BLOOD CELL AN OVERVIEW OF STUDIES IN HEMATOLOGY Edited by Terry E.

Moschandreou

Blood Cell An Overview of Studies in Hematology http://dx.doi.org/10.5772/2979 Edited by Terry E. Moschandreou Contributors Gkhan Cce, Tahsin Murad Aktan, Mahmoud Rafea, Serhiy Souchelnytskyi, Filip Cristia na, Zamosteanu Nina, Albu Elena, Ambreen Shaikh, Deepa Bhartiya, Moneer Faraj, Nihay a Salem, Junko Takahashi, Akiko Takatsu, Masaki Misawa, Hitoshi Iwahashi, Osamu Hayashi, Karen A. Selz, Terry E. Moschandreou, Youngchan Kim, Kyoohyun Kim, YongKeun Park , Hisham Mohamed, A.B. Shrivastav, K.P. Singh, S. Druyan, Kikuji Yamashita, Clemen t E. Zeh, Collins O. Odhiambo, Lisa A. Mills, Nuri Mamak, smail Aytekin Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensur es maximum dissemination and a wider impact of our publications. After this work has been p ublished by InTech, authors have the right to republish it, in whole or part, in any publica tion of which they are the author, and to make other personal use of the work. Any republication, r eferencing or personal use of the work must explicitly identify the original source. Notice Statements and opinions expressed in the chapters are these of the individual co ntributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no res ponsibility for any damage or injury to persons or property arising out of the use of any materi als, instructions, methods or ideas contained in the book. Publishing Process Manager Sandra Bakic

Typesetting InTech Prepress, Novi Sad Cover InTech Design Team First published September, 2012 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from orders@intechopen.com Blood Cell An Overview of Studies in Hematology, Edited by Terry E. Moschandreou p. cm. ISBN 978-953-51-0753-8

Contents Preface IX Section 1 Main Concepts 1 Chapter 1 Platelets 3 Gkhan Cce and Tahsin Murad Aktan Chapter 2 Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 13 Mahmoud Rafea and Serhiy Souchelnytskyi Chapter 3 Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 31 Filip Cristiana, Zamosteanu Nina and Albu Elena Chapter 4 Pluripotent Stem Cells in Bone Marrow and Cord Blood 69 Ambreen Shaikh and Deepa Bhartiya Chapter 5 C-Reactive Protein 89 Moneer Faraj and Nihaya Salem Chapter 6 Whole Blood RNA Analysis, Aging and Disease 101 Junko Takahashi, Akiko Takatsu, Masaki Misawa and Hitoshi Iwahashi Chapter 7 Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 119 Osamu Hayashi Chapter 8 Spontaneous Alternation Behavior in Human Neutrophils 147 Karen A. Selz Chapter 9 RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model 155 Terry E. Moschandreou VI Contents Section 2 Measurement of RBC Deformability and Microfluidics Technology for Cell Separation 165 Chapter 10 Measurement Techniques for Red Blood Cell Deformability: Recent Advances 167 Youngchan Kim, Kyoohyun Kim and YongKeun Park Chapter 11 Use of Microfluidic Technology for Cell Separation 195 Hisham Mohamed

Section 3 Applications in Haematology 227 Chapter 12 Tigers Blood: Haematological and Biochemical Studies 229 A.B. Shrivastav and K.P. Singh Chapter 13 Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Blood Cell 243 S. Druyan Chapter 14 The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 271 Kikuji Yamashita Chapter 15 Laboratory Reference Intervals in Africa 303 Clement E. Zeh, Collins O. Odhiambo and Lisa A. Mills Chapter 16 Principles of Blood Transfusion 321 Nuri Mamak and smail Aytekin

Preface The editor has incorporated scientific contributions from a diverse gro up of leading researchers in the field of hematology and related blood cell research. This boo k aims to provide an overview of current knowledge pertaining to our understa nding of hematology. The main subject areas will include blood cell morphology and functi on, the pathophysiology and genetics of hematological disorders and malignancies, bl ood testing and typing, and the processes governing hematopoiesis. Blood cell physio logy, biochemistry and blood flow are covered in this book. This text is designed for hematologists, pathologists and laboratory staff in tr aining and in practice. Topics include platelets, whole blood RNA analysis, C-reac tive protein, pluripotent stem cells, proliferation and differentiation of hematopoieti c cells, homocysteine in red blood cells metabolism: pharmacological approaches, measurem ent techniques for red blood cells deformability, spontaneous alternation be haviour in human neutrophils, microfluidics technology for cell separation, effects of far infrared radiation on living body, laboratory reference intervals in Africa, RBC-ATP bioc hemistry and modelling, dynamics of RBC and antigens, transfusion principles, ha ematological and biochemical studies in tigers and ascites syndrome in broiler chickens. The work presented in this book will be of benefit to medical studen ts and to researchers of hematology and blood flow in the microcirculation. This book is w ritten primarily for those who have some knowledge of chemistry, biochemistry and gener al hematology. The authors of each section bring a strong clinical emphasis to the

book. I am greatly indebted to many individuals who helped in the preparati on of this book. Those whom I wish especially to thank for their help with this edition include D rs. Dan Goldman and Khoa Nguyen. I am also indebted to Dr. Jiandi Wan, who gave permissi on to use the referenced figure in the chapter contributed by the editor. I also wo uld like to thank all those who took the time to email me offering helpful criti cisms and suggestions. This includes Ms. Sandra Bakic, publishing process manager at InTec h. Dr. Terry E. Moschandreou University of Western Ontario, Middlesex College, London, Ontario, Canada Section 1

Main Concepts

Chapter 1

2012Cce and Aktan, licensee InTech. This is an open access chapter distributed un der the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Platelets Gkhan Cce and Tahsin Murad Aktan Additional information is available at the end of the chapter http://dx.doi.org/10.5772/50969 1. Introduction General information about platelets, origin of plateletes and granule c ontents of platelets were summarized. 2. Platelets These cell fragments are morphologically small scale but functionally v ital under life threatening conditions (1). They originate from megakaryocytes located mainly in the bone marrow, found in circulating blood and stored in spleen (2). Platelets dont conta in a nuclei and during their inactive state they have a discoid morphology with a diameter of 2-4 micrometer (3, 4). But whenever they are active they can change their morphology very rapidly to an irregular branched spread form (5). Currently platelets are being used at wide spread clinic treatments from cosmetic needs to supporting insufficient heart (6

, 7). 2.1. Development of Platelets It is not exactly explained how platelets originate from megakaryocytes . There are several models to explain formation of platelets. Megakaryocytes seem to locate as triple form. With their VEGF secretion capacity they hold vessel endothelial cells close to themselves (8). The most scientifically accept ed three models are mentioned as, 1. Simply blebbing from the cell membrane of megakaryocytes (1). 2. In megakaryocytes there are special cell fields defined as Demarcati on Membrane System where granules of platelets condense and fragments break away (9). 3. The most popular theory seems to be Proplatelet Formation. Here mega karyocytes have long thin branch like extensions at the blood circulating site o f blood vessels of Blood Cell An Overview of Studies in Hematology 4 bone marrow and on these branches there are uprising small bodies where by the h elp of blood shear force platelets enter directly to circulating blood stream. It wa s suggested that the concept of platelet like bodies arise from pseudopods of Meg akaryocytes, the forming platelets were named as proplatelet (10).

Figure 1. Megakaryocyte branches with Platelet Buds (PB) are seen. Proplatelets are released as Dumbell shaped bodies. This image is referenced from Hartwig and Italiano 2003 ( Thanks for the kind permission of John Wiley and Sons to use this image) (11) . Kinetics of platelets; they have a life span as 7-10 days and in 1 liter human blood it is estimated that there are 150-400X10 9 platelets so for a balanced number they are formed 15X10 9 -40X10 9 daily. Megakaryocytes located in the bone marrow sinusoids form a barrier to other bone marrow cells, it forms a physical barrier preventing di rect contact to blood circulation. But there are canallicular openings in megakaryocyte membrane which permits cell migration to other cells to enter blood stream; this is named as Emperipoles is (8). Platelets 5 These small cell fragments have complex properties; 2 cytoplasmic regio ns can be seen in platelets 1. Hyalomere: The light blue homogeneous region of the peripheral cyto plasm is called Hyalomere. Hyalomere includes cytoplasmic filaments and circumferential microtub

ule bundle under the cell membrane. These elements of the cytoskeleton pro vide the movement and the protection of the platelets shapes. 2. Granulomere (Chromomere): This is the central region and tight area . It is ranging in color from blue to purple-staining. Granulomere includes small Golgi complex, sm ooth endoplasmic reticulum, lysosome, scattered granules surrounded by a membrane and a variety of mitochondria (4). Platelets have a simple appearance but carry very complex functional p roperties. By dividing this simple cell fragment to four regions helps for a better understanding of the functions of platelets. 1. Peripheral Zone: This region is composed from unit membrane with open canalicular system. Three p arts are defined as; a. Exterior outer layer: This is a glycocalix membrane with 10-20 nm thickness and thicker than the other blood cells, rich from glycoproteins that are mainly receptors for cell-cell and cell-vessel interactions(1, 8). b. Platelet Unit Membrane: Platelet unit membrane has some similarities and appearance with other unit membranes of cells, it is composed from bilipid layer rich of phospholipids (12 ), it can distribute molecules according to phsico-chemical properties for passing the mem brane. The membrane has anionic and cationic pumps. Platelet unit membrane is an import ant catalyst for liquid phase coagulation. c. Submembrane Zone: Just located under the unit membrane a layer composed of microflament network. T his network is anatomically and functionally related to membrane glycoprotei ns and cytoplasmic filament system. 2. Sol-Jel Zone: This is cytoplasm corresponding part of the cellular fragment, platelet. It is i n soluble or gel phase according to changes of polymerization of the filaments; act in and microtubules(1). Just under the submembrane zone there are microtubules forming a perip heral ring which helps platelet to maintain its discoid shape in inactive form. When activated, the microtubules surround the organelles and with the contribution of other filaments (13), the organelles are tightly contracted. During silent form only 3 0-40 % of actin filaments are polymerized, when platelets are activated the polymerized amount increases(1).

Blood Cell An Overview of Studies in Hematology 6 3. Organel Zone: This is the zone where granules, peroxisomes, lysosomes and mitochondrias are localized. There are enzymes, adenine nucleotids, calcium, serotonin and many other proteins in this region (1). 4. Membrane Zone There is a distinguishing feature of platelets that their plasma membrane contai ns wide spread invaginations that forms a network inside platelet. Finally with pore openings the inner network is directly in contact with outer zone. This system is named a s open canallicular system (OCS) and with this system an extensive amount of surface area stays as potential in silent state. With this system also platelet ga ins a large area for molecular trafficking. A second canal system is composed from endoplasmic reticu lum networks and named as Dense Tubular System (DTS). Here in DTS many enz ymes and calcium ions that are important for activation are located. DTS i s not directly connected to outer membrane (1, 14) but has close connections with OC S. These two systems actively exchange molecules (1). The granulles have diameters ranging between 200 to 500 nm and they are found as spherical or oval structures (15). There are 3 types of granules in platelets, Alfa Granules, Dense granules, lysosomes. Alpha granules are most prominent in terms of material content and majority. These granules include inflammatory molec ules, cytokines, cell-activating molecules, proteins, Growth Factors, adhesion molecules, integrins and other proteins These granules are filled by megakaryocytes (3). 3. Alpha granules It is widely accepted that these granules come from the budding of t rans golgi apparatus organel of megakaryocytes (16, 17). These are 200-400 nm diameter granules widespread in the cytoplasm (16 ) which gives the granular appearance in Romanoski stained smear preparations, each platel et contains around 50-80 of these granules. The content of granules is very diverse; a brief list is given in table 1 (14, 18, 19, 20, 21). When platelets are activated these alpha granules fuse with each other , OCS and plasma membrane. The secretion of alpha granules is mediated by some proteins (such as SNARE) and membrane lipids (19). The secretions effect platelet and cells in the environment (such as endothelial, leukocytes) for migration, adhesion and proliferation(14). A rare syndrome named as Gray Thrombocyte Syndrome (GTS) is both invo lved with the quantity and quality of platelets which cases susceptibility for bleeding. In GT

S the proteins synthesized by megakaryocytes are abnormal and dont enter platelets as they do in normal individuals and additionally the endocytotic mechanisms dont work properl y. As a result the secretions spread to bone marrow and a fibrosis forms (miyelofibrozis)(22, 2 3). Platelets 7 Thrombospondin P-selectin platelet factor 4 beta thromboglobulinler Factors V, XI, XIII fibrinogen von Willebrand factor fibronectin vitronectin high molecular weight complexes kininogen chemokines mitogenic growth factors (platelet-derived growth factor) vascular endothelial growth factor TGF-beta Table 1. Some main components of alpha granules. 4. Dense granules These are smaller granules with 150 nm diameter (24), because of the calcium and phosphate content there image seems dense under electron microscopic (E M) observation (21, 25). Each platelet contains 3-8 of these granules (14). The components of d ense granules are briefly given in Table 2 (10, 14, 19, 20). Ca Mg P pyrophosphate Nucleotides ATP, GTP, ADP, GDP Membrane proteins CD63 (granulophysin) LAMP 2 Serotonin GPIb, GPIIb/IIIa P-Selectin Histamine Epinephrine Table 2. Some main components of dense granules. In activated platelet these granules fuse with plasma membrane and expel their i ngredients to their environment which causes other platelets to aggregate and a local vasoconstriction (especially by serotonin) in the involved vessels. Also the ADP content is a very important participant for homeostasis (14). The importance of the components of dense granules for homeostasis is recognized when the diseases of the deficiency of dense granules was defined as Herma nsky-Pudlak Blood Cell An Overview of Studies in Hematology 8 Syndrome (26, 27, 28) and Chediak Higashi Syndrome. s stoppage of In both syndrome

bleeding is defective based on the impairment in dense granules (14). 5. Lysosomes They have a diameter of 200-250 nm which places them to middle size granule (14). They cant be distinguished from alpha granules under EM observation because of the sim ilarities in dense electron appearance. By the content of acid phosphates and a rylsulphates cytochemical staining techniques can effectively distinguish lysosomes from alph a granules. In an activated platelet they expel their contents to environment as the other two granules by membrane fusing mechanisms. The difference for lysosomes to be involved in ac tivation is that they need a more potent stimulus. The role of lysosomal components in ho meostasis is not well understood as the other granules contribution. They are i nvolved in thrombus formation and extracellular matrix remodeling (8). It seems that lysosomes in platelets dont have any distinguished featur es, they share the common features with other cells lysosomes (29). The components of dense granules are briefly given in Table 3 (8, 18, 30, 31, 32 ). Figure 2. M: Mitochondria, G: alfa-granules, DG: dense granules, Gly: glycogen pa rticles and OCS: open canalicular system. The morphology can be seen in equatorial section of a h uman platelet. This image is referenced from Zufferey 2011 (Thanks for the kind permission of John W iley and Sons to use this image)(33). Platelets 9 PF3 Acid phosphatase Glucose-6 phosphatase Arabinosidase N-Acetyl-galactosominidase ATP = adenosine triphosphate TGF CD63 Cathepsin lysosomal membrane proteins (LAMP-1, LAMP-2) acid hydrolases cathepsins Table 3. Some main components of platelet lysosomes 6. Autologous platelet rich plasma (PRP) The application of growth factors in medical practice is one of the areas where basic clinical research has focused its attention but there are many problems associa ted with their local administration. For example, recombinant human growth factors are not cost effec tive, they have limited shelf life, and ineffectively delivered to target cells a nd in addition, to get efficient therapy, large doses are needed. The use of autologous plate

lets concentrates for tissue regeneration and wound healing has now become an alternative easy and che ap way to obtain high concentrations of these growth factors (34). The autologous blood collected from a patient just before surgery can be prepared as platelet concentrates, platelet-rich plasma (PRP) and platelet gel for the treat ment the patient specifically needs (35). These forms are prepared by gradient density centrifugation techniques to obtain high (x5) concentration of platelets (36). This autologous concentration includes a large amount of growth factors, especially PRP is an easy and inexpensive technique to accelerate the wound healing (37). This quite new field is open for research, there are a lot of techn iques still under development stage such as platelet gels can be obtained by adding thrombin to au tologous platelet-rich plasma. The initiation of fibrin polymerization and the r elease of platelets factors and cytokines can be achieved by the specific activators such as thrombin, glass, freeze-thaw cycle to platelet-rich plasma depending on what is required during t he surgery (35). In spite of the distinct features of platelet-rich plasma (PRP) and its use by d ifferent fields of medicine, no adverse reactions were documented until now(38, 39, 40, 41). Blood Cell An Overview of Studies in Hematology 10 Author details Gkhan Cce * and Tahsin Murad Aktan Deparment of Histology and Embryology, Faculty of Meram Medicine University of K onya Necmettin Erbakan, Turkey 7. References [1] Becker RC. Platelet Biology: The Role of Platelets in Hemostasis, Thrombosis and Inflammation. Platelets in Cardiovascular Disease. In:Bhatt DL. Imperial Colle ge Press. London, 2008:1-3. [2] Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ell is S, Kelly PN, Ekert PG, Metcalf D, Roberts AW, Huang DC, Kile BT. Programmed anuclear cel l death delimits platelet life span. Cell. 2007;128(6):1173-86. [3] Rozman P, Bolta Z. Use of platelet growth factors in treating wo unds and soft-tissue injuries. Acta Dermatovenerol Alp Panonica Adriat. 2007;16(4):156-65. [4] Ovalle WK, Nahirney PC. Netter Essential Histology. Saunders; 2007;166. [5] Klages B, Brandt U, Simon MI, Schultz G, Offermanns S. Activation of G12/G1 3 results in shape change and Rho/Rho- kinase-mediated myosin light chainphosphory lation in mouse platelets. J Cell Biol. 1999;144(4):745-54. [6] Anitua E, Snchez M, Nurden AT, Nurden P, Orive G, Anda I. New insights into a nd

novel applications for platelet-rich fibrin therapies. Trends Biotechnol. 2006(5 ):227-34. [7] Mishra A, Velotta J, Brinton TJ, Wang X, Chang S, Palmer O, Sheikh A, Chung J, Yang PC, Robbins R, Fischbein M. RevaTen platelet rich plasma improves card iac function after myocardial injury. Cardiovasc Revasc Med. 2011;12(3):158-63. [8] Drouin A, Cramer EM. Production of Platelets. Editor: Gresele P, Page CP, Fuster V, Vermylen J. Platelets in Thrombotic and Non-Thrombotic Disorders: Pathop hysiology, Pharmacology and Therapeutics. Cambridge University Press; 2002;25.USA. [9] Schulze H, Korpal M, Hurov J, Kim SW, Zhang J, Cantley LC, Graf T, Shivdasani RA. Characterization of the megakaryocyte demarcation membrane system and its role inthrombopoiesis. Blood. 2006;107(10):3868-75. [10] Italiano JE Jr, Shivdasani RA. Megakaryocytes and beyond: the birt h of platelets. J Thromb Haemost. 2003;1(6):1174-82. [11] Hartwig J, Italiano J Jr. The birth of the platelet. J Thromb Haemost. 2003 ;1(7):1580-6. [12] White JG. Platelet Structure. Editor:Michelson AD. Platelets. Elsevi er: USA, Second Edition, 2007;45 [13] White JG. Views of the platelet cytoskeleton at rest and at work . Ann N Y Acad Sci. 1987;509:156-76. [14] Rumbaut RE, Thiagarajan P. Platelet-Vessel Wall Interactions in Hem ostasis and Thrombosis. Editr: Granger DN, Granger JP. Colloquium Series on Integrated System s * Corresponding Author Platelets 11 Physiology: From Molecule to Function to Disease. Morgan & Claypool Li fe Sciences; 2009-2011:5. [15] Gassling VL, Ail Y, Springer IN, Hubert N, Wiltfang J. Platelet-ri ch plasma and platelet-rich fibrin in human cell culture. Oral Surg Oral Med Oral P athol Oral Radiol Endod. 2009 ;108(1):48-55. [16] King SM, Reed GL. Development of platelet secretory granules. Semi n Cell Dev Biol. 2002(4):293-302. [17] Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev. 2009(4):177-89. [18] McNicol A, Israels SJ. Platelet dense granules: structure, function and implications for haemostasis. Thromb Res. 1999;95(1):1-18. [19] Reed GL. Platelet secretory mechanisms. Semin Thromb Hemost. 2004;30(4):441 -50. [20] Askari AT, Messerli AW, Lincoff M. Thrombosis and Antithrombotics in Vascular Disease. Management Strategies in Antithrombotic Therapy. Editr: Askari AT,

Messerli AW.Wiley; USA. 2008:3. [21] Ma AD, Key NS. Molecler Basis of Hemostatic and thrombotic Disease s. Editr: Coleman WB, Tsongalis GJ, London. Molecular Pathology: The Molecular Ba sis of Human Disease. Academic Press; 1 edition, 2009:258. [22] Di Paola J, Johnson J. Thrombocytopenias due to gray platelet syn drome or THC2 mutations. Semin Thromb Hemost. 2011(6):690-7. [23] Nurden AT, Nurden P. The gray platelet syndrome: clinical spectrum of the disease. Blood Rev. 2007(1):21-36. [24] Rendu F, Brohard-Bohn B. The platelet release reaction:granules constituents , secretion secretion and functions. Platelets. 2001;12(5):261-73. [25] Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense gra nules contain polyphosphate and are similar to acidocalcisomes ofbacteria and unicellu lar eukaryotes. J Biol Chem. 2004;279(43):44250-7. [26] King SM, McNamee RA, Houng AK, Patel R, Brands M, Reed GL. Plat elet densegranule secretion plays a critical role in thrombosis and subsequent vascularrem odeling in atherosclerotic mice. Circulation. 2009;120(9):785-91. [27] Nisal M, Pavord S, Oppenheimer CA, Francis S, Khare M. HermanskyPudlak syndrome: management of a rare bleeding disorder in a twin pregnancy. J Obstet Gynaecol. 2012 ;32(2):185-6. [28] Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol. 2009;10(9):623-35. [29] Skoglund C. Platelets in inflammation. Linkping University Medical D issertations. 2010-Sweden;14. [30] Gerrard JM, Phillips DR, Rao GH, Plow EF, Walz DA, Ross R, Hark er LA, White JG. Biochemical studies of two patients with the gray platelet syndrome. S elective deficiency of platelet alpha granules. J Clin Invest. 1980;66(1):102-9. [31] Nishibori M, Cham B, McNicol A, Shalev A, Jain N, Gerrard JM. The protein C D63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlaksyndrome , and appears identical to granulophysin. J Clin Invest. 1993;91(4):1775-82. Blood Cell An Overview of Studies in Hematology 12 [32] Grau AJ, Reiners S, Lichy C, Buggle F, Ruf A. Platelet function under aspirin, clopidogrel, and both after ischemic stroke: a case-crossoverstudy. Stro ke. 2003;34(4):849-54. [33] Zufferey A, Fontana P, Reny JL, Nolli S, Sanchez JC. Platelet pr oteomics. Mass Spectrometry Reviews, 2011; 31, 331351. [34] Nikolidakis D, Jansen JA. The biology of platelet-rich plasma and its appli cation in oral surgery: literature review. Tissue Eng Part B Rev. 2008 Sep;14(3):249-58. [35] Soffer E, Ouhayoun JP, Anagnostou F. Fibrin sealants and platelet preparati

ons in bone and periodontal healing. Oral Surg Oral Med Oral Pathol Oral Radiol E ndod. 2003;95(5):521-8. [36] Huang Q, Wang YD, Wu T, Jiang S, Hu YL, Pei GX. Preliminary se paration of the growth factors in platelet-rich plasma: effects on the proliferation of human marrowderived mesenchymal stem cells. Chin Med J (Engl). 2009;122(1):83-7. [37] Napolitano M, Matera S, Bossio M, Crescibene A, Costabile E, Almo lla J, Almolla H, Togo F, Giannuzzi C, Guido G. Autologous platelet gel for tissue rege neration in degenerative disorders of the knee. Blood Transfus. 2011;25:1-6. [38] Edwards SG, Calandruccio JH. Autologous blood injection for refract ory lateral epicondylitis. J Hand Surg [Am]. 2003;28(2):272-278. [39] Mishra A, Pavelko T. Treatment of chronic elbow tendinosis with buffered pl atelet-rich plasma. Am J Sports Med. 2006;34(11):1774-1778. [40] Kajikawa Y, Morihara T, Sakamoto H, et al. Platelet-rich plasma e nhances the initial mobilization of circulation-derived cells for tendon healing. Cell Physi ol. 2008;215(3):837-845. [41] Snchez M, Anitua E, Azofra J, Aguirre JJ, Andia I. Intra-articular injection of an autologous preparation rich in growth factors for the treatment of kne e OA: a retrospective cohort study. Clin Exp Rheumatol. 2008;26(5):910-913. Chapter 2

2012 Rafea and Souchelnytskyi, licensee InTech. This is an open access chapter d istributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/li censes/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease Mahmoud Rafea and Serhiy Souchelnytskyi Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48593 1. Introduction The only identifiable function of Red Blood Cells (RBC) is the delive ry of Oxygen. In mammals, RBC is a unique cell because: It does not have cellular organelles like any other cells in the body. It has a very especial protein: Hemoglobin which plays the role of carrying Oxygen to tissues and carries back carbon dioxide to lungs. It has a very especial cell membrane which carries a number of blood groups ant igens systems. Their functions include transporting other proteins and molecules into and out

of the cell, maintaining cell structure, attaching to other cells and molecules, and participating in chemical reactions [1]. Those systems are genetically controlled with blood groups determining genes. The work described in this chapter is based on the function carried out by the c ell membrane antigens which are transporting other proteins and molecules into and out of the cell. The question is what are those proteins that are transported? In fact, th is question identifies the knowledge gap about RBC role in health and disease. In the next sect ion, some hypotheses will be inducted and deduced through analysis of available background knowledge. The experiments that can proof those hypotheses are described in section 3 . This is followed by describing a theory about the role of RBC in health and disease base d on the proved hypotheses and how we can benefit from this theory in diagnosing and treating of patients. 2. Knowledge analysis and hypotheses induction Basically, when an antigen is introduced into a body, the immune syst em (IS) does either one of two reactions: immune tolerance (IT) or immune response (IR). IT-reaction is never Blood Cell An Overview of Studies in Hematology 14 absolute [2]. It is usually accompanied by a weak IR. In normal IR, one cannot identify if there is a degree of IT, because there is no defined laboratory method/test that can measure the degree of IT. Meanwhile, by logical implication, some degree of IT should ex ist with the normal IR. This entails that there is an equivalence relation between IT and IR. Hypothesis I: There is no absolute immune tolerance, if and only if there is no absolute immune response In central IT, immature self-reactive T lymphocytes recognize antigens in the thymus and undergo negative selection (deletion) [3]. Consequently, in normal IR a gainst a particular antigen, measuring the concentration of this antigen in the thymus can be correl ated to the degree of the accompanied IT. The transport mechanism of antigens to the thymus is a critical issue because of the remarkable capacity of IS which can recognize any antigen [4]. In [5], authors claim that Dendritic Cells (DCs) have several function s, not only, in innate and adaptive immunity, but also there is increasing evidence that DCs in situ induce antigen specific unresponsiveness or tolerance in central lymphoid organ s and in the periphery. The evidence that DCs transport antigens to thymus in central toleran ce is very weak while the evidence that DCs have role in peripheral tolerance is more acceptable based on the review article [6]. In conclusion RBC may be vehicles w

hich transport self antigens to induce central IT. The role of RBC in transporting antigens has not been investigated be fore. If RBC are capable of antigen transport to induce IT, this will unveil important knowledge. For instance, in hemolytic disease of fetus and newborn (HDFN), maternal a nti D alloantibody and feto-maternal ABO incompatibility are the two major causes of HDFN, Meanwhil e, with the implementation of Rhesus D immunoprophylaxis, hemolytic disease due to ABO incompatibility and other alloantibodies have now emerged as major caus es of this condition. [7]. In pregnancy, most of delivered infants are normal when there is no anti D alloantibody which means that there is an efficient mechanism that can handle the other incom patibilities. The mechanisms explained in literature explain why ABO incompatibilities , only, do not occur [8], [9] and [10], but these mechanisms do not explain why tho se incompatibilities occur. The mechanism may be based on trapping those antibodies in placenta throu gh RBC catering of ABO and other incompatible blood groups antigens. Consequen tly, the occurrence of HDFN may be due to depletion of those antigens store from RBC. Also , if this RBC transport function is the mechanism a body tolerates his self antigens, this will explain how a pregnant woman is able to tolerate her fetus and placenta, ass uming that they are part of self. Hypothesis II: RBC hide antigens to transport them to target organs. From these hypotheses I & II, if RBC play role in antigen transport, one can ded uce that in any mammal, blood circulating antibodies against self and foreign, either antigens o r tolergens, will react with hemolysate. Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 15 Hypothesis III: There is an injection function (one-to-one) between cir culating antibodies and RBC s hemolysate antigens. To proof that RBC have role in immune reactions (IR and IT), one need to proof t hat there is an inverse correlation between antibodies concentration in plasma and a ntigens concentration in RBC. Hypothesis IV: In immune response, antibodies concentration in plasma a gainst a particular antigen in hemolysate is higher than this antigen concentration in hem olysate. Meanwhile, in immune tolerance, antibodies concentration in plasma against a particula r antigen in hemolysate is lower than this antigen concentration in hemolysate. It should be remarked that Humans expressing a defective form of the transcription factor

AIRE (autoimmune regulator) develop multi-organ autoimmune disease (autoi mmune polyendocrinopathy syndrome type 1) [11]. Liston et all [12] prove tha t this autoimmune syndrome is caused by failure of a specialized mechanism for deleting forbidden T cell clones, establishing a central role for this tolerance mechanism. 3. Experiments The methodology applied will demonstrate the existence of particular se lf tolerogens and particular foreign antigens in RBC (Hypothesis I & II) and show that innumerable antigens exist in RBC which react with innumerable antibodies that exist in pl asma. This partially proves that RBC play a role in immune reaction. To proof Hypothesis IV, it will be demonstrated that the concentration of foreign antigens in RBC varies by time in relation to IR known behavior. The experiments done are the following: 1. RBC of pregnant females transport male spouse ABO blood group antigens 2. RBC of pregnant females transport male spouse HLA antigens 3. RBC transport self HLA antigens 4. RBC transport self Tissue Specific Antigens (TSAs). 5. RBC hemolysate antigens are precipitated by plasma obtained from the same in dividual and cross reacted with plasma from different individuals. 6. RBC transport bacterial antigens. 7. RBC antigens and plasma antibodies concentration vary with time. 3.1. Materials for experiments 1, 2, and 3 Couples that have children, pregnant females, and single females were selected from relatives and friends. The purpose of the experiments was explained to them. Not all the combinations could be found, after blood grouping. The combinations presented, i n Table-1, were used to conduct the experiments. Blood samples were taken on hepa rin. Some of the blood samples were used to prepare RBC and plasma and the rest was used to prepare lymphocytes using the Ficoll hypaque technique [13]. Blood Cell An Overview of Studies in Hematology 16 RBC were washed several times using phosphate buffer saline (PBS). The male RBC were divided into two tubes. The first tube was divided into small aliquot s that were frozen to rupture RBC. The second tube was used to prepare a 5% suspension. The female RBC were divided into small aliquots that were frozen to rupture RBC. Notice t hat we do not need female intact-RBC. Female ABO group Male ABO group O A O B O AB A O A B B A O (SINGLE) Table 1. The ABO blood groups of couples used in the experiments

3.2. RBC of pregnant females transport male spouse ABO antigens To test RBC transport of male spouse ABO antigens, a technique based on competitive inhibition of RBC agglutination was followed. If the hemolysate contain s ABO specific antigens, then those antigens will compete with RBC and prevent their agglutinat ion. Figure 1 illustrates a schematic description of the experiment. Method The experiment was performed, for each couple, as follows: In positive control tubes which represent also reference tubes for comparison w ith test tubes, serial dilutions (up to 1/128) of female spouse plasma were ma de using normal saline. A drop of a male spouse s hemolysate was added before adding his RBCs suspension. In test tubes, serial dilutions of the female spouse s plasma were m ade using normal saline. A drop of the female spouse s hemolysate was added before add ing a drop of her male spouse s RBC suspension. Results Whenever there is ABO incompatibility and the male spouse is not O, ag glutination was inhibited by the female spouse hemolysate and was not inhibited by ma le spouse hemolysate. In most cases, agglutination was inhibited in the first tu be. However, agglutination was never observed in subsequent tubes. The single virgin female R BC do not contain any ABO antigens. 3.3. RBC of pregnant females transport male spouse HLA antigens This experiment was performed using commercial HLA Typing Trays for the identifi cation and definition of HLA Class I Antigens using the microlymphocytotoxicit y assay [14]. It is Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 17 also based on competitive inhibition. Consequently, if typing wells tha t show positive reaction were inhibited in corresponding testing wells by adding hemoly sate, this proves the existence of specific competing antigens. Figure 2 illustrate the experiment steps. Figure 1. Schematic drawing of ABO antigen transport experiment, the upper part shows how the reference positive control is conducted, while the lower part shows how the test is conducted. Figure 2. Re-typing of pete with his lymphocytes Method First, each couple was A hemolysate from sitive control should give positive male spouse but using his female spouse hemolysate to com

HLA typed, and then the following was done: a third person was added to control wells. The po reaction. In this way, we excluded inherent error

s or non-specific reaction. Female spouse hemolysate (diluted 1/16) was added to typing wells Eosin Dye Blood Terasaki plate containing anti HLA Class I antibodies Lymphocytes Compleme Extract RBC Hemolysat Prepare Blood Cell An Overview of Studies in Hematology 18 Male spouse lymphocytes was added and followed by the complement and eosin dye as usual. Wells that gave positive reaction in typing were examined by contrast microscop e. Results It was observed that female spouse hemolysate inhibited the typing reaction whil e the third person hemolysate did not. This indicates the existence of male spouse HLA antigens in female spouse hemolysate. 3.4. RBC transport self HLA antigens This experiment is similar to the previous one. The only difference i s the use of the males own hemolysate instead of his females spouse hemolysate. It was observe d that a male hemolysate inhibited the typing reaction of his lymphocytes indicating the exist ence of self HLA antigens. 3.5. RBC transport Tissue Specific Antigens (TSAs) If RBC transport antigens to central organs of the immune system to induce toler ance, then RBC will definitely transport TSAs. Otherwise this transport function has nothin g to do with tolerance. Consequently, the objective of this experiment was to demonstrate tha t antibodies against TSAs can be prepared through injecting RBC of white mice into rabbits. Figure 3 illustrates the experiment. Figure 3. Preparing antibodies against white mice TSAs in rabbit Materials A number of white mice were slaughtered to collect their blood on sodium citrate and their organs (liver, kidney and spleen) were preserved on 10 % formalin. The separated RBC were Prepare Apply RBC Antiserum Fluorescent Anti Rabbit Ig Inject Mouse Tissue sections Add

Prepare Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 19 washed many times with sodium citrate and then diluted with 3% formol-saline to kill any bacterial contamination. An ordinary rabbit was selected to prepare the antibodi es. Method: A rabbit was injected subcutaneously with one ml of white mice RBC for four tim es on weekly intervals. Blood was collected from ear-vein after 35 days from the first injection. The serum was examined for antibodies against mice RBC using direct agglutination slide test. The serum was examined for antibodies against TSAs of white mice (liver, kidney and spleen) using the sandwich technique in histo-pathology sections. Results: All sections showed florescence. Figure 4 illustrates some of the hist opathology sections taken from a white mouses organs. Figure 4. Histopathology sections from a white mouses organs examined by floresce nt microscope showing florescence due to antigen-antibody reaction, A: kidney tissue, B: liver , and C: spleen. 3.6. RBC hemolysate antigens are precipitated by plasma Ouchterlony immuno-precipitation test of normal serum against self and other normal hemolysate was conducted, Figure 5(a). We confirmed this finding by us ing Western Blot technique, and showed that serum from one individual recognized antigen s in hemolysate from two normal persons, Figure 5(b). Further confirmation was obtained by using twodimensional gel electrophoresis (2-DE) of co-immunoprecipitated hemolysate antigens using self-serum, Figure 5(c). Notice that the number of the immune-precipitated antigens is numerous and many spots were enriched by immune-precipitation because t hose antigens were not detected in 2-DE gel of hemolysate, Figure 5(d). Antigenicity of the separated proteins was confirmed by immune-blotting proteins separated by 2-DE with the sa me selfserum, Figure 5(e). This excluded co-precipitation of non-antigens, as they would not be detected in immune-blotting. 3.7. RBC transport bacterial antigens As TB is a priority disease, trying to find Mycobacterium tuberculosis bacilli protein antigens (MTPAs) in TB-patient hemolysate was conducted through 2D electrophores is, and Blood Cell An Overview of Studies in Hematology 20 then identifying gel spots with mass spectrometry. Fortunately, we discovered f our MTPAs. This motivated us to do the experiments of the next section to ident

ify more MTPAs in hemolysate of TB patients. Identifying MTPAs in TB patients hemolysate The goal is to find the set of antigens, in TB patients hemolysate, which is related to Mycobacterium tuberculosis bacilli. The approach taken follows the follo wing steps Figure 6: 1. The study resources are: [A] Patients [B] Mycobacterium tuberculosis (H37Rv) 2. For each patient: Collect blood sample on anticoagulant (step 1) Separate RBC and wash many times with saline (step 2) 3. Hemolysate [C] is prepared by rupturing RBC with low isotonic solu tion which is the binding buffer in affinity chromatography 4. Prepare hyper immune serum for M. tuberculosis (step 3) 5. Purify antibodies using Protein A Sepharose beads (step 4) 6. The purified antibodies are then used to separate antigens from hemolysate ( step 5) 7. The disease related antigens are identified using in gel trypsin d igestion and MALDI TOF mass spectrometry (step 6) Figure 5. Detection of innumerable antigens in Red Blood Cells. (a) Ouchterlony test showing serum of normal against hemolysate of self and others (b) Western Blot using two normal h emolysate propped with serum of one of them (c) Silver stain of 2-DE of immune-precipitated hemoly sate antigens (d) Silver stain of 2D electrophoresis of hemolysate (e) Western Blot of 2-DE o f hemolysate propped with serum. 3.8. RBC antigens and plasma antibodies concentration vary with time The objective of this experiment is to investigate the dynamics of foreign antig ens in RBC. In effect, antibodies are taken at one instance of time, while RBC are taken at different instances. Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 21 Figure 6. Flowchart depicting the resources and steps for identification of hemo lysate antigens related to Mycobacterium tuberculosis (H37Rv) Figure 7. Precipitated Antigens separated using 2D electrophoresis Gel Materials Bacteria: Escherichia coli O157:H7 strain 1 was inoculated onto SMAC agar (Oxoid). Colonies were tested by E. coli O157 latex kit (Oxoid DR 620) and c onfirmed biochemically. A single colony of E. coli bacterial growth from the plate was in oculated into Brain heart infusion broth (Oxoid) and incubated overnight at 37 C and adjusted

to a concentration of approximately 10 10 CFU. Animals: 1. A rabbit 2. A baladi sheep between 8 to10 months. The rabbit and sheep were tested serologically, to be negative, for Escherichia coli O157:H7. Methods: The first experiment method was done as follows, Figure 8: 1. Rabbits were vaccinated by Escherichia coli. Rabbits were injected subcutane ously with one ml on weekly basis for three weeks. 2. Blood was collected from the ear-vein after 21 days from the first injection . 1 This strain is kindly provided by the serology department, Animal Health Resear ch Institute (AHRI), Giza, Egypt. Blood Cell An Overview of Studies in Hematology 22 3. Rabbits sera were separated and examined for antibodies against E. coli O157 using direct bacteria slide agglutination test. 4. A sheep was infected by oral administration of bacterial suspension. 5. Red blood cells were prepared from anti-coagulated sheep blood collected at 0 time (i.e., before inoculation), 1 st week, 2 nd week, and 3 rd week. The collected blood was C for 25 minutes at 1170 g. Plasma and Buffy coat centrifuged at 4 from each sample were removed. RBC were washed twice in normal saline solution by centrifugation at 4 C for 5 minutes at 2000 g, and then re-suspended in Tris/Saline buffe r pH 7.5 and subjected to lyses by freezing. 6. Nobel agar 1% in Tris/Saline was used as a supportive media for antigen-antibody precipitation, where the central well contained rabbit serum and periph eral wells contained sheep RBC hemolysate. Results The rabbit serum showed high titer (1/160) of antibodies against E. c oli. Antigens of E. coli could be precipitated from sheep RBC of the 1 st and 2 nd week after infection, only, Figure 9. Figure 8. Preparing Antibodies against bacteria and preparing RBC carrying antig ens of this bacteria. The purpose is to precipitate Bacteria antigens from RBC of infected animals usi ng the prepared

antibodies against those antigens. Figure 9. Illustrates the dynamics of RBCs antigens Infect Infect Antiserum Detect antibodies against E. coli RBC Central well: Rabbit antiserum Well 1: Sheeps RBC before infection Well 2: Sheeps RBC 1 st week after infection Well 3: Sheeps RBC 2 nd week after infection Well 4: Sheeps RBC 3 rd week after infection Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 23 4. The role of RBC antigens transport in health and disease The RBC transport function maintains tolerance to self antigens. This function is exploited positively to protect a fetus from the immune system attack using the same mechanism of protecting the self. In effect, a fetus, which is an allograft, is considered pa rt of self. In humans and animals, not all microorganisms are capable of causing disease. Some of those microorganisms are equipped with the machinery that can overcome biological barriers and can cause disease in animals but not in humans and vice versa [15]. The role of RBC antigens transport in inducing tolerance to self-antigens is a fea ture that can be considered as a security-hole, as invaders can exploit this process to escape from the response of the immune system by disguising themselves as self. Tumors and paras ites are negative examples. Notice that this mechanism of tolerance induction does not contradict with all what we know about tolerance. Further, it explains the documented properties of tolerance. For instance, some of the properties that can be explained are: Artificially induced tolerance is of finite duration because antigen stores get depleted. Tolerance to self antigens is a process that continues throughout life but begi ns during fetal development because RBC are transporting self antigens all the time. Notice that the discovered function of RBC fills a gap in the understanding of t olerance. Part of this gap can be expressed in the following questions: 1. Why soluble antigens administered intravenously favor tolerance while particulate antigens injected into the skin favor immunity. 2. Why ingested large doses of soluble proteins induce systemic T lym

phocyte tolerance, whereas the components of vaccines such as the Sabin polio vaccine induce an eff ective local immune response. 3. Why tolerance is easier to induce in prenatal rather than postnatal life. Answer of Question 1 and 2: RBC can easily absorb soluble antigens t hrough pinocytosis while a particulate antigen needs receptor sites on RBC in order to be absorbed, which is the RBC membrane antigens function. Notice that the probability that the i mmune system will react to some processed antigens still exists. That is why the dose of antigens plays an important role. As far as there are enough stores of antigens in RBC , they are effectively tolerated. Answer of Question 3: If antigens are introduced to a fetus while the immune sys tem is still incapable of respond, there is a good chance for those antigens to b e processed by the Antigen-Presenting-Cells (APC) and then absorbed by RBC. When mature ly mphocytes production starts, later in life, antigen stores of RBC are used to induce toler ance. This may explain why tolerance is easier to induce in prenatal life. Further, a pathogenesis mechanism of some autoimmune disease can be postulated. If RBC antigen-transport function is impaired for a particular self-antigen, fo r some reason, the Blood Cell An Overview of Studies in Hematology 24 tolerance to that antigen will eventually vanish. Consequently, an auto immune response will be provoked to that antigen and autoimmune disease is established. 5. How this RBC antigens transport function can be exploited This observed RBC antigens transport function creates an antigens store. This store can be exploited in many directions. The proposed direction is to exploit fun ctional proteomics approach [16] with the following three crucial aspects of the experimental desig n to produce products which are among diagnostic kits, vaccines or treatment components: 1. The strategy used for the selection, purification and preparation o f the antigens to be analyzed by mass spectrometry 2. The type of mass spectrometer used and the type of data to be obtained from it 3. The method used for the interpretation of the mass spectrometry da ta and the search engine used for the identification of the proteins in the different types of seq uence data banks available The aim of this approach is to identify antigens which are relevant to a particular disorder. 5.1. Direct approach for products development This approach is based on using a subset of antibodies which are specific agains t a subset of antigens of a particular disease to enable the use of those antibodie s and those antigens in

preparation of beneficial products. Diagnostic kits can be prepared for all infectious microorganism and a ll tumors. In such disorders, simple kits can be prepared using the following steps: 1. Extract antigens from microorganism/tumor-cell-line cultures in coupling buf fer 2. Prepare hyper immune serum using extracted antigens 3. Build an affinity column 4. Antibodies purification: Use affinity column containing antigens to separate their related antibodies from hyper immune serum 5. Adsorb purified antibodies to latex beads A more advanced kits based on selection of antigen-determinant sites ( epitopes) can be prepared. The problem of such kits, which uses a particular antigen, is in its validation which will be more sophisticated. One can expect that this particular antigen ma y not exist in RBC antigens store of some population who are genetically different from the p opulation used in preparation of the kit. Active vaccines against all infectious microorganism and all tumors can be prepared by using the purified antibodies prepared for diagnostic kits in identifyi ng related antigens existing in RBC antigens store. The identified antigens can be prepared using the technology of recombinant proteins purification. Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 25 5.2. Bioinformatics approach for products development The proposed mathematical model and a data mining algorithm will not only help in identifying proteins (antigens) that can be used in diagnosis and tr eatment of difficult disorders, but also will help in etiological diagnosis of idiopathic d isorders and their treatment. This approach is based on building large databases of RBC antigens store for patients and normal individuals. Consequently, a patient sample is coll ected on anticoagulant. RBC and plasma are separated. The plasma IgG is separated and the n used as ligand in immunoaffinity chromatography to separate hemolysate antigens. The collected antigens are identified by mass spectrometry. The database record consi sts of the diagnosis and the set of identified antigens. 5.2.1. Mathematical model It consists of four main parts; definitions of symbols, model of dise ases caused by microorganisms, tumors, or foreign proteins; model of autoimmune diseases which result as a consequence of missed tissue proteins from RBC antigens store; and model of dis eases of unknown cause (Idiopathic). Definitions Let the assumption of this work be as the following: pi: protein amino acid sequence, where i = 1 .. n

dj: health state, i.e., normal or disease name, where j = 1 .. m P = {p1, , pn}, Set of all proteins of RBC antigens store D = {d1, , dm}, Set of all diseases Pp: patient proteins where Pp P where p is the patient ID Op: (pi , dj), ordered pair of patient presented by protein sequence (i) and he alth state (j). a. Model of Diseases caused by microorganisms, tumors, or foreign proteins Pdj = {Pp}dj Where Pdj is the set which contains all common proteins associated with dj. Pnormal = {Pp}normal Where Pnormal is the set which contains proteins associated with normal. P normal such that p in Pnormal if the number of occurrence of p Pnormal is less than 5% of the total number of p in Pnormal then remove p from Pnormal. P dj = Pdj P normal Where P dj is the set which contains proteins that can be used as b iomarker or vaccines, Figure 10. Blood Cell An Overview of Studies in Hematology 26 Figure 10. Venn diagram depicting set of abnormal protein of disease X (P dj) b. Model of Diseases caused as a result of missed tissue proteins P u dj = {Pp}dj The result is the set which contains all proteins associated with dj P dj = Pnormal - P u dj The result is the set which contains proteins that can be used to d iagnose patients through detecting circulating auto-antibodies and to treat those patients through desens itizing them with the proteins that give positive reaction, Figure 11. Figure 11. Venn diagram depicting set of missed normal proteins of disease Y (P dj) c. Model of Diseases of unknown cause (Idiopathic) There are many diseases that are identified as idiopathic. Those diseases can be caused due to existence of abnormal protein or absence of tissue proteins. Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 27 Applying data mining methods (A and B) can help to identify new dis eases and treat patients appropriately. 5.2.2. Scenario of the system in clinical environment Patients blood samples will be collected on anticoagulant. RBC and plas ma are then separated in different tubes. Plasma is used as a ligand in immune-af finity chromatography to separate hemolysate antigens that can bind to plasma antibodies. The separated antigens are identified by MS and stored in the database indexed by th e patient disorder.

