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  Table of Contents    
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 329-336
Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist

1 Department of Pathology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Thiruvananthapuram, Kerala, India
2 Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India

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Date of Submission27-Jan-2022
Date of Decision18-Feb-2022
Date of Acceptance19-Feb-2022
Date of Web Publication11-May-2022


Over the last three decades, skin punch biopsy has become the gold standard for diagnosis of small fiber neuropathies, including autonomic neuropathies commonly seen in diabetics, patients with HIV, and children with hereditary sensory autonomic neuropathies and toxin-induced neuropathy. Clinical, biochemical, electrophysiological tests are inconclusive, making it difficult to diagnose and initiate treatment. A skin punch biopsy is easy to perform in the outpatient clinic, is highly sensitive, and provides an objective diagnosis. Importantly, it helps avoid performing invasive nerve biopsy in patients with small fiber neuropathy, thereby preventing complications such as non-healing of the biopsy site, which is common in these patients. Secondly, the greatest advantage of skin punch biopsies is that they can be repeated any number of times, unlike a nerve biopsy, and are useful to evaluate disease progression and therapeutic response. More recently, its use has been expanded to the diagnosis of large fiber neuropathies, inherited demyelinating neuropathies, etc., obviating the need for a nerve biopsy. The European Federation of Neurological Societies has published guidelines for evaluation to ensure uniformity with regard to the site of biopsy, processing, and quantification. The evaluation of the skin biopsy involves morphometric assessment of the intraepidermal nerve fiber density using PGP 9.5 immunostained sections by bright-field microscopy. This review focuses on the practical aspects of skin punch biopsy and its utility for the practicing pathologist.

Keywords: Autonomic neuropathy, intraepidermal nerve fiber, morphometry, PGP 9.5, small fiber neuropathy

How to cite this article:
Narasimhaiah D, Mahadevan A. Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist. Indian J Pathol Microbiol 2022;65, Suppl S1:329-36

How to cite this URL:
Narasimhaiah D, Mahadevan A. Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Sep 25];65, Suppl S1:329-36. Available from:

   Background Top

Small fiber neuropathy (SFN) is a painful disease affecting small myelinated fibers, unmyelinated fibers, and autonomic fibers associated with significant neurologic morbidity. The thinly myelinated fibers are responsible for cold temperature and sharp pain sensation, unmyelinated fibers for warm sensation and heat pain, and autonomic fibers include postganglionic fibers innervating internal organs and sweat glands.

Patients with SFN can present with pain or a variety of autonomic symptoms such as palpitations, postural giddiness, dry eyes, and abnormal sweating. About half the cases of SFN are idiopathic, while the remaining are secondary to chronic diseases (diabetes, chronic kidney diseases, hyperlipidemia), infections (HIV, Hepatitis C), endocrine disorders (hypothyroidism), autoimmune diseases (Sjogren syndrome, systemic lupus erythematosus, vasculitis), hereditary (Fabry's disease, Tangier's disease) and amyloidosis, among others.

The diagnosis of small fiber neuropathy cannot be made on the evaluation of clinical features alone as conventional methods used in the evaluation of neuropathies, such as nerve conduction studies, are normal in SFN. The diagnosis of small fiber neuropathy involves functional and structural assessment of small nerve fibers. The functional assessment involves autonomic evaluation using tests like quantitative sudomotor axon reflex testing (QSART), quantitative sensory testing (QST), sympathetic skin responses (SSR), thermoregulatory sweat testing (TST), laser-evoked potentials (LEP), and electrochemical skin conductance (ESC). The structural assessment of small fibers requires skin punch biopsy or corneal confocal microscopy.[1],[2],[3] Amongst these tests, the skin punch biopsy has the highest sensitivity (88–90%) and specificity (89–97%)[3] and is considered the gold standard for diagnosis of small fiber neuropathy.

