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To date, animal models for lymphographic studies mainly focused on dog, while lymphography is rarely reported in cats, and even less involving cutaneous lymphatic territories. This study aims to assess the feasibility of cutaneous lymphography using indocyanine green (ICG) fluorescence in cat cadavers and describe predictable lymphatic pathways from cutaneous regions of head and hind limb anatomical districts. Frozen or refrigerated cadavers of adult cats that died for causes unrelated to the study were included. Twenty cutaneous regions (6 from the head; 14 from the hind limb) were selected using easily assessable anatomical landmarks, and expected draining lymphocentrums were presumed based on canine studies since there is no similar information for cats. For each lymphography, a single selected cutaneous region per anatomical district was assessed. After intradermal ICG injections, lymphatic drainage was favored by massage and/or flexion-extension movements. For each lymphography, all expected and detected lymphocentrums were dissected, and lymph nodes extirpated. Variables regarding cadavers and lymphography characteristics were assessed. ICG-lymphography was repeated in 33 cadavers. Out of the 99 selected cutaneous regions available, 15 were excluded following inclusion criteria, therefore lymphographies were performed for a total of 84 selected cutaneous regions (26 from the head and 58 from the hind limbs). A success was recorded in 63/84 (75%) lymphographies, with a median migration time of 8 (1–30) minutes. The ICG drained to the expected lymphocentrum in 28/63 (44%) lymphographies, and to other ones in 35/84 (56%). ICG-lymphography is feasible in cat cadavers, regardless of technique or cadaver characteristics. The observed difference in lymphatic drainage (56% to unexpected lymphocentrums) highlights the importance of specifically mapping lymphatic territories in cats. ICG-lymphography demonstrated as an effective technique and could be used to improve knowledge of feline lymphatic physiology. Further studies may provide a more complete understanding of superficial lymphatic territories in cats.

Splenic nodular lesions in dogs can be either benign or malignant. They might be discovered incidentally or, in case of rupture, they may lead to hemoabdomen. Nevertheless, splenectomy followed by histopathology is essential for diagnosis and to prevent rupture. Yet, this invasive procedure might be postponed for dogs with benign splenic nodular lesions. Conversely, owners may opt for euthanasia over surgery for malignancies with poor prognosis like hemangiosarcoma. Thus, anticipating diagnosis with non-invasive biomarkers is crucial for proper patient management. In this prospective study, plasma samples were collected from 66 dogs with histologically confirmed splenic nodular lesions. A canine-specific ELISA kit was applied to assess nucleosome concentration, with histopathology of the spleen serving as the gold standard. Nucleosome concentration was found to be significantly higher in dogs with malignant splenic nodular lesions, particularly in those with hemangiosarcoma and other malignancies. The presence of hemoabdomen, more prevalent in dogs with splenic malignancy, also resulted in increased plasmatic nucleosome concentrations. Plasma nucleosomes could serve as a biomarker for detecting malignant splenic nodular lesions in dogs. More research is needed to understand how nucleosome concentration relate to disease stage and prognosis in dogs with hemangiosarcoma.

Background. Healthcare workers’ health can be influenced by physical, psychological, social, emotional, and work-related stress. MountainTherapy Activities (MTAs) are an integrated therapeutic approach that uses nature to enhance their well-being through group activities like hiking. This cross-sectional study examines well-being levels among Italian Departments of Mental Health workers who do or do not participate in MTAs. It hypothesizes that MTAs may reduce burnout, boost psychological resilience, and increase job satisfaction. Methods. The study involved 167 healthcare workers from 11 Italian Local Health Authorities, divided into MTA (who take part in MTA; n = 83) and non-MTA (who have never participated in MTA; n = 84) groups. They completed five validated questionnaires on psychological distress, burnout, resilience, job engagement, and psychological safety. Data were compared between groups, considering MTA frequency and well-being differences during MTAs versus workplace activities. Results. MTA participants scored higher in psychological well-being (t(117.282) = −1.721, p = 0.044) and general dysphoria (t(116.955) = −1.721, p = 0.042). Additionally, during MTAs, they showed greater job engagement (vigor: t(66) = −8.322, p < 0.001; devotion: t(66) = −4.500, p < 0.001; emotional involvement: t(66) = −8.322, p = 0.002) and psychological safety (general: t(66) = −5.819, p < 0.001; self-expression: t(66) = −5.609, p < 0.001) compared to other activities. Conclusions. MTAs can be considered a valid intervention for the promotion of the mental health of healthcare workers.

