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Background: Vesiculobullous disorders (VBD) are heterogeneous group of dermatoses in which the primary lesion is a vesicle or a bulla. These disorders can be extremely debilitating and even fatal. Histopathological evaluation of skin punch biopsy is the gold standard, widely available and cost effective test for the diagnosis of VBD. Aims and objectives: To evaluate the clinico-demographic profiles of patients with vesiculobullous disorders of skin and to study their characteristic histopathological findings. Materials and methods: This is a descriptive study conducted in Department of pathology Government Siddhartha medical college, Vijayawada, Andhra Pradesh, India for 3 years period between March 2021 - February 2024. All the cases presenting with vesiculobullous lesions in the OPD and inpatient department during the study period were included after recording detailed history. Paraffin embedded, Haematoxylin & eosin stained tissue sections of skin punch biopsy were evaluated under light microscope. Results: During the study period a total of 745 skin punch biopsy specimens were received out of which 49 were vesiculobullous lesions which constituted 6.6% of skin biopsy specimens. Bullous pemphigoid (BP) (14 cases; 28.6%) was most frequently diagnosed VBD with male preponderance. Concordance between clinical and histopathological diagnosis was observed in 44 cases (89%). Conclusion: Histopathological evaluation of skin punch biopsy is a cost effective, widely available and reliable diagnostic modality in diagnosis of VBD of skin. Present study showed 89% concordance between clinical and histopathological diagnosis. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Our aim was to investigate structural changes of cutaneous Schwann cells (SCs), including nociceptive Schwann cells (nSCs) and axons, in individuals with diabetic polyneuropathy. We also aimed to investigate the relationship between these changes and peripheral neuropathic symptoms in type 1 diabetes. Methods: Skin biopsies (3 mm) taken from carefully phenotyped participants with type 1 diabetes without polyneuropathy (T1D, n=25), type 1 diabetes with painless diabetic polyneuropathy (T1DPN, n=30) and type 1 diabetes with painful diabetic polyneuropathy (P-T1DPN, n=27), and from healthy control individuals (n=25) were immunostained with relevant antibodies to visualise SCs and nerve fibres. Stereological methods were used to quantify the expression of cutaneous SCs and nerve fibres. Results: There was a difference in the number density of nSCs not abutting to nerve fibres between the groups (p=0.004) but not in the number density of nSCs abutting to nerve fibres, nor in solitary or total subepidermal SC soma number density. The overall dermal SC expression (measured by dermal SC area fraction and subepidermal SC process density) and peripheral nerve fibre expression (measured by intraepidermal nerve fibre density, dermal nerve fibre area fraction and subepidermal nerve fibre density) differed between the groups (all p<0.05): significant differences were seen in participants with T1DPN and P-T1DPN compared with those without diabetic polyneuropathy (healthy control and T1D groups) (all p<0.05). No difference was found between participants in the T1DPN and P-T1DPN group, nor between participants in the T1D and healthy control group (all p>0.05). Correlational analysis showed that cutaneous SC processes and nerve fibres were highly associated, and they were weakly negatively correlated with different neuropathy measures. Conclusions/interpretation: Cutaneous SC processes and nerves, but not SC soma, are degenerated and interdependent in individuals with diabetic polyneuropathy. However, an increase in structurally damaged nSCs was seen in individuals with diabetic polyneuropathy. Furthermore, dermal SC processes and nerve fibres correlate weakly with clinical measures of neuropathy and may play a partial role in the pathophysiology of diabetic polyneuropathy in type 1 diabetes. [ABSTRACT FROM AUTHOR]

