Table 1.

Summary of scientific studies on alcohol-based fixatives and their suitability on histopathology evaluation.

References	Fixative	Type of tissue	Techniques	Main results and comments
Chung et al. J Histochem Cytochem. (2018); 66(2):121–135	10% NBF, 70% ethanol, buffered ethanol (BE70)	Mouse tissues	H&E, IHC, Western Blot, DNA and RNA extraction and quantification, Real Time RT-PCR	Ethanol-based fixatives show a broader time spectrum than NBF, preserving histomorphological features and quantity and quality of biomolecules from paraffin-embedded tissue.
Ghoddoosi and Masir J Med Surg Pathol. (2016); 1:2	NBF, RCL2 diluted in 100% ethanol, RCL2 diluted in 95% ethanol	Benign fresh human surgical specimens	H&E, Perl’s stain, IHC, FISH, SISH, genomic DNA extraction and quantification	RCL2 is potentially a good substitute for formalin.
Perry et al. J Histochem Cytochem. (2016); 64(7):425–440	10% NBF, 70% ethanol, 70% ethanol + 0.5× phosphate-buffered saline, 70% ethanol + 1% glycerol + 0.5 × PBS, 70% ethanol + 0.5% glacial acetic acid + 0.5 × PBS, 70% ethanol + 1% glycerol + 0.5% glacial acetic acid + 0.5 × PBS, buffered ethanol (BE70)	Mouse tissues	H&E, IHC, Western Blot, DNA and RNA extraction and quantification, Real-Time RT-PCR, Multiplex RT-PCR	BE70 fixative may be a potential replacement for NBF in both research and clinical settings, with the benefit of better biomolecule preservation, without the trade-off of impaired histomorphology.
Stefanits H. et al. Clin Neuropathol. (2016); 35(1):3–12	NBF, RCL2, KINFix	Neurosurgical biopsy specimens	Nuclear Magnetic Resonance (NMR) spectroscopy, H&E, Gomori-Trichrome, Alcian blue, Periodic acid Schiff, IHC, DNA and RNA extraction and quantification, PCR, HPLC-MS/ MS	RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin.
Belloni B. et al. J Clin Pathol. (2013); 66(2):124–135	NBF, PAXgene	Human malignant melanoma biopsy specimens	H&E, IHC, DNA and extraction and quantification, qRT-PCR, Real-time RT-PCR, Sanger sequencing	In PAXPE samples, morphology is well preserved but immunohistochemistry requires re-evaluation of markers and staining procedures. PAXPE fixation offers some advantages concerning molecular analysis.
Chieco C. et al. Biotech Histochem. (2013); 88(2):109–119	1. FAA (formalin, acetic acid, 95% ethanol, distilled water, 10:5:50:35, v/v), FineFIX (70% ethanol concentration) 2. 2.5% glutaraldehyde in 0.1 M phosphate buffer; FineFIX (70% ethanol concentration)	Healthy leaves 1. T. cordata Mill. Tiliaceae, P. avium L. Rosaceae and P. persica (D.C.) C.A. Mey Hamamelidaceae 2. C. australis L. Ulmaceae, U. minor Mill Ulmaceae and G. biloba L. Ginkgoaceae	1. Light microscopy: Histological staining (toluidine blue) 2. SEM	FineFIX is a viable alternative to formalin for both histological and SEM studies of vegetative plant tissues.
Groelz D. et al. Exp Mol Pathol. (2013); 94(1):188–194	NBF, PAXgene	Rat tissues	H&E, RNA extraction, microcapillary electrophoresis, Real-time RT-PCR	PAXgene preserves histology similarly to formalin, and does not chemically modify RNA.
Gündisch S. et al. PLoS One. (2013); 8(3):e60638	3.5–3.7% NBF, PAXgene	Non-malignant and malignant human tissue specimens	Western Blot, Two-dimensional SDS-PAGE, ELISA	PAXgene preserves even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis.
