Semi-Automated and Manual Methods for Counting Cells Expressing P75 Receptor in Endometriotic Lesions

There are several limitations to assess immunohistochemical staining in the cells manually such as wide differences in the qualifications of researchers working with histological samples, infeasible to count every single cell, time consuming and experience needed. In order to bridge the gap between experienced and inexperienced researchers, save time, and optimize quantitative accuracy, automated and semi-automated methods for histologic tissue analyses have been introduced. These methods make the interpretation of protein expression in immunohistochemistry (IHC) more objective. One such tool is HistoQuest® software, a semi-automated cell analysis program (TissueGnostics GmbH, Austria) that can be used for counting the number of positive and negative-staining cells as well as for quantitatively assessing the relative staining intensity on a histological slide. The aim of this methodological study was to compare the manual method of IHC with a semi-automated method in evaluating endometriosis lesions with IHC stained with p75 receptor antibody. Methods: Two independent observers were selected, both are experienced gynecologists: Experienced and inexperienced observer. Each received a tutorial about Histoquest® software and then independently set the parameters/tools in Histoquest® prior to independent slide interpretation. The images were arranged using TissueFaxs Plus system (Tissue-Gnostics®, Vienna, Austria) software with the same settings including exposure time and signal amplifications. Quantitative immunohistochemistry analysis was performed by using Histoquest® immunohistochemistry analysis software (TissueGnostics®, Vienna, Austria). Manual counting was done for comparison. Results: Histoquest software was able to identify nuclei stained with hematoxylin perfectly if the nucleus had a ‘normal’ form which is not pale color in the center. If the nucleus appeared irregular with uneven color, the software failed to identify. The discrepancy between semi-automated cell counting and manual counting by intra-experienced observer was smaller than the discrepancy in intra- inexperienced observer in overall result (95% LoA -9.76 to 12.8 vs. 95% LoA -63.39 to 49.25). The two observers had very poor agreement. Conclusion: With IHC, cell counting using Histoquest software benefits the experienced observer/user since it gives highly consistent results compared to the manual method, with the additional benefits of being more objective and very much less time consuming. The software cannot be used by the inexperienced observer, as it cannot overcome that individual's lack of knowledge and produces erroneous results.