Serologic detection of Angiostrongylus vasorum infection in dogs.

Angiostrongylus vasorum, French Heartworm, is a metastrongylid nematode infecting the pulmonary arteries and right heart of wild and domestic canids in various regions of the world. Infection in dogs can result in fatal cardiopulmonary disease. A single endemic focus of A. vasorum in North America occurs in the southeastern portion of Newfoundland, Canada. Dogs are currently diagnosed by detection of first-stage larvae shed in feces using the Baermann technique or fecal flotation. However, these procedures may lack sensitivity due to intermittent fecal larval shedding. The potential for using detection of circulating worm antigen for diagnosis was investigated by developing a sandwich-ELISA using rabbit anti-whole adult worm antiserum. This test detected circulating antigen in sera from 22/24 Baermann positive dogs naturally infected with A. vasorum. Negative results (0/52) were obtained from sera collected from Baermann negative dogs from outside of the endemic region, and from sera (0/30) from dogs from non-endemic regions that were infected with Crenosoma vulpis, the fox lung worm. Receiver operating curve analysis gave a specificity of 100% and a sensitivity of 92% for the sandwich-ELISA at an optical density cut-off of 0.19. Subsequently, 239 dogs from Newfoundland displaying clinical signs of cardiopulmonary disease, were examined using both the Baermann fecal examination and the sandwich-ELISA. Larvae were detected in 10% (24/239) of these dogs by fecal examination, whereas the sandwich-ELISA detected circulating antigen of A. vasorum in serum from 18.8% (45/239) of the dogs. This suggests that fecal diagnostics may have missed approximately half of the A. vasorum infected dogs, and that the sandwich-ELISA may be a useful tool in the diagnosis of this parasite.

Impact of the patient population on the risk for heparin-induced thrombocytopenia.

The frequency of immune heparin-induced thrombocytopenia (HIT) varies among prospective studies. It is unknown whether this is caused by differences in the heparin preparations, the patient populations, or the types of serologic assay used to confirm the diagnosis. Seven hundred forty-four patients were studied from 3 different clinical treatment settings, as follows: unfractionated heparin (UFH) during or after cardiac surgery (n = 100), UFH after orthopedic surgery (n = 205), and low-molecular-weight heparin (LMWH) after orthopedic surgery (n = 439). Both an activation assay and an antigen assay were used to detect heparin-dependent IgG (HIT-IgG) antibodies. By activation assay, the frequency of HIT-IgG formation ranged from a low of 3.2% in orthopedic patients receiving LMWH to a high of 20% in cardiac patients receiving UFH; by antigen assay, the corresponding frequencies ranged from 7.5% to 50%. Both UFH use (P =.002) and cardiac surgery (P =.01) were more likely to be associated with HIT-IgG formation. However, among patients in whom HIT-IgG formed and who were administered UFH, the probability for HIT was higher among orthopedic patients than among cardiac patients (by activation assay: 52.6% compared with 5%; odds ratio, 21.1 [95% CI, 2.2-962.8]; P =.001; by antigen assay: 34.5% compared with 2.0%; odds ratio, 25.8 [95% CI, 3.2-1141]; P <.001). It is concluded that there is an unexpected dissociation between the frequency of HIT-IgG formation and the risk for HIT that is dependent on the patient population. HIT-IgG antibodies are more likely to form in patients who undergo cardiac surgery than in orthopedic patients, but among patients in whom antibodies do form, orthopedic patients are more likely to develop HIT. (Blood. 2000;96:1703-1708)

Activation of platelets by sera containing IgG1 heparin-dependent antibodies: an explanation for the predominance of the Fc gammaRIIa "low responder" (his131) gene in patients with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder caused by heparin-dependent IgG (HIT-IgG) that recognizes a complex of heparin and platelet factor 4 (PF4), leading to platelet activation via the platelet Fc gammaIIa receptors (Fc gammaRIIa). Not all patients who generate HIT-IgG in response to heparin develop HIT, however, possibly because of observed differences in the ability of platelets from healthy individuals to be activated by HIT sera. It is known that a polymorphism in the platelet Fc gammaRIIa plays an important role in determining platelet reactivity to murine platelet-activating monoclonal antibodies of the IgG1 subclass: homozygous arg131 ("high responder" or HR) platelets respond well, and homozygous his131 ("low responder" or LR) platelets respond poorly, respectively, to these murine monoclonal antibodies. We sought to determine whether the differing risk for HIT among patients who receive heparin, as well as the variable platelet reactivity to HIT sera, could be explained by preferential activation by HIT-IgG of platelets bearing a particular Fc gammaRIIa phenotype. We found that the LR Fc gammaRIIa gene frequency was significantly overrepresented among 84 HIT patients, compared with that of 264 control subjects (0.565 versus 0.471; p = 0.03). We studied the subclass distribution of HIT-IgG against its major antigen, heparin/PF4 complexes, and found that 55 of 61 (90%) HIT sera expressed IgG1 antibodies either alone (n = 47) or in combination with IgG2 (n = 5) or IgG3 (n = 3). We then compared the platelet-activating profile of HIT sera with murine platelet-activating monoclonal antibodies. As expected, the murine IgG1 monoclonal antibodies preferentially activated platelets from homozygous HR individuals. In contrast, however, the LR homozygous platelets exhibited the greatest reactivity to HIT sera that contained predominantly anti-heparin/PF4 antibodies of the IgG1 subclass. We conclude that the significant overrepresentation of the LR (his131) gene among patients with HIT may be explained by the preferential activation of LR Fc gammaRIIa platelets by HIT antibodies of the IgG1 subclass, which is the predominant immunoglobulin subclass generated in HIT.

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model).

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.

Production of monoclonal antibodies specific for antigens derived from tissue of chinook salmon (Oncorhynchus tshawytscha) affected with plasmacytoid leukemia.

Two distinct monoclonal antibodies (MAB) were prepared for testing with kidney, spleen, and retrobulbar tissue imprints made from chinook salmon (Oncorhynchus tshawytscha) affected with plasmacytoid leukemia. (PL). Hybridomas were prepared from mice immunized with whole and lysed cells purified from renal or retrobulbar PL-positive tissues, which had been obtained from naturally and experimentally infected fish from British Columbia, Canada. The MAB reacted with at least 4 morphologically different cell types; fluorescence was associated with the plasma membrane and cytoplasm. The MAB also reacted with kidney imprints made from chinook salmon affected with a PL-like lymphoproliferative disease in California, indicating that these 2 diseases might be caused by a similar agent. The MAB did not react with any of the kidney or spleen imprints made from wild chinook salmon collected from a river in Ontario, Canada.