Simple Summary: It is paramount to preserve the genetic material of endangered wildlife species for future generations, including that of the African savanna elephant (Loxodonta africana). By analyzing their genetic information, it is possible to decipher how endangered species such as elephants adapted to live on our planet and how they are related to one another and to their extinct ancestors. This information can further assist us in the protection of endangered animals and the habitats they live in. One way of doing this is through the cryopreservation of elephants' primary cell lines. This protocol presents the successful establishment of primary dermal fibroblast cell lines using a simple explant outgrowth method, starting from a small punch biopsy sample, which is minimally invasive and allows sample collection of free-roaming elephants. The average explant outgrowth, doubling time and further characterization of the dermal fibroblast cells of different elephants have been evaluated and compared. This preliminary method illustrates the potential to establish cell lines from living elephants, which contributes to the conservation of their genetic material and allows further research on the cancer suppressing ability of elephants. Following population declines of the African savanna elephant (Loxodonta africana) across the African continent, the establishment of primary cell lines of endangered wildlife species is paramount for the preservation of their genetic resources. In addition, it allows molecular and functional studies on the cancer suppression mechanisms of elephants, which have previously been linked to a redundancy of tumor suppressor gene TP53. This methodology describes the establishment of primary elephant dermal fibroblast (EDF) cell lines from skin punch biopsy samples (diameter: ±4 mm) of African savanna elephants (n = 4, 14–35 years). The applied tissue collection technique is minimally invasive and paves the way for future remote biopsy darting. On average, the first explant outgrowth was observed after 15.75 ± 6.30 days. The average doubling time (Td) was 93.02 ± 16.94 h and 52.39 ± 0.46 h at passage 1 and 4, respectively. Metaphase spreads confirmed the diploid number of 56 chromosomes. The successful establishment of EDF cell lines allows for future elephant cell characterization studies and for research on the cancer resistance mechanisms of elephants, which can be harnessed for human cancer prevention and treatment and contributes to the conservation of their genetic material. [ABSTRACT FROM AUTHOR]

Development of pharmacological interventions for wound treatment is challenging due to both poorly understood wound healing mechanisms and heterogeneous patient populations. A standardized and well‐characterized wound healing model in healthy volunteers is needed to aid in‐depth pharmacodynamic and efficacy assessments of novel compounds. The current study aims to objectively and comprehensively characterize skin punch biopsy‐induced wounds in healthy volunteers with an integrated, multimodal test battery. Eighteen (18) healthy male and female volunteers received three biopsies on the lower back, which were left to heal without intervention. The wound healing process was characterized using a battery of multimodal, non‐invasive methods as well as histology and qPCR analysis in re‐excised skin punch biopsies. Biophysical and clinical imaging read‐outs returned to baseline values in 28 days. Optical coherence tomography detected cutaneous differences throughout the wound healing progression. qPCR analysis showed involvement of proteins, quantified as mRNA fold increase, in one or more healing phases. All modalities used in the study were able to detect differences over time. Using multidimensional data visualization, we were able to create a distinction between wound healing phases. Clinical and histopathological scoring were concordant with non‐invasive imaging read‐outs. This well‐characterized wound healing model in healthy volunteers will be a valuable tool for the standardized testing of novel wound healing treatments. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Schwann cell clusters have been described at the murine dermis‐epidermis border. We quantified dermal Schwann cells in the skin of patients with small‐fiber neuropathy (SFN) compared with healthy controls to correlate with the clinical phenotype. Methods: Skin punch biopsies from the lower legs of 28 patients with SFN (11 men, 17 women; median age, 54 [range, 19‐73] years) and 9 healthy controls (five men, four women, median age, 34 [range, 25‐69] years) were immunoreacted for S100 calcium‐binding protein B as a Schwann cell marker, protein‐gene product 9.5 as a pan‐neuronal marker, and CD207 as a Langerhans cell marker. Intraepidermal nerve fiber density (IENFD) and subepidermal Schwann cell counts were determined. Results: Skin samples of patients with SFN showed lower IENFD (P <.05), fewer Schwann cells per millimeter (P <.01), and fewer Schwann cell clusters per millimeter (P <.05) than controls. When comparing SFN patients with reduced (n = 13; median age, 53 [range, 19‐73] years) and normal distal (n = 15, median age, 54 [range, 43‐68] years) IENFD, the number of solitary Schwann cells per millimeter (p <.01) and subepidermal nerve fibers associated with Schwann cell branches (P <.05) were lower in patients with reduced IENFD. All three parameters correlated positively with distal IENFD (P <.05 to P <.01), whereas no correlation was found between Schwann cell counts and clinical pain characteristics. Discussion Our data raise questions about the mechanisms underlying the interdependence of dermal Schwann cells and skin innervation in SFN. The temporal course and functional impact of Schwann cell presence and kinetics need further investigation. [ABSTRACT FROM AUTHOR]

