Accuracy of Assessment of Positive Cell Markers in Tissues Prepared With Immunohistochemistry and In Situ Hybridization Techniques

Abstract

The aim of the current study was to evaluate differences in the expression of phenotype and cytokine markers of lymphocytes in tissues prepared by either immunohistochemistry (IHC) or In Situ hybridization (ISH) techniques. Soft-tissue biopsies from inflamed gingival tissues were collected from 11 sites in 9 subjects with generalized or localized forms of severe periodontitis or periimplantitis, that is, inflammatory destructive disease in the tissues surrounding teeth and dental implants. The biopsies were snap frozen in liquid nitrogen and stored in −70°C. Fivemicrometer sections were cut in a cryostat and every second section was prepared for either IHC or ISH techniques. Monoclonal antibodies or biotin-labeled sense and antisense probes for CD3, CD4, CD8, CD19, interleukin-6, and interleukin- 10 were used. The histological quantitative assessments were performed using a Leitz DM-RBE microscope (Leica, Wetzlar, Germany) equipped with a Leica Q-500 MC® Image system. For the identification of positive cell markers, an interference contrast setting at a magnification of ×400 was applied. A point counting procedure was used to determine the percentage of positive cell markers within the immunochromatographic technology (ICT). A lattice comprising 400 light points was superimposed over the tissue area. Cross points that indicated positive cell markers in the compartment to be examined were counted and related to the total counts for the entire tissue area (%). To estimate the error of measurements for each of the two techniques, double assessments were performed with a two months interval. It was demonstrated that assessments of cell proportions using IHC were associated with larger variations and error of measurements than using ISH techniques. The IHC method resulted in general larger densities of phenotype markers and smaller fractions of cells positive for cytokine markers than ISH. It is suggested that the accuracy of the ISH technique is superior to that of IHC when used on comparable tissue samples (The J Histotechnol 30:167, 2007).

Submitted December 14, 2006; accepted with revisions July 17, 2007

Keywords:

• histology
• human
• IHC
• ISH
• monoclonal antibodies
• mRNA
• probe