Physician Education

Overview

This web page addresses the clinical indications and the pathological analysis from skin biopsy specimens of epidermal (intra-epidermal) nerve fiber density (ENFD) for the diagnosis of small-fiber neuropathy (SFN). The assessment of nerve fibers is a component of evaluating peripheral nerve disease. Most nerve disease targets the small nerve fibers, (as opposed to large nerve fibers). This procedure is an objective measure of small fiber neuropathy by identifying a reduction in the density of epidermal nerve fibers.

Small-fiber neuropathy (SFN) is a disease characterized by diminished nerve fiber density in the epidermis (outer layer) of the skin, resulting in painful symptoms, usually, in the extremities. It may occur either independently or as the result of another disease, such as diabetes or alcohol abuse.

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Neuropathy is an abnormal and usually degenerative state of the nervous system or nerves. Typically it affects the lower extremities. It can affect one nerve or many nerves. The form of neuropathy can be broken down by the size of the fiber involvement (that is, small-fiber or large-fiber neuropathy). Small nerve fibers are those near the surface of the skin and the symptoms deal with sensation. The small nerve fibers within the epidermis generally assist with the perception of hot and cold sensation, as well as pain. In addition, small autonomic fibers assist with control of sweating and blood vessel tone.

Small fiber neuropathy is diagnosed clinically but has traditionally been a diagnosis of exclusion based on clinical findings and the absence of large fiber involvement, as determined by electrophysiological studies. SFN is among the least understood of all neuropathies, primarily because standard diagnostic tests for neuropathy such as electromyography (EMG) and nerve conduction studies (NCS) are usually normal in this group of individuals. The disparity between subjective complaints and objective signs increases the difficulty of diagnosis.

SFN generally presents as a painful neuropathy that may rarely become disabling. Symptoms suggestive of this condition may include pain (burning, tingling, shooting, or prickling in character), paresthesia, sheet intolerance, or restless legs syndrome. Leading causes of SFN include diabetes, alcohol abuse, human immunodeficiency virus (HIV) or acquired immune deficiency syndrome (AIDS), amyloidosis, connective tissue disorders, paraneoplastic syndromes, certain neurotoxins, various genetic diseases, as well as an idiopathic form. In addition, conditions other than nerve fiber damage, including venous insufficiency, spinal stenosis, myelopathy, and psychosomatic disturbances may mimic small fiber neuropathy.

There is no treatment to cure small fiber peripheral neuropathy but medications may be provided for pain management, and for some etiologies, treatment of the underlying condition (e.g., glucose control, intravenous immunoglobulin or plasma exchange) may be given to reduce progression of the disease and its symptoms. In fact, our understanding of treatment is changing as new studies are showing reinnervation with disease treatment (Nodera; 2003). If SFN is secondary to another condition, then treatment appropriate for that condition should be administered. In addition, treatment of SFN itself is usually based on symptoms and may include tricyclic antidepressants, anticonvulsants, opioid medications, or local anesthetics applied to the painful areas. Nonpharmacologic treatment may include cool soaks, heat, massage, skin moisturizers, elevation or lowering of the limbs, exercise, spinal cord stimulation, intrathecal morphine, or transcutaneous electrical nerve stimulation.

A specific test to assess epidermal nerve fiber density (ENFD) using skin biopsy and immunostaining of the tissue has been developed that allows the identification and counting of intraepidermal and sudomotor nerve fibers. Assessment of nerve fiber density typically involves a 3-mm punch biopsy of skin from the calf (and sometimes foot or thigh). After sectioning by microtome, the tissue is immunostained with anti-protein-gene-product 9.5 (PGP 9.5) antibodies and examined with immunohistochemical or immunofluorescent methods. This technique has improved research and contributed greatly to the understanding of small fiber neuropathy. Skin biopsy with measurement of ENFD has also been investigated as an objective measure for the diagnosis of small fiber neuropathy.