Fueled by HIV, sub-Saharan Africa has the highest incidence of Kaposi's sarcoma (KS) in the world. Despite this, KS diagnosis in the region is based mostly on clinical grounds. Where biopsy is available, it has traditionally been excisional and performed by surgeons, resulting in multiple appointments, follow-up visits for suture removal, and substantial costs. We hypothesized that a simpler approach - skin punch biopsy - would make histologic diagnosis more accessible. To address this, we provided training and equipment for skin punch biopsy of suspected KS to three HIV clinics in East Africa. The procedure consisted of local anesthesia followed by a disposable cylindrical punch blade to obtain specimens. Hemostasis is facilitated by Gelfoam®. Patients removed the dressing after 4 days. From 2007 to 2013, 2,799 biopsies were performed. Although originally targeted to be used by physicians, biopsies were performed predominantly by nurses (62%), followed by physicians (15%), clinical officers (12%) and technicians (11%). There were no reports of recurrent bleeding or infection. After minimal training and provision of inexpensive equipment (USD 3.06 per biopsy), HIV clinics in East Africa can integrate same-day skin punch biopsy for suspected KS. Task shifting from physician to non-physician greatly increases access. Skin punch biopsy should be part of any HIV clinic's essential procedures. This example of task shifting may also be applicable to the diagnosis of other cancers (e.g., breast) in resource-limited settings.

Background and aims: Small fiber neuropathy (SFN) is increasingly suspected in patients with pain of uncertain origin, and making the diagnosis remains a challenge lacking a diagnostic gold standard. Methods: In this case–control study, we prospectively recruited 86 patients with a medical history and clinical phenotype suggestive of SFN. Patients underwent neurological examination, quantitative sensory testing (QST), and distal and proximal skin punch biopsy, and were tested for pain-associated gene loci. Fifty-five of these patients additionally underwent pain-related evoked potentials (PREP), corneal confocal microscopy (CCM), and a quantitative sudomotor axon reflex test (QSART). Results: Abnormal distal intraepidermal nerve fiber density (IENFD) (60/86, 70%) and neurological examination (53/86, 62%) most frequently reflected small fiber disease. Adding CCM and/or PREP further increased the number of patients with small fiber impairment to 47/55 (85%). Genetic testing revealed potentially pathogenic gene variants in 14/86 (16%) index patients. QST, QSART, and proximal IENFD were of lower impact. Conclusion: We propose to diagnose SFN primarily based on the results of neurological examination and distal IENFD, with more detailed phenotyping in specialized centers. [ABSTRACT FROM AUTHOR]

Objectives: To assess potential correlations between intraepidermal nerve fiber densities (IENFD), graded with light microscopy, and clinical measures of peripheral neuropathy in elderly male subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes (T2DM), respectively. Materials and methods: IENFD was assessed in thin sections of skin biopsies from distal leg in 86 men (71‐77 years); 24 NGT, 15 IGT, and 47 T2DM. Biopsies were immunohistochemically stained for protein gene product (PGP) 9.5, and intraepidermal nerve fibers (IENF) were quantified manually by light microscopy. IENFD was compared between groups with different glucose tolerance and related to neurophysiological tests, including nerve conduction study (NCS; sural and peroneal nerve), quantitative sensory testing (QST), and clinical examination (Total Neuropathy Score; Neuropathy Symptom Score and Neuropathy Disability Score). Results: Absent IENF was seen in subjects with T2DM (n = 10; 21%) and IGT (n = 1; 7%) but not in NGT. IENFD correlated weakly negatively with HbA1c (r = −.268, P =.013) and Total Neuropathy Score (r = −.219, P =.042). Positive correlations were found between IENFD and sural nerve amplitude (r =.371, P =.001) as well as conduction velocity of both the sural (r =.241, P =.029) and peroneal nerve (r =.258, P =.018). Proportions of abnormal sural nerve amplitude became significantly higher with decreasing IENFD. No correlation was found with QST. Inter‐rater reliability of IENFD assessment was good (ICC = 0.887). Conclusions: Signs of neuropathy are becoming more prevalent with decreasing IENFD. IENFD can be meaningfully evaluated in thin histopathological sections using the presented technique to detect neuropathy. [ABSTRACT FROM AUTHOR]

