Effects of red blood cell aggregation, hematocrit and tube diameter on wall shear stress in microtubes

EFFECTS OF RED BLOOD CELL AGGREGATION, HEMATOCRIT AND TUBE DIAMETER ON WALL SHEAR STRESS IN MICROTUBES YANG SHIHONG NATIONAL UNIVERSITY OF SINGAPORE 2010 EFFECTS OF RED BLOOD CELL AGGREGATION, HEMATOCRIT AND TUBE DIAMETER ON WALL SHEAR STRESS IN MICROTUBES YANG SHIHONG (B.Eng (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DIVISION OF BIOENGINENEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENT I would like to express my deepest gratitude and heartfelt thanks to my advisor Dr Kim Sangho for his encouragement and guidance throughout the course of this study His experience, knowledge and patience have proven to be invaluable and vital Without his kind financial support from the grant R-397-000-048-133, the study would not been possible Please pardon me to appreciate him again for bringing me to the wonderful research world I wish to thank all my Microhemodynamics Lab members (Mr Ong Peng Kai, Mr Namgung Bumseok Ms Woo Yeon I, Mr Ju Meong Keun and Ms Jain Swati) who made this pleasant lab like my home in Singapore I am grateful to Mr Ong Peng Kai for his help on the microfluidic experiments and valuable discussions on the study, Mr Namgung Bumseok and Mr Ju Meong Keun for their assistance on the computational data analysis, and Ms Woo Yeon I and Ms Jain Swati for their help in blood cell preparations Acknowledgement is also extended to undergraduate students, Mr Tan Ze Hao, Mr Lim Xuan Yu and Mr Leong Yongzhi Arnold who have assisted me during their undergraduate research projects I also wish to thank Division Officers Mr Tham Mun Chew Matthew, Ms Lee Yee Wei and Ms Teo Mun Mun Jacqueline for helping me purchase essential instruments and consumables for the project, Ms Chong Millie and Ms Low Jenelle for assisting me in administrative tasks i I would like to thank my parents for their continued and unbounded understanding, support and love I am deeply indebted to all the above people who made the study success I will always cherish the friendship and bonding forged in Microhemodynamics Lab in National University of Singapore for my life ii TABLE OF CONTENTS ACKNOWLEDGEMENT i TABLE OF CONTENTS iii SUMMARY vi LIST OF FIGURES viii LIST OF TABLES x NOMENCLATURE xi CHAPTER INTRODUCTION 1.1 Clinical significance of wall shear stress in microcirculation 1.2 Motivation and objectives 1.3 Outline of the dissertation CHAPTER LITERATURE REVIEW 2.1 RBC aggregation 2.1.1 Aggregation formation 2.1.2 Aggregation in diseases 2.2 Hematocrit (Hct) 2.2.1 Hct 2.2.2 Hct in diseases 2.3 Cell-free layer 2.3.1 Cell-free layer formation iii 2.3.2 Effects of cell-free layer 2.4 Tube effect 11 2.4.1 Fahraeus effect 11 2.4.2 Fahraeus-Lindqvist effect 12 CHAPTER WALL SHEAR STRESS 13 3.1 Wall shear stress and its physiological roles 13 3.1.1 Roles of nitric oxide 15 3.2 Pathophysiological effects of abnormal WSS 17 3.3 Methods of measuring WSS 17 3.3.1 Estimation of WSR 18 3.3.2 Estimation of local blood viscosity 19 CHAPTER MATERIALS AND METHODS 20 4.1 Blood preparations and microtubes 20 4.2 Perfusion system 21 4.3 Experimental Procedure 21 4.4 WSS calculation 24 4.5 Statistical analysis 25 CHAPTER RESULTS 26 5.1 Aggregation effect on relative WSS 26 5.2 Tube effect on relative WSS 30 iv 5.3 Hct effect on relative WSS 36 CHAPTER DISCUSSIONS 45 6.1 Aggregation effect on WSS 45 6.2 Tube diameter effect on WSS 47 6.3 Hct effect on WSS 48 6.4 Threshold pseudoshear rates and WSS 50 6.5 Pathophysiological implications 51 CHAPTER CONCLUSIONS 54 REFERENCES 57 APPENDIX A DERIVATION OF WSS 71 APPENDIX B FIGURES OF RELATIVE WSS 73 CURRICULUM VITAE 83 v SUMMARY Wall shear stress (WSS), a tangential stress exerting on the inner surface of blood vessels is an important determinant of endothelial cell structure and function It is also one of the major stimuli for the release of vasoactive substance, nitric oxide (NO), which plays a critical role in the regulation of vascular diameter and maintenance of vascular resistance Abnormally low WSS is found to be associated with atherosclerosis whereas abnormally high WSS is correlated with aneurysm Therefore the study of WSS is of particular importance as any disruptions of WSS in microcirculation might lead to diseases Red blood cell (RBC) aggregation and hematocrit (Hct) are