Assessment of Biochemical Analytes Stability: The Role of Centrifugation and Storage Conditions in the Preanalytical Phase

ABSTRACT

The preanalytical phase represents a critical component of laboratory testing, encompassing all procedures prior to sample analysis and exerting a substantial influence on the quality and reliability of laboratory results. To evaluate changes in the concentrations of commonly measured biochemical analytes in blood samples subjected to different preanalytical conditions, including centrifugation duration, storage temperature and storage time. Blood samples obtained from 20 individuals (960 measurements) were analysed for glucose, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), triglycerides (TG), potassium (K), sodium (Na) and chloride (Cl). Samples were centrifuged for 5, 10 and 15 min, analysed under optimal conditions (within 2 h of collection), and subsequently reassessed after storage for 2, 4, 8, 10 and 14 days at +23, 4 and −20°C. Statistical analysis was performed using the Kruskal–Wallis test. Storage temperature significantly affected HDL, TG, ALT, AST, glucose, albumin, LDL, and TC (p < 0.001) concentrations, with higher HDL and TG concentrations at +23°C ([1.71(1.12‐2.50)]mmol/l] and [1.82(0.73‐10.19)]mmol/l respectively) and significant AST and ALT concentration decrease at 23°C ([16.0 (4‐60)]U/l and [10.0(2‐95)]U/l respectively). Storage duration influenced TC, HDL, TG, albumin, ALT, and AST concentrations (p < 0.001). Centrifugation time had a significant effect only on serum chloride and glucose concentrations (p = 0.018, p = 0.025 respectively). Most biochemical analytes were significantly influenced by storage temperature and duration, whereas centrifugation time had a comparatively limited effect.

Assessing the impact of different preanalytical factors on biochemical analyte concentrations helps to control emerging risks associated with sample handling. In this study, we provide evidence that the stability of serum biochemical analytes was strongly dependent on both storage temperature and duration. These findings highlight the importance of standardised storage protocols to ensure the reliability of biochemical measurements in delayed or repeat analyses.

1. Introduction

The preanalytical phase is widely recognised as the most error‐prone stage of the total testing process in clinical laboratories, accounting for the majority of laboratory‐related inaccuracies [1, 2]. These errors primarily arise from factors such as inadequate patient preparation, improper sample collection, and suboptimal handling and storage conditions. Recent evidence underscores the importance of implementing targeted strategies and tools to reduce the frequency and impact of preanalytical errors and to improve overall laboratory quality [1].

Despite substantial efforts to standardise this phase—particularly through guidelines developed by the Clinical and Laboratory Standards Institute and initiatives led by the European Federation of Clinical Chemistry and Laboratory Medicine—preanalytical variability remains a persistent challenge [3, 4]. Consequently, further investigation into methods and approaches aimed at minimising the impact of preanalytical factors on sample quality is warranted.

Once collected, blood specimens must be handled and transported under carefully controlled conditions to preserve their integrity. Factors such as temperature regulation and timely processing are essential to ensure the suitability of samples for biochemical testing [5]. In the present study, we focused on the effects of specimen handling and storage conditions prior to the analytical phase. A schematic illustration of the study design is presented in Figure 1.

FIGURE 1.

Schematic representation of the research design (C – centrifugation time in min).

2. Materials and Methods

A literature search was conducted in the NCBI database using predefined keywords related to analyte stability and the preanalytical phase. Full‐text articles published within the last 10 years were included. Of 199 identified records (October–December 2025), 19 studies were selected for final analysis.

The experimental study was performed using serum samples obtained from 20 clinically healthy individuals (without diagnosed chronic, metabolic or oncological conditions) attending the Kaunas City Outpatient Clinic (Lithuania) over a 2‐week period (1 October 2025–15 October 2025). All samples were anonymised to ensure patient confidentiality.

A total of 960 measurements were performed according to the predefined study schedule. The analytical panel included: glucose (hexokinase/G‐6‐PDH method), albumin (bromocresol green method), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (NADH method, without pyridoxal‐5'‐phosphate), total cholesterol (TC) (enzymatic method), high‐density lipoprotein (HDL) cholesterol (accelerator selective detergent method), low‐density lipoprotein (LDL) cholesterol (direct liquid selective detergent method), triglycerides (TG) (glycerol phosphate oxidase method), and electrolytes (potassium [K], sodium [Na] and chloride [Cl]) measured using the indirect ion‐selective electrode method.

