1. Bacterial Screening of Platelet Concentrates by Real-Time PCR Theo Cuypers 1 also on behalf of, H.W. Reesink 1,3 I.G.H. Rood 1,2 T. Mohammadi¹, ²P.H.M. Savelkoul² R.N.I. Pietersz¹C.M.J.E. Vandenbroucke-Grauls² ¹ Sanquin, Amsterdam, The Netherlands ² VU University Medical Center, Dept. of Medical Microbiology & Infection Control, Amsterdam, The Netherlands ³ Academic Medical Center, Dept. of Gastroenterology and Hepatology, Amsterdam, The Netherlands

2. Method (1) Extraction (MagNa Pure) -Filtration MagNa Pure DNA isolation chemicals (filtration Gen Elute Plasmid Maxiprep (Sigma)) -200 µL PC -Extraction according to manufacturer Real-Time PCR (ABI-PRISM 7000) - pre-treatment 25 µL Taqman PCR mixture with Sau3A I digestion -amplification -detection Mohammadi et al, 2003, 2004, 2005

3. Method (2) Controls -DNA extraction : HLA – DQA DNA -amplification : Bordetella avium DNA -negative control Standard calibration curve (quantification) -Staphylococcus aureus DNA (ATCC 25923) -serial dilutions with known CFUs Mohammadi et al, 2003, 2004, 2005

4. Standard Curve with DNA Extracted from Serial Dilutions of S. aureus (ATCC 25923) Assay is linear from 1x10 0 -1x10 5 CFU eq/PCR (R 2 :0.999) Log CFU/PCR CTCT Mohammadi et al Transf (2005) 45: 731-6

5. Comparison Real Time-PCR and Automated Culturing Mohammadi et al Transf (2005) 45: 731-6 * representing 10,739 donations BacT/Alert positive result = identified microorganism in culture bottle

6. Comparison Real Time-PCR and BacT/Alert Mohammadi et al Transf (2005) 45: 731-6

7. Analysis of spiked PCs Bacteria spp (B. cereus, E. coli, S. epidermidis, P. acnes, P. aeruginosa) Ten fold serial dilutions plated on agar BacT/Alert culture 20 mL (aerobic and anaerobic bottles) time = 0: < 2 hrs after spiking time = 1, 2, 3, 6 and 7: days after spiking Real Time PCR at all time points BacT/Alert at t=0, and other time points only when earlier samples were negative Mohammadi et al. Vox Sang(2005) 89: 208-214

8. Results BacT/Alert 1 CFU/mL10 CFU/mL100 CFU/mL Bacterium sppdays 0 1 2 3 6 7 B. cereus + ND................. E. Coli - + ND............ + ND................. S. epidermidis + ND................. P. acnes - - - - + ND - + ND............ P. aeruginosa + ND................. All spiked dilutions were tested in triplicate Mohammadi et al. Vox Sang(2005) 89: 208-214

9. Results Real Time PCR 1 CFU/mL10 CFU/mL100 CFU/mL Bacterium sppdays 0 1 2 3 6 7 B. cereus - + + + + + + + + E. Coli - + + + + + + + + S. epidermidis - + + + + + + + + P. acnes - + + + + + + + + P. aeruginosa - ND + + + + + ND + + + + All spiked dilutions were tested in triplicate Mohammadi et al. Vox Sang(2005) 89: 208-214

10. Conclusion Real Time-PCR improvement sensitivity/specificity by filtration and digestion of reagents screening > 2000 PCs (~10,000 donors) indicate equal sensitivity and specificity with BacT/Alert time to detection: 4 hrs (BacT/Alert ± 24 hrs) sensitivity improved when PCs stored for > 24 hrs (after preparation, i.e. 48 hrs after collection)

11. Biological standards to compare NAT test protocols for bacterial contamination Application of both methods to prevent endogenous contamination of reagents; not perfect with all reagent batches. Selection of batches is necessary. Alternative PCR assays and methods in development to get around this problem. Problem is direct comparison of the sensitivity of assays. CFU is to imprecise to determine small differences. Develop ideas to prepare and characterize standards. Biomatrix, panels with dilution series of representative strains?