SIXTH FRAMEWORK PROGRAMME
Contract
nr:
LSHG-CT-2007-037211
Acronym:
IMPACTS
Title:
ARCHIVE’S TISSUES: IMPROVING MOLECULAR
MEDICINE RESEARCH AND CLINICAL PRACTICE
Instrument:
COORDINATION ACTION
Thematic
Priority:
INTEGRATING
AND
STENGTHENING
EUROPEAN RESEARCH AREA
THE
Activity Report
Period covered:
1stMarch 2007 – 28th February 2010
Date of preparation:
April 2010
Start date of project:
1st March 2007
Duration:
36 months
Project coordinator name:
Prof. Giorgio Stanta
Project coordinator
organization name:
Consorzio per l’AREA di Ricerca
Scientifica e Tecnologica di Trieste
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Revision
Project duration: 36 months
Official web site: www.impactsnetwork.eu
Contact person: Giorgio Stanta (coordinator) and Valentina Melita (project assistant), tel. +39
040 399 6203,
e-mail: stanta@impactsnetwork.eu
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INDEX
Publishable executive summary...............................................................................................4
Section 1 – Project objectives and major achievements during the reporting period..............5
Overview of general project objectives........................................................................5
Summarise the objectives, work performed, contractors involved and
the main achievements..................................................................................................6
Section 2 – Workpackage progress of the period...................................................................10
WP1: Management of the Action...............................................................................10
WP2: Technology comparison, validation and standardization.................................11
WP3: Technology dissemination.................................................................................12
WP4: Technology innovation………………………………………………………..13
WP5: Archive tissues bioethics……………………………………………………...13
WP6: Human tissue archives………………………………………………………...13
WP7: Proteomics in archive tissues………………………………………………….14
Table 1: Deliverables List............................................................................................17
Table 2: Milestones List……………………………………………………………...18
Publishable results…………………..………………………………………………………..19
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Publishable executive summary
All the tissues taken from patients for diagnostic or therapy reasons are routinely fixed and paraffinembedded and, after few sections are cut for histopathological diagnosis, those tissues are stored in
archives often for decades. For this reason they are called “archive tissues”. Such tissues can be a
very important resource for molecular analysis in the research and clinical practice. The reason for
this is that in many cases the only tissue available for molecular clinical analysis in a patient is a
small fixed and paraffin-embedded biopsy tissue. Medicine has been improving the possibility to
personalize diagnosis and treatment through the research and application of molecular biomarkers.
The use of archive tissues will enhance the amenability of tissues for research and clinical practice
because of their availability in any hospital.
The coordination action “Archive tissues: improving molecular medicine research and clinical
practice – IMPACTS” involves 20 participants in 11 countries. Among them are the major experts
in molecular analysis in archive tissues and those that first developed these methods. The group has
validated and standardized these molecular methods, in DNA and RNA analysis, and specific
guidelines have been prepared and discussed for clinical application. This step is extremely
important to be able to diffuse the methods at the European level for a clinical application. The
group has also studied innovative techniques such as new fixatives that are preserving
macromolecules, e.g. proteins, in the same way as in fresh frozen tissues. The introduction of
proteomics research and clinical application is also one of the priorities, with the development of
two strategies. The first one is a new method that allows protein analysis in formalin-fixed tissues
(most of the tissues today are formalin-fixed). The second one is the use the new fixatives in order
to obtain a perfect preservation of proteins. These developments will make it possible to perform
molecular analysis at the DNA, RNA and protein level, directly in those tissues in which the clinical
diagnosis was defined, even in a tiny biopsy, with a careful selection of the cells of interest. The
group has also prepared multicentric studies in human archive tissues, especially for the validation
and standardization of molecular analysis and to develop methods for the guidelines.
The activity was also related to the preparation of tissue bio-banking rules specific for archive
tissues, for which specific guidelines are necessary also in archive tissues. A new project to define a
network of archive tissue virtual banking among the participants in the project was developed. This
network was included in the European Biobanking Infrastructure (BBMRI). At the end of the
project, in order to continue the activity of IMPACTS, a Pan-European Archive Tissue Biobanking
Network was begun, starting from the already existing IMPACTS network. This network is being
developed in collaboration with the European Society of Pathology (ESP) and the European
Biobanking Infrastructure (BBMRI).
From the second year of activity bioethics issues were discussed to prepare specific approaches in
the use of archive human tissues for clinical research. An international meeting on bioethics in the
translational research use of archive tissues was organized with the participation of the IMPACTS
groups and the experts of the European Infrastructure (BBMRI). This has given the bases for the
specific management of the archive tissue biobanks in Europe.
The project was supposed to last two years, but a 12-month elongation was granted, until February
2010, to allow the participants to better fulfil their final goals, to improve and accelerate molecular
medicine research and clinical practice using archive tissues.
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Section 1 – Project objectives and major achievements during the reporting
period
Overview of general project objectives
The translation of molecular biology knowledge to a clinically applied molecular medicine is a very
slow process as pointed out by many international organizations (see IMI in Europe and FDA in the
US). The project “Archive Tissues: Improving Molecular Medicine Research and Clinical Practice
– IMPACTS”, is devoted to accelerate this process by promoting the use of archive tissues as
biological material for the clinical application of molecular analysis in medicine. These archive
tissues are the human tissues taken from the human body for diagnostic or surgical therapy reasons.
All these tissues are fixed and then paraffin-embedded to be able to cut few sections for the
definitive histopathological diagnosis. Most of the tissues are then stored in the pathology archives
of any hospital, sometimes for many decades. For this reason, these tissues are called “archive
tissues”. It is important to point out that archive tissues are all the routinely treated tissues in any
hospital in Europe and all over the world. If we are able to perform molecular analysis in these
tissues, we do not have to modify any procedure at the clinical level, but we can just apply the new
molecular methods. This is a crucial point because it is very difficult and demanding and it takes a
very long time to try to change any clinical procedure in hospitals.
The aim of the IMPACTS project is to work on the use of archive tissues at the molecular level in
the clinical practice, with the development of work packages devoted to validation and
standardization of the molecular methods, proteomics in archive tissues, the use of new fixatives
with a high level of molecular preservation, tissue bio-banking, bioethics rules in archive tissues
and diffusion of the new approaches at the European level. The project is also promoting
multicentric research on methods and biomarker validation.
