Background: Diagnostic manufacturers* must ensure/ document metrologically traceable assays. We report on a feasibility study of a split-sample comparison for that purpose. Processed, frozen single-donation sera, assigned target values by candidate reference measurement procedures (cRMPs), were used with immunoassays for total thyroxine (TT4) and triiodothyronine (TT3) as models. Methods: Two serum panels were quantified for TT3 and TT4 with validated cRMPs and measured in parallel with at least 14 immunoassays. The results were interpreted in terms of traceability of calibration (trueness) and of the individual measurement result (accuracy) by linear regression analysis and graphical representation against specifications. The commutability of the sera was investigated by parallel analysis of TT4 in freshly collected but nonfiltered specimens. Results: The TT4 (TT3) concentrations in the sera (according to the cRMPs) were 64-269 nmol/L (0.88-13.7 nmol/L). The method comparison showed that for TT4 on average, the immunoassays produced results in agreement with the cRMPs, whereas for TT3, results were typically higher. It also demonstrated a considerable between-assay divergence in traceability of calibration and accuracy. The evidence of noncommutability of the sera attributable to processing, however, indicates that the interpretation should be treated with caution. Conclusions: Frozen sera can be used for documenting/ validating traceability of total thyroid measurements. The way in which the sera are processed may jeopardize commutability, however, and therefore requires indepth investigation. (c) 2005 American. Association for Clinical Chemistry.