OPTIMIZATION OF IMMUNOHISTOCHEMICAL STAINING PROTOCOL USING PGP 9.5 FOR QUANTITATIVE EVALUATION OF INTRAEPIDERMAL NERVE FIBER DENSITY IN SKIN BIOPSIES
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Abstract
Introduction: Small Fiber Neuropathy (SFN) is a peripheral neuropathy characterized by damage to small myelinated (Aδ) and unmyelinated (C) nerve fibers. Conventional electrophysiological studies are unable to detect these lesions. Quantification of intraepidermal nerve fiber density (IENFD) through skin biopsy using the free-floating immunohistochemistry method with the PGP 9.5 marker has been proven to be a reliable diagnostic tool for SFN. However, a standardized protocol for this technique has not yet been established in Vietnam. This study aimed to develop and optimize an immunohistochemical staining protocol with PGP 9.5 for IENFD assessment in skin biopsy samples.
Methods: The study was conducted at the Department of Histology–Pathology, University of Medicine and Pharmacy at Ho Chi Minh City. Two 4-mm skin punch biopsies were taken from each patient with suspected SFN—one from the distal leg (10 cm above the lateral malleolus) and one from the proximal thigh (10 cm below the greater trochanter)—according to EFNS/PNS guidelines. Samples were fixed in 2% paraformaldehyde–lysine–periodate (PLP) at 4°C for 24 hours, rinsed in 0.1 M PBS (pH 7.4), cryoprotected with 30% sucrose, and longitudinally sectioned at 50 µm using a cryostat. Sections were immunostained with anti-PGP 9.5 antibody using the free-floating technique. Nerve fibers crossing the dermal–epidermal junction were manually counted under a light microscope at ×40 magnification. The IENFD was expressed as the number of fibers per millimeter of epidermal length (fibers/mm) and compared with reference values established by Umapathi et al. (2006).
Results: The study identified several factors influencing staining quality, including fixation duration, primary antibody concentration, incubation time, and antigen detection steps. When optimized, the PGP 9.5 staining produced sharp, high-contrast labeling that clearly visualized epidermal and dermal small nerve fibers. This optimized technique enabled accurate and reproducible quantification of IENFD, supporting its value in diagnosing and monitoring SFN.
Conclusion: A reliable and standardized PGP 9.5 immunohistochemical staining protocol was successfully established, improving the accuracy and reproducibility of intraepidermal nerve fiber density assessment in patients with suspected SFN. This protocol contributes to the standardization of neuropathological techniques in Vietnam and enhances both diagnostic practice and future research in neurology and pathology.
Keywords
Small fiber neuropathy, peripheral neuropathy, skin biopsy, PGP 9.5
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References
2. McArthur JC, Stocks EA, Hauer P, Cornblath DR, Griffin JW. Epidermal nerve fiber density: normative reference range and diagnostic efficiency. Archives of neurology. 1998 Dec 1;55(12):1513-20.
3. Lauria G, Cornblath DR, Johansson O, et al. EFNS/PNS Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. European journal of neurology. 2010 Jul;17(7):903-e49.
4. Devigili G, Tugnoli V, Penza P, et al. The diagnostic criteria for small fibre neuropathy: from symptoms to neuropathology. Brain. 2008 Jul 1;131(7):1912-25.
5. Gibbons CH, Illigens BMW, Wang N, et al. Quantification of sweat gland innervation: a clinical-pathologic correlation. Neurology 2009;72(17):1479-1486.
6. England JD, Gronseth GS, Franklin G, et al. Evaluation of distal symmetric polyneuropathy: the role of autonomic testing, nerve biopsy, and skin biopsy (an evidence-based review). Muscle Nerve 39: 106-115, 2009.
7. Hoitsma E, Reulen JP, de Baets M, et al. Small fiber neuropathy: a common and important clinical disorder. Journal of the neurological sciences. 2004 Dec 15;227(1):119-30.
8. Gøransson LG, Mellgren SI, Lindal S, et al. The effect of age and gender on epidermal nerve fiber density. Neurology 2004;62:774.
9. Goodman BP. Approach to the Evaluation of Small Fiber Peripheral Neuropathy and Disorders of Orthostatic Intolerance. Semin Neurol 2007;27:347-355.
10. Umapathi, T., Tan, W. L., Tan, N. C., & Chan, Y. H. (2006). Determinants of epidermal nerve fiber density in normal individuals. Muscle & Nerve: Official Journal of the American Association of Electrodiagnostic Medicine, 33(6), 742-746.