title: Automated PGP9.5 immunofluorescence staining: a valuable tool in the assessment of small fiber neuropathy?
abstract: 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.
authors.affiliation: Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. nathalie.vanacker@student.uantwerpen.be.|HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium. nathalie.vanacker@student.uantwerpen.be.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Van Acker
authors.affiliation: Institute of Neuroscience, Université Catholique de Louvain, Avenue Mounier 53, B1.53.04, 1200, Brussels, Belgium.
authors.forename: Michael
authors.initials: M
authors.lastname: Ragé
authors.affiliation: HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium.
authors.forename: Ellen
authors.initials: E
authors.lastname: Sluydts
authors.affiliation: HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium.
authors.forename: Michiel W M
authors.initials: MW
authors.lastname: Knaapen
authors.affiliation: HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium.
authors.forename: Martine
authors.initials: M
authors.lastname: De Bie
authors.affiliation: Janssen Research and Development, Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.|Reference Center for Biological Markers of Dementia (BIODEM), Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Timmers
authors.affiliation: StatUa Center for Statistics, University of Antwerp, Antwerp, Belgium.
authors.forename: Erik
authors.initials: E
authors.lastname: Fransen
authors.affiliation: HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium.
authors.forename: Carla
authors.initials: C
authors.lastname: Duymelinck
authors.affiliation: HistoGeneX NV, Pr J Charlottelaan 10, Berchem, 2600, Antwerp, Belgium.
authors.forename: Stefanie
authors.initials: S
authors.lastname: De Schepper
authors.affiliation: Peripheral Neuropathy Unit, Hammersmith Hospital, Du Cane Road, London, W12 0HS, UK.
authors.forename: Praveen
authors.initials: P
authors.lastname: Anand
authors.affiliation: Janssen Research and Development, Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.
authors.forename: Theo
authors.initials: T
authors.lastname: Meert
authors.affiliation: Institute of Neuroscience, Université Catholique de Louvain, Avenue Mounier 53, B1.53.04, 1200, Brussels, Belgium.
authors.forename: Léon
authors.initials: L
authors.lastname: Plaghki
authors.affiliation: Department of Neurology, Antwerp University Hospital, Born Bunge Institute, University of Antwerp, Wilrijkstraat 10, 2650, Edegem, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Cras
mesh_terms: D000328:Adult; D005455:Fluorescent Antibody Technique; D006801:Humans; D008875:Middle Aged; D015588:Observer Variation; D000071075:Small Fiber Neuropathy / Q000175:diagnosis* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*
chemical_list: C482860:UCHL1 protein, human; D043222:Ubiquitin Thiolesterase
keywords: Clinical; Diagnostic; Immunofluorescence; Skin biopsy; Small fiber neuropathy