Candidate reference methods for FT4 first separate the T4 binding proteins (TBG, prealbumin , albumin, thyroid antibodies) by either equilibrium dialysis or ultrafiltration at 37 C. This is followed by measurement of FT4 using either immunoassays or mass spectrometry. Using these approaches the resulting FT4 values correlate very well with log TSH, an important requirement for any FT4 assay.

Unfortunately neither the diagnostic companies nor the FDA have used this important relationship to test the validity of the direct analogue FT4 methods on a variety of chemistry platforms. In a study over several years we showed that the direct analogue FT4 method disagreed with the TSH value approximately 50% of the time in patients with either hypo- or hyperthyroidism. In contrast the FT4 employing ultrafiltration at 37C followed by tandem mass spectrometry was found to consistently correlate well with log TSH.

There are several conclusions that can be drawn from all these studies. You cannot depend on the direct analogue FT4 result in patients with thyroid disease. Instead of recommending that all FT4's be performed by candidate reference methods employing either equilibrium dialysis or ultrafiltration, we suggest that a candidate reference approach employing either immunoassay or mass spectrometry be used whenever the TSH is above the 90th percentile or below the 10th percentile. This approach should optimize the correct diagnosis of these patients.