Over the past decade, clinical laboratory medicine has undergone a marked transformation driven by automation and advances in analytical technology, leading to a substantial reduction in analytical-phase errors and a corresponding improvement in the reliability of laboratory data [1]. However, this progress has shifted attention toward the pre-analytical phase, now recognized as the most vulnerable step in the total testing process. Pre-analytical variables, including specimen collection, handling, transport, and processing, remain insufficiently standardized in many settings and continue to exert a profound influence on the validity of laboratory results, thereby directly affecting clinical decision-making [1].

Among routine biochemical investigations, serum electrolytes (sodium, potassium, and chloride) and glucose occupy a central role in both acute and chronic patient management. These analytes guide critical interventions such as the correction of dyskalemias and the management of dysglycaemia, making their accuracy non-negotiable for patient safety [1]. Yet, the pre-analytical phase contributes disproportionately to laboratory errors, accounting for approximately 40% - 70% of all total testing process inaccuracies, with hemolysis identified as the most frequent and clinically significant source of interference [2]. Hemolysis, defined as the rupture of erythrocytes with subsequent release of intracellular contents into plasma or serum, commonly arises from preventable procedural and mechanical factors during blood collection and processing [3].

The clinical relevance of hemolysis is particularly evident in potassium measurement, where even minimal erythrocyte lysis can cause substantial false elevations due to the markedly higher intracellular potassium concentration relative to plasma. This phenomenon frequently results in pseudohyperkalaemia, which may lead to unnecessary and potentially harmful interventions [4]. While sodium and chloride are generally less sensitive to hemolytic interference, their measured concentrations may still be affected depending on analytical methodology, particularly the use of indirect ion-selective electrodes and variations in plasma composition [5]. Glucose measurement also demonstrates variable susceptibility, with hemolysis-induced release of intracellular enzymes and assay-dependent factors producing device-specific analytical bias in some systems [6].

To mitigate these challenges, laboratories increasingly rely on automated hemolysis indices (H-index) as objective tools for detecting and quantifying hemolysis. These indices, endorsed by international bodies such as IFCC and CLSI, correlate with free hemoglobin concentration and support standardized decision-making regarding sample rejection or result validation [7]. Despite these advances, high hemolysis rates persist in routine practice, particularly in high-throughput clinical environments, leading to repeated sampling, diagnostic delays, increased healthcare costs, and potential patient harm [8]. Importantly, variability in phlebotomy practices, training levels, and infrastructure across settings, especially in low-resource environments, continues to exacerbate the burden of pre-analytical errors [9].

In Cameroon, despite the implementation of national laboratory quality policies and alignment with WHO-AFRO SLIPTA/SLMTA frameworks, hemolysis remains a persistent challenge due to inconsistent adherence to standard operating procedures, limited continuous training, and insufficient infrastructure support [6][7]. Moreover, there is a critical lack of institution-specific experimental data quantifying the magnitude of hemolysis-induced bias on key biochemical analytes using locally available laboratory systems. This gap is particularly evident in Bamenda, where no experimental studies have systematically evaluated the effect of hemolysis on sodium, potassium, chloride, and glucose within teaching and diagnostic laboratories. Therefore, this study, conducted at the Florence Nightingale Higher Institute of Health and Biomedical Sciences, aims to generate locally relevant evidence on hemolysis-related analytical bias, with the goal of strengthening laboratory quality systems, refining sample rejection criteria, and improving both training and patient safety outcomes [10].

