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Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer
Chapters
Summary June 10th, 2022
This protocol outlines the tape method on how to manually construct a tissue microarray using FFPE donor blocks of differing depths.
Transcript
The tissue microarray, or TMA, is an important research tool that assembles small exemplary pieces of tissue known as tissue cores from formalin-fixed paraffin embedded tissue blocks, referred to as donor blocks, into a single paraffin block. The tape method is a fully manual construction method that inverts the construction process by casting the block around upright cores and is thus compatible with non-ideal donor blocks and uses inexpensive, widely available disposable handheld tissue microarrayers. The multi-step workflow of this process will be demonstrated by Mr.Lee Wisner and Dr.Brandon Larsen.
One of the most important concepts to remember when constructing TMA blocks is the fact that tissues in formalin-fixed paraffin embedded tissue blocks are actually three=dimensional structures. We've developed an animation here to help explain this concept. What you're seeing here is an animation depicting a formalin-fixed paraffin embedded tissue block showing the three-dimensional nature of that tissue within the block.
You can see that the tissue is visible at the top of the block and that area is quite large. If we were able to have X-ray vision, as it were, and see through the block, we might see that the tissue is not actually uniformly shaped deeper within that block, almost like an iceberg in the ocean, where what you see on the surface may not actually represent what's below. Within that tissue block there may be additional tissue pieces that are not visible on the surface.
There may be areas of necrosis or tissue death within the center of that tissue deeper down. A pathologist should actually review that two-dimensional section off the top of that tissue block and then annotate that slide to show the areas that contain the tissue of interest and excluding necrosis or other types of tissue that are not actually of interest. To facilitate proper core sampling of the tissue, the pathologist should do three things.
First, using a slide marking pen, the pathologist will mark the tissue of interest on the slide so that other tissues can be avoided. Second, if there are areas within that area of interest that should be avoided, the pathologist can use the same marking pen to blot out those areas that should not be sampled. And lastly, preferably using a different color of pen, the pathologist should mark those areas that are ideal within the area of interest for core sampling and TMA construction.
It's important to keep in mind that when that process is done that the tissue composition may change deeper within from where a core sample is obtained and we'll show this here in the animation, that the location of the core will determine the composition of that core, which will vary by the depth of the tissue and what's present within that block. For example, here the first core that we're illustrating is capturing a portion of the tissue in its upper half but deeper within the block there's no longer any tissue present where that core is obtained from, so only paraffin wax has been sampled. The second core we've illustrated here has captured tissue along its entire length because tissue is present throughout the entire depth of that block at that location.
The third core has captured a portion of tissue and then has captured a portion of paraffin wax in the center where there was no tissue present and then captured a bit more tissue at its bottom end where there was tissue in the bottom portion of that block. Once the pathology review is complete, compile the final list of donor blocks to be used in the TMA construction, then create a TMA map. The TMA map is a schematic outlining where the cores will be located in the completed TMA and slide-mounted tissue sections cut from the resulting TMA.
For orientation purposes, ensure the TMA map avoids placing cores in an even matrix such as a three by three or four by four matrix and includes at least one orientation marker. Because the tape method inverts the construction process by pouring molten wax around the inverted cores, this necessitates the creation of a second map known as a construction map which is a mirror image of the TMA map. The construction map shows where each core must be placed during the construction in order to appear in the correct location in the completed TMA.
Once the maps have been created, prepare the metal TMA base mold. To guide core placement use a disposable paper checkered grid. Cut the paper checkered grid to size and affix a piece of double-stick tape to the back of the grid.
Place the grid and tape into the metal tray and add a second piece of double-stick tape on top of the grid. Overlay the pathology-reviewed H&E slide on the FFPE tissue block to be punched and use the pathologist's markings to identify where the tissue block is to be punched. Using a handheld manual core punch, disposable or reusable, punch the FFPE donor block in the appropriate region.