The project started in March 2007 and its first task was to evaluate the molecular methods used in
archive tissue analysis, because at present each laboratory is using different protocols. This goal
was reached by comparing the techniques used by the participating laboratories and by defining the
preliminary guidelines for molecular analysis in archive tissues. These guidelines are also the
baseline for archive tissue technique dissemination in Europe. In the third year the guidelines were
completed, edited and are going to be published by Springer Verlag in 2010. Many validation tests
were performed by the laboratories of the group, with special attention to pre-analytical
modification of archive tissues and method standardization. Several publications were completed or
submitted.
To approach the clinical use of these methods it is important to develop specific research and
coordination projects. For this reason, the IMPACTS group prepared some proposals for the 7th EP:
three projects proposals were presented to the first LHS call, two proposals to the second LHS call,
four proposals to the third LHS call and one proposal to the fourth LHS call (see results and
comments in the specific objectives and WP description). Moreover, the IMPACTS group
submitted a project proposal for the 7th and 8th COST collection date (see results and comments in
the specific objectives and WP description).
To work on a multicentric project on human tissues it is absolutely necessary to establish and follow
general bioethics rules. One of the groups’ goals was the preparation of some bioethics guidelines at
the European level for research use of archive tissues. This WP was outlined in the IMPACTS
meeting in Munich in February 2008 and started in the second year of activity. It was managed by
the Pisa group and was developed mostly in the third year. In January 2010 a dedicated meeting was
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organized in Pisa, with the participation of many European experts in the legal and scientific aspects
of the bioethics issue.
Any project for validation of methods or clinical biomarkers is related to the amenability of a large
number of human tissues, for this reason one of the goals of the coordination action is also to
develop guidelines and projects for tissue bio-banking. This activity was supposed to be developed
in the second year of activity, but a preliminary organization for tissue bio-banking had already
been prepared at the beginning of the action, as a basis for another European project called “Biobanking Network – Molecular Medicine Evidence groups”. This activity was developed together
with the European Infrastructure for Biobanking (Biobanking and Biomolecular Resources
Research Infrastructure – BBMRI) and it was presented in the proposals submitted for the 1st call of
the 3rd FP7.
The activity of biobanking was further developed after the second year of activity and the
organization of a Pan-European Archive Tissue Biobanking Network was prepared, starting from
the IMPACTS groups and with the collaboration of the European Infrastructures BBMRI and the
European Society of Pathology (ESP).
Specific attention was given to technology innovation. The participants studied in their laboratories
the possibility to use new fixatives that preserve a high level of molecular integrity for nucleic acids
and proteins. Some approaches were also done with groups of nanobiotechnology to define new
methods of protein separation and new types of arrays. Throughout the project, the participants
continued to study at the multicentric level the results obtained with the use of new fixatives that
preserve a high level of molecular integrity for nucleic acids and proteins. This study was also
related to the proposals at the European level to ban formaldehyde from the environment because of
the dangerous consequences on health.
The groups also tested new techniques about high throughput imaging in archive tissues using
nanotechnology approaches.
A specific group within the IMPACTS project dealt with the possible approaches of archive tissue
techniques to proteomics methods. In this field two research lines were developed. The first one
was dedicated to protein extraction from formalin-fixed and paraffin-embedded tissues, for which
the Munich group developed a commercial kit that was multicentrically validated. The second line
of proteomics research is related to new alcoholic fixatives, that perfectly maintain proteins. The
Munich group also prepared a European course about proteomics in archive tissues and continued to
organize multicentric validation studies that are going to be developed also after the end of the
project.
A website of the group was prepared (www.impactsnetwork.eu) and constantly updated and
enriched with information about the BN-MEM (Biobanking Network: Molecular Evidence
Medicine group) project, the scientific production of the groups and the reports of the IMPACTS
meetings.
Summarise the objectives, work performed, contractors involved and the main achievements
In the three years of activity, the following objectives of the IMPACTS coordination action were
fulfilled:
a)To analyze the present knowledge and use about molecular analysis in archive human tissues in
Europe and to propose method validation and standardization: after comparing the molecular
protocols used for the analysis of DNA and RNA in archive tissues (the results of this comparison
were discussed in specific meetings held in Istanbul in September 2007 and especially in Munich in
February 2008), the laboratories participating in the project validated the protocols used for DNA
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and RNA extraction in FFPE tissues. The results of this validation were discussed in the Dubrovnik
and Porto meetings. The further results of the validation performed during the third year were
discussed during the final IMPACTS meeting. To make the connection among the different
institutions more effective, the coordinator visited many of the laboratories throughout the project
period.
In the first Munich meeting the preliminary guidelines for molecular analysis in archive tissues
were presented, to be discussed and improved. Those guidelines for were further developed in the
Dubrovnik and Porto meetings and their preparation was concluded at the end of the third year of
activity.
All the laboratories produced papers published in international peer review journals throughout the
project period (the whole list of publications is reported in the IMPACTS website).
The scientists involved in the IMPACTS project participated in many meetings and congresses
related to the aims of the project.
b)Dissemination in European laboratories of validated and standardized methods with good
practice rules: the guidelines for molecular methods in archive tissues prepared by the participants
are the baseline for the dissemination of these methods in Europe. Moreover, a website
(www.impactsnetwork.eu) was prepared and constantly updated and enriched, to give information
on the project and to diffuse methods and initiatives in the field.
Dissemination is also performed by the organization of meetings within the ESP. The first one took
place during the ESP congress in Istanbul in September 2007 and the other IMPACTS symposiums
were organized within the ESP meetings in Barcelona (May 2008) and Florence (September 2009).
During those meetings the technological approaches to archive tissues were presented to the
European pathologists in a specific session. An IMPACTS technical meeting was held in June 2008
in Dubrovnik, in correlation with the Adriatic Society of Pathology (ASP) meeting.
Moreover, a European Molecular Pathology course was organized in Graz in April 2009 and a
European Protein Analysis workshop was organized in Munich in March 2009.
c)To point out future perspectives of functional genomics and proteomics at the research and
clinical level: during the IMPACTS activity, the participants in the project submitted several
proposals to the 7th EP, to develop research in the field of molecular analysis in archive tissues. The
proposals are listed here as follows:
1. “Bio-banking Network – Molecular Evidence Medicine groups (BN-MEM)”: this project was
presented to develop a very large tissue banking system in 18 different European areas, by using the
virtual collection of the pathology archives of 128 hospitals and the collection of surgical left-over
treated with new fixatives. The proposal received a positive evaluation, but didn’t reach the
threshold to be financed.
