Diagnostic research of multiple myeloma*
 

  •  Efficient Plasma cell isaolation for analysis of malignant cells
  • Advantages of enrichment for cytogenic studies
  •  Flow analysis of malignant plasma cells
* for research purposes only

Application notes and a scientific poster for diagnostic research of multiple myeloma

Application data for multiple myeloma research by workflow step

Plasma cell isolation 
 

Cell isolation solutions for cytogenetic analysis and efficient enrichment of CD138+ plasma cells is a prerequisite for valid cytogenetic analysis of bone marrow samples. Conventional methods for the isolation of CD138+ plasma cells requires laborious density gradient centrifugation and generation of mononuclear cells prior to any plasma cell purification. MACSprep™ Multiple Myeloma CD138 MicroBeads, human allow the isolation of CD138+ plasma cells directly from bone marrow or peripheral blood without the need for gradient centrifugation or erythrocyte lysis. The minimal manipulation of sample ensures high integrity of CD138+ plasma cells, suitable for further cellular analysis (flow cytometry), cytogenetic analysis (FISH), or molecular analysis (sequencing, PCR, microarray).

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MACSprep Multiple Myeloma CD138 MicroBeads.
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Isolation of plasma cells using MACSprep Multiple Myeloma CD138 MicroBeads, human

Frequency of CD138+ plasma cells prior to and after automated enrichment from bone marrow using MACSprep Multiple Myeloma CD138 MicroBeads, human in combination with the autoMACS Pro Separator. Plasma cells were analyzed by flow cytometry. Each data point represents one individual bone marrow sample. Using this method, the frequency of plasma cells can be significantly increased compared to cell isolation without enrichment. Automated cell isolation on the autoMACS Pro Separator minimizes hands-on time, ensures consistency, and delivers highly pure cells. By this anylsis of rare patient materials is ensured and quality of data obtained is enriched.

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Direct cell isolation from bone marrow or blood with the autoMACS® Pro Separator 

With the autoMACS® Pro Separator, CD138+ plasma cells can be automatically isolated from  bone marrow or peripheral blood samples. Take this short instrument tour and see how your target cells are being separated without the need for density gradient centrifugation or red blood cell lysis.

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Cytogenetic and flow analysis of malignant plasma cells
 

Most multiple myeloma studies perform fluorescent in situ hybridization (FISH) analysis with isolated CD138+ plasma cells. More studies are incorporating new molecular methods to diagnose multiple myeloma, such as next generation sequencing (NGS) and single nucleotide polymorphism (SNP) array. These assays require DNA isolation from CD138+ plasma cells, as well as analysis of phenotype of malignant plasma cells, which can be determined by flow cytometry.
 

Cytogenetic studies 

FISH is a standard method for identifying genomic aberrations in multiple myeloma. It can also be performed at the interphase of cells, iFISH. In multiple myeloma applications, iFISH is primarily performed. To analyze the cells at interphase directly, isolation of CD138+ cells is necessary to show the aberrant chromosome/genes are from plasma cells 
 

After separation of CD138+ PCs, two interphase cells were hybridized with the LSI D13S319 Single Color Probe. The normal cell (left) shows the two orange signal patterns. The malignant cell (right) shows only one orange signal pattern, due to a deletion event including locus D13S319 or monosomy of chromosome 13. B) Two interphase cells, one normal (left) and one malignant (right) were subjected to FISH analysis and hybridized with the LSI IGH/FGFR3 Dual Color, Dual Fusion Translocation Probe (t(4;14)(p13;q32)). The normal cell shows the two orange (FGFR3) and two green (IgH) signal patterns, respectively. The malignant cell shows an abnormal signal pattern with one orange (FGFR), one green (IGH) and three fusion signal pattern, resulting from a chromosomal translocation event (t(4;14) FGFR3/IgH).
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CD138+ Plasma cell FISH analysis

Using our Whole Blood CD138 MicroBeads, we have developed an SOP for an automated, reliable, and standardized method, which allows the processing of multiple samples in a single day, while maintaining sample integrity and increasing the sensitivity of FISH analysis and whole genome arrays. 

This technology proved superior to a different automated technology, Including less sample manipulation due to  the avoidance of RBC lysis, and higher recovery and purity. Moreover there is no need for density gradient centrifugation. The detection rate of chromosomal abnormalities per sample in multiple myeloma and plasma cell dyscrasia significantly improves when analysis is performed on purified populations of high-quality CD138+ plasma cells.

 

References:

A. Palumbo et al.; J Clin Oncol; Revised International Staging System for Multiple Myeloma: A Report From International Myeloma Working Group 

P. Martin et al,; Modern Pathology, hMLH1 and MGMT inactivation as a mechanism of tumorigenesis in monoclonal gammopathies
 

Multiparameter flow analysis of malignant plasma cells

To gain more information on the isolated malignant plasma cells flow analysis can be performed. Our flow reagents ensure that the data you generate on your flow cytometer is reliable and consistent:

Build your own B cell flow panel with our  Flow Panel Builder
 

Gating strategy to define malignant plasma cells by flow cytometry
For this application we have designed a specific panel that can help you with the analysis of malignant plasma cells.

Antibody panel design for analysis of myeloma plasma cells: 

Antigen/parameterFluorochrome
CD138 APC
CD38sFITC
CD235a (Glycophorin A) PE-Vio® 700
CD45VioGreen™
CD19VioBlue®
CD28/CD56APC-Vio 770
ViabilityPropidium iodide
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