A-113 Preserving ACTH Integrity: Impact of Different Preanalytical Conditions on Stability
Daisy Unsihuay Vila, Bernard Cook, Nicole Haughey, Layling Fong-RamirezAbstract
Background
Adrenocorticotropic hormone (ACTH) is a peptide hormone produced by the anterior pituitary that plays a crucial role in regulating cortisol secretion. Measuring ACTH levels is essential for evaluating conditions such as Cushing*s syndrome and adrenal insufficiency. However, ACTH is unstable in whole blood due to proteolytic degradation. To minimize this instability, specific collection and processing protocols are recommended, including the use of pre-chilled tubes, immediate centrifugation, ice bath, or transporting samples in a frozen state. These precautions pose some logistical challenges for laboratories. This study aims to assess the impact of storage and processing conditions in ACTH results.
Methods
A total of 71 residual whole blood samples in EDTA tubes from inpatients were centrifuged within 3 hours after collection. Baseline ACTH measurements (median: 18.1, range: 2.1-460) in plasma were performed immediately using a Roche e801 analyzer (AMR 2-200 pg/mL). Condition 1: A subset of n=16 samples were stored at -30°C. ACTH measurements were taken after each freeze-thaw cycle, with a 24-hour interval between cycles, for a total of three cycles. Condition 2: A subset of n=12 samples were stored at -30°C. After the first freeze-thaw cycle, plasma was divided into three aliquots for storage at 4°C and ACTH measurements at 24, 48, and 72 hours. Condition 3: A subset of n=43 samples were divided into three to five aliquots (depending on sample volume) for storage at 4°C and ACTH measurements at 4, 8, 24, 48, and 72 hours. Percentage change from baseline was calculated for all storage conditions, and time points. Changes of more than ±2.0 pg/mL or ±10% from baseline concentration were considered clinically significant.
Results
Plasma ACTH levels remained stable after up to three freeze-thaw cycles (Condition 1), with mean percentage changes of –2.4% (95% CI: –9.0 to 4.2) after the first cycle, –4.7% (95% CI: –12.9 to 3.5) after the second, and –5.6% (95% CI: –14.9 to 3.66) after the third. In ACTH samples subjected to a single freeze-thaw cycle followed by refrigeration at 4°C (Condition 2), ACTH levels exhibited no significant changes at 24 hours (mean change: –3.4%, 95% CI: –9.4 to 2.7). However, at 48 hours, two samples demonstrated negative biases of –12.5%, with an overall group mean change of –6.4% (95% CI: –14.8 to 2.1). By 72 hours, the mean change was –6.6% (95% CI: –27.4 to 14.11), with four outliers exhibiting decreases of up to -28.12%. Lastly, samples that were refrigerated at 4 °C after centrifugation (Condition 3) did not exhibit significant changes until 48 hours (mean change: –4.4%, 95% CI: –12.3 to 3.5), with three outliers showing decreases of up to -11.6%. By 72 hours, the overall mean change was –7.3% (95% CI: –15.9 to 1.4) with four outliers with less than -15% change.
Conclusion
Our findings indicate that ACTH in plasma remains viable for analysis under processing and storage conditions less stringent than commonly practiced, with ACTH stability maintained for up to 48 hours under refrigeration at 4 °C and up to three freeze-thaw cycles without compromising accuracy.