SpOT the Correct Tissue Every Time in Multi-tissue Blocks

The overall goal of this procedure is to construct a tool to aid in tissue identification in multi tissue paraffin blocks. This is accomplished by first combining HistoGel with tissue marking dye to form a brightly colored gel plug. The second step is to process and embed the colored HistoGel plugs following standard tissue processing and embedding techniques.

Next, a biopsy punch is used to remove the small cylinders. These are specimen orientation tags or spot. The final step is to embed the spot in a multi tissue block with the location of the spot and each sample carefully documented for easy reference.

Ultimately, the spot provides a clear visual cue for specimen orientation and identification in tissue microarrays and multi tissue blocks. The main advantage of this technique over existing methods such as sutures or asymmetric cores, is that the spot does not alter the tissue and allows flexibility in the arrangement of the samples for optimal analysis. We first had the idea for this method when we received requests from investigators to combine tissue specimens from multiple animals into a single paraffin block to save money and simplify analysis To prepare the spots start with adding 50 milligrams of biochemical grade BSA to one milliliter of tissue marking dye in a 15 milliliter conical tube.

Vortex the mixture for at least one minute until it is completely dissolved. Next load nine milliliters of hydroxyethyl aros processing gel into a 15 milliliter conical tube and heat it in a microwave at 30%power. Pulse the heat in ten second increments until the gel is completely melted.

Now combine the melted processing gel and dye BSA solution in a single 15 milliliter conical tube and mix it thoroughly with a pipette. Then vortex the solution and transfer it to a refrigerator for a few hours to solidify. Once hardened, remove the solid dilate in gel by gently releasing it with a narrow spatula.

Slice the plug into five millimeter thick sections. Do not use the rounded ends. Transfer three to six sections to a histology cassette and immerse them in 70%Ethanol, change the 70%ethanol solution every two to four hours, and at least three times over 24 hours until the plugs are fully dehydrated.

They will leach some of the marking dye into the ethanol solution. Using changes of fresh ethanol initially helps to flush out some of the excess dye resulting in a cleaner plug that is less likely to leach color. Next, transfer the cassettes to 80%ethanol and then to 95%ethanol for one hour in each bath.

The dye may leak from the gel plug, which could affect other tissues and the liquid reagents in an automated tissue processor. As such, manual dehydration is highly recommended. However, automated processing is equally effective.

Complete the dehydration with three washes in 100%ethanol for an hour each. Clear the samples by immersing in atic hydrocarbons or xylenes three times for one hour in each bath. Now infiltrate the sections with three one hour baths.

In molten paraffin. Embed the processed sections as usual, and when the block is at room temperature, use a dermal punch needle to make spot cores. One block can make 22 millimeter cores or even more 1.5 millimeter cores.

Start by making a diagram to indicate the desired locations and identities of all the individual tissue pieces to be embedded together and the location of the spot. Prepare a copy to keep on hand. Successful use of specimen orientation tags requires a clear and easy to follow biospecimen map that accurately details all biospecimen their location and their position relative to the spot.

After processing the tissue pieces, arrange them on the embedding station. According to the tissue orientation diagram, correct orientation is absolutely critical. It's also vital that the spot core is taller than all tissue pieces that will be embedded in the block.

After completing the tissue arrangement, embed the spots. Hold each spot upright with forceps until the paraffin has solidified so that it will not topple. A minute or two should suffice.

For a manual TMA kit, fill the TMA mold with melted paraffin. As the mold is cooling, attach the spots in a similar manner at the array margins. Now section and stain the sections with the spot.

Using a standard approach, five micron sections are made here. To put the sections on slides, float them in a 40 degree Celsius water bath, and then transfer them to positively charged glass slides. Once attached to the slides, dry them at 60 degrees Celsius for at least 20 minutes.

The slides can now be stained manually or on an automated staining system. For routine h and d staining, special stains or immunohisto chemistry spots appeared as a round brightly colored. in the paraffin block and in all paraffin sections.

Spots allowed for easy orientation and tissue identification in multi tissue blocks containing similar appearing tissues from different individuals and treatment groups. When used in a tissue, microarray spots allowed every core to be utilized for valuable tissue samples and spots could easily be picked out adjacent to a tissue microarray core. While attempting this procedure, it's important to remember that accurate and clear documentation of the biospecimen and the spot in the block is critical to its successful use.

After watching this video, you should have a good understanding of how to easily create and use the specimen orientation tag as a tissue identification aid in multi tissue paraffin blocks.