Recommandations pour la prise en charge des preleveme

Recommandations Sénopath – 28 septembre 2011
Guide de bonnes pratiques émis par le groupe Sénopath pour l’optimisation des
prélèvements en pathologie mammaire – 28 Septembre 2011
Sommaire
•
Préambule et composition du groupe......................................................................... 2
•
Délai de rendu de résultat anatomo-pathologique...................................................... 3
•
o
Biopsies ......................................................................................................... 3
o
Pièces opératoires.......................................................................................... 3
Délai de fixation ......................................................................................................... 4
o
Biopsies ......................................................................................................... 4
o
Pièces opératoires.......................................................................................... 4
•
Orientation des pièces opératoires............................................................................. 5
•
Encrage des pièces opératoires................................................................................. 6
•
Fixateur...................................................................................................................... 7
•
Durée de fixation........................................................................................................ 7
•
Transmission au laboratoire – Renseignements cliniques.......................................... 8
•
Remerciements.......................................................................................................... 8
•
Références ................................................................................................................ 9
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Recommandations Sénopath – 28 septembre 2011
Préambule
Ces recommandations ont été élaborées par un groupe pluridisciplinaire composé de
professionnels impliqués dans la prise en charge des cancers du sein dans le but
d’homogénéiser et d’optimiser la gestion des prélèvements tissulaires (biopsies et pièces
opératoires). Ce travail s’inscrit dans le cadre d’une démarche qualité, soutenue par le
réseau ONCOMIP et le Comité Sein de l’IUC.
Membres du groupe
•
Magali Lacroix-Triki (Pathologiste, Institut Claudius Regaud, Toulouse), coordinateur
•
Paul Caverivière (Pathologiste, SCP des Feuillants, Toulouse), coordinateur
•
Jean-Dominique Bernard (Chirurgien, Clinique St Jean du Languedoc, Toulouse)
•
Geneviève Caruana (Technicienne, SCP des Feuillants, Toulouse)
•
Hélène Charitansky (Chirurgien, Institut Claudius Regaud, Toulouse)
•
Henri Ducoin (Pathologiste, SCP Ducoin/Gabay/Sorbara/Laurent/Palasse, Toulouse)
•
Ghislaine Escourrou (Pathologiste, CHU Rangueil, Toulouse)
•
Jérôme Farnarier (Chirurgien, Médipôle, Toulouse)
•
Viviane Feillel (Radio-sénologue, Institut Claudius Regaud, Toulouse)
•
Pierre Léguevaque (Chirurgien, CHU Rangueil, Toulouse)
•
Jean-Luc Manenc (Chirurgien, Médipôle, Toulouse)
•
Eliane Mery (Pathologiste, Institut Claudius Regaud, Toulouse)
•
Jean-Luc Pages (Radiologue, Clinique de l’Union, Toulouse)
•
Hélène Plana (Technicienne, CHU Rangueil, Toulouse)
•
Marie-Laure Quintyn (Pathologiste, CHU Rangueil, Toulouse)
•
Hélène Sanner (Radiologue, Clinique St Jean du Languedoc, Toulouse)
•
Chantal Silvagni (Technicienne, Institut Claudius Regaud, Toulouse)
•
Bogdan Vierasu (Radiologue, CHU Rangueil, Toulouse)
•
Pascal Wuithier (Pathologiste, Laboratoire rue Carnot, Tarbes)
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Recommandations Sénopath – 28 septembre 2011
En préambule, il est rappelé qu’à partir du moment où il est effectué, le prélèvement
tissulaire est sous la responsabilité du pathologiste. Cette responsabilité inclut notamment
les phases initiales de conditionnement et d’acheminement par le clinicien vers le laboratoire
de pathologie. La fixation optimale du prélèvement est une condition sine qua non pour la
réalisation
de
techniques
histopathologiques
(analyse
morphologique,
étude
immunohistochimique, techniques d’analyse moléculaire) de qualité.
Délai de rendu de résultat anatomo-pathologique
En introduction, il est rappelé par le groupe les délais de rendu de résultat anatomopathologique. En dehors d’équipements spécifiques (type automate Morales), ces délais sont
incompressibles en raison d’impératifs techniques incluant les étapes de fixation, de
déshydratation/imprégnation en paraffine, de coupe, de coloration et d’explorations
complémentaires. Les délais ci-dessous correspondent aux délais minimum, en dessous
desquels il ne peut être rendu de résultat :
Biopsies
-
Prélèvement à J0
-
Première lecture morphologique (type, grade histologique) à J1
-
Evaluation immunohistochimique (récepteurs hormonaux, HER2) à J2
Pièces opératoires
-
Prélèvement à J0
-
Recoupe macroscopique à J1
-
Première lecture morphologique (type, grade histologique) à J2
-
Evaluation immunohistochimique (récepteurs hormonaux, HER2) à J3
A noter qu’en cas de besoin, les techniques d’hybridation in situ nécessitent 2 jours
techniques supplémentaires, auxquels il faut, selon le site technique initial, ajouter le temps
de désarchivage et d’acheminement vers les plateformes d’analyse moléculaire. La
technique d’hybridation in situ est le plus souvent réalisée par séries de prélèvements (3 à 4
séries/mois) afin d’optimiser l’organisation et l’économie des réactifs. Le rendu des résultats
pour ces pièces là est donc différé.
