The levels of evidence (class I-IV) supporting the recommendations and ratings of recommendations (A-C) are defined at the end of the "Major Recommendations" field.
Methods to Perform Skin Biopsy and Choice of Biopsy Location
The Task Force members emphasize that the 3-mm punch skin biopsy is a minimally invasive technique. It requires training and is safe as long as sterile procedures and haemostasis are correctly performed. For diagnostic purposes in peripheral neuropathies, performance of a 3-mm punch skin biopsy is recommended. In polyneuropathies, the Task Force recommends performing skin biopsy at the distal leg for quantification of epidermal innervation density. An additional biopsy from the proximal thigh may provide information about a length-dependent process (level A recommendation).
Methods to Process Tissue and Quantify Intraepidermal Nerve Fibres (IENF)
For diagnostic purposes in peripheral neuropathies, the Task Force recommends bright-field immunohistochemistry or immunofluorescence with anti-protein-gene-product 9.5 (anti-PGP 9.5) antibodies in 2% paraformaldehyde-lysine-periodate (PLP) or Zamboni's fixed sections of 50 micrometer thickness. For methodological issues on bright-field immunohistochemistry the Task Force refers to McCarthy et al. (1995)*, on immunofluorescence to Wang et al. (1990)**, and on confocal microscopy to Kennedy and Weldelschafer-Crabb (1993)***. Intraepidermal nerve fibres (IENF) should be counted at high magnification (i.e., 40x) in at least three sections per biopsy. The Task Force emphasizes that only single IENF crossing the dermal–epidermal junction should be counted, excluding secondary branching from quantification. The length of the section should be measured in order to calculate the exact linear epidermal innervation density (IENF/mm) (level A recommendation).
Further studies are warranted to establish the reliability of the "ocular" method (level B recommendation) and the "blister technique" (level C recommendation) for quantification of IENF density in peripheral neuropathies.
*McCarthy BG, Hsieh ST, Stocks A et al. (1995). Cutaneous innervation in sensory neuropathies: evaluation by skin biopsy. Neurology 45:1848-1855.
**Wang L, Hilliges M, Jernberg T, Wieberg-Edstrom D, Johanson O (1990). Protein gene product 9.5-immunoreactive nerve fibers and cells in human skin. Cell Tissue Res 261:25-33.
***Kenney WR, Weldelschafer-Crabb G (1993). The innervation of human epidermis. J Neurol Sci 115:184-190.
Diagnostic Performances of Skin Biopsy
Diagnostic efficiency and predictive values of skin biopsy with linear quantification of IENF in the diagnosis of peripheral neuropathy were very high (level A recommendation). Immunohistochemical technique does not seem to influence the ability of skin biopsy to demonstrate small fibre sensory neuropathy (SFSN). For diagnostic purposes or as outcome measure in clinical trials the Task Force recommends rigorous quantitative assessment with appropriate quality controls (level B recommendation). Cut-off values for epidermal densities in studies based on immunofluorescence microscopy appeared to be higher than in bright-field microscopy studies. Thus far, only the bright-field microscopy method was used to establish normative reference range and diagnostic performances. For quantitative purposes in evaluating peripheral neuropathies, the Task Force recommends determination of IENF density using either immunohistochemistry with bright-field microscopy or immunofluorescence (level A recommendation). Appropriate normative data from healthy subjects matched for age, gender, ethnicity and anatomical site should be used. Quality control should include all the steps of the procedure, in particular, the aspect of intra- and inter-observer ratings.
Studies comparing the diagnostic yield of bright-field microscopy and immunofluorescence with and without confocal microscopy in homogeneous groups of neuropathy patients are warranted. The Task Force emphasizes that the confocal microscopy technique may be useful to investigate cutaneous nerve fibres in demyelinating neuropathies. Furthermore, the diagnostic yield of dermal nerve fibre quantification needs to be addressed. Confocal microscopy technique applied to glabrous skin allows investigation of dermal receptors and their myelinated endings and might provide morphological information that potentially enlarges the usefulness of skin biopsy in sensory neuropathies.