2. “Tapping the untranslated potential of the human transcriptome in colorectal cancer archive
tissues (UNTAP)”: this project was proposed to study untranslated RNAs, in large case studies of
archive tissues. The project was rejected.
3. “A new fixative offers revolutionary opportunities for cancer biomarkers research (FORCaRe)”:
this proposal was presented as an SME project by Milestone S.r.l., one of the IMPACTS
participants, to develop research about a new fixative that allows preserving proteins just like in
fresh frozen tissues. The proposal was rejected.
4. “International Meeting on Archive Tissues: a shortcut for biomarker validation and biomedicine
clinical application (ATMEET)”: this proposal was submitted as a support action to prepare a world
congress about archive tissues to compare the European work with the experiences done in archive
tissues by other research centres in the world. The proposal had a positive evaluation but did not
rank for financing. For this reason it was resubmitted for the 3rd FP7 call, unfortunately without
success.
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5. “European Project on Improvement and Standardisation of Pre-Analytical Procedures for Modern
Pathology (EURO-IS4PATH)”: this proposal was developed to study the pre-analytical procedure
of tissue treatment to standardize such techniques to a high level of macromolecule preservation.
The proposal had a positive evaluation, but did not rank for financing.
6. “Systems Integration of DNA SEquence changes with RNA and protein QUantitative Alterations in Large
archival case studies (SISEQUAL)”: this proposal was submitted for the 3rd FP7 call. It is related to
the large International Cancer Genomic Consortium project, in which high numbers of tumour
tissues are sequenced. The aim of this project is to compare results of very large archive tissues case
studies at mRNA and protein level. The proposal had a positive evaluation, but did not rank for
financing.
7. “Molecular Omics technologies Validation and standardization In large-scale fixed and Embedded clinical
Samples (MOVIES)”: this proposal was submitted for the 3rd FP7 call. It is related to the use of the
very large collection of archive tissues as biobanking resource, with the validation and
standardization of high throughput molecular methods. The proposal had a positive evaluation, but
did not rank for financing.
8. Protein Technologies for Clinical Tissues (PROTECT): this proposal was submitted for the 3rd
FP7 call by the Technologische Universität of Munich (TUM). It is related to the validation and
standardization of protein analysis in FFPE tissues and new fixatives treated tissues. The proposal
had a positive evaluation, but did not rank for financing.
9. “Molecular Workflow for the Effective Detection of Multiplex HPV Markers in Cervical Cancer
Patients (CERVIFLEX)”: this proposal was a two-step one and was submitted for the 4th FP7 call.
The proponents of this project aim at developing and implementing a new molecular diagnostic
workflow performing a multiplex analysis of HPV biomarkers at the DNA, RNA and protein level
directly in clinical samples from patients with pre-malignant and neoplastic cervical lesions.
Unfortunately this proposal did not pass the first-step evaluation, despite the importance of the issue
and the good potentialities this project could offer in the clinical practice. For this reason, the
IMPACTS group decided to use the redress procedure in order to have it re-evaluated.
Moreover, the IMPACTS group submitted a proposal for the 7th collection date (oc-2009-2) of the
European Cooperation in the field of Scientific and Technical Research (COST). The proposal is:
“European Network for Archive Tissue Analysis and Clinical Application (EN-ATACA)”: it is a
two-step proposal and it’s aimed to disseminate the knowledge acquired during the IMPACTS
project worldwide.
This project was rejected at the first-step evaluation due to its overlapping with the aims of the
IMPACTS project. For this reason, since the IMPACTS project ended on 28th February 2010, the
participants decided to resubmit this proposal (named EN-ATACA2) for the 8th collection date (oc2010-1), unfortunately without success despite the great chance this project could have given to the
IMPACTS group to disseminate all over Europe and even outside the important results reached so
far.
By presenting all these proposals, the IMPACTS group tried to develop a basic system of “Reverse
Translational Research” in Europe, starting from clinical material. This could be the first large
group of European scientists working on archive tissues in the different pathology fields. The
IMPACTS participants were very surprised to see all those proposals rejected by the evaluators, as
this new approach could offer great opportunities to Europe to be in the forefront of international
research. Moreover some of the most experienced European pathologists were involved in those
issues. A better understanding and openness to such new techniques could really help European
research to be competitive worldwide. Therefore we really think that the European scientific
evaluation system needs some adjustments.
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The coordinator of the IMPACTS project was also invited to participate in one of the major
international research projects on cancer, the “International Cancer Genomics Consortium – ICGC”,
that was proposed by the major international organizations, like the European Commission, NCI,
Genome Project, Sanger Institute etc. The project aims at sequencing a high number of major
cancers.
The scientists of the coordination action developed new methods to study molecular markers with
the proteomics techniques. Karl Becker of the Technological University of Munich developed a
method to extract proteins directly from formalin-fixed and paraffin-embedded tissues. This allows
having a more precise quantification of proteins in archive tissues, which can help clinical
prognostic and therapeutic decision. Moreover, the groups of Trieste and Torino showed that with
new alcoholic fixatives it is possible to study proteins just like in fresh-frozen tissues. A specific
group was appointed within the IMPACTS coordination action to study biomarkers at the
proteomics level. The validation of this method was carried on by the IMPACTS groups through
multicentric studies. A multicentric effort was also performed to study the preservation and the
analysis of proteins in tissues fixed with new types of alcoholic fixatives. A specific meeting was
organized in Munich in August 2008. A course on the proteomics techniques in archive tissues was
organized by TUM in Munich in March 2009.
d)To establish bioethical guidelines for the correct use of human archive tissues in research and
clinical practice: the bioethics activity started officially in the second year, although its organization
was decided during the Munich meeting in February 2008. The Pisa group was in charge of the
development of this issue, with the hiring of an expert in law. The topic was broadly discussed
together with well-known European bioethics experts in a dedicated meeting which took place in
Pisa in January 2010. The objective is to create European bioethics guidelines for research use of
archive tissues.
In the first year, a European project called ACUME2 was contacted. This project is dedicated to life
sciences-humanities comparison and a specific activity about bioethics in the use of human tissues
in molecular research was developed. Our cooperation with ACUME2 went on throughout the
project.
e)To explore the possibilities to organize archive tissue banks for future research: this activity
started officially in May 2008, but the first steps for its development had already been taken in the
preparation and organization of the BN-MEM proposal presented for the 1st call of the 7th EP. This
bio-banking project is related to the collection of archive tissues stored in the pathology
departments of 128 European hospitals in 18 European areas. The biobanking initiative of the
IMPACTS groups was developed in the context of the European Infrastructure BBMRI and the
coordinator of the IMPACTS groups was asked to participate as an expert of disease biobanks in the
Work Package 3 of the BBMRI project.
