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Comparison of Formalin-Free Tissue Fixatives: A Proteomic Study Testing Their Application for Routine Pathology and Research [Archives of Pathology & Laboratory Medicine]
[June 11, 2011]

Comparison of Formalin-Free Tissue Fixatives: A Proteomic Study Testing Their Application for Routine Pathology and Research [Archives of Pathology & Laboratory Medicine]

(Archives of Pathology & Laboratory Medicine Via Acquire Media NewsEdge) Context.-Formalin-fixed, paraffin-embedded tissue is the routine processing method for diagnostics practiced in pathology departments worldwide.

Objective.-To determine the potential value of non-cross-linking, formalin-free tissue fixation for diagnostics in pathology and proteomic investigations.

Design.-We tested 3 commercially available, formalinfree tissue fixatives-FineFIX, RCL2, and HOPE-in lung cancer specimens from 10 patients. The fixatives were evaluated for their effects on tissue morphology, protein recovery, and immunoreactivity for a selected panel of proteins differently expressed in lung cancer, using immunohistochemistry and Western blotting.

Results.-Tumor-cell analysis with hematoxylin-eosin worked equally well for all tested fixatives when compared with the standard formalin-fixed, paraffin-embedded procedure. Movat pentachrome stains showed comparable results for the different matrices and cellular proteins analyzed. The RCL2 (P = .01) and HOPE fixatives (P = .03) improved protein recovery when compared with formalinfixed, paraffin-embedded or frozen tissues. Our data clearly show that the fixatives evaluated influenced immunoreactivity to matched, formalin-fixed, paraffinembedded lung cancer tissue. In particular, membranebound proteins, such as epidermal growth factor receptor EGFR, can be detected more efficiently by immunohistochemistry and Western blotting.

Conclusion.-We have demonstrated that formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.

(Arch Pathol Lab Med. 2011;135:744-752) In clinical practice biopsies as well as surgical resection specimens are fixed as soon as possible to avoid autolysis and putrefaction, and embedded in paraffinwax. Formalin-fixed, paraffin-embedded (FFPE) tissue is the routine processing method practiced in pathology departments worldwide. Fixation is a process involving a series of complex chemical modifications of macromolecules present in tissues and cells to preserve the structural and functional components as closely as possible to the living state. Formalin is a cross-linking fixative creating methylene bridges among proteins and, if not adequately buffered, results in nucleic acid fragmentation and degradation due to formic acid.1,2 Although formalin-fixed tissues are well preserved for histopathologic evaluation, the quality of the macromolecules is severely compromised, and that has been assumed to render them unsuitable for proteomic studies. However, this view has been challenged in the past decade through the development of innovative methods coupled with heat-induced extraction approaches to achieve solubilized, nondegraded, immunoreactive proteins from FFPE tissues.3-7 Formalin-fixed, paraffin-embedded tissue processing represents a highly stable, cheap, and easily storable form of tissue; is an invaluable material for research in molecular medicine; and is archived in hospitals and tissue banks worldwide. However, formalin is a toxic fixative and exposure by nasal, oral, or dermal routes is a human health risk.8 The International Agency for Research of Cancer recently classified formaldehyde as a human carcinogen (group 1) that causes nasopharyngeal cancer, and it also concluded there is ''strong but not sufficient evidence for causal association between leukemia and occupational exposure to formaldehyde.'' 9(p1206) For this reason, several European countries already restrict the use and import of formaldehyde because of its carcinogenic properties, and the European Union is considering a complete ban on formaldehyde usage.10 To overcome the problems of formalin fixation, numerous attempts have been made to find formalin-free tissue fixatives with low toxic properties and a similar quality to preserve nucleic acids and proteins.11 Numerous formalin substitutes have been examined and seem to be appropriate for DNA and RNA analysis,12-15 but the related procedures have been poorly tested in routine pathology practice and the proteomic field.16 Unfixed-fresh or snap-frozen tissue is commonly used as the gold standard for proteomic studies because it preserves proteins excellently. However, the histologic architecture on frozen tissue sections is often disrupted, and it will not work with many special stains. Also, frozen tissues are not widely available because of storage problems. Therefore, it is worth investigating whether formalin-free tissue fixation would improve protein preservation. The aim of our study was to assess the potential value of non-cross-linking, formalin-free tissue fixation for routine diagnostics and proteomic investigations. We compared 3 commercially available, formalinfree fixatives-FineFIX, RCL2, and HOPE-with standard formalin-fixed or frozen, lung cancer tissue samples. The fixatives were evaluated for their effects on tissue morphology, immunohistochemistry, and protein recovery and immunoreactivity by Western blotting.