This experimental laboratory-based study was conducted in Bamenda III Subdivision, Mezam Division, North West Region of Cameroon, in the Florence Nightingale Higher Institute of Health and Biomedical Sciences (FLENHIHBS) laboratory, where all sample collection, processing, and biochemical analyses were performed from January 15 to July 13, 2026. The study involved 21 venous blood samples collected from consenting FLENHIHBS students selected through convenience sampling, with inclusion criteria restricting participation to healthy individuals without hemolytic disorders, G6PD deficiency, or active malaria infection, and exclusion applied to those who declined consent. Eligibility was verified through a brief health assessment and participant self-reporting, complemented by review of available medical history, with malaria status confirmed by absence of current clinical symptoms and no evidence of active infection at the time of sample collection. Participants with a known history of hemolytic disorders or G6PD deficiency were excluded based on their reported medical history. Each blood sample was divided into four aliquots comprising one control sample and three test samples (T1, T2, and T3). The control aliquots were processed under optimal pre-analytical conditions, while the test aliquots were subjected to controlled hemolysis-inducing factors such as forcing blood into tubes through the needle (T1), vigorous shaking (T2), and excessive centrifugation (T3). The degree of haemolysis was assessed visually after centrifugation by comparing the colour intensity of the serum with a standardized visual haemolysis scale. Samples were graded according to the extent of visible pink to red discoloration of the serum, where 1+ represented mild haemolysis (faint pink discoloration), 3+ represented moderate haemolysis (distinct red discoloration), and 4+ represented severe haemolysis (deep red serum indicating marked red blood cell lysis). Visual grading was performed consistently by the same investigator under uniform lighting conditions to minimize observer variability. Serum was obtained by clotting and centrifugation at 2500 rpm for 5 minutes, while glucose was analyzed from fluoride oxalate samples. For glucose analysis, blood was collected separately into fluoride oxalate tubes to obtain fluoride oxalate plasma, which was used directly for glucose determination using an enzymatic electrochemical glucometer method (OneTouch Ultra 2). Serum sodium, potassium, and chloride were measured using standard spectrophotometric methods (magnesium uranyl acetate for sodium, sodium tetraphenylboron for potassium, and mercuric thiocyanate for chloride), with strict quality control measures including correct specimen handling, appropriate tube selection, timely processing, prevention of pre-analytical errors in controls, and adherence to storage protocols. Data was collected using structured questionnaires and laboratory result sheets, then analyzed using SPSS version 21, with descriptive statistics summarizing variables. Since each participant contributed measurements under all four experimental conditions (control, T1, T2, and T3), inferential analyses accounted for the repeated-measures study design. Normality of the data was assessed prior to hypothesis testing. Differences between control and individual test conditions were evaluated using paired-samples t-tests, while overall differences across the four related conditions were assessed using repeated-measures analysis of variance (repeated-measures ANOVA), followed by appropriate post hoc pairwise comparisons where significant differences were observed. Statistical significance was set at p < 0.05. Ethical approval was obtained from the North West Regional Delegation of Public Health, and written informed consent was secured from all participants, ensuring voluntary participation, confidentiality, and anonymity throughout the study.

The present study demonstrated that the degree of hemolysis varied according to the type of pre-analytical error induced, with vigorous shaking producing the most severe hemolysis (4+), followed by forcing blood through a needle (3+), while excessive centrifugation speed resulted in only mild hemolysis (1+). These findings confirm that mechanical trauma during specimen collection and handling is a major contributor to invitro hemolysis. Vigorous agitation and the application of excessive pressure during blood transfer are known to disrupt erythrocyte membranes, causing leakage of intracellular constituents into serum. Previous studies have similarly identified inappropriate specimen handling, syringe-to-tube transfer, prolonged or forceful aspiration, and improper mixing as leading causes of hemolysis in clinical laboratories, particularly where phlebotomy practices are not fully standardized [2][3]. The findings further support reports that most laboratory errors occur during the pre-analytical phase and are largely preventable through strict adherence to standard operating procedures, proper staff training, and continuous quality improvement programs [1][7][9][11].

