Evaluating storage conditions for insulin and parathyroid hormone testing

INTRODUCTION

The accurate measurement of hormones, such as insulin and parathyroid hormone (PTH), is critical in both clinical diagnostics and research settings.^[1] Insulin plays a central role in glucose metabolism, while PTH is essential for calcium and phosphate homeostasis.^[1,2] Given their biological importance, precise quantification of these hormones is crucial for diagnosing and managing various metabolic and endocrine disorders, such as diabetes and hyperparathyroidism.^[1,3] However, the stability of these hormones in blood samples can be significantly affected by storage conditions and the type of collection tubes used, potentially leading to inaccurate measurements if not properly managed.^[4,5]

Hormones are sensitive biomolecules that degrade over time, particularly when exposed to ambient conditions. Degradation can lead to significant variability in assay results, complicating clinical decision-making and research outcomes. Therefore, understanding how different storage conditions, such as room temperature, refrigeration at 4°C, freezing at −20°C, and repeated freeze-thaw cycles, impact hormone stability is essential. In addition, the choice of blood collection tubes (serum separator tubes, ethylenediaminetetraacetic acid [EDTA] plasma tubes, and heparin plasma tubes) can further influence hormone preservation, as additives in the tubes may interact with the hormones differently.^[6]

Previous studies have indicated that improper storage and handling of samples can lead to rapid degradation of insulin and PTH, resulting in inconsistent or inaccurate results.^[7] Despite these findings, there is limited research directly comparing the effects of various storage conditions and tube types on the stability of these hormones over time.^[8,9] This study aims to address this gap by systematically evaluating the stability of insulin and PTH under different storage conditions and across various collection tubes, providing practical recommendations for clinical laboratories and researchers to ensure optimal sample preservation.

The objective of this study is to determine the optimal storage conditions for insulin and PTH, as well as to identify which blood collection tube provides the best stability for these hormones over time. By understanding the degradation profiles of insulin and PTH, this research aims to improve the reliability of hormone assays, ultimately enhancing the accuracy of diagnostic and research outcomes in endocrine and metabolic disorders.

MATERIALS AND METHODS

The study was conducted at the Department of Biochemistry, Maulana Azad Medical College, and Lok Nayak Hospital, New Delhi, with the objective of assessing the stability of insulin and PTH across various storage conditions and blood collection tubes. Three types of collection tubes, serum separator tubes, EDTA plasma tubes, and heparin plasma tubes, were used (BD Vacutainer^®, Becton, Dickinson and Company [BD], USA), and samples were stored at room temperature (approximately 25°C), 4°C, and −20°C. In addition, the effect of repeated freeze-thaw cycles on hormone stability was also evaluated.

This study involved healthy adult volunteers who were informed about the purpose and procedures of the sample collection for laboratory analysis. Participation was voluntary, and consent was considered implied through the act of providing the sample. The study was non-interventional, involved minimal risk, and all samples were anonymized before processing. In accordance with routine institutional practices for minimal-risk studies using anonymized biological samples, separate ethics committee approval was not sought.

Following collection, whole blood samples were immediately processed by centrifugation at 3,000 rpm (approximately 2,000 g) for 10 minutes at room temperature to separate serum or plasma. The separated fractions were aliquoted into labeled microcentrifuge tubes and stored under designated conditions. Hormone concentrations were measured at 0, 6, and 24 hours for samples stored at room temperature and 4°C, and at 24, 48, and 72 hours for samples stored at −20°C.

To assess freeze–thaw effects, a subset of −20°C aliquots underwent three cycles, 30 minutes thaw at ~25°C followed by refreezing, with measurements at 24, 48, and 72 hours. Refrigeration (4°C) was evaluated only for 24 hours to model short-term holding, whereas −20°C storage represented medium-term preservation and was monitored for 72 hours; because frozen aliquots must be thawed before analysis, the first −20°C measurement occurred at 24 hours.

Hormone quantification was performed using chemiluminescence immunoassays on the VITROS^® ECiQ Immunodiagnostic System (Ortho Clinical Diagnostics, USA). Both insulin and intact PTH were measured using their respective VITROS^® Immunodiagnostic Products reagent packs, with all procedures carried out in accordance with the manufacturer’s instructions. The reagent kits were stored at 2-8°C, and all samples were processed promptly to minimize any deviation from recommended handling conditions.

The assay precision, based on manufacturer-reported data, showed intra-assay coefficients of variation (CVs) of 2.1-3.5% for insulin and 3.5-4.0% for PTH, while inter-assay CVs were up to 5.0% and 6.0%, respectively. These benchmarks were used to interpret whether observed fluctuations exceeded expected analytical variability, as in-house CV% values could not be generated due to limited sample volume and the pilot nature of the study.

