AbstractBackgroundCentral nervous system (CNS) involvement is associated with relapse in childhood acute lymphoblastic leukemia (ALL) and is a diagnostic challenge.ProcedureIn a Nordic/Baltic prospective study, we assessed centralized flow cytometry (FCM) of locally fixed cerebrospinal fluid (CSF) samples versus local conventional cytospin‐based cytology (CC) for detecting leukemic cells and evaluating kinetics of elimination of leukemic cells in CSF.ResultsAmong 300 patients with newly diagnosed ALL, 87 (29%) had CSF involvement by FCM, while CC was positive in 30 (10%) of 299 patients with available CC data (P < 0.001). Patients with FCM+/CC+ had higher CSF leukemic blast counts compared to patients positive by FCM only (medians: 0.10 vs. 0.017 leukemic blasts/μl, P = 0.006). Patients positive by FCM had higher white blood cell counts in peripheral blood than patients negative by FCM (medians: 45 × 109/l vs. 10 × 109/l, P < 0.001), were younger (medians: 3 years vs. 4 years, P = 0.03), and more frequently had T‐cell ALL (18/87 vs. 16/213, P = 0.001). At treatment day 15, five of 52 patients (10%) who had CSF positive by FCM at diagnosis remained so despite at least two doses of weekly intrathecal chemotherapy.ConclusionsLonger follow‐up is needed to clarify whether FCM positivity has prognostic significance and is an indicator for intensified CNS‐directed therapy.