Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.This study was supported by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education, Youth and Sports of the Czech Republic and by the Charles University Research Fund (Progres Q39). LC, AT, VL, VM and MJ also received support from the Ministry of Education, Youth and Sports under the project FIND No. CZ.02.1.01/0.0/0.0/16_019/0000787 and from the European Regional Development Fund–Project 'Application of Modern Technologies in Medicine and Industry' (No. CZ.02.1.01 /0.0/0.0/17_048/0007280). JH was supported by the Ministry of Health of the Czech Republic (Grant no: 15–29241A) and by the grant SVV–2020- 2022 No. 260 536. MG was supported by the Charles University Grant Agency, project No. 1313420 and SVV 260 536. LC and VL received support from the Charles University grant UNCE/MED/006, and by the grant of Ministry of Health of the Czech Republic –AZV NU20J-08-00009. AT has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement Nº856620. The analysis demonstrated in example O represented a pilot study for the dissertation project of Violeta García-Espín, supervised by Prof. Monika Egerbacher and Prof. Gilles Dupré (all University of Veterinary Medicine Vienna, Austria). The help of Dr. Stephan Handschuh (University of Veterinary Medicine Vienna) with the whole slide image analysis is gratefully acknowledged