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Recommandations Sénopath – 28 septembre 2011
Délai de fixation
Il s’agit du délai entre le moment de prélèvement (heure de prélèvement pour les biopsies,
heure de dévascularisation pour les pièces opératoires) et le moment où le tissu est immergé
dans le fixateur.
Biopsies
-
pour une masse, le délai de fixation est idéalement très court, à moins de 10min,
puisque la fixation est effectuée immédiatement par le radiologue ;
-
pour des microcalcifications, le délai peut être plus long (rarement supérieur à 30min)
en raison de la nécessité de réaliser des radiographies des carottes biopsiques. Dans
ce cas, les biopsies seront humidifiées à l’aide d’une compresse imbibée de sérum
physiologique afin d’éviter leur dessèchement. Le dessèchement peut en effet produire
d’importants artéfacts gênant considérablement les analyses ultérieures.
Pièces opératoires
En fin d’intervention, les pièces opératoires seront :
-
soit acheminées immédiatement au Laboratoire de Pathologie, quand celui-ci est
sur le même site, le délai de fixation optimal étant théoriquement fixé à un maximum
de 1h. En pratique courante, compte tenu de l’éloignement physique des laboratoires
par rapport aux blocs opératoires, ce délai théorique peut être dépassé.
-
Soit fixées au bloc opératoire. Plus que sur le délai théorique d’1h, le groupe insiste
surtout sur l’importance de la qualité et du mode de fixation. En effet, les artefacts de
sous-fixation liés à une fixation inadéquate, telle que l’immersion d’une pièce
volumineuse dans un volume insuffisant de fixateur, sont irréversibles et extrêmement
délétères pour les techniques ultérieures. La fixation sera effectuée par un personnel
formé et sensibilisé à ces techniques (rapport volume de fixateur/pièce classiquement
recommandé à 10, récipient adapté).
-
Soit conservées temporairement à 4°C :
o
Système TissueSAFE (Microm) (cf annexe 1): système qui met les pièces
opératoires sous vide et permet un transport en conditionnement propre et
une bonne conservation temporaire sur une durée maximale de 48h à +4°C
pour une fixation différée. Les pièces sont ensuite fixées au laboratoire et
suivent le process habituel. Système testé à l’ICR (E. Méry) et au CHU de
Rangueil (G. Escourrou) : bonne morphologie, techniques IHC et FISH de
bonne qualité.
o
Conservation temporaire courte à 4°C dans un récipient hermétique avant
fixation différée au laboratoire.
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Recommandations Sénopath – 28 septembre 2011
Orientation des pièces opératoires
En préambule, il est rappelé la nécessité absolue par le chirurgien d’orienter les pièces
opératoires dans les trois dimensions. Le groupe propose une standardisation du mode
d’orientation des pièces opératoires par les chirurgiens (Figure 1) :
-
Tumorectomies :
o
2 clips posés sur fil en externe ;
o
1 clip posé sur fil en interne ;
o
1 fil sans clip en superficiel
-
1 clip côté tumoral pour les recoupes
-
1 fil en externe pour les pièces de mastectomie
Le fait de poser les clips sur les fils présente plusieurs avantages : identification plus aisée
par le pathologiste, non gênant en cas de radiographie de pièce. Cette standardisation est
optimale pour prévenir les erreurs d’orientation des pièces.
Fil sans
clip en
superficie
2 clips en
externe
1 clip en
interne
Figure 1 : Orientation de la pièce.
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Recommandations Sénopath – 28 septembre 2011
Encrage des pièces opératoires
L’encrage des berges d’exérèse doit être effectué avant ouverture de la pièce opératoire,
afin de repérer la berge chirurgicale et de pouvoir par la suite mesurer la marge d’exérèse
(distance entre la tumeur et la berge), information indispensable pour la prise en charge des
patientes en cas de traitement conservateur. Cet encrage peut être effectué soit par le
pathologiste, soit par le chirurgien formé à cette technique. L’encrage par le chirurgien
permet une identification optimale des berges latérales par rapport aux berges profonde et
superficielle (Figure 1), distinction qui peut ensuite être difficile compte tenu de la
déformation de la pièce opératoire au cours du transport. L’encrage s’effectue à l’aide
d’encre de chine classique disponible dans le commerce (papeterie) ou de pâte à tatouer;
pour mémoire, les systèmes d’encre stérile ne sont pas recommandés car radio-opaque et
potentiellement gênant si une radiographie de pièce est nécessaire. Afin d’éviter toute
diffusion de l’encre, la pièce une fois encrée est badigeonnée à l’alcool. Des encres de
couleur différente peuvent être utilisées pour aider à l’orientation de la pièce (Figure 2).
Figure 2 : Encrage de la pièce. Des encres colorées peuvent être sur-appliquées
secondairement au laboratoire pour aider à l’orientation (ici, encre jaune en externe et bleue
en superficiel).
