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Background: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting., Methods: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3)., Results: In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy., Conclusion: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint., (© 2022 International Clinical Cytometry Society.)

Background: Cellular aggregation is a physiological response of lymphocytes to various extracellular stimuli. Currently, lymphocytes aggregation is only evaluated qualitatively or by semiquantitative methods. In this study, we assessed the capacity of flow cytometry to measure lymphocytes aggregation in a quantitative, accurate, and reproducible manner, and examined the significance of aggregation responses in various lymphoproliferative diseases., Methods: Extracellular triggers such as anti-CD19 antibodies or phorbol ester were utilized to induce lymphoid cells aggregation in a concentration dependent manner. Aggregation was quantified by flow cytometry based on the forward or side scatter (SSC), or by dark-field SSC of aggregates measured by ImageStreamX. Accuracy, reproducibility, and limitations of the methodology were evaluated. Aggregation responses were measured in various types of lymphoproliferative diseases, and correlated with immunophenotyping and IGHV mutational status in chronic lymphocytic leukemia., Results: Lymphoid aggregates provoked by extracellular stimuli elevate the forward and SSC signals relatively to the number of cells in each event. Aggregation responses vary among different types of lymphoproliferative diseases. Moreover, elevated levels of CD19-induced aggregation are associated with aberrant chronic lymphocytic leukemia characteristics, but not with IGHV mutational status of the disease, Conclusions: We have demonstrated that flow cytometry can provide accurate and reproducible measurement of both primary as well as T and B cell lines aggregation in response to extracellular stimuli. The use of quantitative evaluation of activation driven or other cellular aggregation may provide an analytical tool to elucidate biochemical and molecular mechanisms associated with lymphoproliferative diseases. © 2015 International Clinical Cytometry Society., (© 2015 International Clinical Cytometry Society.)

Background: ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories., Methods: Intracellular ZAP-70 levels in CLL and normal B cells were assessed by molecules of equivalent soluble fluorochrome (MESF), employing Quantum FITC MESF calibration beads to establish a standard curve relating channel value to fluorescence intensity in MESF units and the QuickCal v. 2.2 program (www.bangslabs.com) and clinical relevance of the data was determined., Results: The average ZAP-70 expression value in the CD19(+)/CD5(+) cells from 35 CLL patients was 103,701 MESF when compared with 12,621 MESF in B cells from 20 normal blood samples. "Low" and "high" ZAP-70 CLL subgroups were defined. Patients with "high ZAP-70 MESF" CLL had a shorter time to disease progression (P = 0.0005) and a more advanced clinical stage (P = 0.0018) when compared with patients in the "low ZAP-70 MESF" CLL subgroup., Conclusions: This quantitative analysis method can be employed to obtain a more specific and highly accurate assessment of ZAP-70 levels in CLL cells. The method can easily be standardized, in any routine flow laboratory, thereby improving reproducibility and reliability of ZAP-70 analysis., ((c) 2006 International Society for Analytical Cytology.)