F-SOLV

HISTOLOGICALLY FIXATIVE - 100% FORMALDEHYDE FREE - READY TO USE
and safe 

1. F-solv does not contain any hazardous products and the result obtained is equal to or better
then a fixation with formaldehyde.  F-solv does not irritate the eyes, throat and nose.
2. F-solv is highly suitable for PCR by means of crosslinking, is selective for the
protein, and will not affect the structure of DNA or RNA
3. F-solv the fixation is firm but still pliable enough to cut.
4. F-solv contains products that prevent the growth of viruses, fungi and bacteria
making the fabric last longer.
5. F-solv has a disinfecting effect.
6. F-solv fixes whole tissues, preserving their structure for more than 1 year.
7. F-solv is environmentally friendly and cost effective. It can be diluted with
tap water can be flushed, while formaldehyde is chemical waste.
8. In the tissue processor, formaldehyde can simply be replaced by F-solv whereby
can fix whole organs.

Dutch tech file

HISTOLOGISCH FIXATIEF – 100% FORMALDEHYDE VRIJ - GEBRUIKSKLAAR
1.VEILIG:

 F-solv bevat geen gevaarlijke producten en het bekomen resultaat is gelijk aan of beter
dan een fixatie met formaldehyde.
 F-solv werkt niet irriterend voor ogen, keel en neus.
2. F-solv is uitermate geschikt voor PCR door middel van crosslinking, is selectief voor het
eiwit, en zal de structuur van DNA of RNA niet aantasten
3. F-solv is de fixatie stevig maar nog plooibaar genoeg om te snijden.
4. F-solv bevat producten die de groei van virussen, schimmels en bacteriën voorkomen
waardoor het weefsel langer houdbaar blijft.
5. F-solv werkt ontsmettend.
6. F-solv fixeert hele weefsels, waardoor hun structuur meer dan 1 jaar behouden blijft.
7. F-solv is milieu vriendelijk en kostenbesparend. Het kan door verdunning met
kraantjeswater doorgespoeld worden, terwijl formaldehyde chemisch afval is.
8. In de weefselprocessor kan men formaldehyde gewoon vervangen door F-solv waarbij men
hele organen kan fixeren

F-solv – Fixatiemiddel in de thanatopraxie

Weg met het toxisch en kankerverwekkende formol , weg met de irriterende geur, tranende ogen, een schurende keel… een werkend alternatief ( zonder formaldehyde) is F-solv.

In het uitvaartwezen ( in de thanatopraxie) wordt gebalsemd/gefixeerd met formaldehyde dat toxisch en kankerverwekkend[1] is ( zie studie). Vanuit onze chemische kennis in de medische sector ( meer bepaald in de pathologie) werd dit vervangproduct ontwikkeld. F-solv ( zijnde een gedenatureerde alcoholoplossing met stabilisatie componenten, geactiveerd met een zeer laag aldehyde derivaat ) is helder, vrij reukloos en zeer aangenaam om mee te werken.

De preservering van het lichaam is minimum 10 dagen of langer, waarbij ook de soepelheid van de weefsels gegarandeerd bleven ( zie praktisch resultaten ).

Prijs :  14,9 Euro / liter excl. Btw en verzendkosten

            Aanbieding geldig tot 31/08/2012 : 10 L + 2 L F-solv gratis

• Onverdund te gebruiken voor de holtebehandeling (Cavity)
•  5 à 7 % te gebruiken voor de slagaders (Arterial)

[1] Zie ook studie Michael Hauptmann (NKI-AVL) in samenwerking met de Amerikaanse National Cancer Institue (25 Nov 2009 – Journal of the National Cancer Institute)

