Utility of Immunocytochemistry in Cerebrospinal Fluid of B‐ ALL Patients for CNS Involvement: A Pilot Study

Background Central nervous system (CNS) involvement in paediatric B‐acute lymphoblastic leukaemia (B‐ALL) significantly impacts relapse risk and survival. Conventional cytomorphology (CM) of cerebrospinal fluid (CSF) is rapid and specific but has low sensitivity because of limited cellularity and morphological overlap with reactive lymphocytes. Objective To evaluate the diagnostic utility and resource efficiency of immunocytochemistry (ICC) as an adjunct to CM in detecting leukaemic infiltration of CSF in paediatric B‐ALL. Methods In Cohort 1 ( n = 45), all CSF samples underwent CM and ICC using CD3, CD79a, and TdT. In Cohort 2 ( n = 44), ICC was applied selectively to samples with pleocytosis (> 4 cells/μL) on the basis of a triage algorithm. Results In Cohort 1, ICC upgraded a subset of “doubtful” CM cases to definitive leukaemic infiltration, particularly with CD79a and TdT. CD3 reliably confirmed reactive T‐cell morphology, enhancing specificity. In Cohort 2, selective ICC use maintained diagnostic yield, detecting blasts in doubtful cases while conserving slides and resources. Together, ICC demonstrated added diagnostic value, especially in morphologically equivocal and higher‐cellularity specimens. Conclusion ICC complements CM in CSF evaluation for paediatric B‐ALL, improving sensitivity and supporting a pragmatic triage strategy. A combination of CD3 and CD79a is optimal when two smears are available; with limited material, marker choice should be guided by morphology. This cost‐effective workflow enhances detection of CNS involvement where flow cytometry or molecular assays are not readily available.