Controls for Blood Septicemia Nucleic Acid Tests Mark Manak BBI Diagnostics, Inc. A Division of SeraCare Life Sciences, Inc. SoGAT XIX Meeting Berne, Switzerland June 15, 2006

Sepsis: Infection of Blood Stream Bacterial infection (Bacteremia); Fungal Infection (Candidemia) Second leading cause of death among patients in non-coronary intensive care units (ICU) Tenth leading cause of death overall in US Usually diagnosed by culture using specialized apparatus to detect and identify any viable microorganisms. Limitations of Blood Culture –Relatively insensitive and time consuming: 24 to 48 hrs to results –>30% of septic patients are false negative by blood culture

Nucleic Acid Tests for Sepsis Roche SeptiFast TM Test Highly specific – able to detect <100 organisms per ml blood Detects microorganisms that cause >90% of all blood stream infections Real Time PCR on Light Cycler Platform 3 parallel reactions: gram positive, gram negative, and fungus Melting curve analysis of end product ensures proper sequence Rapid, automated results in less than 5 hours

ACCURUN 500 Design Specifications Manufactured from P. aeruginosa, S. aureus, and C. albicans Inactivated to reduce viability >6 logs. Diluted in negative human plasma Targeted at low concentration (about 300 CFU/ml ) Intended Use Estimate laboratory testing precision Detect errors in laboratory testing procedures. Controls for the entire workflow

Linearity of C. albicans in SeptiFast Test

100 cfu/reaction Quantification -Channel 640 C. albicans Tm Calling – Channel 640 NHP 100 cfu/reaction NHP IC

Quantification -Channel 670 P. aeruginosa Tm Calling – Channel cfu/reaction NHP

Excellent Reproducibility: Multiple Lots, Test Sites Test Site A Test Site B Mean: SD: %CV: 5% 4% 2% Test Site A Test Site B Lot Number S. aureus P. aeriginosa C. albicans cp

Stability Studies Accelerated Stability 37 o C Freeze-Thaw Stability

ACCURUN 500 Summary Stable, low reactive independent quality control material Made from natural bacteria in plasma diluent to mimic clinical sample Controls for all test steps: extraction, amplification and detection Well suited for the quality control of Nucleic Acid assays for microorganisms in human blood. Gram positive, Gram negative and fungal controls showed amplification peaks only in the respective channels for all lots Negative controls showed Internal Control peaks only Excellent Reproducibility between Control Lots, Test Runs, and Test Sites Use of ACCURUN 500 will allow laboratories to detect immediate testing errors and monitor the long term performance of such assays.