Stability of Serum Total Cholesterol, Triglycerides, and Plasma Glucose of Healthy Individuals at Different Storage Conditions

: Evaluation of the stability of serum and plasma analytes under different temperatures and time points is vital since it significantly affects the pre-analytical errors. The aim of the study was to determine the stability of serum total cholesterol and triglyceride and plasma glucose of healthy individuals under different storage conditions (25-30°C and 2-8°C) and at different time points (1-hour, 4-hour, 6-hour, and 24-hour). Venous blood samples were collected from healthy individuals aged between 20-50 years after a 12-hour fast. Plasma and serum were separated and stored at different temperatures (25-30°C and 2-8°C) and different time points (1-hour, 4-hour, 6-hour, and 24-hour). Results indicated a significant difference between the mean plasma glucose concentration at room temperature (25-30°C) and refrigerated temperature (2-8°C) at 1-hour (0.072%, p = 0.05). However, there were no significant differences in mean serum total cholesterol concentrations (p = 0.633) and mean serum triglyceride concentrations (p = 0.246) between these temperatures at 1-hour time point. Regarding time points (1-hour, 4-hour, 6-hour, and 24-hour), significant differences in plasma glucose concentrations occurred at 24-hour (10.03%, p = 0.001) at room temperature (25-30°C) and 6-hour (8.79%, p = 0.003) at refrigerated temperature (2-8°C) compared to the 1-hour reference values. Additionally, there were significant differences in serum total cholesterol concentration at 4-hour (12.55%, p = 0.000) and serum triglyceride at 24-hour (29.18%, p = 0.001) compared to the reference values obtained at 1-hour at room temperature (25-30°C). No significant differences were observed among the time points (1-hour, 4-hour, 6-hour, and 24-hour) for serum total cholesterol (p = 0.507) and serum triglyceride values (p = 0.761) at refrigerated temperature (2-8°C). Plasma glucose is unstable at both room temperature (25-30°C) and refrigeration (2-8°C), with recommended testing within 6 hours at room temperature (25-30°C) and stored in a refrigerator (2-8°C) if delayed. It is better to estimate serum total cholesterol within 1-hour of collection at room temperature (25-30°C) while serum triglyceride concentration within 6 hours at the same temperature to obtain accurate and reliable results.


INTRODUCTION
The main goals of a health system include disease prevention, early diagnosis, risk assessment, avoiding delays in treatments, providing accurate results, and followup of the treatments (Sikaris, 2017).Clinical laboratories play a significant role in the health care system.The value of clinical laboratory testing is often directed to clinical decision-making to determine patient's health and wellbeing.
Evaluation of a lipid profile is an important component in a clinical setting for several key reasons.It is a tool for the assessment of the risk of cardiovascular disease.Elevated levels of low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) and low level of high-density lipoprotein cholesterol (HDL-C) are known risk factors for cardiovascular diseases.Evaluation of the lipid profile aids clinicians to identify individuals at increased risk for cardiovascular diseases and make appropriate preventive interventions such as lifestyle modifications and lipidlowering medications.Furthermore, the evaluation of the lipid profile is essential to monitor the effectiveness of lipid-lowering therapies.This allows clinicians to adjust the treatment regimen to achieve optimal lipid levels.Serum total cholesterol and triglyceride estimations are used to diagnose mainly hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia. Also, it is often used as a part of routine health screening programs and can provide valuable information about a person's overall health status.Plasma glucose is a crucial and common test performed in clinical laboratories.Plasma glucose testing is a fundamental tool in the diagnosis and monitoring of patients with diabetes mellitus, and impaired glucose tolerance.Regular monitoring of plasma glucose levels is essential to assess the effectiveness of treatment plans such as lifestyle modifications, insulin therapy, or oral medications for patients with diabetes mellitus, the plasma glucose test allows clinicians to monitor changes in glucose levels over time, make-adjustments in the treatment, and helps to assess the glycemic control of the patient.Therefore, the analysis of the stability of serum total cholesterol, triglycerides, and plasma glucose is important to ensure optimal integrity of the sample during the pre-analytical phase.
Samples are usually stored at different storage conditions for various reasons, including the reuse of samples to confirm the accuracy of a test value that was taken previously (delta checking), add new quantifications of missing analytes when samples are transferred to distant places, etc.It is important to ensure the consistency of the sample after collection.Temperature and time during sample storage are critical for quality assurance and play a vital role in preserving analyte stability and measurement precision (Hirigo, 2020).Hence, the stability of analytes following storage should be noted as an important pre-analytical factor that determines the accuracy of test results.Although there are recommendations from the World Health Organization (WHO) and the Clinical Laboratory Standard Institute (CLSI) for sample storage and processing, it is difficult to apply those recommendations for routine laboratory settings in Sri Lanka with limited resources.The samples sent from multiple laboratories are typically stored in a central laboratory for analysis, but various factors such as delays in sample shipping, power outages, or voltage fluctuations may impact the correct value of test results (Cuhadar et al., 2012).Many studies have been published that tend to focus more on the effect of delays between centrifugation and analysis (Lima-Oliveira et al., 2017;Smith and Johnson, 2020), but sometimes the literature search revealed confusing and incomplete evidence.The effect of storage conditions (duration of storage and temperature) on sample stability is poorly investigated in the laboratory setting in Sri Lanka.Hence, it is more beneficial to develop an evidence-based for sample handling and storage for routine clinical laboratory.Thus, storage temperature and duration after separating plasma or serum till the analysis are important to ensure optimal sample integrity during the pre-analytical phase.

