Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples
Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples
Gianni Bussolati 0 1
Laura Annaratone 0 1
Enrico Berrino 1
Umberto Miglio 1
Mara Panero 1
Marco Cupo 0 1
Patrizia Gugliotta 0 1
Tiziana Venesio 1
Anna Sapino 0 1
Caterina Marchiò 0 1
0 Department of Medical Sciences, University of Turin , Turin , Italy , 2 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), IRCCS , Candiolo , Italy , 3 Pathology Division, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino , Turin , Italy
1 Editor: Cesario Bianchi, Universidade de Mogi das Cruzes , BRAZIL
Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by the Italian
Ministry of Education, University and Research
(MIUR ex-60%-2016-17 to CM and PRIN
2015HAJH8E), by AIRC, Associazione Italiana per
la Ricerca sul Cancro (MFAG13310 to CM), and by
5xMille Ministero Salute 2011 - FPRC onlus AIRC
5xMille Molecular Clinical Oncology Extension
program - Ref 9970 (to AS). The funders had no
Fixation of histological specimens in formalin is in practice since over a century [
] and still
represents the procedure of choice for tissue preservation . Over time, additional and
ancillary techniques such as immunohistochemistry (IHC) and molecular analyses have been
optimized in formalin-fixed paraffin embedded (FFPE) tissues, so that a sudden change of fixative
is presently considered as impractical being potentially detrimental to the quality of diagnostic
pathology. On the other hand, environmental authorities are increasingly concerned for the
objective toxicity of this volatile reagent, so that a banning of formalin from 2016 has been
proposed in the European Community. This has been stated by the EC Regulation n.605/2014 of
05.06.2014 that modifies the EC Regulation n.1272/2008 defining formalin as a carcinogen
role in study design, data collection and analysis,
decision to publish, or preparation of the
(category 1B/2) and mutagen. This regulation may exert a heavy impact on diagnostic
pathology. Reaction to this status of affairs is presently limited to adoption of protective procedures,
designed to prevent excessive exposure to formaldehyde vapors.
To be accepted by the scientific community, an ªalternative fixativeº should likely be an
aldehyde, thus acting in a chemical reaction similar to that of formalin and affecting proteins
and nucleic acids in a comparable way. This would avoid dramatic effects on IHC and
molecular procedures and permit their use with minor adjustments, so that the bulk of the acquired
and internationally accepted diagnostic parameters would not be lost. In addition, a possible
alternative fixative should be relatively cheap, ideally in line with formalin, so as not to increase
the final cost of histopathologic examinations. Moreover, it should not be toxic to avoid further
restrictions. Finally, it should be rather fast, without affecting present turn around times.
Glyoxal (aka ethanedial, oxalaldehyde) was proposed in 1943 [
] as a fixative alternative to
formalin since it is a simple di-aldehyde. As reported by Harke & HoÈffler [
] glyoxal does not
appear to evaporate from solution. Indeed, the reported Henry law constant of 3.38 × 10±
4Pa m3/mol [
] indicates that glyoxal is essentially non-volatile with regard to the aqueous
phase. Glyoxal is not classifiable as a human carcinogen [
], nevertheless is irritating to skin
and eyes [
]. Tumor-promoting activity of glyoxal has been reported in rats subjected to
longterm exposure to this agent in drinking water [
Taken together these data show that glyoxal has a very low toxicity even though holding a
similar reactivity to formaldehyde.
Several studies have described the effect of glyoxal on tissues [9±11] and a variety of
fixatives based on this reagent have been proposed. Still, criticisms have been reported,
discouraging the use of this fixative as an alternative to formalin [
]. In particular, it has been
claimed that glyoxal-fixed tissues show clarity of cellular details, erythrocytes are lysed and
microcalcifications are dissolved . In addition, fluorescence in situ hybridization (FISH)
analysis led to technically-compromised results [
] and extraction and sequencing nucleic
acids proved unsatisfactory [12, 14, 16±18].
By taking these phenomena into account and having observed that commercially available
glyoxal is strongly acid, we reasoned that this peculiar acidity may be responsible for the
observed detrimental effect on tissues. Acidification of glyoxal is likely due to its fast oxidation
that leads to formation of acids, mainly glyoxilic acid, a very strong acid [
In this study we aimed to assess whether an acid-free form of glyoxal could represent a
novel tissue fixative by investigating reliability of morphological details, IHC reactions and
molecular analyses in a series of surgical specimens, in which ad hoc parallel sampling was
performed to allow formalin- and glyoxal-fixed samples.
Materials and methods
Glyoxal (40% in water) and basic ion-exchange resins (Amberlite1 IRA-400 chloride form
and Amberlite1 IRA-67) were all purchased from Sigma (Milan, Italy). Addition of
commercial glyoxal (40% in water) to a 0.1 M phosphate buffer pH 7.3 at a final concentration of 2%
resulted in a drop of pH to the acidic side and this solution was in the range of pH 4 after 6
months. To deprive glyoxal of acids and thus to obtain Glyoxal Acid-Free (GAF), 250 g of the
strongly basic ion-exchange resin were moistened with de-ionized H2O, then activated by a
short wash with NaOH 1 M. Following washes in water to remove sodium hydroxide, 150 ml
of glyoxal were added. The resin was allowed to act for 30 min at RT and then removed by
passages through a filter. The resulting, water-clear liquid, whose pH is around neutrality,
represented 40% glyoxal deprived of acids. We adopted a 2% solution of GAF as the fixative of
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choice. The 2% solution was selected by referring to the same amount of aldehyde as present
in 4% formalin.
