NeuroBioBank Best Practices

This page is intended to serve as a guide that integrates the policies and procedures utilized by the Brain and Tissue Repositories (BTRs) participating in the NIH NeuroBioBank (NBB) program. Although the specific and operational procedures employed by the participating BTRs may differ from each other in detail, all of the banks adhere to the general principles and approaches described herein.


Enrollment/Informed Consent

Donations are obtained from individuals who register before death, and/or from next-of-kin who authorize a postmortem donation. Eligibility for postmortem donation of brain and other tissues is determined by trained staff at the individual BTR. BTR staff notification of a request for postmortem donation may come from surviving family members, treating physicians, hospital systems, donor services, organ and tissue banks, disease advocacy groups, specialized residential facilities, and/or collaborating medical examiners.

Although the specific procedures for enrollment and consent may vary between the BTR sites, the policies and procedures of each bank have been reviewed and approved by their respective Institutional Review Boards (IRB) and any additional appropriate oversight committees of the BTR site's home institution. Trained individuals request and document consent for brain tissue donation from the deceased's next-of-kin or legally authorized representative. Individual requests for release of medical records, questionnaires, and/or interviews with individuals knowledgeable of the deceased are obtained according to IRB approved policies and procedures.

Brain Collection

The BTRs serve as biospecimen source sites for the collection of brain, other relevant tissues, and fluids from postmortem donors. Each NBB BTR has trained staff who conduct rapid recovery and processing of postmortem specimens.

The brain is removed from the calvarium, weighed and photographed. After the brain has been removed, the olfactory bulbs and, when possible, cerebrospinal fluid (CSF) and a portion of the cervical spinal cord are retrieved. In addition, some BTRs collect and bank other tissue specimens including skin and samples from other organ systems. Postmortem blood is sampled and submitted for serology and toxicology testing.

Quality control measures are initiated at the time of biospecimen collection and include documentation of agonal state and postmortem interval, standardized sectioning, processing and storage of the brain and related tissues/fluids, completion of a standard neuropathology assessment, and measurement of the RNA Integrity and pH.

The BTRs use comparable collection, processing and storage protocols. In general, one hemisphere of a brain is fixed either with formalin or 4% paraformaldehyde, and the other hemisphere is flash frozen. However, both fixed and frozen tissues are not always available from every brain.

The unique anatomic structures and subdivisions of the brain require reliable and reproducible sampling protocols.

The BTRs use standardized templates for dissection and matching of select anatomic regions by personnel trained in human brain anatomy. Regions of interest are sampled from a series of hemi-coronal slices of fixed or frozen brain tissue for distribution to end-users.

Tissue Processing and Preparation

Brain and tissue specimens are excised as fresh preparations, packed in wet ice, and transported to BTR laboratories within 24 hrs from death. Upon receipt by a BTR, specimens are weighed and photographed and the two cortical hemispheres are bisected and photographed. One hemisphere (or selected bilateral blocks) may be dissected and fixed (fixation protocols may vary modestly among brain banks). From the fixed hemisphere, some tissue blocks are paraffin embedded, sectioned and retained for neuropathology assessment and diagnosis, while other tissue blocks are preserved for distribution. The other hemisphere (or selected bilateral blocks) may be dissected in coronal sections approximately 0.5-1.0 cm thick, photographed, flash-frozen (-150°C) and stored at -80°C.

The BTRs prepare samples of brain and other tissues for the widest use by the neuroscientific community. For some NBB cases, limited quantities of DNA and/or RNA may be available for distribution.

In addition to the preparations described above, end-users may require brain and tissue samples at particular stages of neurological disease (e.g. CERAD or Braak staged Alzheimer's disease or Parkinson's disease cases), and/or may require specific brain, brainstem or spinal cord loci, which require careful microdissection. The BTRs use standardized anatomic procedures guided by established landmarks to ensure regional samplings of the brains are processed with precision and uniformity.

Specimens are generally available as...

  • Cryoprotected formalin or paraformaldehyde fixed tissue blocks
  • Formalin or paraformaldehyde fixed paraffin-embedded (FFPE) blocks or glass slide mounted sections
  • Paraformaldehyde-fixed, cryoprotected sections
  • Flash frozen blocks or frozen tissue aliquots dissected from hemi-coronal slices maintained at -80°C

Neuropathology

All brain specimens donated to the NIH NBB are assessed and reviewed by board-certified neuropathologists. A standard assessment is performed to document possible neuropathologies and to establish diagnosis of disease condition. Gross inspection of the brain includes assessment of regional atrophy and inspection of macroscopic lesions and blood vessels for vascular disease. Brains are photographed in multiple planes to document the gross features and macroscopic abnormalities of each donation.

A two stage logical algorithm-based strategy is followed for microscopic neuropathological assessment of all donor brain specimens.

In the first step, the clinical presentation of each donor is reviewed along with a screening assessment of the brain. Following gross examination of the brain, watershed territories (Brodmann areas 4,3,1,2), basal ganglia and cerebellum are sampled, H&E (hematoxylin-eosin) are stained and microscopically evaluated. For all donors ≥65 years of age the screening protocol is followed by the full neuropathological assessment described below.

