Small Fiber Neuropathy: Is Skin Biopsy the Holy Grail?

Transcription

1 Curr Diab Rep (2012) 12: DOI /s MICROVASCULAR COMPLICATIONS NEUROPATHY (D ZIEGLER, SECTION EDITOR) Small Fiber Neuropathy: Is Skin Biopsy the Holy Grail? Giuseppe Lauria & Raffaella Lombardi Published online: 9 May 2012 # Springer Science+Business Media, LLC 2012 Abstract Small fiber neuropathy (SFN) is characterized by negative sensory symptoms (thermal and pinprick hypoesthesia) reflecting peripheral deafferentation and positive sensory symptoms and signs (burning pain, allodynia, hyperalgesia), which often dominate the clinical picture. In patients with pure SFN, clinical and neurophysiologic investigation do not show involvement of large myelinated nerve fiber making the diagnosis of SFN challenging in clinical practice. Over the last 15 years, skin biopsy has emerged as a novel tool that readily permits morphometric and qualitative evaluation of somatic and autonomic small nerve fibers. This technique has overcome the limitations of routine neurophysiologic tests to detect the damage of small nerve fibers. The recent availability of normative reference values allowed clinicians to reliably define the diagnosis of SFN in individual patients. This paper reviews usefulness and limitations of skin biopsy and the relationship between degeneration and regeneration of small nerve fibers in patients with diabetes and metabolic syndrome. Keywords Diabetic neuropathy. Small fiber neuropathy. Skin biopsy. Neuropathic pain. Intraepidermal nerve fibers. Dermal nerves. Autonomic neuropathy Introduction The term small fiber neuropathy (SFN) commonly refers to a condition characterized by the damage of small caliber G. Lauria (*) : R. Lombardi Neuromuscular Diseases Unit, IRCCS Foundation, Carlo Besta Neurological Institute, Via Celoria, 11, Milan 20133, Italy glauria@istituto-besta.it nerve fibers (thinly myelinated Aδ and unmyelinated C) with somatic function, namely peripheral axons conveying thermal and nociceptive sensation. When there is an isolated or predominant impairment of the subgroup of these small fibers with autonomic function, the term autonomic neuropathy is used. Unmyelinated and thinly myelinated nerve fibers represent the large majority of peripheral sensory nerves in mammals. However, the possibility to detect their selective damage by nerve conduction study is hidden by the intrinsic limitation of this technique allowing the clinician to assess only the damage of large myelinated axons (i.e. reduced sensory action potential amplitude) and myelin sheath (i.e. reduced conduction velocity). Indeed, the physiological properties of small nerve fibers, including their very low conduction velocity, do not allow clinicians to assess their integrity even in healthy subjects and makes the definition of their damage impossible in patients with selective degeneration and normal large sensory nerve functions. On the other hand, the use of other non-conventional neurophysiologic and psychophysical techniques, such as microneurography, nociceptive reflex study, laser and contact heat evoked potentials, and quantitative sensory testing, was limited by their complex setting in clinical practice and reliability in individual patients. Finally, the clinical evidence of thermal and nociceptive hypoesthesia in the feet, which would reflect the damage of small nerve fibers, may be difficult to detect at the bedside because frequently hidden by positive sensory signs, such as hyperalgesia and tactile allodynia. These limitations, which had led to a nonhomogenous approach and an overall underestimation of SFN in clinical practice, were overcome by the availability of skin biopsy that proved to be a reliable tool for diagnosing SFN.

2 Curr Diab Rep (2012) 12: Small Fiber Neuropathy Definition SFN should refer to a condition characterized on clinical and neurophysiologic ground by the exclusive impairment of somatic small nerve fibers. The NeuroDiab expert panel [1 ] has recently proposed a definition of SFN that is graded as follows: 1) possible: presence of length-dependent symptoms and/or clinical signs of small fiber damage; 2) probable: presence of length-dependent symptoms, clinical signs of small fiber damage, and normal sural nerve conduction study; and 3) definite: presence of length-dependent symptoms, clinical signs of small fiber damage, normal sural nerve conduction study, and altered intraepidermal nerve fiber (IENF) density at the ankle and/or abnormal QST thermal thresholds at the foot. Frequency in Diabetes and Impaired Glucose Tolerance Although supposed to be more common among patients with diabetes or metabolic syndrome, there are no epidemiological studies providing definite data on incidence and prevalence of SFN. Moreover, the variability of the diagnostic criteria used do not permit a retrospective analysis. Finally, the prevalence of SFN in case series of patients with symptoms of neuropathy was likely biased by selection criteria. Therefore, the real frequency of SFN in diabetes and impaired glucose tolerance (IGT) is not known. The etiology of SFN was attributed to diabetes or IGT in 6 % and 42 %, respectively, of patients formerly diagnosed with idiopathic SFN [2]. In a larger case series [3 ], SFN was found in about 25 % of diabetic and 11 % of IGT patients. In one further study [4], three of six patients with IGT had definite SFN and one patient probable SFN, whereas among 24 subjects with diabetes nine patients had definite, four patients probable, and three patients possible SFN. These differences might be explained by the different criteria used to define the diagnosis of SFN. Moreover, these studies were performed using different techniques (e.g. bright field immunohistochemistry and indirect immunofluorescence) and, most of all, before the availability of age and gender adjusted normative reference values for IENF density. Therefore, these data need to be confirmed. One prospective and controlled study in patients with idiopathic neuropathy, though not restricted to SFN [5], and another on patients with suspected sensory neuropathy [6] did not find any difference in the prevalence of IGT between patients and controls. Symptoms and Signs Burning feet is the most common complaint of SFN. Patients can show also local dysautonomic disturbances (e.g. abnormal sweating and vascular regulation of leg and feet, and sometimes in calf cramps). The quality of neuropathic pain can differ, though it is most frequently described as spontaneous, such as burning or like a sunburn, paroxysmal, pruritic, or deep. Pain frequently worsens at rest, particularly at night, interfering with sleep. Evoked pain can present with thermal (cold or warm) or dynamic mechanic (i.e. tactile) allodynia. Some patients complain that their feet feel cold though they are warm at touch, or as though they are constricted like in tight gauzes. Allodynia may make bedsheets intolerable or induce pain while wearing certain footwear or walking. Symptoms of restless legs syndrome can coexist and should be identified because of the excellent response to dopaminergic drugs. Sensory examination should be focused to feet and soles in order to detect both negative (thermal and/or pinprick hypoesthesia) and positive (allodynia, hyperalgesia, aftersensation) signs at the most distal sites of the lower extremities, where the degeneration of small fibers is expected to start in a length-dependent neuropathy. Vibratory sensation should be examined using the 64 Hz tuning fork and referred to available normative values [7]. In pure SFN, it should be normal at the toe. Similarly, patients with pure SFN should not have muscle atrophy or weakness and their deep tendon reflexes should be normal. In SFN, autonomic functions mediated by cholinergic and skin vasomotor fibers were reported to be more impaired than those mediated by systemic adrenergic fibers, suggesting that vascular deregulation in the lower limbs is more frequent than cardiovascular autonomic impairment [3, 8]. It should be taken into account that the clinical presentation of SFN in patients with diabetes or IGT does not differ from that of patients with neuropathy of other etiology. Moreover, neuropathic pain that is considered prototypical of SFN can occur also in mixed (large and small fiber) diabetic neuropathy. Therefore, symptoms and signs, including the features of neuropathic pain, should not be considered specific of diabetic SFN and a diagnostic work-up to rule out other causes of neuropathy should be always considered. Questionnaires and Scales The only validated tool available is the SFN Symptom Inventory Questionnaire (SIQ) [9]. It includes 13 questions each having four response options: 00never, 10sometimes, 20often, 30always. It has been proposed to define the diagnosis of SFN in patients with at least two positive answers and evidence of IENF and/or thermal threshold QST abnormalities, after large fiber impairment is ruled out by clinical and nerve conduction examinations [9, 10 ]. However, this tool has never been specifically used in SFN associated with diabetes or IGT. No study has specifically analyzed the relationship between skin biopsy

3 386 Curr Diab Rep (2012) 12: and the most commonly used screening tools for diabetic neuropathy, such as the Michigan Diabetic Screening Instrument and Score [11]. Spontaneous and evoked pain intensity should be always graded in SFN patients in order to have information on the efficacy of treatments at follow-up visits. The Neuropathic Pain Scale (NPS) or the Visual Analogue Pain Scale (VAS) can be used to score pain from 0 (no pain) to 10 (most intense pain imaginable). Skin Biopsy Technique Skin biopsy is most commonly performed using a 3-mm disposable circular punch under sterile technique, after topical anesthesia with lidocaine. No suture is required. The biopsy includes the epidermis and the dermis, and it allows for investigation of sweat glands, hair follicles, and arterovenous anastomosis. To optimize the sampling of these latter structures, it is suggested to include a hair in the specimen. A less invasive sampling method is the removal of the epidermis alone by applying a suction capsule to the skin. With this blister technique there is no bleeding and local anesthesia is not needed [12]. For diagnostic purposes in patients with possible or probable SFN, skin biopsy should be performed at the leg, 10 cm above the lateral malleolus, within the territory of the sural nerve, to evaluate the loss of the most distal sensory endings typical of length-dependent axonal neuropathy. Mechanoreceptors and myelinated fibers can be more easily examined in biopsies from glabrous skin taken at the tip or lateral aspect of index or middle finger. After the biopsy is performed, the specimen is immediately fixed in cold fixative for approximately 24 h at 4 C, then kept in a cryoprotective solution for one night, and serially cut with a cryostat. Paraformaldehydelysineperiodate at 2 % is the fixative used for bright-field microscopy, whereas Zamboni s solution (2 % paraformaldehyde, picric acid) is mainly used for indirect immunofluorescence method with or without confocal microscopy. Formalin fixation should be avoided. Each biopsy yields about 50 vertical sections of 50 μm thickness. The first and the last few sections should not be used for epidermal nerve examination because of possible artifacts. At least three sections should be immunostained using the cytoplasmatic neuronal marker PGP 9.5, an ubiquitin carboxyl-terminal hydrolase, to quantify the density of IENF. Skin biopsy is a minimally invasive and safe technique. Healing occurs within 7 10 days. The estimated frequency of side effects is 1.9:1,000, most commonly mild infections due to improper wound management which recovered with topical antibiotic therapy, or excessive bleeding which did not require suturing [13 ]. Structure and Innervation of Human Skin The epidermis is composed of four layers of keratinocytes which undergo gradual differentiation as they progress from the basal layer to the stratum corneum, with a turnover time of about 30 days. Other resident cells include Langerhans cells, melanocytes, and Merkel cells. The dermal epidermal junction separates the epidermis from the subpapillary dermis in which papillae vascular plexus and capillary loops are located. The superficial dermis includes also autonomic structures such as hair follicles, pilomotor muscles, blood vessels, sebaceous glands, and sweat glands. In the glabrous skin, the apexes of the papillae contain Meissner s corpuscles with a density in the fingertip of about 30 per millimeter [14]. Pacini s and Ruffini s corpuscles reside in the deeper layers of the dermis. IENF are the endings of dorsal root ganglion (DRG) small diameter neurons and arise from nerve bundles which run in the subpapillary dermis. They have three main characteristics: 1) are unmyelinated sensory fibers with exclusive somatic function; 2) lose the Schwann cell ensheathment as they cross the dermal epidermal junction; and 3) widely express the transient receptor potential vanilloid type 1 (TRPV1), also known as capsaicin receptor [15 17]. Also, keratinocytes and other non-neuronal cells (e.g. vascular smooth muscles, endothelium) express members of the TRP family which participate in the homeostasis of temperature sensations and play a role in the pathogenesis of mechanical hyperalgesia and inflammatory pain. Moreover, keratinocytes secrete chemical substances (e.g. neurotrophins, β-endorphin, interleukins) which may influence axon and DRG neurons excitability through ATP and purinergic receptor signaling [18 20]. No synaptic contact between IENF and epidermal cells has been described, and the presence of naked axons is likely to favor communication through paracrine pathways. Therefore, the current view is to consider the whole skin, in particular the epidermis, as a huge polymodal receptor, whose functions are based on the relationship between resident cells and nerves [21 ]. The dermis of hairy and glabrous skin is innervated by bundles of unmyelinated and myelinated fibers. These latter have been the recent subject of investigation following the hypothesis that their measurement could increase the diagnostic yield of skin biopsy in SFN patients [22]. The morphometry of dermal nerve length analyzed throughout the entire skin section within a depth of 200 μm from the dermal-epidermal junction correlated with quantification of IENF and reliably differentiated SFN patients from healthy subjects [23]. Further studies are needed to demonstrate the

4 Curr Diab Rep (2012) 12: contribution in the diagnosis of SFN in symptomatic patients with normal IENF density. Skin biopsy allows the innervation of dermal autonomic structures. It differs in terms of function and can be investigated using markers for adrenergic, noradrenergic, and cholinergic sympathetic and vasodilatory peptidergic fibers [24]. Recently, novel techniques have been standardized to quantify the innervation of sweat glands [25 ] and pilomotor muscles [26 ]. Quantification of IENF The innervation of the epidermis using bright-field immunohistochemistry is based on the assessment of the linear density of nerve fibers (IENF/mm). They are counted in at least three sections, then the length of the epidermis is measured using a software for biological measures and the density per millimeter is calculated (Fig. 1). Using confocal microscope immunofluorescence technique, the density is usually calculated based on evaluation of a stack of consecutive 2 μm optical sections for a standard linear length of epidermis. Technical procedures and methods to assess the innervation density of epidermal nerve fibers are reported in the guidelines of the European Federation of the Neurological Societies and Peripheral Nerve Society [13 ]. Normative reference values adjusted for gender and age decade (using the bright-field method) have been obtained from a cohort of 550 healthy individuals [27 ]. This study definitely demonstrated that the density of IENF declines with aging and that values slightly differ between genders. These values should be used in clinical practice. No study has assessed yet the normative range of IENF density using indirect immunofluorescence with or without confocal microscopy. This is a major limitation for using this method to diagnose SFN. The density of IENF is highest in the paravertebral region of the trunk, and shows a decreasing proximal-to-distal gradient in the limbs, being about 40 % lower in the supramalleolar area than in the thigh [28]. This paradoxical distribution, if considered in terms of relationship between number of receptors and discriminative size of sensory fields, remains unexplained. Fig. 1 Skin biopsies from the distal site of the leg taken in a healthy subject (a, e) and in patients with small fiber neuropathy (b, c, d, f). In a, note the rich distribution of intraepidermal nerve fibers (arrows) with are almost completely lost in b. Image c shows large swellings of intraepidermal nerve fibers (arrowheads) which are considered predegenerative axonal changes. Image d shows fragmentation and weaker staining of a dermal nerve bundle. In e, note the normal innervation of a sweat glands with axons surrounding the ducts. Image f is an example of sweat gland denervation. Bright field immunohistochemical studies with polyclonal antiprotein gene product 9.5 antibodies (Ultraclone, Wellow, Isle of Wight, UK). Original magnification 40x. Bar is 50 μm in all images

5 388 Curr Diab Rep (2012) 12: Somatic Skin Nerve Damage in Diabetes Skin biopsy has been used to demonstrate the involvement of small fibers in patients with diabetic neuropathy [29]. This study showed that both the density of IENF and their length were reduced compared with healthy subjects. However, the real advantage given by skin biopsy has been the possibility to demonstrate the selective degeneration of small nerve fibers in patients without clinical and electrophysiological evidence of peripheral neuropathy. After being initially described in idiopathic SFN [30], this finding was reported in several case series suggesting that the depletion of IENF is common in patients with diabetes or IGT and may even correlate with the degree of neuropathy [2, 31 38]. Overall, skin biopsy showed sensitivity and specificity values above 90 % [39, 40] and a yield higher than that of the psychophysical examination of sensory thresholds [3] for diagnosing SFN. Few studies on SFN have been performed in diabetes, IGT, or metabolic syndrome. One of these with a crosssectional design showed that IENF density progressively declined over time and was not correlated with the degree of metabolic control in patients with diabetic neuropathy, suggesting that hyperglycemia may trigger other mechanisms able to maintain the axonal damage [41]. At 2-year follow-up [3], diabetes or IGT were found in 20 % of patients formerly diagnosed with idiopathic SFN. About 10 % of patients with SFN showed a progression to a mixed (large and small fiber) neuropathy. In most patients with SFN, the clinical picture did not change over time, though about one third experienced a worsening of neuropathic pain intensity. Progression toward the depletion of IENF and an overt neuropathy was observed in patients with diabetic painful neuropathy and diffuse swellings of IENF, which have been therefore considered pre-degenerative axonal changes [42]. The recognition of similar morphological changes also in patients with HIV-related neuropathy [43] strengthened the hypothesis that the abnormalities of IENF might be an early marker of neuropathy, but points to a pathological process that is non specifically related to hyperglycemia. The relationship between SFN and IGT or metabolic syndrome is of particular importance. Indeed, the evidence of early nerve fiber degeneration could put patients who are at risk for diabetes also at risk for the development of mixed neuropathy over time. Since chronic damage of large nerve fibers causes the denervation of Schwann cells, which is an impediment to the axonal regeneration, the recognition of IENF depletion as an independent risk factor for neuropathy might lead to earlier and specific therapeutic behaviors. IGT and metabolic syndrome were reported to be more frequent among patients with SFN assessed by reduced IENF density in the leg [2, 31, 32, 34, 38, 44]. However, data on skin innervation in patients were biased by the lack of control with normative reference values. Indeed, all the studies so far published referred to non-stratified cohorts of healthy subjects. Therefore, it is impossible to estimate the real proportion of IGT and diabetic patients in whom IENF density is lower than that of gender and age matched controls. Moreover, the higher frequency of IGT in idiopathic SFN was denied by two European prospective studies [5, 6]. Finally, there is no data showing that loss of IENF and SFN are independent risk factors for the development of major late complications of diabetic neuropathy, such as foot ulcers. Focused studies should be designed to address these issues, which results would have an extremely important impact on patients. Autonomic Skin Nerve Damage in Diabetes Specialized fibers with different functions provide the innervation to the autonomic structures of the dermis. Among them, sweat glands and piloerector muscles have been more intensely investigated and morphometric methods have been recently proposed. Compared to the examination of the epidermal innervation, the analysis of these structures is more complex due to their three-dimensional conformation. Quantification of sweat gland innervation proved to be reliable when compared with stereological methods and demonstrated that the linear loss of fibers as diabetic neuropathy progressed correlated with the Neuropathy Impairment Score in the Lower Limb (NIS-LL) and the loss of IENF [25]. These findings have been confirmed by other studies which show also a correlation with cardiac autonomic dysfunction as assessed by reduced heart rate variability [45, 46]. Pilomotor nerve fiber density was recently reported to decline in patients with diabetic neuropathy and the loss of noradrenergic fibers assessed by dopamine β-hydroxylase staining correlated with sweating impairment [26]. Correlation with Psychophysical and Nonconventional Neurophysiological Tests Quantitative sensory testing (QST) has been used to determine the functional impairment of small nerve fibres by the detection of warm and cooling thresholds. Several works demonstrated sensory abnormalities in patients with SFN and correlation studies with skin biopsy have been performed. Results were not completely reproducible and it is still unclear whether these two methods are easily exchangeable for diagnostic purposes. QST has several drawbacks and was suggested to be a useful technique in population studies but an unreliable tool for diagnosing SFN in individual patients [47 49 ]. Moreover, QST showed a remarkable phenotypic heterogeneity across the major neuropathic pain syndromes that included an overlap between central and peripheral nervous system diseases in patients with peripheral

6 Curr Diab Rep (2012) 12: neuropathy [50]. In some studies on diabetic neuropathy, IENF density was found to be inversely correlated with thermal and pain thresholds, showing the highest correlation with warm threshold, [36, 41, 51]. However, in others the concordance between these methods was low [3, 52]. This is likely due to the intrinsic properties of the two methods, whose direct comparison is incorrect. Indeed, QST assesses the function of small nerve fibers through patients responses to stimuli which are perceived and elaborated in the somatosensory cortex. Conversely, skin biopsy is a much simpler method that provides the quantification of distal small nerve fibers. No study focused on the correlation between skin biopsy and cutaneous silent period (CSP) or microneurography in patients with diabetic SFN. CSP has a poor specificity and its diagnostic value in SFN is questionable [53]. Conversely, microneurography is a minimally invasive method that allows single Aδ and C fiber activity recording [54] and has significantly contributed to the knowledge on their physiology and mechanisms underlying sensitization in neuropathic pain [55 59]. Laser-generated radiant heat pulses selectively excite free nerve endings in the superficial skin layers and are recorded from the scalp as late and ultralate laser evoked potentials (LEPs) which are generated by Aδ and C fibers, respectively [60]. LEPs provide an accepted method of investigating nociceptive pathways and have been used to detect the damage of small fibers in painful neuropathies [61]. The correlation between LEPs and skin biopsy has been investigated in a limited number of patients [3, 62]. Moreover, LEPS are frequently absent both in SFN and mixed neuropathies [3, 63 ], making this technique non specific to achieve the diagnosis in individual patients. Contact heat-evoked potential stimulators (CHEPs) provide a technique, more recently developed [64], that exploits an extremely rapid heat rising time to evoke Aδ and C-fibre related scalp components. Only a few studies on SFN have been performed using this technique, mainly demonstrating a correlation with Aδ fiber damage and skin denervation also in patients with diabetic SFN [63, 65 68]. Skin Innervation and Neuropathic Pain The distinct characteristics and functional properties of IENF make them the most distal nociceptors of our body. Therefore, the relationship between their density and neuropathic pain has been the subject of several studies [69]. In diabetic neuropathy, patients with pain had lower IENF density values than did asymptomatic patients, but there was no correlation with pain intensity within the group of symptomatic patients [51]. However, in patients with painful diabetic neuropathy, the density of IENF was invariably reduced [4]. In patients with IGT, life-style modifications induced a slight recovery of IENF in the thigh along with a reduction in pain symptoms [32]. Pain recovery correlated with IENF regeneration in patients with diabetic truncal neuropathy [70]. Overall, these findings led to the understanding that the loss of IENF is likely to increase the risk of developing neuropathic pain, which intensity might decrease in parallel with nerve regeneration. Regeneration of Skin Nerves in Diabetic Patients Skin nerves can regenerate after chemical denervation following topical capsaicin application [71]. This method has been used to investigate the ability of IENF to regrowth in patients with diabetes [72]. The rate of IENF regeneration was about 80 % slower in patients with diabetic neuropathy than in healthy subjects and it was reduced by 40 % in diabetic patients without overt neuropathy. These findings indicate that abnormalities in small nerve fibers occur very early and that the recovery from hyperglycemia per se might interfere with the machinery of axonal transport and mitochondrial functions, though there is no clue in support of this view. Therefore, the pathogenesis of small nerve fiber damage in diabetes and IGT remains unclear. Active regeneration of IENF was found to occur after recovery from chronic hyperglycemia in the streptozotocin model of experimental diabetic neuropathy. Rats showed foot pad reinnervation after islet function was regained and near-euglycemia attained [73]. The results of a prospective clinical study seemed in keeping with this observation. Indeed, the improvement of the metabolic status allowed clinicians to achieve neuropathic pain relief and regeneration of IENF density in the thigh of patients with IGT [32]. However, skin nerves undergo dynamic changes whose course over time is not exactly known. Moreover, the criteria to define a clinically meaningful skin reinnervation have not been established yet. Therefore, although possible, the relationship between IENF regeneration and improvement of diabetes remains uncertain [74]. Thus far, the analysis of IENF regeneration has been used as outcome measure only in experimental diabetic neuropathy to assess the neuroprotective effect of insulin-like growth factor-1 and erythropoietin [75, 76]. The excision of the skin using a 3-mm punch, as is commonly done in clinical practice, causes more profound changes in the ability of skin nerves to regenerate [77]. A rapid proliferation and migration of epidermal cells covers the site of biopsy, while the subepidermal area is filled by fibroblasts and penetrated by blood vessels. However, the rate of dermal and epidermal axon regeneration is very slow and impaired in patients with diabetic neuropathy compared with healthy subjects. This was likely explained by the atrophy of Schwann tubes and the lower rate of Schwann cell migration and blood vessel growth in diabetic patients [78 ]. Impaired

7 390 Curr Diab Rep (2012) 12: axon regeneration is a key deficit in diabetic neuropathy. Possibly, the enhancement of blood vessel growth might improve this process and prevent or slow its development. Conclusions: Usefulness and Limitations of Skin Biopsy Skin biopsy has indubitably become a new tool in the neurologist s and diabetologist s toolbox due to its high diagnostic yield. It has contributed to the definition of SFN, a condition that likely represents the earliest phase of peripheral nerve damage in most patients with diabetes, and should be considered when patients complain of painful neuropathy symptoms in the feet and have normal nerve conduction studies. The method is safe, simple, painless, cheap, and minimally invasive. It provides a morphometric analysis of IENF density that proved to have high sensitivity and specificity for diagnosing SFN. The entire procedure has been standardized allowing each laboratory to obtain reliable measurements. Age and gender adjusted normative values allow the clinician to confirm or deny the diagnosis of probable SFN in individual patients. Skin biopsy can provide the opportunity to identify early and even subclinical damage of small nerve fibers. This has been suggested to occur frequently in patients with IGT and metabolic syndrome. However, this hypothesis was based on studies using mean density values of IENF density from non-stratified cohorts of healthy subjects instead of age and gender adjusted normative values. Therefore, the frequency of SFN in pre-diabetic patients remains uncertain and needs to be ascertain by new focused studies. One further limitation of skin biopsy is that it cannot address the etiology of neuropathy. Therefore, other causes of SFN should also be always ruled out in patients with diabetic SFN. Finally, the relationship between skin biopsy, most commonly used clinical screening questionnaires, and major late complication of diabetic neuropathy need to be established. Disclosure No potential conflicts of interest relevant to this article were reported. References Papers of particular interest, published recently, have been highlighted as: Of importance, Of major importance 1. Tesfaye S, Boulton AJ, Dyck PJ, et al. Diabetic neuropathies: update on definitions, diagnostic criteria, estimation of severity, and treatments. Diabetes Care 2010;33: This consensus paper revised definition and grading of diabetic neuropathy, and provides the first definition of small fiber neuropathy for clinical practice and research. 2. Sumner CJ, Sheth S, Griffin JW, Cornblath DR, Polydefkis M. The spectrum of neuropathy in diabetes and impaired glucose tolerance. Neurology. 2003;60: Devigili G, Tugnoli V, Penza P, et al. 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