Abstract 211: Spin coated peripheral blood film reveals tumor heterogeneity in circulation
Authors: Cheng Andy , Xu Leo , Hong Bin 
Published: August 2018

Abstract
Peripheral blood film (PBF) is an important laboratory workup for clinical characterization of haematological malignancies that involves blood smear on microscope slide and cytology of blood cells. PBF preparation, typically manually operated, requires technical training and empirical experience and is not without human errors. Automated blood smearing by traditional spin coating has been an alternative. However, with intended use originally for semiconductor industries, it utilizes volatile organic solvents, employs powerful centrifugation at high speed, which frequently alters morphology and damages cells, and inherently creates radius dependent pattern. In addition, manual blood dispensing could bring contamination and spin-off of excess blood as aerosol can be hazardous. While improvement of traditional spin coating technology to overcome intrinsic defects and compensate operational variations is possible, low-speed spin coating is more predicable and controllable for uniform PBF thickness. An innovative automatic PBF coating system has been developed to smear monolayer of dispersed blood cells for cytopathology and thick PBF of multilayered blood cells as well for identification, enumeration, and examination of rare tumor cells in peripheral blood. Explicitly, whole blood is dispensed through nozzle directly from the blood collection tube that slides radially on rails above substrate, and coated on the spinning substrate at low speed in non-contact mode circle-by-circle with no intra/inter- circular overlaps and negligible centrifugal force. Consequently, there is no spin-off, saving blood samples and preventing potential biohazard. External contamination is also eliminated by dispensing blood directly from the tube. Temperature control of substrate is further enabled to preserve cellular viability and morphology. At speed of 60 rpm, contact angle of 10°, stepper of 0.5 mm, and room temperature, monolayer PBF with intact cellular morphology across the entire substrate was created using healthy fresh whole blood directly from the BD Vacutainer K2-EDTA tube. To study tumor cell heterogeneity in circulation, thick PBFs have been evenly coated on 100 mm diameter discs using 2 mL of pre-stained, cancer patient blood samples at speed of 30 rpm, contact angle of 45°, stepper of 1 mm and elevated temperature of 37°C and fully dried. PBFs were then scanned by our innovative Spinning Disc Imaging system (iSDI) to pinpoint fluorescent nucleated cells of epithelial origin. Images of suspects were automatically displayed with identities of location, area, orientation, width, height, and signal intensity. Results demonstrated tumor cells with variable sizes, shapes and expression levels of EpCAM, in addition to clusters and fragments. Despite advances in genetic and molecular diagnostics, PBF remains an indispensable part of diagnostic jigsaw in unraveling heterogeneity and evolution of tumor in liquid phase.
Citation Format: Andy Cheng, Leo Xu, Bin Hong. Spin coated peripheral blood film reveals tumor heterogeneity in circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 211.