WO2004083369A2 - Composition de fixation tissulaire - Google Patents
Composition de fixation tissulaire Download PDFInfo
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- WO2004083369A2 WO2004083369A2 PCT/FR2004/000608 FR2004000608W WO2004083369A2 WO 2004083369 A2 WO2004083369 A2 WO 2004083369A2 FR 2004000608 W FR2004000608 W FR 2004000608W WO 2004083369 A2 WO2004083369 A2 WO 2004083369A2
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- WIPO (PCT)
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- tissue
- sample
- paraffin
- fixing
- composition
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Definitions
- the present invention relates to the field of the preservation of tissue samples under optimal conditions, so as to be able subsequently to carry out analyzes in molecular biology in particular from the tissues thus preserved for diagnostic or therapy purposes.
- the subject of the present invention is a tissue fixing composition allowing the preservation, within the tissue thus fixed, of proteins and nucleic acids so as to allow their analysis in situ or their subsequent extraction from the tissue in question for analysis purposes.
- tissue samples can provide important information relating to pathophysiological mechanisms such as the inflammatory response, growth or cell differentiation. Advances in the knowledge of such phenomena are at the origin of advances both in terms of diagnosis and treatment. If the freezing of tissue samples (at a temperature lower than or equal to - 80 ° C) remains the standard technique for analysis in molecular biology, its binding nature is recognized by all. Indeed, this technique not only requires appropriate facilities for storing frozen tissue, but also imposes drastic conditions for transporting said tissue so as not to break the cold chain.
- RNA on fixed and paraffin embedded tissues Three essential conditions are required for the analysis of RNA on fixed and paraffin embedded tissues: 1) a good yield of TARN extraction, 2) a good quality of the extracted RNA, and 3) the absence contamination by genomic DNA (Shibutani 2000).
- the extraction and analysis of total RNA from tissues fixed in formalin and included in paraffin have been mainly applied for the detection of viral RNA, in the context of hepatitis C for example (Dries 1999; Guerrero 1997).
- Methacarn has shown its effectiveness (Shibutani 2000), but the main disadvantage of this new fixer is the high toxicity of its constituents. Furthermore, the paraffin inclusion and dewaxing stages do not seem to have a major influence on the quality of protein extraction when the tissues have been fixed using non-bridging fixatives (Shibutani 2000).
- the present invention proposes to remedy the drawbacks of the prior art by proposing in particular a new tissue fixing composition based on non-toxic chemical compounds preserving the cellular and tissue structures.
- the tissue fixing composition of the invention has easy handling because it allows the preservation of tissue samples in paraffin block or in dehydrated form in particular.
- tissue fixing composition of the invention comprises trehalose associated with other compounds. More particularly, this composition comprises: - trehalose at a concentration between 40 and 80 g / 1, preferably between 40 and 70 g / 1 and more preferably still around 60 g / 1,
- - acetic acid in an amount between 0% and 5% (v / v), preferably between 0.5% and 3% (v / v) and more preferably still around 1% (v / v), and
- the inventors have also demonstrated that the preparation of the above tissue fixing composition could be carried out optimally in two stages.
- the first step is to prepare a mixture with only 45% ethanol (stable at 0 ° C) and to add pure ethanol (also stable at 0 ° C) when preparing the final composition in an amount sufficient to obtain the tissue fixing composition of the invention as specified above.
- This makes it possible in particular to stabilize the preparation products of said composition and to store them in a cold environment so therefore to use them already cooled from the start of the fixing.
- tissue-fixing composition of the invention it is possible to prepare, in a first step, a mixture comprising 60 g of trehalose, 10 ml of acetic acid, 300 ml of and
- the above mixture and the pure ethanol must be mixed just before using the final composition, in an amount of 5.5 parts of the mixture and 4.5 parts of ethanol.
- the final tissue fixing composition includes: 60 g of trehalose, 10 ml of acetic acid, 300 ml of water and 700 ml (250 + 450) of ethanol, which corresponds well to the composition indicated above.
- the above tissue-fixing composition additionally comprises glycerol in an amount between 0% and 10% (v / v), preferably between 3% and 8% (v / v ) and more preferably still around 6%.