MATERIALS AND METHODS Tissue Fixation Ten unfixed, lung cancer samples submitted for frozen-section diagnosis were chosen arbitrarily. The patients gave informed consent, and the study was approved by the Ethics Committee of the Medical University of Graz (No. 17-247).

From each tumor, 5 tissue samples were taken. One sample was routinely fixed with 10% neutral-buffered formalin, paraffin- embedded, and dehydrated according to the standard protocols. Another sample was immediately embedded in optimal cutting temperature medium (Tissue-Tek, Sakura Finetek Europe, Zoeterwoude, the Netherlands), snap frozen, and stored in liquid nitrogen until use. The remaining 3 samples were parallel-fixed in FineFIX (Milestone Srl, Sorisole, Torre Boldone, Italy), RCL2 (Alphelys, Plaisir, France), and HOPE (DCS Innovative Diagnostik-Systeme, Hamburg, Germany), dehydrated, and paraffin-embedded, according to the respective manufacturer recommendations. To evaluate the effects of prolonged fixation, different fixation times ranging from 21 to 69 hours were analyzed. The resulting FFPE, paraffin-embedded FineFIX, and paraffin-embedded RCL2 tissue blocks were stored at room temperature, whereas the paraffin-embedded HOPE tissue blocks were maintained at 4°C (manufacturer's recommendation). From each case, representative sections were examined to ensure qualitatively similar degrees of cellularity and the absence of necrosis.

Histologic Staining Sections of FFPE, paraffin-embedded FineFIX, paraffin-embedded RCL2, and paraffin-embedded HOPE tissues (4 µmthick) were dewaxed with xylene and dehydrated with several graded ethanols before routine hematoxylin-eosin (H&E), Movat pentachrome, and immunohistochemical staining were performed.

An H&E-stained reference section of each case and each fixative was histologically verified by experienced pathologists (H.H.P. and E.S.), and tumor areas (with >85% of tumor cells) were indicated with a pen mark on the slide. The H&E reference slides were used to locate the tumor area on the unstained sections for further protein extraction. Five consecutive, unstained sections from the same tissue blocks were used for protein extraction.

In all fixed samples, the following details were evaluated separately: nuclear and nucleolar preservation; chromatin pattern and details; mitosis and chromosomes, if present in the metaphase; cytoplasmic details, such as phagolysosomes in the alveolar macrophages; and mucin in mucinous adenocarcinomas. Both pathologists evaluated the cases on a score sheet containing each of these details: nucleoli present/enlarged; chromatin coarse/fine, granular/reticular; mitosis visible; mitotic figure/chromosomes visible; phagolysosomes visible; and mucin visible (using Harris H&E). After both pathologists had evaluated the cases, those cases with disagreements were reviewed using a multiheaded microscope.

Movat Pentachrome Staining The Movat pentachrome stain was chosen because that staining procedure exposes each tissue section to a variety of acidic and basophilic solutions and also to metal impregnation procedures. In addition, several matrix and cellular proteins are differently colored, which makes this stain very useful in evaluating mesenchymal tissue reactions. The Movat staining was done according to standard protocols.17 The staining pattern of the matrix proteins-elastin, collagen, reticulin fibers-and the myofilaments of smooth muscle cells was evaluated.

Immunohistochemistry Immunohistochemical analysis was performed in parallel on 3- µm-thick FFPE, paraffin-embedded FineFIX, paraffin-embedded RCL2, and paraffin-embedded HOPE tissue sections by routine protocols for FFPE tissue sections for the following antibodies: CK7 (cytokeratin 7), E-cadherin, EGFR (epidermal growth factor receptor), PCNA (proliferating cell nuclear antigen), and STAT1 (signal transducer and activator of transcription 1). The EGFR pharmDx kit (Dako Österreich GmbH, Wien, Austria) was used for EGFR immunohistochemistry. The kit's protocol was strictly followed for all samples. Detailed information about antibody source, antigen retrieval, and detection is listed in Table 1. Immunohistochemical analysis was carried out by 2 pathologists (H.H.P. and E.S.) blinded to the type of fixation. Protein expression was recorded semiquantitatively as described recently. 18 Staining scores were calculated by multiplying the percentage of positive cells (0%-100%) and by the staining intensity (1- 3). The product scores obtained, ranging from 0 to 300, were used for statistical analyses. Both pathologists scored the slides independently and recorded their scores on a separate sheet. Then, the pathologists together evaluated on a multiheaded microscope those cases with a disagreement.

