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.
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.
  
  
  METHODS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Haematology, LabMed Directorate, St. James's Hospital, Dublin, Ireland akdowling@stjames.ie.
authors.forename: Anita K
authors.initials: AK
authors.lastname: Dowling
authors.affiliation: Department of Haematology, LabMed Directorate, St. James's Hospital, Dublin, Ireland.
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Liptrot
authors.affiliation: Department of Haematology, LabMed Directorate, St. James's Hospital, Dublin, Ireland.
authors.forename: David
authors.initials: D
authors.lastname: O'Brien
authors.affiliation: Department of Haematology, LabMed Directorate, St. James's Hospital, Dublin, Ireland.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Vandenberghe
mesh_terms: D000368:Aged; D015703:Antigens, CD / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006801:Humans; D016130:Immunophenotyping; D007494:Ireland; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000628:therapy; D008297:Male; D018365:Neoplasm, Residual; D012680:Sensitivity and Specificity; D016896:Treatment Outcome
chemical_list: D015703:Antigens, CD; D014408:Biomarkers, Tumor
keywords: 8-color assay; B-cell chronic lymphoid leukemia; flow cytometry; minimal residual disease