Skin is a densely innervated and highly sensitive organ with different types of sensory nerves as well as autonomic nerve fibers. Sensory nerves innervate the epidermis, dermis, and subcutis. Nerve trunks enter through the subcutis to form a deep nervous plexus at the dermo-subcutaneous junction and a superficial nervous plexus in the subepidermis. Sensory nerves are of four types: Aα fibers (highly myelinated), Aβ fibers (moderately myelinated), Aδ fibers (thinly myelinated), and C fibers (unmyelinated). The Aδ fibers and C fibers convey the thermal and noxious stimuli, and Aβ fibers are responsible for mechanical sensory perception. The thinly myelinated and unmyelinated fibers branch from the subepidermal plexus and either terminate in the dermis or enter the epidermis crossing the dermo-epidermal junction (free nerve endings). These free nerve endings in the epidermis and dermis are called intraepidermal and dermal nerve fibers, respectively, and the former are affected in SFN.[4],[5],[6] Quantifying the densities of these nerve fibers is useful in the diagnosis of SFN, whereas evaluating the innervation of adnexal structures reflects disorders of the autonomic nervous system.

   Skin Biopsy/Skin Punch Biopsy Top

Skin biopsy is useful for investigating small-diameter sensory nerves, unmyelinated intraepidermal nerves, myelinated dermal nerves, and autonomic nerves. Over the last three decades, skin biopsy has gained widespread acceptance as a method of choice to investigate small fiber neuropathy, including small fiber sensory neuropathy and autonomic neuropathy. It can also be a diagnostic alternative to nerve biopsy in some large fiber neuropathies. Skin biopsy is used to quantitatively and qualitatively evaluate the intraepidermal nerve fibers (IENF) and dermal nerve fibers. In addition, serial skin biopsies can be useful to assess disease progression and response to therapy.[7]

In 2005, the European Federation of Neurological Societies (EFNS) published guidelines for the use of skin biopsies in the evaluation of patients with neuropathy to ensure uniformity with regard to the site of biopsy, processing, and quantification [Table 1]. These guidelines were revised in 2010 by the European Federation of Neurological Societies and the Peripheral Nerve Society.[8]
Table 1: EFNS guidelines for processing of skin punch biopsy for bright-field microscopy

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Skin biopsies are generally performed from the distal leg – 10 cm above the lateral malleolus and from the lateral aspect of the upper thigh, 20 cm below the anterior iliac spine. The biopsy from the upper thigh may provide information regarding a length-dependent process.[7],[8]

The biopsy is performed using a 3 mm disposable punch (hence, also called skin punch biopsy) using local anesthesia. To study epidermal nerves, a superficial biopsy 3–4 mm in depth is sufficient, but a deeper biopsy (6–8 mm deep) is required to study the innervation of hair follicles, sweat glands, and blood vessels.[7],[8]

There are no major complications associated with the skin biopsy. Mild infection and excessive bleeding are two minor complications reported.[8]

Another less-invasive method is the use of a suction cap to remove the epidermis alone. Even though this method does not require local anesthesia, it samples only the superficial layers of the epidermis and dermis, and hence it does not provide information regarding hair follicles and sweat glands.[9]


The skin biopsy should be immediately fixed in the cold fixative for 24 hours at 4°C, and then transferred to a cryoprotective solution overnight, following which sections can be cut either in a freezing microtome or cryostat.

The fixatives used are 2% paraformaldehyde-lysine periodate (2% PLP) for bright-field microscopy and Zamboni's fixative (2% paraformaldehyde, picric acid) for indirect immunofluorescence with or without confocal microscopy.[10],[11]

50 μm sections are used for the examination of the intraepidermal nerves by bright-field microscopy. For each biopsy, at least 3 step sections should be examined. The initial and last few sections should be excluded to avoid artifacts.[7]


For diagnostic purposes, bright-field immunohistochemistry[10],[11],[12] or indirect immunofluorescence with or without confocal microscopy can be used. The sections are stained with anti-protein gene product 9.5 (anti-PGP 9.5) antibodies. PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase and a cytoplasmic neuronal marker.[7]

