Magnetic fluorescent microparticles as markers for particle transfer in extracorporeal blood purification.

The microspheres-based detoxification system (MDS) is a combined membrane-adsorption system for extracorporeal blood purification in which adsorbent microparticles are recirculated in an extracorporeal filtrate circuit. Because the plasma filter represents the only barrier between the adsorbents and the patient's blood, there is the potential risk of particle entrance into the patient in case of a membrane rupture. To guarantee first fault safety of the system required for clinical application, magnetic fluorescent microparticles are added as markers to the adsorbent circuit. Detection of these particles in the venous blood line results in immediate shutdown of the pumps. Magnetic beads were functionalized with cresyl violet and tested with an in vitro setup of the particle detector to assess the detection limit in different matrices (water versus blood) as well as the influence of flow rate and particle size on the signal. In addition, biocompatibility and influence of sterilization on the performance of the particles were assessed. Functionalization of the magnetic particles with cresyl violet yielded fluorescent particles that were stable at 4 degrees C for at least 12 months. No leakage of dye was detectable, and the particles were neither cytotoxic nor mutagenic. The particles could be steam sterilized without significant loss in fluorescence intensity. With an in vitro setup of the particle detector, 0.1 mg and 5 mg of particles were reproducibly detectable in water and blood, respectively.

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility.

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.