N. C. Jain

Quantitative Platelet Disorders

Thrombocytopenia may be caused by abnormal platelet production, accelerated removal owing to immunologic or nonimmunologic reasons, or sequestration of platelets in the spleen. Bleeding associated with thrombocytopenia usually presents as petechial or ecchymotic hemorrhages or epistaxis. Immunologic and nonimmunologic cases of thrombocytopenia may be diagnosed with routine hematology, bone marrow cytology, and platelet specific tests. Thrombocythemia may also be associated with platelet functional abnormalities, contrasting the normal platelet function noted in reactive thrombocytosis.

Autoimmune Hemolytic Anemia in Dogs and Cats

As improved diagnostic reagents become available, greater diagnostic accuracy can be expected. After a thorough clinical and laboratory examination, intensive therapy must be started immediately and maintained until clinical remission is achieved. An effort must be made to determine the existence of associated disease states and to institute appropriate treatment. Prognosis is guarded when AIHA is accompanied by severe hepatic and renal diseases, especially in the older animal. Prognosis is more guarded when AIHA is associated with either AITP or SLE. Remissions are to be expected and necessitate continual laboratory examinations and prolonged therapy. With care and tenacity, careful management can provide long-term benefits to the patient.

Classification of myeloproliferative disorders in cats using criteria proposed by the animal leukaemia study group: A retrospective study of 181 cases (1969–1992)

Bone marrow cytology in Wright or Wright Giemsa stained smears from 181 feline patients were evaluated according to recently published criteria by the animal leukaemia study group for classification of acute] myeloid leukaemas (AML) and myelodysplastic syndrome (MDS). AML was recognised in 107 (59.1%) and MDS in 39 (21.5%) cats. The frequency of acute myeloblastic leukaemia (M1 and M2 combined) was high (30.9%), erythroleukaemia (M6 and M6-Er combined) was relatively less frequent (17.1%), and acute undifferentiated leukaemia (AUL), acute monoblastic leukaemia (M5) and acute myelomonocytic leukaemia (M4) were less common, each with an incidence of less than 5.0%. Most of the M1 cats had greater than 70.0% blast cells in bone marrow compared to only a few of the M2 cats. Some blast cells in occasional AUL cats showed cytoplasmic differentiation to either erythroid or myeloid lineage or rarely to both. Type I (classical) myeloblasts predominated in all Ml cats, but occasional type II myeloblasts were seen in some cats and a few cats had some type III myeloblasts. Rubriblasts were prominent in most of the M6-Er cats.Dysplastic changes were more common in M2 than in M1 cats and included primarily dysgranulopoiesis (giant metamyelocytes and bands) and dyserythropoiesis (megaloblastic rubriblasts and rubricytes) and rarely dysmegakaryocytopoiesis (abnormal nuclear morphology). Dysplastic changes involving primarily the erythroid series were seen in M6 and M6-Er cats. Dysmyelopoiesis, in the form of occasional giant metamyelocytes and band neutrophils, was also seen in most cats considered to have myeloid hyperplasia unassociated with leukaemia or MDS. Two M2 and two MDS cats had high (>10.0%) eosinophils in bone marrow and were designated M2-Eos and MDS-Eos, respectively.Cytochemical staining was performed on bone marrow smears from 65 cases to demonstrate myeloid markers such as alkaline phosphatase (ALP), peroxidase (PO), chloroacetate esterase (CAE), and Sudan black B (SBB) and monocytic markers such as α-naphthyl acetate esterase (NAE) and a-naphthyl butyrate esterase (NBE). NAE and NBE staining reactions were usually complementary. Cytochemical heterogeneity and lineage infidelity were observed in some AML subtypes. Blast cells were positive for myeloid markers in many but not all cases of Ml and M2. Similarly monocytic markers were evident in some but not all cases of M4 and M5. Monocytic markers were also evident in blast cells of one M1 and three M2 cases, myeloid markers were present in two cases of M4, and both myeloid and monocytic markers were demonstrated in one case of AUL. Blast cells were negative for both myeloid and monocytic markers in most cases of AUL and M6. ALP-positive cells were increased distinctly (>10.0%) only in a small number of cats (15.4%) which included cases of M1, M2, M4. M6 and MDS.Prominent haematological abnormalities in most of the AML and MDS cases included moderate to severe thrombocytopenia, normocytic normochromic non-regenerative anaemia, normoblastaemia, leukocytosis accompanied by a left shift, and a variable number of circulating blast cells. Circulating blast cell counts of ⩾30.0% were seen in only 15.3% cats. Mean leukocyte counts were over 100 000/µl in most of the M5 and M4 cats and in an occasional M1 cat, whereas AUL cats generally had leukocyte counts within the normal range. Leukopenia was evident in slightly over half of the MDS-Er cats, in nearly a third of the M2 cats, and in a small number of cats in other categories. Monocytosis was most pronounced in M5 cats and prominent in M4 cats. Other abnormalities seen in a small number of cats included hyperproteinaernia or hypoproteinaemia, hyperfibrinogenaemia, elevated icterus index, and thrombocytosis.

