliable results in PCR analysis, and the results may not precisely reflect the true state of RNA/DNA in vivo.
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Acid formalin possibly reacts slower with proteins than NBF because the amine groups become charged. Acid formalin maintains the proteins responsible for immunorecognition to a superior degree than NBF. (Arnold 1999 see text) The main drawback with this fixative is the formation of a brown-black pigment due to degraded haemoglobin, this is a significant problem is the patient has a blood abnormality such as malaria or sick cell anaemia. Text
Glutaraldehyde is a bifunctional aldehyde which most likely acts in the same manner as formaldehyde. For each group that binds to the protein, there is another free aldehyde group ready to interact and can lead to further crosslinking with proteins or with histochemical reagents such as antibodies and enzymes. This fixative leads to more crosslinking than formaldehyde which is irreversible. The extensive crosslinking results in superior preservation of ultrastructure, but there is a increased detrimental effect on the success of histochemical methods. If the unreacted aldehyde groups are not blocked, they can lead to increased background staining. text
Osmium tetroxide is a toxic solid which is soluble in water and non-polar solvents. Therefore is can react with hydrophobic and hydrophilic sites on proteins potentially leading to crosslinking. This fixative reacts with nucleic acids, prominent clumping of DNA can be seen when fixed and dehydrated, and this is a serious artefact which alters native morphology. Large proportions of proteins and carbohydrates are lost from tissues perhaps due to poor fixative penetration. This fixative reacts with unsaturated bonds within lipids and phospholipids, and the membranes typically stain black. (text)
Acetone and alcohols such as ethanol and methanol are dehydrant coagulant fixatives, they precipitate without chemical additions. These reagents induce the removal and replacement of water molecules from the tissues. This has many effects on the proteins: "removal of water surrounding hydrophobic proteins weakens the bonds, as repulsive forces hold the structure in its native conformation. Also removal of hydrogen bonding in hydrophilic areas of tissues causes instability." (text) Therefore dehydration disrupts the tertiary structure of the structural proteins - causes denaturation and potentially loss of function and insolubility. They generally do not affect nucleic acids, however they extract lipids leading to destruction of ultrastructure due to excessive shrinking. The histology of the tissues is however almost as good as NBF.
Acidic coagulant fixatives such as picric and trichloroacetic acids change the charge on the side chains, causing ionization. This disturbs the electrostatic and hydrogen bonding and can implement a lipophilic anion in a hydrophilic region causing denaturation. Acetic acid coagulates nucleic acids but does not fix or precipitate proteins. Therefore it can be used in addition to other fixatives to preserve nucleic acids.
A fixing system has been specifically developed to preserve the molecular integrity of tissues. HOPE, (Hepes-glutamic acid buffer-mediated Organic solvent Protection Effect) fixative includes a one step dehydration with acetone before embedding in pure paraffin and its results give formalin-like morphology, excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry, good RNA and DNA yields without crosslinking proteins.
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RNAlater® is a RNA Stabilization Solution that does not disrupt the structure of tissues. It acts as an aqueous tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens. It eliminates the need to immediately process or freeze samples; the specimen can simply be submerged in RNAlater Solution and stored for analysis at a later date. Tissues preserved in RNAlater® can be stored indefinately kept at -80°C. http://www.ambion.com/techlib/prot/bp_7020.pdf
The effect of sample type, temperature and RNAlaterâ„¢ on the stability of avian influenza virus RNA Forster,Julie L.; Harkin,Valerie B.; Graham,David A.; McCullough,Samuel J. J.Virol.Methods, 2008, 149, 1, 190-194.
There are numerous studies which have evaluated the effectiveness of chemical fixatives and outlined the pros and cons associated.