The study showed that potassium concentration increased significantly with increasing severity of hemolysis (F = 13.90, p < 0.001), with the highest mean value observed in vigorously shaken samples (5.84 ± 1.05 mmol/L), followed by samples forced through a needle (5.10 ± 0.92 mmol/L), whereas excessively centrifuged samples were comparable to the control group. This observation is consistent with the well-established physiological distribution of potassium, where erythrocytes contain potassium concentrations many times higher than plasma. Consequently, rupture of red blood cells releases intracellular potassium into the extracellular compartment, producing pseudohyperkalaemia rather than reflecting the patient’s true physiological status [4]. Similar findings have been reported in several experimental and clinical studies, which consistently demonstrate that potassium is the electrolyte most susceptible to hemolysis-induced analytical interference and that the magnitude of false elevation correlates with the severity of hemolysis [11]-[13]. These findings reinforce current laboratory recommendations that visibly hemolyzed specimens should be rejected or interpreted with caution when potassium measurement is requested to prevent inappropriate clinical management [7][12].

Sodium concentrations remained remarkably stable across all experimental conditions, with no statistically significant difference between the control and hemolyzed samples (F = 0.11, p = 0.953). The minimal variation observed suggests that sodium measurement is relatively resistant to interference from mechanical hemolysis under the conditions employed in this study. This finding agrees with previous reports indicating that because intracellular sodium concentration is lower than extracellular concentration, erythrocyte rupture contributes very little additional sodium to serum, resulting in negligible analytical bias [5]. Furthermore, several investigations have shown that although severe hemolysis may occasionally produce minor analytical variation depending on the assay principle, particularly with indirect ion-selective electrode methods, such changes are generally not clinically significant [5][11][14]. The present findings therefore support the view that sodium estimation remains reliable despite mild to moderate hemolysis.

Chloride concentrations did not differ significantly among the study groups (F = 0.28, p = 0.840), indicating that chloride measurement is largely unaffected by the degree of hemolysis induced in this experiment. Although slight fluctuations in chloride values were observed, these remained within expected biological variation and were not statistically or clinically meaningful. These findings are consistent with previous studies demonstrating that chloride is relatively insensitive to hemolytic interference because the intracellular and extracellular chloride concentrations are comparatively similar, thereby limiting the impact of erythrocyte rupture on measured serum levels [5][11][15]. Collectively, the results indicate that while hemolysis produces a pronounced and clinically important effect on potassium measurement, its influence on sodium and chloride is minimal. This emphasizes the importance of recognizing hemolysis as a critical pre-analytical variable, particularly when interpreting potassium results, while sodium and chloride measurements can generally be interpreted with greater confidence in mildly hemolyzed specimens [7][12].

The present study found no statistically significant effect of hemolysis on plasma glucose concentration (F = 0.39, p = 0.76), despite minor fluctuations observed among the different hemolysis-induced groups. These findings suggest that the pre-analytical conditions evaluated in this study did not produce sufficient analytical interference to alter glucose measurements significantly. Similar observations have been reported in previous studies, where mechanical hemolysis had little or no clinically significant effect on plasma glucose when samples were processed promptly after collection [6][16]. However, other investigators have noted that prolonged delays in sample processing or severe hemolysis may reduce glucose concentrations due to continued glycolysis and the release of intracellular enzymes, making the extent of interference dependent on specimen handling, storage conditions, and the analytical method employed [6][17]-[19]. Therefore, although glucose measurement appeared relatively stable under the present experimental conditions, adherence to recommended specimen collection and prompt processing remains essential to ensure accurate glucose estimation [2][7].

This study demonstrated that the severity of hemolysis depends on the type of pre-analytical error, with vigorous shaking causing the greatest degree of hemolysis, followed by forcing blood through a needle, while excessive centrifugation produced only minimal hemolysis. Hemolysis had a significant effect on serum potassium concentration, resulting in falsely elevated values, whereas sodium, chloride, and glucose concentrations were not significantly affected under the experimental conditions.

The study was limited by its relatively small sample size and inclusion of participants from a single institution, which may limit the generalizability of the findings. In addition, only three experimentally induced pre-analytical causes of hemolysis were investigated, while other common sources of hemolysis encountered in routine clinical practice were not evaluated.