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A pilot study showing a decrease in androstenedione concentration in serum collected into gel-containing serum tubes (STs) triggered an investigation of the effect of serum collection tube on steroid hormone stability.In the main study, two tube types were examined: BD Vacutainer® SST™II Advance and BD Vacutainer® Serum Tube. Forty-seven serum samples from apparently healthy volunteers were collected and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP) (n=20); and oestradiol (n=27). Primary specimens were centrifuged once, maintained at room temperature and extracted within 2 h for day zero (d0) results. To assess stability following refrigeration (2-8 °C), aliquots were taken from the primary tube on day one (d1) and day five (d5) and analysed immediately. Differences in measurand concentration between tubes at d0 and following storage (d1 and d5) were evaluated for statistical significance.There was a progressive and statistically significant decrease in androstenedione concentration from d0 to d5 (p<0.001) in the SST™II tubes. In addition, there was a statistically significant reduction in testosterone, 17-OHP and oestradiol concentrations at d5 (p<0.01). Interestingly, oestradiol and testosterone concentrations increased with time in plain STs (p<0.01). The only change likely to have a clinical impact was that of androstenedione in serum gel tubes.To optimise conditions and to reduce pre-analytical error we recommend the use of plain serum collection tubes for androstenedione and rapid separation of serum from cells when oestradiol and testosterone are requested.