Autoimmune thrombocytopenia in dogs and cats.

Autoimmune thrombocytopenia has been recognized as a distinct entity in the dog and cat. It is characterized by: (1) clinical signs of thrombocytopenia, such as hemorrhages into the skin and tissues and from body orifices, (2) coagulation defects related to thrombocytopenia, such as prolonged bleeding time and poor clot retraction, (3) hematologic changes such as severe to moderate thrombocytopenia, often blood loss anemia, and signs of increased erythropoiesis, and (4) an absence or decreased number of megakaryocytes in the bone marrow during early phase and increased number during compensatory phase. Megakaryocytes may also show morphologic abnormalities. Serologic diagnosis of AITP in the dog and cat currently involves demonstration of antiplatelet antibody in serum by PF-3 test and/or associated with marrow megakaryocytes by a technique of direct immunofluorescence. Circumstances leading to formation of antiplatelet antibody remain unknown. Immune-mediated platelet destruction is believed to occur in the reticuloendothelial system, primarily in the spleen. Treatment consists primarily of corticosteroids and other immunosuppressive drugs. Dogs with primary AITP generally respond favorably to such therapy.

Activation of bovine neutrophil functions by interferon-gamma, tumour necrosis factor-alpha, and interleukin-1 alpha

Neutrophils are the first line of defence against microbial infections. They are important in protecting the bovine mammary gland against environmental and pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. Neutrophils in the peripheral blood of healthy cows are functionally a heterogeneous population of cells with markedly varied phagocytic and oxidative activities. Several cytokines have been found to augment leucocyte functions in humans and animal studies. Therefore, the main objective of the present study was to determine if recombinant human interleukin-1 alpha (rhIL-1 alpha), tumour necrosis factor-alpha (rhTNF-alpha), and interferon-gamma (rhIFN-gamma) would potentiate the functional activity of bovine blood neutrophils, thereby providing a possible means to manipulate the resistance of cows to microbial infections.Neutrophils were isolated from the peripheral blood of nine Holstein-Friesian heifers, with a purity of 89%–96% and viability greater than 98%. Aliquots of neutrophils were incubated with five concentrations of rhIL-1 alpha (0.001–10 ng/ml), rhTNF-alpha (0.5–1000 ng/ml), and rhIFN-gamma (0.01-100 ng/ml) at 37°C for 1 h. Then, fluorescein-labelled opsonised zymosan particles were added and the neutrophil suspensions were incubated further for 30 min to evaluate phagocytic activity by fluorescence microscopy. Similarly, unlabelled opsonised zymosan particles and nitroblue tetrazolium (NBT) were added and incubation was carried out for 15 min to evaluate oxidative activity by a spectrophotometric method. Chemotactic activity of cytokine-treated neutrophils for E. coli lipopolysaccharide-activated fetal calf serum was evaluated using a modified Boyden chamber method. The results showed that pretreatment of neutrophils with various concentrations of each cytokine enhanced their phagocytic and NBT reductive activities but reduced chemotactic activity. The phagocytic and NBT reductive activities increased significantly (p⩽0.05) with an increase in the concentration of the cytokines.