Neuropathische Schmerzen sind häufig und schränken die Lebensqualität betroffener Patienten erheblich ein. Die Diagnostik basiert in erster Linie auf der ausführlichen Schmerzanamnese und der klinisch-neurologischen Untersuchung zur Ortung der zugrunde liegenden Störung. Verfahren, mit denen (neuropathische) Schmerzen abgebildet oder gemessen werden können gibt es nicht, allerdings wurden insbesondere zur Untersuchung der schmerzleitenden kleinkalibrigen Nervenfasern zahlreiche Methoden etabliert, die bei der differenzialdiagnostischen Abklärung eingesetzt werden können (s. Tabelle). Neben der Anwendung von speziellen Fragebögen, psychophysischen Tests und morphologischen Untersuchungen können neurophysiologische Verfahren wie evozierte Potenziale und Microneurographie zum Einsatz kommen. Im vorliegenden Artikel werden diese Verfahren vorgestellt und in den Kontext der Diagnostik bei neuropathischen Schmerzen gestellt. Neuropathic pain is frequent and leads to relevant impairment of health related quality of life. Diagnostics are primarily based on a detailed pain history and clinical neurological examination to locate the underlying damage. There are no methods to image or measure (neuropathic) pain. However, several novel techniques have been established to investigate small caliber nerve fibers conducting pain. Besides using special pain questionnaires for neuropathic pain, psychophysic tests, and morphological examinations, neurophysiological tests such as evoked potentials and microneurography may be used. These tests are characterized in this article and their impact on the diagnostics in neuropathic pain is discussed. [ABSTRACT FROM AUTHOR]

Objective To demonstrate the prevalence of small fiber polyneuropathy (SFPN) in patients with refractory chronic pelvic pain (CPP). Design Retrospective study of prospective database. Subjects Participants were complex CPP patients recruited from subspecity referral clinics defined as those who were refractory to initial treatment and/or exhibited comorbid pain syndromes at initial presentation. Methods Comprehensive treatment history for CPP was obtained, and participants referred as above; 3-mm punch biopsies were obtained of the lower extremity and sent to diagnostic reference labs to evaluate for SFPN. The reported lab sensitivity and specificity for SFPN are 78–92% and 65–90%, respectively. Results Twenty-five of 39 patients (64%) were positive for SFPN. Comorbid conditions noted in our population included gastroesophageal reflux disease (46%), migraine (38%), irritable bowel syndrome (33%), lower back pain (33%), fibromyalgia (38%), endometriosis (15%), interstitial cystitis (18%), vulvodynia (5%), and other chronic pain syndromes (36%). Conclusions The prevalence of SFPN in our specialty referral patients with complex CPP is remarkably high vs published general population prevalence data (53/100,000). Identification of SFPN in this complex population shifts the focus from undefined syndromes to symptom complexes with linked potentially treatable mechanisms (e.g. SFPN, central sensitization). Most CPP patients with SFPN are undiagnosed. Considering the diagnosis may expand treatment options beyond conventional or so-called adjuvant analgesics. Treatment may expand to therapies such as IV lidocaine, IVIG, or other immunomodulatory options. In addition, the value to the patient of receiving a diagnosis for a multisystem or refractory pain syndrome, often attributed to negative psychologic factors, cannot be underestimated. Identifying SFPN should be contemplated in CPP patients who present with multisystem pain or who have not responded to initial evaluation and management. [ABSTRACT FROM AUTHOR]