Aldehyde fixatives have been found to allow collection of smaller yields of fragmented RNA. (Proof - Goldworthy 1999, masuda 1999, srinivasan 2002, Abrahamsen 2003, Cronin 2004). This RNA can still be used for gene expression studies, however it is of limited use in other technologies which require intact RNA such as cDNA libraries and northern analysis. (cox)
Non-aldehyde based fixatives with best morphology are alcohol based. Ethanol and methanol-based solutions including Carnoy's fixative and methacarn are commonly used during fixation of nucleic acids. These precipitating fixatives have the advantage of minimizing chemical modifications. Gillespie et al. concluded that 70% ethanol and paraffin embedding is a useful method for molecular profile studies, as it produces results similar to that obtained from snap frozen specimens, though the protein quantity was somewhat decreased. Recovery of DNA and mRNA was superior to formalin-fixed samples. The histology is also excellent and the fixatives are non-toxic and relatively inexpensive. (Gillespie paper 2002)
Table 1 showing fixatives and overall rank (Gillespie et al 2002)
70% ethanol:100% methanol (3:1)
95% ethanol:100% methanol (3:1)
Streck molecular biology fixative
10% neutral buffered formalin
(Ranking was based on the evaluation of nuclear morphology, cellular morphology, tissue architecture and staining characteristics. 1=best)
A study by Leiva et al. concluded that ethanol fixation and paraffin embedding allows for histology similar to formalin fixation with much improved preservation of biomolecules. (Leiva)
Cox and colleagues published an assessment of fixatives effect on morphology and RNA integrity (quantity and quality) in rat liver. They concluded that modified methacarn, 70% ethanol and modified Carnoy's solution preserved morphology and RNA quality. Modified methacarn was found to give the most superior outcome when both RNA integrity and morphology need to be assessed in one tissue sample. RNA quality and quantity recoverable do not share a linear relationship. In occasions when high quantities of RNA were obtained, it was usually in smaller fragments. Whereas RNA collected from frozen tissue provided a smaller quantity of RNA but in desirable large fragments. The best RNA quality was obtained using Umfix and methacarn, which provided a high yield but with slightly poorer quality (less intact) than with snap freezing. (COX)
Goldworthy et al. determined the optimal fixative for RNA analysis using LCM, which maintained morphology and mRNA integrity. The fixative they found to be optimal in frozen tissue was 70% ethanol and PLP (Paraformaldehyde/Lysine/Periodate)was optimal with paraffin embedded tissues although inferior to the former.
Effects of Fixation on RNA Extraction and Amplification from Laser Capture Microdissected Tissue
Susan M. Goldsworthy,1,2* Pat S. Stockton,2 Carol S. Trempus,3 Julie F. Foley,2 and Robert R. Maronpot
MOLECULAR CARCINOGENESIS 25:86-91 (1999)
Many other studies also confirm that alcohol based (non-crosslinking) fixatives permit the collection of high quality RNA, superior to formalin based fixatives.
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Maronpot RR (1999). Effects of fixation on RNA extraction
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Shibutani et al., 2000; Methacarn Fixation: A Novel Tool for Analysis of Gene Expressions in Paraffin-Embedded Tissue Specimens LABORATORY INVESTIGATION Vol. 80, No. 2, p. 199, 2000
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UMfix (universal molecular fixative) is a mixture of methanol and polyethylene glycol, that has improved nucleic acid preserving properties and safety aspects when compared with formalin based fixatives
Antimicrobial activity of UMFix tissue fixativeAuthors:Cleary,T.J.; Morales,A.R.; Nadji,M.; Nassiri,M.; Vincek,V.Source:J.Clin.Pathol., 2005, 58, 1, 22-25, England
Vincek et al. assessed common fixatives and the potential value of UMfix, with the aim of deciphering which maintained histomorphology and protected macromolecules. UMfix-exposed tissue gives comparable morphology to FFPE tissues. RNA was preserved with a high molecular weight. There was no significant different between UMfix exposed and frozen tissues when examined in PCR, PT-PCR and expression microarrays. Proteins are similar to the fresh state, whereas in formalin fixation there is considerable degradation. The study concluded it is possible to preserve tissue morphology and macromolecule integrity in the same archival paraffin embedded tissue through the use of novel fixative (e.g UMfix) and a rapid processing system. Lab Invest 2003, 83:1427-1435A Tissue Fixative that Protects Macromolecules (DNA, RNA, and Protein) and Histomorphology in Clinical Samples Vladimir Vincek1, Mehdi Nassiri1, Mehrdad Nadji1 and Azorides R Morales1
One problem with Umfix is that it has inferior microbicidal activity when compared to 10% NBF, it is not effective in killing bacteria which form spores. Therefore bacterial degradation may occur in tissues fixed with Umfix (J Clin Pathol 2005;58:22-25. Antimicrobial activity of UMFix tissue fixative
T J Cleary, A R Morales, M Nadji, M Nassiri, V Vincek)
K. H. Wiedorn et al. investigated the use of HOPE, a protection solution as a potential alternative to conventional fixing techniques which preserves and allows extraction of high molecular weight nucleic acids. "It can be used as an alternative to RNAlater, because it does no exhihibt significant differencies concerning RNA yield and quality" Additionally HOPE permits paraffin-embedding to enable prolonged storage in HOPE solution which is not possible in tissues which have been stored in RNAlater as it must be frozen which is an expensive storage method.
