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Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy. The aim of the study was to determine if the immunophenotype of neoplastic cells predicts survival in canine CLL. Retrospective study. Forty-three dogs with CLL. Records of dogs with a final diagnosis of CLL were reviewed. For each included dog, a CBC, blood smear for microscopic reevaluation, and immunophenotyping data had to be available. Data on signalment, history, clinical findings, therapy, follow-up, as well as date and cause of death were retrieved. Seventeen dogs had B-CLL (CD21+), 19 had T-CLL (CD3+ CD8+), and 7 had atypical CLL (3 CD3− CD8+, 2 CD3+ CD4− CD8−, 1 CD3+ CD4+ CD8+, and 1 CD3+ CD21+). Among the variables considered, only immunophenotype was associated with survival. Dogs with T-CLL had approximately 3-fold and 19-fold higher probability of surviving than dogs with B-CLL and atypical CLL, respectively. Old dogs with B-CLL survived significantly longer than did young dogs, and anemic dogs with T-CLL survived a significantly shorter time than dogs without anemia. Although preliminary, results suggested that immunophenotype is useful to predict survival in dogs with CLL. Young age and anemia are associated with shorter survival in dogs with B-CLL and T-CLL, respectively. [ABSTRACT FROM AUTHOR]

Nodal metastasis is a negative prognostic factor in dogs with mast cell tumours (MCTs), thus early detection enables more informed decision-making and provides valuable prognostic information. The aim of this study is to assess the concordance between histopathologic findings of LNs and cytology and flow cytometry (FC), respectively, and to evaluate the ability of FC to differentiate between metastatic (HN2-HN3) and non-metastatic (HN0-HN1) LNs. Overall, 117 LNs from 64 dogs with first occurring MCTs were submitted for cytology, histology and FC. LNs were cytologically and histologically classified according to Krick and Weishaar systems, respectively. Using FC, mast cells (MCs) were identified as IgE+ CD117+ CD5- CD21- cells and quantified as a percentage. When compared with histologic classification, cytology showed an accuracy of 88.2% in distinguishing between metastatic and non-metastatic LNs but did not detect 25.3% of metastatic cases. FC revealed an increase in the median percentages of MCs across histologic classes, progressing from HN0 to HN3. ROC curves pinpointed 0.3% as the optimal cut-off for distinguishing between metastatic and non-metastatic LNs, with an accuracy of 84.3%. A 1.1% cut-off proved valuable in identifying HN3 LNs. The combined interpretation of cytology and FC increased accuracy to 92.2%. An algorithm for guiding the combined interpretation of cytology and FC is suggested based on these findings. In conclusion, FC proves beneficial in enhancing the early detection of metastatic LNs, particularly when utilised alongside cytology. Histopathology remains essential for confirmation, enabling the discrimination of HN classes or, in doubtful cases, for the detection or exclusion of nodal metastases., (© 2024 John Wiley & Sons Ltd.)

The identification of non-hematopoietic cells in effusions is a diagnostic challenge in cytology. Biopsies from mesothelium or primary lesions are infrequently performed in clinical settings and immunochemistry on smears or immunohistochemistry on cell blocks are the most common ancillary test to refine the cytological diagnosis. Cavitary effusions are an ideal matrix for flow cytometry and the availability of a cytometric panel to describe non-hematopoietic cells would represent a useful tool. Here we present the results of the flow cytometric and immunohistochemical determination of cytokeratin (CK), vimentin (VIM) and desmin (DES) in 36 canine effusions. The concordance between the two methods was perfect for CK (100%), substantial for VIM (77.8%), and almost perfect for DES (97.2%). The panel was interpreted to define the epithelial (CK+VIM-DES-), mesothelial (CK+VIM+DES+), or mesenchymal (CK-VIM+DES-) origin of the cells. Unexpected profiles were considered doubtful and observed patterns were individually discussed. The concordance of the panel interpretation between two methods was 75%. The evaluation of discordant and doubtful cases suggests a lower sensitivity of flow cytometry in detecting VIM expression and revealed a high frequency of VIM+ epithelial cells, variable expression of VIM in mesothelial cells, and an important role of DES in excluding an epithelial origin when positive. Multicentric studies based on histopathological diagnoses are necessary to confirm these findings and evaluate the diagnostic utility of the panel to refine cytological diagnosis. Our results show that flow cytometry can be a timesaving alternative to IHC on cell blocks in clinical settings to detect CK, VIM and DES expression. The interpretation of the panel is similar in most cases; however, occasional discordant results, particularly for VIM, may occur., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Sini, Melega, Cannizzo, Miniscalco, Valenti and Riondato.)

