INFORMATICS AND SYSTEM INTEGRATION68 WTEC Workshop on Biosensing Research and Development in the...

65

SESSION 3

INFORMATICS AND SYSTEM INTEGRATION

Imant Lauks

THE ANALYTICAL SYSTEM

¶ The sensors ¶ The fluidics

Sample prep, sensor calibrations, control measurements, wash steps, reagent additions, sample additions

¶ The measurement interface Thermal control, fluidic actuators, detectors, pre-amplifiers, digitization, signal formatting, transmitter

¶ The rest of the system Computer, display, network, keyboard, memory

INFORMATICS FOR INTERNAL DIAGNOSTICS AND ERROR DETECTION

¶ Extension of factory SQC system to the product in the field

INTEGRATION OF SENSORS AND FLUIDICS

¶ Sample to answer devices (point of use applications) ¶ High density assay systems

ANALYTICAL SYSTEM: IS IT A DEVICE OR A PROCESS?

Lab

¶ Fixed working environment & re-usable subsystems

¶ Predominant error modes are persistent ¶ Laboratory analytical systems are configured

as process equipment ¶ Quality systems are based on pseudo-sample

statistical process control

Point of Use

¶ Variable working environment & unit-use subsystems

¶ Predominant error modes are sporadic ¶ Point of use analytical systems are devices¶ Quality system must be based on internal

diagnostics

WTEC Workshop on Biosensing Research and Development in the United States66

INTERNAL DIAGNOSTICS

¶ Predominant error mode is singular non-conformity without persistence ¶ Pseudo-sample QC is ineffective. Internal diagnostics performed every time a sample is run ¶ Singular non-conformities

Pre-analytical

¶ Diluted,contaminated,clotted, hemolysed, settled, aged or inappropriately pre-treated specimen¶ Improper specimen collection

Analytical

¶ Improper sample introduction to device - Underfill - Overfill - Bubbles/foam - Improper closure

¶ Device- Manufacturing non-conformity - Sensors / reagents - Fluidics - Shipment defect - Storage defects - Environmental abuse at point of use:

mechanical, chemical, thermal, radiation

Pseudo-Sample QC: Traditional Analyzers

Defect rate fraction of reported results with medical error (ME) Defect Rate = f ( 1 + X ) ( 1 – PedPnr )n run length of n samples between Nth and N+1th QC f fraction of runs with MEX persistence of error conditionPed probability of detection of the error within the runPnr probability of not reporting results from erroneous runPed = X / n which is the probability that the error condition has

persisted to the N+1th QC assuming that the QC detects the error condition with a probability of 1

Defect Rate = f ( 1 + X ) ( 1 – XPnr/n )Central Lab using `bracketing’ QCWhen X > n and Pnr = 1 Defect rate = 0When X < n Defect rate = f ( 1 + X )Stat Lab or POCSince Pnr = 0 Defect rate = f ( 1 + X )

persistence

Nth QC N+1th QCError correctedError

occurred

Ana

lytic

al v

alue

50 100 150 Sample number

Pseudo-Sample QC: Traditional Analyzers

Defect rate fraction of reported results with medical error (ME) Defect Rate = f ( 1 + X ) ( 1 – PedPnr )n run length of n samples between Nth and N+1th QC f fraction of runs with MEX persistence of error conditionPed probability of detection of the error within the runPnr probability of not reporting results from erroneous runPed = X / n which is the probability that the error condition has

persisted to the N+1th QC assuming that the QC detects the error condition with a probability of 1

Defect Rate = f ( 1 + X ) ( 1 – XPnr/n )Central Lab using `bracketing’ QCWhen X > n and Pnr = 1 Defect rate = 0When X < n Defect rate = f ( 1 + X )Stat Lab or POCSince Pnr = 0 Defect rate = f ( 1 + X )

persistence

Nth QC N+1th QCError correctedError

occurred

Ana

lytic

al v

alue


Pseudo-Sample QC: Unit-use Analyzers

Defect Rate = f ( 1 + X ) ( 1 – XPnr/n )When X =0 Defect rate = f

Nth QC N+1th QCError occurred

Anal

ytic

al v

alue


Pseudo-Sample QC: Unit-use Analyzers

Defect Rate = f ( 1 + X ) ( 1 – XPnr/n )When X =0 Defect rate = f

Nth QC N+1th QCError occurred

Anal

ytic

al v

alue


3. Informatics and System Integration 67

EXTENSION OF A FACTORY SQC SYSTEM TO THE PRODUCT IN THE FIELD

Process test data

Finished goods test data

Field test data

Data base

Statistical analysis package

In use dataClinical trial dataField QC test data

Stress test

Stability test

Raw materials test data

Product sample test

softwareDS/LIS/HIS

Instrument software

Instrument software

Instrument software

Test engineering

Process engineering

acquire signal

acquire measurement attribute data that might be predictive of a non-conforming measurement

