External quality assessment for the determination of diphtheria antitoxin in human serum

Abstract

Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

FIG. 1.

Interlaboratory comparison of the diphtheria antitoxin levels (IU/ml) of the standard panel tested by NT in lab XI (A), lab VIII (B), and lab VI (C) versus reference lab IX. The slope of the regression (S) and intercept (D), correlation coefficient (R) of the regression line (solid lines), and line of identity (dashed lines) are shown. Vertical and horizontal dotted lines indicate the cutoffs used by the reference laboratory to determine negative (<0.01 IU/ml), equivocal (0.01 to 0.09 IU/ml), and positive (≥0.1 IU/ml) sera.

FIG. 2.

Interassay comparison of the diphtheria antitoxin levels (IU/ml) of the standard panel tested by NT and PHA (A), dDA-DELFIA (B), DAE (C), and ToBI (D). The slope of the regression (S), intercept (D), correlation coefficient (R) of the regression line (solid lines), and line of identity (dashed lines) are shown. Vertical and horizontal dotted lines indicate the cutoffs used by the laboratories to determine negative (<0.01 IU/ml), equivocal (0.01 to 0.09 IU/ml), and positive (≥0.1 IU/ml) sera. dDA-DELFIA used a different cutoff for negative (<0.015 IU/ml).

FIG. 3.

Interassay comparison of the diphtheria antitoxin levels (IU/ml) of the standard panel tested by NT and seven different ELISAs: lab III in-house ELISA (A), lab XI in-house ELISA (B), Serion ELISA (C), Euroimmun ELISA (D), Virotech ELISA (E), NovaLisa ELISA (F), and VaccZyme ELISA (G). The slope of the regression (S), intercept (D), correlation coefficient (R) of the regression line (solid lines), and line of identity (dashed lines) are shown. Vertical and horizontal dotted lines indicate the cutoffs used by the NT reference laboratory to determine negative (<0.01 IU/ml), equivocal (0.01 to 0.09 IU/ml), and positive (≥0.1 IU/ml) sera.