ART
All CFMPB activities are supported by an in house developed web application ‘Application and Request Tool’ (ART). ART is the online tool for e.g. study registration (human biospecimens IRBm and human data IRBd, or combi IRBdm and IRBb for biobanks), Institutional Review Board (IRB) review, lab logistics, track and trace of biospecimens and cost recovery (https://irb.nki.nl/). All the user activities (histology & digital pathology, immunohistochemistry, molecular genetics) are stored in the ART database and by doing so, information about the applications, biospecimen identifiers, succeeding actions and derivatives accumulates and this enriches the data already available.
Histology & digital pathology
The CFMPB provides full service of FFPE sectioning, frozen sectioning, histochemical stains (HE’s), micro/macro dissection of tissue, design and construction of Tissue Micro Arrays (TMA) and slide digitization.
Our Glass slide printer (printing 2D barcode etc.) and our 3DHistec P1000 slide scanner are part of our digital slide logistics. The online platform Slide Score supports the complete organization of our digital slide logistics (intern and extern) like sharing, scoring, TMA grid manipulation and is linked with ART.
We have 2 cryostats for frozen sectioning. For minimal frozen tissue fragments such as needle biopsies, we use the 4FME method using the Presto Chill.
TMAs are constructed using the TMA GrandMaster. When (old) FFPE blocks don’t fit in the TMA GrandMaster, the CFMPB has the ability to construct manual TMAs.
For dissection of very small tumor areas and obtaining tissue as pure as possible, the CFMPB has a laser microdissection (LMD) system from Leica (LMD6500). But this process is very labor intensive. The stereo microscope is also used to obtain specific small tissue from slides when it is not possible to scratch it off without a microscope.
The CFMPB is staffed for two days a week with a highly experienced histological technician mainly for FFPE sectioning and three days a week with a very dedicated research pathologist for scoring a lot of slides.
Histology equipment list:
- Microtome (Thermo Scientific)
- Discussion microscope (Leica)
- Glass printer Tissue-Tek auto write (Sakura)
- Multistainer (ST5020, Leica)
- Coverslipping Instrument (Klinipath)
- P1000 3D Histec scanner Sysmex (see below)
- Grand Master TMA arrayer Sysmex (see below)
- 2x Cryostates (1xLeica, 1xThermo Scientific)
- Prestochill Cryo-embedding system Prestochill (Milestone Medical)
- Laser Microdissection System (LMD6500, Leica)
Immunohistochemistry
The CFMPB has a fully equipped and dedicated immunohistochemistry (IHC) lab. All routine IHC is performed using the BenchMark Ultra (Ventana, Roche) automated stainer, in close collaboration with the human diagnostic pathology department (340 antibody protocols available in total of which 274 are in research setting only). Additionally we have 2 Discovery Ultra (Ventana, Roche) automated stainers with a broad array of tissue testing capabilities for developing new IHC protocols, multiplexing (brightfield brown, yellow, purple red and green and fluorescent), RNA scope and FISH.
All IHC slides are scannend and scored digitally by a pathologist using the Slidescore tool. All data is uploaded in the ART database.
The Vectra 3 scanner (Perkin Elmer) is used for digital pathology multi spectral imaging (coordinated by Erik Hooijberg, pathology department). The Vectra is a microscope/scanner enabling detection of up to six different markers in situ. Fluorescent immunohistochemistry (IF) allows for measurement of the number and position of immune cells, tumor cells, stromal cells and other components of the tumor microenvironment. A multiplex IF (MPIF) antibody panel for breast, head & neck, bladder and melanoma tumors is up and running and the design/optimization of additional (general) MPIF panels is in progress. HALO software (Indica labs) is used for digital image analysis. Ample experience is available for the development of new antibody staining protocols.
IHC equipment list:
- 2x Benchmark Ultra, Ventana stainer (Roche) (see below)
- 2x Discovery Ultra, Ventana stainer (Roche)
In collaboration with the Pathology department:
- Vectra 3 scanner (Perkin Elmer)
Molecular genetics
To guarantee standard high quality, all DNA and RNA isolations from NKI Biobank samples (FFPE, FF, serum, blood etc.) are performed by, or under supervision of, the CFMPB technicians (and in the clinical chemistry department). All samples are handled and stored in the dedicated CF lab (pre-PCR, QC conditions) under supervision of the coordinating technicians according to standard protocols modified and adapted to the specific requirements per project. The CFMPB has five QIAcube systems with which we carry out all molecular isolation requests. With these QIAcubes we mainly perform isolations using the QIAgen AllPrep DNA/RNA kit (FFPE and FF) with which we can effectively isolate DNA and RNA from the same tissue. The Clincal Chemistry Department (AKL) carries out all isolations of samples from the serum biobank and Blood biobank using the Qiasymphony. the CFMPB performs multiple QC measurements on all isolated samples.
A variety of molecular analysis techniques are offered, in close collaboration with the molecular diagnostic department (Sequenom Mass array, PCR, (digital)MLPA, Archer, HPV typing, NGS, Nanostring etc. ).
Molecular genetics equipment list:
- 5x Qiacube DNA/RNA isolation robot (Qiagen) (see below)
- Tapestation (Agilent)
- Nanodrop 2000 (Isogen lifescience)
- Quantas Fluorometer (Promega)
- Qubit 2.0 Fluorometer (Invitrogen)
- Nanostring Flex Analysis system (Nanostring)
In collaboration with and on location of the Pathology department:
- Sequenom (Agena Bioscience)
- Rotorgene (HPV)
In collaboration with and on location of the General clinical laboratory (AKL) department:
- Qia-Symphony (Qiagen)
All studies will be carried out by the CFMPB technicians, in consultation technical support can be provided by the applicant.