A BioSpleen Blood Cleansing Device for Sepsis Therapy and Diagnostics

Abstract

Sepsis is a major cause of deaths of patients in state-of-the-art hospital intensive care units, affecting about 750,000 Americans each year. Pathogens, including bacteria, fungi, and viruses, which have invaded the bloodstream, activate a systematic immune response that often leads to death when the pathogen load is significantly high or the immune system is depressed. Blood cultures, and molecular diagnostic tests that directly detect the presence of live bacteria and pathogen DNA in blood, fail to detect bloodstream infections in most septic patients. Thus, it is a daunting task that can rapidly determine the presence of pathogenic materials in blood to guide therapy, and decrease the incidence of sepsis. Here we describe an biologically inspired blood cleansing device that can remove pathogens and endotoxins from septic blood flowing at high rates and clearance efficiencies without knowing the pathogen type1. This was accomplished using magnetic nanoparticles coated with a generic opsonin–Mannose Binding Lectin (MBL)–that binds various bacteria, fungi, viruses and endotoxin, and by designing a microfluidic configuration that mimics the microarchitecture and cleansing function of the human spleen. We also applied this capability to distinguish septic patients from non-infection trauma patients by measuring PAMPs (pathogen associated molecular patterns) unit in blood. We engineered the biospleen device using computational simulation programs, which allowed us to optimize flow conditions for magnetic separation of pathogens from blood. The core parts of the microfluidic device were fabricated using hot-embossed medical grade polycarbonate materials; the outer surfaces of the machined parts were then bonded with a thin (125 µm) layer of transparent polycarbonate film. The engineered MBL opsonin was produced and immobilized on magnetic nanoparticles (128 nm) to capture a variety of pathogens and endotoxin without pre-identifying the pathogens. To determine the functionality of the device, we spiked various types of pathogens into human whole blood anticoagulated with heparin (1.5 U/mL), and then flowed the blood samples through the device. Blood-cleansing efficiencies were quantified using blood cultures and ELISA. To provide proof-of-concept for our blood cleansing device therapy, we carried out testing in rats. Blood is drawn from a rat infected with pathogens or endotoxin via a jugular catheter at a flow rate of 10 mL/h, and is continuously mixed with magnetic nanobeads coated with FcMBL. The blood and magnetic opsonins are then flowed through an in-line mixer to promote bead binding, and into millimeter-sized fluidic channels of the magnetic separator unit of the biospleen device. Stationary magnets pull the magnetically opsonized pathogens and toxins from the blood through sinusoid-like slits, and into a parallel saline channel; the cleansed blood is then returned back to the rat. The biospleen treatment significantly reduced pathogen levels in blood and lung of rats infected with S. aureus. To prove its diagnostic capability, we measured PAMPs unit in blood collected from animals and patients with sepsis. The FcMBL ELLecSA was able to detect PAMPS present on 85% of clinical isolates representing 47 of 55 different pathogen species. The PAMP assay rapidly detected the presence of active infection in animals, even when blood cultures were negative. It also distinguished infection from non-infection trauma-related inflammation with a higher specificity than the conventional clinical sepsis biomarker, C-reactive Protein (CRP)2.