Rapid and Sensitive Detection of Minimal Plasma Cell Populations Using Six-Colour Flow Cytometry.

Flow cytometry is a powerful tool for diagnosis and detection of minimal residual disease (MRD) in patients with monoclonal gammopathies. Multi-parameter MRD flow analysis is more sensitive than serum immunofixation for the detection of residual disease and is as effective as PCR analysis for predicting outcome after high-dose therapy. Attaining a limit of detection of 0.01% abnormal plasma cells in every patient requires analysis of several neoplastic markers. However, basic immunophenotyping coupled with clonality assessment can be informative in many patients. This approach can also be helpful at diagnosis in MGUS patients to determine whether the primary abnormal component is plasmacytoid or lymphoid. The aim of this study was to develop a single six-colour assay that could be used as a rapid and sensitive screen for the presence of neoplastic plasma cells. Bone marrow aspirate samples were assessed from 83 patients at diagnosis with a suspected plasma cell disorder (median age 69.1 years, 48 male and 35 female) and from 62 patients with myeloma after treatment (median age 61.0 years, 40 male and 22 female). Cells were washed and surface-labelled with CD19 PE, CD38 PE-Cy7, CD138 APC and CD45 APC-Cy7 then fixed, permeabilised and incubated with Kappa FITC and Lambda PE-Cy5. The assay allows assessment of B-cell surface-Ig and plasma cell cytoplasmic-Ig light chain restriction and abnormal plasma cell phenotype according to CD19 and CD45 expression. In presentation cases with more than 10% plasma cells by morphology, neoplastic plasma cells were present in 48/50 samples with one reactive plasmacytosis (32% plasma cells) and one patient with Castlemans disease (11% PC). In the remaining 33 cases with <10% plasma cells, 16/33 (48%) had monoclonal plasma cells, 4/33 (12.1%) showed a low-level of monoclonal B-cells and the remainder had normal B-cells and plasma cells. The assay provided a quantitative MRD result in 44/62 (70%) cases: neoplastic plasma cells represented <1% of leucocytes in 26/44 of the samples (59%) and the assay could detect neoplastic plasma cells when they represented as few as 0.01% of total leucocytes in several patients (4/44, 9.1%). Using this screening approach an extended MRD analysis panel was only required in 18/62 (30%) of cases where CD19- plasma cells were detected on a polyclonal background. Marrow quality was compared with a paired trephine biopsy in 68 samples. Using a algorithm based on the levels of normal plasma cells, B-progenitors, nucleated red cells and myeloid progenitors it was possible to determine whether the samples were representative bone marrow and a quantitative result could be provided. This single tube six-colour approach allows simultaneous assessment of B-cell and plasma cell immunophenotype and clonality. The incorporation of all the relevant markers into a single test makes analysis more straightforward and the gating strategy more reproducible. The assay is more rapid and cost-effective than four-colour analysis as fewer total markers are required. It can be applied to presentation samples to exclude reactive plasmacytoses and B-lymphoproliferative disorders, and to follow up samples to reduce the need for further flow cytometric investigations and optimise any downstream testing that is necessary. In approximately 70% of post-treatment myeloma samples this assay is capable of providing a definitive MRD result up to a maximum sensitivity of 0.01%.