WHO/BS/2012.2190 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL

WHO/BS/2012.2190

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 15 to 19 October 2012

Report of a WHO collaborative study to assess the suitability of a candidate

replacement International Standard for antibody to pandemic H1N1

influenza virus

Diane Major

1, Alan Heath

2 and John Wood

1

Division of Virology1 and Biostatistics

2

National Institute for Biological Standards and Control, Blanche Lane, South Mimms,

Potters Bar, Herts EN6 3QG, UK

Study coordinator: Diane Major1

Phone: +44 (0)1707 641550, Fax: +44 (0)1707 641, Email:[email protected]

This document has been prepared for the purpose of inviting comments and suggestions on

the proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 01 October 2012 and

should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland,

attention: Quality Safety and Standards (QSS). Comments may also be submitted

electronically to the Responsible Officer: Dr Tiequn Zhou at email: [email protected].

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WHO/BS/2012.2190

Page 2

Abstract Haemagglutination-inhibition (HI) and virus neutralisation (VN) serology assays are being used

to assess immunogenicity of pandemic H1N1 (H1N1pdm) vaccines in clinical trial throughout

the world, yet the assays are poorly standardised and previous studies have shown that results of

such assays to be variable between different laboratories. A freeze dried International Standard

(IS) for H1N1pdm antibody was established by WHO in 2010 and this has been distributed to

numerous laboratories conducting H1N1pdm serology. Due to the urgent need to make this

material available it was only possible to prepare a limited number of vials and stocks are now

completely depleted. A freeze dried candidate replacement IS for H1N1pdm antibody was

prepared from pooled plasma of subjects who had received a split pandemic H1N1 vaccine

prepared from the reassortant virus NYMC X-179A (derived from A/California/2009 virus).

Eighteen laboratories from eleven countries tested the candidate IS 10/202 and a panel of human

plasmas from recipients of A/California/7/2009 pandemic vaccine; eighteen using HI assays and

sixteen also using VN assays. A plasma negative for antibody to H1N1pdm was also included in

the panel. In most laboratories the tests were performed on at least three occasions using the

reassortant viruses NYMC X-179A (seventeen laboratories) and/or NYMC X-181(twelve

laboratories). Tests were also performed with A/California/2009 virus (two laboratories),

A/Christchurch/16/2010 virus (one laboratory) and NIBRG-121 virus (one laboratory). For tests

of antibody to NYMC X-179A and NYMC X-181, the % geometric coefficients of variation

(%GCV) for 10/202 between laboratories were 143% and 137% respectively for HI and 441%

and 372% respectively for VN. For tests of all sera, the % GCV ranged from 108-157% for HI

and 189-354% for VN, but for the titres relative to 10/202 the median % GCV was much

reduced (HI 36-144%; VN 29-178%).

H1N1pdm viruses continue to circulate and to date there has been very little antigenic drift

observed in the isolates [1]. Whilst there is no requirement to conduct clinical trials for the

H1N1pdm component of currently licensed influenza vaccines the H3N2 and B components of

seasonal influenza vaccines have been updated and therefore vaccines containing H1N1pdm

continue to be evaluated in clinical trials. Consequently, there is a continuing need to standardise

serology assays for antibody to H1N1pdm virus.

The results demonstrate that the candidate IS 10/202 will be of use in standardisation of HI and

VN assays of antibody to H1N1pdm virus vaccines. It was also shown that the relationship

between HI and VN titres for the 1st IS 09/194 and the candidate IS 10/202 are different.

Consequently it is proposed that 10/202 be established as the second IS for antibody to

A/California/2009 (H1N1) virus with an assigned potency for use in HI assay of 1200

International Units per ampoule ie 2400 IU/ml when reconstituted as directed with 0.5 ml dH2O

and that the consensus VN titre is stated in the Instructions for Use for 10/202.

WHO/BS/2012.2190

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Introduction

Recent WHO collaborative studies to evaluate influenza H3N2, H5N1 and H1N1pdm

serological tests have found a high degree of variability between VN and HI results from

different laboratories. However the use of a standard serum or plasma significantly reduced inter

laboratory variability [ 2, 3, 4]. Difficulties in comparing VN and HI titres include the lack of

standard protocols, differences in sensitivity and specificity of reagents and assay variability. An

International Standard (IS 09/194) for H1N1pdm antibody was established in 2010 and a recent

study in which panels of sera from several clinical trials were retested in a single laboratory

(separate laboratories for HI and VN) has demonstrated that use of the standard significantly

improved agreement in assay results between laboratories. [5]

Stocks of the first IS for antibody to H1N1pdm virus (09/194) are completely depleted and a

proposal by NIBSC to establish a replacement was approved by ECBS in October 2010.

The aims of the collaborative study are:

To evaluate the suitability of a freeze-dried plasma pool to serve as a replacement for

09/194, the first IS for antibody to H1N1pdm virus with an assigned potency in

International Units per ampoule for use in HI and VN assays. There is no international

conventional reference measurement procedure and this measurement is not traceable to

International System of Units (SI) of quantity.

To assess the candidate IS in terms of reactivity in the conventional HI and VN assays

used to detect H1N1pdm antibody.

To assess commutability ie to establish the extent to which the IS is suitable to serve as a

standard for the variety of different samples and viruses being assayed.

Materials

Candidate International Standard code 10/202 This is a batch of ampoules containing a freeze-dried pool of plasma obtained from:

6 human subjects who had received Shanghai Institute of Biological Products

inactivated split influenza vaccine containing the H1N1pdm strain NYMC X-179A

(A/California/7/2009).

Subjects gave informed consent for the use of their plasma which was approved by the ethical

committee of NIBSC.

The donations were tested and found negative for HBsAg, antibodies to HIV-1, 2 and HCV RNA

by nucleic acid amplification tests. After being tested and found positive for antibody to NYMC

X-179A virus by HI and VN, the donations were pooled (volume 900ml) and diluted in human

plasma tested and found negative for HBsAg, antibodies to HIV-1, 2 and HCV RNA and

negative for antibody against H1N1pdm virus (volume 4100 ml). The pooled plasma was filled

in ampoules and freeze-fried at NIBSC on 4 November 2010 following documented procedures.

At the end of processing, the ampoules were back-filled with Nitrogen (from liquid nitrogen

99.999% purity) and sealed by heat fusion of the glass. The integrity of the sealing of the

ampoules was checked visually. This was a 0.5g fill weight with a mean filled weight of 0.5153g

(contents of 396 ampoules weighed) and mean dry weight of 0.0406g (contents of 6 ampoules

WHO/BS/2012.2190

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weighed). The coefficient of variation (CV) for filled weights was 1.33%. 9917 ampoules were

filled and 9655 were available for issue. Residual moisture determined by colorimetric Karl

Fisher titration on 12 samples was 0.4347% with a CV of 18.92%.Oxygen headspace

determined using a Lighthouse laser head space analyser measured in 12 ampoules was 0.18%

with a CV of 48.25%. Bioburden of the contents of four samples was determined by total viable

count. This was 3cfu for one vial, the remaining three were negative. The ampoules were stored

at NIBSC at -20oC. Currently there are 9305 ampoules of 10/202 available for use. A summary

of the production of 10/202 and key parameters tested is given in appendix 3.

Additional coded study samples In order to demonstrate that the candidate IS is suitable for use in a variety of situations; the

following antiserum preparations were included as coded samples along with the first IS:

Sample code Description

A Freeze dried Human plasma negative for antibody to H1N1pdm

E Post vaccination plasma (H1N1pdm) - low titre

B Post vaccination plasma (H1N1pdm) - medium titre

C Post vaccination plasma (H1N1pdm) - high titre

F Freeze dried pool of human plasma candidate replacement

(10/202) for 09/194

D Duplicate of F

Viruses supplied If needed, participants were supplied with the following H1N1pdm viruses:

NYMC X-179A (X-179A) reassortant derived from A/California/7/2009 (used for

pandemic H1N1 vaccine production)

NYMC X-181 (X-181) reassortant derived from A/California/7/2009 (used for

pandemic H1N1 vaccine production)

Participants were expected to grow their own stocks of virus in readiness for serology tests

Design of study

Participants were requested to store sera at -20oC and viruses at -70

oC until use. Participants

were asked to reconstitute freeze dried samples with 0.5 ml distilled water and then to test

09/194 and the coded study samples for antibody to the two H1N1pdm viruses concurrently, on

three separate occasions. It was recommended that wherever possible, new ampoules of the

standards and samples were used for each assay. An excel spreadsheet was supplied for reporting

of the raw data from each test.

Statistical methods The results for the HI and VN tests were provided by participants as absolute titres (reciprocal

end-point dilutions). Within each laboratory, replicate titres were combined as geometric means.

Where a titre was negative, or below the starting dilution, a value of half the starting dilution was

assigned (e.g. a titre of <10 was taken as 5) to allow calculations of geometric means. Similarly,

for values reported as greater than a final dilution, a value of twice the final dilution was

assigned (e.g. >1280 was taken as 2560). Overall mean titres were calculated as the geometric

mean of the individual laboratory means. Variation between laboratories was expressed as the

percentage geometric coefficient of variation (%GCV).

WHO/BS/2012.2190

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To assess within laboratory reproducibility, a comparison was made of replicate tests in each

laboratory and calculating the percentage of tests giving greater than 2-fold or greater than 4-fold

variation in titres for all study samples. Each laboratory was requested to test the 7 samples

using the two viruses, giving 14 sets of replicate titres. The calculations were also performed for

the human plasmas E, B, C, F, and D and 09/194 alone.

To assess the effect of using a common standard, titres were expressed relative to the first IS

09/194 and the candidate replacement standard 10/202 (sample F), by taking the ratio of the

mean titre for a sample to the mean titre for 09/194 or 10/202 obtained by a laboratory, and

multiplying by an “assigned” value for 09/194 or 10/202.

The distribution of geometric mean titres between laboratories for each sample is also displayed

in histogram form, where each box represents the mean obtained by an individual laboratory, and

is labelled with the laboratory code number. Where a laboratory obtained a negative result, the

results are plotted as “-ve” (ie negative) in a position equivalent to a titre of 5.

Participants Although thirty two participants were invited, only eighteen participants from eleven countries

took part in the study. Participants were selected for their previous experience of influenza

serology, for their public health importance and for geographical distribution. The participants

are listed in Appendix 1 and were randomly assigned a laboratory code number, not necessarily

in the order listed.

Assay methods As there are no established standard protocols for HI or VN assays, participants were requested

to use their in-house methods. A checklist was supplied to each participant to indicate the key

parameters used in their methods. One of the benefits of an IS would be to reduce errors

associated with protocol variation.

VN protocols Thirteen of eighteen (72%) laboratories returning VN data also supplied checklists (table 1).

Most VN protocols could be grouped under a single methodology, with three different protocols

being used by a minority of laboratories.

