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In this clinical and neurophysiological study using a novel cold stimulator, we aim at investigating whether cold-evoked potentials (CEPs) may prove to be a reliable diagnostic tool to assess trigeminal small-fibre function. Using a novel device consisting of micro-Peltier elements, we recorded CEPs after stimulating the supraorbital and perioral regions and the hand dorsum in 15 healthy participants and in 2 patients with exemplary facial neuropathic pain conditions. We measured peripheral conduction velocity at the upper arm and studied the brain generators using source analysis. In healthy participants and patients, we also compared CEPs with laser-evoked potentials. In the healthy participants, cold stimulation evoked reproducible scalp potentials, similar to those elicited by laser pulses, although with a latency of about 30 ms longer. The mean peripheral conduction velocity, estimated at the upper arm, was 12.7 m/seconds. The main waves of the scalp potentials originated from the anterior cingulate gyrus and were preceded by activity in the bilateral opercular regions and bilateral dorsolateral frontal regions. Unlike laser stimulation, cold stimulation evoked scalp potential of similar amplitude across perioral, supraorbital, and hand dorsum stimulation. In patients with facial neuropathic pain, CEP recording showed the selective damage of cold pathways providing complementary information to laser-evoked potential recording. Our clinical and neurophysiological study shows that this new device provides reliable information on trigeminal small fibres mediating cold sensation and might be useful for investigating patients with facial neuropathic pain associated with a distinct damage of cold-mediating fibres.

Background and Purpose: We aimed to test the clinical utility of the leg:thigh intraepidermal nerve-fiber (IENF) density ratio as a parameter to discriminate between length-dependent small-fiber neuropathy (SFN) and small-fiber sensory ganglionopathy (SFSG) in subjects with signs and symptoms of small-fiber pathology., Methods: We retrospectively evaluated thigh and leg IENF density in 314 subjects with small-fiber pathology (173 with distal symmetrical length-dependent SFN and 141 with non-length-dependent SFSG). A group of 288 healthy subjects was included as a control group. The leg:thigh IENF density ratio was calculated for all subjects. We used receiver operating characteristic curve analyses to assess the ability of this parameter to discriminate between length-dependent SFN and SFSG, and the decision curve analysis to estimate its net clinical benefit., Results: In patients with neuropathy, the mean IENF density was 14.8 ± 6.8/mm at the thigh (14.0 ± 6.9/mm in length-dependent SFN and 15.9 ± 6.7/mm in patients with SFSG) and 7.5 ± 4.5/mm at the distal leg (5.4 ± 3.2/mm in patients with length-dependent SFN and 10.1 ± 4.6/mm in patients with SFSG). The leg:thigh IENF density ratio was significantly (P < 0.01) lower in patients with length-dependent SFN (0.44 ± 0.23) compared with patients with SFSG (0.68 ± 0.28). The area under the curve of the receiver operating characteristic analysis to discriminate between patients with length-dependent SFN and SFSG was 0.79. The decision curve analysis demonstrated the clinical utility of this parameter., Conclusions: The leg:thigh IENF ratio represents a valuable tool in the differential diagnosis between SFSG and length-dependent SFN., (© 2018 EAN.)

Objective: To assess the involvement of the peripheral nervous system in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) by means of immunofluorescence and confocal analysis of punch skin biopsies., Methods: We recruited 14 unrelated patients with CADASIL (M/F = 9/5; age 53.9 ± 10.5 years) and 52 healthy controls (M/F = 31/21; age 53.8 ± 9.8). Patients underwent clinical and neuroradiologic assessment. Three-millimeter punch skin biopsies were taken from the fingertip, the thigh, and the distal leg and processed using indirect immunofluorescence and a panel of primary antibodies to mark vessels and sensory and autonomic nerve fibers. Intraepidermal nerve fibers (IENF), Meissner corpuscles (MC), and sudomotor, vasomotor, and pilomotor nerves were assessed using confocal microscopy., Results: In patients, compared to controls, we found a severe loss of IENF at the distal leg (p < 0.01), at the thigh (p < 0.01), and at the fingertip (p < 0.01) with a non-length-dependent pattern and a loss of MC (p < 0.01). A severe sudomotor, vasomotor, and pilomotor nerve fiber loss was found by semiquantitative evaluation. Along with nerve loss, a severe derangement of the vascular bed was observed. In our patient population, sensory and autonomic denervation did not correlate with age, sex, type of mutation, or MRI involvement., Conclusions: We found an involvement of the peripheral nervous system in patients with CADASIL through the assessment of cutaneous somatic and autonomic nerves. The neurovascular derangement observed in the skin may reflect, although to a lesser extent, what happens in the CNS., (© 2016 American Academy of Neurology.)