Il est rappelé que seules les berges latérales intéressent le clinicien, puisque lors du geste
chirurgical, le plan superficiel de résection correspond à la peau et le plan profond au muscle
pectoral. Par ailleurs, une résection monobloc est souhaitable dans la mesure du possible
pour garantir l’étude optimale de la qualité de l’exérèse chirurgicale (i.e. en marges saines et
suffisantes).
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Recommandations Sénopath – 28 septembre 2011
Fixateur
Selon les recommandations nationales (Penault-Llorca et al, ann pathol 2010) et
internationales (Wolff et al, JCO 2007 ; Hammond et al, JCO 2010), le fixateur optimal
recommandé est le formol tamponné à 10% (V/V) (soit 4% M/V). En effet, les techniques
complémentaires et les kits commerciaux sont calibrés pour ce fixateur, garantissant une
qualité optimale. Les techniques d’analyse moléculaire (hybridation in situ, séquençage, etc.)
sont réalisées en routine de façon optimale sur tissus fixés en formol.
Dans le cadre d’une démarche d’accréditation et sanitaire, le groupe recommande :
-
l’installation de hottes ventilées dans le laboratoire pour la macroscopie avec système
d’aspiration basse préconisé sur table de recoupe
-
récupération du formol dans poubelle à formol
-
un dosage régulier du formol dans les laboratoires
-
l’installation d’armoires ventilées dans les lieux de stockage (laboratoire +/- bloc
opératoire)
-
mise à disposition de poudre anti-formol (par exemple, référence et notice Labonord en
annexe 2) et de buvards absorbants (par exemple, buvards Labonord D FORMALIZER
280 x 356 ref 11037514 et D FORMALIZER 406 x 502 ref 11037520) dans les blocs
opératoires et les véhicules de transport si applicable. Les buvards absorbants sont à
mettre au fond des seaux de transport et au fond des armoires de stockage.
-
triple emballage si transport en véhicule.
Si la pièce opératoire est fixée au bloc opératoire, elle ne doit pas être ouverte (ouverture
dans un temps secondaire par le pathologiste à l’arrivée au laboratoire), et doit être
immergée dans un volume de fixateur suffisant par rapport à la pièce (rapport volume
fixateur/volume pièce=10).
Durée de fixation
Les durées de fixation optimales suivent les recommandations nationales et internationales
(Penault-Llorca et al, ann pathol 2010 ; Wolff et al, JCO 2007 ; Hammond et al, JCO 2010),
et sont à adapter au volume et au caractère adipeux de la pièce fixée. Les pièces de
mastectomie nécessitent un temps de fixation plus long (24-48h).
A titre indicatif, ces durées sont :
Biopsies : minimum 6h
Pièces opératoires : minimum 24h
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Recommandations Sénopath – 28 septembre 2011
Il est rappelé par le groupe qu’une sous-fixation est beaucoup plus délétère (et non
rattrapable) pour les techniques ultérieures (immunohistochimie, hybridation in situ) qu’une
surfixation. Afin d’éviter une surfixation sur les week-ends, les automates de déshydratation
peuvent être programmés de façon spécifique pour les week-ends, avec des temps d’attente
dans les bains alcooliques plutôt que dans les bains de formol.
Transmission au laboratoire – Renseignements cliniques
Les prélèvements mammaires doivent être transmis au pathologiste accompagnés de
renseignements cliniques (recommandations INCa, Annexe 3) comportant notamment :
-
identification de la patiente
-
date et heure de prélèvement
-
heure de fixation du prélèvement
-
médecin prescripteur
-
siège et latéralité du prélèvement
-
traitement antérieur (chimiothérapie, hormonothérapie, radiothérapie) en cas de
situation néoadjuvante
-
TNM ou données radiologiques si lésion infra-clinique
-
caractère unifocal ou multifocal
-
si une radiographie de la pièce est effectuée, il est souhaitable qu’elle soit transmise
-
si la pièce est complexe, dessin informatif avec les explications et les repérages
d’orientation du chirurgien.
En cas de mastectomie pour microcalcifications, tumeur multifocale avec traduction
mammographique, ou récidive de petite taille, une radiographie de la pièce de mastectomie
est souhaitable avec repérage des lésions par le radiologue (aiguilles ou encrage en regard)
afin d’assurer une identification précise des lésions par le pathologiste. Cette radiographie de
pièce doit être transmise au pathologiste avec la pièce opératoire.
Remerciements
Le groupe remercie chaleureusement Isabelle Boquet et les laboratoires Roche pour le
soutien apporté à l’organisation et la mise en place de ce groupe de travail.
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Recommandations Sénopath – 28 septembre 2011
Références
Hammond, M. E., D. F. Hayes, et al (2010). "American Society of Clinical Oncology/College Of
American Pathologists guideline recommendations for immunohistochemical testing of
estrogen and progesterone receptors in breast cancer." J Clin Oncol 28(16): 2784-95.
Penault-Llorca, F., A. Vincent-Salomon, et al (2010). "[Update of the GEFPICS'
recommendations for HER2 status determination in breast cancers in France]." Ann Pathol
30(5): 357-73.
