A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules. For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules. Significant advances in genomic and proteomic technologies have provided detailed insight into the diversity of human diseases. Consequently, diagnosis of diseases relies more and more on the evaluation of a diversity of parameters both by means of classical diagnostic techniques such as histopathology and by analysis of a broad spectrum of biomolecules for genomic alterations, deregulated gene expression, or protein modifications.1Sawyers C.L. The cancer biomarker problem.Nature. 2008; 452: 548-552Crossref PubMed Scopus (720) Google Scholar To exploit the full capacities of the latest analytical technologies, excellent preservation of biomolecules in biological samples is required. The quality of biological samples analyzed ultimately determines the quality of any analysis performed with these samples.2Naylor S. Overview of biomarkers in disease, drug discovery and development.Drug Discovery World [Internet]. 2005; (Spring)http://www.ddw-online.com/s/2005/p142677/spring-2005-edition.htmlGoogle Scholar However, the availability of fresh-frozen tissue, which represents the best material for molecular analyses, is very limited. Furthermore, the additional collection of fresh-frozen tissue samples is not feasible in several disease conditions, such as small tumors that cannot be divided into several aliquots for research and routine applications or for different preanalytical procedures. Fixation of tissues in buffered formaldehyde solution (formalin) and embedding in paraffin is the gold standard for processing tissues for histopathological diagnosis.3Fox C.H. Johnson F.B. Whiting J. Roller P.P. 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HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues.Pathol Res Pract. 2001; 197: 823-826Crossref PubMed Scopus (117) Google Scholar, 12Stanta G. Mucelli S.P. Petrera F. Bonin S. Bussolati G. A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.Diagn Mol Pathol. 2006; 15: 115-123Crossref PubMed Scopus (85) Google Scholar, 13Buesa R.J. Histology without formalin.Ann Diagn Pathol. 2008; 12: 387-396Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14National Toxicology Program: Final Report on Carcinogens Background Document for Formaldehyde.Rep Carcinog Backgr. 2010; (i–512)Google Scholar, 15Delfour C. Roger P. Bret C. Berthe M.L. Rochaix P. Kalfa N. Raynaud P. Bibeau F. Maudelonde T. Boulle N. RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin-embedded breast carcinoma and microdissected breast tumor cells.J Mol Diagn. 2006; 8: 157-169Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Despite certain advantages, however, the utility of available tissue preservation techniques for achieving high-quality results in classical histopathological evaluation, immunohistochemistry, and molecular analyses from a single paraffin-embedded sample has been limited. We have therefore developed a new tissue stabilization technology (PAXgene Tissue System), for simultaneous preservation of nucleic acids, proteins, and morphological features, and have tested it in comparison with current state-of-the-art techniques: FFPE for routine morphological diagnostics and snap-freezing for molecular analyses. Kap et al16Kap M. Smedts F. Oosterhuis W. Winther R. Christensen N. Reischauer B. Viertler C. Groelz D. Becker K.F. Zatloukal K. Langer R. Slotta-Huspenina J. Bodo K. de Jong B. Oelmuller U. Riegman P. Histological assessment of PAXgene tissue fixation and stabilization reagents.PLoS One. 2011; 6: e27704Crossref PubMed Scopus (66) Google Scholar recently demonstrated that morphology and antigenicity are well preserved in tissue samples treated with PAXgene fixative and then paraffin-embedded (PFPE samples). Here, we provide a detailed description of the development of this new stabilization technology and its ability to preserve biomolecules (and nucleic acids in particular). First, commercially available fixatives described as formalin replacements for preservation of morphology, nucleic acids, or both were tested for their ability to preserve morphology and biomolecules. Additional fixatives were selected based on reports indicating their use for special pathology staining applications or for nucleic acid preservation (Table 1). In addition, 1150 substances and combinations of substances (including mono-, bi-, tri-, and polyvalent alcohols, cross-linkers, soluble salts, and buffers) were screened and classified for their properties to stabilize biomolecules