Fixation on Histology

Creating Tissue Microarrays: Manual vs. Automated Tissue Microarrayer

  

Tissue microarrays (TMAs) are becoming increasingly popular in histology research and for use as a quality control method in both research and clinical labs. A TMA paraffin block is termed the “recipient block” as it contains cylindrical tissue cores from different “donor blocks.” The cylindrical tissue cores are removed from specific areas of interest from the donor blocks. This method allows a large number of tissue samples to be represented on one slide. TMAs allow researchers to study multiple tissue samples simultaneously, reducing cost and time, and providing a more representative sample of the population being studied.

The original method was created in 1986 by Dr. Hector Battifora. The original block was called a “multitumor (sausage) tissue block” and was further improved in 1990 as the “checkerboard tissue block.” With this improvement, the tissue pieces were arranged in an evenly distributed checkerboard pattern. The tissue microarray we know today was developed by Dr. Juha Kononen and his collaborators in 1998. With this latest method, the block has a higher density of cores and the cores are more precisely arrayed.

I currently work in a veterinary research core lab at a cancer research institute and we have used both the manual and automated process to create TMAs. In the past, we manually created TMAs by utilizing a punch biopsy tool. The recipient block would be created by using a special silicone TMA mold. The mold would be placed in the embedding unit to bring it up to temperature and then the liquid paraffin would be dispensed on top of the mold to fully submerge the core rods. Next, the cassette would be placed on top of the mold and the top of the cassette would be filled with more paraffin. The mold would cool to solidify the wax and then the mold is separated from the embedding cassette. Then a punch biopsy tool would be used to punch the areas of interest from donor blocks. A tissue punch plunger is then used to gently push the tissue core out of the punch biopsy and into one of the cores of the recipient block. Lastly, the recipient block is placed on a glass slide faced down and incubated in the oven to allow the cores to adhere to the recipient block. The temperature of the oven is dependent on the type of paraffin wax and the time to leave the block in the oven is dependent on temperature. The main pro to this manual method is cost. The silicone molds that can be reused hundreds of times and the biopsy punches are cheaper than a fully automated machine.

The main con is time and quality. Since a human is punching all the cores from the donor blocks and manually placing the cores into the recipient block, more time is needed and human error is possible. Sometimes if we were not gentle enough while inserting the recipient core into the donor block, the tissue core would break or the wax of the donor block would crack. There were also a few times during the oven incubation step that we forgot about the block in the oven and because the temperature was too high, it melted the block! (Always use a timer!!) Our lab also noticed the cores were not perfectly on the same plane (even after the oven incubation step) which resulted in microtomy issues of having full sections of some cores but not others. 
 

Recently, our lab bought an automated tissue microarrayer called the TMA Grand Master by 3DHISTECH. Instead of creating a mold from a silicone TMA mold, a donor block is created by filling an empty metal mold that does not have recipient cores. Once the block is pulled from the metal mold, there is a solid paraffin block with no holes. The TMA Grand Master has a drill to create the holes on the recipient block and a separate punch to pull the cores from the donor blocks to the recipient block. Once the TMA recipient block is created, the oven incubation step is still needed to allow the cores to adhere to the recipient block (again, do not forget a timer!). The main con of an automated issue microarrayer is cost. These machines are not cheap but if your lab can buy one, there are many pros. Because a machine is performing most of the work, there was way less human error. We had fewer tissue cores break and the wax of the recipient block would not crack. Animal tissue can be very fragile compared to human tissue so occasionally some cores would break apart (specifically mouse liver). It is important to realize that human error is still possible. This could happen during preparation work before the machine performs its protocol. For example, once all the donor blocks are loaded onto the machine, the machine takes a picture of the cut side of each block and the block label. The tech uses the included software to select the areas on each donor block that will be punched. H&E images of the donor blocks can be uploaded to the software. These images can have circle annotations that instruct the tech on what areas of the donor blocks need to be punched. The tech can then line up the annotated H&E image over the image of the cut surface of the block and then select the annotated areas for punching. If the tech does not align the H&E image over the tissue block image correctly, the wrong area could be selected resulting in the wrong area being punched.
 

TMAs that are made manually or from an automated tissue microarrayer are valuable tools. Researchers can perform immunohistochemistry or in situ hybridization to study the expression of specific proteins or genes across multiple samples on one slide. The data obtained from TMA studies can be used to identify potential biomarkers for diagnoses, prognosis, and treatment of diseases such as cancer, as well as to better understand the biology of diseases and how they develop. There can be challenges when creating TMAs but they can be incredibly useful. 

 

References:

Battifora H. (1986). The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Laboratory investigation; a journal of technical methods and pathology, 55(2), 244–248.

Battifora, H., & Mehta, P. (1990). The checkerboard tissue block. An improved multitissue control block. Laboratory investigation; a journal of technical methods and pathology, 63(5), 722–724.

 Kononen, J., Bubendorf, L., Kallioniemi, A., Bärlund, M., Schraml, P., Leighton, S., Torhorst, J., Mihatsch, M. J., Sauter, G., & Kallioniemi, O. P. (1998). Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature medicine, 4(7), 844–847. https://doi.org/10.1038/nm0798-844

Olli-P. Kallioniemi, Urs Wagner, Juha Kononen, Guido Sauter, Tissue microarray technology for high-throughput molecular profiling of cancer, Human Molecular Genetics, Volume 10, Issue 7, 1 April 2001, Pages 657–662, https://doi.org/10.1093/hmg/10.7.657

Ryan W. Askeland, Christine Bromley, Mohammad A. Vasef. (2005) Detection of HER-2/neu Gene Amplification Using Chromogenic In Situ Hybridization and Tissue Microarray: Correlation with HER-2/neu Protein Expression Using Immunohistochemistry. Journal of Histotechnology 28:1, pages 11-14.

Ştefan, A. E., Gologan, D., Leavitt, M. O., Muşat, S., Pleşea, I. E., Stan, L. G. R., Pleşea, R. M., & Militaru, M. (2020). Tissue microarrays - brief history, techniques and clinical future. Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie, 61(4), 1077–1083. https://doi.org/10.47162/RJME.61.4.10

Written By:  Sara Sheppard McCracken, BS, HTL(ASCP)QIHC

Sara would like to give credit to Arizona Ngyuen, HT (ASCP) for creating the TMAs that are shown in the images



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