In the same time, queries are done to verify the diagnosis and get a prognosis and a recommended treatment component. The following formulas describe the usa ge of this model in clinical practice. Let Dp is the set of all discovered P dj Let Dp is the set of all discovered P dj Then P dj Dp , if P dj P, then patient is diagnosed to have dj Else P dj Dp , if P dj P, then patient is diagnosed to have dj 6. Conclusions All the previous work in RBC proteomics neither has identified another function nor has mentioned the finding of: HLA, TSAs, or foreign proteins. The reasons are obviou s. Firstly, it is not expected to find such proteins and consequently the method used for the interpretation of the mass spectrometry data, and the search engines u sed for the identification do not consider the right types of sequence data banks available. Secondly, the amount of most of the antigens which belong to the RBC antigens store is little. This makes those antigens invisible and hence easily missed. The work described is just a pilot study that throws some light on a new theory related to RBC. This theory is based on finding antigens store consisting of self and non-s elf antigens. Although this theory can be related to immune tolerance by logical in duction, the concrete evidence and mechanism need further research. Mainly, the logical induc tion is based on finding all kind of antigens in hemolysate, especially HLA antigens wh ich are related to fetus. This existence of all kinds of antigens, definitely, plays some immunological role which may be immune tolerance. The initial experiment, which shows the existence of ABO antigens in hemolysate of pregnant females, explains the mechanism of how HDFN occurs. Meanwhile, the Blood Cell An Overview of Studies in Hematology 28 experiment which shows that HLA antigens exist in their hemolysates pr oposes a new mechanism by which a pregnant woman is able to tolerate her fetus and placenta. Simply, it is the same mechanism a body tolerates his self antigens. The experiments which use hemolysate against self-serum: Ouchterlony imm uneprecipitation test, Western Blot, and 2-DE of co-immunoprecipitated anti gens demonstrated that RBC have an antigens store. Mass spectrometry of spot s obtained from 2-DE gel demonstrated the finding of all kind of antigens, self and non-self, in hemolysate. This indicates that blood circulating antibodies in any ind ividual will react with his RBCs hemolysate antigens. In effect, there is no absolute imm

une response, too. This directed our attention to use hyper immune serum against Mycobact erium antigens. This will help to get rid of other proteins and do better separation ; and hence better identification. Consequently, we could identify 11 proteins from 60 gel spots belonging to H37Rv strain. The rest of spots are proteins related to bacterial commensals. Co nsequently, purification of specific antibodies from hyper immune serum is recommended to ge t further better separation. In the experiment which investigates the dynamics of foreign antigens in RBC using sheep RBC which has been infected with E. coli, it was shown that the con centration of foreign antigens in RBC varies by time in relation to IR known behavior. This proves tha t RBC have role in immune reactions (IR and IT). Whatever the reason of this existence of antigens in hemolysate this existence can help in designing diagnostic kits for different types of diseases. Further, it will help in discovering, not only, new immunological disorders which are, now, categorized under idiopathic disease, but also, identifying the obscure cause of many immunological disorders, including cancer. The identification of the cause of a disorder will help in its treatment and prevention. Author details Mahmoud Rafea and Serhiy Souchelnytskyi Karolinska Biomics Centre, Department of Oncology-Pathology, Karolinska Institut et, Stockholm, Sweden Acknowledgement We would like to thank Dr. Saleh El-Ayouby, Dr. Essam Nasr, Professor Dr, Mervat El Anary, and Ms. Heba Zaki. Dr. El-Ayouby helped in preparation of anti serum and in conducting Ouchterlony immuno-precipitation test. Dr. Nasr has provided the H37R v strain and helped in the preparation of the antigen extract. Professor El An sary has provided lab facilities and reagents for HLA typing and has examined the typing tr ays. Ms. Zaki Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Ro le in Health and Disease 29 developed a computer program that implements the mathematical model to help in its verification. 7. References [1] Daniels, G. (2007). Functions of red cell surface proteins. Vox s anguinis , 93, 331 340. [2] Burek, C. L. (1998). Autoantibodies test for. In I. Roitt, & P. Delves (Ed. ), Encyclopedia of Immunology (Second Edition ed., pp. 260-265). Baltimore, Maryland, U

SA: Elsevier Inc. [3] Qin, S., Cobbold, S., Benjamin, R., & Waldmann, H. (1989). Induct ion of classical transplantation tolerance in the adult. J. Exp. Med. , 169, 799. [4] Perelson, A. S., & Weisbuch, G. (1997). Immunology for physicists. Rev. Mod . Phys. , 69 (4), 1219--1268. [5] Steinman, R. M., Daniel, H., & Michel, N. C. (2003). TOLEROGENIC DENDRITIC CELLS. Annu. Rev. Immunol , 21, 685-711. [6] Walker, L. S., & Abbas, A. K. (2002). THE ENEMY WITHIN: KEEPING SELFREACTIVE T CELLS AT BAY IN THE PERIPHERY. NATURE REVIEWS IMMUNOLOGY , 2, 11-19. [7] Basu, S., Kaur, R., and Kaur, G. (2011). Hemolytic disease of th e fetus and newborn: Current trends and perspectives. Asian J Transfus Sci. 2011 January; 5 (1): 37. doi: 10.4103/0973-6247.75963 [8] Mollison, P., Engelfriet, C., & Contreras, M. (1997). Blood transf usion in clinical medicine (10th ed.). Oxford: Blackwell Science. [9] Hadley, A., & Soothill, P. (2002). Blood diseases in pregnancy. C ambridge University Press. [10] Tawfik, H. (2005, March). Management of Alloimmune Fetal Anemia. R etrieved April 12, 2008, from ASJOG: http://www.asjog.org/journal/V2Issue1/262%20fetus&newbornFetal%20Anemia%20_dr.pdf [11] Anderson, M. S., Venanzi, E. S., Klein, L., Chen, Z., Berzins, S. P., Tureley, S. J., et al. (2002). Projection of an immunological self shadow within the thumus b y the aire protein. Science , 298 (5597), 1395-1401. [12] Liston, A., Lesage, S., Wilson, J., Pletonen, L., & Doodnow, C. C. (2003). Aire regulates negative selection of organ-specific T cells. Nat. Immunol. , 4 (4), 350--4. [13] Tamul, K., Schmitz, J. L., Kane, K., & Folds, J. D. (1995). Com parison of the Effects of Ficoll-Hypaque Separation and Whole Blood Lysis on Results of Immunophe notypic Analysis of Blood and Bone Marrow Samples from. CLINICAL AND DIAGNOSTI C LABORATORY IMMUNOLOGY , 337342. [14] Hopkins, K. (1990). The basic microlymphocytotoxicity assay. The AS HI laboratory manual. 2nd edition . ASHI Lenexa. Blood Cell An Overview of Studies in Hematology 30 [15] Casadevall, A., & Pirofski, L. A. (2000). Host-Pathogen Interaction s: Basic Concepts of Microbial Commensalism, Colonization, Infection, and Disease. Infection and Immu nity , 12 (68), 6511-6518. [16] Thomson, J. D., Schaeffer-Reiss, C., & Ueffing, M. (2008). Functi onal Proteomics Methods and Protocols. Series: Methods in Mollecular Biology, 484 (XVIII), 115. Chapter 3

 2012 Cristiana et al., licensee InTech. This is an open access chapter distribut ed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches Filip Cristiana, Zamosteanu Nina and Albu Elena Additional information is available at the end of the chapter http://dx.doi.org/10.5772/47795 1. Introduction Red blood cells are responsible for oxygen transport from lung to tissues. Oxyge n transport depends on reduced state of iron (Fe 2+ ) in hemoglobin. To accomplish their delivery function erythrocytes must accommodate to the environment conditions as they cha nge along the vascular branches. Both element oxygen (O2) and iron are able to quic kly shift their oxidative state in response to different external/internal emerging stim uli. Moreover in erythrocyte nitric oxide (NO ) a vasodilalatory messenger is present. All these el ements act in normal condition in well established mechanisms but they may generate alone o r together high reactive species, named free radicals, that damage red blood cell s as well as vascular endothelium. Free radicals may be generated from both oxygen and nitrogen and ar e known as reactive oxygen species (ROS) respectively nitrogen reactive species (RNS). However erythrocytes have intracellular enzyme/non enzyme defense system. When r eactive species are quickly and intensely generated under external/internal stimuli the activity of antioxidant defense system is overwhelmed. Free radicals generation is triggered by normal, adaptive or pathological stimuli such as: superoxide detoxification, dec reasing oxygen saturation in vascular branches, shear stress or atherosclerosis, ischemic attac k and bacterial infections. One of the most potent oxidant agents in living cells is homocysteine (Hcy) a metabolic compound from methionine metabolism. The already mention metabolism requires vit amin B12, B6 and folic acid involvement. Every deficiency in vitamins supplies or enz ymes activity triggers the onset of different diseases and erythrocyte is first affe cted in megaloblastic or Biermer anemia. A secondary consequence of vitamins deficiency is hyperhomocyste inemia. HyperHcy represents a high risk in cardiovascular diseases and not onl y. Nowadays is generally accepted that Hcy disturbs the normal endothelial function, promoting

thrombosis and inhibiting fibrinolysis through many mechanisms which can possibly integrate and are Blood Cell An Overview of Studies in Hematology 32 not mutually exclusive; oxidative processes, decreasing NO bioavailabilit y and specific protein targeting. The already specified free radicals are not all bad. NO can be regarded as a good radical but it is inactivated by many Hcy-dependent Mechanisms that fin ally impair its vasodilatatory function. Thus damaging Hcy effects expands to the envir onment where erythrocytes move and act. As a consequence directly and indirectly Hc y has a big impact on erythrocytes whose deformability in shear stress is crucial for circulatory f unction. Taken into account all these factors pharmacological approaches envisage lower ing homocysteine levels by different ways such as: vitamins B supplementation, antioxida nt drugs, hypotensive agent, antithrombotic drugs etc. Some of these patterns suc h is vitamins supplementation proved to have limited clinical benefits while others a s nitrite/nitrate are still in debates. Because most pathological processes mentioned above i nvolve oxidative pathway mechanism, pharmacological presentation will focus on drugs with antioxidant properties As a conclusion the effect of elevated homocysteine appears multifactorial affec ting both the vascular wall structure, function as well as erythrocytes metabolism. 2. Homocysteine and red blood cells 2.1. Red blood cells oxidative-reducing balance Red blood cells are responsible for oxygen transport from lung to tis sues. Their function depends on reduced state of iron (Fe 2+ ) in hemoglobin (Hb). Both element oxygen and iron are able to quickly shift their oxidative state in response to differ ent emerging stimuli. Literature shows in [1] that hemoglobin may undergo oxidative reaction in the process of releasing oxygen. In this process iron is oxidized to Fe 3+ and reactive oxygen species are generated. The bigger the reactive species release is the higher norma l deformability and flexibility of erythrocytes is disturbed. 2.1.1. Reactive species generation In erythrocyte are found together oxygen (O2), nitrogen oxide (NO ) and iron (Fe 2+ ). All these elements act in normal condition in well established mechanisms but they may generate alone or together reactive species, named radicals, that damag e red blood cells as

well as vascular endothelium. A radical is a chemical species that possesses a single unpaired elec tron in outer orbitals, and is able to independently exist, known also as free radical. Radicals are hig hly reactive in extracting an electron from any neighbor molecule in order to complete theirs ow n orbitals. There are two main groups of free radicals: ROS or reactive species of oxygen, RNS or reactive nitrogen species. ROS and RNS can act together damaging cells and causing nitrosactive stress. Therefore, these two species are often collectively referred to as ROS/RNS. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 33 Transitional metals (Fe belongs to this group) particularly behave. The y have a single unpaired electrons in theirs outer orbitals, but they dont behave as f ree radicals because within cells they are attached to proteins in most cases. However the y are able to catalyze electron transfer in many processes and sometimes generate radicals. 2.1.2. Reactive oxygen species Reactive species of oxygen refers to a group of highly reactive O2 m etabolites, including superoxide anion (O2 ), hydrogen peroxide (H2O2), singlet oxygen ( 1 O2), and hydroxyl radical (OH), that can be formed within cells. Reactive oxygen species are constantly formed as byproducts in normal enzymatic reaction in all human cells through normal aerobic processes as mitochondrial oxidative phosphorylation or as neces sary products in neutrophils in order to kill invading pathogens. The above mentioned p henomena are consuming oxygen processes. Erythrocytes must save oxygen for delivering it to the cells; as a consequence r ed blood cells lack mitochondrion the main oxygen consumer within cell. In this parti cular condition the source of ROS in erythrocyte may be the carried oxygen itself. In order to understand oxygen behavior an inside in its structure is needed. The ground state of oxygen is triplet oxygen meaning that the molecule has two unpaired electrons occupying two different molecular orbitals. Figure 1. The structure of oxygen molecule These electrons cant travel both in the same orbital because they have parallel spin (they spin in the same direction). As a consequence molecular oxygen is par amagnetic and from this feature it was concluded that the structure in the right may be assigned to O2 (figure1). Although O2 is very reactive from thermodynamic standpoint its single electrons cannot

react rapidly with already paired electrons in the covalent bond of o rganic molecules (abundant in living cells). As a consequence it is harmless to these molecules. Instead molecular oxygen can rapidly react with single unpaired electrons from transitory metals (e.g. Fe, Cu, Mn). One mole of properly chelated cooper could catalyze consumpti on of all of the oxygen in an average room within one second in [2]. In fact oxygen O2 is both kinetically stable thus not reactive and very reactive promoting fast reactions, depending the surrounding conditions. Within cells where transitional metals are bounded to proteins (in metal contain ing proteins or enzymes) oxidative attack of O2 tend to be slow, meaning that a first single electron is relatively difficult to add. As a consequence superoxid radical (O2

) will form very slow. Blood Cell An Overview of Studies in Hematology 34 Figure 2. Superoxide radical generation Once an electron acquired additional electrons are easier added, furthe r reactions quickly occur in [3] and reactive species of oxygen are generated. Figure 3. Reactive oxygen species generation Superoxide (O2

) is formed when molecular oxygen (O2) gains an additional electron, producing a molecule with only one unpaired electron (figure 2), generating a ve ry reactive free radical. When accepts an electron, superoxide is reduced to hydrog en peroxide, which is not a radical. In the next one-electron reduction step hydrogen pe roxide generates water and the hydroxyl radical (OH ) which is probably the most reactive fr ee radical. A final electron acceptance (figure 3) reduces hydroxyl radical to water in [4]. Superoxide radical (O2

) is a reactive radical, however it cannot diffuse to far limited lipid solubility. Instead it might react in the presence of with de hydrogen peroxide generating the most potent hydroxyl radical through matic reaction known as Haber-Weiss reaction. The reaction takes place in two steps that involved ferric iron and s follows: O2

having ferric iron a non-enzy superoxide a

+ Fe 3+ O2 + Fe 2+ Fe 2+ + H2O2 Fe 3+ + OH + OH The general non-enzymatic reaction occurring in living cells is: O2

+ H2O2 OH + OH + O2 It appears that superoxide radical is also a source of c iron presence. Hydrogen peroxide (H2O2) is not a free radical s a ROS because it generates the most powerful hydroxyl radical (OH ) in l metals (Fe 2+ , Cu + ), the reaction above. Hydrogen peroxide (H2O2) as a

hydroxyl radical in ferri but still classified a the presence of transitiona

is lipid soluble and

Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 35 consequence it can diffuse through lipid membranes. No matter where it meets proteins (including hemoglobin) that contain transition metals Fe 2+ , Cu + , H2O2 generates OH at the specific site where these metals are located thus damaging protein structure. Another reactive species (but not a radical) derived from molecular ox ygen is singlet oxygen, designated as 1 O2. Singlet oxygen ( 1 O2), a highly excited state created when molecular oxygen absorbs sufficient energy to shift an unpaired electron to a hi gher orbital, can be formed from superoxide radical in [5]: 2 O2

+ 2H +

H2O2 + 1 O2 Singlet oxygen is even more reactive than the hydroxyl radical, although it is n ot a radical. As a conclusion the most reactive radical is hydroxyl (OH ) which indiscriminatel y extracts electrons from any other molecules around it whereas superoxide (O2 ) and hydrogen peroxide (H2O2) are more selective in their reactions with biological molecules [6]. All the above reactions and processes take place in all human cells including er ythrocytes. As for the other cells the main source for free radicals is mitochondrion, in the p articular case of red blood cells the main source for radicals is the carried oxygen. Molecular ox ygen is carried in order to be delivered to tissues and as a consequence it is found, for a shor t period of time, free, thus unbound. In this state it might be prone to generate the above descri bed radicals. Oxygen binds to hemoglobin at the ferrous iron. The ferrous state (Fe 2+ ) of iron is a condition for hemoglobin normal function. However a small percent of Fe 2+ is slowly converted by O2 to ferric form (Fe 3+ ) in resulting methemoglobin. An enzymatic system, methemoglobin reductase quickly restores Fe 3+ to Fe 2+ and reduces methemoglobin back to hemoglobin. Binding of oxygen to the iron in the hem is considered not to change the oxida tion state of the metal. However oxygenated hem has some of the electronic characteristics of a Fe 3+ OO peroxide anion [3]. Misra and Fridovich demonstrate that the Fe 3+ O 2complex is able to generate superoxide radical in [7] during the normal molecular oxygen transport to tissues through the hemoglobin auto-oxidation. Thus hemoglobin auto-oxidation causes sup eroxide formation within erytrocyte. Other researchers show that hemoglobin may undergo oxidative reaction i n the oxygen releasing process. Balagopalakrishna and coworkers demonstrate in [8] that at in termediate oxygen pressure, where hemoglobin partially releases molecular oxygen, t he superoxide

radical production increases. They show that superoxide radical is rele ased in the hydrophobic hem pocket. The process in slow enough thus the formation of superox ide was followed for more than 15 min, and thus detected by low temperature electron paramagnetic resonance technique. Being a radical superoxide reacts fast with other radicals or alternat ively it is efficiently scavenged through the specific superoxide dismutase (SOD) activity. When collides with other radicals O2 gives birth to new reactive species as follows: - O2 reacts with itself generating molecular oxygen (O2) and hydrogen peroxide (H2O2 ) a source for hydroxyl radical. Blood Cell An Overview of Studies in Hematology 36 - O2 reacts fast with NO radical generating toxic peroxinitrite (ONOO ), a reactive nitrogen specie (RNS). At physiological pH, ONOO rapidly protonates to peroxynitrous acid, ONOOH. This powerful oxidizing and nitrating agent can directly damage proteins and lipids. Thus, any system producing O2 and NO can cause biological damage, and erythrocytes make no exception in [6]. Hydrogen peroxide is not a radical because it doesnt have any unpaired electron. The limited reactivity of H2O2 allows it to cross membranes and to become widely dispersed. Even hydrogen peroxide is not a radical it can generates the short-li ved but very active hydroxyl radicals via Harber-Weiss non-enzyme reaction in [9]. The hydr oxyl radical (OH), which is highly reactive, diffuses only a short distance before it reacts w ith whatever biomolecules it collides with. Recent study consider that the high and indiscriminate reactivity of the hydroxyl radical minimizes its ability to diffuse an d makes it more damaging within cell or in the environment where it is generated in [10]. This c onsideration becomes more important when the oxidative events prevail within a spec ific cellular compartment. Hydroxyl radical are especially dangerous because it can i nitiate an autocatalytic radical chain reaction. Being so harmful cells carefully control h ydroxyl radical by limiting the availability of both Fe

2+ and H2O2 in [6]. The non-enzymatic decomposition of hydrogen peroxide described by HaberWiess and especially the mechanism through which hydroxyl radical (OH) acts was highly deba ted. Some researchers consider that hydroxyl radical is responsible for dama ging cellular component on behalf of a radical mechanism. Others consider that ferryl ion (Fe( IV)O 2+ ), an oxidizing species where iron is in high oxidation state (Fe IV) in [ 11,12] is an active intermediate responsible for chain reaction propagation. Another group c onsider that conditions inside cell dictates whether metallo-oxo species or hydroxyl radical (OH) is the main oxidant [in 13]. Some other like Prousek concluded in [14] that both oxidis ing species can be formed in living cells. As a general conclusion in erythrocytes ROS are produced both accident ally and physiologically in different enzyme-catalyzed reactions (figure4). Figure 4. ROS generation in erythrocyte. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 37 2.1.3. Damaging effects of ROS ROS are considered bad radicals because can they indiscriminately interac t with any biological molecule they meet causing DNA, lipids and protein damage. As a missing nucleus cell erythrocyte can undergoes the last two lesional processes but endot helium can undergoes all three mentioned injuries. Erythrocytes are particularly af fected by oxidation of polyunsaturated fatty acids in membrane phospholipids which causes their pero xidation, degradation and fragmentation [15]. During lipid peroxidation other reac tive species as peroxyl radicals are generated in a succession of chain reactions (fig .5). This reactive intermediates amplify the injury at the place were they are formed. Figure 5. Lipid peroxidation and radical and non-radical intermediates formation [taken from 15] Erythrocytes also undergo the amino acids and/or whole proteins oxidati on. Protein oxidation leads to inactivation (if targeted proteins are enzymes), fra gmentation, aggregation of fragments and/or increased susceptibility to proteolysis [15]. In addition if injured proteins belong to erythrocytes skeleton the deformability of e rythrocytes is impaired [16]. ROS attack doesnt limit to erythrocytes it also affect endothelium cells, which in turn influence erythrocyte metabolism. In nucleus containing endothel ium cell beside lipids and proteins oxidation ROS cause DNA injuries. Free radicals can interact

with DNA leading to strand breaks or structural changes such as adduct formation (figure 6) [15]. Figure 6. Over simplifying scheme of ROS damaging activity in erythrocytes and i n endothelium cells Blood Cell An Overview of Studies in Hematology 38 2.1.4. ROS signaling Until recently reactive oxygen species were considered only oxidizing damaging f actors. But it was demonstrated by in [15, 17] that ROS can be also good as they ac t as signaling molecules. In fact both authors show that ROS are neither good nor bad, temporal len gth and intensity of free radicals generation make the difference between phys iological, adaptive or pathological effect. Thus oxidizing molecules is not the end of the road for reactive species alternatively they trigger cellular responses which depending on the int ensity of ROS attack, prepare the cell to survive or on contrary trigger cell death (figure 7) . Figure 7. Cells response under ROS attack (taken from 17) Being highly reactive ROS can intercept cell signaling pathways within successive steps in cascade events modulating the functions of many enzymes and transcripti on factors. Oxidative stress triggers cellular response by activating many signaling pathway s. ROS can directly or indirectly modulate a) the function of different types of enzymes, b) the transcription factors activity and c) the activity of ion-channels. a. Enzymes modulated by ROS include both kinases and phosphatases. The big class of kinase includes both tyrosine kinase as Src, Ras, JAK2, Pyk2, PI3K, a nd the mitogenactivated protein kinase (MAPK). The three best-characterized MAPK subfamilies a re cJun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK) [18]. All these MAPK pathways are structurally similar, but functionally d istinct. Importantly, ERK, JNK and p38 MAPK have all been shown to be activat ed by oxidative stress [19]. ERK and JNK are important in recruiting c-Fos and c-Jun to the nucleus where they activate the transcription factor AP1 (activator prot ein -1), whereas activation of p38 and inhibitory kappa kinases (IKK) is import ant in the transcriptional activation of NF-B. Both of these factors are important in regula ting the diverse genes, which play key roles in the pathogenesis of inflammatio n, and in regulation of cell cycle, proliferation, and apoptosis. ROS may inhibit tyrosine phosphatase activity further contributing to tyrosine kinase activation. b. ROS also influence gene and protein expression by activating transcription f

actors, such as the already mention NFkB and activator protein-1 (AP-1) and hypoxia -inducible factor-1 (HIF-1). c. ROS stimulate ion channels, such as plasma membrane Ca2+ and K+ channels, le ading to changes in cation concentration. The cytosolic Ca 2+ level can be increased by ROS in various cell types, including epithelium cell, through the mobilization of intracellular Ca 2+ stores and/or through the influx of extracellular Ca 2+ [15]. The ROS-mediated increase Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 39 in Ca 2+ concentration contributes to the oxidative stress-mediated activation of PKC and to the transcriptional induction of the AP-1 proteins c-Fos and c-Jun [20] (figure 8). Figure 8. Major pathways activated by ROS generation (modified from 21).MAPK=mit ogen-activated protein, JNK=c-Jun N-terminal kinases, ERK=extracellular signal-regulated kinase s, NFB=nucle r factor B, AP-1=activator protein-1, HIF-1= hypoxia-inducible factor-1, PKC=protei n kinase C More details about the cellular response in ROS and other radical and non-radicals species attack in oxidative events can be found in [15,17,19] 2.1.5. Scavenging ROS Erythrocytes have an impressing antioxidant enzyme and non-enzyme system that deals with an important amount of free radicals. Superoxide dismutase and gl utathione peroxidase are the most efficient antioxidant enzyme in red blood cells. Erythrocytes superoxide dismutase remove O2 by catalyzing its dismutation, one O2 being reduced to H2O2 and another oxidized to O2 (figure 9). Figure 9. Superoxide dismutase activity The dismutation of superoxide O2

by SOD is very efficient having the largest kcat/KM (an approximation of catalytic efficiency) of any known enzyme (~7 x 10 9 M 1 s 1

) [22]. SOD catalyst activity is limited only by the frequency of collision with superoxide. That means the reaction rate is limited only by the diffusion of superoxid radic al. Diffusion limitation becomes canceled in radicals over production thus activating the process. Blood Cell An Overview of Studies in Hematology 40 As seen in upper reaction (fig.9) superoxide dismutase must work with enzymes that remove H2O2. Glutathione peroxidase (GPx) removes H2O2 by coupling its reduction to water (figure10) with oxidation of reduced glutathione (GSH), a thiol-containing tripeptide (glucys-gly). The product, oxidized glutathione (GS-SG), consists of two GSH linked by a disulphide bridge, and can be converted back to GSH by glutathione reductase enzymes in [6]. Figure 10. Glutathion peroxidase activity. Beside enzyme antioxidant systems erythrocytes uses antioxidants agents (fig.4). These agents are preferentially oxidized by reactive species to preserve more important biomolecules and can be reversibly reduced back. For example, GSH and ascorbate can scavenge O2 , OH, and also ONOOH. Tocopherols are good scavengers of peroxyl radicals and help to protect membranes against lipid peroxidation by interrupting the pro pagationchain reaction (figure 5) in [6]. 2.1.6. Reactive nitrogen species Nitrogen compounds found in the body comes from exogenous sources as nitrites/nitrates or from endogen production of nitric oxide (NO ). The group of nitrogen derivatives includes: - NO nitric oxide a natural free radical also named nitrogen monoxide is involved in vasodilatation in mammals - NO2 nitrogen dioxide or nitrite. In organism is found in its corresponding salts nit rites( from nitrous acid HNO2) - NO3 nitrate (from nitric acid HNO3) also found in the body in corresponding salts Figure 11. Nitrogen derivatives Nitrogen derivatives convert into each other forward and backward conti nuously under shifting conditions within cells (figure11). Endogenous NO is synthesized by nitric oxide synthases (NOS) in the en dothelial and other cells, where is involved in vascular physiology.

Endothelial nitric oxide synthases (eNOS) synthesizes NO (figure 12) from L-argin ine with 1,5 consumption of 1.5 NADPH equivalents and two oxygen molecules per NO formed i n [23]. The reaction requires the presence of Ca 2+ -Calmodulin and tetrahydrobiopterin (BH4) as cofactors in[24]. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 41 Figure 12. Nitric oxide generation Generated NO is a gaseous molecule with unpaired electrons and as a c onsequence a radical; it is lipophilic and diffuses rapidly through membranes. NO is a messenger in many physiological processes: endothelial relaxation of the smooth muscl e, inhibition of platelet aggregation, neurotransmission and cytoxicity in [25]. NO pathology incl udes both low and high concentrations as follows: insufficient NO production is i nvolved in hypertension, the activation of platelet aggregation and atherogenesis w hile high NO production generates septic shock, stroke, and carcinogenesis. NO released from endothelial cells diffuses through blood or to the un derlining smooth muscle cells in the media where it triggers vasodilatation. In blood stream NO will affect platelets, leucocytes and erythrocytes. Recent studies show that: a. e-NOS can produce NO not only in normal oxygenation (figure 12) but also dec reased oxygenation gradient across vascular branches. In addition eNOS can som etimes be a source of ROS generating O2 b. endothelial cells are not the only ones able to generate NO , blood erythrocytes are expressing functional NOS c. Hemoglobin itself also produces NO from nitrite in order to modulate vasodilatation. a. It is only recently found that endothelial NOS may be a source o f ROS generating O2 depending the availability of its substrates within cell (figure 13) [23]. Endothelial nitric oxide synthetase activity is regulated by a combinat ion of mechanisms that allow eNOS to modulate its activity under physio-pathological condition in [22]. eNOS contains 2 enzymatic domains, a flavin-containing reductase and a hemecontaining oxygenase domain (Fe 3+ ) connected by a regulatory calmodulin-binding domain. Binding of the Ca 2+ /calmodulin complex orients the other domains in such a position that

NADPHderived electrons generated on the reductase domain flow to the oxygenase domain in [26]. The oxygenase domain of eNOS contains an iron ion (Fe 3+ ) that binds oxygen on reduction Fe 2+ , and this complex finally causes the conversion of L-arginine to NO and L-citrulline. This sequence of events properly rules if the cofactor BH4 provides th e connection Blood Cell An Overview of Studies in Hematology 42 between the two domains. Deficiency of arginine or BH4 causes the red uctase uncoupling from oxygenase. At the oxygenase domain intermediate Fe 2+ -O2 complex dissociates to form superoxide and the original Fe 3+ group of the eNOS)[27]. Thus eNOS releases O2

instead of NO (figure 9). Figure 13. Endothelial NOS differently behaves generating either NO or O2

depending on the substrate availability. Oxygen deficiency is known to halt the L-arginine cycle if the oxygen levels fall below a threshold level of ca [O2] ~ 10 M [28]. However, eNOS is not wholly inactivated i n hypoxia, instead, in the presence of nitrite (figure 14), it shifts again and produces NO in [29]. Figure 14. Endothelial NOS may generate NO in both normal and low oxygenation Stroes et al demonstrate in [30, 31] an intriguing activity for eNOS only, the simultaneous generation of both NO and superoxide, even in the presence of BH4 and L-arginine, under physiological conditions. The consequence is the production of peroxynit rite, a highly reactive molecule, by eNOS (figure 15). Thus eNOS is a source of free radical producing good or bad radicals up on inside cell condition. Figure 15. Peroxinitrite generation as a result of particularly eNOS activity Peroxynitrite anion (ONOO ) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. Recent research indicates in [32] that peroxynitrite is able to cross the erythrocyte membrane by two different mechani sms: in the

anionic form through the anion exchange channel, and in the protonated form by passive diffusion. Entering the erythrocyte peroxynitrite causes nitration of in tracellular hemoglobin, in a process that is enhanced in thiol-depleted erythrocytes. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 43 To summarize NO can be produced by eNOS from either L-arginine in goo d oxygenation physiological state or from nitrite in hypoxia. In vitamins deficiency (low BH4 levels) eNOS produces superoxide radical. Recent studies demonstrate that endothelium cells surprisingly produce ROS under hypoxia. The primary site of reactive o xygen species production was demonstrated to be complex III in electron transport in mitochondria. The paradoxically increase in ROS production under low oxygenation is still not fully understood but it is considered that reactive oxygen species released during hypoxia act as signalling agents that trigger induction of erythropoietin, endothelial growth factor and glycolytic enzymes. Systemically, these responses enhance the delivery o f O2 to cells and facilitate the production of glycolytic ATP instead of mitochondrion. Induction of these genes is mediated by specialized hypoxia inducible factor 1 (HIF-1) [33, 34]. As a conclusion in normal oxygenation NO is produced by eNOS. In hypoxia adaptive respons es are onset; the release of NO from nitrite to sustain normal vascular function is one path. Alte rnatively when mitochondrion senses hypoxia it releases ROS as signaling molecules that activate diverse functional responses, including activation of gene expression that promote cell survival. b. Kleinbongard demonstrates in [35] that red blood cells express func tional eNOS which is located in both the internal side of the plasma membrane and the cyt oplasm with a higher expression in the membrane. The enzyme has a similar activity and regulatory mec hanism as the endothelial-derived NOS. Besides its vasodilatation activity NO also regulate s red blood cells deformability and inhibits platelet activation. In physiological condition where there is a normal supply of L-Arginine and subsequently a normal NO production, nitric oxide sustains red blood cells deformability. On contrary decreased NO levels reduces erythrocytes deformability preventing them to easily pass through microcirculation. T he same effect was observed on platelet aggregation when decreased NO levels promote thrombosis. Ulker demonstrates in [36] that red blood cell-NOS is activated by me chanical factors and that export of NO from erythrocytes is enhanced by mechanical stress thus pointing erythrocyte contribution to the regulation of vascular tonus c. NO is a short life species as a consequence it quickly reacts wit

h any encountered molecules or it is rapidly oxidized by hemoglobin in blood. In fact the general accepted theory is that Hb in the red blood cells is an extremely effective NO scavenger in [37]. Oxigenated-Hb reacts with NO which is rapidly converted into nitrate (f igure 16). After reacting oxygenated hemoglobin is converted to methemoglobin (met-Hb). T his reaction is considered to be limited only by the diffusion. Figure 16. Oxyhemoglobin activity of scavenging the nitric oxide Lundberg shows in [38] that NO can be alternatively produced in hypoxi c condition by deoxi-hemoglobin from nitrite. Inside erythrocyte Hb can interact with NO i n many ways depending on its oxygen saturation, as follows: Blood Cell An Overview of Studies in Hematology 44 - in oxygenated form, oxi-Hb acts as a scavenger removing NO as nitrate. - in deoxygenated form deoxi-Hb acts in two different ways: first it binds NO thus functioning as a transporter and second it reduces nitrite to generate NO (figure17) Recently several authors suggest in [39-41] that this behavior represen ts a mechanism for NO generation in regions of poor oxygenation where deoxy-Hb predominates Figure 17. Hemoglobin regulates the NO use/consumption, from [38] Two recent theories try to explain the Hb involvement in nitric oxide use/consumption in vasorelaxation process in [42]. Stamler and colleagues originally suggested in [43] a role for a thiol (SH) gr oup in Hb as a carrier and releaser of NO . According to this theory, the binding ( formation of Snitrosohemoglobin) and release of NO from Hb are allosterically regulate d so that NO release occurs when Hb is deoxygenated. - Cosby, Crowford et all suggest in [44] that Hb is not a transporter of NO but rather an enzyme dealing with NO depending oxygen saturation as follows: when Hb is fully oxygenated, the primary reaction is oxidation of nitrite into biologica lly inert and supposed pool nitrate. As oxygen saturation falls along the vascular tre e, Hb gradually turns into a "reductase" and starts to reduce nitrite into vasodilator NO (figure 18). The maximal nitrite reduction is observed when Hb is app roximately 50% oxygenated (P50) in [45,46]. Concomitantly, vasodilation is initiated at the P50, ideally suited for the regulation of hypoxic vasodilation under varied physiolo gic and pathologic conditions (figure 19). Figure 18. Hemoglobin behavior depending on oxygen saturation. J.O.Lunderg concluded in [42] that: in just one decade, Hb has gone f

rom being merely a NO scavenger to NO carrier and now NO

generator.

Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 45 Figure 19. Hemoglobin uses NO to generate nitrate a supposed NO2 / NO pool in full oxygenation and releases it with maximal activity at P50 oxygen saturation in hypoxia [modif ied from [38]. To summarize: when there is plenty of oxygen, NO is mainly produced by endothelia l NOS or by erythrocyte own NOS. Thus nitric oxide radical maintains good e rythrocytes deformability and probably oxy-Hb regulates the amount of NO by trappin g its extra amount to form S-nitrosohemoglobin. When oxygen is scarce NO is synthes ized in low amount (as a consequence of low eNOS activity where oxygen is cofactor). In this condition NO is saved through binding on deoxyHb which becomes a source of NO i n vascular circulation, or deoxy-Hb catalyses the NO generation from nitrite. Behavin g this way deoxy-Hb can maintain also red blood cell deformability in hypoxic con dition. As a consequence hemoglobin may regulates membrane deformability along circulati ng branches through the way it uses/produces NO radical. [47] In addition two other supplementary mechanisms, endothelial xantinoxidase activity and blood pH dictate superoxid radical versus NO generation depending on th e blood oxygenation/deoxigenation status in [48,49]. 2.2. Homocysteine metabolism Homocysteine is a metabolic compound formed in methionine and betaine metabolism . The already mention metabolisms require vitamin B12, B6 and folic acid inv olvement and the proper activity of two main enzymes cystathionine synth se (CBS) and methylenetetrahydrofolate reductase (MTHFR). Every deficiency in vitamins supplies or enzymes activity triggers the onset of different disease. Megaloblastic anemia or Biermer disease affect primarily the red blood cells. Secondary to the above mentioned illnesses hyperhomocysteinemia can also install. Blood Cell An Overview of Studies in Hematology 46 Hyperhomocysteinemia is considered to be involved in many diseases from cardiova scular to neurological illnesses. It is generally agreed that two general mec hanisms cause hyperhomocysteinemia: one is low vitamins (B12, B6, folic acid) supplie s and second the main enzymes deficiencies (cystathionine -synthase deficiency and methylenetetrahydrofolate reductase deficiency). Hypehomocysteinemia is tod ay considered a severe risk factor in vascular illnesses. Many approaches envisage lowering

homocysteine levels by vitamin B or oral folic acid supplementation but many rec ent studies show that vitamins administration fail to give a real clinical benefit and suggest that B vitamins might instead increase some cardiovascular risks in [50,51]. H owever not all patients with cardiovascular events or neurodegenerative diseases are enzymes de ficient or poor vitamins supplied. The majority of research works report hyperhomo cysteinemia associated to many diseases but the question what triggers hyperhomocysteinemia is yet to answer. An interesting hypothesis suggests that in fact hyperhomocystein emia is more a secondary effect that amplifies in its turn the initial injury [52]. Brattstrm and Wilcken in [52] consider that impaired renal function due to hypertension and ath erosclerosis is an important cause of the elevated plasma homocysteine found in vascular disease patients. The reasons are as follows. Atherogenesis and elevation of blood press ure commonly develop silently over many years before the emergence of clinically evident vasc ular events. These processes also lead to nephrosclerosis and a degree of deterioration of re nal function, and this is highly relevant to the plasma clearance of homocysteine. For these reasons, the presence of vascular disease itself may contribute to an elevation in circulating homocysteine by leading to a decline in renal function. This means that because of reduced renal function, patients with either occult or clinically evident cardi ovascular disease may have elevated circulating homocysteine concentrations (figure 20). This could also explain the relation between plasma homocysteine and the severity of atherosclerosis. Figure 20. Proposed mechanisms for the causes of hyperhomocysteinemia (taken fro m [52] ) Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 47 2.3. Hyperhomocysteinemia a disturbing factor of the endothelial function Homocysteine refers to all species that contain and can release homocy stein including homocystine (the dimer of homocysteine) and mixed cysteine-homocysteine disulfide or homocysteine bound on proteins. In fact the major form of homocysteine in circulation, around 70% is protein bounded. In early data normal levels of homocysteine were admitted to be aroun d 15M/L. It was found that homocysteine slightly increases with age in [53]. Levels of 15-30 M/L corresponds to mild, 30-100 M/L to moderate and more than 100 M/L to severe hyperhomocysteinemia in [54]. Nowadays it is considered that concentrations below 9 micro mol/L are an appropriate target level for therapy in [55].

Nowadays it is generally accepted that homocysteine promotes thrombosis with simultaneously vasodilatation inhibition. It is considered that homocyste ine triggers its effects by three distinct mechanisms which can possibly integrate and are not mutually exclusive; oxidative processes, decreasing NO bioavailability and specific protei n targeting (figure 21). Figure 21. Homocysteine adverse effects. (+/ represent activation/inhibition proc esses) Even the precise path was not yet established, it seems that Hcys in hibits some good factors and activates same bad factors that finally influence the proces ses of thrombosisfibrinolysis and constrictionvasorelaxation and which are summarized in (figure 2 1) [56]. Amongst good factor can be included: NO , GPx, eNOS, protein C, tissue P lasminogen Activator (tPA), annexin II. Amongst bad factor can be included: ADMA, O2 , H2O2 Homocysteine directly and indirectly influences erythrocytes metabolism. It dire ctly affects the erythrocyte antioxidant enzyme systems promoting free radical genera tion. Indirectly Hcy decreases NO bioavailability and modifies the environment where erythrocytes move and act. Blood Cell An Overview of Studies in Hematology 48 2.3.1. Homocysteine pro-oxidative activity Homocysteine is involved in reducingoxidative processes by reacting eithe r with itself or with different compounds. In other words homocysteine can submit auto-oxidative as well as oxidative processes. Thiols (RSH) can auto-oxidize in the presence of transition metal cata lysts and molecular oxygen, leading to the formation of reactive oxygen species (ROS). Hcy like all containing thiol group undergoes oxidation to disulfide (RS-SR) in O2 presence at normal pH. It was found that cooper catalyses Hcy (noted with general formula RSH) auto-o xidation, even in low homocysteine concentration, yielding hydrogen peroxide and thus prom oting ROS generation in both extra and intracellular compartments through reaction proposed by Starkebaum in [57]: 2 Homocysteine (RSH) + O2 Homocystine (RS-SR) Homocystine (RS-SR) + superoxid (O2 ) H2O2 Hydrogen peroxide generated by the copper catalysed auto-oxidation of homocystei ne was involved in the mechanism of toxicity by the demonstration of the reduction in e

ndothelial damage with the addition of catalase in [58]. Homocysteine was proved to generate superoxide radicals which promote vasoconstr iction. Lang et al. demonstrates in [59] that the inhibitory effect of homocys teine on endotheliumdependent relaxation is caused by an increase of the intracellular lev els of O2 in the endothelial cell and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia. Cysteine is also a thiol circulating aminoacid related to homocysteine and its c oncentration is 20 to 30 times higher than Hcys one. In fact Cys is the main circulatory thio l but there was found no correlation of Cys with free radicals generation. Instead a strong association of hyperhomocysteinemia with F2-isoprostane was found. F2-isoprostane is an indicator of in vivo lipid-peroxidation and its association with Hcy lead to the concl usion that this amino acid is involved in free radicals generation in [60] thus pointing Hcy as pro-ox idative agent. Hcy involvement in ROS generation was also indirectly proved in connec tion with antioxidant enzyme system modulation SOD and GPx. The activity of superoxide dismutase, an important antioxidant enzyme i n vascular tissue, was measured along with homocysteine in homocystinuric patients and fou nd to be positively associated with homocysteine levels. This strong relationship can be regarded as a protective antioxidant response to homocysteine-induced oxidative action and as indirect evidence that Hcy represents a source of free radicals in [61]. In o ur study we found an increased superoxide dismutase activity in red blood cells lysate in e xperimental induced hyperhocysteinemia in rats. We consider this increased response in enzy me activity as evidence for free radicals production in [62]. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 49 Homocysteine may affect glutathione peroxidase activity, thus altering t he microenvironment in the propagation of ROS in [63]. Our study on GPx activity, in installed hyperhomocysteinemia, was consistent with these reported data. GPx activ ity in red blood cells lysate significantly decreases as a consequence of experimental i nduced hypehocysteinemia in rats. We considered that increased amount of free radicals consume the GSH enzyme cofactor which subsequently trigger the enzyme activity decay in [62]. As a consequence GPx activity is lowered in hyperhomocysteinemia thus disturb

ing the detoxification process of H2O2 within cell. Upchurch in[63] demonstrates that homocysteine reduces mRNA levels of g lutathione peroxidase, indicating that the expression of this enzyme is inhibited and/or downregulated. Even it was attributed to different causes such as: a decrease in en zyme activity, a down regulation from high homocysteine levels or an inappropriate gene expre ssion of GPx, the decrease in GPx activity in Hcys presence is generally reported. Homocysteine-induced oxidative stress was proved to be generated within vascular cells in [64]. Our data show that in installed hyperhomocysteinemia the intracellular spa ce is more affected than the extracellular, circulatory one. We found significant changes i n antioxidant enzyme systems within erythrocyte (we worked on erythrocyte lysate) as compared with total antioxidant capacity (TAC) in plasma in [62]. As a conclusion hyperhomocysteinemia by promoting free radical generatio n affects both erythrocytes and endothelial cells as well in [65,66]. 2.3.2. Homocysteine decreases NO bioavailability The second hypothesis considers that Hcy acts to prevent NO bioavailability. This process is considered to have, at least partially, the same oxidative basis. In living organisms, including in human, endothelial-derived nitric oxide performs the follow ing function: regulates vessel tone by promoting vasodilatation, inhibits platelet activation, adhesion and aggregation, limits smooth muscle proliferation and modulates endotheliall eukocyte interactions in [56]. Homoysteine was proved to limits NO bioavailabili ty thus promoting the contrary processes: vasoconstriction, thrombosis and fibrinolysis inhibition . There are proposed many patterns for homocysteine impairing NO bioavaila bility (figure 22). A first process that limits NO bioavailability seems to be more a pro tecting mechanism than a harmful one. Homocysteine reacts with nitric oxide to form S-nitroso-homo cysteine, which has some of the properties of nitric oxide. It markedly inhibits platelet aggregation, is a potent vasodilator and does not support hydrogen peroxide generation. This represents much more a protective mechanism against the adverse effects of homocy steine than a limiting process in NO bioavailability in [56]. Blood Cell An Overview of Studies in Hematology 50 However prolonged exposure to high homocysteine concentrations impairs n itric oxide production. Thus in hyperhomocysteinemia the limited bioavailability of nitric o xide could be due to S-nitrosothiol formation in [67].

Figure 22. Proposed mechanism through which Hcy inhibits some good factors and act ivates same bad factors thus influencing thrombosis-fibrinolysis respectively constrictionvasor elaxation processes. tPA, ADMA represents tissue plasminogen activator/respectively asymme trical dimethyl arginine. A second process that limits NO bioavailability is nitric oxide trappin g/degradation by other radical species. NO is trapped by superoxide to form peroxinitrit e thus being inactivated. This mechanism was confirmed by many experimental data in [63, 68]. Nitric oxide can be alternatively degraded by hydrogen peroxide as a consequence of GPx activity inhibition through Hcy-dependent mechanism. Homocysteine seems to be th e only amino acid amongst all circulating others capable to inhibit glutathione peroxid ase activity in vitro. Cysteine is also capable of generating free radicals and is present in serum at concentrations four times higher than homocysteine but cysteine doesnt p rove inhibiting properties on GPx activity. Experimental data show that Hcy inhibits GPx activit y and also suppresses the cellular GPx expression thus promoting the increase of hydrogen peroxide concentration in [64]. Hydrogen peroxide promotes in its turn free rad icals generation and peroxinitrite production thus decreasing NO availability. A third mechanism that limits NO bioavailability is the decrease in NO synthesis t hrough Hcy-dependent asymmetrical dimethylarginine (ADMA) generation. ADMA is produced by methylation of specific arginine residues of certain cellular proteins. Most of these proteins are found in the nucleus. When the proteins are degraded, ADMA and o ther isomers are released to the extracellular space where especially ADMA acts as pote nt endogenous inhibitors of NOS enzyme in [69]. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 51 Methionine loading was proved to induce hyperhomocysteinemia. In methion ine loading there is S-adenosylmethionine accumulation and as a consequence a high proteins methylation. Asymmetrical dimethylarginine (ADMA) the product of degradat ion from methylated proteins competitively inhibits NO-synthetase activity. Elevate d ADMA levels found in hyperhomocysteinemia are supposed to inhibit NO synthesis thus decreasin g the NO availability in [70]. 2.3.3. Homocysteine own action A third way that homocysteine acts is targeting specific proteins whic h are located within cell, on cell membrane or in the extracellular space. Two important intracellular proteins, already mentioned, targeted by Hcy

are the antioxidant enzyme GPx which activity decreases and SOD which activity increases in homocysteine presence (fig22). Hcy alters other intracellular proteins d isturbing the redox potential of endoplasmic reticulum and Golgi apparatus thus inhibiting the surface expression and secretion of proteins in [71,72]. Hcy targets proteins located on both membrane surface and within cell. Jacobsen considers that circulatory oxidized form of Hcy enters the cell were it is con verted back to reduced Hcy, in reducing environment within cell. Under reduced form Hcy impairs the bin ding of tissue plasminogen activator (tPA), a protein involved in the breakdown of blood clots, to annexin II in [71] by forming a disulfide bridge with Cys9 on annexin II. Thus H cy limits the plasminogen conversion to plasmin. This results in a decreased fibrinolysis. The circulating reduced Hcy acts in the same manner with annexin II on the membrane from the vascular endothelium. Homocysteine was found to be the only circulating thiol t hat impairs the binding of tissue-plasminogen activator. The atherogenic factor lipoprotein (a) [Lp (a)] competitively inhibits the binding of plasminogen to fibrin. Fibrin is a cofactor for plasminogen activation to plasmin, an important enzyme that degrades fibrin clot. Homocysteine was found to interfere in this process. Hcy and lipoprotein (a) seems to act in the same direction: homocystein e promotes lipoprotein(a) binding to fibrin and lipoprotein(a) competitively inhibit s the binding of plasminogen to fibrin. The final effect is the decrease of fibrinolysis. The com bination of Lp (a) plus homocysteine is a possible mechanisms for the occurrence of thrombosis in hyperhomocysteinemia in [73]. Protein C is an example of circulating proteins whose activity is inh ibited by Hcy. The protein C enzyme system appears to be one of the most important anticoagulant pa thways in the blood. Its activation depends on the complex thrombomodulin-trom bin. Thrombomodulin is an integral membrane protein expressed on the surface of endothelial cells where it serves as a receptor for thrombin. The complex thrombo modulin-trombin activates protein C thus raising its activity. Homocysteine inhibits th e function of thrombomodulin. Both thrombomodulin and protein C contain disulfide-rich domains. Reduction of these disulfide bonds by homocysteine may disrupt importan t structures Blood Cell An Overview of Studies in Hematology 52 within these domains, resulting in impaired function in [74]. The result is the promotion of

thrombotic process. Hcy acts on both endothelial cells and smooth muscle where it generates contrast ing effect. On endothelium it promotes injury and impairs DNA repair, in smooth m uscle Hcy stimulates proliferation in [69]. Md S. Jamaluddin considers that Hcys promotes vascular injury through hypomethylation. When Hcys accumulates it uses adenosine, a normal constituent of all cells, to form S-adenosyl-homocysteine (SAH) a potent inhibit or of cellular methylation. By impairing methylation Hcy arrests cell growth, increases cellular SAH concentration in endothelial cells (EC) and decreased DNA synthesis thu s decreasing cellular repair. This chain of events was not found in vascular smooth muscle ce lls in [75]. Erythrocytes are also affected by homocysteine-induced hypometilation. Hi gh intracellular SAH impairs the posttranslational methylation of membrane proteins. Reduction in membrane protein methylation was particularly observed for erythrocyte cytoskeletal component ankyrin, which is known to be involved in membr ane stability and integrity. Because of hypomethylation, structural damages accumulate in eryt hrocyte membrane proteins, and are not adequately repaired thus affecting membr ane physical properties. Erythrocyte deformability is a crucial properties for circul atory function in [76]. As a conclusion the effect of elevated homocysteine appears multifactorial affec ting both the vascular wall structure and the blood coagulation system as well as e rythrocytes metabolism in [77]. 3. The pharmacological influences on the blood cell metabolism Antioxidant drugs in cardiovascular risk status and roll of red blood cell antioxidant defense capacity There are growing evidences on the role of adaptive mechanisms of ery throcyte in pathological processes: atherosclerosis, ischemic attack, bacterial infect ions, etc. All of this processes involve as main mechanism oxidative stress. Erythrocytes have an intracellular enzyme and non-enzyme defense system. In order to remove reactive spec ies of oxygen, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase ac t together. Glutathione (GSH) participates as a co-substrate for GPx in order to detoxify H2 O2 generated by SOD enzyme. GSH is a critical tripeptide that oxidizes to glutathione disulph ide (GS-SG) inactive form after reacting with oxygen radicals. GSH proves to be e ssential for reactive species detoxification as a consequence it is permanently restore in i ts reduced active form by glutathione reductase based on nicotinamide adenine dinucleotide phos phate-oxidase

(NADPH) from Glucose-6-phosphate dehydrogenase (G6P-DH) catalysed reaction in pentose phosphate pathway. When reactive species of oxygen are quickly and intensely generated under external or internal stimulus the activity of SOD, GPx and GSH concentration are severely changed. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 53 When erythrocytes are undergo shear stress in constricted vessels, they release ATP which causes the vessel walls to relax and dilate so as to promote normal blood flow [ 78]. Also, when their hemoglobin molecules are deoxygenated, erythrocytes rel ease Snitrosothiols which acts to dilate vessels, thus directing more blood to areas of the body depleted of oxygen in [35]. Using L-arginine as substrate, erythrocytes can also synthesize nitric oxide enzymatically, just like endothelial cells. The nitric oxide synthase is activated wh en the erythrocytes are exposure to physiological levels of shear stress, thus, nitric oxide i s synthesized, exported and it may contribute to the regulation of vascular tonus [79]. Another mechanism that involves the erythrocytes in relaxing vessel walls is the production of hydrogen sulfide. It works as a signaling gas. It is believed tha t the cardioprotective effects of garlic are due to erythrocytes converting its sulfur compou nds into hydrogen sulfide. [80] The free radicals released by erythrocytes when they are lysed by pathogens brea k down the pathogen s cell wall and cell membrane, and so, they are killing them . This represents the involving of erythrocytes in the body s immune response in [81]. On the other hand, as response of injury after several stressors, inc luding oxidative stress, energy depletion, as well as a wide variety of endogenous mediators a nd xenobiotics, the erythrocytes can initiate the self suicidal death (eryptosis). Eryptosis is char acterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine ex posure at the outer membrane leaflet. This can make the macrophages to recognize d and engulf erythrocyte to be degraded. Eryptosis can be considered a mechanism of defective erythrocytes to escape hemolysis. Conversely, excessive eryptosis favors the dev elopment of anemia. Conditions with excessive eryptosis include iron deficiency, lea d or mercury intoxication, sickle cell anemia, thalassemia, glucose 6- phosphate dehy drogenase deficiency, malaria, and infection with hemolysis-forming pathogens. Inhibitors of eryptosis include erythropoietin, nitric oxide, catecholamine and high concentratio ns of urea in [82, 83]

The red blood cell SOD activity has been found to be useful in evaluating the bi ochemical index of copper, zinc and manganese nutrition. The largest amount of SOD enzyme is found in liver and erythrocytes. There are two forms of SOD in human tissue. One form is present in cytosol and it is a protein containing two atoms each of copper and zinc. The other form is a much larger molecule containing four atoms of manganese and it i s found in mitochondria and cytosol. Significant changes in cellular concentrati on of copper, manganese and zinc have the potential of altering the antioxidant activity of SO D. On the other hands, the correlation between of copper and zinc plasma level, the oxidas e activity of ceruloplasmin in serum, and Cu,Zn-SOD activity in erythrocytes can be a way to investigate involvement of oxidative stress in pathological conditions, as atherosclerosis obliterans [84] Blood Cell An Overview of Studies in Hematology 54 Another element involved in the function of necessary enzyme for cellu lar protection is selenium. Selenium functions primarily as an activator of enzymes neces sary for cellular protection from oxidative damage and maintenance of normal redox potent ials. A primary role of selenium in erythrocytes appears to be the activation of the enzyme glutathione peroxidase whereby glutathione (the critical tripeptide antioxidant/antito xin for all cells) reacts with oxygen radicals. Importantly, selenium catalyzes glutathione reductase, an enzyme that maintains the glutathione in its reduced or active form [85]. Specify participation of erythrocyte enzymatic system as adaptive mechan ism to different pathological processes and specify how nutritional deficiencies and oxid ative drugs can interfere these systems introduces the chapter on pharmacology of eryth rocyte antioxidant system. 3.1. Antioxidant drugs in cardiovascular risk status and roll of red blood cell antioxidant defense capacity 3.1.1. Probucol Probucol has modest lipid-lowering properties. It was used for the tre atment of hypercholesterolemia until more tolerable and effective cholesterol-loweri ng treatments, such as the HMG Co-A reductase inhibitors, or "statins," became available. Probu col lowers the level of cholesterol in the bloodstream by increasing the rate of LDL catabolism. Additionally, probucol may inhibit cholesterol synthesis and delay cholesterol a bsorption in [86]. Another possible mechanism of action of probucol is inhibition o f ABCA1-mediated cholesterol efflux without influencing scavenger receptor class B type Imediated efflux

