Optimizing the management of thyroid specimens to efficiently generate whole slide images for diagnosis.

Transition from optical to digital observation requires an additional procedure in the pathology laboratory, the scanning of glass slides, leading to increased time and digital archive consumption. Thyroid surgical samples often carry the need to collect several tissue fragments that generate many slides to be scanned. This study evaluated the impact of using different inking colours for the surgical margin, section thickness, and glass slide type, in the consumption of time and archive. The series comprehended 40 nodules from 30 patients, including 34 benign nodules in follicular nodular disease, 1 NIFTP, and 5 papillary carcinomas. In 12 nodules, the dominant pattern was microfollicular/solid and in 28 it was macrofollicular. Scanning times/mm2 were longer in red-inked fragments in comparison to green (p = 0.04) and black ones (p = 0.024), and in blue-inked in comparison to green ones (p = 0.043). File sizes/mm2 were larger in red-inked fragments in comparison to green (p = 0.008) and black ones (p = 0.002). The dominant pattern microfollicular/solid was associated with bigger file size/mm2 in comparison with the macrofollicular one (p < 0.001). All scanner outputs increase significantly with the thickness of the section. All scanning outputs increase with the usage of adhesive glass slides in comparison to non-adhesive ones. Small interventions in thyroid sample management that can help optimizing the digital workflow include to prefer black and green inking colours for the surgical margins and 2 µm section in non-adhesive glass slides for increased efficiency.

One nodule-one punction-one slide: Optimizing thyroid fine-needle aspiration for a digital workflow.

OBJECTIVE: Interventional pathologists have expanded their expertise by acquiring proficiency in ultrasound-guided thyroid fine-needle aspiration biopsy (FNAB) and are now required to optimize punction procedures due to low resources and digital workflows. The aim of this study is to compare FNAB sample adequacy in two series with one versus two slides available for cytopathological analysis and its influence on diagnosis categorization, time taken to reach a final diagnosis, scanning time and size of the digital files produced. METHODS: Patients were retrospectively selected based on the sampling of thyroid nodules using either two glass slides (two-slide group) or one slide only (one-slide group) and cytological diagnosis was performed using the second edition of the Bethesda system. For each group, the initial 15 cases were sorted to be scanned. RESULTS: From a total of 713 procedures, 328 were sampled into two slides and 385 on one slide only. No significant differences were found regarding nodule size, location or EU-TIRADS classification between the two groups. The one-slide group did not exhibit a higher prevalence of non-diagnostic or atypia of undetermined significance (AUS) categories. As expected, the mean time taken to finalize diagnoses in cases where only one slide was prepared was 1.2 days faster. Scanning time and total file size were also significantly smaller in the one-slide group. CONCLUSIONS: Adopting the 'one nodule-one puncture-one slide' strategy for thyroid FNAB optimization enhances procedural efficiency in digital workflows, leading to cost savings without compromising diagnostic accuracy.

Inking cell blocks improves scanner detection for diagnosis in pathology.

Cell blocks may be hard to be totally automatically detected by the scanner (ADS), generating incomplete whole slide images (WSIs), with areas that are not scanned, leading to possible false negative diagnosis. The aim of this study is to test if inking the cell blocks helps increasing ADS. Test 1: 15 cell blocks were sectioned, one half inked black (1HB) and the other inked green (1HG). Each of the halves was individually processed to generate a WSI stained by the H&E. 1HBs and 1HGs had similar scanning time (median 59 s vs. 65 s, p = .126) and file sizes (median 382 Mb vs. 381 Mb, p = .567). The black ink interfered less in the observation (2.2% vs. 44.4%; p < .001) than in the green one. Test 2: 15 cell blocks were sectioned, one half inked black (2HB) and the other left unstained/null (2HN). Each of the halves was individually processed to generate three WSIs-one HE, one periodic-acid Schiff (PAS), and one immunostained by cytokeratin AE1&AE3 (CKAE1AE3). HE and PAS WSIs from both 2HN and 2HB groups were all totally ADS and had similar scanning times and file sizes. Concerning immunostaining with CKAE1AE3: ADS (46.7% vs. 93.3%; p = .014), median time for scanning (57 s vs. 83 s; p < .001) and file size (178 Mb vs. 338 Mb; p < .001) were reduced significantly in the 2HN group in comparison with the 2HB. Although increasing scanning time and file size, inking the cell blocks helps increasing ADS after immunostaining, improving the safety and efficiency of the digital pathology workflow.

