Automated PGP9.5 immunofluorescence staining: a valuable tool in the assessment of small fiber neuropathy?

BACKGROUND: In this study we explored the possibility of automating the PGP9.5 immunofluorescence staining assay for the diagnosis of small fiber neuropathy using skin punch biopsies. The laboratory developed test (LDT) was subjected to a validation strategy as required by good laboratory practice guidelines and compared to the well-established gold standard method approved by the European Federation of Neurological Societies (EFNS). To facilitate automation, the use of thinner sections. (16 µm) was evaluated. Biopsies from previously published studies were used. The aim was to evaluate the diagnostic performance of the LDT compared to the gold standard. We focused on technical aspects to reach high-quality standardization of the PGP9.5 assay and finally evaluate its potential for use in large scale batch testing. RESULTS: We first studied linear nerve fiber densities in skin of healthy volunteers to establish reference ranges, and compared our LDT using the modifications to the EFNS counting rule to the gold standard in visualizing and quantifying the epidermal nerve fiber network. As the LDT requires the use of 16 µm tissue sections, a higher incidence of intra-epidermal nerve fiber fragments and a lower incidence of secondary branches were detected. Nevertheless, the LDT showed excellent concordance with the gold standard method. Next, the diagnostic performance and yield of the LDT were explored and challenged to the gold standard using skin punch biopsies of capsaicin treated subjects, and patients with diabetic polyneuropathy. The LDT reached good agreement with the gold standard in identifying small fiber neuropathy. The reduction of section thickness from 50 to 16 µm resulted in a significantly lower visualization of the three-dimensional epidermal nerve fiber network, as expected. However, the diagnostic performance of the LDT was adequate as characterized by a sensitivity and specificity of 80 and 64 %, respectively. CONCLUSIONS: This study, designed as a proof of principle, indicated that the LDT is an accurate, robust and automated assay, which adequately and reliably identifies patients presenting with small fiber neuropathy, and therefore has potential for use in large scale clinical studies

Automated single-trial assessment of laser-evoked potentials as an objective functional diagnostic tool for the nociceptive system

OBJECTIVE: To assess the clinical usefulness of an automated analysis of event-related potentials (ERPs). METHODS: Nociceptive laser-evoked potentials (LEPs) and non-nociceptive somatosensory electrically-evoked potentials (SEPs) were recorded in 37 patients with syringomyelia and 21 controls. LEP and SEP peak amplitudes and latencies were estimated using a single-trial automated approach based on time-frequency wavelet filtering and multiple linear regression, as well as a conventional approach based on visual inspection. RESULTS: The amplitudes and latencies of normal and abnormal LEP and SEP peaks were identified reliably using both approaches, with similar sensitivity and specificity. Because the automated approach provided an unbiased solution to account for average waveforms where no ERP could be identified visually, it revealed significant differences between patients and controls that were not revealed using the visual approach. CONCLUSION: The automated analysis of ERPs characterized reliably and objectively LEP and SEP waveforms in patients. SIGNIFICANCE: The automated single-trial analysis can be used to characterize normal and abnormal ERPs with a similar sensitivity and specificity as visual inspection. While this does not justify its use in a routine clinical setting, the technique could be useful to avoid observer-dependent biases in clinical research

Asymptomatic small fiber neuropathy in diabetes mellitus: investigations with intraepidermal nerve fiber density, quantitative sensory testing and laser-evoked potentials

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(2) laser stimulation [laser-evoked potentials (LEPs)] of hand dorsum and distal leg using small (0.8 mm(2)) and large (20 mm(2)) 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

The time course of CO(2) laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers

OBJECTIVE: To assess the temporal relationship between skin nerve denervation and regeneration (dermal and intra-epidermal fibres, IENF) and functional changes (CO(2) laser-evoked potentials, LEPs, and quantitative sensory tests, QST) after topical cutaneous application of capsaicin. METHODS: Capsaicin (0.075%) was applied to the lateral calf for three consecutive days. QST, LEPs and skin biopsies were performed at baseline and time intervals up to 54days post-capsaicin treatment. Biopsies were immunostained with antibodies for PGP9.5, TRPV1, and GAP-43 (marker of regenerating nerve fibres), and analyzed for IENFs and dermal innervation (for GAP-43). RESULTS: At 1day post-capsaicin, cutaneous thermal sensitivity was reduced, as were LEPs. PGP9.5, TRPV1, and GAP-43 immunoreactive-nerve fibres were almost completely absent. By Day 12, LEPs had fully recovered, but PGP9.5 and TRPV1 IENF continued to be significantly decreased 54days post-capsaicin. In contrast, dermal GAP-43 immunoreactivity closely matched recovery of LEPs. CONCLUSIONS: A good correlation was observed between LEPs and GAP-43 staining, in contrast to PGP9.5 and TRPV1. Laser stimulation is a non-invasive and sensitive method for assessing the initial IENF loss, and regenerating nerve fibres. SIGNIFICANCE: Assessing skin biopsies by PGP9.5 immunostaining alone may miss significant diagnostic and prognostic information regarding regenerating nerve fibres, if other approaches are neglected, e.g. LEPs or GAP-43 immunostaining

Reappraising neuropathic pain in humans—how symptoms help disclose mechanisms

Neuropathic pain--that is, pain arising directly from a lesion or disease that affects the somatosensory system--is a common clinical problem, and typically causes patients intense distress. Patients with neuropathic pain have sensory abnormalities on clinical examination and experience pain of diverse types, some spontaneous and others provoked. Spontaneous pain typically manifests as ongoing burning pain or paroxysmal electric shock-like sensations. Provoked pain includes pain induced by various stimuli or even gentle brushing (dynamic mechanical allodynia). Recent clinical and neurophysiological studies suggest that the various pain types arise through distinct pathophysiological mechanisms. Ongoing burning pain primarily reflects spontaneous hyperactivity in nociceptive-fibre pathways, originating from 'irritable' nociceptors, regenerating nerve sprouts or denervated central neurons. Paroxysmal sensations can be caused by several mechanisms; for example, electric shock-like sensations probably arise from high-frequency bursts generated in demyelinated non-nociceptive Aβ fibres. Most human and animal findings suggest that brush-evoked allodynia originates from Aβ fibres projecting onto previously sensitized nociceptive neurons in the dorsal horn, with additional contributions from plastic changes in the brainstem and thalamus. Here, we propose that the emerging mechanism-based approach to the study of neuropathic pain might aid the tailoring of therapy to the individual patient, and could be useful for drug development.Neuropathic pain - that is, pain arising directly from a lesion or disease that affects the somatosensory system - is a common clinical problem, and typically causes patients intense distress. Patients with neuropathic pain have sensory abnormalities on clinical examination and experience pain of diverse types, some spontaneous and others provoked. Spontaneous pain typically manifests as ongoing burning pain or paroxysmal electric shock-like sensations. Provoked pain includes pain induced by various stimuli or even gentle brushing (dynamic mechanical allodynia). Recent clinical and neurophysiological studies suggest that the various pain types arise through distinct pathophysiological mechanisms. Ongoing burning pain primarily reflects spontaneous hyperactivity in nociceptive-fibre pathways, originating from 'irritable' nociceptors, regenerating nerve sprouts or denervated central neurons. Paroxysmal sensations can be caused by several mechanisms; for example, electric shock-like sensations probably arise from high-frequency bursts generated in demyelinated non-nociceptive Aβ fibres. Most human and animal findings suggest that brush-evoked allodynia originates from Aβ fibres projecting onto previously sensitized nociceptive neurons in the dorsal horn, with additional contributions from plastic changes in the brainstem and thalamus. Here, we propose that the emerging mechanism-based approach to the study of neuropathic pain might aid the tailoring of therapy to the individual patient, and could be useful for drug development. © 2013 Macmillan Publishers Limited