Quality Assurance for Pneumococcal Assays in Europe

Quality Assurance For Pneumococcal Assays in Europe

Background and Aims

The European Primary Immunodeficiencies (PIDs) Consensus Conference in Langen (2006) outlined the need for quality assurance for assays to measure specific antibodies to common pathogens and immunization antigens. In order to make a diagnosis of primary antibody failure, it is important to show that a patient is unable to make antibodies to pathogens; this may be demonstrated using either common exposure antigens or those used for immunization. Streptococcus pneumoniae, a common gram positive bacterium, is one such pathogen that is estimated by the WHO to be responsible for over one million acute deaths worldwide. This includes patients with primary immune deficiencies, who are particularly susceptible to infections caused by encapsulated bacteria.

Assays for antibody responses to S. pneumoniae are of two types: those that measure total pneumococcal IgG antibody to Pneumovax® by enzyme linked immunosorbent assays (ELISA), and those that measure serotype specific antibodies also by ELISA. The current European Society for Immunodeficiencies (ESID) guidelines (de Vries 2005) recommend the use of the unconjugated pneumococcal vaccine (Pneumovax®) for diagnosing PIDs by monitoring IgG responses to booster immunisations.

In recent studies various pneumococcal serotype specific ELISA assays have been used for the same purposes. Akikusa and Kemp found that a low response to serotype 3, post Pneumovax® vaccination, in combination with a low titre to serotype 4/6B, was suggestive of a severe immune deficiency state in children being investigated following recurrent upper or lower respiratory tract infections. They found a significant correlation between a low IgG response to serotype 3 with pneumonia.

Boyle et al. used specific IgG response of children with recurrent respiratory infections to 12 serotypes (1, 3, 4, 5, 6B, 7, 9V, 14, 15, 18C, 19F, 23F) following Pneumovax® vaccination in order to diagnose specific antibody deficiency (SAD). They deemed an adequate response to any one serotype was an IgG value of 1.3 mg/l, or a fourfold increase in the IgG concentration. An unsatisfactory response to more than 50% of the serotypes tested was used to define a SAD. They also found that failure to respond to serotypes 4, 9V, 15 or 23F significantly increased the risk of SAD, suggesting that these serotypes might be sufficient for assessing specific antibody defects.

In order to assess the variability of these antibody measurements in Europe, prior to re-examining diagnostic criteria, a pilot study was undertaken. The aims were to develop quality assurance for pneumococcal assays, particularly for new laboratories in Europe, and to lay the foundation for a comparison of available assays for test immunization in the diagnosis of PIDS, particularly between the Pneumovax® total ELISA assay and the serotype specific assays. Binding Site UK kindly provided total pneumococcal IgG ELISA kits to all participating laboratories, facilitating comparisons of performance between assays.

METHODS

Serotype ELISA assays

In-house ELISA assays for the detection of human immunoglobulin G (IgG) to 7 common pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) were developed. Optimum antigen coating concentration for each serotype (antigens provided by LGC Promochem) was determined, following the Standard Operating Procedure provided by Nahm and Goldblatt (2002). This was also used to establish the optimum working dilution for the enzyme labeled secondary antibody (anti IgG phosphatase BioSource Ltd.) in each serotype assay.

Validation

This was undertaken using the12 pneumococcal WHO calibration sera samples. These are supplied with values for the expected specific pneumococcal serotype IgG concentrations and are obtained from the National Institute for Biological Standards(Colindale, UK). They were tested for specific pneumococcal IgG using the Standard Operating Procedure (Nahm, Goldblatt 2002) and used to validate the developed serotype assays for routine and the QA purposes. The concentrations of serotype specific IgG were determined in 16 control sera samples along with 16 known positive sera samples.

The in-house Pneumovax ELISA assay and the ELISA kit provided by The Binding Site UK were already validated.

Samples

Random serum samples from 16 individuals were tested for total anti-pneumococcal IgG using the in-house Pneumovax® ELISA assay & ELISA kit provided by The Binding Site UK in addition to the serotype specific assays.

