HIV, HCV, and HBV NAT ControlsFormulation, Stability and Performance

Mark ManakBBI Diagnostics, Inc.

A Division of SeraCare Life Sciences, Inc.

SoGAT XX MeetingWarsaw, PolandJune 12-13, 2007

Quantitative viral load testing is used for monitoring disease progression, or response to therapy

Need for reliable quantitation data to ensure proper patient management

Report of the NIH Panel to Define Principles of Therapy of HIV Infections and Guidelines of the Use of Antiretroviral Agents in HIV Infected Adults and Adolescents. MMWR 1998:46 (no.RR-5)

A log 0.5 increase in HIV titer in two sequential samples is considered a significant increase.

Need for Precision of Quantitation

Viral Load False Positive Rate vs. Interassay SD

0

2

4

6

8

10

12

0 0.2 0.4 0.6 0.8

% F

alse

Pos

itiv

e R

ate

• Biological Variation SD 0.24 log (10)

• 3% of patients will falsely be classified as

having significantly increased viral load

Standard Deviation

• Small increases in interassay variation significantly affect both false poistive and false negative rates

• Assays are complex, many

factors may negatively affect

interassay variation

HIV, HBV, and HCV ACCURUN Controls

• Manufactured from whole virus

• Non-infectious cultured HIV (8E5), or

• HBV or HCV positive plasma

• Diluted into Defibrinated Plasma (Basematrix)

• Resemble patient specimen

• Different Series target different viral load levels

• Manufactured under cGMP and ISO 13485

• FDA and CE Mark

Independent Run Control

• Independent of the test kit

(manufacurer, reagents, calibration)

• Designed to perform with a wide number of test kits and/or

procedures

• Control limits are usually set by the individual laboratory

• Intended Use

– Estimate laboratory testing precision

– Detect errors in laboratory testing procedures.

– Controls for the entire workflow

2.00

2.50

3.00

3.50

4.00

4.50

5.00

11/6/06

11/21/06

12/6/06

12/21/06

1/5/071/20/07

2/4/072/19/07

3/6/073/21/07

4/5/074/20/07

+3SD

+2SD

Mean

-2SD

-3SD

Levy Jennings Plot of A315 Values

Log IU/ml

Date

Consistency of A315 HIV ControlsA315 Series

1

10

100

1,000

10,000

100,000

1,000,000

21-Sep 11-Oct 31-Oct 20-Nov 10-Dec 30-Dec 19-Jan 8-Feb 28-Feb 20-Mar

Date

S400

S300

S200

S100

Mean St Dev % CVS400 41,604 11,271 27.1%S300 3,329 740 22.2%S200 720 211 29.3%S100 67 22 32.3%

IU/ml

Real Time Stability of 8E5 Stock

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

Mar-97 Jul-98 Dec-99 Apr-01 Sep-02 Jan-04 May-05 Oct-06 Feb-08

Date

4696N

20297N

20397N

IU/ml

Sources of Variability HIV NATA315 Lot Consistency

10

100

1,000

10,000

1 2 3 4 5Lot Number

A315 S200 Testing on Different Kits

10

100

1,000

10,000

1 2 3 4 5 6

Kit Number

IU/mlIU/ml

IU/ml

Lab to Lab Comparisons of A315 S 200 Results

10

100

1,000

10,000

A B C D E

Lab Number

Mean St Dev % CVKit Lot variability 953 40 4.2%Lab to Lab Variability 707 241 34.1%A315 Lot Variability 810 142 17.5%

100

1,000

10,000

100,000

1,000,000

B C E F G

1 2 4 6 10 11 15 16 17 18 19 20

Lot Number

S400S300S200

Consistency of HCV NAT: Effect of Kit Lot and A325 Lot

ACCURUN Kit Lot

Mean Std Dev % CVSeries 400 89,757 20,433 22.8%Series 300 18,457 4,090 22.2%Series 200 5,337 1,921 36.0%

IU/ml

Real Time Stability HCV A325 S300

1,000

10,000

100,000

1,000,000

10,000,000

Mar-03 Jun-03 Oct-03 Jan-04 Apr-04 Aug-04 Nov-04 Feb-05 May-05

Date

HBV Controls

10

100

1,000

10,000

100,000

1,000,000

A B C D

1 2 3 4 5 6 7 8 9 10 11 12 12 13 13 14 15 16

Lot Number

KHPC

A325

KLPC

ACCURUNKit Lot

IU/ml

Consistency of HBV NAT: Effect of Kit Lot and A345 Lot

Quantitation Controls/Panels Available

• ACCURUN Controls– HIV A315

• Series 100, 200, 300, 400, 500

– HCV A305• Series 100, 200, 300, 400• A405 Series 100 calibrated to IU

– HBV A325• Series 300, 600, 700

• Linearity Panels– Series of 10 Dilutions ranging from 3 x 106 – 10 IU/ml– HIV Linearity Panel PRD801– HCV Linearity Panel PHW804– HBV Linearity Panel PDH801

ACCURUN Controls and PanelsStable, independent quality control material

• Made from Whole virus in Defibrinated Plasma

• Resemble patient specimen

• Long term stability when stored frozen at -70o C

• Controls for all test steps: extraction, amplification and detection

Reliable Performance • Manufactured under audited quality system

• Targeted to decision levels

• Large lot sizes to enable usage over extended periods

• Excellent Reproducibility between Control Lots, Test Runs, and Test Sites

• Access to technical support

Acknowledgements

• SeraCare– Debbie Petit– Pat Garrett– Bharathi Anekella– Joan Thurston

• Quest Diagnostics

• Quintiles

• Dallas VA Medical Center

• NIH Clinical Lab

• Medical Center of New Orleans

• SeraCare Testing Lab