Update on the Update on the P. falciparumP. falciparum Standard & Replacement of Standard & Replacement of the HBV & HCV International the HBV & HCV International

Standards Standards

Sally Baylis, NIBSCSally Baylis, NIBSC

SoGAT XIXSoGAT XIX

NIBSC Proposal for NIBSC Proposal for Production of Standards for Production of Standards for

P. falciparumP. falciparum

For standardisation of NAT assays (qualitative For standardisation of NAT assays (qualitative and quantitative)and quantitative)

Screening of blood for transfusion and tissues: Screening of blood for transfusion and tissues: exclusion of infected donations (run controls); exclusion of infected donations (run controls); determination of levels of parasitaemia where TTIs determination of levels of parasitaemia where TTIs occur; validation of assay sensitivitiesoccur; validation of assay sensitivities Diagnosis and clinical management of malaria: Diagnosis and clinical management of malaria: standardisation of commercial and in-house tests – standardisation of commercial and in-house tests – harmonisation of results to a single reference materialharmonisation of results to a single reference material

Vaccine studies: cross-comparison of studies, parasite Vaccine studies: cross-comparison of studies, parasite loads, efficacy etc.loads, efficacy etc.

Candidate Candidate P. falciparumP. falciparum Standards Standards

Sample AA Freeze-dried blood from patient Sample AA Freeze-dried blood from patient (~10% parasitaemia)(~10% parasitaemia)

Sample BB Liquid preparation of Sample BB Liquid preparation of P. falciparumP. falciparum cultured cultured in vitroin vitro to ~10% ring forms to ~10% ring forms

Sample CC Liquid preparation of blood from Sample CC Liquid preparation of blood from patient (~7% parasitaemia)patient (~7% parasitaemia)

Sample DD Liquid preparation of blood from Sample DD Liquid preparation of blood from patient (~0.007% parasitaemia)patient (~0.007% parasitaemia)

Collaborative Study to Establish an Collaborative Study to Establish an International Standard for International Standard for P. P.

falciparumfalciparum Commenced April 2005Commenced April 2005

14 laboratories participated in collaborative study14 laboratories participated in collaborative study

Participants requested to test samples in four Participants requested to test samples in four independent assaysindependent assays

Initially at 10-fold dilutionsInitially at 10-fold dilutions Subsequently at half log dilutions around the end pointSubsequently at half log dilutions around the end point

20 data sets received, 2 from quantitative assays 20 data sets received, 2 from quantitative assays & the rest from qualitative assays& the rest from qualitative assays

NIBSC collated & analysed dataNIBSC collated & analysed data

Sample CC

0

1

2

3

4

5

6

7

8

9

10

PCR Detectable Units/ml (log10)

6.0 7.0 8.0 9.0 10.0 11.0

11B 03A

14B

01A

02A

02B

07

14A

04

08

10B 09 06

10A

01B 13 12

Sample AA

0

1

2

3

4

5

6

7

8

9

10

PCR Detectable Units/ml (log10)

6.0 7.0 8.0 9.0 10.0 11.0

03A

04

07

02A

02B

06

14B

01A

08

11A

11B

14A

10B 12 09 10A 13

Sample DD

0

1

2

3

4

5

6

7

8

9

10

PCR Detectable Units/ml (log10)

3.0 4.0 5.0 6.0 7.0 8.0

03A 14B 02B

07

11B

14A

02A

11A

06

08

10B

01A

04

09

10A

01B 13 12

Sample BB

0

1

2

3

4

5

6

7

8

9

10

PCR Detectable Units/ml (log10)

6.0 7.0 8.0 9.0 10.0 11.0

03A

14B

11B

14A

01A

02B

07

02A

08

09

10B

11A 01B

04

06

10A

12

13

PCR Detectable Units/ml (logPCR Detectable Units/ml (log1010))

Overall Mean PCR Detectable Units/ml (log10) from End-Point Assays

N - Number of laboratory estimatesSD - Standard Deviation of log10 estimates across laboratories

Sample N Mean Min Max Range SD

AA 17 8.51 7.44 10.46 3.01 0.74

BB 18 8.45 7.62 9.85 2.23 0.69

CC 17 8.35 6.35 9.92 3.57 0.83

DD 18 5.51 4.51 6.86 2.36 0.60

Sample N Mean Range SD

BB 17 -0.09 2.31 0.67

CC 16 -0.22 3.06 0.76

DD 17 -3.04 2.35 0.60

Overall Potencies Relative to AA (log10) from End-Point Assays

Accelerated Degradation Studies Accelerated Degradation Studies of Sample AAof Sample AA

Incubation Time -20°C +4°C +20°C +37°C +45°C

8 months 0.00 -0.04 0.26 0.41 0.44

12 months 0.00 0.06 0.35 0.62 0.62

Relative potencies of accelerated degradation samples with respect to sample AA stored at -20ºC (log10 drop)

Summary & ConclusionsSummary & Conclusions

The mean log10 “equivalents”/ml were 8.51 for sample The mean log10 “equivalents”/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD (NIBSC code 04/112). for sample DD (NIBSC code 04/112).

