**3.4 Immunophenotyping**

The muliparameter flow cytometry (MPF) was performed at the beginning at the Institute for Immunobiology and Human Genetics, Faculty of Medicine-Skopje and then continued at the University Clinic of Hematology-Skopje. Immunophenotyping was done first, by using Cytomation (DAKO-Cytomation) flow-cytometer and than by BD FACSCanto™ II analyzer, on whole blood and/or bone marrow specimens using lysing solutions (BD-Biosciensies, San Jose.CA. USA)(Bain et al., 2002). We prepare the samples for simultaneous detection of three cytoplasmic/nuclear and membrane antigens. The first step involved immunostaining of cell suspension with multiple panels of tree monoclonal antibodies (McAb) labeled with fluorescein (FITC), phycoerythrin (PE) and phycoerythryn-Cy5 tandem complex (Pe-Cy5) as third color (Panovska-Stavridis et al., 2008). The slightly modified panel of monoclonal antibodies (McAb) against myeloid- and lymphoid-associated antigens as suggested by the EGIL was utilized (Bain et al., 2002; Panovska-Stavridis et al., 2008). The antibodies and their manufacturers are listed in Table 1.


All markers were manufactured by BD-Biosciences, except antilysozyme (DAKO); 1.cyt: cytoplasmic; 2.m: membrane \*MPO: myloperoxidase;\*\* SmIg:surface immunoglobulin;\*\*\* TCR:T cell receptor

Table 1. Panel of monoclonal antibodies (McAb) for diagnosis of acute leukemias

For the detection of cytoplasmic and nuclear antigens we used commercially available permeabilization/fixation solutions (FACS Permeabilization solution BDBiosciencies;San Jose.CA.USA) ((Bain et al., 2002).

We incubated 100ml of specimens (peripheral blood or bone marrow) and appropriate McAb for 15min at room temperature, than added the permeabilization solutions and/or lysing solution and repeated the incubation procedure. After the incubation we washed the samples three times with PBS-A, than re-suspended the cell with isotones solution and acquired the data. As control we used a lysed, but unstained sample (Panovska-Stavridis et al., 2008)

Acquired data were analyzed with software by using CD45 gating strategy (Borowitz et al, 1993). This technique involves incubation of all samples with fluorochrome - labeled CD45 McAb and with the McAb for which reactivity needs to be established with an alternative fluorochrome). In the initial step of the analyses, gating was set up on a CD45-positive versus light side-scatter dot plot. The procedure allowed the discrimination between the blast cell population and normal cells and the exclusion of platelets and debris. Thereafter, if necessary, it was possible to perform another gating, to separate the cells positive with the McAb under study (Borowitz et al, 1993). Leukemias were first screened by the primary panel and, if necessary, further characterized by the McAb of the secondary panel (Bain et al., 2002).

Antigen expression was considered positive if 20% or more blast cells reacted with a particular antibody, except reactivity of blasts cells with MPO. It was considered positive if 10% or more of mononuclear cells were MPO positive (Bain et al., 2002; Döhner et al., 2010).

#### **3.5 Molecular analysis**

486 Advances in Cancer Therapy

Clinical data from the patients were collected according the internal protocol that was approved by the University Clinic of Hematology Institutional Review Board. All included

The morphology was analyzed by microscopic examination of >500 nonerythroid cells on May Gruenwald Giemza stained air-dried bone marrow smears (Bennet et al,1997; Döhner

Air-dried bone marrow smears were stained for MPO, non specific esterase (NSE) and periodic acid-Schiff (PAS) according to the manufacturers guidelines. The percentage of positive cells for either stain was assessed by microscopic examination of 200 nonerythroid