Wolff, A. C., M. E. Hammond, et al. (2007). "American Society of Clinical Oncology/College of
American Pathologists guideline recommendations for human epidermal growth factor
receptor 2 testing in breast cancer." J Clin Oncol 25(1): 118-45.
9
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ARTICLE IN PRESS
STOTEN-11941; No of Pages 4
Science of the Total Environment xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Science of the Total Environment
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Vacuum-based preservation of surgical specimens: An environmentally-safe step
towards a formalin-free hospital
Cinzia Di Novi a, Davide Minniti b, Silvana Barbaro b, Maria Gabriella Zampirolo b,
Antonio Cimino c, Gianni Bussolati c,⁎
a
b
c
Dept. Of Public Policy and Choice University of Eastern Piedmont, Italy
Azienda Ospedaliera-Universitaria San Giovanni Battista di Torino, Italy
Dept. Biomedical Science and Human Oncology University of Turin, Italy
a r t i c l e
i n f o
Article history:
Received 2 March 2010
Received in revised form 7 April 2010
Accepted 13 April 2010
Available online xxxx
Keywords:
Formalin
Tissue specimens
Histopathology
Under-vacuum sealing
Pathology
a b s t r a c t
Formalin as a fixative has no practical substitutes, but is toxic and potentially carcinogenic, so caution of its
use in hospitals and elsewhere is mandatory. In our hospital, preservation of surgical specimens into
formalin to be transferred to pathology labs was replaced by under-vacuum sealing (UVS) tissues into plastic
bags and preservation at 4 °C until transfer. Data analysis showed UVS processing to be superior in terms of
staff satisfaction and of gross anatomic preservation; no problems in terms of technical feasibility or
histopathologic preservation were encountered. Formalin was confined to pathology labs while its use on
hospital premises was vastly reduced.
© 2010 Published by Elsevier B.V.
1. Introduction
Formalin, a 4% solution of formaldehyde in water, is a world-wide
and extensively used fixative for histopathological specimens. Since
its discovery at the end of 19th Century (Fox et al., 1985), this
aldehyde has been universally appreciated as a simple reagent that is
a good antiseptic, penetrates tissues quickly (diffusion rate of 1 cm in
24 h) and is easy to handle. In tissues that are formalin-fixed,
morphology is well preserved, as is tissue antigenicity, and immunohistochemical procedures of diagnostic interest have routinely been
adapted to formalin-fixed tissues (Dabbs, 2008).
In addition to multiple industrial applications, the medical use of
formalin as a tissue preserver and fixative is extensive, especially in
pathology laboratories. In fact, the amount used in public hospitals in the
Piedmont region (Italy) alone for the preparation of approximately
300,000 histological exams is in the range of 100,000 liters per year.
Tissues preserved in formalin are even sent by post, in the number
of several thousands per year.
Despite its advantages, formaldehyde has some drawbacks that
demand caution: it is allergenic to the skin and produces irritating
vapors, causing asthma. The International Agency for Cancer Research
⁎ Corresponding author. Department of Biomedical Sciences and Human Oncology,
University of Turin, Via Santena 7, 10126 Turin, Italy. Tel.: +39 011 633 4274; fax: +39
011 663 5267.
E-mail address: gianni.bussolati@unito.it (G. Bussolati).
(IARC, 2006) has declared formaldehyde to be a Class 1 carcinogenic
agent, and statistical evidence has been presented on a possible link
between formaldehyde exposure and lymphohematopoietic malignancies (Beane Freeman et al., 2009).
Several attempts have been made to find a substitute for formalin,
but so far all of the proposed alternatives have failed, being either
ineffective or unreliable (Titford and Horenstein, 2005). A more
practical approach would be to limit the use of formalin to pathology
laboratories, where this toxic agent is carefully handled with hoods
and gloves in safe environmental conditions, and to avoid its use in
other less-protected areas of the hospital, such as in surgical theaters,
where removed tissues are commonly placed in boxes full of formalin
until transfer to the pathology labs. In fact, despite the advantages
linked to this procedure (fixation and anti-sepsis begin immediately
for the removed tissues and organs, and dehydration is avoided)
several disadvantages are also recognized (see Table 1).
To overcome these problems, we proposed an alternative
procedure, which is the under-vacuum sealing of tissues (UVS) in
plastic bags inside the surgical theatre immediately after removal, and
to keep them cooled at 4 °C until transfer to the pathology labs, where
they are routinely processed.
Safety and advantages linked to this UVS procedure have already
been reported elsewhere (Bussolati et al., 2008). This processing was
tested for more than two years in a single surgical theater, and it is
now being extended to the whole hospital.