(ABCA1 = ATP-binding cassette transporter - member 1 of human transpor ter sub-family ABCA, also known as the cholesterol efflux regulatory protein is a protein which in humans is encoded by the ABCA1 gene). The inhibition of ABCA1 translocation to the plasma membrane may in part explain the reported in vivo high-density lipopro teinlowering action of probucol in [89]. Probucol is a powerful antioxidant which inhibits the oxidation of cholesterol i n LDLs; this slows the formation of foam cells, which contribute to atherosclerotic plaques. The major mechanism by which probucol lowers LDL levels relates not t o changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL in [87]. I t has been postulated that the oxidative modification of LDL might contribute to atherogenesis by faci litating lipid accumulation in macrophages (foam cells) and by inhibiting macrophage m otility. LDL resists oxidative modification, however, when probucol is added to in vitro incubations or when the LDL itself is isolated from probucol-treated patients in [88] . Under the treatment with probucol xanthomatous lesions disappear which that suggest a facil itation of cholesterol transferred from tissues to the excretion or catabolic path ways. Compared with other hipolipemiants, probucol is a non hepatotoxic drug and induces a decrease of lithogenic index of bile. Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 55 In recent studies was shown that probucol protect against diabetes-asso ciated and adriamycin-induced cardiomyopathy by enhancing the endogenous antioxidant system including glutathione peroxidase, catalase and superoxide dismutase [90]. 3.1.2. The HMG Co-A reductase inhibitors, or "statins" Specific for hypercholesterolemia status is the high production of free oxygen radicals. These can impair the endothelial function because destroying of nitric oxide (NO) and secondary affecting its beneficial and protective effects on the vessel wall. Mo st of the other cholesterol-lowering therapies present, also, antioxidant effects. There are two way improving antioxidant defence system in hypercolesterolemiant patients: e ither increasing the activities of CuZn-SOD and GSH-Px or preventing the production of the superoxide radicals. Malone dialdehyde (MDA), more than cholesterol plasma level, is conside red a marker of patients with increased risk of coronary heart disease, because MDA is a marker of lipid peroxidation. In individuals who smoke or who have diabetes are partic ularly prone to

oxidative stress that can lead to the formation of oxidized LDL (oxLD L). Oxidatively modified LDL is considered to be highly atherogenic and can be consid ered a biochemical risk marker for coronary heart disease. Oxidative modification of LDL increases their ability to bind to the extracellular matrix, increasing its retention within t he intima and accumulation of oxLDL in macrophages, so, it contributes to the format ion of an atherosclerotic lesion. The oxLDL accumulation within macrophages promotes the chemotaxis of mo nocytes into the vessel wall and initiates the various pro-inflammatory effects by different scavenger receptor pathways: CD36 class B scavenger receptors from human macropha ges (activates nuclear factor kB that regulates the expression of many pro-inflammator y genes), class A scavenger receptors (modify macrophage activation), lectin-like oxidized LDL receptor LOX-1 (the expression of endothelial cell adhesion molecule). On the o ther hands, the accumulation of inflammatory cells can further increase the levels of oxidative stress. Oxidative stress inactivates nitric oxide (NO) and inhibits its synthesis by end othelial nitric oxide synthase (eNOS). On this way, the vasoprotectant effect of NO ( anti-inflammatory, anti-platelets, antioxidant and vasodilator) is affected [92]. Statins inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase the rat e-limiting enzyme in the mevalonate pathway through which cells synthesizes choles terol. On this way, the "statins" increase the resistance of LDL to oxidation. Statins may also exert effects beyond cholesterol lowering. These pleiotropic vascular effects of statins are involved in restoring or improving endothelial function: by increasing the bioavailability o f nitric oxide, promoting reendothelialization, reducing oxidative stress, and inhibiting inflammatory responses. Other effects of statins that explain their involving in preserving no rmal vascular function and blood flow are: inhibition of the uptake and generation of Ox-LDL , decreasing the Blood Cell An Overview of Studies in Hematology 56 vascular and endothelial superoxide anion formation by inhibition of NA DH oxidases via Rho-dependent mechanisms and preserving the relative levels of vitamin E, vitami n C and endogenous antioxidants (such as, ubiquinone and glutathione) in LDL pa rticles. All these mechanisms explain a dual action of statins on oxidative stress, not only decrea sing oxidants but also restoring antioxidants [92]. Statins reduce both extracellular LDL oxidation (by reducing substrate availability) and intracellular oxidative stress (by

cholesterolindependent effects on NO and, indirectly, by reducing Ox-LDL) [91]. Statins themselves may be able to reduce levels of superoxide radicals , an effect that can only partially be explained by a reduction in LDL cholesterol. Rosuvas tatin has been reported to reduce markers of oxidative stress in ApoE (/) mice [93] w hile fluvastatin treatment has been shown to decrease superoxide radical generation and to reduce the susceptibility of LDL to oxidation in cholesterol-fed rabbits [95, 96]. Atorvastatin has been demonstrated to inhibit angiotensin II-induced superoxide formation by NADPH oxidase in isolated rat vascular smooth muscle cells [96] and in rats i n vivo [97]. In addition, statins have been shown to reduce NADPH-dependent superoxi de formation by a monocyte-derived cell line in culture [98]. Another beneficial effect of statins is potentiation the synthesis of tetrahydrobiopterin, which may prevent the uncoupling of eNOS and shift the balance away from NOSgenerated superoxide production to the generation of NO [99]. Statins may also b e influence the endogenous antioxidants other than NO. Atorvastatin has been shown to increase paraoxonase activity and reduce the enhanced cellular uptake of oxLDL of monocytes differentiating into macrophages [100]. Long-term treatment with HMG-CoA reductase inhibitors (statins) appears to upregulate the expression and the activ ity of the vascular endothelial NO synthase (eNOS) pathway and increases nitric oxide availability, resulting in not only a downregulation of oxidative enzymes but also a direct scavenging of s uperoxide anion. As oxygen radical production is increased in various clinical s ettings such as hypercholesterolaemia, diabetes and hypertension, this statin-induced eNOS upregulation may play a foremost role in the vascular protective effects of these drugs. [119]. Moreover, sustained nitroglycerin (NTG) treatment is associated with an increased bioavailability of superoxide anion, likely playing a major role in the development of n itrate tolerance. The triggering events leading to this redox imbalance remain controversial as several cellular enzyme systems have been shown to be impaired by sustained in vivo e xposure to NTG, including membrane bound oxidases in [121] endothelial NOS in [122] an d arginine transporters [123]. Other effects than hipocholesterolemic of statins was described. Lovasta tin or simvastatin has been shown to have anti-inflammatory properties. They reduce monocy te adhesion to endothelial cells, cytokine expression and MCP-1 production [101-103]. B y limiting the influx of inflammatory cells statins may reduce the release of superox

ide radicals and the oxidative modification of LDL. On this way statins increases the resis tance of LDL to oxidation. Macrophage growth stimulated by oxLDL can also be inhibited by statin s [92] Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 57 3.1.3. Fenofibrate Very few data concerning the fibrates are available. In hypercholesterolemic pat ients, it has been shown that bezafibrate is more active than pravastatin in reducing the susc eptibility of LDL oxidation [104]. Moreover, in diabetics, De Leeuw and Van Gaal ha ve found that fenofibrate, but not pravastatin or simvastatin, can reduce the oxidizi bility of LDL and of VLDL [105]. 3.1.4. Beta-adrenergic blockers Beta adrenergic blocking agents have also been shown to have beneficia l effect on atherosclerosis. Several mechanisms of action have been suggested including an a ntioxidant action. All loc ers have in vitro antioxidant activity which appears to be rela ted to their degree of lipophilicity. In patients with CHD, Croft and coworkers sho wed that, while the lag time in patients with CHD is not significantly different from con trols, in patients with CHD who are taking loc ers, the lag time is higher than that observ ed in patients who are not taking loc ers in [106]. When LDL are oxidized in vitro by copper or by macrophages, carvedilol, the most lipophilic -blocker appears more potent than p indolol, labetolol, atenolol and propranolol and this is confirmed in vivo [107]. 3.1.5. Angiotensin-converting enzyme (ACE) inhibitors ACE inhibitors have been shown to have a beneficial effect in atheros clerosis. They reduce the progression of the disease in animals. These beneficial effects of ACE inhibitors have been related to an antioxidant activity against LDL oxidation that has been demo nstrated. In vitro, the lag time was found to be clearly increased by the presenc e of captopril at concentrations close to those that can be achieved therapeutically with large do ses. A similar effect is observed with N-acetylcysteine which contains like captopril, a sulfhydryl group. Quinapril, which lacks the sulfhydryl group, had no antioxidant activit y [108]. In vivo, Aviram and coworkers have shown that the propensity of LDL to oxidation is incre ased in patients with hypertension and is positively correlated with the blood pressure. Giving captopril or enalapril for 3 weeks decreases the oxidizibility of LDL. That sugg ests that the sulfhydryl group, which is absent in enalapril, does not have any influence on t he resistance of LDL oxidation [109]. Actually, the same group gave data suggesting that the a ntioxidant

activity might be related to the decreased production of angiotensin-II (A-II) a s A-II appears to increase the LDL oxidation by macrophages [110]. 3.1.6. Calcium channel blocker All calcium channel blocker are potent antioxidants in vitro and this property is probably related to their interaction with the lipid bilayer of the membranes. Lacidipine has the highest degree of interaction with the membrane Lacidipine inhibits the LDL oxidation produced by several oxidants. [111]. Blood Cell An Overview of Studies in Hematology 58 3.1.7. Metabolic medication - Trimetazidine Trimetazidine (TMZ) is the first in a new class of metabolic agents, available f or clinical use. In conditions of hypoxia or induced ischemia, TMZ maintains homeostasis and cellular functions by selectively inhibiting 3-ketoacyl-CoA-thiolase [112]. As a consequence, fatty acid b-oxidation is reduced and glucose oxidation is stimulated, result ing in decreased cellular acidosis and higher ATP production [113, 114]. In humans, TMZ has been shown to increase the ischaemic threshold and to relieve angina pectoris in pat ients with coronary artery disease. These benefits have been observed without any change i n heart rate, blood pressure, and rate-pressure product at rest, during submaximal and peak exercise in [115,116]. There is also demonstration that TMZ has antioxidant propert ies. During acute and chronic ischemia, TMZ reduces the loss of intracellular K+ induced by oxygen free radicals and also the membrane content of peroxidated lipids [117]. In vivo, pre-treatment with TMZ (4060 mg per day for 7 days) significantly decreases membrane malondialdehyde (MDA) content of red blood cells incubated with superox ide dismutase inhibitor diethyldithiocarbamate [118]. In humans, plasma levels of MDA were decreased after pre-treatment with TMZ during coronary artery bypass surgery [118]. 4. Instead of conclusion Mechanism of action of homocysteine is far from being elucidated. The big number of studies on this subject was gathered a lot of evidences about the ro le of Hcy as a major cardiovascular risk factor. All studied diseases: nephropathies, neurodeg enerative illnesses, osteoporosis, atherosclerosis seems to be tributary to this homocyste ine effect. It is widely accepted that involvement of homocysteine in the pathogenesis of these diseases activates prooxidative mechanisms. Therefore, the initiation of therapy of drug with antioxidant properties in such pathologies is justified. Moreover, there is clin ical evidence to support this point of view. Thus, although the clinicians question the value of

trimetazidine in the treatment of myocardial ischemia or degenerative deafness. [124-128] there are the clinical trials and basic research that support the benefits of th is antioxidant metabolic medication. Scientific arguments exist regarding the use of a torvastatin [129, 130] or nimodipine [131] therapy for antiischemic effects and preventio n of vascular events. Author details Filip Cristiana and Zamosteanu Nina Dept. Biochemistry. Univ.Med. Pharm. Gr.T.Popa, Iasi, Romania Albu Elena Dept. Pharmacology. Univ.Med. Pharm. Gr.T.Popa, Iasi, Romania Homocysteine in Red Blood Cells Metabolism Pharmacological Approaches 59 5. Acknowledgement Our research study to which we referred at the references was supported by grant PNCDII Idei 1225/2007 sustained by Ministry of Education and Research National Authority for Scientific Research UEFISCSU 6. References [1] Rifkind JM, Nagababu E, Ramasamy LB. Redox Rep. Review hemoglobin redox reactions and oxidative stress, 2003, (8): 234-7 [2] Ingraham, L. L., (1966) Compressive Biochemistry 14, 424-446 [3] David E. Metzler, Biochemistry. The Chemical Reaction of Living Ce lls, second edition, 2001, Elsevier Academic Press [4] Michael Lieberman, Allan D. Marks, Colleen Smith, Marks Essentials of Medical Biochemistry. A Clinical Approach.2007, edited by Lippincott Williams&Wilkins [5] T.McKee, J. McKee. Biochemistry: The Molecular Basis of Life. 2004 . 3rd Ed T McKee, J McKee (McGraw Hill) [6] Halliwell B; Plant Physiol. 2006,141, 312-322 [7] Hara P. Misra and Irwin Fridovici, The generation of superoxide r adical during the autoxidation of hemoglobin, The Journal of Biological Chemistry, 1972, 247(21): 69606962 [8] Balagopalakrishna C . Manoharan PT, Abugo OO, Rifkind JM, Production of sup eroxide from hemoglobin-bound oxygen under hypoxic condition, Biochemistry, 1996, 35(20): 6393-8 [9] Koppenol, W.H. (2001). "The Haber-Weiss cycle 70 years later". Redox Report 6 (4): 229 234. [10] Mwebi N.O., Fenton&Fenton-like reaction: the nature of oxidizing in termediates involved (dissertation submitted to the Faculty of the Graduate School of the Un iversity of Maryland, Maryland 2005 [11] Bray W.C. and Gorin M.H., J. Am. Chem. Soc., 1932, 54, 2124-2125, [12] Bogdanova A. Y. and Nikinmaa M., J. Gen. Physiol., 2001,117,181-19 0, Groves J.T., Inorg. Biochem., 2006, 100, 434-447 [13] Krzysztof Barbusinski, Fenton reaction-controversy concerning the che

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2012 Shaikh and Bhartiya, licensee InTech. This is an open access chapter distri buted under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by /3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Pluripotent Stem Cells in Bone Marrow and Cord Blood Ambreen Shaikh and Deepa Bhartiya Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48133 1. Introduction In a short span of few years, the possibility that the human body c ontains cells that can repair and regenerate damaged and diseased tissue has become a reality . Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and ot her non-embryonic sources, and have demonstrated a surprising ability for transformation into othe r tissue and cell types and for the repair of damaged tissues. (Image: Hematopoiesis_ (human) _diagram.png by A. Rad) Figure 1. Hematopoiesis in Bone Marrow Blood Cell An Overview of Studies in Hematology 70 In the 1950s, researchers discovered that the bone marrow contains ste

m cells i.e. hematopoietic stem cells (HSC) with the ability to self-renew and give rise to c ell types in the blood and immune system (Figure1). Multipotent HSCs reside at the apex of hematopoietic hierarchy and they are connected to mature cells by a complex roadmap of progenitor intermediates. The HSC differentiate into two different kinds of progen itors viz. Common Myeloid Progenitors (CMP) and Common Lymphoid Progenitors (CLP), which further differentiate to various blood cells including platelets, granulocytes, lymphocytes and macrophages. As a result, bone marrow transplantation became the standard method of care for most hematopoietic malignancies whereby the HSCs were able to repo pulate bone marrow after any kind of hematopoietic failure. A recent review by Do ulatov et al [1] describes the knowledge gathered over the years on Hematopoiesis. Besides HSC, another stem cell population, the mesenchymal stem cells (MSC) was identified in the bone marrow about 40 years ago [2]. MSCs comprise of the adher ent stem cell population with immune-modulatory properties. Besides bone marrow, MSCs can also be extracted from virtually all post-natal as well as extra-embryonic tissues su ch as amniotic membrane, placenta and umbilical cord. They can differentiate along multiple lin eages and exhibit significant expansion capability in vitro. Co-transplantation wit h MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation [3]. MSCs are also considered useful as vehicles for emerging cell and gene therapies in the field of tissue engineering [4]. Recently it has been postulated that MSC provide the conducive microenvironment for HSCs and thus maintain the stemness and proliferation of HS Cs and support HSC transplantation [3]. 2. Trans-differentiation of bone marrow stem cells Blood is one of the most highly regenerative tissues in our body wit h almost one trillion cells arising daily. Over the last decade several investigators have d emonstrated that BM stem cells not only contribute to development of blood cells but also to the regeneration of various organs and tissues [5, 6]. MSC isolated from various sources can differentiate into diverse cell types, showing a unique ability to cross lineage borders (i.e. are able to differentiate towards ectoderm-, mesoderm- and endoderm-derived cell type s) and do not express the major histocompatibility complex (MHC) class II Human Leuko cyte Antigen (HLA-DR) antigens. This, together with their in vitro proliferative pot ential and their immunoregulatory properties, renders them extremely promising for regenerative m edicine

applications in several diseases [7]. These observations were mainly explained by the hypothesis that the BM stem cells are plastic and thus could dedifferentiate into various cell types of non-he matopoietic organs and tissues [8]. The possibility that HSC/MSC are plastic and able to trans-differentiate raised hope that HSC/MSC isolated from BM, mobilized into the peripheral blood ( mPB) or cord blood (CB) could become a universal source of stem cells for tissue/ organ repair. This was supported by several demonstrations of the remarkable regenerative potential of HSC in animal models, for example after heart infarct [5], stroke [9], spinal cord injury [10], and VSELs in Bone Marrow and Cord Blood 71 liver damage [11] and of MSC in skeletal regeneration [12], cardiac r egeneration [13], diabetes [14] and osteogeneis imperfect [15]. The potential of adult stem cells also resulted in slow growth of research and funding restrictions on ES cells during President Bu sh regime in USA based on the argument that destroying embryos to derive human ES cell lin es was not essential, when better alternatives including adult stem cells are available for regenerative medicine (http://en.wikipedia.org/wiki/Stem_cell_controversy). H owever the excitement over plasticity of HSC reduced when their role in repair o f damaged organs became controversial [16, 17]. Several alternative mechanisms were proposed to explain the trans-differ entiation of bone marrow stem cells [18] including (i) epigenetic changes i.e. factors p resent in the environment of damaged organs may induce epigenetic changes in the gen es that regulate pluripotency of HSCs (ii) cell fusion during which infused HSCs may f use with cells in damaged tissues and form heterokaryons which express markers of both d onor and recipient cells (iii) paracrine effect i.e. HSCs are source of different trophic and angiopoietic factors that may promote tissue/organ repair (iv) microvesicles- depende nt transfer of molecules like receptors, proteins and mRNA between HSC and damaged ce lls and (iv) presence of pluripotent stem cell population in the bone marrow in ad dition to HSC & MSC that may contribute to regeneration. Presence of other stem cells in the BM may also explain the loss of contribution of BM cells to organ regeneration wi th the use of highly purified population of HSC [16]. Of these various possibilities (i) and (ii) are extremely rare and most likely the fact that BM houses heterogeneous and perhaps plu ripotent stem cells may explain transdifferentiation potential of bone marrow. It has been demonstrated that

there are heterogeneous stem cell populations in adult bone marrow com partment. Under appropriate experimental conditions, a certain type of bone marrow stem cells appears to differentiate (or transdifferentiate) into a variety of non-haemopoietic cells of ectodermal, mesodermal and endodermal origins (such as myocytes, neural cells and hepatocytes) [67].Various investigators have reported pluripotent stem cells in the bone marr ow by using varied approaches to demonstrate their presence and are listed in Table 1. The potential relationship of the BM-derived pluripotent stem cells rep orted by various investigators and compiled in Table1 is not clear. It is possible tha t these are overlapping populations of cells identified by slightly different isolation/ expansion strat egies and likely that all of these versatile BM-derived Oct-4+ non-hematopoietic stem ce lls, which were given different names, are in fact very closely related to the same type of BM-residing Pluripotent Stem Cells (PSC). This overlap was elegantly described earlier by Ra tajczak and his group [25] that various investigators are looking from different " keyholes" at the same population of stem cells that are hiding in a "darkroom" of the bone marrow environment. They further suggested that a founder cell may exist in the bone marro w which is responsible for multi-lineage differentiation. Table 2 is a compilation of various markers reported on these differently described PSCs in the bone marrow respon sible for their mobility (CXCR4), pluripotency (Oct-4, Nanog, Rex, Tert), non-hematopoiet ic lineage (CD45), immune status (MHC-1) and their developmental migration similari ty to PGCs (SSEA1). Blood Cell An Overview of Studies in Hematology 72 Stem Cell Functional attributes (in brief) MAPCs Multipotent Adult Progenitor Cells Described first by Verfailles and her group [19] Extracted from bone marrow in mouse, rat and human Plastic in nature and give rise to multiple cell types Single MAPC in early mouse embryo can contribute to all body tissues Ability to transdifferentiate Do not form teratomas SSEA-1+, CD13+, Flk-1low, Thy-1low, CD34, CD44, CD45, CD117(c-kit), MHC I, MHC IIm SSEA1+, OCT-4+ Can reconstitute bone marrow and also give rise to HSCs Many characteristics like ES cells MAPCs maintain telomere length Pluripotent properties even after 50 doublings MIAMI Marrow

Isolated Adult Lineage Inducible Cells Bone marrow derived adult stem cells isolated in humans aged 3- 72 years [20] Pluripotent by nature Capable of differentiating into cells from all three germ layers Positive for OCT-4, REX-1 and telomerase >50 population doublings with no sign of senescence Express markers typically associated with embryonic stem cells RS cells Recycling Stem Cells Are a sub-population of cells present amongst the MSCs [21] Small in size Proliferate rapidly CD45 MACS Multipotent Adult Progenitor Cells Express pluripotent-state-specific transcription factors (OCT-4, Nanog and Rex1[22] Cloned from human liver, heart, and BM-isolated mononuclear cells High telomerase activity Wide range of differentiation potential. MPCs Mesodermal Progenitor Stem Cells Detected in bone marrow and cord blood [23] Exist as a sub-population in MSC culture Fail to divide in culture thus quiescent Multi- to pluripotent by nature Express SSEA-4, OCT-4, Nanog by IF and RT-PCR VSELs Very Small Embryonic-like Stem Cells Homogenous population of rare (~0.01% of BM mononuclear cells) Sca-1+ Lin CD45 cells identified in murine BM [24] Express SSEA-1, OCT- 4, Nanog and Rex-1 & Rif-1 telomerase protein Small size (~3.5 m in diameter) Large nucleus surrounded by a narrow rim of cytoplasm Open-type chromatin (euchromatin) Differentiate into three lineages Do not form teratoma Quiescent population of cells Table 1. Pluripotent Stem Cells Reported in the Bone Marrow VSELs in Bone Marrow and Cord Blood 73 Characteristics Markers MAPC MIAMI MACS RS VSEL Shape and size Form small

colonies in culture Form small colonies in culture Small Small Small CXCR4 + + + + + CD 133 ND ND - - + Sca 1 ND ND ND ND + CD 45 - - - - OCT-4 + + + ND + REX-1 ND + + ND + Nanog + + + + + TERT + + ND + + SSEA 1 + ND ND ND + MHC-1 - ND + ND Quiescent by nature No data available No data available No data available No data available + Teratoma formation Do not form teratoma Do not form teratoma Do not form teratoma Do not form teratoma Do not form teratoma ND-experiment not done; + positive; - negative Table 2. Compilation of Various Markers on BM Pluripotent Stem Cells Besides these pluripotent stem cells, BM also houses Tissue Committed Stem Cells (TCSCs) including Epithelial Progenitor Cells (EPCs). Available literature sugges ts that postnatal neovascularization does not rely on formation of new blood vessels fro m pre-existing ones (angiogenesis) rather on EPCs migrating from the BM to induce neovascu larization. EPCs and HSCs share certain markers like Flk-1, Tic2, Sca-1, and CD34. As a result it has been suggested that they both may arise from a common precursor [26]. Interestingly the trans-differentiation ability of adult BM cells into various TCSCs like hepatocytes, cardiomyocytes, vascular endothelial cells, neuronal cells etc. occ urs only when there is a need i.e. into hepatocytes when damage is inflicted on th e liver by radiation or

chemical damage [27], into cardiomyocytes when myocardial infarction is induced [28], into endothelial cells on inducing ischemia [29] and into neural stem cells on inducing stroke [30]. In the same manner, the BM stem cells have also been shown to trans-differ entiate into germ cells when gonadal function is compromised e.g. by treating with busulphan in female [31] and male [32] mice. Freshly prepared BM may also exhibit early tissue-speci fic markers but are up-regulated several folds when the function of organ is compromised [33 ]. Pluripotent stem cells are expected to be more primitive to TCSCs bas ed on their developmental hierarchy (totipotent - pluripotent - multipotent unipotent stem ce lls). This is also supported by various observations shown below. Blood Cell An Overview of Studies in Hematology 74 Freshly isolated TCSC from the BM express tissue committed markers PSCs in BM acquire these markers after many days in culture TCSCs express c-Kit which is a more differentiated marker not expressed by PSCs . Thus we propose following developmental hierarchy of stem cells in bone marrow ( Figure:2) as opposed to the existing notion that HSC sit at the apex of hematopoietic syst em [1]. Figure 2. Developmental hierarchy of stem cells in bone marrow The existing controversial literature that HSCs and MSCs can trans-differentiate into various lineages can be alternatively explained by the presence of these pluripotent ste m cells. These PSCs interact closely with the MSCs by a process defined as emperipol esis [34]. The MSCs secrete SDF-1(Stromal Derived Factor-1) and other chemo-attractants there by creating a homing environment for these pluripotent stem cells (express CXCR4). Th us isolated BM stem cells have always been contaminated with these PSCs which may ha ve resulted in trans-differentiation and that HSCs/MSCs (being lineage restricted themse lves) possibly do not account for the observed plasticity. 3. Origin & deposition of hematopoietic and non-hematopoietic stem cells in bone marrow Early embryogenesis is the most active period for the developmental mi gration/trafficking of stem cells. With the beginning of gastrulation and organogenesis, s tem cells migrate to places where they establish rudiments for new tissues and organs. At certain points, of development stem cells colonize tissue specific niches, where they reside as a p opulation of self renewing cells supplying new cells that effectively replace senesc ent ones or those undergoing apoptosis. VSELs in Bone Marrow and Cord Blood 75 In mammals the first primitive HSC are found in the yolk sac and first definitiv

e HSC a few days later in the aorta-gonadmesonephros (AGM) region [35]. From the y olk sac and/or AGM region HSC migrate to the fetal liver (FL), which during the sec ond trimester of gestation becomes the major mammalian hematopoietic organ. By the end of the second trimester of gestation, HSC leave the fetal liver and colonize BM tis sue. Signals for the translocation of HSC from the fetal liver to BM are provided by the alpha chemok ine SDF1 that is secreted by osteoblasts lining the developing marrow cavitie s, marrow fibroblasts and endothelial cells. In response to SDF-1, HSC that expresses, SDF-1 receptor-a seven transmembrane-spanning G protein coupled CXCR4 receptor, leave the fetal liver a nd begin to home into BM where they finally establish adult haematopoiesis. It is very likely that at this point BM is also colonized by severa l other nonhematopoietic stem cells that may circulate during organogenesis and rapid foetal gr owth/expansion. In support of this stem cells for different tissues express CXCR4 on their surface and follow an SDF-1 gradient. Thus the SDF-1-CXCR4 axis alone or in combination with other chemoattractants plays a crucial role in the accumulation of non-hematopoietic s tem cells in developing BM [36, 37]. These cells find a permissive environment to survive in BM, and may play an underappreciated role as a reserve pool of stem cells fo r organ/tissue regeneration during postnatal life. The presence of these various populations of stem cells in the BM (Table 1) is a result of the developmental migration of stem cells during ontogenesis and the presence of the permissive environment that attracts them to the BM tissue. HSC and o ther nonhematopoietic stem cells are actively chemoattracted by factors secreted by BM s tromal cells and osteoblasts (e.g. SDF-1), hepatocyte growth factor (HGF)) and colon ize marrow by the end of the second and the beginning of the third trimester of gestation [24]. It is assumed that these non-hematopoietic pluripotent stem cells are deposited in the BM during early embryogenesis and subsequently may be mobilized in stressed situations and circulate in the peripheral blood. Similarly due to the stress of delivery these cells may also be present in cord blood [18]. Interestingly various terminologies like MAPCs, MIAMI, RS cells etc. (Table 1) d isappeared from the literature after initial publications and excitement except VS ELs. Ratajczak and group have made tremendous contribution to the field of VSELs biology. At presen t various laboratories across the world are providing evidence to support the pl uripotent property and potential of VSELs isolated from cord blood and bone marrow [38].

Possible reason being the method to isolate VSELs by flow cytometry described by Rata jczak and group could easily be replicated in various labs across the world. 4. Very small embryonic like stem cells (VSELs) VSELs were identified by Ratajczak and group in 2006 by multi paramet er sorting in adult murine BM. They express several morphological (e.g., relatively large n uclei containing euchromatin) and molecular (e.g. expression of SSEA-1, Oct4, Nanog, Rex 1) markers Blood Cell An Overview of Studies in Hematology 76 characteristic for embryonic stem cells (ESCs) [33]. The morphology of the cells was investigated using transmission electron microscopy which showed their d istinctive morphology and size differentiating VSELs from HSC in particular in terms of siz e (36 m vs. 68 m for HSC), chromatin structure and nucleus/cytoplasm ratio. Based on their small size, presence of PSC markers, distinct morphology (open-type chromatin, large nucleus , narrow rim of cytoplasm with multiple mitochondria) and ability to differentiate into all t hree germ layers, including mesoderm-derived cardiomyocytes, these cells were named very s mall embryoniclike stem cells. The true expression of Oct-4 and Nanog in BM-derived VSELs (BMVSELs) was recently confirmed by demonstrating transcriptionally active chromatin structure s of Oct4 and Nanog promoters. A mechanism based on parent-of-origin-specific reprogramming of genomic imprinting that keeps VSELs quiescent in a dormant state in tissues has been des cribed. VSELs highly express Gbx2, Fgf5, and Nodal, but express less Rex1/Zfp42 transcript as compared to ESC-D3s what suggests that VSELs are more differentiated than ICM-derived ESCs a nd share several markers with more differentiated EpiSCs. VSELs also highly expr ess Dppa2, Dppa4, and Mvh, which characterize late migratory PGCs. The expression of germ line mar kers (Oct4 and SSEA-1) and modulation of somatic imprints suggest a potential developmental similarity between VSELs and germ line-derived primordial germ cells (PGCs) [39, 40]. Developmental Origin of VSELs: VSELs are epiblast-derived PSCs deposited early during embryonic development in developing organs as a potential reserve pool of precursors for TCSCs and thus this population has an important role in tissue rejuve nation and regeneration. VSELs originate from or are closely related to a populat ion of proximal epiblast migratory Stem Cells (EpiSCs) that approximately at embryonic day (E)7. 25 in mice, become specified to PGCs, and egress from the epiblast into extra-embryonic tiss ues (extraembryonic mesoderm) [41]. VSELs follow developmental route of HSCs colonizing to gether

with HSCs first fetal liver and subsequently BM [37]. Thus PGCs, HSCs, and VSELs form all together a unique highly migrator y population of interrelated Stem Cells (SCs) that could be envisioned to be a kind of fourth highly migratory germ layer. [37] Self-renewal and in vitro differentiation of VSELs: VSELs exist in var ious mouse organs [42], have been well-characterized and are capable of differentiating into al l three lineages, supporting their true pluripotent character. Murine VSELs form embryoid body-like structures in co-cultures over C2C12 supportive cell line [24] and cou ld become specified into HSCs after co-culture over OP-9 stroma cells. VSELs-derived HSCs harvested from these co-cultures reconstitute murine bone marrow after total body irra diation [43]. The Umbilical Cord Blood (UCB)-purified VSELs have also been reported to d ifferentiate into neural cells [44] and after co-culture over OP-9 stroma cells were specified int o HSCs similar to murine BM-derived VSELs [45]. Apart from umbilical cord blood and bone marrow, VSELs have also been reported in Whartons jelly and gonadal tissue [46- 51]. Thei r presence amongst the MSCs in the Whartons jelly is in agreement with observation s made by other groups that MSCs contain a sub-population of more primitive stem cells [52] or even as postulated by Taichman and group [53] that VSELs are precursors of MSCs. Various studies VSELs in Bone Marrow and Cord Blood 77 have also reported that VSELs are mobilized into peripheral blood in response to injury/ stress in animal models [27,54-56] as well as in humans [28-30,57] thus suggesti ng a role in regeneration and homeostasis. 5. Our studies on VSELs in cord blood and bone marrow We studied the VSELs in UCB and discarded fraction of BM [46]. Usual ly the buffy coat obtained after Ficoll-Hypaque centrifugation is considered to be rich in stem ce lls and used for various studies over several decades. However, we reported that VS ELs settle along with the RBCs rather than getting enriched in the buffy coat (Figure:3 ). Similarly we found that the discarded RBC pellet obtained during initial processing of bone mar row was also rich in VSELs. These results were explained on the basis of buoyancy. T he adult stem cells have abundant cytoplasm, are relatively larger and thus observed in t he buffy layer whereas the VSELs are the pluripotent stem cells, with high nuc leo-cytoplasmic ratio, minimal cytoplasm and thus sink to the bottom of the tube alo ng with the RBCs. These VSELs exhibited various pluripotent markers, like CD45 -

, CD133 + SSEA-4 + . They also exhibit other primordial germ cell markers like Stella and Fragillis, thus supporting their origin from the epiblast stage embryo at the same time when PGCs mig rate via the dorsal mesentry to the gonadal ridges to become a source of germ cells. Figure 3. Isolation and characterization of VSELs from Cord Blood: A-Separation of cord blood into four layers on Ficoll-Hypaque; B-Description of cells observed in each layer sep arated; CImmunolocalization studies on MNC (A) and VSEL (B) using polyclonal Oct-4 (40X); D-Markers characterized on VSELs using Quantitative PCR and immunofluorescence These studies have several implications e.g. the stem cell biologists should ask themselves what is getting banked in the cord blood banks. VSELs unknowingly get discarded and only adult stem cells (and progenitors) including HSCs and MSCs get banked. Similarly autologus stem cell therapy for various indications other than blood related dis eases have resulted in Blood Cell An Overview of Studies in Hematology 78 minimal improvement. This may be explained since fate restricted progenitors HSC and MSC may have limited trans-differentiation ability. The pluripotent VSELs ha ve maximum plasticity and regenerative potential but are getting discarded unknowingl y. This raises a valid question on the success of BM transplantation to treat blood re lated diseases. This success could be accounted for by the differentiation ability of progenitor cell s into blood cells. While doing immunolocalization studies to detect OCT-4 positive cells, we found the VSELs express nuclear OCT-4 whereas a slightly bigger cell in the buffy coat collected f rom both the cord blood and bone marrow exhibited cytoplasmic OCT-4 (Figure:4). These are possibly the most immediate progenitors descendants from VSELs. We also conducted immunolocalization studies on umbilical cord tissue in the region of Whartons jel ly which is rich in MSCs. Results show that the MSCs had cytoplasmic OCT-4 li ke HSCs and that there was a distinct subpopulation of small cells with nuclear OCT-4 and were the VSELs, based on their size (Figure:4). On a similar note, when we did immunolocalizatio n of mouse bone marrow stem cells, we observed that the MSCs with typical fibroblast like m orphology have cytoplasmic OCT-4 along with VSELs with nuclear OCT-4. The MSCs showed a very heterogeneous staining pattern. Only a sub population of MSCs were positive wher eas other MSCs totally lacked cytoplasmic OCT-4. This possibly shows different di

fferentiation state since as the cell gets more committed, cytoplasmic OCT-4 is no longer required. Figure 4. Immunolocalization of Oct-4 in umbillical cord tissue Thus we concluded that the bone marrow compartment comprises of pluripotent VSEL s and their immediate descendants like HSCs and MSCs. Also that the most primitive ste m cell in the bone marrow is a pluripotent VSEL as shown in Figure 2. Being the most primi tive stem cell in the BM, we hypothesize that VSEL will show best engraftment post transplantation and also will be best vehicle for gene therapy. VSELs possibly undergo asymmetric cell division to self- renew and giv e rise to progenitors which further expand and differentiate to committed cell types. VSELs remain relatively quiescent throughout life, maintain long telomeres and are possibly the normal body stem cells which give rise to cancer stem cells (CSC) under certain unfavo urable conditions. We propose that this transformation of a VSEL into CSC occurs due uniden tified changes in the VSELs in Bone Marrow and Cord Blood 79 microenvironment. Recently it has also been reported that VSELs resist radiother apy (because of their quiescent nature) that destroys all actively dividing stem ce ll population in the bone marrow [43]. The somatic microenvironment is also compromised by the r adiotherapy. Thus although the VSELs persist, they are unable to reconstitute the bone marrow. Existence of two stem cell populations in various adult body tissues is an inter esting concept put forth by Li and Clevers [58]. They proposed that both quiescent (out of cell cycle and in a lower metabolic state) and active (in cell cycle and not able to retain DNA la bels) stem cell subpopulations may coexist in several tissues like gut epithelium, hair follicle , bone marrow etc. We have generated data to show that similar two distinct populations of ste m cells exist in mammalian gonads also. Interestingly similar stem cell biology persists in th e mammalian gonads irrespective of sex and is possibly an evolutionarily conserved phenomenon as we have reported the same in mice, rabbits, sheep, monkey and humans [48, 50]. 6. VSELs in mammalian testis We have reported for the first time the presence of a distinct popul ation of VSELs with nuclear OCT-4 in adult mouse [48] and human [47] testis, located towa rds the basement membrane of the seminiferous tubules. Besides, we also detected a prog enitor stem cell population with cytoplasmic OCT-4, which was slightly bigger and had a bundant cytoplasm. These cells showed extensive proliferation with cytoplasmic bridges a s cords. As these cells differentiated further, the cytoplasmic OCT-4 was gradually lost. In terestingly the

VSELs were found resistant to busulphan treatment which otherwise destroyed the dividing progenitors, haploid cells and damaged the somatic niche. Thus, it is evident that like the earlier report on bone marrow VSELs, gonadal VSELs are also resistant to oncotherapy. VSELs possibly undergo asymmetric cell division to give rise to progenitors, whi ch undergo clonal expansion and may further differentiate into sperm (Figure:5) 7. VSELs in mammalian ovary A gentle scraping of the adult ovary surface (mouse, rabbit, sheep, m onkey and human) with a sterile blade releases stem cells in a Petri dish [50]. On H & E staining, two distinct stem cell populations can be easily detected based on their size and differential OCT-4 staining pattern. The smaller stem cell population are smaller than th e RBCs and exhibit nuclear OCT-4 whereas the slightly bigger population exhibits cytoplasmi c OCT-4. Like cords in the testis, in the ovary we observed the presence of germ cell nests wi th cytoplasmic continuity representing extensive proliferation of progenitor stem cells. These stem cells were present in peri- menopausal human ovary and also persisted in mo use ovary after busulphan treatment. Like in the testis, the functionality of ovarian stem cells is also affected by a compromised niche. Three weeks culture of peri-menopausal ovarian stem cells produces oocyte-like s tructures, embryo-like structures in vitro [50]. Thus the stem cells retain their functionality but are unable to differentiate because of a non-supportive niche. Blood Cell An Overview of Studies in Hematology 80 Figure 5. Revised scheme for premeiotic development of germ cells in adult human testis 8. Significance of somatic microenvironment Niche on VSELs functionality A relatively quiescent VSEL and actively dividing progenitor model that possibly exists in ovary, testis, bone marrow, cord blood and Whartons jelly ensures that the master stem cell undergoes very few rounds of DNA replication to prevent its genome from agerelated changes and acquisition of errors during DNA replication. Table 3 highlights the importance of the somatic niche in controlling the stem c ell fate. It is the same VSEL that exists in different body organs but the niche dictates its fa te [46]. Sr.no Tissue VSEL with nuclear Oct-4 Progenitor stem cells with cytoplasmic Oct-4 (tissue-specific progenitor stem cells) 1 Testis Adark spermatogonia stem cells SSCs (Adark)

2 Ovary Ovarian germ stem cells (OGSCs) 3 Bone marrow, Umbilical cord blood and tissue Hematopoietic stem cells (HSC) Mesenchymal stem cells (MSC) Table 3. Details of Very Small Embryonic-Like Stem CellDerived Progenitors in Ad ult Human Tissues The possible reason why extensive plasticity of VSELs is evident in bone marrow and cord blood (so many different kind of TCSCs have been described) in contra st to testis or ovary may be because the bone marrow niche is more permissive as compared to a gonadal n iche which is more specialized and thus restricted in nature. VSELs in Bone Marrow and Cord Blood 81 9. VSELs and cancer Several years of cancer research suggests that cancers begin with gene tic changes that occur over a period of 15 to 20 years and in few cases a link to chroni c inflammation has been proposed e.g. in case of ovarian cancers, Barretts esophagus etc. However, emergi ng literature suggests that quiescent VSELs distributed in various organs may be a cellular or igin of cancer development. In 1855 Virchow proposed the embryonal rest hypothesis of tumor formation, based on histological similarities between tumors and embryonic tissues. This th eory was later expanded by other pathologist including Julius Conheim, who suggested that tumor s develop from residual embryonic remnants lost during developmental organogenesis [59] Recently identified VSELs in various adult body tissues display morphol ogy and markers characteristics as the pluripotent embryonic stem cells. These cells could suppo rt Virchows concept of an embryonic origin of cancer. Possibly the somatic niche, which keep s the VSELs in a quiescent stage under normal circumstances, undergoes some changes which pu sh the quiescent VSELs to an actively dividing state i.e. the tumor. Wang et al [60] recently reported that persisting embryonic cells in adult mice and humans at the squamo-columnar junction are possibly the source of Barrett s metaplasia and that it does not arise from mutant cells. They proposed that certain precancerous l esions, such as Barrett s, initiate not from genetic alterations but from competitive i nteractions between cell lineages driven by opportunity. Similarly, almost 90% of ovarian cancers arise f rom the ovary surface epithelium which is also the niche for ovarian stem cells. It is being proposed that ovarian niche gets compromised with age leading to menopause [61, 62] and also t o cancer. It

is essential to dissect out age related changes which lead to menopause and how they differ from those which lead to cancer. OCT-4, characteristic marker of VSELs is also a very good marker with high sensitivity and specificity for testicular germ cell tumors as well [63]. Cancer stem cells and VSELs with embryonic characteristics have a lot of similarities in terms of markers, telomere length, and resistance to radiotherapy; thus it may b e proposed that VSELs transform into CSCs when certain not so well understood ch anges occur in the microenvironment. It is possible that inflammation may alter the niche where the VSELs reside. It is highly unlikely that a somatic cell which is relatively senescent and has short telomeres will dedifferentiate and acquire long telomeres to transform into a cancer stem cell. Keeping this in mind, because of a defect in stem cell in the bone marrow due to an altered niche defective stem cell divisions occur and differentiation of such altered cells results in appearance of chromosomal defects in mononuclear cells picke d by standard cytogenetic studies. Identification of VSELs in adult tissues also opens new areas of inve stigation to elucidate how these cells contribute to the development of poorly differentiated tumors. S tudying the biology of normal stem cells may help us to better understand the bi ology of cancer, and explain its resistance to radio-chemotherapy, ability to an unlimited p roliferation and establishment of distant metastases. Blood Cell An Overview of Studies in Hematology 82 10. Conclusions The field of BMT also stands to greatly benefit once VSELs potential are realize d. A review article by Takizawa et al [64] makes an interesting reading and that despite adv ances in the field, timely availability of HLA matched BM is still problematic for patients i ncluding those who require multiple transplantations. In this context, in vitro expans ion of HSC is crucial but not yet achieved. It is still hoped that a single HSC may suffi ce to induce long-term multilineage engraftment. Notta et al [65] reported the possibility of CD49f as a specific marker to isolate HSC. In the chapter we are proposing that VSELs possibly give rise to HSC and may be better cell source to induce engraftment. Using cell surface markers to identify cell types always has associated issues since surface phenotype of a cell can change depending on the activation status of the precursor cells. Danova-Alt et al [66] recently concluded that UCB VSELs neither have embryonic nor adult stem cell-like phenoty pe, are not equivalent to mouse VSELs, have aneuploid karyotype and should not be regard

ed as a stem cell population. However, they have studied Lin-/CD45-/CD34+ cells and not Lin/CD45-/CD133+ cells, which are the VSELs as described by Ratacjzak and group [38 ]. Further it remains to be confirmed whether the aneuploidies they report are a technical artefact, since no cell lineage is expected to be aneuploid. VSELs could potentially be a real therapeutic alternative to the use of human ES cells since they do not form teratomas, are relatively quiescent and can be isolated from an autologous source. The fact that VSELs may differentiate in vitro into cells fro m all three germ layers makes these cells potential candidates in regenerative medicine. Finally , the mechanism by which VSELs could contribute to development of some malignancies could shed more light on origin of tumours. In conclusion it is of vital importance to eva luate if VSELs could be efficiently employed in the clinic. The work on VSELs is on the verge of develop ment and in coming years will bring more answers to the potential of these cells. 11. Key points of the Chapter The trans-differentiation potential of the HSC or MSC from bone marro w is controversial and can be alternatively explained by the presence of pl uripotent stem cells in the bone marrow. MAPC, MIAMI, VSELs, RS are possibly the same pluripotent stem cells, described differently by various investigators. All of these terminologies have d isappeared over time in published literature except VSELs that are being widely studie d by various groups across the world. VSELs are epiblast derived stem cells expressing pluripotent markers with high nucleocytoplasmic ratio and transcriptionally active chromatin. They have been isolate d from various murine organs as well as from human organs including gonadal tissues, cord blood, bone marrow and Whartons jelly. VSELs (with nuclear OCT-4) possibly are the most primitive cell type in BM and umbilical cord and give rise to HSC and MSC (with cytoplasmic OCT-4). VSELs in Bone Marrow and Cord Blood 83 The quiescent nature of VSEL prevent it from tumor formation in vivo but an altered somatic niche may lead to transformation of VSEL to cancer stem cell resulting in cancer. Author details Ambreen Shaikh and Deepa Bhartiya * Stem Cell Biology Department, National Institute for Research in Reproductive He alth (ICMR), Mumbai, India

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wski W, ZubaSurma E (2011) Identification of Very Small Embryonic/Epiblast-Like Stem Cells ( Vsels) Circulating in Peripheral Blood During Organ/Tissue Injuries. Methods Cell Biol. 103:3154. [34] Kucia M, Wysoczynski M, Ratajczak J, Ratajczak MZ (2008) Identification of Very Small Embryonic Like Stem Cells (VSEL) in Bone Marrow. Cell Tissue Res.331(1):125-34. [35] Ivanovs A, Rybtsov S, Welch L, Anderson RA, Turner ML, Medvinsky A (2011) H ighly Potent Human Hematopoietic Stem Cells First Emerge in the Intraembryoni c AortaGonad-Mesonephros Region. J Exp Med. 208(12):2417-27. [36] Kucia M, Zuba-Surma E,Wysoczynski ,Dobrowolska H, Reca R, Ratajczak J, Ratajczak MZ (2006) Physiological and Pathological Consequences of Identification of Very Small Blood Cell An Overview of Studies in Hematology 86 Embryonic Like (Vsel) Stem Cells in Adult Bone Marrow J Physiol Pharmacol.57 Sup pl 5:5-18. [37] Zuba-Surma EK, Kucia M, Rui L, Shin DM, Wojakowski W, Ratajczak J, Ratajczak MZ (2009) Fetal Liver Very Small Embryonic/Epiblast Like Stem Cells Follow Developmental Migratory Pathway of Hematopoietic Stem Cells. Ann N Y A cad Sci. 1176:205-18. [38] Ratajczak MZ, Shin DM, Liu R, Mierzejewska K, Ratajczak J, Kucia M, Zuba-Su rma EK (2012) Very Small Embryonic/Epiblast-Like Stem Cells (Vsels) and their Potential Role in Aging and Organ Rejuvenation - An Update and Comparison to Other Primitive Small Stem Cells Isolated from Adult Tissues. Aging (Albany NY). [Epub ahead of print]. [39] Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kuc ia M (2009) Novel Epigenetic Mechanisms that Control Pluripotency and Quiesce nce of Adult Bone Marrow Derived Oct4(+) Very Small Embryonic-Like Stem Cells. Leukemia . 23:2042-2051. [40] Shin DM, Liu R, Klich I, Wu W, Ratajczak J, Kucia M, Ratajczak MZ (2010) Molecular Signature of Adult Bone Marrow-Purified Very Small Embryonic-Like Stem Cells Supports their Developmental Epiblast/Germ Line Origin. Leukemia. 24:14501461. [41] Hayashi K, de Sousa Lopes SM, Surani MA (2007). Germ Cell Specif ication in Mice. Science 316:394396. [42] Zuba-Surma EK, Kucia M, Wu W, Klich I, Lillard JW Jr, Ratajczak J, Ratajczak MZ. (2008). Very Small Embryonic-Like Stem Cells Are Present in Adult Muri ne Organs: Imagestream-based Morphological Analysis and Distribution Studies. Cytomet

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Shannon M, Oosterhuis JW, Looijenga LH (2011) Expression and Interdepen dencies of Pluripotency Factors LIN28, OCT3/4, NANOG And SOX2 in Human Testicular Germ Cells and Tumours of the Testis. Int J Androl 34e160-74. [64] Takizawa H, Schanz U, Manz MG (2011) Ex Vivo Expansion of Hemato poietic Stem Cells:Mission Accomplished? Swiss Med Wkly 141:w13316 [65] Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I, Dick JE (2 011) Isolation of Single Human Hematopoietic Stem Cells Capable of Long-term Multilineage Engraft ment. Science 333(6039):218-21. [66] Danova-Alt R, Heider A, Egger D, Cross M, Alt R (2012) Very Sma ll Embryonic-Like Stem Cells Purified from Umbilical Cord Blood Lack Stem Cell Character istics. PLoS ONE 7(4): e34899. doi:10.1371/journal.pone. [67] Tao H, Ma DD (2003) Evidence for transdifferentiation of human bone marrowderived stem cells: recent progress and controversies. Pathology 35(1):6-13. Chapter 5

2012 Faraj and Salem, licensee InTech. This is an open access chapter distribute d under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. C-Reactive Protein Moneer Faraj and Nihaya Salem Additional information is available at the end of the chapter http://dx.doi.org/10.5772/47735 1. Introduction C- reactive protein (CRP) was so named because it was first discovered as a subs tance in the serum of patients with acute inflammation that reacted with the C- (c apsular) polysaccharide of pneumococcus [1]. Discovered by Tillett and Francis in 1930[2], it was initially thought that CRP might be a pathogenic secretion as it was elevated in people with a variety of illnesses in cluding cancer [3], however, the discovery of hepatic synthesis demonstrated that it is a native protein [4][5][6][7]. CRP is phylogenetically a highly conserved plasma protein, with homolog in vertebrates and many invertebrates that participates in the systemic response to i nflammation. Its plasma concentration increases during inflammatory states, a character t hat has long been employed for clinical purposes. CRP is a pattern recognition molecule, binding to specific molecular configurations that are typically exposed during cell death o r found on the surfaces of pathogens. Its rapid increase in synthesis within hours af

ter tissue injury or infection suggests that it contributes to host defense and that it is part of th e innate immune response [8]. 2. Molecular structure of CRP Entrez Gene summary for CRP the protein encoded by this gene belongs to the pentaxin family. It is involved in several host defense related functions based on its ability to recognize foreign pathogens and damaged cells of the host and to initi ate their elimination by interacting with humoral and cellular effector systems in the blood . Consequently, the level of this protein in plasma increases greatly during acute phase response to tissue injury, infection, or other inflammatory stimuli[12]. It is induced by IL1/inte rleukin-1 and IL6//interleukin-6 Blood Cell An Overview of Studies in Hematology 90 UniProtKB/Swiss-Prot: CRP_HUMAN, P02741 Size: 224 amino acids; 25039 Da Cofactor: Binds 2 calcium ions per subunit Subunit: Homopentamer. Pentaxin (or pentraxin) have a discoid arrangemen t of 5 noncovalently bound subunits Subcellular location: Secreted Mass spectrometry: Mass=23028; Method=MALDI; Range=19-224; Source=Ref.15; Mass spectrometry: Mass=22930; Method=MALDI; Range=19-223; Source=Ref.15; Function: Displays several functions associated with host defense it promotes ag glutination, bacterial capsular swelling, phagocytosis (CRP initiates the activation of the complement cascade and binds Fc gamma RI (CD64) and Fc gamma RIIA (CD32a) on p hagocytes to activate phagocytic responses) and complement fixation through its calci um-dependent binding to phosphorylcholine. It can interact with DNA and histones an d may scavenge nuclear material released from damaged circulating cells [13]. The CRP Entrez gene cytogenetic band located on the first chromosome: 1q21-q23 Ensemble cytogenetic band: 1q23.2 HGNC cytogenetic band: 1q21-q23. CRP is a 224-residue protein with a monomer molar mass of 25106 Da. The protein is an annular pentameric disc in shape [14][15]. Figure 1. Pentameric structure of CRP viewed down the 5-fold symmetry axis. The effector face of the molecule is on the top, while the calcium- and PCh-binding sites are on the oppo site recognition face [1] C-Reactive Protein 91 3. Methodology and clinical applications CRP is used mainly as a marker of inflammation. Apart from liver fai lure, there are few known factors that interfere with CRP production Measuring and charting CRP values can prove useful in determining disease progre ss or the effectiveness of treatments.

