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Open Access Technical advance

Method validation of circulating tumour cell enumeration at low cell counts

Jeffrey Cummings*, Karen Morris, Cong Zhou, Robert Sloane, Matt Lancashire, Daniel Morris, Stephen Bramley, Matt Krebs, Leila Khoja and Caroline Dive

Author Affiliations

Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester Cancer Research Centre, University of Manchester, Manchester M20 4BX, UK

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BMC Cancer 2013, 13:415  doi:10.1186/1471-2407-13-415

Published: 11 September 2013

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.

Keywords:
Circulating tumour cells; Predictive biomarker; Enumeration; Low cell counts; Method validation; Statistical analysis