For donors below age 65, clinical assessments are reviewed for any indication of brain-related disorders with known physical impact on the brain. If clinical evidence for such disorders is identified, the brain receives a full disease-appropriate neuropathological assessment. If the review of the clinical history does not endorse the presence of discernable neuropathology, then, the neuropathological screening protocol is implemented to rule out unsuspected pathology. If this screen reveals any indication of neuropathology, then a full evaluation is performed using stains for the indicated neuropathology. If there is no clinical history of a brain disorder with identifiable histopathology or systemic disease with neuropathological consequences, and the neuropathology screen confirms the absence of any pathologic process, then the neuropathological assessment is determined to be complete.

Neuropathological assessment of brains from aged persons (≥5) or donors with a clinical diagnosis of neurologic disease are examined with immunohistochemistry performed in multiple brain regions, including: superior and middle frontal gyrus, orbital cortex, watershed territories (Brodmann areas 4,3,1,2), basal ganglia with basal forebrain, amygdala, hippocampus (rostral and caudal levels with adjacent amygdala, parahippocampal and inferior temporal cortex), superior temporal gyrus, parietal cortex (angular gyrus), calcarine cortex, hypothalamus with mammillary bodies, thalamus, substantia nigra, midbrain at the level of the SN, pons including LC, medulla including DMV, cerebellar vermis, lateral cerebellar hemisphere and cervical spinal cord.

In addition to these standardized brain regions, additional sampling is performed based on clinical history or neuropathology review and findings where applicable.

Histochemistry and immunohistochemistry can include, but is not limited to: H&E, modified Bielschowski, thioflavin S, anti-beta-amyloid, alpha synuclein, phosphorylated tau and TDP-43. Cerebrovascular pathology is assessed using an adaptation of the protocols described by Vinters et al., 2000. The vessels of the circle of Willis are photographed and evaluated semiquantitatively. The extent of arteriosclerosis is also assessed semiquantitatively. Congophilic angiopathy is rated using the criteria developed by Vonsattel, et al., 1991. Ischemic lesions are tabulated and mapped as: cystic infarcts; lacunar infarcts; and microinfarcts. The hippocampus is evaluated separately for sclerosis.

Following the completion of the neuropathology evaluation, a report is generated indicating major neuropathological findings. This report is available to researchers and to the donor's family / next-of-kin, by request.

Serology and Toxicology

Postmortem brain and tissue donations received through the BTRs are tested via CLIA certified laboratory assessments for HIV-1/2, Hepatitis B surface antigen (HBsAg), Hepatitis B surface antibody (anti-HBs) and Hepatitis C.

Irrespective of serology findings, all users are advised to exercise universal precaution (CDC-1987) for blood borne pathogens when handling human tissue biospecimens.

Postmortem analysis of nicotine, alcohol, illicit drugs and pharmaceuticals are performed on the majority of cases in the NBB biorepository. Postmortem blood or brain tissues toxicology assays are performed by NMS Labs using the expanded postmortem toxicology panel (8052B/T) unless other tests of psychoactive substances are indicated. If a positive result is obtained, data on further quantitative analyses may be available. Annotated toxicology results are anticipated to be available for brain and tissue samples from the NIH NBB.

Distribution of Brain Biospecimens and Tissues

All requests for brain and tissue specimens are placed through the NIH NBB website. Following review and approval by NIH and NBB staff, one or more of the repositories will fill the request based on specimen availability. If questions arise, the requestor is contacted to clarify and confirm study design.

Although there is no fee for specimens from the NIH NBB, end users are required to provide an account number with a shipping carrier to cover the costs of shipping the approved specimens. Shipping usually occurs between 4-6 weeks following approval; however, large requests may take longer to process. If the shipment is intended for a foreign country, the researcher is responsible for obtaining the required documents for entry of biohazardous material.

By accepting tissue from the NIH NBB, the researcher attests in the signed MTA to acknowledge in all publications and presentations NIH NBB and the specific BTR(s) as the source of tissue (e.g., "Human tissue was received from the NIH NBB at the University of Miami and the Sepulveda Research Corporation"). Recipients are required to submit a progress report no later than 1 year after receipt of tissue.

Requestor's must follow the Tissue Request Instructions including the Tissue Request Guidelines and Tissue Request Standards when submitting a request.

Issues that frequently arise include...

  • Clarification of amount of tissue or number of sections needed
  • Clarification of number of cases and unaffected controls requested
  • Method validation in postmortem human brain
  • Sample size required for pilot vs full study
  • Lack of availability of specific requested cases and the need for substitution

References

  • Hyman BT, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Carrillo MC, Dickson DW, Duyckaerts C, Frosch MP, Masliah E, Mirra SS, Nelson PT, Schneider JA,Thal DR, Thies B, Trojanowski JQ, Vinters HV, Montine TJ. National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease. Alzheimers Dement. 2012 Jan;8(1):1-13.
  • Esiri MM; Perl DP. Oppenheimer's Diagnostic Neuropathology: A practice manual. 3 ed: Oxford University Press; 2006.
  • Vinters HV; Ellis WG; Zarow C; Zaias BW; Jagust WJ; Mack WJ, et al. Neuropathologic substrates of ischemic vascular dementia. JNeuropatholExpNeurol. 2000;59(11):931-45.
  • Vonsattel JP; Myers RH; Hedley-Whyte ET; Ropper AH; Bird ED; Richardson EP, Jr. Cerebral amyloid angiopathy without and with cerebral hemorrhages: a comparative histological study. AnnNeurol. 1991;30(5):637-49.