Construction of tissue‑arraying instrument
To manufacture the tissue‑arraying instrument, a drill chuck, a stainless-steel capillary tube (length, 200.0 mm; outer diameter, 2.4 mm; inner diameter, 2.0 mm), a stainless-steel tube (length 60.0 mm, outer diameter, 6.0 mm; inner diameter, 4.0 mm) and a stainless-steel wire (length, 200.0 mm; diameter, 1.0 mm) were purchased from Taobao Co. (https://www.taobao.com, Hangzhou, China). A stainless-steel handle was purchased from Shengde Co. (Jinan, China). The stainless-steel tube was welded to the drill chuck and the stainless-steel handle. The stainless-steel capillary tube, which was used to obtain tissue cores from donor blocks and insert them into a recipient block, was put through a stainless-steel handle and stainless-steel tube and clamped by a drill chuck.
Recipient block preparation
Three plastic moulds, which were marked as mould A, mould B and mould C, were used to construct the recipient blocks. These moulds were designed by us and processed using a computer numerical control (CNC) milling machine. Mould A was milled into a hollow that was the same as the blank recipient block in the centre of the mould to fix the recipient block. Mould B and mould C had 63 and 63 staggered array holes 2.0 mm in diameter, respectively. The holes in mould B and the holes in mould C were also staggered arrayed, namely, the holes of mould B were located at spacing between the adjacent holes of mould C.
Paraffin wax was purchased from Leica Microsystems, Inc. (Wetzlar, Germany). Blank paraffin blocks prepared for the recipient blocks were made according to routine procedures. Briefly, ordinary stainless-steel embedding moulds and plastic cassettes were placed in melted paraffin and warmed to 62 °C before being filled to avoid the formation of bubbles in the paraffin blocks. The mould was then filled with melted paraffin and covered with a plastic cassette. After pouring was finished, the paraffin blocks were cooled to room temperature and removed from the steel moulds.
The blank paraffin block was put into the hollow of mould A, and mould A was covered by mould B. Then, mould A and mould B were connected and fixed by two screws. Finally, 63 staggered array holes (diameter of holes, 1.9 mm; distance between holes, 1.9 mm) were drilled in the blank paraffin block using a mini electric drill with a drill bit 1.9 mm in diameter that was purchased from Shengde Co. (Jinan, China). The recipient block was removed from mould A to be filled with the tissue cores.
TMA block construction
In order to apply this novel method to construct high-density tissue microarrays and to clearly demonstrate its process of creating microarrays, several breast cancer and uterine leiomyoma paraffin-embedded tissue blocks were collected from the Department of Pathology (The Third Affiliated Hospital of Guangzhou Medical University). The haematoxylin and eosin (H&E)-stained slides of these samples were reviewed, and the areas of interest were marked by a pathologist. Then, the corresponding areas of tissue blocks were circled with a marker pen to prepare the TMAs. To demonstrate the process of creating high-density tissue microarrays clearly, the areas of interest for breast cancer and uterine leiomyoma paraffin-embedded tissue blocks were painted with red and blue inks, respectively. Tissue cores were obtained from breast cancer donor blocks by using the stainless-steel capillary tube of a self-made tissue‑arraying instrument. Tissue cores were gently pressed out of the stainless-steel capillary tube with stainless-steel wires and pushed into the holes of the recipient block until the tissue core was approximately 1 mm higher than the surface of the recipient block. Then, the tissue core above the surface of the recipient block was completely pushed into the holes of the recipient block by a thumb to ensure that the tissue core tips were arrayed at the same surface as the recipient block.
By repeating the recipient block preparation and TMA block construction steps, a high-density TMA containing 126 tissue cores 2.0 mm in diameter could be easily created. Briefly, the TMA block with 63 breast cancer tissue cores was re-placed into mould A and covered by mould C. Then, 63 holes, which were located at the space between these breast cancer tissue cores, were drilled out in the TMA block using the mini electric drill. Finally, 63 tissue cores were obtained from uterine leiomyoma paraffin-embedded tissue blocks and transferred to the TMA block using a self-made tissue‑arraying instrument as described previously.
Cutting and staining
After all holes were filled with breast cancer and uterine leiomyoma tissue cores, the TMA block was removed from mould A and stored in a refrigerator at -4 °C for at least 30 minutes to harden the TMA block. Some 4-µm-thick sections were cut from the TMA block with a microtome (Reichert and Jung, Heidelberg, Germany) and put on electrostatically coated slides according to routine procedures. Four slides were chosen for conventional H&E staining, immunohistochemical staining and fluorescence in situ hybridization. To avoid oxidation, every remanent slide was covered by several “blank” sections cut from an empty paraffin block.
The TMA slide H&E staining was performed routinely. To display this novel method for the staggered construction of high-density TMA, primary antibodies against CK (1:100; cat. no. PA0554, Leica Microsystems, Inc.) and SMA (1:100; cat. no. GT210701) were selected for marking the breast cancer and uterine leiomyoma tissue cores, respectively. Immunohistochemical staining was performed using a Leica automatic immunostaining device (Leica Microsystems, Inc.). The third sections of TMA were also stained by fluorescence in situ hybridization (e.g., HER2 (PathVysion, Abbott-Vysis)), according to the manufacturer’s instructions.
Microphotography
All H&E, immunohistochemistry and FISH slides were scanned using a digital pathology scanner (3DHISTECH Ltd., Budapest, HUN). Images were photographed with a digital slide viewer provided by 3DHISTECH Ltd.