Flow cytometry and the study of cerebrospinal fluid in leukaemic patients: additional facts (2002)

We read with great interest the paper by Subirá et al (2001), which revised the results of 168 cerebrospinal fluid (CSF) samples from 31 patients with acute leukaemia. They studied these CSF samples using morphological and flow cytometric methods, and concluded that flow cytometry was useful in confirming central nervous system (CNS) leukaemia. It seems necessary to compare their results with other studies. We have studied the CSF samples from patients affected with acute lymphoblastic leukaemia (ALL) using similar methods for 8 years. The cytological study has been performed using cytocentrifuge preparations. The cytometric study has been performed using a Cytoron flow cytometer (Ortho Diagnostic), with an antibody panel. The antibodies have been chosen according to the antigens of the leukaemic cells of the bone marrow and the peripheral blood. Three hundred and fifty-two CSF samples from 51 patients (adults and children with ALL) were analysed between January 1993 and March 2001. Two hundred and ninety-one samples were suitable for clinical evaluation (the remainder had a volume < 1 ml or were not red blood cell free). Morphological and flow cytometric results were concordant in 283 samples (97%). Two hundred and sixty-nine CSF samples were identified as negative for CNS involvement and 14 were identified as positive (see Table I). Discrepancies between the two methods were observed in eight samples from five patients. Patients 1 and 2 were negative for CNS involvement at diagnosis (morphology and flow cytometry), but they later experienced CNS relapse. They were treated with intrathecal therapy; post-therapy CSF samples became negative using morphological methods earlier than using flow cytometry. The blast cells of Patient 1 had high expression of CD34, but they resembled reactive lymphocytes in the cytological observation. Patient 3 was positive by cytological observation and negative by cytometric tests at diagnosis, but during follow-up he relapsed in the CNS. Flow cytometry showed blast cells in the initial CSF sample of Patient 4, but they were not observed by morphological study. The clinicians did not change the treatment and he did not relapse. The CSF sample of this patient was considered positive, but only had CD19 expression on the leukaemic cells. Patient 5 was positive by morphology and negative by flow cytometry, but the treatment was not changed and he did not relapse. In our hands, the two methods have showed good agreement. It seems that flow cytometry is slightly more sensitive in detecting residual leukaemic cells in CSF, in patients treated for central nervous system relapse. This fact is probably not fundamental for any therapeutic decisions in these patients, because they usually receive additional intrathecal therapy, after which blast cells disappear from CSF samples, when studied using morphological techniques. We have found ‘false positives’ and ‘false negatives’ using the two methods. We continue to use cytological and flow cytometric methods, but we consider flow cytometry an auxiliary method, secondary to morphological study.