The present study compares the compliance of personnel and the
feasibility of this new procedure in a large regional hospital to the
0048-9697/$ – see front matter © 2010 Published by Elsevier B.V.
doi:10.1016/j.scitotenv.2010.04.022
Please cite this article as: Di Novi C, et al, Vacuum-based preservation of surgical specimens: An environmentally-safe step towards a
formalin-free hospital, Sci Total Environ (2010), doi:10.1016/j.scitotenv.2010.04.022
ARTICLE IN PRESS
2
C. Di Novi et al. / Science of the Total Environment xxx (2010) xxx–xxx
Table 1
A) Tissues immersed in formalin (large specimens)
Disadvantages
• Degradation continues in deep areas
• Tissue banking is hampered
• Formalin-containing vessels are heavy to carry
• Spilling of formalin may occur
• Fumes dispersed while grossing
• Nurses refuse to handle this carcinogen inside the surgical theatre
(without hoods)
• Tissue forgotten by the surgeon because it is “already safe in formalin”
B) Tissues preserved under vacuum
Merits
• No more formalin in surgical theatre (except for small
specimens, where pre-filled tubes are employed)
• No spilling
• No fumes
• No drying of tissues
• Colour, form and consistency are preserved
• Lack of insulating air around tissues allows fast cooling
• Tissues (bags) light and easy to carry
• Structures (RNA, antigens) preserved for days
• Banking (selective) and gene-expression profiling allowed
• Fixation times can be standardized
• Microbiological and viral analysis feasible
traditional process of immersing surgical specimens in formalin. The
survey was conducted with questionnaires and interviews specifically
dealing with the various steps of the processing that were given to all the
staff (nurses, technicians and pathologists) involved.
2. Material and methods
The present study was conducted in the S. Giovanni “Molinette”
hospital of Turin (Italy), a teaching hospital with 1162 beds, 54,560
yearly admissions, and over 40,000 histopathological exams in the
year 2008. The hospital was originally built in 1938 as a pavilion
hospital. As a result, the main pathology laboratories are separated
and located in a different building from the surgical theaters. The
study involved four surgical theaters, all located in different areas, that
produce over 90% of the surgical biopsies.
Biopsies were subdivided into two classes: “small”, i.e., less than
2 cm. in diameter, and “large”, or N2 cm in size. The latter corresponded
to roughly 25% of the total number of specimens. The rationale for such
subdivision is related to the well-known fact (Hewitt et al., 2008) that
the penetration rate of formalin into tissues is in the range of 1 cm in
24 h, thus theoretically assuring fixation of “small” biopsies in
acceptable times. These “small” biopsies are routinely collected in
50-ml containers pre-filled with buffered formalin (Diapath s.r.l.,
Martinengo, Bergamo, Italy).
The present study is concerned with “large” biopsies which, until
now, were transferred from the operating theatre to the pathology
laboratories in large plastic containers (ranging in size from 1000 to
5000 ml) filled with formalin. The volume of formalin varied
according to the size of the specimen, but is recommended to be 20
times the weight of the specimen. It is customary in our hospital that
surgical specimens are collected once a day, in the early afternoon, to
be transferred to the pathology labs. Thus, in cases operated on Friday
afternoon, or before long weekends, the specimens are transferred to
pathology only on Monday.
For UVS processing, specimens were sealed into plastic bags
immediately after removal in the vacuum apparatus (Tissue-safe® ,
Milestone, Bergamo, Italy). The process lasted a few seconds. The bags
were labeled with identification data. The specimens were then kept in a
refrigerator at 4 °C inside the premises of the surgical theater until they
were transferred to pathology. Once the sealed bags, which were kept in
a chilled plastic box, arrived at the pathology labs, the tissue was
removed and routinely processed. This included grossing and then
fixation in buffered formalin (Diapath) under hoods and for a controlled
time, followed by embedding in paraffin.
2.1. Evaluation of staff satisfaction, technical feasibility and the quality of
tissue preservation
A series of questionnaires were distributed in sequence to the
hospital staff, collected and statistically analyzed. Overall, the study
was conducted over a time period of six months (Oct. 2008–April
2009).
2.1.1. Staff satisfaction
The first questionnaire, distributed on October 2008 to all the
personnel of the surgical theaters and to the technical staff of the
pathology laboratories (N = 60 and 58, respectively) enquired about
the satisfaction or dissatisfaction experienced with the traditional
process of tissue handling (following categorical outcomes: very
satisfied, satisfied, not satisfied) and with related problems or
difficulties. One month after the introduction of the UVS processing,
the same questionnaire was distributed to the same 58 technicians of
the pathology laboratories as before and to 28 personnel of the
surgical theatres now equipped with the Tissue Safe apparatus.
Overall, after correcting for the missing values, the sample included
177 observations.
2.1.2. Technical feasibility
A questionnaire analyzed the technical feasibility of the different
sequential steps involved in the transfer, examining either the
traditional procedure employing formalin or the new UVS processing.
The form accompanied single tissue specimens. Requests to fill out the
forms were stopped after collecting 323 forms from senders (staff of
the surgical theaters).
2.1.3. Quality of tissue preservation
Questionnaires enquiring about tissue preservation at either the
gross or microscopic level were collected from 46 members of pathology
staff (24 medically qualified, 22 biologists or technicians). The
questionnaires regarded the quality (form, colour and consistency) of
the gross anatomic preservation of different organs and tissue specimens (esophagus/stomach, colon, kidneys/prostate, breast, thyroid,
liver/spleen), and qualified each as weak, satisfactory or good. They
surveyed the preservation of tissues processed either with formalin or
UVS.
