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Master's Thesis. Size based platelet isolation on a centrifugal microfluidic device. Dong Yeob Ki Department of Biomedical Engineering. Graduate School of UNIST 2018.

Size based platelet isolation on a centrifugal microfluidic device. Dong Yeob Ki. Department of Biomedical Engineering. Graduate School of UNIST.

Size based platelet isolation on a centrifugal microfluidic device. A thesis/dissertation submitted to the Graduate School of UNIST in partial fulfillment of the requirements for the degree of Master of Science. Dong Yeob Ki. 01/04/2017 Approved by _________________________ Advisor Yoon-Kyoung Cho.

Size based platelet isolation on a centrifugal microfluidic device Dong Yeob Ki. This certifies that the thesis/dissertation of Dong Yeob Ki is approved. 01/04/2017. ___________________________ Advisor: Yoon-Kyoung Cho. ___________________________ Yoon-Kyoung Cho. ___________________________ Jiyun Kim. ___________________________ Joo Hun Kang.

Abstract Cardiovascular diseases are one of the leading causes of disability and mortality worldwide and directly associated with the enhanced reactivity of platelets. Platelet is the smallest (1 ~ 4 µm) cells in the circulation, comprising the second largest volume fraction of blood, responsible for the maintenance of the circulatory systems in normal condition. However, platelets can significantly contribute to the formation of thrombus which blocks the blood flow if hyper-functional, or result in bleeding, if dysfunctional. Aside from its hemostatic role, platelets are also involved in other essential and versatile functions of immunity, wound healing, and inflammation. Its pathophysiological involvements in cancer, Alzheimer’s disease, cardiovascular disease, diabetes and viral infections were recently established and have gained much attention for its potential use in both diagnostics and therapy. Conventional platelet isolation uses density-based centrifugation, which lacks global standard, hence, high variations that influence clinical decision have been reported. In addition, platelet activation due to high shear stress during the centrifugation, and WBC contamination limited the use of platelet in bioassays for protein quantification and RNA analysis. The conventional isolation approach also suffers from handling errors, long processing time, and labour intensiveness. Hence, microfluidics based technological interventions have been developed to overcome above limitations but was not able to achieve the optimal platelet isolation of uncompromised high purity, throughput, and recovery with minimal activation in short time from undiluted whole blood. Still, isolation of platelets for molecular diagnosis in small blood volume remains a challenging task. Therefore, we developed a fully automated lab-on-a-disc device to isolate platelets for downstream analysis. By integration of sequential filtration on disc with 3 µm and 600 nm pore size membranes, highly pure platelet isolation was achieved. From our results, the disc based isolation significantly increased platelet count by 3~4 fold, while simultaneously lowering activation even in the absence of inhibitors. The flow cytometry and RT-PCR analysis of isolated platelets revealed that our disc platform results in ~ 99 % pure platelets free from WBC contamination having WBC specific gene undetected. In summary, the experimental result confirmed that disc is capable of separating platelets ideal for downstream analysis having high purity and recovery with minimal activation in time efficient manner. Prior to downstream analysis, platelet function test for screening of multiple platelet related disorders or conditions are commonly requested in clinical settings. As a proof of concept, we demonstrated the potential of full integration of light transmission aggregometry - the reference gold standard of addressing platelet function - on lab on a disc platform as a point of care testing device to improve time efficiency, overall cost, and higher precision without restriction to the staff and facilities. 5.

Table of contents CHAPTER 1 Introduction ................................................................................................................. 13 1.1 Platelet ................................................................................................................................. 13 1.1.1 Characterization of platelets in physiological and pathophysiological aspects ............ 13 1.1.2 Pathological aspects of platelet dysfunction ................................................................. 16 1.1.3 Role of platelet in the progression of chronic inflammatory disease ........................... 16 1.1.4 Conventional platelet isolation method ........................................................................ 18 1.1.5 Platelet function testing ................................................................................................ 21 1.2 Microfluidic systems for isolating platelets ........................................................................ 24 1.2.1 Acoustophoresis ............................................................................................................ 24 1.2.2 Dielectrophoresis .......................................................................................................... 24 1.2.3 Inertial focusing ............................................................................................................ 25 1.2.4 Hydrophoresis............................................................................................................... 25 1.2.5 Microfluidic pattern ...................................................................................................... 25 1.3 Centrifugal microfluidics .................................................................................................... 28 1.3.1 Theory........................................................................................................................... 28 1.3.2 Unit operations ............................................................................................................. 28 1.3.3 Fully integrated lab on a disc system for biomedical applications ............................... 30 1.3.4 Size based filtration on a disc ....................................................................................... 31 1.4 Research outline .................................................................................................................. 32 1.4.1 Objective of the research .............................................................................................. 32 1.4.2 Outline of the thesis ...................................................................................................... 33 CHAPTER 2 Experimental methods & materials ............................................................................. 34 2.1 Sample preparation.............................................................................................................. 34 2.1.1 Chemicals and reagents ................................................................................................ 34 2.1.2 Blood specimen collection and processing ................................................................... 35 6.

2.2 Device fabrication ............................................................................................................... 37 2.3 Operation setting ................................................................................................................. 41 2.3.1 Visualizing machine for centrifugal microfluidic operation system ............................. 41 2.3.2 Imaging and optical measurement system .................................................................... 41 2.3.3 Flow cytometry based platelet analysis ........................................................................ 43 2.3.4 RNA extraction and RT-PCR. ....................................................................................... 44 CHAPTER 3 Platelet isolation on a centrifugal microfluidic device ................................................ 45 3.1 Disc design .......................................................................................................................... 45 3.2 Disc operation protocol ....................................................................................................... 49 3.3 Optimization of device and operation conditions................................................................ 52 3.3.1 RPM & time of plasma separation on disc ................................................................... 52 3.3.2 Filter set ........................................................................................................................ 54 3.3.3 Number of washing steps ............................................................................................. 56 3.3.4 FAST or non-FAST ....................................................................................................... 58 3.4 Comparison with manual separation method ...................................................................... 60 3.4.1 Flow cytometry based comparison for purity, activation and relative count ................ 60 3.4.2 RT-PCR based platelet gene expression ....................................................................... 63 3.5 Conclusion........................................................................................................................... 64 CHAPTER 4 Platelet function test / LTA on centrifugal microfluidic device................................... 66 4.1 Literature surveys on light transmission aggregometry ...................................................... 66 4.2 Integration of LTA on a disc ................................................................................................ 69 4.2.1 Disc design of LTA on a disc ........................................................................................ 69 4.2.2 Flow operation on disc ................................................................................................. 71 4.3 Operation protocol for sample analysis ............................................................................... 72 4.4 Optimization of assay protocol ........................................................................................... 72 4.4.1 Optimization of mixing conditions ............................................................................... 74 4.4.2 Effect of anticoagulant on LTA..................................................................................... 74 7.

4.4.3 Preparation and measurement on disc .......................................................................... 76 4.5 Conclusion........................................................................................................................... 78 CHAPTER 5 Concluding remarks .................................................................................................... 79 5.1 Summary of the work .......................................................................................................... 79 5.2 Strength and limitation ........................................................................................................ 79 5.3 Future prospect .................................................................................................................... 80 Reference .......................................................................................................................................... 84 Acknowledgements ........................................................................................................................... 91 Curriculum Vitae ............................................................................................................................... 92. 8.