Abstract Background Cytopathology is a minimally invasive and convenient diagnostic procedure, often used as a substitute for histopathology to diagnose and characterize lymphoma in dogs. Objectives Assess the diagnostic performance of cytopathology in diagnosing lymphoma and its histopathological subtypes in dogs. Animals One-hundred and sixty-one lymph node samples from 139 dogs with enlarged peripheral lymph nodes. Methods Based only on cytopathology, 6 examiners independently provided the following interpretations on each sample: (a) lymphoma vs nonlymphoma; (b) grade and phenotype; and (c) World Health Organization (WHO) histopathological subtype. Histopathology and immunohistochemistry (IHC) findings were used as reference standards to evaluate diagnostic performance of cytopathology. Clinical, clinicopathologic, and imaging data also were considered in the definitive diagnosis. Results Classification accuracy for lymphoma consistently was >80% for all examiners, whereas it was >60% for low grade T-cell lymphomas, >30% for high grade B-cell lymphomas, >20% for high grade T-cell lymphomas, and <40% for low grade B-cell lymphomas. Interobserver agreement evaluated by kappa scores was 0.55 and 0.32 for identification of lymphoma cases, and of grade plus immunophenotype, respectively. Conclusions and Clinical Importance Cytopathology may result in accurate diagnosis of lymphoma, but accuracy decreases when further characterization is needed. Cytopathology represents a fundamental aid in identifying lymphoma and can be used as a screening test to predict grade and phenotype. However, these results must be confirmed using other ancillary techniques, including flow cytometry, histopathology, and immunohistochemistry (IHC).

Abstract Background Regional lymph nodes are frequently sampled in cats with suspected intestinal lymphoma; however, their diagnostic value has not been explored. Objectives To investigate whether histologic and immunohistochemical analysis of mesenteric lymph nodes correlates with the diagnosis of intestinal lymphoma in cats. Animals One hundred 2 client-owned cats diagnosed with intestinal lymphoma. Methods Retrospective study. The inclusion criteria required a full-thickness biopsy of the small intestine and concurrent excision of mesenteric lymph nodes. Histologic and immunophenotypic analyses were performed on intestinal biopsies and corresponding lymph nodes. Selected nodal samples diagnosed with reactive lymph nodes underwent clonality testing. Results Transmural T-cell lymphomas, encompassing small and large cell types, were predominant (64 cases, 62.7%), with large B-cell lymphomas being more frequently transmural (68.8%) than mucosal (31.2%). Among all lymph nodes examined, 44 (43.1%; 95% CI: 33.9%-52.8%) exhibited neoplastic infiltration. Among cases of small cell lymphoma, 51 out of 72 (70.8%; 95% CI: 59.4%-80.1%) showed no nodal involvement. Clonality results correctly identified 19/30 (63.3%; 95% CI: 45.5%-78.2%) reactive lymph nodes. Concerns were raised regarding clonal identification in the remaining cases and potential misdiagnoses based on phenotypic characteristics. Conclusion and Clinical Importance The study underscores the potential drawbacks of relying solely on mesenteric lymph nodes for diagnosing intestinal lymphomas in cats, particularly small cell subtypes. It emphasizes the importance of full-thickness biopsies for assessing transmural infiltration and recommends caution when utilizing mesenteric lymph nodes for histologic, immunohistochemical and clonality evaluations in mucosal lymphomas. Despite limitations, this research highlights the need for comprehensive diagnostic strategies in cats with intestinal lymphoma.

IntroductionFlow cytometry (FC) has been used recently to assess percentages of infiltration by mast cells in sentinel lymph nodes (SLN) of dogs with mast cell tumors (MCT). SLN mapping often includes the use of different dyes such as methylene blue (MB) and indocyanine green (ICG), which might influence fluorescence assessment by FC. This study aimed to assess whether the color given by mapping dyes might affect the baseline fluorescence of SLN aspirates for FC.MethodsBaseline fluorescence was calculated as Median Fluorescence Intensity (MFI) in 4 channels (FL1, FL2, FL3, FL4, respectively corresponding to 530/30 nm, 585/40 nm, >670 nm, 660/20 nm) with a cytometer equipped with two lasers (488 nm and 638 nm) with constant setting and compensation. SLN aspirates were suspended in RPMI medium. They were classified based on results of mapping techniques in 4 dye classes (blue/fluorescent, fluorescent/non-blue, blue/non-fluorescent, non-blue/non-fluorescent), and possible differences in MFI values among SLN dye classes were assessed.ResultsThirty-five SLNs from 17 dogs were assessed. Considering dye classes, 13 were blue/fluorescent, 16 were fluorescent/non-blue, 4 were blue/non-fluorescent, 2 were non-blue/non-fluorescent. In all fluorescence channels, except FL1, MFI varied among SLN dye classes. Blue/non-fluorescent SLNs showed the highest fluorescence, followed by blue/fluorescent, fluorescent/non-blue, and non-blue/non-fluorescent.DiscussionThese results suggest that the use of dyes for SLN mapping may introduce a relevant bias when MFI is quantitatively assessed via FC.

