Stability of 17 endocrine analytes in serum or plasma after four cycles of repeated freezing and thawing

Publications of systematic studies on analyte stability after repeated freezing and thawing of samples are rare. We examined the stability of 17 endocrine analytes in pooled serum and/or EDTA plasma after one to four cycles of repeated freezing at -80 °C and thawing. Pooled serum and plasma samples were used. Following baseline measurements in fresh samples (T0), four aliquots (T1-T4) were frozen at -80 °C, and subjected to one to four cycles of freezing and thawing before analysis on the same day. Results were compared to baseline measurements (T0) and to samples frozen once (T1), and were adjusted using quality control material to account for analytical variation between the two time points of analysis. Analytes were considered stable based on statistical significance and percent change compared to allowable bias (AB) based on biological variation. According to criteria based on AB, serum 17-OH progesterone, aldosterone, androstenedione, anti-müllerian hormone, cortisol, dehydroepiandrosterone sulphate, proinsulin C-peptide and sexual-hormone-binding-globulin, and plasma aldosterone and cortisol were stable for four cycles of freezing and thawing. Only serum free thyroxine was considered unstable. For serum erythropoietin, estradiol, free triiodothyronine, human chorionic gonadotropin, human growth hormone, insulin-like growth factor-1, prolactin and plasma free thyroxine, human growth hormone, parathyroid hormone results were not conclusive. Based on average results of pooled samples, eight out of 17 analytes appeared stable for four freeze-thaw cycles compared to AB, while free thyroxine in serum increased more than AB.