title: Rapid automated cell quantification on HIV microfluidic devices.
abstract: Lab-chip device analysis often requires high throughput quantification of fluorescent cell images, obtained under different conditions of fluorescent intensity, illumination, focal depth, and optical magnification. Many laboratories still use manual counting--a tedious, expensive process prone to inter-observer variability. The manual counting process can be automated for fast and precise data gathering and reduced manual bias. We present a method to segment and count cells in microfluidic chips that are labeled with a single stain, or multiple stains, using image analysis techniques in Matlab and discuss its advantages over manual counting. Microfluidic based cell capturing devices for HIV monitoring were used to validate our method. Captured CD4(+) CD3(+) T lymphocytes were stained with DAPI, AF488-anti CD4, and AF647-anti CD3 for cell identification. Altogether 4788 (76 x 3 x 21) gray color images were obtained from devices using discarded 10 HIV infected patient whole blood samples (21 devices). We observed that the automatic method performs similarly to manual counting for a small number of cells. However, automated counting is more accurate and more than 100 times faster than manual counting for multiple-color stained cells, especially when large numbers of cells need to be quantified (>500 cells). The algorithm is fully automatic for subsequent microscope images that cover the full device area. It accounts for problems that generally occur in fluorescent lab-chip cell images such as: uneven background, overlapping cell images and cell detection with multiple stains. This method can be used in laboratories to save time and effort, and to increase cell counting accuracy of lab-chip devices for various applications, such as circulating tumor cell detection, cell detection in biosensors, and HIV monitoring devices, i.e. CD4 counts.
authors.affiliation: Bio-Acoustic MEMS in Medicine Laboratory, Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA 02139, USA.
authors.forename: Mohamad A
authors.initials: MA
authors.lastname: Alyassin
authors.forename: SangJun
authors.initials: S
authors.lastname: Moon
authors.forename: Hasan O
authors.initials: HO
authors.lastname: Keles
authors.forename: Fahim
authors.initials: F
authors.lastname: Manzur
authors.forename: Richard L
authors.initials: RL
authors.lastname: Lin
authors.forename: Edward
authors.initials: E
authors.lastname: Hæggstrom
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Kuritzkes
authors.forename: Utkan
authors.initials: U
authors.lastname: Demirci
mesh_terms: D017252:CD3 Complex / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology; D018791:CD4 Lymphocyte Count / Q000295:instrumentation / Q000379:methods; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D015658:HIV Infections / Q000097:blood* / Q000276:immunology; D006801:Humans; D018655:Lymphocyte Count / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D046210:Microfluidic Analytical Techniques / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017252:CD3 Complex; D015704:CD4 Antigens