Cerebrospinal Fluid Monitoring in Childhood Leukemia: Laboratory Medicine’s Central Role in an Ongoing Oncology Success Story

Cure of childhood leukemia is arguably one of the greatest medical achievements of the last century.^1-3 B-lymphoblastic leukemia (B-ALL), the most common cancer of childhood, was previously a uniformly fatal disease. Famously, pathologist Sidney Farber’s^4-6 and others’⁷ introduction of folate antagonists produced cures and led to the recognition of the central nervous system (CNS), specifically the cerebrospinal fluid (CSF), as a sanctuary site for disease.^8,9 Prophylactic intrathecal (IT) chemotherapy remains standard of care today, having largely replaced cranial irradiation.^10-13 Indeed, the largest incremental improvement in leukemia outcomes over the last century, even compared to improvements in supportive therapy, is attributable to CNS therapy.^8,14 Moreover, staging of CSF at initial leukemia is a significant independent prognostic factor¹⁵ that requires accurate manual CSF total nucleated cell (TNC) differential counts and blast identification.¹⁶ 

Skilled clinical laboratory professionals are therefore crucial to accurate CSF staging and monitoring, the development of standardized cytocentrifugation having been fundamental to this practice.^17-21 In the U.S., modern Children’s Oncology Group (COG) clinical trials as well as institutional standard-of-care (SOC) protocols for ALL (B-cell and T-cell) utilize CSF status categories based on the absence or presence of blasts on manual review of cytomorphology on concentrated CSF cytospin slides stained with Wright Giemsa and, when blasts are present, the TNC based on unconcentrated manual count by hematocytometer chamber, which has its roots in older protocols.^22-24 Current categories predictive of clinical behavior are: CNS1 = no blasts present, CNS2 = blasts present in a sample with a TNC <5 cells/mL, and CNS3 = blasts present in a sample with a TNC ³5 cells/mL.²⁵ CSF TNC and cytomorphologic evaluation is repeated at each lumbar puncture (LP) prior to intrathecal chemotherapy administration to ensure CSF clearance or maintained absence of blasts.^26,27 

Lymphoblasts, while often stereotypical and reproducibly identifiable, can show significant morphologic variation, particularly in body fluid preparations such as CSF. Typical lymphoblasts (Figure 1A) are slightly larger than small mature lymphocytes with an increased nuclear:cytoplasmic ratio, smooth to notched nuclear contours, finely dispersed chromatin, and inconspicuous to absent small nucleoli.  The scant cytoplasm may show occasional small pale vacuoles and/or fine granules. However, sample centrifugation, age, temperature and staining as well as interval intrathecal therapy and leukemia genetics may affect cellular morphology of lymphoblasts (Figure 1B, 1C) as well as that of normal CSF cells, which include small mature lymphocytes (Figure 1D) and monocytes. Inherent challenges with CSF blast morphology have led many pathology groups to pair cytospin evaluation with CSF flow cytometry for immunophenotyping and therefore definite cell identification (Figure 1E-H, corresponding right panels). Some groups are utilizing flow only in morphologically challenging cases, while others are running flow by routine.^28,29 Flow may also provide a prognostic staging advantage over conventional cytology.^30,31