An in-depth knowledge of non-neoplastic patterns is fundamental to diagnose neoplasia. In the present study, we described the flow cytometric (FC) cell size (FSC) and fluorescence intensity (MFI) of B- and T-lymphocytes in 42 canine reactive lymph nodes and 36 lymphomas. Proliferative activity (Ki67%) in reactive lymph nodes was also reported. Reactive lymph nodes were composed of a mixed population of small and large T (CD5+) and B (CD21+) cells. Small T-cells were larger in size than small B-cells, and large T-cells were larger than large B-cells. Small T-cells were composed of CD5+CD21− and CD5+CD21+dim subpopulations. Large B-cells were <20% in reactive lymph nodes and >20% in lymphomas and showed a higher FSC in lymphomas than in reactive lymph nodes. Large T-cells were <4% in reactive lymph nodes and >4% in lymphomas and showed a higher CD5 MFI in lymphomas (if expressed) compared to reactive lymph nodes. A subset of CD5+CD21+dim lymphocytes was recognized in addition to CD5+CD21- and CD5−CD21+ cells. In T-zone lymphomas, neoplastic cells had higher FSC and CD21 MFI values than small CD5+CD21+dim cells in reactive lymph nodes. Ki67% values were higher than those reported in normal lymph nodes, and largely overlapped with those reported in low-grade lymphomas and partially in high-grade lymphomas. Our results may contribute to making a less operator-dependent FC differential between lymphoma and reactive lymph nodes.

CD94 is a natural killer (NK) cell receptor that also marks subsets of T cells, referred to as NKT cells. In humans, the role of CD94 as both an immune checkpoint and a potential therapeutic target has gained increasing attention. However, data about its expression in leukemia and lymphoma in dogs remain limited. This study aimed to explore CD94 expression in canine leukemia and nodal lymphoma, using a newly available anti-canine CD94 monoclonal antibody in a multicolor flow cytometry panel. Surplus blood and lymph node aspirate samples from eleven client-owned dogs (leukemia: n = 7, lymphoma n = 4) and two clinically healthy controls, were analyzed. The control dogs as well as most cases showed low CD94 + lymphocyte frequencies, consistent with a non-neoplastic population. However, markedly expanded CD94 + populations were identified in two out of four of the T cell chronic lymphocytic leukemia (T-CLL) cases. In one of them, the neoplastic population was uniformly CD3 + CD8 + CD94 + , while the other showed a heterogeneous mixture of CD3 + CD8 + CD94 + and CD3 − CD8 + CD94 + lymphocytes. Our findings demonstrate that the canine-specific CD94 antibody can be applied to both blood and lymph node samples in a diagnostic flow cytometry setting. While CD94 expression was infrequent overall, its detection in a subset of T-CLL cases highlights the need for larger studies to determine its diagnostic and therapeutic value in canine leukemia and lymphoma.

Phenotypic aberrancies have been reported occasionally in canine lymphomas. Here, we retrospectively collected 310 canine lymphomas with an aberrant phenotype detected via flow cytometry and describe their clinical and clinical pathological features at diagnosis. There were 152 T-cell lymphomas not otherwise specified (T-NOS), 101 T-zone lymphomas (TZL), 54 B-cell lymphomas, and 3 cases with two suspected concurrent neoplastic populations. The most represented aberrancies were: CD5-, CD4-CD8-, and CD3- in T-NOS lymphomas, CD21+, CD4-CD8-, and CD3- in TZLs, and CD34+, CD44-, and CD5+ in B-cell lymphomas. Among T-cell lymphomas, the aberrant expression of CD21 was significantly more frequent in TZL and the loss of CD5 and CD44 in T-NOS. More than 75% of dogs were purebred; males outnumbered females; the mean age at diagnosis was 8–10 years, depending on lymphoma subtype. A few dogs were symptomatic at the time of diagnosis, and 30% had peripheral blood abnormalities, in line with what is already reported for the general population of dogs with lymphoma. Further studies are needed to assess the pathogenetic mechanisms underlying each specific antigen aberrancy, as well as the diagnostic and prognostic role.

Epigenetic deregulation is a hallmark of cancer characterized by frequent acquisition of new DNA methylation in CpG islands. To gain insight into the methylation changes of canine DLBCL, we investigated the DNA methylome in primary DLBCLs in comparison with control lymph nodes by genome-wide CpG microarray. We identified 1,194 target loci showing different methylation levels in tumors compared with controls. The hypermethylated CpG loci included promoter, 5′-UTRs, upstream and exonic regions. Interestingly, targets of polycomb repressive complex in stem cells were mostly affected suggesting that DLBCL shares a stem cell-like epigenetic pattern. Functional analysis highlighted biological processes strongly related to embryonic development, tissue morphogenesis and cellular differentiation, including HOX, BMP and WNT. In addition, the analysis of epigenetic patterns and genome-wide methylation variability identified cDLBCL subgroups. Some of these epigenetic subtypes showed a concordance with the clinical outcome supporting the hypothesis that the accumulation of aberrant epigenetic changes results in a more aggressive behavior of the tumor. Collectively, our results suggest an important role of DNA methylation in DLBCL where aberrancies in transcription factors were frequently observed, suggesting an involvement during tumorigenesis. These findings warrant further investigation to improve cDLBCL prognostic classification and provide new insights on tumor aggressiveness.