Whole blood samples were collected in BD SST II Advance tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and allowed to clot for 30 min. Subsequently, serum samples were centrifuged at 1500 × g for 5, 10 or 15 min using a Digtor 22 ventilated centrifuge (Ortoalresa, Spain).

Analyses were performed on the same samples using an Abbott Alinity ci series analyser (Abbott Laboratories, USA). Measurements were conducted under optimal conditions within 2 h of collection and subsequently after storage for 2, 4, 8, 10 and 14 days according to the study design. Samples were stored at +23°C (thermostatically controlled), 4°C and −20°C under standard laboratory conditions.

No evidence of haemolysis was observed in any of the analysed samples.

Data distribution was assessed using the Shapiro–Wilk test. As the data did not follow a normal distribution, nonparametric statistical methods were applied. The Kruskal–Wallis test was used to evaluate the effects of centrifugation time, storage temperature, and storage duration on analyte stability. Post hoc pairwise comparisons were performed using Bonferroni correction to control for multiple testing. Only Bonferroni‐adjusted p‐values are reported in the results tables. The significance level of p < 0.05 was considered statistically significant. All statistical analyses were conducted using IBM SPSS Statistics (Version 31).

Ethical approval for the study was granted by the Kaunas Regional Biomedical Research Ethics Committee (approval No. BE‐2‐106).

3. Results

The statistical data for each analyte regarding the impact of preanalytical conditions are presented in detail below and in Tables 1, 2, 3.

TABLE 1.

Impact of the storage time on biochemical analyte levels.

		Testing timeline
		Baseline concentration			1 day			4 days			8 days			10 days		14 days
Blood test	Units	Mdn	Min	Max	Mdn	Min	Max	Mdn	Min	Max	Mdn	Min	Max	Mdn	Min	Max	Mdn	Min	Max	p
Albumin	g/L	46	41	54	47	41	55	48	40	60	49	41	58	49	42	60	49	42	78	<0.01
ALT	U/L	25	8	101	23	5	100	19	2	97	16	2	93	15	2	89	16	2	81	<0.01
AST	U/L	24	16	62	23	15	64	23	13	67	22	7	63	20	4	63	20.5	4	64	<0.01
Chloride	mmol/L	103	96	106	105	94	108	106	98	112	106	98	126	108	98	130	109	99	141	<0.01
Glucose	mmol/L	5.1	4.21	6.23	5.11	4.23	6.25	5.2	4.26	6.7	5.21	4.21	6.67	5.25	4.22	6.54	5.21	4.17	7.32	0.076
HDL cholesterol	mmol/L	1.625	1.21	2.01	1.685	1.25	2.25	1.71	1.3	2.45	1.74	1.24	2.5	1.65	1.19	2.47	1.66	1.12	2.37	<0.01
LDL cholesterol	mmol/L	3.905	1.55	6.91	4.035	1.59	6.96	4.07	1.5	7.22	4.04	1.44	7.44	4.04	1.44	7.55	4.06	1.45	7.72	0.966
Potassium	mmol/L	4.31	3.45	5.75	4.345	3.52	5.84	4.48	3.54	6.06	4.57	3.57	6.06	4.52	3.52	6.14	4.64	3.59	6.52	<0.01
Sodium	mmol/L	140	103	144	141	127	148	143	138	158	145	140	169	145	138	175	148	140	189	<0.01
Total cholesterol	mmol/L	6.195	3.36	9.7	6.255	3.39	9.85	6.36	3.44	10.55	6.63	3.5	10.62	6.66	3.48	10.53	6.8	3.49	10.96	<0.01
Triglycerides	mmol/L	1.24	0.62	7.87	1.31	0.68	8.34	1.35	0.71	8.5	1.45	0.71	9.03	1.58	0.69	9.44	1.75	0.71	10.19	<0.01

				p day versus day	↓
Albumin	<0.01 c , d , e , g , h , i ; 0.021 b ; 0.004 f ; 0.027 l ; 0.012 k	Chloride	<0.01 b , c , d , e , g , h , i , k , l , m , n ; 0.004 a ; 0.001 f ; 0.007 j	Sodium	<0.01 b , c , d , e , f , g , h , i , l , m , n ; 0.002 j
ALT	<0.01 c , d , e , g , h , i ; 0.006 k ; 0.015 j	HDL cholesterol	0.011 b ; <0.001 c ; 0.034 m	Total cholesterol	0.048 d ; 0.024 c ; 0.006 e ; 0.016 h ; 0.005 g ; 0.001 i ; 0.027 l
AST	<0.01 h , i ; 0.001 e , l ; 0.011 k ; 0.004 d ; 0.008 g	Potassium	<0.01 e , i ; 0.001 g , l ; 0.018 d ; 0.008 c ; 0.006 h	Triglycerides	<0.01 e , i ; 0.025 c ; 0.012 d ; 0.036 g ; 0.013 h

Mdn – median.