(i) Cell suspension method and short incubation time of assay to endpoint (<26 hr): used

by labs 1, 2, 3, 5, 6, 7, 8, 10, 14, 15 and 17

(ii) Preformed cell monolayer and long incubation time of assay to endpoint (≥3 days): used

by labs 1, 11 and 16

(iii) Preformed cell monolayer and short incubation time of assay to endpoint(<26 hours):

used by lab 13

(iv) Plaque reduction assay: used by lab 18

Another variable was the quantity of input virus, which could affect the VN titre. Labs 1, 2, 7,

10, 11, 14, 15 and 17 used 100 TCID50/well whereas labs 5, 6 and 8 used 200 TCID50/well. Lab

3 calculated input virus based on pfu/ml and lab13 based on ffu/ml. Labs 3 and 10 incubated

virus/serum mixtures at room temperature whereas all the other laboratories which supplied

checklists performed this step at 35-37oC.

WHO/BS/2012.2190

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HI protocols Thirteen of eighteen (72%) returning HI data also supplied checklists (table 2). HI protocols

were generally similar. The variable parameters considered to be critical are:

(i) Red cell type: turkey rbc (used by labs 3, 6, 7, 8, 10, 11, 13, 14 and 17), chicken rbc

(used by labs 1 and 2), guinea pig (used by lab 5), human O (used by lab 15) or goose

(used by lab 16)

(ii) Rbc concentration ranged from 0.4% (lab 1) to 1% (lab5)

(iii) Virus added was either 4 Agglutinating Doses (Ads) (labs 2, 3, 5, 7, 8, 11, 13, 14, 15

and 17) or 8Ads (labs 1, 6, and 10)

(iv) Adsorption of sera with rbcs to remove nonspecific agglutinins (labs 5 and 14)

Lab 14 incubated serum/virus mixtures at 37oC. All other laboratories which supplied checklists

performed this step at room temperature.

Results and data analysis

Data Received

Study samples were sent to twenty one laboratories. Data were received from eighteen

laboratories. Two laboratories only returned data from HI assays, while the other sixteen

performed HI and VN assays. For HI assay seventeen laboratories that returned data used the

X179A virus. In addition X-181 was used by twelve of the laboratories and A/California/7/2009

(A/California), A/Christchurch/16/2010 (A/Chch) and NIBRG-121 by one laboratory each. One

laboratory returned data for A/California only. Nine laboratories provided data for VN assays

using X179A and X181. One of these also returned VN data using NIBRG-121. The remaining

laboratories returned data for one virus only, X-179 (laboratories 6, 7, 13 and 14), X-181

(laboratory 11), A/California (laboratory 8) and A/Chch (laboratory 5). Laboratory 1 provided

two sets of VN assay results using two different methodologies.

In previous studies the definition of titre appeared to differ, depending on whether addition of

virus was considered to be part of the dilution. In this study titres were reported as those prior to

addition of virus or cell suspension by all laboratories that returned assay check lists except one.

This laboratory included the virus volume in the expression of initial serum dilution for both HI

and VN assays. Laboratory 16 reported that the ELISA based VN assay would not work with

X179A and reported data for a CPE based assay as an alternative.

One laboratory returned data for single radial haemolysis assay (SRH) in addition to HI and VN

data. The SRH data for the individual samples reflected the overall pattern of antibody titres

observed for HI and VN assays and reproducibility of the assay within the laboratory was very

good. However the SRH data was not included in the analyses as there was only a single data set.

Assay reproducibility – replicate assays

The within laboratory reproducibility was assessed by comparing the replicate titres within each

laboratory for each sample and virus across replicate tests. The negative control sample A was

not included. The percentages with a range greater than 2-fold, and greater than 4-fold, are

shown in table 3. No analysis of reproducibility could be performed for Laboratory 16, which

only returned a single result for each sample.

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The reproducibility of both HI and VN assays is good, with the majority of laboratories having

no replicate tests varying by more than 2-fold. Laboratory 2 had poorer reproducibility with 50%

of replicate VN tests varying by more than 2-fold. Their HI tests were more consistent, although

for the current standard 09/194 the HI for the 3rd

replicate test was reported as 8-16 compared to

128 for the previous 2 tests (with both X179A and X181). The other samples in the 3rd

test gave

HI titres that were consistent across tests. Laboratory 17 also had some variability with their VN

tests, with 33% giving titres that differed by more than 2-fold, although none were greater than

4-fold.

Laboratory 15 performed repeat series of tests using isolates from different ampoules of virus

(X179A), which gave some differences in titres, particularly for the VN tests. The comparison

between replicate titres was performed separately for each of the repeat sets of tests, and so these

differences in titres resulting from different isolates are not reflected in table 3. If comparisons

were made across the tests with the different isolates, 42% of VN tests would have replicates

greater than 2-fold, and 17% greater than 4-fold.

Assay reproducibility – duplicate samples Samples D and F were duplicates of the candidate replacement IS (10/202). When the laboratory

GMTs for samples D and F were compared, the HI GMTs were within a 2-fold range for all

laboratories. The VN GMTs were within 2-fold for all laboratories except laboratory 2, which

had GMTs of 1345 and 3200 for samples D and F respectively, for both viruses X179A and

X181. Laboratory 16 was not included in the analysis, because of a potential mixing of sample

results, (reported under absolute titres: their results for sample D are all negative).

Comparing titres of samples D and F within individual assays, none of the HI tests had titres that

differed by more than 2-fold. For the VN tests, only laboratory 2 had titres that differed by more

than 2-fold. Laboratory 2 had 4 out of 8 repeat tests with a titre of 6400 for sample F and a titre

of 1600 for sample D. Apart from this, there was good reproducibility within assays for the

duplicate samples.

Absolute Titres Most laboratories obtained negative HI or VN titres for the negative sample A, with the

following exceptions:

Laboratory 1 obtained VN titres of 10 for 5 out of 12 replicate tests of sample A with their cpe

neutralisation assay (1A), but negative (<10) for all HI and all ELISA (1B) neutralisation assays.

Laboratory 2 obtained a titre of 20 for 1 out of 8 replicate tests with their VN assay, and titres of

10 were reported for the remaining replicates.

Laboratory 5 obtained a titre of 10 for 2 out of 8 replicate HI tests, but negative (<10) for all

other replicate HI tests and all VN tests.

Laboratory 16 reported titres of 20 or 40 for sample A in their HI and VN tests, but for sample

D, the candidate replacement IS, they reported negative (<10) or 10. It is possible that there was

a mix up of the samples either during the testing or the reporting of the results.

Laboratory 18 reported titres of 40 for 4 out of 6 replicate plaque reduction VN tests, using a

50% reduction threshold (18A), with the 2 other replicates giving negative results. Using a 100%

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reduction threshold (18B), 5 out of 6 replicates gave negative results, and one replicate gave a

titre of 10. All HI tests were negative.

The laboratory GMTs for the study samples (excluding the negative control sample A) are

shown in tables 4-9 for the HI assays, and tables 10-15 for the VN assays. Results obtained with

X179A and X181 viruses are shown separately in tables 16 (HI) and 17 (VN). For each

laboratory and sample, the overall GMT obtained from pooling results with all viruses is also

shown. The overall GMT (Geometric mean of the laboratory GMTs) and the between laboratory

percentage geometric coefficient of variation (%GCV) are also shown, excluding laboratory 16.

The results for laboratory 15 are generally lower than for other laboratories. The GMT and

%GCVs are also shown excluding laboratory 15, for comparison.

All laboratories obtained positive GMTs for samples C, D, F and 09/194 (with the exception of

laboratory 16, sample D, noted above). For the medium titre sample B, laboratories 6 and 15

obtained negative (<10) HI GMTs for virus X179A. Laboratory 6 did not use virus X181, and

laboratory 15 obtained a low positive HI GMT of 11. The VN tests gave positive titres for

sample B in all laboratories that performed them. For the low titre sample E, laboratories 6, 11,

13, 15, 17 and 18 obtained negative HI titres. Laboratories 1 and 5 also obtained negative HI

titres with X181, but low positives with X179A. Laboratories 10, 11, 15 and 18B obtained

negative VN titres, laboratory 1A a negative with X179A but positive with X181, and laboratory

17 a negative with X181 but a low positive with X179A.

The laboratory GMTs for the different samples are also shown in histogram form in figure 1.

Each box represents the GMT from an individual laboratory, and is labelled with the laboratory

code number. Results obtained with different viruses are shown separately, and colour coded.

The histograms illustrate that there was considerable variation between the GMTs from different

laboratories. There is no apparent consistent difference in laboratory GMTs between the different

viruses used. A paired comparison of results from laboratories that used both X179A and X181

showed no significant differences (p > 0.01) for any sample, for HI or VN tests.

The variation between laboratories is also reflected in the high %GCVs (pooling results from all

viruses), summarised in table 18, and in the fold range (max/min) between labs for the different

samples, summarised in table 19. The variability is generally greater between the VN assays than

the HI assays. The range (max/min) for the HI GMTs is between 10-fold (sample E) and 43-fold

(sample D). For the VN assays, the range is between 32-fold (sample E) and 463-fold (sample

F). These figures would be reduced if the results from laboratory 15 were excluded.

The overall GMTs for the candidate standard (10/202, samples D and F), pooling results from all

viruses, were 263 for HI assays and 381 for VN assays (excluding lab 16). These figures rise to

311 for HI and 601 for VN if the results from laboratory 15 are also excluded.

The overall GMTs for the current IS 09/194, pooling results from all viruses, were 149 for HI

and 520 for VN (excluding lab 16). These figures rise to 167 for HI and 557 for VN if the results

from laboratory 15 are also excluded. The previous collaborative study to establish the 1st IS

obtained “consensus” titres of 183 (HI) and 516 (VN) for 09/194. There is therefore very good

agreement between the two studies, despite the large variation in results between individual

laboratories.

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Use of 09/194 as a standard

Expression in IU The 1

st IS 09/194 was assigned a value of 650 International Units (IU) per ampoule, or 1300

IU/ml on reconstitution. The laboratory results for samples B – F were expressed in IU relative

to 09/194. To express the titre of a sample in IU, the ratio of the GMT obtained for the sample to

the GMT obtained for the concurrently tested IS is calculated, and multiplied by the assigned

value of 1300 IU/ml for the IS. For example, if an assay gives titres of 640 for sample X and 320

for the standard, then the titre of X relative to the standard is 2600 IU/ml ( = 1300 x 640/320), or

twice the assigned value of the IS.

The potencies of the samples B to F are shown in tables 20-29, split by virus, and also the overall

values for each laboratory, pooling the data for all viruses. They are also shown in histogram

form in figure 1 with different viruses are shown separately, and colour coded. Although the

values on the histograms in figure 1 are in IU rather than titres, the same scale has been used,

allowing direct comparison of inert-laboratory variability between absolute titres and potencies

in IU. To achieve this, negative results are plotted as 5 IU. A negative result is defined as a

negative titre (<10 or <8) for the sample before any conversion to IU. A value of 5 IU has also

been used to calculate the overall geometric means shown in tables 4-15. As a result, where there

is a mixture of positive and negative results, as with samples B and E, the overall geometric

mean in IU may be unrealistically low. However, it is difficult to select a more appropriate value

in IU to represent a negative result, as there are positive results (for example, laboratory 5 VN

results for samples B and E) which are as low as 12.

