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