in tissue samples. Finally, a multitude of different alcohols and acids in a great variety of compositions, combined with substances identified in the large screening program as biomolecule preservatives, were tested for biomolecule and morphology preservation. In all, more than 1500 single compounds and combinations were tested.Table 1Examples of Commercially Available or Published Reagents Tested in the Screening ProgramReagentVendor or compositionRNAlaterLife Technologies, Carlsbad, CAMichel'sZeus Scientific, Raritan, NJHistoChoiceAmresco Inc., Solon, OHPreferAnatech Ltd, Battle Creek, MIZ5Anatech Ltd, Battle Creek, MIFineFIXMilestone, Bergamo, ItalyHopeDCS, Hamburg, GermanyRCL2Alphelys, Plaisir, FranceStreck Tissue FixativeStreck, Omaha, NEPreservCytHologic, Bedford, MAAquaPreserveMultiTarget Pharmaceuticals, Salt Lake City, UTRNAsecureLife Technologies, Carlsbad, CAS.T.A.R. bufferRoche Molecular Biochemicals, Penzberg, GermanyUMFixSakura Finetek, Torrance, CAEthanol70% ethanol p.a. (pro analysis)MethacarnMethanol, chloroform, acetic acidCarnoy's fixativeEthanol, chloroform, acetic acidFarmer'sEthanol, acetic acidWolman's solutionEthanol, acetic acidDelaunay'sAcetone, ethanol, trichloroacetic acidFAAFormalin, acetic acid, ethanolFPAFormalin, acetic acid, propionic acidDavidson's fixativeFormalin, ethanol, acetic acid, water Open table in a new tab Rats (rattus norvegicus) were bred, maintained, and sacrificed at the Animal Research Facility of Dusseldorf University (Universitätsklinikum Düsseldorf Tierversuchsanlage) in accordance with the German Protection of Animals Act. Rats were raised until a weight of 500 g. Equal parts of rat liver and kidney were dissected within 5 minutes post mortem, submerged into one of the preservation solutions from the screening program, and stored at room temperature. After fixation and storage, rat liver samples of 10 to 15 mg each were used for RNA extraction using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and were eluted in 80 μL of RNase-free water. Gel electrophoresis was performed with 15 μL of eluate on 1% agarose gels under denaturing conditions, and RNA was by For evaluation of morphology rat kidney samples with a of were submerged in a preservation solution for and then were or on a tissue was performed with and ethanol, by with and embedding in were for minutes with and for with and then were with Germany) for tissue (eg, and tissue (eg, were divided into aliquots and snap-frozen in by in buffered formaldehyde and to or and to in PAXgene Tissue and then into PAXgene Tissue and to at room temperature. After in and ethanol, by and the PAXgene and samples were in paraffin and stored in the at for also at room temperature. samples were using were for minutes with and for with and then were with for sample provided and the was by the of the University of For and analyses, human cancer tissue samples were obtained from analyses were performed for research For assessment of were and treated according to used for diagnosis of FFPE samples. For were for minutes in a at with was performed on a system with minutes of the of snap-frozen samples were with buffered formaldehyde at room by and for 5 minutes at was in a for 10 minutes at using was for minutes at room and was performed with a System were with For RNA 10 to from or FFPE human tissue samples were on a or a The paraffin-embedded samples were by and ethanol RNA was extracted according to the manufacturer's instructions using a PAXgene Tissue RNA Kit for an RNeasy FFPE Kit for FFPE or the Technologies, Germany) for snap-frozen samples. RNA was eluted in a of μL of or RNase-free water and stored at RNA and were using a Scientific, were obtained using an Technologies, with an RNA was used to the RNA integrity each sample, of RNA was into using Kit City, according to the manufacturer's After was stored at of as for using For the of of human the were and samples and were analyzed in for of using a on an System were analyzed using were by and were used for were on a agarose and by For analysis of cancer gene of RNA from and snap-frozen human liver samples was into using the Kit according to the manufacturer's 10 μL of gene was to 10 μL of to a of were analyzed on an System using the Molecular of and from were analyzed using The of was as the of correlation from aliquots of cancer were RNA was in each from using a PAXgene Tissue Kit for tissue and a Kit for FFPE for FFPE tissue, according to the manufacturer's RNA was eluted with μL (PFPE or μL of the RNA from the snap-frozen sample was from 10 mg of tissue with a Mini Kit