The analysis of epidermal nerve fibre spatial distribution improves the diagnostic yield of skin biopsy.

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.

The role of skin biopsy in differentiating small-fiber neuropathy from ganglionopathy.

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.

Non-motor involvement in amyotrophic lateral sclerosis: new insight from nerve and vessel analysis in skin biopsy.

AIMS: To assess cutaneous sensory and autonomic nerves and the vascular bed in amyotrophic lateral sclerosis (ALS). METHODS: We enrolled 41 patients (M = 20, aged 63.5 ± 11.8 years), and 41 age- and gender-matched healthy volunteers (M = 20, aged 63.5 ± 11.8 years). Disease severity and sensory and autonomic symptoms were scored using dedicated rating scales. Skin biopsies obtained from thigh, leg and fingertip were processed using indirect immunofluorescence. Intraepidermal nerve fibres, Meissner corpuscles (MCs), intrapapillary myelinated endings, cholinergic and noradrenergic pilomotor nerves and dermal vessels were quantified on confocal images. Intraepidermal nerve fibres, pilomotor nerves and vessels were also assessed on distal leg skin samples of 10 spinal cord injury patients to compare our findings with those of a chronic hypomobility condition. RESULTS: Compared to healthy controls skin biopsies showed: (i) non-length-dependent loss of intraepidermal nerve fibres (P < 0.01) and loss of MCs (P < 0.01); (ii) reduced (P < 0.01) density of pilomotor nerves involving cholinergic and noradrenergic fibres and (iii) a reduced (P < 0.01) vascular bed. Autonomic nerve and dermal vessel densities were higher in patients with higher disease progression rate (P < 0.01). Moreover, we observed signs of nerve regeneration coexisting with nerve degeneration and increased complexity of the dermal vessels. In patients with posttraumatic spinal cord injury, the density of intraepidermal nerve fibres, pilomotor nerves and of the vascular bed did not differ from controls (P > 0.05). CONCLUSIONS: We demonstrated a cutaneous sensory and autonomic denervation in ALS and a previously undescribed relationship between autonomic and vascular involvement that appeared to be linked to the disease progression rate.

A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg.

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.

Quantification of pilomotor nerves: a new tool to evaluate autonomic involvement in diabetes.

OBJECTIVES: Quantification of the complex, autonomic networks in the skin is difficult. Although sporadic attempts focusing mainly on sudomotor plexus have been reported, an easy and reliable method of quantification has not yet been made available. We developed a method to quantify pilomotor nerve fibers (PNFs), which, compared to sudomotor nerves, have a less complex pattern. We used this procedure on a population of normal and diabetic subjects, and propose it as a new tool to study cutaneous autonomic nerves. METHODS: Skin biopsies were performed from thigh and distal leg in 20 diabetic patients and 20 age- and sex-matched controls. Samples were processed applying indirect immunofluorescence and using pan-neuronal and selective markers for cholinergic and noradrenergic fibers. Pilomotor nerve fiber density was blindly calculated on single 2-µm optical sections selected from confocal z-stacks. Interobserver and intraobserver reliability was evaluated. Results were compared with values obtained by 2 other methods that explored PNFs more extensively. Pilomotor nerve fibers density was compared to epidermal nerve fiber (ENF) density, to pilocarpine-activated sweat gland density, and to the severity of neuropathy as assessed by the modified total neuropathy score. RESULTS: A significant loss of PNFs was found in diabetic subjects' thigh and leg. PNFs density did not correlate with ENF density, disease duration, or total neuropathy score. Noradrenergic PNFs correlated instead with sweating impairment. CONCLUSIONS: A reliable assessment of PNF density is possible. When studying cutaneous innervation, PNF quantification should be done to gain information on autonomic nerves in addition to somatic nerves.

Evaluation of sudomotor function in diabetes using the dynamic sweat test.

BACKGROUND: The study of sudomotor function represents a useful tool to evaluate autonomic disorders. Currently available tests allow either the measurement of sweat output from the whole body or selected small skin locations over time, or quantification of the number and size of sweat drops at a fixed time after stimulation. We devised a dynamic sweat test (DST) that measures at the same time sweat gland density, distribution of active glands, and sweat rate, and applied it to the evaluation of sudomotor function in diabetes. METHODS: The DST was used to evaluate sweating in the forearm of 14 asymptomatic diabetic subjects and 14 age- and sex-matched healthy controls. Distal leg was also tested in 7 patients and 7 controls. The formation of the imprint of pilocarpine-induced sweating was recorded by a digital video camera through a cornstarch-powdered transparent tape used as a contrast-enhancing device. Mean sweat output per gland and per skin area and sweat gland density per cm(2) were evaluated. RESULTS: We observed a significant reduction of sweating in diabetic subjects as compared to controls; sweat gland density per cm(2) (59.7 +/- 18.6 vs 83.7 +/- 17.3; p < 0.05) and mean sweat output (nL/min) per gland (4.7 +/- 0.7 vs 8.3 +/- 2.7; p = 0.01) and per skin area (261 +/- 100 vs 645 +/- 296; p = 0.01) were reduced in the lower limb. Values for the forearm were not significantly different from controls. CONCLUSIONS: Dynamic sweat test is an easy-to-perform, informative method to study sudomotor function. It provides the ability to detect subtle functional changes occurring in the early stages of diabetic neuropathy.

Serological diagnosis of chlamydial abortion in sheep and goats: comparison of the complement fixation test and an enzyme-linked immunosorbent assay employing solubilised proteins as antigen.

A new ELISA for antibodies against chlamydial abortion of ewes which uses detergent solubilised proteins (dsp) of Chlamydia psittaci as antigen (Anderson, I.E., Herring, A.J., Jones, G.E., Low, J.C., Greig, A., 1995. Development and evaluation of an indirect ELISA to detect antibodies to abortion strains of Chlamydia psittaci in sheep sera. Vet. Microbiol., 43, pp. 1-12] was compared with the complement fixation test (CFT) in screening 1000 ovine and caprine sera obtained from selected flocks/herds ('flocks') and submitted to a veterinary diagnostic laboratory. Fifteen of the 17 'flocks' had a history of abortion while the remaining two did not and were classified as 'negative flocks'. Infection with Chlamydia was confirmed during the study period in five 'flocks' using direct immunofluorescence and the modified Ziehl Neelsen stain on pathological material. The dspELISA and CFT identified 37 and 45 positive sera on 158 samples tested from these 'flocks'. Chlamydia antibodies were not detected in one of the two negative flocks, in two other flocks where the cause of abortion was undetermined and in three flocks in which the causes of abortion were diagnosed as Listeriosis and/or Salmonellosis. One of the 'negative flocks' yielded two positive reactors by CFT and five by dspELISA, suggesting infection with a cross-reactive subtype of C. pecorum. Of the five 'flocks' in which a definitive diagnosis from pathological material was not possible, four were positive by both serological tests, suggesting that the abortions were due to Chlamydia. The fifth flock, though negative by dspELISA and marginally positive in two samples by CFT, had experienced confirmed chlamydial abortions in previous lambing seasons, but culling and tetracycline treatment have prevented further abortions in the study period. Overall, the proportions of samples positive by CFT and dspELISA were similar (9.1% and 8.8%). These studies confirmed the value of the dspELISA as a screening test for chlamydial abortion. Furthermore, the dspELISA compared to the CFT is easier to perform, does not require reagent titration at each testing and uses automated assessment of results.