Microsieves for the detection of circulating tumor cells in leukapheresis product in non-small cell lung cancer patients

BACKGROUND: Circulating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard. METHODS: DLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×10(8) leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 µm pore microsieve and subsequently over a 5µm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA. RESULTS: Of 29 patients a median of 1.4 mL DLA product (range, 0.5–4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 µm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 µm but not 7 µm microsieves was positively correlated with fixation time (ρ=0.51, P<0.01). VyCAP detected CTC in 44% (12/27) of DLA products. Median CTC count per mL DLA was 0 [interquartile range (IQR): 0–1]. CellSearch detected CTC in 63% of DLA products (median =0.9 CTC per mL DLA, IQR: 0–2.1). CTC counts detected by CellSearch were significantly higher compared with VyCAP (P=0.05). CONCLUSIONS: VyCAP microsieves can identify CTC in DLA product, but workflows need to be optimized.