using the system for tissue and then was eluted in 80 μL of RNA was with a RNA integrity was with an RNA Kit on an RNA were with on an and and were using the RNA Kit and Carlsbad, with and was performed with 5 RNA in a μL from the was used for In addition, of and and analyses of small and and were performed with a in with and a was performed with RNA in μL from the was used for were obtained from RNA sample and were analyzed in For each miRNA, the of FFPE or and the snap-frozen sample was from paraffin-embedded tissue was in each from using a PAXgene Tissue Kit for tissue and a FFPE Tissue Kit for FFPE was eluted in μL of the was from 10 mg of snap-frozen tissue with a Mini Kit using a system for tissue and then was eluted in μL of was performed with a of each and the Germany) were on 1% buffered agarose gels and with was performed with and using a and were used for of a of human D. S. A. D.J. of large in identified using PubMed Google Scholar were on 1% buffered agarose gels with a For 10 was in with a Kit of human were of protein of protein of of of of of and of gene were on buffered agarose gels with a For to of from and FFPE samples from human cancer was on an with of the human gene or of the human were on and on a using and the provided with the and from according to the manufacturer's human liver tissues were divided into aliquots and with PAXgene fixative or fixation each by of and or formalin-fixed and from and snap-frozen tissues were extracted according to the FFPE Tissue Kit with of tissue in and in the tissues were from using a into extraction and for 15 minutes on by at for with from FFPE tissues were extracted according to the FFPE Tissue Kit Equal of protein were on gels and was performed using from and The proteins were using In more than 1500 single compounds and combinations were tested for simultaneous preservation of morphology and RNA in tissues by using a high-throughput screening First, we the ability of commercially available fixatives and fixatives described in as formalin replacements (Table to preserve biomolecules, morphology, or both A and The results obtained as for screening and of 1150 single substances and combinations (including mono-, bi-, tri-, and polyvalent alcohols, cross-linkers, soluble salts, and buffers) for their ability to stabilize RNA in tissue samples and alcohols and acids in a great variety of compositions, combined with substances identified in the large screening program as biomolecule were tested for their ability to preserve Although to preserve morphology we that affected the integrity and of nucleic acids in applications such as even or were the other alcohols or in and at performed well for biomolecules, but such as ethanol, preserved morphology in tissue (eg, rat but in (eg, rat kidney was to be to RNA from rat liver was to RNA by inferior these were therefore as the tissues for of morphological such as and biomolecule be only with a of alcohols at and the addition of acids in with a soluble identified in the screening The of alcohols, an acid, and a soluble that best the screening was by the best for each The new tissue stabilization the PAXgene Tissue a fixation that tissues and a stabilization of a of different alcohols that the fixation and the sample for or until processing and paraffin embedding and For used for processing and paraffin embedding have to be of such as novel tissue stabilization technology was then and with current state-of-the-art techniques for routine morphological diagnostics and molecular analyses, using of and tissue samples. tissues from different revealed very preservation of morphology and antigenicity, similar to that of the FFPE tissues also at which is in with results recently by Kap et M. Smedts F. Oosterhuis W. Winther R. Christensen N. Reischauer B. Viertler C. Groelz D. Becker K.F. Zatloukal K. Langer R. Slotta-Huspenina J. Bodo K. de Jong B. Oelmuller U. Riegman P. Histological assessment of PAXgene tissue fixation and stabilization reagents.PLoS One. 2011; 6: e27704Crossref PubMed Scopus (66) Google Scholar There was a for to for immunohistochemistry, with FFPE tissues, which is with the of in the PAXgene tissue stabilization system not Importantly, the preservation of in tissues was similar to that in snap-frozen tissues, and the quality from FFPE samples of snap-frozen with tissues similar of RNA Furthermore, obtained by demonstrated similar for both small and large In FFPE tissues to 15 on the with the for the snap-frozen tissue fixation with the PAXgene tissue system was not to or because effect on quality was a