Objective To detect small fiber neuropathy in patients with possible or established diabetic peripheral neuropathy (DPN) by skin punch biopsy and explore clinical characteristics and pathological features in different DPN patients. Methods A total of 37 patients were collected with possible or established DPN from December 2015 to February 2018. All patients underwent skin punch biopsy, intra epidermal nerve fiber density (IENFD) calculation, nerve conduction studies and clinical examination of neurological signs and symptoms. Fifteen healthy subjects were set as control. Chi⁃square test was used to analyze the impact of biopsy on DPN diagnosis rate. Student t test and Wilcoxon rank sum test were utilized to compare the difference of clinical characteristic and pathological feature between two subtypes of DPN patients. Results The average IENFD in control was (13.4±0.8) fiber/mm in calf and (21.1±5.9) fiber/mm in thigh while the diagnostic cutoff point of DPN was 13.0 fiber/mm in calf and 11.4 fiber/mm in thigh, respectively. Among total 37 patients, 35 were diagnosed as small fiber neuropathy by using skin punch biopsy and the proportion of DPN raised from 73% (27/37) to 95% (35/37) (P<0.05), which included 8 patients with primarily small fiber neuropathy and 27 patients with mixed small and large fiber neuropathy. Patients with primarily small fiber neuropathy had lower level of glycated hemoglobin A1c, lower proportion of diabetic retinopathy, nephropathy and cardiovascular autonomic neuropathy and higher IENFD in thigh [(8.8±2.6) vs (4.5±1.8) fiber/mm, t=5.424, P<0.01] than patients with mixed small and large fiber neuropathy. IENFD in calf was not significantly different between groups [(1.5±1.2) vs (2.5±1.3) fiber/mm, t= －1.965, P>0.05]. Conclusion Skin punch biopsy can significantly improve DPN diagnosis rate and is an important tool for identifying diabetic small fiber neuropathy. [ABSTRACT FROM AUTHOR]

We assessed pain characteristics and sensory profiles of a large and extensively phenotyped cohort of patients with polyneuropathies (PNPs) and small fiber neuropathy (SFN) using quantitative sensory testing (QST). Our aim was to detect potentially discriminative QST profiles of patient subgroups determined by pain, etiology, or skin innervation. We prospectively recruited 350 patients with painful and painless PNPs and with SFN at 1 neuromuscular center. After neurological work-up, patients underwent QST at the dorsal foot and 5-mm skin punch biopsy at the lower leg and upper thigh for intraepidermal nerve fiber counts. A healthy control group of 273 volunteers was investigated accordingly. Pain was present in 50% of the patients with PNP with a median intensity of 6/10 on a numeric rating scale, and, by definition, in all patients with SFN, with a median intensity of 5/10 numeric rating scale. Axonal PNP was painful more often than demyelinating PNP (P < 0.01). Patients with PNP mostly had loss of function profiles, whereas most patients with SFN belonged to the gain of function (hyperalgesia) phenotype. In healthy controls, skin innervation positively correlated with sensory thresholds, whereas this correlation was lost in patients with PNP and SFN. Quantitative sensory testing did not distinguish between painful and painless neuropathies regarding small fiber function, but revealed higher mechanical pain (P < 0.01) and detection thresholds (P < 0.05) and lower mechanical pain sensitivity in the group of patients with painful neuropathies. Etiological neuropathy subgroups were not distinguished by QST. [ABSTRACT FROM AUTHOR]

