The MRC London Neurodegenerative Diseases Brain Bank: a resource for neurodegeneration research Claire Troakes and Safa Al-Sarraj Institute of Psychiatry, King’s College London, UK ABSTRACT: Clinically and neuropathologically well-characterised human brain tissue is one of the most important resources for neuroscience research and is essential in the battle to develop new strategies and treatments for neurodegeneration. There has been significant research progress in recent decades and post-mortem tissue has played a major role in enabling advances in diagnosis, characterisation of pathological features, molecular genetics and bioinformatics. The MRC London Neurodegenerative Diseases Brain Bank (LNDBB) is one of the largest brain banks in the UK. Since its establishment in 1989 it has collected over 2000 cases (formalin fixed and frozen samples). We focus our banking on neurodegenerative diseases, including Alzheimer’s disease, Dementia with Lewy Bodies, Motor Neurone Disease and Frontotemporal lobe dementia and agematched controls but also house smaller collections, such as psychosis and paediatric disorders, in order to enhance research in these areas. The LNDBB operates a transparent and open-door policy for provision of central nervous system tissue to researchers. So far we have completed over 1270 requests and provided over 10, 000 samples to national and international institutions. We are part of the MRC UK Brain Banks and Brains for Dementia Research networks which aim to encourage and facilitate both tissue donation and accessibility and use by researchers. The brain bank also carries out studies into the best methods of preservation of tissue and the research potential of archival fixed tissue. We are constantly updating our procedures to ensure tissue is of the best quality for use in current research techniques. 1. Recruitment of cases a) “Diseased” brains Brain donations are accepted in the Brain Bank via two routes: I. Donor programme: The majority of donations from Alzheimer’s disease, MND and movement disorder patients are registered through cohort studies. These donations are ideal and provide not only well-characterised brain tissue but a fully documented clinical profile, sometimes with neuro-imaging and neuropsychological information. Such donations are very useful in many molecular and genetic studies where knowledge of clinical and pathological heterogeneity is important. II. Ad-hoc donation: This has noticeably increased in the last few years and is a very useful source of various types of dementias, other neurological conditions and “normal” control tissue. Brain tissue available Neuropathological diagnosis Stored material 1989 to 2010 Fixed TOTAL 5. Environment and links MRC Centre for Neurodegeneration The MRC Centre for Neurodegeneration Research at the Institute of Psychiatry aims to understand the mechanism of neurodegeneration and to translate this into new treatments by carrying out translational research and facilitating new collaborations. The Brain Bank occupies a key role in this and is a critical resource for the execution of the centre’s research. Brains for Dementia Research (BDR) The Brain Bank is a member of the Brains for Dementia Research network which is funded jointly by the Alzheimer’s Research Trust and the Alzheimer’s Society. King’s College London acts as the coordinating centre and the Institute of Psychiatry is one of 6 component Brain Banks. BDR aims to create and maintain a web based database for all accessible samples in the component brain banks, host training in organ retention, ethics and governance issues and introduce dedicated Brain Bank research workers who will be responsible for liaising with the donor and their families at the time of consent, the time of donation and afterwards. MRC UK Brain Banks Network The Brain Bank is also a member of the new MRC UK Brain Banks Network. This is an independent and coordinated national network of existing brain tissue resources. The network will seek to provide operational efficiency for the benefit of donors, researchers and future patients. Frozen 1716 Steering Committee 1452 EXAMPLES Alzheimer’s disease (AD) 459 AD-familial 33 14 AD & Cerebral Vascular Disease (CVD) 31 66 58 Autism 4 4 Argryophilic grain disease 8 5 Cerebral vascular disease 67 43 Corticobasal degeneration 12 8 Prion disease (CJD) 27 40 Dementia with Lewy Bodies (DLB) 73 57 Down’s syndrome 4 12 Frontotemporal dementia 49 37 Huntington’s Disease 10 8 Motor Neurone Disease 187 180 21 15 Parkinson’s Disease 26 18 Progressive supranuclear palsy 27 19 Rett Syndrome 5 5 Schizophrenia 1 23 Normal adult brain 156 Centre for Neurodegeneration Research IOP, KCL 17 AD & DLB King’s College Hospital Brain Bank Director: Dr Safa Al-Sarraj 368 Multiple system atrophy b) Control brains are collected via - recruiting normal individuals in cohort studies; - liaising with the local Transplant Coordinating centres and Coroners; - expanding on ad-hoc donation. 4. Database information We have a comprehensive database of information on registered donors and on donated brain tissue, detailing clinical and pathological diagnosis, post mortem details of fixed and frozen tissue, CSF etc and non-clinical data such as age, sex and source of donation. Recent donations are also being added to the MRC and BDR network searchable databases. 2. Post mortem and tissue collection a) Post mortem delays Ideally, we seek to minimise the post mortem delay (time between death and obtaining tissue for freezing and fixation) to less than 48 hours, wherever possible. Generally we accept donations with post mortem delay of up to 72 hours (to accommodate deaths that occur over a weekend or Bank Holiday). 