HOW-TO SESSION: CLINICAL PATHOLOGY


were given parenterally, even if new lesions are occurring.2,12 Lesions that are secondarily infected with bacteria or yeast should be treated prior to biopsy so the primary disease process can be identified.2 All biopsies should include the dermis. Do not prepare or clip skin if the lesion is in the epidermis because preparation may remove important compo- nents of the lesion. For deeper lesions hair may be clipped with scissors. Instruments should not re- move any crusts or scales and should not touch the skin. After obtaining the biopsy, specimens should be fixed in 10% buffered neutral formalin (BNF). Formalin containers should have a top as wide as the container (do not use glass), seal completely, and the formalin solution should be no older than 1 year. If any precipitates are present in the formalin it is unsuitable for use.13 Autolysis of biopsy samples begins in less than 5 minutes; thus, biopsies should be placed in fixative immediately after they are ob- tained. If multiple biopsies are obtained, each bi- opsy should be placed in formalin in sequence, rather than waiting until all samples are collected.14 Punch biopsies are useful for lesions of the epider-


mis and dermis, but not if the disease extends into the subcutaneous tissue. A new punch biopsy should be used for each patient. A minimum of a 6-mm biopsy punch should be used, and an 8-mm or larger punch is preferred.15 When obtaining a punch biopsy only abnormal skin should be included and the punch should be centered over the lesion. Us- ing a permanent marker (such as a Sharpie) to mark the desired biopsy site prior to acquisition ensures the biopsy contains the part of the lesion selected. The junctional edge of a lesion should not be sampled with a punch. During processing by the histopathology laboratory, the circular sample is cut down the middle and each half placed face down in the cassette. Sections for slides are made from these cut surfaces. Skin coloration or other fea- tures that may be obvious on clinical examination lose their color and distinction once fixed in formalin and cannot be used for orientation.d If it is impor- tant to examine the junctional edge of a lesion then an elliptical biopsy should be obtained. If a biopsy is obtained from an area of alopecia the direction of the hair in surrounding normal skin should be marked with a permanent marker before taking the biopsy. On the submission form, re- quest that the tissue be cut along the marked line. This will ensure that entire hair follicles are present in the resultant slide and avoid cross sections of hair follicles.15 To ensure the disease process is properly repre-


sented several punch samples should be obtained, preferably from lesions in different stages of devel- opment. A minimum of three samples is recom- mended and some authors recommend five to eight.15,16 Most diagnostic laboratories have a single charge for three biopsies; some laboratories do not


150 2015  Vol. 61  AAEP PROCEEDINGS


limit the number of skin biopsies that can be sub- mitted from a patient on one submission form.e,f Care should be taken to ensure that local anes-


thetic is placed in the subcutaneous tissue under the biopsy site as infiltration of the local anesthetic into the skin will result in artifacts that can obscure pathological changes.16 The biopsy punch should be held perpendicular to the skin and turned in one direction while gentle pressure is applied. The bi- opsy should be carefully lifted using small forceps or a small needle, handling the subcutaneous tissue only, and cut by a scalpel blade (preferably) or small scissors to avoid creating stretching or crushing ar- tifacts. If the crust detaches it should be wrapped in microscope lens paper and included with the bi- opsy.12 The submission form should indicate that the crust is separate and should be cut in. To avoid curling artifact, punch biopsies 8 mm or larger should be placed subcutaneous side down onto card- board or a piece of a tongue depressor for a few seconds prior to being placed upside down in forma- lin for fixation. Any wrapped crust should also be submerged into the formalin for fixation.12,15 An elliptical biopsy is preferable when the junc-


tional area between normal and abnormal skin and/ or evaluation of structures deep to the dermis is needed. The biopsy should include approximately one third normal skin and two thirds abnormal skin and be perpendicular to the lesion edge. Elliptical biopsies should be placed on a tongue depressor or cardboard, cut side down, with gentle pressure ap- plied to adhere the biopsy. Preferably, the sample should be pinned in place (25-gauge needles work well) to avoid curling artifact.12 The entire unit is then placed in BNF, biopsy side down. The ratio of BNF to tissue should be 10:1 or greater to ensure complete fixation. Excisional biopsies may be preferred when a small, circumscribed lesion can be completely removed. A wedge biopsy may be used when a large lesion, such as a tumor or other mass, requires deep tissue for evaluation. To avoid distortion of the tissue with lo- cal anesthetic an “L” or ring block or regional anesthe- sia should be used. Electrocautery or laser should not be used when obtaining the biopsy. Submitted tissue should be no thicker than 0.5–1 cm, as that is the limit of formalin penetration. If a mass larger than 1 cm has been removed, slice through most of the mass at 0.5–1-cm intervals to allow proper tissue fix- ation. If the margins of a lesion must be examined to determine whether a tumor has been completely re- moved, the edges may be marked with India ink (http://www.americanmastertech.com/india_ink.htm) by first submersing the mass in ink prior to formalin fixation. The ink will dry in approximately 60 sec- onds and the sample is then placed in BNF. Alterna- tively, each border of the sample may be marked with different colors of ink (http://www.cancerdiagnostics. com/CDI_Products.aspx?pid25) and a key provided on the submission form.


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