Blood, usually collected in a serum-separating tube, is analyzed in a medical la boratory or at the point of care. Various analytical methods are available for CRP d etermination, such as ELISA (Enzyme-linked immunosorbent assay ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name ca rried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobil ized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes only th e ligand and its specific binding counterparts remain specifically bound or "immunoso rbed" by antigenantibody interactions to the solid phase, while the nonspecific or unbound compo nents are washed away. Unlike other spectrophotometric wet lab assay formats wher e the same reaction well (e.g. a cuvette) can be reused after washing, the ELISA plates hav e the reaction products immunosorbed on the solid phase which is part of the plate and thus are not easily reusable)[19], immunoturbidimetry (Immunoturbidimetric Method This reagent is in tended for the in vitro quantitative determination of CRP concentration in se rum or plasma on automated clinical chemistry analyzers)[20], rapid immunodiffusion(is a d iagnostic test which involves diffusion through a substance such as agar.Two commonly known for ms are Ouchterlony double immunodiffusion and radial immunodiffusion) [21], and visual agglutination [22][23] (quantitative slide method and semi quantitative diluted method) There are two different tests for CRP. The standard test measures a much wider range of CRP levels but is less sensitive in the lower ranges. The high-sensit ivity CRP (hs-CRP) test can more accurately detect lower concentrations of the protein (it is more sensitive), which makes it more useful than the CRP test in predicting a healthy perso n s risk for cardiovascular disease [24]. (hs-CRP) test measures using laser nephelometry. The test gives results in 25 minutes with sensitivity down to 0.04 mg/L [26]. hs-CRP usually is ordered as one of several tests in a cardiovascular risk profile, often along with tests for cholesterol and triglycerides. Some experts say that the best way to predict risk is to combine a good marker for in flammation, like hs-CRP, along with the lipid profile [27]. CRP is one of several proteins that are often referred to as acute phase reactan ts and is used to monitor changes in inflammation associated with many infectious and autoimmune

diseases [28]. We should be healthy at the time of the sample collection, without a ny recent illnesses, infections, inflammation, or other tissue injuries. Since the hs-CRP an d CRP tests measure the same molecule, people with chronic inflammation, such as those wit h arthritis, should Blood Cell An Overview of Studies in Hematology 92 not have hs-CRP levels measured. Their CRP levels will be very high due to the arthritis often too high to be measured or meaningful using the hs-CRP test [29]. Normal concentration in healthy human serum is usually lower than 4.9 mg/L, slightly increasing with aging. Higher levels are found in late pregnant women, active inflammation, bacterial infection, severe bacterial infections, tissue in jury (postoperation), trauma and burns. CRP is a more sensitive and accurate reflection of the acute phase response than the ESR[30] another blood test often ordered in conjunction with CRP (erythrocyte sedimentat ion rate or sed rate known as ESR) both CRP and ESR give similar information abo ut non-specific inflammation. CRP appears and disappears more quickly than changes in ESR. Therefore, your CRP level may drop to normal following successful treatment [31] , whereas ESR may remain elevated for a longer period. The half-life of CRP is constant. Therefore, CRP level is mainly dete rmined by the rate of production (and hence the severity of the precipitating cause). In the first 24 h, ESR may be normal and CRP elevated [32]. CRP and ESR have been used to diagnose postoperative infections after spinal surgery. We did a prospective study in Baghdad [33]. The aim of the study was to determine the duration of the physiological rise in the serum CRP w ithout the development of infection following lumbar laminectomy. Forty patients (1 9 women, 21 men) mean age 44.2, age range 27-60 yrs were included in the study. All patients underwent laminectomy. Additional clinical data relevant to the stu dy included body temperature, duration of surgery & blood transfusion. The indication of surgery established several days to weeks before the surgical procedure. Pathologic findings included: 27 lumber spinal canal stenosis 2 reoperation for stenosis 1 spinal canal hydrated cyst 10 lumber disc herneation. Preoperatively, a single shot antibiotic prophylaxis with cefotaxime 1 gram was give to all cases. All patients were operated under general anesthesia. Duratio n of surgery varied from 60-180 min. (average 80.5 min.). Before surgery no patient receiv ed steroids. Blood

samples were taken on the day of surgery and on each consecutive day after surge ry for10 days. The parameters taken were CRP, ESR, Total white blood cell count. On 1st post op erative day the CRP started to increase in 34 patients (range 12- 96, averag e 27). In the 2nd and 3rd post operative day all the patients had high CRP with an average of 39 &38 respectively. This increase was highly significant (P<0.001). A dramatic decline in the CRP level was noticed to start in the 5th post operative day (average 27 ), then gradual C-Reactive Protein 93 reduction was noticed until a normal ranges at day 9th post operative ly (average 4.8) as shown in figure -2. Figure 2. The CRP level of all patients from day one which is here is the operat ion day to day 11, the average values plotted together [34] Increased CRP values during the first 5 post-operative days did not i ndicate that an infection is ongoing. An infection should be considered with prolonged CRP elevation (more than 5 days) as noticed is one of our patients or when a second rise occur s. Although we did not use steroids or non steroidal anti inflammatory drugs post operativel y, but these medications seems to effect on the level of CRP. Munoz m. et al [35 ] revealed that preoperative treatment with naproxane and famotidine was well tolerated and reduced the acute phase response after instrumented spinal surgery. In this study we cou ld not find any correlation of the raised CRP level they age , sex , ESR , WBC cou nt , body temperature duration of surgery blood transfusion[36] with exception of Orrego LM et al[37] who noticed that more complex surgical procedure had higher CRP level and explaine d due to the amount of tissue trauma. Sugimorik et al [38] showed no correlation b etween the high CRP concentration and the level, type of lumbar disc herneation or the preoperative clinical data. Thelander et al [39] noticed that peak levels were not related to bl eeding, transfusion, operation time, administered drugs, age or sex However, it has not be en demonstrated if resolution of the signs and symptoms of postoperative spinal wound inf ections in patients who are being treated with intravenous antibiotics correlates with thes e markers. CRP is a sensitive marker of pneumonia. A persistently high or rising CRP level suggests antibiotic treatment failure or the development of an infective complication. These results suggest that CRP, rather than TNF- or IL-6, may have a role as a clinical marker

in pneumonia [40]. Most recently CRP has made headlines as it relates to heart disease an associati on between Blood Cell An Overview of Studies in Hematology 94 minor CRP elevation and future major cardiovascular events has been re cognized, leading to the recommendation by the Centers for Disease Control and the Amer ican Heart Association that patients at intermediate risk of coronary heart diseas e might benefit from measurement of CRP. It is yet to be determined if CRP serves as a marker of hear t disease or whether it plays apart in causing atherosclerotic disease (hardening th e arteries)[41]. CRP has been shown to have a close relationship with vascular diseases. C RP is a powerful independent risk factor for atherosclerosis and atherosclerosis-related diseases (Lusic et al., 2006 [42]; Verma et al., 2006)[43]. Elevated high-sensitivity CRP (hsCRP) has be en measured in the blood of patients with essential hypertension (Li et al., 2005) [44] or a bdominal aortic aneurysms (Vainas et al., 2003 [45]; Tambyraja et al., 2007 [46]) wit h enhanced systemic or local arterial strain. Elevated serum hsCRP independently correlates wit h blood pressure (Sung et al., 2003)[47], arterial stiffness (Kim et al., 2007) [48], and aneurys mal size (Vainas et al., 2003) [49]. Although several investigations have demonstrated that aneurysmal tissues and diseased coronary artery venous bypass grafts (Jabs et al., 2003)[50] produc e CRP, little is known about its mechanism. Blood vessels are dynamically subjected to mechani cal strain in the forms of stretch and shear stress that result from blood pres sure and blood flow. Mechanical strain on the vessel wall can increase from 15 to 30% in hypertensive individuals (Safar et al., 1981[51]; Shaw and Xu, 2003)[52][53]. CRP testing is not precise enough to diagnose specific diseases but serves more as a general indicator that more testing may be needed if inflammation or infection is found. The CRP test is therefore useful in assessing patients with the following list[54]: Swelling and bleeding of the intestines (inflammatory bowel disease). Painful swelling of the tissues that line the joints (rheumatoid arthritis). Diseases of the immune system, such as lupus. Pelvic inflammatory disease (PID) Painful swelling of the blood vessels in the head and neck (giant cell arteriti s). Cancer of the lymph nodes (lymphoma). Infection of a bone (osteomyelitis). Connective tissue disease Heart attack Infections Pneumococcal pneumonia Rheumatic fever Tuberculosis 4. Factors that effects on high levels of CRP

Many doctors will ((NSAIDs like aspirin, ibuprofen, blood. Both antiinflammatory drugs us reducing CRP. However, there are in the blood.

prescribe taking non steroidal anti-inflammatory drugs and naproxen) or statins may reduce CRP levels in and statins may help to reduce the inflammation, th natural treatments that can help reduce inflammation

C-Reactive Protein 95 Following are some of the natural treatments for lowering C - reactiv e protein levels and inflammation in the blood: Fish Oil Omega 3 Fatty Acids Doctors and nutritionists have recommended Omega 3 s for years, and recently fish oil has been the most recommended source for Omega 3 Fatty Acids. Fish oil contains two of the most therapeutic Omega 3 Fatty A cids the DHA and EPA. These two fatty acids are the most readily absorbed by the body (much more so than the ALA found in flax seed oil), and can help reduce inflammation in the blood among other benefits. Ginger - Ginger root extract has long been used in Asian cooking, an d has been used for centuries as a digestive aid and motion sickness cure, and more recen tly to lower cholesterol. Ginger can also help reduce inflammation, as it relaxes the muscles surrounding blood vessels and facilitates blood flow throughout the body. MSM - Methyl Sulfonyl Methane, commonly known as MSM, is a naturally occurring s ulfur compound found in some vegetables. MSM is found in many arthritis for mulas, and has strong anti-inflammatory properties. These three nutrients may help reduce CRP levels in your blood. All three are im portant for maintaining heart health as well as general health and wellbeing [55]. There was a study found a significant effect of treatment for 2 mont hs with 1000 mg/day vitamin C on plasma CRP, in non diseased moderately overweight nonsmok ers with baseline CRP 1.0 mg/L. The magnitude of the effect was similar to that of statins . There was no significant effect of vitamin E. These data represent the largest study to date on the effects of vitamins C and E on CRP and extend our previous findings in overweight active and passive smokers. They indicate that vitamin C should be further i nvestigated for its potential for reducing chronic inflammation and its consequences. And t hey identify a threshold concentration above which there is a potential for reduction in CRP. Future studies to determine whether vitamin C can reduce some of the inflamm ation-related adverse consequences of obesity should be considered. Such trials shoul d focus on individuals with elevations (.1.0 mg/L) in CRP, because studies with l

ow-risk persons are less likely to show an effect, resulted in misleading outcomes. If pe rsons with lower CRP levels must be included, separate randomization of those with CRP .1.0 mg/L woul d justify separate examination of this subgroup, assuming adequate power in this stratum. In addition, if the potential independent effect of vitamin C is to be determined, it would be necessary to exclude persons who are taking other anti-inflammatory dru gs (except lowdose aspirin for heart disease prevention) and to exclude users of mu ltiple vitamins (something which has not been done in most large antioxidant trials), because multiple vitamins alone can raise plasma ascorbic acid levels substantially and make the control group insufficiently different from the active treatment group. Finally, it may be prudent to evaluate vitamin C alone, unpaired with vitamin E, as we found a wea ker CRP-lowering effect with the combination than with vitamin C alone in our previous trial [56] . Blood Cell An Overview of Studies in Hematology 96 5. C - reactive protein concentrations in cerebral spinal fluid in grampositive and gram-negative bacterial meningitis Several reports have shown an ability of CRP to discriminate between patients with bacterial meningitis and patients with aseptic (viral) meningitis. Altho ugh a recent Metaanalysis suggested that a negative CRP test in either cerebrospinal fluid (CSF) or serum can be used with a very high probability to rule out bacterial meningitis , a more recent report suggested that serum concentrations are a better screening tool for th is differential diagnosis. The substantial increase in CSF CRP, as well as the trend of an increased CSF/bl ood ratio of CRP, suggests that infection with gram-negative bacteria enhances permea bility of CRP through the blood-brain barrier. It is possible that these findings re flect the ability of the endotoxin lipopolysaccharide-s, present in gram-negative but not in gram-positiv e bacteria, to affect the permeability of the blood-brain barrier [57][58]. CSF ni tric oxide (NO) may be involved in this mechanism because its concentration in CSF is higher in gram-negative meningitis. This possibility is supported by the higher potency of gram-negative bacteria to promote macrophage NO production [59], the enhanced production of NO i n the CSF of septic meningitis [60], and the role of NO in permeability changes of the bloodbrain barrier in LPS-induced experimental meningitis [61]. Another interesting potential explanation for the present observation is that lipopolysaccharide-s produced by gram-negative bacteria could induce local CRP s

ynthesis in the central nervous system. CRP can be produced in neurons [62], and lipopolysaccharide-s can induce CRP in extrahepatic sites [63]. This ma y also explain the increase, albeit nonsignificant, in serum CRP in the gram-negative cases . There is currently no single test to diagnose the etiology of meningitis promptly and accurately. G iven its high sensitivity and easy measurability, CRP may be a useful supplement for rapid dia gnosis and categorization of bacterial meningitis. Author details Moneer Faraj and Nihaya Salem Department of Neurosurgery, Hospital of Neurosciences, Baghdad, Iraq 6. References [1] http://en.wikipedia.org/wiki/C-reactive_protein [2] Tillett WS, Francis T (September 1930). "Serological reactions in pneumonia with a nonprotein somatic fraction of pneumococcus". J. Exp. Med. 52 (4): 56171. [3] Pepys, MB; Hirschfield, GM (June 2003). "C-reactive protein: a criti cal update" (PDF). J Clin Invest 111 (12): 180512. doi:10.1172/JCI18921. PMC 161431. PMID 12813013. C-Reactive Protein 97 http://www.jci.org/articles/view/18921/files/pdf?disposition=attachment. [4] http://en.wikipedia.org/wiki/C-reactive_protein [5] Peter J. Kennelly; Murray, Robert F.; Victor W. Rodwell; Kathleen M. Botham (2009). Harper s illustrated biochemistry. McGraw-Hill Medical. ISBN 0-07-162591-7. [6] Matthew R. Pincus; McPherson, Richard A.; Henry, John Bernard (2007). Henry s clinical diagnosis and management by laboratory methods. Saunders Elsevier. ISBN 1-4160-0 287-1. [7] John J. Ratey MD; Gary A. Noskin MEd MD; MD, Ralph Braun; Edwar d N. Hanley Jr MD; Iain B. McInnes; Shaun Ruddy MD (2008). Kelley s Textbook of Rheu matology: 2Volume Set, Expert Consult: Online and Print (Textbook of Rheumatology (Kelley s)(2 Vol)). Philadelphia: Saunders. ISBN 1-4160-3285-1. [8] http://www.jbc.org/content/279/47/48487 [9] http://www.genecards.org/cgi-bin/carddisp.pl?gene=CRP [10] Thompson, D; Pepys, MB; Wood, SP (February 1999). "The physiologic al structure of human C-reactive protein and its complex with phosphocholine". Structure 7 (2): 169 77. doi:10.1016/S0969-2126(99)80023-9. PMID 10368284. [11] Dhillon, B; Yan, H; Szmitko, PE; Verma, S (May 2005). "Adipokines : molecular links between obesity and atheroslcerosis". Am J Physiol Heart Circ Physiol 288 (5): H2031 41. doi:10.1152/ajpheart.01058.2004. PMID 15653761. http://ajpheart.physiology.org/content/288/5/H2031.full.pdf. [12] http://www.genecards.org/cgi-bin/carddisp.pl?gene=CRP [13] A C-reactive protein promoter polymorphism is associated with type 2 diabetes mellitus in Pima Indians. (PubMed id 12618085) 1, 4, 5 Wolford J.K....Hanson R.L. (2003)

[14] http://www.genecards.org/cgi-bin/carddisp.pl?gene=CRP [15] A C-reactive protein promoter polymorphism is associated with type 2 diabetes mellitus in Pima Indians. (PubMed id 12618085) 1, 4, 5 Wolford J.K....Hanson R.L. (2003) [16] Mantovani A, Garlanda C, Doni A, Bottazzi B (January 2008). "Pent raxins in innate immunity: from C-reactive protein to the long pentraxin PTX3". J. Clin. Immunol. 28 (1): 113. doi:10.1007/s10875-007-9126-7. PMID 17828584 [17] Clyne B, Olshaker JS (1999). "The C-reactive protein". J Emerg Me d 17 (6): 101925. doi:10.1016/S0736-4679(99)00135-3. PMID 10595891 [18] NCBI Entrez Protein #CAA39671 [19] http://en.wikipedia.org/wiki/ELISA [20] http://www.proz.com/kudoz/english_to_spanish/medical_general/1100998immunoturbidimetric_method.html [21] http://en.wikipedia.org/wiki/Immunodiffusion [22] http://en.wikipedia.org/wiki/C-reactive_protein [23] Pepys, MB; Hirschfield, GM (June 2003). "C-reactive protein: a criti cal update" (PDF). J Clin Invest 111 (12): 180512. doi:10.1172/JCI18921. PMC 161431. PMID 12813 013. http://www.jci.org/articles/view/18921/files/pdf?disposition=attachment. [24] Clyne B, Olshaker JS (1999). "The C-reactive protein". J Emerg Me d 17 (6): 101925. doi:10.1016/S0736-4679(99)00135-3. PMID 10595891. Blood Cell An Overview of Studies in Hematology 98 [25] http://labtestsonline.org/understanding/analytes/hscrp/tab/test . [26] http://en.wikipedia.org/wiki/C-reactive_protein [27] http://labtestsonline.org/understanding/analytes/hscrp/tab/test. [28] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832720/ [29] http://labtestsonline.org/understanding/analytes/hscrp/tab/test. [30] http://en.wikipedia.org/wiki/C-reactive_protein [31] http://boneandspine.com/definitions/c-reactive-protein/ [32] http://en.wikipedia.org/wiki/C-reactive_protein [33] The New Iraqi Journal of Medicine 2009 ; 5 (3): http://www.scopemed.org/journal.php?jid=20 [34] The New Iraqi Journal of Medicine 2009 ; 5 (3): http://www.scopemed.org/journal.php?jid=20 [35] Munoz M. , Garcia Vallejo JJ , Sempere JM , Romero R. , Ollala E , Sebastian C acute phase response in patients undergoing lumbar spinal surgery ; modulatio n by per operative treatment with Naproxen and Famotidine . Eur Spine J. 2004 Jul; 13(4): 367-73. [36] The New Iraqi Journal of Medicine 2009 ; 5 (3): http://www.scopemed.org/journal.php?jid=20 [37] Qrrego LM, Perez CM, Perez YM, Cheyre EJ, Mardones PR, Plasma C- reactive p rotein in elective orthopedic surgery Rev. Med Chil. 2005 Nov. 133 (11): 1341 8 . [38] Sugimori K , Kawaguchi Y , Morita M , Kitajima I , Kimura T. High sensitivi ty analysis of serum C- reactive protein in young patients with lumbar disc herniation .J Bo ne Joint surg. Br. 2003 Nov, 85 (8) : 1151-4 [39] Thelander U , Larsson S. Quantitation of C- reactive protein level and ESR after spinal

surgery. Spine 1992 April 17 (4):400-4. [40] http://chestjournal.chestpubs.org/content/108/5/1288.abstract [41] http://www.jbc.org/content/279/47/48487 [42] Lusic I, Radonic V, Pavelin S, and Bilic I (2006) Is C-reactive protein a b etter predictor of recurrent carotid disease following carotid endarterectomy than established risk factors for atherosclerosis? Vasa 35: 221-225. [43] Verma S, Devaraj S, and Jialal I (2006) Is C-reactive protein an innocent bystander or proatherogenic culprit? C-reactive protein promotes atherothrombosis. Circulatio n 113: 2135-2150. [44] Li JJ, Fang CH, and Hui RT (2005) Is hypertension an inflammator y disease? Med Hypotheses 64: 236-240. [45] Vainas T, Lubbers T, Stassen FR, Herngreen SB, van Dieijen-Visser MP, Brugg eman CA, Kitslaar PJ, and Schurink GW (2003) Serum C-reactive protein level is associated with abdominal aortic aneurysm size and may be produced by aneurysmal tissu e. Circulation 107: 1103-1105. [46] Tambyraja AL, Dawson R, Valenti D, Murie JA, and Chalmers RT (20 07) Systemic inflammation and repair of abdominal aortic aneurysm. World J Surg 31: 1210-1214 . C-Reactive Protein 99 [47] Sung KC, Suh JY, Kim BS, Kang JH, Kim H, Lee MH, Park JR, and Kim SW (2003) High sensitivity C-reactive protein as an independent risk factor for essent ial hypertension. Am J Hypertens 16: 429-433. [48] Kim JS, Kang TS, Kim JB, Seo HS, Park S, Kim C, Ko YG, Choi D, Jang Y, and Chung N (2007) Significant association of C-reactive protein with arterial stiffness in treated nondiabetic hypertensive patients. Atherosclerosis 192: 401-406. [49] Vainas T, Lubbers T, Stassen FR, Herngreen SB, van Dieijen-Visser MP, Brugg eman CA, Kitslaar PJ, and Schurink GW (2003) Serum C-reactive protein level is associated with abdominal aortic aneurysm size and may be produced by aneurysmal tissu e. Circulation 107: 1103-1105. [50] Jabs WJ, Theissing E, Nitschke M, Bechtel JF, Duchrow M, Mohamed S, Jahrbeck B, Sievers HH, Steinhoff J, and Bartels C (2003) Local generation of C-r eactive protein in diseased coronary artery venous bypass grafts and normal vascular tissu e. Circulation 108: 1428-1431. [51] Safar ME, Peronneau PA, Levenson JA, Toto-Moukouo JA, and Simon AC (1981) P ulsed Doppler: diameter, blood flow velocity, and volumic flow of the brachi al artery in sustained essential hypertension. Circulation 63: 393-400. [52] Shaw A and Xu Q (2003) Biomechanical stress-induced signaling in smooth muscle

cells: an update. Curr Vasc Pharmacol 1: 41-58. [53] http://jpet.aspetjournals.org/content/330/1/206.full [54] http://www.creactiveprotein.net/c-reactive-protein.html [55] http://www.healthy-heart-guide.com/crp-blood-test.html [56] Vitamin C treatment reduces elevated C-reactive protein.(Gladys Bloc k a, Christopher D. Jensen a, Tapashi B. Dalvi a, Edward P. Norkus b, Mark Hudes a, Patricia B. Crawford a, Nina Holland a, Ellen B. Fung c, Laurie Schumacher c, Paul Harmatz) Free Radical Biology & Medicine 46 (2009) 7077 [57] http://www.clinchem.org/content/48/3/591.full [58] Wispelwey B, Lesse AJ, Hansen EJ, Scheld WM. Haemophilus influenza e lipopolysaccharide-induced blood-brain barrier induced permeability during experimental meningitis in the rat. J Clin Invest 1988;82:1339-1346 [59] Jungi TW, Valentin-Weigand P, Brcic M. Differential induction of N O synthesis by grampositive and gram-negative bacteria and their components in bovine mono cyte-derived macrophages. Microb Pathog 1999;27:43-53. CrossRefMedlineOrder article via InfotrieveWeb of Science [60] Tsukahara H, Haruta T, Hata I, Mayumi M. Nitric oxide in septic and aseptic meningitis in children. Scand J Clin Invest 1998;58:71-80. [61] Boje KMK. Inhibition of nitric oxide synthase attenuates blood-brain barrie r disruption during experimental meningitis. Brain Res 1996;720:75-83. CrossRefMedlineOrder article via InfotrieveWeb of Science [62] Yasojima K, Schwab C, McGeer EG, McGeer PL. Human neurons generate C-reacti ve protein and amyloid P: upregulation in Alzheimers disease. Brain Res 2000;887:80-89. Blood Cell An Overview of Studies in Hematology 100 CrossRefMedlineOrder article via InfotrieveWeb of Science [63] Introna M, Alles VV, Castellano M, Picardi G, De Gioia L, et al. Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic s ites. Blood 1996;87:1862-1872. Chapter 6

2012 Takahashi et al., licensee InTech. This is an open access chapter distribut ed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Whole Blood RNA Analysis, Aging and Disease Junko Takahashi, Akiko Takatsu, Masaki Misawa and Hitoshi Iwahashi Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48226 1. Introduction Microarray techniques allow to detect genome-wide perturbations during v arious treatments and to measure various responses by multitude of gene probes. Toxicog enomics,

in which microarray techniques are specifically used in toxicology test , has been widely recognized as one of standard safety procedures for chemicals [1-3]. G ene expression microarrays have been used particularly for screening of genes involved in specific biological processes of interest, such as diseases or responses to environmental stimuli. Such experiments adopt the healthy state as a control, and identify highly e xpressed or suppressed genes. However, few studies deal with the features of gene expression and its variation at the healthy state to be influenced by species, age, sex, and individual variability. In measuring the state of disease and drug response, mini mally invasive blood sampling, which allows for direct measurement of immune-responsive blood cells, excels other invasive biopsy techniques upon disease diagnostics and assessment of drug response, as well as health monitoring. Blood RNA contains an enormous amount o f information on expression of messenger RNA and non coding functional RNA which remain s without being translated into protein. Thus, blood RNA offers an opportunity t o detect subtle change in physiological state. In this chapter, we discuss the potential of the RNA diagnosis using whole blood, showing a series of whole blood microarray experime nts to evaluate variations of correlation among individuals and ages [4], dietary-induce d hyperlipidemia, and other stresses using specific pathogen-free (SPF) miniature pigs. 2. The use of whole blood RNA analysis Use of whole blood was intended on two accounts. First, RNA expression and degra dation is susceptible to artificial manipulation such as cell separation and extraction. The whole blood manipulation avoids this risk, unlike dealing with extracted whit e blood cells. In addition, whole-blood RNA can be stabilized immediately by using RNA b lood sampling Blood Cell An Overview of Studies in Hematology 102 tube such as PAXgene. This avoids the cell separation process after sampling and minimizes the possibility of RNA denaturation. Usually, peripheral blood mononuclear cells (PBMCs) separation employs the difference of specific gravity between other blo od components, which should be followed immediately after the blood sampling. Such ma nipulation requires a skilled operator to reduce the influence of separation proc edures on gene expression. Second, the whole blood is a heterogeneous population of l ymphocytes (monocytes, T-cells, and B-cells), granulocytes (neutrophils, eosinophils, and b asophils), and platelets. One can expect that representative subpopulations in white b lood cells may vary

depending on the health condition of an individual. When a great alteration occu rs in some subpopulations, the whole blood may also depart from the normal state o f its age, because whole blood is a heterogeneous mixture of such subpopulations. Therefore, identi fication of gene expression characteristics and age-related variation in subpopulatio ns in whole blood are essential issues. 3. The advantage of using miniature pigs Pigs are a useful model animals of humans because they have similar anatomy and digestive physiology to human [5-6]. In particular, miniature pigs are easier to breed and handle than other nonprimates, making them an optimal species for preclinical test [7]. More over, blood samples can be taken repeatedly and human medical devices such as endo scopes and MRI and CT scanners are also applicable. These advantages increasingly allow miniatu re pigs for laboratory animals, with recent progress in upgraded supply systems. In spite of some large-scale microarray studies on pigs, only a limited amount of funda mental data is available for pigs compared to other laboratory species [8-9]. In September 2003 , the Swine Genome Sequencing Consortium (SGSC) was formed by industry, government, and academia, to promote pig genome sequencing under international coordinat ion [10]. In November 2009, since the announcement of completed swine genome map by members of the SGSC, its research environment has been enhanced [11]. 4. Gene expression profiles change related to aging It is particularly important to identify gene expression characteristics and variation of heterogeneous population of cells with age in whole blood. Fractions of lymphocytes, monocytes, neutrophils, eosinophils, and basophils in white blood cells showed insignificant differences with age as a result of ANOVA analysis. This study attempted to identify characteristics of age-related gene expression by taking i nto account of change in the number of expressed genes by age and similarities of g ene expression intensity between individuals. 4.1. Characteristics of study subjects Five males and five females of 12 week old Clawn miniature pigs were housed indi vidually in cages of 1.5 m 2 at the SPF facility of the breeder (Japan Farm Co., Ltd, Kagoshima, Japan) Whole Blood RNA Analysis, Aging and Disease 103 for 18 weeks. Mean body weights of males and females at the beginning of the experiment were 7.0 kg and 6.9 kg respectively. During this period, all animals were fed wi th 450g/day standard dry feed (Kodakara73, Marubeni Nisshin Feed Co., Ltd., Tokyo Japan) with free

access to water. Fetuses were taken out from their mothers on days 77 to the 84 days of the pregnancy by a Caesarean section. The unborn baby s sex was determined based on the shape of the vulva. Table 1. Subject body weight results doi:10.1371/journal.pone.0019761.t001 All blood samples were collected from the superior vena cava at 12, 16, 20, 24, and 30 weeks of age. Blood (EDTA), plasma (EDTA) and serum samples for hematology and biochem ical tests were collected 24 hours after fasting. Hematology and biochemical tests were conducted by Clinical Pathology Laboratory, Inc. (http://www.patho.co.jp/i ndex.html) (Kagoshima, Japan) using standard clinical methods. Body weight change and hematological variation during breeding period are shown Table 1 and Table 2, respectively. One-way ANOVA analysis for age-related variations in red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit value (HCT) showed significant differences for both males and females. However, the mean corpuscula r volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobi n concentration (MCHC) remained unchanged. Differences in platelet count ( PLT) and fibrinogen level (Fbg) were significant only for females. Any significant differ ences were not observed for both males and females for Prothrombin time (PT), activat ed partial thromboplastin time (ATPP), and the white blood cell count (WBC). Simi larly to humans, the ratio of lymphocytes to white blood cells increased with maturation from 16 to 30 weeks of age. However, its difference was statistically insignificant according to ANO VA analysis. From 12 to 30 weeks of age, the ratios of granulocytes (neutrophils, eosinophils, and basophils), lymphocytes, and monocytes to white blood cells were unchan ged, and differences were also insignificant. 4.2. Microarray gene expression profiles - Number of expressed genes To characterize the age-related gene expression in whole blood from mi niature pigs, RNA analysis was conducted on bloods sampled from fetal stage, 12, 20, an d 30 weeks subjects. Each RNA sample was analyzed by an Agilent #G2519F#20109 Porcine Gene Expression Microarray (44K) consisting of 43603 oligonucleotide probes. The change in the number of expressed genes to identify age-related c haracteristics was examined. Microarray gene expressions were divided into two groups; abse nt and j y g Sex n 12 weeks 16 weeks 20 weeks 24 weeks 30 weeks P 0.6 10.7 3.8 12.1 2.6 15.0 1.7 17.7 1.7 < 0. 001 Male 5 7.0 Female 5 6.9 0.5 7.9 3.2 10.1 2.6 13.5 2.1 16.0 2.6 < 0. 001

Values are mean SD.

P values were calculated using one-way factorial ANOVA.

Blood Cell An Overview of Studies in Hematology 104 present, using flag indicators given by the scanner. Background level wa s determined from spot intensities outside the gene probing area. Absent was assigned to the spots whose intensities were less than the background level, while the rests were marked as present. Then each gene was judged as either expressed or unexpressed based on the number of present events. We defined a certain gene as expressed when pre sent exceeds 75% out of replicated events. A threshold of 75% was chosen by considering experimental deviation. Table 2. Subject hematology results doi:10.1371/journal.pone.0019761.t002 j gy Hematological analysis Sex n 12 weeks 16 weeks 20 weeks 24 weeks 30 weeks P RBC, 10 4 72.6 858.0 97.7 894.8 55. 8 919.0 21. 0 866.2 24. 5 < .05 /L Male 5 742.7 Female 5 727.0 20.2 886.6 62.2 921.2 64. 5 901.4 46. 1 838.4 44. 2 < .001 HGB, g/dL Male 5 14. 9 1.6 16.4 1.2 17.3 0.6 18.3 0.4 17.7 0.3 < .00 1 Female 5 14. 9 0.4 17.5 0.8 18.0 0.9 18.4 1.1 17.5 0.6 < .001 HCT, % Male 5 50. 9 5.1 53.6 2.7 54.7 2.1 58.4 2.8 55.3 1.2 < .05 Female 5 49. 0 1.8 56.1 2.2 57.8 4.2 57.9 3.0 54.8 2.8 < .01 MCV, fL Male 5 65. 8 1.0 66.3 2.5 67.3 2.9 65.1 1.4 65.8 2.2 NS MCH, Pg Male 5 19. 8 0.5 20.0 1.1 20.1 0.9 20.5 0.6 20.6 0.8 NS CHC, % Male 5 30. 1 0.4 30.2 1.0 29.9 0.9 31.5 0.9 31.2 0.8 NS PLT, 10 4 /l Male 5 21. 3 0.4 31.6 10.8 18.1 4.4 25.0 8.6 24.9 5.1 NS Female 5 34. 5 2.0 24.8 5.5 19.0 5.0 24.8 8.9 19.7 5.7 < .05 PT, sec Male 5 13. 8 3.2 15.5 0.3 16.5 0.9 15.9 0.7 16.1 0.6 NS Female 5 - 15.8 1.1 16.1 0.5 16.4 0.5 16.0 0.7 NS APTT, sec Male 5 < 20 < 20 < 20 < 20 < 20 Female 5 < 20 < 20 < 20 < 20 < 20 Fbg, mg/dl Male 5 171.3 36.9 185.8 93.8 169.4 39. 4 158.6 9.0 147.8 34. 2 NS Female 5 - 160.2 19.4 145.2 16. 3 176.5 20. 1 123.3 27. 5 < .05 WBC, 10 2 /L Male 5 62. 0 18.7 86.6 12.7 78.8 24.7 79.6 24.0 71.8 13.2 NS Female 5 66. 0 23.4 74.0 13.7 78.0 18.7 72.4 10.4 61.8 11.3 NS Lymphocyte, % Male 5 34. 8 12.1 45.2 7.4 44.6 9.3 36.8 6.9 33.6 7.6 NS Neutrophil, % Male 5 55. 0 10.9 43.1 10.3 44.8 7.4 52.2 7.0 56.2 9.2 NS Eosinophil, % Male 5 3.8 2.2 3.1 1. 4 3. 0 1. 9 5. 0 2.7 4.6 1.7 NS Basophil, % Male 5 0.3 0.5 0.3 0. 4 0. 2 0. 4 0. 0 0.0 0.2 0.4 NS Monocyte, % Male 5 6.3 1.0 8.0 3. 2 7. 4 1. 5 6. 0 2.1 5.4 1.3 NS Biochemical variables for miniature pigs during the experiment are shown. Values are mean SD. RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit value; MCV, mean corpuscular volume;

MCH, mean corpuscular hemoglobin ; MCHC, mean corpuscular hemoglobin co ncentrat ion; PLT, blood platelet count; PT, prothrombin time; ATPP, activated partial thromboplast in time; Fbg, fibrinogen level; WBC, whit e blood cell count; and NS: not significant. P values were calculat ed usi ng one-way factorial ANOVA. Whole Blood RNA Analysis, Aging and Disease 105 The number of expressed genes was less in fetal stage and infancy period but inc reased with age, reaching a steady state of gene expression after 20 weeks of ag e (Figure 1). Expressed genes for male and female were analyzed by one-way factorial ANOVA. T hen TukeyKramers method was applied only to significant groups. Differences betwe en age groups (fetal stage, 12, 20, and 30 weeks of age) were significant for male , female, and mixed subjects of male and female. A Tukey-Kramers multiple comparisons test revealed that differences between fetal stage and other age groups were statistically significant (p<0.001) for both male and female. Also, differences were significant (P<0.05) between 12 and 30 weeks females. Figure 1. Number of genes expressed in whole blood of miniature pigs at differen t ages. In the graph, represents male and represents female. Values are means SD. doi:10.1371/journal.pone.0019761.g001 4.3. Microarray gene expression profiles Correlation of gene expression Variations in correlation coefficients among individuals of the same ag e and different age groups were evaluated. Pearson correlation coefficient was used for cor relation analysis. Correlation coefficients for a total of 31 microarrays were obtained i n normalized signals log-scale after excluding absent spots. A color-coded pairwise correlation matrix is shown in Figure 2. The color scale at the bottom indicates correlation strength. The average correlation coefficient within the same age group is shown in Figure 3. Variations in gene expression were greater for younger subjects, but it diminish ed with age while generating resembling expression patterns. Correlation coefficient within 30 weeks age group was slightly smaller than that within 20 weeks age group. However, this difference is smaller than other distant age groups. Significant differ ences were observed between any age groups according to an ANOVA analysis using Fishers Ztransform. The average correlation coefficient between different age groups is shown in Figure 4. Significant 0 5000 10000 15000 20000 25000

Fetal stage 12 20 30 N u m b e r o f e x p r e s s e d g e n e s Age (weeks) Male Female Blood Cell An Overview of Studies in Hematology 106 differences were observed except between fatal stage vs. 20 weeks and fatal stage v s. 30 weeks, and between 12 weeks vs. 20 weeks and 12 weeks vs. 30 weeks according to an ANOVA analysis using Fishers Z-transform (P < 0.05). These results sugg est that the variation in gene expression intensity within the same age was great in fetal stage and infancy period, but converged with age. Figure 2. Correlation matrix of age-related gene expression. This color-coded co rrelation matrix illustrates pairwise correlations between the levels of gene expression in indiv iduals. Probe sets with normalized signals (log-transformed and scaled) were used to calculate correlati ons between 31 arrays using Pearson correlation coefficient; signals flagged as absent were excluded. doi:10.1371/journal.pone.0019761.g002 4.4. Classification of genes depending on the status of age-related expression All spots on the microarray were divided into 16 categories as shown Table 3 aft er assigning 1 for expressed genes and 0 for unexpressed genes. Here, definitions of e xpressed and unexpressed are described in Materials and methods. Category 1 consists of a tot al of 6,763 genes expressed in the fetal stage, 12, 20, and 30 weeks of age. Catego ry 2 consists of a total of 7,564 genes expressed at 12, 20, and 30 weeks of age. Category 4 cons

ists of a total Whole Blood RNA Analysis, Aging and Disease 107 Figure 3. Age-related correlation coefficients within the same age groups. Corre lation coefficients were calculated between individuals within the same age groups. The bottom and t op of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiske rs denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. * p < 0.05, ** p < 0.01.

Figure 4. Age-related correlation coefficients between the different age groups. Correlation coefficients were calculated between the different age groups. The bottom and to p of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiske rs denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. * p < 0.01. 0.7 0.8 0.9 1.0 1.1 Fetal stage 12 weeks 20 weeks 30 weeks C o r r e l a t i o n c o e f f i c i e n t s 0.87 0.90 0.98 0.95 * **

0.04 0.04 0.00 0.03 **

** ** ** 0.4 0.5 0.6 0.7 0.8 0.9 1.0 F e t a l s t a g e v s . 1 2 w e e k s F e t a l s t a g e v s . 2 0 w e e k s F e t a

l s t a g e v s . 3 0 w e e k s 1 2 w e e k s v s . 2 0 w e e k s 1 2 w e e k s v s . 3 0 w e e k s 2

0 w e e k s v s . 3 0 w e e k s C o r r e l a t i o n c o e f f i c i e n t s * * Blood Cell An Overview of Studies in Hematology 108 of 3,547 genes expressed after 20 weeks of age. Category 8 consists of a total of 827 genes expressed after 30 weeks of age. Sum of the genes expressed at certa in age and those unexpressed (Categories 3, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 15) was 1,051. Its fraction was 5.6% of 18,701 genes (Categories 1, 2, 4, and 8) expressing constantl y once they appeared. Category 16 consists of genes unexpressed throughout the breeding period. Figure 5 shows the ratio of the genes belonging to each category. Table 3. Genes classified into 16 categories according to the status of age-rel

ated expression doi:10.1371/journal.pone.0019761.t005 To characterize gene expression in each category, TC Annotator List (Porcine ver sion 14.0 311-10) was downloaded from the TIGR gene Indices. TC Annotator List i ncludes the gene number and the GO terms. Out of 43,603 probes in the Agilent porcine microarray (#G2519F#20109), 6,019 genes bear GO annotation. Microarray cDNA probes were cla ssified by GO terms of biological processes. Out of all genes, fraction in Categories 1, 2 , 4, 8, and 16 were 31%, 20%, 8%, 2%, and 38% respectively. Then the difference in gene expression between all spots and those in 4 categories (Categories 1, 2, 4, and 8) was examined. GO groups dominantly expres sed in Category 1 relates to mitosis (GO:0000070, GO:0000022, GO:0007052, and GO:0007100) a nd to immune Category Fetal stage 12 weeks 20 weeks 30 weeks Number of genes Definition 1 1 1 1 1 6763 genes expressed from fetal stage to 30 weeks 2 0 1 1 1 7564 genes expressed from 12 to 30 weeks 3 1 0 1 1 49 4 0 0 1 1 3547 genes expressed from 20 to 30 weeks 5 1 1 0 1 14 6 0 1 0 1 80 7 1 0 0 1 7 8 0 0 0 1 827 genes expressed at 30 weeks 9 1 1 1 0 73 10 0 1 1 0 124 11 1 0 1 0 29 12 0 0 1 0 428 genes expressed at 20 weeks 13 1 1 0 0 16 14 0 1 0 0 147 genes expressed at 12 weeks 15 1 0 0 0 84 genes expressed in fetal stage 16 0 0 0 0 23851 genes not expressed from fetal stage to 30 weeks Depending on the status of expression, all spots on the microarray can be divide d into 16 categories. Here, 1 represents an expressed gene and 0 represents an unexpressed gene. Whole Blood RNA Analysis, Aging and Disease 109 (GO:0043161, GO:0045059, GO:0019886), while those highly expressed in Category 2 related to cellular defense and regulation. Figure 5. Ratios of categories for groups of the same age. The ratios of the gen

es in each category were calculated for groups in the fetal stage and at 12, 20, and 30 weeks of age. Cat egories are defined in Table 3. doi:10.1371/journal.pone.0019761.g003 4.5. Age-related changes in gene expression levels for the immune system Expression intensity of immunity gene was examined. Antigen processing and prese ntation (GO:0019882) and T cell selection (GO0045058) include the major histocompatibili ty complex (MHC) genes. By presenting antigens, MHC is involved in elimination of bacterial or viral pathogen, rejection of cancer cells, and rejective response on organ t ransplantation. Also MHC is indispensable in the immune system. Swine leukocyte antigens (SLA) are im portant immunogens for humoral responses and important mediators of the cellula r immune responses through both direct and indirect presentation of peptides to T-cells [12]. SLA includes 6 of classical class I genes (SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, and SL A-8) and 8 of classical class II genes (SLA-DMA, SLA-DMB, SLA-DOA, SLA-DOB1, SLA-DQA, SLADQB1, SLA-DRA, and SLA-DRB1) [13-14]. SLA class II lacks DPA1, DPB1, DRB3, DRB4, and DRB 5 in humans. On the Agilent porcine microarray, all of SLA genes except DOA are mounted on 28 spots. Among these, 11 SLA genes fell under Category 1 , 1 fell under Category 2, and 1 fell under Category 8. Expression of SLA classical class I and class II genes are shown in Figure 6A and 6B, respectively. Both genes expressed in fatal stage , 12, 20, and 30 weeks in an increased manner by age. The Agilent porcine microarray had 7 probes with 7 types of interferon and 7 pro bes for 4 types of interferon receptors. All of 7 interferon genes fell under C ategory 16. Normally these genes remain unexpressed but expressed upon necessity. In contras t, 1 type of interferon receptor gene fell under Category 1, 3 fell under Category 2, and wer e expressed Blood Cell An Overview of Studies in Hematology 110 until 12 weeks of age. Their signal intensities stayed at constant le vels after 12 weeks (Figure 6C). Toll-like receptors (TLRs) are the principal pattern recognition recepto rs. With this innate immunity, the first immune response is mediated into reserved foreign patterns on recognition. TLRs recognize reserved molecular patterns, start rapid response to protect the host upon infection, and produce signals, such as cytokines and co-stimulatory m olecules to activate the adaptive immune system [15-16]. Regulation of the TLR sig naling cascade is important for inflammatory responses, innate host defense, and adaptive immune r esponses

[17-18]. Most mammalian species are estimated to have between 10 and 15 types of TLRs. The Agilent porcine microarray has 10 types of TLRs probes. Among the se TLRs, 5 of TLR genes fell under Category 2 (expressed until 12 weeks of age), 1 und er Category 8, and 4 under Category 16. Their signal intensities remained constant after 12 weeks of age (Figure 6D). Figure 6. Signal intensity of major histocompatibility complex (MHC) genes. (A) Swine leukocyte antigens (SLA) classical class I genes. (B) Swine leukocyte antigens (SLA) class ical class II genes. (C) Interferon receptor genes. (D) Toll-like receptor (TLR) genes. Signal intens ities were normalized using quantile normalization and log-transformed after excluded signals flagged as absent. The category numbers are shown in graph legends. Genes in Categories 1, 2, and 4 are shown in the graph. doi:10.1371/journal.pone.0019761.g005 Whole Blood RNA Analysis, Aging and Disease 111 5. Gene expression profiles change related to hyperlipidemia To examine the usage of whole blood RNA analysis for the early diagn osis of the disease, we showed transitions in dietary induced hyperlipidemia gene expression profiles of whole blood RNA in miniature pigs. Hyperlipidemia is well recognized as a risk factor for cardiovascular disease (C VD). As diet represents the most important determinant of hyperlipidemia, dietary animal mode ls can be useful for the study of CVD progression [19]. High-fat, high-cholestero l, and high-sugar diets have been shown to induce hyperlipidemia, obesity, and insulin resistance in humans and rodents [20-22]. Dietary-induced hyperlipidemia pig models have also been es tablished [23-29]. A high-fat and high-cholesterol diet (HFCD) as a typical dietary treat ment were used for dietary-induced hyperlipidemia miniature pig models, by using specific pathogenfree (SPF) Clawn miniature pigs. Eight 12-week-old, male Clawn miniature pigs were housed individually in cages o f 1.5 m 2 at the breeders specific pathogen-free (SPF) facility (Japan Farm Co., Ltd, Kagoshima, Japan) for 27 weeks. Body weights at the beginning of the experiment were 5.1 (2.6) kg (mean (standard deviation; SD)). During this period, 5 pigs were fed with 450 g/day standard dry feed (Kodakara73, Marubeni Nisshin Feed Co., Ltd., Tokyo Japan), and had unlimited access to water (control group). Five pigs were fed a highfat, high-cholesterol diet containing 15% lard and 2% cholesterol (HFCD group).

Almost no changes were observed in fasting plasma triglyceride levels. Fasting p lasma total cholesterol concentrations had increased in the HFCD group by week 5 of the feeding period (P<0.001) and were maintained between 350 and 1150 mg/dL from weeks 1027. Fasting plasma high-density lipoprotein cholesterol (HDL-C) concentrations increased and showed significant differences (P < 0.001) from weeks 1027. Fasting pla sma low-density lipoprotein cholesterol (LDL-C) concentrations also increased and showed significant differences from weeks 527. Fasting plasma glucose concentrations remained unchan ged. 5.1. Gene expression profiles of dietary-induced hyperlipidemia for whole blood RNA RNA analyses were conducted on blood samples obtained at weeks 10, 19 , and 27 of the feeding periods to characterize the dietary effects on gene expression profiles in whole blood and white blood cells of miniature pigs. Each RNA sample was analyzed by a porcine gene expression microarray consisting of 43603 oligonucleotide probes. Variation in correlation coefficients among individuals on the same diet and bet ween different diet groups was evaluated. Pearson correlation coefficients were used f or the correlation analysis. Correlation coefficients for 23 microarrays in total were obt ained for a normalized signals log-scale after excluding absent spots, definition of absent were described in Materials and Methods. A color-coded pairwise correlation matrix is displayed in Figure 7. Blood Cell An Overview of Studies in Hematology 112 The correlation coefficients of whole blood expression profiles within the same diet groups were 0.97 (0.01) (mean (standard deviation; SD)), and 0.94 (0.05) for the control, HFCD whole blood at 10 weeks, 0.94 (0.03), and 0.93 (0.06) at 19 weeks, and 0.95 (0.02), and 0.95 (0.03) at 27 weeks, respectively. Using Fishers Z-transformation to normalize the correlation distributions, no significant differences in correlation coefficients amo ng dietary groups were observed at any period during the treatments. This indicates unif ormity of dietaryinduced hyperlipidemia for our protocols. The whole blood correlation coefficients among the different diet groups were 0. 95 (0.04) for control vs. HFCD at 10 weeks, 0.93 (0.03) at 19 weeks, and 0.95 (0. 03) at 27 weeks, respectively. 5.2. Assigning known functions to gene expression - Gene ontology annotation Up- and down-regulated genes were identified and classified these accor ding to function using information from the Gene Ontology (GO) Database to understand t he observed differences in whole blood gene expression profiles for the different dietary groups. Top-

ranked genes with fold changes in expression greater than 2.0 (p < 0.05) and les s than 0.5 (p < 0.05) were selected at 10, 19, and 27 weeks. As a result, the GO categories of many genes up-regulated at the end of the 19-week dietary period were related to nucleotide binding (GO: 0000166, GO: GO: 0005524, 0005525, GO: 0017076, GO: 0019001, GO: 00032553, GO: 00032555, GO: 0032561), and catabolic processes (GO: 0009057, GO: 00199 41, GO: 0030163, GO: 0043632, GO: 0044257, GO: 0044265,). Many genes down-regulated after 27 week s were in the GO categories related to biological adhesion (GO: 0007155, GO: 0022610). 5.3. Effect of white blood cells on whole blood gene expression profiles in dietary-induced hyperlipidemia Microarray analyses were conducted from white blood cells at the end of the diet ary period to evaluate the effect of white blood cells on whole blood gene expression profi les (Figure 8). The correlation coefficients of white blood cells expression profiles within the same dietary groups were 0.94 (0.05) and 0.95 (0.03) for the control and HFCD gro ups at 27 weeks. The white blood cells correlation coefficients was 0.94 (0.04) between cont rol and HFCD. The average correlation coefficients between whole blood and white blood cells were 0.83 (0.04) and 0.79 (0.05) for control and HFCD. Using Fishers Z-transformation to normalize the correlation distributions, no significant differences in correlation coefficient s of white blood cells were observed between control and HFCD groups. Up- and down-regulated genes were identified and classified these accor ding to function using information from the Gene Ontology (GO) Database to understand t he observed differences in white blood cells gene expression profiles for the different diet ary groups, as the same as whole blood gene expression profiles. Top-ranked genes wit h fold changes in expression greater than 2.0 (p < 0.05) and less than 0.5 (p < 0.05) were selecte d at 27 weeks. As a result, many genes down-regulated related oxidation-reduction proce ss (GO:0055114) and keg pathways of steroid biosynthesis. Whole Blood RNA Analysis, Aging and Disease 113 Figure 7. Correlation matrix of dietary-related gene expression profiles of whol e blood. This colorcoded correlation matrix illustrates pairwise correlations between the levels of gene expression in individuals. Probe sets with normalized signals (log-transformed and scaled) wer e used to calculate correlations between 23 arrays using Pearson correlation coefficient; signals fl agged as absent were excluded. The color scale at the bottom indicates the strengths of the correlati ons.

