amiodarone, thyroxine) and immunoassay interference, can cause these discrepancies ((6). interference on the Immulite immunoassay analyser ML204 caused by heterophilic antibodies, which were eliminated by protein A/G treatment. We point out the importance of a close collaboration between doctors and the laboratory to avoid unnecessary clinical intervention. Key words: FT4, thyroid function test, diagnostic errors, interference, immunoassay Introduction In most patients euthyroidism, thyrotoxicosis or hypothyroidism is confirmed with the combination of free thyroxine ML204 (FT4) and thyroid stimulating hormone ML204 (TSH) measurements. Predominantly, thyroid function tests (TFT) are easily interpreted and correspond with the patients clinical status. However, in a limited number of cases the TFT and the clinical impression are discordant. A number of factors, including concurrent (non-thyroidal) illness, medication (e.g. amiodarone, thyroxine) and immunoassay interference, can cause these discrepancies ((6). The reference ranges for FT4 were as follows: for Cobas Modular 12C22 pmol/L, for Immulite 2500 12C23 pmol/L, for DXi 8C14 pmol/L and for Vitros 11C25 pmol/L. The FT4 reference ML204 range for the Dialysis method was determined in-house (14C29 pmol/L). FT4 measurements were normalized by dividing the results with the upper ML204 limit of normal (ULN) for each assay (FT4/ULN). Figure 1 schematically presents FT4 measurements on each immunoassay analyzer commonly used in routine clinical chemistry laboratories. FT4 measurements on Cobas Modular E, Immulite 2500, and DXi were performed in duplicate, whereas FT4 measurements on Vitros ECi and equilibrium dialysis was done in singlicate. For the duplicate FT4 measurements, intra-assay variation was determined by taking the 95% confidence interval of the standard error based on the difference between these measurements. Open in a separate window Figure 1 Schematic presentation of the FT4 measurement commonly used in routine clinical chemistry laboratories.(A) one-step labelled T4 analog (Immulite, Siemens) in which the patients serum is simultaneously incubated with the labelled T4 analog and competes for binding with the immobilized anti-T4 antibody. After incubation, the unbound fraction is removed by washing and the signal recorded.(B) The one-step analog two-step incubation (Cobas, Roche Diagnostics) uses a labelled T4 antibody in the first incubation round, followed by a second incubation step in which the T4 analog is added. Only the T4 analog, with or without the labelled T4 antibody, will subsequently bind via biotin and streptavidin and become immobilized. The unbound fraction is removed by washing and the signal is recorded.(C) The two-step labelled T4 analog method (DXi, Beckman Coulter) introduces the T4 antibody in the first incubation round to the patients serum. During this incubation step, the T4 antibody becomes immobilized to the solid phase followed by a wash step. In the second incubation step, the labelled T4 analog is allowed to bind to the vacant anti-T4 antibody binding sites, the unbound labelled T4 analog is washed away and the signal recorded.(D) The one-step labelled antibody method (Vitros, Ortho Clinical Diagnostics) incubates the patients serum with immobilized T4 analog and uses a labelled anti-T4 antibody. The FT4 in the serum competes for binding with the T4 analog followed by a wash step and signal generation.In all these designs, the signal generated is inversely proportional to the FT4 concentration. Treatment of serum with heterophilic blocking tubes (HBT) (Scantibodies Laboratory, Santee, USA) was performed according to manufacturers instructions. The recombinant fusion protein that combines immunoglobulin (Ig) G binding domains of both Protein A and Protein G (protein A/G) agarose beads; Pierce Protein A/G Plus Agarose (Thermo Scientific, Rockford, USA) were washed and incubated at 4 Mouse monoclonal to PBEF1 C overnight with sera. The effect of HBT and protein A/G treatment on IgG and.