F-solv
Door de firma Yvsolab NV uit Beerse werd het nieuw fixeermiddel F-solv ontwikkeld. Het fixeermiddel is 100% formaldehydevrij
en door z'n samenstelling - op basis van een alcoholoplossing met stabilisatiecomponenten en een aldehydederivaat -
nagenoeg onschadelijk. (Yvsolab)
Het blijft echter een chemisch produkt, vandaar als bijlage : 1. Informatieblad F-solv.
2. Veiligheidsinformatieblad F-solv
Er waren vorig jaar nog weinig gegevens beschikbaar ivm de benodigde concentratie van het produkt voor het fixeren van
biologische handnet- en waterbodemstalen. Daarom werd door drie buitendiensten een handnetstaal genomen van het meest
voorkomende substraat ( bvb voor Oo een kleisubstraat ) met waterplanten. Het staal werd verdeeld en gefixeerd met
respectievelijk 100%, 50%, 25% en 10% F-solv . Bij een staal van bvb 1L werd 0.5 L F-solv toegevoegd om een fixatie van
50% te bekomen. Na enkele maanden werden de stalen gespoeld en getriëerd en werd nagegaan vanaf welke minimale
concentratie de organismen goed gefixeerd en geconserveerd waren.
Thierry Vercauteren, PIH Antwerpen, gebruikt F-solv reeds geruime tijd en was bereid zijn ervaringen met het produkt tbv dit
rapport door te geven.
a. Resultaten
- Het staal van Bd Gent (Joost.Mertens) moest reeds een tweetal weken na de staalname verwerkt worden.
De fixatie was voor alle concentraties goed, conserveringsduur te kort om een uitspraak te doen.
- Het staal van Oostende (Erg) was afkomstig van de Keygnaertkreek op 6/05/09 en vijf maand later gespoeld op 4/09/09.
Het staal bevatte veel kleigrijs en kleikorrels en veel detritus.
Bij de 10% verdunning bleken de wormen en vedermuglarven zeer slapjes en geel-beige van kleur, geen bloedzuigers
aanwezig. Ook de Gammaridae en Mollusca waren niet gefixeerd zoals bij fixatie met formaldehyde.
Vanaf verdunning met 25% F-solv is de fixatie veel beter : vastere structuur (vlokreeften iets minder) en ook het lichaam van
grote Lymnaea sp is steviger dan bij een verdunning met 10%. Ook de schelpstructuur van de Mollusca stevig en normaal van
kleur.
- Het staal van Bd Leuven (Saskia .Scheers. & Eric .Coenen.) werd genomen op het meetpunt 426500 ( 01/07/09), substraat
bestond uit stenen, grint en zand Na drie maand werd het staal een eerste maal nagezien, het spoelen en triëren gebeurde na
zeven maand op 3/02/10.
Met de 10% F-solv verdunning bleek de kleur van de MI geler dan bij de andere verdunningen.
Voor de overige verdunningen waren alle MI goed gefixeerd en geconserveerd.
Bij elke verdunning valt op dat de schelp van de slakken veel brozer is dan wanneer er Norvanol werd gebruikt.
- Monster van het PIH ( verslag Th.V.) was afkomstig uit zandige waterloop met veel stenen en bladeren; monster
ondergedompeld in water van waterloop + 1/4 volume F-Solv . Begindatum 22/10/2008 - bekeken 13-14/1/2010 .
In monsters met weinig bladeren: geen opvallende weke of ontkalkte driehoeksmossels, brakwaterhorentjes en grotere
Planorbidae, wel broze randen bij een aantal poelslakken en mutsslakken. Deze broosheid had ik vroeger ook met opslag in
formol. Monsters met veel bladeren: ontkalking bij Planorbidae, poelslakken en erwtenmosseltjes.
Insecten, borstelwormen: meestal iets minder 'stijf' dan bij fixatie in formol.
Schaaldieren (vlokreeften, pissebedden): wisselvallig: sommige exemplaren stijf en broos, andere dan weer slapper.
b. Samenvatting ( VMM-PIH)
- De 10% F-solv fixatie lijkt aanvankelijk voldoende ( staal Gent) maar voldoet na verloop van tijd niet, noch als fixatief noch als
bewaarmiddel . ( stalen Leuven en Oostende).
- Met een 25% F-solv fixatie zijn de organismen voldoende gefixeerd en geconserveerd. Er werd na de fixatieperiode geen
verschil opgemerkt met de organismen uit de 50% - en 100% F-solv fixatie's.
Opmerkingen : - (Erg -Th.V.) Organismen zijn in veel gevallen iets minder stevig dan bij een Formolfixatie. Dit staat vermeld in
het Informatieblad F-solv : ...fixatie is stevig maar nog plooibaar.... .
- ( S.S. -Th.V.) De schelp van sommige Mollusca is broos . Dit is een gelijkaardig probleem als met formol,
bij een "fixatie" met bewaarvloeistof Norvanol kwam dat minder voor (S.S.). Oorzaak van deze ontkalking kan
liggen bij een zuur monster of bij verzuring tijdens fixatieperiode. Misschien kan bekalken van het staal een
oplossing brengen, wordt vervolgd (Th.V.).

