For my second week in the Histology lab, I will describe the processes of tissue processing, embedding and microtomy. During this week, I processed, embedded and cut over 100 tissues.
In my previous blog, the gross description of tissues was explained. After grossing, tissues are put into tissue processor machines. Tissue processing consists of different chemicals entering the tissue for preservation. Tissues are then embedded into paraffin wax using the appropriate orientation. After tissues are embedded in wax and cooled, they can be cut using a microtome to create thin ribbons of tissues. These thin ribbons are then put onto slides.
The first step of tissue processing includes the dehydration of tissues. This dehydration needs to occur in order for all of the water to be removed from the tissue. Dehydration occurs by using a series of graded alcohols (70%, 95% and 100%) to gradually remove the water. Care should be implemented to not overdehydrate the tissue, because this will make it hard and brittle. The next step in tissue processing is clearing, which uses a clearant (typically xylene), to remove the dehydrating alcohols from the tissue. This step must occur in order for the next step, the wax infiltration, to occur. While processing my tissues throughout the week, I also performed tissue processor maintenance, which included replacing reagents and cleaning the machines.
After tissues are processed, they are then ready to be embedded. This step occurs at an embedding station that consists of a paraffin reservoir, storage area, forceps warmer, wax dispensing nozzle, hot orientation plate, cold plate, main cooling plate, control panel and excess wax drawer. Before embedding begins, the cassette must be opened and tissue must be looked at in order to choose a size of mold that is appropriate for the tissue. You do not want to choose a mold that is too large or too small in respect to the tissue, and ideally you should choose a mold that has about 2 mm of space surrounding the edges. Then, the mold is filled with hot wax and the tissue is placed into it using forceps. When placing tissue into the mold, make sure that the tissue lays flat on the bottom. Other considerations for orientation need to be done for specific tissues. For example, longer tissues should be placed diagonally, and tubular tissues should be placed on their end so that their lumen can be seen. If there are many pieces of the same tissue present, they should all be oriented in a line so that their surfaces face in the same direction. However, when dealing with prostate chips that come in multiple pieces, this orientation is not done. Finally, in embedding cysts that are dome-like, the bottom must lay flat while embedding in order for all layers of the cyst to be seen in the final product. It is also important to position tissues in ways that provide the least resistance while cutting, which will prevent compression of the tissue. After a tissue is oriented, the base of the cassette is put on top of the mold, and wax is added on top of that. The cassette and mold are then quickly brought to the cooling plate to solidify. During the embedding process, technologists can embed hundreds of tissues. Therefore, it is important for technologists to wipe their forceps in between each case they embed, in order to prevent carryover of tissue. Below shows a great video on the steps involved in the embedding process!
When the tissue embedded in paraffin wax has cooled and can be removed from the mold, it is then cut using a rotary microtome. When cutting, a coarse trim is first done cutting the tissue at 15-30 microns using the coarse trim knob. This coarse trim exposes the full face of the tissue. Blocks are then placed onto an ice bath that softens the tissue and makes it easier to cut. Then, a fine trim at 4 microns is done to create a ribbon of tissue for the slide. Once a ribbon is obtained, it is moved and floated onto a water bath that is kept at approximately 46 °C. The ribbon must not contain any folds when it is in the water bath or else it won’t be able to be placed on a slide. Then, using a clean slide, the ribbon is picked up and the slide is placed vertically on paper towel to drain water from the slide. Between every slide that is cut, the technologist must clean their water bath with a Kimwipe in order to prevent carryover of tissue in the slide. At the end of every day, the water bath must be thoroughly cleaned and the water changed in order to prevent microbial contamination. Safe work practices must be performed when operating the microtome due to the presence of a blade. For safety purposes, the blade guard must be up and the fine trimming wheel must be locked whenever the microtome is not in use. Finally, when disposing of blades, carefully remove the blade from the microtome and dispose of it in the sharps container. Here is a great video that demonstrates the process of fine trimming a tissue!