European Journal of Cardiovascular Medicine

The accuracy of biochemical serum analyte measurements is critical for clinical diagnostics and research ^[1]. Pre-analytical factors, including storage conditions such as temperature and time, can influence the stability of analytes, potentially leading to erroneous results ^[2,3]. Despite advancements in laboratory technologies, the effects of varying storage durations and temperatures on serum analytes remain a significant concern ^[4,5]. This study explores how these factors impact key biochemical markers like urea, creatinine, AST, ALT, total protein, albumin, sodium, and potassium.

The findings aim to establish guidelines for optimal storage conditions, ensuring the reliability of diagnostic and research outcomes.

Aims: To investigate the impact of varying storage durations and temperatures on the stability of biochemical serum analytes.

Objectives:

1. To establish baseline values for urea, creatinine, AST, ALT, total protein, albumin, sodium, and potassium in serum samples from healthy individuals.
2. To analyze the effects of different storage temperatures (−20°C, 4°C, and 25°C) on the stability of these analytes over time.
3. To evaluate the stability of analytes at multiple time intervals, including 24 hours, 48 hours, 72 hours, and one week.
4. To provide evidence-based recommendations for optimal storage conditions to ensure the integrity of biochemical analytes.

Review of Literature Several studies have documented the effects of storage conditions on serum analytes. For instance, elevated storage temperatures have been shown to accelerate enzymatic degradation, while prolonged storage can lead to protein denaturation ^[6,7].

• Urea and Creatinine: Stability largely depends on enzyme activity and exposure to fluctuating temperatures ^[5].
• AST and ALT: These transaminases are sensitive to freeze-thaw cycles, which may compromise their activity levels ^[4,8,9,10].
• Total Protein and Albumin: These markers are influenced by dehydration and potential protein denaturation during storage ^{[2,6,11,12,13]}.
• Sodium and Potassium: Electrolyte levels can vary due to hemolysis or evaporation under improper storage ^[3,7,14].

The study conducted at the Department of Biochemistry, ShriVasantraoNaik Government Medical College, Yavatmal, seeks to expand on these findings by comprehensively evaluating multiple analytes under varied conditions.

Study Design: A prospective experimental study conducted in the Department of Biochemistry, Shri. Vasantrao Naik Government Medical College, Yavatmal.

Inclusion Criteria:

1. Healthy individuals aged 18–50 years with no known chronic illness or medication use.
2. Individuals with normal baseline biochemical values as determined during preliminary screening ^[14].

Exclusion Criteria:

1. Individuals with known liver, kidney, or electrolyte disorders
2. Samples with evidence of hemolysis or contamination
3. Samples stored under suboptimal conditions before the study

Sample Collection:

• Population: 100 healthy individual, plain vacutainers under all aseptic precautions
• Analytes: Urea, Creatinine, AST, ALT, total protein, albumin, sodium, and potassium.
• Baseline Values: Initial values were measured immediately after collection to establish reference points.^[18-19]

Storage Conditions:

• Temperature Groups: Samples were stored at 4°C (refrigerated), −20°C (frozen), and room temperature (25°C).
• Time Intervals: Analytes were assessed at 24 hours, 48 hours, 72 hours, and one week^[20]

Analytical Methods: Random Access Chemistry analyzer using the following methods

• Urea: Urease GLDH method
• Creatinine: Enzymatic method
• AST: AST reagent IFCC without PLP
• ALT: ALT reagent IFCC without PLP
• Total Protein: Biuret method
• Albumin: Bromocresol green dye binding method
• Sodium: Assayed using ion-selective electrodes.
• Potassium: Assayed using ion-selective electrodes.

Statistical Analysis: Data was analyzed using paired t-tests and ANOVA to compare stability across different storage conditions and time points.

The stability of serum analytes under various storage conditions is summarized in Table 1.

Table 1: Stability of serum analytes

Table 2: Mean, standard deviation, and percentage change across different storage conditions

Figure 1: Percentage changes observed over time in analytes across storage conditions and durations

The stability of serum analytes under various storage conditions is summarized in Table 1. This study highlights the significant impact of storage temperature and duration on the stability of various biochemical serum analytes as shown in table-2. The statistical analysis is detailed in Table 2. Each analyte demonstrated distinct patterns of stability under different storage conditions^[15,16,17]:

1. Urea and Creatinine: As observed in Table 1, urea and creatinine showed exceptional stability across refrigerated and frozen conditions, reflecting minimal degradation over time. However, slight declines in concentration were observed under room temperature conditions, emphasizing the necessity for cooler storage for long-term accuracy [5].
2. AST and ALT: As observed in Table 1, enzyme activities for AST and ALT showed a marked decline, especially at room temperature by the 72-hour mark. This sensitivity underscores the critical need for either immediate processing or storage at low temperatures to maintain enzymatic activity and reliability in diagnostic tests ^[8,9,10].
3. Total Protein and Albumin: As observed in Table 1, both protein markers were moderately affected by room temperature, with evidence of denaturation likely contributing to reduced levels. Refrigeration and freezing effectively preserved these proteins, supporting their utility in diagnostic and research applications when stored appropriately ^{[2,6,11,12,13]}.
4. Sodium and Potassium: As observed in Table 1, electrolyte stability was influenced by hemolysis and evaporation, primarily under room temperature conditions. Significant deviations after 72 hours highlight the importance of refrigerated or frozen storage to ensure reliable measurements for clinical assessments ^[3,7,14].

Overall, these findings underscore the necessity of adhering to specific storage protocols to preserve the integrity of serum analytes. Freezing remains the gold standard for long-term preservation, while refrigeration is suitable for short-term storage. Deviations from these protocols can result in altered analyte concentrations, impacting diagnostic accuracy and research outcomes.

This study highlights the pivotal role of storage temperature and duration in maintaining the integrity of serum analytes. Proper storage practices are not merely logistical considerations but are critical to ensuring diagnostic precision, clinical reliability, and research accuracy. The implications extend beyond laboratory settings, emphasizing the importance of standardized pre-analytical protocols globally.

Refrigeration provides a dependable option for short-term storage, maintaining stability for clinical assays. Freezing emerges as the definitive approach for long-term preservation, minimizing biochemical degradation and ensuring data fidelity for retrospective studies. These findings contribute to the ongoing discourse on best practices in clinical biochemistry and laboratory medicine, offering a framework to optimize pre-analytical handling of samples. By implementing these recommendations, laboratories can enhance the reliability of diagnostic results and uphold the integrity of medical research worldwide.

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