The preanalytical phase is a vital l component of testing process, and most errors (46–68.2%) occur during this stage. Factors like sample collection and handling, patient posture, venous stasis, timing of sample collection, diet, exercise, and medication can affect test outcomes. To make sure of accurate results, both preanalytical and analytical variations must be reduced to levels that do not affect clinical interpretation of the results. The transition between preanalytical and analytical phases is more prone to errors. Time interval between sample collection and serum separation from the clot should be enough for complete clot formation but not more enough to modify the test result due to serum-clot contact. A minimum clotting time of 20–30 minutes is generally recommended. Both bbiological activity of cells and transmembrane diffusion can affect concentrations of specific analytes in serum if there is prolonged contact with the clot. So, serum or plasma be separated after natural clot formationn. Many researchers recommended processing the samples in two hours of collection. Implementing WHO and CLSI guidelines for analyte stability in clinical settings can be challenging. Time duration between blood collection and centrifugation plays main role in the reliability of the results. The serum clot contact time is main optimal interval between sample collection and serum separation from the clot, and serum electrolytes are especially sensitive to this factor.1-3

Sodium is main cation in the extracellular fluid (ECF) that is required for regulating the distribution of water and osmotic pressure in this fluid. It is important for proper nerve conduction and muscle contraction. The impact of hyponatremia and hypernatremia is significant on nervous tissue, where changes in sodium levels can lead to pronounced effects, increased morbidity and mortality.

Potassium is a main intracellular cation. Changes in extracellular potassium levels can cause disturbances in heart rhythm.4,5 Hypokalemia and hyperkalemia are medical emergencies that require immediate management False elevations in these electrolytes, that do not reflect the patient’s actual condition, become more common in laboratory settings. But when specimens are retested and reanalyzed, these values usually normalize. Laboratory services, which constitute only about 5% of a hospital's budget, are important in up to 60–70% of all major clinical decisions, like admissions, discharges, and treatment strategies for patients.6

Delayed processing of samples is caused by factors like heavy sample load, equipment malfunction, or lack of a backup system. As the information on contact time for electrolytes is contradictory this study was done.7

OBJECTIVES

1. To know the effect of clot contact time on serum electrolyte values
2. To know the variation in serum electrolyte levels on analysis within 1hr of sample collection, at 3 hours and at 24 hours.

Study Site

Government General Hospital (GGH), Kurnool.

Study Duration

2 months-January 2025 to February 2025

Source of samples:

900 samples were collected from inpatients (IP) admitted to GGH, Kurnool.

Methodology:

 These samples were analyzed in the 24-hour Clinical Biochemistry Laboratory using the Indirect Ion Selective Electrode (ISE) method on the Sensa core ST Pro 200 Electrolyte Analyzer. Hemolyzed, icteric, and lipemic samples were excluded from the study due to preanalytical errors. The collected samples were stored at 4°C and analyzed at 1, 3, and 24-hour intervals.

Each blood sample was collected into red-topped vacutainer tubes and centrifuged at 3200 rpm for 15 minutes. The serum was then separated for analysis. One set of samples was processed immediately after 30 minutes, and serum was separated for electrolyte analysis. Remaining samples were stored at 30°C for 3 hours after which they were centrifuged, and the serum was separated and analyzed.

The serum electrolytes were measured using ion-selective electrodes (ISE) on the Cobas 6000 integrated analyzer. Sodium was analyzed using the ISE indirect method, while potassium and chloride were analyzed using the ISE direct method.

Inclusion criteria: Normal samples

Exclusion criteria: Lysed samples

Statistical Analysis

Analysis was done using SPSS software 17.0. Paired t-tests were applied to evaluate the values of serum electrolytes at the three different time intervals (1, 3, and 24 hours).

Ethical Aspects

Ethical approval was taken from ethics committee attached to the college.

Informed consent was taken from study patients for collecting their samples and using their information.

MEAN ELECTROLYTE LEVELS AT ALL INTERVALS:

Mean baseline sodium level was 135.60 mg/dL. After 1 hour, sodium levels increased to a mean of 136.60 mg/dL, with an SD  of 3.155 mg/dL. At 6 hours, sodium levels increased to a mean of 137.40 mg/dL, showing a rise in sodium concentration. By 24 hours, sodium levels reached a mean of 145.10 mg/dL, with SD of 3.201 mg/dL, showing a consistent increase over the study period.

Mean baseline potassium level was 37.02 mg/dL

1 hour after sample collection, mean potassium levels increased to 38.10 mg/dL. At 6 hours, mean potassium

Levels increased to39 mg/dL, indicating an increase in concentration. By 24 hours, mean potassium reached 44.9 mg/dL, showing gradual increase over the study period.

The maximum values for both sodium and potassium were seen at 24 hours, with sodium peaking at 150 mg/dL and potassium at 50 mg/dL.

TABLE 1: DESCRIPTIVE STATISTICS

 DIFFERENCES AT VARIOUS TIME INTERVALS:

Sodium Baseline vs. Sodium 1 Hr: The t-value is -42.7 with a p-value of 0.000, showing significant difference between sodium levels at baseline and 1 hour after collection.

Sodium Baseline vs. Sodium 6 Hr: The t-value is -64.5 with a p-value of 0.000, showing significant difference in sodium levels between baseline and 6 hours.

Sodium Baseline vs. Sodium 24 Hr: The t-value is -82.5 with a p-value of 0.000, showing significant difference in sodium levels between baseline and 24 hours.

 Potassium Baseline vs. Potassium 1 Hr: The t-value is -19.9 with a p-value of 0.000, showing significant difference between baseline potassium levels and those at 1 hour.

Potassium Baseline vs. Potassium 6 Hr: The t-value is -37.473 with a p-value of 0.000, showing a significant change in potassium levels between baseline and 6 hours.

Potassium Baseline vs. Potassium 24 Hr: The t-value is -66.246 with a p-value of 0.000, showing significant difference between baseline and 24-hour potassium levels.

TABLE 2: P VALUE AND T VALUES - SHOWING DIFFERENCES

Serum electrolyte testing was commonly done in biochemistry laboratories. Prolonged serum clot contact time can modify the concentration of analytes due to biological activity and transmembrane diffusion.

An increasing trend in sodium and potassium levels was seen with prolonged clot contact time.

A study by Baruah A et al. in New Delhi showed that the stability of sodium, potassium, and chloride is affected by delay in analysis. Sodium and chloride levels increased after 3 hours, and potassium increased within 1 hour. These differences were linked to improper temperature control leading to evaporation from the sample cups. Climatic conditions and uncovered sample cups placed under a fan were responsible for evaporation, which caused falsely increased serum electrolyte values. These findings differ from our study.6

Ono et al.8 studied the effect of serum clot contact time and temperature on various analytes. His study showed changes in sodium and potassium levels when exposed to certain temperature. This implies the need for controlling temperature and storage conditions to ensure accurate results. In our study, we only assessed samples stored at room temperature.

Boyanton et al.9 investigated plasma and serum specimens kept in prolonged contact with cells in uncentrifuged tubes at room temperature (25°C) in the dark. Samples were analyzed at different intervals up to 56 hours. Sodium showed no significant changes, and potassium remained stable for 24 hours.

Donnelly et al.10 analyzed the stability of 25 analytes in serum from healthy donors stored at room temperature, 4°C, and -20°C for varying duration (48 hours, 14 days, and 4 months). The study found that electrolytes (sodium, potassium, chloride) are stable during all temperatures and duration.

In a study by Dash P et al.11 samples left without serum separation at room temperature were subjected to evaporation. The study showed that the difference in potassium concentration was insignificant after 4 hours at room temperature, but after 24 hours, the combined effect of clot contact time and low temperature significantly increased potassium levels.

Kachhawa K et al.12 investigated the stability of 17 biochemical analytes in serum stored at −20°C for 7, 15, and 30 days. They found that most analytes, including sodium and potassium, remained stable under these conditions.

Same findings were reported by Selvkumar et al.,13 where serum sodium levels decreased in lysed samples, and serum potassium levels increased compared to normal samples. Our study focused on normal samples, with lysed samples being excluded from the analysis.

According to our study, both sodium and potassium levels showed an increasing trend over time. The stability of electrolytes is highly sensitive to more contact time with the clot. The stability of sodium and potassium could also be affected by temperature. So, it is important to process the electrolyte samples promptly to reduce preanalytical variations and ensure accuracy of test results.

1. Lee JW. Fluid and electrolyte disturbances in critically ill patients. Electrolyte Blood Press. 2010;8(2):72-81.
2. Burtis CA, Ashwood ER, Bruns DE. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. Philadelphia (PA): WB Saunders; 2006. p. 604-609. 3. Zhang DJ, Elswik RK, Miller WG, Bailey JL. Effect of serum clot contact time on clinical chemistry laboratory results. Am J Clin Chem. 1998;44(6):1325- 33
3. Procedures for the handling and processing of blood specimens; approved guideline. Villanova (PA): NCCLS; 1995. Report No.: H18-A.
4. Dipti Tiwari, Pramod Ingale, Shubhangi Wankhade, Minal Pore. Study of analyte stability time of serum electrolytes in a tertiary care public hospital. MedPulse International Journal of Biochemistry. June 2019; 10(3): 34-37.
5. Zhang, D., Elswick, R., Miller, W., & Bailey, J. (1998). Effect of serum-clot contact time on clinical chemistry laboratory results. Clinical chemistry, 44 6 Pt 1, 1325-33.
6. Baruah A, Goyal P, Simha S, Ramesh KL, Datta RR. Delay in specimen processing major source of preanalytical variation in serum electrolytes
7. Forsman RW. Why is the laboratory an afterthought for managed care organizations?. Clin Chem. 1996;42(5):813-6
8. Ono T, Kitaguchi K, Takehara M, Shiiba M, Hayami K. Stability of biochemical analytes in clinical specimens. Clin Chem. 1981;27(1):35-8
9. Boyanton BL Jr, Blick KE. Stability studies of twentyfour analytes in human plasma and serum. Clin Chem. 2002;48(12):2242-7
10. Donnelly JG, Soldin SJ, Nealon DA, Hicks JM. Stability of twenty-five analytes in human serum at 22°C, 4°C and -20°C. PaediatrPathol Lab Med. 1995;15(6):869-74
11. Dash P, Tiwari R, Nayak S, Mangaraj M. Impact of time delay in the analysis of serum ionized calcium, sodium, and potassium. J Lab Physicians. 2022;14(4):373-6.
12. Kachhawa K, Kachhawa P, Varma M, Behera R, Agrawal D, Kumar S. Study of the stability of various biochemical analytes in samples stored at different predefined storage conditions at an accredited laboratory of India. J Lab Physicians. 2017;9(1):11-5.
13. Selvakumar C, Madhubala V. Effect of sample storage and time delay on analysis of common clinical biochemical parameters. Int J Clin Biochem Res. 2017;4(3):295-8.