Since one of the most important goals of IMPACTS is the development of archive tissue
biobanking systems, the participants decided to develop a Pan-European Archive Tissue
Biobanking Network starting from the IMPACTS network, with the collaboration of BBMRI and
the ESP. The network will be enlarged to all the European countries.
f)To promote technology innovation in archive tissue molecular analysis in collaboration with
biotechnology industries: one of the participants in the coordination action is an SME (Milestone
S.r.l.) which has the patent for a new fixative that preserves proteins well, the same as in freshfrozen tissues, and maintains a high level preservation also for DNA and RNA. Many participants
tested this new fixative by comparing it among laboratories. Validation and standardization projects
were multicentrically developed. Milestone prepared a series of prototypes for vacuum treatment of
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fresh tissues and to obtain precise fixation time. These issues were further discussed during the final
IMPACTS meeting in Munich.
Section 2 – Workpackage progress of the period
WP1: Management of the Action
Deliverables:
D1.1-Consortium management: the consortium management started with the project in March 2007
and in particular with the organization and development of the Kick-off Meeting, the definition of
the Steering Committee, the preparation of the Consortium Agreement and the general meeting in
Munich held in February 2008. The activity was continuous and went on in the second year with the
organization of the IMPACTS technical meeting in Dubrovnik, of the 4th IMPACTS meeting in
Porto, with the gathering of the Steering Committee in Vienna and of the PMB in Porto. In the third
year the consortium management was carried out with the organization of the IMPACTS session in
the ESP meeting in Florence, of Bioethics in Pisa and with the final and PMB meeting in Munich.
D1.2-Administrative management: the administrative management started in the first year and was
continuously carried out throughout the project by our administrative officer, who managed all the
project expenses, kept in touch with the administrative officers of each participating group and, at
the end of each reporting period, prepared the Management Report, collecting information from all
the participants.
Milestones and expected results:
M1.1- Control of WP results: the control of WP results was done by the coordinator at due time.
The coordinator urged the groups that were late with their work in sending information about
protocols, activity and other material for comparison and discussion. The coordinator also visited
some of the participating institutions and developed some direct or e-mail discussions to solve some
problems and to manage the research developments.
M1.2- Diffusion of the WP results: a final diffusion is already being performed with the forthcoming
publication of the European guidelines, with the organization of the meetings, especially those in
connection with scientific societies, and with the organization of technical courses, as well as with
the preparation and updating of the IMPACTS website.
M1.3- Co-organization of the general meetings: the meetings that were organized are:
a. Kick-off Meeting in Trieste from 12th to 14th March 2007, organized by the Area Science Park
and the Trieste ICGEB.
b. Short IMPACTS informal meeting in Istanbul during the 21st ESP-European Congress of
Pathology in Istanbul from 8th to 13th September 2007, organized by the Area Science Park - Trieste
and the Trieste ICGEB.
c. Technical and general meeting in Munich from 7th to 9th February 2008, organized by the
Technologische Universität in Munich, the Area Science Park - Trieste and the Trieste ICGEB.
d. IMPACTS session in the European Society of Pathology meeting in Barcelona from 19th to 22nd
May 2008, organized by the University of Lausanne, and Area Science Park and ICGEB - Trieste.
e. Technical meeting in Dubrovnik, associated with the Adriatic Society of Pathology meeting, on
26th and 27th June, organized by the Ljudevit Jurak University – Zagreb, and Area Science Park and
ICGEB - Trieste.
f. IMPACTS Proteomics Meeting in Munich on 7th and 8th August 2008, organized by TUM –
Munich.
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g. 4th IMPACTS Meeting in Porto from 21st to 23rd November 2008, organized by IPATIMUP –
Porto, and Area Science Park and ICGEB - Trieste.
h. IMPACTS session in the European Society of Pathology meeting in Florence on 9 th September
2009, organized by the University of Lausanne, and Area Science Park and ICGEB-Trieste.
i. Bioethics meeting in Pisa on 29th January 2010, with IMPACTS PMB meeting, organized by the
University of Pisa, and Area Science Park and ICGEB-Trieste.
j. IMPACTS Final Meeting in Munich on 19th February 2010, organized by TUM-Munich, and
Area Science Park and ICGEB-Trieste.
WP2: Technology comparison, validation and standardization
Deliverables
D2.1-Diffusion of technical guidelines: The guidelines were preliminarily presented and discussed in the
technical and general meeting in Munich from 7th to 9th February 2008. They were further
developed and discussed in the meetings held in the second year (Barcelona, Dubrovnik, Munich
and Porto) and the final version was thoroughly discussed during the final meeting in Munich on
19th February 2010. A very intense editing activity was performed by the authors and the editing
group (Giorgio Stanta, Valentina Melita, Isabella Dotti, Renzo Barbazza and Danae Pracella).
Moreover, the index of the guidelines has been published in the IMPACTS project website
(www.impactsnetwork.eu).
Finally, two courses were organized:
a. Workshop: “Protein Analysis of Tissues” – Current Views and Clinical Perspectives, Munich, 6th
and 7th March 2009, organized by TUM – Munich.
b. Postgraduate Course on Molecular Pathology of Colon Cancer, Graz, 16th - 18th April 2009,
organized by Medical University of Graz.
D2.2-Presentation of research proposals for multicentric translational researches on archive tissues for the
7th EP: the following proposals were presented to the 7th EP:
1. Bio-banking Network – Molecular Evidence Medicine groups (BN-MEM)
2. Tapping the untranslated potential of the human transcriptome in colorectal cancer archive tissues
(UNTAP)
3. A new fixative offers revolutionary opportunities for cancer biomarkers research (FORCaRe)
4. International Meeting on “Archive’s Tissues”: a shortcut for biomarker validation and
biomedicine clinical application (ATMEET)
5. European Project on Improvement and Standardisation of Pre-Analytical Procedures for Modern
Pathology (EURO-IS4PATH)
6. Systems Integration of DNA SEquence changes with RNA and protein QUantitative Alterations in Large
archival case studies (SISEQUAL)
7. Molecular Omics technologies Validation and standardization In large-scale fixed and Embedded clinical
Samples (MOVIES)
8. Protein Technologies for Clinical Tissues (PROTECT)
9.Molecular Workflow for the Effective Detection of Multiplex HPV Markers in Cervical Cancer
Patients (CERVIFLEX).
Moreover, the IMPACTS group submitted a proposal for the 7th collection date (oc-2009-2) of the
European Cooperation in the field of Scientific and Technical Research (COST). The proposal is:
European Network for Archive Tissue Analysis and Clinical Application (EN-ATACA).
Milestones and expected results
M2.1-Technical guidelines proposal: during the meeting in Munich in February 2008 the preliminary
technical guidelines were presented and discussed. In the second year, they were further improved with
many new chapters and with the results of the technical validation multicentric tests. The proposed
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guidelines were discussed in the meetings in Dubrovnik, Munich and Porto. The different
participating groups exchanged scientists and technicians, and continuously shared information
among laboratories. The final version of the guidelines was discussed during the final meeting in
Munich on 19th February 2010. A very intense editing activity of the guidelines was developed
during the third year.
M2.2-Definition of future research proposals on archive tissues: as reported in D2.2, nine proposals were
presented to the 7th EP and one to COST.
WP3: Technology dissemination
Deliverables
D3.1-European meetings for dissemination of the standardized protocols: The following European
meetings and courses were organized:
a. Kick-off Meeting in Trieste from 12th to 14th March 2007.
b. Short IMPACTS meeting in Istanbul during the 21st ESP-European Congress of Pathology in
Istanbul from 8th to 13th September 2007.
c. Technical and general meeting in Munich on 8th and 9th February 2008.
d. IMPACTS session in the European Society of Pathology meeting in Barcelona from 19th to 22nd
May 2008.
e. Technical meeting in Dubrovnik, associated with the Adriatic Society of Pathology meeting, on
26th and 27th June 2008.
f. IMPACTS Proteomics Meeting in Munich on 7th and 8th August 2008.
g. 4th IMPACTS Meeting in Porto from 21st to 23rd November 2008.
h. Workshop: “Protein Analysis of Tissues” – Current Views and Clinical Perspectives, Munich, 6th
and 7th March 2009, organized by TUM – Munich.
i. Postgraduate Course on Molecular Pathology of Colon Cancer, Graz, 16th - 18th April 2009,
organized by Medical University of Graz.
j. IMPACTS session in the European Society of Pathology meeting in Florence on 9 th September
2009.
k. Bioethics meeting in Pisa on 29th January 2010, with IMPACTS PMB meeting.
l. IMPACTS Final Meeting in Munich on 19th February 2010.
The IMPACTS project website has been prepared (www.impactsnetwork.eu) and two courses (one
at basic and one at advanced level) were organized in 2009.
D3.2-Diffusion of guidelines: the final version of the guidelines was edited and will be published by
Springer Verlag in 2010.
Milestones and expected results
M3.1-Standardized protocols guidelines diffusion: the final version of the guidelines was edited and
will be published by Springer Verlag in 2010.
M3.2- European meetings organization together with ESP: three symposia within the ESP meetings
were organized:
a. Short IMPACTS meeting in Istanbul during the 21st ESP-European Congress of Pathology in
Istanbul from 8th to 13th September 2007.
b. IMPACTS symposium in the ESP meeting in Barcelona from 19th to 22nd May 2008.
c. IMPACTS symposium in the ESP meeting in Florence from 5th to 9th September 2009.
Moreover, two sessions of the IMPACTS group are going to be prepared for the ESP Congress in
Krakow in September 2010.
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WP4: Technology innovation
New fixation procedures were recently developed to shorten the time of histopathology diagnoses,
these methods have shown also a major improvement in nucleic acids and protein preservation. The
objective is to test the possibility for these new procedures to be used as routine treatment of tissues
in hospital pathology departments and to test the opportunities of better molecular analyses at DNA
and RNA level
Deliverables
D4.1 - Proposal of new fixation procedures with better molecular preservation: the study of new
fixatives started from the beginning of the project and many participating laboratories tested
different fixatives. Peer-review publications were prepared.
Milestones and expected results
M4.1 - Validation of new fixation procedures with better molecular preservation: a validation study
of new fixatives was proposed in the Munich meeting in February 2008. Molecular preservation
standards in new fixatives were tested by many laboratories on new fixative treated tissues.
WP5: Archive tissue bioethics
There are no common bioethics rules in the European countries for research use of human tissues
and this is complicated by the “subsidiarity principle”. It was decided in Munich in 2008 that the
activity would start with the collaboration of experts defined by each participating institution to
discuss and evaluate the possibility of a common frame of bioethics rules necessary for the
multicentric studies. Transdisciplinary collaboration should ensure to obtain results consistent with
research opportunities but also with social concerns.
In the first year of activity a European project called ACUME2 was contacted. This project is
dedicated to life sciences-humanities comparison and some specific activities about bioethics in the
use of human tissues in molecular research were developed in association with IMPACTS. The
cooperation with this project went on throughout the project. To this regard, a brainstorming
meeting about “Bioethics and Biobanks” was organized in Bologna on 23rd and 24th May 2009 by
ACUME2.
In the last year, a specific activity about bioethics was organized by the Pisa group with a final
meeting held in Pisa in January 2010, with the participation of international experts.
Deliverables
D5.1- Diffusion of proposal of common bioethics guidelines for human tissues multicentric
research: guidelines for multicentric studies in Europe on archive tissues were prepared by the Pisa
group with the collaboration of the Trieste group. A summary of the bioethics guidelines will be
published in a peer-review journal.
Milestones and expected results
M5.1-Proposal of bioethics guidelines for human tissues multicentric research: the guidelines for
bioethics in archive tissues will be disseminated by the participating groups and they will be
diffused among pathologists and other researchers interested in molecular analysis in archive
tissues.
WP6: Human tissue archives
A very large number of tissues specimens with any kind of diseases are stored in the pathology
archives of any European hospital. This is very valuable material for molecular research in human
pathology. All these tissues represent an already existing tissues bank for molecular studies in AT.
13
Possible management of this material among the hospital institutions that participate in the proposal
is a specific objective of the Action.
Although this activity was supposed to start officially in May 2008, the first steps for its
development were taken in the first year, with the preparation and organization of the BN-MEM
proposal presented to the 7th EP.