Background and Purpose: Quantification of intraepidermal nerve fibers (IENFs) in skin biopsies is now the tool of choice to diagnose small fiber neuropathies. An adequate normative dataset, necessary to assess normality cutoffs, is available for brightfield microscopy but not for immunofluorescence., Methods: Intraepidermal nerve fiber density data in distal leg skin samples processed with immunofluorescence were collected from 528 healthy individuals from four experienced laboratories worldwide. In all laboratories skin samples were collected, processed and analyzed according to standard procedures. Quantile regression analysis was employed to tailor the fit of the 5° percentile as the normal cutoff value and to test and measure the effect of age, gender, body mass index, race, biopsy site (lateral distal lower leg or medial posterior mid-calf) and participating laboratory as possible influential variables., Results: Age, gender and biopsy site showed an independent linear correlation with IENF density. For each decade the 5° quantile IENF cutoff showed a 0.54 fibers/mm decrease, whilst females exhibited a 1.0 fiber/mm cutoff greater than males. Compared to the lateral distal lower leg, biopsies from the calf showed a 3.4 fibers/mm lower 5° percentile cutoff, documenting a variation linked by site., Conclusions: An age- and gender-adjusted normative dataset for IENF density at the lateral distal lower leg obtained with indirect immunofluorescence is presented for the first time by sharing data from four experienced laboratories worldwide. This dataset can be used as reference for laboratories processing skin biopsies with this technique., (© 2015 EAN.)

We investigated the agreement between simple indirect immunofluorescence (IF) and bright-field immunohistochemistry (BFI) on free-floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty-five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland-Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age-adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland-Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut-off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values., (© 2015 Peripheral Nerve Society.)

Introduction: Intraepidermal nerve fiber (IENF) linear density is a reliable tool to detect small-fiber neuropathies. In immunofluorescence, the IENF count is performed typically by computer-assisted nerve tracing on confocal images. Alternatively, the count can be performed directly through the oculars of a standard epifluorescence microscope. We specifically compared measures obtained using the 2 methods., Methods: We compared measures of IENF density in the same 50 skin samples using computer-assisted image analysis and direct count., Results: There was excellent agreement between the 2 methods. Linear regression showed a slope between paired measures virtually equal to 1 (β = 0.99). Bland-Altman analysis showed a mean difference (offset) between the measures of 0.46 ± 0.91 fibers/mm., Conclusions: Direct observation with epifluorescence microscopy proved as reliable as the more time-consuming 3-dimensional computer-assisted analysis of confocal digital images for determining IENF density., (© 2014 Wiley Periodicals, Inc.)

Objective: To assess the involvement of small nerve fibers in Charcot-Marie-Tooth type 1A (CMT1A)., Methods: We used indirect immunofluorescence and confocal microscopy on punch biopsies from glabrous (fingertip) and hairy (thigh and leg) skin of 20 unrelated patients with CMT1A to quantify somatic and autonomic nerve fibers. In particular, we quantified epidermal nerve fibers (ENF), Meissner corpuscles (MC), intrapapillary myelinated endings (IME), and sudomotor nerves. We correlated morphologic data with findings from quantitative sensory testing, sudomotor output, sympathetic skin response, and cardiovascular reflexes. A control population of healthy age- and sex-matched controls was included with a matching ratio of 1:2., Results: We found a length-dependent loss of ENFs that worsened with aging. We also observed a loss of MCs, IMEs, and sudomotor nerves. The loss of ENF at distal leg correlated with the increase in heat-pain thresholds (p < 0.05) and with tactile thresholds (p < 0.05). Sudomotor nerve fiber loss correlated with ENF density (p < 0.05) and sweating output (p < 0.001)., Conclusions: We demonstrated through morphologic, physical, and psychophysical testing that small somatic and autonomic fibers are abnormal and cause symptoms in patients with CMT1A. Awareness of such symptoms by the clinician could lead to better treatment., (© 2014 American Academy of Neurology.)

