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

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

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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

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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.

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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

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Whole Blood WBF2 Pall filter
Whole Blood
WBF2
AS
-
3
PPP
Platelet Poor Plasma
Leukoreduced
Whole Blood
RBC
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Platelets ATS and Red Cell RCM1 Pall filters
Leukoreduced
Leukoreduced
AS-3
Platelet Concentrate
RBC
PC
PPP
PL filter
Whole
Blood
RC filter
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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
  cells.

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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.

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Leukoreduction whole blood filtrationcell
recovery
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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
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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

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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

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(No Transcript)
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Titration results566 days post inoculation
Corrected with Poisson distribution
(post/pre) 58
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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.

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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.

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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
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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
Filtrate
4 - 30
affected mice
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.
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PrPsc removal by whole blood leukofilters
Removal of spiked PrPres and Leukocytes by
Filtration
PrPres
Leukocyte
Removal
Whole Blood Filter
Log10
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
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