Selection Of Biopsy Site And Processing Technique

Generally, skin biopsies are very well tolerated and result in negligible scarring in individuals without a predilection to keloid formation. Discoloration at the biopsy site tends to be more prominent among darker pigmented individuals. The rate of infection even among neuropathic populations is small, approximately 1:500. Biopsy sites

Keloid Magnified Photos

Fig. 1. Example of innervation of dermal appendages. Examples of dermal appendages stained with PGP 9.5. (A) Sweat gland from a normal control that is robustly innervated. (B) Denervated sweat gland, as evidenced by the relative paucity of PGP 9.5 staining. (C) Example of a normally innervated arrector pili muscle. (D) Example of a denervated arrector pili muscle from a distal leg biopsy in a patient with small fiber neuropathy. From ref. 43.

Fig. 1. Example of innervation of dermal appendages. Examples of dermal appendages stained with PGP 9.5. (A) Sweat gland from a normal control that is robustly innervated. (B) Denervated sweat gland, as evidenced by the relative paucity of PGP 9.5 staining. (C) Example of a normally innervated arrector pili muscle. (D) Example of a denervated arrector pili muscle from a distal leg biopsy in a patient with small fiber neuropathy. From ref. 43.

generally heal through a process of granulation without a need for cautery of suturing. Selection of the biopsy site depends on the clinician's intent. If the intent is to diagnose small fiber neuropathy, the availability of normative data is important. These data are available for several locations in the lower extremity by different processing techniques and to a lesser extent in the arm (7,8). Areas of trauma or where scar formation is present should be avoided as these can artificially lower epidermal nerve fiber densities. In general, a distal location where there are abnormalities on examination, particularly decreased sensibility to pin prick or thermal sensation, or where the patient has symptoms is best. Biopsies from the dorsum of the foot, distal, or proximal calf often have reduced nerve fiber densities or are denervated altogether in patients with neuropathy. In our experience, sites within the foot are prone to trauma and this can limit interpretation of the biopsy. In addition, sites within the foot are more prone to infection and for these reasons the distal leg for a caudal biopsy site is prefered. Additional biopsies from more proximal locations can provide additional information allowing the severity of the nerve fiber loss to be assessed as well as providing an internal control. If the intent of the biopsy is to follow a patient longitudinally for neuropathy progression or to monitor a treatment effect, the site chosen should have proximity to the symptomatic area, but should retain enough innervation to provide a substrate for nerve regeneration or

Fig. 2. Example of crush. Panel C shows an entire skin section at low magnification. The crushed region appears pale and has reduced PGP 9.5 staining. Panels A and B show region without (A) and with (B) crush artifact. Arrows indicate epidermal nerve fibers.

Fig. 2. Example of crush. Panel C shows an entire skin section at low magnification. The crushed region appears pale and has reduced PGP 9.5 staining. Panels A and B show region without (A) and with (B) crush artifact. Arrows indicate epidermal nerve fibers.

allow documentation of further degeneration. Future biopsies should be performed adjacent to the original biopsy at a distance of 5-10 mm. One distinct advantage of the technique is that nearly any site can be assessed in contrast to electrophysiology where testing is limited to specific nerves at specified sites. In patients being evaluated for asymmetric, focal symptoms in sites where normative data are not available, biopsies can be performed bilaterally using the asymptomatic site as an internal control.

Two biopsy techniques have been described: punch skin biopsy and skin blister formation (14). Punch biopsy is the most widely used and is performed with a 3-mm diameter circular biopsy instrument. Generally biopsies are performed to a depth of 2-3 mm. This facilitates the removal of the tissue plug and allows innervation of dermal appendages to be assessed. If one is interested only in epidermal innervation it is possible to perform a shallower biopsy. It is crucial to avoid crushing or pinching the biopsy tissue, which can produce artifact resembling denervation (Fig. 2). An alternative biopsy procedure is achieved by application of 300 mmHg negative pressure to a 3 mm blister capsule. This approach has the advantage of being less invasive though blister formation is dependent on maintenance of a tight seal for 20-40 minutes. Application of a heating pad can reduce the time needed and is necessary to achieve blister formation in younger subjects. Blisters are removed with microscissors or superficial skin punches and processed as whole mounts. Both forms of biopsy tissue are immediately placed into refrigerated fixative solution for 12-18 hours at 4°C. Zamboni (2% paraformaldehyde, picric acid), Lana (4% formaldehyde, picric acid), and PLP (paraformaldehyde, lysine, periodate) fixatives all preserve antigenic integrity and are routinely used. If these fixatives are not available, an acceptable alternative is 10% formalin; however this produces a more fragmented appearance of the epidermal nerves and is suboptimal. Glutaraldehyde destroys the antigens and should not be used. Following fixation, the biopsy tissue is transferred to 20% sucrose in phosphate buffered saline cryoprotectant where it can remain for up to 1 month at 4°C or frozen for longer periods. In multicenter trials, or in instances where biopsies need to be sent to a processing center, they should be shipped after fixation and in cryoprotectant overnight on wet ice.

