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The early identification of septically infected newborn infants is important for ensuring good outcomes. Blood cell differentiations are helpful, but they are often time consuming and inaccurate. In this study, we evaluated the use of automatic white blood cell differentiations by flow cytometry for the diagnosis of neonatal sepsis. Episodes of suspected infection in neonates were retrospectively classified into two groups, unlikely infection (UI, levels of Interleukin-6 < 400 pg/mL or CRP within 48 h < 10 mg/L), n = 101 and probable infection (PI, Interleukin-6 ≥ 400 pg/mL or CRP within 48 h ≥ 10 mg/L), n = 98. Complete blood cell counts were performed by Sysmex XN-9000® using flow cytometry. Relative and absolute proportions of immature granulocytes were evaluated. Unexpectedly, the absolute count of immature granulocytes was significantly lower in the group of PI compared to UI neonates. Similar results were found when analysing the relative proportion of immature granulocytes among all neutrophil granulocytes. On the other hand, manually counted immature to total (I/T) ratios of granulocytes were higher in PI than in UI infants. Therefore, we conclude that differentiations of granulocytes by Sysmex XN-9000® can be used to distinguish between infected and uninfected neonates if the results are interpreted according to our findings. A low count of immature granulocytes as determined by Sysmex XN-9000® may indicate neonatal infection. [ABSTRACT FROM AUTHOR]