RESULTS

The stability of insulin and PTH was evaluated under three temperature conditions – room temperature, 4°C, and −20°C, over a period of 72 hours, using three types of blood collection tubes: Serum separator, EDTA plasma, and heparin plasma tubes. Detailed analyses of insulin and PTH concentrations were conducted to evaluate the effects of time and temperature on hormone stability. The following sections outline the key findings.

Time-dependent degradation of insulin and PTH

Figure 1 displays time-dependent degradation of insulin (μIU/mL) and PTH (pg/mL) under three storage conditions: Room temperature, 4°C, and −20°C. Hormone concentrations were measured at 0, 6, and 24 hours for samples stored at room temperature and 4°C and at 24, 48, and 72 hours for samples stored at −20°C. Baseline (0 h) values were available for room temperature and 4°C, since these aliquots could be assayed immediately after separation. For −20°C, however, measurements could only be made after thawing, so the first meaningful time point was 24 hours. Refrigeration was assessed only up to 24 hours to model short-term holding; −20°C represented medium-term storage, so measurements were extended to 72 hours.

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Figure 1:

Time-dependent degradation of (a) insulin (μIU/mL) and (b) parathyroid hormone, (pg/mL) under different storage conditions (room temperature, 4°C, and −20°C). For −20°C, measurements begin at 24 hours after thawing. Points show mean concentrations at each time point. PTH: Parathyroid hormone.

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At room temperature, both insulin and PTH showed significant degradation within the first 24 hours. Hormonal stability improved notably at 4°C, while storage at −20°C provided the highest level of preservation throughout the 72-hour period.

Insulin concentrations declined markedly by 6 hours at room temperature [Figure 1]. In contrast, refrigeration at 4°C and freezing at −20°C preserved insulin levels more effectively, with minimal degradation observed at −20°C. Similarly, PTH levels decreased rapidly at room temperature but remained more stable under cold storage, particularly at −20°C [Figure 1].

Comparison of tube types: Serum separator, EDTA plasma, and heparin plasma tubes

Absolute baseline concentrations differed among tube types, likely due to matrix effects and tube additives, as reported in prior studies. Therefore, comparisons were made in terms of relative stability (percentage degradation from each tube’s baseline) rather than direct comparison of absolute values [Figure 2].

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Figure 2:

(a) Stability of insulin and (b) parathyroid hormone concentrations at room temperature across different collection tube types (serum separator, ethylenediaminetetraacetic acid plasma, heparin plasma). Values represent mean concentrations. EDTA: Ethylenediaminetetraacetic acid.

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Figure 2 illustrates mean insulin concentrations (μIU/mL) measured at 0, 6, and 24 hours in samples stored at room temperature across the three tube types.

Figure 2 shows a similar comparison for mean PTH concentrations (pg/mL) at the same time points and under the same storage conditions.

Because baseline concentrations differed markedly by tube (matrix/additive effects), we focus on within-tube change over time: Across RT measurements, serum and EDTA showed broadly comparable relative stability for insulin, while for PTH, EDTA and heparin changed little in relative terms despite very different baselines. Thus, absolute values are not comparable across tubes, and stability should be interpreted within each matrix.

Hormone concentration at 24 hours

At the 24-hour mark, insulin and PTH concentrations were compared across the three storage conditions (pooled across tube types) [Figure 3]. This figure provides a snapshot of hormone levels at 24 hours only, while baseline (0 h) values and detailed time-course trends have been presented in Figure 1. As shown, storage at −20°C preserved the highest concentrations, whereas room temperature led to the greatest degradation. Considerable variability was noted across replicates – particularly for insulin. Bar heights represent mean concentrations at 24 hours.

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Figure 3:

(a) Mean concentrations of insulin (μIU/mL) and (b) parathyroid hormone (pg/mL) at 24 h across storage conditions (room temperature, 4°C, −20°C), pooled across tube types. Baseline (0 h) values. PTH: Parathyroid hormone.

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Insulin: At 24 hours, insulin concentrations were highest under −20°C storage, followed by 4°C, with the lowest values recorded at room temperature [Figure 3].

PTH: At 24 hours, concentrations were highest at 4°C, followed by −20°C, and lowest at room temperature [Figure 3].

Figure 3 highlights the critical importance of low-temperature storage, especially at 4°C and −20°C, for maintaining the stability of both insulin and PTH.

Variability and distribution of insulin and PTH concentrations

The variability in insulin and PTH concentrations under different storage conditions was assessed using box plots [Figure 4]. Both hormones exhibited the least variability at 4°C and −20°C, indicating enhanced stability under cold storage. In contrast, room temperature storage resulted in greater variability and wider distribution, reflecting the destabilizing effects of ambient conditions.

