Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular-signal-regulated kinase and DNA in circulating tumor cells

A simple, selective, and sensitive multiparameter fluorescence activated cell sorting method utilizing density gradient centrifugation and magnetic antibody cell sorting was developed and validated for the determination of phosphorylated extracellular-signal-regulated kinase (pERK) and DNA in circulating tumor cells (CTCs). Cell preparation tubes (CPT) were used for peripheral blood collection and density gradient centrifugation, followed by phosphorylation of ERK with epidermal growth factor (EGF). After fixation with formaldehyde and methanol, magnetic anti epithelial cell adhesion molecule (EpCAM) micro-beads were used for the selective isolation of CTCs from the background, consisting of peripheral blood mononuclear cells and platelets. Subsequently, samples were stained with Hoechst 33342, and fluorescent antibodies against EpCAM, CD45, and pERK. Flow cytometry was used for identification and enumeration of CTCs and determination of their pERK and DNA content. The validation parameters included specificity, recovery, linearity, precision, sensitivity, and stability. The lower limit of quantification was two CTCs per 8 ml peripheral blood. Samples were stable for 4 months in storage at -80°C. The applicability of the method was demonstrated by successful enumeration of CTCs, and the determination of DNA, and pERK before and after stimulation with EGF in 8 ml peripheral blood samples from patients with metastatic cancer.