Staining Procedure (Mahadevan A, et al. - unpublished data)

The sections are floated (in Nunc multi-well plates) in staining reagents containing Triton X-100 to promote penetration of antibodies. Sections are incubated overnight at 4°C in mouse anti-PGP 9.5 antibody (Serotec, 1:5000) after blocking with 3% skimmed milk powder. Sections are washed in three changes of wash buffer on the next day, followed by overnight incubation in HRP-tagged secondary. The antigen visualization is carried out using the streptavidin-biotin-HRP method with SG substrate (Vector Labs, immunoenzymatic method). The peroxidase reaction product is then visualized by bright-field microscopy. Stained sections are transferred to chrome alum gelatin-coated slides and counter-stained with eosin.

Intraepidermal nerve fiber morphometry

Quantification using the bright-field technique

For quantification of intraepidermal nerve fiber density by bright-field immunohistochemistry, the IENF are counted on anti-PGP 9.5 immunostained sections under a light microscope using 40X objective. Counting can also be done using image analysis software. Only single nerve fibers crossing the dermal-epidermal junction should be counted, avoiding secondary branches.[13] The length of the section should be measured in millimeters (mm) using image analysis software such as Image J. The intraepidermal nerve fiber density is expressed as a number of IENF per mm (IENF/mm). For each biopsy, IENF is counted in at least three 50 μm sections, and the average of the counts is taken.

Nerve fiber counting rules:[13]

  • Only nerves crossing the basement membrane into the epidermis are counted.
  • Nerves that branch before crossing the basement membrane are counted as two fibers.
  • Nerves that branch within the basement membrane are counted as two fibers.
  • Nerves that branch after crossing the basement membrane are counted as one fiber.
  • Nerve fragments that cross the basement membrane are counted.
  • Nerve fibers that approach but do not cross the basement membrane are not counted.
  • Epidermal nerve fragments that do not cross the basement membrane are not counted.

The nerve counting rules are shown in [Figure 1].
Figure 1: Cartoon illustrating the nerve counting rules[13] The numerical value mentioned above each fiber indicates the number of intraepidermal nerve fibers counted

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Quantification using confocal immunofluorescence technique

For confocal microscopy, 80–100 μm thick sections are cut. The intraepidermal nerve fiber density estimation is performed on images based on a Z-stack of about 16 consecutive optical sections, each at 2 μm intervals.[7] Confocal image acquisition is performed on four epidermal areas (two each from two different sections).[8] Confocal microscopy is more expensive, complicated, and time-consuming.

The IENF density does not vary with the methodology, and there is a good correlation between simple indirect immunofluorescence and bright-field immunohistochemistry.[14] For diagnostic purposes, bright-field immunohistochemistry is considered sufficient.[7]

Normative reference values for bright-field immunohistochemistry

The normative reference values are based on IENF density in healthy controls. Based on published literature, the normative value for healthy subjects in distal leg ranged from 9.8 ± 3.6/mm to 13.8 ± 6.7/mm (mean ± standard deviation).[8] The normative value for healthy subjects in the proximal thigh is 21.1 ± 10.4/mm (mean ± standard deviation).[7] In a large multicenter study with 550 subjects, comprising laboratories from the USA, Europe, and Asia, the IENF density cut-off for skin biopsy from distal leg was calculated at the fifth percentile separately for males and females.[15] IENF density below the lower fifth percentile is considered abnormal. Another study by Collongues, et al.,[16] which included 298 healthy volunteers of the Caucasian population, found some difference with respect to IENF density in females when compared to the multicenter study by Lauria, et al.,[15] which may be due to the heterogeneity of the study population in the latter. The intraepidermal nerve fiber density decreases in the leg from proximal thigh to ankle. In addition, the IENF density is lower in men compared to women and decreases with age.[15],[16] The gender-related difference in IENF density may be related to hormonal status.[17] A few studies have also indicated ethnic differences in IENF density in distal leg.[18],[19] These aspects should be kept in mind while evaluating skin biopsies.