Introduction

Neuropathy is an abnormal and usually degenerative state of the nervous system or nerves. Typically it affects the lower extremities. It can affect one nerve or many nerves. The form of neuropathy can be broken down by the size of the fiber involvement (that is, small-fiber or large-fiber neuropathy). Small nerve fibers are those near the surface of the skin and the symptoms deal with sensation. The small nerve fibers within the epidermis generally assist with the perception of hot and cold sensation, as well as pain. In addition, small autonomic fibers assist with control of sweating and blood vessel tone.

Small fiber neuropathy is diagnosed clinically but has traditionally been a diagnosis of exclusion based on clinical findings and the absence of large fiber involvement, as determined by electrophysiological studies. SFN is among the least understood of all neuropathies, primarily because standard diagnostic tests for neuropathy such as electromyography (EMG) and nerve conduction studies (NCS) are usually normal in this group of individuals. The disparity between subjective complaints and objective signs increases the difficulty of diagnosis.

SFN generally presents as a painful neuropathy that may rarely become disabling. Symptoms suggestive of this condition may include pain (burning, tingling, shooting, or prickling in character), paresthesia, sheet intolerance, or restless legs syndrome. Leading causes of SFN include diabetes, alcohol abuse, human immunodeficiency virus (HIV) or acquired immune deficiency syndrome (AIDS), amyloidosis, connective tissue disorders, paraneoplastic syndromes, certain neurotoxins, various genetic diseases, as well as an idiopathic form. In addition, conditions other than nerve fiber damage, including venous insufficiency, spinal stenosis, myelopathy, and psychosomatic disturbances may mimic small fiber neuropathy.

There is no treatment to cure small fiber peripheral neuropathy but medications may be provided for pain management, and for some etiologies, treatment of the underlying condition (e.g., glucose control, intravenous immunoglobulin or plasma exchange) may be given to reduce progression of the disease and its symptoms. In fact, our understanding of treatment is changing as new studies are showing reinnervation with disease treatment (Nodera; 2003). If SFN is secondary to another condition, then treatment appropriate for that condition should be administered. In addition, treatment of SFN itself is usually based on symptoms and may include tricyclic antidepressants, anticonvulsants, opioid medications, or local anesthetics applied to the painful areas. Nonpharmacologic treatment may include cool soaks, heat, massage, skin moisturizers, elevation or lowering of the limbs, exercise, spinal cord stimulation, intrathecal morphine, or transcutaneous electrical nerve stimulation.

A specific test to assess epidermal nerve fiber density (ENFD) using skin biopsy and immunostaining of the tissue has been developed that allows the identification and counting of intraepidermal and sudomotor nerve fibers. Assessment of nerve fiber density typically involves a 3-mm punch biopsy of skin from the calf (and sometimes foot or thigh). After sectioning by microtome, the tissue is immunostained with anti-protein-gene-product 9.5 (PGP 9.5) antibodies and examined with immunohistochemical or immunofluorescent methods. This technique has improved research and contributed greatly to the understanding of small fiber neuropathy. Skin biopsy with measurement of ENFD has also been investigated as an objective measure for the diagnosis of small fiber neuropathy.

What Are Small Fibers?

Small fibers are small narrow diameter myelinated (Aδ) and unmyelinated (C) nerve fibers of the peripheral nervous system (McGlone; 2010). Somatosensory Aδ-fibres and C-fibres innervating skin pass through the dermis where they innervate cutaneous structures; both groups of fibers end as free nerve endings in the epidermis (the Aδ-fibres lose their myelin sheath as they cross the dermo-epidermal junction).

Aδ-fibres are responsible for conveying cold input and nociceptive input. C-fibres convey innocuous warm sensations and possibly innocuous cold sensations (Hensel; 1960), and noxious input from a variety of high threshold mechanical, thermal and chemical stimuli.

Small fibers play an important role in the autonomic nervous system because thinly myelinated fibers contribute to preganglionic fibers and C-fibres contribute to postganglionic fibers, innervating structures such as sweat glands, blood vessels and the heart.

