Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs.

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.

Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes.

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.

Alterations in normal canine platelets during storage in EDTA anticoagulated blood.

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.

Platelet size, platelet surface-associated IgG, and reticulated platelets in dogs with immune-mediated thrombocytopenia.

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.