Figure 8. Correlation matrix of dietary-related gene expression profiles of whol e blood and white blood cells. This color-coded correlation matrix illustrates pairwise correlatio ns between the levels of gene expression in individual at feeding period at week 27. Probe sets with norm alized signals (logtransformed and scaled) were used to calculate correlations between 15 arrays us ing Pearson correlation coefficient; signals flagged as absent were excluded. The color scale at the botto m indicates the strengths of the correlations. Blood Cell An Overview of Studies in Hematology 114 6. Gene expression profiles change with other stresses Furthermore, a possibility was shown that whole blood RNA analysis is applicable to evaluation of physiological state. The degree of stress can be comparable according to the numbers of u p-regulated and down-regulated genes, even if the stress is different in quality from the others . Sodium azide was given orally to the miniature pigs over 20 weeks. T here were no significant changes of hematological and biochemical properties for admi nistrated dose of 300g/kg, one hundredth of LD50. On the other hand, gene expression pro files were obviously changed. Anesthesia group showed a slight degree, but the on e week fasting group showed a significant difference. This can be clearly noticed whe n the contents of stress is classified by the function of up-regulated and down-regulated genes. C onsequently, grade of the stress can be estimated according to the expression state of genes. Stresses P<0.05, Fold change>2 total up regulation down regulation sodium azide 300g/kg ; LD50 1/100 893 339 554 blood removal 150ml after 6 hours 1747 227 1520 Fasting a week 3136 1840 1296 anesthesia after 6 hours 160 87 73 non treatment (blood removal 20ml) 73 14 59 Table 4. Summery of gene expression condition of several types of stress Number of genes 7. Effects of white blood cells on whole blood gene expression profiles Whole blood contains a variety of cell types as red blood cells, gra nulocytes, lymphocytes, and platelets. Most of the nucleated cells in blood are white blood cells such a s neutrophils, T-cells, B-cells, and monocytes. The number of white blood cells in h umans is known to decrease steadily from infancy to adulthood, and its composition (i.e. lymphocytes, granulocytes) also changes with age [30]. In study of the gene expres sion profiles change related to aging, hematological data of the fetal stage was unavailable because

the amount of collected blood was insufficient for the analysis. From 12 to 30 week s of age, ANOVA analysis indicated no significant differences in the fractions of lymph ocytes, neutrophils, eosinophils, basophils, and monocytes. In addition, these compositions were almo st equal to those in human adults. The above result suggests that the gene expression profil e change of age-related whole blood RNA is not due to the composition of white b lood cell subpopulations. The intraclass correlation between Staphylococcus enterotoxin B-stimulated and unstimulated blood from healthy subjects was significantly higher in le ukocyte-derived samples the in whole blood, suggesting that the method of RNA isolation from who le blood Whole Blood RNA Analysis, Aging and Disease 115 can be a critical step in blood RNA assay [31]. Although PBMCs do not contain ne utrophils, eosinophils, basophils, nor platelets, Min et al. reported highly corre lated results (r 2 = 0.85) for 8,273 genes expressed between the whole blood RNA, by using the PAX gene Blood RNA system, and peripheral blood mononuclear cell (PBMC) RNA samples i solated from healthy volunteers by using a Ficoll-Paque gradient and TRI Reagent (S IGMA) [32]. Other workers conducted a large scale genome-wide expression analysis of whit e blood cells subpopulations. This study indicates that correlation coefficients for T -cells and monocytes among different healthy subjects were 0.98 0.01 and 0.97 0.01, respectively. However, for the same subjects (n=5), correlation coefficients between T-cells and m onocytes was 0.88 0.01, indicating varied correlation between white blood cells subpopu lations. In addition, gene expression analysis were showed a varying dependence on the isolation method such as PAXgene, Buffy coat, and lysis. The correlation coeffic ients between isolation methods were 0.89 0.04, 0.91 0.04, 0.96 0.06, for PAXgene vs. lysis , PAXgene vs. Buffy coat, and Buffy coat vs. lysis, respectively [33]. In order to ensure the reliability for to clinical use of whole blood RNA diagnosis, the development of standard method and measurement standards needs to be sought. The Gene Ontology (GO) Database was used to categorize gene expression profiles functionally to conduct the effects of white blood cells on whole blood gene exp ression profiles in our study of hyperlipidemia. As a result, the GO term, related to white blood cell function (GO: 0006954, 0007166), had a high correlation coefficient. In contrast, GO term

s related to the repair of damaged organs, including translation (GO: 0006412), positive regulati on of growth rate (GO: 0040010), and growth (GO: 004007), showed low correlation co efficients. We, therefore, conclude that the difference in the gene expression profiles between the whole blood and white blood cells are not only caused by differences in experimental protoco ls, but also by differences in RNA origin [34]. 8. Conclusion Whole blood RNA is easy to handle compared to isolated white blood cell RNA and can be used for health and disease monitoring and animal control. In addition , whole blood is a heterogeneous mixture of subpopulation cells. Once a great change occur s in composition and expressing condition of subpopulations, their associated change will be reflected on whole blood RNA. Whole blood microarray analyses were conducted to evaluate variations o f correlation among individuals and ages using specific pathogen-free (SPF) Clawn min iature pigs. The characteristics of age-related gene expression by taking into account o f change in the number of expressed genes by age and similarities of gene expression intensity between individuals were identified. As a result, the number of expressed gene s was less in fetal stage and infancy period but increased with age, reaching a steady state of gene expression after 20 weeks of age. Variation in gene expression intensity within the same ag e was great in fetal stage and infancy period, but converged with age. The variation between 20 and 30 Blood Cell An Overview of Studies in Hematology 116 weeks of age was comparable to that among 30 weeks individuals. These results indicate that uniformity of laboratory animals is expected for miniature pigs after 20 we eks of age. In dietary-induced hyperlipidemia study, feeding treatments commenced when the p igs were 12 weeks old, RNA analysis was conducted on whole blood sampled after 10, 19, an d 27 weeks of the feeding period. Variation in whole blood gene expression intens ity among individuals within the HFCD group was in the same range as that of the controls at any perio d, indicating uniformity of dietary-induced hyperlipidemia expression profiles in minia ture pigs. Dietaryinduced transitions of gene expression profiles for genes bearing GO t erms were examined. Major changes included an induction of proteins involved in catabolic processes and protein metabolism after a 19-week dietary period, and a reduced expression of proteins involved in steroid metabolism and lipid biosynthesis after a 27-week dietary period. In several kinds of stress study, the degree (extent) of stress can be comparabl

e according to the gene number of up-regulate, or down-regulate, even if the stress is different in kind from the others. A possibility was shown that whole blood RNA analysis is applicable t o evaluation of physiological state. By considering variation in gene expression profile s of miniature pigs, whole blood RNA analyses can be used in practical applications. The b lood RNA diagnostics under development may eventually be useful for monitoring human heal th. Author details Junko Takahashi * and Akiko Takatsu National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan Masaki Misawa Human Technology Research Institute, National Institute of Advanced Industrial S cience and Technology, Tsukuba, Ibaraki, Japan Hitoshi Iwahashi Health Research Institute, National Institute of Advanced Industrial Science and Technology, Takamatsu, Kagawa, Japan Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan 9. References [1] Williams-Devane CR, Wolf MA, Richard AM (2009) Toward a public to xicogenomics capability for supporting predictive toxicology: survey of current resou rces and chemical indexing of experiments in GEO and ArrayExpress. Toxicol Sci 109(2):358 -371. [2] Pennie W, Pettit SD, Lord PG (2004) Toxicogenomics in risk assessment: an o verview of an HESI collaborative research program. Environ Health Perspect 112(4):417-419. * Corresponding Author Whole Blood RNA Analysis, Aging and Disease 117 [3] Tong W, Cao X, Harris S, Sun H, Fang H, et al. (2003) ArrayTra ck--supporting toxicogenomic research at the U.S. Food and Drug Administration National Center for Toxicological Research. Environ Health Perspect 111(15):1819-182 [4] Takahashi J, Misawa M, Iwahashi I (2011) Oligonucleotide microarray analysis of agerelated gene expression profiles in miniature pigs. PLoS ONE 6(5):e19761. [5] Lunney JK (2000) Advances in Swine Biomedical Model Genomics. Int J Biol Sci 3(3):179184. [6] Simon GA, Maibach HI (2000) The Pig as an Experimental Animal Mo del of Percutaneous Permeation in Man: Qualitative and Quantitative Observations An Overview. Skin Pharmacol Appl Skin Physiol 13(5):229-234. [7] Vodicka P, Smetana K Jr, Dvornkov B, Emerick T, Xu YZ, et al. (2005) The mini

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microarray analysis of dietary-induced hyperlipidemia gene expression pro files in miniature pigs. PLoS ONE 7(5):e37581. Chapter 7

2012 Hayashi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions Osamu Hayashi Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48322 1. Introduction A limited number of pluripotent stem cells are mainly located in the bone marrow , and give rise to all blood cell 1ineages. Because of their relatively short lifespan, cir culating cells must be continually replaced in living body throughout the life. The task performed by hematopoietic stem cells is shared in two main features, that is, the capacity o f regeneration which prevents depletion of the cells and the ability of preservation of blood homeostasis. The mechanisms behind the critical choice between lineage-commitment and maintenance of the stem-cell pool involve a number of complex interactions between hematopoietic progenitor cells at different stages of maturation, stromal cells and their extracellular matrix, as well as a variety of stimulatory or inhibitory cytokines p rovided by the microenvironment. Hematopoietic growth factors were first identified in the 1960s as sol uble agents produced in spleen, uterus or lung, and found to maintain the formation of di fferentiated colonies from hematopoietic progenitor cells in semisolid culture systems. Hence they were named colony-stimulating factors, CSFs (Schneider and Dy, 1999). Most of thes e molecules have been purified and their genes have been sequenced. They are currently available in recombinant form and have been used with success in clinical trials. Hematopoietic growth factors or CSFs can be divided into two categories, accordi ng to their target cell specificity (Figure 1). One group comprises the factors whose activi ty is relatively restricted to particular cell types, such as macrophage colony-stimulating facto r (M-CSF) for macrophages, granulocyte colony-stimulating factor (G-CSF) for neutrophils , interleukin-5 (IL-5) for eosinophils and B cells, and thrombopoietin (Tpo) for megak

aryocytes and erythropoietin (Epo) for the erythroid lineage. The second category of growth factors has a Blood Cell An Overview of Studies in Hematology 120 relatively wide spectrum of activities, such as IL-3 and granulocyte-ma crophage colonystimulating factor (GM-CSF). The factors target a heterogeneous populati on of cells, including both primitive and lineage-committed progenitors. Action of these two molecules can be modulated by a number of cytokines which are not essentially growth factors. Among these, IL-1, IL-6, IL-9, IL-1l and leukemia inhibitory factor (L IF) are involved. An interleukin 6 class cytokine or stem cell factor (SCF) plays a partic ularly important role in the amplification of early stem cell commitment. IL-7 is also noteworthy in this context, with respect to its crucial role in lymphopoiesis, as evidenced by the strong lymphop enia in IL7deficient mice. Hematopoiesis can be also regulated negatively by a he terogeneous set of molecules, such as interferon, tumor necrosis factor-alpha (TNF-), transf orming growth factor beta (TGF) and compounds like prostaglandins, ferritin and lactoferrin. The precise function of cytokines during constitutive hematopoiesis in a healthy organism is still unclear, although much evidence has been accumulated from the st udy using genetically modified mice. The purpose of hematopoiesis, however, is no t only the maintenance of homeostasis, but also a rapid and controlled response t o stress situations. The immune response induced by infection, the number of circulating white blood cells can be remarkably increased (Schneider and Dy, 1999). In the process, the cytokines generated by sensitized lymphocytes and activated cells of the immune system pla y a crucial role in the recruitment and the differentiation of hematopoietic cells. Figure 1. Simplified haematopoietic differentiation scheme and cytokines (modif ied from Elk and Dy, 1999) Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 121 In the chapter, we focus on relations and networks of cytokines, induced by inge sting in-vivo study of Spirulina and by in-vitro cultured cells, to differentiation of hematop oietic cells and preservation of immune functions, and discuss the possibility of their medicinal application for sustaining a healthy state. 2. Spirulina Spirulina platensis is a helicoidal filamentous blue-green alga (cyanoba cterium) and has a history of being used as food for over a thousand years, and has be en commercially produced for more than 40 years as a food supplement (Ciferri, 1983; G

ershwin and Belay, 2008). Spirulina platensis is prokaryote and belongs to the class Cyan ophyceae, or Cyanobacteria. In its commercial use, the common name, Spirulina, refer s to the cyanobacterium, Arthrospira platensis, and is a whole product of biolog ical origin. In its taxonomic use, Spirulina is a name used to describe mainly two specie s of Cyanobacteria, Arthrospira platensis and A. maxima, which are commonly used as food, dietary su pplement, and feed supplement (Vadiraja et al., 1998) . These and other Arthros pira species forming helical trichomes were once combined and classified into a single genu s, Spirulina (Geitler, 1932). Before Geitler, on the basis of the presence of septa or division in the trichomes, the two genera were placed separately, that is, the Spirulina species being wi thout septa and the Arthrospira species with septa. Recent morphological, physiological, and biochemical studies have shown that these two genera are distinctively different and that the edible forms commonly referred to as Spirulina platensis have little in common with other much smaller species. This distinction has been also based on results from the complete seque nce of the 16S ribosomal RNA gene and the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes determined for two Arthrospira strains and one Spirulina strain (Nelissen et al., 1992) showing that the two Arthrospira strains formed a close cluster distant from the Spirulina strain. Blue green algae Spirulina platensis (Arthrospira platensis) is gaining more and more attention as a nutraceutical and source of potential pharmaceuticals. Spirulina is known to h ave nutritional advantages of high-quality protein contents and other components such as vitamin s; minerals, and essential fatty acids, including linolenic acid, and c rotene (Belay et al., 1993) , and has been approved its safety in the report from United Nations Internation al Development Organization, UNIDO (Chamorro-Cevallos, 1980). Moreover, sulfated polysacc harides, called calcium-spirulan (Ca-Sp) and isolated from a hot-water extract of Spiru lina, exhibit immunomodulatory activity and inhibit metastasis of melanoma cells to the lungs (Mishima et al., 1998) , and can also inhibit virus entry (Hayashi et al., 1996). Immolina, a high-molecularweight polysaccharide fraction of Spirulina, promotes chemokine expressio n in human monocytic THP-1 cells (Grzanna et al., 2006). Spirulina contains phycoc yanin (CPC; Cphycocyanin), a blue, 270-kDa photosynthetic pigment protein, which acco unts for approximately 15% of the dry weight of Spirulina (Ciferri, 1983). Recently, more attention has been given to the study of the therapeutic effects of Spirulina. In

addition to its effectiveness in reducing hyperlipidemia, diabetes, and high blood pressure in humans and animals, anti-viral and anti-cancer effects of orally ad ministered S. platensis involving immune functions have also been reported (Belay, 2002). Blood Cell An Overview of Studies in Hematology 122 3. Structure of phycocyanin The biological and pharmacological properties of Spirulina were attribut ed mainly to Cphycocyanin (CPC). CPC is a major light-harvesting or photosynthetic pi gment protein present in the antenna rods of Spirulina platensis. S. platensis also contains allophycocyanin (APC) as a minor component present at the core of the antenna rods. CPC and APC including phycoerythrin (PE) are the principal classes of phycobiliprote ins which form supramolecular complexes known as phycobilisomes assemblies in cyanobacte ria (Figure. 2a). In phycobiliproteins, a linear tetrapyrrole (bilin) as the chrom ophore is covalently attached to the apoprotein by thioether bonds to cysteine residues. CPC molecule is composed of two kinds of subunits, and su units to form trimeric ag gregation 33 (Figures. 2b and 2c). Padyana et al. (2001) solved the crystal structure of CPC by molecular replacement technique (Figures. 2d and 2e). The and su unit polypeptides exhib it high affinity for one another and associate into () monomers, which in turn aggregate into ()3 trimers and ()6 hex mers (Figure. 2f). The medicinal and pharmacological properties of CPC have been reported earlier ( Romay et al., 1998). Recent studies have demonstrated the antioxidant (Vadiraja et al., 1998; Wu and Annie Ho, 2008), anti-inflammatory (Reddy et al., 2000), and hepatoprot ective properties (Vadiraja et al., 1998), in addition to anticancer, anti-allergic, immune-enhanc ing (Hayashi et al., 2008), blood-vessel-relaxing and blood-lipid-lowering effects of CPC (Gershwin and Belay, 2008).

Rod Rod Cor a. b. c. Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 123

A phycobilisome (a) has six antenna rods with a three-cylinder core of allophyco cyanin, APC (circles), two of the core

cylinders lie on the thylakoid membrane, while the third one does not. Each rod has four hexameric disk-like structures, two of phycoerythrin, PE (red), and two of phycocyanin, CPC (blue) (MacColl, 2004). CPC consists of and su unit polypeptides to form trimeric aggregation 33 and nine phycocyanobilin moieties as a chromophore shown in closed circles (b). c shows chemical structure of phycocyanobilin (Li et al., 2006). d and e show ribbon representation of CPC su unit and CPC su unit, respectively, with chromophores s hown in ball and stick representation (Padyana et al., 2001). f shows coil representation of the two ()6 hex mers in the crystal asymmetric unit, and the box drawn at the center highlights the close proximity of phycocya nobilins at the position 155 on each su unit in the region between the adjacent hexamers (Padyana et al., 2001). Figure 2. Schematic representation of one type of phycobilisome (a), and various representations of Cphycocyanin (b - f). Preparation of phycocyanin solution in the experiments Phycocyanin was extracted from spray-dried Spirulina platensis with 50 mM sodium-phosphate buffer (pH 6.0). The crude extract was partially purified by DE-52 ion-exchange chromatography. The eluate was dialyzed against distilled water and lyophilized. Phycocyanin contents o f the resultant powder were over 80%, and the recovery from the crude extract was approximately 6%. The d. e. f. Blood Cell An Overview of Studies in Hematology 124 phycocyanin powder was dissolved in distilled water to a concentration of 0.05%, centrifuged in a refrigerated machine for 10 min at 1,500 g, and the supernatant was sterilized by filtration through a 0.20-m-pore filter (Hayashi et al., 2006).

a. CPC molecule composed of two kinds of subunits( and su units) to form 33 and ni ne phycocyanobilin moieties as a chromophore (closed circles). b. Chemical structure of phycocyanob ilin. Phycocyanobilin was covalently bound to polypeptide chain of CPC by ways of thioetherlinkage to cysteine residu es, one on the ch in and two on the ch in (Li et al., 2006). Figure 3. Structure of CPC ()3 trimer and phycocyanobilin Figure 4. Ribbon representation of (a) CPC su unit (b) CPC su unit. Chromophores are shown in ball and stick representation (Padyana et al., 2001). Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 125 The figure illustrates the arrangement of chromophores at various locations with in the hexamers. The box drawn at the center highlights the close proximity of phycocyanobilins at the position 155 on each su unit in the region between the adjacent hexamers (Padyana et al., 2001).

Figure 5. Coil representation of the two hexamers in the crystal asymmetric unit , a view through the approximate central axis of hexamers. 4. Enhancement of proliferation and differentiation of bone marrow cells stimulated with Spirulina and its extracts Immunomodulation properties of Spirulina have been widely studied in ch ickens, prawns and fish, other animals, and humans. Generally, Spirulina and its extracts, such as hot-water extracts and phycocyanin, tended to enhance immune functions including mucosal o r innate immunity through macrophage and secretions of the related cytokines (Be lay, 2002; Hirahashi et al., 2002; Nemoto-Kawamura et al., 2004). Mao et al. (Ma o et al., 2000) demonstrated that Spirulina stimulated the secretion of IL-1 and IFN- in human peripheral blood mononuclear cells (PBMC) examined to nearly 2.0 and 3 .3 times basal levels, respectively, and suggested that Spirulina helped balance the pr oduction of Th1 and Th2 cytokine stimulation. Phycocyanin, a characteristic photosynthesis pigment p rotein and an antioxidant in Spirulina, has been known to promote the growth of a human mye loid cell line, RPMI 8226 (Shinohara et al., 1988). Liu et al. (2000) reported that phycoc yanin inhibited growth of human leukemia K562 cells and enhanced the arrest of the c ell growth at G1 phase, suggesting enhancement of differentiation of the cells. We have reported that Spirulina and its extracts enhanced immune respo nses in mice, mainly through increased production of interleukin-1 (IL-1) in macrophages (Haya shi et al., 1994; Hayashi et al., 1998). In the mice which ingested phycocyanin for 6 week s, a marked increase of OVA antigen-specific IgA, as well as total IgA level was observed in the Peyers patches, mesenteric lymph nodes and intestinal mucosa, as well as in the spleen cells Blood Cell An Overview of Studies in Hematology 126 (Nemoto-Kawamura et al., 2004). These findings suggest that Spirulina o r its components such as phycocyanin, affects immune functions by promoting immune compe tent-cell proliferation or differentiation in lymphoid organs. We first investigated the effects of Spirulina and its extracts on proliferation of hematopoietic cells of mice and induction of colony-forming activity. Colony-formation of bone marrow cells in in-vitro study Spirulina extracts such as a hot-water extract (SpHW), phycocyanin (Phy c), and cell-wall fraction (SpCW) recovered from Spirulina treated with 0.1 % sodium dod ecyl sulfate to remove cytoplasmic material were used in this study. Culture supernatants of spl een (SP), Peyers patch (PP), and peritoneal-exudated (PE) cells cultured with 20 g /mL of the Spirulina extracts significantly enhanced proliferation of bone marrow c

ells (Figure 3). Each of the Spirulina extracts, SpHW, Phyc, and SpCW, itself, also di rectly enhanced proliferation of bone marrow cells in the concentration of 100 g/mL of culture medium. In addition to that, colony- and cluster-formations of the bone marrow cells sup plemented with culture supernatants of the spleen cells stimulated with Spirulina extracts, 50400 g/mL, were measured by soft agar method. The supernatants of cells cultured with Phyc and SpCW significantly increased the colony- and cluster-formations of the bone marrow cells in comparison to that of control or of the smallest concentrati on of each extract (Figure 4a). Culture supernatants of PE cells, which consisted of macr ophages and lymphocytes in a ratio of about 50 % each and a small ratio of mast cells and ne utrophils, also enhanced colony- and cluster-formations (Figure 4b). The numbers of these c olonies, however, were almost the same as that by each other culture supernata nt. Furthermore, Both granulocyte macrophage-colony stimulating factor (GM-CSF) and interl eukin 3 (IL-3) contents in the culture supernatant or the serum as colony-form ing activities were measured by commercially supplied ELISA assay kits. High amounts of GM -CSF or IL-3 were detected in the culture supernatants of the spleen and peritoneal -exudates cells stimulated with the Spirulina extracts, especially those with SpCW (Table 1). Th e amounts of IL-3 in the culture supernatants of the cells stimulated with SpHW and Phyc were relatively high, although colony formation by the supernatant was not so high. Culture supernatant of the cells stimulated with SpCW contained high amounts o f GM-CSF but not of IL-3. stimulated with Colonies/well GM-CSF pg/mL of CS IL-3 pg/mL of CS SP PE SP PE SP PE 0.7 2.0 2.3 <4 <4 47.3 4.0 <3 Control 0.5 SpHW 2.8 2.6 33.0 7.1 <4 7.1 76.7 8.0 10.7 Phycocyanin 14.0 5.9 37.3 9.3 9.2 0.7 4.3 94.7 10.8 11.0 SpCW 28.2 5.5 32.2 4.6 1,206 333 104.7 481.7 144.4 <3 Table 1. GM-CSF and IL-3 contents in the culture supernatants (SC) of the splee n (SP)and the peritoneal-exudates (PE) cells stimulated with Spirulina extracts (values are me an SD, N = 3) Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 127

Figure 6. Bone marrow-cell proliferation by Spirulina extracts, SpHW, Phy, and S pCW, and by culture supernatant (CS) of lymphoid organ, spleen (SP) and Peyer s patch (PP), and peri toneal-exudates (PE) cells stimulated with the Spirulina extracts (Hayashi et al., 2006) (values are mean SD, n = 6) Figure 7. Bone marrow-cell colony and cluster formation in soft agar assay with the culture supernatant of the spleen cells (a) and peritoneal-exudates cells (b) stimulated with Spirul ina extracts. Spleen cells were stimulated with 0.5, 1.0, 2.0 and 4.0 mg Spirulina extract/mL. Peritonealexudates cells were stimulated with 2.0 mg Spirulina extract /mL (Hayashi et al., 2006) (values are mean SD, n = 3). M e d i u m S p H W P h y c S p C W C o n t . S p H W P h y c S p C W C o n t . S p H W

P h y c S p C W C o n t . S p H W P h y c 0 2000 4000 6000 8000 10000 12000 F l u o r e s c e n c e i n t e s i t y SP cs PP cs PE cs C o n t . P W M S p

H W 0 . 5 S p H W 1 . 0 S p H W 2 . 0 S p H W 4 . 0 P h y c 0 . 5 P h y c 1 . 0 P h y c 2 . 0 S p C W 1

. 0 S p C W 2 . 0 S p C W 4 . 0 0 5 10 15 20 25 30 35 40 N u m b e r o f c o l o n i e s & c l u s t e r s p e r

w e l l extracts as a stimulant (mg mL -1 ) Colony Cluster * * * * * * A. Spirulina Cont. LPS SpHW Phyc SpCW 0 10 20 30 40 50 N u m b e r o f c o l o n i e s & c l u s t e r s p e r w

e l l PE cell culture supernatant Colony Cluster B. Blood Cell An Overview of Studies in Hematology 128 Colony-formation activity in the mice fed Spirulina in in-vivo study As a preliminary experiment for in-vivo study, we measured next colony forming a ctivity in the mice fed with the Spirulina extracts, SpHW, Phyc and SpCW, for 5 consecutive days or in the mice treated with an intra-peritoneal single injection of the extracts. The sera from the mice which ingested Phyc or SpCW (1 mg/0.2 mL) for 5 consecuti ve days with feeding catheter enhanced colony formation of bone marrow cells ( Figure 5). The serum from Phyc-feeding group significantly increased it in comparison to contro ls in which normal serum was added. All of the sera obtained from the mice which were treated with intra-peritoneal single injection of the Spirulina extracts (10 mg/0.5 mL) also showed significantly high colony formation in comparison to control of normal serum, although levels of the activities of the sera were almost the same each other (data were not shown). Colony-stimulating factors, GM-CSF and IL-3, in the sera from the mice which were either fed or intra-peritoneally injected with the extracts, however, were under detect ion limit (<4 and 3 pg/mL serum, respectively). Concentration of GM-CSF in the LPS serum obtai ned by i.p. injection was 50.1 pg/mL. For longer-period experiment in in-vivo study, colony- and cluster-forma tion in the bone marrow cells with culture supernatants of the spleen (SP), Peyers patch (PP), and peritoneal-exudated (PE) cells from the mice, which ingested the Spirul ina extracts, SpHW, Phyc, and SpCW, for 5 weeks were then measured to confirm the former results. Culture supernatants of each lymphoid-organ, SP, PP, and PE cells from the gro ups were prepared under stimulation with or without phycocyanin. Colony formation by the culture supernatant of SP cells from the mice of SpHW group, as well as by that of PE cells from Phyc or SpCW group, under stimulation with phycocyanin, was significant ly higher than that by each culture supernatant of cells from control group, and thu s colony-forming activity was also significantly induced in the blood, spleen, and Peye rs patch cells in the mice which ingested Spirulina extracts for 5 weeks although neither si gnificant amount of GM-CSF nor IL-3 was detected in the blood (data not shown). On the other hand, ratios of

neutrophils in the SpHW-ingesting group and of lymphocytes in the SpCW-ingesting group were significantly higher than in controls, while ratios of lymphocytes , neutrophils, and monocytes in the peripheral blood of control group were in the normal range. A s ignificant increase in ratio of lymphocytes was also observed in bone marrow cel ls in Phyc-ingesting group, although the number of cells was small. In addition, increased ratio of reticulocytes was observed in the bone marrow of the mice fed with SpHW. In the mice ingested 0.05% phycocyanin solution for 6 weeks, a marked increase in the antigen-specific IgA antibody level as well as the total IgA antibody level was observed in the intestinal mucosa, the Peyers patches and mesenteric lymph nodes, w hich comprise a major part of the gut-associated lymphoid tissues (GALT), whereas neith er IgG1 nor IgE was affected in the spleen cells (Nemoto-Kawamura et al., 2004). Phycocyanin ing estion for 8 weeks, on the other hand, suppressed the production of antigen-speci fic IgG1 and IgE antibody in the serum. Further, we investigated the effect of Spirulin a on salivary IgA antibody level of the subjects who customarily ingested the Spirulina tablets as health food Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 129 in various period of usage in their daily life, and measured correlation between the salivary IgA level and the amount of Spirulina ingested (Ishii et al., 1999). Total S-IgA level of the group ingesting Spirulina for more than one year was significantly inc reased (p < 0.01) in comparison to the group ingesting Spirulina for less than half a year , and statistically significant correlation between S-IgA levels in the saliva and total a mount of Spirulina ingested by the subjects was observed (correlation coefficient R = 0.288, n = 72 , p < 0.05). Figure 8. Bone marrow-cell colony and cluster formation in soft agar assay with the serum from the mice fed with Spirulina extracts for 5 consecutive days (Hayashi et al., 2006) ( values are mean SD, n = 3). *; p<0.05 compared to Normal It is known that multi-potent colony-stimulating factors such as G- an d GM-CSF and IL-3, which are produced by a variety of cells including monocytes and lymphocytes can support proliferation of immature hematopoietic cells (Ihle, 1992). Liu et al. (2000) reported that phycocyanin from Spirulina platensis inhibited growth of human leukemia K562 cells in a dose-dependent manner, arresting them at the G1 phase with increased l evel of c-myc expression, suggesting that phycocyanin may enhance differentiation of t

he leukemia cells. Seya et al. (Hirahashi et al., 2002; Akao et al., 2009) reported tha t hot-water extract of Spirulina when taken orally in adult human enhances NK activation thro ugh the MyD88 pathway via Toll-like receptor (TLR) 2 and TLR4 on myeloid dendritic cells. From these findings, it appeared that Spirulina, including its components such as phycocyan in can affect enhancing proliferation or differentiation of immune competent-cells incl uding bone marrow cell, which may cause normally sustaining or enhancing immune f unctions. Colony-stimulating activity other than IL-3 or GM-CSF, for example arginase and G-CSF, in the serum may contribute to the cell differentiation, although this is still not clear. Zhang et al. (Zhang, 1994) found that C-phycocyanin and polysaccharide isolated from Spirulina increased leukocyte and bone marrow nucleated cell counts as well as colony formation of colony forming unit-granulocyte and macrophage (CFU-GM) in the gammaray irradiated mice, and also found that C-phycocyanin possessed high erythropoietin activity. Some institution facilities have reported the potential radiation prot ection effects of Spirulina against radiation-induced membrane damage and cellular dysfunction by reactive Normal serum OVA SpHW Phyc SpCW 0 5 10 15 20 25 30 N u m b e r o f c o l o n i e s & c l

u s t e r s p e r w e l l serum samples Colony Cluster * Blood Cell An Overview of Studies in Hematology 130 oxygen species in mice and against reduced levels of the leukocytes i n the blood and nucleated cells in the bone marrow in dog (Zhang et al., 2001; Verma et al., 200 6). Doctors in Belarus reported that ingestion of 5 g of Spirulina a day resulted in the reduct ion of Cesium137 in urine by 50%, in children subjected to low level of radiation over a long period of time (Loseva and Dardynskaya, 1993). Rahadiya and Patel in India (Rabadiya and Patel, 2010) also reported radiation-effect-reducing activity of Spirulina in their review. A nti-oxidant and anti-inflammatory effects as well as proliferation and differentiation a ctivity of Spirulina possibly contribute to the radiation protection effects. 5. Effect of phycocyanin on differentiation of human myeloid leukemia cell lines A study of aerosolized GM-CSF demonstrated tolerance and possible efficacy in pa tients with malignant metastases to the lungs, possibly through upregulation of ant igen-specific cytotoxic T-cells (Rao et al., 2003). It is known that various food compounds and the metabolites involving phycocyanin can influence the processes in cellula r differentiation, apoptosis, and proliferative potential, and there is considerable eviden ce that vitamins and micronutrients are able to regulate gene expression of cancer cells, r esulting in influence on the carcinogenic process (Sacha et al., 2005). All-trans-retinoic acid and vitam in D3 are known as one of the physiologic agents which can modulate the proliferation and diffe rentiation of hematopoietic cells (Collins, 2002). The vitamin plus interferon- (IFN) t reatment and enrichment with polyunsaturated fatty acids such as arachidonic acid, eicosapent aenoic acid or docosahexaenoic acid also significantly enhanced immunoregulatory effe cts, or enhanced

the expression of monocytic surface antigens CD11b and CD14 on human premonocyti c U937 cells (Obermeier et al., 1995). In this section, we investigated the effects of phycocyanin on differentiation and morphological and cytochemical changes of human myeloid leuk emia cell lines, U937 and HL-60 cells, generally used for the studies of cell differentiat ion. A human hematopoietic cell line, U-937, was derived from a patient wi th generalized histiocytic lymphoma. The histiocytic origin of the cell line was show n by its capacity of lysozyme production and the strong esterase activity (naphtol AS-D acet ate esterase inhibited by NaF) of the cells (Sundstrom and Nillson, 1976). The cel l line was morphologically identical to that of the tumor cells in the pleural effusion, an d is known to be functionally differentiated to phagocytic macrophage by cytokines fro m lymphocytes (Koren et al, 1979). A continuous human myeloid cell line, HL-60, was derived from the per ipheral blood leukocytes of a patient with acute promyelocytic leukemia and establish ed. The predominant cell type is a neutrophilic promyelocyte with prominent nuc lear/cytoplasmic asynchrony (Gallangher et al., 1979). HL-60 cells lack specific markers for lymphoid cells, but express surface receptors for Fc fragment and complement (C 3), which have been associated with differentiated granulocytes. They exhibit phagocytic activity and responsiveness to a chemotactic stimulus commensurate with the prop ortion of mature cells. Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 131 Preparation of Conditioned Medium (CM) of peripheral blood mononuclear cells cultured with phycocyanin Human peripheral blood mononuclear cells (PBMCs) from 7 healthy volunte ers were separated by density centrifugation (800 g, 30 mins) using a Lymphopre p (Density 1.077 g/mL, NYCOMED) under approving by the Institutional Review Board of ou r University. Cells from each subject were suspended in RPMI-FBS medium and adjusted to 1 x 10 6 cells/mL and were cultured with and without 2 mg/mL of phycocyanin (Phyc) using 24-well cultured plates. The conditioned mediums (CMs), both with and without Phyc (Phyco-CM and Cont-CM, respectively), were harvested on the 7th day and filtered through an 0.45 m filter to remove cell debris. Cell growth of U937 as well as HL-60 cells supplemented with Phyco-CM resulted in

significant inhibition during the 7-day culture in comparison with those of control without supplementation, while supplementation with 2 mg/mL Phyc itself had no effect on growth in these cells. Both U937 and HL-60 cells stimulated with Phyc and P hyco-CM were more than 80% viable, as were those cells stimulated with phorbol-12-myristate-13-ace tate (PMA) 0.02 g/mL as a positive control of differentiation. Morphology and flow cytometric assay of cell surface antigens on U937 and HL60 Cells U937 cells cultured in the medium supplemented with Phyco-CM morphologi cally changed into the cells with large cytoplasm with vacuoles, non-condensed nuclea r chromatin, and a persistence of nucleolus that resembled to those stimulated with PMA a s a positive control (Figure 6a) while control U937 cells, without stimulation, were promonocyte-like with variable nuclear shapes and regular indentations and comprised moderate cytoplasm containing numerous small eosinophilic granules and a few vacuoles. Cont-CM or co nditioned medium of lymphocytes cultured without phycocyanin, only partially changed U937 cells into monocytic cells comprising moderate cytoplasm with large indented nucleus (Figur e 6a). U937 cells stimulated with Phyco-CM and Cont-CM consisted of monocytes/macrop hages in the ratio of 57% and 21%, respectively, and each ratio was significantly higher than that of control (1.4%) without stimulation. Stimulation by Phyc changed U937 cells partially to promonocytes with indents on the nuclei. The ratio of monocytes/macrophages was only 3.4 %. Control HL-60 cells, without stimulation, was predominantly promyelocytes with azurophilic granules, large round nuclei, and prominent nucleoli. Morpho logical classification of the cells, especially those stimulated with Phyco-CM, was rela tively difficult because various features of promyelocytes coexisted. The Phyco-CM-stimulated HL60 cells showed a morphologically matured monocytic cell lineage (about 15.4%), that is, with decreased nuclear/cytoplasmic ratio and a paler cytoplasm with vacuoles (Figure 6b). The cells (about 80%) other than monocytic cells consisted of granulocytes, including promyelocytes and myelocytes, with large nuclei, less prominent cytoplasmic gran ules and a marked decrease or complete disappearance of nucleoli. Almost of all HL-60 cells Blood Cell An Overview of Studies in Hematology 132 stimulated with Phyc and Cont-CM were promyelocyte-like, while all-trans retinoic acid (ATRA) induced cells to differentiate into granulocytes (Figure 6b). Cell morphology was measured under light microscopy. U937 cells (a) and HL-60 ce

lls (b), 0.5 x 10 6 cells/mL of medium, were cultured for 3 days with RPMI-FBS (Cont.) (1); with Phyc (2); with PMA (3); with Phyco-CM (4); with Cont-CM (5); with ATRA (6) (Ishii et al., 2009). (Original magnification x 1000) Figure 9. Morphology of U937 and HL-60 cells stimulated with Phyco-CM and others . Flow cytometric assay was carried out using a Flow Cytometer (FCM; EP ICS ALTRA, Beckman Coulter, Inc., Fullerton, CA). The expression of cell surface antigens, CD14, CD11b, CD66b and CD15, on U937 and HL-60 cells stimulated with variou s CMs, as described above, were determined by direct immunofluorescence method using appro priate fluorescent labeled monoclonal antibodies. Typical patterns of FCM analy sis for CD14 antigen in both U937 and HL-60 cells stimulated with Phyco-CM we re shown in Figure 7. Ratio of CD14-antigen positive cells in U937 cells stimulated with Phy co-CM, 53%, was significantly high in comparison with those of Cont-CM and Cont without stimulat ion, 30% and 15%, respectively, while ratio of CD14-positive cells in HL-60 cells was origina lly low but was significantly increased by Phyc and Phyco-CM stimulations, about 20% (F igure 8a). Ratio of FcR positive cells in U937 cells was originally high and those of the cells stimulated with Phyco-CM and Cont-CM were almost the same as Cont without stimulation, 65%. In H L-60, on Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 133 the other hand, Phyco-CM increased FcR positive cells significantly, 41%, compare d with Cont and Cont-CM, 24% and 20%, respectively (Figure 8b). Further, a final concentration of 0.01 mg/mL of Phyc significantly increased the population of CD11b-antigen posit ive cells in U937 cells compared with Cont and Cont-CM (Figure 9a). Although the fluorescence inte nsity of antiCD14 antibody per cell in U937 cells stimulated with Phyc was marginally higher than that of Cont (Figure 7a), the ratio of CD14-antigen positive cells was low (F igure 8a) and morphologically comprised 3.4% of monocytes/macrophages. This suggests th at phycocyanin stimulates U937 cells to some extent to differentiate or express some CD antigens such as CD11b and CD14. CD11b as well as CD66b is specific in monocytes and granulocytes. Expression of CD11b is known to be up-regulated during granulocytic an d monocytic differentiation, and is used as a marker of differentiation of myelomonocytic li neage (Lubbert et al., 1991). It has been also recognized that morphological changes of

differentiating U937 cells are accompanied by cellular adherence and are paralleled by an expression of the 2 integrins, CD11a, CD11c, CD18, and particularly CD11b (Hass et al., 1989). CD11b glycoprote in represents the su unit of a heterodimeric association with the common su unit CD18 in 2 inte grin, an adhesion molecule. Their extracellular domains with the CD11b/CD18 (CR3/Mac-1) 2 integrin contribute to adhesion to adjacent cells, for example, the regulation of leukocy te-endothelial cell interactions (Ebnet et al., 2004). In the study using stably-transfecte d U937 cells with a vector containing the 2 integrin gene in antisense orientation, Otte et al. (2011) sugge sted that induced adherence predominantly mediated by a functional CD11b/CD18 integrin con tributed to cell cycle regulation and apoptosis during monocytic maturation. Concerning apoptotic cell death, photodynamic therapy (PDT) for tumors which is based on the tumor-selective accu mulation of protoporphyrin IX (PpIX), as a photosensitizer after addition of 5-amin olevulinic acid (ALA) followed by irradiation with visible light has been demonstrated by some investi gators, and it was reported that ALA-based photodynamic action (PDA) induced apoptotic cell dea th in U937 cells through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors (Amo et al., 2009). Furthermore pulsating electromagnet ic field (PEMF) can affect cancer cells proliferation and death (Kaszuba-Zwoinska et al ., 2010). They reported that U937 cells exposed to a pulsating magnetic field 50Hz, 45 5 mT th ree times for each 3 h with 24 h intervals induced cells death in higher cell density and c onversely prevented puromycin-induced cell death. We could take advantage of the halfway d ifferentiated U937 cells induced by phycocyanin as a cell culture model of cell differen tiation and apoptotic cell death to investigate the molecular mechanism of these various tumoricidal treatm ents. Both Phyc and Phyco-CM significantly increased the ratio of CD11b-antigen positi ve cells in U937 cells, about 30%, in comparison with those of Cont and Cont-CM, 12% and 22%, respectively, while in HL-60 neither CD11b nor CD66b cells showed significant in creases in ratio when stimulated with Phyco-CM (16%, 22%) or Cont-CM (10%, 18%) cells. In c ontrast, the ratio of CD15-antigen positive cells in the U937 cells was low r egardless of stimulation (Figure 9a). CD15-antigen is generally characteristic of granulocytes an d monocytes. The ratio of CD15-antigen positive cells in the HL-60 cells showed insigni ficant changes when stimulated with Phyc, Phyco-CM and other CMs (Figure 9b). Blood Cell An Overview of Studies in Hematology 134

Patterns of U937 and HL-60 cells (0.5 x 10 6 cells/mL of medium) stimulated with Phyc (1), Cont-CM (2), Phyco-CM (3) and PMA (4) were shown in solid lines and that of Cont was shown in dotted lines (Ishii et al., 2009). Figure 10. Typical patterns of CD14-positive cells in U937 and HL-60 cells. Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 135 Data analysis was based on examination of 5000 cells/sample. ++; p < 0.01, +++; p < 0.001 to each Cont, **; p < 0.01, ***; p < 0.001 to each Cont-CM, ###; p < 0.001 to each Cont and Cont-CM. Each value shows mean SD, n=3~7 (Ishii et al., 2009) Figure 11. Percentage of CD14 and FcR positive cells in U937 and HL-60 cells stim ulated with PhycoCM and other stimulants. Data analysis was based on examination of 5000 cells/sample. Each value shows me an SD, n= 3 (Ishii et al., 2009) Figure 12. Percentage of CD11b-, CD15- and CD66b-antigen positive cells in U937 and HL-60 cells stimulated with Phyco-CM and other stimulants. Phagocytic activity and TNF- production, cytochemical analysis Differentiation of both HL-60 and U937 cells was also assessed by cyt ochemical analysis with specific and non-specific esterase (SE/NSE) double staining method and Nitro-blue tetrazolium (NBT) reducing activity which is characteristic of phagocytic cells (Table 2). 0 10 20 30 40 50 60 70 80 90 100 CD14+ FcR+ CD14+ FcR+ p o s i t i v e c e l l s ( %

) Cont Phyc Cont-CM Phyco-CM PMA a. U937 cells b. HL-60 cells # # # # # # # # # # # # # # # # # # + + + + + 0 10 20 30 40 50 60 70 80 90 100 CD11b CD15 C11b CD15 CD66b p o s i t i v e c e l l s ( %

ATRA

) Cont Phyc Cont-CM Phyco-CM # # # # # # # # # # # # # # # # # # # # # # # # # # # a. U937 cells b. HL-60 cells + + + + *

PMA

ATRA

Blood Cell An Overview of Studies in Hematology 136 Although U937 cells were originally NSE positive, the Phyco-CM stimulat ed cells showed equally high ratio, while most HL-60 cells were SE positive under all stimulants . Phyco-CM stimulation significantly increased the ratio of NBT-positive cells in both U937 and HL-60 cells, while that of Cont-CM was almost the same as Cont. In additio n to that, phagocytic activity in U937 cells stimulated with Phyco-CM was significantly highe r than that of the cells stimulated with Cont-CM. In HL-60 cells, both Phyco-CM and ContCM increased phagocytic activity, as compared with Cont. Increased levels of TNF- in both U937 and HL-60cells stimulated with Phyco-CM, were relatively high in comparison with Cont-CM but not significant, and were not synergistically increased by suppleme ntation with LPS (1000 ng/mL) in the culture. Level of TNF- in the cells stimulated with Phyc was under the detection limit. Stimulators Phagocytic activity (%) TNF- (pg/mL) NBT reducing activity (%)

U937 Cont 18 5.2 ND 1.3 0.3 Phyc 23 5.1 < 15.6 5.0 3.0 Cont-CM 32 5.5 ++ 46 31 1.3 0.8 Phyco-CM 81 13 +++, *** 66 15 7.5 4.1** PMA 96 4.6 +++, *** 968 150 *** 5.2 2.9 HL-60 Cont 17 6.2 ND 0.7 0.3 Phyc 16 3.8 < 15.6 2.0 1.0 Cont-CM 35 12 ++ 32 29 2.1 0.7 Phyco-CM 36 13 ++ 71 18 5.3 1.9 +++, ** PMA 84 13 +++ 585 39 1.7 0.8 ATRA 72 5.2 +++ ND 10.8 3.3 +++, *** Phagocytic activity was determined by ingestion of opsonin-treated latex beads. NBT reducing activity was measured as percentage of the positive cells, which contained intracellular blue-black fo rmazan deposits. Positive cells out of 200 cells were counted under light microscopy. ++; p < 0.01, +++; p < 0.001 compared to Cont, **; p < 0.01, ***; p < 0.001 compared to Cont-CM, < ; under detection limit (15.6 pg/mL), ND; not detected (v alues are mean SD, n=3) Table 2. Cytochemical analysis of U937 and HL-60 cells stimulated with Phyco-CM Phyco-CM and Phyc quite significantly increased the population of CD14antigen positive cells in HL-60 cells, although the level was lower than that in U937 cells (Figure 8). In addition to that, Phyco-CM increased also the NBT reducing activity of HL-60 cells. Tamagawa et al. (1998) reported that NBT reducing activity was used as a marker of HL-60 cell differentiation into granulocytes and monocytes. Fontana et al. (1 981) suggested that HL-60 cells were able to commit themselves to the development of two different p rogram of hematopoietic differentiation, that is, either myeloid or macrophage depending o n cytokine or stimulant. In fact, both monocytic and granulocytic cells coexisted in HL-60 cells stimulated with Phyc and Phyco-CM as the result of differentiation in

two types of directions. However, NSE and SE ratios in HL-60 cells did not necessa rily correspond to morphological observation. Normally NSE has been thought to be specific for Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 137 monocyte/macrophage activity (Tanaka et al., 1983), and Chiao et al. ( 1981) reported that conditioned medium of normal human peripheral blood lymphocytes induced HL-60 cells into macrophage and monocyte-like cell lines, but most HL-60 cells stimulated wi th PhycoCM in the present study were SE positive, and only about 15% of mon ocytic matured cells were found in the cells after Phyco-CM stimulation. It appeared that the effects of PhycoCM on HL-60 cells may insufficiently induce to matured cells. CD14 antigen has been reported to be a receptor for the complex of LPS and LPS-binding protein (LBP). It is known that LPS and Gram negative bacteria as triggers (Beut ler, 2000; Lu et al., 2008) can cause TNF- release in human monocytes through TLR4 (Tudhope et al., 2008). The U937 cells stimulated with Phyco-CM, that showed high ratio of CD14-positive cells, are expected to express TLR4 in addition to CD4 because expres sion of TLR4 needs CD14 and LBP in response to the binding of LPS with LBP. Phyco-CM i nduced TNF- production in the culture supernatants of U937 and HL-60 cells. A hig h molecular weight polysaccharide, Immulina, from Spirulina was a potent activator of nucl ear factor-kappaB (NF-B) and induced both IL-1 and TNF- mRNAs in THP-1 human monocytes (P ugh et al., 2001), and expression of TLR2 and CD14 probably contributed to t he NF-B activation and immune enhancing activity of the Immulina in mice (Balachandran et al., 2006). The levels of TNF-, however, were not further increased with LPS stimulation (1000 ng /mL) in the U937 cells stimulated with phyco-CM. Phagocytic activity in the st imulated U937 cells was significantly higher than that of the cells stimulated with Cont-C M, and there was no stimulatory effect in the existence of LPS. Phyc alone did not induce TNF- in U93 7 and HL60 cells. 6. Age-related changes in intestine intraepitherial lymphocyte subsets and their functional preservation by Spirulina in mice Age-related immune dysfunction has been reviewed by many researchers (S olana et al., 2006). The complex age-related changes in the immune system, collective ly termed immunosenescence, have been demonstrated in diverse species, including hum ans, and have been recognized as contributing to morbidity and mortality due to greater i ncidence of

infectious diseases, autoimmune diseases, and cancer. The concept of ag e-related immunosenescence is in agreement with numerous data such as the change of cytokine balances, the decrease of interleukin (IL)-2 contrary to the increase of IL-6, and nutritional imbalance or malnutrition (Miquel, 2001; De la Fuente, 2002). It was reported that antigenspecific secretory immunoglobulin A titer in the intestinal lumen decli ned in senescent animals (Koga et al., 2000). Some studies have also reported that red uced bioavailability of key conditionally essential nutrients might limit immune response in ag ing (CunninghamRundles, 2004) and that well-nourished elderly people appear to have l ess significant or minimal changes in immune response (Krause et al., 1999). It is generally accepted that the development of age-associated alterat ions occurs earlier in the mucosal immune system than in the systemic immune compartment (Sch mucker et al., 2003). The mucosal immune system of the intestinal epithelia contains a functionally Blood Cell An Overview of Studies in Hematology 138 specialized T-cell population known as intraepithelial lymphocytes (IELs) . Because of their unique location in the mucosal epithelium, IELs are recognized as a f irst-line mucosal barrier against infectious diseases and food-borne allergens (Hayday et al., 200 1). We have reported that ingestion of phycocyanin enhanced the antigen-spe cific immunoglobulin A response in the intestinal mucosa of mice (Nemoto-Kawa mura et al., 2004). In this section, we investigated age-related changes in intestine IEL sub sets in mice by flow cytometric (FCM) analysis and their functional preservation after the anima ls were fed Spirulina. Characterization of IELs of adult and aged mice IELs possess phenotypic features distinct from those of lamina propria lymphocytes in intestine. Lamina propria lymphocytes consist of predominantly activated T cells and are mainly CD4 + and CD8 + single-positive T cells in proportions of about 70% and 30%, respectively. The phenotype of lamina propria lymphocytes, in general, is simila r to that of the cells in the peripheral lymphoid tissues and in the circulating blood, that is, over 95% of the cells possess a surface phenotype of T-cell receptor + (TCR + ), whereas less than 5% possess TCR

+ . These cells are known to be matured in the thymus (Lydyard and Gr ossi, 1998). IELs, on the other hand, possess TCR + in a greater percentage (3060%) and TCR + in a percentage of 4070%, which might be related to their state of activation (E wijk et al., 1999). In adult mice bred in a conventional environment, about half of t he IELs have a phenotype of surface CD antigen similar to that of most peripheral T lymphocytes, that is, Thy-1 + , TCR + , and either CD4 + or CD8 + , which are made up of heterodimers of CD8 and chains (CD8 + ). These cells were matured in a thymus-dependent manner (Kaminogawa and Nanno, 2004). Another major IEL population possesses the surface p henotype TCR + or TCR + , which expresses CD8 homodimeric chains (CD8 + ) but does not express CD4 or CD8 heterodimeric molecules (Rocha et al., 1994). These cells are known to be of extrathymic origin. Small percentages of the TCR + and TCR + but no TCR cells are CD8 CD4 . TCR + IELs co-expressing both CD4 and CD8 molecules are rare but bear high levels of TCR and CD8 (Lefrancois, 1991). Our preliminary experiment showed that the number of CD45 + (leukocyte-common antigen-positive) cells as IELs was significantly lower in aged mice than in adult mice. Either the proportion or the number of CD8 + cells in addition to CD45 + cells of aged mice was significantly lower than that of adult mice,

corresponding to the previous article by Komuro et al (1990). The proportion and number of CD4 + CD8 + double-positive cells in the aged mice, on the other hand, were higher than tho se in adult mice. It has been reported that CD4 + CD8 + T cells bearing TCR in the epithelium, which were derived from thymus-dependent populations, expanded with agin g at a local site of the intestine under the influence of intestinal microflora, contributing to the first line of defensive barrier in the epithelium (Takimoto et al., 1992). Overall, increased or decreased levels of these surface antigen-positive cells o bserved in the aged mice tended to be restored by the ingestion of SpHW for 5 weeks in the aged -SP group. In fact, significant decreases of CD45 + CD8 + cells and increases of CD8 CD4 and Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 139 CD45 + TCR + cells were observed in the aged mice, whereas neither an increase n or a decrease was observed in the aged-SP group fed with SpHWthat is, the levels were similar to those in adult mice. In particular, the proportions of CD4 5 + CD8 + cells and CD45 + TCR + cells in the aged-SP group significantly increased in comparison to the aged group. CD8 + T cells expressing TCR (T cells) are engaged in antigen-specific cell cytotoxicity mediated by major histocompatibility complex (MHC) molecules , whereas T

cells expressing TCR (T cells) often manifest preliminary target cell kill ing without MHC restriction (Cruse and Lewis, 1995). T cells have also been shown to be associated with regulation of the generation and differentiation of IELs (Komano et al., 1995). These results suggest that ingestion of SpHW in the aged-SP group may contr ibute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against infectious agents through retaining the numbers of certain IELs. Decreased levels of RBCs, especially the level of hematocrit, Ht, in the aged gr oup, were also restored after ingestion of SpHW in the aged-SP group. Significant decreases in WBCs in the aged-SP group, in contrast to the increase in the aged group, may be ascribed to the antiinflammatory activity of Spirulina (Vila et al., 2008) and/or to the restoration of immunological function by ingesting Spirulina. Some reports indicated th at phycocyanin and the polysaccharide isolated from Spirulina increased bone marrow nu cleated cell and erythrocyte counts in the gamma-ray irradiated mice or dog (Zhang, 199 4; Zhang et al., 2001; Verma et al., 2006). Many studies have demonstrated that Spiruli na including phycocyanin possesses antioxidant activity, as well as an anti-inflammatory acti vity (Romay et al., 1998; Remirez et al., 2002), which scavenges peroxyl radicals, and also acts as an inhibitor of cyclooxygenase, like nonsteroidal anti-inflammatory drugs. In addit ion, a downregulation of pro-inflammatory cytokines, such as TNF- and -, was observed in the aged animals on the Spirulina-enriched diet (Vila et al., 2008). Overexpress ion of MHC class Irelated chain A in the intestine of experimental transgenic mice resul ted in a clonal expansion of CD4 + CD8 + IELs and attenuated acute colitis in an experimental model of inflammatory bowel disease induced by dextran sodium sulfate administration (Par k et al., 2003). CD8 + IELs developed along an extrathymic pathway may work as antiinflammatory regulator T cells to sustain the mucosal intranet formed by intesti nal epithelial cells and IELs and to diminish the expansion of enterotoxigenic Escher ichia coli (Kim et al., 2001). Although ingesting SpHW did not significantly increase the level of CD4 + CD8 + IELs in the present study, these facts, in addition to our present results, suggest t

hat ingestion of Spirulina appears to be effective for protecting immune functions or i mproving immune systems vulnerable to age, thereby reducing the risk of infectious and autoimmun e diseases. However, additional detailed study is needed. 7. Conclusions Spirulina and its extracts enhanced proliferation of hematopoietic cells and col ony formation of bone marrow cell, as a marker for cell differentiation activity, in i n-vitro and in-vivo study using mice. Phycocyanin, a light-harvesting pigment of Spirulina, also induced cell differentiation of human leukemia cell lines, U937 and HL-60 cells, into monocyt e/macrophage Blood Cell An Overview of Studies in Hematology 140 and granulocyte, respectively, to some extent directly and indirectly t hrough enhancing cytokine production in human peripheral blood lymphocytes stimulated wit h phycocyanin. These distinguished activities of Spirulina as well as other certain f unctional foods can be preferably emphasized to be used, especially for elderly people. Recent intervention study showed that 6- and 12-week supplementation of Spirulina increased mean hemoglobin level and indoleamine 2,3-dioxygenase activity, as a sign of immune function, in the elderly subjects, suggesting that Spirulina may ameliorate anemia and immunosene scence in elderly people (Selmi et al., 2011). Pentn-Rol et al. (2011) demonstrated that phycocyanin triggered preventing or downgrading experimental autoimmune encephalitis (EAE) expr ession in rats, and that ingestion of phycocyanin induced a regulatory T cell (Treg) response in peripheral blood mononuclear cells from the patients with multiple sclerosis (MS). The auth ors suggested that phycocyanin may act as a neuroprotector and thereby may restore the functio nal damage in neurodegenerative disorders of the central nervous system (CNS). Another anim al model in rats showed that Spirulina promoted stem cell genesis and protected ag ainst LPS-induced declines in neural stem cell proliferation, and that cytokines did appear capabl e of regulating several phases of the neurogenesis process, supporting their hypothesis that a diet enriched with Spirulina may help protect the stem/progenitor cells from insults (Bachstet ter et al., 2010). These studies including reports summarized in this chapter show that S pirulina is useful in providing complementary nutrients for modulating or maintaining the immu ne system and that is also may have potential therapeutic benefits for improvement of immune d ysfunctions caused by, for example, radiation, chemotherapy using anti-cancer and a nti-infectious drugs, and certain microorganisms such as human immunodeficiency virus (HIV) i

tself, other than ageing. Further research along these lines is needed to validate these evidences . Author details Osamu Hayashi Kagawa Nutrition University, Japan 8. References Akao, Y, Ebihara, T, Masuda, H, Saeki, Y, Akazawa, T, Hazeki, K, Hazeki, O, Mats umoto, M, and Seya, T (2009). "Enhancement of antitumor natural killer cell acti vation by orally administered Spirulina extract in mice", Cancer Sci, 100, 1494-1501. Amo, T, Kawanishi, N, Uchida, M, Fujita, H, Oyanagi, E, Utsumi, T, O gino, T, Inoue, K, Shuin, T, Utsumi, K, and Sasaki, J (2009). "Mechanism of cell death by 5-aminole vulinic acid-based photodynamic action and its enhancement by ferrochelatase inh ibitors in human histiocytic lymphoma cell line U937", Cell Biochem Funct, 27, 503-515. Bachstetter, AD, Jernberg, J, Schlunk, A, Vila, JL, Hudson, C, Cole, MJ, Shytle, RD, Tan, J, Sanberg, PR, Sanberg, CD, Borlongan, C, Kaneko, Y, Tajiri, N, Gemma, C, and Bick ford, PC (2010). "Spirulina promotes stem cell genesis and protects against LPS induced declines in neural stem cell proliferation", PLoS One, 5, e10496. Proliferation and Differentiation of Hematopoietic Cells and Preservation of Imm une Functions 141 Balachandran, P, Pugh, ND, Ma, G, and Pasco, DS (2006). "Toll-like re ceptor 2-dependent activation of monocytes by Spirulina polysaccharide and its immune enha ncing action in mice", Int Immunopharmacol, 6, 1808-1814. Belay, A (2002). "The potential application of Spirulina (Arthorspira) as a nutritional and therapeutic supplement in health management", J Am Nutraceutical Association, 5, 27- 48. Belay, A, Ota, Y, Miyakawa, K, and Shimamatsu, H (1993). "Current knowledge on p otential health benefits of Spirulina", J Appl Phycol, 5, 235-241. Beutler, B (2000). "TLR4: central component of the sole mammalian LPS sensor", Current Opinion in Immunology, 12, 20-26. Chamorro-Cevallos, G (1980). "Toxicological research on the alga Spiruli na", Report: United Nations International Development Organization (UNIDO) UF/MEX/78/048, Vienna. Chiao, JW, Freitag, WF, Steinmetz, JC, and Andreeff, M (1981). "Changes of cellu lar markers during differentiation of HL-60 promyelocytes to macrophages as induced by T lymphocyte conditioned medium", Leukemia Research, 5, 477-489. Ciferri, O (1983). "Spirulina, the edible microorganisms", Microbiol Res, 47, 55 1-578. Collins, S (2002). "The role of retinoids and retinoic acid receptors in normal hematopoiesis", Leukemia, 16, 1896-1905. Cruse, JM, and Lewis, RE (1995). Illustrated Dictionary of Immunology. Florida: CRC Press, Inc.

