Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Quantitative assessment of informative immunophenotypic markers increases the diagnostic value of immunophenotyping in mature CD5-positive B-cell neoplasms.

BACKGROUND: The data on the clinical utility of the quantitative assessment of immunophenotypes in distinguishing mature CD5-positive B-cell neoplasms is limited. The study aim was to assess the diagnostic value of the quantitative assessment of a panel of 18 markers and to identify the most informative ones. METHODS: The immunophenotype of the neoplastic population was determined in diagnostic specimens from 188 patients. BD FACSCanto II flow cytometer and FACSDiva software were used to analyze the positivity/negativity and mean fluorescence intensity (MFI) of the surface expression of 18 markers. Advanced data mining methods were used to define the key differential diagnostic features of CLL/SLL (chronic lymphocytic leukemia/small lymphocytic lymphoma), MCL (mantle cell lymphoma), and CD5+ MZL (marginal zone lymphoma). RESULTS: The most informative markers for the distinction of CLL/SLL, MCL, CD5+ MZL, including atypical cases, were the MFI values of CD79b, CD20, CD23, CD43, CD38, CD11c, FMC7, CD200, kappa light chain, and their combinations. CD23 and CD200 were the most discriminant between CLL/SLL and MCL and CD23 plus CD79b between CLL/SLL and CD5+ MZL. The quantitative analysis of the most informative markers failed to accurately distinguish MCL and CD5+ MZL. The study highlights the data mining methods for the analysis and selection of the most informative immunophenotypic markers and for the design of a predictive model (diagnostic classifier), minimizing the subjectivity of expert-based assessment. CONCLUSIONS: Our data confirmed that the quantification of the expression of informative markers increases the diagnostic value of immunophenotyping in mature CD5+ B-cell neoplasms. © 2017 International Clinical Cytometry Society.

Optimizing antibody panels for efficient and cost-effective flow cytometric diagnosis of acute leukemia.

BACKGROUND: Differentiating acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) determines effective patient management and often depends on flow cytometry. Antibodies used in flow cytometry are costly, and the expenses are not always reimbursed. Having observed that AML and ALL have distinct patterns in the CD45/SSC panel, we set to analyze more leukemia cases and establish an algorithm for the efficient diagnosis of acute leukemia. METHODS: We retrospectively analyzed 127 consecutive cases of acute leukemia within the last 2 years and correlated the blast distribution patterns in the CD45/SSC panel, with the morphology and the detailed immunophenotype. RESULTS: Our results show that all the acute leukemias can be initially triaged into AML, ALL, and Indeterminate provisional groups based on the blast distribution patterns in the CD45/SSC panel and morphology. Each group was then further analyzed with tailored AML, ALL, and Indeterminate flow panels. Using this approach, we have efficiently and correctly diagnosed almost all the acute leukemias. Our analysis also determined the minimal numbers of immunological markers needed for the lineage assignment of acute leukemia. CONCLUSION: The algorithmic approach with tailored subsequent antibody selection could maintain diagnostic accuracy while significantly reducing reagent use, labor, and time. With a shrinking reimbursement for flow cytometric studies, an increase in laboratory efficiency without compromising diagnostic accuracy or turnaround time will contribute to preserving revenue and optimizing clinical service.

The technicon H6000 analyzer discriminates chronic lymphocytic leukemia from other B-cell leukemias through automatic assessment of large unstained cells.

The separation of chronic lymphocytic leukemia of B-cell origin from other chronic B-cell leukemias is subjective, being largely based on the morphologic features of the lymphoid cells in the peripheral blood. The percentage of large unstained cells determined with a Technicon H6000 analyzer (an automated blood cell differential analyzer, Technicon Instruments Corp, Tarrytown, NY) was used as a cell volume variable in an investigation of 70 cases of chronic lymphocytic leukemia of B-cell origin and of other chronic B-cell leukemias. The significant degree of correlation between the percentage of large unstained cells and morphoimmunophenotypic diagnosis, although obtained for a relatively small number of cases, suggests that this method of cell volume analysis can be used to improve diagnostic reproducibility in chronic B-cell leukemia.