PB2166 SUPERIOR AUTOMATED MAGNETIC ISOLATION OF PLASMA CELLS FROM MYELOMA BONE MARROW YIELDING HIGH‐QUALITY CELLS FOR DOWNSTREAM PROCESSING
Authors: Vorholt D. , Morrissey‐Wettey F. , Huang Y. J. , Hartmann K. , Nebe C. T. , Dzionek A. 
Published: June 2019

Background:

Multiple Myeloma is the 2nd most common blood cancer in the western world that forms in terminally differentiated, clonal plasma cells (PCs). These malignant PC accumulate in the bone marrow, which is typically accompanied by secretion of monoclonal immunoglobulins detectable in the serum or urine, thereby skewing the adaptive immune system. Despite remarkable improvements in diagnosis and treatment over the last decade, multiple myeloma remains incurable with a median 5‐year survival rate of just 49%. Hence, there is a strong need for sensitive diagnostics as early therapeutic intervention benefits treatment outcome, particularly when combined with long term monitoring. Therefore, the isolation and pre‐enrichment of PC from patient samples, especially when at low abundance, facilitates subsequent diagnostics and their functional analysis by for instance cytogenetic methods such as FISH or molecular methods as NGS.

Aims:

To optimize our MACS™ technology‐based automated isolation of PC from myeloma patients’ bone marrow in regards to PC yield and purity as well as their performance in downstream applications.

Methods

Bone marrow from multiple myeloma patients or a cell line‐based model system were used as source to isolate CD138+ Plasma cells with MACSprep™ Multiple myeloma CD138 MicroBeads, human in an automated manner using the autoMACS® Pro Separator (aMP). For subsequent analysis and performance assessment the cells were measured by flow cytometry using an in‐house developed panel of antibody‐conjugates.

Results:

The optimization of our workflow strongly improved the PC isolation performance from both, primary multiple myeloma bone marrow samples as well as a cell line‐based model system. This model system is based on a plasma cell line, whose CD138 expression can be set to mimic that of primary PC more closely. Additionally, our workflow isolated CD138+ PCs to considerably higher purity than another separation technology using column‐free magnetic isolation, particularly at lower starting PC frequencies.

Noteworthy, the aMP workflow yielded significantly higher cell recoveries over a range of starting PC frequencies. Interestingly, plasma cells isolated with the aMP displayed the expected normal PC morphology as analyzed by flow cytometry, phase contrast microscopy and Pappenheimer staining, whilst those from the other separation technology looked in part abnormal, not uniform and somehow distorted. This, in addition with the large aggregates that were formed during this other separation technology, resulted in clustered cells and hybridization artefacts in downstream FISH analysis, rendering the dispersed normal PC isolated with the aMP much easier to analyze. Furthermore, cells isolated with this other separation technology were contaminated with erythrocytes as examined by flow cytometry and microscopy, despite their obligatory use of a red blood cell lysis reagent. An advantage of the optimized aMP workflow is that it does not require sample preparation or red blood lysis resulting in virtually no hands‐on time.

Summary/Conclusion:

The fast and virtually no hands‐on aMP workflow significantly improves the performance in regard to obtained cell purity and cell yield when isolating PC from myeloma patient material. Noteworthy, it performs superior to another tested separation technology in regards to downstream analyses, thus facilitating the unambiguous analysis and study of myeloma plasma cells in research as well as routine analysis settings.