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2012 Selz, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Spontaneous Alternation Behavior in Human Neutrophils Karen A. Selz Additional information is available at the end of the chapter http://dx.doi.org/10.5772/47851 1. Introduction SAB: Spontaneous alternation behavior (SAB) generally refers to the ten dency of animals, even single-celled organisms, to alternate their non-reinforced (Dember & Richman, 1989) choices of T- or Y-maze arms on subsequent trials, following an initi al trial or turn. First described over 80 years ago (Tolman, 1925), the phenomenon has been a scribed to the operation of a variety of mechanisms including Hullian reactive inhibition (Solo

mon, 1948), stimulus satiation (Glanzer, 1953), action decrement (Walker, 1958), cur iosity (Dember and Earl, 1957), habituation to novelty (Carlton, 1969), foraging strategies (Estes and Schoeffler, 1955) and spatial working memory (Sarter, et al., 1988). Studies have suggested that the primary cue for alternation among invertebrates to be is the body tur n. Vertebrates rely primarily on directional and odor cues. The fitness benefits associated with sti mulus seeking and behavioral exploration, foraging, remain the most compelling explana tion of why SAB is found ubiquitously and reliably (Richman, et al.1986). Although the underlying mechanism of SAB is open to study, there is general agreement that th e ability to alternate choices requires that the organism remember its previous choice (Hughes, 2004). Because SAB implicates future behavior which is statistically dependent on prior behavior and accompanied by memorially-dependant loss of degrees of freedom, SAB has been used to suggest the presence of a functional short-term memory. A left or right turn in a T- or Ymaze is a statistical function of the presence and direction of the previous tur n, when such a prior turn exists. While theories of memory are many and not the focus of this chapter, SAB is generally suggested to be recent memory dependent in animals complex enough to possess the structures postulated to underlie recent memory, and to be dependent o n a more basic sensory/membrane/receptor depletion time-limited memorial mechanism in sim ple Blood Cell An Overview of Studies in Hematology 148 organisms and cell systems that have also demonstrated SAB. Recent memory decays , some suggest exponentially in time (Eukaszewska and Deawichowska, 1982; Lalonde, 2002 ). This decay or, alternative, interference assumptions are explicit in many theori es of shortterm memory (e.g., Thorndike, 1914; Oberauer and Lewandowsky, 2008), so that the SAB effect would be expected to also diminish in the mean with the extension of the long leg of the T. Figure 1. A general layout for T-mazes. In fact, when the length of the vertical leg is treated as an exper imental variable, using distance as a proxy for time, SAB consistently decreases as vertical leg length increases (Hughes, 1989). Similarly, with two-trial SAB, increasing the intertrial interval, and so the time between successive choice opportunities, has been shown to reduce alternation frequencies (Dember and Richman, 1989). This inverse relationship follows from a memory-

based view of SAB, has been used to suggest that the longer the distance between successive turns, the greater the probability that the organism will forget the direction of its previous turn and choice will return to a statistically equiprobable condition (Hughes, 1989). No particular mechanism of memory is implied in this context. That is, any mechanism that causes current actions to be influenced by previous actions could potentially le ad to SAB in general and to the loss of SAB with increased distance/time between t he forced and choice turns. PMN: Human polymorphonuclear leucocytes, PMN, are highly motile cells averaging 12 to 15 m in diameter, exhibiting prominent, lobular nuclei. They emerge fro m bone marrow stem cells and are the most populous of the white blood cell categor y. PMNs are essential for host defense participating in both acute and chronic inflammatory processes. Other related properties include the regulation of immune responses, angiogene sis, and in physical interactions with neoplasms. Making this wide variety of functions poss ible is their dynamically adaptive behavior exemplified by the range of intrinsic pat terns of motility. Following my recent work demonstrating phase transition scenarios in PM N morphodynamics (Selz, 2011), I developed a new method to assess the a utonomous behavior of these human PMNs and whether single constituent cells exhibit SAB. Spontaneous Alternation Behavior in Human Neutrophils 149 Recall that whereas the natural physiological environment for autonomous motion in human neutrophils contains the PMNs endogenous tropic peptide attractant, the classical "spontaneous alteration", SAB design of these studies requires that the observed behavior, the cell s spontaneous motion, be unperturbed. The necessary absence of these chemoattractants, leads to a multitude of required trials for the acqu isition of a single data point. It was for this reason that I recorded hundreds of PMNs, incr easing the number of trials until n>50 left/right decisions at the T-intersections in each of the thr ee types of mazes will have been observed. 2. Methods and procedures Three similar T-mazes were developed; 1) a control T-maze, 2) a forced-turn T-ma ze, and 3) a long-leg forced turn T-maze. As SAB is the tendency of an organism to alternate successively turning left and then right in a maze, the control maze is the one in which the first turn is the only turn. Cells in control mazes are expected to turn to the right or left with roughly equal probability. However, in the second, forced turn maze, in which th e cell must

always make a right turn before reaching the experimental left versus right choice point of the T, the statistical expectation is that more cells will turn left at the T. I n the third maze, the SAB effect can decay in the mean with the extension of the long leg of the T. The mazes were constructed on standard glass slides, inside a 12mm diameter, 25m deep well. The specifications are indicated in the figures 2. Figure 3 is an electron micrograph of a set of the nano-structured mazes on a finished slide. Tolerances we re 5%. Sidewall angles of mazes were 88-90 0 . Surface variation (roughness) was in the neighborhood of 10nm.

Figure 2. A rough schematic of the mazes on each slide. Blood Cell An Overview of Studies in Hematology 150 Figure 3. An EM image of one set of mazes on a slide. Note the taper at the entr ance of each maze and the flow-through gap at the terminal ends of the mazes (0um at the base to 3um a t the top of the maze walls). These allow the passage of medium, but not the entry or exit of PMN. After gentle sedimentation, PMNs (along with some other white cells an d platelets) were removed from the buffy coat by micropipette and, along with associated plasma. T he 5.7 m gap of standard slides and coverslips is smaller than the average diameter of PM Ns, leading to some mechanical compression of the cells and contributing to their activation , as well as allowing the cell to move along the slide substrate and cover slip simultaneousl y (Malawista and deBoisfleury Chevance, 1997). The slides used in this study do no t suffer from these deficits. All samples were drawn from 5 healthy, adult volunteers (3F, 2M, ages 25-47). All slides were pre-filled with Lactated Ringer s solution, that is isotoni c with blood. This was done to avoid cells being deposited into the maze ends, as well as to avoid trap ped air in the mazes, and to dilute the PMN sample. Figure 4. Shows a typical trial, in which a PMN (with evident lobular nuclei) tr anslocates up the vertical leg of a control T-maze while another lingers outside the maze. Spontaneous Alternation Behavior in Human Neutrophils 151 As implied by the word spontaneous, SAB studies require that the cell s motion be observed without manipulating exogenous agents. Lactated Ringer s solutio n provided a uniform, undoped medium base. Without the activating influence of chemoattractants or physical activators (e.g. heat, pressure, sheer), many trials were required for

the acquisition of each data point. In total, 102 PMN navigated the control maze past the decisi on point, 57 navigated the forced-turn maze, and 82 PMN completed the long-legged f orced-turn Tmaze. PMN autonomous motions were observed through an Olympus BX41 microscope with a mixed CytoViva dark field and fluorescent optical illumination system. This inco rporates a high-aperture, cardioid annular condenser (www.scitech.com.au) unique to this sy stem and which makes possible visualization of objects below 90nm in diameter i n real time, including cellular samples in an unfixed, living, active state (Samoylov, et al. , 2005; Vainrub, et al. 2006). Because PMNs were treated gently, avoiding perturbations of column separation and elution, it became possible to reliably study a PMN co ntinuously for extended times without the onset of granular clumping, membrane blebbing and oth er signs of impending apoptosis (Lodish, 2005). Whenever a PMN moved to the left or right beyond the choice point of a maze, its choice was recorded. 3. Results Left-Right choices in the control maze were not significantly different from equ iprobability (Left=49.02%; Right=50.98%; 2 (1)=0.039, =0.843). However, in the forced-turn T-maze, 63.16% of the cells that had been forced to turn Right previously, t urned Left at the decision point ( 2 (1)=3.947, =0.0469) . Contrary to initial expectation of the disappearance of the SAB effect with the increased length of the vertical leg of the maze, this e ffect persisted and even appeared to be strengthened in the long-legged forced-turn ma ze, in which 67.07% ( 2 (1)= 9.561, =0.0021) of completing cells turned Left at the decision p oint, long after a forced Right turn. 4. Conclusions This is the first demonstration that SAB is present in PMN, a constitu ent cell of the human body. While SAB has been shown in human spermatozoa (Brugger, et. al., 2002), sp erm cells are required to function in the world exterior to the body, while PMN are not. The apparent strengthening of SAB in the long-legged forced-turn T-maze compared to the standard forced-turn maze, may be partially attributable to the higher n in the former and natural variation in data, but observation of hundreds of PMN in the maze environments suggests it might also result from a sort of practice effect of frustrated turn at tempts in the channel, in a dose-response like paradigm. Length of time/distance spent in foil

ed attempts to turn may then serve as an order parameter. The persistence of SAB in this con dition may also suggest multiple time scale, memory-like mechanisms operating in PMN. Blood Cell An Overview of Studies in Hematology 152 There are, in fact, established physiological mechanisms and behavior t hat are consistent with this speculation and with my qualitative microscopic. PMNs are known to osc illate on multiple temporal and spatial scales, from 7sec, 70sec, and 260sec mem brane potential fluctuations (Jger, et al., 1988) and 25sec calcium flux oscillations, to the ~8sec G-F-actin oscillations (Marks and Maxfield, 1990), to 21.6sec and 230sec glycolytic cycles that produce NAD(P)H oscillations (Jger, et al., 1988), and 10sec and 20sec pericell ular proteolysis fluctuations (Marks and Maxfield, 1990), among many others. The time s eries in my recent study of PMN morphodynamics demonstrated scaling, board band power spec tra with multiple resonances, suggesting a constellation of motility times (Selz, 2011). While there is debate over the specific fitness value(s) and mechanism(s) underlying S AB, it is clearly a phylogentically highly conserved behavior (Richman, et al.1986), and so, is assumed to be valuable. It follows that multiple mechanistically diverse time scales of function could be recruited to its service. This suggests the possibility of decentralize d control in a highly interconnected, living dynamical system through local feedback. The nece ssary and sufficient conditions for a stable macro-system composed of multiple sm aller systems operating on a variety of temporal and spatial scales are known in n on-biological contexts (Ramakrishna and Viswanadham, 1982). Work is currently underway, adaptin g these constraints to a PMN model. Because, with the prior forced turn, systems statistically deviate from equiprobability in a later choice circumstance, SAB represents a reduction in population behavioral e ntropy, and a situation in which a reduction in the behavior degrees of freedom and reduced statistical behavioral entropy is favored evolutionarily. This finding is contrary to some findings of increased entropic states being associated with greater biological health and/or function (e.g. Mandell, 1987; Paulus et al., 1980). Author details Karen A. Selz Franklin-Fetzer Laboratory, Cielo Institute, Asheville, NC, USA 5. References Brugger, P, Macas, E., and Ihlemann, J. (2002) Do sperm remember? Behavl Brain R es., 136(1), 325-8. Carlton PL. (1969). Brain-acetylcholine and inhibition. In: Tapp JT, editor. Rei

nforcement and behavior. New York: Academic Press,p. 286327. Dember WN, Earl RW.(1957). Analysis of exploratory, manipulatory and cu riosity behaviors.Psychol Rev, 64:916. Dember, W. N., & Richman, C. L. (Eds.) (1989). Spontaneous alternation behavior. New York: Springer. Spontaneous Alternation Behavior in Human Neutrophils 153 Estes WK, Schoeffler MS. (1955). Analysis of variables influencing alte rnation after forced trials. J Comp PhysiolPsychol, 48:35762. Eukaszewska, I., Deawichowska, (1982). How long do rats remember the s patial arrangement of visual stimuli? ActaNeurobiol.Exp, 42, 127-133.; Glanzer M. (1953). Stimulus satiation: an explanation of spontaneous alternation and related phenomena. Psychol Rev, 60:25768. Hughes, R. N. (1989). Phylogenetic comparisons. In W. N. Dember & C. L. Richman (Eds.), Spontaneous alternation behavior (pp. 38-57). NewYork: Springer. Hughes, R. N. (2004). The value of spontaneous alternation behavior (S AB) as a test of retention in pharmacological investigations of memory. Neuroscience & Bi obehavioral Reviews, 28, 497-505. Jger U, Gruler H, Bltmann B (1988) Morphological changes and membrane p otential of human granulocytes under influence of chemotactic peptide and/or echo-vi rus type 9. Klin Wochenschr 66: 434. Lalonde, R. (2002). The neurobiological basis of spontaneous alternation, Neuros ci&Biobehav Rev, 26(1), 91-104.; Lodish HF (2005) Molecular and Cell Biology. W.H. Freeman, New York. Malawista SE, deBoisfleury Chevance A (1997) Random locomotion and chem otaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations. PNAS 94:11577-82. Mandell AJ (1987) Dynamical complexity and pathological order in the c ardiac monitoring problem Physica D 27 235-42. Marks PW, Maxfield FR (1990) Local and global changes in cytosolic fr ee calcium in neutrophils during chemotaxis and phagocytosis. J Cell Biol 110:43. Oberauer K, Lewandowsky S (2008) Forgetting in immediate serial recall: decay, temporal distinctiveness, or interference? Psychology review, 115(3), 544-576. Paulus MP, Geyer MA, Gold LH, Mandell AJ (1990) Application of entrop y measures derived from the ergodic theory of dynamical systems to rat locomotor behavior. Proc.Natl.Acad.Sci. 87: 723-727. Ramakrishna A, Viswanadham N (1982) Decentralized Control of Interconnected Dyna mical Systems. IEEE Transactions on Automatic Control, 27(1), 159-164. Richman, R., et al.(1986). Spontaneous alternation behavior in animals:

A review. Current Psychology, 5(4), 358-91. Samoylov AM, Samoylova TI, Pustovyy OM, Samoylov AA, Toivio-Kinnucan MA, et al. (2005) Novel metal clusters isolated from blood are lethal to cancer cells. Cells Tissues Organs 179:115-124. Sarter M, Bodewitz G, Stephens DN. (1988). Attenuation of scopolamine induced impairment of spontaneous alternation behaviour by antagonist but not inverse ag onist and agonist beta-carbolines. Psychopharmacology, 94:4915. Selz KA, (2011).A Third Measure-Metastable State in the Dynamics of Sp ontaneous Shape Change in Healthy Human s White Cells.PLoSComputBiol 7(4): e1001117. doi:10.1371 . Blood Cell An Overview of Studies in Hematology 154 Solomon RL. (1948). The influence of work on behavior. Psychol Bull;45 :140.502; R.N. Hughes (2004)Neuroscience and Biobehavioral Reviews 28, 497505 Thorndike EL, The Psychology of Learning, N. Y., Teachers College, 1914 Tolman EC. (1925). Purpose and cognition: the determiners of animal learning. Ps ychol Rev, 32:28597. Vainrub A, Pustovyy O, Vodyanoy V (2006) Resolution of 90 nm (lambda/ 5) in an optical transmission microscope with an annular condenser. Optics Lett 31, 2855-2857 Walker EL. (1958). Action decrement and its relation to learning. Psychol Rev, 6 5:12942. Chapter 9

2012 Moschandreou, licensee InTech. This is an open access chapter distributed u nder the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model Terry E. Moschandreou Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48580 1. Introduction It is known that red blood cells release ATP when blood oxygen tension decreases . ATP has an effect on microvascular endothelial cells to form a retrospective conducted v asodilation to the upstream arteriole. Local metabolic control of coronary blood f low due to vasodilation in microvascular units where myocardial oxygen extraction i s relatively high occurs due to ATP.[5] Arterioles dilate or constrict in response to c hanging intravascular pressure.[6]

It is well known that myogenic responses, ow-dependent vasodilation, loca l metabolic effects, and propagation all contribute to blood ow regulation. Primaril y responsible for carrying oxygen in blood, red blood cells (RBCs) may also act as oxy gen sensors and thus play a role in the communication of metabolic demand [3,7]. The mechanisms for re lease of ATP from the RBC in response to lowered oxygen saturation have been studied. [8] . Jagger et al. [9] measured the ATP release at low O2 levels in the presenc e and absence of CO to demonstrate that the release of ATP from RBCs may be connected to th e change of the hemoglobin molecule from its relaxed state to its deoxygenated state. Upon release, ATP binds to P2Y purinergic receptors on the luminal surface of the endot helium, starting the conducted response [10].An in vitro microfluidic experimental study to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolutio n was conducted by Wan et al. [11].Conclusively it was shown that there is a sizable delay time between the onset of increased shear stress and the release of ATP. "It was seen that this response time decreases with shear stress, but does not depend signifi cantly on membrane rigidity. It was shown that even though the RBCs deform significantly in short c onstrictions (duration of increased stress <3 ms), no measurable ATP is released."[11] Blood Cell An Overview of Studies in Hematology 156 ATP is short for adenosine triposphate, which is a currency of biologic al energy. There exists an adenosine group with 3 phosphate groups attached to it. Hyd rolizing this bond detaches one of the phosphate groups and produces ADP, which is adenosine di-pho spahte plus a phosphate group and energy. Through chemical reaction if you pop off a ph osphate group of ATP, energy will be generated for general heat or one can couple this r eaction with other reactions that require energy. This chemical reaction of ATP to ADP involves going from stored energy to used energy. ADP can be recharged back to ATP by processes in the mitochondria. Figure 1. Adenosine Triphosphate[12] The same part(adenine) that makes up DNA is that which makes up these energy cur rency molecules known as ATP molecules. Adenine makes part of adenosine which makes pa rt of ATP. The other part of ATP is known as ribose from RNA, which is a five carbon s ugar. ATP drives biological reactions. In terms of electrons when one pops off the phospha te group the electrons enter a lower energy state between phosphate and oxygen atoms which ge nerates

energy. RBCs have no nucleus or mitochondria. As a result RBCs obtain their en ergy using glycolysis to produce ATP. There are both advantages and disadvantages to this. An advantage is due to the biconcave disk shape which optimizes the cell for the exchange of oxygen with its surroundings and optimizes space for the hemoglobin. T he RBCs are deformable and flexible so that they can move through the tiny capillaries where oxygen is released. The disadvantage is that because of the absence of nuclei a nd organelles, mature RBCs do not contain DNA and cannot synthesize any RNA, and cannot div ide or repair themselves. The Mitochondria enables cells to produce 15 times more ATP than usu al. Lack of mitochondria means that the cells use none of the oxygen they tra nsport. Instead they produce the energy carrier ATP by means of fermentation, via glycolysis of gluco se and by lactic acid production. RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model 157

Figure 2. Ball and Stick Model of ATP based x-ray diffraction data[13] Blood Cell An Overview of Studies in Hematology 158

Figure 3. Space-Fill Representation of ATP[14] RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model 159 2. Structural features erythrocyte and the erythrocyte membrane Lacking organelles as nucleus, mitochondria, or ribosomes, the red cell does not synthesize new proteins, carrying out the oxidative reactions associated with mitochondria, or undergo mitosis. The RBC consists of a membrane surrounding a solution of protein and electrolyte s. About 95% of the protein is the oxygen-transport protein, hemoglobin. The re mainder of the protein includes enzymes required for energy production and for mainten ance of hemoglobin. 3. Shape of RBC In immobile state, the normal human RBC is shaped as a biconcave dis c. The disc shape is important to erythrocyte function. The ratio of surface to volume is

optimized so that oxygen transfer is possible. Also the biconcave disc is more deformabl e than a sphere and undergoes the change in shape necessary for optimal movement in microvasculature . The four possible forces to maintain the shape described are (1) elas tic forces within the membrane, (2) surface tension, (3) electrical forces on the membrane surface, an d (4) osmotic or hydrostatic pressures. The maintenance of RBC shape is dependent on the struc ture of the cell as well as in the external environment. If these are changed, t he cell may become spherical. When RBCs are suspended in hypotonic solutions andosmotic swelling occur s. This can make the cell spherical. These changes are associated with an increase in volume while the cell surface area remains the same or changes only slightly. When sph erical shape is attained, the cell diameter decreases, and this shows the elastic properties of the membrane. Discocyte-echinocyte transformation takes place when ATP is depleted, wh en intracellular calcium is increased, when the cell is exposed to plasma, anionic det ergents, high pH, lysolecithin or fatty acids. 4. Dimensions of RBC Photomicrographs are used to measure the dimensions of RBCs. Average va lues for the mean cellular volume in normal subjects are from approximately 85 to 91 fl. Ninety-five percent of normal red cells are between about 60 and 120 fl in volume. Various r esults have yielded an average normal value for red cell diameter of 7.2 to 7.4 microns. 5. Present objective In this work we model the time delay of release of ATP as supporting work shows by Wan et al. [11] for shear-induced ATP release from red blood cells. A re lease rate which is a function of time and introduces a delay mechanism is introduced to sh ow how the concentration of ATP is thus affected. Blood Cell An Overview of Studies in Hematology 160

Figure 4. [11] RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model 161 6. Method of solution By conservation of mass, the decline in oxygen flux must equal the r ate of oxygen consumption, giving the following equation for the change in oxygen saturation, S(x), with distance, x, along each arteriole: ( )

o D d Qc H S x dx =

(1) where Q is volume flow rate in an individual vessel, o c is the carrying capacity of RBCs at 100% saturation. D H is the discharge hematocrit, and 2 2 0 1, ( ) j j q M r r t = [ 3]. The release rate of ATP from an RBC, R[S(x)], is defined by a decreasing linear function of oxyhemoglobin saturation based on experimental data. ATP release from h uman erythrocytes in response to normoxia and hypoxia was observed in in v itro experiments.[3]. A linear fit of experimental values defines the ATP release function of saturati on : 0 1 ( ) 1 ( ) R S x R R S x =

(2) In general,the rate of change in plasma ATP concentration, C(x,t), is given by t he difference between the rates of ATP release and degradation: 2 2 [ (1 ) ] [(1 ) ( )] [ ( , )] 2 ( , ) T D T d R H C H QC x R H R S x t k RC x t t x t t t c c + = c c (3) where T H is tube hematocrit, R is radius of vessel and d k is a concentration rate constant.([3]) We assume that there is no convection in the vessel and there is no x-dependence . Equation (3) simplifies to the following equation: 2 ( ( )) 1 1 d T T T

k H R S dt =

dC t C H R

In this chapter a model is developed which predicts tissue ATP concentrations as a variation of time and depth into the tissue due to changing oxygen tensions. The ATP concentration within plasma as a variation of time due to ch anges in oxygen tension at the tissue surface is related to the release and degradati on of ATP, by the following equation: ( ) ATP dC R t C dt n o = (4)

Blood Cell An Overview of Studies in Hematology 162 is some constant of degradation. where 2 1 d T k R H [Related to the RBC fraction], R is the release of ATP from the RBC and C is ATP concentration. In this model the ATP concentration maximizes at some constant value, depending on the oxygen saturation. This model can be used to predict the plasma ATP concentration based on different oxygen saturations. 7. Discussion From Equation (4), with varying degradation constant, related to the R BC fraction, and release rate , ( ) ( 3) ATP R t H t where H is the Heaviside function as a function of t, we show results in Figure 5 for concentration of ATP, ( M ), versus time (ms). The val ue in the shifted Heaviside function corresponds to the experimental results of Wan et al [11] for time t< 3 ms. This is the time interval where there is no ATP release and can be confirmed in Figure 4 where there is no ATP release before and throughout the stenosis of the microflu idic device. In fact there is ATP release in the low shear expansion on the right of the micr ofluidic device. This can be seen in the increase in concentration of ATP released by RBC as sho wn in Figure 4. The parameter represents the rate of increase of ATP release an d for large , the

concentration of ATP increases rather steeply, wheras for smaller the concentrat ion of ATP increases less steeply as can be seen in Figure 6 for varying values of degradation constant. Also our model is consistent with the experimental results of Wan et al.[11] in that the greatest increase in ATP occurs approximately 29 ms after the onset of increased shear stress. (See Figure 6 for 0.1 at time t=30ms.) It is noteworthy to see that these resu lts for shearinduced ATP release can also be extended to ATP release due to a decrease in sat uration. Figure 5. RBC-ATP Theory of Regulation for Tissue Oxygenation-ATP Concentration Model 163

Figure 6. 8. Summary and conclusion In this work we have outlined the importance of ATP as a signaling molecule in the microcirculation and have discussed the biochemical aspects of ATP and ADP as well as introduce a model for ATP release as used in microfluidic devices where RBCs are subject to compression and deform thus resulting in ATP release. It is shown by Wan et al. [11 ] that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is in proportion with a characteristic membrane relaxation ti me determined from observations of the cell deformation by using high-speed video[11] . The results suggest a model wherein the retraction of the spectrin-actin cytoskelet on network triggers the mechano-sensitive ATP release and a shear-dependent membrane viscosi tycontrols the rate of release.[11] It is noteworthy to see that these results for shear-induced ATP release can als o be extended to ATP release due to a decrease in saturation.[3] Author details Terry E. Moschandreou Department of Medical Biophysics, University of Western Ontario, London Ontario, Canada Blood Cell An Overview of Studies in Hematology 164 9. References [1] Segal S.S. (2005) Regulation of blood flow in the microcirculation, Microci rculation 12(1): 33-45. [2] M.L.EllsworthM.L.,Ellis C.G., Goldman D., Stephenson A.H., Dietrich H.H., Sprague R.S.(2009)Erythrocytes: oxygen sensors and modulators of vascular tone. Physiology (Bethesda)24: 107-16. [3] ArcieroJ.C.,Carlson B.E., Secomb T.W.(2008) Theoretical model of met

abolic blood flow regulation: roles of ATP release by red blood cells and conducted res ponses, Am. J. Physiol. Heart Circ. Physiol. 295(4): H1562-71. [4] Bergfeld GR, Forrester T. (1992) Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovas Res 26: 40-47. [5] Farias Martin. III, Gorman Mark W., Savage Margaret V. and Feigl. Eric O. ( 2005) Plasma ATP during exercise: possible role in regulation of coronary blood flo w AJP Heart April 1, vol. 288 no.4: H1586-H1590. [6] Johnson PC. (1980)The myogenic response.In:Handbook of Physiology.The Cardiovascular System.Vascular Smooth Muscle. Bethesda, MD: Am.Physiol. Soc., se ct. 2, vol. II: p. 409 442. [7] Ellsworth ML.(2000)The red blood cell as an oxygen sensor: what is the evidence? Acta Physiol Scand 168: 551559. [8] Buehler PW, Alayash AI.(2004)Oxygen sensing in the circulation: cros s talkbetween red blood cells and the vasculature.Antioxid Redox Signal6:1000 1010. [9] Jagger JE, Bateman RM, Ellsworth ML, Ellis CG.(2001)Role of erythr ocyte in regulating local O2 delivery mediated by hemoglobin oxygenation. Am J Physiol Hear t CircPhysiol280: H2833H2839. [10] Gorman MW, Ogimoto K, Savage MV, Jacobson KA, Feigl EO(2003)Nucleo tide coronary vasodilation in guinea pig hearts.Am J Physiol HeartCircPhysiol285: H10 40 H1047. [11] Wan Jiandi, Ristenpart William D., and Stone Howard A. (2008) Dyn amics of shearinduced ATP release from red blood cells. PNAS: 1-7. Section 2

Measurement of RBC Deformability and Microfluidics Technology for Cell Separation

Chapter 10

2012 Park et al., licensee InTech. This is an open access chapter distributed un der the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Measurement Techniques for Red Blood Cell Deformability: Recent Advances

Youngchan Kim, Kyoohyun Kim and YongKeun Park Additional information is available at the end of the chapter http://dx.doi.org/10.5772/50698 1. Introduction Human red blood cells (RBCs) or erythrocytes have remarkable deformabil ity. Upon external forces, RBCs undergo large mechanical deformation without ruptu re, and they restore to original shapes when released. The deformability of RBCs pl ays crucially important roles in the main function of RBCs - oxygen transport throu gh blood circulation. RBCs must withstand large deformations during repeated passages through the microvasculature and the fenestrated walls of the splenic sinusoids (Wa ugh and Evans, 1979). RBC deformability can be significantly altered by various pathop hysiological conditions, and the alterations in RBC deformability in turn influence pathophysiology, since RBC deformability is an important determinant of blood viscosity and thus blood circulation. Hence, measuring the deformability of RBCs holds the key to understanding RBC related diseases. For the past years, various experimental techniqu es have been developed to measure RBC deformability and recent technical advances re volutionize the way we study RBCs and their roles in hematology. This chapter reviews a variety of tools for measuring RBC deformability. For each technique, we seek to provid e insights how these deformability measurement techniques can improve the study of RBC pathophysiology. 2. Deformability of RBCs RBCs are the most deformable cell in the human body. RBC deformabilti y is an intrinsic mechanical property determined by (1) its geometry, (2) cytoplasmic vis cosity, mainly attributed to hemoglobin (Hb) solution in the cytoplasm, and (3) visco elastic properties of RBC membrane cortex structure. Blood Cell An Overview of Studies in Hematology 168 2.1. RBC geometry Mature human RBCs have a biconcave disc shape and they do not contai n nucleus or subcellular structures but mainly consist of Hb solution in the cytoplasm (Fig. 1a). A typical human RBC has a thickness of 2-3 m, diameter of 6-8 m, cell volume of 90 fl, and s urface area of approximately 136 m 2 (Kenneth,2010). Depending on species, RBC shape and size vary. In mammals, RBCs develope from nucleated progenitor cells in bone mellow b ut RBCs discard their nucleus as they mature, whereas RBCs of other vertebrate s have nuclei. Throughout their life span of 100-120 days, human RBCs circulate the

body delivering oxygen from the lungs to tissues. RBCs gradually lose their deformabil ity with age and eventually rupture in spleen. The biconcave shape of normal RBCs has advantages in having deformability. Compared to a spherical shape, RBCs with biconcave shape h ave less volume for a given surface area, which can decrease bending energy as sociated with the membrane (Canham,1970). 2.2. Membrane cortex structure The unique deformability of RBCs is mainly determined by the structures of RBC m embrane cortex. The membrane of human RBC is a multicomponent structure compri sed of three layers: (1) an external carbohydrate-rich layer, (2) the phospholipid b ilayer with 4-5 nm thickness, embedded with transmembrane proteins, and (3) a 2-D triangul ar mesh-like spectrin cytoskeleton network attached to the surface bilayers. The mesh size of the spectrin network is 60-80 nm. The spectrin network is anchored to the phosphol ipid bilayer via juntional complexes and ankyrin proteins. Junctional complexes and ankyr in proteins can diffuse in the lipid membrane. (c) (b) (a) ~ 8 m ~ 2 m Volume ~ 90 fl Surface area ~ 136 m 2 Figure 1. (a) RBC morphology. (b) Spectrin network measured by high resolution n egative staining electron microscopy. (b) Schematic model of the red cell membrane. Reproduced, w ith permission, from (Liu, Derick et al.,1987; Tse and Lux,1999). 2.3. Viscoelastic properties of RBC In a view of classical mechanics, soft biomaterials can be characteriz ed by viscoelastic properties - exhibiting both energy-storing elastic and energy-dissipatin g viscous characteristics. RBC is a typical soft biomaterial showing unique visco elastic properties (Hochmuth and Waugh,1987). Measurement Techniques for Red Blood Cell Deformability: Recent Advances 169 2.3.1. Elastic property Elastic property chracterizes deformability of a material when a force is applie d. Since RBC cytoplasm mainly consists of Hb solution, the elastic properties of RB C is determined by RBC membrane cortex structures. RBC membranes are only a few molecules thick, an d they can be treated with a 2-D continuum model. Although the deformation of RBC membr ane is highly complex, it can be simply explained by three fundamental deformat ion modes: area

expansion, shear, and bend of the membrane (Fig. 2). Area expansion Shear Bend Figure 2. Schematic illustrations of area expansion, shear, and bend modes of a 2-D membrane. The elastic property of a 2-D membrane cortex is characterized by thr ee mechanical elastic moduli: area expansion modulus K, shear modulus , and bending modulus B. The deta iled explanations for three elastic moduli are described as follow: Area expansion modulus. The area expansion (or compression) modulus K reflects the elastic energy storage produced by an isotropic area dilation or compr ession of the membrane surface. The area expansion modulus K is described as , t o A T K A (1) where Tt, A0, and A correpond to the isotopic tensile force, the orig inal surface area, and the increase in surface area, respectively (Hochmuth and Waugh,1987). The area e xpansion modulus of RBC membranes is mainly dominated by the elasticity of the bilayer. Interestingly, the lipid bilayer itself is highly inextensible; the stand-alone lipid bilayer area compression modulus was given in the range of 200-300 mN/m (Rawicz, Olbrich et a l.,2000). However, RBC membranes exhibit significant area extensibility. There is a wide range of measured values for K of RBCs that fall into two groupings. (1) Valu es reported from micropipette-based studies are in the range of 300-500 mN/m (Evans,1973 ; Waugh and Evans, 1979). (2) Recently, measurements based on dynamic membrane fluct uations report K of RBC membranes in the range of 10-100 N/m (Gov, Zilman et al.,2003; Betz, Len z et al., 2009; Park, Best et al.,2010; Park, Best et al.,2011; Byun, Higgins e t al., in press). These two techniques sample mechanical responses of the RBC under very different loading c onditions and they involve different components of the cell; micropipette-based studies ma inly probes lipid-bilayer dominated behavior while membrane fluctuation measurements primarily analyze spectrin network dominated behavior. In addition, the area expansion mod ulus K of RBC membranes can be changed by temperature; the micropipette asiperati on techniques measured K at 25 C is 450 mN/m and the temperature dependency of K was found to b e -6 mN/mC. (Waugh and Evans,1979). Blood Cell An Overview of Studies in Hematology 170

Shear modulus. The shear modulus of a 2-D structure reflects the ela stic energy storage associated with extension of the membrane surface with the same membrane area. T he shear modulus is described as 2 2 , 2 s T

(2) where Ts is the shear force and is the the extension ratio (Evans,1 973). Shear modulus of lipid bilayers is essentially zero due to its fluidity nature; shear modulus of RBC is mainly contributed from the spectrin network. The shear moduli of RBC membran es have been extensively measured by micropipette aspiration; the values for are in the range of 6-10 N/m (Evans and La Celle,1975; Chien, Sung et al.,1978; Waugh and Evan s,1979; Evans, Mohandas et al.,1984). Techniques based on optial tweezers (Lenormand, Hnon et al .,2001; Dao, Lim et al.,2003), magnetic twisting cytometry (Puig-de-Morales-Marin kovic, Turner et al., 2007), and dynamic membrane fluctuation measurements (Park, Best e t al.,2010; Park, Best et al.,2011) have also reported consistent values for . The shear modulus is sensitive to the environment condition of the membrane. The shear modulus decrea sed as temperature increased from 5 to 45 C (Waugh and Evans,1979). Decreasing pH signifi cantly increase the shear modulus of RBC membranes, but increasing pH above 7.2 does no t cause a significant change (Crandall, Critz et al.,1978). More interestingly, bimodal distributions in the values for were observed in independently reported data (Lenormand , Hnon et al., 2001; Park, Best et al.,2010), suggesting the nonlinear stiffening of spectrin network (Park, Best et al.,2011). Malaria invasion cause significant increases in shear moduli values (Mills, Diez-Silva et al.,2007). Bending modulus. Bending modulus (or fluxural modulus) B of a membrane is determ ined by the energy needed to deform a membrane from its original curvature to some other curvature. The bending modulus B of a 2-D membrane is described as 1 2 3 M B C C C (3) where M is the bending momemt. C1 and C2 are two principle curvature s, and C3 is the

curvature in the stress-free state (Helfrich,1973; Evans,1974). Bending of a 2-D structure involves both area compression and expansion. For a lipid bilayer stru cture, the bending modulus, area expansion (or compression) modulus, and the thickness of the bilayer are related by B=h2K/4, where h is the bilayer separation distance, and K is the com pressibility of the bilayer (Helfrich,1973; Evans,1974). The elastic bending moduli B of lipid bilayer is determined by chemical compositions of the lipids, and there is a bro ad range of reported bending moduli for lipid bilayers (Boal,2002). The elastic bending modu li B of RBC membranes have been measured with various techniques. The values for t he bending modulus measured by micropipette-based studies are in the range of 50 kbT (~ 10-19 Nm) where kb is Boltzmann constant, and T is the temperature (Evans,1983). The bending modulus B of RBCs does not significantly change with temperature (Nash and Meiselman, Measurement Techniques for Red Blood Cell Deformability: Recent Advances 171 1985) or cell Hb concentration for both normal and sickle cells (Evan s, Mohandas et al., 1984). The recent experiments (Betz, Lenz et al., 2009; Yoon, Hong et al., 2009) have also measured that the bending modulus of RBCs is of the order of 50 kbT . However, several other techniques have measured lower bending moduli of RBC membranes. Studies based on measurements of RBC membrane fluctuations reported membrane bending moduli in the range of 10 kbT (~ 10-22 J) (Brochard and Lennon,1975; Zilker, Engelhardt et al. ,1987; Zilker, Ziegler et al.,1992; Park, Best et al.,2010; Park, Best et al.,2011). 2.3.2. Viscous property While the elasticic property of RBC membranes characterizes its resista nce to deformation, the viscous property characterized its resistance to a rate of deforma tion (Hochmuth and Waugh, 1987). The viscous properties of RBC membranes can be determine d by 3-D cytoplasmic viscosity and 2-D membrane viscosity. Cytoplasmic viscosity. The values for the 3-D viscosity of blood plasm a and cytosolic Hb solutions are ~ 1 mPas and ~ 5 mPas, respectively (Cokelet and Meiselman,1968). Cy tosolic viscosity depends on the concentration and viscosity of Hb. By measuri ng the dynamic contour fluctuations of RBC membrane, the cytoplasmic viscosity has bee n obtained in the range of 2-5 mPas (Yoon, Hong et al.,2009). Recently, the dynamic memb rane fluctuation measurements retrieved the cytoplasmic visocity of the RBCs at physiolo gical osmotic pressure as 5-6 mPas, and the cytosol viscosity increases monotonically from with increasing osmolality (Park, Best et al.,2011).

Membrane viscosity. The major source of viscous dissipation in RBC mem branes is the membrane viscosity. During the recovery process after large deformation of RBCs, 2-D membrane viscosity dominates energy dissipation (Evans and Hochmuth,1976) . The 2-D viscosity of lipid membranes n2D can be qualitatively related to a 3D bulk visosity of phospholipid n3D as n2D ~n3D d where d is the thickness of the 2D str ucture. For a typical lipid bilayer, n3D ~ 10 3 mPas and d ~ 1-10 nm, and thus n2D ~ 10 -10 -10 -9 Ns/m. Reported surface viscosities for lipid bilayers are of the order of 10 -10 -10 -9 Ns/m (Waugh,1982; Evans and Yeung,1994). Considering viscous dissipation due to a 2-D membrane viscosity, the modified version of the shear force from Eq. (2) is described as ( 2 2 2 ln 2 , 2 s D T t u c = c )

n +

(4) where t is time (Evans and Hochmuth,1976). Assuming the RBC membrane follows Kel vinVoigt model, Eq. (4) can be simply expressed as 2 / . c D t n u = (5) where tc is the recovery time after large deformation of RBC membrane s (Evans and Hochmuth,1976). Typically, tc ~ 0.06 s at 37C (Hochmuth, Buxbaum et al.,1980), a nd thus if Blood Cell An Overview of Studies in Hematology 172 ~ 0.06 0.6 Ns/m. The 2-D surface viscosity of RBC membranes has been ~ 1-10 N/m, measured by several experiments. Tether experiments performed on model membrane systems, where cytoskeleton structure was absent, obtained a resultant upper bou

nd of 510 3 Ns/m for

2D (Waugh,1982). The diffusion constant of membrane-bound proteins can be

used to calculate the membrane viscosity (Saffman and Delbrck,1975). Using this m ethod, the 2-D membrane viscosity values of RBC membranes have been reported in the range of (0.5-14)x10 -9 Ns/m with various technqiues including fluorescence photobleaching reco very (Golan and Veatch,1980), fluorescence photo-bleaching technique (Kapitza and Sackmann, 1980), and restriction of the lateral motion of membrane embedded prot eins (Tsuji and Ohnishi,1986). 2.4. Mathematical models and simulations Using mathematical models, the mechanics of the membrane cortex structu res has been simulated. Using a worm-like-chain model with surface and bending energ y, the forcedisplancement relations for the spectrin network of RBCs have been des cribed (Discher, Boal et al.,1998; Dubus and Fournier,2006). The viscoelastic properties of the R BC membrane was described using an effective continuum membrane model that simulate s a finitethickess 2-D continuum plane model with in-plane shear modulus and ben ding modulus (Dao, Lim et al.,2003). Recently developed numerical models accurately describes the complex viscoelastic properties of RBCs deformabilty (Fedosov, Caswell et al.,20 10). 3. RBC deformability and blood microcirculation The RBC deformability can influence blood microcirculation since viscosity and f low can be significantly changed by the viscoelastic properties of RBCs. 3.1. Blood viscosity Viscosity of liquid characterizes its resistance to flow under certain deforming force, especially shear stress. Under laminar flow conditions where particles move parallel to adjacent neighbors with minimal turbulence, the fluidity is classified by the dependence of viscosity to shear strain or shear stress: (1) Newtonian fluid, if the v iscosity is independent of shear stress or shear strain so that shear stress is linearly proportional to shear strain, (2) non-Newtonian fluid whose viscosity either decrease (shear-thinning) or increase (shearthickening) depending on the changes of shear stress (Merrill,1969). Blood is non-Newtonian fluid which exhibits shear-thinning behavior. Blo od viscosity decreases at high shear stress due to the deformation of RBCs, while it increase s at low shear stress because RBCs aggregate with each others and form stacked coin structure, called rouleaux (Shiga, Maeda et al.,1990). For normal blood at 37C, blood viscosity at

high shear rate (100~200 s -1 ) is measured as 4 ~ 5 cP, while it increases rapidly up to 10 cP as shear stres s decreases less than 10 s -1 (Rand, Lacombe et al.,1964). Measurement Techniques for Red Blood Cell Deformability: Recent Advances 173 Whole blood is a two-phase liquid consisting of a liquid medium (plas ma) and formed elements such as RBCs, white blood cells, and platelets. Thus, its vi scosity is mainly determined by (1) viscous properties of plasma, (2) the fraction of R BCs in the blood (hematocrit, normal range is 42 47%), and (3) viscoelastic properties of the formed elements. Hematocrit (%) V i s c o s i t y ( m P a s ) 0 20 40 60 80 100 120 10 20 30 40 50 60 0 0.11 s -1 0.51 s -1 2.0 s -1 20 s -1 (b) (a) Aggregation Deformation Elongation and orientation 0.1 1 10 100 1000

1 10 100 Shear rate (s -1 ) V i s c o s i t y ( m P a s ) Normal plasma Normal red cells in plasma Hardened red cells in plasma Normal red cells in fibrinogen-free plasma VC-Veins Ven. AO-Art. Arterioles Cap. Figure 3. (a) Apparent viscosity of blood as a function of shear rates. (b) Hema tocrit effects on blood as a function of shear rates. Modified, with permission, from (Somer and Meiselman, 1993; Baskurt,2007) Plasma is a Newtonian fluid which viscosity in normal condition varies 1.10 ~ 1 .35 cP at 37C, while the viscosity of pure water is 1.0 cP at 20C (Lowe, Drummond et al.,198 0). Plasma proteins such as fibrinogen are thought to cause RBC aggregation by facil itating binding between RBCs. Elevated levels of fibrinogen concentration in pl asma enhance RBC aggregation and thus it increases blood viscosity. Formed elements in the stream lines of laminar flow of blood can be considered as the source of turbulence which significantly increases blood viscosity. Amon g formed elements, RBCs cause the most significant effects since RBCs concentrat ion is the highest among the formed elements in blood. The blood viscosity increa ses as hematocrit increases; the hematocrit effect becomes more severe when sh ear stress decreases since more aggregation of RBCs takes place (Dormandy,1970; Baskurt,200 7). 3.2. Blood flow in microcirculation Microcirculation transports blood to the small vessels in the vasculatu re embedded within organs. The arterial side of vessels in the microcirculation, surrounde d by smooth muscle cells, has the inner diameter of ~ 10 100 m. Capillaries, parts of the microcircu

lation, have only one RBC thick, having the diameter of ~ 5 10 m. Blood flow in microcirculat ion has low Reynold number and thus it is governed by Stokes law (Baskurt,2007). Flow dyn amics in microcirculation requires deep consideration of (1) fluid dynamics i n capillaries, (2) interaction between formed elements with vessel walls, and (3) the structure and network of microvessels. Blood flow in microcirculation is not only determined by the geometric features of blood vessels and hydrostatic blood pressure, but also affected by t he rheological properties. RBC deformability can significantly alter blood flow in microcircula tion (Chien, Blood Cell An Overview of Studies in Hematology 174 1987). The reduction in RBC deformability under certain physiological o r pathological conditions results into the retardation of blood-flow thourgh the micro circulation, which plays important roles in the stages of peripheral vascular insufficiency (Reid, Dormandy et al.,1976); reduced RBC deformability in sickle cell disease and malaria results into occlusions in the microcirculation. 4. Measurement techniques for individual RBCs 4.1. Micropipette aspiration Micropipette aspiration techniques have been extensively used to measure the mechanical properties of RBC membranes (Evans and La Celle,1975; Shiga, Maeda et al.,1990; Hochmuth, 2000). Micropipette aspiration uses a glass micropipette, having inner diameter of 1~3 m, to apply negative pressure onto RBC membranes. When negative pressure is applied, RBC membrane is aspirated into the micropipette and the amoun t of aspiration depends on the viscoelastic properties of cell membrane. Detailed measurement te chniques vary depending on the mechanical property of interest (Fig. 4): (1) m easuring pressure necessary to aspirate the membrane when the aspirated distance is equal to the r adius of the pipette; (2) measuring the ratio between aspirated length of membrane and the ra dius of the pipette in given pressure; (3) measuring pressure required to aspirate whole RBC inside the micropipette (Evans,1973; Evans and La Celle,1975). The area expansion modulus of RBC membranes can be measured by using micropipette aspiration based on Eq . (1); the measured value for K for normal RBCs at room temperature was 450 mN/ m (Evans and Waugh, 1977). In order to measure the shear modulus of RBC membranes, the second method (Fig. 4b) can be used and the shear modulus of the RBCs can be related to the aspirated length (or tongue length) of membrane Dp as, / ~ / ,

p p p D R pR (6) where Rp is the radius of the micropipette, p is the applied pressur e (Evans,1973; Chien, Sung et al.,1978). Using micropipette aspiration, the value for was m easured as 9 1.7 N/m (Evans, Mohandas et al.,1984). (A) (C) (B) Figure 4. Various methods for micropipette aspiration. (A) Measuring pressure P to aspirate the distance same with the micropipette radius. (B) Measuring the ratio between the aspirated length of membrane D and the micropipette radius at a certain negative pressure. (C) Measu ring pressure Pt necessary to aspirate a whole RBC into the pipette. Reproduced, with permissi on, from (Evans and La Celle,1975) Measurement Techniques for Red Blood Cell Deformability: Recent Advances 175 Micropipette asperation technique can measure the bending elastic modulu s B of RBC membranes (Evans,1983; Shiga, Maeda et al.,1990). The value for B depends direc tly on the magitude of the aspiration pressure when RBCs start to buckle and inversely on t he pipette area; measuring negative pressure with varying radius of the pipette c an measure B of RBCs. The measured value for B was 43.5 kBT (Evans,1983). By measurin g the time for recovering original shape from releasing negative pressure, the 2-D vis cosity of RBC membranes can also be obtained by Eq. (5). 4.2. Atomic force microscopy Atomic force microscopy (AFM) is a tip-scanning technique that images topographies of materials in atomic or molecular scale (Binnig, Quate et al.,1986). It uses a ca ntilever with a sharp tip as a probe. Depending on the amount of force to apply or sensitivity, diverse tip shapes are used such as triangular, parabolic, or cylindrical shapes (Weisenhorn , Khorsandi et al.,1993). As a tip scans over a sample with physical contact, the vertical m otion of the tip is monitored by photodiodes which precisely detect small changes in la ser beam position reflected from the tip. As shown in Figs. 5a-b, the topographic image s of RBCs can be obtained in high spatial resolution; cytoskeleton structure of membrane can even be revealed (Kamruzzahan, Kienberger et al.,2004). (a) (b) 10 m 3 m (c) (d) Figure 5. AFM measures RBC topography and deformability. (A) Topogram of normal RBCs. (B) Detailed texture of the RBC membrane surface. (C) Indentation depth measurement. (D) Different forceversus-indentation depth curves of RBCs in various conditions: a. anisocytosis;

s. hereditary spherocytosis; d. G6PD deficiency; and n. normal condition. Reproduced, with per mission, from (Kamruzzahan, Kienberger et al.,2004; Dulinska, Targosz et al.,2006) Since AFM can apply forces to sample surfaces at the nN scales, it can measure m echanical properties of soft materials such as RBCs. The displacement of the st age required for the same deflection of the tip is different between solid- and soft-materials, from which applied forces can be calibrated. For a parabola-shaped or a spherical tip ha ving the radius of curvature Rc, the indentation depth z relates an applied force F and a relative Youngs modulus E* (Weisenhorn, Khorsandi et al.,1993): * 3/ 2 4 ( ) . 3 c R F E z (7) The relative Young s modulus E* is defined as: Blood Cell An Overview of Studies in Hematology 176 2 2 1 2 1 2 * 1 2 1 1 for E E E 2 1 1 1 1 , E E E

(8) where E1, E2, 1, and 2 are the Youngs moduli and Poisson ratios for the simple and the tip, respectively. Since typical value of E2 (~150 GPa for Si3N4 tip) is much greater than that of biological samples (1 ~ 100 kPa), the rightmost equation is valid for biological samples (Radmacher,1997). The Poisson ratio is 0.5 for a perfectly incompressible and el astic material deformed elastically; the Poisson ratio of soft tissues varies with 0. 490 ~ 0.499 (Fung,1993). Youngs moduli of RBCs at various pathophysiological conditions have been measured using AFM. Youngs moduli of healthy RBCs have been obtained to be is 4.4 0.6 kPa (Dulinska, Targosz et al.,2006). RBCs from hereditary spherocytosis, thalassemia (Dulinska, Targosz et al.,2006) and diabetes mellitus (Fornal, Lekka et al.,2006), and sickle cell traits (Maciaszek and Lykotrafitis,2011) have measured. 4.3. Optical tweezers Optical tweezers use highly focused laser beams that transfer linear or angular momentum

of light, in order to optically trap m- and nm-sized dielectric spheri cal particles (Ashkin, 1970). Light refraction at a sample induces linear momentum change, resulting in to trapping forces (Fig. 6). High numerical aperture (NA) objective lens is used to generate a tightly focused optical trap, and its trapping force is governed by the refra ctive indices of sample and surrounding medium, laser power, and sample size; optical force to trap part icles much smaller than laser wavelength can be described by Rayleigh scattering theory, while trapping samples much larger than laser wavelength belongs to Mie scat tering regime (Ashkin, Dziedzic et al.,1986; Svoboda and Block,1994). Optical tweezers have been widely used in many fields such as biophysics and soft matter sciences, where manipulat ion of m sized particles (e.g. cells or microspheres) with a small force (pN s cale) is required (Grier, 2003; Lee and Grier,2007). Gradient Profile Dielectric Sphere focus bright ray dim ray Momentum Change Momentum Change out in out in Light in Light in Light out Light out 5 m (a) (b) (c) Figure 6. Principles of optical tweezers. (A) Laser beam with gradual intensity transfers linear momentum to a microsphere to escape from the beam center. (B) Focused Gaussian beam exert s trapping force. (c) Deformation of a RBC by exerting various optical forces to microspheres attached on the RBC membrane. The change of diameter D in response of optical force F is converted to shear mo dulus of the RBC. Modified, with permission, from (Svoboda and Block,1994; Henon, Lenormand et al. ,1999). Measurement Techniques for Red Blood Cell Deformability: Recent Advances 177 Since optical tweezers can apply forces at the pN level, it has been employed fo r measuring the deformability of RBCs. Measurements of the mechanical properties of RBCs wit h optical tweezers can be done either by applying optical force to microspheres attached to RBCs (Henon, Lenormand et al.,1999; Dao, Lim et al.,2003) or stretching RBCs by diver ging beams

from opposite directions (Guck, Ananthakrishnan et al.,2001). In the fo rmer approach, two silica beads are attached to the opposite sides of a RBC, then these beads are t rapped with a Nd:YAG laser beam ( = 1064 nm) with maximum power of ~ 605 mW, corr esponding maximum optical force is 80 pN (Henon, Lenormand et al.,1999). The ch ange in the projected diameter of the RBC in response of optical force is converted to shear modulus of the RBC using mathematical membrane models. The shear modulus of disco tic RBCs were measured as ~ 10 N/m (Dao, Lim et al.,2003). Using optical twezers sy stem with a high power laser, the shear modulus values of RBCs under large deformation (correspon ding to 400 pN) was measured as 11-18 N/m while initial values were 19-30 N/m, showing hyperelastic constitutive response (Lim, Dao et al.,2004). Optical stret cher, a variant of optical tweezers, uses two diverging laser beams from opposite directio ns (Guck, Ananthakrishnan et al.,2001). Linear momentum changes by two laser beam s apply stretching force to the RBC along the optical axis, and the RBC deformations und er varying optical force are measured from which mechnical properties are retrieved. Optica l tweezers can also be used for detecting membrane fluctuation dynamics of RBCs by imposing a deformation (Yoon, Kotar et al.,2011). 4.4. Magnetic twisting cytometry Magnetic twisting cytometry (MTC) applies both static and oscillating m agnetic field to ferromagnetic microbeads attached to the surface of cell membrane (Wang , Butler et al.,1993). Depending on the applied magnetic field, the microbeads exhibit both translational and rotational motion, which applies torques to the cell membrane. The motion o f beads is recorded by a CCD camera, and the stiffness G and loss modulus G of the membrane c an be obtained by analyzing the displacement of bead in response to appl ied torque. By varying oscillating frequency (0.1 to 100 Hz) and the magnitude of ap plied magnetic field (~ 1 - 10 Gauss), the stiffness and loss modulus of RBC membranes are measured a t different driving frequencies (Puig-de-Morales-Marinkovic, Turner et al.,2007). (a) (c) (b) Figure 7. Magnetic Twisting Cytometry (a) Bright field and (b) Scanning electron microscopy images of RBCs with ferromagnetic beads attached. (c) Principles of magnetic twisting cyto metry. Reproduced, with permission, from (Puig-de-Morales-Marinkovic, Turner et al.,2007) Blood Cell An Overview of Studies in Hematology 178 The torsional stiffness modulus is independent of frequency, whose value is ~ 10 -3

Pa/nm at sinusoidal magnetic field of 1 G, while the loss modulus increases as frequency increases; these values correspond to the bending moduli in the range of 0.2 - 0.8 pNm and th e shear moduli in the range of 6-12 N/m (Puig-de-Morales-Marinkovic, Turner et al.,2007). MTC technique also revealed dramatic increases in the stiffness of malariainfected RBC at the febrile temperature (41C) (Marinkovic, Diez-Silva et al.,2009). 4.5. Quantitative phase imaging Quantitaitve phase imaging technqiues measure the electric field, i.e. amplitude and phase images whereas conventional brightfield microscopy only images light int ensity (Fig. 8) (Popescu, 2011). Employing the principle of laser interference, electric field information of target sample is modulated onto intereferograms recorded by a CCD came ra. By using appropriate field retrieval algorithms, the field information can be re trieved from the measured holograms (Debnath and Park, 2011). Typical interferogram and quantitative phase image of a RBC are shown in Fig. 8b-c. Quantitative phase imag ing techniques can measuring dynamic membrane fluctuations of RBCs (Popescu, Ikeda et al., 2005; Popescu, Park et al.,2008; Park, Best et al.,2011) as well as cellular dry-mass (Popescu, Park et al.,2008). Dynamic membrane fluctuation, consisting of submicron displacement of th e membrane, has a strong correlation with deformability of RBCs and can be altere d by biochemical changes in protein level (Waugh and Evans,1979). By measuring membrane fluctuation of RBCs, bending modulus and tension factor of RBCs were calcualated (Popescu, Iked a et al., 2006). (a) 20 pixels 1.5um [rad] 0 0.5 1 1.5 2 2.5 (b) (c) Figure 8. Quantitative phase imaging. (A) Schematic of the principle of quantita tive phase imaging. (B) Measured interferogram and (C) retrieved phase image of a RBC using quantitative phase imaging. Reproduced, with permission, from (Park, Best et al.,2011). Diffraction phase microscopy (DPM), a highly stable technique for quant itative phase imaging, has been widely used for investigating the deformability of R BCs. Employing common-path laser interferometry, DPM provides full-field quantitative ph

ase imaging with unprecedented stability (Park, Popescu et al.,2006; Popescu, Ikeda et al.,2006). DPM measured spatiotemporal coherency in dynamic membrane fluctuations (Popes cu, Park et al.,2007), shear modulus for the RBCs invaded with malaria-inducing par asite Plasmodium falcifarum (Pf-RBCs) (Park, Diez-Silva et al.,2008), and effective visco elastic properties of RBCs (Wang, Ding et al.,2011). Recently, integrated with a mathematical model, DPM provide the mechanical properties of individual RBCs from membrane fluc tuations: shear modulus, bending modulus, area expansion modulus, and cytoplasmic viscosity (Par k, Best Measurement Techniques for Red Blood Cell Deformability: Recent Advances 179 et al.,2010). Several alterations in the deformability of RBCs have been studied using DPM, including the effects of ATP (Park, Best et al.,2010; Ben-Isaac, Park et al.,201 1), the nonlinear behavior of RBC deformability in response to different osmotic pressure (Park, Best et al., 2011), and malaria egress mechanism (Chandramohanadas, Park et al.,2011) . Employing spectroscopic quantitative phase imaging, cytoplasmic Hb concentration th at is tightly related to the cytoplasmic viscosity, can also be simultaneously quantified (Par k, Yamauchi et al.,2009; Jang, Jang et al.,2012). In addition, polarization-sensitiv e quantitative phase microscopy will be potentially used for the study of sickle cell disease and its implications to RBC deformability (Kim, Jeong et al.,2012). 4.6. Dynamic light scattering Dyanmic light scattering signals provide rheological information about R BCs (Tishler and Carlson, 1987; Amin, Park et al.,2007). Although dynamic light scatteri ng have been extensively used in combination with ektacytometry, it provides averaged signals from many RBCs. Thus it is difficult to access the deformability of individual RBCs. Fourier transform light scattering (FTLS) provides both static and dyna mic light scattering signal from individual cells. Light field, measured by quantitative pha se microscopy or digital holographic microscopy, contains both amplitude and phase inform ation, and thus far-field light scattering pattterns can be directly calculated by numerically p ropagating the measured field technically applying Fourier transformation (Ding, Wang et al.,20 08). FTLS technique can provide both morphological and rheological information abo ut individual biological cells. By analyzing dynamic light scattering signals measured by FTLS, one can qualitatively access the membrane surface tension and viscosity of indi vidual RBCs (Park, Diez-Silva et al.,2010). Due to its capability of measuring light scat

tering signals from individual cells with high signal-to-noise ratio, FTLS has been employe d to study several pathophysiological effects to the deformabiltiy of RBCs, including malar ia infection (Park, Diez-Silva et al.,2010), depletion of ATP (Park, Best-Popescu et al.,20 11), and sickle cell disease (Kim, Higgins et al.,2012). 5. Measurement techniques for blood rheology 5.1. Blood viscometer and ektacytometry Blood viscometer measures the viscosity of blood over a wide range of shear rates. Blood viscometer controls either shear stress or shear rate of blood using rational objects. Stresscontrolled blood viscometer applys a constant torque which corresponds to constant rotational speed in a well-designed rotational rheometer. In a rate-con trolled system, applied torque is controlled by a stress-sensing device so that a constant rotat ional speed is achieved. Viscometers can be classified by the cylinder shape: a conce ntric cylinder, a cone plate, and a parallel plate viscometer (Fig. 9). Cylinder-type viscometer uses two concentric cylinders: a rotational inn er cup and a stationary outer cylinder. Time-independent shear rate can be precisely measured by Blood Cell An Overview of Studies in Hematology 180 concentric cylinder viscometer (Nguyen and Boger,1987). Cone and plate viscomete r rotates an inverted cone having very shallow angle (~ 5); the shear rate unde r the plate is maintained consistently and independent of a flow curve. Parallel plate viscometer is a simplified version of the cone and plate viscometer and has a advanta ge of flexible space between two parallel plates. The viscous fluid can confined and rotate d in narrow space between two circular parallel plates (Gent,1960). Figure 9. Schematic diagrams of typical viscometers. (a) Concentric cylinder vis cometer (or Couette viscometer), (b) cone and plate viscometer, and (c) parallel plate viscometer. ( d) Experimental setup of ektacytometer. Ektacytometer employes a laser diffraction technique with blood viscomet er in order to measure RBC deformabiltiy. Conventional blood viscometer applys controlle d shear stress to the RBCs in the blood viscometer, and deformability of RBCs can be measured f rom laser diffraction pattern. Ektacytometer consists of a concentric rotational o uter cup and a stationaly inner cylinder; outer cup produces varying shear stress fiel d on blood (Fig. 9d). Through the measurement of diffraction patterns of the laser passing t hrough the blood, RBC deformability can be obtained. The RBC deformation is quantitatively calcula ted from

the scattered laser beam intensity pattern. Under a certain shear rate, isointe nsity curves in the intensity pattern of the scattered beam will show elliptical shape s, which represent elliptically deformed RBC population (Bessis, Mohandas et al.,1980). Fro m the measured isointensity curves, a deformaion index (DI) of RBCs is calculated as , l s DI l s

(9) where l and s are distances along the long- and short- axes in the elliptical isointensity curves. DI values are measured at different angular velocities (and thus differe nt shear rate) of the outer cyliner in the ektacytometer. Ektacytometer is a simple and effecti ve technique to measure the deformability of RBC population, and it has been widely used for the study pathophysiology of RBCs. Abnormal deformability in RBCs from patients w ith hereditary pyropoikilocytosis, hereditary spherocytosis, and Hb CC disease were stu dies by ektacytometer (Mohandas, Clark et al.,1980). 5.2. Microfluidic device technique Microfluidic device has emerged as a promising tool to precisely contr ol fluids with small volumes of fluid containing samples and reagents in channels with dime nsions of 10-100 Measurement Techniques for Red Blood Cell Deformability: Recent Advances 181 m. Microfludic device reduced space, labor, and measurement time on num erous experiments, and also enabled precise control and manipulation of the small volume of samples. Microfludic device has been used to study the deformabiltiy of RBCs. Mi crofluidic channel with a few micrometer diameter mimics micro-capillary structure in blood circulation system. Rheological behaviors of Pf-RBCs were studies in mi crofludic devices (Shelby, White et al.,2003). Microfludic device was used to induce lar ge deformation of RBCs and its mechanical behavior was studied (Fig. 10) (Li, Lykotrafitis et al., 2007) . 0 s 0.4 s 0.8 s 1.4 s Figure 10. (a-d) Snapshot showing the fluidization of a healthy RBC when it pass es through a microfluidic channel. Reproduced, with permission, from (Li, Lykotrafitis et al. ,2007). For the study of sickle cell disease, microfluidic device has been us ed to measure the resistance change rate of blood flow under the sudden change of oxyge n concentration

(Wang, Ding et al.,2011). Recently, microfludic channels with obstracles have me asured the deformabiltiy of malaria infected RBCs in high throughput (Bow, Pivkin et al.,2011; DiezSilva, Park et al.,in press). 5.3. Filtration test Filtration test measures RBC deformabiltiy using a membrane filter with holes of diameter of 3-5 m (JANDL, SIMMONS et al.,1961). By applying a negative pressure, whole blo od is subject to pass through holes in the membrane filter. The deformabilit y of RBCs can affect the speed of flow. RBC deformabiltiy can be calculated from either the flow time or the volume of blood filteres in a certain amount of time (~1 min). Since the f ilteration test requires for a relatively simple instrument and provides clinically relevant results with high reproducibility, it has been widely used in various studies related to RBC deformability, includi ng the effects of diabetes (Juhan, Buonocore et al.,1982), spesis (Baskurt, Gelmont et al.,1998), sickle cell disease (JANDL, SIMMONS et al.,1961), and oxygen radical (Srour, Bilto et al.,20 00). 6. Pathophysiological coditions affecting RBC deformability Mechanical properties of RBCs is crucial for cell physiology of RBCs. This essential deformability is in turn affected by various physiological and pathological cues . 6.1. Temperature Temperature plays important roles in RBC deformabilty. The elastic prop erties of RBC membrane were investigated as function of temperature using the micropi pette aspiration Blood Cell An Overview of Studies in Hematology 182 technique (Waugh and Evans,1979). Over the temperature range of 2-50 C, both the shear modulus and the area expansion modulus decrease as temperature increase d; the changes were -6 10 -2 N/m C and 6 10 3 N/m C, respectively. Due to the structual transitions of proteins occuring at certain critical tempertures, RBC deformabiltiy exh ibits complex behaviors. At the transition temperature, RBCs undergo a sudden change from blocking to passing through a micropipette with a diameter of ~ 1 m (Artmann, Kel emen et al.,1998). Body temperature or febril temperature are particularly important in various pat hophysiology of RBCs. Membrane fluctuation measurements using DPM revealed that the shear mod ulus of Pf-RBCs significantly increases as temperature increases from body tempe rature to febrile temperature whereas healthy RBCs do not show noticible changes (Park, Diez-Silva et al., 2008). MTC study also reported that Pf-RBCs becomes significantly stiffened with

temperature compared to the healthy RBCs (Marinkovic, Diez-Silva et al.,2009). 6.2. Morphology RBCs exhibit diverse morphological features depending on pathophysiologic al conditions (Diez-Silva, Dao et al.,2010). A healthy human RBC shows a smooth and biconcave disc shape (discocyte). However, atypical shapes of RBCs can be found under abnormal pathophysiological conditions, including acanthocyte, stomatocyte, schizocy te, and tear drop cells (Kenneth,2010). Our understanding of what determines RBC mor phology and how RBC morphologies are related to the mechanics of RBCs still remains incomple te. One of the hypotheses describing RBC morphology is the bilayer-couple hypothesis (Sh eetz and Singer,1974); small changes in the relaxed area difference between two layers of phospholipids. Later, this model can be used for explaning stomatocytedi scocyte echinocyte morphological transitions (Lim HW, Wortis et al.,2002). (a) (c) (d) (e) (f) (b) Figure 11. (a-c) Topographies of (a) discocyte, (b) echinocyte, and (c) spherocy te. (d-f) Retrieved mechanical properties: (d) bending modulus , (e) shear modulus , and (f) area modu lus KA of discocytes (DCs), ATP-depleted discocytes [DCs (-ATP)], echinocytes (ECs), and s pherocytes (SCs). Reproduced, with permission, from (Park, Best et al.,2010) Measurement Techniques for Red Blood Cell Deformability: Recent Advances 183 Increased deformability of RBCs in abnormal shapes has been reported w ith various experimental methods. Ektacytometer measured increased DI values for SCs and ECs that were induced by 2,4-dinitrophenol treatment (Meiselman,1981). Recently, u sing DPM, the mechanical properteis of RBCs in different morphologies were quantified from dynamic membrane fluctuations (Park, Best et al.,2010). Bending modulus and are a expansion modulus of ECs and SCs showed significantly high values compared to n ormal DCs. The shear moduli values show bimodal distributions (Fig. 11e), suggesting t wo independent conformations of the spectrin network: a soft configuration ( ~ 7 N/m) and a stiff one ( ~ 12 N/m). Aging of RBCs also cause significant morphological alterations: aged RBCs exhibit decreased surface area and volume (Waugh, Narla et al.,1992). The aged R BCs were found by ektacytometry to have decreases shear modulus mainly because of decreased surface area and increased cytoplasmic viscosity. 6.3. Osmotic pressure

Different osmolalities of extracellular medium can bring significant cha nges in RBC shape and thus deformability. At normal physiological condition (295mOsm/kg), RBCs maintain their biconcave shapes. In hypotonic medium, RBCs are swollen due to water intak e. At the osmotic pressure less than 100mOsm/kg, most of RBCs are lysed. In the hypertonic case, RBCs lose its volumes, which result in significant cell shrinkage. Although the total amount of Hb molecules in RBCs, or the mean corpuscular Hb (MCH), does not significantl y change at different osmolality, Hb concentration can be considerably changed d ue to water influx and efflux. RBCs exhibit the maximum deformability at physiological con dition; under either hypertonic or hypotonic condition, the deformability of RBCs dec reases (Mohandas, Clark et al.,1980). 0 100 200 300 400 500 0 20 40 60 80 100 D I ( % ) Osmolarity (mOsm/kg) 40 50 60 70 0 100 200 300 400 Osmolarity (mOsm/kg) M e m b r a n e f l u c t u a t i o n

500

( n m ) 0 200 0 5 10 15 0 20 40 60

400

600

800

[ m P a . s ] , K A [ m N m 1 ] [ m N / m ] , [ x k B T ] K A (a) (b) (c) Osmolarity (mOsm/kg) Figure 12. (a) DI of RBCs as a function osmolality, measured by ektacytometer. M odified, with permission, from (Mohandas, Clark et al.,1980). (b) Membrane fluctuations of RBC s as a function of

osmotic pressure, measured by DPM. (c) Retrieved mechanical properties of RBCs f rom membrane fluctuations. 20 individual RBCs were measured at each osmotic pressure. Modifie d, with permission, from (Park, Best et al.,2011). A recent study, based on membrane fluctuation measurements, retrieved m echanical properties of RBC membrane under diffferent osmolarities (Park, Best et al.,2011 ). Although membrane fluctuation or deformability decreases either in hypotonic or hypertoni c case; the Blood Cell An Overview of Studies in Hematology 184 reasons for the decreased deformability are different. Under hypotonic cases, both shear moduli and area expansion moduli increase, suggesting nonlinear stiffeni ng in streached membrane structure. Under hypertonics cases, other mechanical parameters are not significantly changed except that cytoplasmic viscosity increases. 6.4. ATP effect The presence of adenosine 5-triphosphate (ATP) is important in maintaining the bi concave shape of RBCs and also significantly affects the RBC deformability. In the absence of ATP, RBCs loss its biconcave shapes and become flattened echinocytes (Sheetz and Singer,1977). The metabolic state of RBCs, determined by the level of ATP, is cruc ial for maintaining cellular deformability. When celullar ATP level decreases, the stored RBCs signi ficantly lose the deformability (Weed, LaCelle et al.,1969). Micropipette aspiration t echnqiue measured mechanical properties of RBCs upon ATP depletion; shear modulus and el astic area compression modulus increase by 17% and 14%, respectively (Meiselman, Evans et a l.,1978). Decreased membrane fluctuation in the absence of ATP was first observe d by using darkfield microscopy (Tuvia, Levin et al.,1998). Membrane fluctuation measurements s tudied the effects of ATP to the mechanical properties of RBCs (Betz, Lenz et a l.,2009; Park, Best et al.,2010). Analysis on dynamic membrane fluctutions further showed non-G aussian dynamics in the presence of ATP, suggesting the metabolic remodelling in the lipid membrane and spectrin network structure (Park, Best et al.,2010). ATP-d ependent RBC deformability has been also studied using theoretical models (Gov and Safran,2005; BenIsaac, Park et al.,2011). 6.5. Malaria: Parasite invasion Pathogenesis of malaria causes structural and mechanical modifications t o the host RBCs. During intra-erythrocytic development, the malaria-inducing parasite expor ts proteins that interact with the host cell membrane and spectrin cytoskeletal network (Simmons, Woollett

et al.,1987). Parasite-exported proteins modify material properties of host RBCs, resulting in altered cell circulation. Despite the genetic and biochemical approaches identif ied, proteins exported by parasites have remained elusive as well as the mechanism and effect of these proteins on the host cells. Pf-RBCs exhibits significantly decreased deformability. Microfluidic techn ique demonstrated the occlusion of small channels by infected RBCs (Shelby, White et al .,2003). Optical tweezers technique measured that membrane shear modulus continuously inc reases as the disease progesses during the intraerythrocytic cycle (Suresh, Spatz et al.,2005). Employing genetic knock-out assay, the effects of RESA protein to the host RBC deformabilt iy has been studied (Mills, Diez-Silva et al.,2007). Membrane fluctuation measurement also showed increased shear modulus of malaria-invaded RBCs (Park, Diez-Silva et al .,2008). Recently, the loss of deformability in the malaria-invaded RBCs has been simulat ed using multiscale numerical models (Fedosov, Lei et al.,2011). Measurement Techniques for Red Blood Cell Deformability: Recent Advances 185 (a) (b) Healthy RBC Pf-RBC (Schizont stage) sporozites Inside the liver Liver merozoite Inside Red Blood Cells Ring gamatocyte gamates Ookinete Oocyst Salivary gland sexual cycle Inside the mosquito Schizont Hemozoin Parasitophorus vacuoles Trophozoite Figure 13. Malaria parasite life cycle in human body. Reproduced, with permissio n, from (Cho, Kim et al.,2011). (b) Optical images of a healthy RBC and a Pf-RBC (schizont stage) str etched by optical tweezers. Reproduced, with permission, from (Suresh, Spatz et al.,2005). 6.6. Genetic diseases: sickle cell disease Sickle cell disease, characterized by abnormal rheological properties an d a sickle-shape of

RBCs, is an autosomal recessive inherited blood disorder. A point mutation in -gl o in gene encoding Hb results in the production of sickle Hb (HbS) instead of normal Hb (HbA) (Barabino, Platt et al.,2010). Under deoxygenated conditions, HbS molecu les becomes selfassembled and grows to fibers inside RBCs up to a few micrometer len gths. Due to these highly stiff HbS fibers, sickle RBCs have elongated- and crescent-shape at deoxygenated conditions and the deformabiltiy of sickle RBCs significantly decreases. Sickle RBCs have different morphologies depending on its density (Kaul, Fabry et al.,1983; Evans, Mohandas et al.,1984). After repeated sicklings, a fraction of RBCs becomes irreversibly sickled cells and they exhibit the most significant loss in deformability. While Hb concentrations does not affect static rigidity of normal RBCs, stat ic rigidity of sickle RBCs depends on Hb concentration (Evans, Mohandas et al.,1984). Earlier studies using ektacytometry and filteration techniques reported decreased deformability of sickle RBCs even under oxygenated conditions (Chien, Usami et al.,1970; Klug, Lessin et al., 1974). Quantitative phase microscopy measured decreased membrane fluctuations for sickl e RBCs (Shaked, Satterwhite et al.,2011). FTLS showed significantly altered ela stic and viscous membrane properties in sickle RBCs (Kim, Higgins et al.,2012). Recently , four important mechanical properties of sickle RBCs were retrieved with memebrane fluc tuations measurements (Byun, Higgins et al.,under review). Using AFM technique, decresed deformability was measured in sickle RBCs (Maciaszek, Andemariam et al. ,2011). RBCs in sickle cell trait, having only one abnormal allele of the Hb beta gene, also exh ibit decreased deformability compared to healthy RBCs (Maciaszek and Lykotrafitis,2011). Blood Cell An Overview of Studies in Hematology 186 (b) (c) (d) 5 m 0 1 2 3 [m] (a) Spectrin Membrane Cytoskeleton Normal Cell membrane Unexposed transmembrane segment Abnormal cell membrane Hb

polymer Exposed transmembrane segment PS flip PS Transmembrane protein Transmembrane protein Figure 14. (a) Illustration showing structural modifications inside a sickle RBC . Modified, with permission, from (Barabino, Platt et al.,2010). (b-d) Typical morphologies of si ckle RBCs measured by DPM; (b) echinocyte, (c) discocyte, and (d) crescent-shaped irreversibly sickled cell. Reproduced, with permission, from (Kim, Higgins et al.,2012). 6.7. Other conditions altering RBC deformability There are still many pathophysiological conditions that affect the defo rmabiltiy of RBCs, which are not covered in the above sections. Several hereditary disord ers associated with formation of RBC membrane structures and Hb protein can result into a ltered RBC deformability. Thalassemias, causing the formation of abnormal Hb molecu les due to the limited synthesis of the globin chain, results into loss of RBC defor mability. Thalassemia is thus often accompanied by the destruction of a large number of RBCs in spleen, accompanying with the enlargement of spleen. In addition, abnormal Hb molecules in thalassemia often caues the formation of Heinz bodies, inclusions within RBCs co mposed of denatured Hb, and it causes the local rigidification of RBC membrane (Reinhart, Sung et al., 1986). Ektacytometer study measured that RBCs in hereditary spherocytosi s showed markedly diminished deformability while their surface/volume ratio was n ormal (Nakashima and Beutler,1979). RBCs from the patients with homozygous he reditary elliptocytosis exhibits marked abnormalities in deformability and membrane fragi lity; these changes are closely related to the reduced levels of band 4.1 proteins (Tchernia , Mohandas et al.,1981). Since band 4.1 plays an important role in the modulation o f spectrin-actin interaction, it has been suggested to be closely related to the maint enance of normal Measurement Techniques for Red Blood Cell Deformability: Recent Advances 187 membrane shape and deformability. In addition, it will be possible to study RBC deformability in vivo in the near future, by directly imaging and man ipulating RBCs through highly scattering skin tissues. Recent works have demonstrated that it is indeed possible to control and suppress multiple light scattering (Vellekoop,

Lagendijk et al.,2010; Vellekoop and Aegerter,2010; Mosk, Lagendijk et al.,2012; Park, Park et al.,2012 ; Park, Park et al.,2012). In diabetes mellitus, RBCs exhibit reduced deformability (McMillan, Utterback et al.,1978), which has been attributed to the changes in lipid composition of t he membranes. This impaired RBC deformability in diabetes occurs before significant h istological vascular changes (Diamantopoulos, Kittas et al.,2004). RBCs from the patients with diabet es mellitus undergoes substantial alterations in the lipid composition, membrane pro teins, and Hb molecules. Saturated fatty acid levels in diabetes mellitus were signif icantly elevated compared to normal RBCs while the amount of polyunsaturated fatty acids were dec reased in diabetes (Prisco, Paniccia et al.,1989). 7. Conclusion and outlook We have highlighted techniques for studying RBC deformabilty. Due to v arious deformability test techniques developed in the last years, our understa ndings on pathophysiology of RBCs have been significantly improved. Recent advances have e nabled the precise measurements of various biomechanical properties of RBCs un der systemically controlled conditions that mimic complex in vivo physiological environme nts. However, three major technical issues should be resolved in order to bring a much signifi cant impact. First, the molecular mechanisms on RBC deformability should be directly accessed and studied. Employing biochemical assays such as molecular imaging and gen etic knock-out methods, the relation between molecule-level changes and cellular-level deformability alterations can be studied. Second, such measurements should be perform ed at individual cell levels. Profiling mechanical, chemical, and biological properties at the ce llular levels and their correlations may allow accessing to unexplored regimes of disease s mechanisms. Third, interactions between cell-to-protein, cell-to-cell, and cell-to-ves sel should be considered, since these interactions can be affected and in turn modif y RBC deformability. As more knowledge is gained about the pathophysiology of RBCs and the ir circulation through biomechanical studies, the potential for the development of nov el diagnostic and treatment strategies for various RBC-related disease will become real a nd answer to important questions in hematology. Author details Youngchan Kim, Kyoohyun Kim and YongKeun Park Department of Physics, Korea Advanced Institute of Science and Technology, Daeje on, South Korea Acknowledgement The authors wish to acknowledge supports from KAIST, KAIST Institute for Optical

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2012 Mohamed, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Use of Microfluidic Technology for Cell Separation Hisham Mohamed Additional information is available at the end of the chapter http://dx.doi.org/10.5772/50382 1. Introduction 1.1. Motivation for cell sorting The cell is the basic functional unit within a tissue or an organ. Methods that can be used to probe the cell, so as to understand, or even manipulate its interrelated process es, pathways, and/or overall functioning, are of great scientific and commercial value . Research efforts in molecular biology, biochemistry, and biotechnology over the last two decades hav e created high demand for efficient, cost-effective, cell enrichment, isolation, a nd handling methods. Cell studies can be performed on continuously growing cell lines, many of which are commercially available, in tissue culture, or on cells obtained from intact tiss ues or isolated from blood [1-3]. Mammalian cells are highly heterogeneous in structure, function, and ch aracteristics. However, many types of biochemical, pharmaceutical, and clinical studies , such as immunophenotyping, studies of the cell cycle, cell proliferation, or ap optosis, and other specialized cell analyses require a homogenous population consisting of a single cell type; as the analyte. Only then can the results deemed accurate and specific t o the cell type under investigation [4, 5]. Accordingly, techniques to separate cell types in a heterogeneous cell population are of immense practical value. Any such efforts are furthe r complicated when the target cell is rare within a population such as in many cancer

and prenatal diagnosis applications. The more stringent the requirements for specific and prec ise cell separation, the greater the degree of accuracy and reproducibility required in the technology that underlies the separation method [6-8]. Recent progress in microfabrication, technologies developed and utilized by the integrated circuits (ICs) industry, is being exploited to biomedicine, spawning a relativel y new field of research that has become known as BioMEMS. Microfabricated devices have already had a Blood Cell An Overview of Studies in Hematology 196 broad range of biomedical and biological applications [9, 10]. These d evices can be manufactured with a reproducible accuracy of less than 1 micrometer (1/ 100 th the diameter of a human hair). In the last decade, microchips have been used in a huge range of devices and contexts: microscale sensors for surgical instruments, monitoring of physiological activities, drug discovery and delivery, DNA amplification, and electrop horesis, as well as cell sorting, the application discussed in this chapter [11-16]. Microfluidic technology, a subcategory of BioMEMS, is a set of techniques and pr ocesses for making devices to precisely control and manipulate fluid in a geometrically smal l channels; sub- to few hundred- micrometers in size. Microfluidic is multi-discipl inary; developing a device with biological utility requires the integration of knowledge and techniq ues from the fields of engineering, biology, physics, and chemistry. Such microfabric ated devices are used to study biological systems and to generate new insights into how these sys tems work. Conversely, the biological knowledge gained through micro/nano - scale analyses can lead to further improvements in device design. BioMEMS is a challenging fie ld because the materials, and chemistries, important for biological microfluidics applications are so diverse [17, 18]. The objective of the present chapter is to introduce the principals o f cell sorting by microfluidic technology, and to discuss its strengths, current limitatio ns, and current and potential applications, with illustrative examples from the literature a nd from the authors laboratory. 1.2. Challenges in cell sorting Cells of different types have characteristic sizes, shapes, densities, and arrays of surface molecules that can be exploited for sorting. For example, red blood cells (RBCs) are the cells responsible for delivering oxygen to the tissues. RBCs have to squeeze through capillaries

and therefore are relatively small, approximately 6-8 m in diameter, an d flexible. Mature RBCs are anucleate; the maturing cells lose their nuclei before leavin g the bone marrow. Loss of the nucleus leads to cell membrane collapse, conferring the characterist ic biconcave shape of RBCs, and giving the cell a greater surface area to volume ratio than of spherical cells. These physical features allow easier movement of oxygen (O2) an d carbon dioxide (CO2) through the membrane. RBCs are composed mainly of hemoglobin, whose iron a toms temporarily bind O2 molecules in the lungs, and then release the molecules throu ghout the body. RBCs have the highest density of any cell type in blood. All of the above characteristics can be used for separation: for example size can be u sed to separate RBCs since they are smaller and more flexible than white blood cells (WBCs). RBCs high density causes them to spin down to the bottom of a test tube after density gradient cen trifugation. The high iron content gives RBCs intrinsic magnetic properties and can be used f or magnetic separation [19]. In other cases, a cell changes its shape or size as a result to a disease or cha nge in function. In sickle-cell disease, a genetic blood disorder, RBCs assume an abnor mal, rigid, sickle Use of Microfluidic Technology for Cell Separation 197 shape. The change in shape and the increased rigidity of the RBCs le ad to obstruction of capillaries, thereby restricting blood flow to organs, causing anemia a nd other sickle cell crisis, and decreasing life expectancy. Cancer is a second example of such changes [20]. Malignant tumor cells differ from benign tumor cells in structure, growth rate, invasiveness, and their ability to metastasize. Benign tumor cells grow slowly, push ing neighboring tissues away while staying well encapsulated. In contrast, malignant tu mor cells grow rapidly, invade neighboring tissues, and may metastasize [21]. Furthermore, they generally are irregular in shape, and exhibit a more rugged or ruffled surface appearance than do normal cells [22, 23]. Cancer is detected and diagnosed based on physical change s present in tissues and cells. Nuclear changes such as an increase in size, deformation, and a change in internal organization are among the most universal criteria for detecting malign ancy [24-25]. These changes may reflect alterations in the nuclear matrix and the c onnections of the nuclear matrix to or via the cytoskeleton. Cancer cells modify their morphology, principally by increasing the size of the nucleus, before they become invasive, i .e., in dysplasia and carcinoma in situ. The nucleus of a dysplastic cell can be up to four times as l

arge as that of a non-dysplastic cell. Accordingly, light scattering microscopy has been u sed to distinguish normal, dysplastic, and cancerous epithelial cells in a range of tissues includi ng esophagus, colon, bladder, and oral cavity [26]. However, this technique can only succeed i f a sufficient number of cells are available for analysis. Additionally, most cell types have characteristic complements of surface molecul es. Among the most useful for identification is the cluster of differentiation ( CD), or cluster of designation. CD molecules have roles in signaling and adhesion processes, and th e specific compliments of CD molecules is a determinant of the specific function of the cell. Cell populations are usually denoted by a pattern of + or a -scorings; indicating the pre sence or absence of specific CD molecules. For example, a nomenclature of CD34 + , CD31 denotes a cell that expresses CD34 but not CD31. Use of more than one marker can make the cell selection very specific. However, not all cell types have a known spe cific surface marker [27]. For some cells such as cancer cells, not many cell-surface markers ha ve been identified. In such a situation, some techniques have used a surface marker directed against surface membrane antigens that are expressed in tissue of origin, which is mo st often epithelial. Thus, detection of these tissue-specific surface markers in the blood stream is suggesting that cancer cells have detached from the tumor. Some techniques attemp ting to isolate circulating tumor cells (CTCs) from whole blood have used EpCAM, an e pithelial cell adhesion molecule that is overexpressed in epithelial carcinomas such a s colon and breast. However, published studies have shown inconsistent frequency of EpCAM e xpression in breast cancer, from as low as 35% to as high as 100% [28-31]. Thus, EpCAM cannot be considered a CTC specific marker. Additional bio-molecules such as DAPI (nucleic acid stain) and antibodies against cytokeratin (expressed on the epithelial cell membrane) and CD45 (expressed on the majority of hematopoietic cells) must be used if captured cells are to be positively identified as CTC. Therefore a cell with the phenotype EpCAM + , DAPI + , CK + ,

and CD45 is considered a CTC. Blood Cell An Overview of Studies in Hematology 198 The number of available cells of interest poses and additional challen ge; in some applications such as the isolation of CTCs from cancer patient or fet al nucleated red blood from maternal circulation, only 1-2 cell are available per milliliter (mL) of whole blood. Specificity is problematic for either method, due to the lack of a c ell-specific surface marker/antibody to exclusively detect CTCs or fetal cells [32]. Despite great successes, cell sorting techniques are not ideal and therefore rem ain an active area of research. In addition to sensitivity and specificity requiremen ts, an ideal technique should not be overly labor intensive, should be automated and quantita tive, the results should predict clinical outcome, and help the physician personalize the rapeutic options. Automating sample preparation and handling would minimize human errors. Integrat ion of preparation, cell sorting, and post processing will lead to more costeffective instruments, and alleviate the need for trained personnel and infrastructure. Microf luidic technology enables the precise control over the cell microenvironment during separ ation, scales down the analyses to very small volume of blood, and has the potential for high-throu ghput to cell separation and analyses. 2. Microfluidic technology 2.1. What microfluidic has to offer? Microfabrication enables the deposition and etching of thin layers (angstrom to micrometer) of different materials on silicon or glass substrates. These layers ca n be patterned with accuracy and high resolution, down to the nanometer level using lithography. Lit hography is the technique used to transfer a pattern from a mask to the subs trate to control the location of the deposition of the next layer or the etching of an e xisting layer on the substrate. Microfabricated devices have been used in a broad range of biomedical and biological applications. In the last decade, microchips have been used in microsc ale sensors for surgical instruments, to monitor physiological activities, in microfluidi cs applications such as drug discovery and delivery, cell sorting, DNA amplification, electr ophoresis, etc. Of relevance to this chapter are the microchips for blood fractionation and cell so rting. Micromachining consists of a series of robust, well controlled, and we ll characterized processes that enable the fabrication of microfluidic devices. Such devices can be made cost-

effective by the use of any of a wide range of biocompatible polymer s or plastics and bulk processing (mass production). The miniaturization of reactions and assay s confers many advantages over macro scale techniques beyond the obvious reduction in q uantities of reagents and materials required per test. The scaling down of volumes results in higher surface to volume ratio: a cube with side length L will scale down by a factor o f L 3 , while the surface area will scale down by only a factor of L 2 . Thus, miniaturization results in higher reactivities, shorter diffusion distances, smaller heat capacities, faster heat exchange, shorter assay times, and better overall process control, as well as the capability to in tegrate multiple steps and to achieve massive parallelization on-chip. Additionally, microfluidic devices are Use of Microfluidic Technology for Cell Separation 199 safer than macro platforms due to the smaller chemical quantities used and hence the lower stored energies. A microfluidic device that performs one assay is typicall y referred to as Lab-on-a-chip, while a device that integrates more than one step i s referred to as micro total analysis system (TAS) [18]. 2.2. Introduction to microfabrication This section will briefly describe the basic concepts in the microfabr ication of microfluidic devices. Microfabrication is the already subject of many textbooks and the inter ested reader can consult one of these for more in-depth details [33, 34]. Microfabrication is the technology developed by ICs industry to make devices and circuits with feature sizes as sma ll as 14 nm in research, and 45 nm in production. Among these are the microproces sors and the electronic components found in computers, smart phones, television sets, and major other electronic products. Microfabrication is also used for MEMS devices (mi cro-electro mechanical systems), devices that include a movable part and can be used for sen sing. The airbag sensor used to deploy an airbag in a vehicle, the pressure sensors inside car tires, and the electronic compass in a smart phone are all examples of MEMS dev ices that we unknowingly use every day. BioMEMS, microfluidic devices, and TAS are a ll microfabricated devices similar to MEMS but customized for biological a nd chemical applications. Creation of a a new microfluidic device includes design of the channel(s), fluid inlet(s) and outlet(s), using a CAD (computer-aided design) software. These softwares , such as

CoventorWare [35], ANSYS CFD [36], COMSOL Multiphysics [37], can be also used for simulation of the various design parameters such as device dimensions, heat transfer, and flow conditions, therefore narrowing the design space range in which o ptimum performance should be obtained. The pattern of channels is laid out with the CAD software; this is the 2D design of the device. The depth of the channel will be determined by the etching time. The drawings are transferred to a mask, typically a gla ss or quartz plate (transparent to UV light), covered with chrome. The chrome is etched (removed) w here the UV light will expose the photoresist. The mask, similar to a stencil, transfers the pattern to the photoresist. The device is built on a substrate, which is a silicon, glass o r quartz wafer, or a regular glass slide. After substrate cleaning, the photoresist, a ph otosensitive polymer, is applied. Photoresist is dispensed onto the substrate and it is spun a t high speed (20004000rpm) to create a thin (1-100 micrometer), uniform and smooth layer. The mask is placed in contact with the substrate and exposed to UV lights on a mask aligner. The ph otoresist is developed in a developer solution specific for it, and is removed from areas exp osed to UV light (positive photoresist). The channels can be etched, with substrat e material being removed from areas unprotected by the photoresist. Etching can be eith er wet (using chemicals) or dry (plasma etching). Deep reactive ion etching (DRIE) i s a plasma etching technique typically used to achieve deep channels with vertical side w alls. Use of DRIE is necessary if the etched substrate will serve as a template for moldin g devices in polymer such that the polymer mold can be peeled off the substrate. At this stage, the d evice can be Blood Cell An Overview of Studies in Hematology 200 sealed by the bonding of a top piece to the substrate. Top pieces are typically transparent, to permit observation under the microscope. Silicon can be bonded to glas s by thermal or anodic bonding technique. Usually, however, microfluidic devices are con structed of polymer. Polymers are more cost effective, transparent, and biocompatible materi als, many polymer devices can be molded from one silicon master, this is a key advantage because microfluidics devices are hard to clean and hence, can only be used for one or a few experiments. Polydimethysiloxane (PDMS), PMMA, polyurethane, and polystyrene are all polymeric materials used for microfluidic devices. PDMS, the most frequently used, comes i n the form of two liquid components that are mixed (1:10, w/w) and poured onto

the substrate. The PDMS is degassed to remove all air bubbles and ensure that the liqui d fills the smallest feature of the mold. PDMS is cured in a 60-80 o C oven for 20 to 45 min to solidify. Once solid, it can be peeled off the master substrate. PDMS devices are sealed with glass co ver slips to form the channels. Several alternatives to the above described processe s exist that can accelerate fabrication. Masks can be printed on transparencies using hi gh-resolution printers; this method is suitable for feature sizes of 100 m or large r. SU8 is a photoresist that is used to create deep structures for molding eliminating the need for deep etching such as the utilization of DRIE. Utilization of a chrome mask and a wellequipped clean-room facility are necessary for making devices with very small features; sub-micromet er to a few micrometers. The development cycle from concept to prototype can take few weeks. For less fine featured devices, the use of transparencies, polymers, and/or SU8 can be re duce the cost and developmental time to one to few days from concept to prototype [38]. Figure 1a and 1b illustrates the typical processes involved in making channels in silicon and mol ding devices in PDMS respectively. Figure 1. a: Schematic summary of the processes involved in making channel in a hard substaret such as silicon. b: Schematic summary of steps involved in making a PDMS mold from a hard master. Use of Microfluidic Technology for Cell Separation 201 2.3. Types of flow: laminar versus turbulent Two modes of fluid flow exist: laminar and turbulent. In laminar flow, the fluid moves with slow velocity and each particle of fluid moves in a straight trajectory parallel to the channel walls in the flow direction; and the velocity, pressure, and other fl ow properties at each point in the fluid remain constant. There are no cross-currents perpen dicular to the flow direction, no eddies or swirls of current in laminar flow. Examples a re oil flowing slowly through a tube, and blood flowing through a capillary. Turbulent flow in contras t is chaotic, with rapid, spatial and temporal variations of pressure and velocity. Examples o f turbulent flow are the blood flow through in arteries, the flow of water through pumps and turbines, and the flow eddying seen in boat wakes and around the wing tips of aircraft [17 , 33]. The relative turbulence of a flow can be determined by the dimensionless Reynold s number (Re), which is the ratio of inertial forces to the viscous forces: e L

R v p u = Where: p is the density of the fluid (kg/m ), V is the mean velocity of the object relative to the fluid (SI units m/s), L is a characteristic linear dimension, (travelled length of the fluid, and is the dynamic viscosity of the fluid (Pas or Ns/m or kg/(ms)). Below a certain Re value the flow is laminar; above this threshold t he flow becomes turbulent. For macroscopic structures such as pipes with a circular cr oss-section, the transition from laminar to turbulent flow has been empirically determin ed at Re of approximately 2300. For most microstructures, in contrast, the Re number is usua lly low (10 1 to 100) thus only laminar flow is relevant for microfluidic devices. 3. Fluid transport process 3.1. Poiseuille flow A pressure-driven laminar flow inside a tube with a circular cross-sec tion, away from the entrance, is commonly known as Hagen-Poiseuille flow or simply Poiseuille flow [ 17, 33]. This flow is governed by the Navier-Stokes equations, which are nonlinear, second-order, partial differential equations for describing incompressible fluids. These equati ons are derived from motion and conservation of mass equations. Poiseuille flow is a solution to the NavierStokes equation, and describes the fluid velocity at any point as a function of the viscosity, pressure, and radius of the tube: ( ) 2 2 1 4 z p V r R z u | | c = | c \ . Blood Cell An Overview of Studies in Hematology 202 Where: Vz is the velocity distribution, is the fluid viscosity, ( p/ z) is the z component of the pressure gradient, R is the distance from the center of the tube, and R is the radius of the tube. This equation reveals that the velocity distribution is parabolic, with the maxi mum velocity occurring at the center of the tube. Figure 2a 3.2. Electrokinetic Flow

Electrokinetic flow is the underlying basis of electro-osmosis, electrop horesis, streaming potential, and dielectrophoresis. In electro-osmosis, fluid is made to flow through, by the application of an electric field. The field induces the formation of an electric double layer (EDL). This EDL consists of (1) a compact liquid layer adjacent to t he channel surface that has immobile balancing charges, and (2) a second diffuse liquid layer, composed of mobile ions. Most solid-liquid, and many liquid-liquid, interfaces have an electrostat ic charge, and hence an electric field exists there. In the presence of an electric field, molecules of many dielectric materials, such as the glass or polymer used for making the microf luidic devices, will become permanently polarized, since the material has a dipole tha t comprises two opposite, but equal, charges, due to the asymmetrical molecular structure. The e lectrostatic charges on the channel surface, mainly negative charges in the case o f glass or polymer, attract counter-ions from the liquid. This attraction creates the channel double layer; the first has immobile charges that balance the charges on the channel surface. The second diffuse layer has a higher concentration of counter-ions near the channel surface than d oes the bulk of the fluid. The net charge density gradually decreases to zero in the bulk liquid. The diffuse layer will move under the electric field. Surrounding molecules are pull ed along by a viscous effect, resulting in bulk fluid motion or electro-osmotic flow. The diffuse layer is several nm to 1 or even 2 m thick, depending on the ionic concentrat ion and electrical properties of the liquid. The Poisson-Boltzmann equation describes the ion and potential distribution in the diffuse layer. The electro-osmotic flow velocity can be quan tified by Lis equation [39]: av z r o V E

Where, Vav is the average elelctro-osmotic flow velocity, z is the applied electric field (V/m), r is the Dielectric constant of the medium, o is the permetivity of the vacuum, is the zeta potential at the shear plane, and Use of Microfluidic Technology for Cell Separation 203 is the Viscosity. This equation reveals that the velocity is linearly proportional to th e applied electric field, such that all travel at the same speed inside the channel Figure 2b. This situat ion contrasts

with the pressure-driven flow, in which the middle has the greatest v elocity resulting, in a parabolic migration. In conclusion, the velocity distribution being parabolic or flat can i mpact the microfluidic device performance. The electrokinetic flow is favored in applications such as DNA separation, proteomics, rapid mixing, and time dependent applications wh ere sample homogeneity and reaction time need to be stringently controlled. The f low pattern is typically not an issue for cell separation since it happens in contin uous flow without time dependence. Figure 2. a: schematic of velocity distribution in Poiseuille flow. b: schematic of velocity distribution in Electro-osmosis flow. 4. Technologies and on-chip mechanisms of separation Cells are separated either in bulk or individually. In individual cell sorting, each cell is analyzed, and then the cells of choice are individually selected. This technique is rarely used, due to its very low throughput. Thus cells are generally sorted by bulk se paration, in which a large number of cells are selected on the basis of shared c haracteristics such size, density, or the affinity of a receptor for a specific cell-surface target. The r esult of such bulk separation is enrichment rather than a true purified population [9]. The cells of interest are first identified, than separated, and finally collecte d. The initial step is to screen the cells of interest to identify one or more common specific chara cteristics to be used as the basis of sorting. Specific characteristics can be intrinsi c such as size, density, response to electrical or magnetic fields, or resistance to chemical l ysis. Alternatively, cells can be labeled using a specific cell surface target that binds to a monoclonal antibody conjugated to a fluorophore, or to magnetic particles for flow cytometric cell s orting. Once the cells of interest have been identified, they can be separate d from the other cell types. In some methods, the identification and separation occur simultaneously, e.g., affinity capture. Blood Cell An Overview of Studies in Hematology 204 In other methods such as fluorescence-activated cell sorting, cells are identifi ed on the basis of the presence or absence of one or more fluorescent tags then are then sorted into two or more separate containers. Sorting can either use negative or positive selection. In positive selection, the target cell itself is labeled; in negative selection, i t is the background cells or other cells in the solution that are labeled. 4.1. Separation of cells on the basis of affinity Affinity chromatography or separation of cells on the basis of their affinity is the

fractionation of a cell mixture based on the use of specific immunologic targets . One or more of these specific targets are immobilized on a chip surface; there, t hey capture the cells of interest with high purity (positive selection), or alternatively they c apture large number of background cells so as to enrich the remaining sample (negative select ion). Microfluidic devices provide a high surface area to volume ratio, which is necessary for chro matography; to keep processing times manageable, many devices can be used in parallel. Toners group demonstrated affinity-based separation of cells based on tw o specific immunologic targets; CD5 and CD19 [40]. Two microfluidic devices were used for affinity separation: a Hele-Shaw device and a parallel flow chamber device. The chamber i n the HeleShaw device was 57 m deep and 50 mm long, and the inlet was 5 mm wide. The device was fabricated using PDMS and sealed with a glass cover slip. The chip surface was t reated with silane solution in ethanol, followed by GMBS in ethanol (a water-solub le amine-tosulfhydryl crosslinker with a short spacer arm), flushed by Neutravidin (strong affinity to biotin) solution in PBS, and incubated overnight. Antibody, anti-CD5 or anti-CD1 9, solution stocks were diluted in BSA and sodium azide, flowed through the chamber, incubat ed for 15 min and then flushed with PBS to remove unattached antibodies. Flow e xperiments were performed with human immature T-lymphoblasts (MOLT-3 cells) and human m ature Blymphocyte (Raji cells). MOLT3 cells, which express CD5 but not CD19, were stained with green cell-tracker dye. Raji cells, which express CD19 but not CD5, were stained with orange cell-tracker dye. Cells were flowed over the coated chip surface with a syringe pump, at flow rate of 30 or 50 L/min. Flow rates were optimized for efficient capture to allow dynamic cell adhesion, shear stress was minimized in the axial directi on permitting sufficient interaction between the cell and the coated surface so that binding can occur. A device coated with anti-CD19 was used to deplete a 50:50 MOLT-3/Raji cell mixtur e of Raji cells, producing a 100% pure MOLT-3 in less than 3 minutes. Obviously , this level of enrichment is exceptional and cannot be achieved with a heterogeneous (real world) blood sample. Nevertheless enrichment by 10- to 2000-fold have been obtained usi ng similar devices [41]. Sin and coworkers also used an alternative parallel flow serpentine chamber design, the effective increase in device length was intended to increa se the number of cell capture and also to ensure constant shear stress throughout the device. In summa ry, Sin and coworkers have demonstrated the possibility of using dynamic cell attachment to

antibodycoated microfluidic chambers in shear flow to enrich mixtures of MOLT-3 and Raji cells. To further increase the chip surface area, Toners group has developed a microflui dic platform the CTC-chip [42]. This device is composed of a two-dimensional array of round pillars. Use of Microfluidic Technology for Cell Separation 205 These pillars are 100 m in diameter and 100 m deep; they are separated by 50 m and each three rows are shifted from the previous three rows by 50 m to maximize interacti on (Figure 3). The chip was etched in silicon with DRIE to produce smooth straight pillars; the result was 78,000 pillars within a surface of 970mm 2 . The pillars were coated with antibody (EpCAM). The chip is enclosed by a manifold. The group had optimized the flow rate allowi ng adequate cell-pillar interaction time, and to minimize shear forces, ensuring at tachment of maximal number of cells to the pillars. The chip successfully identified CTCs in the per ipheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 (99%), with a range of 51,281 CTCs per mL and approximately 50% purity (Figure 3). Similarly, Chang and coworkers produced two chips with an array of pillars coate d with Eselectin IgG chimera, to test the interaction of HL-60 and U-937 cell s with these structures [43]. In one device, the square pillars were separated by 25 m; each pillar was 2 5 m wide by 25 m long by 40 m deep. In the second device, pillars were 10 m wide by 35 m long by 40 m deep, and spaced 30 m apart. In this second device each row of pillars was offset from the previous one by 15 m so as to allow more cells to ne interrogated. Dev ices were fabricated in silicon by DRIE and were sealed with thin pyrex glass using anodic bonding. In the first device, HL-60 cells were enriched 400 fold over to the original concentration, while the second device achieved only 260-fold enrichment. Offsetting t he pillars on the second device had been expected to improve the cell capture, however smaller siz e resulted in the flow being faster in the channels, thereby increasing shear st ress on the cells and making them less likely to be captured on the surface. It is possible to further increase cell surface interaction if the cells passed through a packed bed of antibody-conjugated beads. However, such a design is no longer attractive since cell crossing takes as long as 2 hr due to the retardation of the flow rate [44]. Figure 3. Isolation of CTCs from whole blood using a microfluidic device a, The workstation setup for CTC separation. The sample is continually mixed on a rocker, and pumped through the chip using a

pneumatic-pressure-regulated pump. b, The CTC-chip with microposts etched in sil icon. c, Whole blood flowing through the microfluidic device.d, Scanning electron microscope im age of a captured NCI-H1650 lung cancer cell spiked into blood (pseudo coloured red). The inset sh ows a high magnification view of the cell (Reprinted with permission from Nature Publishing Group). Blood Cell An Overview of Studies in Hematology 206 Malmstadt and coworkers developed an innovative approach that could ben efit from the increased bed of beads area while avoiding its limitation [45]. The g roup used beads that were modified twice: (i) with the affinity moiety biotin, which binds streptavid in to function as chromatographic affinity separation matrix, (ii) with the temperature -sensitive polymer poly(N-isopropylacrylamide). At room temperature, a suspension of these beads flows through the channel. When the temperature is raised above a certain t hreshold, the beads aggregate. The sample is then introduced and the beads remain stable during samp le flow, and the device functions as a chromatography affinity matrix. The temp erature is then lowered below the threshold to allow the beads to dissolve and elute from the ch annel. 4.2. Separation of cells by flow cytometry Flow cytometry is a technique that uses light scattering to measure various phys icochemical characteristics of suspended cells. Cells are stained and then confined to flow in a single file through a fluidic system. The stream of cells passes at high speed t hrough a focused laser beam. Light scattering and fluorescence data are collected from the in dividual cells and analyzed. A laser of one wavelength such as blue can be used for excitation; the instrument records fluorescence at multiple wavelengths such as red, orange, and green, in addition to the blue light scattering in the forward direction and right angle to the laser beam. This information is processed by a computer attached to the cell analytical instrumentation. Alternatively, flow cytometers can be equipped to sort cells of intere st based on scattering properties, by use of an electric field, flow switching, or optical trapping. The first key aspect of flow cytometry is the focusing of the cells in a single file so they can be individually interrogated by the optical detection system. This step is usually achieved by hydrodynamic focusing [46, 47]. A sheath flow surrounds the samples from both sides; the flow rate can be adjusted to make the middle stream as thin as required to c arry cells in a single file (Figure 4). This also reduces cellular aggregation that ca n clog the device. Microfabricated channel structures are capable of stably delivering samp les to a detection

