SoGAT 24-05-2005

24 May 2005

Are conversion factors to international units assay related?

Marco Koppelman, Henk Reesink, Nico Lelie, Theo Cuypers

SoGAT 24-05-2005

NAT test system for comparison

• NucliSens Extraction Cobas Amplicor HCV

• Procleix Ultrio (Chiron/Gen-Probe) HIV-1, HCV, and HBV

• Multiprep Cobas AmpliScreen HBV

• AmpliPrep TNAI Cobas AmpliScreen HIV vrs 1.5

• AmpliPrep TNAI Cobas AmpliScreen HCV vrs2.0

• AmpliPrep TNAI Cobas AmpliScreen HBV

SoGAT 24-05-2005

NucliSens Extraction Cobas Amplicor HCV

Generic silica extraction‘Boom’ Technology

Cobas AmplicorNuclisens

SoGAT 24-05-2005

Procleix Ultrio HIV-1, HCV, and HBV

ResultsLuminometer

DetectionWater bath

TMAWater baths

PipettingTECAN 2

SampleProcessing

TCS

Specific Capture of Target

SoGAT 24-05-2005

AmpliPrep TNAI Cobas AmpliScreen HIV/HCV/HBV

Generic magnetic silica extraction

AmpliPrep Cobas Amplicor

SoGAT 24-05-2005

Reference materials

Serial dilutions of the WHO International standards for:

HBV genotype A * (NIBSC 97/746)

HIV-1 genotype B* (NIBSC 97/656)

HCV genotype 1* (NIBSC 96/790)

*IU: Calibrated in WHO Collaborative Studies.

PeliCheck reference panels for

HBV genotype A# (S2184)

HIV-1 genotype B# (S2092)

HCV genotype 1# (S2088)#Geq: Calibrated with Bayer bDNA

SoGAT 24-05-2005

Analytical sensitivityWHO International Standard HBV DNA (97/746)

IU/mL Multi-Prep

Ampli-Prep

Ultrio

n=24 n=24 n=63

40 100% 100% 100 %

20 100 % 100 % 100 %

10 100 % 100 % 98 %

5 90 % 96 % 76 %

2.5 86 % 71 % 58 %

1.25 71 % 50 % 27 %

0.63 33 % 46 % 13 %

0.31 36 % 13 % 6 %

0.16 9 % 13 % 8 %

0.08 0 % 0 % 0 %

log (dilution)

50%

% positive

log (dilution)

50%

% positive

log (dilution)

50%

% positive

SoGAT 24-05-2005

Analytical Sensitivity Procleix Ultrio test*

Reference material

50% detection limit 95% detection limit

WHO-IS N1 IU/mL 95%CI IU/mL 95% CI HIV-1 IS2 48 3.4 2.4 - 4.7 26 16 - 58 HCV IS 63 0.9 0.7 - 1.0 4.6 3.7 - 6.5 HBV IS 63 1.9 1.5 - 2.6 11 7.3 - 22 PeliCheck ref. panel

N geq/mL 95%CI geq/mL 95%CI

PeliCh. HIV-1 48 5.7 4.3 - 7.6 65 41 - 119 PeliCh. HCV 48 13 9.0 - 20 86 50 - 204 PeliCh. HBV 48 34 26 - 44 328 213 - 586 1 Number of repeats per dilution 2 Discriminatory HIV-1 test

*Multicenter study; Assal (BC-Tours), Chudy (PEI), Garcia de Villaescusa (BC-Madrid),

Koppelman (Sanquin). Transfusion in press.

SoGAT 24-05-2005

0 2 4 6 8log (dilution)

Conversion Factor between two data-sets

95%

50%

% positivePotency calculated with SPSS

SoGAT 24-05-2005

Conversion factors IU to Geq

AmpliScreen

 WHO

studies Ultrio1 AmpliPrep2 MultiPrep2 AmpliCor3

HIV-1 1 1.7 (1.2-4.3) 1.6 (0.9-2.7) - -

HCV 3.8 17.0 (5.8-84) 9.4 (3.3-43) - 5.1 (3.1-9.1)

HBV 7.4 20.7 (9.1-60) 12.5 (5.4-40) 11.0 (4.9-33) -

1Koppelman et al. Transfusion 2005 in press.

2Koppelman et al. 2005 Submitted.

3Cuypers et al.; Biologicals. 1999 27 303-14.

SoGAT 24-05-2005

Conversion factors between IU and copies/ml are assay specific

• Conversion factors of the WHO collaborative studies; the mean of different nucleic acid extraction techniques and amplification/detection systems (considerable interlaboratory variation)

• Molecular composition of the reference material

• Physical composition reference materials. Liquid versus lyophilized reference materials

Commutability of reference materials; knowledge of molecular characteristics of preparations is necessary

Preliminary conclusion: conversion factors from IU to VQC-geq assay specific

SoGAT 24-05-2005

Conversion factor Dutch guilder to Euro

What was the conversion factor between Euro and Guilder in 2001?

It makes no difference !

SoGAT 24-05-2005

Analytical Sensitivity AmpliScreen HIV-1/HCV/HBVAmpliPrep MultiPrep

Standard Nucleic acid extraction

method 50% limit of detection 95% limit of detection

HBV-IS AmpliPrep 1.0 IU/mL (0.7-1.3) 6.7 IU/mL (4.3-13) MultiPrep 0.7 IU/mL (0.5-1.0) 5.2 IU/mL (3.3-10) MultiPrepa 1.3 IU/mL (0.9-1.7) 5.5 IU/mL (4.0-10) HCV-IS AmpliPrep 2.9 IU/mL (1.7-5.5) 23 IU/mL (11-106) MultiPrepa 7.3 IU/mL (2.2-10) 29 IU/mL (21-86) HIV-1-IS AmpliPrep 18 IU/mL (13-26) 187 IU/mL (108-422) MultiPrepa 22 IU/mL (13-29) 78 IU/mL (60-137) HBV-PeliCheck AmpliPrep 12 geq/mL (8.2-18) 123 geq/mL (68-301) MultiPrep 8.2 geq/mL (5.4-13) 101 geq/mL (52-295) MultiPrep 8.0 geq/mL (6.0-13) 100 geq/mL (55-291) HCV-PeliCheck AmpliPrep 36 geq/mL (17-65) 289 geq/mL (142-1061) MultiPrepb 9.9 geq/mL (6.3-15) 77 geq/mL (43-203) HIV-1-PeliCheck AmpliPrep 28 geq/mL (20-41) 183 geq/mL (108-434) MultiPrepb 6.6 geq/mL (4.4-10) 92 geq/mL (49-238) a data from kit insert. b data from Lelie et al. Transfusion et al. 2002; 42: 527-536.

SoGAT 24-05-2005

Conversion factors IU to Geq

AmpliScreen

 WHO

studies Ultrio1 AmpliPrep2 MultiPrep2 AmpliCor3

HIV-1 1 1.7 1.6 - -

HCV 3.8 17.0 9.4 - 5.1

HBV 7.4 20.7 12.5 11.0 -

1Koppelman et al. Transfusion 2005 in press.

2Koppelman et al. 2005 Submitted.

3Cuypers et al.; Biologicals. 1999 27 303-14.