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Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9-36.7%, but was eliminated by the use of the F(abЈ) 2 fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs') test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (Ͻ30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.Key words: Antibody classes; Coombs' test; Direct immunofluorescence; Erythrocyte antibody; Flow cytometry. Immune-mediated hemolytic anemia (IMHA) is an immunohematologic disorder in which destruction of red blood cells is accelerated by the attachment of antibody, with or without complement, to the erythrocyte membrane. Antibodies may be directed against unaltered red blood cells (primary or idiopathic) or against erythrocytes that have been antigenically altered through interaction with secondary causes, including drugs, neoplasia, and infectious diseases.1-6 The relative frequencies of primary and secondary IMHA in dogs are 43% and 57%, respectively. 7The diagnosis is confirmed by the presence of spherocytosis, autoagglutination, or by a positive direct antiglobulin (Coombs') test that has been validated for use in the species of interest. [8][9][10][11][12] The Coombs' test is based on detection of agglutination or clumping of erythrocytes after addition of an anti-species polyvalent mixture of antibody to immunoglobulin M (IgM), IgG, and complement protein C 3 . Serial dilutions of the polyvalent Coombs' reagent are prepared and tested against patient erythrocytes to provide the proper concentration equivalence between antiglobulin and the antibody-coated erythrocytes at which agglutination occurs. Because of the...

Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni. Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24-72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-infected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.

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Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.

Fusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. Its major virulence factor is leukotoxin, a high-molecular-weight secreted protein, primarily toxic to ruminant leukocytes. In this study, bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin and the cytotoxicity was analyzed by flow cytometry and scanning and transmission electron microscopy. At very low toxin concentrations, polymorphonuclear leukocytes (PMNs) showed activation, as indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability of F. necrophorum leukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.

Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24-72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4 degrees C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6- to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8- to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4ϩ) or cytotoxic (CD8ϩ) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested agerelated changes in the cell-mediated immune system in horses. Absolute circulating CD4ϩ and CD8ϩ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.