+

calculateanalyticalvalue

Is attribute data

acceptable

abort

Display result

Display failure mode

yes

no

compare the instant value of the attribute to the historical batch data or the time series data

USLLCL UCL LSL

accept rejectmaybe reject

maybe reject

reject

We know with high probability that the measurement is abnormal.Is it safe to deliver a result?.


¶ Small scale biosensor array + fluidics

INTEGRATION OF SENSORS AND FLUIDICS

A sample-to-answer device

LAB-ON-A-CHIP DEVICES

¶ Not quite a lab on a chip yet

Lab-glassware-on-a-Chip


INTEGRATION OF SENSORS AND FLUIDICS IN HIGH DENSITY BIOASSAYS:

¶ the fluidics i/o problem

INTEGRATED ELECTROKINETIC DEVICES

Epocal 2002

¶ `Solid-state’ fluidics for controlled transport and reaction of sub pL volumes of fluid and sub fmol quantities of reagents

¶ Self- contained micro-analytical systems with integral dry reagents microfabricated into hydrophilic-matrix circuits enclosed by a gas permeable insulator

1. Apply aqueous sample over chip. The circuit is wet up by vapor transport through insulating cap ( 0.1 mm/s/mm ). Ddry reagents dissolve to form aqueous chemical reservoirs and conducting elements. Dissolved chemicals move through conducting elements by electrokinesis powered by electrodes

2. Chemicals electrokinetically pumped into / out of circuit through orifice 3. Chemicals electrokinetically pumped through circuit elements

INTEGRAL REAGENT INJECTOR


e

O2O2

H2O H2OAir

-+ ++ eHOOH 442 22-- ++ OHeOHO 4422

H2O

nhCOinoxyluciferPPiAMPOATPluciferin luciferase ++++½½½ ½++ 22

ATP

MICRO-REACTOR ARRAY WITH INTEGRAL INJECTORS

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0.0018

0.002

0 200 400 600 800 1000 1200

seconds

light

inte

nsity

(V)

0

1

2

3

4

5

6

7

8

9

10

pum

p vo

ltage

0.01

0.1

1

10

100

1E-14 1E-13 1E-12 1E-11 1E-10 1E-09

moles ATP

Peak

are

a V

sec

A high density microarray with integral fluidic i/o

•array of micro-reactors with integral electrokinetic reagent injectors


WTEC – 12/3/02

Rapid, Reliable, Confident PCR

for Bio-Detection

Kurt Petersen, PhDCepheid

Sunnyvale, CA

RAPID, RELIABLE, CONFIDENT PCR FOR BIO-DETECTION

Kurt Petersen, Ph.D

Some controversy has recently surrounded the utilization of PCR for the detection and identification of bio-threat agents. Disadvantages which have been cited are 1) sensitivity to inhibitors, 2) operator errors, 3) unverified, unvalidated DNA target sequences, 4) unstable reagents, and 5) the overall complexity of the PCR procedure. These problems have resulted in false positives and false negatives when PCR is used in a a casual and thoughtless manner. This presentation will show how all these issues can be addressed and solved through a careful and thorough system design and implementation. The application of meticulous sample preparation methodologies, internal reaction controls, multiple target sequences before calling a positive, lyophilized, single-dose reagents, and complete protocol automation to eliminate operator errors is now allowing rapid PCR to be used reliably and with confidence for the detection and identification of bio-threat organisms.