^a 0 versus 1.

^b 0 versus 4.

^c 0 versus 8.

^d 0 versus 10.

^e 0 versus 14.

^f 1 versus 4.

^g 1 versus 8.

^h 1 versus 10.

ⁱ 1 versus 14.

^j 4 versus 8.

^k 4 versus 10.

^l 4 versus 14.

^m 8 versus 14.

ⁿ 10 versus 14.

TABLE 2.

Impact of the storage temperature on biochemical analyte levels.

Temperature (°C)
		Baseline concentration			Room temperature (23°C)			Refrigerated (4°C)			Frozen (−20°C)
Blood test	Units	Median	Min	Max	Median	Min	Max	Median	Min	Max	Median	Min	Max	p	p temperature versus temperature
Albumin	g/L	46	41	54	47	41	78	49	40	58	48	41	60	<0.01	0.021 a ; <0.01 b , c ; 0.038 d ; 0.001 e
ALT	U/L	25	8	101	10	2	95	22	5	98	19	4	100	<0.01	<0.01 a , d , e ; 0.037 b
AST	U/L	24	16	62	16	4	60	23	14	63	24	16	67	<0.01	<0.01 a , d , e
Chloride	mmol/L	103	96	106	106	98	141	106	98	120	107	94	116	<0.01	<0.01 a , b , c
Glucose	mmol/L	5.1	4.21	6.23	5.08	4.17	7.32	5.25	4.23	6.67	5.21	4.24	6.7	<0.01	0.008 d ; <0.01 e
HDL cholesterol	mmol/L	1.625	1.21	2.01	1.71	1.12	2.5	1.68	1.25	2.17	1.67	1.27	2.28	<0.01	0.026 c ; <0.01 a ; 0.015 d
LDL cholesterol	mmol/L	3.905	1.55	6.91	3.81	1.44	6.87	4.185	1.62	7.72	4.12	1.6	7.44	<0.01	<0.01 d , e
Potassium	mmol/L	4.31	3.45	5.75	4.51	3.52	6.52	4.52	3.54	6.3	4.48	3.52	6.06	0.08	0.016 b ; 0.008 a ; 0.005 c
Sodium	mmol/L	140	103	144	144	138	189	145	138	167	144	127	161	<0.01	<0.01 a , b , c
Total cholesterol	mmol/L	6.195	3.36	9.7	6.56	3.43	10.96	6.61	3.4	10.74	6.48	3.39	10.62	0.059	0.047 c
Triglycerides	mmol/L	1.24	0.62	7.87	1.82	0.73	10.19	1.36	0.68	8.64	1.33	0.68	8.5	<0.01	<0.01 a , d , e

^a 1 versus 23.

^b 1 versus 20.

^c 1 versus 4.

^d 23 versus 20.

^e 23 versus 4.

^f 20 versus 4.

TABLE 3.

Impact of the centrifugation mode on biochemical analyte levels.

		Centrifugation 5 min			Centrifugation 10 min			Centrifugation 15 min
Blood test	Units	Median	Min	Max	Median	Min	Max	Median	Min	Max	p	p minutes versus minutes
Albumin	g/L	48	40	64	48	41	58	48	41	78	0.991
ALT	U/L	18	2	97	18	2	98	19	2	100	0.888
AST	U/L	22	4	67	22	4	62	22	4	63	0.971
Chloride	mmol/L	107	98	141	106	94	119	106	98	130	0.018	0.020 a
Glucose	mmol/L	5.24	4.27	6.71	5.185	4.29	6.49	5.15	4.17	7.32	0.025	0.027 a
HDL cholesterol	mmol/L	1.69	1.12	2.46	1.695	1.17	2.42	1.69	1.19	2.5	0.972
LDL cholesterol	mmol/L	4.06	1.44	7.66	4.03	1.44	7.72	4.06	1.45	7.3	0.971
Potassium	mmol/L	4.46	3.52	5.76	4.565	3.55	6.07	4.51	3.52	6.52	0.068
Sodium	mmol/L	145	133	189	144	127	162	144	132	175	0.214
Total cholesterol	mmol/L	6.56	3.43	10.69	6.545	3.39	10.74	6.51	3.47	10.96	0.941
Triglycerides	mmol/L	1.45	0.69	9.65	1.43	0.69	10.19	1.42	0.68	9.42	0.970

^a 15 versus 5.

