title: Parallel affinity-based isolation of leukocyte subsets using microfluidics: application for stroke diagnosis.
abstract: We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as S100A9, TCRB, and FPR1, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated.
authors.affiliation: Department of Chemistry, Louisiana State University , Baton Rouge, Louisiana 70803, United States.
authors.forename: Swathi R
authors.initials: SR
authors.lastname: Pullagurla
authors.forename: Małgorzata A
authors.initials: MA
authors.lastname: Witek
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Jackson
authors.forename: Maria A M
authors.initials: MA
authors.lastname: Lindell
authors.forename: Mateusz L
authors.initials: ML
authors.lastname: Hupert
authors.forename: Irina V
authors.initials: IV
authors.lastname: Nesterova
authors.forename: Alison E
authors.initials: AE
authors.lastname: Baird
authors.forename: Steven A
authors.initials: SA
authors.lastname: Soper
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D000475:Alkenes / Q000737:chemistry; D015703:Antigens, CD / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D058851:GPI-Linked Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D016131:Lymphocyte Subsets / Q000737:chemistry*; D044085:Microfluidics / Q000379:methods*; D009504:Neutrophils / Q000737:chemistry; D011108:Polymers; D019904:Polymethyl Methacrylate / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D012972:Sodium Hydroxide / Q000737:chemistry; D020521:Stroke / Q000175:diagnosis* / Q000473:pathology
chemical_list: D000475:Alkenes; D015703:Antigens, CD; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D007202:Indicators and Reagents; D011108:Polymers; D012333:RNA, Messenger; D012972:Sodium Hydroxide; D019904:Polymethyl Methacrylate; D019840:2-Propanol