WTEC – 12/3/02

Difficulties of PCR

FALSE NEGATIVES

• PCR is sensitive to inhibiting chemicals– Can be as simple as humic acid in dirt

• System must properly prepare sample– Wash away and remove inhibitors– Lyse organisms to release internal DNA– Concentrate organisms from dilute samples

• System must include internal control(s)– Assures PCR reaction was not inhibited

WTEC – 12/3/02

Difficulties of PCR

FALSE POSITIVES

• Bacteria exchange DNA sequences among each other– DNA sequences must be validated– Detection of a single sequence can be a

false positive• System must be able to detect multiple

unique DNA sequences simultaneously– This is the standard method in microbiology

to have confidence in a positive result


WTEC – 12/3/02

Difficulties of PCR

FALSE POS and NEG

• PCR is a VERY complex chemistry– Even experienced operators make mistakes

• Estimates are up to 2-5%– Cross-contamination is a common problem

• System must be totally automated– Reagents, including internal controls must

be single-dose lyophilized pellets – This strategy eliminates mistakes

WTEC – 12/3/02

PCR, “the right way”

Perform proper sample prep(remove inhibitors, lyse spores, concentrate)

Include internal control(s) in each reaction

Monitor multiple DNA targets simultaneously

Automate entire process

Supply single-dose reagents


WTEC – 12/3/02

GO

DNA test results -where and when they are needed

DNA Analysis : Vision for the Future

WTEC – 12/3/02

WHY DNA Detection ?

• The genome of humans, as well as most other organisms, are becoming readily available – even over the internet

• Other assays are not sensitive enough for many applications– Immuno-assays LOD ~ 104 -105 organisms

• Many false negatives and false positives– PCR LOD ~10 or fewer organisms

• Other assays cannot determine factors such as virulence or antibiotic resistance

• DNA assays are extendible to future bio-agents• Can be performed rapidly using new technologies


WTEC – 12/3/02

Detection of and surveillance for bio-threat agents

Testing for foot-and-mouth disease in livestock where rapid, on-site test results are urgently needed.

WTEC – 12/3/02

A surgeon must determine whether a lymph node is cancerous—

while the patient is still in surgery.

Testing for salmonellain poultry and E.coli in beef


WTEC – 12/3/02

Disease-Free Survival from Esophageal Cancer Surgery

Rapid PCR is the strongest independentprognostic factor in esophageal cancer patients

Time (months)806040200

Dis

ease

Fre

e Su

rviv

al1.11.0.9.8.7.6.5.4.3.2.1

PCR negative

PCR positive

From Dr. Tony GodfreyU. of Pittsburgh

WTEC – 12/3/02

The needle in a haystack.

• Bacteria in blood – sepsis <10/mL(volume = 1 part in 1011)

• HIV in blood <100/mL• Chlamydia in urine 100/mL• Listeria, salmonella in food 1/25 gr• Cryptosporidium in water 1/liter• Bio-threat agents in air <100/liter

( US Postal Service Application )

• FMD in pigs or cattle 1000/swab


WTEC – 12/3/02

10-18 10-15 10-12 10-9 10-6 10-3 1L

milli-liter:(1 cm)3

micro-liter:(1 mm)3

nano-liter:(100 mm)3

pico-liter:(10 mm)3

Less thanone moleculeper sample

DNAProbe

Assays

COPIES

6x1020/mL

6x1017/mL

6x1014/mL

6x1011/mL

6x108/mL

6x105/mL

600/mL

6/mL

PERFECTDETECTION &STATISTICALCONFIDENCE

SAMPLE VOLUME

Clinical Chem

Immuno Assays

Nanolitersand picoliters

are notfor everyone !

Concentrationof Target Molecule

Andreas Manzoriginal paper

Micro-fluidicsfor molecular diagnostics?

WTEC – 12/3/02

Raw Sample Prep

2 - 4 hours

PCR Amplification

1.5 - 2.5 hours

Fluorescent Detection

1 - 2 hours

Existing DNA Test Methods :the Problems

operator errors;reproducibility;

consistency


WTEC – 12/3/02

Next stepfor rapid DNA

analysis:

