title: Comparative Analysis of Fluorescence In Situ Hybridization and Real Time Polymerase Chain Reaction in Diagnosis of Chronic Myeloid Leukemia.
abstract: OBJECTIVE
  To compare the sensitivity and specificity of fluorescence in situ hybridization (FISH) with real time polymerase chain reaction (RT-PCR) in the diagnosis of Chronic Myeloid Leukemia (CML).
  
  
  METHODS
  A cross-sectional, analytical study.
  
  
  METHODS
  Haematology Department, Armed Forces Institute of Pathology, Rawalpindi, from January 2012 to February 2014.
  
  
  METHODS
  A total number of 87 patients of CML were studied. The diagnosis was made on the basis of clinical history, peripheral blood and bone marrow aspiration. These patients were tested for the presence of BCR-ABL1 fusion gene by RT-PCR and FISH. About 5 ml of venous blood was collected, half was taken in heparin for FISH and half in ethylenediamine tetra-acetic acid (EDTA) for CBC and PCR. For FISH, cells were cultured for 24 hours in RPMI 1640 medium and evaluated using BX51 fluorescence microscope for dual fusion signal of yellow colour. Samples having 20 or more interphases positive for dual fusion signals were taken as positive. For PCR, RNA extraction was done by Tri-Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. RT-PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of RT-PCR and FISH were compared.
  
  
  RESULTS
  Out of the 87 patients, 85 (97.7%) were PCR positive and 2 (2.3%) were PCR negative, whereas in FISH 83 (95.4%) were positive and 4 (4.5%) were negative. Sensitivity and specificity of FISH was 97.6% and 100%, respectively.
  
  
  CONCLUSIONS
  FISH is a reliable supplementary method to PCR for detection of BCR-ABL1 fusion gene in the diagnosis of CML.
authors.affiliation: Department of Pathology, Combined Military Hospital, Pano Aqil.
authors.forename: Javaid
authors.initials: J
authors.lastname: Ali
authors.affiliation: Department of Pathology, Army Medical College, Rawalpindi.
authors.forename: Saleem Ahmed
authors.initials: SA
authors.lastname: Khan
authors.affiliation: Department of Pathology, Combined Military Hospital, Thal, FATA.
authors.forename: Shan -E-
authors.initials: S-
authors.lastname: Rauf
authors.affiliation: Department of Pathology, Armed Forces Institute of Pathology, Rawalpindi.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ayyub
authors.affiliation: Department of Pathology, Combined Military Hospital, Peshawar.
authors.forename: Nadir
authors.initials: N
authors.lastname: Ali
authors.affiliation: Department of Pathology, Combined Military Hospital, Gilgit.
authors.forename: Nabeel Khan
authors.initials: NK
authors.lastname: Afridi
mesh_terms: D000328:Adult; D016009:Chi-Square Distribution; D015331:Cohort Studies; D003430:Cross-Sectional Studies; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity