Plasma Cell Phenotyping and DNA Content Analysis by 8-Color Flow Cytometry: A Highly Sensitive Assay That Simultaneously Measures Clonality, Ploidy, and Proliferation
William G Morice II, Michael M Timm, Dragan Jevremovic, Rhett P Ketterling, Shaji Kumar, Kaaren K Reichard. Mayo Clinic, Rochester, MN
Background: New plasma cell (PC) proliferative disorder therapies have created the need for highly sensitive assays for clone detection and prognostic factor assessment including PC ploidy, PC proliferation, and proportion of normal PCs. An 8-color PC flow cytometry (FC) assay including DNA content analysis was designed to simultaneously measure these parameters; it was compared to a similar 6-color assay lacking DNA measurement combined with pulse labeling and cytogenetic analysis.
Design: 6-color FC with antibodies to CD19, CD38, CD138, CD45, and cytoplasmic kappa and lambda Ig light chains (3x105 events) and 8-color FC with these antibodies and DAPI DNA staining (5x105 events) were performed on 30 normal bone marrows and 202 clinical specimens (BD FACSCanto II instruments, BD FACSDiva software). PC proliferation was measured by BrDU PC pulse label in 129 cases; 108 had cytogenetic studies.
Results: 8-color FC was more sensitive for PC clone detection (detection limit 0.003%). The 17 cases missed by 6-color FC had a median of 140 clonal events, and 11 were aneuploid. Normal PCs were also always detected and their proportion relative to clonal PCs was stable.
|8-color FC Pos (n=159)||8-color FC Neg (n=43)|
|6-color FC Pos (n=142)||142 (70%)||0|
|6-color FC Neg (n=60)||17 (8%)||43 (22%)|
|FC Diploid||FC Aneuploid|
|Cyto Diploid (n=59)||42||17|
|Cyto Aneuploid (n=49)||0||49|