TSE Advisory Committee October 14, 2004, Silver Spring, MD Removal of blood-borne TSE infectivity by leukoreduction filters Luisa Gregori V.A. Medical Center and University of Maryland - Baltimore

Distribution of infectivity in blood components Plasma Buffy Coat RBC Component Volume Infectivity Recovered Infectivity Normalized 25% 35% 20% 30% 45% 25% 70% WBC 60% platelets 14% plasma 7-10% RBC

Infectivity in blood TSE infectivity is not associated with platelets. Infectivity unlikely to be associated with red cells. Is infectivity uniquely associated with white cells? Significant proportion of infectivity is found in plasma.

Experimental tests of leukoreduction High speed centrifugation does not remove all plasma infectivity. Filtration experiments with TSE infectivity –Brown et al., 1999 –C.V. Prowse & A. Bailey, 2000

Summary Infectivity was not removed by the leukoreduction plasma filter tested. Spiked PrP res was not removed by the leukoreduction whole blood filters tested. Leukofiltration cannot be presumed to remove TSE infectivity without proof.

Leukoreduction conditions Endogenous TSE infectivity in blood No scale down - Full unit of scrapie hamster whole blood Use of the same protocol and equipment used in the blood centers in Canada Leukoreduction of two in-line Leukotrap filtration systems: whole blood and RC-PL

WBF2 AS-3PPP [RBC] Whole Blood Leukoreduced Whole Blood Platelet Poor Plasma Whole Blood WBF2 Pall filter

Whole Blood PL filter RC filter Leukoreduced PPP Leukoreduced PC AS-3 RBC Platelet Concentrate Platelets ATS and Red Cell RCM1 Pall filters

Hamster blood leukoreduction using human blood parameters Collection of a full unit (~ 450 mL) of scrapie infected hamster blood in a few hours. Blood processed within 8 hr from collection. At least 3 log of white cells must be removed by the filter. Cell recovery and level of white cells contamination within specifications. A unit of leukoreduced RBC must contain at least 85% of the original red cells and < 5 X 10 6 white cells. AABB specifications

Leukofiltration validation Measured total blood cell count with cell counter calibrated for hamster blood. Measured white cell count with manual count and by flow cytometry. Did not measure cell fragmentation or microvesicles generation. - Prowse et al

Leukoreduction whole blood filtration cell recovery > 85% < 5 X 10 6 ~ 3-log

WBF2 AS-3PPP [RBC] Whole Blood Leukoreduced Whole Blood Platelet Poor Plasma Whole Blood WBF2 Pall filter Titration 1 pre filtration Titration 2 post filtration

Titration studies Titration of whole blood pre and post leukoreduction filtration Limiting dilution titration method ~ 100 animals for titration (~5 mL) Titration was completed after 566 days post inoculation Brain from every animal was analyzed by Western blot

Limiting Dilution Titration of Blood Inoculation of 5 mL total 50  L each  44 infected of 100 Inoculated  44 infected of 5 mL Inoculated  Approx. 44/5 = 8.8 ID/mL  Poisson Correction 11.6 ± 0.7 ID/mL Inoculate DonorBleed Incubate

Titration results 566 days post inoculation (post/pre)% ~ 58% *Corrected with Poisson distribution

Conclusions ~ 40% of infectivity was retained by the filter. Same percentage of infectivity found in the buffy coat fraction. Consistent with removal of TSE infectivity associated with white cells. Post leukoreduction infectivity is most likely in plasma. TSE infectivity is present in at least two forms: associated with white cells and in plasma. Infectivity did not “wash off” during filtration and filtration did not liberate infectivity.

Implications Leukoreduction is a necessary but not sufficient to remove all blood-borne TSE infectivity ID/unit of whole blood  3400 ID/unit of leukoreduced whole blood. Post leukoreduction infectivity is not cell associated. Need for additional methods to remove cell-free infectivity.

Acknowledgment Rohwer Lab Ellen Elliott Renee Kahn Claudia MacAuley Justin Duzsa Dwayne Moore Sean Carter Fidel Gillin Daryl Butler Lee Preston Johari Barnes Health Canada and Canadian Blood Services Tony Giulivi Nancy McCombie Doug Palmer Paul Birch Pall Corp. US and Canada Sam Coker Bonnie Quanbury-Duern

Filtration of endogenous infectivity in Plasma Filtration of plasma through 28 mm Pall Corp. PLF1 Experiment Conditions SpecimenID/mL Challenge Filtrate Challenge Filtrate Challenge Filtrate Frozen/thawed plasma from clinically affected mice Frozen/thawed plasma from pre- clinical infection Fresh plasma from symptomatic mice Brown, P., Cervenakova, L, McShane, L.M., Barber, P., Rubenstein, R., Drohan, W.N. (1999) Transfusion 39,

PrP sc removal by whole blood leukofilters Removal of spiked PrP res and Leukocytes by Filtration Whole Blood Filter PrP res Removal Log 10 ControlBrain* Baxter/Asahi Fresnius Macopharma Pall Leukocyte Removal Log 10 Prowse C.V. and Bailey A. Vox Sang (2000) 79, 248 * 500 mL of whole blood spiked with 10 mL of microsomal fraction of 10% brain from 263K scrapie hamster