Breakpoint cluster region rearrangement in CML
This test is done to detect Breakpoint Cluster Region rearrangement in chronic myeloid leukemia patients. It can be detected using conventional karyotyping or FISH.
To carry out the conventional karyotype, one ml of peripheral blood is drawn intravenously from the candidate. This is transferred into a sterilized culture vial containing growth media, serum and PHA (a mitogen). The vial is incubated at 37ºC in 5% CO2 atmosphere for 72 hours after which 0.1mL colchicine was added to arrest the cells at 72 hours.
The contents of the vial are transferred to a centrifuge tube, suspended in a hypotonic solution (0.075M KCl ) and spun at 500xg for 5 minutes. This last step is repeated. The supernatant is removed and the sediment is suspended in ice-cold fixative (1 part acetic acid to 3 parts methanol). Refrigerate for ten minutes. Centrifuge again and re-suspend the cells in about 0.5ml of fixative. Take a drop of this suspension and drop onto a clean slide and allow it to air dry. Stain slide using Giemsa stain. Observe under microscope. In case of FISH, fluorescent DNA probes are used. Sometimes, even PCR is employed to detect the break point cluster gene rearrangement.
CML is characterized by reciprocal translocation between chromosomes 9 and 22 resulting in the Philadelphia chromosome.
The translocation that generates the Philadelphia chromosome involves a gene rearrangement between the breakpoint cluster region (bcr) located on chromosome 22(22q11 and c-abl oncogene on chromosome 9(9q34). As a result of this translocation a fusion gene BCR –ABL is formed on chromosome 22.
The Philadelphia chromosome was one of the original chromosomal abnormalities ever detected in malignancies and it is still a very important marker for cancers involving the myeloid cells.
Normal Range :
Philadelphia chromosome has been found in 20% to 25% of patients with acute lymphoblastic leukemia and 2% of patients with acute myelogenous leukemia (AML)Interpretation :
Philadelphia chromosome has been found in the vast majority of chronic myeloid leukemia (CML) patients and hence, is a useful marker to confirm CML diagnosis.
20% to 25% of patients with acute lymphoblastic leukemia and 2% of patients with acute myeloid leukemia (AML) harbor a Philadelphia chromosome.
Test Method :
Nucleic acid technology