A Mill Based Instrument and Software System for Dissecting Slide-mounted Tissue That Provides Digital Guidance and Documentation

Nils Adey; Dale Emery; Derek Bosh; Steven Callahan; John Schreiner; Yang Chen; Ann Greig; Katherine Geiersbach; Robert Parry

Disclosures

BMC Clin Pathol. 2013;13(29) 

In This Article

Results

Mesodissection Capabilities and Performance Metrics

Four performance metrics were established to test the mesodissection technology: resolution, accuracy, efficiency, and purity. The resolution (Figure 5A) is the minimum dissectible area determined using point dissections and is primarily dependent on the diameter of the cutting blade but is also dependent on any non-concentricity of the blade (runout). Currently, the minimum resolution is a circle about 170 microns in diameter. xScisors with 100 micron plastic cutting blades have been made but the wear rate was excessive; a steel tip blade is currently under development. A second metric, accuracy (Figure 5B), is a measure of how well the user can guide stage movement using the joystick (transverse movement). The average accuracy from this test, performed by a single user, was 60 microns. Most users with practice can achieve 100 micron accuracy, and point dissection accuracy is often better as the user can precisely position the blade before lowering it onto the slide surface. The third metric, recovery efficiency (Figure 5C), is determined by Pico Green quantitation of DNA following Proteinase K digestion of the recovered tissue. The recovery efficiency of both mesodissection and manual macrodissection are similar and also appear to be near 100% by visualization. The fourth metric, purity (Figure 5D) is a measure of potential contamination from neighboring undissected tissue (for example, if DNA were to leach out of the surrounding tissue and be picked up by the milling solution). Purity was determined by dissecting immediately adjacent human and mouse 5 micron liver tissue sections at variable distances from the intersection. The recovered samples were treated with Proteinase K, then subjected to multiplex endpoint PCR/gel electrophoresis, and single amplicon quantitative PCR. The PCR primer pairs (amplicons) were directed to either the human or mouse Cox1 mitochondrial sequence. As seen in the gel image, while both bands are present from a control containing an equal amount of each tissue (H + M), only the expected band is detectable from the dissected samples indicating little to no contaminating template. For the qPCR titration experiments, the entire section of human and mouse tissue were dissected separately, treated with Proteinase K, and then mixed at the ratios shown. While the human Cox1 amplicon amplified earlier than the mouse, the response to relative template ratios is clearly visible. In the single sample dissections, very little contaminating template (the titration experiments indicate it is significantly less than 1%) is recovered even from dissections immediately adjacent to the tissue intersection. The results from both multiplex endpoint PCR and real time qPCR indicate purity is greater than 99%, but this result could potentially be sample dependent.

Applications

The mesodissection system was applied to two common techniques in molecular diagnostics, RNA expression analysis and anatomical FISH (Fluorescent In Situ Hybridization) analysis in order to investigate capability. Because RNA is labile, many labs prefer to leave the tissue sections paraffinized during dissection to minimize exposure to moisture and atmospheric oxygen. Therefore, mineral oil was used as the milling solution, as it can aspirate paraffinized tissue and maintain a non-aqueous environment (used in Figure 5C). The recovered mineral oil/tissue fragment mixture was added directly to an equal volume of aqueous Proteinase K reaction solution where it formed a separate upper phase. When the tube was subject to heating and shaking, the tissue fragments migrated to the lower aqueous phase and were digested by the Proteinase K, which also digests RNase. Following the digestion, the organic phase was removed and RNA isolation, cDNA synthesis and quantitative PCR were performed using commercial kits. The results indicate the mesodissected material produced a larger quantity of RNA, as assayed by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), but sample loss during the additional centrifugation steps used for deparaffinization of the manual dissected sample likely accounts for the difference (Figure 6A). A link to a poster describing an endpoint RT-PCR analysis of these same samples is available at http://www.avanscibio.com

Tissue fragments that had been mesodissected from slide-mounted tissue sections were re-adhered to slides and used for FISH analysis (Figure 6B). The majority of the fragments were present in a single thickness (not folded or stacked) with minimal obvious damage. The tissue fragments remained bound to the slides throughout the subsequent FISH processing steps and produced good quality FISH chromosomal signals. Testing using a variety of human tissue samples (described above, all 5 microns thick) and dissection conditions found that fragment size (determined by visual inspection using a scale bar) was variable ranging from just a few cells to over a millimeter in diameter. However, for a given dissection condition, over 90% of the total tissue area was within a 2–3 fold size range. Dissection of well adhered tissue (for example paraffinized tissue dissected using mineral oil) produced small fragments (2–10 cells in diameter) whereas dissection of less well adhered tissue (for example deparaffinized pre-moistened tissue) produced larger fragments (half to one millimeter in diameter). No obvious tissue type specific effects on tissue fragment size were noted, but we did observe that the tissue tended to tear along natural boundaries. For example, cross sections of kidney tubules remained intact as small rings of cells. We also noted some physical dissection and handling effects on fragment size. For example, larger slower rotating xScisor blades and gentle use of pipet tips with larger openings in the subsequent handling steps tended to produce larger tissue fragments.

An FFPE tissue block was mounted onto the mesodissection instrument stage using tape and the Z-axis contact position of the xScisor adjusted using potentiometers on the electronics board (an earlier version instrument). The tissue block was then dissected using mineral oil and overhead visual guidance (Figure 6C). The depth of the cut appeared to be related to the depth of the blade as the end of the inner syringe barrel appeared to ride on or very near the upper surface of the tissue block. Tissue fragments along with the expected volume of mineral oil were recovered suggesting sufficient dissection liquid fluid flow exists, possibly through the path cut in the tissue block. A significant problem with this approach is difficulty visualizing the dissection process in real time; the tissue block is opaque making visualization from beneath the block very difficult.

Figure 6.

Applications. (A) qPCR analysis (using a mouse cyclin D1 gene amplicon) of cDNA prepared from RNA isolated from paraffinized FFPE mouse olfactory tissue. The same AOIs from serial tissue sections were either mesodissected using mineral oil, or manual (hand) dissected where the recovered tissue was deparaffinized in limonene and recovered by centrifugation (loss during the centrifugation steps probably account for the lower yield of the manual protocol). Two samples were isolated using each method and two qPCR reactions performed for each sample. Similar results were obtained using a mouse Bcl-2 gene amplicon (not shown). (B) FISH imaged fragments of tissue sections. FFPE tissue sections from human placenta (not shown: liver, bowel, and kidney performed similarly) were deparaffinized, mesodissected, and re-adhered to charged glass slides. These slides were processed using standard tissue FISH protocols and assayed with a chromosome 17 centromeric probe, then stained with DAPI. (C) Paraffinized tissue block milled with the mesodissection instrument.

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