Formalin-fixed paraffin-embedded or FFPE tissues are often admixtures of tumor and non-tumor materials, where the presence of non-tumor material is frequently unwanted and can, if present at a high proportion, significantly impact the results of genomic analysis, thus the main advantage of macro-dissection compared to working with unresected tissues is that the resulting deparaffinized resected tissue sections can be collected for RNA and DNA extraction, which can then be confidently used in downstream genomic analysis. Knowing that the extracted materials are derived from tissues with enhanced tumor content with minimal contributions from contaminating non-tumor tissues. The multi-step workflow of this process will be demonstrated by Mr.Lee Wisner, Dr.Brandon Larsen, and myself In preparation for tissue sectioning, presoak the paraffin blocks on iced water for 10 to 15 minutes.
Chilling the blocks hardens the wax while the water moistens the tissue, which together makes cutting the tissue easier. While the blocks are on ice, label the microscope slides appropriately. Fill and heat the water bath to 39 degrees Celsius and set the microtome to the desired cutting thickness.
Carefully load the blade onto the microtome. Lock the blade in place and place the safety guard up until you're ready to begin sectioning the blocks. While the safety guard is in place, insert the FFPE block in the microtome chuck.
To begin, section the block slowly until a full face section is obtained. This is known as facing the block. Once this is achieved, the block can be in section faster to obtain a ribbon of sections.
Once cut, transfer the ribbon of tissue sections to the warm water bath. Use forceps to separate a single tissue section away from the ribbon. To do so, place the back of the closed curved forceps at the joint between the two sections and allow the forceps to gently open to separate the sections.
Collect the isolated tissue section on a microscope slide. To avoid trapping bubbles, insert the microscope slide into the water at an angle. Gently shake the slide to remove excess water before placing on rack to air dry.
Once dry, select at least one of the freshly cut tissue sections for hematoxylin and eosin staining, also known as H and E.Once stained, submit the H and E slide for pathology review. During pathology review, an expert pathologist reviews the H and E stain slides to determine where the tissue of interest lies in each tissue. The pathologist first reviews the entire tissue to determine where the tissues of interest resides within the tissue.
Once the initial histology and morphological review is complete, the pathologist uses a slide marking pen to annotate each H and E stained slide. Using the marker, the pathologist draws a circle around the tissue of interest, excluding the tissue that is not of interest. This review process is then repeated for all H and E stained slides in the project.
Set up the macro-dissection bench workspace that permits each sample to be handled sequentially and efficiently. For each sample, the pathologically-reviewed H and E, the corresponding unstained slide mounted tissue sections, as well as the pre-labeled micro tubes containing the digestion buffer for tissue collection should be at hand. Load the unstained slides into a rack and proceed to the fume hood for deparaffinization.
Deparaffinize the slides by immersing the slides in two sequential washes of undiluted D-limonene or CitriSolv followed by a final wash in 100%ethanol for two minutes per wash. At the beginning of each wash, gently agitate the wash for about five to 10 seconds. To minimize carryover between washes, allow the slides to drain briefly and dab the rack on the paper towel.
After the three washes, the tissues are now highly visible on the microscope slides. Allow the slides to air dry for 10 minutes. Once dry, the H and E is then placed face down on the bench and the deparaffinized tissue placed on top, also face down, lining up the deparaffinized tissue with the H and E.Once correctly aligned, the tumor area marked by the pathologist on the H and E is traced on the back of the deparaffinized tissue section slide.
The now marked unstained slides are dipped in a 3%glycerol solution to minimize static and aid tissue collection. Remove the dipped slides slowly to minimize the amount of glycerol solution that clings to the dipped slide. Once removed, wipe the back of the slide and any tissue-free areas on the front of the slide to remove excess glycerol.
Next, using a clean blade, trace the pathology markings with the blade to break the connection between the tissue of interest and the rest of the tissue. Noting that this step can be done prior to glycerol dipping, particularly if the pathological markings are complex as cutting dry tissue can be easier and can also minimize the risk of tissue drag. The tissue outside the pathology markings are not of interest.
These are removed using the flat edge of the blade, making sure the corner of the blade hooks the outside of the pathology markings. Once, the unwanted tissue is removed, a new blade is used to collect the tissue of interest. The collected tissue is then removed from the blade using the flat end of a wooden stick and then transferred into a pre-labeled micro tube with digestion buffer, thus completing macro-dissection mediated tumor tissue enrichment, which are now ready for nucleic acid extraction.
To demonstrate how macro-dissection can affect downstream genomic analysis, tissue sections from five diffuse large B-cell lymphomas, or DLBCL, with different tumor contents were macro-dissected or not macro-dissected. Nucleic acid extractions were performed on the collected tissues and the resulting RNA examined using the DLBCL90 digital gene expression profiling assay, which is also known as the double-hit signature assay. DLBCL is comprised of three distinct molecular subtypes, namely GCB, ABC, and their intermediate subtype denoted unclassified, which can be determined using the DLBCL90 assay.
This assay is also capable of identifying DLBCL tumors that harbor double-hit translocations involving the BCL2 gene. The DLBCL 90 results are shown in this table. Macro-dissected samples were run twice.
Once using their RNA stock concentration and once using the RNA stock diluted to match the concentration of their respective non-macro-dissected counterparts. The results show that macro-dissection resulted in subtype or double-hit call changes in three of the five samples. Macro-dissection of sample A had no effect on the subtype call, but changed the double-hit call from negative to unclassified, and this change was observed irrespective of the macro-dissected sample RNA input.
In contrast, macro-dissection of sample C had no effect on the double-hit call, but changed the subtype call from GCB to unclassified, which was also observed irrespective of macro-dissected sample RNA input. Similar to sample A, macro-dissection of sample E had no effect on the subtype call, but changed the double-hit call. However, for sample E, the call changed from unclassified to negative, and again, did so irrespective of the macro-dissected sample RNA input.
Notably, this call changed to double-hit negative makes biological sense given that sample E was found to be ABC and double-hit translocations involving BCL2 have been reported to be exclusively observed in GCB tumors. Together, these results highlight the importance of tumor purity and genomic assays and macro-dissection as a reliable tool to achieve this. After watching this video, you should have a better understanding of what macro-dissection is, the importance of pathological review and the study benefits gained by including macro-dissection into your tissue handling workflow.