Protein Extraction From Fixed Tissue Sections Protein extraction from each case of the parallel-fixed and paraffin-embedded tissue blocks for FFPE, FineFIX, RCL2, and HOPE was performed using Qproteome FFPE tissue kit (Qiagen, Hilden, Germany), as previously reported by Becker et al,4 for the application with FFPE tissue sections. Briefly, the 10-µm thick tissue sections from each case (n = 5) of the FFPE, paraffinembedded FineFIX, paraffin-embedded RCL2, and paraffinembedded HOPE blocks were cut and placed onto glass slides (SuperFrost, Menzel-Gläser, Braunschweig, Germany). After deparaffination of the tissue sections, the tumor areas were scratched from the unstained slide with a needle. The microdissected tissue was transferred into the Qproteome kit buffer, and proteins were extracted according to manufacturer recommendations and stored frozen at -20°C. From the frozen tissue specimens, multiple sections were cut; the first section was used for reference H&E staining, and consecutive 10-mm-thick sections were cut onto glass slides and stored at -80°C until protein extraction was performed as described above.

Protein Yield Protein concentrations were determined in 3 replicates using the Pierce Micro BCA Protein Assay Kit (Thermo Scientific, Waltham, Massachusetts) according to the protocol provided by the supplier for 96-well formats. To take measurements of the tumor area, the H&E reference slides with the marked tumor areas were scanned on a flatbed scanner, JPEG files were generated, files were imported into the FixFoto software program (version 2.91, 2008), and the data were used for calculating the tumor area in square millimeters. Protein yield was then expressed as micrograms per square millimeter of tissue section. The protein yield from all tested tissue fixatives and frozen tissues was evaluated.

Western Blot Analysis Twenty micrograms of total protein from the protein extracts was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride-type membranes (Hybond- P, GE Healthcare Europe GmbH, Freiburg, Germany) using a semidry blotting apparatus (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Blocking was performed in Trisbuffered saline containing 0.1% Tween-20 and the blocking substance for 2 hours at room temperature. The blocked membrane was incubated with the primary antibodies for detecting ß-actin, E-cadherin, EGFR, PCNA, and STAT1 for 16 hours at 4°C. Additional information about the source of the antibodies and their blocking and dilution is in Table 2. After incubation with the primary antibody, the membrane was washed 3 times for 10 minutes with Tris-buffered saline containing 0.1% Tween-20 and then incubated with the secondary horseradish peroxidase-conjugated antibody for 1 hour at room temperature. The membrane was washed 3 times for 10 minutes with Tris-buffered saline containing 0.1% Tween-20 and developed using ECL Western Blot Detection Reagents (GE Healthcare Europe) and Kodak film (T-MAT G film, Eastman Kodak Company, Rochester, New York). Quantitative analysis of the Western blot band intensities was performed using Scion Image forWindows (beta 4.0.3, Scion Corporation, Frederick, Maryland). Films were scanned on a flatbed scanner; images were saved as 8- bit TIFF files and imported to the imaging software.

Statistical Analyses To avoid the problems in testing multiple fixations with multiple immunochemistry and Western blots measurements, we adapted the Goeman global test,19 after which, it was possible to test whether any of the fixation methods caused any difference in any of the immunohistochemistry or Western blot measurements by 1 P value. To reduce the influence of outliers, each measurement (immunohistochemistry scores, Western blot, protein yield) was rank-transformed. In addition, the mean rank of each specimen was subtracted to remove the block effect of the specimens. The global test was applied using the permutation test criterion with 10 000 replications. Pairwise differences among fixation methods were tested by Wilcoxon signed rank test. Values of P < .05 were considered statistically significant. The R 2.9.0 ( and R-package global test (bioconductor package 4.10.0) software, as well as the R-code provided by the Department of Medical Statistics at the Georg- August-University at Göttingen, Germany (http://www.ams., were used.

RESULTS Histologic/Morphologic Findings Sections stained with H&E and Movat pentachrome were compared in each of the 10 lung cancer cases after fixation and paraffin embedding using either standard formalin or FineFIX, RCL2, and HOPE fixatives. On frozen tissue sections, only H&E staining was performed, but those sections were not used for the morphologic evaluation.