Three serum samples were selected for QA distribution on the basis of having high, medium and low titres of anti-pneumococcal IgG. These were distributed to 16 collaborating centres (table 1) in Europe and were tested using both their established pneumococcal assay (whether for total pneumococcal or serotype specific antibodies) and a Binding Site kit.

Contact / Location / Assay / B Site received
1 / Mr Daniel Harrison, Dr Helen Chapel / Oxford - UK / 4, 6B, 9V, 14, 18C, 19F, 23F serotypes. Pneumovax total IgG ELISA and Binding Site kit. / Yes
2 / Dr Vojtech Thon / Brno - Czech Rep / Binding Site Kit / Yes
3 / Dr Rasa Duobiene / Vilnius - Lithuania / No
4 / Dr Michael Schlesier / Freiburg - Germany / 4,5,6B,7F,9V,14,18C,19F, 23F serotypes and Pneumovax total IgG ELISA / Yes
5 / Dr Ulrich Baumann / Hannover - Germany / Pneumovax total IgG ELISA / Yes
6 / Pr Arnaldo Caruso / Brescia - Italy / No
7 / Dr Andy Gennery / Newcastle - UK / No
8 / Mrs Lynn Day / Slough - UK / Binding Site Kit
9 / Dr Françoise Mascart, Julie Smet / Brussels - Belgium / 1,7F,14,19,23F serotypes / Yes
10 / Dr Anna Sediva, Dr Ales Janda / Prague - Czech Rep / Binding Site Kit / Yes
11 / Dr Ray Borrow / Manchester - UK / 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F serotypes (mulitplex assay) / No
12 / Mrs.Rajee Goswami, Dr.Tony Williams / Southampton-UK / 4,6B,9V,14,18C,19F,23F serotypes, Pneumovax total IgG ELISA and Binding Site Kit / Yes
13 / Dr Paul Herbrink / Delft - Netherlands / 1,3,4,5,9N,23F serotypes. / No
14 / Robert K. Hallam, Mrs Kate Campbell / Papworth - UK / No
15 / Fiona Alcock / Birmingham - UK / Binding Site Kit / Yes
16 / Xavier Bossuyt, Margaretha Charlier / Leuven - Belgium / 3,4,9N,18F,19F serotypes / No
17 / Peter Ciznar, MD / Bratislava - Slovakia / Binding Site Kit / No

Table 1. Table of participants in the pilot study. Those contacts who did not receive the

Binding Site assay failed to confirm the delivery address.

Protective Level

Although normal ranges for age have been determined for the total pneumococcal assay (Windebank et al 1987), protection levels of these antibodies have not been established in large groups of patients, only in those with lymphoid malignancies from whom a value of 30 U/ml was derived (Griffiths et al 1992). For serotype specific assays, the value of 0.2 mg/l was used as the putative protection level, based on data from Black et al. and Henckaerts et al. Black et al. demonstrated that more than 95% of their pneumococcal conjugate recipients developed >= 0.15 mg/l of serotype specific IgG after a third dose of vaccine compared with the non-vaccinated state (Figure 1).

The 22F serotype was used as an absorbent, to block non-specific responses and cross-reactive antibodies. This slightly decreases the antibody concentration observed and will alter the protection level as demonstrated in Henckaerts et al. They showed that levels of serotype specific IgG post vaccination were greater than 0.35 mg/l in 78.9% of individuals. This percentage corresponded to a threshold of 0.20 mg/l compared with the non-22F absorbed sera, seen in Fig 2. Therefore, in the presence of absorption with 22F in a routine assay, 0.2 mg/l is the correct protective level. Since the WHO recommended assay includes absorption with 22F material, this level was used.

Fig 1. Cumulative distribution curves of post-Dose 3 antibody Fig.2. Shows cumulative distribution curves concentrations. (from Black et al) of the 30 post-vaccination samples for the non-22F ELISA and the 22F inhibition ELISA. Curves were calculated from antibodies to serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

Statistical Analysis

Serotype specific IgG results were compared to results obtained from the existing total Pneumovax® ELISA assay using Kappa coefficients (Dr Martin Lee, University of California at Los Angeles).

RESULTS

Validation of serotype specific ELISA Assays for comparison with other laboratories

The results shown in Figure 3 suggest that the assays in our laboratory are detecting similar concentrations of specific IgG with the calibration sera. However some assays appear to be performing better than others; for example, the assay using 9V material gives less satisfactory validation. However, in many instances the sensitivity of the assays appears better for the observed values than for the expected.

Fig 3. Graphical comparison between the expected WHO IgG concentrations (in red) for 8 calibration sera with the observed IgG concentrations (in blue) using the developed serotype ELISA assays.

QA of European Serotype ELISA assays

The three selected QA samples gave similar serotype results between laboratories, except for thesample with the higher titers (Figures 4, 5 and 6). In particular Figure 6 shows good reproducibility between laboratories for the lower titer sample 3 (except for serotype 14). Figure 4 shows that reproducibility for sample 1 (highest levels of antibodies) was less good in quantitative terms, though all laboratories gave results above the protective level. Figure 5 shows that there was good reproducibility for sample 2, but that for serotypes 14, 19F and 23F was particularly poor. However, despite there being poor reproducibility for the higher titre sera, there was good agreement in terms of protection throughout.

The results for individual laboratory performance for the 7 serotypes are shown alongside. It is evident from these graphs that although there was agreement in terms of protection, the sensitivity of these serotype assays varied hugely, explaining the large range of results especially for the higher titre sample.

Table 2 demonstrates that serotype results from most laboratories correspond well in terms of protection since there was almost always a majority outcome of a positive or negative response. The level of agreement was not as consistent for the lower titre sample due to the number of serotype concentrations that were borderline (close to 0.20 mg/l).

Sample 1 / Sample 2 / Sample 3 / Overall agreement for
for serotype
Serotype / +ve / -ve / +ve / -ve / +ve / -ve
4 / 66.7 / 33.3 / 66.7 / 33.3 / 0 / 100 / 77.8
6B / 100 / 0 / 75 / 25 / 25 / 75 / 83.3
9V / 100 / 0 / 100 / 0 / 0 / 100 / 100
14 / 80 / 20 / 100 / 0 / 100 / 0 / 93.3
18C / 100 / 0 / 80 / 20 / 80 / 20 / 86.7
19F / 100 / 0 / 100 / 0 / 83.3 / 16.7 / 94.4
23F / 100 / 0 / 100 / 0 / 50 / 50 / 83.3

Table 2. Table shows the level of agreement, as a percentage, between European labs for the comparison samples, in terms of a positive or negative response to the particular serotypes. The overall agreement highlights the serotype assays which are most likely to give a unanimous verdict, whether positive or negative. The putative protective value of 0.2mg/l was used to judge a positive or negative outcome.

Total Pneumococcal ELISA assays

The laboratories also received a Binding Site kit to test alongside their serotype specific assay or their in-house total pneumococcal assay. The results from the laboratories using this kit are shown in Figure 7.

Fig 7. Range of total pneumococcal IgG for the 3 QA samples Fig 8. Range of total pneumococcal IgG for the 3 QA samples

calculated by labs using the Binding Site assay.calculated by labs using their own in house Pneumovax® assay.

This shows that overall results from the Binding Site kit correlated well. As with the serotype assays, the higher titre comparison serum (sample 1) tended to give more varied results whereas the lower titre sera were closely matched between laboratories. Only 3 laboratories gave results for their in-house Pneumovax® ELISA method (Figure 8) but since there is no international reference preparation yet for this type of assay, the results were given in local units and were not convertible to standard unitage for comparison.

Pneumovax
Serotype / ELISA
4 / 0.24
6B / 0.35
9V / 0.26
14 / N/A
18C / 0.16
19F / N/A
23F / 0.60

Fig 8. Diagram showing the frequency of assay usage Table 3. Kappa coefficient correlation between

for the European labs.serotype specific and total pneumococcal ELISA assays.