Valid and reliable biomarkers can play an important role in clinical trials as indicators of biological or pathogenic processes or as a signal of treatment response. Currently, there are no biomarkers for pain qualified by the U.S. Food and Drug Administration or the European Medicines Agency for use in clinical trials. This article summarizes an Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials meeting in which 3 potential biomarkers were discussed for use in the development of analgesic treatments: 1) sensory testing, 2) skin punch biopsy, and 3) brain imaging. The empirical evidence supporting the use of these tests is described within the context of the 4 categories of biomarkers: 1) diagnostic, 2) prognostic, 3) predictive, and 4) pharmacodynamic. Although sensory testing, skin punch biopsy, and brain imaging are promising tools for pain in clinical trials, additional evidence is needed to further support and standardize these tests for use as biomarkers in pain clinical trials.Perspective: The applicability of sensory testing, skin biopsy, and brain imaging as diagnostic, prognostic, predictive, and pharmacodynamic biomarkers for use in analgesic treatment trials is considered. Evidence in support of their use and outlining problems is presented, as well as a call for further standardization and demonstrations of validity and reliability. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study was to determine the diagnostic usefulness of skin punch biopsies with emphasis on visualization and quantification of T-cells and macrophages in patients with polyneuropathies.Methods: We quantified inflammatory cells in skin samples (lower leg, upper thigh) in 187 patients and compared data with counts in their sural nerve biopsies and with skin biopsies from 32 healthy volunteers.Results: Vessel-bound T-cells and macrophages were increased in proximal and distal skin samples of neuropathy patients compared with controls (P < 0.001 in both). Patients with vasculitic neuropathy had increased T-cell and macrophage counts in distal skin compared with controls (P < 0.01; for scattered macrophages/mm2 diagnostic sensitivity 71% and specificity 79%). In patients with vasculitic neuropathy, distal skin perivascular inflammatory cell counts also correlated with those in sural nerve biopsies (P < 0.001).Conclusion: Neuropathy per se may lead to skin inflammation. In cases of possible vasculitic neuropathy, skin biopsy may be an additional tool to support the diagnosis. Muscle Nerve 55: 884-893, 2017. [ABSTRACT FROM AUTHOR]

Background: The oncologic outcomes of patients diagnosed with inflammatory breast cancer (IBC) based on clinical exam only versus those with dermal lymphatic invasion on skin punch biopsy may be different and are worth further investigation., Methods: Patients diagnosed from 2006 to 2021 with IBC at our institution were grouped according to clinical diagnosis or skin biopsy performed. Oncologic and survival outcomes among groups were compared., Results: A total of 72 IBC patients were identified and grouped into 3 categories based on method of diagnosis: skin biopsy positive (n = 24), skin biopsy negative (n = 10) and no biopsy performed (n = 38). Skin biopsy positive patients had a higher incidence of lymphovascular invasion identified on final pathology and were more likely to experience a chest wall recurrence. At 5.1 yrs of follow-up, 40% of patients experienced recurrence, with 61% overall survival., Conclusion: Clinical diagnosis remains diagnostic for IBC, but skin punch biopsy allows for improved oncologic insight., Competing Interests: Declaration of competing interest Valente, DO, ElSherif, MD, Tran, DO, Tu, MS, Kruse, MD have no conflicts of interest related to this manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Growth associated protein-43 (GAP-43) is one of the neural proteins associated with nerve injury that is upregulated after nerve injury. To investigate whether GAP-43 quantification in skin biopsies would differentiate subtypes of peripheral neuropathies, we analyzed GAP-43 expression in skin from the lateral thigh and the distal leg. We prospectively enrolled 130 patients with peripheral neuropathies and compared data with healthy controls. Intraepidermal nerve fiber density (IENFD) was determined using antibodies against protein gene product 9.5 (PGP 9.5); anti-GAP-43 antibodies were applied to visualize regenerating nerve fibers. PGP 9.5 and GAP-43 gene expression was analyzed using qRT-PCR. Patients with neuropathies had a generalized reduction of IENFD and GAP-43 immunoreactive fibers compared to controls (p < 0.01). In contrast, cutaneous GAP-43 gene expression was increased in proximal skin in patients (p < 0.05), particularly when disease duration was short (< 3 years; p < 0.01). While fiber density for both markers decreased with age in healthy skin (p < 0.01), age-dependent reduction of skin innervation was absent in neuropathies. Diagnostic subgroups and neuropathic pain had no influence on skin innervation. We conclude that peripheral neuropathies lead to an initial increase in GAP-43 gene expression as a potential mechanism of regeneration, which is not sustained in neuropathies of long duration. [ABSTRACT FROM AUTHOR]