A final questionnaire, distributed to the same staff, was related to the
quality of the histo-pathological and immuno-histochemical preservation of surgical biopsies processed with the new UVS procedure.
3. Estimation method
A general linear regression model was used to test whether there
exists a positive and significant correlation between the tissue
handling procedure and the hospital staff satisfaction or the tissue
quality conservation indicators (detailed description of the method
can be found in the Appendix A).
4. Results
4.1. Hospital staff satisfaction
In order to test if the operator acceptance of the tissue handling
procedure was positively correlated with the alternative procedure in
which tissues are sealed under-vacuum in plastic bags, this study used a
cross-sectional survey design in which hospital staff (nurses, pathology
laboratory technicians, biologists, and physicians) from the San
Giovanni Battista University Hospital (Turin, Italy) completed a
questionnaire (reported in the Appendix A). The sample included 177
Please cite this article as: Di Novi C, et al, Vacuum-based preservation of surgical specimens: An environmentally-safe step towards a
formalin-free hospital, Sci Total Environ (2010), doi:10.1016/j.scitotenv.2010.04.022
ARTICLE IN PRESS
C. Di Novi et al. / Science of the Total Environment xxx (2010) xxx–xxx
respondents. We create a binary variable that takes the value one if
respondents work with the under-vacuum procedure and zero
otherwise (that is, if they practice the fixation of tissues with formalin).
Other than the tissue handling procedure, the following factors that may
influence respondent satisfaction were measured using self-report
questionnaires and included as explanatory variables: demographic
variables (age, sex), professional activity indicators (whether respondents are nurses or physicians, pathology laboratory technicians and
biologists), symptom perception (whether the respondents from the
hospital staff perceive symptoms such as cough, chest pain, shortness of
breath, and wheezing deriving from the use of tissue conservation
procedure), difficulties (whether respondents encounter difficulties in
using the tissue conservation procedure), and time of experience with
the conservation procedure. The sample was divided into two
categories: the first sub-sample includes personnel who were experienced with the traditional processing with formalin, and the second
sub-sample includes personnel who were experienced with the new
preservation method.
The most important statistic concerns the indicator of satisfaction,
which increased with the UVS procedure (statistics reported in
Supplementary Table 1 in the Appendix A, which provides descriptive
statistics including means, standard deviations (SDs) and percentages
for all relevant sample variables, as well as responses to the
questionnaire concerning staff satisfaction). Briefly, 67% of respondents reported that they were very satisfied with the UVS based
preservation mechanism, whereas for those who were using formalin,
only 39% report satisfaction with the method of preservation. For the
UVS procedure, 24% answered that they are satisfied, versus 41% of the
sub-sample who used formalin. Finally, 8% of the sub-sample who
preserve tissue with the UVS processing answered that they were
dissatisfied with the preservation method, versus 39% of those who
use the formalin fixation method. It is worth noting that respiratory
symptoms such as cough, chest pain, shortness of breath, and
wheezing increased with formalin use (34% versus 4% for UVS).
Among personnel who used the UVS processing, 10% reported that
they encountered difficulties with the preservation procedure, versus
39% of personnel who operated with formalin.
Supplementary Table 1c shows coefficients for hospital staff
satisfaction calculated with the conservation procedure equation
estimated using the ordered probit specification. From our empirical
results, it arises that suffering from respiratory symptoms and having
difficulties in using the tissue conservation procedure both have a
strong negative impact on the satisfaction with the conservation
procedure employed. The most interesting results concern the undervacuum procedure: this technique in fact has a double effect on
hospital staff satisfaction: a direct effect and an indirect one. The
under-vacuum procedure directly improves operators' satisfaction
and decreases the probability of suffering from respiratory symptoms,
which, in turn, decreases the negative influence on operators'
satisfaction.
4.2. Technical feasibility of the procedure
Our data indicate that no technical inconveniences were encountered when using the new UVS procedure (data available on request).
4.3. Tissue preservation quality
We evaluated the gross anatomic preservation of different organs
and tissue specimens, including esophagus/stomach (1), colon (2),
kidneys/prostate (3), breast (4), lung (5), endocrine/thyroid (6), and
liver/spleen (7), each to be qualified as 1 = weak, 2 = satisfactory or
3 = good (see Supplementary Tables 2a and 2b in the Appendix A,
which show a simple descriptive analysis that presents sample means
and standard deviations for the questionnaire). In order to make
comparisons between the formalin and UVS procedure, the pathology
3
samples were divided into two categories based on the method of
tissue conservation, either formalin fixation or UVS preservation.