Why Do a Skin Biopsy for ENFD

When to consider ENFD testing…

Skin biopsy should be considered in patients with symptoms of small-fiber neuropathy when nerve conduction studies do not reveal abnormalities. Innervation density should be assessed using a standardized quantitative method, the protocol for which has been recently published (Kennedy, 2005).

A qualitative, clinical examination of epidermal innervation density could be misleading, and should not be used alone to diagnose small-fiber neuropathy. Once small- fiber neuropathy has been diagnosed, focused screening (e.g. glucose tolerance test) and treatment of neuropathic pain can begin. Skin biopsy provides an opportunity to identify subclinical involvement of autonomic nerve fibers and degeneration of somatic nerves in neuropathies otherwise considered exclusively autonomic, leading to a better comprehension of symptoms and awareness of potential complications.

Why choose Nova Labs…

Nova Labs LLC is a College of American Pathologists-accredited laboratory and is dedicated to improving diagnosis and research in small fiber sensory neuropathy. Techniques to measure ENFD have led to an improved understanding of the relation between the loss of small nerve fibers and symptoms of peripheral neuropathy. Small fiber sensory neuropathy is a common neuromuscular disorder associated with many medical conditions, including diabetes mellitus, amyloidosis, HIV infection, connective tissue diseases and pharmacological neurotoxicity.

The clinical presentation usually consists of cutaneous pain, sensory loss and autonomic dysfunction, which can lead to functional impairment. Some patients may present with pain as the primary or only symptom, but the pain is inherently subjective and dif cult to measure or quantify. A sensitive and specific diagnostic tool is thus essential for making a correct diagnosis and providing appropriate subsequent management.

Small caliber nerve fibers cannot be evaluated by nerve conduction studies, but histological examination of skin allows quantitation of intra-epidermal nerve ber density to confirm a diagnosis of small fiber sensory neuropathy. It can be safely repeated to monitor disease progression or treatment response.

3-mm punch biopsies are easy-to-learn, minimally invasive, reimbursable procedures that can be performed in an outpatient office setting. An outline of the collection process can be found below.

Our new specimen collection protocol allows you to request free kits in advance, keep them in your of office
at room temperature (constituents included are stable for up to one year), and then ship specimens overnight directly to our lab. This revised protocol will allow you to perform the biopsy during the same clinic visit, making it more convenient for you and your patients. Alternatively, you may send your specimen to your pathology lab to have specimens interpreted.

Sensitivity and Specificity

Coming Soon

ENFD Testing for Diabetes

Measurement of cutaneous innervation has proven to be particularly useful in the study of diabetic neuropathy. Abnormalities in epidermal innervation have been demonstrated to be more sensitive for the diagnosis of diabetic neuropathy than clinical or electrophysiological methods.

Several investigators have demonstrated that dermal PGP immunoreactivity was reduced in subjects with diabetes testing normal on clinical examination, electrophysiology, and quantitative sensory testing (QST) when compared with healthy control subjects. This likely reflects the observation that small unmyelinated nerve fibers are vulnerable early in diabetes (Levy; 1992).

Evaluation of epidermal nerve fibers appear to be even more sensitive, perhaps because of their further distance from the cell body, absence of a Schwann cell or collagen covering sheath, and the avascular nature of the epidermis that increase their susceptibility to disease. Kennedy demonstrated that subjects with diabetes had reduced innervation densities and nerve fiber lengths with many subjects being completely denervated (Kennedy; 1996).

IENF density has been shown to be inversely related to diabetes duration in people with type 2 diabetes, but not to HbA1C levels (Shun; 2004). The latter might represent an effect of historical glycemic control or “metabolic memory” as has been demonstrated for other diabetes end-organ complications (EDIC Study; 2003).

