Evaluation of removal of prion infectivity from red blood cells with prion reduction filters using a new rapid and highly sensitive cell culture-based infectivity assay.

摘要

BACKGROUND: The clearance of infectious prions from biologic fluids is usually quantified by bioassays based on intracerebral inoculation of hamsters or mice; these tests are slow, cumbersome, imprecise, and very expensive. In the present study we describe the use of a new and highly sensitive cell culture-based infectivity assay to evaluate the performance of several prion removal prototype filters. STUDY DESIGN AND METHODS: Five units of 1- to 2-day-old ABO-compatible human red blood cells (RBCs) in saline-adenine-glucose-mannitol were obtained from an AABB-accredited blood bank. The 5 units were combined to create a homogenous pool. Scrapie-infected mouse brain homogenate of a Rocky Mountain Laboratory strain was added to the pooled RBCs. The pooled RBCs were divided into 300-mL aliquots, which were filtered with either standard leukoreduction filter or four prototypes of prion reduction filter. The levels of prion infectivity in the pre- and postfiltration samples were measured with a cell culture-based standard scrapie cell assay (SSCA). RESULTS: All the 22-layer prion reduction filters removed prion infectivity below the limit of detection of the SSCA (reduction in prion infectivity > or =2.0 log(10)LD(50)/mL) while the 10-layer variant showed some residual infectivity. CONCLUSIONS: These results demonstrate the utility of a highly sensitive cell culture-based infectivity assay for screening prion reduction filters. The use of this type of in vitro infectivity assay will substantially help expedite the screening and discovery of devices aimed at reducing the risk of variant Creutzfeldt-Jakob disease transmission through blood transfusion.