Inter-laboratory variability In figure 1, the histograms of absolute GMTs and the corresponding potencies expressed in IU

relative to the 1st IS 09/194 are shown together for both HI and VN tests, allowing direct

comparison of the inter-laboratory variability. As noted above, some of the results from

laboratory 16 appear anomalous, possibly as a result of a mix-up in reporting of sample results.

Expression of results in IU does not alter this. The results from laboratory 15, which were

generally much lower in absolute titres than other laboratories, for all samples, are more

consistent with other laboratories when expressed in IU relative to 09/194. Apart from the results

from laboratory 15, for the HI tests there is little apparent improvement in agreement between

laboratories from expressing results in IU relative to 09/194. There is reasonable agreement

between laboratories (excepting 15 and 16) for the absolute GMTs. For samples B and E, the

histograms show an apparent increased separation between the negative and positive results.

However, this is an artefact of plotting the negatives as 5 for both absolute titres (derived from

negative being typically <10 with doubling dilutions) and for IU, as noted above.

For the VN tests, there is greater variability between laboratories for absolute GMTs, and

expressing results in IU relative to 09/194 does improve the agreement between laboratories.

The variation between laboratories for results relative to 09/194 is also summarised as %GCVs

(pooling results from all viruses), in table 18, and in the fold range (max/min) between

laboratories for the different samples, summarised in table 19. The calculations are shown

excluding laboratory 16, and also excluding both laboratories 15 and 16.

The %GCVs and fold ranges for results relative to 09/194 were calculated using “corrected

titres”, rather than IU. The corrected titre is calculated in a similar way to the IU, but rather than

using an assigned IU for 09/194, the consensus titres obtained from the original collaborative

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study are used. These were 1:183 for HI tests and 1:516 for VN tests. For example, if an assay

gives an HI titre of 640 for sample X and 320 for 09/194, then the corrected titre of X relative to

09/194 is 366 ( = 183 x 640/320), or twice the consensus titre of 09/194. Where there is a

mixture of negative and positive results across laboratories, as for samples B and E, the choice of

a value to assign negative results has a large impact on the resulting %GCVs and fold-ranges.

Taking a value of 5 for a negative (<10) appears more appropriate for titres than for potencies in

IU, where the most appropriate assignment of an IU value to a negative result is not clear, as

discussed above. For this reason, the calculations were based on corrected titres. For all other

samples, where the results were positive for each laboratory, there is no difference in GCV or

fold range using either IU or titres, as there is simply a scale difference between the different

units.

From the table of %GCVs, looking at the results for the HI tests, excluding laboratory 16, there

is some reduction in inter-laboratory variability, particularly for samples D & F. However,

looking at the results excluding laboratory 15, it appears that this reduction is entirely due to the

low titres from laboratory 15 for these samples being brought into line by expressing them

relative to 09/194. There is no discernible improvement in inter-laboratory agreement for the HI

tests for the other laboratories. This pattern is mirrored with the fold-ranges between

laboratories, and from the histograms.

The results for the VN test show greater variability than the HI tests. For the current IS 09/194

the between laboratory %GCV of absolute titres is 195% for VN compared to 129% for HI. For

the candidate replacement standard, samples D and F, the %GCVs are 311% and 354% for VN,

and 141% and 127% for HI. Excluding laboratory 15, the values for D and F are 226% and

253% for VN and 81% and 54% for HI. The corresponding fold-ranges are 80-90 fold for VN

compared to 4-10 fold for HI. Expressing results relative to 09/194 does lead to a reduction in

inter-laboratory variation, particularly for the higher titre samples C, D and F, where the GCVs

(excluding laboratory 15) drop from 170%, 226% and 253% to 112%, 94% and 94%

respectively. The improvement in inter-laboratory agreement for the VN tests is most clearly

seen from the histograms.

The VN titres obtained by laboratory 5 using the A/ChCh virus are low for all samples, but the

laboratory only used this virus in VN tests, so it is not clear whether it is something specific to

the virus. Their HI results with this virus are generally lower than their HI results with other

viruses however. Apart from this observation, there were no apparent trends indicating

differences in results with the different viruses used.

Use of Candidate Replacement Standard (10/202)

Expression in IU The laboratory results for samples B, C, D, E and 09/194 were expressed in IU relative to sample

F (10/202). For the purpose of these calculations, Sample F was assigned a candidate unitage of

2400 IU/ml, based on the calibration against 09/194 from the HI tests.

The potencies of the samples B, C, D, E and 09/194 are shown in tables 30-39, split by virus, and

also the overall values for each laboratory, pooling the data for all viruses. They are also shown

in histogram form in figure 2 with the different viruses are shown separately, and colour coded.

As with the calculations and histograms relative to 09/194, negative results were plotted as 5 IU,

to maintain identical scales allowing direct comparisons.

WHO/BS/2012.2190

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Inter-laboratory variability Figure 2 shows the histograms of absolute GMTs and the corresponding potencies expressed in

IU relative to sample F together for both HI and VN tests, allowing direct comparison of the

inter-laboratory variability. Comments on the results from laboratories 15 and 16 are as before.

The variation between laboratories for results relative to sample F is also summarised as %GCVs

(pooling results from all viruses), in table 18, and in the fold range (max/min) between

laboratories for the different samples, summarised in table 19. The calculations are shown

excluding laboratory 16, and also excluding both laboratories 15 and 16. As before, the %GCVs

and fold ranges for results relative to 09/194 were calculated using “corrected titres”, rather than

IU.

For sample D, the duplicate of sample F, expressing results relative to sample F leads to

excellent agreement between laboratories for both HI and VN tests, which can be clearly seen

from the histograms. This is not unexpected, as essentially it is expressing the candidate standard

against itself. The %GCVs are reduced from 81% to 25% for HI tests, and from 226% to 29%

for VN tests.

For the other samples, any improvement in agreement is less marked. For the higher titre

samples C and 09/194 there is some improvement, mainly for the VN tests, but for samples B

and E there is less impact. For sample C the %GCV drops from 59% to 35% for HI tests and

from 170% to 80% for VN tests. In contrast for sample B the %GCV drops from 136% to 101%

for HI tests and from 200% to 160% for VN tests.

Using sample F as a candidate standard appears to give better improvement in inter-laboratory

agreement for sample C compared to using 09/194 (%GCVs of 35% and 84% respectively for

HI, and 80% and 112% for VN). For the other samples, apart from the duplicate D, there is little

difference in the effectiveness of the candidate standard compared to 09/194.

Calibration of Candidate Replacement Standard 10/202 The candidate replacement standard was included in duplicate as samples D and F. From tables

22 and 24, the overall means of the HI tests across laboratories (excluding laboratories 15 and

16), when expressed in IU/ml relative to 09/194, were 2309 and 2531 (geometric mean 2417).

For the VN tests the means were 1321 and 1488 (geometric mean 1402). These values represent

good agreement between the duplicate samples. However, they demonstrate considerable

differences in the calibration of the candidate replacement standard against the current IS 09/194

depending on whether HI or VN tests are used. For HI tests, an assigned value of 2400 IU/ml

would be appropriate for the candidate standard, but for VN tests, 1400 IU/ml would be

appropriate.

The relationship between the HI titres and the VN titres is different for the current IS and the

candidate replacement, which leads to this difference in calibrations. For the current IS, 09/194,

the overall consensus titres from the original collaborative study were 1:183 for HI and 1:516 for

VN. These were matched closely in the current study with values of 1:167 for HI and 1:557 for

VN. The VN titres are therefore approximately three times the HI titre. For the candidate

replacement standard, the corresponding means of the absolute HI titres were 297 and 326 for D

and F, with a mean of 311, and for VN titres the means were 567 and 638, with a mean of 601.

The VN titres are therefore approximately two times the HI titres for the candidate replacement

standard, compared to three times for the current IS.

WHO/BS/2012.2190

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Stability studies on 10/202 Samples of the candidate standard, NIBSC code 10/202, were stored at temperatures ranging

from -150˚C to +45˚C for 18 months. They were assayed concurrently at NIBSC, using X-181

virus in both haemagglutination-inhibition (HI) assays and a virus neutralisation (VN) assay.

Three different ampoules stored at each temperature were tested.

The samples stored at 45˚C could not be reconstituted, and could not be tested. The individual

assay results for the other samples are shown in table 40, along with the geometric mean titre.

For the HI tests, it is impossible to distinguish between the sample stored at +37˚C for 18 months,

and the baseline sample stored at -150˚C. For the VN tests there is an apparent drop in titre for

the +37˚C sample, but no apparent differences between the other samples, given the assay

variability present. With the problems in reconstitution experienced with the +45˚C samples, it is

possible that the apparent drop in titre at +37˚C may also be partly due to problems in

reconstitution, although the HI results have not been affected.

There is insufficient data to apply the usual Arrhenius model to predict the rate of degradation at

different temperatures. However, applying the ‘rule of thumb’ that the degradation rate will

double for every increase of 10˚C in temperature, the lack of any observed degradation after 18

months at +20˚C in either HI or VN tests is equivalent to non-observable degradation for a

minimum of 12 years when stored at -20˚C.

The stability studies will continue. With the current data there is no indication of any issues with

stability and 10/202 appears suitable to serve as an International Standard.

Discussion and conclusions There was considerable variation observed between participating laboratories in the level of

titres obtained for the same serum samples. Differences of up to 43-fold between laboratories

were observed for HI and up to 463-fold for VN tests. This level of variability is consistent with

that seen in previous collaborative studies [1,2,3]. Consensus HI and VN titres for IS 09/194

from this study are similar to those obtained in the previous collaborative study to establish the

IS showing very good agreement between the two studies.

In a previous study [3] most of the laboratories, recorded higher titres for tests using the

reassortant X-179A virus than those using the wild type A/California virus. In this study there

were no apparent trends associated with the use of different viruses however; most laboratories

only used X179A and X181 viruses so any differences related to the use of A/California, A/Chch

or NIBRG-121 are limited to observations of single laboratory comparisons.

Expressing titres relative to the IS 09/194 gives a reduction in the variation between laboratories

in nearly all cases. However as discussed previously the improvement in agreement for the HI

data is entirely due to bringing low titres from laboratory 15 in line by expressing them relative

to 09/194. For VN there is a clear improvement in inter laboratory agreement when titres are

expressed relative to 09/194.

Expressing titres relative to the candidate replacement IS 10/202 gives a reduction in the

variation between laboratories similar to that observed with the use of 09/194 for samples B and

E and greater than using 09/194 for samples C and D. As discussed previously this is not

unexpected for sample D which is a duplicate of 10/202. Sample C is an aliquot of one of the

WHO/BS/2012.2190

Page 13

plasma donations used to prepare 10/202 and consequently an improved reduction in variability

against 10/202 compared to 09/194 is not surprising.

The candidate replacement IS was included in the study as duplicate sera F and D and the overall

means for the duplicates were in good agreement for both HI and VN assay. However when the

absolute titres were converted into IU by calibration against the IS 09/194 there was a

considerable discrepancy in value depending on whether HI or VN titres were used.

Previous studies [2, 3] have demonstrated that there is no consistent correlation between HI and

VN titres either between laboratories or to a lesser extent for individual sera.

This study has demonstrated that the relationship between HI and VN titre is different for the IS

09/194 and candidate replacement IS 10/202. VN titres are approximately two times the HI titres

for the candidate replacement standard, compared to three times for the current IS.