Milcheva et al. Acta Histochem. (2013); 115(3):279–289	10% NBF, ethanol + glacial acetic acid (EtAc), methanol + glacial acetic acid (MetAc)	Mouse tissues	H&E, IHC, RNA and extraction and quantification, quantitative real-time PCR analysis	Alcohol-based fixatives are an excellent tool for storage of tissue samples designed for IHC and mRNA expression studies.
Staff S. et al. J Clin Pathol. (2013); 66(9):807–810	10% NBF, zinc-based Z7 fixative, RCL2, PAXgene, Allprotect, RNAlater	Human tissue specimens	H&E, IHC, DNA and RNA extraction and quantification, qRT-PCR, Real-time RT-PCR, FISH, CISH	PAXgene provides the best alternative to both liquid nitrogen and formalin, enabling high quality molecular analyses, IHC and sufficient morphological examination.
References	Fixative	Type of tissue	Techniques	Main results and comments
Masir et al. Histopathology. (2012); 60(5):804–815	10% NBF, RCL2 diluted in 100% ethanol, RCL2 in 95% ethanol	Benign fresh surgical human specimens	H&E, Periodic acid-Schiff with diastase, Prussian blue and Masson trichrome, IHC, FISH, SISH, DNA extraction and quantification	RCL2 is a potential formalin substitute suitable as a fixative for use in routine histopathological examination.
Turashvili et al. Exp Mol Pathol. (2012); 92(1):33–43	10% NBF, Molecular Fixative (MF)	Non-cancerous human tissues	H&E, DNA and RNA extraction and quantification, PCR, RT-PCR	The Molecular Fixative were able to preserve large DNA and RNA fragments in paraffin blocks.
Zanini et al. Environ Health. (2012); 11:59	NBF, Cell-Block, Neo-Fix, RCL2, ZBF, Z7, PAGA, PAGA-T, FineFixx, Carnoy’s fixative, B5	Human surgical pathology specimens	H&E, Giemsa, trichromic stain, Alcian blue, PAS, IHC, RNA extraction and quantification	These fixatives are suitable for routine use for surgical pathology diagnostic work.
Arzt L. et al. Exp Mol Pathol. (2011); 91(2):490–495	10% NBF, FineFix, RCL-2, HOPE	Human lung cancer specimens	H&E, RNA extraction and quantification, miRNA amount, real-time RT-PCR analysis	Formalin-free fixatives are in general not superior for RNA studies.
References	Fixative	Type of tissue	Techniques	Main results and comments
Kap et al. PLoS One. (2011); 6(11):e27704	4% NBF, PAXgene	Human surgical pathology specimens	H&E, Periodic acid Schiff, resorcin fuchsin, sirius red, Gomori, IHC, CISH	Results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue.
Kothmaier et al. Arch Pathol Lab Med. (2011); 135(6):744–752	10% NBF, FineFIX, RCL2, HOPE	Human lung cancer specimens	H&E, The Movat pentachrome stain, IHC, protein quantification, Western Blot	Formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.
Moelans C.B. et al Am J Clin Pathol. (2011a),2011b; 136(4):548–56	4% NBF, F-solv, FineFIX, RCL2	Fresh surgical human tissue specimens	H&E, periodic acid–Schiff (PAS) without and with diastase (PASD), alcian blue, azan, elastin van Gieson (EvG), and Gordon-Sweet (G&S) and Jones silver stains, IHC	None of the alcohol-based fixatives was comparable overall to NBF with regard to macroscopy, morphologic examination, and immunohistochemical studies.
Moelans C.B. et al Am J Clin Pathol. (2011a),2011b; 64(11):960–967	4% NBF, F-solv, FineFIX, RCL2	Fresh surgical human tissue specimens	DNA and RNA extraction and quantification, epidermal growth factor receptor sequence analysis, microsatellite instability (MSI), qPCR, CISH, FISH	FineFIX and RCL2 performed better than F-solv and NBF with regard to DNA and RNA yield, quality and applicability in molecular diagnostics.
