Impact of in vitro acidosis on leukocyte cell population data measured by automated hemocytometry

Objectives: Acidosis is a frequent and clinically significant abnormality in critically ill patients, known also to affect leukocyte metabolism, morphology, and immune function. Cell population data (CPD), generated by modern hematological analyzers, provide quantitative information on leukocyte size, complexity, and nucleic acid content, but their sensitivity to acidosis has not been fully characterized. Methods: Thirty-five whole blood samples collected in K2EDTA, referred for routine hematological testing, were divided into paired aliquots and subjected to in vitro acidification with hydrochloric acid or an equivalent volume of saline, serving as control condition (hereafter referred to as "untreated"). Samples were analyzed at baseline and after 30 min on Sysmex XN-10. Results: Acidification induced rapid and sustained reduction in pH, from a median value of 7.30 to 6.90. After 30 min of incubation, compared to the untreated aliquots, acidified samples displayed increased monocyte and lymphocyte size indices (MO-Z, LY-Z) and lymphocyte complexity (LY-X), along with reduced neutrophil complexity and fluorescence (NE-SSC, NE-SFL), reduced neutrophil size (NE-FSC), and decreased DNA/RNA-related fluorescence in monocytes and lymphocytes (MO-Y, LY-Y). LY-Y showed the highest accuracy (area under the curve [AUC=0.93]), followed by MO-Y (AUC=0.84) and LY-Z (AUC=0.82) for discriminating acidosis. Conclusions: In vitro acidosis induces consistent and biologically plausible alterations in leukocyte CPD, reflecting changes in cell size, granularity, and nucleic acid content, mostly indicative of leukocyte dysfunction. These findings support the hypothesis that CPD could be used as sensitive markers of acid-base-related leukocyte dysregulation, with clinical significance in conditions characterized by severe acidosis.