op basis van prijsoffertes maart 2009
Formaldehyde : 18 €  per 5 Liter
F-solv : 55 €  per 5 Liter
Praktisch : - voor een staal van 1 Liter dient 0.1 L Formaldehyde ter fixatie toegevoegd = kostprijs van 0€27
- voor een staal van 1 Liter dient 0.25 L F-solv ter fixatie toegevoegd = kostprijs van 1€75

3. Besluit
Door z'n zware toxiciteit, zowel voor de monsternemer als voor het milieu, is Formaldehyde geen optie meer om algemeen als
fixeermiddel te worden gebruikt.
Alhoewel gevoelig duurder in gebruik is F-solv een kwaliteitsvol alternatief voor Formaldehyde. VMM, PIH en KBIN doen verder
testen met F-solv in verband met de bewaring van organismen op langere termijn.
Fixeren met F-solv en nadien de organismen bewaren in Norvanol voor alle handnet- en waterbodemstalen vereenvoudigt
tevens de taak van het KBIN als collectiebeheerder

Formaldehyde Substitute Fixatives

Analysis of Macroscopy, Morphologic Analysis,
and Immunohistochemical Analysis
Cathy B. Moelans, PhD, Natalie ter Hoeve, Jan-Willem van Ginkel, Fiebo J. ten Kate, MD, PhD,
and Paul J. van Diest, MD, PhD
Key Words: Fixative; Formalin; Formaldehyde; FineFIX; RCL2; F-Solv; Fixation
DOI: 10.1309/AJCPHH1B0COCBGOM
Abstract
Because formaldehyde is toxic and creates crosslinks that may hinder immunohistochemical studies, we
tested 3 new cross-linking (F-Solv [Adamas, Rhenen,
the Netherlands]) and non–cross-linking (FineFIX
[Milestone, Bergamo, Italy] and RCL2 [Alphelys,
Plaisir, France]) alcohol-based fixatives for routine
staining in comparison with neutral buffered formalin
(NBF) as the “gold standard.” Fresh tissue samples
were divided into 4 equal pieces and fixed in all
fixatives for varying times. After paraffin embedding,
H&E staining, 7 common histochemical stains, and 9
common immunohistochemical stains were performed.
RCL2 fixation resulted in soft and slippery tissue,
causing sectioning difficulties. F-Solv and FineFIX
led to partial tissue disintegration during fixation.
F-Solv performed morphologically similar to NBF but
needed considerable protocol adjustments before being
applicable in daily histologic and immunohistochemical
practice. FineFIX did not necessitate major protocol
changes but caused shrinkage artifacts, degranulation,
and lysis of RBCs. RCL2 generated morphologically
overall good results without major protocol changes
but caused pigment deposition, degranulation, and
RBC lysis. The alcohol-based fixatives had positive
and negative attributes and environmental drawbacks,
and none was overall comparable to NBF with regard
to macroscopy, morphologic evaluation,
and immunohistochemical studies.
An optimal fixative should be nontoxic and allow
for detailed morphologic analysis, high-quality special
histochemical and immunohistochemical staining, and
good preservation of DNA and RNA at a reasonable price.
Unfortunately, such a universal fixative does not exist, and
it is important to assess the advantages and drawbacks of
existing and new fixatives for each platform.
In surgical pathology, neutral buffered formalin
(NBF, aqueous solution of 4% buffered formaldehyde) has
been the “gold standard” fixative for decades. It is cheap,
enables long-term storage of surgical material, preserves
morphologic features well, allows special histologic stains,
and, in combination with antigen retrieval, allows for reliable
immunohistochemical analysis. However, formaldehyde
was classified as “carcinogenic to humans” (group 1) by
the International Agency for Research on Cancer1 and,
therefore, represents a risk to anyone handling the solution.
Furthermore, its cross-linking masks antigens, which may
hamper immunohistochemical analysis,2-7 and fragments
nucleic acids, which impairs the extraction efficiency and
