Amsterdam, Netherlands
NBB - Participant 10: Netherlands Brain Bank

Participant 10: Netherlands Brain Bank    
Meibergdreef 47
1105 BA Amsterdam
The Netherlands

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Project Leader


Dr. Inge Huitinga
Phone: +31 (20) 566 5499 
Fax: +31 (20) 691 8466 
 
Dr. Inge Huitinga


Project Staff


Ing. M. Kooreman (BSc)
Technical coordinator 

A. Van den Berg (MSc)
Technician, stainings for quality control 

P. Evers (BSc)
Technician, stainings for quality control 

M.C. Rademaker (MA)
Management assistant  

E.P. Fritschy (MA)
Donor registration, tissue user applications  

N.M. Klioueva (LLM)
Inventarisation of the legislation, ethical guidelines and donor register in the various member states 

C. van Eden (Phd)
Scientific staff member  

Dr. A. Rozemuller
Pathology Department of the VUmc
Neuropathologist, quality control, diagnostic criteria 

Prof. Dr. P. van der Valk
Pathology Department of the VUmc
Head of pathology department 

Pathology assistants/General assistance
Pathology Department of the VUmc 


Institute Presentation


The Netherlands Brain Bank

The Netherlands Brain Bank (NBB) is a department of the Netherlands Institute for Neuroscience, an institute of the Royal Netherlands Academy for Arts and Sciences (KNAW). The NBB has a close cooperation with the VUmc (the medical centre of the Vrije Universiteit). The VUmc takes care of the autopsies and diagnosis. The NBB (NBB) was established in 1985 to support neurobiological and medical research by supplying clinically and neuropathologically well-characterized post-mortem specimens from patients who suffered from neurological and psychiatric disorders.
The NBB provides specimens upon research protocols submitted in advance, specifying a variety of requirements, such as the age of patients and controls, total number of patients and controls needed, agonal state, post-mortem delay, exact anatomical boundaries of the brain region, type of fixation and other treatment requirements of the tissue. Each applicant has to send us an approval of their research grant application for which the tissue is requested and a summary of the project. Investigators who did not perform any work on human post-mortem tissue are requested to try to develop the techniques by performing animal experiments or they are offered to perform a small-scale pilot study on NBB specimens.

Due to the large variability of the collected material, there are many drawbacks in the use of post-mortem specimens. Therefore, brain banks should put special emphasis to the development of rapid autopsy systems, i.e. as practiced by the NBB (Ravid and Swaab 1993) and guarantee the quality of the specimens by proper matching for the various ante and post-mortem factors and by measuring the pH (Ravid et al., 1992).

The NBB has three unique features:

(1) The bank has established a national donor-program, consisting of approximately 1800 registered donors. All donors and their next of kin are requested to sign an informed consent, when joining the program. In addition to the donation of brain, spinal cord and body fluids for scientific research, the donors are able to donate some tissues for transplantation and whole body for the anatomy.
The NBB has collaboration with Euro-transplant through the national transplantation coordinators for tissues.

(2) Human brain tissue is obtained by means of rapid autopsies with a very short post-mortem delay, ranging between two to eight hours.

(3) A fresh brain dissection procedure is used. The latter is a difficult regime to establish, requiring qualified staff at inconvenient times, but is a prerequisite for an increasing range of technical procedures, such as cell culture, and for neurochemical, immunocytochemical and metabolic procedures. At autopsy, the pH of the cerebrospinal fluid (CSF) is determined as a measure for agonal state, which is of importance for, e.g., the yield of mRNA.

Once the brain has been removed it is macroscopically examined and immediately dissected following a standard protocol into 80 different structures. In addition to brain samples we also collect ventricular CSF in each rapid autopsy for measuring the pH of the brain and for subsequent investigation of biological markers and other compounds present in the CSF in various disorders. After centrifugation, the CSF is aliquotted into 1ml small tubes and then slowly frozen and stored in - 80 C. The freezers of the brain bank have a liquid nitrogen backup system and are carefully monitored to assure continuous temperature maintenance. Automatic signal alarm systems are used to maintain the right temperatures.

