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| Formalin-free Tissue Fixative |
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| RCL2®, Formalin-free tissue fixative |
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| RCL2®, a true solution
for formalin-free tissue fixation for a better service to Patient |
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| Tissues fixation has been for decades using
formaldehyde based reagents such as formalin or AFA (alcohol+formalin+acetic
acid). Formaldehyde reacts with the bio specimen by creating
covalent bounds between biological macromolecules, thus
stabilizing them and inhibiting enzymatic activities.
While of massive use in all pathology labs of the world
whether in human or animal histology, formalin and its
variations demonstrate two major well known for ages
disadvantages:
(1) Formalin is a highly toxic reagent. Classified carcinogenic
of class I by WHO since October 2006, it is demonstrated
to be at the proven beginning of respiratory pathologies,
allergies and respiratory tract cancers.
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(2) The chemical action of
formalin binds severely DNA, RNA and proteins and makes
them difficult or even impossible to extract in adequate
quantitiesand qualities for expanding molecular predictive
tests to new targeted treatments.
Developed since 1999 by Dr Philippe ROCHAIX, pathologist
at the Department of Pathology of Professor VOIGT, Cancer
Research Centre Claudius Regaud, Toulouse, France, RCL2®
is a new, truly innovative tissue fixative, preserving
tissue structures and cells. |
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| Make molecular diagnosis
on each and every tissue specimen for a better service
to the Patient thanks to RCL2® |
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Specimen formalin fixation is a major obstacle to medical
research and the development of molecular techniques
for patient profiling in the identification of targeted
therapies. RCL2® fixes tissues while preserving
histological and molecular components at a high level
of quality and yield.
By changing to RCL2®, you facilitate Patient access
to most modern and efficient therapeutic methods to
better cure without altering unnecessarily Patient quality
of life and while preserving Health costs.
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Adopt RCL2® and move your
lab to the molecular pathology era. Change for a better
service to Patient.
ALPHELYS will bring you all the required technical support
in protocols optimization to speed up the transition process. |
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| RCL2® allows you to
combine histology and molecular techniques on the exact
same specimen for a higher efficiency in your Research |
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The use of formalin in tissue
fixation for diagnosis and histology obliges laboratories
to deep-freeze part of each specimen for molecular research
applications (read below). Thus, molecular biology techniques
are achieved on frozen specimen different from the fixed
tissues which were used to obtain the lesion type and
the diagnosis.
Most of tissue and frozen tumour banks do not have the
required massive resources to do a systematic quality
control on each frozen tissue and guarantee that it corresponds
to the correct lesion type.
Using RCL2® allows the researcher to work exactly
on the same exact tissue to used for diagnosis dramatically
improving efficiency and accuracy of your Research projects.
RCL2® is the only tissue fixative that produces specimens
of quality compatible with molecular techniques even after
room temperature traditional fixation, without changing
your procedures. |
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RCL2® makes tissue and tumour banks accessible
to every lab
Degradations caused by formalin fixation on tissues oblige labs
to deep-freeze and store tissues specimens in costive environment
at investment as well as in maintenance.
The exceptional fixation properties of RCL2® allow you to
avoid deep-freezing. In a single regular fixation procedure,
you fix tissues which will be compatible with diagnosis as well
as molecular research and all techniques used to check tumour
responsiveness to new targeted therapies.
Using RCL2® simplifies laboratory procedures and reduces
your expenses.
Specimen collected from private labs and clinics constitute
a large part of the total recruitment of specimens. These specimens
can now be fixed with RCL2® and thus become available without
extra costs to Research.
RCL2® fixed tissues can be stored at room temperature or
at -20°C when a ultra high molecular quality is required.
Thus, required budgets for storing tissues are strongly reduced.
Exploitation is risk-less and requires only regular equipments
of cheaper maintenance costs.
Without changing your procedures, the use of RCL2® to fix
your tissues is a source of simplification, a source of money
savings and availability of specimens to medical Research. |
| RCL2®, the only truly
non toxic tissue fixative for Health and Environment |
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RCL2® components, which
preparation is patent protected, are demonstrated without
risks for Human health and Environment since decades in
compliance with European or American regulations and are
even authorized for Human food.
RCL2® Tissue Fixative has thus absolutely no risk to be one
day in the future classified as dangerous unlike some
other formalin-free fixatives that still contain dialdehydes,
molecule of similar activity to formaldehyde and that
can show dangers for Human health.
Using RCL2® preserves your staff and the efficiency
of your lab by eliminating any pathogenic and carcinogenic
risks of formalin.
Are you ready to invest efforts to change your tissue fixative
and take the risk that it could be classified dangerous
in the next future and that a new regulation obliges you
to change again? Make the right choice at once, not a
compromise, adopt RCL2®.
Download RCL2® MSDS here. |
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Achieve budget savings with RCL2®
You think that the new fixatives to substitute formalin are
more expensive than formalin?
Make a more global assessment of your costs.
Adopting specialized bench equipment to protect your staff from
formalin vapours is expensive. You have to maintain a permanent
audit of your environment to prevent contamination of your staff
or even worse a temporary interruption of your activity by medical
inspectors.
The management of toxic wastes, their collect and treatment
by specialized contractors is also costive and involve your
liability.
Make your cost assessment more globally and you will realize
that RCL2® allows you to save budgets while ensuring safety
and simplicity of use for you, your staff and your Environment.
Purchase serenity for yourself with RCL2®. |
| RCL2® IN IMAGES |
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| + RCL2® & Morphology |
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RCL2® preserves tissues
and cells morphology.
H&E staining displays morphology and colours similar
to those obtained with formalin fixed tissues. Morphology
is even preserved after many years of room temperature
storage as demonstrated by tests.
Unlike formalin which has a progressive chemical action
that continues even after fixation and during storage,
RCL2® action is immediate and complete at time of
contact with tissue. There is no degradation of tissue
after prolonged immersion giving more flexibility to lab
organization.
RCL2® produces a hardness that is pleasant for cutting
in macroscopy, it does not dilute Chinese ink that can
therefore be used to identify resection limits. |
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Breast cancer RCL2® fixed tissue section
stained with H&E. |
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| + RCL2® & Immuno-Histochemistry |
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RCL2® preserves the antigen
content. Numerous antigens have been tested with sucess
as shows the list below. Don't hesitate to contact us
to learn if your antigen has been tested also.
On many antigens, signal intensity can be superior allowing
for increasing antibody dilution for cost savings. You
will also observe that frequently antigens may not require
retrieval procedures, simplifying work at the lab and
providing extra costs savings.
As with any change in fixative, it is quite expected to
have some fine tuning to achieve.
ALPHELYS will bring you the necessary technical support
to speed up the transition to RCL2®. |
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TTF1 immunostaining with clone SPT24 RCL2®
fixed tissue, objective 10X. |
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Her2neu immunostaining on RCL2® fixed
tissues, objective 10X. |
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CD 30 immunostaining on RCL2® fixed
Hodgkin's without antigen retrieval, objective 40X. |
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- List of validated antigens on RCL2®fixed
tissues -
| Antigene |
Clone |
Antigene |
Clone |
| ACE |
polyclonal |
Epithelial Membrane Antigen |
E29 |
| Actine |
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Estrogen receptor |
6F11 |
| B cell |
DBB42 |
Galectine 3 |
9C4 |
| BCL2 |
124 |
GFAP |
polyclonal |
| Calcitonine |
polyclonal |
Hairy cell |
DBA44 |
| CD3 |
F7.2.38 |
HER2 |
DA0485 |
| CD5 |
DK23 |
HMB45 |
HMB45 |
| CD15 |
80H5 |
Ki67 |
MIB1 |
| CD20 |
L26 |
Kerzin 3 |
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| CD23 |
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Kerzin 5 |
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| CD30 |
BerH2 |
Mammaglobine |
304-1A5 |
| CD31 |
JC/70A |
MDM2 |
MDM2 |
| CD34 |
QBEND10 |
Melan A |
A103 |
| CD45 |
2B11 PD7/26 |
Mic-2 |
1,20E+08 |
| CD57 |
NC1 |
Myogenine |
F5D |
| CD68 |
KP1 |
Neurofilament |
2F11 |
| CD68 |
PGM1 |
NSE |
BBS |
| CD 79 |
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PNL2 |
PNL2 |
| CD 138 |
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Progesteron receptor |
PgR636 |
| CD 791 |
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Protein S100 |
DAKO |
| CDK4 |
DSC-31 |
P16 |
E6H4 |
| Cycline D1 |
SP4 |
P21waf |
SX118 |
| Chromogranine |
DAK-A3 |
P27 |
SX53G8 |
| C-Kit |
CD117 DAKO |
P53 |
DO-7 |
| Collagen IV |
CIV22 |
Smooth muscle actin |
1A4 |
| Cytokeratines |
AE1/AE3 |
Smooth muscle actin |
HHF35 |
| Cytokeratines |
KL1 |
Thyroglobulin |
DAKO |
| Cytokeratine 5/6 |
D5/16B4 |
TTF-1 |
SPT24 |
| Cytokeratine 7 |
OVTL12/30 |
Vimentin |
V9 |
| Cytokeratine 8/18 |
CK8/18 |
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| Cytokeratine 19 |
RCK108 |
| Cytokeratine 20 |
K20.8 |
| D2 40 |
D2 40 |
| Desmine |
D33 |
| EGFR |
Kit DAKO |
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| + RCL2® & In
Situ Hybridization |
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| Excellent results in CISH and
FISH are obtained on RCL2® fixed tissues. Expression
signals are high and specific, background noise remains
low. |
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Her2neu in FISH on RCL2® fixed tissues,
objective 40X. |
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Her2neu in CISH on RCL2® fixed tissue,
objective 40X |
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| + RCL2® & DNA |
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| DNA in good quality
and quantity can be extracted from RCL2® fixed tissues.
DNA displays a high molecular weight and authorizes long
length PCR. PCR products obtained can be easily sequenced. |
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Genomic DNA
Lane 1: RCL2®
Lane 2: Frozen tissue
Lane 3: Lambda marker 20,000 bp max |
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PCR on 2,4 kpb BRCA-1 gene
T- Negative control
R1 RCL2® fixed tissue
F1 Frozen tissue
R2 RCL2® fixed tissue
F2 Frozen tissue
Lambda marker |
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Similar RNA extraction yield
can be obtained from RCL2® fixed and paraffin embedded
tissues in comparison to frozen tissues and 100 times
higher than those obtained from formalin fixed tissues.
Even higher quality RNA can be obtained if you comply
with optimal conditions of preservation of RNA i.e. reduce
as much as possible time between resection and fixation,
fix your tissues in 4°C cooled RCL2®, paraffin
embedding in slightly reduced temperature and cycle time
in the tissue processor and storage at -20°C.
Don't hesitate to contact us to obtain a technical support
for your transition to RCL2®. |
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Frozen tissue |
+4°C RCL2® fixed tissue |
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| + RCL2® & Proteins |
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| Protein extraction yield up
to 1,000 higher can be obtained in RCL2® fixed and
paraffin embedded tissues compared to formalin fixed tissues.
The yield is comparable to those obtained from frozen
tissues. Proteins profile in acrylamide gel in 1D, 2D
and Western blot are similar to those obtained from frozen
tissues. SELDI-TOF profiles are also comparable. |
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SDS-PAGE gel
Proteins extracted with Tris 10 mM, SDS 2%, 95°C buffer.
Lane 1 : Marker
Lane 2 : RCL2® fixed tissues
Lane 3 : Frozen tissues
Lane 4 : Formalin fixed tissues |
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SDS-PAGE gel
Proteins expressed by various genes and extracted Tris
10 mM, SDS 2%, 95°C buffer.
Lane 1 : Frozen tissue
Lane 2 : RCL2® fixed tissue |
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SELDI-TOF profile
Upper: Frozen tissue
Lower: RCL2® fixed tissue |
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| + RCL2® & Stability |
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| Stability tests have been run
on RCL2® fixed, paraffin embedded specimens. Observation
shows no detectable modifications of morphology on specimens
stored at room temperature for over 6 years. |
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A : Lymph node fixed with RCL2®
: cellular morphology siùilar to the one
obtained with formolated fixatives.
B : Thyroïd fixed in december 1999,
cut and stained in january 2007 : excellent morphological
preservation.
C : CD 20 immunostaining on lymph node RCL2®
fixed in december 1999, cut and stained in january
2007 : excellent antigens preservation.
D : CD 3 immunostaining on lymph node fixed
in RCL2® in december 1999, cut and stained in
january 2007 : excellent antigens preservation. |
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| + RCL2® : No changes
in your method of fixation |
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RCL2® substitutes to your
regular formalin fixative without major changes in your
methods.
