title: Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization.
abstract: BACKGROUND
  Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH).
  
  
  METHODS
  RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH.
  
  
  RESULTS
  The reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH.
  
  
  CONCLUSIONS
  Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Shigeto
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan. Electronic address: kmatsuda@shinshu-u.ac.jp.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Core Technology Development Center, Seiko Epson Corporation, Suwa, Japan.
authors.forename: Akemi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Sueki
authors.affiliation: Core Technology Development Center, Seiko Epson Corporation, Suwa, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Uehara
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Sugano
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uehara
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Honda
mesh_terms: D000328:Adult; D000368:Aged; D005260:Female; D050939:Gene Fusion / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics*; D000071156:Promyelocytic Leukemia Protein; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D000072482:Retinoic Acid Receptor alpha; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human
keywords: APL; Droplet-RT-PCR; FISH; IQ-FISH; PML-RARA