Immunohistochemical Stain for Small Fiber Neuropathy Diagnosis
Use anti-protein gene product 9.5 (PGP 9.5) antibodies for immunohistochemical staining of skin biopsy specimens when diagnosing small fiber neuropathy. 1
Standard Technique and Staining Protocol
PGP 9.5 immunohistochemistry is the validated gold standard stain that allows identification and quantification of intraepidermal nerve fibers (IENF) with excellent intra- and inter-observer reliability. 1, 2
Technical Specifications
Perform a 3 mm punch biopsy from the distal leg (typically 10 cm above the lateral malleolus). 1, 3
Fix tissue in 2% PLP (periodate-lysine-paraformaldehyde) or Zamboni's solution to preserve nerve fiber architecture. 3
Section at 50 μm thickness using a microtome for optimal visualization of the three-dimensional epidermal nerve fiber network. 1, 3
Stain with anti-PGP 9.5 monoclonal antibodies using either bright-field immunohistochemistry or immunofluorescence methods. 1, 3
Quantify linear IENF density in at least three sections per biopsy to ensure reproducibility. 3
Why PGP 9.5 Is the Preferred Stain
PGP 9.5 is a panaxonal marker (neuron-specific ubiquitin hydrolase) that reliably demonstrates both the dermal plexus of myelinated and unmyelinated fibers and the unmyelinated C-fibers that ascend vertically through the epidermis. 4 This stain has been specifically validated by the European Federation of Neurological Societies (EFNS) as the most sensitive and reliable method for IENF density determination. 1, 3
Diagnostic Performance
The sensitivity of decreased IENF density ranges from 45-90%, while specificity is very high at 95-97%. 1, 5 This means that finding reduced IENF density markedly increases the likelihood of polyneuropathy, though normal density does not completely rule it out. 1, 5
A cutoff of ≤8.8 fibers/mm at the ankle demonstrates good diagnostic accuracy for distinguishing small fiber neuropathy from normal controls. 2
Critical Pitfalls to Avoid
Never rely solely on nerve conduction studies or electromyography when evaluating for small fiber neuropathy—these tests only assess large myelinated fibers and will be normal in pure small fiber disease, leading to missed diagnoses. 2, 5 Small fiber damage often precedes large fiber involvement, particularly in diabetic neuropathy. 2
Do not dismiss patient symptoms when electrodiagnostic studies are normal—this is the expected finding in isolated small fiber neuropathy and should prompt skin biopsy rather than reassurance. 2
Additional Morphologic Features
Beyond IENF density, look for axonal swellings on PGP 9.5-stained sections, as these predict progression of polyneuropathy and reflect active axonal degeneration. 1, 5 However, the diagnostic accuracy of axonal swellings requires further validation. 1
Alternative and Adjunctive Methods
While PGP 9.5 remains the gold standard, confocal microscopy may be particularly useful for investigating dermal myelinated nerve fibers, dermal receptors, and dermal annex innervation. 3 Corneal confocal microscopy provides a non-invasive alternative for assessing corneal nerve fiber density. 2
Automated immunofluorescence staining with PGP 9.5 has been validated as an accurate and robust alternative to manual methods, with sensitivity of 80% and specificity of 64%, making it suitable for large-scale clinical studies. 6