Rapid Bacterial Detection Rapid Bacterial Detection SystemSystem

Fully Automated 4 minute CFU/ml response Fully Automated 4 minute CFU/ml response systemsystem

SUBC Inc.

Rochester MN

MII Inc. ConfidentialMII Inc. Confidential

Principle and Sequence of Principle and Sequence of Rapid DetectionRapid Detection

1. Platelets and cellular debris removed from 1. Platelets and cellular debris removed from platelet concentrate sampleplatelet concentrate sample

2. Present Bacteria are isolated and immobilized 2. Present Bacteria are isolated and immobilized in isolation chamberin isolation chamber

3. Isolation chamber rinsed with buffer, leaving 3. Isolation chamber rinsed with buffer, leaving isolated bacteriaisolated bacteria

4. Lyse solution in combination with localized 4. Lyse solution in combination with localized heat provide rapid bacterial membrane lyseheat provide rapid bacterial membrane lyse

5. Bacterial ATP containing lysate is mixed with 5. Bacterial ATP containing lysate is mixed with bioluminescent firefly extractbioluminescent firefly extract

6. Generated light reaction is detected by photon 6. Generated light reaction is detected by photon counter. Burst of light is correlated to CFU/mlcounter. Burst of light is correlated to CFU/ml

Fluidic Analysis for Fluidic Analysis for Detection of BacteriaDetection of Bacteria

Sampling Sample Processing Detection

Carrier Stream

DilutionMixingEnrichmentChemistry

Waste

Pump

1 2 3

4

5

678

9

10 Selectionvalve

Carrier

Detector

ReactorHoldingcoil

Sample

Reagent

SIA

Sequential Injection Sequential Injection Analysis for Bacterial Analysis for Bacterial

DetectionDetection

CFURapid

Platelets

RinseLyse

+Heat

Platelet removalSystem

Bacteria Isolation Chamber

wasteReactor

Rapid Fluidic Engine Rapid Fluidic Engine SequenceSequence

3 Minute Result

BioluminescentReagent

Firefly Luciferace Firefly Luciferace ReactionReaction

ATP +D-luciferin + O2 AMP+pyrophosphate+oxyluciferin+CO2 + Light

TheThe quantum efficiency is very high resulting in quantum efficiency is very high resulting in almost one photon per ATP molecules consumed almost one photon per ATP molecules consumed in the reactionin the reaction

Intensity of the emitted light is proportional to Intensity of the emitted light is proportional to the ATP concentrationthe ATP concentration

If the luciferase level is low the intensity will be If the luciferase level is low the intensity will be essentially constantessentially constant

If the luciferase level is high the light will decay If the luciferase level is high the light will decay rapidly since ATP is consumed in the reaction (the rapidly since ATP is consumed in the reaction (the initial peak light is proportional to the decay rate)initial peak light is proportional to the decay rate)

If decay rate t If decay rate t 1/2 1/2 = 139 minutes sensitivity range = 139 minutes sensitivity range 1010-15-15 - 10 - 10-9-9

If decay rate t If decay rate t 1/2 1/2 = 235 minutes sensitivity range = 235 minutes sensitivity range 1010-14-14 - 10 - 10-18-18

Prototype Device #1Prototype Device #1

BPAC December 2002 BPAC December 2002 Bacterial Required for Bacterial Required for

DetectionDetection

Bacillus cereusBacillus subtilisClostridium perfringensCornybacterium speciesEcherichia coliEnterobacter cloacaeKlebsiella oxytoca

Propionibacterium acnesPsudomonas aeruginosaSerratia marcesensStaphyloccocus aureusStaphylococcus epidermidisStreptococcus pyogenesStreptococcus viridans

Bacteria verified and Bacteria verified and validated for bead validated for bead

isolationisolation

AeromonasBacillusCampylobacterCitrobacterClostridiumE.coliEnterobacterEnterococcusHafniaKlebsiella

LactobacillusListeriaMorganellaProvidenciaProteusSalmonella(Serratia)ShewanellaShigellaVibrioYersinia

BordetellaBorreliaChlamydiaClostridiumCorynebacterium(E. coli)EnterobacterHaemophilusHelicobacterKlebsiellaListeria

MicrococcusMycobacteriumNeisseriaPropionebacteriumProteusPseudomonas(Salmonella)SerratiaStreptococcusStaphylococcusYersinia

GloBac™GloBac™

GloBac™GloBac™

Fully AutomatedFully Automated Touch Screen Touch Screen

DisplayDisplay Microprocessor Microprocessor

DrivenDriven Internal Reagent Internal Reagent

cartridgecartridge Top loading sample Top loading sample

portport

University of Minnesota University of Minnesota Field Evaluation TestingField Evaluation Testing

Dr. McCulloughDr. McCullough Dr. BowmanDr. Bowman Dr. Gundu Rao Dr. Gundu Rao Mary Clay: Project ManagerMary Clay: Project Manager Shelley Pulkrabek: Blood Bank CoordinatorShelley Pulkrabek: Blood Bank Coordinator Debbie Cocking Johnson: Related lab supportDebbie Cocking Johnson: Related lab support Nancy Ward: Technical SupervisorNancy Ward: Technical Supervisor

Bacterial Contam ination Detection in PlateletsUniversity of M innesota

N egative G lucose C onc entra te sto Pla te le t Pools

Pla te le t Poolsn=420 0

n=200 ne ga tive sto M ic ro la b

All N e ga tive s to T ransfusion

S BA + IDan d

Ba cT + ID

All Positives to M icro la b

G loBa c T estingn=420 0

N ega tive to Pla te le t Pools Positive to M ic ro lab

Positive or N e ga tivePerform G loBac

T estingn=21 ,000

Positives to M ic ro la b

G lucose M e te r testingn=21 ,000

Pla te le t C once ntra tesn=21 ,000

India StudyIndia Study

Bangalore Rotary Blood Bank StudyBangalore Rotary Blood Bank Study Expected contamination rate of over Expected contamination rate of over

6%6% Study size 2000 unitsStudy size 2000 units Expected positives greater than180Expected positives greater than180 Discussion with FDA on accepting Discussion with FDA on accepting

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