Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

One of the most critical components of the TMA construction process is pathology review of FFPE donor blocks from which the TMA cores will be obtained⁴. During the review, a board-certified pathologist examines a representative H&E stained tissue section from each donor block. It is imperative that the H&E is generated using a freshly cut tissue section so that it is the best representation of its corresponding donor block. The use of older H&Es is not recommended given that FFPE tissues are 3-dimensional structures whose tissue profile can change significantly with block depth and extensive sectioning; this may have occurred since the H&E was generated, potentially making its representation of the FFPE block inaccurate. The review process is essential for the selection of suitable cases and identification of tissue areas from where cores should be obtained, as well as identification of areas that should be avoided when collecting cores. In the absence of pathological review, the probability of including unsuitable tissues significantly increases. Inclusion of such tissues has the potential to render the constructed TMA ineffective and unsuitable for its intended purpose. Importantly, unknowingly using such ineffective TMAs has enormous potential to result in false and misleading data. This combined with the knowledge that the profile of FFPE tissues, and thus their derivative cores, can change significantly with increasing depth highlights the importance of continued pathology review throughout the lifetime of a constructed TMA block. Ideally H&Es should be generated using every 15^th or 20^th section to ensure any changes in the tissue profiles of the cores are captured and recorded. At a minimum, H&Es should be generated and reviewed at the start and end of a project to monitor these potential changes. In light of these points and the importance of the TMA as a research tool, it is imperative that the pathological review is firmly embedded in the TMA construction process and throughout the life of the TMA block.

FFPE blocks are often extensively sectioned during routine diagnostic processing before being released for research purposes. As a result, donor block depth and thus donor block core lengths are often less than the recipient method ideal of 4 mm. Here we have demonstrated how to construct TMAs using the tape method construction protocol, the principal advantage of which is its compatibility with cores from non-ideal FFPE tissue blocks. Although the tape method is of great research value and offers an inexpensive, convenient, and accessible method for constructing TMA blocks, it is not without its challenges and limitations. Compared to both automated and manual recipient methods, which can accommodate 100-1,000s of cores in single TMA block, a maximum of 40 cores is recommended for TMAs constructed using the tape method⁹. Another limitation is with respect to the ease of construction. In the recipient method, punched cores are merely inserted into a precast mold, which provides core stability by encasing each core in its own individual well, thereby preventing core migration as well as promoting highly regular core placement and separation²². Moreover, the recipient method offers the optional convenience of being fully manual, semi-manual, and fully automated. In contrast, the manual tape method requires careful, gentle placement of each core by hand using a needle pick. Although the absence of a precast mold in the tape method precludes the highly regular placement and separation experienced with the recipient method, this deficiency is overcome through the inclusion of a checkered grid. It is important that the checkered grid is affixed to the center of the metal tray in order to avoid block-edge placement, which increases the risk of core loss if there is insufficient paraffin holding the core in place. It must also be noted that the small core separations possible with the recipient method cannot be achieved with the tape method due to manual core placement and the need for the needle pick to fit between adjacent cores. Cores are placed in a freestanding erect fashion with the smallest surface area or footprint of the core contacting the DSST covered grid. This setup provides significantly less core stability than the recipient method and confers an enhanced risk for core toppling and or migration when pouring the molten paraffin. Indeed, one of the most critical steps in the protocol is pouring the molten paraffin. It is essential that this is done quickly upon removal from the oven to ensure the paraffin is completely liquid and that the pour is performed gently with minimal turbulence. Interestingly, Chen et al. developed a highly novel auxiliary device, akin to a stencil with 7 x 11 evenly distributed 2 mm diameter holes, that is placed on top of a blank paraffin block to guide needles when creating the recipient block and when inserting the donor block cores²³. Although designed to aid recipient block construction, such a device could easily be adapted to the tape method to guide placement, regulate separation, and increase core stability during the construction process.