Demonstrating the interference of tissue processing in the evaluation of tissue biomarkers: The case of PD-L1.

The immunohistochemical (IHC) expression of PD-L1 in cancer models is used as a predictive biomarker of response to immunotherapy. We aimed to evaluate the impact of the usage of 3 different tissue processors in the IHC expression of PD-L1 antibody clones: 22C3 and SP142. Three different topographies of samples (n = 73) were selected at the macroscopy room: 39 uterine leiomyomas, 17 placentas and 17 palatine tonsils. Three fragments were collected from each sample and were inked with a specific color that represented their separate processing in a different tissue processor (A, B or C). During embedding, the 3 fragments with distinct processing were ensemble in the same cassette for sectioning of 3 slides/each: hematoxylin-eosin, 22C3 PDL1 IHC staining and SP142 PD-L1 IHC staining, that were blindly observed by 2 pathologists under digital environment. All but one set of 3 fragments were considered adequate for observation even in the presence of artifacts associated with processing issues that were recorded as high as 50.7 % for processor C. The occurrence of background non-specific staining and the presence of false positive results appear to be unrelated with the PD-L1 clone or the type of tissue processing. 22C3 PD-L1 was more frequently considered adequate for evaluation than SP142 PD-L1 that, in 29.2 % of WSIs (after tissue processor C) were considered not adequate for observation due to lack of the typical pattern of expression. Similarly, the intensity of PD-L1 staining was significantly decreased in fragments processed by C (both PD-L1 clones) in tonsil and placenta specimens, and by A (both clones) in comparison with those processed by B. This study demonstrates the need to standardize the tissue processing in pathology to cope with the growing needs of precision medicine quantifications and the production of high-quality material necessary for computational pathology usage.

Artificial intelligence-assisted cancer diagnosis improves the efficiency of pathologists in prostatic biopsies.

Paige Prostate is a clinical-grade artificial intelligence tool designed to assist the pathologist in detecting, grading, and quantifying prostate cancer. In this work, a cohort of 105 prostate core needle biopsies (CNBs) was evaluated through digital pathology. Then, we compared the diagnostic performance of four pathologists diagnosing prostatic CNB unaided and, in a second phase, assisted by Paige Prostate. In phase 1, pathologists had a diagnostic accuracy for prostate cancer of 95.00%, maintaining their performance in phase 2 (93.81%), with an intraobserver concordance rate between phases of 98.81%. In phase 2, pathologists reported atypical small acinar proliferation (ASAP) less often (about 30% less). Additionally, they requested significantly fewer immunohistochemistry (IHC) studies (about 20% less) and second opinions (about 40% less). The median time required for reading and reporting each slide was about 20% lower in phase 2, in both negative and cancer cases. Lastly, the average total agreement with the software performance was observed in about 70% of the cases, being significantly higher in negative cases (about 90%) than in cancer cases (about 30%). Most of the diagnostic discordances occurred in distinguishing negative cases with ASAP from small foci of well-differentiated (less than 1.5 mm) acinar adenocarcinoma. In conclusion, the synergic usage of Paige Prostate contributes to a significant decrease in IHC studies, second opinion requests, and time for reporting while maintaining highly accurate diagnostic standards.

The impact of different coverslipping methods in the quality of the whole slide images used for diagnosis in pathology.

Digital pathology workflow aims to create whole slide images (WSIs) for diagnosis. The quality of the WSIs depends primarily on the quality of the glass slides produced by the pathology laboratory, where the coverslipping method plays an important role. In this study we compare the glass, the film, and the liquid coverslipping methods to evaluate which ones are suitable to create WSIs for diagnosis. The study included 18 formalin-fixed paraffin-embedded tissue blocks. Of each block, 3 consecutive sections were covered using 1 of the 3 methods. The slides were scanned and evaluated for quality criteria by 2 pathologists experienced in digital pathology. The coverslipping method interferes with the quality of the WSIs, as well as with the scanning time and the file size of the WSIs. All coverslipping methods were found suitable for diagnosis. The glass and liquid methods were manual and had similar results concerning the presence of air bubbles/polymer accumulation, air drying artefacts, tissue exposed, and staining alterations. The glass method was the one with more air bubbles. The liquid method was associated with more alterations on the WSIs, but with the lowest file sizes. Automation of coverslipping and calibration of the scanner for the coverslipping method chosen by the pathology laboratory are relevant for the final quality of the WSIs.

