NINDS – HD - Initial brain processing Four options 1.Thorough processing (laboratories specialized in brain banking) 2.Half brain fixed / half brain sliced fresh -> frozen 3.Half brain fixed / half brain frozen en bloc 4.Whole brain fixed 1.Vonsattel JPG, Amaya MdP, Cortes EP, Mancevska K, Keller CE. Twenty-first century brain banking: Practical prerequisites and lessons from the pasts: The experience of New York Brain Bank, Taub Institute, Columbia University. Cell Tissue Banking 2008;9: DOI: /s y /s y 2.Keller CE, Amaya MdP, Cortes EP, Mancevska K, Vonsattel J-PG. Electronic tracking of human brain samples for research. Cell Tissue Banking 2008;9: DOI: /s /s Vonsattel JPG, Amaya M, Keller C. Twenty-first century brain banking. Processing brains for research: the Columbia University methods. Acta Neuropathologica 2008;115: DOI: /s /s

“#1” Thorough processing -One half immersed in 10 percent buffered formalin solution - Standardized series (minimum 18) of blocks for neuropathological evaluation - These blocks are then available for further studies, as is the rest of the fixed half brain One half processed fresh 1 - Fresh frozen blocks (each about 3.0 x 2.5 x 0.5 cm) 1.1) standardized series, as above (fixed); 1.2) extra blocks, as needed 2- Fresh parenchyma pulverized in liquid nitrogen Processed locally or shipped to the closest processing laboratory

“#1” Advantage -Improve their anatomic specificity and quality -Samples are ready for immediate disbursement once categorized diagnostically, reducing the time between the receipt of request and disbursement of samples -Prevents thaw-refreeze cycles & carving out of regions of interest from frozen tissue, which is cumbersome and deleterious to both samples and source brains -Accurate quantitative data on stored samples according to anatomical regions and distributive diagnosis -Monitoring freezer compartments made available upon sample disbursement Dissection and harvest of samples at the fresh state

“#1” Disadvantage -Requires prosector with reliable command of the protocol and neuropathological experience to assess samples while harvesting and processing them -Labor intensive upon receipt of brain (average processing time 2h30) -Appropriate facilities and laboratory consumables including electronic tracking of samples (bar code labels) -Compartmentalization of freezers and storage boxes each compartment with unique identifier -Currently few sites in existence with manpower, supports, and equipped (Massachusetts General Hospital; Harvard Brain Tissue Center, New York Brain Bank) Dissection and harvest of samples at the fresh state

Formalin fixed Diagnosis Blocks -> Microscopical examination Thoroughly processed fresh Frozen blocks Pulverized parenchyma “#1”

Fresh Diagnosis Gross & microscopical evaluations Two standardized reports Two standardized reports Text Medical record Medical record Quantitative data Data management Data management Fixed Clinicopathological correlations Pulverized “#1”

“#1” Laterality -If degenerative changes are bilateral and symmetric, and without unilateral concomitant lesion on external examination (e.g., infarct) -Even days of reception of brain: left half immersed in formalin -Odd days of reception of brain: right half immersed in formalin -If a lesion is found on gross examination of the external surface, or if there is suspicion of a lesion on one side, or if there was clinical evidence of a focal lesion, the half brain with the putative abnormality is immersed in formalin -In the absence of focal lesion or symptoms indicative of a focal pathologic process, it is assumed that the findings in one half brain reflect the parenchymal state of the contralateral half, which is verified while processing the other half fresh

“#2” Half fixed / half sliced -One half immersed in 10 percent buffered formalin solution - Standardized series of blocks (minimum 18) for neuropathological evaluation - These blocks are then available for further studies, as is the rest of the fixed half brain One half processed fresh -1.0 to 1.5 cm thick slices -> frozen on dry ice (cooling device) -Series of coronal slices from the cerebral hemisphere (16) -Series of transverse slices from the brainstem -Series of sagittal slices from the cerebellum Vonsattel J-PG, Aizawa H, Ge P, et al. An improved approach to prepare human brains for research. Journal of Neuropathology and Experimental Neurology 1995;54: PMID:

1/2 in formalin1/2 Sliced fresh 18 or more paraffin embedded blocks Fixed tissue saved Diagnostic classification Neuropathological examination Clinical data 16 standardized coronal slices from the cerebral hemisphere & Transverse slices from brainstem & Sagittal from cerebellum -> Standard blocks harvested -> Then, blocks and slices Frozen & banked “#2”