fixation to or a fixation excellent preservation of by the PAXgene tissue stabilization system was demonstrated in more than analyses of different tissues a variety of fixation Furthermore, analysis of cancer a correlation of the gene from and snap-frozen tissue In results from FFPE tissue not only but also to with the snap-frozen samples studies of preservation and revealed a correlation of tissues and snap-frozen samples for FFPE samples a correlation was from tissues was of similar molecular as from snap-frozen tissues, and the was well for and as well as for not and reactions at In samples of FFPE tissues not of large In addition to nucleic acids, proteins can also be from tissues. The proteins were for a variety of protein analysis protein and B. S. Langer R. Kap M. Viertler C. C. U. Riegman P. Zatloukal K. A. Becker K.F. analysis of Res. 2010; PubMed Scopus Google Scholar extracted from tissues their and revealed preservation of several an for the analysis of J.A. Y. Y. N. of fixative is to of in paraffin-embedded tumor Histochem Cytochem. 2009; PubMed Scopus Google Scholar, V. C. K. M. Tissue is new technologies are to the of protein biomarker 2009; PubMed Scopus Google Scholar, V. C. in clinical tissue and its for protein Mol 2011; PubMed Scopus Google Scholar, C. K.H. C. E. C. R. S. C. V. preservation of and tissue morphology at room for diagnostic and research One. 2011; 6: PubMed Scopus Google Scholar In the extraction of certain proteins from FFPE tissues was (eg, of and the preservation of was different (eg, of in FFPE tissues, with snap-frozen tissues The PAXgene tissue stabilization system is not to be a formaldehyde but it analysis of disease in which collection of tissue for molecular analyses is or not with analysis of FFPE samples in with snap-frozen samples. Furthermore, we to in of FFPE on the which in of different I. Slotta-Huspenina J. R. N. H. F. to formalin fixation: for direct comparison of in paraffin-embedded tissues.Diagn Mol Pathol. 2006; 15: PubMed Scopus Google Scholar, I. A. M. A. D. R. R. P. M. E. J.A. of gene by and formalin-fixed, paraffin-embedded breast cancer 2010; PubMed Scopus Google Scholar and were by PAXgene Comprehensive studies using a of different analytical techniques demonstrated that RNA quality and of gene were similar in and snap-frozen samples. the fixation from to a of these were used to the preservation of morphology and biomolecules. In addition, we were in the of the new fixation technique in a routine clinical a of fixation We RNA as one of the Fixation and stabilization of to effect on RNA quality and analysis and fixation the to and stabilization for to at room for or feasible without the quality of molecular analyses. The excellent preservation of RNA, molecular weight DNA, and in with the morphological quality of are the of the new fixation The high-throughput screening used for the development of PAXgene fixation of the currently fixatives, and demonstrated that the properties on the of its any in the or chemical of the or even with formalin, we of the PAXgene Tissue System to be The properties of the PAXgene tissue stabilization system demonstrated in the the for analysis of a broad spectrum of biomolecules from the tissue samples as for histopathological the that molecular analyses with routine and that molecular can be with PAXgene technology markedly of molecular alterations in tissues that are to in a routine and preservation such as processing human tissue samples according to different and snap-freezing in are to in the are and are a of in clinical The tissue stabilization technology we have developed and tested therefore to the of medical research in as well as to the development of nucleic N. B. The of diagnostics in the development and use of cancer 2006; PubMed Scopus Google Scholar, the use of biomarkers in cancer drug Res. 2010; PubMed Scopus Google Scholar, C.H. H. of for biomarkers used in drug Res. 2008; 14: PubMed Scopus Google Scholar We and for excellent and for for the Kap for the and the for Research at the University of for in sample for the and of the for their and with of morphology in different and FFPE and tissues. staining of different human tissue samples in in formaldehyde for (FFPE) or in PAXgene fixative for by PAXgene tissue stabilization FFPE and are carcinoma of the and with of and and by from and FFPE samples from human cancer were used for of in and of the human gene and of the human gene using and of results FFPE and samples from with a to in of and FFPE and samples from with a to in of was performed in a from to The are by