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

Abstract

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 extracellularsignal-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 2808C. 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. ' 2012 International Society for Advancement of Cytometry

Bevacizumab combined with docetaxel, oxaliplatin, and capecitabine, followed by maintenance with capecitabine and bevacizumab, as first‐line treatment of patients with advanced HER2‐negative gastric cancer: A multicenter phase 2 study

“…Blood for the detection of CTCs was collected before treatment for patients enrolled at the Netherlands Cancer Institute. A fully validated fluorescence‐activated cell sorting assay was used to enumerate CTCs . The lower limit of quantification of the assay is 2 CTCs per 8 mL of blood .…”

“…A fully validated fluorescence-activated cell sorting assay was used to enumerate CTCs. 14 The lower limit of quantification of the assay is 2 CTCs per 8 mL of blood. 13 Therefore, samples with <2 CTCs per 8 mL of blood were considered CTC negative, and samples with 2 CTCs per 8 mL of blood were considered as CTC positive.…”

Circulating tumor cell technologies

“…This method uses specific biomarkers expressed on the cell surface (e.g., EpCAM and CD45) to capture cells. The antibodies used for selection are typically tethered to either the device surface or a magnetic substance (i.e., (Miltenyi et al, 1990;Pluim et al, 2012) MagSweeper EpCAM High purity, can process WB, 9 mL/h throughput Kim et al, 2014;Lohr et al, 2014;Powell et al, 2012;Talasaz et al, 2009 (Harb et al, 2013). mF, IM CTC-iChip EpCAM, Size Pos/neg enrichment, size-based separation debulks WB, inertial focusing aids in magnetic deflection, 8 mL/h (Karabacak et al, 2014;Ozkumur et al, 2013) IM, in vivo GILUPI CellCollectorä EpCAM Can process large volumes of blood (Saucedo-Zeni et al, 2012) Immunoaffinity e Negative Enrichment Antibodies targeting leukocyte-associated antigens are tethered to magnetic particles or the device surface deplete unwanted background cells.…”

“…The unique design of MACS technology, which involves strong magnetic fields generated across materials with a large surface area to volume ratio, allows efficient capture of desired cell populations. Studies have used MACS to capture CTCs from metastatic cancer patients to study p-ERK expression following ex vivo EGF stimulation (Pluim et al, 2012). In another clinical study both enrichment and depletion strategies were employed to capture and profile CTCs from HER2-positive breast cancer patients for EMT-related gene expression (Giordano et al, 2012).…”

Measurement of lymphocyte aggregation by flow cytometry–physiological implications in chronic lymphocytic leukemia

“…In CLL, flow cytometric approaches are utilized to assess genetic abnormalities as well as prognosis . Originally, flow cytometry was designed to analyze single cells under flow in a robust manner, but its uses have expanded during the last few decades to include the enumeration of microorganisms , measurements of soluble factors such as cytokines , identification of intracellular aberrant proteins , and the assessment of signaling responses . As we show herein, flow cytometry can address the essential need for the quantitative measurement of lymphocyte aggregation, pending on adequate working protocols and recognition of the limitations.…”