The candidate replacement IS has been prepared exclusively from donations from immunized

subjects, whereas the IS 09/194 was prepared from donations mainly from convalescent subjects.

It is plausible that convalescent sera contain a higher proportion of antibodies directed against

antigenic determinants that function in VN assay but not in HI assay compared to antibodies in

post immunisation sera.

Based on results of this collaborative study, different unitages would apply depending on

whether HI or VN assays were used. However there is no consistent relationship between HI and

VN titres in different laboratories [3, 4] and it therefore does not appear appropriate to assign a

unitage based on VN titres. This belief is consistent with the approach taken to assign unitage to

09/194 where linkage to the 1st IS for antibody to H5N1 antibody clade 1 07/150 by HI and not

VN assays was used (see below).

Due to the pandemic situation, 09/194 was prepared, evaluated and distributed before evaluation

by ECBS could take place and was assigned a consensus HI titre of 1:183 and VN titre of 1:516

(obtained by using X-179A virus). Subsequently it was evaluated by ECBS and assigned a

unitage of 1300 IU/ml linked with the value assigned to the first established IS for influenza

serology; 1st IS for antibody to H5N1 antibody clade 1 07/150. The value was based on a

consensus of HI titres.

Stocks of 09/194 are completely depleted; consequently 10/202 has been evaluated and

distributed before evaluation by ECBS. It has been assigned an interim consensus HI titre of 311,

based on comparison with the 1st IS 09/194.

Proposal 10/202 should be established as the 2

nd IS for antibody to influenza A/California/7/2009

(H1N1)pdm virus with an assigned potency for use in HI assay of 1200 IU per ampoule (2400

IU/ml on reconstitution). This potency has been linked to the potency of the 1st IS for antibody to

(H1N1)pdm 09/194 using the HI titre of 09/194 (1:183), to give equivalent conversion factors

from IU to consensus HI titre for both standards. It is also proposed that the consensus HI titre of

1:310 and VN titre of 1:600 be declared in the “Instructions for Use” that accompany 10/202.

(titres rounded to simplify conversion calculations)

The material is suitable for use in HI and VN assays of human sera for antibody to

A/California/7/2009 (H1N1)pdm like viruses.

WHO/BS/2012.2190

Page 14

Comments from participants A draft of this report was sent to participants who were invited to check their data for errors and

asked if they agreed with the conclusions and recommendations of the report.

Twelve of the eighteen participants responded. Of these, four indicated agreement with the report

content, three stated agreement with the conclusions and one also stated agreement with the

recommendation of the report. None of the respondents indicated any specific direct

disagreement with either the conclusions or the proposal.

Laboratory 1 Correction to participant information – text corrected

Minor corrections to text – text corrected

In agreement with report

Laboratory 2 No errors in presentation of own data

In agreement with report and conclusions.

Laboratory 3 In agreement with presentation of own data and conclusions of report.

Correction to participant information – text corrected

Laboratory 5 Query regarding the method used to calculate GMTs. – methods checked and verified.

Explanation of method used sent to laboratory.

Laboratory 6 Correction to participant information – text corrected

Laboratory 7 Under HI protocols. We titrate the virus at 8 HA, but we add 4 HA into the assay itself. –

text corrected

Question under Results and data analysis/Data Received. Are we the only laboratory that

reports an initial serum dilution that includes the virus volume? – this was the only

laboratory that reported including virus volume in initial serum dilution

Laboratory 8 In agreement with the conclusions and recommendation.

Laboratory 13 Responded, but had no comments

Laboratory 14 Some minor editorial changes – text corrected

Sentence in section VN protocols. Thirteen of eighteen (72%) laboratories returning VN

data also supplied checklists. Comment ‘Information is listed for 14 laboratories’. -

WHO/BS/2012.2190

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Thirteen laboratories returned checklists, laboratory 18 did not return a checklist but

specified the use of a plaque reduction assay on their result sheet.

Sentence in section Inter-laboratory variability. Using sample F as a candidate standard

appears to give better improvement in inter-laboratory agreement for sample C compared

to using 09/194. Comment: ‘Is that possible to discuss this a bit further in the discussion

section? Why such high range of difference were seen since sample F (replacement IS) is

calibrated off the 09/194’ – sentence added to discussion. Sentence in section Inter-laboratory variability: For the other samples apart from the

duplicate D, there is little difference in the effectiveness of the candidate standard

compared to 09/194. Comment ‘Also, is that possible to discuss this non-linear impact

among different samples after converting to IU/ml – will this non-linear normalization

change the interpretation of the trial results?’ - The results are showing that both the

candidate standard 10/202 and the existing standard 09/194 are similar in their

effectiveness. It is not expected that switching from using 09/194 to 10/202 will have an

impact on the interpretation of trial results. The use of IU rather than absolute titres

should facilitate the comparison of results between different trials.

Sentence in section Calibration of Candidate Replacement Standard 10/202: The VN

titres are therefore approximately two times the HI titres for the candidate replacement

standard, compared to three times for the current IS. Comment ‘It would be interesting to

see the relationship between VN and HI, as expressed in original reported titers and the

converted or corrected IU/ml as calculated from both 09/194 and 10/202 separately, for

each sample from each individual lab. It seems very different pictures can be derived –

the relationship can be reverted when converted to IU/ml …’- It has been established in

previous studies that there is no consistent relationship between HI and VN titres

between laboratories and since the proposal is that 10/202 is for use as an IS for HI

assay only it was not considered that detailed analysis of VN/HI ratios was relevant to

this report. A detailed analysis of the relationship between VN and HI titres in this study

is however of great interest generally and will be included in a subsequent publication.

Sentence in Discussion and conclusions: It is plausible that convalescent sera contain a

higher proportion of antibodies directed against antigenic determinants that function in

VN assay but not in HI assay compared to antibodies in post immunisation sera.

Comment ‘It is possible that Abs with different avidities were generated from

convalescent sera as compared to the Abs generated from the post immunization. Lab 14

uses the condition of incubating virus/serum mixture at 37oC to enhance the Ab and virus

binding (interaction), high VN/HI tier ratio were seen for all test samples without losing

the assay specificity (Sample A remain negative for both HI and VN)’- We agree with the

suggestion that populations of antibody with different avidities may be generated in post

infection and post immunisation sera. However, most laboratories use 37oC for the

virus/serum mixture incubation step, so this is unlikely the reason that lab 14 records

higher titres in VN assay. Information on virus/serum incubation conditions added to

protocol sections.

It is valuable to use the 10/202 candidate 2nd

H1N1 International Standard (IS) to

evaluate the variability of the HI and VN within and among laboratories. However, the

10/202 IS should be used with caution. The relationship between the HI titer and the VN

titer for the 1st H1N1 IS (09/194) and the candidate 2

nd H1N1 IS is different, making it

challenging to propose a consensus VN titer to 10/202. In addition, the 10/202 candidate

2nd

H1N1 IS would be most valuable if the assigned potency was determined with data

from HI and VN assays with certain inclusion criteria; for example, assigned IS potency

with data generated from assays measuring same type of immune response(s) and with no

extreme outliers (outside the 2SD range of the GMT) contributing to the assignments.

WHO/BS/2012.2190

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The current assigned value of 10/202 was established from labs with variable assay

reproducibility or measuring different types of immune responses, which may

misrepresent the true value of the 10/202 2nd

H1N1 candidate IS. - We accept that the

various serology assays may be measuring different antibody populations, but in this

study the candidate IS 10/202 reduced inter-laboratory variability for all the assays used.

The issue of the consensus titre being derived from a variety of labs each with different

assay sensitivities and validity criteria is difficult to address. It would be difficult to select

which labs’ results to use, or be able to justify it, as the IS needs to be accepted for use by

all labs. However if the potency of 10/202 is described in IU, then the actual consensus

GMT is not important.

Laboratory 15 Responded but had no comments

Noted low titres and are investigating cause.

Correction to participant information – text corrected

Laboratory 16 Confirmed the mix up in reporting of their data and provided corrected data sheets – It is

too late to modify the data analysis for this report and the data for laboratory 16 has not

been included in the assignment of unitage for reagent 10/202. The corrected results

appear to agree with data from other laboratories, in particular individual titres are

close to overall study GMTs for most samples and assays. Consequently, the inclusion of

the data would not have a significant impact on the assignment of unitage for reagent

10/202.

Data analysis will be updated to include lab 16 for subsequent publications.

A completed protocol checklist was supplied – information added to study documents.

Assay parameters all fall within ranges in comparison tables, no corrections required.

Lab 16 did perform replicate assays, however there was a misunderstanding regarding the

way in which titres should be reported and only a single data set was originally

submitted. A full data set has now been submitted. - Data analysis will be updated for

subsequent publications

Laboratory 18

Responded but had no comments

WHO/BS/2012.2190

Page 17

References

1. Recommended composition of influenza virus vaccines for use in the 2012-2013 northern

hemisphere influenza season.

WHO Weekly epidemiological record 2012; No 10, 87: 83-95

2. Stephenson I, Das R, Wood J, Katz J. Comparison of neutralising antibody assays for

detection of antibody to influenza A/H3N2 viruses: an international collaborative study.

Vaccine 2007; 25: 4056-65.

3. Stephenson I, Heath A, Major D, Newman R, Hoschler K, Wang-Junzi, Katz J, Weir J,

Zambon MC, Wood JM. Reproducibility of serologic assays for influenza virus A (H5N1).

Emerging Infectious Diseases 2009;15: 1250-1259

4. Wood JM, Major D, Heath A, Newman RW, Höschler K, Stephenson I, Clark T, Katz JM,

Zambon MC. Reproducibility of serology assays for pandemic influenza H1N1: collaborative

study to evaluate a candidate WHO International Standard.

Vaccine 2012; 30(2):210-7

5. Wagner R, Göpfert C, Hammann J, Neumann B, Wood J, Newman R, Wallis C, Alex N,

Pfleiderer M. Enhancing the reproducibility of serological methods used to evaluate

immunogenicity of pandemic H1N1 influenza vaccines-An effective EU regulatory approach.

Vaccine 2012; 30(27):4113-22

Acknowledgements We are grateful to Dr Wang Junzhi (NIFDC) and Dr Chaggui Li (NIFDC) for donation of the

plasma used in production of the candidate IS.

We are also grateful to Dr. Paul Metcalf (NIBSC), Dr Dorothy Xing (NIBSC) and Dr Jackie

Katz (CDC) for assistance in design and co-ordination of the study.

We thank Kate Guilfoyle, Sarah Roseby and Paul Jefferson for organisation of the study at

NIBSC and the study participants.

http://www.ncbi.nlm.nih.gov/pubmed?term=Wood%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Major%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Newman%20RW%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=H%C3%B6schler%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Stephenson%20I%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Clark%20T%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Katz%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Zambon%20MC%5BAuthor%5D&cauthor=true&cauthor_uid=22100887

http://www.ncbi.nlm.nih.gov/pubmed?term=Wagner%20R%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=G%C3%B6pfert%20C%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Neumann%20B%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Wood%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Newman%20R%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Wallis%20C%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Alex%20N%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

http://www.ncbi.nlm.nih.gov/pubmed?term=Pfleiderer%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22446639

WHO/BS/2012.2190

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Table 1. Comparison of variable parameters in VN assay protocols

Parameter or variable Most frequent variables used Range

A. Stock virus preparation

Cell substrate for virus

growth

10-11 day old embryonated

eggs

10-11 day old embryonated eggs

MDCK

Conditions of virus growth Various used Eggs 2-3 days @32-37oC

MDCK 2-5 days @33oC

Stock virus infectivity and

method of determination

~106 TCID50/ml ELISA based

assay

105.23

-108.2

TCID50\pfu\ffu/ml

B. Serum preparation

Storage of sera following

receipt from NIBSC

various +4oC - -70

oC 0-2 freeze thaw cycles

Pre-assay treatment of sera Heat treatment, 56oC for

30mins

56oC for 30-40mins. No treatment

Initial serum dilution 1:10 (10µl in 90µl diluent) 1:5 to 1:40

Serum diluent Various used PBS or assay diluent

Serial dilution steps 1:2 dilution steps 1:2

Range of serum dilutions Various used 1:5 to 1:1280; 1:10 to 1:10240

C. virus preparation

Virus conc added per well 100 TCID50 100-200 TCID50;

105.3

ffu ; 102.57

- 103.03

pfu

Dilution of stock virus to

achieve assay virus conc

Various X179A 1/10 -1/2000

X181 1/50 - 1/63000

Vol of virus solution added 50µl 50-100µl

Virus diluent Assay diluent PBS or assay diluent

Virus/serum mix

incubation

1hour, room temp 1-2 hour, room temp - 37 oC

Calculated starting serum

dilution

1:10 expressed as start dilution

of serum without addition of

virus or cells

1:10- 1:20 (either include virus volume

or not)

D. Cell preparation

Preparation of cells and

number of cells added

Cell suspension method Cell suspension or preformed

monolayer method

Cell type used MDCK MDCK

Assay diluent DMEM + BSA DMEM or MEM or DCoons or 199 or

Ultra +/- BSA or FCS +/-trypsin

E. Assay set up

Total assay volume per

well

200µl 200µl

Incubation time of assay to

endpoint reading

18-20 hours 16 hours - 6 days

Incubation conditions 37 O

C in 5% CO2 35-37 O

C in 5% CO2

F. Endpoint estimation and

calculation

Endpoint determination

Viral antigen detection by

ELISA using anti-nucleoprotein

antibody

Detection of viral antigen by ELISA/

CPE (light microscopy)/HA/plaque

reduction

Endpoint titer calculation

method

50% neutralization 50% neutralization or highest serum

dilution showing no CPE or 50%/100%

plaque reduction

WHO/BS/2012.2190

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Table 2. Comparison of variable parameters in HI assay protocols

Parameter or variable Most frequent variables

used

Range

A. Stock virus preparation

Cell substrate for virus

growth


eggs


eggs, MDCK

Conditions of virus growth 3 days at 34-35oC Eggs 2-3 days @32-

37oC;MDCK 5 days @33

oC

Stock virus

haemagglutination units

titre

various X179A 8->2048

X181 16 - >2048

B. Serum preparation

Storage of sera following

receipt from NIBSC

various +4C to -70C 0-2 freeze thaw

cycles

Treatment of sera 3 RDE to 1 sera, 18hours

37°C, Heat for 30-60 min

56OC

3-4 parts RDE to 1 part sera,

18hrs/overnight 37°C. Heat for

30-60 min 56OC +/- adsorption

with rbcs

Serum diluent PBS PBS; 0.85% saline

Initial serum dilution 1:10 1:4 to 1:10 (1 lab included

virus vol in start dilution)

Serial dilution

steps/volume

1:2 dilutions in 25µl vol 1:2 dilutions in 25-50µl vol

Range of sera dilutions 1:10 to 1:1280 1:8 to 1:16384;

1:10 to 1:1024 or 20480

C. Cell preparation

Cell type Turkey Turkey; chicken; guinea pig;

human O; goose

Preparation of rbcs Within 72 hrs of bleed Within 2 hrs to 2 weeks of

bleed

Red cell diluent PBS PBS +/- 1% BA or NaCl +

0.05%BSA

Red cell suspension

concentration

0.5% v/v 0.4-1% v/v

D. Virus preparation

Virus HA titration 4 HA units 4-8 HA units

Volume of virus added 25µl 25-50µl

Virus/serum mix

incubation conditions

30 min at room temp 15-60 min room temp or 37oC

E. HI Assay set up

Total volume per well various 75-200µl

Incubation conditions to

HI endpoint

various 30-60 min room temp

F. Endpoint estimation and

calculation

Endpoint determination Reciprocal of last well giving

complete inhibition shown by

streaming of RBC button

Reciprocal of last well giving

complete inhibition as shown

by streaming of RBC button;

Reciprocal of last well giving

complete inhibition as shown

by RBC button

WHO/BS/2012.2190

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Table 3: Within Laboratory Reproducibility of VN and HI Titres

% > 2-fold % > 4-fold

Lab HI VN HI VN

1 0 - 0 -

1A - 0 - 0

1B - 0 - 0

2 17 50 17 8

3 0 0 0 0

4 0 - 0 -

5 0 17 0 0

6 0 0 0 0

7 0 0 0 0

8 0 0 0 0

9 0 - 0 -

10 0 0 0 0

11 0 0 0 0

12 0 0 0 0

13 0 17 0 0

14 0 0 0 0

15 0 0 0 0

16* - - - -

17 8 33 0 0

18 0 - 0 -

18A - 17 - 0

18B - 0 - 0

* - only returned a single result for each sample

WHO/BS/2012.2190

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Table 4 HI - Laboratory GMTs by Virus: Sample B

Sample B

Lab X179A X181 A/Cal/7/09 A/ChCh/16/10 NIBRG121 GMT

1 80 45 60

2 45 57 51

3 64 64 59 62

4 160 160

5 40 40 40 13 26

6 <10 <10

7 63 63

8 80 80

9 90 71 80

10 127 127 127

11 40 40 40

12 80 80 80

13 40 40

14 202 202

15 <10 11 <10

16 320 320 320

17 32 28 30

18 80 80 80

Overall Excluding 16

52 50 51

Overall Excl 15 & 16

59 58 57

%GCV Excluding 16

170 91 157

%GCV Excl 15 & 16

138 55 136

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Table 5 HI - Laboratory GMTs by Virus: Sample C

Sample C


1 226 320 269

2 456 512 483

3 256 256 174 225

4 640 640

5 320 1280 320 160 349

6 160 160

7 640 640

8 508 508

9 359 320 339

10 640 640 640

11 320 320 320

12 640 640 640

13 508 508

14 806 806

15 28 45 36

16 320 320 320

17 254 320 285

18 640 640 640


349 375 367


413 464 425

%GCV Excluding 16

127 134 112

%GCV Excl 15 & 16

63 65 59

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Table 6 HI - Laboratory GMTs by Virus: Sample D

Sample D


1 320 180 240

2 256 256 256

3 256 256 256 256

4 640 640

5 320 320 160 101 174

6 80 80

7 320 320

8 403 403

9 320 359 339

10 508 508 508

11 160 254 202

12 640 640 640

13 202 202

14 806 806

15 14 25 19

16 10 <10 <10

17 202 160 180

18 320 320 320


252 239 253


306 299 297

%GCV Excluding 16

161 134 141

%GCV Excl 15 & 16

81 53 81

WHO/BS/2012.2190

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Table 7 HI - Laboratory GMTs by Virus: Sample E

Sample E


1 13 <10 <10

2 8 8 8

3 16 9 16 13

4 20 20

5 10 <10 10 13 10

6 <10 <10

7 50 50

8 10 10

9 13 11 12

10 10 10 10

11 <10 <10 <10

12 40 40 40

13 <10 <10

14 20 20

15 <10 <10 <10

16 320 320 320

17 <10 <10 <10

18 <10 <10 <10


11 <10 10


11 <10 11

%GCV Excluding 16

114 84 108

%GCV Excl 15 & 16

114 86 108

WHO/BS/2012.2190

Page 25

Table 8 HI - Laboratory GMTs by Virus: Sample F

Sample F


1 320 254 285

2 256 256 256

3 256 256 256 256

4 640 640

5 320 320 226 127 207

6 160 160

7 320 320

8 403 403

9 320 285 302

10 508 508 508

11 320 320 320

12 640 640 640

13 254 254

14 640 640

15 14 22 18

16 320 320 320

17 202 190 195

18 320 320 320


275 248 274


335 315 326

%GCV Excluding 16

143 137 127

%GCV Excl 15 & 16

52 43 54

WHO/BS/2012.2190

Page 26

Table 9 HI - Laboratory GMTs by Virus: Sample 09/194

Sample 09/194


1 160 101 127

2 57 57 57

3 256 219 256 243

4 320 320

5 160 160 160 202 174

6 80 80

7 218 218

8 320 320

9 226 160 190

10 254 160 202

11 160 160 160

12 320 320 320

13 80 80

14 640 640

15 22 22 22

16 160 80 113

17 63 40 49

18 160 127 143


151 111 149


171 130 167

%GCV Excluding 16

128 120 129

%GCV Excl 15 & 16

96 65 100

WHO/BS/2012.2190

Page 27

Table 10 VN - Laboratory GMTs by Virus: Sample B

Sample B


1A 320 254 285

1B 160 254 213

2 269 320 293

3 119 108 209 139

4

5 12 12

6 40 40

7 180 180

8 80 80

9

10 26 27 27

11 25 25

12 40 40 40

13 304 304

14 423 423

15 13 20 15

16 320 640 453

17 50 52 51

18A 320 254 285

18B 63 80 71


106 83 87


125 96 97

%GCV Excluding 16

205 186 219

%GCV Excl 15 & 16

163 170 200

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 28

Table 11 VN - Laboratory GMTs by Virus: Sample C

Sample C


1A 1140 403 678

1B 1016 2560 1612

2 1903 3200 2468

3 795 1175 176 548

4

5 113 113

6 640 640

7 905 905

8 202 202

9

10 152 235 211

11 254 254

12 160 160 160

13 776 776

14 3345 3345

15 22 80 34

16 320 320 320

17 201 359 269

18A 806 1280 1016

18B 320 320 320


507 495 451


645 594 529

%GCV Excluding 16

256 223 222

%GCV Excl 15 & 16

155 189 170

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 29

Table 12 VN - Laboratory GMTs by Virus: Sample D

Sample D


1A 285 202 240

1B 3225 4561 3835

2 1345 1345 1345

3 1467 1339 400 923

4

5 190 190

6 640 640

7 1613 1613

8 254 254

9

10 278 254 260

11 320 320

12 80 80 80

13 408 408

14 6457 6457

15 13 40 19

16 <10 <10 <10

17 587 508 552

18A 1016 1280 1140

18B 160 202 180


528 411 463


704 519 567

%GCV Excluding 16

392 302 311

%GCV Excl 15 & 16

240 238 226

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 30

Table 13 VN - Laboratory GMTs by Virus: Sample E

Sample E


1A <10 40 14

1B 51 63 57

2 95 80 87

3 36 31 59 40

4

5 12 12

6 20 20

7 57 57

8 16 16

9

10 <10 <10 <10

11 <10 <10

12 10 10 10

13 47 47

14 160 160

15 <10 <10 <10

16 320 320 320

17 13 <10 11

18A 58 80 68

18B <10 <10 <10


24 18 22


26 20 23

%GCV Excluding 16

213 211 189

%GCV Excl 15 & 16

210 204 184

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 31

Table 14 VN - Laboratory GMTs by Virus: Sample F

Sample F


1A 226 180 202

1B 2873 4063 3417

2 3200 3200 3200

3 1740 1350 438 1010

4

5 190 190

6 640 640

7 2281 2281

8 320 320

9

10 200 273 252

11 320 320

12 80 80 80

13 650 650

14 7348 7348

15 13 25 16

16 320 320 320

17 570 508 538

18A 1016 1280 1140

18B 202 202 202


588 420 513


791 557 638

%GCV Excluding 16

441 372 354

%GCV Excl 15 & 16

277 270 253

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 32

Table 15 VN - Laboratory GMTs by Virus: Sample 09/194

Sample 09/194


1A 285 570 403

1B 1613 1810 1709

2 1600 1600 1600

3 734 507 1049 731

4

5 1280 1280

6 320 320

7 1613 1613

8 320 320

9

10 169 162 164

11 254 254

12 80 80 80

13 795 795

14 4191 4191

15 127 320 173

16 320 640 453

17 279 285 282

18A 806 1280 1016

18B 160 160 160


498 408 520


553 418 557

%GCV Excluding 16

222 177 195

%GCV Excl 15 & 16

215 191 194

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 33

Table 16: HI - Laboratory GMTs by Sample and Virus

Sample B Sample C Sample D Sample E Sample F 09/194

Lab X179A X181 X179A X181 X179A X181 X179A X181 X179A X181 X179A X181

1 80 45 226 320 320 180 13 <10 320 254 160 101

2 45 57 456 512 256 256 8 8 256 256 57 57

3 64 64 256 256 256 256 16 9 256 256 256 219

4 160 640 640 20 640 320

5 40 40 320 1280 320 320 10 <10 320 320 160 160

6 <10 160 80 <10 160 80

7 63 640 320 50 320 218

8

9 90 71 359 320 320 359 13 11 320 285 226 160

10 127 127 640 640 508 508 10 10 508 508 254 160

11 40 40 320 320 160 254 <10 <10 320 320 160 160

12 80 80 640 640 640 640 40 40 640 640 320 320

13 40 508 202 <10 254 80

14 202 806 806 20 640 640

15 <10 11 28 45 14 25 <10 <10 14 22 22 22

16 320 320 320 320 10 <10 320 320 320 320 160 80

17 32 28 254 320 202 160 <10 <10 202 190 63 40

18 80 80 640 640 320 320 <10 <10 320 320 160 127

Overall Excluding

16

52 50 349 375 252 239 11 <10 275 248 151 111

%GCV Excluding

16

170 91 127 134 161 134 114 84 143 137 128 120

WHO/BS/2012.2190

Page 34

Table 17: VN - Laboratory GMTs by Sample and Virus

Sample B Sample C Sample D Sample E Sample F 09/194

Lab X179A X181 X179A X181 X179A X181 X179A X181 X179A X181 X179A X181

1A 320 254 1140 403 285 202 <10 40 226 180 285 570

1B 160 254 1016 2560 3225 4561 51 63 2873 4063 1613 1810

2 269 320 1903 3200 1345 1345 95 80 3200 3200 1600 1600

3 119 108 795 1175 1467 1339 36 31 1740 1350 734 507

4

5

6 40 640 640 20 640 320

7 180 905 1613 57 2281 1613

8

9

10 26 27 152 235 278 254 <10 <10 200 273 169 162

11 25 254 320 <10 320 254

12 40 40 160 160 80 80 10 10 80 80 80 80

13 304 776 408 47 650 795

14 423 3345 6457 160 7348 4191

15 13 20 22 80 13 40 <10 <10 13 25 127 320

16 320 640 320 320 <10 <10 320 320 320 320 320 640

17 50 52 201 359 587 508 13 <10 570 508 279 285

18A 320 254 806 1280 1016 1280 58 80 1016 1280 806 1280

18B 63 80 320 320 160 202 <10 <10 202 202 160 160

Overall Excluding

16

106 83 507 495 528 411 24 18 588 420 498 408

%GCV Excluding

16

205 186 256 223 392 302 213 211 441 372 222 177

WHO/BS/2012.2190

Page 35

Table 18: Between Laboratory %GCV

%GCV

HI VN

09/194 B C D E F 09/194 B C D E F

Absolute

Titres

Excluding

Lab 16 129 157 112 141 108 127 195 219 222 311 189 354

Excluding

Labs 15 &

16 100 136 59 81 108 54 194 200 170 226 184 253

Potency

relative to

09/194

Excluding

Lab 16 168 82 60 97 62 164 127 132 132 143

Excluding

Labs 15 &

16 124 84 57 98 56 168 112 94 125 94

Potency

relative to

Sample F

Excl

Lab 16 62 144 36 24 95 143 178 85 29 143

Excl

Labs 15 &

16 56 101 35 25 96 94 160 80 29 135

WHO/BS/2012.2190

Page 36

Table 19: Fold Range (max/min) Between Laboratories

Fold Range (max/min) between laboratories

HI VN

09/194 B C D E F 09/194 B C D E F

Absolute Titres

Excluding

Lab 16 29 40 23 43 10 36 52 36 98 349 32 463

Excluding

Labs 15 & 16 13 40 5 10 10 4 52 36 30 81 32 92

Potency relative to

09/194

Excluding

Lab 16 33 9 5 8 6 76 23 21 13 22

Excluding

Labs 15 & 16 33 9 4 8 4 76 23 15 13 13

Potency relative to

Sample F

Excl

Lab 16 6 21 2 3 10 22 25 8 3 16

Excl

Labs 15 & 16 4 21 2 3 10 13 25 8 3 16

WHO/BS/2012.2190

Page 37

Table 20 HI – Potency relative to IS 09/194 by Virus (IU): Sample B

Sample B

Lab X179A X181 A/Cal/7/09 A/ChCh/16/10 NIBRG121 GM

1 650 581 614

2 1026 1298 1154

3 325 379 301 334

4 650 650

5 325 325 325 81 193

6 -ve -ve

7 379 379

8 325 325

9 516 581 547

10 650 1032 819

11 325 325 325

12 325 325 325

13 650 650

14 409 409

15 -ve 650 -ve

16 2600 5200 3677

17 650 916 798

18 650 819 729


283 587 289


371 581 372

%GCV Excluding 16

402 65 391

%GCV Excl 15 & 16

247 69 245

WHO/BS/2012.2190

Page 38

Table 21 HI - Potency relative to IS 09/194 by Virus (IU): Sample C

Sample C


1 1838 4127 2754

2 10404 11678 11023

3 1300 1517 884 1204

4 2600 2600

5 2600 10400 2600 1032 2600

6 2600 2600

7 3819 3819

8 2064 2064

9 2067 2600 2318

10 3276 5200 4127

11 2600 2600 2600

12 2600 2600 2600

13 8254 8254

14 1638 1638

15 1638 2600 2064

16 2600 5200 3677

17 5200 10400 7601

18 5200 6552 5837


3011 4411 3209


3136 4651 3298

%GCV Excluding 16

80 101 82

%GCV Excl 15 & 16

79 104 84

WHO/BS/2012.2190

Page 39

Table 22 HI - Potency relative to IS 09/194 by Virus (IU): Sample D

Sample D


1 2600 2315 2454

2 5839 5839 5839

3 1300 1517 1300 1369

4 2600 2600

5 2600 2600 1300 650 1300

6 1300 1300

7 1909 1909

8 1638 1638

9 1841 2919 2318

10 2600 4127 3276

11 1300 2064 1638

12 2600 2600 2600

13 3276 3276

14 1638 1638

15 819 1459 1093

16 81 -ve -ve

17 4127 5200 4788

18 2600 3276 2918


2176 2808 2210


2322 2998 2309

%GCV Excluding 16

63 57 60

%GCV Excl 15 & 16

54 52 57

WHO/BS/2012.2190

Page 40

Table 23 HI - Potency relative to IS 09/194 by Virus (IU): Sample E

Sample E


1 102 -ve -ve

2 182 182 182

3 81 56 81 72

4 81 81

5 81 -ve 81 81 75

6 -ve -ve

7 301 301

8 41 41

9 72 91 81

10 51 81 64

11 -ve -ve -ve

12 163 163 163

13 -ve -ve

14 41 41

15 -ve -ve -ve

16 2600 5200 3677

17 -ve -ve -ve

18 -ve -ve -ve


32 20 27


36 23 30

%GCV Excluding 16

373 403 368

%GCV Excl 15 & 16

360 412 361

WHO/BS/2012.2190

Page 41

Table 24 HI - Potency relative to IS 09/194 by Virus (IU): Sample F

Sample F


1 2600 3273 2917

2 5839 5839 5839

3 1300 1517 1300 1369

4 2600 2600

5 2600 2600 1838 819 1546

6 2600 2600

7 1909 1909

8 1638 1638

9 1841 2315 2064

10 2600 4127 3276

11 2600 2600 2600

12 2600 2600 2600

13 4127 4127

14 1300 1300

15 819 1300 1032

16 2600 5200 3677

17 4127 6180 5198

18 2600 3276 2918


2373 2913 2401


2547 3158 2531

%GCV Excluding 16

62 63 62

%GCV Excl 15 & 16

49 53 56

WHO/BS/2012.2190

Page 42

Table 25 VN - Potency relative to IS 09/194 by Virus (IU): Sample B

Sample B


1A 1460 579 919

1B 145 182 162

2 219 260 238

3 210 276 259 247

4

5 12 12

6 163 163

7 145 145

8 325 325

9

10 200 218 214

11 129 129

12 650 650 650

13 497 497

14 131 131

15 129 81 111

16 1300 1300 1300

17 235 237 236

18A 516 258 365

18B 516 650 579


277 265 217


294 298 226

%GCV Excluding 16

110 93 164

%GCV Excl 15 & 16

109 75 168

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 43

Table 26 VN - Potency relative to IS 09/194 by Virus (IU): Sample C

Sample C


1A 5202 920 2187

1B 819 1839 1227

2 1546 2600 2005

3 1407 3013 219 975

4

5 115 115

6 2600 2600

7 730 730

8 819 819

9

10 1169 1881 1670

11 1300 1300

12 2600 2600 2600

13 1268 1268

14 1038 1038

15 230 325 258

16 1300 650 919

17 940 1635 1239

18A 1300 1300 1300

18B 2600 2600 2600


1325 1578 1126


1516 1848 1235

%GCV Excluding 16

110 89 127

%GCV Excl 15 & 16

76 47 112

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 44

Table 27 VN - Potency relative to IS 09/194 by Virus (IU): Sample D

Sample D


1A 1300 459 773

1B 2600 3276 2918

2 1093 1093 1093

3 2597 3433 496 1641

4

5 193 193

6 2600 2600

7 1300 1300

8 1032 1032

9

10 2138 2034 2059

11 1638 1638

12 1300 1300 1300

13 667 667

14 2003 2003

15 129 163 139

16 -ve -ve -ve

17 2739 2317 2543

18A 1638 1300 1459

18B 1300 1638 1459


1380 1311 1158


1656 1615 1321

%GCV Excluding 16

122 143 132

%GCV Excl 15 & 16

53 79 94

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 45

Table 28 VN - Potency relative to IS 09/194 by Virus (IU): Sample E

Sample E


1A -ve 91 46

1B 41 46 43

2 77 65 71

3 63 80 73 72

4

5 12 12

6 81 81

7 46 46

8 64 64

9

10 -ve -ve -ve

11 -ve -ve

12 163 163 163

13 77 77

14 50 50

15 -ve -ve -ve

16 1300 650 919

17 62 -ve 50

18A 93 81 87

18B -ve -ve -ve


33 23 32


38 27 36

%GCV Excluding 16

259 342 228

%GCV Excl 15 & 16

234 336 206

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 46

Table 29 VN - Potency relative to IS 09/194 by Virus (IU): Sample F

Sample F


1A 1032 409 650

1B 2316 2918 2600

2 2600 2600 2600

3 3080 3462 543 1796

4

5 193 193

6 2600 2600

7 1838 1838

8 1300 1300

9

10 1538 2182 1999

11 1638 1638

12 1300 1300 1300

13 1063 1063

14 2279 2279

15 129 102 119

16 1300 650 919

17 2661 2317 2480

18A 1638 1300 1459

18B 1638 1638 1638


1536 1341 1283


1859 1734 1488

%GCV Excluding 16

121 180 143

%GCV Excl 15 & 16

43 83 94

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 47

Table 30 HI – Potency relative to Sample F by Virus (IU): Sample B

Sample B


1 600 426 505

2 422 534 474

3 600 600 556 585

4 600 600

5 300 300 424 238 300

6 -ve -ve

7 476 476

8 476 476

9 673 602 636

10 600 600 600

11 300 300 300

12 300 300 300

13 378 378

14 756 756

15 -ve 1200 -ve

16 2400 2400 2400

17 378 356 368

18 600 600 600


269 484 276


351 442 354

%GCV Excluding 16

%GCV Excl 15 & 16

WHO/BS/2012.2190

Page 48

Table 31 HI - Potency relative to Sample F by Virus (IU): Sample C

Sample C


1 1696 3026 2266

2 4276 4800 4531

3 2400 2400 1632 2100

4 2400 2400

5 2400 9600 3394 3024 4036

6 2400 2400

7 4800 4800

8 3024 3024

9 2695 2695 2695

10 3024 3024 3024

11 2400 2400 2400

12 2400 2400 2400

13 4800 4800

14 3024 3024

15 4800 4800 4800

16 2400 2400 2400

17 3024 4039 3509

18 4800 4800 4800


3046 3635 3208


2955 3535 3128

%GCV Excluding 16

%GCV Excl 15 & 16

WHO/BS/2012.2190

Page 49

Table 32 HI - Potency relative to Sample F by Virus (IU): Sample D

Sample D


1 2400 1698 2019

2 2400 2400 2400

3 2400 2400 2400 2400

4 2400 2400

5 2400 2400 1697 1905 2018

6 1200 1200

7 2400 2400

8 2400 2400

9 2400 3026 2695

10 2400 2400 2400

11 1200 1905 1512

12 2400 2400 2400

13 1905 1905

14 3024 3024

15 2400 2694 2543

16 75 -ve -ve

17 2400 2019 2211

18 2400 2400 2400


2201 2314 2209


2188 2279 2190

%GCV Excluding 16

%GCV Excl 15 & 16

WHO/BS/2012.2190

Page 50

Table 33 HI - Potency relative to Sample F by Virus (IU): Sample E

Sample E


1 94 -ve -ve

2 75 75 75

3 150 88 150 126

4 75 75

5 75 -ve 106 238 116

6 -ve -ve

7 378 378

8 60 60

9 94 94 94

10 47 47 47

11 -ve -ve -ve

12 150 150 150

13 -ve -ve

14 75 75

15 -ve -ve -ve

16 2400 2400 2400

17 -ve -ve -ve

18 -ve -ve -ve


33 18 29


37 21 32

%GCV Excluding 16

%GCV Excl 15 & 16

WHO/BS/2012.2190

Page 51

Table 34 HI - Potency relative to Sample F by Virus (IU): 09/194

09/194


1 1200 953 1070

2 534 534 534

3 2400 2056 2400 2279

4 1200 1200

5 1200 1200 1697 3810 2018

6 1200 1200

7 1634 1634

8 1905 1905

9 1695 1347 1511

10 1200 756 952

11 1200 1200 1200

12 1200 1200 1200

13 756 756

14 2400 2400

15 3810 2400 3024

16 1200 600 849

17 756 505 600

18 1200 952 1069


1315 1071 1300


1225 988 1233

%GCV Excluding 16

%GCV Excl 15 & 16

WHO/BS/2012.2190

Page 52

Table 35 VN - Potency relative to Sample F by Virus (IU): Sample B

Sample B


1A 3395 3395 3395

1B 150 150 150

2 202 240 220

3 163 191 1145 330

4

5 150 150

6 150 150

7 189 189

8 600 600

9

10 312 240 256

11 189 189

12 1200 1200 1200

13 1123 1123

14 138 138

15 2400 1905 2222

16 2400 4800 3394

17 212 246 229

18A 756 476 600

18B 756 952 849


433 474 406


379 412 365

%GCV Excluding 16

%GCV Excl 15 & 16

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 53

Table 36 VN - Potency relative to Sample F by Virus (IU): Sample C

Sample C


1A 12100 5394 8078

1B 848 1512 1133

2 1427 2400 1851

3 1096 2088 966 1303

4

5 1427 1427

6 2400 2400

7 952 952

8 1512 1512

9

10 1824 2069 2005

11 1905 1905

12 4800 4800 4800

13 2865 2865

14 1093 1093

15 4276 7620 5184

16 2400 2400 2400

17 848 1694 1198

18A 1905 2400 2138

18B 3810 3810 3810


2070 2824 2106


1957 2558 1991

%GCV Excluding 16

%GCV Excl 15 & 16

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 54

Table 37 VN - Potency relative to Sample F by Virus (IU): Sample D

Sample D


1A 3024 2695 2855

1B 2694 2694 2694

2 1009 1009 1009

3 2024 2380 2191 2193

4

5 2400 2400

6 2400 2400

7 1697 1697

8 1905 1905

9

10 3336 2237 2472

11 2400 2400

12 2400 2400 2400

13 1505 1505

14 2109 2109

15 2400 3810 2800

16 -ve -ve -ve

17 2471 2400 2461

18A 2400 2400 2400

18B 1905 2400 2138


2155 2346 2166


2138 2235 2131

%GCV Excluding 16

%GCV Excl 15 & 16

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 55

Table 38 VN - Potency relative to Sample F by Virus (IU): Sample E

Sample E


1A -ve 535 168

1B 42 38 40

2 71 60 65

3 49 56 321 96

4

5 150 150

6 75 75

7 60 60

8 119 119

9

10 -ve -ve -ve

11 -ve -ve

12 300 300 300

13 173 173

14 52 52

15 -ve -ve -ve

16 2400 2400 2400

17 56 -ve 49

18A 136 150 143

18B -ve -ve -ve


37 28 48


43 34 55

%GCV Excluding 16

%GCV Excl 15 & 16

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 56

Table 39 VN - Potency relative to Sample F by Virus (IU): 09/194

09/194


1A 3024 7625 4802

1B 1347 1069 1200

2 1200 1200 1200

3 1013 901 5744 1737

4

5 16145 16145

6 1200 1200

7 1697 1697

8 2400 2400

9

10 2028 1430 1561

11 1905 1905

12 2400 2400 2400

13 2936 2936

14 1369 1369

15 24190 30478 26127

16 2400 4800 3394

17 1173 1346 1258

18A 1905 2400 2138

18B 1905 1905 1905


2031 2327 2432


1678 1799 2096

%GCV Excluding 16

%GCV Excl 15 & 16

Lab 1A – cpe

Lab 1B – ELISA

WHO/BS/2012.2190

Page 57

Table 40 – Stability Studies for 10/202. Titres (HI and VN) after 18 months

storage at elevated temperatures

HI VN

Run 1 2 3 GMT 1 2 3 GMT

Temperature

-150˚C 320 320 320 320 184 267 266 236

-70˚C 320 640 640 508 335 329 177 269

-20˚C 640 640 320 508 200 215 236 217

+4˚C 320 320 320 320 233 187 228 215

+20˚C 320 320 160 254 194 216 231 213

+37˚C 320 320 320 320 145 134 124 134

WHO/BS/2012.2190

Page 58

Figure legends

Figure 1 Histograms showing the distribution of geometric mean titres across laboratories.

Each box represents the GMT for one laboratory, and is labelled with the laboratory code

number. Results for X179A are shown in white, and for X181 in light grey. The results for

A/Chch, NIBRG-121 and the wild type A/California virus are all shown in dark grey. The results

for the HI and VN tests are shown side by side. The respective titres relative to 09/194 are shown

below the absolute titres.

Figure 2 Histograms showing the distribution of geometric mean titres across laboratories.

Each box represents the GMT for one laboratory, and is labelled with the laboratory code

number. Results for X179A are shown in white, and for X181 in light grey. The results for

A/Chch, NIBRG-121 and the wild type A/California virus are all shown in dark grey. The results

for the HI and VN tests are shown side by side. The respective titres relative to sample F (10/202

the candidate replacement for IS 09/194) are shown below the absolute titres.