Introduction: Programmed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated., Methods: Samples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration., Results: Considering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21-2.03, range 1.01-3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07-1.32, range 1.02-2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03-1.46, range 1.02-6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas ( p  = 0.023)., Discussion: PD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ubiali, Cesar Conti, Dall’Ara, De Maria, Aresu, Moretti, Sini, Riondato, Stefanello, Comazzi and Martini.)

Myeloid sarcoma (MS) is a solid tumor of granulocytic origin with extramedullary localization. This tumor is rare in humans and animals. The diagnostic approach is heterogeneous, and the definitive diagnosis may be difficult to achieve. Primary MS has never been described as a spontaneous neoplasm in companion dogs. Two purebred and 1 mixed-breed dogs, 6- to 11-year-old, developed round cell tumors in the mediastinum, lymph nodes (LNs) and tonsils, and LNs, respectively. Granulocytic origin and exclusion of lymphoid lineage were confirmed by flow cytometry, supported by immunohistochemistry or immunocytochemistry. Pivotal to the diagnosis were positive labeling for myeloid (CD11b, CD14) and hematopoietic precursors (CD34) markers, along with negative labeling for lymphoid markers. Blood and bone marrow infiltration were not detected at initial diagnosis, excluding acute myeloid leukemia. The behavior of these tumors was aggressive, resulting in poor clinical outcomes, even when chemotherapy was attempted., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Mast cell tumor (MCT) is a common skin cancer in dogs that has a wide range of clinical behaviors. The purpose of this study was to develop a novel multicolor flow cytometry (FC) panel that will enable the quantification of candidate prognostic markers (Ki-67 and pKIT) in fine needle aspirate (FNA) samples prior to surgical removal of the tumors. FNA of canine MCTs and the NI-1 cell line were utilized to develop a FC panel that includes a viability dye (FVS620, BD Biosciences; 7-AAD, Invitrogen) and the following primary conjugated antibodies: CD117-PE (ACK45, BD Biosciences), pKIT-A647 (polyclonal bs-3242R, BIOSS) and Ki-67-FITC (20Raj1, eBioscience; MIB-1, DAKO). A total of nine FNA samples of canine MCTs were collected, seven out which produced sufficient cells for FC analysis. The Ki-67 antibody clone 20Raj1 produced a positive signal when applied to blood leukocytes but failed to provide robust labeling of neoplastic mast cells. The Ki-67 antibody clone MIB-1 delivered a superior staining quality in both the NI-1 cells and primary MCT cells. CD117-PE signal was adequate post fixation and permeabilization and in the combination of 7-AAD. pKIT produced non-specific staining and was not suitable for this multicolor FC panel. In conclusion, FNA samples of canine MCTs can often yield adequate cell numbers for FC analysis, and a multicolor FC panel was developed that can detect Ki-67 in canine mast cells. This would permit further studies into the potential use of this panel for canine cutaneous and subcutaneous MCT prognostication purposes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Wu, Lejeune, Affolter, Iamone, Riondato and Kol.)

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.

