Assay-dependent variability in free thyroxine (FT4): differential interference related to immunoassay design in a patient with subclinical hypothyroidism

In clinical practice, thyroid function tests (TFTs) usually comprise thyroid-stimulating hormone (TSH) and free thyroxine (FT4), with free triiodothyronine (FT3) measured less frequently. We report a 76-year-old woman with autoimmune thyroiditis presenting with raised TSH and incongruously elevated FT4. The discordance between biochemical findings and clinical status prompted further investigations for assay interference. TFTs were assessed using four different immunoassay platforms and equilibrium dialysis. Additional studies included heterophilic antibody blocking and polyethylene glycol (PEG) precipitation. PEG precipitation demonstrated a high-molecular-weight interferent, consistent with thyroid hormone autoantibodies. FT4 measured by two-step immunoassays (Abbott Alinity and Beckman DxI) aligned with FT4 reported by equilibrium dialysis, whereas one-step assays (Roche Cobas and Siemens Atellica) reported spuriously elevated FT4. One-step immunoassays are particularly susceptible to interference from thyroid hormone autoantibodies, which may yield misleading results. Recognition of potential TFT assay interference and the use of alternative methodologies are essential for accurate diagnosis and appropriate patient management.