3.1. Lipid Profile

Storage temperature did not significantly affect TC concentrations across measurements. However, storage duration had a significant impact (p < 0.001), with higher TC levels observed in samples stored for more than 8 days compared with baseline values measured within 2 h after blood collection (Figure 3).

FIGURE 3.

Alterations in biochemical analyte concentrations in response to different sample storage conditions and at predetermined measurement intervals.

HDL concentrations were significantly influenced by storage temperature (p < 0.001). Samples stored at room temperature (+23°C) and under refrigerated conditions (+4°C) showed higher HDL concentrations (p < 0.001: 1.71 [1.12–2.50] mmol/L and p = 0.026: 1.68 [1.25–2.17] mmol/L, respectively) compared with baseline values (1.62 [1.21–2.01] mmol/L). In addition, HDL levels were significantly lower in frozen samples (−20°C) compared with those stored at room temperature (p = 0.015).

Storage duration also significantly affected HDL concentrations (p < 0.001), with a statistically significant increase observed on day 4 compared with baseline values (p = 0.011; 1.71 [1.30–2.45] vs. 1.62 [1.21–2.01] mmol/L) (Figure 3).

LDL concentrations in samples stored at room temperature (3.81 [1.44–6.87] mmol/L) were significantly lower than those stored under refrigerated (+4°C; 4.18 [1.62–7.72] mmol/L) or frozen conditions (−20°C; 4.12 [1.60–7.44] mmol/L) (p < 0.001 for both comparisons). Storage duration did not significantly affect LDL concentrations over the 14‐day observation period (p = 0.96).

TG concentrations were significantly affected by storage temperature (p < 0.001). Samples stored at +23°C showed higher TG levels (1.82 [0.73–10.19] mmol/L) compared with baseline (1.24 [0.62–7.87] mmol/L) and samples stored at +4°C (1.36 [0.67–8.64] mmol/L) or −20°C (1.33 [0.68–8.50] mmol/L) (p < 0.001).

Storage duration also significantly influenced TG concentrations (p < 0.001), with a significant increase observed on day 8 compared with baseline values (1.45 [0.71–9.03] vs. 1.24 [0.62–7.87] mmol/L).

Centrifugation time did not significantly affect any lipid profile parameter. None of the centrifugation modes had a significant effect on any lipid profile analyte.

3.2. Glucose

Storage temperature had a significant effect on serum glucose concentrations (p < 0.001). Lower glucose levels were observed in samples stored at +23°C (5.08 [4.17–7.30] mmol/L) compared with those stored at +4°C (5.25 [4.23–6.67] mmol/L, p < 0.001) or −20°C (5.21 [4.24–6.70] mmol/L, p = 0.008). No significant difference was observed between refrigerated and frozen conditions (p > 0.05).

Centrifugation time also had a statistically significant effect on glucose concentrations (p = 0.025). The highest glucose levels were observed after 5 min of centrifugation (5.24 [4.27–6.71] mmol/L), with slightly lower values after 10 min (5.19 [4.29–6.49] mmol/L) and 15 min (5.15 [4.17–7.32] mmol/L). A significant difference was observed between 5‐ and 15‐min centrifugation durations (p = 0.027), whereas other comparisons were not statistically significant.

No significant changes in glucose concentrations were observed over the storage period (p = 0.076) (Figure 3).

3.3. Albumin

Serum albumin concentrations varied significantly across storage temperatures (p < 0.001). Higher albumin levels were observed in samples stored at +4°C (49.0 [40–58] g/L), +23°C (47.0 [41–78] g/L) and −20°C (48.0 [41–60] g/L) compared with samples analysed within 2 h after collection (46.0 [41–54] g/L).

Albumin concentrations were significantly lower in samples stored at room temperature compared with those stored at +4°C (p = 0.01) and −20°C (p = 0.038).

Storage duration also had a significant effect on albumin levels (p < 0.001). An increase was first observed on day 4 (48.0 [40–60] g/L, p = 0.021), reaching 49.0 [41–58] g/L by day 8 (p < 0.001). No further significant changes were observed between days 8, 10 and 14 (p > 0.05), indicating stabilisation after the first week.

