Biopsy with Cutaneous Nerve Fiber Evaluation


Fig. 3.1

3-mm punch skin biopsy for diagnosing small fiber neuropathy. (a) After cleaning the biopsy site, a 3-mm punch is placed on the site perpendicular to the skin surface and twisted down. (b) The skin biopsy should be picked up by a forceps to pinch the subcutaneous layer but not the top epidermis. (Reprinted by permission from Zhou [28])



Skin Biopsy Specimen Processing


The biopsy specimen should be placed into a tube filled with special fixative solution immediately after the biopsy is taken. The tube should be labeled with the patient’s identification and the biopsy side and site. The normative values of small fiber densities at different sites are different [11, 12]. The normative values are also influenced by age and gender [32]. Therefore, these pieces of information should be clearly provided to pathologists. The specimens should be submitted to a cutaneous nerve laboratory, not a routine reference laboratory, as a special technique is used for processing. It is very important to contact a specialized cutaneous nerve laboratory regarding the fixative and specimen handling before planning a biopsy.


Immunohistochemical assays are used to detect an antigen expressed by nerve axons to visualize cutaneous nerve fibers for morphometric and morphological evaluation. Two methods of immunostaining have been used, the bright-field immunohistochemistry [8] and the immunofluorescence with [7] or without [9, 33] confocal microscopy. Since most diagnostic cutaneous nerve laboratories use the bright-field immunohistochemistry, this immunostaining method is briefly reviewed here.


After a skin biopsy is removed, it should be fixed immediately in fixative solution for approximately 24 hours. Two types of fixatives can be used, 2% paraformaldehyde-lysine-periodate (2% PLP) and Zamboni (2% paraformaldehyde and picric acid). Formalin, which is commonly used by routine histopathology laboratories, should be avoided because it may cause fragmented appearance of nerve fibers [11]. The skin specimen is then cryoprotected for at least 6 hours using 20% glycerol in 0.1 M Sorrensons phosphate buffer. After freezing, the specimen is sectioned at 50 μm. The wavy nerve fibers can be better viewed in thick 50-μm sections than in routine 5-μm sections. About 45–55 skin sections can be obtained from each specimen. Four non-adjacent sections from each specimen are chosen for immunostaining, and the rest can be stored in antifreeze solution (30% ethylene glycol) at −20°C for future use when needed.


Immunostaining is done manually under a dissecting microscope using free-floating skin sections and 96-well plates (Fig. 3.2). The primary antibody used in our lab for the immunostaining is a polyclonal antibody against protein gene product 9.5 (PGP9.5). PGP9.5 is an ubiquitin carboxyl-terminal hydrolase [34], which is a neuronal cytoplasmic marker. It is found in all types of efferent and afferent nerve axons [35, 36], so it is a useful pan-axonal marker to highlight all the nerve fibers . After the primary antibody incubation, sections are incubated with a biotin-conjugated secondary antibody which binds to the primary antibody. This is followed by incubation with avidin-conjugated horseradish peroxidase, and avidin can bind to biotin. The immunostaining signal is then developed using an SG kit (blue chromogen/peroxidase substrate) which produces a blue-gray reaction product [8].

../images/462719_1_En_3_Chapter/462719_1_En_3_Fig2_HTML.jpg

Fig. 3.2

Skin biopsy specimen processing . PGP9.5 immunostaining is done manually under a dissecting microscope using free-floating skin sections and 96-well plates


Small Cutaneous Nerve Fiber Evaluation


Intraepidermal Nerve Fiber Density Evaluation


Skin consists of three layers which are firmly attached to one another: the outer epidermis, the deeper dermis, and the subcutaneous layer. The cutaneous innervation was initially thought to mainly consist of a plexus of nerve fibers in the reticular dermis and a more superficial plexus of nerve fibers in the papillary dermis parallel to the skin surface. Rich innervation of epidermis was not demonstrated until late 1980s and early 1990s by immunostaining using PGP9.5 antibodies [7, 9, 37]. The intraepidermal unmyelinated C fibers originate from sensory nerves as they express substance P and calcitonin gene-related peptide (CGRP) [38, 39]. In addition, these fibers arise entirely from dorsal root ganglions (DRG) as they disappear from skin after experimental dorsal root ganglionectomy, but not after dorsal rhizotomy, ventral rhizotomy, or sympathectomy [40]. Before reaching the epidermis, the unmyelinated C fibers are arranged in Remak bundles which also consist of non-myelinating Schwann cells. Axons exchange among Remak bundles as they pass from DRG to skin [41]. The Remak bundles lose their Schwann cells, and the S-100 staining signal of Schwann cells ends at the dermal-epidermal junction [8]. The unmyelinated C fibers ascend vertically through the epidermis between adjacent keratinocytes as free nerve endings [42] (Fig. 3.3).

