Multiple myeloma (MM) is characterized by the clonal proliferation of terminally differentiated plasma cells. These dyscrasias range from the phenotypically benign monoclonal gammopathy of unknown significance (MGUS), to the clinically significant form, multiple myeloma (MM). Molecular cytogenetic abnormalities are one of the measures that has been used to stratify patients. As the clinical cut-off values for individual fluorescence in situ hybridization (FISH) probes can exceed the total percentage of abnormal plasma cells within a specimen, the International Myeloma Workshop Consensus Panel recommends analysis for genetic abnormalities by FISH, preferably after plasma cell sorting, as one means to stratify patients into standard risk or high risk categories.
We present a novel method for plasma cell isolation in which antibody coated para-magnetic immunobeads are used in a two-step separation process that deposits an enriched population of CD138+ plasma cells directly onto a predefined area of a microscope slide in a manner suitable for subsequent staining and FISH analyses.
The clinical materials utilized were appropriately anonymized extraneous volumes of bone marrow aspirates obtained after all routine clinical testing requests had been fulfilled. Briefly, 100ml of bone marrow is incubated with a blocking buffer and paramagnetic immunobeads covalently conjugated to anti-CD138 antibody (WaveSense, Irvine, CA) for 45 minutes at room temperature with gentle agitation. Primary enrichment is achieved when the tube is placed in a magnetic dock for 15 minutes allowing the paramagnetic immunobead-plasma cell-complexes to sediment on the side of the reaction tube. Unbound cells remain in the supernatant solution which is gently removed. The cells from the supernatant are still suitable for additional analysis for hematologic disorders not involving a CD138+ lineage. The residual CD138+ cell population is resuspended in blocking buffer. This enriched cell suspension is pipetted into an EpiSep® (WaveSense, Irvine, CA) chambered slide. As the solution diffuses through the unit, the suspended plasma cell-paramagnetic bead complexes are immobilized at the slide's surface as they pass close to a fixed magnet in the slide dock while the residual supernatant solution is eluted into adjacent chambers containing compact absorbent pads. A small volume of Carnoy's fixative is introduced through the EpiSep port to affect an initial fixation of the captured plasma cells deposited on the microscope slide's surface. The unit is then manually separated from the microscope slide. Samples were analyzed with a panel of FISH probes to assess copy number changes for chromosomes 5, 7, 13, 1p/1q, 17p and t(4;14), t(11;14) and t(14;16).
Bone marrow from 32 patients with indications of possible or known plasma cell disorders were enriched for CD138+ plasma cells. Conventional non-enriched FISH analysis detected reportable abnormalities in 11 (34%) of patients. With enrichment, abnormalities were detected in 21 (66%) of patients. When compared to abnormalities detected by FISH on non-enriched marrow, new abnormalities were detected in 14 (44%) of cases. Of the14 cases, 5 (36%) cases had abnormalities that shifted the prognosis from standard risk to high. An additional 3 cases (21%) had patterns consistent with an uncharacterized IGH rearrangement which may also hold a higher risk. The increased rate of abnormal cells ranged from 1.2x to 44.0x with an average increase of 9.7x. Cut-off values for the FISH analysis with this enrichment method were comparable to other methods of plasma cell enrichment.
We evaluated the use of a novel method to isolate and present only the target cell lineage. Deposition of a sorted plasma cell suspension via a slide chamber limits loss of plasma cells and confines these cells to a defined area for analysis. This method preserves cell morphology and avoids loss of cells that can occur with manual slide dropping methods. We found this strategy significantly increased both the sensitivity of detection levels and accuracy for determinations of these low-level, clinically prognostic genetic alterations while reducing the amount of bone marrow required for complete FISH analysis.
Asterisk with author names denotes non-ASH members.