The effects of repeated freeze–thaw cycles on endocrine parameters in plasma and serum

A recent study in the Annals examined the stability of endocrine parameters following repeated freeze–thaw cycles. ¹ Apart from plasma renin activity, the authors found no effect of multiple freeze–thaw cycles, thus challenging alarmist notifications in assay manuals.

I wish briefly to highlight aspects of the physical process of freezing plasma/serum and its potential impact on immunoassays of haptens or macromolecules, whether they are glycoprotein, lipoprotein or just protein. (Binding site(s) for the same analyte vary from one immunoassay to another.)

The molecular integrity of binding sites is paramount for the specificity and affinity of all immunoassays. ² The binding reaction involves an epitope on the antigen and a complementary antigen-combining site (paratope) on the antibody. Even the simplest antigen has multiple epitopes, and the binding reaction involves multiple paratopes. Each individual epitope has a linear and continuous primary structure made up of amino acids in secondary and tertiary configuration.^3,4 The binding reaction also involves an array of matrix constituents the structure of which may or may not be affected by freezing.

Freezing at −20℃ is slow, while ‘snap-freezing’ using dry ice or liquid nitrogen at −70℃ is rapid. The latter is known to maintain integrity of samples, raising the question of how slow-freezing affects the samples from different individuals, with different matrices, using different antibodies and different immunoassay formats. Transformation of water to ice is a well-studied process. Speed is important: in slow-freezing, large, solid abrasive ice crystals are formed; in snap-freezing, ice crystals are small and amorphous. ⁵ Additionally, when snap-freezing to temperatures below −50℃, not all water molecules are converted into solid ice; some remain in liquid form.

It appears impossible to predict a priori the impact of slow freeze–thaw cycles on the analyte itself and/or other serum constituents from patients with different matrices. A practical alternative, which has stood the test of time, is to freeze multiple aliquots of plasma/serum, to thaw one at a time, to use for immunoassay analyses and to discard after single use. When snap-freezing was also initially used, subsequent storage at −20℃ allowed accuracy to be maintained on re-analysis over period of months.