Quantitative flow cytometry can distinguish between normal and leukaemic B‐cell precursors

Abstract

The immunological detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia (ALL) has been hampered by the fact that the leukaemic cells represent the malignant counterparts of normal haemopoietic precursors expressing terminal deoxynucleotidyl transferase (TdT), CD10 and CD19. We have used quantitative double-labelling flow cytometry with standard fluorescent beads to convert the mean fluorescence to the number of antigen molecules per cell. The number of TdT, CD10 and CD19 molecules per cell was determined in normal B-cell precursors from 22 healthy donors and eight regenerating marrows from patients with various malignancies and in 20 cases of B-lineage ALL. In normal bone marrow we characterized two different B-cell populations: TdT+/CD10+/CD19+ and TdT-/CD10+/CD19+. We demonstrated a major difference in the level of expression of TdT, CD10 and CD19 between normal bone marrow and B-lineage ALL blasts. Normal TdT+ precursors have significantly higher number of TdT (> 100 x 10(3)) and lower number of CD10 (< 50 x 10(3)) and CD19 (< 10 x 10(3)) molecules per cell than B-lineage ALL blasts (< 100, > 50, > 10 x 10(3) molecules per cell respectively); these differences were statistically highly significant. Furthermore, regenerating marrows had a significantly higher percentage of B-cell precursors than healthy donors. This increase was at the expense of the TdT-/CD10+/CD19+ population which, in the context of B-lineage ALL, could be wrongly interpreted as evidence of relapse if TdT is not included in the analysis. Therefore the quantitative analysis of TdT combined with CD10 and CD19 may allow a clear distinction between normal precursors and minimal residual leukaemia in B-lineage ALL and avoid the pitfall of misinterpreting regenerating B-cells as evidence of relapse.

Quantitative flow cytometry of ZAP‐70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome

“…The MESF unit provides a powerful tool to compare flow cytometry analysis of intracellular protein levels in an entire cell population, in a precise quantitative manner over extended periods of time and across instruments (24,25). MESF has been used to assess the number of TdT, CD10, and CD19 molecules per normal B-cell precursors and in B-CLL, with a major difference in the level of expression of TdT, CD10, and CD19 between normal bone marrow and B-lineage ALL blasts (32) and to monitor changes in the amount of CD5, CD19, CD20, CD23, and HLA DR in treated CLL patients when compared with untreated patients at diagnosis (33). MESF has also been used to quantitate expression of T-cell antigens in normal peripheral blood lymphocytes (34) and EGFP, CD4, CD8, and CD45 in Molt T cells (28).…”

Quantitative multiparametric immunophenotyping in acute lymphoblastic leukemia: correlation with specific genotype. I. ETV6/AML1 ALLs identification

“…For that purpose we decided to use a multiparametric objective approach for the immunophenotypic analysis in which, prior to their phenotypic characterization, B-lineage leukemic cells were specifically identified on the basis of their reactivity for the CD19 B cell-associated antigen. In order to increase the ability to discriminate leukemic from normal B cell precursors and avoid misinterpretation of positive and negative antigens, [42][43][44][45] the immunological characterization of the leukemic cells was performed using objective criteria based on the evaluation not only of the presence/absence of reactivity for a certain antigen, by which low sensitivity and specificity were previously obtained as recently underlined by Hrusak et al, 46 but also on the quantitative levels of antigen expression and its distribution on the whole leukemic cell population. In order to provide reproducible results at various times in the same laboratory and in different laboratories, the absolute levels of antigen expression were translated from arbitrary units (fluorescence channels) into molecules equivalent to soluble fluorochrome (MESF), specific information being provided on each fluorochrome-conjugated reagent used.…”

Correlation between karyotype and quantitative immunophenotype in acute myelogenous leukemia with t(8;21)

“…13 Recent reports have suggested that quantitative immunophenotyping may enhance the diagnosis of leukemias with specific cytogenetic abnormalities [15][16][17] and help define aberrant immunophenotyping patterns that could be useful for monitoring minimal residual disease. [17][18][19] It has also been suggested that quantitative immunophenotyping can contribute to our understanding of the pathogenesis of acute leukemia. 13,15,20 We undertook this prospective study of quantitative immunophenotyping to define whether there is a distinctive pattern in patients with AML and t(8;21).…”