Clinical immunology
Multicolor flow cytometric immunophenotyping for diagnosis of childhood precursor-B-ALL and monitoring of minimal residual disease

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent cancer in childhood. Current treatment strategies induce complete remission in almost all patients. However, approximately
one-third of patients suffer from relapse and most of these children die of this disease. Flow cytometric immunophenotyping is of high value both at diagnosis of BCP-ALL and for treatment monitoring
– detection of minimal residual disease (MRD). In the current study, 70 bone marrow samples from childhood BCP-ALL patients were immunophenotyped with 3-color flow cytometry based on the BIOMED-I Concerted Action approach. Five triple monoclonal antibody combinations were used: TdT/C10/CD19 (I), CD10/CD20/CD19 (II), CD34/CD38/CD19 (III), CD34/CD22/CD19 (IV) and CD34/CD45/CD19 (V). Areas devoid of normal bone marrow cells (so called “empty spaces”) were defined for each of the triple staining dot plots. In addition, further stainings were performed to investigate possible T-lineage (CD1a, CD2, CD5, CD7) and myeloid lineage (CD13, CD15, CD33) antigen co-expression. Every triple staining revealed different efficiency in detection of aberrant leukemic phenotypes ranging from about 44% for combination III and IV to as high as 96% for the combination II. The study confirmed relatively high incidence of myeloid marker coexpression - CD13 (24%) and CD33 (7%). The T-lineage markers (CD2, CD5 or CD7) were coexpressed rarely (6%). In conclusion, we could confirm the capability of the “empty spaces” method to detect abnormalities in antigen expression in BCP-ALL patients and to discriminate between normal and leukemic differentiation patterns in virtually all cases, which could be applied for MRD monitoring.