The latest generation of 25-OHD immunoassays has improved performance compared to previous assays. However, some immunoassays can still give discrepant results and this is most apparent when immunoassays are evaluated with a range of samples that challenge their analytical performance.
Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.
We would like thank Donnelly and his colleagues from Siemens for their comments [1] on our recent study that evaluated the impact of assay design on 25-hydroxyvitamin D (25-OHD) immunoassay performance [2]. In their Letter to the Editor they question some statements and conclusions in the original paper that we would like to comment on. The first point raised is the sequence in their assay in which vitamin D is liberated from vitamin D binding protein (DBP). Donnelly and colleagues claim that the Siemens assay releases and captures 25-OHD simultaneously. The various immunoassays tested in our study liberate vitamin D at different points in the assay sequence. For example, the DiaSorin LIAISON XL assay adds the patient sample to a cuvette which already contains both DBP-dissociation reagent and capture antibody and incubates this mixture; hence simultaneously treating the sample with dissociation and capture reagents. In contrast, the Siemens Centaur assay incubates the patient sample with DBP-dissociation reagent alone for 4.5 min and then subsequently adds the capture antibody. These different assay sequences cannot be assumed to exhibit equivalent kinetics. Differences in the ability of the different assay to equally maintain both 25-OHD 3 and 25-OHD 2 in the free form throughout the assay sequence may contribute, along with the binding properties of the capture antibody, to the significant over-recovery of 25-OHD 2 by the Siemens assay observed by us and others [3][4][5]. Therefore, the statement by Donnelly et al., that an assay is considered to not release and capture 25-OHD simultaneously would need to have a wash step between the liberation of 25-OHD from its binding protein and the addition of capture antibody is incorrect and in fact such a design is not employed by any current 25-OHD immunoassay, and would be a rather unfavorable strategy.The second point raised related to our conclusions regarding the precision of the assays employing a 'backfill' design, Abbott and DiaSorin. The Abbott and DiaSorin assays showed the lowest logarithmic fit of the precision data at the highest concentrations. While the DiaSorin assay continued to show superior precision across the concentration range generally, the Abbott assay showed deteriorating precision at concentrations below 16.0 ng/mL (39.9 nmol/L). A backfill design is only one aspect of an assay that may affect precision. The relative amount of signal generated per molecule of conjugate as well as the size of the conjugate will also influence precision; the latter via steric exclusion effects on separation to the solid phase. As the details of these additional parameters
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