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Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D

  • Christopher-John L. Farrell , Joshua Soldo , Brett McWhinney , Sushil Bandodkar and Markus Herrmann EMAIL logo
Published/Copyright: May 29, 2014
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 52 Issue 11

Abstract

Background: Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice.

Methods: 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD2 recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD3 cross-reactivity and precision of the automated assays were evaluated.

Results: The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD2 recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD2 under-recovery, a trend consistent with 3-epi-25-OHD3 cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD3 cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations.

Conclusions: 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.

Keywords: 25-hydroxyvitamin D; immunoassay; liquid chromatography-tandem mass spectrometry
Corresponding author: Associate Professor, Dr. med. habil. Markus Herrmann, Department of Clinical Pathology, District Hospital of Bolzano, 39100 Bolzano, Italy, Phone: +39 0471 909675, E-mail:

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Supplemental Material

The online version of this article (DOI: 10.1515/cclm-2014-0111) offers supplementary material, available to authorized users.

Received: 2014-1-31
Accepted: 2014-5-5
Published Online: 2014-5-29
Published in Print: 2014-11-19

©2014 by De Gruyter

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https://doi.org/10.1515/cclm-2014-0111
Keywords for this article
25-hydroxyvitamin D; immunoassay; liquid chromatography-tandem mass spectrometry

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. From symptoms to biomarkers: a change of paradigm
  4. Reviews
  5. Biochemical markers in early diagnosis and management of systemic amyloidoses
  6. The accuracy of the anti-mitochondrial antibody and the M2 subtype test for diagnosis of primary biliary cirrhosis: a meta-analysis
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  12. Adopting European Network for Health Technology Assessments (EunetHTA) core model for diagnostic technologies for improving the accuracy and appropriateness of blood gas analyzers’ assessment
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