If using a reusable core punch, ensure that it is cleaned before and after each tissue punch. Eject the core from the core punch. Use a needle pick to place the ejected core on the crosshairs of the double-stick tape-covered grid.
Ensure the core is placed in an inverted, upright manner such that the tissue end of the core contacts the tape and is in the correct location as denoted by the construction map. Repeat until all donor blocks are cored and the cores are placed at their appropriate positions. Once all the cores are in place, label a plastic cassette and place it on top of the metal base containing the cores.
The height of the cores should not exceed the depth of the metal tray as tall cores will be tilted or toppled when the cassette is put in place. Gently pour melted paraffin through the cassette into the tray of cores. Allow the molten paraffin to overflow to ensure that there are no air bubbles in the body of the TMA.
Ensure that the paraffin fills to the top of the cassette such that the cassette is embedded and firmly bound to the paraffin block once it is solidified. Allow the block to cool at room temperature for 30 minutes and do not move the block during this time. Refrigerate the block at four degrees Celsius for an additional 30 minutes to completely solidify.
Once completely set, gently separate the metal base mold and remove the double-stick tape from the paraffin block. Once the TMA is constructed, use a microtome to section the new TMA. Once the block has been appropriately faced, collect full-face tissue sections.
Transfer the sections to a pre-warmed water bath and slide-mount the tissue sections. Once dry, submit representative sections for H&E staining and any additional immunohistochemical stains that may be required. Submit the stained TMA sections for pathological review.
For TMA validation, the pathologist should review the H&E slide from the TMA block and review each circular spot under the microscope to see if the tissue of interest is present. A critical component of the construction process is identification of tissues of interest in a given donor block from where the TMA core should be obtained. The first column of this figure shows exemplary donor H&Es with the pathologist's markings.
The second column shows the punched area of the H&E stained tissues. The third column shows the H&E stained tissue of the TMA cores that were harvested from the donor blocks in the areas shown in columns one and two. The pathologist can view the matched donor block and TMA core H&Es together to validate that the area of interest was collected and that the tissue composition is the same.
There are two principle metrics for successful completion of a TMA by the tape method. The first is the presence of tissue core dots at the expected positions and distance apart from one another, which is assessed by visual inspection. The images shown in A and B depict two successfully completed tape method TMAs and the corresponding H&E slides.
Visual inspections of these TMA blocks shows that the cores are present and regularly spaced in each TMA and their corresponding sections. Some of the principal construction issues that can arise during the tape method construction process include separation of the block and cassette due to premature removal of the metal base prior to solidification, as shown in image C.Core toppling and/or placement drift of the embedded cores as shown in image D can occur while excessively turbulent pouring of the molten paraffin, which may be worsened by poorly adhesive double-sided stick tape. This figure shows the H&E stain for each of the cores is an exemplary TMA.
All but one of the core spots is present in the H&E. The figure also shows that some of the cores are present as complete circular tissue dots, while others are not completely present. Such tissue loss is not uncommon and may be due to insufficient block facing to reveal all cores in full.
Alternatively the presence of incomplete or the total absence of tissue may stem from poor tissue quality of that core, which can result in tissue loss during the staining process. In this next figure immunohistochemistry and RNA ISH staining was performed on the TMA sections to further validate the TMA. As shown here, a variety of stains can be used depending on the tissue used to construct the TMA.
Vimentin is used as a quality control. U6 is an indicator of RNA quality. EBER is used to determine the EBV status.
And CD20 is a B-cell marker to indicate the presence of B cells. These are a few stains that can be used to help validate your TMA. After watching this video, you should understand how to make a tissue microarray using the tape method and why TMAs are important time-and resource-saving research tools.
You should also understand that TMA construction is not an ideal process and that continued pathological guidance and review are critical components not only during the construction process but also for immunohistochemical studies performed on TMA sections in order to ensure the tissue of interest is in fact present in the TMA section.
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