Ethical Clearance
Ethical approval was granted from the Ethics Review Committee of the Faculty of Allied Health Sciences, University of Ruhuna, Sri Lanka (Ref.No:241.05.2023).The sample size was calculated using a formula involving variables such as δ (0.52, derived from a previous study by Marjani, 2008), z (1.96), and d (0.2). Applying the formula N= (δ 2 × z 2 )/d 2, where N represents the minimum sample size, resulted the required sample size of 30 individuals for the study.

Study Design
The study focused on healthy individuals, encompassing both males and females aged between 20-50, who were affiliated with the Faculty of Allied Health Sciences, University of Ruhuna, Sri Lanka.Participants underwent a 12-hour fast before venous blood collection (5 mL), performed by trained phlebotomy personnel.Blood was drawn into NaF/Potassium Oxalate anticoagulated and gel separation tubes obtain plasma and serum respectively.The collected samples were labeled with unique identifiers for anonymity.The blood samples were centrifuged at 2500 rpm for 10 minutes.Subsequently, plasma and serum were separated into aliquots and stored (Table 1).

Biochemical Assays
Plasma glucose concentration was estimated by the glucoseoxidase method described by Trinder (1969) using an enzyme assay kit (Biorax, USA).The concentration of serum total cholesterol was estimated by the enzymatic method described by Allain et al. (1974) using an enzyme assay kit (Stanbio, USA).The enzymatic method of Wahlefeld (1974) was followed using the spectrophotometric enzyme assay kit (Stanbio, USA).There can be several reasons for changing plasma glucose concentration in different storage temperatures such as room temperature and refrigerated temperature.Some biochemical reactions can be temperature-sensitive.Enzymatic reactions may proceed at different rates at room temperatures when compared to the refrigerated temperatures, leading to differences in glucose concentration (Peterson et al., 2007).Glucose may undergo chemical reactions more rapidly at high temperatures, leading to degradation or changes in concentration.Refrigeration can slow down these reactions, preserving the glucose concentration (Leitzen et al., 2021).The results of the present study corroborate published literature, observing a significant change when plasma glucose was analyzed at room temperature (20-25°C) and refrigerated temperature (2-8°C).No significant changes were observed in the analysis of total cholesterol and triglycerides under similar conditions (Kughapriya & Elanchezhian, 2019).Similarly, Franca et al. (2018) revealed that the storage temperature did not affect the analysis of total cholesterol and triglycerides.At the same time, another study demonstrated that triglycerides remained stable for up to 7 days when stored in a standard refrigerator when compared to the room temperature (Pahwa, et al., 2015).
There were statistically significant differences in the concentration of plasma glucose obtained at 24-hour at room temperature (25-30°C) (10.03%, p = 0.001) and 6-hour at refrigerated temperature (2-8°C) (8.79%, p = 0.003) compared to the reference values obtained at 1-hour (Table 2 and 3).Blood samples collected for plasma glucose estimation are treated with anticoagulants.Over time, anticoagulants may lose their effectiveness allowing an alteration in the plasma glucose concentration.Prolonged exposure to room temperature can cause evaporation of the serum and plasma, leading to a change in the concentration of analytes.In contrast to the findings of the present study, Kughapriya & Elanchezhian (2019), examined a significant change in the analysis of plasma glucose at 4-hour, compared to the 2-hour in both room temperature (20-25°C) and refrigerated temperature (2-8°C) (Kughapriya & Elanchezhian, 2019).A study revealed that glucose was the least stable analyte in plasma for 48 hours at room temperature, supporting the results of the present study (Marjani, 2008).