The muliparameter flow cytometry (MPF) was performed at the beginning at the Institute for Immunobiology and Human Genetics, Faculty of Medicine-Skopje and then continued at the University Clinic of Hematology-Skopje. Immunophenotyping was done first, by using Cytomation (DAKO-Cytomation) flow-cytometer and than by BD FACSCanto™ II analyzer, on whole blood and/or bone marrow specimens using lysing solutions (BD-Biosciensies, San Jose.CA. USA)(Bain et al., 2002). We prepare the samples for simultaneous detection of three cytoplasmic/nuclear and membrane antigens. The first step involved immunostaining of cell suspension with multiple panels of tree monoclonal antibodies (McAb) labeled with fluorescein (FITC), phycoerythrin (PE) and phycoerythryn-Cy5 tandem complex (Pe-Cy5) as third color (Panovska-Stavridis et al., 2008). The slightly modified panel of monoclonal antibodies (McAb) against myeloid- and lymphoid-associated antigens as suggested by the EGIL was utilized (Bain et al., 2002; Panovska-Stavridis et al., 2008). The antibodies and their

B-lineageT-lineageMyeloid markers

CD117,CD13, CD33,CD14, CD15, anti-MPO, anti-lisosyme

> CD41, CD61, CD42,CD71

Anti-glycophorin A

1

CD 2, CD7, cyt CD3 3

CD1a,mCD31, CD4,CD5,CD8, anti TCR anti TCR

All markers were manufactured by BD-Biosciences, except antilysozyme (DAKO); 1.cyt: cytoplasmic; 2.m: membrane \*MPO: myloperoxidase;\*\* SmIg:surface immunoglobulin;\*\*\* TCR:T cell receptor Table 1. Panel of monoclonal antibodies (McAb) for diagnosis of acute leukemias

Non-lineage restricted

TdT, CD34, HLA-DR, CD 56

CD38

6

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

**3.1.1 Clinical data from the patients** 

patients had to sign a written consent.

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

**3.2 Morphology** 

**3.3 Cytochemical analysis** 

**3.4 Immunophenotyping** 

manufacturers are listed in Table 1.

CD19, cyt CD79b, cyt CD22

SmIg (kappa/lambda)\*\*, cytIgM2,CD138

First line e

Second line

Mononuclear cell preparation, RNA isolation, and cDNA synthesis were performed at the Department of Molecular Biology, Immunology and Pharmacogenetics, Faculty of Pharmacy-Skopje, according to standard procedures. Aliquots of 5 μL of cDNA (100 ng RNA equivalent) were used for Real-time Quantitative polymerase chain reaction (RQ-PCR) with primers and dual-labeled probes as described by Gabert et al. Positions and nucleotide sequences of the primers and probes are shown in Table 2.

The RQ-PCR reaction was performed in a 25-μl reaction volume using 12.5 μl (1x) Master Mix (Applied Biosystems), 300nM primers and 200nM probes on a Mx3005P(TM) QPCR System (Stratagene) under the following conditions: 95C for 10min, followed by 50 cycles of 95C for 15s, 50C for 1min. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript was amplified in parallel to the fusion gene (FG) transcript. Since ABL (Abelson) gene transcript expression did not differ significantly between normal and leukemic samples, ABL was used as a control gene in this study (24). Positions and nucleotide sequences of the primers and probe are shown in Table 2.

#### **3.6 Statistical analysis**

Statistical analyses were performed by using the statistical analyses software SPSS 18.0 and by applying the Descriptive statistics (cross tabulation, frequencies, descriptive ratio statistics), bivariate statistics (means, t-test, correlation (bivariate, partial, distances), nonparametric tests and linear regression statistical methods. The Level of probability for obtaining the null hypothesis, in accordance with the international conventions for biomedical sciences was 0.05 or 0.01 (Armitage et al., 2002).

Immunophenotyping of the Blast Cells in Correlations with

**4.1 Results from the cytochemical analyses** 

respectively.

reactivity of the blasts cells.

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 489

Results from the cytochemical analyses and the images of peripheral smears with positive examples from different cytochemical staining are presented at Figure 2 and Photo 1

A

B

C Fig. 2. Distribution of patients according to reactivity with different cytochemial staining. A:Patient distribution according to MPO reactivity of the blasts cells. B: Patient distribution according to PAS reactivity of the blasts cells. C: Patient distribution according to NSE


Table 2. Sequences and positions of the RQ-PCR primers and probes