- composition of the invention may in this case comprise 40 g / 1 of trehalose, 70% ethanol (v / v), 6% glycerol (v / v) 1% acetic acid (v / v) and 23% water (v / v).
- Glycerol is in fact added to the tissue fixing composition of the invention only in cases where a higher alcohol concentration is necessary, in particular for the purpose of retaining very good nucleic acids.
- the tissue fixing composition of the invention comprising glycerol makes it possible in particular, by the osmotic action of glycerol, to reduce the amount of trehalose used and to avoid its precipitation in a medium of higher alcoholic concentration.
- the subject of the present invention is also a method of tissue fixation consisting in immersing the fresh tissue samples in the tissue fixation composition of the invention, at a temperature between 0 and 6 ° C., of preferably between 0 ° and 4 ° C and more preferably still at a temperature of approximately 1 ° C and this, for at least 12 hours, preferably at least 16 hours and more preferably still for approximately 20 hours, depending on the fabric thickness.
- fresh tissue sample is meant any tissue sample resulting in particular from an animal sample, including a human sample, produced under standard conditions well known to those skilled in the art, it being understood that the fixation must occur as quickly as possible. possible after tissue devascularization in order to best preserve the quality of nucleic acids and in particular RNA.
- tissue fixing composition of the invention makes it possible to carry out a finer analysis and giving rise to better results and / or yields than those obtained by the use of 'a fixer of the prior art, but they have further improved the method of processing a tissue sample, with the aim of further improving the quality of subsequent biological analyzes.
- a tissue sample fixed for the purpose of subsequent analysis can be prepared in the context of the present invention according to a treatment method comprising the steps of: a) fixing the sample by means of the tissue fixing composition of the invention, b) dehydration of the sample obtained in a), c) preservation of the sample obtained in b), i) in the dehydrated state, ii) within a resin, or iii) by inclusion in paraffin.
- the inventors have demonstrated that the conservation of said sample in dehydrated form after it has been fixed by means of the composition of the invention makes it possible not only to carry out analyzes in situ of quality at least equivalent to that of the other known techniques but also made it possible to carry out extractions of nucleic acids of much better quality and more easily than by the implementation of said techniques of the prior art. It is in fact the combination of the implementation of the tissue fixing composition of the invention and the preservation of the sample thus fixed in the dehydrated state which has made it possible to obtain such results.
- RNAs extracted from tissue samples fixed by the composition of the invention and kept dehydrated have a better quality than when said samples, even fixed with the composition of tissue fixation of the invention were included in the paraffin.
- a tissue sample can be prepared in accordance with a treatment process comprising the steps of: a) fixing the sample using the tissue fixing composition of the invention, b) dehydrating the sample obtained in a) , c) imbibition of paraffin from the sample obtained in b), d) inclusion in the paraffin of the sample obtained in c), e) obtaining “cuts” from the inclusion block obtained in d) (by cutting the inclusion block into extremely fine “slices” a few microns thick), f) spreading a section obtained in e) on a glass slide and bonding this section, g) dewaxing of the section thus glued, h) coloring of said section.
- the biological analyzes are then carried out from this section both from the point of view of the visualization of certain constituent elements of the tissue thus prepared and from the extraction of nucleic acids or proteins.
- the blade is said to be “white blade” because not colorful.
- the dewaxing of the cut is carried out using successive xylene baths.
- the tissue sample is then gradually rehydrated using successive alcohol baths at 100 ° C, 80 ° C then 50 ° C, and finally in water. After this rehydration step, the tissue sample is colored.
- the inventors have surprisingly determined that it is possible to obtain even better results in terms of preserving the morphology of the tissue sample if the white slide, before the dewaxing step, was immersed in a bath comprising 75% (v / v) alcohol, 2% (v / v) formalin, 5% (v / v) acetic acid, 1% (v / v) Tween 20® and 17% (v / v) water for about 5 minutes at room temperature.
- the present invention relates to any type of tissue sample to be fixed and / or treated, in particular animal tissue samples, including human, pathological or not.
