Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the alpha-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment.

Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the ␣-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment. Am. J. Hematol. 61:53-61, 1999.

ABSTRACT:Oxygen is essential therapy for neonates with acute respiratory failure, including those with infections. However, high oxygen levels may be counterproductive for overcoming infections because hyperoxia may kill cells, including dendritic cells that are essential to the emergence of the pulmonary immune system and pivotal in mounting immune responses to infections. We studied the impact of hyperoxia on developing dendritic cells from neonatal cord blood and adult blood monocytes, comparing viability, development of maturation, and endocytic function. Our data suggest that cord blood-derived dendritic cells may be more resistant to hyperoxicinduced cell death than adult blood-derived cells. Moreover, the surviving cells in either group are those that maintain an immature phenotype. This may impair their ability to perform optimal immune function. I nfection is one of the most important causes of mortality and morbidity among newborns with acute respiratory failure (1). Inhaled oxygen is a vital therapy for these infants, despite the potential risks of hyperoxia to the lung, including affecting the DNA integrity of type II pneumocytes (2), and normal immune responses in the lungs (3,4). Hyperoxia alters the pulmonary tissue immune response by up-regulating proinflammatory cytokines and inducing neutrophil infiltration in the alveolar spaces (5). It also increases alveolar macrophage apoptosis (4), further weakening the immune response (3). We investigated whether hyperoxia might likewise interfere with the development of DC, immune cells that are more important for presenting antigen to T cells than are macrophages.While the majority of term infants exposed to hyperoxia recover, occasionally with some residual morbidity, adults under similar conditions do not survive beyond a few days (6,7). To investigate whether this differential response to oxygen is reflected in DC function and viability, we compared the hyperoxic effect on developing DC from neonates versus adults. MATERIALS AND METHODSCell culture and hyperoxia. Pulmonary dendritic cells, which are exposed to the highest levels of oxygen, are predominantly of myeloid origin (8) and resemble monocyte-derived DC from cord or adult peripheral blood (9). After institutional board review and informed consent, cord blood from normal term pregnancies was collected into sterile collection bags and processed within 24 h. Some cord blood was supplied by the St. Louis Cord Blood Bank and solely used for determining the optimal duration of hyperoxia exposure. Although the blood from preterm cord blood would be more reflective of the clinical setting underlying this study, limited volume and, therefore, smaller cell numbers made it impractical. Adult blood was collected by venipuncture from healthy volunteers. The dendritic progenitor cells were extracted from mononuclear cells as described previously (10). Briefly, the mononuclear cells were separated from red cells by sedimentation with a solution of hydroxyethyl starch followed by density centrifugation over ...

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