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- Author or Editor: D. S. McVey x
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Abstract
Objective
To develop a flow cytometric assay for detection of platelet-bound IgG in dogs.
Sample Population
Negative-control platelets were obtained from 5 clinically normal Greyhounds. Positive-control platelets were platelets from 1 clinically normal dog, sensitized with dog anti-canine platelet alloantibodies.
Procedure
Washed platelets were incubated with mouse anti-canine IgG conjugated to fluorescein isothiocyanate and analyzed by flow cytometry. Optimal dilution of antibody reagent and dose-response were determined, as were effects on platelet-bound IgG detection of storage time and temperature of K3EDTA-anticoagulated blood samples, variable platelet numbers, and variable filling of K3EDTA evacuated tubes.
Results
A 1:128 dilution of antibody reagent was optimal. There was a linear increase in platelet-bound IgG when normal canine platelets were incubated with increasing concentrations of positive-control serum. Variable numbers of positive-control platelets tested and variable filling of K3EDTA evacuated tubes had no significant effect on platelet-bound IgG concentration. Platelet-bound IgG concentration increased with storage time at room temperature (P = 0.0003), but not when blood was kept cool. Sufficient platelets for assay were able to be isolated from 3 ml of blood from 5 dogs with < 10,000 platelets/μl.
Conclusions
This assay for platelet-bound IgG in dogs is simple, repeatable, and practical. The assay is not affected by platelet count or variable filling of evacuated tubes, and requires only 3 ml of K3EDTA-anticoagulated blood. Blood samples for testing require packaging on ice and overnight delivery but, after arrival at the laboratory, can be refrigerated and analyzed within 72 hours of collection.
Clinical Relevance
Assays for platelet-bound IgG may help in assessing causes and treatment of thrombocytopenia.
Summary
The lipoxygenase metabolites of arachidonic acid have an important role in lymphocyte activation. We used a specific 5-lipoxygenase inhibitor, A-63162, to examine the role of 5-lipoxygenase (5-lo) in equine blood mononuclear cell (bmc) proliferation and leukotriene B4 (ltb 4) synthesis after stimulation with mitogen (phytohemagglutinin, pha) or calcium ionophore (A23187). The A-63162 inhibited pha-induced equine bmc proliferation and, at the same concentration, also inhibited A23187-induced ltb 4 synthesis. The presence of exogenous interleukin 2 (il-2) or the cyclooxygenase inhibitor indomethacin, failed to reverse the immunosuppression caused by A-63162. Further, we found that A-63162, at the concentration that inhibited bmc proliferation and ltb 4 synthesis, had no effect on bmc viability. The addition of the specific protein kinase C inhibitor, H-7, did not inhibit A23187-induced ltb 4 synthesis. Results indicate that 5-lipoxygenase metabolites may have an important role in equine lymphocyte activation and that protein kinase C has no role in regulating ltb 4 production after A23187 stimulation.
SUMMARY
Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbgm-csf)/ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxocity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbgm-csf significantly increased (P < 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbgm-csf-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.
