Colchicine

Two milliliters of peripheral blood was drawn into green-top heparinized vacutainer from each CML patient for cytogenetic monitoring at 3 and 6 months, and 1 year of imatinib therapy as per the cytogenetic analysis protocol followed by us in our earlier study [10 ]. Peripheral blood karyotypes were obtained from cultures of such samples, initiated in duplicates as 1 ml each of peripheral blood was inoculated in 5 ml each of RPMI 1640 (Cell Clone) culture medium with 10% FBS (Gibco) at 37°C for 48–72 h. Cell cultures were treated with Colchicine (Loba Chemie, 1 mg/10 ml) along with Ethidium Bromide (1 mg/10 ml) in the final hour of incubation. Cells were subsequently harvested by subjecting them to hypotonic shock with 0.075 M potassium chloride and fixed in 3:1 proportion of methanol and acetic acid. GTG banding was performed as described by Seabright [11 (link)] and chromosomes were identified and arranged according to the International System for Human Cytogenetic Nomenclature (ISCN) [12 ] with the help of a computerized work station – ‘Cytovision’. The number of cells or metaphase spreads investigated for each patient at each analysis ranged from 20 to 30. A representative karyotype with translocation t(9;22)(q34;q11.2), a hallmark in CML, is shown in Figure 2.