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About the Tissue Bank
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The Tissue Bank Laboratories (Text Version)
The "Tissue Procurement" part of the Office tour covered all the arrangements that must be made for tissue collection. This section takes up from where that left off: when the brain (and, in many cases, spinal cord) tissue arrives at charing Cross Hospital.
Earlier, it was mentioned that there are two cut-off times for retrieving tissue. If it can be retrieved within 24 hours, it is taken "fresh". (This means that it is transported to the hospital without any additional preserving steps.) Between 24 and 48 hours, it must be "fixed", by being immersed in formalin straight-away. This process prevents the tissue from degrading any further, but means that it becomes unsuitable for certain techniques that can be carried out on fresh tissue.
First of all, the brain is photographed from four aspects: superior (from the top), inferior (from the bottom) and left and right hemispheres. Digital photographs are taken using a computer-operated camera set-up. (These are later transferred to the online database, along with all other relevant (anonymised) case information.) The brain is then cut in half. One half (left or right is decided according to whether the donor's year or birth is odd or even) is fixed in formalin, so it can be used for neuropathological characterisation. For more information about this, see "Fixed Dissection", below.
The remaining half is cut into 1cm thick anterior slices, which are photographed on both sides before being cut into 2cm by 2cm blocks, using a labelled grid. The blocked slices are photographed once more, and then the tissue blocks are snap-frozen in isopentane before being placed into 8-well plates. Along the top of each plate is written the following information: case number, plate number, slice(s) and "Snap frozen". Each well is labelled with the grid reference of the block in question and, for the first block of a slice, the slice number. The plates are sealed into labelled bags (two plates to a bag), which are stored in a -85oC freezer until the tissue is requested.
As mentioned above, half of the brain (and spinal cord, if it is available) is fixed in formalin. After about four to six weeks, it is dissected in a similar manner to the fresh tissue. "Whole-fixed" brains cut in half at the start of the fixed dissection. One half is stored in formalin for future anatomical studies. The other half is processed in the same way as the fixed hemisect from a "fresh" brain. The fixed hemibrain is cut into slices and examined by a neuropathologist, who takes samples for more detailed analysis. The sampling protocol is the same for every case, and involves taking blocks from eighteen specific regions. These blocks are placed in labelled processing cassettes and stored in formalin until processed (see below).
As with the fresh dissection, the slices are photographed on both sides, cut into blocks and then photographed again. Before being frozen in isopentane, the tissue blocks must be dehydrated by immersion in sucrose solution for a week. The tissue blocks are logged and stored in the freezer.
Tissue blocks taken from eighteen sampling regions during the fixed dissection must be processed before the tissue can be stained for analysis. This is done using a processing machine. Briefly, the blocks (still in their processing cassettes) are placed in a metal cage, which is then put in the chamber. The machine then floods the chamber with a series of liquids, according to the program selected. (For routine processing, a 24-hour program is used.) The final step of the procedure is to infiltrate the tissue blocks with wax, ready for embedding. When the processor has finished, the blocks are placed in embedding troughs, which are then filled with molten wax. Labelled embedding cassettes are placed over the top, and the wax is topped up before being allowed to set. Processing and embedding preserves the morphology of the tissue and makes the anatomy easier to see when the tissue is stained. |
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About the Tissue Bank
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Sectioning TissueSo, by this stage, the tissue for analysis has been fixed in formalin, infiltrated with paraffin wax and embedded in a wax block. The next step is to cut extremely thin sections of tissue for staining.
Tissue sections are cut on a microtome and then floated out on a water bath. Once the sections have flattened out, they are collected onto slides and allowed to dry. The sections are then ready for staining with a variety of different dyes and/or antibodies.
There are two main types of staining techniques employed by the Tissue Bank for diagnostic work. The first uses dyes which bind to a particular class of structures within the tissue. For example, haematoxylin stains nuclei blue, while eosin colours cytoplasmic constituents pink and red blood cells a glassy orange-red. These two dyes are commonly used together in a routine diagnostic stain (referred to as haematoxylin and eosin, or H&E). Eosin binds to Lewy bodies, one of the main pathological features of Parkinson's disease. The second technique is called immunohistochemistry, and makes use of antibodies that bind to specific molecules. The Tissue Bank routinely uses antibodies against alpha-synuclein, ubiquitin, tau and beta-amyloid. The first two are the main components of Lewy Bodies, while the second two are indicative of Alzheimer's disease pathology. When sections have been cut, all regions are stained with H&E, and two with Luxol fast blue/periodic acid Schiff (LFB/PAS). (LFB is a myelin stain, while PAS shows up carbohydrates and other substances as a deep magenta colour.) The stained slides are examined by a neuropathologist to assess the quality of the tissue, and to determine whether analysis of the pathology will require anything more than the standard screen. Following the primary screen, the next step is to carry out the immunohistochemical staining, plus any other techniques the neuropathologist thinks necessary. The standard secondary screen involves staining all regions for alpha-synuclein and selected regions for tau, beta-amyloid and ubiquitin.
When all the work for a case has been completed, the case is passed on to the neuropathologists for diagnosis.
The aim of the Tissue Bank is to supply tissue to researchers investigating Parkinson's disease. We have received requests for fresh-frozen, fixed-frozen and paraffin-embedded tissue. First, the clinical and neuropathological information is used to select cases with the appropriate profile. Then, the relevant type and amount of tissue is retrieved as described below. Paraffin-embedded sections are cut from the diagnostic blocks using the microtome, as described above. For frozen sections or blocks, the region of interest must first be identified using the photographs taken at the dissection. Once the relevant blocks have been retrieved, sections are cut on a cryostat. (A cryostat is simply a special type of microtome contained within a refrigerated unit.) Tissue blocks or sections are transported to the requesting scientist by courier, using packaging appropriate for the tissue type. |
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