Case study: contamination of heparin with oversulfated chondroitin sulfate.

In late 2007 and early 2008, a cluster of adverse events in patients receiving Heparin Sodium Injection occurred in the United States and in some countries in Europe. The adverse events were reported as being "allergic type" reactions, chiefly characterized by acute hypotension, nausea, and shortness of breath. The root cause of the cluster of adverse events was determined to be a contamination of the heparin by oversulfated chondroitin sulfate. The isolation and structure determination of this contaminant was accomplished by an FDA-led consortium of academic and government laboratories and independently by Baxter Healthcare, whose vial products were first identified in the USA as being associated with the adverse events. Oversulfated chondroitin sulfate was shown to produce acute hypotension in animal models, demonstrating that it was most likely the causative agent responsible for certain of the reported adverse events in patients receiving the contaminated heparin products.

Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.

Review of immobilized antimicrobial agents and methods for testing.

Antimicrobial surfaces for food and medical applications have historically involved antimicrobial coatings that elute biocides for effective kill in solution or at surfaces. However, recent efforts have focused on immobilized antimicrobial agents in order to avoid toxicity and the compatibility and reservoir limitations common to elutable agents. This review critically examines the assorted antimicrobial agents reported to have been immobilized, with an emphasis on the interpretation of antimicrobial testing as it pertains to discriminating between eluting and immobilized agents. Immobilization techniques and modes of antimicrobial action are also discussed.

Structure elucidation and biological activity of the oversulfated chondroitin sulfate contaminant in Baxter heparin.

From late December 2007 to February 2008, the number of adverse responses to heparin infusions rose noticeably above baseline levels in North America, ultimately resulting in a widespread recall of all heparin vial products made by Baxter Healthcare. Using various analytical techniques and the de novo synthesis of a fully sulfated chondroitin sulfate (FSCS) derivative, the authors have confirmed the identity of the contaminant as an oversulfated chondroitin sulfate (OSCS) and have also defined the heterogeneity and concentration of this contaminant in various lots of heparin. Using both contaminated heparin products and the synthetically produced derivative, the authors have shown that the OSCS produces a dose-dependent hypotension in both pigs and rats and that the response in rats can be abrogated with bradyzide, a rodent-selective B(2) bradykinin receptor antagonist. The no observed effect level (NOEL) for this contaminant appears to be approximately 1 mg/kg, corresponding to a contamination level in finished lots of heparin of approximately 3%. Using human plasma, the OSCS derivative was shown to activate kallikrein. These data provide insight into the etiology of the adverse events, particularly refractory hypotension, observed in patients who were exposed to heparin contaminated with OSCS.

Identification of a simple and sensitive microplate method for the detection of oversulfated chondroitin sulfate in heparin products.

Heparin is a commonly implemented anticoagulant used to treat critically ill patients. Recently, a number of commercial lots of heparin products were found to be contaminated with an oversulfated chondroitin sulfate (OSCS) derivative that could elicit a hypotensive response in pigs following a single high-dose infusion. Using both contaminated heparin products and the synthetically produced derivative, we showed that the OSCS produces dose-dependent hypotension in pigs. The no observed effect level (NOEL) for this contaminant appears to be approximately 1mg/kg, corresponding to a contamination level of approximately 3%. We also demonstrated that OSCS can be identified in heparin products using a simple, inexpensive, commercially available heparin enzyme immunoassay (EIA) kit that has a limit of detection of approximately 0.1%, well below the NOEL. This kit may provide a useful method to test heparin products for contamination with oversulfated GAG derivatives.