Development of a Fully Monolithic Microfluidic Device for Complete Blood Count by John Nguyen A thesis submitted in conformity with the requirements for the degree of Master of Applied Science (M.A.Sc.) Graduate Department of Mechanical and Industrial Engineering University of Toronto c Copyright 2014 by John Nguyen Abstract Development of a Fully Monolithic Microfluidic Device for Complete Blood Count John Nguyen Master of Applied Science (M.A.Sc.) Graduate Department of Mechanical and Industrial Engineering University of Toronto 2014 This thesis describes a monolithic microfluidic device capable of complete blood constituent enumeration from whole blood. For the first time, on-chip sample processing (e.g. dilution, lysis, and filtration) and downstream single cell analysis were fully integrated on device to enable complete blood cell count. The microfluidic device consists of two parallel sub-systems that perform sample processing and electrical analysis for simultaneous measurement of red (RBC) and white blood cell (WBC) parameters. The system provides a modular and adaptable environment capable of handling solutions of various viscosities and mixing ratios and features a new offset filter configuration for increased experimental duration. RBC concentration, mean corpuscular volume (MCV), cell distribution width, WBC concentration and differential are determined by electrical impedance measurements. Experimental characterization results of 97,305 WBCs and 104,735 RBCs from 10 patient blood samples demonstrate that the system is capable of performing high volume enumeration and complete blood count with accuracy. ii Acknowledgements I would like to thank Professor Sun for his guidance, perspective on life and passion for research. I would like to thank all the members of the Advanced Micro and Nanosystems Laboratory (AMNL), both past and present, for their support and company. I would also like to give special acknowledgments to all the members of the microfluidic group: Mark, Yi and Yuan for all of their experimental help, advice and long discussions. I would like to acknowledge the financial support I have received from the NSERC Create Program in Microfluidic Applications and Training in Cardiovascular Health (MATCH) and the Ontario Graduate Scholarship (OGS). I would like to thank my parents for everything they've provided me through the years. Lastly, thank you to my partner and best friend Jenny, for her never ending support, encour- agement and love. iii Contents List of Figures vi List of Tables x 1 Introduction 1 1.1 Background.......................................1 1.2 Blood separation on microfluidics...........................4 1.3 Microfluidic coulter counter..............................7 1.4 Fluidic Sample Processing for Blood.........................8 1.4.1 Dilution.....................................9 1.4.2 Lysis....................................... 10 1.5 Motivation....................................... 11 1.6 Thesis outline...................................... 13 2 System Overview 14 2.1 System Flow...................................... 15 2.2 Device Design...................................... 19 2.3 Filter Design and Implementation.......................... 22 3 Fabrication and Experimental Methods 28 3.1 Fabrication....................................... 28 3.2 Experimental Procedure................................ 30 3.3 Signal Analysis..................................... 30 iv 4 Experimental Results and Discussion 35 4.1 Device Characterization................................ 35 4.1.1 Pressure Independence............................. 35 4.1.2 Flowrate Control................................ 37 4.2 Red Blood Cell Analysis................................ 38 4.2.1 Red Blood Cell Enumeration......................... 38 4.2.2 Red Blood Cell Characterization....................... 39 4.3 White Blood Cell Analysis............................... 44 5 Conclusion 52 6 Future Directions 53 Bibliography 55 A Circuit Simulation 61 B Previous Revisions of Device Design 63 v List of Figures 1.1 Relative concentrations of blood constituents in a single µL of blood. Red blood cells - RBCs, white blood cells - WBCs and platelets are highlighted........4 1.2 Various techniques to isolate/separate blood cells..................5 1.3 System schematics from various microfluidic devices utilizing Coulter counter technology........................................9 1.4 Overview of existing microfluidic technologies with on-chip sample processing and integration with measurement units........................ 11 2.1 System Flow Chart. Whole blood is siphoned into two streams for RBC and WBC measurement. WBC analysis requires lysis and quench to eliminate trou- blesome RBCs before measurement.......................... 16 2.2 Device Overview. 