by Christos Evangelou, MSc, PhD – Medical Writer and Editor
When pathologists examine tissues to diagnose a disease, some samples come from body fluids, such as bronchial washes, rather than from solid tissues. To analyze these liquid cytology samples, pathologists concentrate the cells and embed them in a paraffin block, called a cell block. However, the transparent matrix and sparse cells in cell blocks can make them challenging for digital slide scanners to fully capture in focus.
In a recent study, researchers at the Institute of Molecular Pathology and Immunology of the University of Porto investigated the effect of cell block inking on scanner detection. The team found that simply inking the edges of cell blocks enhanced scanner detection, particularly for immunostained samples.1 This may improve the accuracy of diagnosis by reducing incomplete scans. The trade-off of having complete digital images was a slightly longer scanning time and a larger file size.
“Our results show that a simple procedure, such as inking cell blocks, can prevent the loss of tissue or cells and may enhance digital pathology workflows for challenging specimens. This can be particularly useful in cell blocks, core needle biopsies of the breast, and low-contrast tissues, such as adipose tissue, where automatic detection by the scanner often fails, and segments of the tissue or cells are frequently left unscanned,” said first author Catarina Eloy, MD, PhD, who is a professor at Porto University.
She added that minimizing scanning errors in digital pathology workflows “will certainly prevent loss of time,” helping to preserve turnaround time and prevent false-negative results.
The report was published in Diagnostic Cytopathology.
Study Rationale: Improving Scanner Detection of Cell Blocks
Digital pathology relies on scanners to create high-resolution digital images of tissue slides, allowing pathologists to make diagnoses from digital slides on a computer screen, rather than directly viewing glass slides under a microscope. However, scanner failure can lead to incomplete digital images that do not fully represent everything on the original glass slide.
“Cell blocks are hard to be totally captured by the scanner if they are paucicellular, generating incomplete whole slide images with areas that are not scanned, leading to possible false negative diagnosis,” said Dr. Eloy.
In addition, the production of incomplete digital images is one of the reasons for repeated scanning during quality control, which decreases the efficiency of pathology workflows.
When scanners fail to properly capture all areas of a specimen, it creates the risk that pathologists could miss pathological tissue regions that are present on the original slide but absent from the digital image. According to Dr. Eloy, this possibility of misdiagnosis was the motivation behind testing ways to optimize cell blocks for digital scanning.
“After chatting with the lab team, we understood that we had to come up with some sort of highlighting technique to help detect cell blocks,” she said.
Inking the Edges to Enhance Scanner Detection
“The procedure we followed was incredibly simple: after the cell block was solidified, we inked its surface with a brush, using the inking material that was already present in the macroscopy workstations for inking margins in surgical specimens. After letting the ink dry, we proceeded with tissue processing,” Dr. Eloy explained.
She added that this was the first time this procedure was reported in the literature and represents how the technical team needs to interfere in tissue management to guarantee the production of a more complete whole slide image.
To determine whether inking cell blocks would aid scanner detection, the researchers performed two tests. First, they split 15 cell blocks from bronchial washes in half, inking one half black and the other green before creating digital images of each half stained with hematoxylin and eosin (H&E).1
This test showed that black ink and green ink resulted in similar scanning times (median time: 59 sec vs. 65 sec; P = 0.126) and file sizes (median size: 382 Mb vs. 381 Mb; P = 0.567). Nevertheless, the black ink interfered less with cell observation than the green ink (2.2% vs. 44.4%; P < 0.001).
In the second test, another 15 cell blocks were split in half, and one half was inked black while the other was left unstained. Each of the half blocks was processed to generate three whole slide images (WSIs): one for H&E staining, one for periodic acid-Schiff (PAS) staining, and one immunostained for cytokeratin AE1&AE3.1
This test showed that inking did not influence the scanning time and file sizes for H&E- and PAS-stained slides. For the immunostained slides, 93% of the inked halves were fully captured by the scanner, compared to only 47% of the uninked halves (P = 0.014). The researchers suggest that the enhanced detection of immunostained cell blocks after inking is likely because the ink delineates tissue regions for the scanner, even in areas with little staining.
However, inking significantly increased scanning time (median time: 57 sec vs. 83 sec; P < 0.001) and file size (median size: 178 Mb vs. 338 Mb; P < 0.001). The extra time and storage needed to scan the inked blocks is a small price to pay for more complete images, reducing the risk of missing diseased areas and false-negative diagnoses.
“The longer scan times and larger file sizes of inked slides pay off because it reflects the increased information being captured,” Dr. Eloy noted.
Based on these initial findings, Dr. Eloy believes that inking the cell blocks before scanning should become a standard practice to improve clinical diagnosis with digital pathology. Nonetheless, further optimization of the ink color is needed to make the approach even more efficient. In addition, only one scanner model was tested in this study, and testing cell block inking with other scanners is an important next step.
“We certainly need to refine the way we treat our samples in the lab. We understand that the color of the ink may affect the final result of the cell block preparation, so it is a natural path to test which is the best color to use,” Dr. Eloy said.
- Eloy C, Neves B, Vale J, Campelos S, Curado M, Polónia A. Inking cell blocks improves scanner detection for diagnosis in pathology. Diagn Cytopathol. September 2023. doi:10.1002/dc.25224