List of figures Figure 1 Stages of platelet mediated hemostasis................................................................................... 15 Figure 2 Workflow of conventional platelet isolation method in schematic illustration ...................... 19 Figure 3 The representative illustration of the two major principles of classical methods of PFT....... 21 Figure 4 Examples of platelet separation in microfluidic chip based technologies .............................. 26 Figure 5 Operation method of three different types of valves .............................................................. 29 Figure 6 Fully integrated lab on a disc system for diagnostic applications .......................................... 30 Figure 7 Size based liquid biopsy sample preparation on lab on a disc platform ................................. 31 Figure 8 Machines for disc layer and parts fabrication ......................................................................... 38 Figure 9 Exploded view of filter membrane integration on the fabricated and assembled Disc. ......... 39 Figure 10 Post-milling surface polishing methods and proper tool selection ....................................... 40 Figure 11 Photograph of customized instruments for on disc operation with labels ............................ 42 Figure 12. Schematic diagram of the detailed features of the device for platelet isolation .................. 47 Figure 13 Illustration of the disc assembly layers for platelet isolation................................................ 48 Figure 14 Flow operation of platelet isolation and purification on disc with CCD image ................... 51 Figure 15 Effect of time exposure at constant angular velocity for plasma separation ........................ 55 Figure 16 Effect of different filter sets on platelet isolation on a disc .................................................. 53 Figure 17 Effect of number of washing step on platelet isolation ....................................................... 57 Figure 18 Effect of FAST in platelet isolation on disc. ......................................................................... 59 Figure 19 Comparison data of manual and disc method on platelet isolation. ..................................... 61 Figure 20 Comparison of two methods using acquired FACS data ...................................................... 62 Figure 21 Difference in the coefficient of variation in platelet count for 7 sets ................................... 63 Figure 22 The RT-PCR of extracted RNA from isolated platelets ........................................................ 65 Figure 23 Representative illustration of workflow in sample preparation steps for LTA ..................... 68 Figure 24 Exploded view of disc assembly of LTA on a disc ............................................................... 70 Figure 25 Schematic diagram of flow operation of LTA on disc in order. ............................................ 71 9.

Figure 26 The LTA on 96 well plate with different concentration of ADP ........................................... 73 Figure 27 Optimization of mixing condition for positive control ......................................................... 75 Figure 28 Effect of anticoagulant on platelet aggregation .................................................................... 77 Figure 29 Effect of PRP preparation method (disc and manual method) for LTA ................................ 77 Figure 30 Testing of on-disc optical measurement with spectrophotometer. ........................................ 78 Figure 31 Future application of total platelet profiling using lab on a disc .......................................... 81. 10.

List of tables Table 1 Examples and comparison of platelet function tests ................................................................ 23 Table 2 Summary of different microfluidic methodologies applied for platelet isolation .................... 27 Table 3 Characterization of different types of valve in centrifugal microfluidic platform ................... 29 Table 4 The operation program for the disc-based platelet isolation. ................................................... 50 Table 5 List of four forward and reverse primer sets used for RT-PCR of platelet RNA ..................... 64. 11.

List of abbreviations Abbreviation. Definition. ADP. Adenosine diphosphate. APC. Allophycocyanin. CVD. Cardiovascular Disease. CTC. Circulating Tumor Cells. CV. Coefficient of variation. CD. Cluster of differentiation. DCM. Dichloromethane. EDTA. Ethylenediaminetetraacetic acid. FBS. Fetal Bovine Serum. FAST. Fluid Assisted Separation Technology. FACS. Fluorescence-Activated Cell Sorting. FITC. Fluorescein Isothiocyanate. FSC. Forward Scatter. GP. Glycoprotein. LOAD. Lab on a Disc. LTA. Light Transmission Aggregometry. MACE. Major Adverse Clinical Outcomes. MFI. Mean Fluorescence Intensity. OD. Optical Density. PBS. Phosphate-Buffered Saline. PE. Phycoerythrin. PFD. Platelet Function Disorders. PFT. Platelet Function Testing. PRP. Platelet Rich Plasma. PPP. Platelet-Poor plasma. POCT. Point of Care Testing. PC. Polycarbonate. PGE1. Prostaglandin E1. RT-PCR. Reverse Transcription Polymerase Chain Reaction. RPM. Revolutions per minute. RNA. Ribonucleic acid. SSC. Side Scatter. TRAP. Thrombin-Receptor-Activated Protein. TXA2. Thromboxane 2. TEPC. Track-Etched Polycarbonate 12.

CHAPTER 1 Introduction 1.1 Platelet. Normal closed circulation of the blood is critical and essential for several physiological functions in human health, and this vascular integrity is maintained by the versatile crucial hemostatic role of platelets. Platelet, also called thrombocyte, was first discovered by Bizzozero1 in the 1880s, known for its major contribution in blood clot formation by generating hemostatic plug called “thrombi”. The thrombotic disorder derived from either hyper-function or dysfunction of platelet activities hold significant value in treatment and diagnosis.2. 1.1.1 Characterization of platelets in physiological and pathophysiological aspects Physical characteristics of platelets Under physiological conditions, circulating platelets are small, discoid, anuclear cell fragments 14 µm in diameter (average of 2 µm) and 0.5 µm in height. Platelets are present in concentrations of 150,000 ~ 400,000 cells per microliter of blood and occupy the second largest volume fraction of blood. They originate from megakaryocytes in bone marrow and lung,3 each of which can produce 10 million platelets per hour and circulate for 8-10 days in the bloodstream.4 Platelet reactivity depends on the active surface receptors including platelet specific surface markers like CD41, CD61, CD42a and CD42b of which alters the expression cue to different disease induced signaling. Key features of the platelet's structure and function include a variety of externally presented surface receptors as well as internally stored clotting factors. Platelet cytoplasm contains three major types of granule: -granules, dense granules, and lysosomes.5 Alpha ( -) granules (50-80 per platelet; 200~500 nm), the most abundant granules in the platelets, contains more than 300 biomolecules like cytokines, growth factors. The main protein cargoes are platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), platelet factor 4 (PF4), von Willebrand Factor (vWF), fibrinogen, thrombospondin (TSP1)/CD36, glycoprotein (GPIIbIIIa), thromboxane 2 (TXA2) etc., including P-selectin (CD62P) that is translocated onto the platelet surface membrane once it is activated and alpha granule is released.6 Dense granules (3~8 per platelet; ~150 nm) contain small molecules like calcium, nucleotides (ADP, ATP) and serotonin (5-HT), that participate in vasoconstriction, inflammation and platelet aggregation. It also contains several membrane proteins like CD63 that is expressed on platelet surface membrane upon activation2, 7. 13.

Lysosomes (0~3 per platelet; 200~250 nm) contain acid hydrolases and proteases for digestion of phagocytic comoponents including pathogens, degradation of extracellular matrix, and clearance of fibrin and platelet thrombi2, 7. Hemostasis Hemostasis, a physiological response to stop the bleeding from blood vessels, is critically influenced by the key participation of platelets.8-9 The participation of platelets in hemostasis can be subdivided into three major steps: adhesion, activation, and aggregation. Adhesion/ Initiation: Platelets circulate near the healthy endothelium of vessel wall in a resting state unless the damaged endothelial cells consequentially release and expose sub-endothelial matrix proteins like tissue factor, collagen, and vWF that provide attachment site of platelets by the external surface receptors such as GPIb and GPIIb/IIIa (Figure 1.1). Activation/ Extension: Once platelets adhere, triggered platelets get activated by the agonists also secreted by activated platelets. Following adhesion, agonists either stationary like collagen or mobile like thrombin, adenosine diphosphate (ADP), and TXA210 from the activated platelets, amplify hemostatic response by increasing the number of activated platelets near the damaged site in the autocatalytic positive feedback loop. Multiple singaling pathways exist in agonist-induced platelet activation with specific receptors. The N-terminal peptide of the cleaved thrombin, for example, binds to protease activated receptor 1 and 4 (PAR1 and PAR4)11 while ADP binds to P2Y1 and P2Y12 receptors. TXA2 – is converted from arachidonic acid by cyclooxygenase (COX)- binds to thromboxane receptors (TP-α and TP-β)12. Once platelets get activated in response to a cumulative interaction of agonists, their morphology change from discoid to pseudopod (spreading) and secrete granular contents. CD63, P-selectin (CD62P) and phosphatidylserine (PS) expression, and conformational change of integrin GPIIb/IIIa (upon activation allows binding of PAC-1 and fibrinogen) are expressed on the activated platelet membrane13 (Figure 1.2). Antiplatelet therapy drugs usually target the receptors to prevent the serial activation of platelets. The most common antiplatelet agent, aspirin (also called acetylsalicylic acid (ASA)) inhibits the biosynthesis of TXA2 by disrupting the COX enzyme responsible for the conversion process. Since the COX enzyme is not produced in platelets, the effect of drug permanently maintained in aspirin treated platelet. The second most commonly known oral antiplatelet drug, clopidogrel, blocks the action of ADP on the P2Y12 receptors.14 Blockage of activated GPIIb/IIIa by abciximab, a monoclonal antibody, also inhibit the platelet aggregation.. 14.