“#2” Medial Aspect / 16 CS A B C D E F G H I J K L M N O CUTS SLICES Details on landmarks of the slices are available in the Journal of Neuropathology and Experimental Neurology 1995;54: PMID:

“2” Cooling Plates (optional) Bolt 2 Coronal slice 3 Lower plate 4 Upper plate * Dry ice Frozen, slice #6. Posterior aspect

“#2” Advantage -Anatomic specificity relatively preserved -Standardized fresh frozen samples (from frequently requested areas identified as per past experience) are ready for immediate disbursement once categorized diagnostically, reducing the time between the receipt of request and disbursement of samples -Prevents thaw-refreeze cycles & carving out of regions frequently requested for research -Methods relatively easy to apply (average processing time about 1h.) One half fixed – one half sliced fresh -> standard blocks -> blocks and slices frozen and banked

“#2” Disadvantage -The number of fresh frozen blocks available for immediate disbursement is limited to the standard series identified as per past experience (at least 18 blocks) -A set of frozen slices stored as source blocks undergoing thaw- refreeze cycles -With uninitiated prosector: Yield of irregular slices confounding anatomical accuracy -Complex records for identifying eligible source slice, part disbursed, and keeping inventory up to date -Dual compartmentalization of freezer cabinets suitable for either standardized blocks or slices One half fixed – one half sliced fresh -> standard blocks -> blocks and slices frozen and banked

“#2” Laterality -If degenerative changes are bilateral and symmetric, and without unilateral concomitant lesion on external examination (e.g., infarct) -Even days of reception of brain: left half immersed in formalin -Odd days of reception of brain: right half immersed in formalin -If a lesion is found on gross examination of the external surface, or if there is suspicion of a lesion on one side, or if there was clinical evidence of a focal lesion, the half brain with the putative abnormality is immersed in formalin -In the absence of focal lesion or symptoms indicative of a focal pathologic process, it is assumed that the findings in one half brain reflect the parenchymal state of the contralateral half, which is verified while processing the other half fresh

“#3” Half fixed / half frozen en bloc -One half immersed in 10 percent buffered formalin solution - Standardized series of blocks (minimum 18) for neuropathological evaluation - These blocks are then available for further studies, as is the rest of the fixed half brain - One half frozen fresh, en bloc

1/2 in formalin1/2 FROZEN, EN BLOC Molecular biology Regional studies Biochemistry 18 or more paraffin embedded blocks Fixed tissue saved DNA storage cerebellum mRNA screening / pH Diagnostic classification Neuropathological examination Clinical data Figure 2M.L. Shelanski “#3”

“#3” Advantage vs. Disadvantage -Minimal effort required at the initial step of banking brains -Neuropathological assessment of the half frozen en bloc is curtailed -Erratic artifacts due to the formation of ice crystals within cells and within the neuropil -Repeatedly subjected to a wide range of harmful temperature fluctuations -Harvest of parts are labor intensive, anatomically often inaccurate -Time consuming – belated fulfillment of requests Half fixed / half frozen en bloc

“#3” Laterality -If degenerative changes are bilateral and symmetric, and without unilateral concomitant lesion on external examination (e.g., infarct) -Even days of reception of brain: left half immersed in formalin -Odd days of reception of brain: right half immersed in formalin -If a lesion is found on gross examination of the external surface, or if there is suspicion of a lesion on one side, or if there was clinical evidence of a focal lesion, the half brain with the putative abnormality is immersed in formalin -In the absence of focal lesion or symptoms indicative of a focal pathologic process, it is assumed that the findings in one half brain reflect the parenchymal state of the contralateral half, which is verified while processing the other half fresh

Those brains that cannot be processed fresh or that are inadequate for research 10% Buffered formalin phosphate solution “#4” Whole brain fixed -One half processed for neuropathological evaluation -18 standardized blocks -> histology sections -Then blocks and rest of the half brain kept for research, if appropriate -Contralateral half kept intact -Morphometric studies, such as stereological evaluation (Dr. Saper)

“#4” Laterality -If degenerative changes are bilateral and symmetric, and without unilateral concomitant lesion on external examination (e.g., infarct) -Even days of reception of brain: left half immersed in formalin -Odd days of reception of brain: right half immersed in formalin -If a lesion is found on gross examination of the external surface, or if there is suspicion of a lesion on one side, or if there was clinical evidence of a focal lesion, the half brain with the putative abnormality is immersed in formalin -In the absence of focal lesion or symptoms indicative of a focal pathologic process, it is assumed that the findings in one half brain reflect the parenchymal state of the contralateral half, which is verified while processing the other half fresh