# Presentation: 388992

## 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**

## Whole Blood WBF2

**WBF2**

**AS**

**-**

**3**

**PPP**

**[RBC]**

**Whole **
**Blood **

**Leukoreduced **

**Whole Blood**

**Platelet Poor Plasma**

**Whole Blood WBF2**
** ****Pall filter**

## Platelets ATS and 

**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. 1999.**

## Leukoreduction whole blood filtrationcell recovery

## Titration  1

**WBF2**

**AS**

**-**

**3**

**PPP**

**[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 Donor**

**Bleed**

**Incubate**

## Slide 15

## 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. **
**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.**

## 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**

**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 PrP****res**** and Leukocytes by Filtration**

**Whole Blood Filter**

**PrP****res**

**Removal **

**Log****10**

**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 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 **