Aggregation/ Stabilization: Activated platelets with a converted GPIIb/IIIa receptor having high affinity and stable binding toward fibrinogen, VWF, and fibronectin accumulate and pile up as an aggregate to form platelet plug (primary hemostasis). The aggregate forms a gradient of densely packed fully activated platelets in the core and transition zone covered by decreasing number of activated platelets in layers. The packing of activated platelets is attributed to their significant morphological rearrangement from discoid shape to dendritic or spread “fried egg” morphology with a highly exposed surface area (Figure 1.3). The aggregate catalyzes thrombin by the cascade plasma coagulation. In the process of coagulation, fibrin bound to activated GP IIb/IIa stabilizes the aggr egate and stop the bleeding (secondary hemostasis) by forming clot.. Figure 1 Stages of platelet mediated hemostasis. The sequential steps of platelet based primary hemostatic response cascade is comprised of: Adhesion (initiation) of platelet onto the endothelial. damage. site. (binding. with. subendothelial matrix proteins to integrin exemplified. by. the. collagen).. Activation. (extension) upon binding and release of granular contents, the soluble agonists (ADP and TXA2) to induce activation of nearby platelets involving change of shape, and aggregation (stabilization) formed by pack of recruited activated platelets to plug the bleeding injury site. *During the activation, the granular proteins like P-selectin (CD62P) and CD63 are translocated to the surface membrane of the platelets as well as conformational change of integrin like GP IIb/IIIa that enhance binding to the fibrinogen and stabilizes the clot.. 15.

1.1.2 Pathological aspects of platelet dysfunction Platelet dysfunction may arise from several defects in morphology, receptors, granule and cytoskeleton required for normal platelet function and can be categorized as either inherited or acquired with a heightened risk of bleeding. Inherited platelet disorders Inherited platelet function disorders (PFDs) generally categorized by the defective compartment of the platelets resulting in a different degree of bleeding. Abnormalities of the receptors for adhesive proteins due to the mutations in glycoprotein receptors are rare but well-defined disorders like Glanzmann thrombasthenia (GPIIb/IIIa) and Bernard-Soulier syndrome (GPIb-IX-V). The abnormalities of platelet granules also called inherited platelet secretion disorders (IPSD) or storage pool disorders are noted by the defective release of alpha granules and/or dense granules. For instance, δ-storage pool disease and gray platelet syndrome (GPS) causes impaired synthesis of platelet granules.15-16 The defective platelets eventually result in mild to severe bleeding that is critical for neonates and children as well as adults with accidents or surgery. Acquired platelet disorders Platelet dysfunction can be acquired by inappropriate medications, medical conditions, or other pathological conditions. In cases of significant cardiovascular risks, the patients are prescribed with antiplatelet therapy for primary prophylaxis of vascular events and recurrent thrombotic events. The antiplatelet therapy is commonly administered as a combination of aspirin, and the thienopyridines (clopidogrel or prasugrel) that inhibit ADP-induced platelet aggregation particularly for the patients with secondary prevention of cardiovascular diseases (CVD), atrial fibrillation, and coronary artery stents.17 However, the decrease in ischemic strokes with dual antiplatelet therapy also resulted in major bleeding issues. Finding optimal dosage for long-term treatment remains a challenge in consideration to insensitivity and acquired resistance towards antiplatelet therapy and personal factors like age, sex race contributing to the difference of platelet reactivity among the patients. To monitor anti-platelet therapy, a number of platelet function tests has been developed and these examine the ability of platelets in forming aggregate for CVD severity after drug intake.18-19. 1.1.3 Role of platelet in the progression of chronic inflammatory disease Platelet hyperreactivity inclusive of thrombocytosis (increase in the number of platelets) is known to critically participate in the progression of a variety of inflammatory diseases and the major leading causes of deaths like CVD and cancer as well as neurological disorders.20 The potentials of 16.

platelet as a promising diagnostic and therapeutic target as well as drug delivery tool for a variety of pathophysiology are currently under development for clinical use. Cardiovascular disease (CVD) CVD is the leading cause of mortality worldwide, representing 31%, (estimated 17.7 million people) of death globally in 2015 and about 10 million new cases are diagnosed annually.21 CVD is a group of heart and blood vessel related disorder, in which heart attack or myocardial infarction comprises 7.4 million death and stroke 6.7 million. The most common cause of the CVD lies in either vascular occlusion by a formation of atherosclerotic plaque preventing blood flow or bleeding from the blood vessel, called hemorrhage, both of which closely related and influenced by platelet function. Atherosclerosis is characterized by deposition of lipid (mainly low-density lipoprotein (LDL)) followed by leukocyte infiltration and the accumulation of dysregulated platelets under high shear stress. Moreover, platelet hyperreactivity, having increased activation and aggregation, enhances the risk of atherothrombotic diseases.22 Cancer progression and metastasis Metastasis comprises the major cause of cancer-associated mortalities and is strongly assisted by platelets that surround circulating tumor cells (CTCs) to void immune surveillance and deliver biologically active molecules to mediate epithelial mesenchymal transition (EMT).23 In addition, the depletion of platelets demonstrated in pulmonary metastasis mouse model resulted in total blockage of cancer progression.24 Tumor cell induced platelet activation (TCIPA) according to previous studies on platelet-cancer crosstalk denotes three significant features that are deeply involved in cancer survival: 1. Cancer cell mimic the platelet-specific surface integrin proteins, especially integrin αIIbβ3, P-selectin glycoprotein ligand 1 (PSGL-1), C-type lectin-like receptor-2 (CLEC-2), and P-selectin, to facilitate adhesion and interaction with platelets. 2. Secretion of the biological factors for angiogenesis through VEGF, dopamine, serotonin, etc., 3. The release of stored chemokines to augment tumor growth and promote invasive behavior by transforming growth factor beta 1 (TGFB1), lysophosphatidic acid (LPA), platelet derived growth factors (PDGF) and platelet factor 4 (PF4). Platelet has been demonstrated as a liquid biopsy marker for cancer diagnosis through RNA,25 alteration of ultrastructure,26 and quantitative diffrerence of protein among the healthy controls, the benign and the metastatic cancer patients.27 Alzheimer’s disease Alzheimer's disease (AD), the worldwide neurodegenerative disorder affecting nearly 45 million people, caused by the deposition of misfolded amyloid- β (Aβ) and intracellular neurofibrillary tangles in brain parenchyma and cerebral vessels.28 The strong correlation of alterations in the 17.

vascular system among AD patients has recently been investigated. Amyloid- β peptides are generated from amyloid precursor protein (APP) of neurons (695 amino acid) and non-neuronal cells like platelets (751 & 770 a.a). Platelets are known to contain more than 90% of circulating APP and enzymes (α -, β -, and γ -secretases.) required for cleaving APP.29 Alteration of platelet function and structure and significant modifications in platelet APP isoform ratio, ADAM10 (αsecretase), BACE1 (β -secretase) have been reported and distinctively differed from healthy control.30 The major Aβ (1-40) peptide produced from platelets deposited in cerebral vessel resulting in cerebral amyloid angiopathy (CAA).31 Interestingly, patients with Glanzmann’s thrombasthenia, which inhibits platelet aggregation due to the absence or mutated GPIIb/IIIa, do not suffer from neurodegenerative disease including AD and this phenomenon suggests the essential participation of platelet in the progression of the neurological disorder.32. 1.1.4 Conventional platelet isolation method The conventional way of isolating platelet by density-based double centrifugation is widely accepted and utilized, however, the platelets exposed to high centrifugal force are damaged and activated in the time-consuming labor intensive process of isolation, limiting the utilization for further studies and applications in diagnosis. Especially for the platelet gene expression studies, conventional isolation method may post three major problems in isolating pure platelet population from whole blood: 1. Platelet activation; 2. Infiltration of WBCs, which contains ~10,000 times RNA content than platelets and RBCs; 3. Time-dependent mRNA degradation.33 The procedure of conventional platelet isolation follows general protocol of first soft spin (generally 100 g ~ 500 g) for 10 ~ 20 min to retrieve platelets in plasma called platelet rich plasma (PRP) without touching the buffy coat after sedimenting RBCs and WBCs and subsequently induce hard spin (generally 800~2500 g) to pellet down platelets34 (Figure 2). The supernatant plasma is discarded and platelet pellet is resuspended to appropriate buffer solution. The centrifugal force and time in the conventional method of platelet isolation can vary significantly between laboratories and not well standardized to remove discrepancies.35 Furthermore, platelets are sensitive to inappropriate sample handling complicated with a lot of pre-analytical variables and may result in artifactual activation. To prevent platelet activation in the process, inhibitors like apyrase, nitric oxide, prostaglandin E1 or prostacyclin are added and the inhibitors increase the level of synthesized cyclic nucleotides that stimulate cAMP and cGMP dependent protein kinase to phosphorylate a lot of substrate proteins and eventually block platelet activation.36 Platelets with defective or less sensitive cyclic nucleotide 18.

signaling in ischemic heart disease, diabetes, rare genetic abnormalities that may result in abnormal cAMP level and remain insensitive to inhibitors. With uncertainty of the percentage of the activated platelets, quantative analysis may mislead the results in detecting platelet biomarkers and measuring platelet function. To overcome the obstacles in isolating purified platelets, conventional method of platelet isolation should be further developed to systematize a rapid, cost-effective, and gentle isolation resting platelets from a small volume of blood without any residual leukocytes and red blood cells and without sample treatment (such as adding inhibitors) or processing33 in a fully automated manner to ensure less handling error. Since platelets are sensitive to the properties of the binding substrate such as stiffness and surface coating, and shear stress generated by centrifugal force as well as temperature and time-dependent degradation of cellular content, these factors may also have to be considered as pre-analytical factors affecting the status of isolated platelets in conventional method.. Figure 2 Workflow of conventional platelet isolation method in schematic illustration. After blood collection, the centrifugation based platelet isolation method generally follows the scheme of first spin to separate PRP and transfer of PRP without touching buffy coat layer followed by the second spin to pellet down platelets and remove supernatant plasma. Each steps require centrifugation of 10~20 min and consistently exposed to pre-analytical factor like handling error. Lastly, suspend the platelets in elution solution (e.g. HEPES Tyrode’s buffer, or RNA stabilization solution) preferably after washing. 19.

Factors influencing the platelets during isolation process Centrifugal force induced shear and pressure Platelet aggregation at the site of vascular injury driven by rheological changes of blood flow parameter was proven using a microfluidic model in which rapid acceleration of shear generated by stenosis.37 Change in shear and acceleration results in membrane tethering of discoid platelets that in subsequent deceleration, enhances binding strength and stabilizes the aggregate formed by tether restructuring.38 Shear rate in normal veins and arteries is maintained below 2000 s-1 equivalent wall shear stress of 0.35-70 dynes/cm2 under normal conditions but may reach up to 40,000 s-1 with severe stenosis vasculature in atherosclerotic arteries and above 1500 dynes/cm2.39 It is reported that increasing centrifugal force and the duration of centrifugation linearly increases the degree and rate of platelet aggregation by generating shear.40 Time The time dependent degradation of cellular content in human platelets has been proven with the changes in the minute amount of RNA predominantly comprised of vestigial RNA from the megakaryocyte and surrogate RNA uptaken in the circulation. Time dependent platelet RNA degradation has been demonstrated by staining platelet with Thiazole orange (TO) which binds to RNA witnessing half of the RNA content of ribosomal RNA and beta-actin RNA within 6 hr and more than 98% in 24 hr.41 Temperature Effect of the temperature on platelet has been investigated to understand the morphological changes and activation upon transition of temperature from the body temperature of 37°C, room temperature ~20°C, and cold temperature 4°C. Platelets exposed to cold temperature undergoes significant morphological changes of actin rearrangement forming pseudopods, increase of intracellular calcium level and functionality.42 For ~20 °C, changes of platelet have also been observed in aspects of morphology, ultrastructure, surface protein expression and serotonin level.43-44 The morphological changes are reported to be reversible when incubated at 37°C within 6 hr and alpha granule exocytosis didn’t occur by the activation at room temperature.45 Properties of binding substrate Mechano-sensing of platelet allows response towards microenvironmental cues46 demonstrated by alteration of the stiffness of the fibrin/fibrinogen substrate resulted in the increased platelet adhesion, higher activation (increase in PS exposure, granule secretion, and aggregation) and spreading.47 Platelets are known for binding onto surface of a lot of materials especially on polymer surface hence, pre-coating by antithrombotic substance is required to prevent unwanted binding of platelets.48 20.

1.1.5 Platelet function testing Functionality of platelets holds a critical significance in the clinical aspect of hemostasis and platelet-related disorders. If platelet is unexpectedly low in count or defective to form a stable thrombus, mild to severe bleeding may occur. In contrast, if upregulated in count or reactivity, unnecessary thrombus formation may increase the risk of CVD. Therefore, understanding and modulating platelet function is a promising circulating biomarker and, at the same time, therapeutic target. Over decades, a variety of platelet function tests were developed from two classical platelet function tests’ principles (agonist induced aggregation measurement and occlusion time) to further investigate platelet defect and enhance the efficiency of the testing method (Figure 3). Bleeding Time Bleeding time, the oldest assay (not anymore recommended for PFT) measure the time for the cessation of bleeding from wound site made by a small incision or cut in standardized size on the skin (completely done in vivo). However, due to its poor sensitivity, reproducibility, and invasiveness as well as uncontrollable variabilities such as skin temperature, thickness, depth of wound, subjective measure of cessation, etc. limited its reliability for clinical application. Light transmission aggregometry (LTA) LTA developed by Born’s in 1962 has been widely regarded as the gold standard test for detecting platelet function disorders and monitoring antiplatelet therapies.51 Supported by long term studies of LTA, It still remains the most useful reference method for the diagnosis of platelet function defects. The details of LTA will be further discussed in chapter 4.. Figure 3 The representative illustration of the two major principles of classical methods of PFT49-50 21.

Impedance aggregometry Impedance aggregometery utilizes whole blood to measure changes in impedance when platelets bind onto the two electrodes upon administration of specific agonists. Upon loading the sample into the chamber, first consistent baseline value without agonist is measured. Followed by adding specific agonist that induce platelet activation and adhesion to the electrode under shear generated by a stirrer, the rise in impedance in accordance to the degree of platelet attachment is measured. Three parameters for the measurements are lag time (the time required for impedance to reach > 2 Ohms), amplitude (the maximum change in impedance), and area under the curve (the integral of the impedance over time).52 This assay was successful in measuring congenital and acquired platelet disorders as well as monitoring response to antiplatelet therapy.53 The aggregation dynamics of platelets in response to multiple agonists can be measured in whole blood (influenced by hematocrit and platelet count)54 but the sensitivity of the test heavily relies on the electrodes.55 VerifyNow VerifyNow (Accumetrics, California, USA), fully automated POCT device, is a modified version of LTA allowing usage of whole blood through a disposable cartridge consisting of four chambers containing polystyrene beads coated with fibrinogen and specific agonists. Once vacutainer containing blood (< 1mL) is inserted to inlet and aspirated blood fills the chamber, the beads allow platelets to bind upon activation and initiate aggregation increasing light transmission within 2~5 min.56 The test results are reported as high or normal platelet reactive units (PRU) in reference to a cutoff value of 240 PRU. Currently, the VerifyNow developed different types of cartridges to provide platelet function in response to antiplatelet drugs like aspirin/ASA (Arachidonic acid) and clopidogrel (ADP and PGE1)57 and changes in GP IIb/ IIIa (TRAP). The disadvantages of this assay are limited use in a defined range of hematocrit and platelet count with low shear as well as inflexibility in choosing agonists and the concentration. The biggest challenges remaining in the platelet function tests are inconsistencies of results caused by sample handling, running conditions, and a shortage of supportive clinical evidence especially in predicting MACE alongside with antiplatelet therapy.58 Therefore, current systems need a user friendly and robust point of care device that can be done in low volume, high throughput, fast and accurate generation of results having minimal sample handling with valid clinical evidence for monitoring thrombotic disorders and corresponding therapy. Such device may provide a breakthrough allowing large-scale studies on platelet hyperreactivity in CVDs and required the optimal dose of antiplatelet therapies. Table 1 compared the existing PFT especially those having the principle of agonist induced aggregation for fair comparison with our lab on a disc device.. 22.

Table 1 Examples and comparison of platelet function tests Type of tests. Bleeding Time. LTA. Impedance Aggregometry. VerifyNow. Lab on a Disc. Sample Type Sample Preparation Target / Analytes Detection method. WB (Whole Blood). Plasma Rich Plasma (PRP). WB. WB. WB. Not necessary. PRP separation. Not integrated. Not integrated. PRP separation step is integrated. Cessation time. Agonist-induced aggregation. Agonist-induced aggregation. Agonist-induced aggregation. Agonist-induced aggregation. Timer. % Transmission Spectrophotometer. Impedance Electrodes. % Transmission Spectrophotometer. % Transmission Spectrophotometer. 59. 60. 61. 62. On spot. Benchtop. Benchtop. POCT. POCT. ~ 5 min. 4 ~ 7 hr. 10 min. 10 min. 20 ~ 25 min. Gold standard. Small blood volume Multiplex testing. Fully automated Small blood volume. Fully automated Small blood volume Multiplex testing. Device. Use Time Advantage. Disadvantage. Automation. No instrument is required in vivo condition - Not accurate and uncontrolled preanalytical factors. X. - Low reproducibility, - Time consuming and labor intensive sample preparation - Large blood volume, X. - Hematocrit and platelet count can influence the result. - Expensive instruments, reagent storage X. - Hematocrit and platelet count can influence the result.. -. O. O. * Images of bleeding time, LTA, impedance aggregometry, and VerifyNow are derived from the respective company websites.. 23.

1.2 Microfluidic systems for isolating platelets Researches on platelet still heavily rely on density based centrifugation as a common method for its separation: generally performing soft spin to make PRP, and heavy spin to sediment platelets and remove plasma but, limitations of this conventional method should be considered. High speed and repeated centrifugation endows platelets increased chances to aggregate and activate by shear stress.40 Additionally, the WBC infiltration cannot be prevented due to proximity to plasma layer. These problems were addressed in microfluidic platforms for platelet isolation with or without the aid of external field. Acoustic, electric, hydrophoretic, antibody-based, and inertial microfluidic chip platforms are discussed in this section and criteria for comparing these microfluidic approaches of platelet isolation are summarized (Table 2).. 1.2.1 Acoustophoresis Acoustophoresis, arrangement of particles in the flow induced by standing wave field, on the microfluidic platform (acoustomicrofluidics) has been utilized to precisely align cells by applying acoustic pressure waves onto the laminar flow in a microchannel. Pressure waves of equal frequency and magnitude from opposite direction result in the single band at the positions of the pressure nodes of a certain size range of cells. The acoustic strength and flow rate of the suspension can be tuned to fractionate desired platelets from the whole blood. Standing surface acoustic waves (SSAWs) has been integrated on the microchip with its generator interdigital transducers (IDTs) to align the flow stream of cells in the fluid and isolated platelets with 98% purity and 74.1% of yield in the flow rate of 0.25 µL/min.63 Shear-induced activation was neglected for the maximal shear generated on-chip was 333.3 s-1 lower than the threshold shear (2200 s-1) to activate platelets. The separation occurs when larger cells (e.g., leukocytes and erythrocytes) experience greater acoustic forces than platelets leading to a different outlet. So far, only one microfluidic chip succeeded in isolating platelets with high throughput (10 mL / min) in compensation to reduced recovery and purity of approximately 85% and 80%, respectively, by novel vertical positioning of transducer as acoustic separation platform with the increased channel width of the chip64 (Figure 4a). 1.2.2 Dielectrophoresis Dielectrophoresis (DEP) activated cell sorting (DACS) has demonstrated another label-free cell isolation methods in microfluidics for platelet isolation by applying AC electric field in a microchannel with purity of 95%, recovery of 18%, and 50% activation.65 Micron size cells or particles in the fluid channel are exposed to non-uniform electric field and separated according to polarization by its dielectric property or size (DEP force either pulls or repels but proportional to 24.

the cell volume). DEP forces exerted on cells can be manipulated by the amount of input voltage or medium conductivity. The size-based separation of platelets from red blood cells by dielectrophoresis field-flow fractionation (DEP-FFF) on a single stage low voltage (applied due to the patterned micro-electrodes) device resulted ~98% purity and recovery using pre-centrifuged plasma diluted with the buffer solution as the sample66 (Figure 4b).. 1.2.3 Inertial focusing Differential inertial focusing of asymmetric curves that result to streams of particles formed by inertial lifts and Dean drag force had been demonstrated as another hydrodynamic sorting method for platelets. Size-dependent location of stream flow by positioning equilibrium set by the ratio of lift to the dean drag in the curving channel segregated platelets into the cascading separation outlet67. Elasto-inertial effect of non-Newtonian fluids on microfluidic chip resulted to size-dependent lateral migration flow of platelets under shear-thinning effect in viscoelastic fluid flow along microchannel resulting 92.3 % purity and 98% recovery with the flow rate of 4.5 µL / min68 (Figure 4c).. 1.2.4 Hydrophoresis Without the aid of external field, platelets can also be separated by hydrophoretic size filtration as demonstrated in the 10-stack wide-channel ridge-induced hydrophoresis device to yield purity of 78% platelets after two rounds of sorting under the flow rate of 20 µL / min69 (Figure 4d). The hydrophoretic filtration requires lateral pressure gradients formed in a microchannel. According to the array of micropattern and the width of the channel for certain characteristic, (e.g. deformability and size) cells can be separated as the pressure gradient flow. 1.2.5 Microfluidic pattern Another approach for passive platelet isolation that doesn’t even require flow generation is based on binding of the surface antigen of platelets with micropatterns of immobilized protein surfaces. This interfacial platelet cytometry (iPC) developed can separate platelets with high purity from the unprocessed whole blood in a single step.70 It has advantages of addressing innate platelet disorders by characterizing platelets adhesion as shown by its interaction on printed spots of fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody, as well as morphological changes and spatial distribution. But capture based platelet isolation is limited by the binding efficiency and affinity of the immobilized ligands in terms of yield and integrity of platelets. 25.

In order to isolate platelets with full integrity and functionality, microfluidic approaches such as filtration, acoustics, and dielectrophoretic methods were carried out yet, not many of these approaches had achieved high throughput in isolation without dilution and compensation in biocompatibility, purity, and recovery of platelets separation from WBC and RBC infiltrates.. Figure 4 Examples of platelet separation in microfluidic chip based technologies: (a) acoustophoresis having vertical positioning of transducer for high throughput platelet sorting,64 (b) dielectrophoresis field-flow-fractionation (DEP-FFF) for sorting platelets66 (c) elasto-inertial effect based sorting in non-Newtonian fluid flow microchannel68 (d) hydrophoretic sorting of platelets in microfluidic chip with arrays of slanted ridges.69. 26.