quality of DNA and RNA.8-11
Other less toxic alcohol-based cross-linking fixatives
(such as the aldehyde-containing F-Solv [Adamas, Rhenen,
the Netherlands]) and non–cross-linking fixatives (such as
FineFIX [Milestone, Bergamo, Italy] and RCL2 [Alphelys,
Plaisir, France]) have been proposed as NBF alternatives.
Alcohol-based non–cross-linking fixatives exert their effect
by protein precipitation. Reported advantages of this type of
fixation include faster fixation; elimination of carcinogenic
vapors; better preservation of glycogen, DNA, and RNA;
Upon completion of this activity you will be able to:
• list the advantages and disadvantages of formalin as a universal
fixative.
• discuss the effects of alcohol-based fixatives on tissue morphology
and immunohistochemistry.
• compare the effects and costs of crosslinking and non-crosslinking
alcohol-based fixatives in pathology.
The ASCP is accredited by the Accreditation Council for Continuing
Medical Education to provide continuing medical education for physicians.
The ASCP designates this journal-based CME activity for a maximum of 1
AMA PRA Category 1 Credit ™ per article. Physicians should claim only the
credit commensurate with the extent of their participation in the activity.
This activity qualifies as an American Board of Pathology Maintenance of
Certification Part II Self-Assessment Module.
The authors of this article and the planning committee members and staff
have no relevant financial relationships with commercial interests to disclose.
Questions appear on p 654. Exam is located at www.ascp.org/ajcpcme.
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Am J Clin Pathol 2011;136:548-556 549
549 DOI: 10.1309/AJCPHH1B0COCBGOM 549
© American Society for Clinical Pathology
Anatomic Pathology / Original Article
greater staining avidity; and no need for enzyme predigestion
for immunohistochemical analysis. Disadvantages are
variability of tissue staining, tissue shrinkage and hardening,
artifactual pigment deposition in bloody specimens, partial or
complete lysis of erythrocytes, and increased flammability.12,13
Another component increasingly used in alternative fixatives
is acetic acid (such as in RCL2). It complements the action of
other ingredients such as alcohol, makes collagen fibers swell,
precipitates nucleoprotein, and may have a solvent action on
cytoplasmic granules.14
Use of formaldehyde substitutes has been restricted to a
few laboratories, and publications about them are scarce. We
report on the influence of F-Solv (cross-linking), FineFIX
(non–cross-linking, non–acetic acid), and RCL2 (non–
cross-linking, acetic acid) fixation on tissue morphologic
features and the quality of special histologic stains and
immunohistochemical stains in comparison with NBF as the
gold standard.
Materials and Methods
Fixatives
All fixatives were stored and used at room temperature.
NBF 4%, FineFIX, and RCL2 were prepared fresh from
stock solutions before use according to the manufacturers’
instructions. F-Solv was received in a ready-to-use form.
Specimens and Fixation
Fresh surgical tissue samples from placenta, liver, brain,
esophagus, stomach, duodenum, colon, omentum, lung, breast,
adrenal gland, kidney, lymph node, thyroid, tonsil, spleen, and
gallbladder were collected at the Department of Pathology,
University Medical Center Utrecht, Utrecht, the Netherlands.
Procedures followed were in accord with the ethical standards
established by the University Medical Center Utrecht. Each
tissue sample was divided into 4 equal pieces and fixed in one
of the fixatives for 24 hours (n = 16), 2 to 4 days (n = 11), 1
to 2 weeks (n = 5), or 1 to 2 months (n = 7).
To compare shorter and standard fixation times, 10