Assessment of Morphological Changes
Quantification of IENF swellings at the lower limb could have a predictive value to the progression of neuropathy, especially if large (level B recommendation). Further studies are warranted to establish whether increased IENF swellings could support the diagnosis of sensory neuropathy and whether this morphological change occurs prior to decreasing IENF density. Further studies are also needed to verify whether increased branching is an early diagnostic finding in peripheral neuropathy.
Quantification of Sweat Gland Innervation
Data on sweat gland innervation density in healthy subjects and in patients with peripheral neuropathy as well as data on correlation between sweat gland nerve fibre density and autonomic assessment are limited (class III evidence). Although part of the neuropathological examination of skin biopsy, assessment of sweat gland innervation still lacks extensive validation.
Correlation between IENF Density and Clinical, Neurophysiological, Psychophysical, Autonomic, and Sural Nerve Biopsy Examinations
Correlation between IENF density and the severity of neuropathic pain needs extensive validation. Decrease in IENF density might represent a further index to predict poorer outcome in patients with Guillain-Barre syndrome (GBS).
Quantification of IENF density can better assess the diagnosis of SFSN (level A recommendation) than sural nerve conduction study (NCS) and sural nerve biopsy. Concordance between IENF quantification and medial plantar sensory nerve action potential (SNAP) amplitude in patients with normal sural NCS suggests that distal sensory nerve recording might be more sensitive than sural NCS in the diagnosis of sensory neuropathy.
The inverse correlation between IENF density and warm threshold assessed by quantitative sensory testing (QST) in patients with SFSN demonstrates that both methods can reliably assess the impairment of unmyelinated nerve fibres in peripheral neuropathies (level A recommendation). Correlation with heat-pain and cooling thresholds as well as measures of autonomic dysfunction needs more extensive validation (level C recommendation).
Studies of Skin Reinnervation
Skin biopsy with quantification of IENF density can be used to assess the regeneration rate of sensory axons in peripheral neuropathies and could represent a potential outcome measure in clinical trials (level B recommendation).
European Union Standards
Skin biopsy is a reliable technique to assess loss and regeneration of sensory nerve fibres in peripheral neuropathies. For diagnostic purposes, the Task Force endorses 3 mm punch skin biopsy at the distal leg, and quantification of linear epidermal innervation density in at least three 50-micrometer thick sections per biopsy, fixed in 2% paraformaldehyde-lysine-periodate or Zamboni's solution, by immunohistochemistry using anti-PGP 9.5 antibodies and bright-field microscopy or immunofluorescence with or without confocal microscopy.
The Task Force strongly recommends training in an established cutaneous nerve laboratory before performing and processing skin biopsies in the diagnosis of peripheral neuropathies. Appropriate normative data from healthy subjects matched for age, gender, ethnicity and anatomical site should be always used. Quality control should include all the steps of the procedure, in particular, the aspect of intra- and inter-observer ratings for qualitative assessments and for quantitative analysis of epidermal densities.
Definitions:
Evidence Classification Scheme for a Diagnostic Measure
Class I: A prospective study in a broad spectrum of persons with the suspected condition, using a "gold standard" for case definition, where the test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy
Class II: A prospective study of a narrow spectrum of persons with the suspected condition, or a well-designed retrospective study of a broad spectrum of persons with an established condition (by "gold standard") compared to a broad spectrum of controls, where test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy
Class III: Evidence provided by a retrospective study where either persons with the established condition or controls are of a narrow spectrum, and where test is applied in a blinded evaluation
Class IV: Any design where test is not applied in blinded evaluation OR evidence provided by expert opinion alone or in descriptive case series (without controls)
Rating of Recommendations
Level A rating (established as useful/predictive or not useful/predictive) requires at least one convincing class I study or at least two consistent, convincing class II studies.
Level B rating (established as probably useful/predictive or not useful/predictive) requires at least one convincing class II study or overwhelming class III evidence.
Level C rating (established as possibly useful/predictive or not useful/predictive) requires at least two convincing class III studies.
