[1463] 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 Detects PC Clones with High Sensitivity
 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%)
4x4 table comparing method sensitivities

8-color FC was 28% more sensitive than metaphase analysis for detecting PC aneuploidy (table 2). Of the 17 aneuploid cases detected by FC only, the median number of clonal PCs was less than 1%, and those cases with >2% clonal PCs were all hypodiploid.

FC is More Sensitive than Metaphase Studies for Detecting Aneuploid PCs
 FC DiploidFC Aneuploid
Cyto Diploid (n=59)4217
Cyto Aneuploid (n=49)049
Cyto=Cytogenetic metaphase analysis

When comparing the % S phase PCs and including all cases, the correlation coefficient between FC and BrDU pulse label was 0.59. Restricting the comparison to cases with >5% PCs improved the R-value to 0.75.
Conclusions: Combining FC phenotyping and DNA content analysis improves the sensitivity of abnormal PC detection and discrimination. This also powerfully identifies aneuploid (especially hypodiploid) PCs, and improves the accuracy of S phase determination when the PC burden is low. These attributes make this a powerful tool for the evaluation of newly diagnosed and treated PC proliferative disorders patients.
Category: Hematopathology

Wednesday, March 6, 2013 1:00 PM

Poster Session VI # 221, Wednesday Afternoon


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