The availability of cross-reacting antibodies and/or of antibodies working in flow cytometry is a major issue in the veterinary field. One of the main problems is the availability of certain positive controls. With this brief communication, we report an method to quickly screen a wide number of products without the need to look for positive biological samples. We propose this approach as a first step to select the best antibodies to test on biological specimens., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Introduction: Few cases of myelomonocytic leukemia associated with neurological signs have been described in dogs; none have been related to intraparenchymal spinal cord infiltration by neoplastic cells. This short communication describes a case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. A 3-year-old male Golden Retriever was presented with a history of hyperthermia, lymphadenomegaly, leukocytosis with circulating blast cells, anemia and thrombocytopenia, and acute onset paraplegia. Immunophenotyping of peripheral blood by flow cytometry was consistent with acute myelomonocytic leukemia subtype M4. The dog was euthanized because of clinical deterioration and unfavourable prognosis. Postmortem examination revealed multi-organ neoplastic infiltration, including the spinal cord. To our knowledge, this is the first case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. Our findings hold importance for including myelomonocytic leukemia in the differential diagnosis of patients with neurological signs due to spinal cord localisation.

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)., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

B cell lymphoma (BCL) is a heterogeneous group of lymphoid malignancies which comprise the majority of canine lymphomas. Diffuse large B cell lymphoma is the most common lymphoma subtype in dogs but other subtypes (e.g., marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, and others) have been described. This review aims to explore the use of flow cytometry to refine the diagnosis of canine BCL. Particular emphasis will be given to the possible identification of peculiar immunotypes, putative prognostic markers, staging and minimal residual disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Riondato and Comazzi.)

Tumor growth depends on both proliferative and apoptotic rate of neoplastic cells. High proliferation index is a well-known negative prognostic factor in canine lymphomas, whereas little is known about apoptotic activity. We describe proliferative and apoptotic rates in different canine lymphoma subtypes at diagnosis. Flow cytometry (FC) was used to assess the percentage of proliferating cells (Ki67%) and of apoptotic cells (AnnV%) in 128 lymph node (LN) aspirates from dogs with lymphoma. Proliferation/apoptosis ratio (PAR) and turnover index (TI; Ki67% + AnnV%) were then calculated for each case. High-grade B-cell lymphomas showed high values for both Ki67% and AnnV%, low-grade B-cell lymphomas showed low Ki67% and high AnnV%, high-grade T-cell lymphomas showed high Ki67% and low AnnV%, and low-grade T-cell lymphomas showed low levels of both parameters. Lymphoblastic lymphomas had the highest PAR values. High-grade B-cell lymphomas had the highest TI values while small clear cells lymphomas the lowest. The panorama of proliferative and apoptotic activity widely varies among lymphoma subtypes. Our results lay the ground for future clinical and pharmacological studies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Cell blocks are alternative preparations of fluid cytological specimens. They can be used for immunochemical studies as complementary tools or when other techniques (eg, immunocytochemistry, flow cytometry) are not available., Objectives: We aimed to provide comparative morphologic, immunohistochemical, and technical features of agar-based cell blocks (ACBs) and cell tube blocks (CTBs) from cavitary effusions., Methods: Agar-based cell blocks and CTBs were obtained from canine and feline effusions with neoplastic/atypical cells or with packed cell volumes ≥3%. Cellularity, RBC separation, and cellular features were evaluated on digitalized H&E slides with evaluators blinded to the method. The immunohistochemical intensity and nonspecific background were assessed on pan-cytokeratin and vimentin-stained slides. Overall yield was calculated, and morphologic and immunohistochemical features were compared among paired samples. Technical and cellular features were also described., Results: Agar-based cell blocks and CTBs yielded evaluable sections in 100% (52/52) and 98% (51/52) of the cases, respectively. Cellularity and RBC separation scores were significantly higher in CTBs. Similar staining intensities were observed, and background staining was more frequently seen in pan-cytokeratin-stained ACBs. Only basic materials and equipment were required for both methods. Agar-based cell block preparations were more operator dependent and difficult to standardize, whereas CTBs were easier to prepare, but laboratory processing was more demanding., Conclusions: Both methods can be used to produce good sections for immunohistochemistry staining with no significant differences. Cell tube blocks are beneficial for RBC-rich samples, and little additional training is required to prepare the blocks. Both types of cell blocks are reliable, cost-effective methods that could be introduced in diagnostic laboratories to further characterize canine and feline effusions., (© 2020 American Society for Veterinary Clinical Pathology.)