WHO/BS/2012.2190

Page 59

Figure 1: Sample B

Sample B - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

15

05

15

17 05

02

05

11

13

17

01

05

11

08

03

01

03

07

09

12

18

02

03

09

12

18

04

10

14

10

16

16

X 179A X 181 O ther V iruses

Sample B - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05

15

10

10

11

15

06

12

17

12

17

08

18B

03

18B

03

01B

03

07

01A

02

13

14

16

18A

01A

01B

02

18A

16


Sample B - HI relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI relative to 09/194 in IU

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

15

05 05

08

03

03

05

07

11

12

14

03

05

11

12

01

04

09

10

13

17

18

01

09

15

18

02

02

10

17

16 16


Sample B - VN relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN relative to 09/194 in IU

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05 15 01B

02

03

06

07

10

14

15

01B

10

11

08

03

17

02

03

17

18A

12

13

18A

18B

01A

12

18B

01A

16

16


WHO/BS/2012.2190

Page 60

Figure 1: Sample C

Sample C - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15

15

05

03

01

06

05

03

05

09

11

16

17

01

03

09

11

16

17

08

02

04

07

10

12

13

14

18

02

10

12

18

05


Sample C - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15 15 08

05

03

10

12

17

12

16

18B

01A

10

11

16

17

18B

03

06

07

13

18A

01A

01B

03

18A

02

14

01B

02


Sample C - HI relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

03 05

03

14

15

03

05

08

01

04

05

06

09

10

11

12

16

09

11

12

15

07

17

18

01

10

16

18

02

13

02

05

17


Sample C - VN relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05

03

15

15

08

01B

07

16

02

03

10

13

14

16

17

18A

01A

11

17

18A

06

12

18B

01B

02

03

10

12

18B

01A


WHO/BS/2012.2190

Page 61

Figure 1: Sample D

Sample D - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16 16 15

15

05

06

05

11

13

17

01

17

08

03

01

02

03

05

07

09

18

02

03

05

09

11

18

04

10

12

14

10

12


Sample D - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16

16

15 15 12

12

05

18B

01A

18B

08

03

01A

10

13

10

11

06

17

17

02

03

07

18A

02

03

18A

01B 14

01B


Sample D - HI relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16 16 05

15

05

08

03

03

06

11

14

03

15

01

04

05

07

09

10

12

13

18

01

05

09

11

12

18

02

17

02

10

17


Sample D - VN relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16

16

05

15

15

03

13

01A

08

01A

02

07

12

18A

18B

02

11

12

18A

18B

01B

03

06

10

14

17

01B

03

10

17


WHO/BS/2012.2190

Page 62

Figure 1: Sample E

Sample E - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

11

13

15

17

18

01

05

11

15

17

18

05

08

05

01

02

05

09

10

02

03

09

10

03

03

04

14

07

12

12

16

16


Sample E - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

01A

10

15

10

11

15

05

12

17

18B

12

17

18B

08

06

01B

03

13

01A

03

03

02

07

18A

01B

02

18A

14 16

16


Sample E - HI relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

11

13

15

17

18

01

05

11

15

17

18

08

10

14

03

05

05

03

01

03

04

05

09

09

10

02

12

02

12

07 16 16


Sample E - VN relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

01A

10

15

18B

10

11

15

17

18B

05 01B

07

14

01B

08

03

02

03

06

13

17

18A

01A

02

03

18A

12

12

16 16


WHO/BS/2012.2190

Page 63

Figure 1: Sample F

Sample F - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15

15

05

06

17

17

05

08

03

01

02

03

05

07

09

11

13

16

18

01

02

03

05

09

11

16

18

04

10

12

14

10

12


Sample F - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15 15 12

12

05

01A

10

18B

01A

18B

08

03

16

10

11

16

06

13

17

17

03

18A

03

18A

01B

02

07

02

01B 14


Sample F - HI relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05

15

08

03

03

14

03

15

05

01

04

05

06

07

09

10

11

12

16

18

01

05

09

11

12

18

02

13

17

02

10

16

17


Sample F - VN relative to 09/194 (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15 05

15

01A 03

16

08

01A

10

12

13

16

18A

18B

11

12

18A

18B

01B

02

03

06

07

14

17

01B

02

03

10

17


WHO/BS/2012.2190

Page 64

Figure 1: 09/194

09/194 - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15

15

17 02

06

13

17

01

02

16

05

05

01

05

07

09

11

16

18

03

05

09

10

11

18

08

03

03

04

10

12

12

14


09/194 - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

12

12

10

15

18B

10

18B

08

01A

06

16

17

11

15

17

03

13

18A

01A

03

16

05

03

01B

02

07

01B

02

18A

14


WHO/BS/2012.2190

Page 65

Figure 2: Sample B

Sample B - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

15

05

15

17 05

02

05

11

13

17

01

05

11

08

03

01

03

07

09

12

18

02

03

09

12

18

04

10

14

10

16

16


Sample B - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05

15

10

10

11

15

06

12

17

12

17

08

18B

03

18B

03

01B

03

07

01A

02

13

14

16

18A

01A

01B

02

18A

16


Sample B - HI relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI relative to sample F in IU

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

15

05

05

02

05

11

12

13

17

01

05

11

12

17

08

03

01

03

04

07

09

10

14

18

02

03

09

10

18

15 16

16


Sample B - VN relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN relative to sample F in IU

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

05

01B

02

03

06

07

14

17

01B

03

11

10

02

10

17

08

18A

18B

18A

03

12

13

12

18B

01A

15

16

01A

15

16


WHO/BS/2012.2190

Page 66

Figure 2: Sample C

Sample C - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15

15

05

03

01

06

05

03

05

09

11

16

17

01

03

09

11

16

17

08

02

04

07

10

12

13

14

18

02

10

12

18

05


Sample C - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

15 15 08

05

03

10

12

17

12

16

18B

01A

10

11

16

17

18B

03

06

07

13

18A

01A

01B

03

18A

02

14

01B

02


Sample C - HI relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

03

01

05

08

05

03

04

05

06

09

10

11

12

14

16

17

01

03

09

10

11

12

16

02

07

13

15

18

02

15

17

18

05


Sample C - VN relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

01B

17

08

05

03

02

03

07

14

01B

17

06

10

13

16

18A

02

03

10

11

16

18A

12

15

18B

01A

12

18B

01A

15


WHO/BS/2012.2190

Page 67

Figure 2: Sample D

Sample D - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16 16 15

15

05

06

05

11

13

17

01

17

08

03

01

02

03

05

07

09

18

02

03

05

09

11

18

04

10

12

14

10

12


Sample D - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16

16

15 15 12

12

05

18B

01A

18B

08

03

01A

10

13

10

11

06

17

17

02

03

07

18A

02

03

18A

01B 14

01B


Sample D - HI relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16 16 05

06

11

01

08

05

03

01

02

03

04

05

07

09

10

12

13

14

15

17

18

02

03

05

09

10

11

12

15

17

18


Sample D - VN relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

16

16

02

07

13

02

08

05

03

01A

01B

03

06

10

12

14

15

17

18A

18B

01A

01B

03

10

11

12

17

18A

18B

15


WHO/BS/2012.2190

Page 68

Figure 2: Sample E

Sample E - Absolute HI titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

HI Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

11

13

15

17

18

01

05

11

15

17

18

05

08

05

01

02

05

09

10

02

03

09

10

03

03

04

14

07

12

12

16

16


Sample E - Absolute VN titres

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

VN Titre

-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

01A

10

15

10

11

15

05

12

17

18B

12

17

18B

08

06

01B

03

13

01A

03

03

02

07

18A

01B

02

18A

14 16

16


Sample E - HI relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


-ve 10 20 40 80 160 320 640 1280 2560 5120 10240

06

11

13

15

17

18

01

05

11

15

17

18

10

10

05

08

01

02

04

05

09

14

02

03

09

03

03

12

12

05

07

16

16


Sample E - VN relative to sample F (IU)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30


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WHO/BS/2012.2190

Page 69

Figure 2: 09/194

09/194 - Absolute HI titres

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WHO/BS/2012.2190

Page 70

Figure 2: Sample F (10/202)

Sample F - Absolute HI titres

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WHO/BS/2012.2190

Page 71

Appendix 1 Participants

Yan Li

Canadian Science Centre

Influenza and Respiratory Viruses

1015 Arlington

Winnipeg

R3E 3R2

Canada

Irma Lopez Martinez

Instituto de Diagnostico y Referencia

Department of Virology

Carpio 470

Col. Santo Tomas

Del. Miguel Hidalgo

Mexico D.F. 11340

Mexico

Kathy Hancock

WHO Collaborating Centre for Influenza,

Centers for Disease Control

Influenza Branch

Mailstop G-16, 1600 Clifton Road

Atlanta,, GA 30333

USA

Monica Malone McNeal

Cincinnati Children’s Hospital Medical Center

Division of Infectious Diseases

619 Oak Street, Room 2803

Cincinnati, OH 45206

USA

Wayne Hogrefe/Keith Gottlieb

Focus Diagnostics, Inc.

5785 Corporate Avenue

Cypress, California 90630

USA

Jerry Weir/Falko Schmeisser

Chief, Division of Viral Products

Center for Biologics Evaluation and Research

US Food and Drug Administration

1401 Rockville Pike

Rockville

Maryland 20852

USA

WHO/BS/2012.2190

Page 72

Stephen W. Hildreth

Senior Director

Global Clinical Immunology

sanofi pasteur

Discovery Drive

Building 53

Swiftwater, PA 18370

USA

Wilina Lim/Janice Lo

Virology Division

9/F Public Health Laboratory Centre

382 Nam Cheong Street, Shek Kip Mei

Kowloon, Hong Kong, SAR

China

Pilaipan Puthavathana

Dept of Microbiology, Faculty of Medicine

Siriraj Hospital

Mahidol University

Bangkok

Thailand

Robert Shaw/Ian Barr

WHO Influenza Centre for Influenza

45 Poplar Road,

Parkville

Melbourne, 3052

Australia.

Sue Huang

Head, WHO National Influenza Centre

Institute of Environmental Science and Research

66 Ward Street,

Wallaceville,

Upper Hutt 5018 PO Box 40158,

Upper Hutt 5140,

New Zealand

Thomas R. Kriel

Senior Director, Viral Vaccines & Global pathogen Safety

Baxter BioScience

Biomedical Research Center

Uferstrasse 15

A-2304 Orth/Donau

Austria

WHO/BS/2012.2190

Page 73

Roger Bernhard

Manager Serology

GlaxoSmithKline Biologicals

Branch of SmithKline

Zirkusstrasse 40

D-01069 Dresden

Germany

Nikolaos Gaitatzis

Novartis Vaccines and Diagnostics

Emil-von-Behring-Str. 76

Clinical Serology

35041 Marburg

Germany

Emanuele Montomoli

Prof. of Public Health

Dept. of Physiopathology Experimental Medicine and Public Health

University of Siena

Via Aldo Moro 3

53100 Siena

Italy

John Macauley/Rod Daniels

WHO Collaborating Centre for Influenza

National Institute for Medical Research,

The Ridgeway

Mill Hill

London NW7

UK

Katja Höschler

Respiratory Virus Unit

Virus Reference Department

Health Protection Agency

Microbiology Services (Colindale)

61 Colindale Avenue

London

NW9 5HT

UK

Diane Major

National Insitute for Biological Standards and Control

Blanche Lane

South Mimms

Potters Bar

Herts EN6 3QG

UK

WHO/BS/2012.2190

Page 74

Appendix 2 Draft Instructions for Use

WHO/BS/2012.2190

Page 75

WHO/BS/2012.2190

Page 76

Appendix 3 Summary of production of 10/202

NIBSC Code 10/202

Product Name

WHO 2nd

International

Standard for antibody to

influenza H1N1pdm virus

Presentation lyophilised

Appearance Straw coloured powder

Filling machine Bausch & Strobel AFV5090

(CBRM/06/03 Ampoule)

Date of Filling 4 November 2010

No. of ampoules filled 9917

Mean fill weight (g) 0.5153

CV of fill weight (%) 0.5199

Freeze dryer CS100

Date of completion of

lyophilisation 9 November 2010

Mean dry weight (g) 0.0406

CV of dry weight (%) 1.33

Mean residual moisture (%) 0.4347

CV of residual moisture (%) 18.92

Mean oxygen content (%) 0.18

CV of oxygen content (%) 48.25

No. of ampoules available to

WHO 9305

Documents

WHO/BS/2012.2190 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL