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

Abstract

BackgroundIn 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.ResultsWe 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.ConclusionsThis 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.

Small‐fiber neuropathy: Expanding the clinical pain universe

“…amyotrophic lateral sclerosis, [44][45][46] critical illness, 47 and peripheral arterial disease. 48 New techniques to determine the IENFD with indirect immunofluorescence, 49 automated PGP9.5 immunofluorescence staining (laboratory developed test), 50 and 3D analysis 51 have been reported. One study investigated the global spatial sampling in order to determine the epidermal nerve fiber length density (ENFLD) taking into account its biologic complexity.…”

HIV-Associated Sensory Neuropathy

“…Immunohistochemical staining techniques recommended are bright-field immunohistochemistry and indirect immunofluorescence [28]. PGP9.5 immunofluorescence allows nerves to be visualized using a confocal microscope [29]. Smaller intraepidermal nerve fiber densities (IENFD) in HIV-SN patients correlated with the clinical and electrophysiological severity [30].…”

Innervation Changes Induced by Inflammation in the Murine Vagina

“…The polyclonal PGP9.5 antibody (Doran et al, 1983) labels a band at 26 -28 kD on Western blot (Krimm et al, 2006, Van Acker et al, 2016, and labels neurons and nerve fibres of peripheral and central nervous system and some neuroendocrine cells (Wilson et al, 1988). The monoclonal SP antibody (Millipore), produced using a hybridoma (Cuello et al, 1979) has shown specificity in radioimmunoassay using recombinant substance P or tissue extracts (Cuello et al, 1979).…”