Determining the efficacy of disinfectants at nucleic acid degradation.

AIMS: Nucleic acids, particularly antibiotic resistance genes, are commonly found on surfaces within healthcare environments, with levels not reducing following cleaning. Within the UK, there are no regulations for testing disinfectants against nucleic acids. METHODS AND RESULTS: A series of commonplace in vitro methods were used to determine disinfectant-induced physical and functional damage to various nucleic acids; RNA (10 µg), genomic DNA (2 µg), and plasmids (1 µg). Using these methods, the optimal residence time (10 minutes) and working concentration (10%) were determined for a new disinfectant. Furthermore, comparison of disinfectants with different active ingredients including lactic acid (LA), sodium hydroxide (NaOH), chloroxylenol (PCMX), and quaternary ammonium compounds (QACs), were compared to controls. All disinfectants showed greater degradation by gel electrophoresis of genomic DNA and RNA than of purified plasmids. Functional analysis using quantitative polymerase chain reaction (qPCR) and polymerase chain reaction (PCR) demonstrated that no disinfectant tested (apart from control) could damage DNA to the level where PCR amplification was not possible, and only the NaOH reagent could achieve this for RNA. CONCLUSIONS: The set of methods described herein provides a platform for future standardization and potential regulation regarding monitoring cleaning solutions for their activity against nucleic acids.

Abnormal clot microstructure formed in blood containing HIT-like antibodies.

INTRODUCTION: Thrombosis is a severe and frequent complication of heparin-induced thrombocytopenia (HIT). However, there is currently no knowledge of the effects of HIT-like antibodies on the resulting microstructure of the formed clot, despite such information being linked to thrombotic events. We evaluate the effect of the addition of pathogenic HIT-like antibodies to blood on the resulting microstructure of the formed clot. MATERIALS AND METHODS: Pathogenic HIT-like antibodies (KKO) and control antibodies (RTO) were added to samples of whole blood containing Unfractionated Heparin and Platelet Factor 4. The formed clot microstructure was investigated by rheological measurements (fractal dimension; df) and scanning electron microscopy (SEM), and platelet activation was measured by flow cytometry. RESULTS AND CONCLUSIONS: Our results revealed striking effects of KKO on clot microstructure. A significant difference in df was found between samples containing KKO (df = 1.80) versus RTO (df = 1.74; p < 0.0001). This increase in df was often associated with an increase in activated platelets. SEM images of the clots formed with KKO showed a network consisting of a highly branched and compact arrangement of thin fibrin fibres, typically found in thrombotic disease. This is the first study to identify significant changes in clot microstructure formed in blood containing HIT-like antibodies. These observed alterations in clot microstructure can be potentially exploited as a much-needed biomarker for the detection, management and monitoring of HIT-associated thrombosis.

Artificial shear stress effects on leukocytes at a biomaterial interface.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device-related infections and provide insight into the development of pump thrombosis. Biomaterials-DLC: diamond-like carbon-coated stainless steel; Sap: single-crystal sapphire; and Ti: titanium alloy (Ti6 Al4 V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s-1 or 1000 s-1 ). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro-inflammatory cytokines. L-selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L-selectin expression. Sap and Ti blunted the LPS-stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis.