Tissue microarray is a method for organizing and analyzing tiny amounts of tissue samples on a solid support structure. These microarrays consist of paraffin blocks made by excising small cylindrical tissue samples obtained from FFPE donor blocks. After extraction, the samples are re-embedded into a recipient microarray block. The recipient block is annotated in a grid pattern with defined coordinates.
In the initial step, the donor FFPE blocks are sectioned into microscopic slides that are stained with hematoxylin and eosin. These slides are then examined by a pathologist to denote areas of interest for further analysis at the molecular level. Areas of interest often involve cancer. Defining which research questions will be addressed by tissue array is the most important step in the method’s multi-step process. These questions need to be defined prior to staining so that sampling may be targeted to appropriate locations. Molecular heterogeneity and inconsistent gene expression among tumor cells pose challenges that can be addressed by extracting multiple samples from each region of interest.
Manual and automated arraying instruments are used to obtain tissue cores from the donor blocks. Arraying instruments use a hollow needle to extract tissue cores and then inject them into the recipient paraffin block. The tissue cores are cylindrical and have diameters of 0.6 mm to 2 mm. The maximum number of samples per recipient block equals approximately 1000 when 0.6 mm cores are embedded into a 25 mm x 45 mm block. A spreadsheet is used to record the placement of each tissue sample on the X-Y coordinate grid of the recipient block. This process enables parallel analysis of samples from hundreds of patients. Once the recipient block is filled, the block is cut with a microtome to generate 5 um thick slides for molecular analysis.
When setting up the layout of a recipient block, a protection wall of tissue cores lines each outer edge of the block and acts as a frame that will not be analyzed. The source of tissue cores for the protection wall is irrelevant so any tissue available in large quantities can be used. Introducing gaps in the recipient block by intentionally leaving core positions empty enables macroscopic identification of each block and helps researchers visualize sample orientation. Cores from the same donor block are grouped to expedite the workflow. Positioning a group of tumor cores next to the group of normal tissue cores they will be compared to is a common practice.
Air bubbles in recipient blocks are a common problem. After pouring fresh paraffin into a microarray metal mould, the newly formed recipient block should undergo X-ray analysis to screen for the presence of air bubbles that will distort analysis.
Once a recipient block has been screened via X-ray, a magnet is used to hold the recipient block in place while a two-needle microarraying instrument extracts cores from the sample, extracts paraffin from the recipient and then injects each tissue core into the empty paraffin cores. The arraying instrument needles are accurately positioned along the X-Y axis by a manually controlled micrometer. After each recipient hole is filled, the needles are moved down the recipient block to fill the desired grid pattern.
Once the recipient block is completely filled with tissue cores, it is placed upside down on a glass slide and incubated overnight in a 40*C oven to facilitate bonding between the sample cores and the paraffin wax. After the block is cooled, it is ready to be sectioned by a microtome and submitted for array. Sectioning a recipient block is a delicate procedure that should be done by experienced technicians to avoid distortion or heat-induced disintegration of the grid.
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