3.4. AST and ALT

Both AST and ALT concentrations were significantly affected by storage temperature (p < 0.001). AST levels remained comparable to baseline (24.0 [16–62] U/L) when stored at +4°C (23.0 [14–63] U/L) and −20°C (24.0 [16–67] U/L), whereas storage at +23°C resulted in a marked decrease (16.0 [4–60] U/L; p < 0.001 vs. all other groups).

ALT concentrations showed a similar but more pronounced pattern, decreasing from 23.0 (8–101) U/L at baseline to 22.0 (5–98) U/L at +4°C, 19.0 (4‐100) U/L at −20°C, and 10.0 (2–95) U/L at +23°C. Significant differences were observed between room temperature and all other conditions (p < 0.001), as well as between −20°C and baseline samples (p = 0.037).

Storage duration significantly affected transaminase activity (p < 0.001). ALT levels declined notably by day 8 (from 25.0 [8–101] to 16.0 [2–93] U/L, p < 0.001) and remained stable thereafter. AST levels decreased more gradually, with a significant reduction observed by day 10 (from 24.0 [16–62] to 20.0 [4–63] U/L, p < 0.001), with no further changes thereafter. These findings indicate that ALT degradation occurs earlier than AST.

3.5. K, Na and Cl

K, Na and Cl concentrations were significantly affected by storage temperature (p < 0.001). K levels differed significantly between samples analysed immediately after collection and those stored at +4°C (4.52 [3.54‐6.30] mmol/L), −20°C (4.48 [3.52–6.06] mmol/L) and +23°C (4.51 [3.52–6.52] mmol/L) (p = 0.005, p = 0.016 and p = 0.008, respectively).

Na concentrations also varied significantly across storage conditions (p < 0.001), with higher values observed in samples stored at +4°C (145 [138–167] mmol/L), +23°C (144 [138–189] mmol/L) and −20°C (144 [127–161] mmol/L) compared with those analysed immediately after collection.

Similarly, Cl concentrations were significantly elevated in all stored samples compared with baseline values (p < 0.001), with median values of 106 (98–120) mmol/L at +4°C, 106 (98–141) mmol/L at +23°C and 107 (98–141) mmol/L at −20°C. No statistically significant differences were observed between the storage temperature groups (p > 0.05).

Centrifugation time had a modest but statistically significant effect on serum Cl concentrations (p = 0.018). Cl levels were highest after 5 min of centrifugation (107.0 [98–141] mmol/L) and slightly lower after 15 min (106.0 [98–130] mmol/L). Pairwise comparisons revealed a significant difference only between the 5‐ and 15‐min centrifugation durations (p = 0.020), whereas other comparisons were not statistically significant (p > 0.05).

Storage duration significantly influenced electrolyte concentrations (p < 0.001). K levels began to increase from day 8, rising from 4.31 (3.45–5.75) mmol/L in samples analysed within 2 h after collection to 4.57 (3.57–6.06) mmol/L (p < 0.001), and remained elevated thereafter. Na concentrations increased significantly from day 4, rising from 140 (103–144) mmol/L at baseline to 143 (138–158) mmol/L (p < 0.001), and subsequently stabilised.

In contrast, Cl concentrations showed a gradual and continuous increase throughout the storage period, rising from 103 (96–106) mmol/L immediately after collection to 105 (94–108) mmol/L by day 1 (p < 0.01), and continuing to increase progressively over subsequent days.

These findings indicate that K and Na exhibit relatively early changes during storage, whereas Cl demonstrates a progressive time‐dependent increase. Collectively, the results underline the importance of appropriate storage conditions and timely analysis for maintaining the reliability of electrolyte measurements.

The graphic representation of data below provides a detailed depiction of the dynamics of the pre‐analytical phase effects (see Figures 2 and 3).

FIGURE 2.

Alterations in biochemical analyte concentrations in response to different centrifugation conditions and measurement time intervals.

4. Discussion

This study evaluated the effects of centrifugation time, storage temperature, and storage duration on the stability of key biochemical analytes—glucose, albumin, ALT, AST, TC, HDL, LDL, TG, K, Na and Cl—in serum samples. The findings demonstrate that storage temperature and duration are the primary determinants of analyte stability, whereas centrifugation time at 1500 × g exerts only a minor influence. Overall, the observed temperature‐ and time‐dependent changes are consistent with previously reported data on analyte stability [6, 7, 8, 9].

