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 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 reexamining 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 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 the 12 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 Mr Daniel Harrison, Dr Helen Chapel Oxford - UK Dr Vojtech Thon Dr Rasa Duobiene Dr Michael Schlesier Brno - Czech Rep Vilnius Lithuania Freiburg Germany 4, 6B, 9V, 14, 18C, 19F, 23F serotypes Pneumovax total IgG ELISA and Binding Site kit Binding Site Kit Dr Ulrich Baumann Pr Arnaldo Caruso Dr Andy Gennery Mrs Lynn Day Dr Franỗoise Mascart, Julie Smet Dr Anna Sediva, Dr Ales Janda Dr Ray Borrow Hannover Germany Brescia Italy Newcastle UK Slough - UK Brussels Belgium Mrs.Rajee Goswami, Dr.Tony Williams Southampto n-UK Dr 1 Paul Prague Czech Rep - Yes No 4,5,6B,7F,9V,14,18C,1 9F, 23F serotypes and Pneumovax total IgG ELISA Pneumovax total IgG ELISA Yes Yes - No - No - Binding Site Kit 1,7F,14,19,23F serotypes Yes - Binding Site Kit Yes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F serotypes (mulitplex assay) 4,6B,9V,14,18C,19F,2 3F serotypes, Pneumovax total IgG ELISA and Binding Site Kit 1,3,4,5,9N,23F No Manchester UK Delft B Site received Yes - Yes No 3 Herbrink Robert K Hallam, Mrs Kate Campbell Fiona Alcock Netherlands Papworth UK serotypes Birmingham - UK Leuven Belgium Binding Site Kit No Yes Xavier Bossuyt, 3,4,9N,18F,19F No Margaretha serotypes Charlier Peter Ciznar, Bratislava - Binding Site Kit No MD Slovakia Table 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 nonvaccinated 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 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 Cumulative distribution curves of post-Dose antibody curves concentrations (from Black et al) samples for the the 22F inhibition ELISA were calculated from antibodies to serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F Fig.2 Shows cumulative distribution of the 30 post-vaccination non-22F ELISA and Curves 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 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 Graphical comparison between the expected WHO IgG concentrations (in red) for 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 the sample with the higher titers (Figures 4, and 6) In particular Figure shows good reproducibility between laboratories for the lower titer sample (except for serotype 14) Figure shows that reproducibility for sample (highest levels of antibodies) was less good in quantitative terms, though all laboratories gave results above the protective level Figure 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 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 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) Serotype 6B 9V 14 18C 19F 23F Sample +ve 66.7 100 100 80 100 100 100 -ve 33.3 0 20 0 Sample +ve 66.7 75 100 100 80 100 100 -ve 33.3 25 0 20 0 Sample +ve 25 100 80 83.3 50 -ve 100 75 100 20 16.7 50 Overall agreement for for serotype 77.8 83.3 100 93.3 86.7 94.4 83.3 Table 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 Binding Site assay 200 IgG mg/l 150 100 50 Sample tested Fig Range of total pneumococcal IgG for the QA samples QA samples calculated by labs using the Binding Site assay Pneumovax® assay Fig Range of total pneumococcal IgG for the calculated by labs using their own in house 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 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 Serotype assays Binding Site ELISA 1 In House Pneumovax ELISA Serotype 6B 9V 14 18C 19F 23F Pneumova x ELISA 0.24 0.35 0.26 N/A 0.16 N/A 0.60 Fig Diagram showing the frequency of assay usage between for the European labs pneumococcal ELISA assays Table Kappa coefficient correlation serotype specific and total 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 th Framework policy EURO-POLICY-PID [SP23-CT-2005006411] We are indebted to Dr William Egner at UKNEQAS for agreeing to take on this scheme once the pilot phase was completed References de Vries, E 2005 Patient-centred screening for primary immunodeficiency: a multi-stage diagnostic protocol designed for non-immunologists Clinical and Experimental Immunology 145: 204-214 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) www.vaccine.uab.edu 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: 143148 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 10 ... 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... 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... kit are shown in Figure Binding Site assay 200 IgG mg/l 150 100 50 Sample tested Fig Range of total pneumococcal IgG for the QA samples QA samples calculated by labs using the Binding Site assay