Simple method for preparing a concentration gradient of serum components by freezing and thawing.

We created a simple method for obtaining a series of successively more-concentrated samples from a serum without changing the ratio of its components. We froze a pooled serum and then allowed it to thaw undisturbed. The serum components formed a gradient of increasing concentration from the top of the sample to the bottom. We found that (a) in test results, each fraction of serum in the gradient showed almost the same relative concentrations of components (i.e., inorganic and organic compounds, proteins, metals, and hormones), irrespective of atomic or molecular mass; (b) the concentration gradient depended on the thawing temperature but not on the freezing temperature; (c) when we thawed the frozen sample with centrifugation, the slope of the concentration gradient increased with increasing centrifugal force; (d) when the thawed sample was fractionated into 10 fractions from the top to the bottom, the original serum concentration was always maintained between the sixth and seventh fractions from the top; and (e) the concentration gradient became steeper with repeated freezing and thawing. By using this method, one can easily prepare serum samples at gradients of concentration useful in the clinical laboratory, although the mechanism of gradient formation is still unclear.