An Analysis of Parvo B 19 In Source Plasma Andrew Conrad Ph.D.

1. An Analysis of Parvo B 19In Source PlasmaAndrew Conrad Ph.D.

2. INTRODUCTION • We are currently testing for Parvo B 19 in an effort to remove the high titer samples from pools for manufacture. • 40% of individual samples tested were IgG positive 75% of those had low level viremia. • Removal of all Parvo B 19 is not feasible and would cause dramatic shortages of plasma.

3. OBJECTIVE • We developed a mechanism to eliminate the high titer, antibody negative, (Infectious?) Parvo B19 samples from plasma pools for manufacture.

4. Robotic Pooling Device

5. Steps to Build Pool a (8x8x8)512 Member Pool The 8 Tecan pipettes draw from all (colored) ROWS in COLUMN Y1 in LAYER X1 and makes 3 deposits into secondary pool vials: A) 8 individual aliquots into ROW (Z1-Z8=colors) pool vials B) all 8 pipettes aliquots into the single COLUMN Y1 pool vial C) all 8 pipettes aliquot into the single LAYER X1 pool vial

6. “Three Dimensional” Plasma Pool Sample Matrix 8X8X8 (512) X1 Z3 Y3 Primary Pool X1 Y3 Z3 Found Positive

7. “Three Dimensional” Plasma Pool Matrix • Master Pools are diluted 1:1000 (512,000 fold dilution) Sensitivity 20 copies per/Ml =10 6 • Result = Negative conclude that all component samples are below the cutoff i.e. “Not Implicated” and will not cause high level contamination of the pool. • Master Pool Result = Positive resolve through testing of diluted primary pools and the implicated sample

8. Resolution of the Individual Sample • Primary pools must be diluted or quantitative analyzed or low titer samples will begin to confuse the resolution. • Up to 30% of the samples could have low level viremia. • Recommended cutoffs.

9. Parvo Investigation Algorithm Retrieve samples of prior and subsequent bleeds for individual Parvo PCR analysis Determine Parvo antibody status Determine Parvo DNA levels over time Determine prevalence of the high titer samples.

10. Negative Donor Positive 0 4 8 12 16 0 4 8 12 16 Week Composite Results

11. Ideal Subject determine viremic period and shape of curve for each donor and examine antibody status (IgG vs. IgM) throughout viremic window

13. Summary of 20 donors: First appearance of IgM First appearance of IgG 0

14. Summary Points • IgM Appears within 14 days of Viremia • persists for several months • IgG Appears after about 3 weeks • Titer: IgG remains elevated for years • Viremia : There is an immediate burst in viral load . The Average peak viral load is > 1011 • Virus persists for years • Viral Titers peak at about two weeks and immediately prior to IgM. Viral load then drops rapidly and remains at low levels for years. Highest titers can be above 10 10

15. Incidence and Prevalence of Parvo B19 • Incidence of high titer infections 2002 was 1/3281 donations. We feel this truly represents new infections because of the short duration of high viremia. (>10 6 copies /ML) • Prevalence of donors with some viremia is 1/3 • Breakdown according to season.

18. 2002 Incidence of Parvo B19 High Titer Samples