the main hemorheological factors and contributors to the blood viscosity and vascular resistance However, little quantitative information of RBC aggregation and Hct on WSS is available The objectives of the project are to investigate effects of two hemorheological factors RBC aggregation and Hct, and a geometric factor, tube diameter (tube size) on WSS The study was performed with special reference to the levels of RBC aggregation and Hct found in normal and diseases states in microtubes of inner diameter (ID) 30 µm, 50 µm and 100 µm relevant to microcirculation Non-aggregating medium of phosphate buffer saline (PBS), normal aggregating medium (Dextran 500-PBS solution of concentration 7.5 mg/ml) and disease aggregating medium (Dextran 500-PBS solution of concentration 12.5 mg/ml) were used in the study Blood samples at Hct level of 40% mimicking physiological conditions and Hct level of 20% and 60% mimicking clinical levels were utilized Relative WSS (WSS of blood suspension normalized by the WSS of suspending medium) was used to isolate the medium viscosity effect on the WSS vi The results showed that RBC aggregation was effective in reducing the WSS in low pseudoshear rates corresponding to venular flows, but insignificant in affecting WSS in high pseudoshear rates corresponding to arteriolar flows However, increased Hct and tube diameter led to significant elevations in WSS in most pseudoshear rates except at pseudoshear rate of about s-1 corresponding to reduced venular flows At low pseudoshear rate of approximate s-1, insignificance difference in WSS between Hct 20% and 40% and between microtubes of ID 30 µm and 50 µm was found in aggregating mediums and this could likely be attributed to the enhanced cell-free layer formation The results suggested that effects of RBC aggregation could likely be more dominant over effects of Hct and tube diameter in contributing to WSS at low pseudoshear rates whereas the effects of Hct and tube diameter might be more prominent at high pseudoshear rates The comprehensive quantitative information on effects of RBC aggregation, Hct and tube diameter on WSS with special reference to normal and disease level of RBC aggregation and Hct obtained from the study would lead to an advanced understanding of WSS in vivo and possibly to new therapeutic approaches to the WSS related diseases vii LIST OF FIGURES Figure RBC aggregates Figure Cell-free layer formation 10 Figure Micrographs of the flow-induced alignment of cultured endothelial cells in vitro 14 Figure Schematic diagrams of flow-induced dilation showing changes of WSS that occur during a period of increased blood flow velocity 16 Figure Schematic drawing of the experimental set-up 22 Figure Relative WSS of blood samples at 40% Hct over pseudoshear rates in microtube of ID 50 µm in non-aggregating blood suspended in PBS, normal aggregating blood and disease aggregating blood 27 Figure Comparison of relative WSS of blood samples at 40% Hct in microtube of ID 50 µm in non-aggregating blood suspended in PBS, normal aggregating blood and disease aggregating blood at four typical pseudoshear rates 29 Figure Relative WSS of blood samples at 40% Hct over pseudoshear rates in microtubes of ID 30 µm, 50 µm and 100 µm in three aggregating mediums 33 Figure Comparison of relative WSS of blood samples at 40% Hct in microtubes of ID 30 µm, 50 µm and 100 µm in normal aggregating blood 34 Figure 10 Relative WSS of blood samples at Hct of 20%, 40% and 60% over pseudoshear rates in microtube of ID 50 µm in three aggregating mediums 38 Figure 11 Comparison of relative WSS of blood samples at Hct level of 20% VS 40% VS 60% in microtube of ID 50 µm at four typical pseudoshear rates in three aggregating mediums 41 Figure B1 Comparison of relative WSS of blood samples at 40% Hct in microtubes of ID 30 µm, 50 µm and 100 µm in non-aggregating blood 73 Figure B2 Comparison of relative WSS of blood samples at 40% Hct in microtubes of ID 30 µm, 50 µm and 100 µm in disease aggregating blood 74 Figure B3 Relative WSS of blood samples at Hct of 20%, 40% and 60% over pseudoshear rates in microtube of ID 100 µm in three aggregating mediums 76 viii 111 Lipowsky, H.H., S Kovalcheck, and B.W Zweifach, The distribution of blood rheological parameters in the microvasculature of cat mesentery Circ Res., 1978 43: p 738–749 112 Lipowsky, H.H., S