Characterization of blue-green blood leukocyte inclusions and accompanying clinical, hematologic, and serum biochemical changes in dogs.

BACKGROUND: Lipofuscin-like cytoplasmic inclusions have been reported in human blood neutrophils and monocytes but have not been described in dogs. In people, these "green granules of death" have been associated with moderate to severe hepatocellular injury and high mortality. OBJECTIVES: To describe clinicopathologic abnormalities, diagnoses, and outcomes of dogs with greenish inclusions in blood neutrophils or monocytes, and to determine if the inclusions have features of lipofuscin. METHODS: Clinical cases were identified prospectively through routine evaluation of CBC samples. Leukocyte inclusions were characterized with routine staining and assessed for iron and autofluorescence. Additional cases were identified by examination of archived blood smears from dogs meeting search criteria for hepatocellular injury, and clinicopathologic findings were recorded. RESULTS: All 7 prospectively identified dogs with inclusions had inflammation and moderate to marked increases in serum alanine aminotransferase (ALT) activity, as did the 4 dogs identified from the 97 meeting retrospective search criteria. The inclusions were Prussian blue-negative (5/5) with broad-spectrum autofluorescence (5/5) and the appearance of lipofuscin with and without Wright staining. Most clinical diagnoses involved hepatic disorders (5/7 prospective and 3/4 retrospective cases) or pancreatitis (3/7 prospective and 2/4 retrospective cases), and some involved both; 8 of 11 dogs died within 7 days of admission. CONCLUSIONS: Blue-green cytoplasmic inclusions uncommonly found in blood neutrophils ± monocytes of routine canine blood smears have stained and unstained properties of lipofuscin and suggest the presence of hepatocellular injury, often severe. Reporting these inclusions is recommended to guide clinical management.

Multiorgan neutrophilic inflammation in a Border Collie with "trapped" neutrophil syndrome.

Trapped neutrophil syndrome is a rare congenital disease recognized in Border Collies and is characterized by persistent neutropenia with myeloid hyperplasia. The mechanism of neutropenia has not been described. We document the case of a young Border Collie diagnosed with trapped neutrophil syndrome based on clinical features, blood and bone marrow evaluation, and presence of the associated homozygous mutation. Results from flow cytometric and storage studies suggested lower neutrophil survival time. The dog had substantial neutrophilic inflammation in multiple organs, indicating that neutrophils could leave the marrow and enter tissues, making the term "trapped" neutrophil syndrome a misnomer.

Immunoglobulin G and phosphatidylserine in regenerative and nonregenerative immune-mediated anemias of dogs.

BACKGROUND: Although precursor-targeted immune-mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA. HYPOTHESIS: Dogs with PIMA have increased nRBC IgG and PS, and dogs with immune-mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs. ANIMALS: Blood from 20 healthy dogs and from dogs with IMHA (11) or other (non-IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non-IMHA, non-PIMA) conditions (7). METHODS: Marrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS. RESULTS: Red blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non-IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non-IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid- or late-stage fractions, whereas all 7 non-PIMA dogs were negative. Mid- and late-stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity. CONCLUSIONS: Immunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.

Blood and bone marrow findings in two pups with mucopolysaccharidosis type VII.

Routine blood smear findings in two of four 11-day-old mixed-breed dog littermates were suggestive of a lysosomal storage disease (LSD) that was documented to be mucopolysaccharidosis type VII (MPS VII) by molecular testing. In this condition, a functional ß-glucuronidase deficiency results in the accumulation of glycosaminoglycans (GAGs) in cells and tissues where ß-glucuronidase is important in GAG degradation. Most neutrophils and a moderate number of lymphocytes within the blood had atypical cytoplasmic magenta inclusions. The bone marrow assessment from one of the two affected pups at 24 days of age revealed similar magenta granulation in myeloid precursor cells that was most prominent in promyelocytes and myelocytes. Moreover, atypical magenta material was present within vacuoles as well as extracellularly in some osteoblasts and macrophages. Histologic bone marrow sections revealed prominent vacuolation of osteoblasts, and some osteoclasts appeared separated from the bone by layers of osteoblasts or hematopoietic cells. At 2 months of age, the second affected dog showed moderate growth retardation and had similar but more prominent hematologic findings that extended to monocytes, eosinophils, and eosinophil precursors. It had an increased number of bone marrow macrophages with many vacuoles that could be seen cytologically to contain magenta material, and there was mild nonselective phagocytosis of hemic cells. Of the hematologic cells, inclusions were most prominent in promyelocytes, myelocytes, and macrophages, cells with relatively high ß-glucuronidase activity, and GAG exposure within lysosomes or lysosome-like primary granules of granulocyte precursors.

Bone marrow, blood, and clinical findings in dogs treated with phenobarbital.

BACKGROUND: Cytopenias have been reported in dogs treated with phenobarbital, but detailed descriptions of bone marrow findings and response to treatment are lacking. OBJECTIVES: We aimed to characterize the hematologic findings and clinical outcomes of dogs that had been receiving phenobarbital at the time of marrow evaluation. METHODS: Archived bone marrow slides and clinicopathologic data were reviewed in dogs undergoing marrow evaluation for any hematologic problems that developed while receiving phenobarbital (2008-2020). Dogs were excluded if marrow samples lacked diagnostic value, phenobarbital was withdrawn >1 day before marrow collection, a same-day complete blood count (CBC) was lacking, or dogs had concurrent illness or therapy known to cause cytopenias. RESULTS: Thirteen dogs met inclusion criteria: eight pancytopenic, three anemic/thrombocytopenic, one neutropenic/thrombocytopenic, and one nearly neutropenic. Neutropenia was marked (<700/µL) in eight dogs; all neutrophil concentrations were low or low-normal. Of the 11 anemic dogs (Hct = 12%-42%, median = 29%), three had mild reticulocytosis (eight were tested). One dog had erythroid dysplasia in blood and marrow. All nine neutropenic dogs had evidence of ineffective neutropoiesis: neutrophilic hyperplasia with left shift (9) ± neutrophagocytosis (5). Eight of the 11 anemic dogs had evidence of ineffective erythropoiesis: erythroid hyperplasia (7), left shift (3), and/or rubriphagocytosis (6). No thrombocytopenic dog had megakaryocytic hypoplasia; seven dogs had megakaryocytic hyperplasia. One anemic/thrombocytopenic dog had marked collagen myelofibrosis. The noncytopenic dog had equivocal myeloid hypoplasia with neutrophagocytosis. Median maximal responses and resolution times for neutropenia (n = 6) were 14 days. CONCLUSIONS: Phenobarbital-induced cytopenias should be considered in dogs with multilineage ineffective hematopoiesis, particularly when neutropenia and myeloid hyperplasia are present. However, findings in dogs with immune-mediated neutropenia or precursor-targeted immune-mediated anemia might be indistinguishable.

Clinical features of precursor-targeted immune-mediated anemia in dogs: 66 cases (2004-2013).

OBJECTIVE: To characterize the clinical features of dogs with precursor-targeted immune-mediated anemia (PIMA). ANIMALS: 66 dogs with PIMA. PROCEDURES: Electronic record databases of a teaching hospital were searched to identify dogs with a diagnosis of nonregenerative anemia between 2004 and 2013. Inclusion criteria included persistent nonregenerative anemia (Hct ≤ 30% and reticulocyte count < 76,000 reticulocytes/µL), cytologic findings supportive of ineffective bone marrow erythropoiesis, and absence of underlying disease. Information regarding clinical signs, clinicopathologic findings, treatment, and outcome was extracted from records of eligible dogs. A regenerative response was defined as a reticulocyte count > 76,000 reticulocytes/µL or sustained increase in Hct of > 5%. Remission was defined as a stable Hct ≥ 35%. RESULTS: The median Hct was 13%, and reticulocyte count was 17,900 reticulocytes/µL. Rubriphagocytosis was identified in bone marrow aspirate samples from 61 of 66 dogs. Collagen myelofibrosis was detected in bone marrow biopsy specimens obtained from 31 of 63 dogs. Immune-mediated targeting of mature erythrocytes was uncommon. All dogs received immunosuppressive therapy. Fifty-five dogs developed a regenerative response at a median of 29 days, and 40 of those dogs went into remission at a median of 59 days after PIMA diagnosis. Thromboembolic events were confirmed for 9 dogs and were associated with a decreased survival time. Median survival time was 913 days for all dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that most dogs with PIMA responded to prolonged immunosuppressive therapy. Studies to determine optimal immunosuppressive and thromboprophylactic protocols for dogs with PIMA are warranted.

Dynamics of a capsule flowing in a tube under pulsatile flow.