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Figure 4:

(a) Distribution of insulin and (b) parathyroid hormone concentrations across storage conditions (room temperature, 4°C, −20°C). Box plots show median, interquartile range, and outliers. PTH: Parathyroid hormone.

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Figure 4 shows broader dispersion at room temperature and narrower interquartile ranges at 4°C and −20°C for both hormones, consistent with improved stability under cold storage. Both insulin and PTH displayed the greatest variability at room temperature and more consistent values under refrigerated and frozen storage.

Insulin: At room temperature, insulin displayed increased variability, with a broader range of concentrations. This variability was substantially reduced at −20°C, highlighting the advantage of cold storage [Figure 4].

PTH: A similar pattern was observed for PTH, with the highest variability at room temperature and the lowest at −20°C, underscoring the need for low-temperature conditions to maintain hormone integrity [Figure 4].

Figures 1-3 display mean concentrations; Figure 4 shows distributions (median, interquartile range, whiskers/outliers). Values are from 10 biological replicates. Given the exploratory sample size, we did not perform inferential statistics; trends should be interpreted as indicative.

DISCUSSION

This study evaluated the stability of insulin and PTH across various storage conditions and blood collection tubes. The results showed rapid degradation of both hormones at room temperature, particularly within the first 24 hours, with insulin degrading more quickly. This aligns with the known susceptibility of proteins like insulin to denaturation in ambient conditions, reinforcing the need for cold storage.

At 4°C, both insulin and PTH displayed better stability, but some degradation still occurred, particularly beyond 24 hours. This suggests that while 4°C storage is suitable for short-term preservation, it is insufficient for extended storage. Room temperature storage, on the other hand, is not acceptable due to the rapid degradation of these hormones. The most stable condition was −20°C, supporting its role as the optimal choice for short- to medium-term storage. In frozen aliquots, two additional thaw–refreeze cycles beyond the initial thaw produced only small changes (−6.3% from 24 to 48 hours; +1.3% from 24 to 72 hours), within expected analytical variability. Nevertheless, minimizing repeated freeze–thaw cycles remains advisable in practice.

It is important to note that baseline concentrations differed between tube types, which likely reflect matrix and additive effects rather than true biological differences. Our analysis, therefore, emphasized relative stability (percentage degradation within each tube type) rather than direct comparison of absolute concentrations. The comparison of serum separator tubes, EDTA plasma tubes, and heparin plasma tubes revealed that serum separator tubes provided the most consistent stability for both hormones across storage conditions. However, EDTA tubes preserved insulin more effectively at early time points, and heparin tubes showed higher initial PTH levels. Taken together with matrix effects, these patterns suggest that serum tubes may offer practical consistency when processing is delayed, but absolute cross-matrix comparisons are not appropriate.

The variability in hormone concentrations was most pronounced at room temperature and decreased as storage conditions improved, indicating that cold storage not only stabilizes hormones but also reduces variability in results. This finding is critical for ensuring accuracy in clinical and research assays of insulin and PTH.

A limitation of our study is that frozen samples were assessed only up to 72 hours at −20°C; although sufficient to demonstrate short- to medium-term stability, this does not capture longer storage durations (e.g., ≥1 week) commonly used in clinical laboratories. In addition, in-house CV% values for insulin and PTH were not generated because of limited sample volume; therefore, manufacturer-reported precision was used as a reference benchmark, which may limit direct extrapolation to our laboratory setting. Finally, we did not perform matrix-equivalency studies on this platform; accordingly, absolute insulin and PTH concentrations should not be compared across tube types and should be interpreted only within the same matrix.

The study emphasizes the necessity of cold storage, particularly at −20°C, for maintaining insulin and PTH stability over time. Serum separator tubes offer better hormone preservation compared to EDTA and heparin tubes. These insights have important implications for clinical laboratories and research, where proper sample handling is vital for reliable hormone analysis.

CONCLUSIONS

This study highlights the importance of storage conditions for insulin and PTH stability. Both hormones degraded rapidly at room temperature, making ambient storage unsuitable. Short-term preservation was better at 4°C, while −20°C provided the best stability for up to 72 hours, with only minor effects from freeze–thaw cycles. Serum separator tubes preserved hormone levels more consistently than EDTA or heparin tubes. These findings support the use of −20°C storage and serum separator tubes to ensure reliable hormone measurements in clinical and research laboratories.

Author contributions

AC: Contributed to conceptualization, initial drafting of the manuscript, and literature review, supported experimental planning and coo-rdination; AS: Conceived and designed the study, led statistical analysis and result interpretation, prepared figures, wrote and revised the manuscript, and handled journal correspondence; SS: Assisted in data curation, critically reviewed the methodology, and contributed to manuscript revision; PG: Coordinated laboratory work and supported referencing and data tabulation; SHG: Provided intellectual input including initial data acquisition and assisted in final proofreading.