The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an
anticoagulant into blood collection tubes.
Heparin,
ethylene diamine tetraacetic
acid (
EDTA), and
citrate are the most commonly used
anticoagulants. Novel
anticoagulants such as
hirudin are also available but are not broadly used. Despite the notion that certain
anticoagulants may influence assay performance, a systematic comparison between traditional and novel
anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared
hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of
anticoagulant did not influence the performance of the
hemolysis assay. However,
hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional
anticoagulants citrate and
heparin were more appropriate for the coagulation and
cytokine secretion assays. The results also suggest that traditional immunological controls such as
lipopolysaccharide (LPS ) are not reliable for understanding the role of
anticoagulant in the assay performance. We observed differences in the test results between
hirudin and traditional
anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each
anticoagulant and consider individual nanoparticles on a case-by-case basis.