MOLECULAR DIAGNOSIS OF THE PHILADELPHIA-CHROMOSOME TRANSLOCATION - A COMPARISON OF ISOTOPIC AND CHEMILUMINESCENT SOUTHERN BLOTTING

Southern blotting using radiolabeled probes is a well established tech nique for the detection of the Philadelphia translocation in the diagn osis of chronic myelogenous leukemia (CML). However, the use of radioi sotopes in the clinical setting is often problematic. Because of this we investigated the use of a digoxigenin-labeled probe and chemilumino graphy in the detection of the Philadelphia translocation. In this stu dy DNA was extracted from 19 bone marrow or blood samples from patient s with CML or other malignancies and subjected to Southern blotting wi th a probe specific for the Philadelphia translocation, Phl/bcr3. The probe was labeled with either P-32 or digoxigenin to determine the rel ative sensitivity and specificity of autoradiography and chemiluminogr aphy in the molecular diagnosis of the BCR/abl fusion gene. All 19 sam ples were tested by both methods. All blots were performed and interpr eted by individuals blind to the initial patient diagnosis. In additio n, 12 samples (6 positive for CML, 6 negative for CML as determined by Southern blotting with P-32-labeled probe) were subjected to triplica te Southern-blot analyses, with three separate lots of digoxigenin-lab eled probe to assay batch to batch variability in the efficacy of the probe. Radiolabeled and digoxigenin-labeled probes resulted in identic al diagnoses in all cases. All results obtained by molecular analysis correlated perfectly with the clinical diagnoses of the patients from whom the samples had been obtained. Reanalysis of patient samples with different batches of digoxigenin-labeled probe gave highly reproducib le results. With digoxigenin-labeled probe, diagnostic results were ob tained after exposure times of less than 1 h at room temperature. Comp arable results with radiolabeled probe required the use of enhancing s creens and a 3-4-day exposure at -70-degrees-C. Overall, we have found the use of the digoxigenin-labeled probe Phl/bcr3 and chemiluminescen ce using the substrate CSPD to be at least as good and in some respect s superior to the use of radiolabeled probe and autoradiography in the molecular diagnosis of CML.