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  • Title: Optimization and Validation of an 8-Color Single-Tube Assay for the Sensitive Detection of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia Detected via Flow Cytometry.
    Author: Dowling AK, Liptrot SD, O'Brien D, Vandenberghe E.
    Journal: Lab Med; 2016 May; 47(2):103-11. PubMed ID: 27069028.
    Abstract:
    OBJECTIVE: The European Research Initiative on chronic lymphocytic leukemia (ERIC) has designed a single-tube, 8-color chronic lymphocytic leukemia CLL-MRD flow cytometric assay to standardize testing in patients with B-CLL. This study aims to optimize and validate the 8-color CLL-MRD assay, with the desired outcome of it being implemented in St. James's Hospital Dublin and potentially other hospitals worldwide as the most accurate flow-based B-CLL-MRD detection currently available. METHOD: The single-tube assay incorporates 8 antibodies, namely, CD5, CD3, CD19, CD20, CD22, CD43, CD79b, and CD81. We tested a combination of 52 peripheral blood and bone marrow specimens with the antibodies to develop a sequential gating strategy that uses the typical phenotype of B-CLL cells to enumerate small residual B-CLL populations after treatment, effectively distinguishing them from the normal polyclonal and regenerating B-cells. We performed sensitivity assays via a set of serial dilutions and compared the assay with the current International Standardized Approach (ISA) method currently in use for MRD testing in B-CLL. RESULTS: The 52 specimens that we analyzed displayed MRD levels from 0.004% through 78.000%. Dilutional studies demonstrated detection of disease to a level of 0.00702%, and an excellent correlation was achieved against the current ISA method (R(2)= 0.991). CONCLUSION: The 8-color CLL-MRD assay provides a more informative approach than its predecessors for the assessment of MRD in B-CLL by functioning as an important disease biomarker, with MRD negativity acting as an indicator to achieving a clinical endpoint.
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