TSE Advisory Committee October 14, 2004, Silver Spring, MD

1. TSE Advisory CommitteeOctober 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

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

3. 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.

4. 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

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

6. 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

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

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

9. Hamster blood leukoreduction using human blood parameters AABB specifications • 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 106 white cells.

10. 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. 1999.

11. > 85% < 5 X 106 ~ 3-log Leukoreduction whole blood filtrationcell recovery

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

13. 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

14. Limiting Dilution Titration of Blood Incubate Inoculate Donor Bleed Inoculation of 5 mL total 50 mL 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

16. Titration results566 days post inoculation *Corrected with Poisson distribution (post/pre)% ~ 58%

17. 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.

18. Implications • Leukoreduction is a necessary but not sufficient to remove all blood-borne TSE infectivity. • 5800 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.

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

20. Filtration of endogenous infectivity in Plasma Filtration of plasma through 28 mm Pall Corp. PLF1 Experiment Specimen ID/mL Conditions Frozen/thawed plasma from clinically Challenge 18 - 58 1 affected mice Filtrate 4 - 30 Frozen/thawed plasma from pre- Challenge 0 - 11 2 clinical infection Filtrate 16 - 65 Fresh plasma from symptomatic mice Challenge 8 - 47 3 Filtrate 6 - 46 Brown, P., Cervenakova, L, McShane, L.M., Barber, P., Rubenstein, R., Drohan, W.N. (1999) Transfusion 39, 1169-1178.

21. PrPsc removal by whole blood leukofilters Removal of spiked PrPres and Leukocytes by Filtration PrPres Leukocyte Removal Whole Blood Filter Removal Log10 Log10 Control Brain* Baxter/Asahi 4.0 2.7 -0.4 Fresnius 3.6 3.1 0.2 Macopharma 4.3 2.5 0.3 Pall 4.5 3.5 0.2 * 500 mL of whole blood spiked with 10 mL of microsomal fraction of 10% brain from 263K scrapie hamster Prowse C.V. and Bailey A. Vox Sang (2000) 79, 248