3D and 2D schematic of different fluidic modules required for RBC and WBC analysis................................. 17 2.3 Raw RBC and WBC Impedance Peaks. Two 500 ms segments raw data show- ing detection threshold (green), enumerated cells circled red and highlighting effective electrical volume difference between WBCs (left) and RBCs (right)... 18 2.4 Simulation Results: Expected Impedance Change. Simulation results highlight- ing the impedance change of spherical cells of different diameters for various channel dimensions................................... 19 2.5 Fluidically Analogous Circuit Model. Model used to determine channel geome- tries and configuration to ensure precise fluidic mixing ratios. Subscripts denote fluidic resistors in various modules........................... 20 vi 2.6 Types of Debris.Images of various types of debris which can occlude constriction measurement channels and limit experimental throughput.............. 23 2.7 Summary of simulation results comparing unwanted fluidic resistance change as increasing percentage of orifices becomes clogged for existing and our new offset filter configurations................................... 24 2.8 Reconfigured Plug-In Electrodes. Schematic highlighting reconfiguration of con- ventional plug-in electrodes. Use of the new electrode placement as a reservoir for debris. Fluid flow can intermittently be adjusted to clear obstructions and increase device throughput............................... 26 2.9 Electrode Configurations. Schematic showing various electrode configurations used in previous microfluidics.............................. 27 3.1 Schematic of multi-layered fabrication sequence. Two SU-8 feature layers are used to generate channel heights of 15 µm and 40 µm............... 29 3.2 Images of device operation and device schematic highlighting key modules for RBC (right) and WBC (left) measurements. Device dimensions, RBC lysis, filtration and measurement are highlighted...................... 31 3.3 Limits of detection from RBC Analysis. Typical non-gated RBC histogram high- lighting limits of detection for low electrical volume cells. Platelets or particles sized lower than the limit of detection are ignored from analysis due to difficulties in distinguishing them from baseline noise....................... 32 3.4 Image of mixing channel for incubation post-lysis and quench.RBC ghosts and cellular components are visible and can contribute electrical interference. WBCs are circled........................................ 33 3.5 Baseline Correction Histogram. RBC size histograms for raw and post base- line correction of the windowed sample are presented.Corrected size histogram improves size accuracy and produces expected RBC distribution.......... 34 vii 4.1 Pressure Independence: Mixing Ratios. Relative ratios of the fluidic widths ( W1 ) were directly correlated to their relative volumetric flow rate. The use of WT an inlet-outlet configuration for serial dilution is used to enable the device to behave consistently independent of the applied pressure............... 36 4.2 Flow Metering. Effects of varying flow rates on basal impedance are observed. Linear trend indicates the possibility of using observed impedance change to approximate volumetric flow rate............................ 38 4.3 Effect of dilution ratio on RBC enumeration accuracy. Both maximum values and distribution shape of the size histogram are evidence of incorrect enumeration of clustered cells at lower dilution ratios. Concentration results for varying dilution ratios, compared with reference concentration measured by hematology analyzer. 40 4.4 RBC Deformability and its effect on MCV. Size histograms change in terms of µ and distribution for various pressures due to increasing RBC deformation.... 41 4.5 RBC Characterization Results: Size histograms highlighting specific measured RBC indices: distribution width (RDW-SD) and mean corpuscular volume (MCV). Correlation plot for reference hematology analyzer vs. microfluidically measured MCV........................................... 43 4.6 Concordance and Bland-Altman Plots for RBC Enumeration............ 44 4.7 WBC Subtype Size Difference. Optical confirmation of size difference of different WBC subtypes (e.g. lymphocytes, neutrophils and monocytes)........... 45 4.8 Sample Multi-Frequency Measurements for WBC Analysis. 10 kHz to 990 kHz signals were used for WBC analysis. Lower frequency provides purely size infor- mation, while higher frequencies give insight on intracellular properties...... 46 4.9 Sample Scatter Graphs from WBC Analysis. Shown are opacity and electri- cal volume measurements from healthy patient samples of various lymphocyte populations (8.3%-31.8%)............................... 47 4.10 Concordance and Bland-Altman