Dogs with Diffuse Large B-Cell Lymphoma (DLBCL) benefit from the addition of active immunotherapy to traditional chemotherapy. We hypothesized that immune cells within neoplastic lymph nodes (LNs) may play a role in the tumor pathobiology and treatment response. The present study describes the composition and prognostic role of non-neoplastic lymphocytes in LNs of 59 dogs with treatment-naive DLBCL receiving chemo-immunotherapy. The percentage of small non-neoplastic cells and of CD5+, CD21+, CD4+ and CD8+ small cells was recorded via flow cytometry. CD4+/CD8+ and CD5+/large CD21+ cell ratios were calculated. The likelihood of progression significantly diminished with increasing percentage of small cells, CD5+ and CD8+ small cells, and CD5+/large CD21+ cell ratio, with decreasing CD4+/CD8+ ratio and in non-anemic dogs. Active immunotherapy is more effective in dogs with higher percentage of non-neoplastic lymphocytes at diagnosis. We lay the ground for future studies assessing the role of the immune system in the pathobiology of canine DLBCL., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011., Methods: To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety., Results: Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P <  0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P <  0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P <  0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events., Conclusions: Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.

Most dogs with large B-cell lymphoma (LBCL) that undergo chemotherapy and achieve clinical complete remission (CR) eventually relapse. However, time to relapse (TTR) is unpredictable. The aims of this prospective study were to assess the influence of post-chemotherapy lymph node (LN) infiltration by large CD21+ cells using flow cytometry (FC) on TTR, and to establish a cut-off value of prognostic significance. Dogs with newly-diagnosed, completely staged LBCL in CR after treatment were enrolled. Minimal residual disease (MRD) analysis by FC was performed on LN aspirates. TTR was calculated between MRD and relapse. Thirty-one dogs were enrolled: 4% had stage V disease, and diffuse large B-cell lymphoma was the most common histotype (74%). Based on LN infiltration at MRD evaluation, three groups were created: (a) acellular samples, (b) ≤0.5% infiltration and (c) >0.5% infiltration. Overall median TTR was 154 days (range, 31-1974): 22 (71%) dogs relapsed during the study period, whereas 9 (29%) dogs did not. The difference among the three groups was significant (P = 0.042 log-rank test): median TTR was not reached for dogs with LN infiltration ≤0.5% (range, 195-429 days), 164 days (range 63-1974) for dogs with acellular LN samples, and 118 days (range, 31-232) for dogs with LN infiltration >0.5%. These results demonstrate that MRD assessment by FC on LN aspirates in dogs with LBCL in clinical CR predicts TTR. LN infiltration by >0.5% large CD21+ cells after treatment is an unfavourable prognostic factor., (© 2018 John Wiley & Sons Ltd.)