Table 2 Summary of different microfluidic methodologies applied for platelet isolation Platelet isolation technique. Purity. Yield. Platelet activation. Dilution. Hydrophoresis [68]. 76.8%. N/A. Not enough shear to cause. 1:9. activation, stacking up (~10). Sample : PBS. Elasto-inertial effect [67]. 92.3%. 99.8%. No activation observed (CD63). Sanding surface acoustic waves (SSAWs) [63]. 98%. 74.1%. Slight shift of peak (CD63). No dilution. High-throughput vertical transducer [64]. >80%. > 85%. 7.8% increase CD62P. 10% dextrose in. 60.5 morphology score. PBS buffer. 98% WBC. 89% from. recovery. PBMC. ~ 20%. PBS buffer. DEP-FFF (field-flow-fractionation) [66]. 98.8%. 98%. -. DEP Activated Cell Sorting (DACS) [65]. ~95%. ~18%. ~50% CD62P positive. PEO in PBS buffer ~ 1 : 45. F. 20 µL / min. 4.5 µL / min. Acoustofluidics. Acoustophoresis chip [70]. 0.25 µL / min. 10 mL / min. 20 µL / min. Dielectrophoresis. Interfacial cytometry [69]. -. ~10,000. Depending on the substrate. cells 27. PBS + sucrose in PRP 7 : 100 Sample : LEC buffer. No dilution. 134 µm / s. 150 µL/h. -.

1.3 Centrifugal microfluidics 1.3.1 Theory Centrifugal microfluidic platform allows multiple fluidic manipulations of mixing, metering, pumping by controlling spin condition of a disc that generates centrifugal force exerted on the fluid to be pumped radially outward from the center of the disc. On disc fluid dynamics is primarily driven by the geometry of channels, the positioning of reservoirs and channels, rotational speed, and fluid properties.71 Centrifugal force (Fc) created by rotation of disc is defined as72: =. ̅. (1). where ρ is the density of the fluid, ω is the angular velocity, r is defined as average distance of fluid from the disc center (( ̅=( 1+( 0− ))/2),), and △r is the radial extent of the fluid ((Δ = 1−( 0− ))73. The average velocity (U) of fluid in channels based on centrifugal force or spin speed can be derived72: ̅. U= where. !". (2). Dh is the hydraulic diameter of the channel, µ is the viscosity of the fluid, and L is the. length of the liquid in the channel. With the average velocity of fluid, the flow rate (#) of fluid in the channel can be derived by:. (3). #=$∙& where A is the cross sectional are of channel.. 1.3.2 Unit operations Valving Different type of valve has been introduced to the centrifugal microfluidic platform for manipulation of fluid flow on disc especially for sequential loading of reagents on a disc. Without external actuation cues to operate valves, the passive valve (Figure 5a) using the surface properties (hydrophobic valve), radial position and channel dimension (capillary valve and siphoning valve) had been utilized as passive valves, however, limited by the insensitive blocking to vapor, difficulties in selective actuation as well as irreversible properties.71, 74 Laser actuation valve using paraffin wax containing ferrofluid as a sacrificial material called laser irradiated ferrowax microvalves (LIFM) served to block fluid as well as vapor and open independently from rotation program (Figure 5b).75 However, LIFM generated local heating and thermodynamically instable. 28.

To overcome the thermal instability and limited reversible actuation, individually addressable diaphragm valves that can be open and closed by simple mechanical actuation (Figure 5c) was developed for different biomedical applications like PCR and ELISA.73 Table 3 summarized the comparison of currently available valve system on centrifugal microfluidic platform. Metering Metering in centrifugal microfluidics easily, rapidly, and precisely distribute the desired volume of fluid that fills the chambers correspondingly to the radial extent and overflows the excess fluid to waste under appropriate spin condition. Subsequent transfer of multiple metering of fluid allows aliquoting of samples and reagents into appropriate wells or chambers. Mixing Mixing of reagents required for homogeneity of reaction mix in a lot of bioanalytical assays, however, limited in other microfluidics systems due to laminar flow-based diffusive mixing without convection. In centrifugal microfluidics, fast acceleration rate owing to oscillation or repetitive rotation in both clockwise and counter clockwise direction, generate inertial force and damping to contact surface facilitates rapid and efficient mixing on a disc.76 Table 3 Characterization of different types of valve in centrifugal microfluidic platform. Criteria. Passive valve. Sacrificial valve. Diaphragm valve. Individual valve control. X. O. O. Robustness. X. O. O. Reversibility. X. △. O. Thermal stability. X. X. O. Actuation. Appropriate spin speed. Laser heating. Mechanical cue. Figure 5 Operation method of three different types of valves: (a) passive valve (capillary valve as an example)71 (b) sacrificial valve (LIFM)75 and (c) diaphragm valve73 29.

1.3.3 Fully integrated lab on a disc system for biomedical applications The centrifugal microfluidic platform holds unique and strong advantages as POCT device compared to microfluidic systems requiring additional external equipment.72 A simple set of the instrument based on the single rotary motor to generate centrifugal forces for fluid actuation. The lab on a disc (LOAD) can fully integrate and automate the complex multi-step detection tools by pumping, mixing, and metering implemented by altering spin program with appropriately positioned valves required for fluid control.71 This platform enhances the assay by reducing error derived from handling and the time cost efficiency. Potentials of lab-on a disc platform in the versatile diagnostic application has been demonstrated upon full integration of multiple steps of bioassays including sample preparation step (plasma separation, cell lysis, washing), loading of reagents for biochemical assays,77 immunoassays,77, 79 nucleic acid extraction and amplification,8083. and detection of target biomarker.84-86 (Figure 6). Figure 6 Fully integrated lab on a disc system for diagnostic applications. (a-b) disc layout design of fully integrated and automated biochemical assay77 and immunoassay16 (c) molecular assay and paperstrip detection for foodborne pathogen detection on the disc17 (d) disc for secondary PCR in automated operation78 30.

1.3.4 Size based filtration on a disc Selective porous membrane filtration can be accommodated in centrifugal microfluidic devices for size based fractionation of micro to nanometer particles or cells. Label-free isolation of liquid biopsy markers, circulating tumor cell (CTC) and exosomes (Figure 7), on centrifugal microfluidic platform has been demonstrated with the integration of porous filter membrane and fluid assisted separation technology (FAST)87 that provide a reduced pressure drop required for more efficient and gentle isolation of CTC88 (Figure 7a). After the integration of porous membrane fixated on the disc, prime fluid in the bottom of filter was filled to realize FAST on a disc. As demonstrated by isolation of exosomes with two filter system integrated on lab on a disc89 (Figure 7b), size selective isolation of particles or cells within the preferred size range can be achieved on lab on a disc by changing the filter sets with desired pore size. On top of isolation, the filter assist integration of fast and easy washing and elution step for purification of desired samples.. Figure 7 Size based liquid biopsy sample preparation on lab on a disc (LOAD) platform. FAST system based label free isolation 87 was demonstrated by (a) isolation of circulating tumor cells from whole blood of cancer patients 88 and (b) exosome from urinary samples with the two filter system integrated on disc 89 31.