Skin biopsy with a 3mm disposable circular punch is easy to perform and allows, after proper processing, the visualization of epidermal, dermal, and sweat gland nerve fibers. A technique of sampling the epidermis alone by applying a suction capsule, the "blister" technique, has also been developed. It is most common to stain immunohistochemically for the pan-axonal marker protein gene product 9.5 (PGP 9.5), an ubiquitin C-terminal hydroxylase. The sections are then observed and analyzed with bright-field microscopy or with indirect immunofluorescence with or without confocal microscopy. Most studies report quantification of intraepidermal nerve fiber density displayed in bright-field microscopy. Normative values have been established, particularly from the distal part of the leg, 10cm above the external malleolus. In diabetes mellitus early degeneration of intraepidermal nerve fibers is induced and there is slower regeneration even when there is no evidence of neuropathy. Skin biopsy is of particular value in the diagnosis of small fiber neuropathy when nerve conduction studies are normal. It may also be repeated in order to study the progressive nature of the disease and also has the potential of studying regeneration of nerve fibers and thus the effects of treatment. Inflammatory demyelinating neuropathies may also involve loss of small-diameter nerve fibers and IgM deposits in dermal myelinated nerve fibers in anti-MAG neuropathy. In some cases the presence of vasculitis in skin may indicate a nonsystemic vasculitic neuropathy and in HIV neuropathy intraepidermal nerve fiber density is reduced in a length-dependent manner. In several hereditary neuropathies intraepidermal nerve fiber density may be reduced but other abnormalities can also be demonstrated in dermal myelinated fibers. Some small swellings and varicosities may be present in the distal leg skin biopsy of healthy individuals but large axonal swellings are considered as evidence of a pathological process affecting the normal structure of nerves. The indirect immunofluorescence technique with confocal microscopy provides the opportunity to study the complex structure of sensory receptors and cutaneous myelinated fibers and the innervation of sweat glands, arrector pilorum muscles, and vessels., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although the involvement of large myelinated sensory fibers in Friedreich's ataxia (FA) is well documented, an impairment of unmyelinated fibers has not been described. We demonstrate an involvement of cutaneous unmyelinated sensory and autonomic nerve fibers in FA patients. We performed a morphological and functional study of cutaneous nerve fibers in 14 FA patients and in a population of control subjects. We used immunohistochemical techniques and confocal microscopy applied to punch skin biopsies from thigh, distal leg, and fingertip, and compared the density of epidermal nerve fibers (ENFs) with the results of mechanical pain sensation and thermal and tactile thresholds performed on hand dorsum, thigh, distal leg, and foot dorsum. We observed in our patients a statistically significant loss of ENFs, a reduced innervation of sweat glands, arrector pilorum muscles and arterioles, and an impairment of thermal and tactile thresholds and mechanical pain detection.