We analyze numerically the behavior of a deformable micro-capsule confined in a pipe under a pulsatile flow. The capsule moves and is deformed by the action of a pulsatile flow inside the tube with a non-null mean velocity. This configuration can be found in the nature and in many bioengineering systems where artificial capsules are driven by micro-pumps through micro-channels. The capsule is considered as a thin hyperelastic membrane, which encloses an internal fluid. As it has been demonstrated in the literature, this model represents a wide range of artificial capsules, for example, the alginate-based capsules, typically used in bioengineering applications. A hybrid isogeometric finite element method and boundary element method based on a T-spline discretization and formulated in the time domain is used to solve the mechanical and hydrodynamical equations. The influence of the relative rigidity of the membrane, frequency and amplitude of the pulsatile flow is studied. Results show that the behavior of the capsule differs from steady flows and it depends strongly on the frequency of the flow and mechanical characteristic of the capsule.

Analytic characterization of flow cytometric assays for detection of immunoglobulin G on canine erythroid cells, including detection of dog erythrocyte antigen 1 on erythroid precursors.

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors. SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs. PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti-dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs. RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.

Histologic and cytologic bone marrow findings in dogs with suspected precursor-targeted immune-mediated anemia and associated phagocytosis of erythroid precursors.

BACKGROUND: Precursor-targeted immune-mediated anemia (PIMA) has been suspected in dogs with nonregenerative anemia and bone marrow findings varying from erythroid hyperplasia to pure red cell aplasia. Phagocytosis of erythroid precursors/rubriphagocytosis (RP) reported in some affected dogs suggests a destructive component to the pathogenesis of PIMA. OBJECTIVES: The purpose of the study was to characterize laboratory and clinical findings in dogs with suspected PIMA and RP, with emphasis on cytologic and histologic bone marrow findings. METHODS: Dogs with PIMA and RP were identified by review of paired bone marrow aspirate and core biopsy slides collected over a 4-year period. Samples were systematically assessed and characterized along with other pertinent laboratory data and clinical findings. RESULTS: Twenty-five dogs met criteria for PIMA and had RP that was relatively stage-selective. Erythropoiesis was expanded to the stage of erythroid precursors undergoing most prominent phagocytosis, yielding patterns characterized by a hypo-, normo-, or hypercellular erythroid lineage. A 4th pattern involved severe collagen myelofibrosis, and there was a spectrum of mild to severe collagen myelofibrosis overall. Evidence of immune-mediated hemolysis was rare. Immunosuppressive therapy was associated with remission in 77% of dogs treated for at least the median response time of 2 months. CONCLUSIONS: Bone marrow patterns in dogs fulfilling criteria for PIMA were aligned with stage-selective phagocytosis of erythroid precursors and the development of collagen myelofibrosis, common in dogs with PIMA. Recognition of these patterns and detection of RP facilitates diagnosis of PIMA, and slow response to immunosuppressive therapy warrants further investigation into its pathogenesis.

Tissue-scale, personalized modeling and simulation of prostate cancer growth.

Recently, mathematical modeling and simulation of diseases and their treatments have enabled the prediction of clinical outcomes and the design of optimal therapies on a personalized (i.e., patient-specific) basis. This new trend in medical research has been termed "predictive medicine." Prostate cancer (PCa) is a major health problem and an ideal candidate to explore tissue-scale, personalized modeling of cancer growth for two main reasons: First, it is a small organ, and, second, tumor growth can be estimated by measuring serum prostate-specific antigen (PSA, a PCa biomarker in blood), which may enable in vivo validation. In this paper, we present a simple continuous model that reproduces the growth patterns of PCa. We use the phase-field method to account for the transformation of healthy cells to cancer cells and use diffusion-reaction equations to compute nutrient consumption and PSA production. To accurately and efficiently compute tumor growth, our simulations leverage isogeometric analysis (IGA). Our model is shown to reproduce a known shape instability from a spheroidal pattern to fingered growth. Results of our computations indicate that such shift is a tumor response to escape starvation, hypoxia, and, eventually, necrosis. Thus, branching enables the tumor to minimize the distance from inner cells to external nutrients, contributing to cancer survival and further development. We have also used our model to perform tissue-scale, personalized simulation of a PCa patient, based on prostatic anatomy extracted from computed tomography images. This simulation shows tumor progression similar to that seen in clinical practice.

Failure to thrive and life-threatening complications due to inherited selective cobalamin malabsorption effectively managed in a juvenile Australian shepherd dog.

A juvenile Australian shepherd dog exhibited failure to grow, inappetence, weakness, nonregenerative anemia, neutropenia, and cobalamin deficiency. DNA testing confirmed homozygosity of an amnionless mutation (AMN c.3G > A). Clinical signs resolved with supportive care and parenteral cobalamin supplementation. Inherited selective intestinal cobalamin malabsorption requiring lifelong parenteral supplementation should be considered in Australian shepherds, giant schnauzers, border collies, and beagles that fail to thrive.

