(132) Histometric Measurement of CD117 and PGP9.5 Immunopositive Staining in Excised Vestibular Specimens from Patients with Neuroproliferative Vestibulodynia
Authors: Drian A , Yee A , Kim N , Goldstein A , Kady S , Goldstein S , Goldstein I 
Published: May 2023

Abstract

Introduction
In 2004, Bornstein reported using immunohistochemical (IHC) analysis of stained sections of vestibular specimens, defining diagnostic criteria for neuroproliferative vestibulodynia (NPV). Criteria included presence of 8 or more mast cells manually counted in a standardized microscopic field or finding total area of subepithelial nerve fibers was 10 times higher than controls. Replicating such criteria is difficult, in part, because of variability in manual counting, IHC staining and magnification. A more reproducible methodology may be estimating percentage of immunopositive stained area using computer-assisted histometry.

Objective
Utilizing both manual counting and computer-assisted histometry measurements using ImageJ at a standardized magnification in specimens from vestibulectomy and control tissue published in the literature.

Methods
Tissue samples were collected from 17 patients with NPV. Samples were stained with CD117, a marker of mast cells, and PGP9.5, a marker of nerve endings. Twenty-five photomicrographs were each obtained using 200x and 400x magnification and all were analyzed using ImageJ. Images were converted to 8-bit grayscale and threshold manually adjusted to measure immunohistochemically stained tissue with three measurements averaged. Averages and variability between measurements in each photomicrograph and from each patient were determined. This methodology was also applied to images of tissues from patients with and without NPV (Tympanidis 2003; Goetsch 2010). For comparison, the method of analysis by manually counting CD117 immunopositive cells, described by Bornstein et al, was applied to our tissue samples by an independent observer. Clusters of immunopositive granules positively stained and separated from an adjacent cell membrane were counted as a single immunopositive cell.

Results
Using Image J, tissues stained with CD117 averaged 2107 ± 1339 μm2 of immunostained area, representing 0.68 ± 0.38% (range = 0.11 – 1.15%) of total area analyzed. Tissues stained with PGP9.5 averaged 1123 ± 695 μm2 of immunostained area, representing 0.40 ± 0.27% (range = 0.34 – 1.81%) of total area analyzed. Standard deviations of repeated measurements for the same area within a tissue section ranged from 0.04 – 0.7% (mean = 0.17%) for CD117 immunostaining and 0.01 – 0.78% (mean = 0.10%) for PGP 9.5. Counting cells, CD117 stained tissue averaged 30.6 ± 15.3 (range = 12 – 72) immunopositive cells per standardized field (200x magnification). These are all greater than the 8 mast cells per standardized field reported by Bornstein. In PGP9.5-stained tissue, immunostained area was 2.6-fold greater than that reported by Bornstein for controls (425 μm2). Applying our methodology to photomicrographs from Goetsch and Tympanidis, 2.48% and 2.87% of the control areas were immunopositive. Tissues from control patients without vestibulodynia had 8-fold lower (0.31%, from Goetsch) and 6.4-fold lower (0.45%, from Tympanidis) immunostained area than tissues from vestibulodynia patients using computer-assisted histometry.

Conclusions
NPV is a diagnosis of exclusion without routine pathology confirmation. Our 17 patients had increased density of CD117-immunopositive cells and increased PGP9.5-immunopositive nerves, confirming their NPV diagnosis. To facilitate more consistent, widespread and rapid quantification of staining, we propose using computer-assisted histometry for future studies.

Disclosure
No