All fixatives yielded a comparable, good H&E-staining quality (Figure 1, A through D). All nuclear details were clearly visible, and cytoplasmic differentiation products could also be seen. However, there were a few differences; FFPE, FineFIX, and RCL2 were found to be excellent at preserving nuclear and cytoplasmic details, whereas the HOPE-stained sections were ranked visible but not clear regarding the nuclear features. Also mucin and surfactant inclusions were more difficult to see in the HOPE-stained sections than in the FFPE-, FineFIX-, and RCL2-stained sections. However, for tissue analysis of complex tissue reactions, such as desmoplastic stroma or vascular abnormalities, all tissues fixed by different processes were equally well suited. Movat stains showed comparable results for the different matrix proteins (not shown). Tumor cell analysis was equally possible for all 3 formalin-free fixatives, as compared with the routine, standard formalin fixative.

Immunohistochemical Analyses Representative immunohistochemical stains for antibodies detecting CK7, STAT1, PCNA, E-cadherin, and EGFR for parallel-stained FFPE, paraffin-embedded Fine- FIX, paraffin-embedded RCL2, and paraffin-embedded HOPE tissue sections are shown in Figure 2 (A through T). Each antibody measurement from the 3 formalin-free fixatives and the standard FFPE process were matched, and the resulting differences are listed in Table 3; all antibody measurements are summarized in Figure 3 (A and B). Cytoplasmic staining with CK7 showed no differences among the standard FFPE and the formalinfree tissue fixatives, and no differences could be detected among the formalin-free fixatives, respectively. Some proteins like STAT1 (cytoplasmic and nuclear) and PCNA (nuclear) could not be detected in the formalin-free fixatives when the staining intensity was low in the FFPE tissue sample. Interestingly, STAT1 was better preserved in FFPE (P=.005) than it was with the FineFIX- and RCL2- fixed lung cancer tissue. On the contrary, formalin-free fixatives were superior to formalin in preserving surface proteins, such as the membrane-receptors EGFR and Ecadherin we used. We found significantly higher staining for EGFR with FineFIX (P = .005) and RCL2 (P = .005), whereas E-cadherin could be more strongly detected in paraffin-embedded RCL2 (P = .007) and paraffin-embedded HOPE (P = .02) than in FFPE tissues.

The comparison among the formalin-free fixatives (Table 3) revealed that CK7, STAT1, and PCNA could be detected equally well in paraffin-embedded FineFIX, paraffin-embedded RCL2, and paraffin-embedded HOPE, whereas paraffin-embedded RCL2 and paraffin-embedded FineFIX gave excellent staining results for membranous E-cadherin (P = .01) and EGFR (P = .03). Overall, there was less protein preservation in paraffin-embedded HOPE tissues, likely because of digestion by the proteinase pretreatment and the retrieval procedures. In general, FineFIX and RCL2 worked equally well and were most often comparable to formalin, although they yielded lessintense stained cells with STAT1 and PCNA; the HOPE fixative yielded the least-satisfying results.

The global comparison among the fixatives (includes all antibody measurements) showed that paraffin-embedded FineFIX, paraffin-embedded RCL2, and paraffin-embedded HOPE highly influenced the efficiency of the protein preservation for the selected antibody panel (P = .02) when compared with standard FFPE tissue (global test; Table 3).

Western Blot Analyses To evaluate different tissue fixatives for possible effects on protein detection, proteins with different cellular localizations and molecular weights, such as ß-actin (42 kDa), STAT1 (84/91 [ß/a isoforms] kDa), PCNA (36 kDa), E-cadherin (120 kDa), and EGFR (175 kDa), were selected for Western blot analyses (Figures 4 and 5, A). The quantitative image analyses of the Western blot band intensities were evaluated globally for the influence of the fixatives and for individual proteins by Wilcoxon pair testing (Table 4). The latter indicated no difference in the immunoreactivity for ß-actin and PCNA between FFPE and alternative tissue fixatives, whereas STAT1 could be more strongly detected in the paraffin-embedded HOPE tissue samples (P = .01). In addition, we found b-actin and STAT1 were better preserved in frozen tissues than in fixed tissue, whereas PCNA seemed better preserved in the fixed tissue samples (Table 4).