Modulation of phagocytic and oxidative burst activities of bovine neutrophils by human recombinant TNF-α, IL-1-α, IFN-γ, G-CSF, GM-CSF

In vitro experiments were conducted to determine the effects of recombinant cytokines on phagocytic and oxidative burst activities of bovine neutrophils. Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated blood from healthy Holstein-Friesian heifers. Aliquots of neutrophils (10 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5–1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001–10 ng/ml), interferon-gamma (rhIFN-γ, 0.01–100 ng/ml), granulocyte colony-stimulating factor (rhG-CSF, 25 ng/ml), and granulocyte-macrophage colony-stimulating factor (rhGM-CSF, 10 ng/ml). Then, the percentage phagocytosis and average number of intracellular bacteria per cell were evaluated by flow cytometry and/or fluorescent microscopy using FITC-labelled opsonised bacteria (Escherichia coli 0111:B4). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H2O2 production, a measure of oxidative burst, by flow cytometry. The results showed that all five cytokines significantly (p <0.05) increased percentage phagocytosis (52.4–86.1%), number of intracellular bacteria per cell (24.9–47.9%), and H2O2 production (31.3–58.2%) when compared to untreated neutrophils. A gradual increase in mean channel fluorescence but not in percentage phagocytosis was consistently seen with increasing concentrations of rhTNF-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.

Long-term haematological changes in cats experimentally infected with feline immunodeficiency virus (FIV)

Haematological changes of experimental feline immunodeficiency virus (FIV) infection in six inoculated and six control cats were studied over a 98 week period. Acute infection was characterised by moderate to severe leucopenia, neutropenia, and eosinopenia between weeks 5 and 13 post-inoculation (PI). Normal myeloid activity or mild myeloid hyperplasia with a left shift to promyelocytes accompanied neutropenia. Chronic infection was characterised by intermittent neutropenia in three of six cats beginning after week 50 Pl and lymphopenia in two cats starting on week 66 Pl. The severity and duration of neutropenia and lymphopenia varied in individual cats. In contrast to natural FIV infections, anaemia and thrombocytopenia did not develop in either acute or chronic experimental infection. Age-related haematological changes such as increasing packed cell volume and plasma protein concentration as well as decreasing neutrophil and total leucocyte counts were noted as both control and inoculated cats reached maturity.The mechanisms of neutropenia and eosinopenia remain unknown. Viral infection of myeloid cells may be involved in the pathogenesis of cytopenias, and further studies are needed to elucidate the mechanisms. This study also demonstrated that FIV infection in cats may be a useful model in studying haematological abnormalities associated with other immunodeficiency-causing lentiviruses.

Functional competence and monoclonal antibody reactivity of neutrophils from cows injected with Escherichia coli endotoxin

Neutrophils of cows injected with endotoxin were evaluated for their functional competence and monoclonal antibody binding in relation to morphological maturity. Six clinically healthy lactating Holstein cows were each injected intravenously with 100 μg Escherichia coli endotoxin. Blood was collected at 0, 6, 30, 54, 78 and 150 hours postinjection for total and differential leucocyte counts and to isolate neutrophils for functional assays. Marked leucopenia, neutropenia with left shift to band neutrophils, lymphopenia and monocytopenia were evident at 6 h postinjection. Neutrophil and lymphocyte counts normalised by 30 hours, while band neutrophils remained elevated until 54 hours postinjection.Neutrophils isolated at 6, 30 and 54 hours postinjection revealed significantly increased relative phagocytic index, reduced chemotactic activity, and increased binding of a bovine neutrophil monoclonal antibody (36H10). Ingested bacteria per cell were increased at 6 hours. Phorbol myristate acetate-induced oxidative product formation was significantly reduced at 30 hours, but chemiluminescence activity did not change significantly. In vivo binding of autologous IgG2 and IgA to neutrophils significantly decreased at 30 and 54 hours and IgM binding was reduced at 78 hours postinjection.Band neutrophils were chemotactically and phagocytically less active than segmented neutrophils. The number of band neutrophils in blood correlated directly with the relative phagocytic index and the percentage of cells binding monoclonal antibody 361110 and inversely with the chemotactic activity of neutrophils. These observations indicate that functional heterogeneity of bovine neutrophils can be attributed, at least in part, to morphological maturity of cells and to differences in expression of surface antigens.

