Removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter.

BACKGROUND: Two recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In the absence of a preclinical screening test, removal of the infectious agent by processing is the only means by which risk to recipients of blood from donors with inapparent vCJD infections can be eliminated. STUDY DESIGN AND METHODS: In the endogenous infectivity study, a pool of 500 mL of whole blood was collected into CP2D anticoagulant from 263K-strain scrapie-infected hamsters, processed into 300 mL of red cells (RBCs), and then passed through a prion removal filter. Pre- and postfiltration samples were tested for PrP(sc) by Western blot and for infectivity by inoculation of healthy hamsters. In the exogenous (spiking) infectivity study, 30 mL of 10 percent (wt/vol) scrapie-infected brain homogenates was added to 270 mL of human RBCs and then filtered. Levels of PrP(sc) and infectivity were determined by Western blot and bioassay. RESULTS: In the endogenous infectivity study, the prefiltered RBCs transmitted disease to 6 of 43 animals, whereas the postfiltered RBCs did not transmit disease to any of 35 animals, and a barely visible prefiltration PrP(sc) Western blot signal was reduced below the level of detection in the postfiltration sample. In the exogenous (spike) study, infectivity was reduced by 3.7 log LD50 per mL, from 9.2 to 5.5 log LD50 per mL. CONCLUSION: The new filter was effective in removing both infectivity and PrP(sc) from RBCs. The use of this type of filter should reduce the risk of vCJD transmission through blood transfusion.

Polymerase chain reaction method to identify Down syndrome model segmentally trisomic mice.

The Ts65Dn segmentally trisomic mouse possesses an extra copy of a segment of chromosome 16 translocated to chromosome 17. This segment includes the mouse homolog of the Down syndrome critical region of human chromosome 21. The Ts65Dn mouse serves as a useful model to study the developmental regulation of the Down syndrome phenotype. To identify mice bearing the extra chromosome 16 segment, we developed a polymerase chain reaction (PCR) method as an alternative to karyotyping. Conditions under which segments of genes on chromosome 16 (App and Dyrk1a) could be coamplified with a control gene on chromosome 8 (Acta1) so that the yield of each PCR product was proportional to the amount of its template were determined. The amplification products were resolved and quantified by two methods. In the first method, the DNA segments were separated by agarose gel electrophoresis and stained with ethidium bromide. The fluorescence yields were quantified by photodensitometry. In the second method, the fragments were resolved and quantified by the high-performance DNA analysis system, a high-throughput, multichannel, microcapillary electrophoresis instrument. The results of both methods were within 10% of the expected ratio of 1.5. Application of these methods has allowed the maintenance of a Ts65Dn breeding colony through six generations and should permit the precise and efficient identification of trisomic and disomic animals at any developmental stage with minimally invasive procedures.