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 Major1, Alan Heath2 and John Wood1 Division of Virology1 and Biostatistics2
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].
© World Health Organization 2012
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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.
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
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 -70oC 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).
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.
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.
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%
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.
Use of 09/194 as a standard
Expression in IU
The 1st 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
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.
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.
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
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 2nd 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.
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’. -
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 2nd 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.
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 commentsReferences
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.
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 - -70oC 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;
105.3ffu ; 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 OC in 5% CO2 35-37 OC 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
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
10-11 day old embryonated eggs
10-11 day old embryonated eggs, MDCK
Conditions of virus growth 3 days at 34-35oC Eggs 2-3 days @32-
37oC;MDCK 5 days @33oC 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
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
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
Table 5 HI - Laboratory GMTs by Virus: Sample C
Sample C
Lab X179A X181 A/Cal/7/09 A/ChCh/16/10 NIBRG121 GMT
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
Overall Excluding 16
349 375 367
Overall Excl 15 & 16
413 464 425
%GCV Excluding 16
127 134 112
%GCV Excl 15 & 16
63 65 59
Table 6 HI - Laboratory GMTs by Virus: Sample D
Sample D
Lab X179A X181 A/Cal/7/09 A/ChCh/16/10 NIBRG121 GMT
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
Overall Excluding 16
252 239 253
Overall Excl 15 & 16
306 299 297
%GCV Excluding 16
161 134 141
%GCV Excl 15 & 16
81 53 81