- the invention applies for example to the fixation and / or treatment of tumor tissues, in particular in the context of the constitution of a tumor library.
- the fresh tissue samples are immersed in the tissue fixing composition of the invention at about + 1 ° C for at least 12 hours. They are then dehydrated in 3 or 4 successive baths of absolute alcohol for one hour each at a temperature between 0 and 4 ° C and then in an acetone bath of about 1 h at about 4 ° C in 2 baths of acetone of approximately 1 hour each at room temperature.
- the samples are cut in a microtome ribbon and then dewaxed by 2 successive baths of xylene and 2 baths of absolute ethanol.
- the extraction uses a lysis buffer (Tris HC1 100 mM pH 7.4, 2% SDS, protease inhibitor TM Sigma) at 95 ° C for 5 minutes and then sonication.
- a lysis buffer Tris HC1 100 mM pH 7.4, 2% SDS, protease inhibitor TM Sigma
- Second extraction method The extraction uses a lysis buffer (Tris HC1 50 mM pH 7.5, 150 mM NaCl, Nonidet P40 1%, protease inhibitor TM Sigma) at 4 ° C and grinding.
- a lysis buffer Tris HC1 50 mM pH 7.5, 150 mM NaCl, Nonidet P40 1%, protease inhibitor TM Sigma
- the protein samples are resolved on a 10% polyacrylamide gel and transferred to a PVDF membrane.
- the membrane is stained by incubation for one hour in a solution of AMIDOBLACK TM (0.1% AMIDOBLACK TM Sigma, 45% ethanol, 10% acetic acid).
- the membrane is incubated with a primary antibody, then a secondary antibody, then is revealed by chemiluminescence (ECL + TM Pierce) according to the manufacturer's recommendations. 3. DNA extraction
- Trizol TM extraction After dewaxing or cutting frozen samples.
- Tissue sections 5 ⁇ m thick were produced, spread on slides, dewaxed and then stained with hemalun-eosin for a morphological study.
- the inventors obtained a very good histological and cytological morphology, both in terms of the epithelial and connective constituents, comparable to that observed in current technique.
- a post-fixing step (optional) can be carried out: after spreading the sections on slides, the slides are immersed before dewaxing for 5 minutes in AFA supplemented with 1% of Tween 20, then rinsed with water.
- the preservation quality of the antigenic sites was evaluated by immunohistochemical study using a large battery of monoclonal antibodies (Table 1). This study was carried out on paraffin sections, spread on slides and dewaxed, without reactivation of the antigenic sites (no microwave, nor enzymatic digestion), even if it was recommended by the supplier of the antibody.
- the peroxidase activity was revealed using the LSAB kit (Dako). For each antibody, the marking obtained on the same tissue type was analyzed in a comparative manner between the usual fixative (AFA) and the fixative composition of the invention (Table 1).
- the immunoreactivity observed with the tissue fixing composition of the invention is higher than that observed with the usual fixer (AFA) for all of the antibodies tested.
- AFA usual fixer
- the use of the composition of the invention makes it possible to suppress the pretreatment, intended to unmask the antigenic sites, even if this is recommended by the supplier laboratory.
- the extraction yields differ according to the type of extraction buffer: the fixative used.
- the composition of the invention always keeps yields lower than those of freezing but gives better results than AFA.
- DNA extraction tests were carried out on tissue samples fixed according to the technique of the invention, fixed in AFA or frozen, a few days after fixing the samples and then repeated a year later.
- the amount of DNA extracted from the tissues fixed by the composition of the invention was similar to that of frozen tissues, much greater than that of tissues fixed in AFA (Table 3).
- the DNA extracted during these manipulations was of very high molecular weight.
- the inventors were able to amplify by PCR a 2800 bp sequence of the BRCA-1 gene.
- the tissue fixation technique of the invention is therefore compatible with the extraction and analysis of DNA from tissues thus fixed and included in paraffin. Migration of total DNA on agarose gel to
- RNA extraction tests were carried out on tissue samples fixed according to the technique of the invention, fixed in AFA or frozen, a few days after fixing the samples and then repeated a year later.
- the amount of RNA extracted from the tissues fixed by the composition of the invention is similar to that of frozen tissues, much greater than that of tissues fixed in AFA (Table 4).
- the bands corresponding to the 28S and 18S ribosomal RNAs are visible, demonstrating good preservation of the RNAs.
- the use of the fixing composition of the invention made it possible to obtain the same RNA profile as that obtained by freezing, before inclusion in paraffin.
- RNA obtained and the absence of contaminating DNA were evaluated by amplification of the Raf gene by RT-PCR.
- Tissue samples were fixed using the tissue fixing composition of the invention and then dehydrated.
- RNAs were then extracted from these different samples, resolved on 0.8% agarose gel and compared with those extracted from frozen samples. It turns out that the RNAs extracted from paraffin blocks give rise to an attenuation of the bands corresponding to the ribosomal RNAs and the appearance of a slight “smear” suggestive of partial degradation and / or contamination by l DNA. Identical results are obtained after one year of conservation of the samples. However, the RNAs extracted from the samples preserved in dehydrated form have a better quality in the sense that they appear in particular more clearly on the gel (data not shown).
- paraffin samples are accompanied by a partial degradation of the messenger RNAs and a possible contamination by genomic DNA. Therefore, when the study of nucleic acids is essential, it appears preferable to keep the fixed samples in dehydrated form in an anhydrous medium, without including them in paraffin.
- TGO hepatic enzyme
- the formula of the tissue fixing composition of the invention comprises trehalose, ethanol, acetic acid and water (called T6 A7 A01) and may be declined in two forms, one comprising glycerol but no acetic acid (called T4 G6 A7) and the other devoid of acetic acid (called T6 A7).
- T6 A7 A01 trehalose, ethanol, acetic acid and water
- T4 G6 A7 acetic acid and water
- T6 A7 A01 trehalose 60 g / 1; 1% acetic acid (V / V); ethanol 70% (V / V); water 29% (V / V), - T4 G6 A7: trehalose 40 g / 1; glycerol 6% (V / V); ethanol 70% (N / N); water 24% (N / N),
- T6 A7 trehalose 60 g / 1; ethanol 70% (V / V); water 30% (V / V). • three other fixing compositions of the same formulation but whose trehalose has been replaced by another diose: sucrose:
- S4 G6 A7 sucrose 40 g / 1; glycerol 6% (V / V); ethanol 70% (N / N); water 24% (V / N), 6.
- S6 A7 sucrose 60 g / 1; ethanol 70% (V / V); water 30% (V / V).
- a fresh nude mouse liver was dissected into six equivalent fragments and each of them was fixed in one of the above six fixing compositions according to the protocol reported in the description above.
- the samples were dehydrated and stored at 4 ° C.
- This protocol was carried out four times, on different days, from different mouse livers.
- the proteins were extracted using a weak denaturing extraction buffer (Tris HC1 50 mM pH 7.5, ⁇ aCl 150 mM, ⁇ onldet P40 1%, protease inhibitor TM Sigma) at 4 ° C and assayed according to the technique of Bradford.
- a weak denaturing extraction buffer Tris HC1 50 mM pH 7.5, ⁇ aCl 150 mM, ⁇ onldet P40 1%, protease inhibitor TM Sigma
- the measurement of the enzymatic activity of the TGO was carried out by a Konelab 5.0.5 automaton. The results were weighted by the amount of protein and reported as international imity / microgram of protein (IU / ⁇ g of protein).
- Rupp GM Locker J. Purification and analysis of RNA from paraffin-embedded tissues. Bio Techniques 1988; 6: 56-60.
- RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. Bio Techniques 1991; 11: 304-8.