Wiedorn KH, Olert J, Stacy RA et al. HOPE - a new fixing technique enables preservation and extraction of high molecular weight DNA and RNA of â‰¥ 20 kb from paraffin-embedded tissues. Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect. Pathol Res Pract 2002; 198: 735-40
Goldmann T, Flohr AM, Murua Escobar H et al. The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues. Pathol Res Pract 2004; 200: 511-5
RNAlater was evaluated in a study by Hoffman et al. in comparison to formalin and Boonfix it was found that the best RNA quality was obtained from RNAlater preservation followed by RNAeasy mini kit extraction.
( Comparison of different methods to obtain and store liver biopsies for molecular and histological research. Gaby Hoffmann1, Jooske Ijzer1,2, Bas Brinkhof1, Baukje A Schotanus1, Ted SGAM van den Ingh3, Louis C Penning*1 and Jan Rothuizen1 Comparative Hepatology 2009, 8:3)
The fixatives used in the diagnostic laboratories will be changed to allow improved molecular preservation. However the vast archive of FFPE tissues from clinical samples is an extremely valuable resource for testing and discovering biomarkers and scientists are striving to find a way to improve the quality of the macromolecules that are retrievable. Although the DNA, RNA and protein retrieved from FFPE tissues is fragmented it has been found to give remarkably similar results to fresh tissue in biomarker research.
(Am J Pathol 2001, 158:419-429) Quantitative Gene Expression Analysis in Microdissected Archival Formalin-Fixed and Paraffin- Embedded Tumor Tissue Katja Specht,* Thomas Richter,â€ Ulrike Mu¨ ller,*
Axel Walch,â€ Martin Werner,â€ and Heinz Ho¨ fler*â€
Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples Source: The journal of histochemistry and cytochemistry [0022-1554] Scicchitano yr:2006 vol:54 iss:11 pg:1229 -1237
Title: Unlocking the molecular archive: the emerging use of formalin-fixed paraffin-embedded tissue for biomarker research in urological cancer Source: BJU international [1464-4096] Gnanapragasam yr:2010 vol:105 iss:2 pg:274 -278
As new technologies are being developed to enable molecular analysis, molecular studies are becoming increasingly important. They provide the means to discover the molecular basis for many diseases. Medical scientists are searching for molecular markers to diagnose and monitor medical conditions and to develop targeted treatments. Therefore the molecular integrity of tissue samples is of critical importance and therefore a reassessment of standard tissue fixation techniques has been underway for many years now.
Formalin fixation seriously affects the molecular structure of tissues and although RNA isolation is possible it is significantly degraded, and there is structural alterations of templates that makes amplification methods unreliable and hard to reproduce.
The ideal fixative would interact minimally with the tissue biochemically, preserving the biomolecules in their native in vivo state and maintain excellent tissue histomorphology. The same tissue biopsy or sample would preferably be used for histology and molecular studies which could then be handled and stored easily, economically and safely. Unfortunately to date, the selection of a fixative is still a compromise and they must be chosen on the basis of what the intended analysis on the tissue will be. One important aspect must be sacrificed to enable optimal study of the other. There are many novel fixatives which have the potential to be used in the diagnostic laboratory but further research on a larger scale is required.
Improved results have been observed with alcohol based fixatives, and this is a potential replacement to the gold standard formalin fixation. The main concern is the altered histology when compared to cross-linking formalin and therefore pathologists will need to adjust their visual analysis accordingly.
Molecular studies of the genome hold the key to innumerable future biomedical discoveries and this discipline will be the basis of future diagnostics and treatment for all human disease.