Optimization of immunomagnetic selection of myeloma cells from bone marrow using magnetic activated cell sorting

Abstract

Plasma cells (PCs) enrichment from bone marrow samples of multiple myeloma (MM) patients is frequently performed by immunomagnetic separation (magnetic activated cell sorting, MACS) using anti-CD138 MicroBeads. The aim of our work was to find optimal strategy for immunomagnetic separation of PCs and determine optimal algorithm of separation techniques for samples with various percentage of neoplastic cells. From 2007 to 2008, selection of PCs using separation programs Possels and Posseld(2) was carried out on 234 bone marrow samples obtained from 208 MM patients. In 2008, an optimal algorithm for separation programs was introduced based on the analysis of the previous experiments. The Possels program is applicable for samples with >10% PCs in the mononuclear fraction, while the Posseld(2) program is used for samples with 5-10% PCs in the mononuclear fraction. Median purity of 92.6% for the positive fraction of cells (range 14.5-99.6%) and median recovery of 60.4% (range 25.7-99.5%) were obtained when the Possels program was applied (n = 45). A total of 80% (36/45) of processed samples had purity of >70%. Median purity for the positive fraction of 83.7% (range 14.3-99.7%) and median recovery of 14.3% (range 3.6-50.0%) were achieved using the Posseld(2) program (n = 99). A total of 68% (67/99) of processed samples reached >70% purity. This separation strategy enabled us to obtain sufficient amounts of highly purified PCs required for subsequent research purposes. The MACS method has been unsuccessful if the percentage of PCs in the initial sample was <5%. These samples were processed by fluorescence activated cell sorting (FACS).

Genome-Wide Screening of Cytogenetic Abnormalities in Multiple Myeloma Patients Using Array-CGH Technique: A Czech Multicenter Experience

“…The enriched samples of PCs were obtained by either anti-CD138+ immunomagnetic beads (AutoMACS Pro, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or by fluorescent-activated cells sorting (FACS Aria, BD Biosciences, San Jose, CA, USA). Detailed protocol of sorting algorithm used in our center was described elsewhere [13]. Briefly, cutoff level of 5% for CD138+ PCs infiltration in the bone marrow was established, and sorting technique (<5% FACS, >5% MACS, resp.)…”

Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells

“…4,10,11 Although significantly more karyotypic aberrations can be identified in MM and MGUS when CD138 + PC are enriched rather than when unsorted whole bone marrow is studied, with the major fraction of CD138 + PC in clonal plasma cell disorders. 18 Finally, the 85%-95% median purity of magnetically enriched CD138 + PC limits detailed evaluation of clonal profiles in cases carrying one or multiple cytogenetic changes, particularly as regards identification of a fraction of non-altered aPC, 19 as also described for other B-cell neoplasias. 20 Simultaneous usage of the FISH technique and May-Grünwald-Giemsa staining or immunophenotyping (e.g., staining for CD138 or cytoplasmic light chains) through the so-called FICTION technique has occasionally been described.…”

Incidence of cytogenetic aberrations in two B lineage subpopulations in multiple myeloma patients analyzed by combination of whole-genome profiling and FISH

“…Detailed protocols and sorting algorithms were published elsewhere [17,18]. Briefly, the PCs from MM patients in mononuclear cell fraction were enriched by anti-CD138+ immunomagnetic beads and sorted using AutoMACS Pro (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).…”