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Abstract
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Priority Applications (5)
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NZ542908A NZ542908A (en) | 2003-03-12 | 2004-03-12 | Tissue fixing composition based on non-toxic chemical compounds that preserve cell and tissue structures, and provides easy handling as it allows tissue samples to be conserved in a paraffin block or in dehydrated form |
EP04720046A EP1601950A2 (fr) | 2003-03-12 | 2004-03-12 | Composition de fixation tissulaire |
US10/548,567 US20070072167A1 (en) | 2003-03-12 | 2004-03-12 | Tissue binding composition |
AU2004221625A AU2004221625B2 (en) | 2003-03-12 | 2004-03-12 | Tissue binding composition |
CA002518981A CA2518981A1 (fr) | 2003-03-12 | 2004-03-12 | Composition de fixation tissulaire |
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FR0303043A FR2852392B1 (fr) | 2003-03-12 | 2003-03-12 | Composition de fixation tissulaire |
FR03/03043 | 2003-03-12 |
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WO2004083369A2 true WO2004083369A2 (fr) | 2004-09-30 |
WO2004083369A3 WO2004083369A3 (fr) | 2005-01-20 |
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PCT/FR2004/000608 WO2004083369A2 (fr) | 2003-03-12 | 2004-03-12 | Composition de fixation tissulaire |
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US (1) | US20070072167A1 (fr) |
EP (1) | EP1601950A2 (fr) |
AU (1) | AU2004221625B2 (fr) |
CA (1) | CA2518981A1 (fr) |
FR (1) | FR2852392B1 (fr) |
NZ (1) | NZ542908A (fr) |
WO (1) | WO2004083369A2 (fr) |
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EP2202320A1 (fr) | 2008-12-24 | 2010-06-30 | Agendia B.V. | Procédés et moyens pour le typage d'un échantillon comprenant des cellules colorectales cancéreuses |
WO2011157678A1 (fr) | 2010-06-14 | 2011-12-22 | Qiagen Gmbh | Procédé de détermination de cellules ou de tissu cibles pour l'extraction de biomolécules à partir d'échantillons biologiques fixés |
WO2012044167A2 (fr) | 2010-09-28 | 2012-04-05 | Agendia N.V. | Procédés et moyens de typage d'un échantillon comprenant des cellules cancéreuses basés sur les voies de transduction du signal oncogène |
WO2012087144A2 (fr) | 2010-12-23 | 2012-06-28 | Agendia N.V. | Procédés et moyens de classification moléculaire des cancers colorectaux |
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US11313772B2 (en) * | 2015-11-13 | 2022-04-26 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Fixatives and methods of use |
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US20020076445A1 (en) * | 2000-02-10 | 2002-06-20 | Crowe John H. | Eukaryotic cells and method for preserving cells |
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2003
- 2003-03-12 FR FR0303043A patent/FR2852392B1/fr not_active Expired - Fee Related
-
2004
- 2004-03-12 AU AU2004221625A patent/AU2004221625B2/en not_active Ceased
- 2004-03-12 WO PCT/FR2004/000608 patent/WO2004083369A2/fr active Application Filing
- 2004-03-12 US US10/548,567 patent/US20070072167A1/en not_active Abandoned
- 2004-03-12 NZ NZ542908A patent/NZ542908A/en not_active IP Right Cessation
- 2004-03-12 CA CA002518981A patent/CA2518981A1/fr not_active Abandoned
- 2004-03-12 EP EP04720046A patent/EP1601950A2/fr not_active Withdrawn
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US4839194A (en) * | 1985-07-05 | 1989-06-13 | Bone Diagnostic Center | Methods of preparing tissue samples |
EP0311035A2 (fr) * | 1987-10-09 | 1989-04-12 | Dr. Barry A. Siegfried | Fixateur histologique |
US5849517A (en) * | 1991-05-08 | 1998-12-15 | Streck Laboratories, Inc. | Method and composition for preserving antigens and nucleic acids and process for utilizing cytological material produced by same |
US5648222A (en) * | 1994-07-27 | 1997-07-15 | The Trustees Of Columbia University In The City Of New York | Method for preserving cells, and uses of said method |