The Brain Bank has at its disposal a growing collection of body fluids (CSF, blood and plasma) obtained from a wide range of disorders as well as a large series of formalin fixed brain tissue. Donor tissues that are acquired for storage include also spinal cord, cerebral arteries, olfactory bulb, pituitary and optic nerves.
The majority of the brain specimens is rapidly frozen in liquid nitrogen and stored at -80 C. On request, the specimens can be obtained fresh, before freezing, or placed in special media or fixatives according to the research protocol.

A standard number of approximately 16 samples are fixed in formalin, further processed, and stained by various histological or immunocytochemical methods for diagnostic purposes. To prevent contamination of the dissected tissue and to make all specimens suitable for isolation of high yield m-RNA we use semi-septic methods including sterilization of instruments and working surfaces and use of protective gloves for personnel during tissue handling.
To support the clinical and neuropathological documentation, the Tau and Amyloid load are both graded (according to Braak) on the sections used for the neuropathological diagnosis and sent to the researchers. In collaboration with several groups we classify the genetic typing of the samples. All NBB material is routinely ApoE genotyped. In cases of fronto-temporal dementia and familial Huntington's disease, genetic analysis of the various families is given as well.

The NBB collects a comprehensive medical history from each donor after autopsy and we try to include as much relevant information available on chronic and acute diseases, their course and medication used, alcohol and drug abuse and smoking habits. All medical records are secured with informed consent of the donor or next of kin requested in advance together with the informed consent for performing the autopsy.

Shipped samples are always accompanied by a D-base printout specifying the major items such as age, gender, brain weight, post-mortem delay, pH (agonal state), clock time of death, types of tissue and anatomical structures acquired from the donor and a summary of each donor's medical history.

The final diagnosis is established after an extensive neuropathological examination and relates the findings to the clinical diagnosis. The definite diagnosis is sent to the patient's physician as well as to the various research groups. The neuropathology reports are produced in a highly standardized format, which on its turn increases significantly the utility of such reports for international investigators. The conclusions and summary of the neuropathology report are also sent to the investigators on request.

Once the diagnosis is complete the specimens are shipped on dry ice (for frozen samples) or in vacuum-sealed plastic (for fixed samples) to the various investigators. All the data regarding the material collected by the brain bank are stored in a database and the confidentiality of the patient data is maintained at all times. The existence of such a database provides instant access to relevant tissue, patient and autopsy data which are registered in a standardized manner. This also facilitates the matching task for both the NBB in supplying the samples and the investigators in setting up their experiments and interpreting the results correctly.

Standardization of protocols for collecting brain bank material:
Operating in the framework of the European Brain Bank Network (EBBN and later Brain-Net Europe) and in collaboration with other European Brain Banks, the NBB has managed to set up standardized protocols for the clinical and neuropathological diagnosis as well as standard procedures for the way human brain tissue is made available for research. These include protocols for sampling, dissection, tissue preparation and matching. In the current routine of brain banking it is of crucial importance to match samples for various factors. Ante-mortem factors include age, sex, circadian and seasonal variation, lateralization, medication and agonal state of the patient. Post-mortem factors include the post-mortem delay, freezing and fixation procedures, duration of fixing the tissue and storage time.

The samples provided by the bank are well documented and are controlled for quality in order to permit users to draw the accurate meaningful conclusions from their studies. Standardizing the documentation, collection, dissection, processing and storage procedures can ensure these demands. To assure the specimens quality, the bank pays special attention to monitoring the agonal state of the deceased prior to death. Measuring the pH of the brain in CSF collected during rapid autopsies makes it possible to match the samples for agonal state and has therefore been introduced as a routine procedure in the Netherlands Brain Bank. From observations made on human autopsy material collected by the NBB it became evident that the pH value measured in CSF is not affected by the post-mortem delay (Ravid et al., 1992). Tissue pH has been also reported to be a fair indicator of mRNA preservation in human post-mortem brain (Kingsbury et al., 1995). Measuring pH provides a simple means of screening and is thus a crucial measure for agonal state that has to be included in brain banking routine procedures.
The various enzymes, transmitter systems and other active substances in the brain have their specific localization. Therefore, data obtained by biochemical analysis in homogenates or tissue extracts have only a limited value and brain banks collecting human brain specimens for research should strive to develop techniques which leave the morphology of the tissue intact (Swaab et al., 1986, 1987; Swaab and Uylings, 1988).