It is worth noting that the quality of gross anatomic preservation
increases with the UVS procedure. Those samples present better colour,
form and consistency for all different organs and tissue specimens. The
average colour, form and consistency of the esophagus/stomach, colon,
kidneys/prostate, thyroid, and liver/spleen sealed under-vacuum in
plastic bags were in the good range, versus the satisfactory range for
tissue fixed with formalin. These gross anatomical parameters are
pertinent for pathological evaluation and diagnosis, especially in hollow
organs such as stomach and colon, where formalin-induced discoloration, hardening and retraction of the mucosa may hamper the proper
recognition of ulcerative and infiltrative foci, or the evaluation of the
correct distance of the lesion from resection margins. The evaluation
was rather subjective, hence we opted for a 3 scale evaluation into weak,
satisfactory or good quality. The final data (as extensively reported in
Supplementary Tables 2a and b in the Appendix A) stress the
importance of sealing and cooling conditions for a good preservation
of both structure and consistency in esophagus, stomach and colon. In
solid organs as kidney, liver and prostate the improvement of gross
anatomical features, linked to the use of UVS as compared to formalin
preservation, was lower, though still significant.
Thus, we can conclude from our empirical results that the UVS
procedure is more effective than formalin for preserving the quality of
tissue. The age and sex of the involved personnel had no influence on
the results. In each estimation model, we have tested for multicollinearity by using the Variance Inflation Factor (VIF) and Tolerance
(1/VIF) (Wooldridge, 2002). We found that VIF for all the independent
variables in both the equation models were quite low. Therefore, we
can safely assume that there are no problems of multicollinearity.
Finally, data of the questionnaire collected from members of the
pathology staff concerning the quality of histo-pathological and
immuno-histochemical preservation of tissues processed with the
UVS procedure were categorized as either no damage or damaged. No
damage affecting histopathological reporting, including tumor classification, grading and staging, was ever noticed in the period of use of UVS
processing. We therefore concluded that the procedure was safe and
reliable. Moreover, a neat improvement in the quality of histological
features was noticed in solid organs such as kidney, liver, prostate and
breast when kept for several hours, or even over the week-end, in UVS at
4 °C instead of formalin. In fact, in the latter conditions at variance with
the former, areas deeper than 1 cm. (the penetration front of formalin)
underwent autolysis resulting in poor structural preservation.
In our Institution the number of immunohistochemical and FISH
(fluorescent in situ hybridization) procedures performed for diagnostic purposes amounted in year 2009 to 40.995 and 554, respectively,
but no reports of damage affecting results was ever related to UVS
processing. However, for a more objective evaluation, we checked the
immunohistochemical values reported for breast cancer prognostication in 375 consecutive cases diagnosed between 06/2005 and 06/
2007, before adoption of UVS processing, and the same number of
cases in the two years after. Percentages of positivity for ER, PgR and
Ki67 (N21%) were respectively 83.2, 84 and 40.8% before, and 86.9,
81.06 and 39.7%, after (non significant difference). In addition, we
observed that UVS processing of breast cancer specimens facilitates
gene expression profiling, since in 40 consecutive breast cancer
specimens collected at times ranging from 1 to 72 h after removal, the
quality of RNA was optimal in all cases (RIN value N7) (Sapino et al.,
2009).
5. Conclusion
Formalin, a buffered solution of formaldehyde, is extensively used
for histopathological preservation, and its substitution with alternative
fixatives cannot be foreseen at present (Dabbs, 2008; Hewitt et al.,
2008). Concerns of carcinogenic activity of this mutagenic aldehyde
Please cite this article as: Di Novi C, et al, Vacuum-based preservation of surgical specimens: An environmentally-safe step towards a
formalin-free hospital, Sci Total Environ (2010), doi:10.1016/j.scitotenv.2010.04.022
ARTICLE IN PRESS
4
C. Di Novi et al. / Science of the Total Environment xxx (2010) xxx–xxx
have been raised (IARC, 2006), and evidence has recently been
presented on its possible association with leukemia (Beane Freeman
et al., 2009), an observation that might fit with data reporting an excess
of deaths due to cancer of the lymphatic and hematopoietic systems
among British pathologists (Hall et al., 1991). Still, the major concern is
linked to the production of toxic, irritating and allergenic vapors.
Indeed, a positive relationship between formalin and respiratory
symptoms has been reported not only in workers in match factories
(Vaughan and Black, 1939), but also in hospital staff members
professionally exposed to this substance (Hendrick and Lane, 1975).
Accordingly, our results support a positive and significant relationship
between formalin and the probability of reporting distressing respiratory symptoms.
The ultimate goal of our approach was to reduce the use of
formalin outside protected areas (i.e., fume hoods in pathology labs
and pre-filled containers for small biopsies). Thus, we focused our
attention to the practice of immersing surgical biopsies in large
containers filled with formalin inside the surgical theater and their
transfer to the pathology labs in due time. This process is endowed
with several inconveniences (see Table 1a). An alternative process,
whereby specimens are sealed under-vacuum into plastic bags and
kept at 4 °C until transfer for grossing in the pathology labs was
originally proposed by our group (Bussolati et al., 2008) and has been
tested for over two years. In our experience, this process offers merits
(see Table 1b) in terms of simplicity, feasibility and preservation of
the original characteristics of tissues.
The extension of UVS processing to the whole hospital (a large,
regional hospital) required a series of validation tests, to be checked
with questionnaires analytically and statistically analyzed.