There is no normative data available for intraepidermal nerve fiber density in the distal leg using immunofluorescence with or without confocal microscopy.

Inter and intra-observer variability of IENF quantification

Assessment of reliability of IENF quantification is an important quality control step in using skin punch biopsy for diagnostic purposes in neuropathies. It is essential for each laboratory to compare inter and intra-observer variability of normative reference values. Intraepidermal nerve fiber density quantification in skin punch biopsies is generally considered a reliable technique with good inter and intra-observer correlation.[20],[21] The staining quality and the presence of a well-delineated basement membrane are also important since the presence of a poorly delineated basement membrane can lead to significant inter-observer variability.[22]

Quantification of sweat gland innervation

The quantification of sweat gland innervations is impeded by technical challenges such as the three-dimensional structure of sweat glands, variation in the number and size of glands in each section, increased background with PGP 9.5 staining in sweat glands, and lack of established guidelines for assessment. Gibbons et al.,[23] have described a novel technique to overcome these difficulties.

Qualitative/Morphologic changes in IENF

The morphological changes described in intraepidermal nerve fibers of patients suffering from small fiber neuropathy include axonal swellings and branching.[7] Axonal swellings are defined as axonal enlargement above 1.5 μm in diameter or a two-fold increase in axonal diameter. They are further divided into small (1.5–4 μm) and large (> 4 μm). These swellings are considered as indicators of axonal damage and higher numbers are associated with the presence of neuropathies and their progression.[24] Multiple small swellings are associated with aging, and larger ones are indicators of axonal damage.[16] Another morphological change described in IENF is increased branching with an increased number of branch points associated with sensory neuropathy. In addition, increased complexity of branching is considered a pre-degenerative change preceding the loss of nerve fibers.[11]

Automation in PGP 9.5 staining and quantification of intraepidermal nerve fiber density

The immunostaining for PGP 9.5 is a labor-intensive procedure and challenging for testing large batches of samples in a standardized manner for clinical trials.[25] Likewise, estimation of intraepidermal nerve fiber density by manual counting is time-consuming, observer-dependent, and can be subject to interobserver variability.[26] There is a role for automation for immunostaining[25] as well as IENF quantification.[26],[27] The automated methods are based on fluorescence immunostaining and not immunoperoxidase.[25] The use of automation for staining is less labor-intensive and reduces variation between batches, but there are limitations such as decreased sensitivity for detection of complex branching of epidermal nerve fibers and axonal swellings. The use of automation for IENF quantification requires motorized microscopes and other image acquisition software, which increases the cost. The technical aspects of image acquisition and processing require standardization.[26] Studies on larger cohorts and the establishment of guidelines for clinical application are required before automation can become part of routine diagnostics.

Diagnostic utility of skin biopsy

Skin biopsy in small fiber neuropathies

Skin biopsy is a useful technique to confirm the diagnosis of small fiber neuropathy [Figure 2]. Loss of intraepidermal nerve fibers is considered a histologic hallmark of small fiber neuropathy. Small fiber neuropathy can be idiopathic or secondary [Table 2] with each category accounting for about 50% each. However, with continuous updating of causes of small fiber neuropathy, the idiopathic category is considered closer to 25%.[3]
Figure 2: Skin biopsies in small fiber neuropathy. Biopsy from right ankle stained with PGP 9.5 showing marked reduction in IENFD (arrows) of 1.6/mm (a) compared to age-matched control with IENFD of 8.2/mm (b) Skin biopsy from the ankle (c) showing a reduction in IENFD (3.6/mm) and thigh (d) showing the normal density of 11.2/mm with preserved adnexal innervations (e). [magnification = scale bar]

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Table 2: Causes of small fiber neuropathy

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A novel way of looking at SFN based on the clinical phenotypes includes four main categories[28]:

  • small fiber sodium channel dysfunction - present with paroxysmal neuropathic pain
  • small fiber-mediated painful neuropathy
  • small fiber-mediated widespread pain (including fibromyalgia)
  • small fiber-mediated autonomic dysfunction.