Storage temperature had a significant impact on most analytes. Glucose concentrations varied depending on storage conditions but remained relatively stable over the 2‐week observation period. The decrease observed in samples stored at room temperature can be attributed to ongoing glycolysis in residual blood cells, a well‐established preanalytical phenomenon [10]. In contrast, refrigeration and freezing effectively inhibited this process. The absence of significant differences between samples stored at 4 and −20°C suggests that refrigeration alone is adequate to maintain glucose stability, provided that prompt serum separation from the clot is ensured.

Centrifugation time had a statistically significant, albeit small, effect on glucose concentrations, with slightly higher values observed after shorter centrifugation periods. This may reflect incomplete separation of cellular components, allowing continued metabolic activity. Although the magnitude of this effect was limited, these findings underscore the importance of standardised centrifugation protocols to minimise preanalytical variability [11].

Albumin concentrations were slightly higher in stored samples compared with freshly analysed specimens. Although these differences were statistically significant, the magnitude of variation was small and unlikely to be clinically relevant, supporting the well‐recognised stability of albumin under a wide range of storage conditions [12]. The observed increases may reflect preanalytical or analytical artefacts, such as evaporation, concentration effects, or assay‐related variability [13].

Both AST and ALT exhibited pronounced sensitivity to storage temperature and duration. Storage at room temperature resulted in a marked decline in enzyme activity, while refrigeration and freezing largely preserved baseline levels. These findings are consistent with the known instability of aminotransferases under suboptimal storage conditions [14, 15]. The more pronounced decrease observed for ALT compared with AST suggests greater susceptibility of ALT to denaturation or conformational changes during storage.

The lipid profile demonstrated both expected and divergent patterns. TG and TC increased with storage time, particularly under room temperature conditions, consistent with previously reported findings [16]. In contrast, HDL and LDL concentrations decreased under similar conditions. These discrepancies may be explained by structural alterations in lipoprotein particles, oxidative processes or assay‐specific sensitivity to changes in lipid composition [17, 18].

Electrolyte concentrations (K, Na and Cl) increased in stored samples compared with baseline measurements. As serum separation was performed within 2 h, these changes are unlikely to reflect active cellular metabolism. Instead, they are more plausibly attributed to passive ion leakage from residual cellular material and minor evaporation effects. Storage temperature had a limited influence on these changes, whereas storage duration emerged as the main determinant. K increased after the first week, Na increased within the first few days, and Cl gradually throughout the entire storage period. These results indicate that electrolytes remain relatively stable in the short term but become progressively altered during prolonged storage, even under controlled conditions [19].

Centrifugation time had only a minor impact overall, affecting Cl concentrations slightly. This effect likely reflects small variations in the efficiency of serum separation rather than true biochemical changes.

Taken together, these findings highlight the critical importance of controlling preanalytical conditions, particularly storage temperature and duration, to ensure reliable measurement of biochemical analytes. While many analytes remain relatively stable under optimal handling conditions, prolonged storage and ambient temperatures may introduce systematic changes that compromise analytical accuracy, especially for enzymatic and lipid parameters.

5. Conclusion

The stability of serum biochemical analytes is strongly influenced by both storage temperature and duration. Lipid parameters and liver enzymes—particularly HDL, TG, ALT and AST—exhibited the greatest variability, whereas glucose and K remained comparatively stable under controlled conditions.

Among the 11 analytes evaluated, nine (81.8%) demonstrated significant time‐dependent changes, 10 (90.9%) were affected by storage temperature, and only two (18.2%) were influenced by centrifugation conditions. These findings underscore the dominant role of storage conditions in preserving sample integrity.

Overall, strict standardisation of preanalytical procedures—particularly with regard to temperature control and timely analysis—is essential to ensure the accuracy and reliability of biochemical measurements, especially in settings involving delayed or repeated testing.

Author Contributions

Zivile Jacike: conceptualisation, investigation, writing – original draft, writing – review and editing, data curation, supervision and project administration. Rugile Skripkauske: writing – review and editing, formal analysis and investigation. Zivile Sauciuniene: investigation, data curation, formal analysis and writing – review and editing. Ema Rajackaite: writing – review and editing, visualisation and data curation. Paulius Kibisa: funding acquisition and resources. Vaiva Lesauskaitė: writing – review and editing. Ingrida Grabauskyte: software, formal analysis and data curation. Gajdová Marie: methodology. Ramune Sepetiene: writing – review and editing.

Ethics Statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.