In the treatment of the surgical origin large specimens, adjunctive tissue fragments can be easily
collected. New fixation procedures allow a very good preservation of nucleic acids and proteins,
similar to that of fresh frozen tissues (WP4 and 7). The aim is to study the possibility of a
multicentric collection of tissues that can be constituted by the anonymous collection of well
documented specimens for large European research studies as already proposed in the BN-MEM. In
this way, it will be possible to compare formalin-fixed tissues with frozen tissues and new fixative
treated tissues. This new procedure will also consider fast processing of tissues using microwave
techniques.
In the final meeting in Munich the IMPACTS group decided to develop a pan-European network of
archive tissue biobanking. This network will start from the already prepared BN-MEM and then
diffused to other European pathologists, with the purpose to develop a very extensive network
throughout Europe. This network will be on voluntary and collaborative basis. The different
institutions will refer to hubs specialized in molecular analysis in this kind of tissues, that will
respect specific standardized methods and high level controls. The diffusion of the network
throughout Europe will be started during the next ESP meeting that will be held in Krakow in
September 2010.
Deliverables
D6.1- Proposal of guidelines for multi-hospital archive tissues collections for European
multicentric projects: bio-banking guidelines for archive tissues were discussed in the Dubrovnik
and Porto meetings. They were further defined in the Florence, Pisa and Munich meetings and will
be further developed in the Krakow ESP meeting.
D6.2- Proposal of multicentric collection of tissues treated with new fixatives with nucleic acids
and protein preservation: the use of new fixatives was tested in many tissue samples by several
participating laboratories and their use was in the projects presented in the 3rd call of the 7th FP
project. In the final meeting in Munich in February 2010 it was decided to start the Pan-European
Network of Archive Tissue Biobanks. This initiative will be further developed in the ESP meeting
in Krakow in September 2010.
Milestones and expected results
M6.1- Guidelines for archive tissue multicentric collection: bio-banking guidelines for archive
tissues were discussed in the Dubrovnik and Porto meetings and finalized in Pisa and Munich in
2010. The issue was also developed in the context of the European Infrastructure devoted to
biobanking (BBMRI).
WP7: Proteomics in archive tissues
There is a need for better protein analysis tools in archive tissues. This WP aims to evaluate novel
protein extraction technologies developed by European researchers and to explore new fixation
procedures compatible with subsequent protein analysis.
The influence of histological procedures on protein yield from archive tissues was not known
before. We started our work showing that histological techniques influence the efficiency of protein
recovery from archival tissues. A suggestion, therefore, was made by the IMPACTS members that
protein extraction for downstream analysis should be performed using unstained tissue sections. In
addition, it was evaluated whether proteins could be extracted from very small pieces of tissues,
such as punch biopsies. Indeed, it was shown that high quality protein samples could be obtained
from punch biopsies of archival tissues. Furthermore, transcription factors which are usually present
14
in minute amounts in archive tissues, such as the E-cadherin repressor Snail, could be quantitatively
analyzed.
As the project went on, we were able to demonstrate that clinically relevant proteins, such as
estrogen receptor, HER2 and EGFR can be extracted from formalin-fixed tissues as well as from
tissues fixed with novel fixatives and analyzed by Western blot and protein lysate microarray
technology. These results were reported at the IMPACTS meeting in Porto.
Moreover, five members of the IMPACTS consortium focused on the evaluation of protein analysis
in tissues fixed with formalin and FineFix, a new fixative. Tissue samples were fixed in parallel
with formalin (FFPE) and FineFix and inspected histologically. Five sections from three matched
samples of each tissue specimen (lung, colon, breast) were distributed within the consortium to
evaluate efficiency of protein recovery. Selected marker proteins were compared between the two
fixation procedures using protein lysate microarray technology. An optimized protocol allows
proteins to be extracted successfully from FFPE and FineFix tissues, even when processed in
different European laboratories. Further, we found remarkable differences for two large membrane
proteins, EGFR and HER2. Signal intensities for EGFR (P=0.007) and HER2 (P=0.02) were
significantly higher in the FineFix samples compared to the FFPE samples, whereas no differences
were observed for β-Actin, E-cadherin, and ER. Although long-term studies for formalin substitutes
such as FineFix are still lacking, formalin-free tissue fixation represents a reliable alternative to
FFPE fixation for protein preservation as far as quality and quantity are concerned.
These results were reported at the European Congress of Pathology, Florence, 2009. In addition, a
manuscript with the data was prepared and submitted for publication.
There was a delay in the deliverables and milestones. The reasons for the delays included problems
with the spotting device and delays in collecting and shipping tissue samples. However, the results
are complete now.
Protein expression in archive tissues can be studied by IHC and tissue arrays, but also by protein
extraction as shown recently by European scientists involved in the action, directly from formalinfixed tissues. These techniques are very important to be used for translational research proteomics
analysis also in AT: at the Technological University of Munich a method to extract proteins directly
from formalin-fixed and paraffin-embedded tissues was developed. This allows having a more
precise quantification of proteins in archive tissues, which can help clinical prognostic and
therapeutic decision. Multicentric validation tests have been performed and the results are going to
be published.
Recently new rapid fixation procedures that can allow to prepare a final histological slide in a short
time have been developed. These methods have shown that DNA, RNA and proteins are preserved
as in fresh frozen tissues. The objective is to explore the technical availability and reproducibility of
these new methods for possible research and clinical applications of protein analysis: new fixatives
allow maintaining a higher level of preservation of proteins; in particular FineFix, patented and
produced by Milestone S.r.l., has very good results, comparable to those obtained in fresh tissues. A
specific group of five laboratories tested these and other new fixatives to obtain the best conditions
for proteomics analysis in archive tissues. Many participating laboratories developed experience in
the field and the results are going to be published.
Deliverables
D7.1-Proposal of proteomics protocols in formalin-fixed archive tissues: the protocol for the
analysis of proteins in formalin-fixed and paraffin-embedded tissues was tested in a multicentric
study by five participating laboratories, and specific protocols were prepared.
15
D7.2-Proposal of proteomics protocols in new fixatives treated tissues: the new alcoholic fixatives
were multicentrically tested to establish the molecular preservation and the morphology level in the
tissues. Papers are in preparation.
Milestones and expected results
M7.1-Proteomic analysis protocols in archive tissues for research and clinical applications:
protocols for analysis of biomarkers at the proteomics level have already been tested and future
experiences are going to be developed by the participating laboratories, to define validated and
standardized protocols.