Gestion efficace d'un retard de croissance et de complications potentiellement mortelles attribuables à une malabsorption de cobalamine sélective héréditaire chez un jeune chien Berger australien. Un jeune chien Berger australien a manifesté une absence de croissance, de l'inappétence, de la faiblesse, une anémie non régénérative, de la neutropénie et une déficience de cobalamine. Des tests d'ADN ont confirmé l'homozygotisme d'une mutation des récepteurs amnionless (AMN c.3G > A). Les signes cliniques se sont résorbés avec des soins de soutien et des suppléments de cobalamine parentéraux. Une malabsorption intestinale sélective héréditaire de cobalamine exigeant des suppléments parentéraux à vie devrait être considérée chez les Bergers australiens, les Schnauzers géants, les Border-Collies et les Beagles qui manifestent des problèmes de croissance.(Traduit par Isabelle Vallières).

Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Skunk musk causes methemoglobin and Heinz body formation in vitro.

BACKGROUND: A captive Red Panda developed a regenerative anemia with Heinz bodies after being sprayed by a skunk. A definite cause-and-effect relationship between skunk musk and oxidative erythrocyte damage has not been reported, but it was suspected in one reported case of a dog with Heinz body hemolytic anemia. OBJECTIVE: The objective was to determine whether skunk musk induces oxidative HGB damage in vitro. METHODS: Plasma and RBC were harvested from heparinized blood of 3 dogs, 3 cats, and a Red Panda. Skunk musk was solubilized in ethanol and mixed with plasma from each species to make stock solutions of 4% musk and 4% ethanol. Aliquots of RBC were resuspended in autologous stock solutions and solvent controls to yield musk concentrations of 0%, 0.04%, and 0.4% (by volume). Aliquots were incubated at 37°C for 4-72 hours and assessed for oxidative damage by visual inspection, optical absorbance spectroscopy, transmission electron microscopy, and light microscopy after Wright and vital New Methylene Blue staining. RESULTS: Dose-dependent brown color and absorption changes characteristic of methemoglobin were present by 4 hours and increased over 24 hours (Red Panda) and 72 hours (dog and cat). Similarly, there were time-dependent (all species) and dose-dependent (dog and cat) increases in the number of Heinz bodies, which were present by 4 hours and numerous by 24 hours. CONCLUSIONS: In vitro, skunk musk causes Heinz body and methemoglobin formation in canine, feline, and Red Panda RBC, supporting the clinical association between Heinz body hemolytic anemia and skunk spray exposure.

Effects of a curricular revision on learner outcomes in veterinary clinical pathology.

A mixed-methods evaluation was conducted to study learner attitudes and knowledge about clinical pathology across a curricular change that instituted a stand-alone clinical pathology course in place of content within a previously integrated pathology course structure. Groups of pre- and post-change students were assessed three times across the two semesters leading up to graduation. At each time, rank-ordered and open-ended response items probed attitudes, and multiple-choice items assessed knowledge. Data about student clinical pathology performance were also collected from clinical pathology instructors and supervising clinicians. Student rank-ordered items were evaluated by factor analysis; resulting factor-scale scores, multiple-choice scores, and rank responses from study cohorts were statistically assessed between groups and within each group over time. Intraclass correlations were calculated for the coding of student open-ended responses, and all coded responses were compared among groups. Analysis revealed that students in the revised curriculum had greater satisfaction with their training and greater confidence in data interpretation compared to students without exposure to an independent clinical pathology course. Although differences in knowledge of clinical pathology were not detected, it was also apparent that the independent clinical pathology course filled a student-perceived curricular need without raising criticisms related to diminished integration with anatomic pathology. Secondary study outcomes included formative feedback for course improvement, evidence of clerkship efficacy, and baseline data for further studies.

The role of high-throughput technologies in clinical cancer genomics.

Cancer is a genetic disease driven by both heritable and somatic alterations in DNA, which underpin not only oncogenesis but also progression and eventual metastasis. The major impetus for elucidating the nature and function of somatic mutations in cancer genomes is the potential for the development of effective targeted anticancer therapies. Over the last decade, high-throughput technologies have allowed us unprecedented access to a host of cancer genomes, leading to an influx of new information about their pathobiology. The challenge now is to integrate such emerging information into clinical practice to achieve tangible benefits for cancer patients. This review examines the roles array-based comparative genomic hybridization and next-generation sequencing are playing in furthering our understanding of both hematological and solid-organ tumors. Furthermore, the authors discuss the current challenges in translating the role of these technologies from bench to bedside.