Predictions of the stability of the freeze-dried Predictions of the stability of the freeze-dried preparation AA indicate that it is extremely stable and preparation AA indicate that it is extremely stable and suitable for long term use.suitable for long term use.

Propose that AA be established as the 1Propose that AA be established as the 1st st International International Standard for Standard for P. falciparumP. falciparum DNA NAT assays, NIBSC code DNA NAT assays, NIBSC code 04/176.04/176.

Proposed potency is 10Proposed potency is 1099 International units per ml. Each International units per ml. Each vial contains the equivalent of 0.5 ml of material, and vial contains the equivalent of 0.5 ml of material, and the content of each vial would be 5 x 10the content of each vial would be 5 x 1088 IU/ml. IU/ml. Submission to ECBS by JulySubmission to ECBS by July

Replacement of the 1Replacement of the 1stst International International Standard for HBV DNA NIBSC Code Standard for HBV DNA NIBSC Code

97/74697/746 The 1The 1stst International Standard for HBV DNA was International Standard for HBV DNA was

established by the WHO ECBS in October 1999established by the WHO ECBS in October 1999

Assigned a concentration of 10Assigned a concentration of 1066 IU/ml IU/ml

Standard made from a dilution of Standard made from a dilution of the Eurohep the Eurohep reference 1, genotype A, HBsAg subtype adwreference 1, genotype A, HBsAg subtype adw

Stocks of 97/746 are extremely lowStocks of 97/746 are extremely low

Study proposed to evaluate replacement at Study proposed to evaluate replacement at SoGAT 2005SoGAT 2005

Candidate 2Candidate 2ndnd International International StandardStandard

Materials coded AA (97/746) & BB (97/750) Materials coded AA (97/746) & BB (97/750) showed no significant difference in potency in the showed no significant difference in potency in the collaborative studycollaborative study

ECBS noted that BB (made from the same stock ECBS noted that BB (made from the same stock as AA) could be reserved for potential future use as AA) could be reserved for potential future use as a replacement standardas a replacement standard

Current study designed to demonstrate the Current study designed to demonstrate the equivalence of the candidate replacement (BB) to equivalence of the candidate replacement (BB) to AAAA

Real-time data on samples AA & BBReal-time data on samples AA & BB Accelerated degradation data for samples AA & BBAccelerated degradation data for samples AA & BB

Collaborative Study to Establish Collaborative Study to Establish 22ndnd International Standard for HBV International Standard for HBV

DNADNA 6 laboratories participated in study6 laboratories participated in study

The 1The 1stst International Standard and candidate BB were International Standard and candidate BB were recoded as samples 1 and 2 respectivelyrecoded as samples 1 and 2 respectively

Participants requested to test samples in four Participants requested to test samples in four independent assaysindependent assays Initially at 10-fold dilutionsInitially at 10-fold dilutions Subsequently at half log dilutions around the end pointSubsequently at half log dilutions around the end point

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

Estimated IU/ml (log10) From Quantitative Assays

Laboratory numberSample

97/746 97/750

1 5.99 5.97

2A 6.08 5.99

2B 6.06 5.92

4 5.94 5.86

Mean* 6.00 5.93

*combining results of lab 2 to give a single lab mean prior to calculating overall mean of laboratories

Estimated PCR-detectable units/ml Estimated PCR-detectable units/ml (log(log1010) )

Laboratory numberSample

97/746 97/750

3A 6.48 6.58

3B 6.90 6.68

3C 6.56 6.35

5 6.49 6.25

6 6.51 6.59

Accelerated Degradation Studies Accelerated Degradation Studies of 97/746 and 97/750 of 97/746 and 97/750

Storage temperature Sample

97/746 97/750

-20°C 6.02 5.92

+4°C 5.92 5.91

+20°C 5.94 6.03

Samples incubated at -20ºC, +4 ºC and +20 ºC for ~4.5 yearsEstimated IU/ml (log10)

Proposal for 2Proposal for 2ndnd International International Standard for HBV DNAStandard for HBV DNA

Real-time & accelerated degradation data Real-time & accelerated degradation data indicate 97/746 & 97/750 are very stable & indicate 97/746 & 97/750 are very stable & suitable for long term usesuitable for long term use

No significant differences found in estimated No significant differences found in estimated IU/ml or PCR-detectable units/ml for 97/746 & IU/ml or PCR-detectable units/ml for 97/746 & 97/75097/750