1.4 Research outline. 1.4.1 Objective of the research Isolation of platelets has most frequently been carried out by the double centrifugation method, however, not been standardized to optimally isolate them. Platelets are affected by centrifugation force induced shear stress and may behave differently in the separation process. In general, low gforce (100~500 g) based PRP formation and hard spin down of the platelets from plasma based on high g force within a concurred range of approximately 500 ~ 3000 g. However, there are few drawbacks in this conventional method, which are difficulty in isolating platelets from low blood volume, activation of platelets, WBC and RBC infiltration eventually resulting to low reproducibility and exposure to pre-analytical factors like pipetting errors. In order to obtain a reproducible result from the clinical application or medical researches, platelets should be prepared as it is in vivo resting state as possible and in a reproducible manner with high purity. Maintaining high purity of platelet isolation is especially important in investigating platelet RNA because WBC contains 10,000 times more RNA compared to platelet. The lab on a disc technology has demonstrated a proof of concept in platelet isolation with a mild g-force to lessen activation without applying inhibitors by integrating two filters to remove WBCs and RBC infiltrates and to wash the platelets from plasma proteins and extracellular vesicles without artificial pellet formation, in fully automated platform. The platelet isolation on disc aim on achieving high yield and purity of platelet suspension without WBC for downstream molecular analysis with minimal activation. In addition, either isolated purified resting state platelets in buffer or PRP is prepared for addressing platelet dysfunction driven by the potential biomedical utilization of inherited and acquired platelet disorder, monitoring anti-platelet therapy, pre-surgery screening test, and drug research tool. Our approach of full integration of traditional LTA, clears the following limitaions set by the requirements of the procedure: specialized laboratory, experienced staff, large blood volume, long turnaround time, and labor intensive process with low reproducibility. The goal of this thesis is to develop a size-selective centrifugal microfluidic system for the rapid and efficient platelet isolation and demonstration of isolated platelet-based diagnostic system. The detailed objectives of this research project include: I.. Design and fabrication of a lab-on-a-disc tool for fully integrated label-free robust platelet isolation process using porous membrane filter for size-selective platelet separation. 32.

II. Evaluation of the device performance in comparison with the conventional platelet isolation method by subsequent flow cytometry analysis and real time PCR. III. Performance evaluation of miniaturized and automated LTA on a Disc for simplification of sample preparation steps and multiplex absorbance measurement of resting platelets reacting to agonists and antagonists as a function of time under constant shear induced by angular mixing / shaking.. 1.4.2 Outline of the thesis In Chapter 1, basic concepts of the platelets and its clinical significance are explained. Multiple platelet isolation methods and commercialized platelet based diagnostic systems are briefly discussed, followed by a short literature review of microfluidic-based platelet studies. The concept of a lab-on-a-disc platform and previously developed systems are discussed at the end of Chapter 1. In Chapter 2, the process of sample preparation, reagents, protocols, device fabrication, experimental set-ups for platelet separation experiments are explained in detail. Chapter 3 discuss experimental procedures and gathered data with analysis related to the device fabrication process, optimization, and performance evaluation of platelet separation experiments in our customized lab on a disc device in comparison with the manual method. Chapter 4 proposes the application of the on-disc prepared platelets, specifically testing platelet function by integration of automated process of LTA. Finally, conclusions and future prospects are summarized in Chapter 5.. 33.

CHAPTER 2 Experimental methods & materials This chapter describes the sample preparation protocols, disc fabrication process, operation systems and procedure for analysis in detail. 2.1 Sample preparation 2.1.1 Chemicals and reagents For preparing HEPES-Tyrode’s buffer, following reagents were used: 140 mM sodium chloride (4.091 g), 5.5 mM dextrose (0.495 g), 3 mM potassium chloride (0.112 g) and 1mM magnesium chloride (0.048g) purchased from Sigma-Aldrich (St. Louis, MO, USA); 12 mM NaHCO3 (0.504 g) purchased from Samchun Chemicals, (Gyeonggido, Korea); 0.35 mM NaH2PO4(0.021 g) purchased from Yakuri Chemicals (Kyoto, Japan); 500 mL of 1X phosphate buffered saline (PBS) and 10mM HEPES Buffer (diluted from 1mM) solution purchased from Gibco Invitrogen, (Grand Island, NY, USA) For cell staining and flow cytometry, following reagents were used: PBS tablets purchased from Ameresco (Framingham, MA, USA); Pluronic F127, bovine serum albumin (BSA) purchased from Sigma-Aldrich (St. Louis, MO, USA); APC mouse IgG1, κ Isotype control (FC) Antibody, APC mouse IgG1, κ Isotype control (FC) antibody, PE mouse IgG1, κ Isotype control antibody FITC anti-human CD41 antibody, APC anti-human CD62P (P-Selectin) antibody, and PE anti-human CD45 antibody purchased from Biolegend (San Diego, CA, USA); OneComp eBeads® purchased from eBioscience (San Diego, CA, USA); BD FACS flow sheath fluid, and calibration beads purchased from BD Bioscience (San Jose, CA, USA); 35 % formaldehyde solution purchased from Samchun Chemical (Gyeonggido, Korea) For RT-qPCR and RNA analysis, following reagents and kits were used: Purelink RNA mini kit, SuperScript® VILO™ cDNA Synthesis kit, and RNAlater® Stabilization Solution purchased from Ambion, Life technologies (Waltham, MA, USA.), RNeasy mini kit purchased from Qiagen (Hilden, Germany), RNA 6000 Pico Kit, Agilent (Santa Clara, CA, USA), ethyl alcohol (ACS grade) Sigma-Aldrich (St. Louis, MO, USA); For platelet aggregation assay, following reagents were used: prostaglandin E1 (PGE1), adenosine 5′-diphosphate (ADP), thrombin receptor-activating peptide (TRAP) / SFLLRN, epinephrine, arachidonic acid, acetylsalicylic acid, fibrinogen from human plasma, apyrase were purchased from 34.

Sigma-Aldrich (St. Louis, MO, USA) and prepared according to the manufacturer’s instructions. In disc fabrication, isopropyl alcohol (IPA) and dichloromethane (DCM) purchased from Samchun Chemical (Gyeonggido, Korea) were used. 2.1.2 Blood specimen collection and processing Blood collection from volunteers Blood samples were collected by venipuncture through a 21-gauge needle with vacutainer tubes (BD, Franklin Lakes, NJ, USA) containing EDTA (Ethylenediaminetetraacetic acid) for evaluation of blood cell counts using Sysmex XP-300 (Sysmex, Kobe, Japan), RNA analysis, and flow cytometric assessment or 3.2% (0.109M) trisodium citrated blood (10% vol/vol) or acid citrate dextrose (ACD) solution B as the anticoagulant for PFT. The samples were processed within 6 hr after collection to prevent cellular degradation and maintain viability and aggregation of the platelets. All samples originated from healthy volunteers. Manual platelet preparation: serial centrifugation For the manual method of retrieving platelets from 600 µL of EDTA anticoagulated whole blood, PRP was prepared by centrifuging at 300 g for 10 mins and 240 µL transferred into 1% Pluronic coated 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany). Transferred PRP was centrifuged at 2000 g for 10 mins to collect platelet suspension. The supernatant plasma was removed and PBS or FACS buffer (2% fetal bovine serum in PBS) was added to suspend the platelets for further assay. On disc platelet isolation: centrifugation with sequential filtration For disc-based platelet isolation from 600 µL of whole blood in EDTA tube, detailed operation protocol is described in chapter 3. Briefly, the whole blood loaded into the disc is separated by density-based spinning (3000 RPM for 5 min) and plasma layer rich in platelets is transferred and serially filtered. The first filtration was integrated for removal of WBCs and RBCs of size bigger than 5 µm and the second filtration to remove the plasma contents while retaining platelets on top of the 0.6 µm filter that is collected after washing. Following the first collection of platelet suspension, bottom fluid is removed and elution buffer fills and retrieved with remaining platelets. The final volume of platelets suspension was 300 µL. PRP / platelet suspension preparation for aggregation assay For aggregation assay, whole blood in citrated tubes was centrifuged 250 g for 15 mins without brakes to collect PRP. The PRP generated is directly used for aggregation assay while for platelet suspension based aggregation assay required additional steps in the preparation. PRP was 35.