../images/462719_1_En_3_Chapter/462719_1_En_3_Fig3_HTML.jpg

Fig. 3.3

Cutaneous innervation and denervation . (a) The epidermis is well-innervated by intraepidermal nerve fibers (arrows). (b) The epidermis is devoid of intraepidermal nerve fibers. (c) The sweat glands are well-innervated by sudomotor autonomic fibers. (d) The sweat glands are largely denervated


Intraepidermal nerve fibers are quantified using a light microscope with 40x objective. A counting rule has been established [43] and recommended to use by EFNS/PNS [29, 30]. Briefly, the nerve fibers that cross the dermal-epidermal junction into the epidermis are counted. The nerve fibers that do not cross the dermal-epidermal junction are not counted. If a nerve fiber branches within epidermis, count as one fiber. If a nerve fiber branches below or within the dermal-epidermal junction, count as two fibers. According to the EFNS/PNS guideline, the nerve fragments within epidermis that do not cross the dermal-epidermal junction are not counted due to the concern that these fragments may be the extension of adjacent fibers on the same skin section that are visualized to cross the dermal-epidermal junction and already counted. Counting these fragments may result in overcounting. However, the original fibers that cross the dermal-epidermal junction may not be shown on the same section due to the wavy nature of the nerve fibers, so excluding these fragments may result in undercounting. Some cutaneous nerve laboratories do count these individual fibers that are within epidermis but without crossing the dermal-epidermal junction [8, 12, 20, 44, 45].


The diagnosis of SFN is made based on the reduction of IENFD. To calculate the linear density of IENF, the length of the epidermal surface is measured [30]. The IENFD is expressed as the number of IENF per length of section (IENF/mm). An alternative “ocular” method has been described and used [46–48], in which special sections are chosen for immunostaining with the assumption that the length of the epidermal surface of these sections is close to 3 mm. So the IENFD is calculated simply by dividing the number of IENF by 3. It has been shown that the IENFD obtained by this “ocular” method significantly correlate with the IENFD obtained from the quantification by measuring the length of the epidermal surface [46]. Further studies are deemed warranted to establish the reliability of the “ocular” method [29].


IENFD measurement is highly reproducible. Reproducibility is the highest when four sections from each biopsy specimen are counted [44]. After reviewers are trained to use the same counting rule, the interobserver and intraoberserver reliabilities are high [8, 12, 44, 49, 50]. There is no significant difference in IENFD when skin sections are stained by different cutaneous nerve laboratories as long as an identical methodology is used by these laboratories to process skin specimens and measure IENFD [44].


The technique of 3-mm punch biopsy with IENFD evaluation using the PGP9.5 immunostaining was standardized and first utilized to evaluate patients with SFN by University of Minnesota [7] and Johns Hopkins University [8]. In 1995, the Johns Hopkins group published the method of the bright-field PGP9.5 immunostaining and IENFD quantification [8]. The majority of the diagnostic cutaneous nerve laboratories adopted this method. By using this method, the Johns Hopkins group showed that the IENFD at the distal leg was lower in patients with HIV-seropositive and HIV-seronegative sensory neuropathy than in normal controls. They subsequently developed normative reference ranges at the distal leg and proximal thigh in 98 healthy subjects with age ranging from 13–82 years [12]. They showed a significantly higher IENFD in the youngest age decile (10–19 years) [11, 12]. By using the cut-off derived from the fifth percentile of the normative range at the distal leg to evaluate 20 patients with sensory neuropathy, they showed that the technique had a diagnostic efficiency of 88%. The high diagnostic efficiency of this technique was also demonstrated by other laboratories [10, 13]. By studying the cutaneous innervation at 5 sites, including distal leg, proximal calf, distal thigh, proximal thigh, and trunk in 10 healthy controls (ages 23–75 years), the Johns Hopkins group showed a normal rostral-to-caudal gradient of IENFD with a linear relationship to the distance from the spine [11]. IENFD at a proximal site was higher than that at a distal site. The IENFD at the proximal thigh was higher than that at the distal leg by about 60% [12].


Several laboratories studied normative reference values at the distal leg and found a decline of the IENFD with age [17, 46, 48–52]. A multicenter study developed the normative values of IENFD at the distal leg by evaluating 550 healthy subjects recruited from eight cutaneous nerve laboratories in Europe, USA, and Asia [32]. The study confirmed the age-related decline of IENFD. IENFD was also found to be influenced by gender but not height or weight. The study developed worldwide age- and sex-adjusted IENFD normative values for clinical use. However, the sensitivity, specificity, and diagnostic efficiency have not been fully determined. Our recent small-scale study suggested that the IENFD at the distal leg appeared influenced by the ethnicity, as the diagnostic sensitivity of using the worldwide age- and sex-adjusted normative reference values was lower in Chinses Americans than in non-Chinese Americans who had pure small fiber sensory neuropathy based on the clinical and electrodiagnostic evaluations [53]. Future large-scale studies are needed to fully address the ethnic differences in IENFD at the distal leg. The normative values may need to be adjusted in certain ethnic groups to improve the diagnostic sensitivity.