The resin, once used to remove acids from glyoxal, can be re-generated (with a bath of 1M
NaOH) and re-used.
A 2% GAF solution in 0.1 M phosphate buffer pH 7.3 is stable for a few weeks then
gradually undergoes oxidation producing an acidic reagent with sub-optimal fixation properties. To
overcome the stability problem, we produced a stock solution containing 20% GAF in 50%
ethanol (Carlo Erba, Milan, Italy) added with 0.1 g insoluble calcium carbonate
(SigmaAldrich) in 100 ml of the solution (stock solution). The final (working) solution employed as
GAF fixative was obtained by diluting the stock solution (exempted of calcium carbonate) 1:10
in 0.11 M phosphate buffer pH 7.3.
Human tissue samples
Thirty human surgical samples (25 colorectal adenocarcinomas and 5 gastric
adenocarcinomas) harboring a lesion of adequate dimensions (>2 cm) to allow multiple sampling in parallel
were sampled according to standard practice and fixed in parallel in PBF and in GAF (working
Following overnight fixation at RT, dehydration in alcohol and paraffin embedding
followed standard procedures to paraffin embedding with an automatic processor (Leica ASP
300, Leica Microsystems, Wetzlar, Germany). Sections were stained in Haematoxylin and
Eosin (H&E). Samples were subjected to i) immunohistochemical staining (whole cohort, 30
cases); ii) FISH analysis (five gastric carcinomas); iii) molecular analyses (eight cases of
The study was approved by the Ethic Institutional Review Board (IRB) responsible for
"Biobanking and use of human tissues for experimental studies"ÐDepartment of Medical Sciences,
University of Turin.
Three μm thick sections were cut from tissue blocks and IHC was performed using an
automated platform (Ventana BenchMark AutoStainer, Ventana Medical Systems, USA). Positive
and negative controls (omission of the primary antibody and IgG-matched serum) were
included for each immunohistochemical run. Optimized IHC conditions, assessed following
experimental trials, are reported in Table 1. Antigen Retrieval was performed with Cell
Conditioning Solution 1 and 2 (CC1 and CC2, both from Ventana Medical Systems, Inc.). CC1 is a
tris based buffer with a slightly basic pH, while CC2 is a citrate buffer at a slightly acidic pH.
Both solutions work at a controlled temperature of 95ÊC. We also tested a Buffer pH 8.6 and
heat-induced epitope retrieval at 125ÊC in pressure cooker [
Fluorescence in situ hybridization
DNA FISH was performed using probes for HER2/CEP17, EGFR/CEP7 and the break apart
ALK probe (all from Abbott Laboratories, PathVysion). FISH experiments followed i) a
standard protocol as previously described [
]; ii) the standard protocol modified with a
preliminary passage in Tris-HCl 0.01 M at pH 8.5 for 3 min at RT before undergoing the standard
protocol. Analysis was performed as previously described [
]: 10 invasive areas on each slide
were selected and automatically acquired at 40X magnification with the motorized Metafer
scanning system (Zeiss, Germany) and Axio Imager epifluorescence microscope. The
PathVysion V2 software was used to analyze the results.
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CC1, 36min (F)
CC2, 60min (G)
Protease 1, 4min
CC1, 60min (F)
CC1, 92min (G)
CC1, 36min (F)
CC1, 90min (G)
CC1, 36min (F)
CC1, 60min (G)
Protease 1, 4min (F)
CC1, 60min (G)
Primary Ab Incubation
Extraction, quantification and quality assessment of DNA and RNA
Nine sections (5 μm-thick) were obtained from paraffin-embedded tissue blocks of eight
colorectal adenocarcinomas processed in parallel, fixed in GAF and in PBF. Sections were
deparaffinized using 1 ml of xylene. After over-night incubation at 56ÊC with proteinase K, DNA was
isolated from five sections using the MagCore Genomic DNA FFPE kit on the MagCore
automatic extractor instrument (RBC Bioscience, Taiwan) according to manufacturer's protocol.
RNA was obtained using the remaining four sections with RecoverAll Total Nucleic Acid
Isolation Kit for FFPE (ThermoFisher Scientific, USA) following the manufacturer's protocols.
Both DNA and RNA extracts were quantified by Qubit BR assay on Qubit Flourometer
(Invitrogen, Carlsbad, CA, USA) and NanoDrop Spectophotometer (ThermoFisher Scientific).
DNA and RNA integrity was evaluated with Agilent 2100 Bioanalyzer (Agilent
Technologies, USA). DNA integrity was evaluated using High Sensitivity DNA Analysis Kit (Agilent
Technologies, Santa Clara, CA) on DNA HS chip. Samples were diluted at 2 ng/μL and DNA
length analysis was performed according to manufacturer's instruction. The average of the
DNA fragment size of GAF and PBF samples was assessed using 5000 nt as the threshold for
the longer DNA fragments (>5000 nt). Their distribution in respect to this threshold was
statistically compared by Chi-square test.
