Linearity PanelsHIV RNA, HCV RNA,
HBV DNA, and CMV DNA
Mark Manak, Ph.D.BBI Diagnostics, Inc.
A Division of SeraCare Life Sciences, Inc.
SoGAT XVIIIBethesda, MD
May 24-25, 2005
Linearity Panel Design
• Mimic clinical specimen • Whole virus in defibrinated plasma diluent
• Challenges the entire analytical process
• One negative, and Nine positive members
• Series of sequential one log or half log dilutions
• Results expressed as specified by kit manufacturers
• Serial dilutions of the WHO International Standards analyzed simultaneously to assign IU values, where appropriate
Intended Use
Assay Effectiveness– Evaluate sensitivity and dynamic range of quantitative viral nucleic acid assays– Identifying consistency over a broad linear range– Establish calibrations vs. IU Standards– Verification of performance at kit lot changes
Training– Operator training program– Operator validation
Part of Laboratory Quality Assurance program
HCV RNA Linearity Panel (PHW804)
Diluent: – Normal human plasma – Negative for antibody to HIV-1/2, HBsAg, HCV, HTLV-I/II– Negative for HBsAg, HIV-1 RNA, HBV RNA and HCV DNA
Virus Spike: – High titer HCV RNA positive human plasma – HCV Genotype 1 by Innogenetics InnoLIPA Assay
Results on Data Sheet: – Roche COBAS Amplicor HCV Monitor v2.0– Bayer Versant HCV 3.0 bDNA Assay– Abbott LCx HCV Quantitative Assay– QuantaSure Plus HCV Roche Taqman ASR
HCV Linearity Panel (PHW804)
10
100
1,000
10,000
100,000
1,000,000
10,000,000
1101001,00010,000100,0001,000,000
Dilution
IU/m
l
Roche COBAS
Bayer Versant
Abbott LCx
Roche Taqman
Power (RocheTaqman)Power (AbbottLCx)Power (RocheCOBAS)Power (BayerVersant)
HCV RNA Unit Conversions to IU
Roche COBAS: 2.2 copies/IU
Bayer Versant: 5.2 copies/IU
Abbott LCx: 4.3 copies/IU
Taqman: 0.6 copies/IU
HIV RNA Linearity Panel (PRD801)
Diluent: – Normal human plasma – Negative for anti-HIV-1/2, anti-HBsAg, anti-HCV, anti-HTLV-I/II– Negative for HBsAg, HIV-1 RNA, HBV RNA and HCV DNA
Virus Spike: – Culture supernatant: HIV-1 Type B 8E5 virus– Intact, but defective virus genome
Results on Data Sheet:– Roche COBAS Amplicor HIV Monitor v1.5– Roche COBAS Amplicor HIV Monitor v1.5 Ultra– Bayer Versant HIV-1 RNA bDNA 3.0 Assay– Chiron/Genprobe Procleix HIV-1 RNA Discriminatory Qualitative– BioMerieux Nuclisens HIV-1 QT
HIV RNA Linearity Panel (PRD801)
10
100
1,000
10,000
100,000
1,000,000
10,000,000
1101001,00010,000100,000
Dilution
Cop
ies/
ml
Roche COBAS
Roche COBAS Ultra
Bayer Versant
Nuclisens
Power (Roche COBASUltra)Power (Nuclisens)
Power (Nuclisens)
Power (Nuclisens)
HBV DNA Linearity/Sensitivity Panel (PHD801)
Diluent: – Normal human plasma – Negative for antibody to HIV-1/2, HBsAg, HCV, HTLV-I/II– Negative for HBsAg, HIV-1 RNA, HBV RNA and HCV DNA
Virus Spike: – High titer HBV DNA positive human plasma– HBV DNA:Type A virus by Roche Taqman and Innogentics InnoLIPA– Serotype ad
Results on Data Sheet: – Roche COBAS Amplicor HBV – Digene HBV DNA Quantitative Assay – Bayer Versant HBV DNA Quantitative bDNA Assay– Taqman HBV DNA Quantitative Assay
HBV DNA Unit Conversions to IU
Roche COBAS: 1.2 copies/IU
Bayer Versant: 0.9 copies/IU
Taqman: 3.9 copies/IU
Digene: 4.6 copies/IUDigene: 1,200,000 IU/pg
CMV DNA Linearity/Sensitivity Panel (PTC801)
Diluent: – Normal human plasma – Negative for antibody to HIV-1/2, HBsAg, HCV, HTLV-I/II– Negative for HBsAg, HIV-1 RNA, HBV RNA and HCV DNA– Negative for anti-CMV and CMV DNA
Virus Spike: – Culture supernatant from CMV cultures
Results on Data Sheet: – Roche COBAS Amplicor CMV Monitor Test
Conclusions
BBI Linearity panels are made from natural virus in plasma diluent
Nine positive members spanning a broad range of virus concentrations and a negative member
Calibrated against WHO standards and expressed in IU/ml
Data from available commercial assays
Useful for monitoring linearity and dynamic range of assays