Canine nodal marginal zone lymphoma (nMZL) is infrequent and is typically diagnosed at an advanced disease stage. However, it is currently unknown whether different levels of peripheral blood (PB) and bone marrow (BM) infiltration may provide prognostic stratification in dogs with nMZL. The aims of the present prospective study were to assess the influence of PB and BM infiltration detected by flow cytometry (FC) on time to progression (TTP) and lymphoma-specific survival (LSS) in dogs with newly-diagnosed multicentric nMZL, and to establish a cut-off value of prognostic significance. Forty-five completely staged and treatment-naïf dogs with histologically-confirmed nMZL were enrolled. After staging, dogs received chemo-immunotherapy or chemotherapy. PB infiltration was significantly associated with TTP (p=0.001): dogs with PB infiltration <30% had a median TTP of 186 days, whereas dogs with PB infiltration ≥30% had a median TTP of 43 days. Additionally, vaccinated dogs had a significantly (p=0.012) longer TTP (399 days) compared with dogs receiving chemotherapy only (211 days). BM infiltration was significantly associated with LSS (p<0.001): dogs with BM infiltration <1% had a median LSS of 1403 days, those with BM infiltration 1-20% of 337 days, and those with BM infiltration ≥20% of 188 days. Normal LDH levels and the administration of chemo-immunotherapy also significantly improved LSS (560 vs 211 days, and 399 vs 211 days, respectively; p<0.001). PB and BM flow cytometric evaluation is an integral part of staging work-up in dogs with nMZL and has prognostic relevance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Feline Mesenchymal Nasal Hamartoma (MNH) is a rare benign tumor-like lesion of the sinonasal tract affecting young cats., Objectives: This study aimed to determine the diagnostic significance of osteoblast-like (OB-L) and osteoclast-like cells (OC-L) in squash preparation cytology from endoscopic biopsies., Methods: A 5-year database was retrospectively reviewed and included 109 cases of which 24 were diagnosed as MNH by histopathology. Slides were examined by two cytologists (one experienced and one inexperienced in nasal and squash cytology) in a double-blind study. The inexperienced cytologist counted OB-L and OC-L in 500 intact nucleated cells. The experienced cytologist assigned samples to four categories for OB-L (0, 1-5, 6-10, >10/field) and OC-L (0, 1-2, 3-5, >5/field)., Results: The presence of OB-L and OC-L was significantly associated (P < 0.001) with the histologic diagnosis of MNH. Receiver operating characteristic curves from the counts by the inexperienced cytologist revealed 3/500 OB-L and 2/500 OC-L as the best cut-offs for the diagnosis of MNH. Those of the experienced cytologist evaluation revealed that all the MNHs presented more than 10 OB-L/field and 3 or more OC-L/field. Both cytologists detected each cell type in all MNHs with an overall concordance of 0.93., Conclusions: The presence of OB-L and OC-L is a consistent finding in MNH, and thus, represents a reliable cytologic diagnostic criterion. The described methods are applicable in routine in-clinic laboratory settings and are easy to apply at any expertise level. Further prospective studies are needed to assess the accuracy of the proposed cut-off values., (© 2018 American Society for Veterinary Clinical Pathology.)

Flow cytometry (FC) is widely applied to characterize and stage nodal lymphomas in dogs because it has a short turnaround time, requires minimally invasive sampling, and allows contemporary evaluation of neoplastic cells in the primary lesion and of blood and marrow involvement. We investigated advantages and limitations of FC in suspected extranodal lymphomas in dogs. The likelihood of obtaining a suitable FC sample was significantly lower for aspirates of extranodal lesions than for lymph node aspirates. However, we noted no differences among different extranodal lesion sites. We also describe FC results for 39 samples compatible with extranodal lymphoma. A dominant population of large cells was easily identified on morphologic FC scattergrams in many cases. Phenotypic aberrancies were frequently present, mainly in T-cell lymphomas. Lymphoma cells were distinguishable from normal residual lymphocytes in >85% of cases, facilitating the quantification of putative blood and marrow involvement by FC. Despite the high percentage of non-diagnostic samples (32 of 73, >40%), we support the inclusion of FC in the diagnostic workup of suspected extranodal lymphomas in dogs, in conjunction with histopathology. Histopathology is the gold standard for diagnosing lymphoma, provides relevant information, including tissue invasion and epitheliotropism, but has a longer turnaround time.