Dotti et al. Diagn Mol Pathol. (2010); 19(2):112–22	NBF, methacarn, FineFIX	Cell line-based model	RNA extraction and quantification; rRNA and mRNA integrity, Northern Blot, Real-Time RT-PCR	Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation.
Ergin B. et al. J Proteome Res. (2010); 9(10):5188–5196	NBF, PAXgene	Mouse tissues, non-malignant human specimens	One-dimensional SDS-PAGE, Western blot, reverse-phase protein microarrays (RPPA), MALDI Imaging MS, RNA extraction and quantification, PCR, electrophoresis	PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.
Nykänen and Kuopio Exp Mol Pathol. (2010); 88(2):265–271	NBF, LN-FIX, FineFIX	Breast cancer cell lines MCF7 (HTB-22) and T-47D (HTB-133)	H&E, IHC, RNA extraction and quantification, Real-Time PCR	Formalin or LN-FIX can be used as a fixative for molecular diagnostics preserving both morphology and nucleic acids; whereas FineFIX proved to be most unsuitable for gene expression analysis.
Paavilainen L. et al. J Histochem Cytochem. (2010); 58(3):237–246	NBF, Glyo-fixx, Zink formalin, FineFIX, HOPE, NEO-FIX, and Zinc-based fixative	Fresh surgical human tissue specimens, cancer cell lines (RT-4, U-251, PC-3)	H&E, tissue microarray, IHC, protein concentration, SDS-PAGE, Western-Blot	Morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde–based fixatives can be advantageous in obtaining high protein yield for Western blot analysis.
Preusser M. et al. Brain Pathol. (2010); 20(6):1010–1020.	4.5% NBF, RCL2	Human brain tumor specimens	H&E, IHC, DNA and RNA extraction, quantification and quality, multiplex PCR, electrophoresis, quantitative MGMT MSP	RCL2 fixation does not seem to significantly compromise histological tumor typing or IHC and preserves nucleic acids at a better quality than formalin fixation.
References	Fixative	Type of tissue	Techniques	Main results and comments
van Essen H.F. et al. J Clin Pathol. (2010); 63(12):1090–1094	4% NBF, RCL2, Boonfix	Fresh human tissue samples	IHC	Tissues fixed in non-crosslinking alcohol based fixatives like RCL2 and Boonfix can successfully be immunohistochemically stained for most antibodies following the usual NBF based protocols.
Balbi T. et al.Am J Forensic Med Pathol (2009); 30(3):242–245	Ethanol-based fixation	Human tissue section	SEM	The details are clearer with respect to those obtainable with formalin fixatives.
Lassalle et al. Thyroid. (2009); 19(11):1239–1248	10% NBF, Glyo-Fixx, FineFIX, ExcellPlus, RCL2, liquid nitrogen	Human thyroid specimens	H&E, periodic acid Schiff, trichromic Masson, and Sweet-Gordon staining, IHC, DNA, miRNA and RNA extraction, quantification, integrity	All the formalin substitute fixatives tested provided good histomorphologic quality for the different stained thyroid tissues, but individually, some fixatives performed better for immunohistochemical and molecular biological procedures for different thyroid pathologies.
Nassiri et al.BMC Clin Pathol. (2008); 29;8:1	10% NBF, UMFIX	Human breast cancer specimen	H&E, IHC, FISH, CISH, RNA and DNA extraction, quantification and integrity, PCR	The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.
Lykidis D. et al. Nucleic Acids Res. (2007); 35(12):e85	NBF, zinc-based Z2 fixative, HOPE, zinc-based Z7, Z8, Z16, Z17, Z18, Z19 fixatives; replacement of zinc solutions with manganese, magnesium, gallium or vanadium solutions; addition of chemicals to the standard zinc-based (Z2) fixation recipe (Z3, Z4, Z5, Z6, Z9, Z10, Z11, Z12, Z13, Z14, Z15)	Mouse tissues	H&E, IHC, DNA and RNA extraction and quantification, PCR, RT-PCR, Real-Time PCR, Real-Time RT-PCR, Two-dimensional (2-D) polyacrylamide gel electrophoresis	Z7 provides significantly improved preservation of DNA, RNA and proteins and allows improved PCR, Real-Time PCR and protein analysis, which may provide an excellent alternative to NBF for contemporary molecular pathobiology research.
