CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia and is distinctly low in KMT2A-rearranged cases.

BACKGROUND: CD49f (integrin α6) is a useful marker for minimal residual disease (MRD) detection in B lymphoblastic leukemia and has recently been suggested to mediate infiltration of the central nervous system by leukemic B lymphoblasts. However, data regarding expression of CD49f protein in B lymphoblastic leukemia are limited, and whether CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia is unknown. METHODS: CD49f protein expression was characterized by flow cytometry in a series of 40 cases of B lymphoblastic leukemia, which included the genetic subgroups: KMT2A-rerranged, BCR-ABL1+, ETV6-RUNX1+, hypodiploidy, and hyperdiploidy. RESULTS: Expression of CD49f differed significantly among the five genetic subgroups studied, whether assessed by percentage of blasts positive for the antigen (p = .0001, Kruskal-Wallis) or median fluorescence intensity (MFI) (p = .0001, Kruskal-Wallis). Moreover, the percentage of CD49f+ blasts and MFI of CD49f were significantly lower in KMT2A-rearranged cases than in cases without KMT2A rearrangement (p = .0002 for both, Mann-Whitney). CONCLUSIONS: CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia, and is distinctly low in KMT2A-rearranged cases.

Participation in the College of American Pathologists Laboratory Accreditation Program Decreases Variability in B-Lymphoblastic Leukemia and Plasma Cell Myeloma Flow Cytometric Minimal Residual Disease Testing: A Follow-up Survey.

CONTEXT.­: Minimal residual disease (MRD) testing by flow cytometry is ubiquitous in hematolymphoid neoplasm monitoring, especially B-lymphoblastic leukemia (B-ALL), for which it provides predictive information and guides management. Major heterogeneity was identified in 2014. Subsequently, new Flow Cytometry Checklist items required documentation of the sensitivity determination method and required lower level of detection (LLOD) inclusion in final reports. This study assesses Laboratory Accreditation Program (LAP) participation and new checklist items' impact on flow cytometry MRD testing. OBJECTIVES.­: To survey flow cytometry laboratories about MRD testing for B-ALL and plasma cell myeloma. In particular, enumerate the laboratories performing MRD testing, the proportion performing assays with very low LLODs, and implementation of new checklist items. DESIGN.­: Supplemental questions were distributed in the 2017-A mailing to 548 flow cytometry laboratories subscribed to the College of American Pathologists FL3 Proficiency Testing Survey (Flow Cytometry-Immunophenotypic Characterization of Leukemia/Lymphoma). RESULTS.­: The percentage of laboratories performing MRD studies has significantly decreased since 2014. Wide ranges of LLOD and collection event numbers were reported for B-ALL and plasma cell myeloma. Most laboratories determine LLOD by using dilutional studies and include it in final reports; a higher proportion of LAP participants used these practices than nonparticipants. CONCLUSIONS.­: Several MRD testing aspects vary among laboratories receiving FL3 Proficiency Testing materials. After the survey in 2014, new checklist items were implemented. As compared to 2014, fewer laboratories are performing MRD studies. While LLOD remains heterogeneous, a high proportion of LAP subscribers follow the new checklist requirements and, overall, target LLOD recommendations from disease-specific working groups are met.

Immunophenotyping of Acute Lymphoblastic Leukemia.

Immunophenotyping by flow cytometry is an important component in the diagnostic evaluation of patients with acute lymphoblastic leukemia. This technique further permits the detection of minimal residual disease after therapy, a robust prognostic factor that may guide individualized treatment. We describe here laboratory methods for both the initial characterization of lymphoblasts at diagnosis, and the detection of rare leukemic lymphoblasts after treatment. In addition to antibody combinations suitable for diagnosis and detection of minimal residual disease, we describe procedures for peripheral blood and bone marrow sample preparation, procedures for labeling of cell-surface and intracellular proteins with fluorochrome-conjugated antibodies, and approaches to analysis of immunophenotypic data.

PhenoGraph and viSNE facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data.

BACKGROUND: Flow cytometric identification of neoplastic T-cell populations is complicated by the wide range of phenotypic abnormalities in T-cell neoplasia, and the diverse repertoire of reactive T-cell phenotypes. We evaluated whether a recently described clustering algorithm, PhenoGraph, and dimensionality-reduction algorithm, viSNE, might facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data. METHODS: We applied PhenoGraph and viSNE to peripheral blood mononuclear cells labeled with a single 8-color T/NK-cell antibody combination. Individual peripheral blood samples containing either a T-cell neoplasm or reactive lymphocytosis were analyzed together with a cohort of 10 normal samples, which established the location and identity of normal mononuclear-cell subsets in viSNE displays. RESULTS: PhenoGraph-derived subpopulations from the normal samples formed regions of phenotypic similarity in the viSNE display describing normal mononuclear-cell subsets, which correlated with those obtained by manual gating (r2 = 0.99, P < 0.0001). In 24 of 24 cases of T-cell neoplasia with an aberrant phenotype, compared with 4 of 17 cases of reactive lymphocytosis (P = 1.4 × 10-7 , Fisher Exact test), PhenoGraph-derived subpopulations originating exclusively from the abnormal sample formed one or more distinct phenotypic regions in the viSNE display, which represented the neoplastic T cells, and reactive T-cell subpopulations not present in the normal cohort, respectively. The numbers of neoplastic T cells identified using PhenoGraph/viSNE correlated with those obtained by manual gating (r2 = 0.99; P < 0.0001). CONCLUSIONS: PhenoGraph and viSNE may facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data. © 2017 Clinical Cytometry Society.

Improved compensation of the fluorochrome AmCyan using cellular controls.

BACKGROUND: Implementation of polychromatic flow cytometry in the clinical laboratory often requires the use of newer fluorochromes, with which experience may be comparatively limited. In the course of implementing polychromatic flow cytometry in our laboratory, we have observed significant differences in compensation values derived for the violet-excited dye, AmCyan, when cells rather than a commercially available set of polystyrene microparticles (BD CompBeads) are used as compensation controls. METHODS: Compensation values were calculated for AmCyan and several other fluorochromes using the BD CompBeads Set according to the manufacturer's protocol, and using unstained and singly stained lymphocytes as compensation controls. RESULTS: When the BD CompBeads Set was used to determine compensation values, spillover from AmCyan into V450 was overcompensated, while spillover from AmCyan into FITC was undercompensated. In contrast, when compensation values were calculated using unstained and singly stained lymphocytes, spillover into V450 and FITC from cells stained brightly with AmCyan-conjugates was compensated appropriately. Although significant differences were observed in the compensation of spillover from AmCyan into V450 and FITC using cells rather than the BD CompBeads Set as compensation controls (P < 0.0001, two-tailed Wilcoxon signed-rank test), such differences were not observed in control experiments using fluorochromes excited by the blue (FITC and PE) or red (APC) lasers. CONCLUSION: Improved compensation of the violet-excited dye, AmCyan, is obtained when cells rather than the BD CompBeads Set are used as compensation controls.