Intraepidermal Nerve Fiber Morphology Evaluation


IENFD can be normal at the early stage of SFN, which makes the disease difficult to diagnose because the skin biopsy diagnosis of SFN is based on the reduction of IENFD. However, in this setting, skin biopsy often shows prominent morphological changes of small fibers, including swellings, increased branching and fragmentation, and tortuous appearance (Fig. 3.4) [11, 14, 16, 47, 54–56]. Two studies investigated the diagnostic implication of IENF swellings in SFN [16, 47]. Both found a higher prevalence of IENF swellings at the distal leg in neuropathy patients than in healthy controls. Increased IENF swellings at the distal leg correlated with impaired heat-pain threshold, development of symptomatic neuropathy, and progression of neuropathy. In patients with small fiber sensory symptoms but normal IENFD, the presence of the large swellings of intraepidermal C fibers was found to be able to identify those who subsequently developed epidermal denervation [54]. Therefore, the abnormal morphological changes, especially the large swellings of intraepidermal nerve fibers, may represent small fiber degeneration. If these changes are prominent but IENFD are still normal, a repeat biopsy in 12 months may detect the reduction of IENFD and reach a final diagnosis of SFN.

../images/462719_1_En_3_Chapter/462719_1_En_3_Fig4_HTML.png

Fig. 3.4

Abnormal morphological changes of intraepidermal nerve fibers . (a) Abundant nerve fiber swellings of varying size (red arrows) are noted in epidermis, papillary dermis, and dermal-epidermal junction. (b) Many small IENF swellings are seen (red arrows). (c) Intraepidermal fibers are fragmented (red arrows) as compared to continuous fibers in a (yellow arrows). (d) Tortuous (red arrow), branched (yellow arrow), and horizontal (white arrow) fibers are present. (Reprinted with permission from Zhou et al. [45])


Cutaneous Autonomic Nerve Fiber Evaluation


There are several types of autonomic C fibers in the dermis that innervate blood vessel wall (vasomotor fibers), sweat gland (sudomotor fibers), and arrector pilorum smooth muscle (pilomotor fibers) (Fig. 3.5). A few reports have described the reduction of dermal autonomic fiber densities in patients with idiopathic SFN [57] or SFN and dysautonomia associated with diabetes [58], multiple system atrophy [59], and CADASIL [60]. Several studies have attempted to establish standard and reproducible methods to quantify dermal autonomic nerve fiber densities [58, 61–63] to facilitate clinical evaluation and research of autonomic dysfunction associated with SFN. Gibbons et al. have developed an automated method to quantify sudomotor fibers, and the sudomotor fiber density correlates well with the Neuropathy Impairment Score in the Lower Limb (NIS-LL) and the symptoms of reduced sweat production [62, 63]. Some cutaneous nerve laboratories include the measurements of sudomotor fiber densities in their skin biopsy reports. It remains to be determined whether the sudomotor fiber density correlates with the sudomotor function gauged by quantitative sudomotor axon reflex testing (QSART) . Nolano et al. have developed a method to quantify pilomotor nerve fiber density (PNFD) , and by using this method they have found that the PNFD is significantly reduced in diabetic patients as compared with normal controls. However, PNFD does not correlate with IENFD or total neuropathy score [58]. Future studies are needed to refine the measurements of dermal autonomic fibers and to fully determine the diagnostic utility of detecting dermal autonomic denervation [3, 30].

../images/462719_1_En_3_Chapter/462719_1_En_3_Fig5_HTML.png

Only gold members can continue reading. Log In or Register to continue

Related posts:

Default Thumbnail61-Year-Old Woman with Progressive Distal Limb and Deltoid Muscle Weakness Default Thumbnail63-Year-Old Man with Progressive Limb Weakness and Slurred Speech Default Thumbnail54-Year-Old Man with Progressive Lower Limb Weakness and CK Elevation Default Thumbnail59-Year-Old Woman with Subacute Lower Limb Weakness and Painful Paresthesia Default Thumbnail64-Year-Old Woman with Progressive Pain, Numbness and Weakness in the Right Lower Limb 6-Year-Old Girl with Muscle Pain and Swelling in the Thighs

Stay updated, free articles. Join our Telegram channel
Tags:
Apr 21, 2020 | Posted by in NEUROLOGY | Comments Off on Biopsy with Cutaneous Nerve Fiber Evaluation

Full access? Get Clinical Tree
Get Clinical Tree app for offline access