Effect of sample dilution on measurements of free (unbound) hormones

We describe the influence of sample dilution on the measurement of free or non-protein-bound hormones in serum or plasma. The concentration of free hormone in the sample will alter after dilution or addition of binding reagents. The extent of this equilibrium perturbation depends on the binding properties of the endogenous carrier proteins, which vary from sample to sample and from sample to reference standard. As examples, we demonstrate the different changes in concentrations of free thyroxin and free cortisol after dilution, both experimentally and by theoretical modeling of the hormone-protein distribution. We also show how this systematic error can be minimized in the design of free hormone assays.