A Single-Tube Seven-Colour Flow Cytometry Assay for Detection of Minimal Residual Disease In Myeloma.

Abstract 1688

The eradication of minimal residual disease (MRD) in myeloma predicts for improved outcome. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Flow cytometric assessment of MRD may be preferable in practice because of lower cost and easier feasibility.

Myeloma MRD flow cytometry requires at least three markers for plasma cell identification (CD38, CD138 and CD45) and combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117. Also, assessment of immunoglobulin light-chain restriction (cytoplasmic K and L) combined with myeloma-associated phenotypic plasma cell abnormalities, is very important.

Four-tube four-colour flow cytometry combine markers CD38/CD138/CD45 with markers for plasma cell phenotypic abnormalities and clonality. Six –colour flow cytometry combines the same markers (markers for plasma cell identification) plus clonality markers; it potentially increases the sensitivity of the method through coincident multiparameter analysis.

However, the single-tube six-colour flow cytometry, proposed by others studies, excludes the myeloma-associated phenotypic plasma cell abnormalities and consequently decreases specificity of the assay.

We propose a new single-tube seven-colour flow cytometry, including plasma cell identification antigens, clonality markers and myeloma-associated phenotypic plasma cell abnormalities markers.

In this new method, PCs are stained with antibodies: (i) CD38, CD138, CD45 used for identified plasma cells and percentage plasma cells to total leucocytes. (ii) CD19 and CD56+CD28 used to identify normal and abnormal plasma cells; and (iii) cy-IgK and cy-IgL, for confirm the plasma cells clonality.

We analysed normal bone marrow provided from healthy individuals. Our results showed a presence myeloma-associated phenotypic plasma cell abnormalities at low levels in healthy individual. The monotypy studies confirm polyclonality of this normal plasma cells.

Then we compared MRD assessement with single-six colour flow cytometry assay (plasma cells markers, clonality markers and exluding myeloma-associated phenotypic markers) and seven-colour flow cytometry assay (including myeloma-associated phenotypic markers).

Six –colour flow cytometry has a better sensitivity and showed efficacy for quantification MRD in myeloma patients. However, the single-tube six-colour flow cytometry excluded the myeloma-associated phenotypic plasma cell abnormalities and in some cases the seven-colour flow cytometry will be more informative because it detected myeloma-asociated phenotypic marquers combined with clonality marquers.

Finally, the single-tube seven colour flow cytometry assay provides reduction in antibody cost and increases sensitivity and specificity of the method through coincident multiparameter analysis.