**2. Flow Cytometry in MGs — Past, present and future**

The basic principle of flow cytometry has not changed from the past, it is used for identification of cell subtypes according to their functional and structural properties. Flow cytometers are

© 2013 Rihova et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

usually equipped with 2-3 lasers allowing excitation of 6 or more standard fluorochromes and the term multiparametric and/or polychromatic flow cytometry is used for this approach [7]. The classical immunophenotypisation identifies cells based on their size and granularity/ complexity as well as by the "visualization" of antigen-antibody binding. More than 360 antigens are currently known and commercial monoclonal antibodies conjugated with different fluorochromes are widely available.

affinity antibodies. The second group differentiate into long-lived memory B cells

Different maturation stages of B cells give a rise to a variety of B cell lymphoproliferations including post-germinal centre (post-GC) neoplasms [26-28]. Knowledge of B and PC pheno‐ type is thus important for determination of PCD diagnosis and its discrimination from other

with cytoplasmic κ expression; clone of myeloma cells (red dots) are typical CD38+CD138+CD56+ PCs with cytoplasmic λ

Syndecan-1 (CD138) is a specific marker of PCs expressed on the surface of both, normal and malignant PCs from their early stages [29]. Expression of CD138 is usually missing and/or is not very intensive on circulating PCs and/or plasmablasts in peripheral blood as well as on immature PCs and/or lymphoplasmacytic cells in bone marrow. Another important marker is CD38, a non-specific marker, whose bright expression (brighter on normal than on abnormal PCs) was used to identify PCs for a long time period. Together with CD138 helps in precise identification of PCs. An important marker for pathological PCs identification is also CD45 which is usually missing on PCs. These surface antigens are still used in analyses, but adding

marrow. Relative number of PCs (determined by morphology and/or flow cytometry) corresponds to type of MG, although results could be distorted by dilution of aspirated bone marrow with peripheral blood. Lower amount of PCs is characteristic for MGUS, aMM and/or amyloidosis, on the other hand higher PC infiltration occurs in MM. There is no

CD45-

IgM-/+IgD-/+) [21-23]. Besides these GC derived memory B cells also

Immunophenotyping in Multiple Myeloma and Others Monoclonal Gammopathies

CD38+/-CD20+

CD27-

http://dx.doi.org/10.5772/55938

CD138- B cells (turquoise dots)

PCs are available in MM bone

IgM+/-IgD-

)

95

(CD19+

expression.

CD38+/-CD20+

haematological malignancies (Fig 1).

CD27+

exist memory B cells lacking their typical marker CD27 (CD19+

**Figure 1. Coexistence of B-CLL and MM.** Clone of B-CLL is represented by CD19+CD38-

**4. Identification and immunophenotype of PCs**

Mostly terminally differentiated clonal CD38++CD138+

of other antigens is necessary [30,31].

[24], which likely arise independently from the germinal center reaction [25].

Flow cytometry has been used in diagnostics of MM since 90th years of the 20th century. Mostly ploidy and proliferative characteristics were analysed, but also the combination of DNA analysis with cytoplasmic immunoglobulin detection was done [8-10]. Discovery of new monoclonal antibodies (MoAb) against PCs helped in the development of immunophenotyp‐ isation in MGs [11,12].

It is well known that MFC underestimate the number of PCs when compared to routine morphological evaluation. However, the sensitivity of MFC is similar to light microscopy, results obtained using both approaches correlate and the percentage of PCs provided by MFC is also an independent prognostic factor affecting the overall survival of patients [13]. MFC is precise in detecting even a small number of PCs and together with analysis of expression of selected markers, normal and abnormal PCs could be easily discriminated [4]. So MFC is helpful method for clinical analyses of MGs.

Development of flow cytometry, including powerful instruments with the possibility to analyze many fluorochromes, availability of new dyes and antibodies, together with accessible specific software for complex phenotype analysis, require reviewing of current settings in MG analyses. The shift towards polychromatic analyses should be associated with standardisation and validation of this method as it is necessary to be consistent in providing analyses and reporting results. Recently, the European Myeloma Network (EMN) started to use the Euroflow settings which led to the development of a uniform protocol for the analysis of biological material of MG cases [14].