Capsaicin applied topically to human skin produces itching, pricking and burning sensations due to excitation of nociceptors. With repeated application, these positive sensory responses are followed by a prolonged period of hypalgesia that is usually referred to as desensitization, or nociceptor inactivation. Consequently, capsaicin has been recommended as a treatment for a variety of painful syndromes. The precise mechanisms that account for nociceptor desensitization and hypalgesia are unclear. The present study was performed to determine if morphological changes of intracutaneous nerve fibers contribute to desensitization and hypalgesia. Capsaicin (0.075%) was applied topically to the volar forearm four times daily for 3 weeks. At various time intervals tactile, cold, mechanical and heat pain sensations were assessed in the treated and in contralateral untreated areas. Skin blisters and skin biopsies were collected and immunostained for protein gene product (PGP) 9.5 to assess the morphology of cutaneous nerves and to quantify the number of epidermal nerve fibers (ENFs). Capsaicin resulted in reduced sensitivity to all cutaneous stimuli, particularly to noxious heat and mechanical stimuli. This hypalgesia was accompanied by degeneration of epidermal nerve fibers as evidenced by loss of PGP 9.5 immunoreactivity. As early as 3 days following capsaicin application, there was a 74% decrease in the number of nerve fibers in blister specimens. After 3 weeks of capsaicin treatment, the reduction was 79% in blisters and 82% in biopsies. Discontinuation of capsaicin was followed by reinnervation of the epidermis over a 6-week period with a return of all sensations, except cold, to normal levels. We conclude that degeneration of epidermal nerve fibers contributes to the analgesia accredited to capsaicin. Furthermore, our data demonstrate that ENFs contribute to the painful sensations evoked by noxious thermal and mechanical stimuli.

Aim: Small fibre neuropathy (SFN) diagnosis represents a challenge for neurologists. The diagnostic gold standard is intraepidermal nerve fibre (IENF) density, but in about 10–20% of patients with symptoms/signs and abnormalities on functional tests, it remains within normal range. We propose an adjunctive parameter to improve the efficiency of skin biopsy diagnosis. Methods: We recruited 31 patients with SFN symptoms/signs, normal nerve conduction study, abnormal quantitative sensory testing and normal IENF density. We also included 31 healthy controls and 31 SFN patients with reduced IENF density as control groups. Results: We measured the distance between consecutive IENFs in the three groups. Mean inter‐fibre distances did not differ between patients with normal counts and healthy controls (66.7 ± 14.5 μm vs. 76.7 ± 13.4 μm; P = 0.052), while the relative standard deviation was significantly (P < 0.001) higher in patients (79.3 ± 29.9) compared to controls (51.6 ± 12.2). Using ROC analysis, we identified an inter‐fibre distance of 350 µm as the measure that better differentiated patients from controls (AUC = 0.85, sensitivity: 74%, specificity: 94%). At least one such segment was also observed in all patients with reduced IENF count. Conclusion: Irregular spatial distribution is an SFN intrinsic feature preceding actual nerve loss. The presence of a stretch of denervated epidermis longer than 350 µm is a parameter able to increase the diagnostic efficiency of skin biopsy. [ABSTRACT FROM AUTHOR]

Skin biopsy has become a widely used tool to investigate small calibre sensory nerves including somatic unmyelinated intraepidermal nerve fibres (IENF), dermal myelinated nerve fibres, and autonomic nerve fibres in peripheral neuropathies and other conditions. Different techniques for tissue processing and nerve fibre evaluation have been used. In March 2004, a Task Force was set up under the auspices of the European Federation of Neurological Societies (EFNS) with the aim of developing guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathies. We searched the Medline database from 1989, the year of the first publication describing the innervation of human skin using immunostaining with anti-protein-gene-product 9.5 (PGP 9.5) antibodies, to 31 March 2005. All pertinent papers were rated according to the EFNS guidance. The final version of the guidelines was elaborated after consensus amongst members of the Task Force was reached. For diagnostic purposes in peripheral neuropathies, we recommend performing a 3-mm punch skin biopsy at the distal leg and quantifying the linear density of IENF in at least three 50- μm thick sections per biopsy, fixed in 2% PLP or Zamboni's solution, by bright-field immunohistochemistry or immunofluorescence with anti-PGP 9.5 antibodies (level A recommendation). Quantification of IENF density closely correlated with warm and heat-pain threshold, and appeared more sensitive than sensory nerve conduction study and sural nerve biopsy in diagnosing small-fibre sensory neuropathy. Diagnostic efficiency and predictive values of this technique were very high (level A recommendation). Confocal microscopy may be particularly useful to investigate myelinated nerve fibres, dermal receptors and dermal annex innervation. In future, the diagnostic yield of dermal myelinated nerve fibre quantification and of sweat gland innervation should be addressed. Longitudinal studies of IENF density and regeneration rate are warranted to correlate neuropathological changes with progression of neuropathy and to assess the potential usefulness of skin biopsy as an outcome measure in peripheral neuropathy trials (level B recommendation). In conclusion, punch skin biopsy is a safe and reliable technique (level A recommendation). Training in an established cutaneous nerve laboratory is recommended before using skin biopsy as a diagnostic tool in peripheral neuropathies. Quality control at all levels is mandatory. [ABSTRACT FROM AUTHOR]

Focuses on the structure and function of epidermal nerve fibers. Effects of topical capsaicin on epidermal nerves; Identification of the morphological proof of peripheral nerve regeneration; Study on the nerves contained in skin biopsies.

