A quality histological slide contains tissue that was properly sampled, fixed, processed, embedded, microtomed, and stained without artifacts. The production of a quality slide requires work be conducted according to the highest standards during each phase of histologic processing.
The first and most important steps are precise necropsy techniques (e.g., postmortem interval, tissue sample size and handling, etc.) and adequate fixation. The choice of fixative is not as important as ensuring that adequate time has been allowed for the tissue sample to properly fix to avoid autolysis. If the tissue sample is excessively thick at the time of collection, it should be further trimmed or opened in a manner that allows the fixative to penetrate the tissue in a reasonable length of time. Some organs (e.g., lung, urinary bladder, intestines) additionally benefit from being infused or flushed with fixative.
As tissue samples are grossly trimmed for processing, they should be no more than 3 mm thick. Anything thicker may prevent the processing solutions from adequately penetrating the tissue. The tissue processing schedule can be challenging, as multiple tissue types and sizes routinely need to be processed simultaneously, which can leave some tissues over processed while others are under processed. Finding the right balance for each laboratory varies with the type of processor, features utilized (pressure/vacuum, stirring, temperature, etc.), and reagents selected. Vendors and colleagues are great resources for establishing a starting point, but the best processing schedule for a particular laboratory needs to be validated via test runs.
An online tutorial for organ sampling and trimming in rats and mice can be found here. This source is an excellent guide for standardizing routine processes for determining the Localization (anatomic site), Number of Samples (sections), Direction (plane of section) and Sample Size. Illustrative image examples are included along with descriptions and directions.
The embedding process is also critical. All tissues within a block should be embedded at the same level and oriented or positioned in a manner that enhances microtomy efforts rather than making it more difficult to get a quality section.
Everyone recognizes the importance of section quality as it pertains to the histopathologic evaluation. It is imperative to avoid knife lines, folds, thick and thin (Venetian blind affect) areas, or floaters, in addition to other artifacts. Floaters are extraneous pieces of tissue or debris that can be inadvertently incorporated into the section during multiple steps along the process (i.e., trimming, embedding, microtoming, staining, etc.). The microtomist should cut deep enough into the block to ensure that a complete section of each tissue is present without exhausting the tissue. If every tissue is not on the same plane within a block, the tissue may need to be re-embedded prior to sectioning.
The H&E (hematoxylin and eosin) stain is the backbone of the histology laboratory, yet there can be a wide range of staining results. Because laboratories use different stainers, stain formulations, reagents, staining protocols and personal preferences, variations in tinctorial quality can be almost limitless. Ultimately, one needs to have a stain that demonstrates excellent nuclear detail, with differential cytoplasmic staining as signified by three shades of eosinophilic coloration for red blood cells, collagen, and muscle, respectively.
Once staining and coverslipping have been completed, a quality control (QC) check of each slide should be performed. The QC check should confirm each tissue type is correct, each section is complete, all organ components are present, sections are void of artifacts (knife lines, folds, etc.), and that the quality of the staining is acceptable. If the required tissue elements are not present, a recut (either from the wet tissue or block) may be needed. A QC check will also ensure all macroscopic observations seen at necropsy or during gross trimming of tissues are also represented on the slide. A final QC check is a slide block match to ensure every block has a corresponding slide and every slide has a corresponding block before sending the slides to the pathologist; this can substantially reduce the number of recuts requested by the pathologist, thereby shortening the slide reading time.
Quality begins with the first step and must be maintained at each step along the process. That is the goal and expectation for each and every slide created in EPL’s laboratories.
Kerry Crabb, BS, HTL (ASCP)
Manager of NC Laboratory Operations