Two similar protocols for processing and imaging fixed biopsy tissue have emerged (15,16). Frozen 50-100 ^M skin sections are cut perpendicular to the skin surface. Sections are immunohistochemically stained as free floating sections in reagents containing the detergent Triton-X-100. The free floating technique and use of a detergent are critical to achieve antibody penetration into the thick sections. After blocking, sections are incubated with a primary anti-PGP 9.5 antibody overnight. After washing, sections are incubated with a secondary antibody directed against the Fc region of the primary

Antibody Pgp Skin

Fig. 3. Normal human epidermal and dermal innervation visualized with confocal microscopy. Nerves are localized with antibody to PGP 9.5 and basement membrane is demarcated with antibody to type IV collagen. Vasculature is labeled with Ulex europaeus agglutinin type I. Epidermal nerve fibers appear aqua and lie within the blue epidermis. The subepidermal neural plexus appears green or yellow. The dermal epidermal junction appears as a red ribbon. Capillaries appear magenta. Nerve fibers (green and aqua) course in bundles through the dermis and branch in the papillary dermis to form the subepidermal neural plexus. Fibers arise from this plexus and penetrate the epidermal basement membrane to enter the epidermis. Some non-neuronal fibrob-lasts appear green as a result of nonspecific PGP9.5 binding. Figure courtesy of William Kennedy and Gwen Wendelschafer-Crabb.

Fig. 3. Normal human epidermal and dermal innervation visualized with confocal microscopy. Nerves are localized with antibody to PGP 9.5 and basement membrane is demarcated with antibody to type IV collagen. Vasculature is labeled with Ulex europaeus agglutinin type I. Epidermal nerve fibers appear aqua and lie within the blue epidermis. The subepidermal neural plexus appears green or yellow. The dermal epidermal junction appears as a red ribbon. Capillaries appear magenta. Nerve fibers (green and aqua) course in bundles through the dermis and branch in the papillary dermis to form the subepidermal neural plexus. Fibers arise from this plexus and penetrate the epidermal basement membrane to enter the epidermis. Some non-neuronal fibrob-lasts appear green as a result of nonspecific PGP9.5 binding. Figure courtesy of William Kennedy and Gwen Wendelschafer-Crabb.

antibody species (e.g., goat antirabbit secondary with rabbit anti-PGP 9.5 primary antibody). If the sections are imaged using confocal microscopy, the secondary antibody must be labeled with a stable fluorescent marker, whereas imaging by light microscopy uses a peroxidase labeled secondary. Multiple individual sections from each biopsy should be stained in order to address concerns of sampling error. Confocal imaging has the advantage that multiple targets can be visualized simultaneously provided secondary antibodies with different fluorochromes are used. Different imaging planes or stacks can be compressed allowing innervation occurring within three dimensions to be viewed in two dimensions. These images are particularly convenient for publication and can facilitate quantification of epidermal nerves though it is possible that overlapping nerve fibers at different depths will be mistakenly viewed as a single fiber once the perspective of the tissue depth is removed (see Fig. 3). The time and expense required to produce such images make such an approach less practical in the setting of clinical trials or longitudinal studies, where large numbers of samples need to be processed quickly. Light microscopy-based techniques are better suited toward such applications where the focus is on determining epidermal nerve fiber densities for which only a signal antigenic determinant is stained (Fig. 4). Recently developed software programs such as Helicon Focus allow multiple light microscopy focal planes to be compressed into a single image—in effect producing a "poor man's" compressed confocal z series.

Most attention has been directed toward staining nerve fibers with antibody directed against PGP 9.5 though other markers have been identified that stain all epidermal nerve fibers such as TuJ1 (17), Ga0 (18), and TRPV1 (19). Specialized stains have also demonstrated IgM deposits in dermal nerve fibers from glabrous skin biopsies in patients with anti-myelin associated glycoprotein (MAG) neuropathy. This suggests that skin biopsies may also be used to assess demyelinating conditions. Interestingly, the deposits of IgM were greatest in the most distal skin biopsies correlating with the electrophysiological hallmark of prolongation of distal motor latencies in anti-MAG neuropathy. Analysis of skin biopsy samples from patients with inherited demyelinating conditions also reproduced the observations from sural nerve biopsies suggesting that skin biopsies will be helpful in the study of these patients as well. Myelinated nerve fibers from skin biopsies have not yet been studied systematically in patients with diabetes.

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