Microscopic examination of formalin fixed, paraffin embedded (FFPE) tissue sections mounted on glass slides is a cornerstone of clinical histopathology. Often, ancillary molecular testing of these samples is required for further diagnosis, risk stratification, and treatment planning, particularly for cancer samples. This molecular testing typically involves mutation or expression analysis of nucleic acids or protein recovered from FFPE tissue sections. Due to the tissue heterogeneity of most slide-mounted tissue sections, regions enriched for the cell types of interest are often dissected directly off these tissue sections. In one of the most commonly used clinical techniques, an Area Of Interest (AOI) is hand annotated by a pathologist on an H&E stained, cover slipped, slide-mounted tissue section. This section is then sent to a laboratory where a technician manually aligns and traces the AOI onto the back of a second non-cover slipped slide containing a serially cut tissue section from the same tissue block. Manual macrodissection is then performed on the second slide using a scalpel or razor blade [1–3]. The process of identifying the AOI on an H&E stained and cover slipped slide and dissecting from a serial section allows for superior visualization of tissue morphology without the need to remove the coverslip, and dissection using a scalpel or razor blade is relatively quick and inexpensive. However, there are a number of drawbacks with this approach. Precision is significantly compromised due to the collective inaccuracy of the initial hand notation of the AOI, the thickness of the marking pen line, the tracing process, and the subsequent hand dissection such that the borders of the actual dissected region can vary by greater than +/- 1 mm from the borders of the region initially identified by the pathologist. This can be a significant problem when the area to be dissected is just a few millimeters wide. The process also tends to be poorly documented as the AOI and dissection notes are often hand written on the slide or accompanying paperwork. Moreover, the process typically requires the slides to be transported to multiple locations, and it is common practice to first remove the tissue surrounding the AOI in order to more efficiently recover the AOI with the straight edge of a razor or scalpel, leaving no record of the actual area dissected.
Two common alternatives to manual macrodissection that provide improved precision are manual microdissection and Laser MicroDissection (LMD). Manual microdissection has been used for decades [4, 5] and is typically performed with a dissecting microscope and a hand held device such as needle or scalpel, which typically limits accuracy to +/- 200 microns, or a micromanipulator, where recovery of small groups of cells can be achieved [6–9]. Manual microdissection is tedious and time consuming, particularly when using micromanipulators, tissue retrieval on a needle can be tricky and unreliable, and process documentation is suboptimal . Also, because it is difficult to physically annotate the areas to dissect with accuracy better than a few hundred microns, higher precision dissections require the operator to have training in order to select the appropriate cells. Alternatively, LMD instruments have been available for over a decade and have become highly sophisticated, providing automation, single cell resolution, and digital annotation and documentation. While LMD systems have many capabilities, they are very expensive and complex to operate, and many systems require the tissue sections to be mounted on special slides. If the areas to be dissected are indicated on a serial section, the variability between serial tissue sections will also limit the absolute precision.
A number of reports have investigated the level of dissection precision required for a variety of downstream assays [4, 11–17]. The current consensus is manual macrodissection is adequate when the area to be dissected is at least a few millimeters in diameter and when the downstream assays are sufficiently sensitive to detect a mutant allele population of at least 20%. This is often the case for tests that assay specific genomic loci such as KRAS codon 12 and 13, and common variants in EGFR and BRAF, using technologies such as qPCR and pyrosequencing. Expression assays and more complex mutational assays typically benefit by some degree of microdissection, and while the precision of laser dissection is generally not necessary for most clinical assays [4, 12, 14, 15], it can have significant benefits for discovery focused research studies [16, 17]. Unfortunately, analysis of the degree of manual microdissection precision achieved by many of these comparison studies is difficult because the process is usually not well documented. The above information along with statements from many clinicians and researchers suggests there is a need for a relatively low cost dissection instrument with better precision than manual methods that also provides digital guidance and documentation of the dissection process. This report describes a novel instrumentation and software system designed to meet these needs. Such a system could improve clinical laboratory workflow, documentation, and help standardize clinical dissection precision requirements and terminology.