Propose that 97/750 be established as the 2Propose that 97/750 be established as the 2ndnd International Standard for HBV DNA with a International Standard for HBV DNA with a unitage of 10unitage of 1066 IU/ml – submission to ECBS by July IU/ml – submission to ECBS by July

Replacement of the 2Replacement of the 2ndnd International Standard for HCV International Standard for HCV

RNA (96/798)RNA (96/798)

Proposal made at SoGAT 2005 to replace the HCV Proposal made at SoGAT 2005 to replace the HCV RNA International Standard as requested by WHORNA International Standard as requested by WHO

Agreement that HCV 1a genotype would be Agreement that HCV 1a genotype would be sourced & would be anti-HCV negative and sourced & would be anti-HCV negative and diluted in plasma rather than cryosupernatant diluted in plasma rather than cryosupernatant

Progress in Replacing 96/798 Progress in Replacing 96/798

3 anti-HCV negative window period genotype 1a 3 anti-HCV negative window period genotype 1a donations have been obtaineddonations have been obtained

The genotype of each has been confirmed by LiPA The genotype of each has been confirmed by LiPA & DNA sequencing& DNA sequencing

Absence of other vial markers confirmed in these Absence of other vial markers confirmed in these stocksstocks

Titres determined by qPCR and Roche Monitor Titres determined by qPCR and Roche Monitor assay by comparison to 96/798 assay by comparison to 96/798

Progress in Replacing 96/798 Progress in Replacing 96/798 contd.contd.

Material has been freeze-dried in two batchesMaterial has been freeze-dried in two batches

Batch 1, 2085 vials, fill CV = 0.77%Batch 1, 2085 vials, fill CV = 0.77%

Batch 2, 2100 vials, fill CV = 1.02%Batch 2, 2100 vials, fill CV = 1.02%

Titre of new freeze-dried preparations is Titre of new freeze-dried preparations is at least at least double that of the 2double that of the 2ndnd HCV RNA HCV RNA

After freeze-drying, the activity of the After freeze-drying, the activity of the preparations is ~70% that of the original bulkpreparations is ~70% that of the original bulk

Call for collaborative study participantsCall for collaborative study participants

AcknowledgementsAcknowledgements

David Padley, Alan Heath & Nita Shah, NIBSCDavid Padley, Alan Heath & Nita Shah, NIBSC

Peter Chiodini, Hospital for Tropical Diseases, Peter Chiodini, Hospital for Tropical Diseases, LondonLondon

Claire Swales, LSHTM, LondonClaire Swales, LSHTM, London

Collaborative study participantsCollaborative study participants

Collaborative Study to Establish an Collaborative Study to Establish an International Standard for International Standard for P.falciparumP.falciparum

ParticipantsParticipants

A CalderaroA Calderaro Università degli Studi di Parma, Università degli Studi di Parma, ItalyItaly P ChiodiniP Chiodini Hospital for Tropical Diseases, London, UKHospital for Tropical Diseases, London, UK A da SilvaA da Silva CDC, USACDC, USA C DeferC Defer EFS Nord de FranceEFS Nord de France I FelgerI Felger Swiss Tropical InstituteSwiss Tropical Institute K KainK Kain Centre for Travel & Tropical Medicine, CanadaCentre for Travel & Tropical Medicine, Canada S KrishnaS Krishna St. George’s Hospital, London, UKSt. George’s Hospital, London, UK R LeeR Lee Institute of Clinical Pathology & Medical ResearchInstitute of Clinical Pathology & Medical Research, Australia, Australia D PadleyD Padley NIBSC, UKNIBSC, UK F F PerandinPerandin University of Brescia, ItalyUniversity of Brescia, Italy G PisaniG Pisani ISS, ItalyISS, Italy T RuckesT Ruckes Qiagen GmbHQiagen GmbH R SauerweinR Sauerwein UMC St Radboud, NetherlandsUMC St Radboud, Netherlands S SauledaS Sauleda Laboratori de Seguretat Transfusional, SpainLaboratori de Seguretat Transfusional, Spain

Collaborative Study to Establish 2Collaborative Study to Establish 2ndnd International Standard for HBV DNAInternational Standard for HBV DNA

ParticipantsParticipants

S Baylis/ D PadleyS Baylis/ D Padley NIBSC, UKNIBSC, UK M Chudy M Chudy PEI, GermanyPEI, Germany W GerlichW Gerlich University of Geissen, University of Geissen,

GermanyGermany S KerbyS Kerby CBER, USACBER, USA A Klotz/M GessnerA Klotz/M Gessner Baxter, AustriaBaxter, Austria G PisaniG Pisani ISS, ItalyISS, Italy