additional tissue samples were cut and divided into 8 pieces
(4 for 4-hour fixation and 4 for 24-hour fixation). After
fixation, tissues were dehydrated and paraffin impregnated
using an automated Peloris Rapid Tissue Processor (Leica,
Valkenswaard, the Netherlands). In short, the Peloris ran
overnight starting with 3 dehydrating ethanol 70% baths
(60°C for a total of 1 hour, 30 minutes), followed by two
80/20 ethanol/isopropyl alcohol (a xylene replacement)
baths at 60°C for a total of 1 hour, 30 minutes), 3 isopropyl
alcohol (100%) baths (at 60°C for 3 hours), and paraffin
impregnation (2 baths at 85°C for 2 hours and 1 bath at 65°C
for 40 minutes). After paraffin embedding, blocks were
stored at room temperature.
H&E Staining and Histochemical and
Immunohistochemical Studies
H&E Staining and Microscopic Evaluation
Paraffin sections were cut at 2 μm, mounted on glass
slides, dried for at least 10 minutes on a hot plate, and
processed with an HMS740 (for F-Solv-, FineFIX-, and
RCL2-fixed tissue sections) and an HMS760 (for NBFfixed tissue sections, along with diagnostics) Robot Stainer
(Microm, Walldorf, Germany). H&E staining protocols for
F-Solv, FineFIX, and RCL2 were adjusted to avoid bias of
the pathologist in microscopic evaluation because staining
according to the protocol for NBF specimens was generally
darker. For F-Solv, the hematoxylin step was reduced from 2
minutes, 15 seconds to 1 minute, 15 seconds; eosin staining
was reduced from 1 minute to 5 seconds; and the subsequent
alcohol steps were increased from 2 minutes, 30 seconds to 4
minutes, 30 seconds. For FineFIX and RCL2, the hematoxylin
step was also reduced from 2 minutes, 15 seconds to 1 minute,
15 seconds; eosin staining was reduced from 1 minute to 10
seconds; and the subsequent alcohol steps were increased
from 2 minutes, 30 seconds to 3 minutes. All other steps were
kept identical to those for the NBF protocol.
A blinded evaluation of H&E staining was carried out by
an experienced pathologist (F.J.tK.). The physical quality of the
sections (disruption, adhesion, cracking, and section thickness),
the quality of tissue preservation (nucleus, cytoplasm,
extracellular components, special tissue-specific features, and
zonal fixation), and the quality of staining (uniformity, nuclear,
cytoplasmic, and extracellular components or muscle) were
separately evaluated. The staining quality for these aspects was
graded as 0 (inadequate for diagnostics), 1 (quality reasonable
for diagnostics, but adjustments in protocol needed), or 2
(quality good for diagnostics). For each fixative, a total
percentage was calculated based on the ratio of the sum of
all scores (given to different tissue samples) relative to the
maximum score possible.
Histochemical Evaluation
Paraffin blocks were sectioned at 2 to 4 μm for staining
with 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. Alcian blue,
PAS, and PASD stains were done in an HMS740 Robot
Stainer. The other stains were done manually.
For alcian blue, slides were deparaffinized in xylene,
rehydrated through graded ethanol, stained with alcian blue
for 15 minutes, rinsed, stained with nuclear fast red for 10
minutes, rinsed again, and dehydrated through graded ethanol
and xylene.
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550 Am J Clin Pathol 2011;136:548-556
550 DOI: 10.1309/AJCPHH1B0COCBGOM
© American Society for Clinical Pathology
Moelans et al / Formaldehyde Substitute Fixatives
For PAS, slides were deparaffinized in xylene, rehydrated
through graded ethanol and incubated in periodic acid (1%) for
15 minutes. After a short rinsing step, slides were incubated in
Schiff reagent for 30 minutes, rinsed, incubated in hemalaun