RNA integrity was assessed using Agilent RNA 6000 Nano Kit. The size distribution of the
RNA fragments was calculated from Agilent 2100 Bioanalyzer readings using a Smear Analysis
with a 200 nt threshold: the percentage of RNA fragments > 200 nt in size (DV200 metric)
was recorded [
DNA sequencing analysis
Direct sequencing. Fifty ng of DNA was amplified for the exon 2 of KRAS (246 bp) using
the following PCR condition: 1x buffer, 2.5 mM MgCl2, 0.4 μM of forward and reverse primers
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(forward: 5’-GGTGGAGTATTTGATAGTGTATTAACC-3’ and reverse: 5’-AGAATGGTCCTG
CACCAGTAA-3’), and 0.2 unit of Taq Polymerase in a final volume of 25 μL. PCR reactions
were carried out with the following touch-down program: 94ÊC for 2 minutes, followed by 3
cycles of 94ÊC for 15 seconds, 64ÊC for 30 seconds and 70ÊC for 30 seconds; 3 cycles of 94ÊC
for 15 seconds, 61ÊC for 30 seconds and 72ÊC for 30 seconds; 3 cycles of 94ÊC for 15 seconds,
58ÊC for 30 seconds and 72ÊC for 30 seconds; 35 cycles of 94ÊC for 15 seconds, 57ÊC for 30
seconds and 72ÊC for 30 seconds, with a final extension of 70ÊC for 5 minutes. PCR templates
were visualized by electrophoresis on 3% agarose gel, and purified with illustra ExoProStar
(GE Healthcare, Italy). A final 15 ng of PCR products were purified with the ExoProStar and
used for sequencing analyses. A cycle-sequencing PCR reaction was set up using the Big Dye
Version 3.1 Terminator cycle-sequencing kit (ThermoFisher Scientific), with the same
amplifying primer added to a final concentration of 5 pmol/μL in a volume of 20 μL. The cycling
conditions were: 25 cycles at 96ÊC for 10 seconds, 50ÊC for 5 seconds, and 60ÊC for 4 minutes;
the reaction was terminated at 4ÊC. The cycle sequencing products were purified using
Agencourt CleanSEQ (Beckman Coulter, USA), and the DNA was sequenced using an automated
16 capillary sequencer (3730 DNA Analyzer, Applied Biosystems, USA).
Pyrosequencing. KRAS exon 2 was amplified and sequenced in order to evaluate the
status of codon 12 and 13 by pyrosequencing, which is a method based on ªsequencing by
synthesisº principle, using PSQ 96 (Qiagen, Germany). DNA amplification was performed with
the following primers: forward 5’-GGCCTGCTGAAAATCACG-3’, reverse 5’ biotin–
GCTCTATCGTCATGGCTCT-3’ (size 80 bp). After denaturation at 94ÊC for 5 minutes, DNA
samples underwent to 40 cycles at 94ÊC for 45 seconds, 57ÊC for 45 seconds and 72ÊC for 1
minute, and a final elongation at 72ÊC for 5 minutes; the 5'-biotinylated PCR products were
bound onto streptavidin-coated paramagnetic beads (GE Healthcare), denaturized by 0.1 mol/
l NaOH and released according to the manufacturer's instructions using PyroMark Vacuum
Prep Workstation (Qiagen). These reactions were performed in a 96-wells plate using Pyro
Gold Reagents (Qiagen). The primed single-stranded DNA templates were subjected to
realtime sequencing of the region including codon 12 and 13 by using the sequencing primer
CTTGTGGTAGTTGTAGCT-3’. The obtained pyrograms were analyzed by using PyroMark
ID Software v 1.0 (Qiagen).
Mass spectrometry. Samples were analyzed for KRAS, BRAF, NRAS and PIK3CA gene
mutational status by using Myriapod1 Colon status kit (Diatech Pharmacogenetics, Italy).
This methodology uses the MassARRAY1 strumental system (Sequenom1 Inc, USA) based
on mass spectrometry MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time Of
Flight) method. DNA samples were first amplified with a multiplex-PCR with Labcycler
following the manufacturer's instructions. Amplified samples were then treated with Shrimp
Alkaline Phosphatase (SAP reaction) to remove the excess of nucleotides and primers from
PCR reactions. Each purified reaction was then subjected to a single base extension reaction
(iPLEX1) with modified mass nucleotides. For each polymorphic site, we obtained one or
more analytes with a specific mass. Mass spectrometer analysis produces an expected mass
peak for each analyte, which will be associated to a wild type or mutant genotype of analyzed
Targeted next generation sequencing (NGS). Samples were subjected to targeted next
generation sequencing on an Illumina MiSeq following validated protocols using the
Myriapod1 NGS-IL 56G Onco-panel (NG032, Diatech Pharmacogenetics), which covers clinically
relevant mutational hot-spots of 56 bona fide cancer genes (S1 Table).
First, a Real Time PCR that simultaneously amplifies two highly conserved regions, thus
generating two PCR products of different length, was used to assess sample quality. The degree
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of fragmentation was derived from the ratio between the two DNA fragments (larger
fragment/shorter fragment) differently labeled with fluorescent probes.
Second, 50 ng of DNA were amplified and processed according to the manufacturers'
instructions. Currently, the protocol does not include a step with uracil-DNA glycosylase
(UDG) treatment of DNA samples. Sequencing was performed on an Illumina MiSeq
(Illumina1 Inc.) and data were processed exploiting the proprietary bioinformatics software
associated to Myriapod NGS kits (Diatech Pharmacogenetics).
cDNA synthesis. A total of 1 μg of RNA was reverse transcribed to cDNA by the Reverse
Transcription System Kit (Promega, USA) using 5 mM MgCl2, 1X Buffer, 1 mM dNTPs, 1U/
μL Recombinant RNasin Ribonuclease Inhibitor, 1.25U/μL AMV Reverse Transcriptase and a
mix of 120 ng of both oligo(dT) and random primers provided by the kit. The cDNA synthesis
was carried out incubating at 42ÊC for 1 h, 95ÊC for 5 minutes and 2.5ÊC for 5 minutes. cDNA
was quantified with the Qubit ssDNA HS assay kit (ThermoFisher Scientific).
