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.

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.

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.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1.

BACKGROUND: In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). OBJECTIVE: The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. METHODS: Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. RESULTS: Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. CONCLUSIONS: Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes.

Uso do estimulante de colônia de granulócitos nas neutropenias em cães e gatos / Granulocyte colony-stimulating factor use in neutropenias in dogs and cats

As neutropenias persistentes podem ser decorrentes de alterações na granulopoiese, causadas por efeitos supressivos ou tóxicos à medula óssea, predispõem o paciente a infecções comprometendo sua sobrevida. As neutropenias intensas decorrentes de toxicidade por quimioterápicos podem requerer a suspensão temporária ou permanente do medicamento, podendo gerar resistência das células neoplásicas ao tratamento. O uso de fatores de crescimento hematopoiético recombinantes em animais tem aumentado muito nos últimos anos, devido a sua crescente disponibilidade na medicina humana. O fator estimulante de colônia para granulócitos recombinante humano (rhG-CSF) age aumentando o número de neutrófilos circulantes e possui grande potencial para amenizar ou reverter quadros de neutropenia associada a condições de mielotoxicidade e mielosupressão em cães e gatos.

Persistent neutropenias can occur after granulopoiesis disturbances caused by myelosupressive or myelotoxic effects, and predispose patients to infections and impairs their survival. Furthermore, severe chemotherapy-induced neutropenias must require temporary or definitive treatment interruption, what may lead to drug-resistance of neoplastic cells. The use of recombinant stem cell factors in animals has been increasing due to its bigger disponibility for human beings. The human recombinant granulocyte colony-stimulating factor (rhG-CSF) increases neutrophil numbers in peripheral blood and has great potential to alleviate or revert myelotoxic or myelosupression neutropenias in dogs and cats.