16
Table 1: Deliverables List
Del.
no.
Deliverable name
WP
no.
Date due
Actual/For
ecast
delivery
date
1 - 36
1 - 36
25
Estimated
indicative
personmonths
37.5
36
92.25
Used
indicative
personmonths
14.45
17.75
34.03
Lead
contracto
r
D1.1
D1.2
D2.1
Consortium Management
Administration Management
Standard Protocols Publication
1
1
2
1 - 36
1 - 36
14
D2.2
Multicentric Proposals
2
7th EP
Calls
34
92.25
40.53
D3.1
Technical Dissemination
Meetings
3
5;11; 20
77.25
36.93
D3.2
Publication of Technical
Guidelines
Technical Innovation: New
Fixatives
Guidelines for Human Tissues
Research Bioethics
Tissues Archives
3
36
7;12;15;16;
21; 25; 26;
31; 35; 36
36
AREA TS
AREA TS
Inst Path
FM UNIV
LJ
Inst Path
FM UNIV
LJ
UNITO
77.25
38.63
UNITO
4
36
36
43.5
15.95
ICGEB
5
34
35
150
71.35
UNIPI
6
28
36
42.75
20.23
6
28
28
42.75
21.23
D7.1
Archives of New Fixatives
Treated Tissues
Proteomics in Archive Tissues
7
16
25
15
3.15
D7.2
Proteomics in New Fixatives
7
28
32
36
15.4
Pathology
TUM
Pathology
TUM
Pathology
TUM
Pathology
TUM
D4.1
D5.1
D6.1
D6.2
17
Table 2: Milestones List
Milestone
no.
M1.1
M1.2
M1.3
M2.1
M2.2
M3.1
M3.2
M4.1
M5.1
M6.1
M7.1
Milestone name
Control of WP results
Diffusion of WP
results
Co-organization of the
general meetings
Technical guidelines
proposal
Definition of future
research proposals on
archive tissues
Standardized protocols
guidelines diffusion
European meetings
organization together
with ESP
Validation of new
fixation procedures
with better molecular
preservation
Proposal of bioethics
guidelines for human
tissues multicentric
research
Guidelines for archive
tissues multicentric
collection
Proteomic analysis
protocols in archive
tissues for research
and clinical
applications
Workpackage
no.
1
1
Date due
Actual/Forecast
delivery date
8; 36
36
Lead contractor
8; 36
36
1
5; 12; 20
2
14
1;7; 12; 15; 16; 21;
31; 35; 36
36
2
22
22
3
32
32
UNITO
3
11;30
7; 15;31
UNITO
4
36
36
ICGEB
5
36
36
UNIPI
6
28
35
Pathology TUM
7
28
32
Pathology TUM
AREA TS
AREA TS
AREA TS
Inst Path FM
UNIV LJ
Inst Path FM
UNIV LJ
18
Publishable results
-For each meeting held within the IMPACTS project, a detailed report was prepared. All the reports
can be accessed by the participants only through the IMPACTS website (www.impactsnetwork.eu,
documents section). They were also sent to the Commission with the periodic reports.
-As for the courses held in Graz and in Munich, all the participants received specific handbooks to
follow the course properly.
-On the specific issue the participants have produced a large number of publications that are
reported in the website (www.impactsnetwork.eu). The full list cannot be reported here because the
number of publications about archive tissues produced by the IMPACTS participants throughout
the three years of activity amounts to more than 500 references.
-As a result of the European Consensus Meeting on Bioethics and Human Tissues held in Pisa on
28 and 29 January 2010 within the IMPACTS project, a final detailed report will be produced and
publish as a paper.
-The final version of the guidelines for molecular analysis in archive tissues has been prepared and
edited, and will be sent to the publisher by the end of July 2010.
The index of the guidelines is as follows:
GUIDELINES FOR
MOLECULAR ANALYSIS IN ARCHIVE TISSUES
1. INTRODUCTION
2. ARCHIVE TISSUES
3. PRE-ANALYTICAL TREATMENT OF TISSUES
3.1. PRE-ANALYTICAL TIME INTERVAL (PATI) AND FIXATION
3.1.1. Pre-Analytical Time Interval (PATI)
3.1.2. Fixation
3.1.3. Tissue Processing
3.2. FORMALIN-FREE FIXATIVES
4. DISSECTION OF TISSUE COMPONENTS
4.1. MANUAL MICRODISSECTION
4.2. TISSUE MICROARRAY (TMA)
4.3 LASER CAPTURE MICRODISSECTION (LCM)
5. NUCLEIC ACID EXTRACTION AND QUANTIFICATION
5.1. DNA EXTRACTION FROM FORMALIN FIXED PARAFFIN EMBEDDED TISSUES
5.2. DNA EXTRACTION FROM FORMALIN FIXED PARAFFIN EMBEDDED TISSUES
(particularly for DNA extraction from SMALL FRAGMENTS of tissues or
microdissected cells)
5.3. FAST PROTOCOL FOR DNA EXTRACTION FROM FFPE TISSUES
5.4. DNA EXTRACTION FROM BLOOD AND FORENSIC SAMPLES
5.4.1. DNA Isolation from Blood Samples
5.4.2. DNA Isolation from Forensic Samples
5.5. RESTORATION AND RECONSTRUCTION OF DNA LENGHT FOR PCR
ANALYSIS IN ARCHIVAL AUTOPSY TISSUES
5.6. RNA EXTRACTION FROM FORMALIN FIXED PARAFFIN EMBEDDEDTISSUES
5.7. RNA TEMPERATURE DEMODIFICATION
19
5.8. microRNA EXTRACTION FROM FORMALIN FIXED PARAFFIN EMBEDDED
TISSUES
5.9. QUANTIFICATION OF NUCLEIC ACIDS
5.9.1. Spectrophotometric Quantification
5.9.2. Quantification With A Fluorescent Dye
5.9.3. Quantification By Realtime PCR
5.10. INTEGRITY ASSESSMENT OF NUCLEIC ACIDS
5.10.1. Agarose Gel Electrophoresis for DNA
5.10.2.. Denaturing Agarose Gel Electrophoresis For RNA
5.10.3. PCR Amplification Of DNA Fragments Of Increasing Length
5.10.4. RT-PCR Amplification Of mRNA Fragments Of Increasing Length
5.10.5. 5. 5’/3’ mRNA Integrity Assay
5.10.6. Agilent Bioanalyzer 2100
5.11. DNase TREATMENT OF RNA
5.11.1. DNase Digestion Followed By Phenol/Chloroform Extraction
5.11.2. DNase Digestion Followed By Heat Inactivation
6. REVERSE TRANSCRIPTION (RT)
6.1. REVERSE TRANSCRIPTION
6.1.1. RT With MMLV-RT And Random Hexamers
6.1.2. RT With AMV-RT And Specific Reverse Primer
7. NUCLEIC ACID AMPLIFICATION
7.1. GENERAL PROTOCOL FOR END-POINT PCR
7.2. QUANTITATIVE END-POINT PCR
7.3. NESTED PCR
7.4. GENERAL PROTOCOL FOR DOT-BLOT
7.5. PCR-ELISA
7.6. QUANTITATIVE REALTIME RT-PCR
7.6.1. Sybrgreen Chemistry
7.6.2. Taqman Chemistry
7.6.3. General Guidelines For Optimization Of The qRT-PCR Assay
7.6.4. General Guidelines For Data Analysis
7.6.5. Determination of The Most Appropriate Reference Gene/Panel Of Genes For
Normalization
7.6.6. Relative Quantification by qRT-PCR
8. DNA SEQUENCING
8.1. DNA SEQUENCING FROM PCR PRODUCT
8.2. PYROSEQUENCING FOR DETECTION OF KRAS MUTATIONS
8.2.1. DNA extraction from FFPE
8.2.2. PCR Amplification
8.2.3. Immobilization of PCR Product to Streptavidin Sepharose High Performance Beads
8.2.4. Sample preparation (Separation of the DNA Strands and Release of the Samples in
Q24 Plate)