The EGFR and E-cadherin, 2 important protein markers for pathologic diagnosis and often involved in therapeutic decisions, gave different results. We found EGFR immunoreactivity significantly increased by the formalin-free tissue fixation (P = .007), when compared with FFPE tissue section, whereas by Western blotting, E-cadherin showed no difference among fixation methods. The global comparison indicated a strong improvement for the paraffin-embedded FineFIX (P < .001), paraffin-embedded RCL2 (P < .001), paraffin-embedded HOPE (P < .001), and the frozen tissues (P < .001) as compared with the FFPE tissue processing method (Table 4).

Protein Yield From Fixed and Frozen Lung Cancer Tissue Proteins can be efficiently extracted from fixed and unfixed lung cancer tissues by applying the same extraction method. Alternative fixation methods improve protein recovery when compared with FFPE tissues or frozen tissue (Table 5). In detail, the comparison of the protein yield from matched FFPE, paraffin-embedded FineFIX, paraffin-embedded RCL2, paraffin-embedded HOPE, and frozen lung cancer tissue samples demonstrated (Figure 3, C) the highest protein-recovery results were from paraffin-embedded RCL2 tissues (P = .01), when compared with FFPE tissues and when compared with frozen-tissue, paraffin-embedded RCL2 (P = .02), and the paraffin-embedded HOPE tissues (P = .03) showed higher protein yields. Interestingly, the protein yield obtained from frozen tissues samples was lower than that from the fixed and paraffin-embedded tissues, regardless of which fixative was used (Figure 3, C).

COMMENT The potential of archival, fixed- and paraffin-embedded tissue collections as an alternative to frozen tissues for biomarker discovery is only just beginning to be recognized. Frozen tissue banking for tissue archiving represents a cost-intensive practice and is, therefore, usually restricted to large pathology departments or universities. The aim of our study was to evaluate the suitability of non-cross-linking, formalin-free tissue fixation for routine pathology diagnostics and proteomic investigations.

The pathologist Karl Weigert realized the better quality of the tissue sections after formalin fixation in 1893, and the rest is history. Most tissues stored in hospitals and pathology departments across the world are fixed in formalin and embedded in paraffin wax. Almost all histology techniques and antibody manufacturers have optimized their products for FFPE tissues, and even the US Food and Drug Administration has approved some procedures only for FFPE tissue processing. The disadvantage of formalin-besides being toxic and carcinogenic-is its limited effectiveness as a fixative for molecular tests, which has driven the quest for formalin substitutes for tissue fixation. Under those circumstances, and because of the large potential market involved, many scientists and companies have developed numerous new formalin-free fixatives.20 Parallel-fixed and paraffin-embedded lung cancer specimens were evaluated for differences in histomorphology staining, immunohistochemistry, protein recovery, and Western blotting. The use of formalin-free tissue fixation can be recommended in the processing of routine pathology specimens and should give good results for routine H&E stains as well as for special stains. However, for the immunohistochemical laboratory, the protocols for each antibody in use need to be optimized. In particular, the proteinase pretreatment should be abolished completely or used only for seconds, and the heat, steam, and buffer-retrieval methods need adaptations. Because proteins in non-cross-linked tissues might more easily dissolve and leak out, false-negative values could result, but the better preservation of membranous proteins by these alternative fixatives opens up new opportunities in diagnostic pathology because these molecules, such as EGFR, HER2, and interleukin receptors, are hard to evaluate in FFPE tissues. The findings of our study clearly show that membrane-bound proteins, like EGFR and Ecadherin, can be detected more efficiently by immunohistochemistry and Western blotting (Figures 3, A and B, and 5, A through I) when the samples were fixed in FineFIX, RCL2, or HOPE. We, therefore, believe that increased immunoreactivity is a consequence of better protein or antigen preservation and easier release of the proteins through the non-cross-linking characteristics of the fixatives tested.

The overall staining intensity of EGFR using the formalin-free fixatives was greater than in FFPE fixatives in lung cancer samples (Tables 3 and 4). A recent study by Nassiri et al21 showed a similar discrepancy for HER2/neu immunohistochemistry among matched UMFIX (a universal molecular fixative marketed as Tissue- Tek Xpress Molecular Fixative; Sakura Finetek USA Inc, Torrance, California) and FFPE breast cancer samples, whereas they found no difference in the immunohistochemical reaction for the estrogen receptor. Based on our data and recent studies with the same or closely related methods, we are in favor of immunohistochemistry for different therapy-associated markers, such as EGFR, HER2/neu, and estrogen receptor, which will need to be optimized and completely validated regarding the definition of scores when those tissue samples are fixed in formalin substitutes.