Fibromyalgia is a clinical syndrome that currently does not have any specific pathological finding to aid in diagnosis. Therefore, fibromyalgia is most likely a heterogeneous group of diseases with similar symptoms. Identifying and understanding the pathological basis of fibromyalgia will allow physicians to better categorize patients, increasing prospective treatment options, and improving potential therapeutic endeavors. Recent work has demonstrated that approximately 50 % of patients diagnosed with fibromyalgia have damage to their small unmyelinated nerve fibers. A skin punch biopsy is a sensitive and specific diagnostic test for this damage as a reduction in nerve fiber density allows for the diagnosis of small fiber neuropathy. Small fiber neuropathy is a disease with symptoms similar to fibromyalgia, but it often has a definable etiology. Identifying small fiber neuropathy and its underlying cause in fibromyalgia patients provides them with a succinct diagnosis, increases treatment options, and facilitates more specific studies for future therapeutics. [ABSTRACT FROM AUTHOR]

We describe the development of a sub-millimetre skin punch biopsy device for minimally invasive and suture-free skin sampling for molecular diagnosis and research. Conventional skin punch biopsies range from 2-4 mm in diameter. Local anaesthesia is required and sutures are usually used to close the wound. Our microbiopsy is 0.50 mm wide and 0.20 mm thick. The microbiopsy device is fabricated from three stacked medical grade stainless steel plates tapered to a point and contains a chamber within the centre plate to collect the skin sample. We observed that the application of this device resulted in a 0.21 ± 0.04 mm wide puncture site in volunteer skin using reflectance confocal microscopy. Histological sections from microbiopsied skin revealed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites. Longitudinal observation in microbiopsied volunteers showed that the wound closed within 1 day and was not visible after 7 days. Reflectance confocal microscope images from these same sites showed the formation of a tiny crust that resolved by 3 weeks and was completely undetectable by the naked eye. The design parameters of the device were optimised for molecular analysis using sampled DNA mass as the primary end point in volunteer studies. Finally, total RNA was characterized. The optimised device extracted 5.9 ± 3.4 ng DNA and 9.0 ± 10.1 ng RNA. We foresee that minimally invasive molecular sampling will play an increasingly significant role in diagnostic dermatology and skin research.

We describe the development of a sub-millimetre skin punch biopsy device for painless and suture-free skin sampling for molecular diagnosis and research. Conventional skin punch biopsies range from 2-4 mm in diameter. Local anaesthesia is required and sutures are usually used to close the wound. Our microbiopsy is 0.50 mm wide and 0.20 mm thick. The microbiopsy device is fabricated from three stacked medical grade stainless steel plates tapered to a point and contains a chamber within the centre plate to collect the skin sample. We observed that the application of this device resulted in a 0.21 ± 0.04 mm wide puncture site in volunteer skin using reflectance confocal microscopy. Histological sections from microbiopsied skin revealed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites. Longitudinal observation in microbiopsied volunteers showed that the wound closed within 1 day and was not visible after 7 days. Reflectance confocal microscope images from these same sites showed the formation of a tiny crust that resolved by 3 weeks and was completely undetectable by the naked eye. The design parameters of the device were optimised for molecular analysis using sampled DNA mass as the primary end point in volunteer studies. Finally, total RNA was characterized. The optimised device extracted 5.9 ± 3.4 ng DNA and 9.0 ± 10.1 ng RNA. We foresee that minimally invasive molecular sampling will play an increasingly significant role in diagnostic dermatology and skin research.

