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