The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

Background: The primary aim was to evaluate by means of thromboelastometry (ROTEM) the effects of hydroxyethyl starch (HES) 130/0.4 administered as a constant rate infusion (CRI) on hemostasis in hypoalbuminemic dogs. The second aim was to use ROTEM analysis to detect whether all hypoalbuminemic dogs of our population were hypercoagulable., Results: The study sample was 20 hypoalbuminemic dogs (albumin < 2 g/dl) with normal perfusion parameters and requiring intravenous fluid therapy. In order to support plasma colloid osmotic pressure, in addition to crystalloid, HES 130/0.4 was administered as a constant rate infusion at 1 ml/kg/h (group 1, n = 11) or 2 ml/kg/h for 24 h (group 2, n = 9). Blood samples were collected at baseline (T0) and 24 h postinfusion (T1); coagulation was assessed by standard coagulation profile (prothrombin time, activated partial thromboplastin time, and fibrinogen), and ROTEM analysis (in-TEM®, ex-TEM® and fib- TEM® profile). No statistically significant differences in ROTEM values in group 1 were observed (P > 0.05), whereas in group 2 statistically significant differences (P < 0.05) were found at T1 in the in-TEM® profile [decrease in clot formation time (P = 0.04) and increase in α angle (P = 0.02)] and in the ex-TEM® profile [increase in maximum clot firmness (P = 0.008) and α angle (P = 0.01)]; no changes were identified in the fib-TEM® profile. In both groups, a statistically significant decrease (P = 0.007) in hematocrit was noted, whereas no statistically significant differences in platelet count and standard coagulation profile were found. In group 2, a statistically significant increase in TS values (P = 0.03) was noted at T1. ROTEM tracings indicating a hypercoagulable state were observed in 7/20 dogs at T0 (5/11 in group 1 and 2/9 in the group 2)., Conclusion: Our findings suggest that HES 130/0.4 administered as CRI does not cause hypocoagulability in hypoalbuminemic dogs. A trend toward hypercoagulability, probably related to the underlying diseases, was observed in group 2 at T1. Although all dogs were hyoalbuminemic, only 7/20 were hypercoagulable at T0, confirming the lack of correlation between albumin level and prothrombotic state.

Stage V lymphoma is defined as the presence of neoplastic cells in peripheral blood (PB), bone marrow, or any other non-lymphoid tissue. Still, official guidelines do not specify which technique should be used to assess infiltration. We assessed the agreement among flow cytometry (FC), blood smear evaluation, and ADVIA120 (LUC and BASO) to quantify PB infiltration in 100 dogs with large B-cell lymphoma (LBCL). Significant errors were found for all methods compared to FC. A moderate agreement was present between FC and blood smear evaluation, whereas LUC and BASO had excellent specificity but unsatisfactory sensitivity in detecting FC infiltrated PB samples. The different techniques should not be used alternatively. We support the use of LUC/BASO as a speedy preliminary test to detect infiltrated samples, and the joined use of blood smear evaluation and FC to quantify definitively the infiltration. Our results are valid only within canine LBCL staging workup, once the diagnosis has been confirmed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Lymph node (LN), peripheral blood (PB), and bone marrow (BM) samples are commonly analyzed by flow cytometry (FC) for the immunophenotyping and staging of canine lymphomas. A prognostic value for FC BM infiltration in dogs with large B-cell lymphoma (LBCL) was demonstrated. Aim of this study was to define the analytical performances of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and noninfiltrated PB and BM samples., Methods: Large B-cells were added to control PB and BM samples, to achieve twelve different large B-cells concentrations, ranging from 0 to 50%. The percentage of large B-cells was recorded for each dilution, using a BD Accuri C6 FC. Accuracy was evaluated by Passing-Bablok regression analysis. Intra-assay precision was assessed at 0%, 1, 3, and 10% dilutions evaluating the CVs of 10 repeated acquisitions. ROC curves were drawn to identify the cutoffs most suitable to discriminate between 25 infiltrated (PARR-positive) and 25 noninfiltrated (PARR-negative) PB and BM samples, respectively., Results: Optimal analytical accuracy and precision were achieved. Almost all CVs were <10%. Negative controls had up to 0.5% large B-cells, with 50 and 22% CV in PB and BM samples, respectively, 0.56 and 2.45% cutoffs were selected based on the ROC curves for PB and BM samples, respectively., Conclusions: Quantification of large B-cells in PB and BM samples by FC is reliable and analytical performances met the acceptance criteria. Assessment of performances of different instruments and protocols is warranted. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Background: Serum protein electrophoresis (SPE) provides useful information in ruminants, but reference intervals (RI) are different from other species. There have been no reports of SPE RI for dairy sheep using agarose gel electrophoresis (AGE)., Objective: The purpose of the study was to evaluate the serum concentration of total protein (TP) and protein fractions determined by AGE in mid-lactating dairy ewes, to establish RI, and to assess potential differences between Lacaune (L) and Sarda (S) sheep breeds., Methods: Blood samples were collected from healthy, mid-lactating ewes. SPE was assessed using a semi-automated AGE system. Reference intervals (90% confidence intervals) for TP and each protein fraction were determined using the nonparametric method for combined data, and the robust method for data from the single breeds. Data from S and L sheep were compared using the Mann-Whitney U test., Results: The 172 sheep included 116 L and 56 S ewes, 2-6 years old. There were significant differences between S and L breeds, and RI were calculated for TP, albumin, α1 -globulin, α2 -globulin, β1 -globulin, β2 -globulin, γ1 -globulin, and γ2 -globulin concentrations, and for the Albumin/Globulin ratio. Group S showed higher concentrations of TP, α2 -, β1 -, β2 -, and γ1 -globulins, whereas L was higher for albumin and γ2 -globulin concentrations, and A/G ratio (P < .05)., Conclusions: The resolution with AGE was excellent, allowing standardization of 7 protein fractions, detection of differences between S and L ewes, and determination of RI for French (Lacaune) and Italian (Sarda) dairy sheep., (© 2015 American Society for Veterinary Clinical Pathology.)

Diffuse large B-cell lymphoma (DLBCL) is the most frequent canine lymphoid neoplasm. Despite treatment, the majority of dogs with DLBCL experience tumour relapse and consequently die, so practical models to characterise dogs with a poor prognosis are needed. This study examined whether the lymphocyte/monocyte ratio (LMR) can predict outcome in dogs with newly diagnosed DLBCL with regard to time-to-progression (TTP) and lymphoma specific survival (LSS). A retrospective study analysed the prognostic significance of LMR obtained at diagnosis by flow cytometry (based on morphological properties and CD45 expression) in 51 dogs that underwent complete staging and received the same treatment, comprising multi-agent chemotherapy and administration of an autologous vaccine. Dogs with an LMR ≤ 1.2 (30% of all cases) were found to have significantly shorter TTP and LSS, and it was concluded that LMR was a useful independent prognostic indicator with biological relevance in dogs with DLBCL treated with chemoimmunotherapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A 2-year and 6-month-old female neutered Labrador Retriever with Horner syndrome, megaesophagus, and a mediastinal mass was referred to the Queen Mother Hospital for Animals of the Royal Veterinary College. A large granular lymphocyte (LGL) lymphoma was diagnosed on cytology; flow cytometric analysis revealed a γδ T-cell phenotype (CD3+, CD5+, CD45+, TCRγδ+, CD4-, CD8-, CD34-, CD21-). Chemotherapy was started with a combination of lomustine, vincristine, procarbazine, and prednisolone, followed by bleyomicin. Euthanasia was elected by the owners, due to progressive deterioration and lack of quality of life, 28 days after diagnosis. This is the first cytologic and immunophenotypic characterization of a canine γδ T-cell lymphoma with LGL morphology and probably of mediastinal origin. The role of chemotherapy in delaying the disease progression remains unknown., (© 2015 American Society for Veterinary Clinical Pathology.)