fully automatedDNA detection

at the point of need

< 5 minutes

SAMPLE PREP

< 25 minutes

AMPLICATION AND DETECTION - PCR

WTEC – 12/3/02

How GeneXpert AutomatesSample Preparation

Raw Biological Sample Loaded into Cartridge

Up to 5mL

Target Organismsare Concentrated,

Isolated, and Washed

Cells and Organisms Lysed

to Release their DNA

DNA Molecules Captured, Purified

and Concentrated

MixtureDelivered to Integrated

Reaction Tube for Amplification and Detection

DNA Molecules Mixed with Amplification and

Detection Chemicals


WTEC – 12/3/02

Cartridge Bodywith Fluid Reservoirs

CapPCR

ReactionTube

Syringe Barrel

Ultrasonic InterfaceRotary Valve

Cartridge Foot

GeneXpertCartridge

-micro-fluidics-

WTEC – 12/3/02

Mechanically Clamped Filter

Solid Interface

Syringe Barrel

Valve Body AssemblyFilter Capture/Lysis Technique

Glass Beads

Before

After

Ultrasonic Energy


WTEC – 12/3/02

I-CORE ModuleBuilding Block for the GeneXpert

Fan

Optic Blocks

Reaction Tube

I-CORE Board

Heater• Rapid thermal cycling– 8-10C/sec heating– 2-3C/sec cooling

• Real-time optical fluorometric reading

• Independently programmable

• Over 16K shipped to date• Licensed from LLNL

WTEC – 12/3/02

I-CORE Optical Blocks

Excitation Detection

4 LED Light Sources

RodLenses

Filters andlenses

4 Photodiode DetectorsFilters and

lenses


WTEC – 12/3/02

Total Internal Control: GBS/IC/SPC Triplex Assay

TRIPLEX ASSAY No GBS

-100

10203040506070

1 5 9 13 17 21 25 29 33 37 41 45

Cycle number

Fluo

resc

ence GBS

GBSS PC 1e2 sp oresS PC 1e2 sp oresIC 100 cop iesIC 100 cop ies

No GBS; Normal IC/SPCTRIPLEX ASSAY

1 cfu GBS

-50

0

50

100

150

200

1 5 9 13 17 21 25 29 33 37 41 45

Cycle number

Fluo

resc

ence GB S

GB SSPC 1e2 s poresSPC 1e2 s poresIC 100 copiesIC 100 copies

Low GBS

TRIPLEX ASSAY 1e5 cfu GBS

-100

0

100

200

300

400

500

1 5 9 13 17 21 25 29 33 37 41 45

Cycle number

Fluo

resc

ence GBS

GBSSPC 1e2 sporesSPC 1e2 sporesIC 100 cop iesIC 100 cop ies

High GBSGBS/IC/SPC T riplex Assay

05

101520

2530

3540

0 1 2 3 4 5 6 7

Log10 GBS cfuC

t

GBS (FAM )

GBS (FAM ) SP C (C Y3 )

SP C (C Y3 )

IC (T R)

IC (T R)

GBS Titration

WTEC – 12/3/02

Detection of Bacillus anthracis (Sterne) spores with GeneXpert System

Cycle

Fluo

resc

ence

-50

0

50

100

150

200

250

300

350

0 5 10 15 20 25 30 35 40

303030150150150100010001000100001000010000

GeneXpert Anthrax Assay


WTEC – 12/3/02

GeneXpertReagent Configuration

• Dual bead lyophilized physical format:– Room temperature stability for easy materials

management– Pre-defined, single-dose reaction mixture

• Minimize possibility of operator errors

– Rapid dissolution in microfluidic cartridges

UMMPol

HS abdNTPs

Buffer salts

P1 - P4IC

Mg 2+

ASR

3 mm

WTEC – 12/3/02

Smart Cycler® System

Almost 1000 Systems Shipped

GeneXpert“Forerunner”Is successful

Used by LLNLBiosensing

Fly-Away Team


WTEC – 12/3/02

Status of GeneXpert

• Systems at USAMRIID and CDC– Under evaluation for sensitivity and specificity– (have been using Smart Cyclers for 2 years)

• 12 systems operating since July at a US postal facility inside automated Biological Detection Systems (BDS) – uses liquid aerosol collector– Cepheid collaboration with Northrup Grumman and

Smiths Industries

• Over 5,000 cartridges consumed since July• Potential USPS application – 1-3M cart/year• Becoming the “standard” for bio-threat detection

WTEC – 12/3/02

Cepheid Mission :“Providing DNA/RNA test resultswhen and where they are needed”


WTEC – 12/3/02

Funding Sources

• DARPA• USAMRIID (Ft. Detrick)• SBCCOM (Aberdeen)• LLNL• VCs

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