title: Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D.
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.
authors.forename: Christopher-John L
authors.initials: CJ
authors.lastname: Farrell
authors.forename: Joshua
authors.initials: J
authors.lastname: Soldo
authors.forename: Brett
authors.initials: B
authors.lastname: McWhinney
authors.forename: Sushil
authors.initials: S
authors.lastname: Bandodkar
authors.forename: Markus
authors.initials: M
authors.lastname: Herrmann
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D000906:Antibodies / Q000276:immunology; D001331:Automation; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D053719:Tandem Mass Spectrometry; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D014809:Vitamin D-Binding Protein / Q000737:chemistry / Q000378:metabolism
chemical_list: D000906:Antibodies; D014809:Vitamin D-Binding Protein; D014807:Vitamin D; D015652:25-Hydroxyvitamin D 2; C104450:25-hydroxyvitamin D