US20020094577A1 (en) * | 1998-06-30 | 2002-07-18 | Guirguis Raouf A. | Cytological and histological fixative composition and methods of use |
Cited By (21)
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EP1895287B1 (fr) * | 2006-08-29 | 2009-12-23 | Biosepar -Gesellschaft für Medizin- und Labortechnik mbH | Utilisation de hexaméthylènetétramine pour la fixation de matériaux biologiques |
EP2202320A1 (fr) | 2008-12-24 | 2010-06-30 | Agendia B.V. | Procédés et moyens pour le typage d'un échantillon comprenant des cellules colorectales cancéreuses |
WO2010074573A1 (fr) | 2008-12-24 | 2010-07-01 | Agendia B.V. | Procédés et moyens de typage d'un échantillon comprenant des cellules de cancer colorectal |
US8921051B2 (en) | 2008-12-24 | 2014-12-30 | Agendia B.V. | Methods and means for typing a sample comprising colorectal cancer cells |
WO2011157678A1 (fr) | 2010-06-14 | 2011-12-22 | Qiagen Gmbh | Procédé de détermination de cellules ou de tissu cibles pour l'extraction de biomolécules à partir d'échantillons biologiques fixés |
WO2012044167A2 (fr) | 2010-09-28 | 2012-04-05 | Agendia N.V. | Procédés et moyens de typage d'un échantillon comprenant des cellules cancéreuses basés sur les voies de transduction du signal oncogène |
EP3257950A1 (fr) | 2010-09-28 | 2017-12-20 | Agendia N.V. | Procédés et moyens de typage d'un échantillon comprenant des cellules cancéreuses basés sur les voies de transduction du signal oncogène |
WO2012087144A2 (fr) | 2010-12-23 | 2012-06-28 | Agendia N.V. | Procédés et moyens de classification moléculaire des cancers colorectaux |
US10036070B2 (en) | 2010-12-23 | 2018-07-31 | Agendia N.V. | Methods and means for molecular classification of colorectal cancers |
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US10072301B2 (en) | 2011-07-13 | 2018-09-11 | Agendia N.V. | Means and methods for molecular classification of breast cancer |
WO2014058317A1 (fr) | 2012-10-10 | 2014-04-17 | Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis | Procédés et moyens de prédiction de résistance à un traitement anti-cancer |
WO2014080381A1 (fr) | 2012-11-26 | 2014-05-30 | Ecole Polytechnique Federale De Lausanne (Epfl) | Classification de cancer colorectal à l'aide de pronostic différentiel et de réponses thérapeutiques personnalisées |
WO2015080585A1 (fr) | 2013-11-28 | 2015-06-04 | Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis | Méthodes pour la classification moléculaire du cancer du sein et/ou de l'ovaire de type brca |
WO2016148573A1 (fr) | 2015-03-17 | 2016-09-22 | Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis | Méthodes et moyens de sous-typage du carcinome lobulaire infiltrant |
WO2016153344A1 (fr) | 2015-03-20 | 2016-09-29 | Iq Products B.V. | Nouveau marqueur du diabète gestationnel |
WO2016153355A2 (fr) | 2015-03-26 | 2016-09-29 | Microbiome Limited | Nouvelle régulation de processus d'isolation et d'amplification |
WO2018128544A1 (fr) | 2017-01-06 | 2018-07-12 | Agendia N.V. | Biomarqueurs pour la sélection de groupes de patients, et leurs utilisations |
WO2018151601A1 (fr) | 2017-02-17 | 2018-08-23 | Stichting Vumc | Diagnostic et sélection de thérapie améliorés par l'intelligence en essaim pour le cancer à l'aide de plaquettes éduquées contre les tumeurs |
WO2020022891A2 (fr) | 2018-07-26 | 2020-01-30 | Stichting Vumc | Biomarqueurs pour fibrillation auriculaire |
WO2021221500A1 (fr) | 2020-04-27 | 2021-11-04 | Agendia N.V. | Traitement du cancer du sein her2 négatif, mammaprint high risk 2. |
Also Published As
Publication number | Publication date |
---|---|
NZ542908A (en) | 2008-02-29 |
WO2004083369A3 (fr) | 2005-01-20 |
FR2852392B1 (fr) | 2005-07-08 |
FR2852392A1 (fr) | 2004-09-17 |
CA2518981A1 (fr) | 2004-09-30 |
US20070072167A1 (en) | 2007-03-29 |
AU2004221625A1 (en) | 2004-09-30 |
EP1601950A2 (fr) | 2005-12-07 |
AU2004221625B2 (en) | 2009-07-09 |
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