1. Standardization of NAT Extraction Methods Paul D. Neuwald, PhD AcroMetrix Corporation, Benicia, CA SoGAT XVIII Bethesda, MD, USA May 24, 2005

2. Standardization of NAT Extraction Methods Overview Potential sources of variation in NAT assays:  Specimen type  Platform / Procedure  Operator / Laboratory  Calibrators / Standards  Assay reagents  Extraction method

3. Standardization of NAT Extraction Methods Overview Potential sources of variation in NAT assays:  Specimen type  Platform / Procedure  Operator / Laboratory  Calibrators / Standards  Assay reagents  Extraction method

4. Example: CMV Assay Comparison Study Study Design Each participating laboratory:  Extracted CMV positive reference panel members using its standard method for clinical specimens  Tested the panel members as unknowns in replicates of 4 using its standard CMV DNA assay method and calibrators  Returned quantification results for analysis and comparison to results from other labs (N = 23)

5. Example: CMV Assay Comparison Study Reference Panel OptiQuant™ CMV DNA Panel Panel MemberNominal CMV DNA Copies/mL* 00 1500 25,000 350,000 4500,000 *Value assignment based upon quantification using the COBAS AMPLICOR CMV MONITOR ® Test

6. CMV DNA Assay Comparison Study Interlaboratory Comparison Overall Results for 23 Laboratories

7. CMV DNA Assay Comparison Study Interlaboratory Comparison Examples from 3 Individual Laboratories

8. Methods used in CMV DNA Assay Study

9. CMV DNA Assay Comparison Study Interlaboratory Comparison LightCycler: Different Extraction Methods

10. CMV DNA Assay Comparison Study Interlaboratory Comparison ABI 7000 Series: Different Extraction Methods

11. Parvovirus B19 DNA Assay Extraction Comparison

12. Standardization of NAT Extraction Methods Future Considerations  Need for better controlled collaborative studies to compare extraction methods for: Different specimen types / calibrators Different platforms / instruments / procedures Different assay methods / reagents Different operators / laboratories  Initially, conduct a study that focuses on comparing extraction methods for one specimen type, on one platform, using common calibrators and reagents  AcroMetrix is willing to coordinate such a study

13. Acknowledgments AcroMetrix Corporation Association for Molecular Pathology Laboratories Participating in the CMV DNA Assay Comparison Study: Advocate Lutheran General Hosp Children’s Mercy Hosp Baylor University Med Center Carolinas HealthCare System Children’s Hosp Loma Linda Mercantile Mayo Clinic Medical College of Virginia Medical University of South Carolina Medicine University of Rochester Med Center Molecular Pathology Network, Inc Oregon Health & Sciences University Sharp Memorial Hosp Spectrum Health St Barnabas Med Center The Methodist Hosp The Nebraska Med Center UMDNJ/NJ Medical School University of Florida University of North Carolina University of Pitts/Magee Women’s Hosp University of Texas SW Med Center University of Washington Med Center Washington Hosp Center