The results unanimously indicate a high degree of satisfaction for
the new procedure (as compared to the traditional use of formalin)
by both nurses from the surgical theater and technicians of the
pathology labs. Not only did the UVS procedure avoid exposure to the
distressing vapors of formalin, but it was also found to be easy and
practical.
Further series of questionnaires specifically dealt with the
feasibility and possible intricacies linked to the use of the UVS machine
for different tissues and organs to be transferred from the surgical
theater to the pathology labs. No specific difficulties were noted, and
the evaluation of gross anatomic features was improved with the UVS
procedure (as opposed to fixation in formalin) in terms of preservation
of form, colour and consistency of the specimens. Finally, an inquiry
among 46 members of pathology staff (24 medically qualified, 22
biologists or technicians) after several months of use of the UVS
procedure did not reveal any difficulties in the diagnostic process
linked to the use of the new procedure.
The conclusion of the present survey on the feasibility, compliance
and quality assurance of a new procedure for transferring surgical
specimens is positive. The UVS procedure was met with favor by the
staff and did not present specific problems of practical or diagnostic
interest. As a result, the new procedure has been adopted as the
standard in our hospital.
Additional bonuses are linked to the possibility of standardizing
fixation times and of implementing tissue banking. In fact, we can
now determine the starting time of fixation in formalin, thus avoiding
over-fixation, which can cause detrimental effects on immunophenotyping of the specimen, an issue that is presently regarded as
mandatory for breast cancer processing (Goldstein et al., 2003; Wolff
et al., 2007). An additional bonus of the novel U–V procedure is the
preservation of RNA, which is enhanced by the storage at 4 °C (van
Maldegem et al., 2008), thus permitting tissue banking and gene
expression profiling.
In conclusion, the present study shows a pathway towards a
progressive reduction of the exposure of nurses, pathologist and
technical personnel to formaldehyde vapors. The use of formalin has
been restricted to dedicated areas in the pathology laboratory, and
transfer of large boxes filled with fixative throughout the hospital was
cancelled. In addition, the simple UVS processing offered advantages in
terms of staff satisfaction, tissue preservation and cost. Complete
elimination of formalin is still out of reach, but its substantial reduction
from hospital premises is attainable and meets requests of environmental safety.
Acknowledgements
This work was supported by grants from Ricerca Sanitaria Finalizzata
2008 Regione Piemonte, European Commission Project IMPACTS —
contract no: 037211 and Ministero per lo Sviluppo Economico (MISE,
Rome) within the frame of the MISE-ICE-CRUI Project.
The active collaboration of the staff personnel of nurses and
technicians of surgical theatres and pathology laboratories is gratefully
acknowledged.
We would like to thank Dr. C. Marchiò and Mrs A. Davello for
careful review and formatting of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.scitotenv.2010.04.022.
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Notice d’utilisation Neutraliseur de formol
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DENOMINATION DU PRODUIT
Q path Neutraliseur de formol.
Référence
Désignation
00699030 Q path Neutraliseur de formol 5 kg
Nature de l’emballage externe
Polyéthylène de haute densité (HDPE)
USAGE DU PRODUIT
Le Q path Neutraliseur de formol réagit avec les solutions d’aldéhydiques usées (4% m/v ou 10% v/v) à l’aide d’un
agent réducteur puissant, pour les transformer en déchets liquides non dangereux.
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La manipulation de ce produit est réservée à des professionnels.
1. Le produit est prêt à l’emploi.
2. Les récipients, de récupération et traitement, doivent être soigneusement fermés et étiquetés
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COMPOSITION
Métabisulfite de sodium
CONDITIONS DE STOCKAGE
Stockage dans un endroit avec une ventilation adéquate.
EQUIPEMENT SPECIAL SUPPLEMENTAIRE
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1. Utiliser un contenant adapté.
2. 125 g de Q path neutraliseur suffit à neutraliser 1L de solution aldéhydique usée.
3. Fermer le contenant.
4. Laisser en agir 24 h.
5. Les résidus peuvent être éliminés avec les eaux usées conformément aux réglementations locales relatives aux
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METHODOLOGIE
• PRINCIPE DE LA METHODE
Le Q path Neutraliseur de formol est un composé fortement réducteur qui, en combinaison avec une solution
d’aldéhyde donne des esters non toxiques. Les composants ainsi formés, ester et acide, sont considérés comme ne
présentant aucun danger. Les esters sont formés par interaction avec les radicaux du formaldéhyde. L’acide réduit le pH
de 7 à 6, ce qui ne présente aucun problème, puisque que l’eau du robinet et l’eau désionisée présentent elles-mêmes un
pH de 6.
• CARACTERISTIQUES DE LA METHODE ET LIMITES DES PERFORMANCES
Le temps de mise en contact est de 24 h minimum pour garantir une transformation efficace du produit.
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Notice d’utilisation Neutraliseur de formol
PREPARATION DES REACTIFS
Le Q path neutraliseur de formol est prêt à l’emploi.
REFERENCES BIBLIOGRAPHIQUES
« Mesure d’efficacité sur un neutraliseur de formol » CREID Centre de Ressources sur l’environnement Industriel
Dunkerque Septembre 2002.
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