title: Detection of neuroblastoma cells during clinical follow up: advanced flow cytometry and rt-PCR for tyrosine hydroxylase using both conventional and real-time PCR.
abstract: OBJECTIVE
  Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary.
  
  
  METHODS
  We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC).
  
  
  RESULTS
  Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression.
  
  
  CONCLUSIONS
  TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.
authors.affiliation: Pädiatrische Hämatologie und Onkologie, Universitätsklinik Frankfurt, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Esser
authors.forename: W
authors.initials: W
authors.lastname: Glienke
authors.forename: K
authors.initials: K
authors.lastname: Bochennek
authors.forename: S
authors.initials: S
authors.lastname: Erben
authors.forename: A
authors.initials: A
authors.lastname: Quaiser
authors.forename: C
authors.initials: C
authors.lastname: Pieper
authors.forename: A
authors.initials: A
authors.lastname: Eggert
authors.forename: A
authors.initials: A
authors.lastname: Schramm
authors.forename: K
authors.initials: K
authors.lastname: Astrahantseff
authors.forename: M L
authors.initials: ML
authors.lastname: Hansmann
authors.forename: D
authors.initials: D
authors.lastname: Schwabe
authors.forename: T
authors.initials: T
authors.lastname: Klingebiel
authors.forename: U
authors.initials: U
authors.lastname: Koehl
mesh_terms: D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000235:genetics; D001853:Bone Marrow / Q000473:pathology; D045744:Cell Line, Tumor; D002648:Child; D003937:Diagnosis, Differential; D005189:False Positive Reactions; D005434:Flow Cytometry; D005500:Follow-Up Studies; D005729:Ganglioneuroma / Q000175:diagnosis / Q000235:genetics; D020869:Gene Expression Profiling; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D009367:Neoplasm Staging; D018365:Neoplasm, Residual / Q000473:pathology; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D009447:Neuroblastoma / Q000175:diagnosis* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
chemical_list: D020410:Activated-Leukocyte Cell Adhesion Molecule; D005819:Genetic Markers; D014446:Tyrosine 3-Monooxygenase