Many patients with idiopathic small fiber predominant neuropathy symptoms have been found to have either occult diabetes or impaired glucose tolerance after rigorous assessment with an oral glucose tolerance test (Singleton; 2001). The diagnosis of diabetes was missed in these patients because of the inappropriate use of glycated hemoglobin as a screening test for diabetes. Using fasting 75 g oral glucose tolerance testing (OGTT), nearly 60% of patients were found to have diabetes or impaired glucose tolerance (IGT) (Sumner; 2003). It is possible, but unlikely that the association between neuropathy and abnormalities on OGTT represents a spurious overlap of two common conditions, as the prevalence of IGT in the neuropathy populations was two- to threefold more than what would have been predicted by the National Health and Nutrition Examination Study (NHANES) study (Sumner; 2003).

Furthermore, there was a dose-response relationship between the pathological and electrophysiological severity of neuropathy and the degree of hyperglycemia. In this study, the ENFD testing was the most sensitive measure of neuropathy and was abnormally low compared with control subjects in both subjects with diabetes and IGT. This observation is consistent with the clinical impression that patients with idiopathic small fiber neuropathy are indistinguishable to those with early diabetic neuropathy. Longitudinal follow-up of subjects with IGT- associated or de-novo diabetes-associated neuropathy suggests that the rate of neuropathy progression is slower in those with IGT-associated neuropathy compared with de novo diabetes.

ENFD Testing for Fibromyalgia

Coming Soon

Requesting a Biopsy Kit 

You may call us at 877.230.1518 to notify us of your desire to obtain a skin biopsy kit.

The kit will be shipped to you and may be stored at room temperature (for up to one year) until you are ready to perform specimen collection.

Skin biopsy kits contain a 3-mm biopsy punch tool, a scalpel, forceps and specimen tubes filled with fixative solution, an ice pack (to be kept frozen until used), a patient referral form and a return shipping label. You can see the video

Method to Perform Skin Biopsy

Skin biopsy is most commonly performed by means of a 3-mm disposable circular punch under sterile technique, after topical anesthesia with lidocaine. No suture is typically required. It is up to physician discretion if lidocaine injection is warranted.

  • Place the patient in a lateral position.

  • Identify one to three standard biopsy sites in one leg: distal leg (10 cm proximal to the lateral malleolus), distal thigh (7 cm proximal to the lateral knee) and proximal thigh (7 cm distal to the lateral greater trochanter). You may collect and send fewer specimens, but the interpretation is optimal when all three specimens are collected.

  • Clean the biopsy sites with alcohol swabs/iodine and prepare the areas with local anesthesia.

  • Perform the biopsy using a 3-mm punch.

  • Remove biopsy specimens using a surgical blade and forceps technique. (Avoid pinching the epidermis.)

  • Put each specimen into a tube containing fixative solution immediately after removal.

  • Label the tube with the patient’s name, DOB, and biopsy side and site (e.g., “left distal leg”).

  • Bandage the sites. No stitch is needed. The patient may shower after 24 hours.

Click here to see the videos NovaLabs 3D Animated Tutorial and ENFD Patient Prep Materials!

Nova Labs also offers on-site training for punch biopsy technique, please ask!

Choice of Biopsy Location

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 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 and upper extremity by different processing techniques. Areas of trauma or where scar formation is present should be avoided as these can artificially lower epidermal nerve fiber densities. 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 preferred.

In general, a distal location where there are abnormalities on examination particularly decreased sensibility to pinprick or thermal sensation, or where the patient has symptoms is best.  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 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.

Skin Biopsy Shipping

Specimens should be returned the same day that they are taken in the original kit (with the provided ice pack). Ship via overnight express mail with the provided label to:

 Nova Labs, LLC
1050 Las Tables Rd., Suite 14
Templeton, CA 93465

The completed request form should also be included. After specimen collection, please call 877.230.1518 to notify us of the shipment.

Specimens must be returned immediately (the same day they are taken) for quality-control purposes.

Do not ship specimens on Fridays, weekends or holidays. If the same-day return is not possible, please contact us for further instructions. Call 877.230.1518 with questions.

