Abstract
Formalin is toxic and has recently been classified as carcinogenic leading to a proposed European formalin ban. But, the pathology use of formalin has however been completely overlooked, and this is proving to be a relevant issue, as no alternative, reliable, tissue fixative is available. Various systems have been proposed to reduce formalin use and exposure; long-term storage and disposal of formalin is also a problem. With this in mind, under vacuum sealing (UVS) systems have been proposed for transportation/storage, however, for how long tissue retains its characteristics (morphological and molecular) is unknown. This study aims to compare histology specimens stored by formalin immersion (FI) and specimens stored after fixation with UVS technique with no additional formalin, at different time periods. Twenty tissue samples (10FI; 10UVS) were stored for different time periods (15 days, 1–2–3–6–12 months) for a total of 120 samples, compared with regard to their morphology, histochemistry, immunoreactivity (24 specific antibodies) and DNA status. All samples showed well-preserved morphology and overlapping staining quality. A significant reduction in immunoreactivity was however identified in the various time periods, particularly for heat pre-treated nuclear antigens, and this commenced earlier (1 month) for FI. UVS storage showed higher DNA content than FI but slightly poorer DNA integrity. These results add important knowledge to the use of UVS in daily practice, as long-term storage of pre-fixed tissue in UVS is not detrimental to the quality of tissue while having the boon of using very little formalin with less operator exposure and lower disposal costs.
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References
Baan R, Grosse Y, Straif K, Secretan B, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V, WHO International Agency for Research on Cancer Monograph Working Group (2009) A review of human carcinogens—part F: chemical agents and related occupations. Lancet Oncol 10:1143–1144. https://doi.org/10.1016/S1470-2045(09)70358-4
Bussolati G, Chiusa L, Cimino A, D’Armento G (2008) Tissue transfer to pathology labs: under vacuum is the safe alternative to formalin. Virchows Arch 452:229–231. https://doi.org/10.1007/s00428-007-0529-x
Bussolati G, Annaratone L, Maletta F (2015) The pre-analytical phase in surgical pathology. Recent Results Cancer Res 199:1–13. https://doi.org/10.1007/978-3-319-13957-9_1
Bussolati G, Annaratone L, Berrino E, Miglio U, Panero M, Cupo M et al (2017) Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples. PLoS ONE 12(8):e0182965. https://doi.org/10.1371/journal.pone.0182965
Di Novi C, Minniti D, Barbaro S, Zampirolo MG, Cimino A, Bussolati G (2010) Vacuum-based preservation of surgical specimens: an environmentally-safe step towards a formalin-free hospital. Sci Total Environ 408:3092–3095. https://doi.org/10.1016/j.scitotenv.2010.04.022
European Parliament and Council of European Union (2008) Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. Off J Eur Union L 353:1–1355
Gambella A, Porro L, Pigozzi S, Fiocca R, Grillo F, Mastracci L (2017) Section detachment in immunohistochemistry: causes, troubleshooting, and problem-solving. Histochem Cell Biol 148:95–101. https://doi.org/10.1007/s00418-017-1558
Grillo F, Pigozzi S, Ceriolo P, Calamaro P, Fiocca R, Mastracci L (2015) Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections. Histochem Cell Biol 144:93–99. https://doi.org/10.1007/s00418-015-1316-4
Grillo F, Bruzzone M, Pigozzi S, Prosapio S, Migliora P, Fiocca R, Mastracci L (2017) Immunohistochemistry on old archival paraffin blocks: is there an expiry date? J Clin Pathol 70:988–993. https://doi.org/10.1136/jclinpath-2017-204387
Grizzle WE (2009) Special symposium: fixation and tissue processing models. Biotech Histochem 84:185–193. https://doi.org/10.3109/10520290903039052
IARC Working Group on the Evaluation of Carcinogenic Risk to Humans (2006) Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol IARC monographs on the evaluation of carcinogenic risks to humans, vol 88. International Agency for Research on Cancer, Lyon
Kiernan JA (2008) Theory and practice. Histological and histochemical methods. Scion Publishing, Bloxham, pp 12–44, 431–437
Kristensen T, Engvad B, Nielsen O, Pless T, Walter S, Bak M (2011) Vacuum sealing and cooling as methods to preserve surgical specimens. Appl Immunohistochem Mol Morphol 19:460–469. https://doi.org/10.1097/PAI.0b013e318214e523
Metovic J, Bertero L, Musuraca C, Veneziano F, Annaratone L, Mariani S, Cassoni P, Bussolati G, Papotti M (2018) Safe transportation of formalin-fixed liquid-free pathology specimens. Virchows Arch 473:105–113. https://doi.org/10.1007/s00428-018-2383-4
Richter KN, Revelo NH, Seitz KJ, Helm MS, Sarkar D, Saleeb RS, D’Este E, Eberle J, Wagner E, Vogl C, Lazaro DF, Richter F, Coy-Vergara J, Coceano G, Boyden ES, Duncan RR, Hell SW, Lauterbach MA, Lehnart SE, Moser T, Outeiro TF, Rehling P, Schwappach B, Testa I, Zapiec B, Rizzoli SO (2018) Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J 37(1):139–159. https://doi.org/10.15252/embj.201695709
Shi SR, Liu C, Taylor CR (2007) Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem 55:105–109. https://doi.org/10.1369/jhc.6P7080.2006
Smith JE, Reese TS (1980) Use of aldehyde fixatives to determine the rate of synaptic transmitter release. J Exp Biol 89:19–29
Sompuram SS, Vani K, Messana E, Bogen SA (2004) A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol 121:190–199. https://doi.org/10.1309/BRN7-CTX1-E84N-WWPL
Tanaka KA, Suzuki KG, Shirai YM, Shibutani ST, Miyahara MS, Tsuboi H, Yahara M, Yoshimura A, Mayor S, Fujiwara TK, Kusumi A (2010) Membrane molecules mobile even after chemical fixation. Nat Methods 7(11):865–866. https://doi.org/10.1038/nmeth.f.314
Veneroni S, Dugo M, Daidone MG, Iorio E, Valeri B, Pinciroli P, De Bortoli M, Marchesi E, Miodini P, Taverna E, Ricci A, Canevari S, Pelosi G, Bongarzone I (2016) Applicability of under vacuum fresh tissue sealing and cooling to omics analysis of tumor tissues. Biopreserv Biobank 14:480–490. https://doi.org/10.1089/bio.2015.0093
Vincek V, Nassiri M, Nadji M, Morales AR (2003) A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples. Lab Investig 83:1427–1435. https://doi.org/10.1097/01.LAB.0000090154.55436.D1
Zanini C, Gerbaudo E, Ercole E, Vendramin A, Forni M (2012) Evaluation of two commercial and three home-made fixatives for the substitution of formalin: a formaldehyde–free laboratory is possible. Environ Health 11:59. https://doi.org/10.1186/1476-069X-11-59
Zarbo RJ (2015) Histologic validation of vacuum sealed, formalin-free tissue preservation, and transport system. Recent Results Cancer Res 199:15–26. https://doi.org/10.1007/978-3-319-13957-9_2
Acknowledgements
We wish to thank all of the technical staff at the Anatomic Pathology laboratory, IRCCS Policlinico San Martino, Genoa, Italy and Mrs Sonia Massa for her contribution to the Quality Assurance Program.
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Mastracci, L., Gambella, A., Bragoni, A. et al. Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin. Histochem Cell Biol 151, 501–511 (2019). https://doi.org/10.1007/s00418-018-1765-7
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DOI: https://doi.org/10.1007/s00418-018-1765-7