Serum and other blood products must frequently be stored in laboratories due to technical malfunctions or to preserve samples for use in future studies. When serum samples are stored, information on the measured concentration of serum analytes is frequently conflicting and lacking. This study is sought to ascertain if storage conditions in sera from people who appeared to be in good health had an impact on the stability of biochemical analytes. Laboratory testing consists of three stages: pre-analytical, analytical, and post-analytical. While the majority of errors occur during the pre-analytical phase (46–68.2%) and the post-analytical phase (18.5–47%), a sizable portion (4–32%) occurs during the intra-analytical phase of the testing process.1 Numerous preanalytical factors such as specimen storage duration and temperature, can be tracked and managed which lowers error magnitude and boosts test accuracy.2 The analyte stability times provided are frequently incompatible with the time required to transfer blood samples from the site of collection to the laboratory, making it challenging to implement some of the guidelines from the WHO 3 and CLSI 4 in everyday practice. Because of this, there is usually a delay before plasma separates from red blood cells which might alter the stability of analytes WHO. For instance, WHO indicates that the stability of potassium and phosphate in whole blood is less than one hour. 5 Bias is primarily an analytical characteristic in which reported results differ from the actual value. Imprecision or lack of reproducibility is due to both physiological and analytical factors.6 the conditions under which a blood sample is stored can alter its physical and biological characteristics. These changes in the sample are referred to as storage lesions. The most common cause of storage lesions is hemolysis which can affect a sample in a number of ways. Hemolysis can impact a blood sample by a number of different ways including haemodilution, the direct impact of haemoglobin concentration on the specific analyte concentration and erythrocyte rupture and release of intracellular contents into the serum.7 For most routine assays in a clinical laboratory serum is the preferred sample.6 Maintaining analyte stability in blood samples is a typical issue both throughout sample transportation from clinical departments to the central laboratory and in the event that the analysis is delayed after centrifugation. A sample is considered stable when held under established conditions, maintaining its original analyte characteristic within predefined ranges for a given amount of time. Keeping serum analytes stable during sample storage is a frequent issue in clinical laboratories.

Samples are typically kept in a deep freezer (−20°C) for extended periods of time or in the refrigerator door (4–8°C). As a result in the clinical biochemistry laboratory context sample storage temperature is a crucial preanalytical variable that could influence analysis outcomes.8 Blood collection and serum separation times need to be regulated in order to get test results that are both clinically relevant and credible. Numerous researchers have examined similar alterations in some analytes, but their findings are debatable.9

Therefore using the previously described standard guidelines for blood sample handling and separation the present study investigated the stability of 10 routine chemistry analytes in immediately cell- separated serum after storage at a designated temperature (−20°C) for different periods (0, 1, 3, 7, 14, 21, and 30 days).

A cross-sectional study was undertaken in the Department of Biochemistry, Government Medical College, Miraj, Maharashtra, India. 40 Healthy persons (age group between 25 – 40 years of either gender) visiting master health checkup OPD in the hospital were selected for the study after getting proper informed consent. The s

Study design: cross-sectional study

Place of study: Hospital based study-Tertiary care hospital Duration of study: 2 months

Sampling method and sample size:

1) 6ml of blood sample was drawn from antecubital vein with the standard aseptic precautions and measurement of serum Glucose, Total protein, Sodium, Potassium, Urea, Creatinine, Chloride, AST, ALT, ALP from the subjects during the study period were included as per inclusion criteria.

Sample size: Duration based

Inclusion criteria:

1. Healthy volunteers who were ready to give consent after proper discussion and explanation.
2. Subjects among age group between 25 – 40 years of either gender who were free from medications for 6 months were included in the study.

Detailed procedure of the study conduct:

About 6ml of fasting venous blood was collected from 40 volunteers. Among them there were 17 females and 23 males. Blood was collected in sterile tubes and the samples were allowed to clot for 30 minutes at room temperature followed by centrifugation at 2000 rpm for 10 minutes. Samples with visible hemolysis were excluded from the study. Serum was separated as early as possible within two hours from sample collection and made into six aliquots and analyzed differently using XL640 autoanalyzer and Electrolyte analyzer.