There were statistically significant differences in the concentration of serum total cholesterol values at 4-hour (12.55%, p = 0.000) and concentration of serum triglyceride values at 24-hour (29.18%, p = 0.001) compared to the reference values obtained at 1-hour at room temperature (25-30°C) as shown in Table 2.However, there were no statistically significant differences among the time points (1-hour, 4-houe, 6-hour, and 24-hour) for both concentrations of serum total cholesterol (p = 0.507) and serum triglyceride (p = 0.761) obtained at refrigerated temperature (2-8°C) as presented in Table 3. Lipids can be sensitive to environmental conditions.Prolonged exposure to room temperature may lead to lipid degradation, causing a decrease in cholesterol concentration over time.Serum samples are generally more stable than plasma samples due to the absence of anticoagulants.Serum triglycerides are susceptible to enzymatic breakdown by lipases, even at room temperature.Over 24 hours, this enzymatic activity might lead to a decrease in triglyceride concentration.Even at room temperature, slight fluctuations in temperature and humidity can impact the stability of serum samples.The findings of the present study were supported by a study conducted by (Franca et al., 2018).Total cholesterol and triglycerides were analyzed within one hour from collection at room temperature, after 4-5 hours at room temperature, and after 2-3 hours of refrigeration (2-8°C).The results showed that the time and storage conditions did not affect the measured analytes (Franca et al., 2018).
The present study provides insights on how different storage conditions affect the stability of plasma and serum  Further, the study assumed homogeneity among participants between 20 -50 years age group, but there could be significant differences in the analytes even within the selected age range.

CONCLUSION
The study revealed that the concentration of plasma glucose is not stable both at room temperature (25-30°C) and refrigerated temperature (2-8°C).In contrast, the concentration of serum total cholesterol and triglycerides are relatively stable at room temperature (25-30°C) and refrigerated temperature (2-8°C).It is better to estimate plasma glucose concentration within 6-hour at room temperature (25-30°C) to maintain stability.The plasma sample is recommended to be stored in the refrigerator (2-8°C) if there is any delay in performing the assay.The serum sample is recommended to be analyzed within 1-hour of collection at room temperature (25-30°C) in the estimation of serum total cholesterol concentration, the while serum triglyceride concentration is recommended to be analyzed within 6-hour at the same temperature to obtain accurate and reliable results.

Table 1 :
Experimental design for storing serum and plasma samples at different temperature intervals and time points.

Table 2 :
Concentrations of plasma glucose, serum total cholesterol, and triglycerides at room temperature (25-30°C) at different time points.

Table 3 :
Concentration over various time intervals.It indicates that keeping at refrigerated storage (2-8°C) generally leads to better stability for these biomarkers.However, the study has few limitations.The inclusion of a more diverse range of participants, including different age groups and possibly various health conditions is recommended to enhance generalizability.The stability of samples under specified storage conditions for longer durations should be assessed.Data accuracy should be assured through the implementation of quality control measures, instrument calibration, and test replication.Environmental factors that could impact sample integrity, such as temperature and humidity during transportation should be focused on.The study participants were limited to healthy individuals between 20-50 years of age.This might not be a representative sample of the entire population as it excluded younger and older age groups.Contaminations are potential limitations in any of the laboratory procedures.Commercially available test kits were used for the estimation of glucose, cholesterol, and triglyceride concentrations in the present study.The results might influence by the accuracy and reliability of these kits.The study did not use equipment calibration which was crucial for the accuracy of laboratory measurements.The testing procedure did not use a replicating procedure.
The values are represented as mean ± SD. a p<0.05 vs value at 1-hour.biomarkers