If you have worked in the histology field for some time, as I have, you may have noticed that the specimens you receive in your laboratory are getting smaller. This is not due to our failing eyesight. Instead, the advances in immunohistochemistry (IHC) and molecular techniques now enable the pathologist to make diagnoses on smaller and smaller fragments of tissue obtained during biopsy procedures. This fact, along with the emphasis on greater patient care quality, requires that each and every specimen received in your laboratory must be routed to the correct area, and handled appropriately.
In non-specialty hospital and private reference histology laboratories, the vast majority of specimens are received in 10% neutral buffered formalin for “routine histology”. This means specimens will undergo surgical grossing procedures and routine processing through graded alcohols, xylene and paraffin embedding. Initial hematoxylin and eosin (H&E) stained slides will be prepared from paraffin blocks. Upon review by a pathologist, a diagnosis is rendered. Some cases may require additional IHC, molecular and/or special stains to confirm the diagnosis.
Some specimens may be received in formalin into the routine lab; however, they may require “non-routine” handling. Skin specimens submitted for “Slow Mohs” processing are such specimens. These are pieces of skin that are carefully marked with colored inks to indicate exact orientation. Laboratory personnel who receive the specimens, and who perform the surgical grossing techniques must recognize these “up front”. Failure to recognize such specimens may result in changing/destroying the orientation, resulting in the pathologist’s inability to render an accurate diagnosis with regard to tumor location with respect to surgical margins.
Similarly, a two millimeter punch specimen of skin may be mishandled by laboratory staff, if not identified “up front” at the time of receipt. These specimens should be identified for embedding and microtomy personnel. The microtomy personnel must pick up the very first sections, and observe the unstained slide under the microscope to ensure correct dermal-epidermal orientation. If the specimen has been mis-embedded, there is enough tissue to melt down and re-embed correctly. Failure to follow this procedure may result in the production of initial and level slides that are mis-embedded. The final result may be that the specimen cannot be diagnosed.
An inherent danger exists in the minority of specimens that may be received for unique, specialized procedures. If these specimens are received in the incorrect fixative, or routed into the “routine histology” workflow, they may be unable to be used in the specialized procedures for which they were sent to the laboratory.
Specimens for direct immunofluorescence (DIF) are usually skin and kidney biopsies. Clinicians must be educated to ensure that these specimens are not fixed in formalin. Instead, these specimens must be submitted in “Michel’s transport medium”, sometimes labelled “Immuno transport fluid”. This solution contains ammonium sulfate, which acts to precipitate proteins at their precise site of localization. This enables the frozen sections prepared to be stained with fluorescein labelled antibody preparations, in order to pinpoint the exact histological location of the proteins in question. Use of formalin fixation causes crosslinking of these proteins, which subsequently changes the antigenicity, making the proteins unable to be recognized by the antibodies.
Two specimen types may be received for urate crystal evaluation: both tissues and fluids may be submitted by clinicians. Joint fluids obtained by fine needle aspiration may be submitted – still contained within the syringe. These specimens must not be fixed in formalin or cytology fixatives. Instead, the fluid should be dispensed on to a clean, labelled microscope slide, using ventilation and Universal Precautions (i.e. the fluid may contain active, blood borne pathogens). After air drying, the slide is cover slipped with Permount and observed under polarizing light.
Similarly, a tissue specimen must be submitted in either Carnoy’s fixative (chloroform and absolute ethanol) or simply absolute ethanol. Fixation cannot be in formalin, as formalin contains water which may dissolve any urate crystals that are present. Once received, the specimen must be processed from 100% ethanol, skipping over the formalin and graded alcohol series in the tissue processor. Once processing and embedding are complete, a slide for H&E and an unstained slide should be prepared. The unstained slide should be rinsed in xylene to remove the paraffin, and then cover slipped. As described above, the unstained slide is viewed under polarized light. Urate crystals will appear as bright white needles on a black background.
In summary, it is excellent practice to identify any and all specimens that may be received by your laboratory for “non-routine” histology. Once identified, procedures should be developed for proper receipt and handling of the specimens. This is the only way to ensure the highest patient care quality.