Comparison of total pneumococcal assay and serotype specific assays

Direct comparison between serotype specific assays and total pneumococcal assays, using all samples found that a good IgG response to most serotypes generally indicated a high total pneumococcal result. Statistical analysis of the 16 random samples was carried out using Kappa coefficient correlations, to look for associations between individual serotype results and the total pneumococcal result. This was achieved by allocating each serotype result and each total Pneumovax result to a positive or negative status, based on the putative protection concentration of 0.2 mg/l (Black et al. and Henckaerts et al.) and a putative protection level of >20 U/ml for our total Pneumovax in house assay. A correlation was found between the Pneumovax assay and the 23F serotype assay only [Table 3]. For Kappa coefficients, a value of 0.6 is indicative of a significant result, but more samples are required for confirmation.

Exact comparison of pre and post immunization samples between the total Pneumovax assay and the serotype assays was not possible as too few samples were available.

Recommendations and Future

The most widely used assay currently is the Binding Site total pneumococcal IgG kit, though some laboratories are using pneumococcal specific antibodies. This study demonstrates that once assays are validated, a reference preparation is essential to cover all the various serotypes tested in order to convert the results in the three different types of methodology. Such a reference preparation should be available throughout Europe.

Antibody responses to the Prevenar vaccine, in order to look for a ‘protective’ response, form the basis of diagnostic assays in children. In regard to test immunization, the definition of an “adequate” response as an IgG value of 1.3 mg/l or a fourfold increase in the IgG concentration for a particular serotype is arbitrary (Boyle et al.). The question of how many such positive responses are required in the serotype specific assays to exclude a diagnosis of immune deficiency remains uncertain, though the criteria quoted above provide the best evidence so far. One approach is to select suitable serotypes, as in other studies, that give the best indication of response and in terms of PID diagnosis (Kemp et al.). From the present study, the 9V and 23F serotypes look to be good candidates, based on the high level of agreement observed between laboratories (Table 2) and the high statistical correlation with the total pneumococcal ELISA assay (Table 3).

Standardisation of both types of assays used is also important. Using 22F as an absorbent to block unspecific responses and cross-reactive antibodies, as recommended by the WHO, is appropriate.

Acknowledgements:

We are grateful to Dr Martin Lee, University of California at Los Angeles for statistical analysis and to The Binding Site Ltd for their generous offer of kits for all participants. This study was only possible due to funding from the EU 6th Framework policy EURO-POLICY-PID [SP23-CT-2005-006411]. We are indebted to Dr William Egner at UKNEQAS for agreeing to take on this scheme once the pilot phase was completed.

References

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Nahm, M. H., and D. Goldblatt. 2002. Training manual for Enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (Pn PS ELISA).

Black, S., H. Shinefield, B. Fireman, E. Lewis, P. Ray, J. Hansen, L. Elvin, K. M. Ensor, J. Hackell, G. Siber, F. Malinoski, D. Madore, I. Chang, R. Kohberger, W. Watson, R. Austrian and K. Edwards. 2000. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr. Infect. Dis. J. 19:187-95

Henckaerts, I., D. Goldblatt, L. Ashton, and J. Poolman. 2006. Critical differences between pneumococcal polysaccharide enzyme-linked immunosorbent assays with and without 22F inhibition at low antibody concentrations in pediatric sera. Clinical and Vaccine Immunology. Mar:356-360.

Akikusa, J. D., and A. S. Kemp. 2001. Clinical correlates of response to pneumococcal immunization. J. Paediatr. Child Health. 37:382-387

Boyle, R. J., C. Le, A. Balloch, and M. L-K. Tang. 2006. The clinical syndrome of specific antibody deficiency in children. Clinical and Experimental Immunology. 146:486-492

Windebank, KP., Faux, J., Chapel H 1987 ELISA determination of IgG antibodies to pneumococcal capsular polysaccharides in a group of children. J Immunolog Methods 104: 143-148

Hargreaves, RM., Lea, JR., Griffiths, H, Faux, JA., Holt, JM., Reid,C., Bunch, C., Lee, M., Chapel, H. 1995 Immunological factors and risk of infection in plateau phase myeloma. J. Clin. Path. 48: 260-266

Griffiths, H. Lea, J. Bunch, C. Lee, M. Chapel H. 1992 Predictors of infection in chronic lymphocytic leukaemia (CLL) Clin Exp Immunol. 89, 374-377

Daniel Harrison

Helen Chapel

16/03/2008

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