Sensory symptoms in CMT1A are generally attributed to large fiber. However, an involvement of small fibers has been recently brought up. We performed an extensive clinical, morphological and functional study of cutaneous somatic and autonomic innervation in 10 patients affected by CMT 1A, in order to assess small fiber involvement in this condition. We used a 13 items questionnaire and QST to evaluate symptoms and function of somatic small fibers, DST, SSR and Ewing tests to evaluate autonomic function. Morphological analysis of cutaneous innervation included lENF density and sudomotor nerves assessment. Twenty age-and-sex-matched healthy subjects were used as controls for morphological data. All patients complained of at least 2 symptoms related to small fiber dysfunction. Nine out of ten patients had an increase in thermal/pain thresholds indicating an impairment of both C and A-delta fibers. DST revealed hypoidrosis in all patients (41.8 ± 22.8 vs 83.7 ± 17.3 sweat drops/cm 2 ). SSR amplitude at feet was significantly lower ( p < 0.05) in patients compared to controls (1.6 ± 0.7 versus 4.2 ± 3.2 mV). One patient showed a cardiac dysautonomia. Density of ENFs sudomotor nerves was significantly lower ( p < 0.01) compared to age-and-sex-matched control subjects. Our findings confirm a sensory and autonomic small fiber damage in CMT 1A patients. [ABSTRACT FROM AUTHOR]

The assessment of epidermal nerve fiber density (ENF) in skin biopsy has been accepted as an objective and reliable technique to diagnose SFN. Bright-field microscopy and immunofluorescence with or without confocal microscopy, are currently used to quantify ENF. The objective of this study was to evaluate the agreement between the two techniques of immunofluorescence ENF analysis, with and without confocal microscopy. Twenty biopsies were obtained from distal leg of controls and neuropathic patients and immunostained for immunofluorescence according to standard procedures. Four sections double stained with PGP and COLIV were analyzed at immunofluorescence microscope and ENF along the entire section length counted directly throughout the oculars. Epidermal length was measured by means of an intraocular calibrated grid. We assessed the accuracy of this method by calculating, inter and intra-rater reliability. ENF were then counted in the same samples using confocal images and Neurolucida software. ENFs were traced on a stack of 16 consecutive 2μm optical sections following published rules. We found a significant agreement between the two methods (p <0.001). Our study demonstrates that the two techniques are comparable, and that the direct count through the oculars is a reliable and faster method for routinary quantification. [Copyright &y& Elsevier]

Our aim was to study peripheral nerve involvement in CADASIL, a hereditary cerebral microangiopathy due to mutations in the Notch 3 gene. We studied skin innervation in 14 patients with CADASIL (6 women, 8 male, mean age 57.6±5.4) by means of 3mm-punch biopsy from thigh, distal leg and fingertip. Samples were processed using indirect immunofluorescence techniques and an extensive panel of primary antibodies to mark both myelinated and unmyelinated somatic and autonomic nerve fibers. Vascular and neural signs of remodeling were present in all patients. We found, compared to 40 age and sex matched controls, a lower density of ENF/mm in patient thigh (12.7±4.3 versus 23.4±3.8), leg (6.7±1.3 versus 13.3±3.0) and fingertip (1.8±0.8 versus 6.9±3.1). In fingertip we found also a marked loss of Meissner corpuscles with a lower density/mm2 (8.3±2.2 versus 27.3±7.4). In addition autonomic innervation appeared severely affected with aspect of structural derangement of dermal annexes. Vascular bed appeared very abnormal with dilated and complex vessels. The involvement of last endings of sensory and autonomic nerve fibers is part of the neuropathological picture of CADASIL. [ABSTRACT FROM AUTHOR]

