Introduction: Monitoring CD3+, CD4+ and CD8+ T lymphocytes count is used in patients with known HIV infection, to determine efficacy of antiretroviral medication. Sometimes, due to the long distance, more time is needed for the sample to reach a more equipped laboratory. The aim of our study was to evaluate the impact of prolonged pre-analytical storage of blood at temperature, 96 h at room temperature, on the quality of results for the three parameters. Methods: The analysis of 60 EDTA-anticoagulated blood samples, stored at room temperature (15-25 degrees C) after sampling, was conducted after 24 h and 96 h, respectively. The BD FACSLyric (TM) system was used to identify and enumerate CD3+, CD4+, and CD8+ T lymphocytes. Results: Following a 96-hour period, no notable discrepancies were observed in the data for CD3+, CD4+, and CD8+ T lymphocytes. Passing-Bablok regression analysis showed no significant difference in y-intercept and slope. The Pearson correlation coefficient (r) demonstrated a significant and strong correlation with rho values of 0.994, 0.992, and 0.996, respectively. The analytical agreements demonstrated that all results fell within the total allowable margin of total error. Conclusion: The results of this study demonstrated that diagnostic samples, monitored for CD3+, CD4+ and CD8+ T lymphocytes, could be stored for up to 96 h without compromising the quality of the results.