References	Fixative	Type of tissue	Techniques	Main results and comments
Cox M.L. et al. Exp Mol Pathol. (2006); 80(2):183–191	10% NBF, modified Davidson’s solution II, 70% ethanol, UMFIX, modified Carnoy’s solution, modified methacarn, Bouin’s solution, PBS, 30% sucrose	Rat tissues	H&E, RNA extraction and quantification, laser capture microdissection, Taqman qRT-PCR	Modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens
Delfour C. et al. J Mol Diagn. (2006); 8(2):157–169	4% NBF, FAAM, methacarn solution, RCL2	MCF-7 cells, human breast carcinoma specimens	H&E, IHC, CISH, DNA and RNA extraction, laser capture microdissection, PCR, Real-Time RT-PCR	Methacarn and RCL2 have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection
Stanta et al. Diagn Mol Pathol. (2006); 15(2):115–123	FineFIX	Human biopsy or surgery tissues	DNA and RNA extraction, PCR, RT-PCR, Western Blot, two dimensional electrophoresis	FineFIX fixed tissues preserved DNA and RNA better than formalin. Proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues.
Nadji M. et al. Appl Immunohistochem Mol Morphol. (2005); 13(3):277–82	NBF, UMFIX	Human neoplastic and non-neoplastic specimens	IHC	IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens.
Titford and Horenstein Arch Pathol Lab Med. (2005); 129(4):502–506	NBF, Glyo-Fixx, STF, Omnifix II, Histochoice, Histofix	Human surgical pathology specimens	H&E	Formalin fixation provided the highest histomorphologic quality for tissue stained with hematoxylin-eosin and examined for diagnostic surgical pathology.
Uhlig U. et al. Pathol Res Pract. (2004); 200(6):469–472	NBF, HOPE	Human lung cancer	Western Blot	HOPE fixation maintains the antigenicity of proteins better than formalin fixation.
References	Fixative	Type of tissue	Techniques	Main results and comments
Soukup J. et al. Neoplasma. (2003); 50(4):300–304	10% NBF, 75% ethanol, formalin-ethanol fixation	Human B-cell lymphomas	DNA and RNA extraction, PCR, RT-PCR	The ethanol fixed samples retained a high quality of both DNA and RNA and provided reproducible PCR products similar to frozen samples and significantly better than those extracted from formalin fixed samples
Vincek V. et al. Lab Invest. (2003); 83(10):1427–35	NBF, UMFIX	Mouse tissue, human tissues	H&E, DNA and RNA extraction, PCR, RT-PCR, Real-Time PCR, Protein extraction, Western Blot, histochemistry, IHC	The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays.
Gillespie et al. Am J Pathol. (2002);160(2):449–457	70% ethanol, 95% ethanol, 70% ethanol: 100% methanol (3:1), 95% ethanol: 100% methanol (3:1), SafeFix, Streck, 10% NBF, Omnifix	Human prostate and kidney specimens	H&E, IHC, One-Dimensional PAGE, immunoblot, Two-Dimensional PAGE, Layered Expression Scanning, DNA and RNA extraction, Agarose Gel Electrophoresis, RT-PCR	70% ethanol fixation is a useful method for molecular profiling studies.
Vince D.G. et al. Anal Cell Pathol. (1997);15(2):119–129.	10% NBF, Histochoice	Human tissue specimens	IHC	Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin
Boon M.E. et al. Pathol Res Pract. (1992); 188(7):832–5	NBF, Kryofix	Human tissues samples	IHC	All markers studied showed enhanced staining in the Kryofix blocks after 4 hours of fixation, whilst in some cases the immunostaining of the formalin blocks was even negative.