title: Method validation of circulating tumour cell enumeration at low cell counts.
abstract: BACKGROUND
  Circulating tumour cells (CTC) are receiving increasing attention as prognostic, predictive and pharmacodynamic biomarkers in cancer patients. However, their clinical significance can be dependent on an accurate determination of CTC around cut-off values at low cell counts (<10 cells/7.5 ml). Consequently, we have conducted method validation of the CellSearch™ system focusing on clinical samples containing CTC in the cut-off region.
  
  
  METHODS
  Analytical accuracy was first assessed employing quality controls (QC) and spiked healthy volunteer blood specimens. Results were analysed by β-expectation tolerance intervals (BETI). Inter-operator error (6 different readers) was then characterised in 38 different patient samples, 68% of which had ≤5 CTC and data were analysed by β-content γ-confidence tolerance intervals (BCTI).
  
  
  RESULTS
  Results from QCs and spiked blood confirmed a 3-4-fold higher degree of imprecision at the low (48 cells, BETI = + 0.288/-0.345, β = 95%) compared to the high QC (987 cells, BETI = +0.065/-0.140, β = 95%). However, when data for individual analysts were interrogated characteristic systematic errors were detected. In the analysis of patient samples again individual analysts introduced a highly specific error into the interpretation of CTC images, which correlated to the level of training and experience. When readers were selected based on BETI and BCTI results, the high level of between-operator error (up to 170%) observed at CTC of ≤ 5 was reduced to < 30%.
  
  
  CONCLUSIONS
  Inter-operator variability in enumeration of CTC at low cell counts can be considerable, but is also potentially avoidable by following simple guidance steps.
authors.affiliation: Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester Cancer Research Centre, University of Manchester, Manchester M20 4BX, UK. jcummings@picr.man.ac.uk.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cummings
authors.forename: Karen
authors.initials: K
authors.lastname: Morris
authors.forename: Cong
authors.initials: C
authors.lastname: Zhou
authors.forename: Robert
authors.initials: R
authors.lastname: Sloane
authors.forename: Matt
authors.initials: M
authors.lastname: Lancashire
authors.forename: Daniel
authors.initials: D
authors.lastname: Morris
authors.forename: Stephen
authors.initials: S
authors.lastname: Bramley
authors.forename: Matt
authors.initials: M
authors.lastname: Krebs
authors.forename: Leila
authors.initials: L
authors.lastname: Khoja
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
mesh_terms: D014408:Biomarkers, Tumor; D002452:Cell Count / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009369:Neoplasms / Q000175:diagnosis / Q000473:pathology*; D009360:Neoplastic Cells, Circulating; D011786:Quality Control; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor
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