1. Aliquot: The separated serum is analyzed at zero hour at room temperature (20–25˚ C) and they act as a baseline value.
2. Aliquot: Serum is stored in a refrigerator at -20° C and analyzed after 24hours. Study was approved by the institutional ethical committee (IEC) of the institute.
3. Aliquot: Serum is stored in a refrigerator at -20° C and analyzed on 3rd day.
4. Aliquot: Serum is stored in a refrigerator at -20° C analyzed on 7th day.
5. Aliquot: Serum is stored in a refrigerator at -20° C analyzed on 14th day.
6. Aliquot: Serum is stored in a refrigerator at -20° C analyzed on 21st day.
7. Aliquot: Serum is stored in a refrigerator at -20° C analyzed on 30th day.

Data were analyzed using standard statistical methods and the results were expressed in percentage. Ten different biochemical parameters were analysed using the following methods.

Table 1: Analytes performed with method name.

Statistical analysis: Statistically significant changes were determined for each analyte by repeated‑measures ANOVA. Data analysis was conducted using Microsoft Excel and SPSS software.

In present study significant changes as compared with the initial time values were noticed for Glucose, AST, ALT, Creatinine and Potassium levels. No statistically significant changes were noted in the levels of analytes till 48 hours when the samples are stored at –20°C, but eloquent changes were noticed after 72 hours in some parameters.

Table 2: Assay values in pooled serum immediately after collection at 0 hour, 24 hours, 3rd, 7th, 14th, 21st and 30th day

There was significant decrease in Glucose concentration (-18.3%) (p<0.001) on 3rd day which got further decreased by 28.9% on 30th day of storage. According to Jandl, serum glucose concentration decreases with time, with resultant increase in lactate concentration.10 Similarly decreased trend in glucose after storage were noted by Tanner et all.11 Utilization by glycolysis might lead to decrease in Glucose as well glucose is required for cellular metabolism and the rate at which glucose is depleted is dependent on temperature and time. The metabolic rate increases with increased temperature and glucose is depleted quickly, whereas at lower temperatures it decreases more slowly.12

Table 5: ALT levels in pooled serum immediately after collection at 0 hour, 24 hours, 3rd, 7th, 14th, 21st and 30th day.

 Significant increase was observed in AST (22%) (p<0.001) by the end of 3rd day which got further increased by 27.9% by the end of 30th day, as well ALT showed significant increase by (10.8%) (p<0.001) by the end of 3rd day which got further increased by 66.1% by the end of 30th day. As storage has the effect of increasing the activities of enzymes apparently, storage of specimens might lead to increased serum concentration of ALT and AST. 13 Increased lactate concentration after 72 hours may also have interfered with the methodology

Significant increase was noted, in the levels of Creatinine (71.6%) (p<0.001) by the end of 3rd day which got further increased by 185.1% by the end of 30th day. It was found that the increase in creatinine concentration during storage could be due to non-specific formation of pseudocreatinine with kinetic Jaffe’s reaction.

Significant rise was found in Potassium (52%) (p<0.001) by the end of 3rd day after which it peaked by 251.8% on day 30. The percentage increase in potassium is greatest after storage because the lower temperature induced inhibition of Na+, K+ ATPase pump that leads to increased release of potassium from cells. After 6 h of serum-clot contact at room temperature the change became clinically significant which is in agreement with earlier researches by Oddoze C, Laessig RH, Zhang DJ.5, 15, 16 Adias et al also observed hyperkalemia in their study but they did not find any significant change in Na+ which goes in line with the present study.17 Whereas no significant variations were noted in sodium, Total protein, ALP, Urea and Chloride levels. There is disagreement on the best type of specimen to use for analyzing many biochemistry analytes.

In conclusion, this study will help to determine which analyte should be assayed till what time period, when prolonged storage occurs inadvertently or unavoidable. Furthermore, the parameters should be assayed as soon as the sample is received to get valid laboratory results and to prevent the misinterpretation of results.

Findings from this study assisted us in identifying the analytes that, in spite of exposure to varying storage conditions yield reliable results. Therefore, with this information it is said that accuracy and precision of the diagnostic techniques can be enhanced.

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