Protein extraction (performed with the same extraction protocol as used for FFPE tissue blocks) with paraffinembedded RCL2, paraffin-embedded FineFIX, paraffinembedded HOPE, and frozen lung cancer tissue enabled us to detect a selected panel of 4 proteins differently expressed in lung cancer, including cytoplasmic STAT1, nuclear PCNA (Figure 4), membranous EGFR, and Ecadherin (Figure 5, A through I) proteins by Western blotting. Furthermore, the protein yield from the formalinfree fixatives was higher than that from FFPE and frozen tissues (Figure 3, C; Table 5). In this respect, our results differ from a previously published report,22 in which the authors found that protein yield and immunoreactivity by Western blotting in paraffin-embedded RCL2 tissue samples were clearly decreased when the protein extraction was performed with the same protein-extraction method we used in this study. At present, we do not know why the protein yield in the fixed tissue samples was significant higher than that from the frozen tissue samples. One explanation could be that the tissue or protein density was increased in fixed tissue samples by the tissue shrinking and dehydrating, whereas in the frozen tissue samples, the lower protein yield was a result of the much greater aqueous phase diluting the protein content. The protein yield from frozen tissue sections, ranging between 0.9 and 2 µg/mm2 (Table 5), was in close agreement with those obtained by Ahram et al,23 who calculated 1 to 1.8 µg/mm2 for protein extraction from frozen tissue sections.

Our study, like others, was limited by evaluations that were mostly focused on one tissue type, such as fixation using FineFIX for colon cancer,12 effusions and fine-needle aspirates,24 HOPE in placenta,25 lung tissue,26 soft tissues,27 RCL2 for normal colonic mucosa samples,22 and breast carcinomas.14 Therefore, further comparisons (ring trials, multicenter studies) among formalin substitutes and standard formalin for several tissue types are needed urgently. It would be of great interest to optimize and compare archival tissue fixation linked with extraction and current leader methodologies for clinical tissue specimens processed in different laboratories to rule out the less viable and cost-effective options for a replacement of good old formalin.

The present study (which was not supported by any commercial company) leads us to the conclusion that the formalin-free fixatives tested are capable of being integrated into routine pathology procedures and research. If, however, formalin is to be replaced by another fixative, all established standard processing methods will have to be adapted because they are currently all optimized for the use of FFPE tissue. Furthermore, for most formalin substitutes, the companies will not disclose the ingredients, components, or formulations, and all are more expensive than formalin. Nevertheless, according to the results presented here and in previously published data, we believe that the formalin-free fixatives tested have the potential to replace formalin in histomorphology and protein preservation.

We gratefully thank Iris Halbwedl, Margit Gogg-Kamerer, and Elisabeth Grygar, Institute of Pathology, Medical University of Graz, and Christa Schott, Institut für Pathologie, Technische Universität München, for excellent technical assistance. This study was supported in part by grant 332 (Dr Kothmaier) and grant 295 (Dr Popper) from the Franz Lanyar-Stiftung at the Medical University Graz, Graz, Austria, and grant BE 1501/3-2 from the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany (Dr Becker). This manuscript was written thanks to the support and discussion developed in the European project FP6, IMPACTS.

References 1. Puchtler H, Meloan SN. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry. 1985;82(3):201-204.

2. Lykidis D, Van Noorden S, Armstrong A, et al. Novel zinc-based fixative for high quality DNA, RNA and protein analysis. Nucleic Acids Res. 2007;35(12):e85.

3. Nirmalan NJ, Harnden P, Selby PJ, Banks RE. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using Western blotting. J Pathol. 2009;217(4):497-506.

4. Becker KF, Schott C, Hipp S, et al. Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol. 2007;211(3):370-378.

5. Ikeda K, Monden T, Kanoh T, et al. Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem. 1998;46(3):397-403.

6. Chu WS, Liang Q, Liu J, et al. A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section. Lab Invest. 2005;85(11):1416-1428.

7. Shi SR, Liu C, Balgley BM, Lee C, Taylor CR. Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem. 2006;54(6):739-743.

8. Morgan KT. A brief review of formaldehyde carcinogenesis in relation to rat nasal pathology and human health risk assessment. Toxicol Pathol. 1997;25(3): 291-307.

