Thinprep preservcyt solution

De-identified blood samples were obtained from newly diagnosed advanced NSCLC patients before treatment with informed consents according to a protocol approved by Institutional Review Board (IRB) at Augusta University. All blood samples were collected into vacutainer tubes (BD, Franklin Lakes, NJ,) containing the anticoagulant K₂EDTA and were processed within 3 hours of blood draw. In a typical process, every 1 mL of whole blood was lysed by 10 mL of RBC lysis buffer for 5 minutes at room temperature. WBCs were then collected by spinning down the solution at 800×g for 5 minutes and the pellet was suspended in 1 mL of ferrofluid containing 0.1% (v/v) Pluronic F-68. The sample was then loaded into a 10-mL syringe (BD, Franklin Lakes, NJ,) followed by processing with the FCS device at a flow rate of 6 mL h⁻¹. A stainless-steel sphere (BC Precision, Chattanooga, TN) with a diameter of 1.6 mm was also loaded into a syringe. A magnet was used to gently agitate the sphere to prevent blood cells from settling down every 5–10 minutes. After separation, the FCS device was flushed by PBS or ThinPrep PreservCyt solution (Hologic, Marlborough, MA) at 30 mL h⁻¹ for 20 minutes to remove any cells in outlet reservoir. During the separation, the cells from outlet 6 of a FCS device were directly preserved in ThinPrep PreservCyt solution for further analysis.