Evaluation and optimization for liquid-based preparation cytology in whole slide imaging

Abstract

Background: Cytology poses different obstacles in whole slide imaging compared to surgical pathology slides. A single focal plane suffices for most of the latter, but cytology slides are thicker, potentially requiring multiple focal planes for adequate diagnostic information. Multiple focal planes adversely impact scanning time per slide, evaluation times, and file sizes. In this pilot study, we evaluated and compared the multilayer stack method to the extended focus algorithm as an alternative which collapses multiple focal planes into a single image, retaining only focused areas from each plane.

Materials and methods: 10 SurePath(®) cervical cytology slides were scanned at three thickness settings: 18, 24, and 30 μm. Three scanners were used: (1) Hamamatsu Nanozoomer 2.0-HT, (2) 3DHISTECH Mirax scan, and (3) Bioimagene iScan Coreo Au. The Nanozoomer and iScan utilized multilayer stacking, while the Mirax files were composited by extended focus. Scan times and file sizes were recorded, and image quality compared.

Results: The Nanozoomer stacks averaged 1.58 gb and around 25 min for each slide, while the iScan stacks ranged from 6.23 to 9.3 gb and took 34-50 min to scan. The Mirax images averaged 210 mb and took 13-20 min to scan. Multilayer stack image quality from both Nanozoomer and iScan was fairly comparable. The iScan revealed significant mechanical issues that did not correspond to user settings. The Mirax images showed worrisome loss of crisp focus detail, worsening with increasing focal planes and impacting assessment of nuclear contours and chromatin detail.

Conclusions: The optimal number of focal planes remains unknown for cytology. Multilayer stacks require excessive scanning time, network bandwidth, and file storage. Extended focus was evaluated as an alternative, but significant image quality issues were revealed. Further large-scale studies are needed to assess their clinical impact.

Keywords: Cytology; cytopathology; digital; pathology; whole slide imaging.

Figure 1

Schematic illustrating differences between (a) surgical pathology slides which are usually 4-6 μm in thickness and (b) cytology slides (bottom diagram) which can range upwards of 30 μm from glass to coverslip. Cells can be positioned anywhere from glass to coverslip in cytology

Figure 2

Multilayer stack from Hamamatsu Nanozoomer (40× view)

Figure 3

Extended focusing algorithm. This picture corresponds to the same area on the same slide as shown in Figure 2′s multilayer stack. All areas are brought into improved focus; however, extracellular debris are accentuated (worse with increasing focal planes), and crisp detail seen in the multilayer stack is lost

Figure 4

Extended focus. Extracellular debris appears to become accentuated with increasing layers

Figure 5

Sequential comparison of extended focus images varying in number of focal planes, with a Multilayer stack image for comparison. With extended focus, intranuclear digital artifacts become more prominent with more focal planes. In all cases, sharp detail is significantly less than with multilayer stack method. (a) Multilayer stack image for comparison, (b) extended focus using 10 focal planes, (c) extended focus using 13 focal planes, (d) extended focus using 16 focal planes

Figure 6

Side by side comparisons between extended focus and multilayer stacking. At high power (40×), (a) extended focus improves focus (black arrow) for areas out of the plane of focus but sharpness and detail appears to be adversely affected. Nuclear contours (red arrow) and chromatin detail (blue arrow) are harder to assess, and white intranuclear digital artifacts are seen only in extended focus (green arrows). 16 focus planes were used in this example. (b) Multilayer stack counterpart to (a). At low power (10×), (c) loss of sharp detail can still be seen in extended focus compared to its multilayer stack counterpart. Nuclear detail is blurred compared to the sharply focused, corresponding area within the multilayer stack version (black arrowhead), and cell borders are difficult to assess (red arrow). (d) Multilayer stack counterpart to (c).

Figure 7

Example of total autofocus failure on a chondrosarcoma slide with two attempts using different scanners. A third attempt using a third scanner is unlikely to fix the problem. Both images are low power (5×) views, and both unusable for neither education nor diagnosis. (a) NanoZoomer, (b) Mirax