(132) Histometric Measurement of CD117 and PGP9.5 Immunopositive Staining in Excised Vestibular Specimens from Patients with Neuroproliferative Vestibulodynia

Abstract Introduction In 2004, Bornstein reported using immunohistochemical (IHC) analysis of stained sections vestibular specimens, defining diagnostic criteria for neuroproliferative vestibulodynia (NPV). Criteria included presence 8 or more mast cells manually counted in a standardized microscopic field finding total area subepithelial nerve fibers was 10 times higher than controls. Replicating such is difficult, part, because variability manual counting, IHC staining and magnification. A reproducible methodology may be estimating percentage immunopositive computer-assisted histometry. Objective Utilizing both counting histometry measurements ImageJ at magnification specimens from vestibulectomy control tissue published the literature. Methods Tissue samples were collected 17 patients with NPV. Samples CD117, marker cells, PGP9.5, endings. Twenty-five photomicrographs each obtained 200x 400x all analyzed ImageJ. Images converted to 8-bit grayscale threshold adjusted measure immunohistochemically three averaged. Averages between photomicrograph patient determined. This also applied images tissues without NPV (Tympanidis 2003; Goetsch 2010). For comparison, method by CD117 described et al, our an independent observer. Clusters granules positively separated adjacent cell membrane as single cell. Results Using Image J, averaged 2107 ± 1339 μm2 immunostained area, representing 0.68 0.38% (range = 0.11 – 1.15%) analyzed. Tissues PGP9.5 1123 695 0.40 0.27% 0.34 1.81%) Standard deviations repeated same within section ranged 0.04 0.7% (mean 0.17%) immunostaining 0.01 0.78% 0.10%) PGP 9.5. Counting 30.6 15.3 12 72) per (200x magnification). These are greater Bornstein. PGP9.5-stained tissue, 2.6-fold that controls (425 μm2). Applying Tympanidis, 2.48% 2.87% areas immunopositive. had 8-fold lower (0.31%, Goetsch) 6.4-fold (0.45%, Tympanidis) Conclusions diagnosis exclusion routine pathology confirmation. Our increased density CD117-immunopositive PGP9.5-immunopositive nerves, confirming their diagnosis. To facilitate consistent, widespread rapid quantification staining, we propose future studies. Disclosure No