1. TOPIC II Potential Screening Assays to Detect Blood and Plasma Donors Infected with TSE Agents: Possible Criteria for Validation Pedro Piccardo, MD LBPUA, DETTD, OBRR, CBER, FDA FDA TSE Advisory Committee 19 th Meeting 19 September 2006

2. Issue FDA seeks advice from TSEAC regarding potential approaches and issues to consider when validating and evaluating candidate screening tests for vCJD and other TSE infections in donors of blood, plasma and human cells and tissues.

3. Background Concern Three presumptive transfusion-transmitted cases of vCJD infections in UK (2 clinically apparent, 1 preclinical) reported in a small group of individuals receiving non-leukoreduced red blood cell concentrates from 3 separate donors who later developed vCJD.  Red blood cell concentrates transmitted vCJD efficiently.  Other blood components might also pose a risk.

4. Background Unknowns  Concentration of vCJD infectivity in human blood (as human intravenous infectious units)  Time during incubation period when infectivity appears in blood before onset of vCJD (implicated blood donated 18 to 42 mo before donors showed signs of vCJD)  Prevalence of vCJD in blood and plasma donors

5. Aim Discuss desirable performance characteristics and a potential approach to validation of candidate tests for detection of PrP TSE in blood. Note on Nomenclature Consultants to WHO recently recommended designating all abnormally folded forms of the prion protein associated with TSEs as PrP TSE (PrP TSE  PrP Sc and PrP res ).

6. TSEAC 31 Oct 2005 Validation criteria for devices to remove TSE infectivity from blood components ≥ 2 animal models and 2 strains of TSE agent ≥ 1 agent strain derived from cow with BSE or human vCJD Reproducibility of results from separate studies Small animal models most accessible

7. Analytical Studies Exogenous Spikes (I) Potential Use of Exogenous TSE Spike Materials (animal-derived and human-derived) Proof of Principle Detection: test serial dilutions of material Intrinsic Variability of Method (test repeated spikes with same material) Blind Studies (except during early test development, optimization)

8. Analytical Studies Exogenous Spikes (II) Animal-derived Exogenous Spike Materials Demonstrate that TSE spiking materials contained both infectivity and PrP TSE and controls did not. Discriminate between samples of normal human blood spiked with suspensions of solid tissues obtained from TSE-infected animals (brain, other) and blood spiked with similar tissue suspensions from matched uninfected control animals.

9. Potential animal models  Rodents with scrapie, human vCJD, other TSE  Sheep with scrapie, BSE  Cattle with BSE

10. Analytical Studies Exogenous Spikes (III) Human-derived Exogenous Spike Materials Discriminate between samples of normal human blood spiked with suspensions of solid tissues from human TSE-infected and matched control tissues.  Brain  Spleen  Other

11. Analytical Studies Endogenous Infections (I) Animal-derived Endogenously TSE-infected Blood Demonstrate that blood from animals with TSE contained both infectivity and PrP TSE and matched controls did not. Discriminate between blood samples from TSE- infected and matched uninfected control animals.

12. Potential animal models  Rodents with scrapie, vCJD, other TSE  Sheep with scrapie, BSE  Primates with BSE, vCJD if/when available  Two models better than one FDA acknowledges logistical difficulties with non-rodent models.

13. Analytical Studies Endogenous Infections (II) Use of Animal-derived Endogenously TSE-infected Blood Periods of Concern  Overt illness  Latter part of incubation period Confirmation  Bioassays using number of animals sufficient to detect expected levels of infectivity Limit of Detection  Serial dilution of endogenously TSE-infected blood in uninfected blood, assaying by bioassay for infectivity and candidate method for PrP TSE Intrinsic Variability  repeated tests of same material Blind Studies (except early test development, optimization)

14. Potential Clinical Validation Studies (I) Blood samples  vCJD Patients  Patients with other TSEs (if detection to be claimed)  Control Patients Other neurological diseases Aged-matched individuals not having neurological disease  Confirmations All diagnoses confirmed by biopsy or autopsy  Detection during Incubation Period Ideal but probably not logistically feasible

