Characterization of Rare Clonal Abnormal Plasma Cells in Monoclonal Gammopathy of Undetermined Significance (MGUS) by Flow Cytometry: Diagnostic Utility and Optimization of Technique
Dalia Salem, Prashant Tembhare, Raul Braylan, Carl O Landgren, Neha Korde, Adriana Zingone, Rene Costello, Elisabet Manasanch, Mary Kwok, Mark Roschewski, Irina Maric, Katherine Calvo, Constance Yuan, Maryalice Stetler-Stevenson. CCR, NCI, NIH, Bethesda, MD; Clinical Center, NIH, Bethesda, MD
Background: Monoclonal Gammopathy of Undetermined Significance (MGUS) exhibits low level involvement (<10% of cells) of the bone marrow (BM) with abnormal plasma cells (PCs). Immunohistochemisty (IHC) on BM biopsy is optimal in estimating % PCs but evaluation of clonality and immunophenotypic abnormalities by IHC is more challenging. Flow cytometry (FC) is ideal for detection of monoclonality and immunophenotypic abnormalities; however, many laboratories encounter difficulties in evaluating MGUS due to low numbers of PCs in aspirates. We have optimized FC detection of rare PCs in MGUS BMs.
Design: Eighty patients ultimately diagnosed with MGUS were evaluated for % PCs by IHC of BM biopsy using CD138 and light chain expression, and for clonality and abnormal immunophenotype using FC of BM aspirate. Aspirates were treated with ammonium chloride to lyse red cells and cell concentration adjusted to 3-5x106 cells/100mL staining volume. Eight-color cell surface staining (CD19, CD27, CD28, CD38, CD45, CD56, CD117, CD138) and intracellular kappa and lambda were performed. At least 2,500 PCs or up to 3x106 total events were acquired per tube (CANTOII flow cytometer), and data analyzed (FCS Express software) according to the European Myeloma Network Criteria.
Results: The mean % PCs estimated by IHC on BM biopsy was 8% (3 - 10%). FC definitively identified monoclonal PCs with abnormal immunophenotype (MAPCs) in 75% (60/80) of BM samples (1/80 suspicious, 19/80 negative), while IHC only detected MAPCs in 16% (13/80) of BM biopsies (18/80 suspicious, 47/80 negative, 2/60 inadequate). The % PCs detected in the BM aspirates by FC varied from 0.01- 0.83% (median 0.06%) of all acquired cells. The % of PCs that were MAPCs varied between 8-100%. The lowest detection limit of MAPCs using FC was 0.0008% of all acquired cells.
Conclusions: FC is highly sensitive in detecting rare MAPCs in MGUS. While IHC is crucial for determining % PCs in BM, FC is superior in demonstrating clonality and abnormal immunophenotype for diagnosis. Whole volume/bulk lysis prior to staining, concentration of cells and acquisition of a large number of events are crucial for optimal FC evaluation of PCs in this disease.
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 218, Wednesday Afternoon