Usami, and S Chien, In vivo measurements of hematocrit and apparent viscosity in the microvasculature of cat mesentery Microvasc Res., 1980 19: p 297-319 113 Ong, P.K., et al., Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles Am J Physiol Heart Circ Physiol., 2010 298(6): p 1870-1878 114 Bishop, J.J., et al., Erythrocyte margination and sedimentation in skeletal muscle venules Am J Physiol Heart Circ Physiol., 2001 281: p 951-958 115 Weaver, J.P., A Evans, and D.N Walder, The effect of increased fibrinogen content on the viscosity of blood Clin Sci., 1969 36: p 1-10 116 Bishop, J.J., et al., Relationship between erythrocyte aggregate size and flow rate in skeletal muscle venules Am J Physiol Heart Circ Physiol., 2003 286: p 113-120 117 Butler, A.R., I.L Megson, and P.G Wright, Diffusion of nitric oxide and scavenging by blood in the vasculature Biochim Biophys Acta , 1998 1425: p 168–176 118 Lamkin-Kennard, K.A., D Jaron, and D.G Buerk, Impact of the Fahraeus effect on NO and O2 biotransport: a computer model Microcirculation 2004 11: p 337–349 69 119 Vaughn, M.W., L Kuo, and J.C Liao, Effective diffusion distance of nitric oxide in the microcirculation Am J Physiol Heart Circ Physiol., 1998 274: p 1705– 1714 120 Freedman, J.E., et al., Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis J Clin Invest., 1996 97: p 979 –987 121 Shen, W., et al., Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise Med Sci Sports Exerc., 1995 27: p 1125-1134 122 Chang, H.Y., M.E Ward, and S.N Hussain, Regulation of diaphragmatic oxygen uptake by endothelium-derived relaxing factors Am J Physiol Heart Circ Physiol., 1993 265: p 123-130 123 Iwamoto, J., et al., N omega-nitro-L-arginine influences cerebral metabolism in awake sheep J Appl Physiol, 1992 73(6): p 2233-2240 124 Brezis, M., et al., Role of nitric oxide in renal medullary oxygenation, studies in isolated and intact rat kidney J Clin Invest., 1991 88: p 390-395 125 Bishop, J.J., et al., Effects of erythrocyte aggregation and venous network geomnetry on red blood cell axial migration Am J Physiol Heart Circ Physiol., 2001 281: p 939-950 70 APPENDIX A DERIVATION OF WSS Considering a fluid flowing in a cylinder, illustrated in the figure below: r z PO τ rz Pz Pz+ Δ z PL z+ Δ z z L Assumptions: Flow is fully developed as length of tube L is much greater than tube radius R, L>>100R Thus entrance effects can be neglected Flow is incompressible Flow is isothermal No external forces acts on the fluid Radial velocity vr=0 as there are no holes in the cylinder for fluid to escape radially Angular velocity vθ=0 is there are no swirls in the fluid No slip condition implies v=0 at the walls With the above assumptions, the mass continuity equation: ∂ρ + ∇ ⋅ ( ρv ) = ∂t (A.1) reduces to the volume continuity equation: 71 ∇⋅v = (A.2) Thus only an axial velocity component exists and it will be a function of tube radius only: vz=vz(r) (A.3) The Navier-Stokes equation is an application of Newton's second law to a continuum and is then expressed as follows: ρ Dv = −∇p + ∇ ⋅ Τ + f Dt where ρ (A.4) Dv is the acceleration of the fluid; Dt − ∇p is pressure gradient and arises from normal stresses; ∇ ⋅ Τ is the viscous forces; for incompressible flow, this is only a shear effect; f represents "other" forces, such as gravity Applying with the assumptions and obtained: ρ Dv = Dt (A.5) ∇ ⋅ Τ = τ rz 2пr Δ z (A.6) − ∇p = пr2 (Pz+ Δ z - Pz) (A.7) Substituting the above back into Navier-Stokes equation, dividing by Δ z, and taking the limit as ΔzỈ0, the following equation is obtained: τ rz (r ) = − r dP (Po − PL )r = dr 2L (A.8) It is important to note that no assumptions have been made regarding the relationship between shear stress and shear rate Thus, Equation (A.8) holds for all fluids regardless of Newtonian fluid or non-Newtonian fluid 72 APPENDIX B FIGURES OF RELATIVE WSS Figure B1 Comparison of relative WSS of blood samples at 40% Hct in microtubes of ID 30 µm, 50 µm and 100 µm in non-aggregating blood (A): at pseudoshear rate of about 200 s-1; (B) at pseudoshear rate of about 80 s-1; (C) at pseudoshear rate of about 10 s-1; (D) at pseudoshear rate of about s-1 Values are means ± SD * P < 0.05 ** P < 0.01 *** P < 0.001 73 Figure B2 Comparison of relative WSS of blood samples at 40% Hct in microtubes of ID 30 µm, 50 µm and 100 µm in disease aggregating blood (A): at pseudoshear rate of about 200 s-1; (B) at pseudoshear rate of about 80 s-1; (C) at pseudoshear rate of about 10 s-1; (D) at pseudoshear rate of about s-1 Values are means ± SD * P < 0.05 ** P < 0.01 *** P < 0.001 ns = not significant 74 75 Figure B3 Relative WSS of blood samples at Hct of 20% (○), 40% (∆) and 60% (□) over pseudoshear rates in microtube of ID 100 µm in three aggregating mediums (A): in nonaggregating medium; (B) in normal aggregating medium; (C): in disease aggregating medium Values are means ± SD 76 77 Figure B4 Relative WSS of blood samples at Hct of 20% (○), 40% (∆) and 60% (□) over pseudoshear rates in microtube of ID 30 µm in three aggregating mediums (A): in nonaggregating medium; (B) in normal aggregating medium; (C): in disease aggregating medium Values are means ± SD 78 79 Figure B5 Comparison of relative WSS of blood samples at Hct level of 20% VS 40% VS 60% in microtube of ID 100 µm at four typical pseudoshear rates observed in normal arteriolar flow (200 s-1) , reduced arteriolar flow (80 s-1), normal venular flow (10 s-1) and reduced venular flow (3 s-1) in three aggregating mediums (A): in non-aggregating medium; (B): in normal medium; (C): in disease medium Values are means ± SD * P < 0.05 ** P < 0.01 *** P < 0.001 80 81 Figure B6 Comparison of relative WSS of blood samples at Hct level of 20% VS 40% VS 60% in microtube of ID 30 µm at four typical pseudoshear rates observed in normal arteriolar flow (200 s-1) , reduced arteriolar flow (80 s-1), normal venular flow (10 s-1) and reduced venular flow (3 s-1) in three aggregating mediums (A): in non-aggregating medium; (B): in normal medium; (C): in disease medium Values are means ± SD * P < 0.05 ** P < 0.01 *** P < 0.001 ns = not significant 82 CURRICULUM VITAE Education National University of Singapore • Master of Engineering (August 2008-present) • Bachelor degree in Engineering (Hons) (Major: Bioengineering; Minor: Mathematics) (August 2003- July 2007) Working Experience Research Engineer in the Microhemodynamics Lab, Division of Bioengineering, National University of Singapore (September 2007- present) • Conducted research on blood rheology in microcirculation, including red blood cell aggregation, hematocrit, plasma viscosity and blood viscosity and tube diameter • Supervised undergraduate research o Tan Zehao, Urop project, Effects of red blood cell aggregation on wall shear stress in tube system, (Academic year 08/09) o Lim Xuan Yu, Vacation internship project, Blood viscosity and wall shear stress in microtubes: effect of tube size, (Academic year 08/09) o Leong Yongzhi, Arnold, Final year project, Effects of hematocrit on wall shear stress in a microfluidic system (Academic year 09/10) • Worked as a teaching assistant and provided consultation to undergraduates o BN2101 Principles of bioengineering (Academic year 07/08) o BN2202 Introduction to biotransport (Academic year 08/09) o BN2203 Introduction to bioengineering design-heart pump (Academic year 09/10) Publications Academic Journal Papers Sangho Kim, Shihong Yang and Dohyung Lim (2009), Effect of dextran on rheological properties of rat blood, Journal of Mechanical Science and Technology, Vol 23, 868-873 Academic Conference Paper Shihong Yang, Xiaotao Pan, Thorsten Wohland and Partha Roy (2007), Red blood cell velocity profile in microchannel flow using fluorescence correlation spectroscopy, BES-4SM, Singapore Page Shihong Yang and Sangho Kim (2008), Effect of Dextran on rheological properties of rat blood, 13th International Conference on Biomedical Engineering, Singapopre, page 216 Shihong Yang and Sangho Kim (2010), Effect of erythrocyte aggregation on blood viscosity and wall shear stress in 50 µm tubes, 6th World Congress on Biomechanics, Singapore, page 473 83 .. .EFFECTS OF RED BLOOD CELL AGGREGATION, HEMATOCRIT AND TUBE DIAMETER ON WALL SHEAR STRESS IN MICROTUBES YANG SHIHONG (B.Eng (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING... RBC Red Blood Cell SD Standard Deviation T Experimental Temperature In Kelvin Scale WSR Wall Shear Rate WSS Wall Shear Stress WSSexp Experimental Wall Shear Stress WSSmed Medium Wall Shear Stress. .. levels of 20% and 60% The same three suspending mediums corresponding to non-aggregating blood, normal aggregating blood and disease aggregating blood and the microtubes of ID 30 µm, 50 µm and 100µm