However, skin biopsy only confirms the presence of SFN but is usually not helpful in identifying the etiology of SFN.

The commonest cause of small fiber neuropathy is diabetes mellitus. The neuropathy may be present before the metabolic syndrome or may develop acutely following glycemic control.[1] The IENF loss does not correlate with the presence of signs and symptoms of neuropathy.[29] Skin biopsy is also a useful tool to monitor the progression of SFN in diabetes.[30] The deficiency of vitamin B12 leads to peripheral neuropathy in about 25% of cases.[31] It is also considered a leading cause of small fiber neuropathy after diabetes mellitus. Skin biopsies from patients with vitamin B12 deficiency show a decrease in intraepidermal nerve fiber density, irrespective of the presence of neuropathic pain.[31]

In HIV infection, distal leg IENF density is associated with a clinical transition to symptomatic distal sensory neuropathy. An IENF density of ≤10/mm was associated with a 14-fold-higher risk of developing neuropathy.[32]

Systemic autoimmune and inflammatory disorders can also cause SFN. The degree of IENF loss varies in patients with systemic lupus erythematosus, Sjogren's syndrome, and rheumatoid arthritis.[33]

Skin biopsies from the distal leg and proximal thigh can be useful in differentiating length-dependent small fiber neuropathy from non-length-dependent sensory ganglionopathy. The leg to thigh IENF density ratio was significantly lower in the former than the latter.[34] This feature may be helpful in the differential diagnosis since length-dependent SFN points towards a toxic or metabolic etiology, and SFN-ganglionopathy indicates a dysimmune etiology.[30]

Skin biopsy in large fiber neuropathies

Skin biopsy can be a useful alternative to nerve biopsy in large fiber neuropathies to study the myelinated dermal nerve fibers and mechanoreceptors. In addition, skin biopsy provides an opportunity to study the nodes of Ranvier and internodal architecture. For the study of myelinated fibers, a skin punch biopsy is applied to glabrous skin at the fingertip or lateral aspect of the finger. The utility of skin biopsies is particularly well-studied in demyelinating disorders such as Charcot-Marie-Tooth (CMT) disease. In CMT1A, skin biopsy showed decreased dermal nerve fiber and IENF densities, as well as shorter internodes. Skin biopsies are also useful in demonstrating tomacula in cases of hereditary neuropathy with pressure palsies (HNPP). Moreover, they are useful in studying the expression of myelin proteins (P0, PMP22, MBP) in CMT.[6],[35],[36],[37],[38] Besides their utility in hereditary neuropathies, skin biopsies are also useful in acquired demyelinating conditions such as Guillian-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy. GBS patients showed decreased epidermal nerve density with associated active nerve degeneration in the dermis. The nerve density in skin biopsies is also correlated with the presence of dysautonomia, disturbances in thermal sensation, and severity of pain in GBS and CIDP.[39],[40],[41]

Skin biopsy in autonomic neuropathies

Skin biopsy shows loss of sudomotor, pilomotor, and vascular nerves, besides IENF loss, and provides information regarding the extent of autonomic involvement. It is also a useful tool for follow-up degeneration of sensory axons.[42] Skin biopsy is also useful in acquired and inherited autonomic neuropathies such as diabetic neuropathy, hereditary transthyretin amyloidosis, Fabry disease hereditary sensory-autonomic neuropathy (HSAN). HSAN is a rare disorder affecting mainly Aδ and C-fibers. Genetic testing and skin biopsy have replaced the need for nerve biopsy in HSAN. In Ross syndrome, a disorder of sweating, skin biopsy showed severe loss of sudomotor and arrector pilorum muscle innervations and decreased IENFs.[43] The skin biopsy may show amyloid deposits and glycosphingolipidic deposits in amyloidosis and Fabry disease, respectively.[30] Besides these, disorders such as antiphospholipid syndrome can also have autonomic dysfunction as the initial manifestation.[44] Skin biopsy in such patients shows reduced sweat gland nerve fiber density, in addition to decreased intraepidermal nerve fiber density.[44]