One of the most significant effectors of core stability is the number of cores included in a tape method TMA. This is because as the number of cores increase, the diameter of the core must reduce in order to accommodate the increasing number of cores, which in turn reduces core footprint adhering to the DSST. A minimum core diameter of 1 mm is recommended for tape method TMA construction, as we have found that cores with smaller diameters are particularly unstable and prone to toppling even with very gentle paraffin pouring. A recent study investigating two different in-house methods that used 16 G needles (core diameter of 1.1 mm) and a 4 mm diameter punch experienced substantial tissue losses with the 1.1 mm (26.5%) but not the 4 mm cores²⁴. This appears to indicate that small cores can be problematic to work with and not just during construction. Moreover, smaller diameters may not represent the original donor block as well as larger cores, making pathological interpretation difficult and increasing the probability of inaccurate donor tissue representation.

The inclusion and placement of orientation blocks is of profound importance in TMA construction. However, this is of particular importance for tape method constructed TMAs. This stems from the fact that the tape method inverts the construction process thereby increasing the risk for spatial disorientation. We advise including up to three orientation cores in every block, and that they are placed away from the sample cores in order to best orient the block. Orientation cores can be cores taken from tissue blocks containing distinctly different tissues from the theme of the constructed TMA or tissue-free-colored orientation tools²¹, where the latter is particularly helpful for non-pathologists. Combined with non-regular matrix patterned core placement, orientation cores minimize the risk for disorientation.

The pronounced difference in core length between TMAs constructed using the tape and recipient methods stems from inclusion of donor block depth in the decision-making process when selecting the construction method. The protocol outlined here employs a threshold where TMAs are constructed using the tape and recipient methods when the donor blocks have depths of <4 mm and 4 mm, respectively. It is important to note that inclusion of donor block depth in construction method choice is not universal. Although it is possible for TMAs to be constructed using either method irrespective of donor block depth, taller cores can interfere with, and or be toppled or tilted by, the placement of the plastic cassette during TMA construction using the tape method. The choice to include or omit criteria in the decision-making process depends on the amenities available to the laboratory, cost, and the desired final product. Under the parameters of this protocol, the number of slide mounted TMA sections that can be obtained from a tape method constructed TMA is significantly less than that obtained from a recipient method constructed TMA. Although it is possible to re-block FFPE tissues to increase the donor block depth and make them compatible with recipient method, the probability of achieving the same tissue orientation within the re-block is low. In turn, this may require extensive block facing to obtain a full-face section, which would likely include significant tissue loss. After block facing, a tape method constructed TMA yields approximately 50 slide mounted TMA sections with all cores present. However, the exact number will vary from block to block and depends on the length of the cores used to construct the TMA and the thickness of the sections being cut (5 µm versus 4 µm). Moreover, it must also be noted that because of their differing core lengths, the cores will exhaust at different times as the TMA is progressively sectioned; an attribute that reemphasizes the need for continued pathological review.

Although the recipient method offers significant benefits and advantages over the tape method, including a less tedious and more expeditious construction processes, the tape method is not aimed at experienced high throughput laboratories. It is aimed at the average laboratory, particularly those in resource limited settings, with access to donor blocks of variable depths but not to TMA construction services. However, future applications could see automation of this method in order to enhance the pool of eligible samples in high throughput laboratories and eliminate the need for re-blocking of donor blocks. In conclusion, the TMA tape method construction protocol described can be easily established at non-specialized laboratories without the need for expensive equipment. However, it is advised that new users should employ FFPE tissue blocks of no value, tissue free colored orientation tools²¹ or even colored paraffin blocks with no tissue at first in order to familiarize themselves with the tape method technique before advancing to TMA construction using precious tissues. Although their construction is not without potential pitfalls, which both those constructing and using TMA blocks should be aware of, this seemingly unpolished “homemade” TMA construction method can yield high quality, biologically relevant TMAs for research. Indeed, TMA sections stemming from tape method constructed TMAs are among one of the most requested tissue samples in the ACSR biorepository.