transferred into P3 coated Eppendorf tubes containing prostaglandin E1 (PGE1; concentration of 1 µM) that inhibits activation of platelets and centrifuged again for 2000 g for 10 mins. The platelet pellet was washed and resuspended in HEPES-Tyrode’s buffer solution (NaCl 140 mM, KCl 3 mM, MgCl2 1mM, glucose 5.5 mM, NaHCO3 12mM, NaH2PO4 0.35 mM, and HEPES 10 mM, pH 7.4 ± 0.15) after removing the plasma and kept in room temperature for 30 min before starting the aggregation assay. Platelet-poor plasma (PPP) was prepared by centrifuging the remaining whole blood at 2500 g for 10 min and collecting the supernatant without touching the buffy coat. (Figure 2) Immunostaining of human platelet in suspension The platelet suspension were prepared by either disc method or manual method as previously described and approximately one-sixth of the final elution volume (generally, only 50 µL from 300 µL eluted platelet suspension was transferred to FACS tube (3 mL polystyrene tube)) was utilized for flow cytometry based analysis. The prepared platelets in suspension was immuno-stained with a cocktail of fluorescent antibodies (FITC anti-human CD41 Antibody, PE anti-human CD45 Antibody, and APC anti-human CD62P (also called P-selectin) Antibody (1 µL each resulting in 5 µg/mL) in 100 µL of PBS for 30 min in dark at room temperature. All antibodies were purchased from Biolegend, (San Diego, CA, USA.). The stained platelets were washed with 1mL of FACS buffer (2% fetal bovine serum (FBS) in PBS buffer) twice and fixed with 0.5% Formaldehyde in FACS buffer (~200 µL final suspension) for a minimum of 30 min at room temperature and stored at 4°C until use. The prepared samples were run with the high flow rate for 100 s or 20 s multiplied by five. Labeling human platelets in whole blood Whole blood (20 µL) was incubated with 100 µL PBS buffer containing CD41-FITC, CD45-PE, and CD62P-APC (1 µL each) for 25 min at room temperature. After washing twice with FACS buffer 3 min at 2000 g, whole blood was fixed with 0.5 % formaldehyde solution for more than 1 hour prior to running the sample in FACS machine. Light transmission based platelet aggregometry Following the above-mentioned protocol, prepared platelet suspension or plasma was equally added into individual wells of Corning flat bottom 96-well plates purchased from Sigma-Aldrich (St. Louis, MO, USA). 10% of the sample volume of original PRP or suspension was filled with or without agonist in PBS added to individual wells. Platelet agonists used in the experiments were TRAP (10~100 µM ), and ADP (1 µM ~1mM). To observe the change in aggregation compared with untreated positive and negative control, PRP and PPP samples were processed in the same 36.

manner and conditions. Upon testing the manually prepared samples, 90 µL of PRP / PPP was added to wells and 10 µL of PBS buffer with or without agonists were added. Mixing is done by vortexing 1500 RPM (condition 7) using vortex genie 2 or pulse (Scientific industries, New York, USA) for 5 min90 and absorbance was measured using Infinite M200 Pro Tecan plate reader (Männedorf, Switzerland) at 600 nm for plasma sample and 400 nm for washed platelet suspension. The results are reported as % aggregation calculated by the optical density (OD) of each values having PRP as 0% aggregation and PPP as 100%.. 2.2 Device fabrication The fabrication method of lab on a disc device for isolation of platelet, including the operation and visualization, is briefly explained in this section. Details of the disc design is further discussed in chapter 3. Disc fabrication The disc framework of the body and top cover is designed with Solidworks 3D CAD design software (Waltham, MA, USA). Designed 3D structure is translated into code by software EDGECAM 2014 (Forest Lake, MN, USA) for fabrication using CNC milling machine (ProMill smart-3530, PROTECH, Korea) (Figure 8a). The disc parts were milled with single flat end mill tools of 3 mm, 2 mm, 1 mm and 0.8 mm (JJtools, Seoul, Korea) correspondingly on the width of channel and diameter of hole structures. The polycarbonate plates and sheets (PC: I-Components Co. Ltd, Pyongtaek, Korea) of different thickness (0.5 mm, 2mm, and 5mm) were used to make 1 bottom layer, 1 body frame, and 1 top layer, respectively, that comprise the platelet isolation disc. Two double sided pressure sensitive adhesive layers (DFM 200 clear 150 POLY H-9 V-95; FLEXcon, Spencer, MA, USA) that attached in between PC layers were designed into 2D CAD image of the adhesive layers using AutoCAD 2012 (San Rafael, CA, USA) and cut using a cutting plotter (CE3000 & 6000; Graphtec Corp., Japan) (Figure 8b). PC body frame prepared by milling was further treated with vaporized DCM to smoothen the surface, and cleansed with isopropyl alcohol. The prepared PC device layers were assembled with pressure sensitive adhesive layers in between two PC layers using mechanical pressurizer (MC Power Vise ALQ-200 G HV, AUTOWELL, Taiwan.). 37.

3D printed individually addressable diaphragm valves (ID Valve) with Actuator The micro ID valve previously developed by our laboratory73 were 3D printed by Projet 3510 HD Plus (3D systems, Rock Hill, SC, USA) (Figure 8c) with VisiJet M3 Crystal as the material and printed 3D valve and actuator was placed in an oven at 78˚C overnight to melt away the supporting material. After heating the 3D printed parts in the oven, the parts were placed in oil and sonicated at 60 ˚C by Branson 2800 ultrasonic cleaner (Branson Ultrasonics, Danbury, CT, USA) to remove remnant supporting material. The oil was cleansed with commercially available detergents and all the parts are washed with distilled water. The 3D printed parts are shortly sonicated with IPA for ~15 mins and dried immediately after the last cleansing. The ID valves are bonded onto designated sites on the top cover previously filled with polydimethylsiloxane (PDMS, Dow Corning, USA) mixed with the curing agent (w/w; 10:1) and heated at 60 ˚C overnight. The Live Cell Instrument (LCI, Seoul, Korea) company developed 3D printed pushpin valve with detachable polysulfone sealing were used for leakage free valving.. Figure 8 Machines for Disc layer and parts Fabrication. (a) Polycarbonate (PC) plastic Fabrication by CNC Milling (ProMill smart-3530, PROTECH, Korea), (b) double-sided pressure sensitive adhesive (PSA) cutting by cutting plotter (CE3000 & 6000; Graphtec Corp., Japan), and (c) printing of micro ID valve by 3D printer Projet 3510 HD Plus (3D systems, Rock Hill, SC, USA) 38.

Track-etch polycarbonate (TEPC) membrane integration Nucleopore polycarbonate track-etched membranes (WhatmanTM, Florham Park, NJ, USA) with the diameter of 13 mm, and the pore sizes of 5 µm, 3 µm, and 1 µm including 600 nm (SPI, West Chester, PA, USA) were integrated with 0.25 mm gasket Swinnex Millipore (Kenilworth, NJ, USA) and 1.0 mm silicone O-ring onto the assembled disc by 4 mm PC holder (ring) and 2 mm PC holder (plate) fastened by bolts and nuts with an interlayer of PDMS to prevent leakage (Figure 9.) This integration method was applied in our laboratories previous works 87, 89 Surface treatment The surface of polycarbonate was treated with < 1% Pluronic solution (0.45 g of Pluronic F-127 from Sigma-Aldrich (St. Louis, MO, USA) dissolved in 50 mL of triple distilled water). The surfactant, pluronic triblock copolymer, PEO-b-PPO-b-PEO, has been utilized to coat plastic surface attributing antithrombotic property by its bioinert PEO segments suppresses plasma proteins adsorption, and platelet adhesion onto PC surface as well as the porous membrane. After coating the PC surface for 2 hr at room temperature, the PC surface was cleansed with PBS solution twice.. Figure 9 Exploded view of filter membrane integration on the fabricated and assembled disc. Components are arranged in orderly manner. 39.