Accuracy of Free Thyroxine Measurements Across Natural Ranges of Thyroxine Binding to Serum Proteins

Systemic inaccuracies, proportional to the concentrations of serum proteins and the thyroxine (T₄) they carry, have been reported in direct free T₄ immunoassays. However, analytical recoveries of free T₄ have not been carefully examined in most current methods, and they have not previously been examined across the pathophysiological range of serum T₄ binding. In the present study we investigated ranges of serum T₄ binding using free and total T₄ measurements from 1359 individuals. Carefully characterized, gravimetrically calibrated, serum-based free T₄ test solutions were then prepared with a constant normal free T₄ concentration (12 ng/L) and varied serum T₄ binding (approximately 300:1 to 24,000:1, ng protein bound T₄: ng free T₄). These standardized test solutions were analyzed using five T₄ analog based free T₄ methods. Analytical recoveries were calculated as ratios of actual free T₄ measurements to the target value, and expressed as a percent of the target. Analytical recoveries were directly proportional to the extent of serum T₄ binding and ranged 2% to 155%, 25% to 131%, 53% to 106%, 37% to 93%, and 37% to 73%, lowest to highest, in different methods. These systemic inaccuracies will confound interpretations of free T₄ test results in clinical conditions with altered T₄ binding. Future investigations into free T₄ status must examine the analytical recovery of the free T₄ method(s) used, as they relate to the extent of serum T₄ binding in the clinical condition(s) studied.