Reverse transcriptase-PCR. A total of 50 ng of cDNA was amplified for the exon six of
Cytocheratin 20 (KRT20) using the following PCR condition: 1x buffer, 2.5 mM MgCl2,
0.4 μM of each primer (forward 5’- AGAGGAGACCAAGGCCCGTTACAG-3’, reverse
CTTCCAGAAGGCGGCGGTAAGTAG -3’) and 0.2 unit of Taq Polymerase in a final volume of
25 μL. PCR reaction was carried out with: 94ÊC for 3 minutes, followed by10 cycles of 94ÊC for
15 seconds, 64ÊC for 30 seconds and 72ÊC for 30 seconds; 25 cycles of 94ÊC for 15 seconds,
61ÊC for 30 seconds and 72ÊC for 30 seconds with a final extension of 70ÊC for 5 minutes.
PCR templates were visualized by electrophoresis on 3% agarose gel to verify the amplicon
Stability of the reagents
To overcome the acidification of the fixative the solution here adopted was linked to the
addition of ethanol and insoluble calcium carbonate to GAF. This stock solution remained stable
for several months. The final (working) solution was obtained by diluting the stock solution
1:10 in 0.11 M phosphate buffer pH 7.3. The resulting 2% GAF in phosphate buffer 0.11 M pH
7.3 (hereafter GAF) is stable for at least one month.
Structure of cells and tissues
Morphology of the 30 tissues included in the cohort fixed in PBF or in GAF was considered by
analyzing nuclear features such as nuclear shape and distribution of chromatin, staining
characteristics, shrinkage around glandular structures or cellular aggregates.
Tissues fixed in commercially available 2% glyoxal in 0.1 M phosphate buffer (an acidic
solution) showed lysis of erythrocytes, shrinkage of stromal components, clarity of cytoplasmic
components and no evidence of eosinophils. Tissues fixed in GAF presented a degree of
structural, cellular and nuclear preservation comparable to that provided by PBF (Fig 1A and 1B, S1
Fig). We observed preservation of erythrocytes and no signs of lysis or of loss of staining of
eosinophils (Fig 1C).
IHC was employed to assess antigen preservation. The selected target proteins represented a
broad spectrum of proteins distributed in different subcellular compartments (cytoplasm, cell
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Fig 1. Histology (H&E staining) of tissues fixed either in GAF or in PBF. A colorectal adenocarcinoma
fixed in GAF (A, 20X) or in PBF (B, 20X), where the similarity in preservation of structural and cell components
can be appreciated. A detailed examination revealed preservation of erythrocytes and no signs of lysis or loss
of staining of eosinophils, as shown here in a GAF-fixed colorectal adenocarcinoma (C, 20x and inset, where
an area enriched of eosinophils is captured at a higher magnification).
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Fig 2. Preservation of antigens in tissues fixed either in GAF or in PBF. (A) CDX2 expression in a GAF-fixed
colorectal adenocarcinoma; (B) CDX2 expression in a PBF-fixed sample of the same colorectal adenocarcinoma
illustrated in A; (C) HER2 overexpression in a GAF-fixed gastric adenocarcinoma; (D) HER2 overexpression in a
PBFfixed gastric sample of the same adenocarcinoma illustrated in C; (E) proliferation index assessed by Ki67 in a
GAFfixed colorectal adenocarcinoma; (F) proliferation index assessed by Ki67 in a PBF-fixed sample of the same colorectal
adenocarcinoma depicted in E.
membrane, nucleus). No discrepancies in subcellular localization of protein expression were
observed in the differently fixed samples (Fig 2). Nuclear antigens required an optimization of
the antigen retrieval procedure, i.e. longer duration of the antigen retrieval (60/90 min versus
30 min, Table 1). Following optimization, all nuclear antigen except Ki67 gave results
superimposable to the reactions performed on PBF fixed samples. Ki67 expression was observed to be
less pervasive in GAF fixed samples compared to corresponding PBF samples. A 60 min long
antigen retrieval procedure for Ki67 reaction gave better results, however Ki67 indices were
lower (mean: 6%) than in formalin fixed samples here analyzed.
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For nuclear antigens we also tested antigen retrieval treatment at high temperature (125ÊC)
in a highly basic buffer (pH 8.6). Such treatment did not yield significant improvements
compared to longer duration of standard antigen retrieval procedures.
For all of the remaining cytoplasmic and membrane markers, time of antigen retrieval was
the same as for standard FFPE tissues.
Fluorescence in situ hybridization analysis
FISH analysis following the standard protocol led to a mild autofluorescent background in
GAF fixed tissues, which prevented a reliable scoring of signals (Fig 3). The diffuse
fluorescence disappeared in sections treated with a short wash in Tris-HCl 0.01 M at pH 8.5 (Fig 3B).
Following this pre-treatment, the FISH procedure in the five GAF fixed tissues here analyzed
produced results matching those obtained in PBF fixed tissues from the same cases. Mean
HER2 and CEP17 copy numbers, as well as EGFR and CEP7 copy numbers were comparable
between GAF- and PBF-fixed samples, as per automated scoring by Metafer. Signal intensity
was comparable between corresponding samples when pre-treatment with Tris-HCl was
performed (Fig 3) and allowed a proper assessment also of fused signals in the experiments using
the break apart ALK probe.
DNA sequencing analysis and RNA analysis
To evaluate the preservation of nucleic acids, DNA and RNA were extracted from GAF-fixed
and PBF-fixed colorectal adenocarcinomas samples. Agilent Bioanalyzer, providing
information about the size range of fragments, was used to assess DNA quality. In terms of DNA
fragmentation profile, the fragment size distribution of the 8 analyzed GAF samples was
significantly enriched for less fragmented DNA ( 5.000 bp) compared with the matching PBF
specimens (p<0.001, Chi-square test, Fig 4A and 4B). These results were corroborated by the Real
Time PCR performed as a quality control for targeted NGS: this assay also identified a higher
degree of fragmentation in DNA extracted from PBF-fixed samples than in DNA extracted
from GAF-fixed samples.
RNA quality, determined by Agilent Bioanalyzer traces, was satisfactory in both PBF- and
GAF-fixed samples: the percentage of RNA fragments greater than 200 nt (DV200) was
superior to 90% (Fig 4C). The RT-PCR study for KRT20 expression was carried out in parallel on
PBF- and GAF-fixed samples. RNA amplification was successful at 185 bp in parallel samples.
The PBF-fixed samples of the 25 colorectal adenocarcinomas collected in the study had
been routinely subjected to Sequenom MassARRAY1 to screen for KRAS/NRAS/BRAF/
PIK3CA mutations. Six samples were found to harbor a KRAS codon 12 mutation and two
were mutated on KRAS codon 117 and 146, respectively. In addition, one sample carried both
KRAS codon 12 and PIK3CA codon 542 mutations. The DNA corresponding to parallel
PBFand GAF-fixed samples of 8 colorectal cancer cases (6 KRAS codon 12 mutant and 2 KRAS
codon 117 and 146 mutant according to the previous test) were then subjected to direct
sequencing, pyrosequencing and Sequenom MassARRAY1. The pathogenic KRAS mutations
previously identified in the PBF-fixed specimens were all confirmed in the GAF-fixed samples
by direct sequencing, pyrosequencing and Sequenom MassARRAY1.
Finally, the eight samples pairs (PBF-fixed and GAF-fixed samples) were subjected to
targeted NGS analysis with a panel including 56 bona fide cancer genes. The NGS analysis was
performed at a comparable mean depth in PBF-fixed (1921X) and GAF-fixed (1934X) samples.
Details about the pattern of non-synonymous single nucleotide variants in each sample pair
are reported in S2 Fig. The KRAS/NRAS/BRAF/PIK3CA mutations detected by Sequenom
MassARRAY1 were all confirmed in the pairs, however differences in mutant allele
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Fig 3. DNA FISH for HER2 and CEP17 in parallel tissue samples fixed in GAF and in PBF.
Representative fields of a breast gastric carcinoma where we performed HER2/CEP17 FISH analysis. (A)
FISH analysis in a GAF -fixed specimen. (B) The same sample as in A in which sections were treated with a
short wash in Tris-HCl 0.01 M at pH 8.5 leading to disappearance of the mild fluorescent background
observed in A. (C) Sampling of the same specimen parallel to (A), but fixed in PBF.
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Fig 4. Preservation of nucleic acids in GAF-fixed tissues. Bioanalyzer results of DNA samples extracted from 8
GAFfixed and matching PFB-fixed colorectal carcinoma specimens. (A) Fragment size distribution of the analyzed DNA
samples (threshold for longer fragments: 5000 bp). GAF samples were significantly enriched for less fragmented DNA
compared with PBF specimens (Chi-square test). (B) Fragmentation analysis example of two DNA specimens. In both
cases the difference of the fragment size average between GAF and PBF samples was statistically significant. (C) RNA
quality, determined by Agilent Bioanalyzer traces, was satisfactory in both GAF- and PBF-fixed samples.
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Fig 5. Representative sample pair showing a better DNA preservation in GAF-fixed versus PBF-fixed tissues. Representative
fragmentation analysis and sequencing results from different methods of two DNA specimens, GAF- and PBF- fixed, respectively (sample
pair 4±12). At Bioanalyzer analysis a higher degree of DNA fragmentation was observed in the specimen fixed in PBF (B) compared to the
corresponding parallel GAF-fixed sample (A). In addition, the allele frequency of the KRAS p.G12D mutation was considerably higher in GAF
fixed specimen with all the employed molecular techniques: panel C shows results of the GAF-fixed sample (Direct sequencing,
Pyrosequencing, Mass Spectrometry, from left to right), whereas panel D shows corresponding results in the PBF fixed sample (Direct
sequencing, Pyrosequencing, Mass Spectrometry, from left to right). Of note, by NGS this mutation showed a mutant allele frequency of 32%
in the GAF-fixed sample versus 7% in the PBF-fixed sample.
frequencies were observed (S2 Fig). Interestingly, in one case (sample pair 4±12) we found that
with all the employed molecular techniques, including NGS, the allele frequency of KRAS p.
G12D mutation was considerably higher in GAF fixed specimen (32.4% vs 7%) (Fig 5). Of
note, three pairs (sample pairs 6±14, 7±15, 8±16) presented a series of single nucleotide
variants harboring a mutant allele frequency <5%, which were uniquely found in PBF-fixed
samples. Formalin fixation can lead to artifactual changes in the DNA double strand [23±25], such
as for instance deamination of cytosine to uracil leading to a C:G>T:A change. It is interesting
to note that some of these low frequency mutations showed a C:G>T:A call (S2 Fig).
The present study demonstrates that fixation with glyoxal produces a preservation of structural
and macro-molecular properties of cells and tissues similar to that obtained by PBF, provided
that this non-toxic dialdehyde is used in acid-free conditions (GAF solution).
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Previous studies analytically comparing the suitability of glyoxal as a fixative alternative to
] concluded on the superiority of the latter, given the unsatisfactory histology,
unreliable immunohistochemistry and suboptimal nucleic acid preservation in glyoxal-fixed
tissues [13±18]. Based on our observations, the reported deleterious effects of glyoxal on the
architecture and chemical integrity of tissues are likely linked to the acidity of the fixing
solutions. Indeed, commercial solutions of glyoxal are strongly acidic, due to the presence of
variable concentrations of glyoxilic, glycolic, formic, acetic and oxalic acids . This leads to the
tendency of the dialdehyde to oxidize and progressively acidify in solution, down to pH 4. The
main acidic component, i.e. glyoxylic acid, shows a 10 times stronger acidity than acetic acid,
with an acid dissociation constant of 4.7 × 10−4 (pKa = 3.32). As a consequence, all these acids
are bound to exert hydrolytic effects on tissue structure, proteins and nucleic acids. Moreover,
previous studies [
3, 13, 14, 17, 26, 27
] only refer to the use of commercially available
formulations having glyoxal as the main component, in addition to other reagents, partly undisclosed.
All of these property fixatives (Glyo-Fix, Shandon; Histo-Fix, Bioworld; Histo-CHOICE,
Amresco; Prefer, Anatech and Safe-Fix II, Fisher Scientific) are reportedly acidic (in the range
of pH 4) and this is likely to represent the reason of the observed drawbacks, especially on
nucleic acid preservation.
To guarantee the use of glyoxal in acid-free conditions, we adopted a two-fold procedure.
First, a substantial decrease of acids was obtained by the use of ion-exchange resins. Second,
long-term (up to 6 months) stable neutral conditions were obtained by the addition of ethanol
and calcium carbonate, which is known to react with acids by producing neutral calcium salts
By using this GAF solution as a histological fixative we obtained a satisfactory preservation
of tissue and cell components. Specifically, unlike what previously reported [
erythrocytes and eosinophils were properly stained. Immunohistochemical tests performed in
GAFfixed material gave results basically matching the tests performed on PBF-fixed on parallel
Of note, the present results on antigen detection by IHC are different from those reported
by previous groups [12±14] using acidic glyoxal solutions such as Prefer or Glyo-Fix, where
the immuno-histochemical results were generally regarded as sub-optimal. It has been
reported that some antigens may require treatment at high temperature (125ÊC) in a highly
basic buffer (pH 8,6) [
]. Such treatment was not needed in our material and, when tested,
did not yield significant improvement. We should point out however that Ki67 staining gave
satisfactory results only following a longer treatment (60' versus 30') using an acidic buffer.
Under these conditions and in the limited number of samples tested, the intensity of the
staining was satisfactory, however the number of Ki67 positive nuclei was lower than those
detectable in the parallel PBF-fixed tissues. More extensive, detailed and comparative studies, also
including systematic automatic scoring of Ki67 reactions are warranted to evaluate
quantitative differences between GAF- and PBF-fixed tissues as this may have an impact in
subclassification of lesions and their related malignant potential.
In terms of molecular pathology analyses, the mild autofluorescent background with FISH
testing is likely due to the link of glyoxal to the DNA bases, mainly to guanine and cytidine
], which results in cross-links most likely responsible of the observed nuclear
auto-fluorescence. In line with previous observations by Hutton and Wetmur [
] on the reversibility of
the binding of glyoxal to DNA in alkaline pH, the autofluorescence disappeared following a
short passage in an alkaline buffer (pH 8.6).
Finally, the three sequencing platforms here tested (Sanger sequencing, Pyrosequencing,
Sequenom MassARRAY1) gave comparable results for KRAS testing on both PBF- and GAF
fixed samples suggesting a DNA/RNA fragmentation not lower than that induced by formalin
13 / 16
crosslinking. Of note, we observed an enrichment of the GAF-fixed samples for longer DNA
fragment size. In our study, the performance of the NGS analysis was comparable between
GAF-fixed ad PBF-fixed samples, nevertheless the platform here employed relied on a robust
targeted NGS assay designed for DNA extracted from FFPE samples. One could hypothesize
that whole exome sequencing approaches are likely to yield better results in GAF-fixed samples
showing a lesser degree of fragmentation/degradation. It is interesting to note that some
mutations detected at low mutant allele frequency (<5%) in PBF-fixed samples showed the typical
C:G>T:A call, which may represent an artifactual change most readily explained as a
consequence of cytosine being deaminated to uracil, a known consequence of formalin fixation [23±
25]. Larger studies are warranted to ascertain whether GAF fixed samples could reliably
minimize C:G>T:A changes as well as other artefactual calls induced by formalin fixation.
In conclusion, the data here presented indicate that fixation in a pH 7.2±7.4 GAF solution
results in a histological, immunohistochemical and nucleic acid preservation not inferior to
that permitted by fixation in PBF. Importantly, DNA appears to be better preserved by GAF
rather than by PBF fixation.
Substitution of formalin as the reference fixative in histopathology is recommended because
of its toxicity, but cannot be presently practiced since this might jeopardize the invaluable
array of common morphological, immunophenotypic and molecular parameters currently
derived from PBF-fixed tissues and playing a pivotal role in personalized therapies.
Adoption of GAF as a non-toxic histological fixative of choice would therefore require a
process of validation, but the present study indicates that it represents a reliable candidate.
S1 Fig. Parallel tissue samples, fixed in GAF and PBF, of normal colonic mucosa showing
comparable preservation of tissue architecture.
S2 Fig. Non synonymous single nucleotide variants (SNVs) detected by using the
Myriapod1 NGS-IL 56G Onco-panel in each sample pair (GAF- and corresponding PBF-fixed
samples for the eight cases subjected to molecular assays). Ranges of mutant allele
frequencies are color-coded according to the legend on the right hand side. Squares with black borders
identify those variants with a mutant allele frequency (MAF) <5% showing a C:G>T:A call.
S1 Table. List of genes represented in Myriapod1 NGS-IL 56G Onco-panel (NG032,
Diatech Pharmacogenetics, Jesi, Italy) and number of amplicons.
This work was supported by the Italian Ministry of Education, University and Research
(MIUR ex-60%-2016-17 to CM and PRIN 2015HAJH8E), by AIRC, Associazione Italiana per
la Ricerca sul Cancro (MFAG13310 to CM), and by 5xMille Ministero Salute 2011ÐFPRC
onlus AIRC 5xMille Molecular Clinical Oncology Extension programÐRef 9970 (to AS). We
would like to thank Mrs. Maria Stella Scalzo for support in IHC and FISH assays and dr.
Gianluca Barbieri (Diatech Pharmacogenetics, Jesi, Italy) for support in NGS data processing.
Conceptualization: Gianni Bussolati, Anna Sapino, Caterina Marchiò.
14 / 16
Data curation: Gianni Bussolati, Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara
Panero, Marco Cupo, Patrizia Gugliotta, Tiziana Venesio, Anna Sapino, Caterina Marchiò.
Formal analysis: Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara Panero, Patrizia
Gugliotta, Tiziana Venesio, Caterina Marchiò.
Funding acquisition: Anna Sapino, Caterina Marchiò.
Investigation: Gianni Bussolati, Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara
Panero, Marco Cupo, Patrizia Gugliotta, Tiziana Venesio.
Methodology: Gianni Bussolati, Tiziana Venesio, Caterina Marchiò.
Project administration: Anna Sapino, Caterina Marchiò.
Resources: Gianni Bussolati, Anna Sapino, Caterina Marchiò.
Software: Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara Panero, Marco Cupo,
Supervision: Anna Sapino, Caterina Marchiò.
Validation: Gianni Bussolati, Anna Sapino, Caterina Marchiò.
Visualization: Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara Panero, Tiziana
Venesio, Caterina Marchiò.
Writing ± original draft: Gianni Bussolati, Laura Annaratone, Anna Sapino, Caterina
Writing ± review & editing: Laura Annaratone, Tiziana Venesio, Caterina Marchiò.
15 / 16
1. Blum F. Der formaldehyde als hartungsmittel . ZWiss Mikrosc . 1893 ; 10 : 314 .
2. Fox CH , Johnson FB , Whiting J , Roller PP . Formaldehyde fixation . J Histochem Cytochem . 1985 ; 33 ( 8 ): 845 ± 53 . Epub 1985/08/01. https://doi.org/10.1177/33.8.3894502 PMID: 3894502 .
3. Buesa RJ . Histology without formalin? Ann Diagn Pathol. 2008 ; 12 ( 6 ): 387 ± 96 . Epub 2008/11/11. https://doi.org/10.1016/j.anndiagpath. 2008 . 07 .004 PMID: 18995201 .
4. Wicks LF , Suntzeff V . Glyoxal, a Non-Irritating Aldehyde Suggested as Substitute for Formalin in Histological Fixations . Science . 1943 ; 98 ( 2539 ): 204 . Epub 1943/08/27. https://doi.org/10.1126/science.98. 2539.204 PMID: 17843715 .
5. Harke HP , HoÈffler J. UÈ bergang antimikrobieller Wirkstoffe von der FlaÈche in die Luft . Hygiene und Medizin . 1984 ; 9 : 259 ± 60 .
6. Betterton EA , Hoffmann MR . Henry's law constants of some environmentally important aldehydes . Environ Sci Technol . 1988 ; 22 ( 12 ): 1415 ±8. https://doi.org/10.1021/es00177a004 PMID: 22200466 .
7. TOXNETÐToxicology Data Network . GLYOXAL (CASRN: 107-22-2) . https://toxnet.nlm.nih.gov/cgibin/sis/search/a?dbs+hsdb:@term+@DOCNO+ 497 .
8. World Health Organization/International Programme on Chemical Safety. Concise International Chemical Assessment Document No. 57 Glyoxal . 2004 .
9. Sabatini DD , Bensch K , Barrnett RJ . Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation . J Cell Biol . 1963 ; 17 : 19 ± 58 . Epub 1963/ 04/01. PMID: 13975866 .
10. Hopwood D. The elution patterns of formaldehyde, glutaraldehyde, glyoxal and alpha-hydroxyadipaldehyde from sephadex G-10 and their significance for tissue fixation . Histochemie . 1969 ; 20 ( 2 ): 127 ± 32 . Epub 1969/01/01. PMID: 4901926 .
11. Dapson RW . Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval . Biotech Histochem . 2007 ; 82 ( 3 ): 161 ± 6 . Epub 2007/11/08. https://doi.org/10.1080/10520290701488113 PMID: 17987441 .
12. Marcon N , Bressenot A , Montagne K , Bastien C , Champigneulle J , Monhoven N , et al. Le glyoxal: un possible substitut polyvalent du formaldeÂhyde en anatomie pathologique? [Glyoxal: a possible polyvalent substitute for formaldehyde in pathology?] . Ann Pathol. 2009 ; 29 ( 6 ): 460 ± 7 . Epub 2009/12/17.
13. Umlas J , Tulecke M. The effects of glyoxal fixation on the histological evaluation of breast specimens . Hum Pathol . 2004 ; 35 ( 9 ): 1058 ± 62 . Epub 2004/09/03. PMID: 15343506 .
14. Tubbs RR , Hsi ED , Hicks D , Goldblum J . Molecular pathology testing of tissues fixed in prefer solution . Am J Surg Pathol . 2004 ; 28 ( 3 ): 417 ± 9 . Epub 2004/04/24. PMID: 15104311 .
15. Willmore-Payne C , Metzger K , Layfield LJ . Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization . Appl Immunohistochem Mol Morphol . 2007 ; 15 ( 1 ): 84 ± 7 . Epub 2007/06/01. PMID: 17536313 .
16. Lassalle S , Hofman V , Marius I , Gavric-Tanga V , Brest P , Havet K , et al. Assessment of morphology, antigenicity, and nucleic acid integrity for diagnostic thyroid pathology using formalin substitute fixatives . Thyroid . 2009 ; 19 ( 11 ): 1239 ± 48 . Epub 2009/11/06. https://doi.org/10.1089/thy. 2009 .0095 PMID: 19888862 .
17. Gillespie JW , Best CJ , Bichsel VE , Cole KA , Greenhut SF , Hewitt SM , et al. Evaluation of non-formalin tissue fixation for molecular profiling studies . Am J Pathol . 2002 ; 160 ( 2 ): 449 ± 57 . Epub 2002/02/13. https://doi.org/10.1016/S0002- 9440 ( 10 ) 64864 -X PMID: 11839565 .
18. Foss RD , Guha-Thakurta N , Conran RM , Gutman P. Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls . Diagn Mol Pathol . 1994 ; 3 ( 3 ): 148 ± 55 . Epub 1994/09/01. PMID: 7981889 .
19. Zhang Z , Zhao D , Xu B. Analysis of glyoxal and related substances by means of high-performance liquid chromatography with refractive index detection . J Chromatogr Sci . 2013 ; 51 ( 10 ): 893 ± 8 . Epub 2012/ 11/28. https://doi.org/10.1093/chromsci/bms186 PMID: 23180757 .
20. Dapson RW , Feldman AT , Wolfe D. Glyoxal Fixation and Its Relationship to Immunohistochemistry . Journal of Histotechnology . 2006 ; 29 ( 2 ): 65 ± 76 .
21. Sapino A , Maletta F , Verdun di Cantogno L , Macri L , Botta C , Gugliotta P , et al. Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method . Oncologist . 2014 ; 19 ( 11 ): 1118 ± 26 . Epub 2014/10/18. https://doi.org/10. 1634/theoncologist.2014-0195 PMID: 25323485 .
22. Illumina . Expression analysis of FFPE Samples. Illumina Technical Note: RNA Sequencing . 2014 .
23. Do H , Wong SQ , Li J , Dobrovic A . Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates . Clin Chem . 2013 ; 59 ( 9 ): 1376 ± 83 . https://doi.org/10.1373/clinchem. 2012 .202390 PMID: 23649127 .
24. Do H , Dobrovic A . Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization . Clin Chem . 2015 ; 61 ( 1 ): 64 ± 71 . https://doi.org/10.1373/clinchem. 2014 .223040 PMID: 25421801 .
25. Kim S , Park C , Ji Y , Kim DG , Bae H , van Vrancken M, et al. Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine . J Mol Diagn . 2017 ; 19 ( 1 ): 137 ± 46 . https://doi.org/10.1016/j.jmoldx. 2016 . 09 .006 PMID: 27840062 .
26. Titford ME , Horenstein MG . Histomorphologic assessment of formalin substitute fixatives for diagnostic surgical pathology . Arch Pathol Lab Med . 2005 ; 129 ( 4 ): 502 ± 6 . Epub 2005/03/30. PMID: 15794674 .
27. Prento P , Lyon H . Commercial formalin substitutes for histopathology . Biotech Histochem . 1997 ; 72 ( 5 ): 273 ± 82 . Epub 1997/12/31. PMID: 9408588 .
28. Brooks BR , Klamerth OL . Interaction of DNA with bifunctional aldehydes . Eur J Biochem . 1968 ; 5 ( 2 ): 178 ± 82 . Epub 1968/07/01. PMID: 5667353 .
29. Hutton JR , Wetmur JG . Effect of chemical modification on the rate of renaturation of deoxyribonucleic acid. Deaminated and glyoxalated deoxyribonucleic acid . Biochemistry . 1973 ; 12 ( 3 ): 558 ± 63 . Epub 1973/01/30. PMID: 4683497 .