Multilaboratory Assessment of Threshold versus Fold-Change Algorithms for Minimizing Analytical Variability in Multiplexed Pneumococcal IgG Measurements

Abstract

dPneumococcal vaccination is frequently used to assess a patient's humoral immune function. The comparison of pre-and postvaccination levels of antipneumococcal antibodies is widely held to be the gold standard for documenting a response. However, many of the published criteria for defining an adequate response are based on assays that are no longer widely available. We compared the clinical classification of patient response by multiplex pneumococcal assays currently performed at three large reference laboratories using a variety of published criteria for defining responses in adults. The classification of responders agreed for 79% of the patients when using a threshold-based algorithm compared to 57 to 96% of the patients when using various fold-change-based algorithms. The highest rate of discordance was seen when the most stringent criteria for response were used (4-fold increase postvaccination in 70% of serotypes). The discordant samples tended to show similar patterns of response across all three assays, with small variations in the final number of serotypes converting postvaccination. We conclude that the use of published cut points for documenting response to pneumococcal vaccination can be affected by interlaboratory differences in pneumococcal assays, particularly for algorithms that require large fold changes for a response to be documented. However, the overall patterns of response were similar in virtually all samples, regardless of the assay used. P neumococcal vaccination is often used to evaluate a patient's response to polysaccharide antigens during a workup for deficiencies in antibody production. The adequacy of the specific vaccine response can be assessed by measuring postvaccination levels of antipneumococcal antibodies and by comparing these levels against predetermined cut points for either the absolute antibody level or the fold change relative to the baseline value. Measurements can be performed by a variety of analytical methods, including enzyme-linked immunosorbent assays (ELISAs) for total pneumococcal antibody level (1), ELISAs for antibodies against specific pneumococcal serotypes (2), and multiplex assays which measure the levels of a panel of serotype-specific antibodies (3-5). Because the vast majority of assays used for this purpose are lab-developed tests (LDTs) that were created and characterized by individual laboratories, the potential for interlaboratory variation in results exists. This may lead to difficulties when trying to interpret analytical results compared to the literature-based definitions for a therapeutic response to vaccination.The assessment of a patient's response when only a single (postvaccination) sample is available is usually done by comparing serotype-specific results against a "protective" threshold (6). However, the use of paired pre-and postvaccination samples is preferred because of the additional information provided for evaluating humoral immune function, which is the primary objective of these assays. Approaches for evaluating response from pa...

Specific Antibody Deficiency: Controversies in Diagnosis and Management

“…Variation in results from different laboratories also creates challenges for diagnosis based on the measurement of vaccine responses (57). Further clinical studies are needed to clearly define antibody responses that are indicative of SAD.…”

Use and Clinical Interpretation of Pneumococcal Antibody Measurements in the Evaluation of Humoral Immune Function

“…Given the variability of the various assays, including the radioimmunoassays, ELISA-type assays, and multianalyte assays currently in use by most of the major reference laboratories, it is reasonable to assume that long-term protection probably does result from a 1-month postvaccine concentration of between 1 and 1.5 g/ml. Arguably, the minor differences between 1.0, 1.3, and 1.5 seem irrelevant given the inherent variability of the most commonly utilized assays, especially for comparisons of results from different reference laboratories (18). Moreover, true responders usually have much higher responses to most serotypes than the minimal 1.0 to 1.5 g/ml, in our experience.…”

“…Quantitative comparisons of PnAb levels measured by multiplex assays were not always consistent with results from WHO-based ELISA methods (15), and the performance characteristics of these assays often did not meet the criteria set in the original WHO document developed for ELISA testing. Intralaboratory comparisons of assays from different laboratories showed substantial variation in quantitative results even when the same basic technology was being utilized (16), although clinical classifications of patients were relatively unaffected by these quantitative variations using published algorithms (17,18). As a result, interpretation of multiserotype test results could be a challenge for clinicians trying to apply pneumococcal serotype analysis to their practice in different clinical scenarios.…”

Improving vaccines against Streptococcus pneumoniae using synthetic glycans

“…Modifications could be implemented down to the atomic level to increase both immunogenicity and protective effects as has been illustrated for ST5 where a ketone was replaced by a hydroxyl group to improve stability while maintaining immunogenicity and antigenicity. One dominant criterium used to assess an adequate vaccination response in adults is a two-to fourfold change in specific antibody levels after vaccination as measured by ELISA (36,37). The semisynthetic pentavalent conjugated vaccine sPCV5 reached this standard for all serotypes except ST14 CPS.…”