Evaluation of epidermal nerve fiber (ENF) density by skin biopsy is nowadays considered as the gold standard for the diagnosis of small fiber neuropathies. ENFs show in normal subjects quite a regular distribution between the keratinocytes while in patients with small fiber neuropathies we can find, besides a loss of ENFs, evident anomalies of their distribution. Different patterns of nerve fiber dispersion can express different pathophysiological processes. The aim of this study was to define a tool capable of describing the distribution of ENF in the epidermis. For this purpose, we measured the distance between subsequent epidermal fibers on skin samples of healthy and diabetic subjects. We calculated the standard deviation of the interfiber length as a parameter (the dispersion index – DI) to evaluate the variability of the distance between consecutive ENFs. We selected thigh skin samples from five healthy subjects (age range 30–50 years) and five age and sex matched diabetic patients. Samples were processed using immunohistochemical techniques. Sections were PGP 9.5 and Collagen IV double stained to show nerve fibers and basement membrane. One randomly selected section for each subject was acquired for its entire length (usually seven 20x confocal images obtained from a z-stack of 16 two micron optical sections) using a confocal microscope (CARV, Atto Biosciences, Rockville MD, USA). We measured the distance between consecutive ENFs along the entire epidermal length, using dedicated software (ScionImage, Scion Corporation Frederick, Maryland USA). In diabetic patients the DI resulted significantly higher than in normal subjects. We suggest that DI along with the ENF density can be useful to better characterize epidermal innervation. [ABSTRACT FROM AUTHOR]

Intraepidermal nerve fiber (IENF) density at distal leg is considered sufficient to confirm the clinical suspect of small fiber neuropathy. Extensive normative datasets demonstrate a reduced nerve density with ageing and in male compared to female subjects at this site. An additional sample from thigh is often analyzed to calculate leg/thigh IENF density ratio and assess the length dependence of the neuropathy. Nonetheless, only small populations have been published reporting thigh IENF density. We applied standard techniques to assess IENF density from thigh and leg of 207 healthy subjects, (M/F = 88/119), age 49.1 ± 14.2 years (range 18–82) and to calculate leg/thigh density ratio. IENF density was 14.3 ± 3.5 at the leg and 22.5 ± 4.2 at the thigh. IENF density decreased with age at both sites. Female subjects showed a higher density of IENF compared to male subjects at the distal site only. Average leg/thigh density ratio was 0.64 ± 0.13. This value decreased with age and differed between female (0.66 ± 0.13) and male (0.62 ± 0.13) subjects. Lower limb IENF analysis shows that age affects nerve density in both proximal and distal sites. Leg/thigh ratio is slightly higher in female compared to male subjects. The gender related IENF density differences observed distally are not evident at the proximal site. [ABSTRACT FROM AUTHOR]

We created an age and gender adjusted normative dataset for intraepidermal nerve fiber (IENF) density at distal leg in skin samples processed with immunofluorescence. We collected from four experienced laboratories worldwide IENF density data of 528 healthy individuals. Skin samples were collected, processed and analyzed according to standard procedures. We employed quantile regression analysis to tailor the fit of the 5° percentile as the normal cut-off value and to measure the effect of age, gender, body mass index (BMI), and race as possible influential variables. Age and sex showed an independent linear correlation with IENF density. For each decade, the 5° quantile cut-off showed a 0.54 fibers/mm decrease, while females exhibited a 1.0 fibers/mm cut-off greater than males. BMI didn’t affect IENF counts and we did not find significant differences between Caucasian and non-Caucasian subjects although our cohort was not suitable to draw definitive conclusions on this issue. In conclusion, we presented for the first time an age and gender adjusted normative dataset for intraepidermal nerve fiber density at distal leg obtained with indirect immunofluorescence by sharing data from four experienced laboratories worldwide. This dataset can be used as reference for laboratories processing skin biopsies with this technique. [ABSTRACT FROM AUTHOR]