20 b) We have a wide range of national and international collaborations and are considered to be a major resource of brain tissue. We have responded to over 2, 230 requests for tissue since our establishment in 1989 with 209 completed requests between 2006 and 2010. Cardiff 7. Tissue Quality Research Brain banks have large collections of fixed human post-mortem tissue stored as FFPE blocks and as wet tissue in formalin solution. Recent advances have enabled genetic material to be extracted from this tissue, however it is important to establish the quality of this DNA/RNA and its suitability for use in the most current research techniques. The effect of storage in liquid fixative on immunohistochemical reactivity is also important to investigate. We have therefore recently been studying the research potential of this archival tissue. 6 1 27 8 1 36 4 10 38 2 3 19 17 3 1 28 35 39 36 4 44 30 32 33 40 45 41 15 42 7 47 49 DNA extraction was carried out from a range of fixed tissues, of various disease classes, stored for a range of time (between 0 and 20 years) as blocks or in formalin (a total of 57 cases). The quantity and quality of DNA extracted from original FFPE blocks (taken at time of autopsy), tissue stored in liquid fixative, and new blocks processed from the wet tissue was examined. The effect of long-term storage in formalin on the reactivity of tissue to a number of diagnostic and research-relevant antibodies was investigated by comparing staining intensity in original and newly processed blocks from the same cases, using tissue microarray technology. 29 1 98 46 37 Bristol 6. Public Engagement The MRC London Brain Bank is committed to openness and transparency in its regulatory procedures and the research it supports. It regularly submits articles to patient support networks and newsletters and participates in public engagement exercises such as Research Open Days. Lay members and charity reps sit on the Steering committee. 14 2 26 25 4 23 MRC UK Brain Banks Network Newcastle 49 11 50 48 5 48 1 2 Overall, original blocks produced a higher quantity and better quality of DNA than either wet tissue or newly processed blocks. There was no direct correlation between time of storage in formalin and quality of DNA. Extracted DNA was of high enough quality to carry out PCR reactions but showed poor results in a gel electrophoresis run. Newly processed blocks showed a decreased level of reactivity with several currently used antibodies, including HLA-DP and GFAP. 13 Quality/purity of DNA extracted from Dementia with Lewy bodies cases. An increase in quality of extracted DNA was seen in the old blocks compared to new blocks and wet tissue. DNA quality (A 260/280 level) in all cases combined showed no significant correlation with length of storage time. Recent tissue provision 3. Tissue dissemination a) The Brain Bank has a transparent and open-door policy for providing services and brain tissue to requestors from any institution without prejudice, on the condition that ethical requirements are satisfied and that a scientifically sound case underlies the application. The researchers are asked to complete a request application providing an abstract of the project, source of funding and other aspects which is assessed by an approval committee. 43 Brain Net Europe 19 Brain Banks Brains for Dementia Research Coordinating Centre Manchester 130 22 21 b) Brain tissue dissection and diagnosis Sets of standard protocols are in use for tissue sampling of fresh and formalin fixed material. The brain is divided along the midline in the sagittal plane: one half to be fixed, while the other is freshly sliced. 50 blocks (1 cm x 1 cm) are sampled from the slices from predefined regions, snap frozen and stored at -80 C. The formalin fixed half of the brain is examined by the team of senior neuropathologists to provide diagnosis according to a comprehensive protocol which not only reaches a definitive diagnosis, but also includes details of the load and staging of pathology and other information important for researchers. Oxford London Neurodegenerative Diseases Brain Bank 1989 -2010 2006 2007 2008 2009 2010 Total completed requests 18 40 45 49 57 209 Total samples distributed 539 1401 2325 2204 3570 10, 039 Frozen 267 944 1515 1711 2967 7404 Fixed 272 457 810 493 603 2635 Disease categories requested A B GFAP immunohistochemistry (1: 4000) : tissue cores from old FFPE blocks show a higher intensity of staining than those taken from newly embedded blocks, suggesting an adverse effect of prolonged storage in formalin. The study shows that DNA can be extracted from FFPE blocks and wet formalin-stored tissue and that this DNA is of a suitable quality to be used in a number of current research techniques. However the best quality DNA is obtained from the original blocks, suggesting as many as possible should be taken at autopsy. The effect of storage in formalin on immuno-reactivity indicates that this variable should be routinely recorded and included in analysis. Contact Details The MRC London Brain Bank practises an unbiased policy towards all peer-review tissue request applications; however proof of ethical research approval for the research project may be required. Tissue request application forms can be requested from Dr Claire Troakes, PO 65, Institute of Psychiatry, King’s College London, De Crespigny Park, London SE 5 8 AF. Claire. troakes@kcl. ac. uk. All applications are treated confidentially. Acknowledgements: We thank the Medical Research Council-UK for supporting the Brain Bank. We thank the staff of the Clinical Neuropathology Department, including Dr Istvan Bodi, Dr Andrew King and Dr Tibor Hortobagyi, for advice and technical support. We also thank Vassiliki Spandoni and Richard Hudspith for technical and administrative help.
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