1. C B E R E R Vostal 10/2005 Labeling Claims for TSE Reduction Studies with Blood Processing Filters Jaro Vostal, M.D., Ph.D. Division of Hematology, OBRR

2. C B E R E R Vostal 10/2005 Normal prion protein (PrPc) Conversion of PrPc to PrPsc Pathologic prion protein (PrPsc) Protease resistant less soluble Accumulated PrPsc and neurotoxicity Altered conformations of PrPc TSE infectivity present

3. C B E R E R Vostal 10/2005 spleen stomach CNS neuron CNS Transport of TSE from peripheral inoculum to the CNS

4. C B E R E R Vostal 10/2005 Asymptomatic BSE infected sheep Healthy sheep BSE infected sheep Blood transfusion BSE transmission by blood transfusion Houston, F. et al. Lancet 2000 Hunter, N. et al. J. Gen Virology, 2002 X X

5. C B E R E R Vostal 10/2005 Epidemiological evidence for vCJD transmission by blood transfusion in humans Llewelyn, C.A. et al, Lancet 363: 417-421, 2004 Peden, A.H. et al. Lancet 364: 527-529, 2004 The UK National CJD surveillance system identified individuals (48 total, 17 alive) who received blood products from 15 donors who later became diagnosed as vCJD cases 2 living recipients were subsequently diagnosed with vCJD –1 developed and died from symptoms of vCJD –1 died for unrelated cause and was unsymptomatic for vCJD but had PrPres identified in spleen Both received non-leukoreduced red cells

6. C B E R E R Vostal 10/2005 Issues to consider for validation of devices and processes used to reduce TSE infectivity in human blood Distribution of TSE infectivity in blood Cell-associated –Intracellular vs extracellular Free floating in plasma –Physical attributes of infectivity (aggregates, fibrils, microvesicles) Interaction of the individual units of infectivity with the devices Distribution during and after processing –(microparticle generation)

7. C B E R E R Vostal 10/2005 Steps to validate TSE reduction efficacy claims In vitro spiking studies of TSE infectivity into human blood –Detection of infectivity by surrogate markers (PrPres) or by bioassay Endogenous TSE infectivity in an animal model –Detection of infectivity by bioassay

8. C B E R E R Vostal 10/2005 Predictive value of animal models for human situation Comparability of animal blood to human blood –Cell type number, size and physical properties of blood cells –Interaction of animal and human blood cells with different materials Transmissibility or infectivity of a TSE agent may be influenced by –Strain of agent used –The dose of agent –Distribution of infectivity in blood –Distribution of normal prion ?

9. C B E R E R Vostal 10/2005 Hematologic values for various species SpeciesRBC count x 10 6 / uL RBC MCV (fL) WBC Count x 10 3 / uL Platelet x 10 3 / uL Sheep10-13354-12500 Golden Hamster 6-760-705-10500-800 Mouse9-11507-12250-500 Human4-586-984-11150-450 Schalm, O.W. Veterinary Hematology, 2 nd ed. Lea & Febiger, Philadelphia 1965 Harrison, Principles of Internal Medicine, 17 th ed. McGraw-Hill, 1994

10. C B E R E R Vostal 10/2005 PrPc expression on blood cells of different species Vostal, J.G. and Holada, K. Transf Med Rev. 15: 268-281, 2001

11. C B E R E R Vostal 10/2005 Validation of TSE infectivity reduction is only one part of the evaluation process TSE reduction devices also need to be evaluated for their impact on transfusion product quality Evaluation of red cells, platelets or plasma FDA follows a standardized evaluation approach to each transfusion product based on previous experience with devices that process transfusion products (e.g. leukoreduction filters)

12. C B E R E R Vostal 10/2005 Leukoreduction: the process of reducing the total number of leukocytes in a transfusion component Methods:filtration of blood products or collection of products by apheresis Use of leukoreduced blood products has been associated with reduction of –febrile non hemolytic transfusion reactions –Alloimmunization –Cytomegalovirus (CMV) No claims by manufacturers for their devices beyond meeting the criteria for leukoreduction –US <5x10 6 leukocytes per transfusion product –Europe <1x10 6 leukocytes per transfusion product

13. C B E R E R Vostal 10/2005 Validation of Leukoreduction Filters-Efficacy Quantitation of leukocytes in particular blood product before and after filtration –Whole blood, red cells, platelets, plasma Define timing of leukoreduction from time of collection Explore effects of temperature on filtration efficacy (room vs cold temperatures) Validation specific for particular anticoagulant

14. C B E R E R Vostal 10/2005 Validation of Leukoreduction Filters-Safety Biocompatibility and integrity of materials Effect on cellular products –In vitro recovery (85%) –Hemolysis at end of storage (<1%) –In vivo recovery of radiolabelled cells (platelets and red cells) Plasma – –Levels and function of plasma proteins –Complement activation

15. C B E R E R Vostal 10/2005 FDA proposal for validating a claim of reducing TSE infectivity in human blood products Demonstrate reduction of endogenous TSE infectivity by bioassay in two animal models (rodent and sheep) Use full scale blood unit and leukoreduction filter TSE infectivity from BSE or vCJD strain Reduction of PrPsc in blood product will be considered supportive but not sufficient for a claim Study performed at two separate sites to minimize issues of cross contamination and differences in laboratory practice Study size sufficient to support statistically valid conclusion

16. C B E R E R Vostal 10/2005 Question 1:Are the FDA’s proposed minimal criteria for validation of TSE infectivity reduction by filtration adequate and appropriate? Please comment on the following points A) Rationale for the use of specific animal models to study the properties of blood-borne TSE infectivity (Are experiments in rodents sufficient, or should experiments also be done in sheep?) B) Is it necessary that each experiment should be done at two separate laboratory sites (i.e. to ensure reproducibility, and accuracy of clearance)? C) General description of informative scaled-down processes for reducing TSE infectivity in blood

17. C B E R E R Vostal 10/2005 Question 1:Are the FDA’s proposed minimal criteria for validation of TSE infectivity reduction by filtration adequate and appropriate? Please comment on the following points D) Levels of clearance acceptable for claims of reduced TSE infectivity in blood components as used in clinical settings E) Estimated logs of clearance of TSE infectivity required to conclude that blood filters have effectively removed infectivity from blood components F) Methodology appropriate to use in evaluating TSE agent clearance (bioassays for infectivity, Western blot or other assay for prion proteins, other methods)

18. C B E R E R Vostal 10/2005 Question 2: Does the FDA’s proposed labeling for a filter meet the appropriate criteria for a claim of reduction of TSE infectivity in blood or blood components? A) This filter (device) has been shown to reduce TSE infectivity in blood from an infected animal model. or B) This filter (device) has been shown to reduce transmission of TSE infectivity by transfusion in an animal model. and (A+C or B+C) C) Due to lack of feasibility, studies have not been performed to validate this claim in a human population.