xviii sogat washington 24 may 2005 sogat and hiv nat standards - 2 nd international standard for...

XVIII SoGAT Washington 24 May 2005

SoGAT and HIV NAT Standards -SoGAT and HIV NAT Standards -22ndnd International Standard for HIV-1 RNA International Standard for HIV-1 RNA

Harvey Holmes*, Clare Davis* and Alan Heath**Harvey Holmes*, Clare Davis* and Alan Heath**

Divisions of Retrovirology* and Informatics**Divisions of Retrovirology* and Informatics**

National Institute for Biological Standards and National Institute for Biological Standards and Control (NIBSC)Control (NIBSC)

UKUK


SoGAT and NAT Standards SoGAT and NAT Standards available from NIBSCavailable from NIBSC

• SoGAT and NIBSC have collaborated in establishing:

International Standards:– 1997: 1st International Standard for HCV RNA (96/790)– 1999: 1st International Standard for HIV-1 RNA (97/656)– 1999: 1st international Standard for HBV DNA (97/746)– 2000: 1st International Standard for B19 DNA (99/800)– 2002: 1st International Standard for HAV RNA (00/560)– 2003: 1st International Reference Panel for HIV-1 Genotypes (01/466)

– genotypes A-H – 2003: 2nd International Standard for HCV RNA (96/798)

Working Reagents:• HIV-1 RNA PWS-1, PWS-2, PWS-3• HCV RNA• HBV DNA• HAV RNA• B19 DNA• Multiplex (HCV, HBV, HIV, HAV, B19)• HCV Genotype Panel (genotypes 1 -6)


Retrovirology Standards and Working ReagentsRetrovirology Standards and Working Reagents


Replacement of 1Replacement of 1stst International Standard for International Standard for HIV-1 RNA (NIBSC Code 97/656)HIV-1 RNA (NIBSC Code 97/656)

• Sample YY established as 1st International Standard (code 97/656) in 1999

• Assigned a concentration of 100,000 IU per vial/ml

• Standard made from HIV-1 PCR-positive, antibody-negative, plasmapheresis donation diluted in defibrinated plasma

• 2300 vials freeze-dried and stored at -200C

• SoGAT 2003 - agreement to replace standard due to:– low Stocks (~ 450)– presence of HBV DNA – may affect suitability for multiplex assays

• Interim report on collaborative study presented by Clare Davis at SoGAT 2004


Candidate 2Candidate 2ndnd International Standard International Standard

• SoGAT recommended that sample XX from first collaborative study be re-evaluated as candidate 2nd International Standard (IS)

• Characteristics:

– HIV-1 subtype B low passage field isolate (CCR5/NSI) from Edinburgh (P Simmonds)

– Propagated on human PBMCs - stock stored under LN

– Virus diluted in human cryosupernatant to suitable concentration

– 2200 vials of material freeze-dried

– Fill CV = 0.2%


International Collaborative Study to Establish International Collaborative Study to Establish 22ndnd International Standard for HIV-1 RNA International Standard for HIV-1 RNA• Aims of study:-

– Calibrate XX against 1st International Standard (1st IS)– Confirm stability of XX– Determine values in current assays

• 10 laboratories participated

• Candidate XX and the 1st International Standard recoded (samples 1 and 2 respectively)

• Participants asked to test samples in 3 independent assays.• Initially at 10-fold dilutions• Subsequently at half log dilutions around the end point

• 8 data sets received, 5 from quantitative assays and 3 from qualitative assays

• NIBSC collated and analysed data


0

1

2

3

4

5

6

7

8

9

10

Estimated RNA Copies (log10/ml)3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

43 43 3

41

44

19

14

47

16

17

18

2

22

33

48

10

6

5

45

24

46

28

34

4

9IH MQ IH

IH

M

AS

B3

B3

IH

M

M

M

M

B3

B3

IH

IH

M

A

M

M

NU

NU

NU

M

Monitor assays Nuclisens bDNA 3 Abbott LCx In-House Ampliscreen

Original Collaborative Study

1st International Standard for HIV-1 RNA (YY)

0

1

2

3

4

5

6

7

8

9

10


16 3

41

43

19

44

43

34

33

47

48

10

17

18

2

24

5

28

45

14

6

22

46

9IH IH

IH

IH

AS

M

MQ

NU

B3

B3

B3

IH

M

M

M

M

M

NU

A

B3

IH

M

M

M


Sample XX


0

1

2

3

4

5

6

7

8

9

10

Estimated IU (log10/ml)