15. Potential Clinical Validation Studies (II) Cadaveric blood samples useful but ideally need comparison with ante-mortem samples in test performance, including false-positive and false-negative results, endpoint dilutions, etc. As for other donor screening tests, the possible effects of potentially interfering substances in blood, including hemolysis, bile, lipemia, etc. evaluated Coded, replicate, randomized samples tested in multiple runs

16. FDA is attempting to assemble or obtain access to collections of TSE biological and control reference materials

17. Potential Clinical Validation Studies (III) The number of TSE blood samples (sCJD or fCJD) available to investigate clinical sensitivity probably limited  Smaller numbers available from patients with vCJD (incubation period samples not feasible) Controls  Samples from subjects with other neurological diseases or non-neurological diseases,  those from TSEs

18. TSE Donor Screening Tests Possible Test Performance Characteristics: Sensitivity (I) Caveat Regarding failure to detect TSEs other than vCJD Unlike those with vCJD, donors who later became ill with other forms of CJD have not been implicated as a source of transfusion-transmitted TSE. It is possible that only in persons with vCJD does infectivity reach levels in blood sufficient to transmit TSE infection. Failure of a candidate test to identify patients with other TSEs would not necessarily rule out a possible value of a test to screen donors for vCJD infection.

19. TSE Donor Screening Tests Possible Test Performance Characteristics: Sensitivity (II) FDA Precedents  Clinical evaluation and validation of screening methods suitable to identify donors with infections require detecting samples from persons with known infections.  Screening tests for blood and tissue donors provide additional safety but generally have not replaced deferral policies.

20. TSE Donor Screening Tests Possible Test Performance Characteristics: Specificity (I) Specificity of an FDA-regulated donor screening test has generally been determined by testing a large number of samples (usually at least 10,000) from healthy suitable donors presumed to be at low risk for the infection of concern. A target specificity of 99.5% or greater has generally been sought.

21. TSE Donor Screening Tests Possible Test Performance Characteristics: Specificity (II) Should a validated TSE test have a lower specificity, its suitability for donor screening would be determined based on evaluation of probable overall risk (predicted adverse consequences of repeatedly reactive results that are false positives) vs benefit (interdiction of blood components and plasma derivative infected with TSE).

22. TSE Donor Screening Tests Confirmation (I) False-positive results especially problematic for TSE screening tests - Incubation periods of TSEs are very long. -Intervention during the TSE incubation period is not available. -Prognosis of TSEs after onset of clinical disease is dire. -Treatments for TSEs are ineffective.

23. TSE Donor Screening Tests Confirmation (II) Notification of donors with unconfirmed repeat-reactive screening test results would have predictable adverse consequences for notified donors.

24. TSE Donor Screening Tests Confirmation (III) The expected adverse public health consequences of notifying a large number of persons having repeatedly reactive false-positive results is problematic. Reliable confirmatory tests detecting PrP TSE or other marker would be desirable, possibly obligatory, for every donor with a repeatedly reactive initial screening test result.

25. TSE Donor Screening Tests Confirmation (IV) The ideal confirmatory test might be a sensitive bioassay for infectivity.  Bioassays for TSE infectivity in human blood are not currently feasible in a donor setting. A possible confirmatory TSE assay might be a supplemental test for PrP TSE based on a principle somewhat different from that of the original screening test but at least as sensitive and specific.

26. TSE Donor Screening Tests Counseling Deferred Donors The predictable adverse consequences of informing deferred donors whose screening tests for TSE infection were repeatedly reactive—with or without reactive confirmatory assays—should be carefully considered in advance.

27. Questions 1.Please comment on pre-clinical analytical studies needed to validate candidate donor screening tests for vCJD and other TSEs. 2. Please comment on clinical studies needed to validate candidate donor screening tests for vCJD and other TSEs. 3.Please discuss the relative merits of feasible technical options to confirm screening test results for vCJD and other TSEs, e.g., bioassays, alternate immunoassays, PrP amplification techniques, etc.