Signal transduction pathways involved in phagocytic and oxidative burst activities of cytokine-treated bovine neutrophils

In vitro studies were conducted to determine the relative importance of various signal transduction factors involved in the phagocytic and oxidative burst activities of cytokine-primed bovine neutrophils. These neutrophil functions were assayed in the presence of known signal transduction pathways inhibitors which included nicotinamide, staurosporine, genistein, pertussis toxin, RO 20-1724 and U-73122.Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated jugular blood from five healthy Holstein-Friesian heifers. Freshly isolated neutrophils (6 ml, 10 x 106 cells/ml) were incubated separately for 1 h at 37 °C with equal volumes of recombinant human cytokines such as tumour necrosis factor-alpha (500 ng/ml), interleukin-l-alpha (1 ng/ml), granulocyte colony-stimulating factor (25 ng/ml), granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interferon-gamma (10 ng/ml). Aliquots (1.8 ml) of various cytokine-treated neutrophils were exposed to each signal transduction pathways inhibitor for 20 min at 37 °C in the dark. Then, percentage phagocytosis and average number of intracellular bacteria per cell were evaluated microscopically using FITC-labelled opsonised bacteria (Escheria coli 0111:134). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H2O2 production, a measure of oxidative burst, by flow cytometry.Phagocytic activity and H2O2 production by bovine neutrophils treated with various cytokines were increased by 52.4–86.1% and 31.3–58.2%, respectively.These functional activities were significantly (p <0.05) reduced after exposure to different inhibitors of signal transduction pathways. The reduction in phagocytic activity of cytokine-primed neutrophils varied greatly depending on the site of action of various inhibitors, with pertussis toxin and U-73122 being the most inhibitory. In comparison, H2O2 production decreased moderately, with pertussis toxin and U-73122 being the most inhibitory and other inhibitors inducing minimal variations. It was concluded that G-inhibitory proteins (pertussis toxin-sensitive) and phospholipase C play a major role, whereas tyrosine kinase plays a minor role in the phagocytic activity and H2O2 production by cytokine-primed bovine neutrophils.

Haematological response of dogs to canine recombinant granulocyte colony stimulating factor (rcG-CSF)

Three beagle dogs were given 5 μg canine recombinant granulocyte colony stimulating factor (rcG-CSF)/kg/day for 42 days. One day after the first dose the neutrophil count exceeded the pretreatment counts by 3- to 4-fold. A steady and rapid increase occurred during days 1 to 12. In two of the dogs the counts continued to increase, but at a slower rate, from day 14 to 28. The neutrophil count in the third dog decreased steadily from day 14 to 28, although the count remained above those of the presample period and exceeded the reference range for dogs established at the Veterinary Medical Teaching Hospital, University of California, Davis. After day 28 that dog had a rapid increase in neutrophil count that reached similar numbers to the other two dogs at 34 days. At no time was a significant left shift found, although an occasional band neutrophil was observed. In addition, a moderate increase in lymphocyte and monocyte counts were found. The degree of these increases was much less than that of the neutrophils. The leukocyte counts decreased rapidly after the last dose, and 10 days later the counts were similar to those of the pretrial period.Concomitant with the marked neutrophilia, bone marrows showed myeloid hyperplasia. The myeloid: erythroid ratios increased from 1.03–1.65 to 2.77–7.03, and the marrow cellularity increased from approximately 30%–50% to about 85%–100%. Clinical evaluation and serum chemistry panels revealed no adverse affects. We conclude that rcG-CSF effectively sustains an increased neutrophil count without producing significant adverse effects.