PMC:4996397 / 9182-14717 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996397","sourcedb":"PMC","sourceid":"4996397","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996397","text":"3. Results and Discussion\nIn the present work, we constructed two new TMAs taking the tissue punches from normal FFPE donor blocks as well as from previous arrays constructed with different instruments. The experiment was carried out with punches of 1 mm and 0.6 mm diameter, respectively, these two sizes being the most commonly used in ongoing translational research TMA approaches. \nAs shown in Figure 2 the resulting new TMAs were successfully built. The tissue spots on H\u0026E slides of the new TMAs were compared to the ones of the original TMAs. The re-punched tissues retained histological characteristics independently from the organs. Even hemorrhagic adipose tissue was successfully transferred (Figure 2A-3). \nSome cores appear slightly compressed (Figure 2B-2,3); the H\u0026E staining revealed that some spots were less round compared with the one of the original TMA. This rare observation occurred exclusively in tissues with 0.6 mm diameter independently from the machine used for the original TMA construction and the age of the TMA donor blocks. Notably, these small variations do not affect the intrinsic histopathological tissue characteristics and can be in part due to the cutting of the TMA slide. Thus the usefulness of the “transplanted” tissue cores is guaranteed.\nFigure 2 Result of re-punched TMA with 1mm core diameter (A) and 0.6 mm core diameter (B), respectively. Distance between cores is 0.6 mm in both cases. The circles indicate tissue provenance. In the TMA with 1mm core diameter (A), red circles represent tissues originally punched with TMA-M (Figure 1A), and green circles represent tissues originally punched with TMA-GM (Figure 1B). In TMA with 0.6 mm diameter (B), red circles represent tissues originally punched with original TMA instrument (Figure 1C), and green circles represent tissues originally punched with TMA-GM (Figure 1D). Example cores for both tissue arrays are shown in detail. Dotted line arrows point from images of the donor block to the core on the recipient TMA. Full line arrows connect the re-punched core to its magnified image. In the 1 mm TMA: A-1. spleen; A-2. pancreas; A-3. hemorrhagic adipose tissue; A‑4. lymph node. In the 0.6 mm TMA: B-1. renal medulla; B-2. liver; B-3. epithelial tumor; and B-4. striated skeletal muscle. \nFigure 3 illustrates the successful IHC staining obtained using five different tissue markers. The examples reported are completely representative for all transferred cores. The obtained similarity in staining of all type of tissues independently from the size of the cores, the original machine used, and the age of the block is impressive. These results underline the precision of the tissue transfer and the fact that the epitopes remain unchanged after a second overnight incubation at 42 °C.\nFigure 3 Comparison of protein expressions in the same tissue cores before (1) and after re-punching (2): (A) Pan-keratin in epithelial carcinoma cells (1mm diameter core); (B) Ki67 in epithelial carcinoma cells (1 mm diameter core); (C) Actin positivity of smooth muscle (0.6 mm diameter core); (D) Colon with blood vessels positive for CD34 (0.6 mm diameter core); and (E) Thyroid carcinoma with CD45 positive lymphocytes (1 mm diameter core). The tissues were in general completely transferred. The TMA-GM can precisely center a tissue carrot even in case of 0.6 mm cores. In Figure 4 the spots on the TMAs of origins are shown before and after the tissue transfer. The holes after tissue transfer are completely empty in all cases. \nThe only limiting factor of this re-punching process is the distance between the tissue cores in the donor TMA. We tested TMA with 0.6, 1 and 2 mm cores’ diameters and distances between cores ranging of 0.5, 0.6 and 0.7 mm. The TMA-GM is very precise and can export any kind of tissue carrots even when these lay very close to each other. However, if the gap between the cores is less than 0.6 mm and many cores in a row have to be re-punched, the bridges between the empty cores lose stability. Therefore, we recommend designing new arrays with an intra-core distance of 0.6 mm. However, since many existing TMAs are constructed with distance less than 0.6 mm, we took inspiration from previously described practical methodologies [26,27] and tested manually refilling of empty holes with paraffin by means of an appropriate syringe. In order to fulfill the aim of stabilizing the donor TMA, the manual refilling of holes in a row or otherwise adjacent to each other must occur after the export of each single tissue. That way, the paraffin helps stabilizing partially the thin bridge that otherwise would result between two empty holes. Subsequently, the next tissue specimen will remain vertical allowing a precise transfer. Nevertheless, this manual refilling requires time, depends on personal ability and, the inserted paraffin will not guarantee the complete stable filling of the empty hole. In this case the usefulness of re-punching and the risk of destabilizing an existing TMA should be carefully evaluated.\nFigure 4 Donor array, before and after tissue transfer, showing unimpaired remaining cores. (A) TMA performed with TMA-M (core diameter is 1 mm, and distance between cores is 0.5 mm); (B) TMA performed with TMA-GM (core diameter is 1 mm, and distance between cores is 0.7 mm); (C) TMA performed with old machine (core diameter is 0.6 mm, and distance between cores is 0.6 mm); and, (D) TMA performed with TMA‑GM (core diameter is 0.6 mm, and distance between cores is 0.6 mm).\n\n4","divisions":[{"label":"Title","span":{"begin":0,"end":25}},{"label":"Figure caption","span":{"begin":1284,"end":1875}},{"label":"Figure caption","span":{"begin":2795,"end":3243}},{"label":"Figure caption","span":{"begin":5055,"end":5534}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600223-19413642-69477665","span":{"begin":4268,"end":4270},"obj":"19413642"},{"id":"27600223-15923162-69477666","span":{"begin":4271,"end":4273},"obj":"15923162"}],"attributes":[{"subj":"27600223-19413642-69477665","pred":"source","obj":"2_test"},{"subj":"27600223-15923162-69477666","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93a6","default":true}]}]}}