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TSE Advisory Committee October 14, 2004, Silver Spring, MD
Removal of blood-borne TSE infectivity by leukoreduction filters
Luisa GregoriV.A. Medical Center and
University of Maryland - Baltimore
Distribution of infectivity in blood components
Plasma
BuffyCoat
RBC
ComponentVolume Infectivity
Recovered
InfectivityNormalized
25%
35%
20%
30%
45%
25%
70% WBC 60% platelets 14% plasma7-10% 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 PrPres 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-3 PPP
[RBC]
Whole Blood
Leukoreduced Whole Blood
Platelet Poor Plasma
Leukoreduction Whole Blood Filtration
Whole Blood WBF2 Pall filter
Whole Blood
PL filter
RC filter
Leukoreduced
PPP
Leukoreduced
PC
AS-3
RBCPlatelet
Concentrate
Leukoreduction RC-PL Filtration
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 106 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. 1999.
Leukoreduction whole blood filtrationcell recovery
Volume (ml) WBC % rec
Log10
reduct RBC % rec Platelets % rec
WB pre 448.5 2.1E+09 100 0 3.7E+12 100 1.4E+11 100
WB post 424.2 3.0E+06 0.15 2.9 3.6E+12 100 1.5E+11 100
PPP 179 3.0E+05 0.02 3.8 0 0 1.1E+10 8RBC +AS3 305.9 2.0E+06 0.15 3 3.1E+12 86 1E+11 71
> 85%< 5 X 106
~ 3-log
WBF2
AS-3 PPP
[RBC]
Whole Blood
Leukoreduced Whole Blood
Platelet Poor Plasma
Leukoreduction Whole Blood Filtration
Whole Blood WBF2 Pall filter
Titration 1pre filtration
Titration 2post 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
Inoculationof 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 Donor Bleed
Incubate
Days post inoculation
0 100 200 300 400 500 600
Nu
mb
er o
f an
imal
s
Whole blood
Leukoreduced whole blood
Titration results566 days post inoculation
(post/pre)% ~ 58%
*Corrected with Poisson distribution
Sample volume
inoculated mL
total animals
inoculated
total animals infected
Titer* ID/mL
standard deviation
fractional distribution
of infectivity
Whole Blood
5.2 104 50 13.1 1.6 1
Leukoreduced Blood
5.4 108 34 7.6 1.2 0.58
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.
• 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.
AcknowledgmentRohwer 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 Specimen ID/mL
Challenge 18 - 58
Filtrate 4 - 30
Challenge 0 - 11
Filtrate 16 - 65
Challenge 8 - 47
Filtrate 6 - 46
Frozen/thawed plasma from clinically
affected mice
Frozen/thawed plasma from pre-
clinical infection
Fresh plasma from symptomatic mice
1
2
3
Brown, P., Cervenakova, L, McShane, L.M., Barber, P., Rubenstein, R., Drohan, W.N. (1999) Transfusion 39, 1169-1178.
PrPsc removal by whole blood leukofilters
Removal of spiked PrPres and Leukocytes by Filtration
Whole Blood FilterPrPres Removal
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
Leukocyte
Removal Log10
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