Rapid Bacterial Detection System Fully Automated 4 minute CFU/ml response system SUBC Inc. Rochester MN

Principle and Sequence of Rapid Detection 1. Platelets and cellular debris removed from platelet concentrate sample 2. Present Bacteria are isolated and immobilized in isolation chamber 3. Isolation chamber rinsed with buffer, leaving isolated bacteria 4. Lyse solution in combination with localized heat provide rapid bacterial membrane lyse 5. Bacterial ATP containing lysate is mixed with bioluminescent firefly extract 6. Generated light reaction is detected by photon counter. Burst of light is correlated to CFU/ml

Fluidic Analysis for Detection of Bacteria SamplingSample Processing Detection Carrier Stream Dilution Mixing Enrichment Chemistry Waste

Pump Selection valve Carrier Detector ReactorHolding coil Sample Reagent SIA Sequential Injection Analysis for Bacterial Detection

CFU Rapid Platelets Rinse Lyse + Heat Platelet removal System Bacteria Isolation Chamber waste Reactor Rapid Fluidic Engine Sequence 3 Minute Result Bioluminescent Reagent

Firefly Luciferace Reaction ATP +D-luciferin + O2 AMP+pyrophosphate+oxyluciferin+CO2 + Light The quantum efficiency is very high resulting in almost one photon per ATP molecules consumed in the reaction The quantum efficiency is very high resulting in almost one photon per ATP molecules consumed in the reaction Intensity of the emitted light is proportional to the ATP concentration Intensity of the emitted light is proportional to the ATP concentration If the luciferase level is low the intensity will be essentially constant If the luciferase level is low the intensity will be essentially constant If the luciferase level is high the light will decay rapidly since ATP is consumed in the reaction (the initial peak light is proportional to the decay rate) If the luciferase level is high the light will decay rapidly since ATP is consumed in the reaction (the initial peak light is proportional to the decay rate) If decay rate t 1/2 = 139 minutes sensitivity range If decay rate t 1/2 = 139 minutes sensitivity range If decay rate t 1/2 = 235 minutes sensitivity range If decay rate t 1/2 = 235 minutes sensitivity range

Prototype Device #1

BPAC December 2002 Bacterial Required for Detection

Bacteria verified and validated for bead isolation

GloBac™

GloBac™ Fully Automated Fully Automated Touch Screen Display Touch Screen Display Microprocessor Driven Microprocessor Driven Internal Reagent cartridge Internal Reagent cartridge Top loading sample port Top loading sample port

University of Minnesota Field Evaluation Testing Dr. McCullough Dr. McCullough Dr. Bowman Dr. Bowman Dr. Gundu Rao Dr. Gundu Rao Mary Clay: Project Manager Mary Clay: Project Manager Shelley Pulkrabek: Blood Bank Coordinator Shelley Pulkrabek: Blood Bank Coordinator Debbie Cocking Johnson: Related lab support Debbie Cocking Johnson: Related lab support Nancy Ward: Technical Supervisor Nancy Ward: Technical Supervisor

India Study Bangalore Rotary Blood Bank Study Bangalore Rotary Blood Bank Study Expected contamination rate of over 6% Expected contamination rate of over 6% Study size 2000 units Study size 2000 units Expected positives greater than180 Expected positives greater than180 Discussion with FDA on accepting data Discussion with FDA on accepting data