Skin biopsy in other neurological disorders

In addition to its role in neuropathies, a skin biopsy is also useful in other conditions such as alpha-synucleinopathies, particularly Parkinson's disease (PD),[45] amyotrophic lateral sclerosis (ALS),[46] and fibromyalgia. Alpha-synuclein is a well-characterized biomarker of PD. The phosphorylated isoform of alpha-synuclein (PASN) has been demonstrated to accumulate in peripheral nerve terminals of skin, myenteric plexus, and salivary glands, among others.[45] The minimally invasive nature of the skin punch biopsy makes it an attractive diagnostic tool for the detection of PASN. Alpha-synuclein accumulates mainly in the dermal autonomic nerves innervating the blood vessels, pilomotor muscles, and sweat glands. The sensitivity of skin biopsy in the diagnosis of PD also depends on the site of the biopsy, with proximal sites such as the posterior cervical region demonstrating increased sensitivity when compared to the more distal sites.[47] In addition, a skin biopsy may be useful in distinguishing multiple system atrophy parkinsonism type (MSA-P) from PD with orthostatic hypotension (PD + OH) as both diseases have a similar clinical picture. In MSA-P, PASN deposits are seen in the somatic nerves of the subepidermal plexi, while in PD-OH, the PASN deposits are seen in the autonomic nerves.[48] Amyotrophic lateral sclerosis can be associated with pain, and there is evidence of sensory system involvement in ALS. Skin biopsies from patients with spinal onset ALS show reduced intraepidermal nerve fiber density, while in those with bulbar-onset ALS, the IENF density is normal.[49] However, the intraepidermal nerve fiber loss does not correlate with the onset, severity, or course of the ALS and cannot be used as a diagnostic or prognostic marker.[50] Fibromyalgia is a chronic pain disorder with associated non-specific symptoms and uncertain pathophysiology.[46] Skin biopsies from patients with fibromyalgia show decreased IENF density, indicating the presence of a small fiber neuropathy in about 50% of cases.[51] In addition, decreased IENF density is associated with higher pain intensity.[52] Skin biopsies can also be used for disease surveillance and assessment of response to treatment in fibromyalgia.[53]

Advantages and limitations of skin biopsy


  • Simple and minimally-invasive procedure
  • Can be applied to any site in the body
  • Can be repeated to assess disease progression and response to therapy


  • Labor intensive and time-consuming
  • Not readily available in most Centers
  • Dependence on observer accuracy in distinguishing intraepidermal nerve fibers from dermal ones and identifying false-positive staining
  • Bias due to the heterogeneous nature of the neuropathy[54]
  • Skin biopsy results may not always correlate with other neuropathy endpoints[55]
  • Not helpful in identifying the etiology of neuropathy

Data on skin punch biopsy in peripheral neuropathies from India

The normative data on intraepidermal nerve fiber density from India using postmortem samples (Mahadevan A, et al. unpublished data) is shown in [Table 3]. Lower values of IENF compared to other published studies[56],[57],[58] could be due to the use of postmortem controls for normative data; however, ethnic differences also need to be ruled out.
Table 3: Comparison of results of IENF normative data with published studies

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   In Conclusion Top

  • Quantification of intraepidermal nerve fiber density in skin biopsy is a useful technique to confirm the diagnosis of small fiber neuropathy.
  • A skin biopsy should be performed and processed as per EFNS recommendations.
  • Quality control at all levels is mandatory.
  • Normative reference values adjusted for age and gender should be established using healthy volunteers.
  • Intra and interobserver variability should be assessed while establishing the normative reference values.
  • The diagnosis of small fiber neuropathy should be based on a comparison with established normative reference values.
  • Training in an established Cutaneous Nerve Laboratory is recommended.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Anita Mahadevan
Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bengaluru - 560 029, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_92_22

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