Mitotic count (MC) is an important element for grading canine cutaneous mast cell tumors (ccMCTs) and is determined in ten consecutive high-power fields with the highest mitotic activity. However, there is variability in area selection between pathologists. In this study, the MC distribution and the effect of area selection on the MC were analyzed in ccMCTs. Two pathologists independently annotated all mitotic figures in whole-slide images of 28 ccMCTs (ground truth). Automated image analysis was used to examine the ground truth distribution of the MC throughout the tumor section area, which was compared with the manual MCs of 11 pathologists.Computerized analysis demonstrated high variability of the MC within different tumor areas. There were 6 MCTs with consistently low MCs (MC<7 in all tumor areas), 13 cases with mostly high MCs (MC ≥7 in ≥75% of 10-hpf-areas) and 9 borderline cases with variable MCs around 7, which is a cut-off value for ccMCT grading. There was inconsistency among pathologists in identifying the areas with the highest density of mitotic figures throughout the three ccMCT groups; only 51.9% of the counts were consistent with the highest 25% of the ground truth MC distribution. Regardless, there was substantial agreement between pathologists in detecting tumors with MC≥7.Falsely low MCs below 7 mainly occurred in 4/9 borderline cases that had very few ground truth areas with MC≥7. The findings of this study highlight the need to further standardize how to select the region of the tumor in which to determine the MC. Keywords: Area selection, high-power field, mitotic activity, mitotic figure distribution, tumor grading, tumor periphery. 3 Aubreville M, Bertram CA, Klopfleisch R, Maier A. Field of interest proposal for augmented mitotic cell count: Comparison of two convolutional networks.

SummaryAn 11‐year‐old Trakehner gelding was presented for evaluation of lethargy, decreased appetite, mild icterus, and elevated hepatic enzyme activities. Physical examination, serum chemistry results, and liver biopsy histopathologic findings were supportive of Theiler's disease. Polymerase chain reaction (PCR) testing results of serum and liver tissue were positive for nonprimate (equine) hepacivirus (NPHV) and a novel equine parvovirus‐hepatitis virus (EqPV‐H). PCR testing of the lot of tetanus antitoxin administered to the gelding 3 months previously also yielded positive results for NPHV and EqPV‐H. Treatment included supportive care and clinical signs resolved within 1 week, although hepatic enzyme activities remained elevated for several months. The horse successfully returned to work as a hunter/jumper for about 1 year until it developed a forelimb lameness and progressive atrophy of shoulder musculature (sweeney), prompting a decision for euthanasia 20 months after initial evaluation. Serial PCR testing of serum revealed persistent infection with both NPHV and EqPV‐H and necropsy examination revealed chronic active hepatitis, mild liver atrophy, and positive PCR results for NPHV and EqPV‐H in liver tissue. This case highlights the possible risk of administering potentially contaminated biologics of equine origin and the importance of screening for recently identified hepatic viruses in donors from which blood products are prepared.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Preparing the skin of rodents for surgery often involves multiple applications of antiseptic agents. However, fewer applications may achieve the same antiseptic outcome. We evaluated the antimicrobial efficacy and effects on intraoperative body temperature of various surgical scrub agents, including novel waterless alcohol-based (WAB) options. Prior to ventral laparotomy, female C57BL/6 mice were treated with 0.9% saline (control); 70% ethanol; 10% povidone-iodine alternated with saline or 70% ethanol; 2% chlorhexidine digluconate alternated with saline or 70% ethanol; or 1 of 3 WAB products-commercial surgical scrub A, commercial surgical scrub B, or a common commercial hand sanitizer. Core temperatures were recorded, and aerobic culture swabs were collected from the surgical site at multiple time points. Intraoperative temperature trajectories for animals treated with scrub B, 10% povidone-iodine with saline, or hand sanitizer did not differ from saline (control). Temperature trajectories of mice treated with other scrub agents did differ significantly from saline. Bacteria were not detected at the operative site after 3 scrubs of 70% ethanol or 10% povidone-iodine alternated with ethanol, 2 scrubs of scrub A or B, 1 scrub of hand sanitizer, and both 1 and 3 scrubs of 2% chlorhexidine alternated with ethanol. Scrub B and 2% chlorhexidine-ethanol demonstrated prolonged antibacterial efficacy. Histology of corresponding haired skin sections revealed no differences in postoperative healing between groups, and no postoperative infections occurred. These results indicate that various novel WAB disinfectants, particularly scrub B (61% ethanol and 1% chlorhexidine gluconate), mitigate intraoperative temperature effects associated with several traditional agents and combinations. Furthermore, reduction of skin bacterial load without adverse effects on healing was seen with fewer than triplicate applications of most tested agents. Ultimately effective skin preparation can be achieved by using only 1 or 2 applications of scrub, thus rendering the triplicate skin-prep method unnecessary in laboratory mice.

Canine oral malignant melanomas (OMMs) exhibit a variety of morphologic phenotypes, including a spindloid variant. The microscopic diagnosis of spindloid OMMs is based on junctional activity and/or the presence of melanin pigment. In the absence of these features, spindloid OMMs are difficult to differentiate from soft tissue sarcomas (STS). An antibody cocktail (MDX) that includes Melan-A, PNL2, and tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) is the current gold standard for identifying amelanotic OMMs by immunohistochemistry (IHC). However, MDX is less sensitive for diagnosing spindloid amelanotic OMMs. This raises concern for biopsy specimens that lack overlying epithelium, making it potentially difficult to differentiate OMM from STS by IHC. The goal of this study was to identify additional markers to help differentiate between STS and OMMs that lack pigment and junctional activity. SOX-10 has recently been proposed as a sensitive marker for melanocytes in humans but has not been validated in dogs. Similarly, RNA expression for various genes has been analyzed in humans, but not in the context of diagnosing canine melanocytic neoplasms. For this retrospective study, formalin-fixed, paraffin-embedded tissues from 20 OMMs, 20 STS, and 20 oral spindle cell tumors (OSCTs) that lacked junctional activity and pigmentation were selected. IHC for MDX, SOX-10, and laminin, in parallel with RT-qPCR of TYR, SOX10, CALD1, CD34, DES, and LAMA1, was performed in all cases. TYR, CD34, and CALD1 were the most discriminatory genes in differentiating between OMM and STS, all having 100% specificity and 65, 95, and 60% sensitivity, respectively. While all 20 OMMs were immunohistochemically labeled for SOX-10, two STS were also labeled (100% sensitivity and 90% specificity). MDX IHC labeled all 20 OMMs and no STS. Surprisingly, none of the 20 OSCTs expressed TYR RNA above the cutoff, and 14/20 OSCTs expressed CALD1 or CD34 RNA above the cutoff, thereby confirming them as STS. Four OSCT were suspect STS, and no OSCTs were confirmed as OMMs based on IHC and RNA expression patterns. In conclusion, the RNA levels of TYR, CD34, and CALD1 should be evaluated in suspected amelanotic OMMs that are negative for MDX to accurately differentiate between OMM and STS.