Anti-neutrophil cytoplasmic autoantibodies (ANCA) are in the circulation of most patients with pauciimmune necrotizing vasculitis and pauci-immune crescentic glomerulonephritis. The current study demonstrates an effect of these autoantibodies on neutrophil function in vitro. ANCA cause normal human neutrophils to undergo an oxidative burst and degranulate. Both ANCA phenotypes (i.e., cytoplasmicpattern ANCA and myeloperoxidase-specific ANCA) induce neutrophil activation. ANCA sera and purified immunoglobulins significantly increase the release ofreactive oxygen species when compared with controls. ANCA, in a dose-dependent manner, induce the release of primary granule contents. These effects are markedly enhanced by priming neutrophils with tumor necrosis factor. Flow cytometry studies demonstrate the presence of myeloperoxidase on the surface of neutrophils after cytokine priming, indicating that primed neutrophils have ANCA antigens at their surfaces to interact with ANCA. These observations suggest an in vivo pathogenetic role for ANCA. We propose that, in patients with necrotizing vasculitis, ANCA-induced release of toxic oxygen radicals and noxious granule enzymes from cytokine-primed neutrophils could be mediating vascular inflammation.

Polymorphonuclear leukocyte (PMN) respiratory burst was stimulated by heterologous antibodies against PMN granule proteins but not by control antibodies. Fluorescence-activated cell sorter (FACS) analysis of activated PMN demonstrated the presence of two primary granule proteins, proteinase 3 (PR-3) and cationic protein 57 (CAP-57) at the membrane surface. The presence of myeloperoxidase (MPO) at the cell surface of primed and unprimed PMN was confirmed by immunoelectron microscopy. Priming doses of recombinant tumor necrosis alpha (rTNF alpha) enhanced the rate of superoxide (O2-) production by these antibodies and increased the amount of surface protein accessible to these antibodies. Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificities for PMN granule proteins are present in patients with Wegener's granulomatosis, polyarteritis nodosa, and idiopathic and crescentic glomerulonephritis. The demonstration that antibodies against granule proteins activate PMN supports the hypothesis that the vasculitis seen in these diseases is due in part to PMN mediated oxidative injury following PMN stimulation by ANCA.

To cite this article: Ingerslev J, Jankowski MA, Weston SB, Charles LA, the ReFacto Field Study Participants. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays. J Thromb Haemost 2004; 2: 623-8.Summary. Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing onestage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a Bdomain deleted recombinant FVIII (BDDrFVIII, ReFacto Ò ). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard TM (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the onestage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL )1. FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in onestage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the onestage clotting and the chromogenic substrate assay for ReFacto.

ObjectiveSeptic shock has a clinical mortality rate approaching fifty percent. The major clinical manifestations of sepsis are due to the dysregulation of the host's response to infection rather than the direct consequences of the invading pathogen. Central to this initial immunologic response is the activation of leukocytes and microvascular endothelium resulting in cardiovascular instability, lung injury and renal dysfunction. Due to the primary role of leukocyte activation in the sepsis syndrome, a synthetic biomimetic membrane, called a selective cytopheretic device (SCD), was developed to bind activated leukocytes. The incorporation of the SCD along an extracorporeal blood circuit coupled with regional anticoagulation with citrate to lower blood ionized calcium was devised to modulate leukocyte activation in sepsis.DesignLaboratory investigation.SettingUniversity of Michigan Medical School.SubjectsPigs weighing 30-35 kg.InterventionsTo assess the effect of the SCD in septic shock, pigs were administered 30×1010 bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in an extracorporeal circuit containing SCD.Measurements and Main ResultsIn this animal model, the SCD with citrate compared to control groups without the SCD or with heparin anticoagulation ameliorated the cardiovascular instability and lung sequestration of activated leukocytes, reduced renal dysfunction and improved survival time compared to various control groups. This effect was associated with minimal elevations of systemic circulating neutrophil activation.ConclusionsThese preclinical studies along with two favorable exploratory clinical trials form the basis of an FDA-approved investigational device exemption for a pivotal multicenter, randomized control trial currently underway.