standardization of nat extraction methods paul d. neuwald, phd acrometrix corporation, benicia, ca...
TRANSCRIPT
Standardization of NATExtraction Methods
Paul D. Neuwald, PhD
AcroMetrix Corporation, Benicia, CA
SoGAT XVIIIBethesda, MD, USA
May 24, 2005
Standardization of NAT Extraction MethodsOverview
Potential sources of variation in NAT assays:
Specimen type
Platform / Procedure
Operator / Laboratory
Calibrators / Standards
Assay reagents
Extraction method
Standardization of NAT Extraction MethodsOverview
Potential sources of variation in NAT assays:
Specimen type
Platform / Procedure
Operator / Laboratory
Calibrators / Standards
Assay reagents
Extraction method
Example: CMV Assay Comparison StudyStudy 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)
Example: CMV Assay Comparison StudyReference Panel
OptiQuant™ CMV DNA Panel
Panel Member Nominal CMV DNA Copies/mL*
0 0
1 500
2 5,000
3 50,000
4 500,000
*Value assignment based upon quantification using the COBAS AMPLICOR CMV MONITOR® Test
y = 0.9078x + 0.4889
R2 = 0.9996
2.00
3.00
4.00
5.00
6.00
7.00
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Expected Log10 copies/mL
Ob
serv
ed L
og
10 c
op
ies/
mL
CMV DNA Assay Comparison StudyInterlaboratory Comparison
Overall Results for 23 Laboratories
y = 1.0156x - 0.083
R2 = 0.9949
y = 1.0423x + 1.2479
R2 = 0.9979
y = 0.6933x + 1.6347
R2 = 0.9744
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Expected Log10 copies/mL
Ob
serv
ed L
og
10 c
op
ies/
mL
CMV DNA Assay Comparison StudyInterlaboratory Comparison
Examples from 3 Individual Laboratories
Methods used in CMV DNA Assay Study
Lab # Instrument / Platform Extraction Method
1 ABI 7000 bioMerieux NucliSens
2 ABI 7500 Qiagen M48 Automated
3 ABI 7700 QIAamp DNA Mini Kit
4 ABI 7700 Qiagen Biorobot
5 ABI 7900HT Gentra Capture Column
6 ABI 7900HT Roche MagnaPure
7 COBAS Amplicor Amplicor CMV Monitor
8 iCycler QIAamp Blood Mini Kit
9 iCycler Qiagen Viral Kit
10 LightCycler QIAamp Blood Mini Kit
11 LightCycler QIAamp DNA Mini Kit
12 LightCycler QIAamp DNA Mini Kit
13 LightCycler QIAamp MinElute
14 LightCycler QIAamp MinElute
15 LightCycler QIAamp UltraSens
16 LightCycler Roche HighPure
17 LightCycler Roche HighPure
18 LightCycler Roche MagnaPure
19 LightCycler Roche MagnaPure
20 LightCycler Roche MagnaPure
21 LightCycler Roche MagnaPure
22 SmartCycler QIAamp Blood Mini Kit
23 SmartCycler QIAamp MinElute
CMV DNA Assay Comparison StudyInterlaboratory Comparison
LightCycler: Different Extraction Methods
QIAamp DNA Mini Kity = 0.8132x + 0.2965
N = 2
Roche MagnaPurey = 1.0071x + 0.4014
N = 4
QIAamp Blood Mini Kity = 0.96x + 0.3623
N = 1
Roche HighPurey = 0.9419x + 0.4305
N = 2
QIAamp UltraSensy = 0.626x + 2.2561
N = 1
QIAamp MinElutey = 0.9234x + 0.5753
N = 2
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00
Expected Log10 copies/mL
Ob
se
rve
d L
og
10 c
op
ies
/mL
CMV DNA Assay Comparison StudyInterlaboratory Comparison
ABI 7000 Series: Different Extraction Methods
bioMerieux NucliSensy = 1.0117x - 0.0575
N = 1
Qiagen Bioroboty = 0.9925x + 0.0296
N = 1
Roche MagnaPurey = 1.0467x - 0.3311
N = 1QIAamp DNA Mini Kit
y = 1.1622x - 1.113N = 1
Gentra Capture Columny = 0.649x + 1.0628
N = 1
Qiagen M48 Autoy = 1.0415x + 1.2836
N = 1
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00
Expected Log10 copies/mL
Ob
se
rve
d L
og
10 c
op
ies
/mL
Parvovirus B19 DNA AssayExtraction Comparison
Extraction Comparison Study: Parvovirus B19 DNA Assay
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
A B C
Parvovirus B19 Positive Sample
Lo
g o
f C
op
ies/
mL
Qiagen Blood Mini Kit QIAamp MinElute
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
AcknowledgmentsAcroMetrix Corporation
Association for Molecular Pathology
Laboratories Participating in the CMV DNA Assay Comparison Study:
Advocate Lutheran General HospChildren’s Mercy HospBaylor University Med CenterCarolinas HealthCare SystemChildren’s HospLoma Linda MercantileMayo ClinicMedical College of VirginiaMedical University of South CarolinaMedicine University of Rochester Med CenterMolecular Pathology Network, IncOregon Health & Sciences University
Sharp Memorial HospSpectrum HealthSt Barnabas Med CenterThe Methodist HospThe Nebraska Med CenterUMDNJ/NJ Medical SchoolUniversity of FloridaUniversity of North CarolinaUniversity of Pitts/Magee Women’s HospUniversity of Texas SW Med CenterUniversity of Washington Med CenterWashington Hosp Center