p40/PD-L1 and TTF1/PD-L1 Immunohistochemical Double Staining Contributes to Intraindividual Variability Associated With PD-L1 Staining Interpretation in Lung Cancer Samples.

The important developments achieved in recent years with a consequent paradigm shift in the treatment of non-small cell lung cancer (NSCLC), including the latest immune checkpoint inhibitors, have led to an increasing need to optimize the scarce material usually available in the diagnosis of these tumors. In this sense, this study intends to evaluate the performance of double immunohistochemistry (IHC) in comparison to simple IHC for programmed death-ligand 1 (PD-L1) evaluation with 22C3 clone for selection to therapy with pembrolizumab. For that, 38 histologic samples of NSCLC small biopsies sent to our laboratory were selected. Double IHC were performed with the doublets TTF1/PD-L1 and p40/PD-L1, after all the usual diagnostic routine and molecular study was performed. The slides were interpreted by 2 independent pathologists and the results obtained were compared with each other and with the results obtained at diagnosis. A perfect agreement was observed when comparing the immunoexpression of TTF1 and p40 in double IHC in relation to single IHC. Although the agreement was substantial in the analysis of the positive/negative PD-L1 IHC (81.6% to 92.1%; κ=0.610 to 0.829) and in the analysis of the 50% cut-off (86.8% to 89.5%; κ=0.704 to 0.759), it fell short of the expected and desirable agreement for a biomarker such as PD-L1, since this result will have a major role in the institution of a treatment. In conclusion, this small series does not allow us to recommend this methodology for the evaluation of the PD-L1 biomarker in double staining IHC with the 22C3 clone for therapy selection.

Digital Pathology Workflow Implementation at IPATIMUP.

The advantages of the digital methodology are well known. In this paper, we provide a detailed description of the process for the digital transformation of the pathology laboratory at IPATIMUP, the major modifications that operate throughout the processing pipeline, and the advantages of its implementation. The model of digital workflow implementation at IPATIMUP demonstrates that careful planning and adoption of simple measures related to time, space, and sample management can be adopted by any pathology laboratory to achieve higher quality and easy digital transformation.

What to expect from the 2018 ASCO/CAP HER2 guideline in the reflex in situ hybridization test of immunohistochemically equivocal 2+ cases?

To evaluate the effect of the 2018 ASCO/CAP guideline in the identification of HER2-positive breast carcinomas (BC) in reflex in situ hybridization (ISH) test. A total of 592 primary invasive BC cases from before and after the publication of the updated ASCO/CAP guideline were evaluated for HER2 amplification by silver ISH according to the 2013 and 2018 guidelines. Cases were mostly (95%) HER2 equivocal by immunohistochemistry (IHC), not centrally reviewed. Other reasons for referring cases were IHC confirmation, IHC discordancy (either between needle-core-biopsy (NCB) and surgical excision specimen (SES) or between different laboratories) and IHC result unexpected for histopathologic features. Cases evaluated with the 2013 guideline (1st cohort) were 14.6% HER2-positive, decreasing significantly after the reclassification with the 2018 guideline due to the exclusion of group 2 cases without HER2 protein overexpression. Cases studied after the implementation of the 2018 guideline (2nd cohort) were 8.7% HER2-positive, a frequency that was not significantly different from the reclassification of the 1st cohort with the 2018 guideline. All cases referred for IHC confirmation had the expected ISH result. Cases with IHC discordancy between NCB and SES were ISH concordant. Only one out of 14 cases with an IHC score 3+ and classified as histological grade 1 or with a Ki67 below 10% was classified as ISH HER2-positive. The 2018 ASCO/CAP guideline resulted in a decrease of HER2-positive cases in reflex ISH test, selecting less patients for anti-HER2-targeted therapy.