Instability of corticotropin during long-term storage – myth or reality?

Introduction

Corticotropin (adrenocorticotropic hormone, ACTH) is a 39-residue polypeptide tropic hormone secreted by the anterior pituitary gland following hypothalamic corticotropin-releasing hormone release. ACTH is cleaved from precursor hormone pro-opiomelanocortin, and plays an important role in the hypothalamic–pituitary–adrenal axis by stimulating the production and secretion of cortisol from the adrenal cortex. Cortisol regulates ACTH release via a negative feedback loop. Measurements of plasma ACTH are crucial for differential diagnosis of endocrine disorders including Cushing’s syndrome and adrenal insufficiency but can be complicated by both preanalytical and analytical factors.

ACTH is notorious for its instability. To prevent proteolytic degradation, it is generally advised to store collected specimens on ice immediately after venepuncture collection, to process specimens in a cooled centrifuge within 1 h, and to prevent freeze-thaw cycles [1, 2]. Hemolysis of the blood specimen and choice of blood collection tube type may also influence ACTH measurements [3, 4]. Recent reports however question ACTH’s instability and the true necessity of these cumbersome pre-analytical actions [5], [6], [7]. For long-term storage, it is generally advised to store specimens at −70 °C. This may, however, not always be possible in daily practice (for clinical diagnostics or research) because of limited −70 °C freezer space. Not only may the storage temperature be critical, but also the storage time [8]. The longer the storage time, the higher the chance of degradation of unstable analytes [9]. To our knowledge, long-term stability studies on ACTH are lacking. Therefore, we designed a study to investigate the preanalytical stability of ACTH by measuring ACTH levels in neat and spiked specimens stored at both −20 °C and −70 °C for one and a half years, and also examined stability of ACTH in neat specimens stored at −20 °C for up to 4 and 6 years.

Materials and methods

Results

Experiment 1: Storing human plasma specimens for up to one and a half years at −20 °C or −70 °C had limited influence on the ACTH levels in these specimens measured by the ARCHITECT ACTH assay. In both neat and spiked specimens at the two storage conditions, ACTH levels remained relatively stable over time with only minimal changes in ACTH levels, as depicted in Figures 1 and 2. After storing neat specimens at −20 °C or −70 °C for one and a half years, ACTH levels showed a minimal relative shift of 5.5% at −20 °C and −4.4% at −70 °C storage, which are within the assay imprecision. Spiked specimens stored at −70 °C showed more instability of ACTH at one and a half years which was highest for specimens spiked at the middle (500–1,000 pg/mL) and higher level (1,500–1,800 pg/mL). Although absolute changes in ACTH levels were substantial at this high concentration level, relative % shifts at one and a half years were limited to 10.6%, as depicted in Table 1. Experiment 2: Storing specimens for up to 4 or 6 years did significantly reduce detectable ACTH levels in native patient plasma specimens as depicted in Table 2 and Figure 3. After 4 years of storage at −20 °C, ACTH levels were 74.8%, whereas after 6 years of storage ACTH levels were only 46.2% of the original ACTH levels measured using the Liaison immunoassay.

Figure 1:

ACTH levels in individual neat and spiked human EDTA plasma specimens during 18 months of storage. Each line represents the mean (of five replicates) ACTH (pg/mL) results of a single donor. Specimens were stored at −20 °C (left panel) and −70 °C (right panel), neat (upper panel) or spiked (lower panel) at different levels. ACTH was measured using the Abott ACTH immunoassay.

Figure 2:

Relative ACTH levels in neat and spiked human EDTA plasma specimens during 18 months of storage. ACTH was measured using the Abbott ACTH immunoassay. Error bars indicate the 95% confidence interval.

Figure 3:

ACTH levels in human EDTA plasma specimens at baseline and following long-term storage. Samples were stored for 4 (A) or 6 (B) years at −20 °C. Relative change in ACTH levels with error bars indicating the 95% confidence interval are depicted in (C). ACTH was measured using the Diasorin ACTH assay.

Discussion

Long-term specimen stability information is important for a variety of purposes that may be related to clinical management and clinical trials that generally utilize aged specimens. The long-term frozen storage stability of ACTH is unknown. This may result in incorrect medical decision making or false scientific conclusions based upon ACTH measurements in specimens stored for several months or even years. Here we show that ACTH levels in EDTA plasma aliquots from healthy controls are remarkably stable when stored at −20 °C or −70 °C for up to one and a half years. We observed only small changes of ACTH levels in neat specimens over time; these changes in ACTH levels were within confirmed assay imprecision and are therefore not critically different nor considered clinically relevant. In spiked specimens, changes in ACTH levels were larger, but did not increase beyond 11% (Experiment 1). ACTH levels in patient specimens, however, showed significant reductions following continued storage at −20 °C (Experiment 2). These experiments suggest that ACTH degradation is a continuing process and increases over time.