Fixation time is similar to formalin fixation whether
at room temperature or at 4°C.
You can even gain in flexibility for your lab as specimens
can be let in RCL2® in extended periods of time without
consequences on tissue quality unlike with formalin.
Biopsies fixation can be achieved into most tissue processors.
Specimens fixed in RCL2® and formalin can be mixed
in tissue processors for dehydration and paraffin embedding. |
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| + RCL2® : Preservation
of your ARNs during laser microdissection |
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Recovery of RNA of high yield
and quality from frozen sections requires significant
precautions.
Indeed, it is demonstrated that once section deposited
on slide, it tends to get moisture which reactivates RNAses.
Some reagents are proposed to inhibit RNAses but they
usually strongly alter morphology which severely makes
tissue structure and cell recognition difficult while
it is a crucial argument for laser microdissection.
A research team at the National Institute for Research
in Agronomics (Jouy-en-Josas, FRANCE) has demonstrated
that a drop of RCL2® poured on a frozen section protects
RNAs during laser microdissection and even beyond at room
temperature with the major benefit to preserve tissue
and cell morphology facilitating microdissection targeting. |
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| Frozen section |
Frozen section
treated with RNAse inhibitor |
Frozen section
protected with RCL2® |
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A true alternate solution for formalin-free
fixation is now available and validated.
RCL2® brings numerous benefits for Research, Healh economy,
Environment and quality of service to Patient.
Contact us to try RCL2® and move your lab into the era of
molecular histo-pathology. |
Publications
1. DELFOUR C, ROGER P, BRET C, BERTHE ML, ROCHAIX P, KALFA N, RAYNAUD P, BIBEAU F, MAUDELONDE T, BOULLE N. RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin-embedded breast carcinoma and microdissected breast tumor cells. J Mol Diagn. 2006;8:157-69.
2. LACROIX-TRIKI M, LAMANT L, ALLAL CUIDER B, DECHA A, VOIGT J-J, ROCHAIX P. RCL2® a new non toxic fixative allowing extraction of high quality DNA, mRNA and proteins from paraffin embedded tissues.
3. Bellet V, Boissière F, Bibeau F, Desmetz C, Berthe M, Rochaix P, Maudelonde T, Mangé A, Solassol J., Proteomic analysis of RCL2 paraffin-embedded tissues , J Cell Mol Med . 2007 Dec 10.
4. V. Bellet, P. Rochaix, A. Mange, J. Solassol , Étude d'un nouveau fixateur pour l'analyse du protéome tissulaire, Study of a new tissue fixative for tissue proteom analysis, Ann Biol Clin 2008; 66 (4): 401-7
5. Belief V, Boissière F, Bibeau F, Desmetz C, Berthe ML, Rochaix P, Maudelonde T, Mangè A, Solassol J., Proteomic analysis of RCL2 paraffin-embedded tissues, J Cell Mol Med . 2008 Oct;12(5B):2027-36.
6. Sophie Le Guellec, Magali Lacroix-Triki, Jean-Pierre Delord, Cuider Ben Allal, Philippe Rochaix , Les nouveaux fixateurs tissulaires, New Tissue Fixative, RFL - Revue francophone des laboratoires, Vol 38, N° 408 - janvier 2009, pp. 25-32. |
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Warm thanks to
Dr Philippe ROCHAIX, Research Cancer Institute Claudius Regaud, Toulouse, France, for its novelty in the invention of this genius product.
Research Cancer Institute Claudius Regaud and its General Management for having offered to ALPHELYS this exceptional opportunity to develop and market RCL2®.
Ms Claudia BEVILACQUA, Unit of Functional and Physiological Genomic of milk production, National Institute of Research in Agronomics INRA, Jouy-en-Josas, France, for her work on RCL2® and for having let us use her images and data.
Dr Nathalie BOULLE, National Institute for Health and Medical Research INSERM Unit U540, Hôpital Arnaud de Villeneuve, Montpellier, France, for her work on RCL2® and for having let us use her images and data. |
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Ferme des Ebisoires, Impasse Paul Langevin, 78370 Plaisir - France
Tel +33 (0)1 30 07 52 95 - Fax +33 (0)1 30 07 51 56 - Email info@alphelys.com
Copyright © 2001/2008 ,
all rights reserved.
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