This study aimed at evaluating the performance of a battery of morphological and functional tests for the assessment of small nerve fiber loss in asymptomatic diabetic neuropathy (DNP). Patients diagnosed for ≥10 years with type 1 ( n = 10) or type 2 ( n = 13) diabetes mellitus (DM) without conventional symptoms or signs of DNP were recruited and compared with healthy controls ( n = 18) and patients with overt DNP ( n = 5). Intraepidermal nerve fiber density (IENFd) was measured with PGP9.5 immunostaining on punch skin biopsies performed at the distal leg. Functional tests consisted of quantitative sensory testing (QST) for light-touch, cool, warm and heat pain detection thresholds and brain-evoked potentials with electrical (SEPs) and CO laser stimulation [laser-evoked potentials (LEPs)] of hand dorsum and distal leg using small (0.8 mm) and large (20 mm) beam sizes. Results confirmed a state of asymptomatic DNP in DM, but only at the distal leg. Defining a critical small fiber loss as a reduction of IENFd ≤−2 z scores of healthy controls, this state prevailed in type 2 (30%) over type 1 DM (10%) patients despite similar disease duration and current glycemic control. LEPs with the small laser beam performed best in terms of sensitivity (91%), specificity (83%) and area-under-the ROC curve (0.924). Although this performance was not statically different from that of warm and cold detection threshold, LEPs offer an advantage over QST given that they bypass the subjective report and are therefore unbiased by perceptual factors. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is an X-linked lysosomal storage disorder that leads to cellular globotriaosylceramide (Gb3) accumulation due to mutations in the gene encoding α-galactosidase A. Trigger-induced acral burning pain is an early FD symptom of unknown pathophysiology. We aimed at investigating the potential role of skin fibroblasts in nociceptor sensitization. We enrolled 40 adult FD patients and ten healthy controls, who underwent a 6-mm skin punch biopsy at the lower leg. Dermal fibroblasts were cultivated and analyzed for Gb3 load. Fibroblast electrical activity was assessed using patch-clamp analysis at baseline and upon incubation with agalsidase-α for 24 h. We investigated gene expression of C C motif chemokine ligand 2 (CCL2), Ca2+activated K+-channel 1.1 (K Ca 1.1), interferone-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and transmembrane receptor notch homolog 1 (Notch1) using quantitative real-time-PCR, and protein levels of K Ca 1.1 by ELISA. Gene expression was determined at baseline and after fibroblast stimulation with tumor necrosis factor-α (TNF), modeling inflammation as a common pain trigger in FD. Total Gb3 load was higher in FD fibroblasts than in control fibroblasts (p <.01). Upon increase of intracellular Ca2+ concentrations, we detected differential electrical activity of K Ca 1.1 in fibroblasts obtained from patients with FD. Gene expression (p <.05) and protein levels of K Ca 1.1 (p <.05) were higher in fibroblasts from FD patients compared to control fibroblasts, whereas electric channel activity was lower in FD fibroblasts. After incubation with agalsidase-α, we observed an over-proportionate increase of K Ca 1.1 activity in FD fibroblasts reaching 7-fold the currents of control cells (p <.01). Gene expression studies revealed higher mRNA levels of CCL2, INF-γ, and Notch1 in FD fibroblasts compared to controls at baseline and after TNF incubation (p <.05 each), while TGF-β1 was higher in FD fibroblasts only after incubation with TNF (p <.05). Gb3 deposition in skin fibroblasts may impair K Ca 1.1 activity and activate the Notch1 signaling pathway. The resulting increase in pro-inflammatory mediator expression may contribute to cutaneous nociceptor sensitization as a potential mechanism of FD-associated pain. • Gb3 load is elevated in Fabry dermal fibroblasts • Gb3 induces immediate reduction of K ca 1.1 channel activity • Notch1 pathway is activated in fibroblasts of male Fabry patients with pain • Cytokine gene expression is elevated in fibroblasts of male Fabry patients with pain [ABSTRACT FROM AUTHOR]