There are however several limitations. The data described above originate from two experiments, differing in design, study population, and the specific immunoassay used. Therefore, findings from both experiments cannot be directly compared. Findings from Experiment 1 indicate that ACTH levels are remarkably stable in human plasma specimens up to one and a half years. Although in general long-term storage at −70 °C is preferable to −20 °C [9], we now showed that storing ACTH specimens in a −70 °C freezer for up to one and a half years is not beneficial over storing specimens in a more frequently available −20 °C freezer. This is important information for researchers, medical doctors and laboratory specialists dealing with concerns about storage of plasma specimens for ACTH measurements future (batch) analysis [8].

Theoretically, instability of ACTH may have been masked by potential instability of the calibrator used in Experiment 1. If the calibrator degraded over time, instability of the specimens could have been missed. Although the same calibrator was used during the 18 months study, degradation of the calibrator is highly unlikely because the calibrator is a stable formulation in a synthetic matrix (not comparable to a plasma matrix with proteases), and accelerated stress stability studies at a series of temperatures above freezing with use of an Arrhenius equation to model calibrator stability supported that calibrators were stable (data not shown). The findings from Experiment 2 show that prolonged storage at −20 °C of native specimens from patients resulted in a significant reduction of ACTH levels, with specimens stored for 6 years being more affected than specimens stored for 4 years. Specimens were collected from patients visiting Amsterdam UMC for whom ACTH levels were requested as part of routine diagnostics of primary adrenal insufficiency, pituitary insufficiency or Cushing syndrome, including sinus petrosal samplings (explaining the high ACTH levels in the 6 years storage group). The reduction of ACTH levels was not related to the reason for referral (data not shown).

In theory, the changes in ACTH levels over time could have been caused by changes in the standardization of the Liaison ACTH assay over time. This is however unlikely as trend analyses based upon internal and external quality control data in the Amsterdam UMC laboratory show no indications for standardization changes (explaining the >25% reduction in ACTH levels) over time. Unfortunately, specimens were only stored at −20 °C and not also at −70 °C; therefore, we cannot deliberate on the effect of prolonged storage time (4–6 years) at different storage temperatures.

As mentioned above, studies were performed using two different ACTH immunoassays; a newly developed ARCHITECT^® i2000_SR (Abbott) automated immunoassay (Experiment 1) and a routine ACTH CLIA (Diasorin) test (Experiment 2). These ACTH assays, like other endocrine immunoassays, suffer from standardization differences due to absence of a commutable reference material and internationally accepted reference method [1, 13, 14]. Both assays are standardized using internally produced working references or standards. Therefore, findings obtained with the Abbott and Diasorin assay in this study may not be compared. Furthermore, the use of different immunoassays may cause differences in detection of possible ACTH fragments, which may increase (e.g. by oxidation) during long-term storage. For the Abbott assay, no cross-reactivity was observed for ACTH_(1–10), ACTH_(11–24), ACTH_(1–24), ACTH_(1–17) and ACTH_(18–39) (all tested after spiking 500, 5,000 and 50,000 pg/mL) [11]. The Diasorin ACTH assay showed no evidence for cross-reactivity of ACTH_(1–24) (1,400 pg/mL), ACTH_(11–24) (1,600 pg/mL) and ACTH_(18–39) (9,800 pg/mL) [12]. Finally, the plasma specimens from Experiment 2 were stored in polystyrene tubes. Polystyrene is a thermoplastic polymer which is more porous than polypropylene (used in Experiment 1). In addition, tube sizes were different; 2 mL (Experiment 1) vs. 5 mL (Experiment 2). The bigger head-space in the 5 mL tubes as well as the more porous tube material and cap type may have caused additional degradation of ACTH in Experiment 2.

This article describes two experiments performed in different settings; the first during manufacturer evaluation and the second during routine workload conditions. Despite the limitations mentioned above, we conclude that ACTH concentrations are influenced by storage time. We showed that up to one and a half years ACTH levels measured using the Abbott research only assay are stable when stored at −20 °C but that with longer storage times at −20 °C significant reductions in ACTH levels can be expected. We recommend consideration of storage time and storage temperature and standardization differences when planning long-term storage of plasma specimens for future ACTH measurements.