Optimal z-axis scanning parameters for gynecologic cytology specimens

Abstract

Background:The use of virtual microscopy (VM) in clinical cytology has been limited due to the inability to focus through three dimensional (3D) cell clusters with a single focal plane (2D images). Limited information exists regarding the optimal scanning parameters for 3D scanning.Aims:The purpose of this study was to determine the optimal number of the focal plane levels and the optimal scanning interval to digitize gynecological (GYN) specimens prepared on SurePath™ glass slides while maintaining a manageable file size.Subjects and Methods:The iScanCoreo Au scanner (Ventana, AZ, USA) was used to digitize 192 SurePath™ glass slides at three focal plane levels at 1 μ interval. The digitized virtual images (VI) were annotated using BioImagene's Image Viewer. Five participants interpreted the VI and recorded the focal plane level at which they felt confident and later interpreted the corresponding glass slide specimens using light microscopy (LM). The participants completed a survey about their experiences. Inter-rater agreement and concordance between the VI and the glass slide specimens were evaluated.Results:This study determined an overall high intra-rater diagnostic concordance between glass and VI (89-97%), however, the inter-rater agreement for all cases was higher for LM (94%) compared with VM (82%). Survey results indicate participants found low grade dysplasia and koilocytes easy to diagnose using three focal plane levels, the image enhancement tool was useful and focusing through the cells helped with interpretation; however, the participants found VI with hyperchromatic crowded groups challenging to interpret. Participants reported they prefer using LM over VM. This study supports using three focal plane levels and 1 μ interval to expand the use of VM in GYN cytology.Conclusion:Future improvements in technology and appropriate training should make this format a more preferable and practical option in clinical cytology.

A method for quantitative analysis of clump thickness in cervical cytology slides

“…Another study reported that cervical specimens scanned with 21 focal planes at a 1.5µm interval achieved better diagnostic accuracy than those scanned with 5 focal planes at 1µm interval [8]. A more recent work argues that scanning only three focal planes (with 1µm interval) is able to achieve diagnostic performances close to that of a conventional glass slide [6]. However, a majority of pathologist participated in this work reported that focusing over cell clusters were not as good as that in conventional microscope, and they did not prefer to use the virtual microscopy for the future diagnosis.…”

Methods for rapid and high quality acquisition of whole slide images

“…The optimal Z interval should also be decided by the requirements for data storage space and the need of end users (e.g. the pathologists) in terms of diagnostic performance (Donnelly et al 2013). …”

“…More importantly computer programs can be developed to asisit diagnosis, in particularly the automated screening algorithms can be easily integrated into this digitised work-flow because the digital image of the specimen has already been created. Many previous studies had demonstrated that WSI based manual screening could give equivalent performance as that using the glass slide, but only if the glass slide has the adequate quality as discussed below (Donnelly et al 2013). …”

“…However, in BF based slide scanners for WSI, it is extremely slow and hence not practical to acquire images at spacing close to optical resolution limit (0.25µm), because hundreds of images are required for thick specimens like cytology specimens. The distance between the slices are normally set to be approximately equal to the DOF of the optics, which is around 1 micron for the experimental microscope system, because within this range the cellular objects are in clear and sharp (Donnelly et al 2013). The effects of increasing the distance between the 3D stack slices on the proposed correction method are investigated by down-sampling the stack along the Z-axis to be multiples of the DOF.…”

“…Another study reported that cervical specimens scanned with 21 focal planes at a 1.5µm interval achieved better diagnostic accuracy than those scanned with 5 focal planes at 1µm interval (Evered and Dudding 2011). A more recent work argues that scanning only three focal planes (with 1µm interval) is able to achieve diagnostic performances close to that of a conventional glass slide (Donnelly et al 2013). However, a majority of pathologist participated in this work reported that focusing over cell clusters were not as good as that in conventional microscope, and they did not prefer to use the virtual microscopy for the future diagnosis.…”