Changes in freezing procedures, fixation and storage time may affect many of the parameters used to assess changes in the brain and the potentialities of staining procedures considerably. Tissues used for biochemical studies are usually rapidly frozen and slowly thawed. Synaptosomal preparations from post-mortem tissue examined by electron microscopy showed they were only slightly less pure than preparations from fresh tissue (Hardy et al., 1982). However, to isolate synaptosomes which are morphologically well preserved and have retained their metabolic performance, one should use the opposite procedure as snap-freezing generally yields metabolically and functionally inactive preparations (Hardy et al., 1983).
The fixation procedure used and storage of the fixed tissue prior to the application of immunocytochemical procedure may affect the staining. When using the proper fixation procedures, sufficient structural integrity is retained in the tissue to allow morphological and morphometrical studies. Formalin fixation causes increase in brain weight and subsequent washing in water introduces a systemic error in brain weight.
Brain banks have to standardize the fixation procedures and subsequent washing and embedding in order to minimize the systematic errors and variables. We introduced a standard fixation procedure in 10% buffered formalin for 1 month at room temperature for all brain bank samples (Fliers et al., 1985).

Safety measures:
Safety issues are of great concern for protection of NBB employees and investigators using brain bank specimens. Human fresh post-mortem tissues and fluids may contain highly infectious agents and have potential risks of diseases that are highly communicable to other humans. All specimens are treated as being a risk for such transmission and handled carefully as certain extremely hazardous infectious agents (viruses, prions) are very stable. The Creutzfeldt-Jakob (CJ) agent, for example, has been shown to be active in tissue, which has been fixed for over 30 years and could still be transmissible. It is resistant to formalin and withstands the conventional autoclaving (Asher et al., 1986). Dissection of fresh tissue is performed under a biohazard hood and discarded according to the policy for handling contagious material. To prevent use of contagious tissues/fluids we never ship tissue of unknown neuropathological diagnosis (see Appendix). As the prevalence of HIV grows, the risk of dealing with infectious material is not negligible, even in patients who are not supposed to be affected (Hauw et al., 1989).
The NBB does not perform rapid autopsies of either HIV or Creutzfeldt-Jakob patients. These autopsies are performed under special strict protocol, including the disinfection of the autopsy rooms and tools and fixing the tissues. All investigators receiving specimens from the NBB are clearly informed of the possible risk of infection, in all cases and asked to apply all necessary safety precautions when processing the samples.

User survey response and outcome assessment:
To be able to co-ordinate the growing number of diverse research projects performed on NBB material, we ask the investigators to report to us once per year about the progress in their work. This feedback is of extreme importance for the NBB to be able to evaluate the output of our efforts both qualitatively and quantitatively. We ask each group to submit a short abstract and a list of publications generated from NBB specimens. We also request the users to acknowledge the NBB in the 'Materials and Methods' and 'Acknowledgements' sections of their publications. Publications generated by users are a very important outcome assessment of brain banking but one should bear in mind that publishing results on human brain specimens is a slow process on itself, because of the time it takes to collect the relevant samples, the complexity of the procedures and techniques and the novelty of some of the methods used. We might be missing publications in our Progress Report due to a lag period of up to 2 years in publishing results generated from NBB material.

Facts and Figures
The NBB operates from the very beginning as an infrastructural organization whose aim is to supply investigators with specimens for high quality scientific research. We supply tissue to all applications provided the project has a high scientific quality and is well designed. We put emphasis on recruiting, collecting and distributing the collected tissue to various research groups in the Netherlands, other European countries, and to the international scientific community. As research progresses, the demands of neuroscientists change accordingly and the NBB needs to have a continuous update of standardized tissue collection, dissection protocols and tissue processing and preservation. As an outcome of these developments, our overall workload is growing; the number of requests and shipments increases and the same holds for the complexity of the requests from the investigators.

So far the Netherlands Brain Bank has provided brain material from 2806 autopsies for 600 research projects in countries all over the world (The Netherlands, other European countries, North America, Middle and Far East). Over 600 papers have been published in the international scientific press as an outcome of research using these specimens. The autopsies were performed on patients who suffered from various neurological and psychiatric disorders.


APPENDIX

Autopsies 1985 - 2006
Number of registered donors
Overview of tissue supply in the last two years

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