Click here to see the video NovaLabs ENFD Shipping Instructions!

Coding & Reimbursements

Medically Necessary:

Pathological analysis of intra-epidermal nerve fiber density for the diagnosis of small-fiber neuropathy is considered medically necessary when all of the following conditions are met:

  • Individual presents with painful sensory neuropathy; and

  • Physical examination shows no evidence of findings consistent with large-fiber neuropathy, such as reduced or absent muscle-stretch reflexes or reduced proprioception and vibration sensation; and

  • Electromyography and nerve conduction studies are normal and show no evidence of large-fiber neuropathy; possibly (depending on plan)

  • Some plans may state that there is no history of a disorder known to predispose to painful neuropathy (e.g., diabetic neuropathy, toxic neuropathy, HIV neuropathy, celiac neuropathy, inherited neuropathy).

Investigational and Not Medically Necessary:

Pathological analysis of intra-epidermal nerve fiber density for the diagnosis of small-fiber neuropathy is considered investigational and not medically necessary in all other cases.

CPT Billing Coding

Coming Soon

Processing & Reporting

Our staff will do a gross examination of skin biopsy specimens, which will be processed per our protocol. Damaged, over-fixed or poorly labeled specimens will not be processed and the referral office will be noticed.

The biopsy will be interpreted and a biopsy report generated within two to three business days after processing is complete. The original copy of the biopsy report will be mailed and/or faxed to the referring physician, generally within two weeks after the biopsy was taken.

References

  • Hensel H, Boman KK. Afferent impulses in cutaneous sensory nerves in human subjects. J Neurophysiol 1960; 23:564–78.

  • Hoeijmakers JG, Faber CG, Lauria G, et al. Small-fibre neuropathies-advances in diagnosis, pathophysiology, and management. Nat Rev Neurol 2012; 8:369–79.

  • Kennedy AJ et al. (2005) Pathology and quantitation of cutaneous innervation. In Peripheral Neuropathy, 869–895 (Eds Dyck PJ and Thomas PK) Philadelphia: Elsevier Saunders

  • Kennedy WR, Wendelschafer-Crabb G, Johnson T. Quantitation of epidermal nerves in diabetic neuropathy. Neurology 1996;47(4):1042–1048.

  • Kremeyer B, Lopera F, Cox JJ, et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 2010; 66:671–80.

  • Levy DM, Terenghi G, Gu XH, Abraham RR, Springall DR, Polak JM.Immunohistochemical measurements of nerves and neuropeptides in diabetic skin: relationship to tests of neurological function. Diabetologia 1992;35(9):889–897.

  • McGlone F, Reilly D. The cutaneous sensory system. Neurosci Biobehav Rev 2010; 34:148–59.

  • Waxman SG. Painful Na-channelopathies: an expanding universe. Trends Mol Med 2013; 19:406–9.

  • Nodera H, Barbano RL, Henderson D, Herrmann DN. Epidermal reinnervation concomitant with symptomatic improvement in a sensory neuropathy. Muscle Nerve. 2003;27:507–9.

  • Shun CT, Chang YC, Wu HP, et al. Skin denervation in type 2 diabetes: correlations with diabetic duration and functional impairments. Brain 2004;127(Pt 7):1593–1605.

  • Singleton JR, Smith AG, Bromberg MB. Increased prevalence of impaired glucose tolerance in patients with painful sensory neuropathy. Diabetes Care 2001;24(8): 1448–1453

  • Sumner CJ, Sheth S, Griffin JW, Cornblath DR, Polydefkis M. The spectrum of neuropathy in diabetes and impaired glucose tolerance. Neurology 2003;60(1):108–111.

  • Sustained effect of intensive treatment of type 1 diabetes mellitus on development and progression of diabetic nephropathy: the Epidemiology of Diabetes Interventions and Complications (EDIC) study. Jama 2003;290(16):2159–2167.