3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

16 19

41

24

28

34

4

45

33

10

3

46

5

14

47

48

6

17

18

2

44

9

43

22

43

IH AS

IH

M

NU

NU

NU

A

B3

IH

IH

M

M

B3

B3

B3

IH

M

M

M

M

M

IH

M

MQ


Original Collaborative Study

Sample XX relative to 1st IS in IU


0

1

2

3

4

5

6

7

8

9

10


70 58

71

4

72

7 22 73IH B3

IH

NU

P

T M A

Monitor assays Nuclisens bDNA 3 Procilex In-House Amplicore TMA

Sample XX in Current Study (coded Sample 1)

0

1

2

3

4

5

6

7

8

9

10


71 58 70 22 73

4

72

7

IH B3 IH M A

NU

P

T


Current Collaborative Study

1st IS (coded Sample 2)


0

1

2

3

4

5

6

7

8

9

10

Estimated IU (log10/ml)

3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

70

4

72

7 58 73

22

71

IH

NU

P

T B3 A

M

IH


Current Collaborative Study Potency of XX (Sample 1) relative

to 1st IS (IU)


Method Type No. Labs Mean

Amplicor Quantitative 1 5.64

bDNA V3 Quantitative 1 5.61

Monitor Quantitative 1 5.70

Nuclisens Quantitative 1 5.09

Taqman Quantitative 1 5.27

Overall Quantitative 5 5.46

In House Qualitative 1 5.00

NGI Qualitative 1 6.25

Procleix Qualitative 1 4.91

Overall Qualitative 3 5.39

Overall 8 5.44

Current Study: Relative Potency of XX to 1st IS – Log10 IU/ml


Method Type N Labs Mean Min Max

Abbott Quantitative 1 5.52

bDNA v3 Quantitative 4 5.71 5.62 5.80

In-House Quantitative 2 5.67 5.59 5.76

Monitor Quantitative 9 5.65 5.30 5.90

Nuclisens Quantitative 3 5.22 5.16 5.32

Overall Quantitative 19 5.59

All Labs Qualitative 5 5.66 5.35 6.10

Overall 24 5.60

Relative Potency of XX to 1st IS

Original Study (Log10 IU/ml)


22ndnd International Standard for HIV-1 RNA International Standard for HIV-1 RNA

• No evidence of any drift in values between the two studies - no stability issues

• Four options for assigning value to XX:

– 1: Use results from original study (5.60 log10 IU/ml)

– 2: Use results from current study (5.44 log10 IU/ml)

– 3: Pool data from both studies (5.56 log10 IU/ml)

– 4: Determine overall mean for each method and obtain compromise between methods – pragmatic rather than statistically based

• Option 3 favoured by NIBSC statistician


International Collaborative Study to Establish International Collaborative Study to Establish 22ndnd International Standard for HIV-1 RNA International Standard for HIV-1 RNA

ParticipantsParticipants

• T Cuypers Sanquin – CLB, The Netherlands

• M Chudy Paul Ehrlich Institute, Germany

• J Docker Gen Probe, USA

• K Fransen Institute of Tropical Medicine, Belgium

• I Hewlett CBER, FDA, USA

• F Intelmann Baxter, Austria

• D Jardine National Reference Laboratory, Australia

• J Turczyn Bayer Testing lab, USA

• Richard Smith NGI, USA

• C Davis/H Holmes NIBSC, UK


HIV-1 Variability – Genotype panelsHIV-1 Variability – Genotype panels• WHO 1st International Reference Panel for HIV-1 Genotypes

(Code 01/466) established in 2003

• Contains 11 members:

– Group M – Subtypes A - G, and AG-GH recombinant

– Group N and O representative

• HIV diversity increasing, with greater frequency of unusual subtypes, outlier groups and circulating recombinant forms (CRFs) being encountered especially in cities eg London, Paris

• Greater diversity presents continuing challenge to NAT technology

• Would a second genotype panel be of value containing:

– More diverse subtypes, group N, group O representatives?

– More CRFs?

– HIV-2 representatives?


ConclusionsConclusions

• Report on candidate 2nd International Standard for HIV-1 RNA to WHO ECBS

• Recommendation on unitage – option 3??

• Need for 2nd HIV Genotype Panel containing more diverse field isolates??

xviii sogat washington 24 may 2005 sogat and hiv nat standards - 2 nd international standard for...

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