8.2.5. Running of the PyroMark Q24
9. MICROSATELLITE ANALYSIS
9.1. MICROSATELLITE INSTABILITY (MSI)
9.1.1. DNA extraction from FFPE for Microsatellite analysis.
20
9.1.2. MSA - Basic method
9.1.3. MSA - DHPLC method
9.1.4. MSA – capillary electrophoresis method
9.2. GENERAL PROTOCOL FOR LOSS OF HETEROZIGOSITY (LOH)
9.2.1. DNA extraction from FFPE for LOH analysis
9.2.2. LOH Analysis – Basic Method
9.2.3. LOH Analysis – Capillary Electrophoresis Method
10. METHYLATION ANALYSIS
10.1. QUALITATIVE METHYLATION STATUS ASSESSMENT
10.1.1. DNA extraction from FFPE for methylation analysis
10.1.2. Bisulfite modification of DNA from FFPE
10.1.3. BM-Specific, Nested MSP For Methylation Detection Of The MGMT Gene
Promoter
10.1.4. Quality control after MSP
10.1.5. Methylation detection by MS-MLPA
10.2. QUANTITATIVE METHYLATION STATUS ASSESSMENT IN DNA FROM FFPE
with bisulfite modification, and Real-Time Quantitative MSP (Methylight)
10.2.1. DNA Extraction from FFPE for Quantitative Methylation Analysis
10.2.2. M.SssI Modification
10.2.3. Bisulfite Modification
10.2.4. Methylight Reaction Setup and Methylation Assessment
11. ANALYSIS OF COPY NUMBER VARIATIONS
11.1 COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
11.1.1. Extraction of Genomic DNA
11.1.2. Metaphase Chromosome Preparation
11.1.3. DNA Labelling by Nick Translation (indirect labelling)
11.1.4. Hybridization
11.1.5. Detection
11.1.6. Image Processing
11.2. MULTIPLEX LIGATION–DEPENDENT PROBE AMPLIFICATION (MLPA)
11.2.1. DNA Extraction
11.2.2. MLPA Procedure
11.3. FLUORESCENT IN SITU HYBRIDIZATION (FISH) ON FFPE TISSUE SECTIONS
(FISH)
12. MICROARRAYS
12.1. INTRODUCTION ON GENOME-WIDE EXPRESSION PROFILING FROM
FORMALIN FIXED AND EMBEDDING TISSUES USING MICROARRAYS
12.1.1. Clinical Applications
12.1.2 Basics on the FFPE Technologies
13. PROTEIN EXTRACTION IN ARCHIVE TISSUES
13.1. PROTEOMICS IN ARCHIVE TISSUE: INTRODUCTION
13.2 PROTEIN EXTRACTION FROM FORMALIN FIXED EMBEDDED TISSUES
13.2.1. Deparaffinization and Protein Extraction from Slide-Mounted Ffpe Sections
13.2.2. Deparaffinization and Protein Extraction from Ffpe Samples Cut Directly from a
Block
13.3. RAPID PROTEIN EXTRACTION FROM FROM FORMALIN FIXED EMBEDDED
TISSUES SAMPLES
21
13.4. PROTEIN EXTRACTION PROTOCOL FROM TISSUES TREATED WITH
FORMALIN FREE FIXATIVES
13.5. TCA PRECIPITATION OF PROTEINS
14. PROTEIN ELECTROPHORETIC SEPARATION
14.1. ONE-DIMENSIONAL SODIUM-DODECIL-SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS
14.2. VISUALIZATION OF PROTEINS
14.2.1. Coomassie Brilliant Blue staining
14.2.2. Silver Staining
15. PROTEIN IMMUNODETECTION
15.1. WESTERN BLOTTING
15.2 DOT BLOT
15.3. MEMBRANE STRIPPING
15.4. REVERSE PHASE PROTEIN MICROARRAYS
15.4.1. Protein Spotting
15.4.2.Protein Detection on Reverse Phase Protein Microarray
15.4.3. Estimation of Total Protein with Sypro Ruby Staining
16. MASS SPECTROMETRY
16.1 MASS SPECTROMETRY ANALISYS: INTRODUCTION
16.2 SAMPLE PREPARATION AND IN GEL TRYPTIC DIGESTION FOR MASS
SPECTROMETRY EXPERIMENTS
16.3. MALDI IMAGING MASS SPECTROMETRY ON FORMALIN FIXED PARAFFIN
EMBEDDED TISSUES
17. ELEMENTS OF GOOD LABORATORY PRACTICE
17.1. INTRODUCTION
17.2. INTERNAL QUALITY CONTROL (IQC) ORGANIZATION
17.2.1. Sample related procedures
17.2.2. Laboratory related procedures
17.3. STANDARDS FOR EQUIPMENT AND REAGENTS
APPENDIX A
APPENDIX B
22