**1.3.5 Molecular genetics**

480 Advances in Cancer Therapy

and it consists of classic cytogenetics, molecular cytogenetics, molecular genetics and

First steps in the diagnostic work-up of a patient with suspected AML is a morphological evaluation of a classical bone marrow aspirate and a peripheral blood smear by using a May-Grunwald-Giemsa or a Wright-Giemsa stain. It is recommended that at least 200 leukocytes on blood smears and 500 nucleated cells on marrow smears to be counted. For a diagnosis of AML, a marrow or blood blast count of 20% or more is required, except for AML with t(15;17), t(8;21), inv(16) or t(16;16), and some cases of erythroleukemia. Myeloblasts, monoblasts, and megakaryoblasts are included in the blast count. Erythroblasts are not counted as blasts except in the rare instance of pure erythroid leukemia (Bennet et

Lineage involvement could be identified with cytochemistry by using myeloperoxidase (MPO) or Sudan black B (SBB) and nonspecific esterase (NSE) stains. Detection of MPO (if present in > 3% of blasts) indicates myeloid differentiation, but its absence does not exclude a myeloid lineage because early myeloblasts and monoblasts may lack MPO. SBB staining parallels MPO but is less specific. NSE stains show diffuse cytoplasmic activity in monoblasts (usually 80% are positive) and monocytes (usually 20% positive). In acute erythroid leukemia, a periodic acid-Schiff (PAS) stain may show large globules of PAS

Application of immunophenotyping together with the cytomorpohology and cytohemistry has a crucial role in the initial diagnosis of all cases with a suspected or proven diagnosis of acute leukemias. Immunophenotyping allows the discrimination of different cell population on the basis of their size, granularity, and antigen expression patterns. Flow cytometry is a powerful technology for characterization and analysis of cells. It simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they move in a fluid stream through a beam of light through an optical and/or electronic detection apparatus. Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5nm to 40nm diameter (Panovska-Stavridis et al., 2008). The applied methodology detects cell surface antigens in a suspension of viable cells and cytoplasmic and nuclear antigens in previously fixed and stabilized cell suspension with the application of monoclonal antibodies conjugated with different fluorochromes. It permits simultaneous detection (multiparameter analyzes) of more than two membrane and nuclear or cytoplasmic antigens by means of double or multiple immunostaining (Bain et al., 2002;

Chromosome abnormalities are detected in approximately 55% of adult AML. Conventional cytogenetic analyses are part of the standard diagnostic approach of a patient suspected with AML. This allows the identification of genetics entities that deserve targeted treatments

positivity (Bennet et al,1997; Döhner et al., 2010, Panovska-Stavridis et al., 2008).

Bene et al., 1995; Döhner et al., 2010; Panovska-Stavridis et al., 2008).

immunophenotyping by multi-parameter flow cytometry (Döhner et al., 2010 )**.** 

al,1997; Döhner et al., 2010; Panovska-Stavridis et al., 2008).

**1.3.1 Cytomorphology** 

**1.3.2 Cytochemistry** 

**1.3.3 Immunophenotyping** 

**1.3.4 Cytogenetics** 

Numerous genetic abnormalities that escape cytogenetic detection like gene mutations and gene expression abnormalities are more recently discovered among CN-AML. Molecular diagnosis by reverse transcriptase- polymerase chain reaction (RT-PCR) for the frequent gene fusions, such as AML1/ETO, CBFMYH11, MLLT3/MLL, DEK/NUP214, can also be useful in certain circumstances. RT-PCR, for which standardized protocols are already published, is also an excellent option to detect recurrent cytogenetic rearrangements, if chromosome morphology is of poor quality, or if there is typical marrow morphology but the suspected cytogenetic abnormality is not present. (Gabert et al., 2003; Beillard et al, 2003).