9. Cogliano VJ, Grosse Y, Baan RA, Straif K, Secretan MB, El Ghissassi F. Meeting report: summary of IARC monographs on formaldehyde, 2-butoxyethanol, and 1-tert-butoxy-2-propanol. Environ Health Perspect. 2005;113(9):1205- 1208.

10. World Health Organization; IARC Working Group. Formaldehyde, 2- Butoxyethanol and 1-tert-Butoxypropan-2-ol. Lyon, France: IARC Press; 2006. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; vol 88.

11. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161(6):1961- 1971.

12. Stanta G, Mucelli SP, 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(2):115-123.

13. 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 > 20 kb from paraffin-embedded tissues: HEPES-glutamic acid buffer mediated organic solvent protection effect. Pathol Res Pract. 2002;198(11): 735-740.

14. Delfour C, Roger P, Bret C, et al. 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(2):157-169.

15. Gillespie JW, Best CJ, Bichsel VE, et al. Evaluation of non-formalin tissue fixation for molecular profiling studies. Am J Pathol. 2002;160(2):449-457.

16. Titford ME, Horenstein MG. Histomorphologic assessment of formalin substitute fixatives for diagnostic surgical pathology. Arch Pathol Lab Med. 2005; 129(4):502-506.

17. McElroy DA. Connective tissue In: Prophet BM, Mills B, Arrington JB, Sobin LH, eds. Laboratory Methods in Histotechnology. Washington, DC: American Registry of Pathology, Armed Forces Institute of Pathology; 1992:127-131.

18. Kothmaier H, Quehenberger F, Halbwedl I, et al. EGFR and PDGFR differentially promote growth in malignant epithelioid mesothelioma of short and long term survivors. Thorax. 2008;63(4):345-351.

19. Goeman JJ, van de Geer SA, de Kort F, van Houwelingen HC. A global test for groups of genes: testing association with a clinical outcome. Bioinformatics. 2004;20(1):93-99.

20. Buesa RJ. Histology without formalin? Ann Diagn Pathol. 2008;12(6):387- 396.

21. Nassiri M, Ramos S, Zohourian H, Vincek V, Morales AR, Nadji M. Preservation of biomolecules in breast cancer tissue by a formalin-free histology system. BMC Clin Pathol. 2008;8:1-10.

22. Bellet V, Boissiere F, Bibeau F, et al. Proteomic analysis of RCL2 paraffinembedded tissues [published online ahead of print December 10, 2007]. J Cell Mol Med.

23. Ahram M, Flaig MJ, Gillespie JW, et al. Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications. Proteomics. 2003;3(4): 413-421.

24. Gazziero A, Guzzardo V, Aldighieri E, Fassina A. Morphological quality and nucleic acid preservation in cytopathology. J Clin Pathol. 2009;62(5):429-434.

25. Blaschitz A, Gauster M, Dohr G. Application of cryo-compatible antibodies to human placenta paraffin sections. Histochem Cell Biol. 2008; 130(3):595-599.

26. Uhlig U, Uhlig S, Branscheid D, Zabel P, Vollmer E, Goldmann T. HOPE technique enables Western blot analysis from paraffin-embedded tissues. Pathol Res Pract. 2004;200(6):469-472.

27. Olert J, Wiedorn KH, Goldmann T, et al. HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract. 2001;197(12):823-826.

Hannelore Kothmaier, PhD; Daniela Rohrer, MD; Elvira Stacher, MD; Franz Quehenberger, PhD; Karl-Friedrich Becker, PhD; Helmut H. Popper, MD Accepted for publication August 17, 2010.

From the Institute of Pathology, Research Unit for Molecular Lung and Pleura Pathology (Drs Kothmaier, Rohrer, Stacher, and Popper), and the Institute for Medical Informatics, Statistics and Documentation (Dr Quehenberger), Medical University of Graz, Graz, Austria; and the Institut für Pathologie, Technische Universität, München, Germany (Dr Becker).

Dr Becker is a named inventor on a patent related to protein extraction from FFPE tissues and receives royalties associated with this patent (European patent DE102005023011). Qiagen, the owner of the patent, did not assert control over the authors' right to publish. The other authors have no relevant financial interest in the products or companies described in this article.

Reprints: Helmut H. Popper, MD, Institute of Pathology, Research Unit for Molecular Lung and Pleura Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria (e-mail:

(c) 2011 College of American Pathologists

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