for 8 minutes, rinsed again, and dehydrated to xylene.
For PASD, slides were deparaffinized up to 96% alcohol,
then incubated for 5 minutes in a 1/1 mixture of 37%
formalin/96% alcohol, rinsed thoroughly, and incubated in
0.15% diastase for 90 minutes at room temperature. After
rinsing, slides were further processed as described for PAS.
For EvG, slides were deparaffinized and rehydrated up
to 70% alcohol, incubated for 45 minutes in Lawson solution,
differentiated in 100% and 96% alcohol, rinsed, stained
with Mayer hemalaun for 5 minutes, rinsed for 10 minutes,
incubated with van Gieson picrofuchsin for 5 minutes, and
dehydrated in graded ethanol and xylene.
For G&S staining, slides were deparaffinized and
rehydrated, oxidated in acidified potassium permanganate for
5 minutes, rinsed, bleached in 1% oxalic acid for 2 minutes
until colorless, and rinsed and treated with 2% iron alum for 15
minutes. After rinsing in distilled water and impregnation with
ammoniacal silver solution, slides were rinsed and reduced
in 3.7% aqueous formalin, rinsed again, incubated in 0.1%
gold chloride for 5 minutes, and fixed in 5% aqueous sodium
thiosulfate for 5 minutes. Finally, slides were counterstained
with nuclear fast red for 5 minutes and dehydrated.
For Jones staining, slides were deparaffinized and
rehydrated, treated with 1% periodic acid for 15 minutes,
rinsed, and incubated for 40 to 60 minutes in a methenamine
silver working solution at 56°C. After rinsing, the slides were
incubated in 0.1% gold chloride for 5 minutes and in 2%
sodium thiosulfate for 5 minutes, followed by a 5-minute
Mayer hemalaun stain and bluing. Finally, slides were stained
with eosin and dehydrated.
For azan (Heidenhain), slides were deparaffinized
and rehydrated, stained with azocarmine G for 5 minutes,
quickly rinsed, and differentiated in aniline-ethanol. After
rinsing in acetic acid–ethanol, slides were incubated in a 5%
phosphotungstic acid solution for 15 minutes, rinsed, and
stained with an aniline blue–orange G solution for 5 minutes
before dehydration.
A blinded evaluation of the histologic stains was carried
out by an experienced pathologist (F.J.tK.). Staining quality
was graded as 0 (inadequate for diagnostics), 1 (quality
reasonable for diagnostics, but adjustments in protocol needed),
or 2 (quality good for diagnostics). For each fixative, a total
percentage was calculated based on the ratio of the sum of
all scores (given to different tissue samples) relative to the
maximum score possible.
Immunohistochemical Evaluation
Paraffin sections were cut at 4 μm, mounted on silanecoated slides, dried for 10 minutes or more on a hot plate,
and processed with a Bond-Max automated staining machine
(Vision Biosystems, Newcastle, England) using the Bond
polymer refine detection kit (catalog No. DS9800, Vision
Biosystems), as previously described.15 ❚Table 1❚ shows the 9
primary antibodies tested, sources, dilutions, and antigen
retrieval methods. For each antibody, stains were done as usual
for NBF, and 2 samples were tested with and without heatinduced antigen retrieval or pepsin pretreatment. A blinded
evaluation of the immunostaining was carried out by an
experienced pathologist (F.J.tK.). Staining quality was graded
as 0 (inadequate for diagnostics), 1 (quality reasonable for
diagnostics, but adjustments in protocol needed), or 2 (quality
good for diagnostics). For each fixative, a total percentage was
calculated based on the ratio of the sum of all scores (given to
different tissue samples) relative to the maximum score possible.
Results
Macroscopy
All fixatives had different penetration speed and resulted
in different tissue color and texture. Tissue color after F-Solv
fixation was much darker than with NBF.