area with higher accuracy and better flow control than are glass capi llary-based fluidic systems used in conventional flow cytometric equipment; this superior p erformance is a function of the small channel dimensions possible in microfluidics. The hydrodynamic focusing typically constrains the cells on both sides, but not in the z directio n [48]. Miyake and coworkers have developed a multilayered sheath flow chamber that can generate a three-dimensionally focused narrow stream. The channel system was for med by the lamination of five separate silicon and glass plates; defining the three-dimensional geometry of a sample injection nozzle and a detection microchannel. A simpler, albeit less flexible or programmable, approach would be to use a shallow channel that confined the cells in the z direction to remain within the analysis window [49]. Sheath liquid-based hydrodynamic focusing, while being the standard tech nology in microfabricated flow cytometers, require continuous pumping of a large volume of sheath liquid at a high flow rate to pinch the middle stream down to singl e-cell width. Sheath Use of Microfluidic Technology for Cell Separation 207 liquid volume required can be up to 1000 time the sample volume. New types of microfluidic systems have been developed to minimize or eliminate the sheath liquid requirements and to further miniaturize flow cytometry. Huh and coworkers demons trated the use of ambient air as an alternative sheath fluid in a disposabl e airliquid two-phase microfluidic system [50]. The system produces a thin (>100 m) liquid s tream transporting cells focused by air-sheath flows in a rectangular microfluidic channel . To achieve this focusing, the authors had to conduct a detailed study of the PDMS surface wettab ility, and of the flow conditions so as to overcome the two-phase (air-liquid) instabilitie s. In a contrasting approach, a V-shaped groove device was developed by Altendorf and coworkers to transport blood cells in a single file. The groove micro channel was fabricated by anisotropic wet etching of silicon [51]. The top of the groove was 2025 m, wide and the constriction channel geometry allowed the generation of a single-file s tream of blood cells moving through the channel without the need for sheath fluid. Light s cattering caused by the flow of the individual blood cell through the device was measured by optics based on a photomultiplier tube, photodiode detector, and laser source. The device was capable of differentiating between various cell populations such as RBCs, platelets , lymphocytes, monocytes and granulocytes, based on the intensity of scattering signal

s. This study demonstrated the potential utility of such a device for differential counting of blood cells. The second aspect of a flow sorter system that is pertinent to our discussion is the system capability to sort cells at high speed. The requirement of rapid defl ection after the cell has been identified by the optical system, can be achieved through redirec tion of the flow via high-speed valving or reverse electrokinetic flow, dielectrophoresis, ult rasonic transducer, or optical trapping. Kruger and coworkers achieved switching by use of pressure-driven flow systems [ 46]. As a liquid sample stream that was hydrodynamically focused along the center of an input channel approach a junction, a small amount of buffer liquid is injec ted into or withdrawn from the side stream along a switch channel, causing the focused samp le stream to be deflected and to flow into a collection channel. Fu and coworkers demonstrated the use of electric field to quickly switch the fl ow from the waste to the collection channel, to isolate a cell of interest [52]. The disposa ble activated cell sorter consisted of three channels joined at a T shaped junction. Electro-osmoti c flow drives cells or particles from the inlet to the junction where the flow is diverted to the waste or collection channels. The diversion is achieved by a switching of the electric field at the T junction to control the flow in a rapid manner. This device does not use sheath flow but rather a small-diameter channel, to constrain the cells in single file. When the fluorescently tagged bacterial cell of interest is detected, the sample stream flowi ng from an inlet to a waste port is quickly switched by reversed electric field, such that the flow is diverted direction to a collection port, selectively delivering the labeled targ et particles to a sample collector. Using this fluidic switching-based sorting technique, the aut hors demonstrated sorting of fluorescent microbeads and E. coli cells at a throughput of 10 beads s1 and 20 cells s1. The device was fabricated by softlithography in PDMS. Blood Cell An Overview of Studies in Hematology 208 Similarly, Oakey and coworkers demonstrated the use of electric field for divert ing the flow rather than switching since their device did not have hydrodynamic focusing [53] . Johansson and coworkers developed an ultrasound transducer that was pla ced in the middle of the channels at the intersection between the waste and collection chan nels. In the absence of ultrasound wave, cells migrated to the waste channel. When a cell of interest was identified, the transducer produced a radiation force acting on a dens ity interface that

caused fluidic movement, and the particles or cells on either side of the fluid interface were displaced in a direction perpendicular to the standing wave direction toward the collection channel [54]. Chun and coworkers demonstrated a polyelectrolytic salt bridge-based ele ctrode, placed across the channel to replace the laser used for cell fluorescence an alyses. The salt bridge produced impedance signals in proportional to cells size to be used as basis for sorting to eliminate the need for the optical system that typically exist off-chip [55]. Figure 4. Left: Image of prototype device in operation. Right: Computational mod elling of fluid dynamics in a microfluidic cell sorter structure. The performance of this struct ure was simulated using FlumeCAD (Microcosm Technologies), a finite element modelling (FEM) software pac kage that uses full NavierStokes equations. (Printed with permission from Institute of Physics Publis hing). 4.3. Separation of cells using immunologic targets Magnetic separation can be achieved by using the cells intrinsic magneti c properties or by attachment of a magnetic particle to a specific surface antigen that can later b e exposed to a magnetic column for separation. Magnetic particles are typically compose d of large numbers of superparamagnetic nanoparticles packed inside micrometer sized sphere made of polymer. The surface of each magnetic bead is then functionalized with antibo dy specific to an antigen found on the surface of one type of cell. The cells are incubated with the Use of Microfluidic Technology for Cell Separation 209 magnetic beads to allow interaction; the magnetic bead attaches specifically to the cell by the antibody-antigen recognition. The cells are transferred to the magnetic column of a separation device. The cells are then manipulated via a magnetic field generated by an internal patterned electromagnet or external macroscopic magnets. The cells atta ched to the magnetic beads stay on the column, while other cells, not expressing the antigen , and hence not attached to beads, flow through it. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s). Positive sel ection results in the binding of the cells of interest to the column and they then need to be wash ed off of the column. In negative selection, the cell of interest do not bind, and are passed through the column enriched [56]. Han and coworkers have exploited the intrinsic differences in the magnetic prope rties of the RBCs and WBCs without use of magnetic beads, to separate the cel l types in a onestage or three-stage magnetophoretic microseparator [57]. Their single-st

age device was able to separate 91% of RBCs out of whole blood; the three-stage device improved on this performance to separate 93.5% of RBCs and 97.4% of WBCs from the who le sample at 5 L/hr flow rate. Qu and coworkers demonstrated identical separation effic iency [58]. Hans channels were 50 m deep created by hydrofluoric acid etching of borofloat gla ss slides. The wire area, included to deform the magnetic field inside the channel and hence generated a high field gradient, was defined using photolithography, an d a Ti/Cu/Cr seed layer was deposited by e-beam evaporation, followed by microelectr oforming (a process for making thick metal structure) of the ferromagnetic nickel wire. The device was sealed with a second glass slide bythermal bonding. An external m agnet provided the magnetic field. Adams and coworkers developed a multi-target magnetic cell sorter to pur ify two types of target cells. They simultaneously sorted (i) multiple magnetic tags ach ieving >90% purity and >5,000-fold enrichment, and (ii) multiple bacterial cell types achieving >90 % purity and >500-fold enrichment, with a throughput of 10 9 cells per hour [59]. Their device incorporates microfabricated ferromagnetic strips (MFS) to generate large and reprodu cible magnetic field gradients within its microchannel and utilizes a multistream laminar flow architecture to accurately control the hydrodynamic forces. This design enables continuous so rting to of multiple target cells into independent spatially addressable outlets wit h high purity and throughput. The chip was fabricated in three layers: glass-PDMS-glass. The channel was 50 m d eep and 500 m wide, and contained two sets of 20-200 nm thick nickel patterns that compose the MFS structures. (Figure 5) The two sets of MFS arrays are aligned at different angles with respect to the flow direction. The result is that two magnetophoretic forces that differ is amplitude and direction, act on the labeled cells. The labels are dif ferent in size and magnetization and thus require different forces to deflect them out of the strea m of laminar flow. The magnetic field is created with a magnet made of a custom stack of neodymiumiron-boron (NeFeB) and placed underneath the chip.The MT-MACS sorting c hip was used to sort two subtypes of Escherichia coli MC1061 cells. One type was labeled with label 1 with Blood Cell An Overview of Studies in Hematology 210 tag 1 (r = 2.25 m, M =14 kA/m) and tag 2 (r =1.4m, M = 30 kA/m), and was fluoresc ently

labeled for observation under a fluorescent microscope. Using 5 mL/hr flow rate, each of the tags was enriched several thousand fold at its respective outlet after a single round of purification. At outlet 1, the population with tag 1 was enriched from 0.020% to 95.876% of the total population corresponding to a 5,000-fold enrichment. The impurities in this fraction consisted of 2.974% tag 2 and 1.150% nontarget beads. Similarly, the population with tag 2 in outlet 2 was enriched 15,000-fold, with contamination of 3.125% tar get 1 and 6.358% nontarget beads. The waste output consisted almost entirely of nontarge t beads (99.997%), and contamination of 0.002% of tag 1 and 0.001% of tag 2. Figure 5. MT-MACS separation architecture. (A) (Step A) The sample contains an e xcess of nontarget cells and 2 different target cells (target 1 and target 2) that are labeled with 2 different magnetic tags (tag 1 and tag 2) by specific surface markers. (Step B) The sample is continuously pu mped into the device where the 2 target cell types are sorted into spatially-segregated independent o utlets. Separation occurs in 2 regions of high magnetic field gradient generated by the microfabricated fe rromagnetic strip (MFS) 1 and MFS 2. (Step C) After sorting, the eluted fractions from each outlet are a nalyzed via flow cytometry. (B) A free-body diagram showing the balance of forces at the MFS stru ctures. At MFS 1 (_1 _ 15), tag 1-labeled target 1 cells are deflected and elute through outlet 1 because Fm1 _ Fd1 sin(_1). This is not the case for tag 2-labeled target 2 cel ls, which are instead deflected at MFS 2 (_2 _ 5) because Fm2 _ Fd2 sin(_2), and elute through outlet 2 . Nontarget cells are not deflected by either MFS and elute through the waste outlet. (C) Optical micr ographs (magnification _ 100_) of the tags being separated at the 2 MFS structures at a total flow rate of 47 mL/h (sample _ 5 mL/h, buffer _ 42 mL/h). (Left) Tag 1 is deflected by the steep angled MFS 1. (R ight) Tag 2 is deflected by MFS 2 (Reprinted with permission from PNAS). Use of Microfluidic Technology for Cell Separation 211 Xia, Modak and coworkers used similar have demonstrated similar magneti c cells sorting design with the structure concentrating the magnetic field being adjace nt to the flow channel but not exposed to the sample solution [60, 61]. Lee and coworkers demonstrated the use of patterned channels to magnetically man ipulate single cell. Lees device had a matrix of two separate layers of strai ght gold wires, each addressed independently, aligned perpendicular to each other, and covere d with an insulating layer. A magnetic field was created by passing electrical c urrent through the wires creating a programmable magnetic field to control the motion of individual cells in the fluid. Lees device demonstrated the separation of viable from nonviable yeast cel

ls attached to magnetic beads [62]. Saliba and coworkers demonstrated a 2D array of dots by deposited by microcontact printing of a magnetic ink acting as magnetic traps. Antibody-coated m agnetic beads were injected in the channels and were submitted to a Brownian motion in the absence of any field. The application of an external vertical magnetic field induced the antibody-coated magnetic beads to assemble on the patterned dots creating columns [63]. Kose and coworkers demonstrated a novel device to use colloidal suspen sion of nonfunctionalized magnetic nanoparticles for manipulation and separation of microparticles. It is a size-based separation mediated by angular momen tum transfer from magnetically excited ferrofluid particles to microparticles. The na nocytometer is capable of rapidly sorting and focusing two or more species, with up to 99% separation efficiency [64]. 4.4. Separation of cells using chemotaxis phenomena Chemotaxis is the process whereby a single cell, or multicellular organ ism, moves away or toward a certain chemical. This movement can be away from a poison ( chemorepellent or negative chemotaxis) or toward food (chemoattractant or positive chemotaxis). Fo r example, neutrophils leave the blood vessel and migrate toward the smell produce d by bacteria in a cut of the skin in an effort to defend the body against infection. The influence of these gradients of molecules and cues cellular behavior in the surrounding m icroenvironment is an important biomedical focus of study. Chemotaxis, in particular, play s an important role in many biological and physiological processes such as creation of new tissues, wound healing, cancer metastasis, and embryogenesis. To test a cell response to a certain chemical gradient, the cells are typically placed in a well, and the test substance or chemical is placed in a second well. The two wells are separated by a barrier such as a weir structure (Dunn chamber) or a filter (Boyden chamber), to ensure that the cell movement is due only to the signaling only and not random motion or dif fusion. The development of microfluidic devices for chemotaxis assays was motivated by t he need to produce the gradient in a controllable and reproducible manner, and to minimize the quantities of test substance and cells required per assay [65]. Blood Cell An Overview of Studies in Hematology 212 Abboodi and coworkers have developed a hydrogel-based microfluidic chip for chem otaxis studies [66]. The device consists of three chambers in this study, the middle ch amber is filled with a 3D porous hydrogel structure that contains the cell culture; h

ere fibrosarcoma cell line HT1080 was used as an invasive cancer cell model. First, the le ft-hand chamber was filled with a cellulose enzyme solution and the right-hand one was fi lled with cell culture medium containing fetal bovine serum (FBS) (Figure 6). The cellulose e nzyme solution diffuses into the hydrogel and degrades it, creating large pores in the structur e closer to the left-hand reservoir. Pore size is progressively smaller as the solution migrates toward the right-hand reservoir. The result is that the cells, stained for observ ation by fluorescence microscopy, moved toward the large pores adjacent to the left-hand reservoir. Af ter 3 days, the FBS containing medium-FBS in the right-hand reservoir was replaced with pure FBS i.e., full strength chemoattractant. The cellulose enzyme solution in t he left-hand reservoir was also replaced with a FBS-containing medium. In response, the cells reversed their motion and moved toward the right-hand reservoir containing the pure F BS, confirming that FBS is a chemoattractant for this cell type. The negative structure of the chambers of this device was fabricated with a 3D printer and a UV-curable polymer. The device was molded in PDMS from the polymer structure and sealed with a glass slide. Agrawal and coworkers, in an effort to explore the sepsis complication s that occur in burn patients, presumably as the result of improper activation of neutrophils, develo ped an assay on an advanced switching gradient device for monitoring the migration behavior of these cells following thermal injuries [67]. The device (i) integrates the i solation of neutrophils from whole blood, (ii) the provision of a controlled combinatorial chemotactic e nvironment, and (iii) the monitoring of real-time migration of the captured neutro phils over different substrates all on-chip, thereby eliminating the need for preprocessing of the blood. The device was made with PDMS and was fabricated with standardized soft l ithographic techniques. In a first trial, the capture was performed by a coating of the microfluidic cell chambers with P-selectin, E-selectin, or fibronectin substrate. A 10 L drop of bl ood/heparin solution was loaded in the cell-capture device, and the cells were al lowed to settle for 10 min; then flow was initiated (0.5 L/min) and most of the unwanted cel l population was washed away. In experiments repeated in the migration device, captured cells wer e exposed to a linear chemotactic gradient of the chemokines IL8 and fMLP. Migr ation patterns for both chemokines over each substrate were recorded with time lapse micr oscopy and were then compared. The two chemoattractants, IL8 and FMLP, were used to create a gradien

t across each binding substrate. Neutrophils over P-selectin reacted similarly for the IL8 and fMLP gradients. However, for E-selectin, average y displacements of 50 m and 70 m were observed within 30 min in the gradients of fMLP and IL8 respectively. For fibronectin, the difference in migration was more significant: the cells mig rated about 65 m in fMLP gradient, but only 35 m in the IL8 gradient over similar t ime courses. This system offers an efficient approach to the development of a simple di agnostic tool suitable Use of Microfluidic Technology for Cell Separation 213 for a variety of applications in addition to chemotactic studies such as genomic and proteomic analyses. Chen and coworkers demonstrated a cell migration chip which can monito r chemotaxis at single cell resolution [68]. The chip is composed of weirs that captu red individual cells. Once a cell is captured, the hydrodynamic force will push other cells to the next weir through a serpentine channel. A high capture rate over 94% is achieve d by optimizing the geometry of capture sites and the length of serpentine structures. Aft er capturing, cell migration experiments induced by chemotaxis were carried out using the fabricated platform, and the behavior of each single cell was successfully traced. Englert, Walker, and coworkers focused their effort on generating repro ducible gradients [69, 70]. Engelrt and coworkers device created reproducible chemoeffecto r gradients. Two gradients, to simulate competing conditions in nature, were created usi ng a Laminar flowbased diffusive mixing and were tested on Escherichia coli. The sample containing the fluorescently stained Escherichia coli, for observation by microscopy, w as introduced in a middle channel. Two side channels introduced the two gradients: quorum-sensing m olecule autoinducer-2 (AI-2) and stationary-phase signal indole were introduced, one on each side of the sample. Results showed that the Escherichia coli was attracted by the AI-2 and repelled by the stationary-phase signal indole. Figure 6. (A) Schematic for the device. (B) Image for each region in the main st orage. (C) Average fluorescent intensity of FITC- BSA at four regions from left to right through th e porous hydrogel in the SD (Reprinted with permission from SPIE and A. Al Abboodi). main storage 4.5. Separation of cells using optical methods and light traping Optofluidics technology, which is the mating of optical trapping, switc hing, and microfluidic has been recently introduced as a new manipulation scheme. It has been Blood Cell An Overview of Studies in Hematology 214

motivated by its high selectivity, ability to maintain sterility, and how it allowed programmable manipulation of particles or fluids in microenvironments based on o ptically induced electrokinetics. While optical switching is often used with flo w cytometry as the switching or sorting mechanism, it is here presented in a separate section to de monstrate its unique capabilities and potential. Typically, a focused laser beam provides a fo rce to hold or move a microscopic dielectric particle. The dielectric particle is attr acted to the strong electric field gradient at the narrowest point of the focused beam. This force i s small, in the order of piconewtons, depending on the refractive index mismatch, and can be att ractive or repulsive. MacDonald and coworkers introduced a bow-tie like chip with four reservoirs link ed in the middle by a flow channel (Figure 7). The two reservoirs on one side are for buff er (top) and sample (bottom) respectively. The two reservoirs on the other side are for waste (top) and sample collection (bottom). In the absence of any force, the cells mi grated from their reservoir to waste, while the buffer passed to the collection reservoir. The two flows shared the channel but no mixing due to laminar conditions. When optical forces were fo cused on the flow of mixed particles as it passed through the lattice, selecte d particles were strongly deflected from their original trajectories into the collection reservoir , while others passed straight through, depending upon their sensitivity to the optical poten tial. The interaction with optical fields provided a selective means of removing material ma tching specific criteria from an otherwise laminar stream. The optical force was appli ed by the mean of a five-beam interference pattern created by a 1,070-nm laser beam that p assed through a diffractive beam splitter, producing four beams diverging from the central, nondiffracted , in a cross shape. Collimating optics provided a parameter space to in dependently control the phase and amplitude of each of the five beams before being co-fo cused through an aspheric lens to produce a large, three-dimensional optical lattice thr ough multibeam interference [71]. Kovac and coworkers introduced a microwell array that is passively loa ded with mammalian cells via sedimentation. These cells were inspected using mic roscopy. After inspection, cells of interest were levitated from the well using a focused infra red laser into a passing stream to the collection reservoir. This is a simple device m ade of PDMS for the channels and the wells were sealed with glass slide [72]. Shirasaki and coworkers used optics to control a thermoreversible gelat

ion polymer (TGP) as a switching valve. The chip has Y-shaped microchannels with one inlet and two outlets. The sample containing fluorescently labeled cells was mixed with a solution cont aining the thermoreversible sol-gel polymer. The fluorescently labeled target cells were introduced in the channels and observed using fluorescence microscopy. In the absence of a fluorescence signal, the collection channel was plugged through laser irradiation of the TGP and the specimens were directed to the waste channel. Upon detection of a fluorescence s ignal from the target cells, the laser beam was then used to plug the waste ch annel, allowing the fluorescent cells to be channeled into the collection reservoir. The r esponse time of the solgel transformation was 3 ms, and a flow switching time of 120 ms wa s achieved. The TGP did not affect cell viability [73]. Use of Microfluidic Technology for Cell Separation 215 Lin and coworkers have demonstrated a microfluidic system based on a computer controlled digital image processing (DIP) technique and optical tweezers for automatic cell recognition and sorting in a continuous flow environment. In this syst em, the cells are focused electrokinetically into a narrow sample stream and are introduced into t he channel where they are recognized and traced in real time. Synchronized contro l signals generated by the DIP system are then used to actuate a focused IR laser beam to displace the target cells from the main sample stream into a neighboring sheath flow, whi ch carries them to a downstream collection [74]. In summary, Optoelectrofluidic technology, which has been recently intro duced as a new manipulation scheme, allows programmable manipulation of particles or fl uids in microenvironments based on optically induced electrokinetics. The behavio r of particles or fluids can be controlled by inducing or perturbing electric fields on demand in an optical manner, which includes photochemical, photoconductive, and photothermal effects [75, 76]. Figure 7. The concept of optical fractionation. Low Reynolds number flows will b e laminar: without an actuator all particles from chamber B would flow into chamber D. Chamber A would typically introduce a blank flow stream, although this could be any stream into which the se lected particles are to be introduced. By introducing a three-dimensional optical latticein this case a body-centred tetragonal (b.c.t.) latticeinto the fractionation chamber (FC), one species of pa rticle is selectively pushed into the upper flow field. The reconfigurability of the optical lattice a llows for dynamic

updating of selection criteria. For weakly segregated species, the analyte can b e either recirculated through the optical lattice or directed through cascaded separation chambers. Th is latter option also allows the use of multiple selection criteria in a single integrated chip. The f low volume in our current sample cells is 100 mm thick; scale bar, 40 mm (Reprinted with permission from N ature Publishing Group). 4.6. Separation of cells using electrophoresis and dielectrophoresis Dielectrophoresis (DEP), first described by Pohl in 1951, is a phenomenon in whi ch a force is exerted on a dielectric (insulator that can be polarized) particle whe n subjected to a nonuniform electric field. This can take place in either direct (DC) or alternating (AC) electric fields. The strength of the force depends on the frequency of the ele ctric field, the medium and particle electrical properties, and the particles size and shape. Varying the fields frequency can manipulate particles with different sizes with great selectivity, which is used for manipulating cells and nanoparticles. Blood Cell An Overview of Studies in Hematology 216 Pommer and coworkers have used DEP phenomenon to separate platelets di rectly from diluted whole blood in microfluidic channels. Since platelets are the smallest cell type in blood, DEP-activated cell sorter (DACS) was used to perform size based fractionation of blood samples and continuously enrich the platelets [77]. Hu and coworkers used a comparable device to Pommer, but the differen ce in size-based separation was attributed to the size of the antibody-conjugated beads attached to the target instead of the cells intrinsic size [78]. Pommers device is composed of two identical purification stages, in each stage a buffer is introduced in the midd le of the channel while the sample is loaded from both sides. The channel electrodes are at angle with the flow forming a funnel shape where the sample flows from the wide to the narrow side. The force exerted by the electric field on the cells has a cubic dependence on the radius ( R 3 ) and can be controlled by varying the applied voltage. The hydrodynamic drag fo rce under laminar flow has a linear dependence on the particle radius ( R 1 ) and is controlled by varying the flow rate. Therefore, the resulting forces exerted on the cells has a square dep endence ( R 2 ) and is used to deflect large cells (RBCs and WBCs) into the middle buffer stream to the waste collection reservoir while the platelets remain in the side flow

and migrate to the collection reservoir. Post sorting cytometric analysis revealed that a single pa ss through the two-stage device yields 95% purity of platelets with minimal platelet activation. Two Borosilicate glass wafers were used to fabricate the top and the bott om of the device, the titanium-gold (20nm and 200nm respectively) electrodes were patterned an d evaporated using e-beam, a polyimide layer was spun and patterned to form 20m ch annel depth between the top and bottom wafer. The two wafers were aligned and bonded using t hermal bonding (300 o C) then 375 o C to cure the ployimide. Vahey et al have extended DEP to multiple electrodes creating an elec tric conductivity gradient to separate cells and particles. This is similar to using is oelectric focusing in analytical chemistry and proteomics with the conductivity replacing the pH gradi ent. Vahey et al have used this device to achieve label-free separation of multi ple (>2) subpopulations from a heterogeneous background. The channel was 1mm wide and 20 m deep molded in PDMS and sealed with pyrex wafer that has the evaporated electrodes. The six electrodes, 200nm gold on top of 10nm titanium (adhesion layer) are 60m wide, separated by 1 5 m, and are patterned at an angle with the flow direction. The electrical gradient was used to separate 1.6, 1.75, and 1.9 m polystyrene beads from a mixture. Additi onally the device was successfully used to separate similar size beads based on surface conductance due to different coating such as COOH modified and unmodified, as well as so rting nonviable from viable cells of the budding yeast Saccharomyces cerevisiae [79]. Lapizco-Encinas et al have used insulator-based (electrodeless) dielectro phoresis (iDEP), in which the nonuniform electric field needed to drive DEP is produced b y insulators, avoiding problems associated with the use of electrodes [80]. This channel was 1 0.2mm long and contained a two dimensional array of 10m deep pillars etched in b orosilicate glass. The channel was thermally bonded with a drilled cover plate for fluid access. Tw o platinum wires, the only electrodes in the device, were placed in the inlet a nd outlet reservoirs, Use of Microfluidic Technology for Cell producing mean electric fields of up to lator posts disturbed the electric field lines, This created higher field strength between the pillars. Separation 217 200 V/mm across the insulators. The insu squeezing them between the pillars. Cell trapping and release were con

trolled by modifying the relative responses of electrokinetics and DEP by adjusting the magnitude of the applied voltage. Dead cells had significantly lower dielectrophoretic mobility t han live cells but similar electrokinetic mobilities. Therefore, live cells were concentrated betwe en the pillars. Cells were labeled with Syto 9 and propidium iodide for observation through a fl uorescent microscope (Figure 8). Figure 8. Schematic representation of the experimental setup: (a) top view, show ing the manifold, glass chip, an enlargement of the flow microchannels; (b) cartoon showing the electric field lines being squeezed between the insulating posts; (c) side view showing the manifold and gl ass chip on the microscope stage (Reprinted with permission from ACS Publications). 4.7. Separation of cells by size Size-based sorting has been our works focus for a few years; we have successfully demonstrated the separation of WBCs, RBCs, or circulating tumor cells from whole blood, and fetal nucleated red blood cells (fNRBCs) from cord blood. Size- a nd density-based sorting has been demonstrated in open flow channels by Sekis group using pinched f low fractionation [81]. Enrichments up to 20 fold have been accomplished by the chip shown in Blood Cell An Overview of Studies in Hematology 218 Figure 9. The sample is loaded into the 20 m wide open channel, and a second solu tion, a buffer, is loaded from a second channel that is 200 m wide. The buff er pinches the flow width against the channel wall down to 15 m, forcing the cells to al ign along the channel wall, before the channel opens to a 350 m wide area with 12 outlet ports. The separation occurs in the wider channel; the smaller, lower-density cells segregate and are dragged along the channel wall, while the larger, denser cells, tended to sed iment earlier in the pinched region and occupy the outer stream that empties to a differen t outlet port. The device was fabricated by standard photolithographic techniques and was molded in PDMS, from an SU8 master. Wildings group used weir-like structure to create a 3.5 m space between the silicon bottom and a glass top of a device to separate WBCs from who le blood, they could then use the cells DNA for amplification [82-84]. Austins group has demonstrated cell sorting based on asymmetric bifurcation of a laminar flow around a periodic array of micrometer-scale pillars [85, 86]. Each row of p illars is offset horizontally with respect to the previous row by a fraction of the d istance separating the pillars. This offset between successive rows forces particles to navigate around the obstacles

and induces a lateral displacement proportional to their size. Therefore, cells or particles of different sizes exit the array at different locations and can be collected separ ately. The main advantage of such a device is that it never clogs, since the distanc e between the pillars is always larger than the cells or particles they separate. This device was used to fractionate a mixture of different-diameter beads; 0.8, 0.9, and 1.03 m into three d istinct streams. Additionally, this device was used to separate a mixture of DNA molecules, 61 an d 158 kb, in two separate trajectories. In this case an electric field of 12V/c m was used to drive the flow. Additionally, Austin group has used a variation of this chip to separ ate RBCs, WBCs, and platelets from blood plasma [87]. Our group has employed pillar configuration to create multiple sieving devices for sizebased separation. With these devices, we have demonstrated the isolation of cult ured cancer cells spiked into whole blood [88]. Our devices have successively narr ower gap widths between the columns in the direction of flows with 20, 15, 10, and 5 m spacings a ll on one device (Figure 10). The first 20 m wide segment disperses the cell sus pension and creates an evenly distributed flow over the rest of the device, whereas the other segments were designed to retain successively smaller cells [89]. The channel depth is constant across the device. Two types of devices were constructed, the first type was 10 m deep and the second type was 20 m deep. As cells traversed the device, they contin ued through each region until they were stopped at a gap width that prohibited passage due to their size. Experiments with human whole blood, from healthy individual, proved that channel s 5 m wide and 10 m deep permitted all normal cell types to cross without resistance un der our experimental flow conditions. Cultured cancer cell lines, mixed with wh ole blood and applied to the device, were retained inside the device while all othe r cells migrated to the output reservoir. Use of Microfluidic Technology for Cell Separation 219 Figure 9. Schematic illustrations showing the separation mechanism of sedimentat ion pinched-flow fractionation. Images a and b show enlarged views of areas (a) and (b) of the up per image, respectively. In the pinched segment, particles are focused onto the upper sidewall regardless of size. By applying sedimentation force to the flowing particles in the curved channel, particles wi th a higher density (black) migrate beyond the streamline, achieving density-based sorting. (Reprint ed with permission

from Springer publisher.) Eight different cancer cell line, brain neuroblasts (SK-N-MC, SK-N-AS, SK-N-SH, BE(2)M(17), and SH-SY5Y), breast epithelial cells (MDA231), colon epithelial cells (SW620), and kidney epithelial cells (HEK293), were successfully isolated from whole blood by our device. Additionally, either intact cells, or DNA, could be extracted for mole cular analysis. DNA was extracted by in-situ cell lysis. After the cancer cells had been retained, the device was flushed for 20 min, with medium used to remove all non-retained cells, followed by water to lyse the retained cells and release their DNA. DNA was collected from the output reservoir for 20 min (approximately 0.33 mL), purified, and tested, as a demons tration that the captured DNA was from the cancer cell line and not a contaminant DNA from the blood used for spiking. Alternatively, retained intact cells were recovered by reversing the di rection of the flow, using medium after allowing 20 min for them to migrate towards the outlet. Once flow was reversed, cells retained at the first row of transition at the 10 m wide channels detached easily and traveled through the 15 m and then the 20 m segments, thro ugh the inlet reservoir, and into a collection tube. Cells were cultured from the co llection tube and were able to proliferate, thus demonstrating the viability of the extracted cells. Our device was made using standard photolithography techniques and was molded in PDMS, polystyrene, or polyurethane [90]. Blood Cell An Overview of Studies in Hematology 220 Figure 10. (top) Schematic showing flow direction and channel structure for the Second generation device having varying channel gap widths (20, 15, 10, and 5 m), cells separate bas ed on size and deformability. Channel depths, constant over a single device are: 20 or 10 m. (Bot tom) Adult blood cells spiked with MDA231 cells. All cells flow freely through the device except for th e MDA231 cells, which are retained at the start of the 10m wide channels A derivative of the same design was utilized to separate fetal cells from cord blood [91]. The device has four segments of successively narrower channels along the flow ax is; these have 15, 10, 5, and 2.5 m spacings. Each segment is 30 mm wide and 15 mm long and has 375 rows of channels of the same width. Therefore, the entire device is 30mm wide and 60mm long, giving rise to over 3.5 million channels. The channels are formed between pillars separated by gaps rather than a continuous structure. This des ign was favored to allow the cells to deform and resume their normal shape as they trav erse the device;

further, if the cell flow is locally slowed or if a channel becomes clogged, a cell is able to migrate around the affected area. Currently, a one-step centrifugation is required for sample preparation, and only the mononuclear layer is used in the dev ice. This step not only separates most of the mature RBCs before sample loading, but it also enriches for fNRBCs since mononuclear cells and fNRBCs have similar densities. To i dentify fNRBCs, we stained the buffy coat with fluorescein isothiocyanate-labeled monocl onal antibody to HbF (green) and SYTO red. Thus, fNRBCs should fluoresce green and red. Double-st ained samples were applied to the device. When the mononuclear layer was tested in the device, WBCs were retained consistently at the start of the 2.5 m wide segmen t, while fNRBCs and mature RBCs migrated to the output reservoir. Cells were removed from the output reservoir, and the DNA was extracted with DNA purification kit and was tested fo r X and Y chromosomal sequence by PCR. We used cord blood from mothers who de livered male babies, so the X and Y chromosome could be used to demonstrate that the isolated cells Use of Microfluidic Technology for Cell Separation 221 came from the baby and not the mother. Mature RBCs do not have a nucleus, and he nce do not contaminate the DNA obtained [92]. 5. Conclusion In the last two decades, we witnessed many advances in microfluidic devices, and many of these devices are in advanced development stages or are commercially a vailable. Microfluidic technology offers superior capability to precisely engineer and control the microenvironment to sort cells. Micro- and nano-fluidic technology will fulfill the sensitivity, specificity, and reproducibility requirements to bring cell sorting into clinical utility. The challenge remains to demonstrate that information acquired using microfluidic devices would change the way physicians diagnose, treat, and/or monitor diseases . Author details Hisham Mohamed Egypt Nanotechnology Center (EGNC), Egypt 6. References [1] Fisher D, Francis GE. and Rickwood D. Cell Separation. New York: Oxford; 19 98. [2] Wheeler AR, Throndset WR, Whelan RJ, Leach AM, Zare RN, Liao YH, Farrell K, Manger ID,

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899. Section 3

Applications in Haematology

Chapter 12

2012 Shrivastav and Singh, licensee InTech. This is an open access chapter distr ibuted under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by /3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Tigers Blood: Haematological and Biochemical Studies A.B. Shrivastav and K.P. Singh Additional information is available at the end of the chapter http://dx.doi.org/10.5772/50360 1. Introduction Tiger (Panthera tigris tigris) population in their historic ranges is critically endangered owing to habitat destructions, ruthless poaching and retaliatory killing. The tiger population now remains in few thousands located in about 150 fragments in 13 countri es (Karanth and Gopal, 2005). However, declination is also associated with health relat ed problems such as nutritional deficiencies and infectious diseases (Prater, 2005). Therefor e, health monitoring and scientific health management, disease diagnosis and treatment should be made mandatory for conservation of wildlife as the tiger is a key stone s pecies and important member of forest ecology (Shrivastav, 2001). Haematological and biochemical studies are important tool for health ev aluation and their interpretations to know the status of physiological functions of variou s organs. The concentration of biochemical constituents in tissues as well as in bod y fluid is fixed and during adverse conditions, it may be elevated or decreased (Douglas an d Nelson, 1991). However, qualitative and quantitative analysis of corpuscles and chemica l constituents of plasma or serum are closely linked with functional unit of the cell and their assessments may reflect the physiological disorders (Harvey, 1997). Nevertheless, several factors involved to transmit infectious diseases either me chanically or biologically through contaminated water, food or vectors (Lice, Flea, T icks and Mites) and the pathogens may alter the normal physiology (Shah, 1983). Viral, bac

terial and parasitic diseases are very common in tigers which can affect the haematological and biochemical normal values (Rao and Acharyjo, 2002). Types of anaemia and significa nt blood loss may be estimated through complete blood count (CBC) and physiological funct ion of different organs by biochemical parameters (Jain, 1986). Qualitative and quantitative redu ction in the blood commonly observed in captive felid particularly in cubs those ma intaining on milk alone. The values of liver function test, elevated on repeated immobil ization by sedative Blood Cell An Overview of Studies in Hematology 230 drugs. It has been experienced that the values of serum enzymes incre ased after 72 hrs interval of 2nd immobilization by Ketamine and Xylazine mixture (person al communication, Shrivastav 2012). Sign of anemia such as pale mucous me mbranes weakness, fatigue and tachycardia may be observed depending on the severity of a nemia. A variety of abnormalities may be noticed by analysis of blood, bone marrow cytolo gy, serum chemistry and urine analysis. Wild felids are commonly injured in territorial fight or sometimes ser ious injuries and internal hemorrhages occur during hunting. If blood loss is above the 50% of tot al volume in short period may be fetal and tiger may die due to hypovolumeic shoc k. Information on haematology and blood biochemistry is meagre in wild animals. However, several studies on selected haematological parameters of exotic species of captive Feli ds have been reported. Currier and Russell (1982) studied the higher pack cell volume in wild and captive mountain lions (Felis concolor) and Fowler (1986) has reviewed the hae matological and biochemical profile of Felids including captive tigers whereas Jain (19 86) reviewed the information of the genera Panthera, Felis, Uncia and Acinonyx concluded that blood parameters were almost similar to that of domestic cat with exception of higher concentration of plasma protein and Pack cell volume (PCV). Seal et al. (1987) h ave studied the haematological and biochemical profile of captive Bengal tigers wit h emphasis of anaesthetic effect on blood parameters. Chandranaik et.al. (2006) also studied the haematology of physically restrained tigers that were kept in squeeze cages without using anaesthetics. However, the haematological and biochemical studies were m ade in twelve apparently healthy tigers in free ranges of Central India (Shrivastav et.al. 201 1). Health monitoring, assessment of health during treatment and disease di agnosis in free

range tigers needs baseline data on haemato-biochemical parameters. This baseline data is important especially for comparative health assessment of felids during out breaks of diseases between sylvatic and domestic cycle vice versa. It is also required, as the tiger is on top of the sylvatic food chain and to be protected for maintaining balances in ecosystem (Gopal, 1993). 2. Blood collection and investigation The collection of blood for laboratory investigations is comparatively difficult in both free range and captive tigers and only possible when animal is sedated or restrained properly in squeeze cage. Withstanding facts, chemical capture is comparatively safe, if accomplish ed by trained and experienced wildlife veterinarians. There are several drugs available for sedati on. Each drug works in a different manner and is more suited to some species only. The time re quired for a drug to have an effect depends upon the factors such as route of administration, absorption rate, concentration and physiological status of the animal while it is difficult to generalize the choice of drug and doses (WII, 1985). It depends upon circumstances like spe cies of the animal, age, sex, weight, location, temperature regimes in season, time of the day and Tigers Blood: Haematological and Biochemical Studies 231 emotional state. Shrivastav et.al.(2011) have used Xylazine hydrochloride + Ketamine hydrochloride as sedative drugs with the help of Tel-inject projectile syringe to immobilize the tigers of free range while Yohimbine hydrochloride was used as reversal drug . Prior to collection of blood from immobilized animal, it is an essent ial protocol to obtain normal values only through free flow of the collected blood drawn from the anima l either at rest or under conditions of least excitement to minimize the physiological varia tions in cell count (Jain, 1986). Normally the cephalic saphenous, femoral and jugular veins a re used for collection of blood from dog, cat and non human primates while in tigers these s ites are not convenient because the blood collector remained in front of face of the tiger. T he caudal vein is convenient and safer site for blood collection (Shrivastav et.al.2011). From sedated free range tigers, 2-5 ml of blood is drawn by venipuncture of the caudal vein through 18 no gauge disposal syringe in a tube containing Ethylenediam inetetraacetate (EDTA at 2 mg/ml of blood) as the anticoagulant (Shrivastav et.al. 2011). The bl ood samples should be processed as soon as possible after collection. If a delay is anticipa ted, it should be refrigerated at 4 o

C (Jain, 1986).The blood sample should be mixed several times before a portion is removed for test procedure (Shrivastav and Sharma, 2000). A utomatic devices providing a continuous rocking or circular motion have been found sati sfactory, but prolonged mixing should be avoided, particularly on a device with circ ular motion, to prevent a mechanical trauma to various blood cells, especially erythroc yte. In any event, blood smear must be made immediately after blood collection, either di rectly from fresh blood or after anticoagulation. Blood films should be dried quickly and protecte d from dust and flies till stained (Shrivastav and Sharma, 2005). Blood films can be made on glass slides and on cover slips.The haematological analysis needs precautionary measu res and blood smear is stained with Romanowsky stains and at least 200 white cells should be examined for the differential leukocytes count. Simultaneously, the blood smears must be screened for parasitic blood protozoa, flagellates and rickettsial infections. 3. Haematology of Tiger 3.1. Erythrocytes 0.45 m in size; appears The morphology of erythrocytes varies with 2 to 7.6, 7.3 circular, discoid, central pallor with slight anisocytosis whereas the rouleaux f ormation(Plate- 2) is common in tigers blood. Chandranaik, et.al. (2006) also reported the mi ld anisocytosis in physically restrained tigers. However, the range and mean (with one standard de viation) of total erythrocyte count (TEC) was 4.66 to 9.15, 7.9 1.42 million /l. L ikewise haemoglobin concentration (Hb) was obtained 9.8 to13.5, 12.8 1.65 mg/dl in male and 7.8 to11. 5, 10.8 1.05 mg/dl in female tigers (Shrivastav et.al. 2011). Jain (1986) defined that the rouleaux formation is associated with erythrocyte s edimentation rate (ESR) and useful for evaluation of the disease status. Shrivastav et. al. (2011) encountered ESR (14 to 26, 21 4.21 /hour) and PCV (36 to 45, 38 4.45 %) in free range tigers (Table 1). The consequences of ESR and PCV up and downs mostl y confined to Blood Cell An Overview of Studies in Hematology 232 erythrocyte osmotic fragility that increased in case of immune-mediated hemolyti c anemia. Taketa et. al. (1967) have assessed the oxygen affinity of the haemoglobin is mu ch lower in felines than that of other mammals including humans. 1 2 3 4 5 Haematology Unit Range Mean SE ( ) Red blood corpuscles (TEC) 106/l 4.669.15 7.90 1.42 Total Leukocytes Count (TLC) 103/l 6.211.05 8.50 1.42 Haemoglobin (Hb) g/dl 7.813.8 12.8 1.65 Haematocrit (PCV) Ratio 3645 38 2.54

Erythrocyte sedimentation rate (ESR) Hours 1426 21 4.21 6 Icterus index (II) u/l 2 5 2 1.51 7 Differential leukocyte count % i Neutrophils 5775 60 5.08 ii Lymphocytes 1835 30 4.56 iii Monocytes 2 6 0.5 1.21 iv Eosinophils 26 0.4 1.30 v Basophils 04 0.1 1.21 Blood plasma biochemistry 1 Albumin (ALB) g/dl 2.14.6 3.50 0.99 2 Total protein ( TPROT) g/dl 3.78.7 6.40 1.88 3 Total bilirubin TBIL) mg/dl 0.43.2 1.90 1.21 4 Creatinine (CRE) mg/dl 1.64.6 2.90 1.03 5 Blood urea nitrogen (BUN) mg/dl 6.548.2 27.90 13.77 6 Alanine Aminotransferase (ALT) IU/L 21.2109.0 67.88 27.84 7 Aspertate Aminotransferase (AST) IU/L 14.484.0 57.96 17.27 Table 1. Haematological and Biochemical Values of Bengal tigers (Panthera tigri s tigris) Jain (1986) reviewed the haematological parameters of big cats including Panther a, Felis, Uncia and Acinonyx and found that the blood composition were almost similar. Among all cats few erythrocytes had single refractile structure (Heinz body) when stained with new methylene blue stain. The Heinz body appearance in erythrocytes is the unique f eature of the family Felidae (Plate -1) while they are not visible usually in blood films with Romanowsky stain (Jain,1986). The reduction in erythrocyte count (TEC) and haemoglobin concentrat ion (Hb) are generally associated with anaemia and classified on the basis of eryth rocyte morphology, Tigers Blood: Haematological and Biochemical Studies 233 pathogenetic mechanism and bone marrow erythroid response ( Jain, 1986) .In wild animals the clinical signs and their magnitude depends on habitat and availabi lity of nutritive materials. Prolonged nutritional deficiencies of protein vitamins and mi nerals essential for erythrocytes production lead to anaemia. The type of anaemia varies wi

th the nutritional deficiency, blood loss and the animal species involved. Despite the nu tritional consequences the blood loss may be encountered through traumatic injuries, complication in bl ood vascular system, thrombocytopenia, and coagulation disorders. A normocytic nonchr omatic, non responsive anaemia is commonly found in association with chronic in fections, chronic infectious inflammatory conditions and some type of malignancies though microcytichypochromic is the sign of iron deficiencies (Jain and Kono 1975). Several blood sucking parasites produce blood loss anaemia in tigers l ike Ancylostomes, Toxoscaris that may cause haemolytic anaemia while Trypanosomes, Babesia and Haemobartonella (Mycoplasm haemofelis) may alter the total blood as well as plas ma volumes with acute blood loss. Chronic blood loss may lead to gastrointestinal lesions, ulcers, heavy parasitism like coccidiosis, neoplasm with bleeding into body cavity, de ficiency of Vitamin K and prothrembin etc.

Plate 1. Tiger Blood smear stained with Modified Wright Stain x1000. Blood Cell An Overview of Studies in Hematology 234 Plate 2. Tiger Blood smear stained with Modified Wright Stain x1000. 4. Leukocytes The total leukocytes count (TLC) and differential leukocytes count are important parameters to judge the body response against diseases. The TLC was 6.2 to 11.05, 8.5 1.42 t housand/l in free range tigers while differential leukocyte counts (DLC) reflect the information of infectious manifestations. A leukocytosis may be physiologic mediated by endogenous release of epinephrine or corticosteroids or it may be pathologic resp onse to a diseases process (reactive leukocytosis) or a result of a neoplastic change in the haematopoiesis (proliferative leukocytosis) while leucopenia is always pathologic event. Quantitative and qualitative changes in a particular type of leukocyte indirectly reflec t the nature of disease process and the body response to it. Jain (1986) reported physiologic factors such as fright and emotional di sturbances as an immediate effect on TLC and DLC and may confined to interpretation of conditions. The normal response to the stress is decrease in lymphocytes and eosinophi l numbers. In emotional leucocytosis, lymphocyte numbers are increased and equal or exc eed Tigers Blood: Haematological and Biochemical Studies 235 neutrophil numbers while eosinophil commonly not affected. Meyers-Wallen

et. al. (1984) observed the young cats normally have high lymphocyte counts and hence a greater tendency to develop lymphocytosis than the adults. This observation may also be attributed in the case of tigers as they belong to the member of same family with wild habitat as an escape behavior. Increases in neutrophil numbers due to physiologic inf luences are more pronounced in felines than in canines because of the difference in th e intravascular distribution of neutrophils. Prasse et. al.(1973) have observed 3 times mean mar ginal pool of neutrophils of clinically healthy cats than the circulating pool whereas in dog it was about equal or slightly greater. 4.1. The neutrophils Neutrophils considered as first line of defense against microbial infec tions and are important participants in inflammatory reactions. Shrivastav, et.al. (2011) enco untered 57 to 75, 60 5.08 % with multi-lobed nuclei of 3-5 lobes while sometimes mono-lobed nuc lei with pale to slightly pink granules in the cytoplasm in free range tigers( Plate3). Chandranaik, et.al. (2006) has also reported the segmented or multi lobed nuclei while Jain ( 1986) studied the sex chromatin in few neutrophils as the drumstick lobe in the female cats. The changes in blood neutrophil differential count (Haden, 1935) is as sociated with many consequences related to infectious diseases. Several functions have been suggested for the contents of granules, as neutrophils are phagocytic cells and regulatin g adhesiveness and aggression hydroxyl radical formation and generation of compliment deriv ed chemotactic factors while azurophilic granules are involved in modulation of inflam matory process (Gallin, et. al.1982). Condensation of nuclear chromatin leads to formation of d arker-staining plaques separated by delicate, light-staining areas with slight brown colour cyt oplasm. 4.2. The eosinophils Shrivastav et.al. (2011) observed eosinophils contained small, uniformly round bright eosinophilic granules almost occupying the entire and clear cytoplasm (Plate 3). These cells were encountered 2 to 6, 4 1.21 % in free range tigers (Table 1). The nuclei of t he cells were generally less lobulated than those of the neutrophils. The eosinophils are slightly larger than neutrophils. Chandranaik, et.al. (2006) also observed the larger eosinophil s larger than neutrophils and lobulated nuclei with orange cytoplasm in tigers. Jain (1986) reported the granules of the eosinophil are rod-like in domestic cats and Cheetah (Acinonyx j ubatus) while round granules in the eosinophils of Lion and Leopard. The eosinophils are commo nly seen in prolonged parasitic infections or allergic disorders.

4.3. The basophiles The basophile is a numerically insignificant but functionally important leukocyte that resemble with mast cells and it believed to share similar function as it is associated with allergic reaction, inflammatory process and immunocompitancy to the body fluids. Galli et. Blood Cell An Overview of Studies in Hematology 236 al. (1982) reported basophiles of cats have a limited capacity to pha gocytised. Chadranaik et.al. (2006) have reported smaller basophiles than eosinophils with pa le lavender pink stained cytoplasmic granules in physically restrained tigers. Jain (1986) observ ed the mature basophiles contains numerous small, round, lightly stained (pinkish or orangish) granules in light gray cytoplasm in experimental cats. The basophiles were rarely observed up to the size with 0 to 0.4 0.1 1.21 5 % in free range tigers. 4.4. The lymphocytes The lymphocytes are comparatively smaller than eosinophils with round t o oval nucleus occupying most space with spherical nucleus (Plate 3). Small and large lymphocytes were also seen in the blood smear. Some lymphocytes contained a few azurop hilic granules in their cytoplasm.Jain (1986) reported small lymphocytes is common in cat s with patchy nucleus and dense clumps of heterochromatin. In tigers, Shrivastav et.a l.(2011) have report lymphocytes from 18 to 35, 30 4.56 % (Table 1& Plates 2). Plate 3. Tiger Blood smear stained with Modified Wright Stain x1000. Tigers Blood: Haematological and Biochemical Studies 237 4.5. The monocytes The monocytes are usually larger than lymphocytes. Shrivastav et.al. (2 011) encountered 2 to 6, 5 1.21% monocytes in free range tigers with distinguishing feature of the reddish grey nucleus and well defined vacuoles, the nucleus of the monocytes report ed amoeboid and some time noticed horseshoe shaped nucleus while cytoplasm stained slig htly blue and appeared foamy vacuolated. (Plate 3). Jain (1986) has also observed s imilar monocytes in experimental domestic cats. The monocytes are associated with phagocytosis principally against intra cellular bacteria, viruses, fungi and protozoa. The cells perform regulation of the immun e response, phagocytic removal of tissue debris (affected cells, antibody coated ce lls and other foreign materials) as scavenger ( Jain, 1986). Rao and Acharjyo, (2002) have emphasized that macrophages, B-lymphocytes and bone marrow precursor cells are targeted cells for viral replication and co mmonly observed in Feline Pan-leucopenia (FPL), Feline Viral Rhinotracheitis (FVR), Immunode

ficiency Syndrome (FIDS), Canine Distemper (CD) and Inclusion Body Hepatitis (IBH) etc. T he body immune system is badly affected and gradually reduced. 4.6. The platelets Platelets are abundant in blood smear and usually distributed in small to large clumps. Shrivastav et.al.(2011) reported that individual platelets are pleomorphi c with rounded to elongated shapes with a central cluster of azurophilic granules (Pl ate 3). Jain (1986) has observed the clumping platelets in cat blood and emphasized that the platele ts of the cats clump readily during excitement of 3 minutes caused a sudden inc rease in platelet counts. A slight decrease occurred in sympathectomized cats and a some what greater decrease reported in splenectomized cats. 4.7. Blood biochemistry The concentration of biochemical compounds in tissues and body fluid c an be measured in a colorimetery, as it is capable of absorbing light of a particul ar wave length (Singh, 2004). Thus the health status of animal can be assessed by evaluation of Blood g ases, acid base balances, electrolytes, metabolic intermediates, inorganic ions, enz ymes and hormones. Shrivastav, et.al. (2011) have conducted blood biochemical analysis of free range tigers for Albumin, Total protein, Total bilirubin, glucose, creatinine, Blood urea nitrogen (BUN), Glutamic oxalo-transaminase (GOT/AST), Glutamic pyruvic transaminase (GPT/ ALT) by using an ERBA Chem-5 plus auto-analyzer (Transasia Bo-medicals Ltd.) wi th standard ERBA reagent kits for respective plasma constituents. The statistical a nalysis of obtained data is expressed in range, mean and standard deviation. Blood Cell An Overview of Studies in Hematology 238 4.8. Icterus index Jain (1986) reported an increase in the values of Icterus index in plasma is an indicative of an absolute increase in bilirubin concentration due to removal of aged er ythrocytes from the circulation by the reticuloendothelial and liver. Shrivastav, et.al. (20 11) reported 2 to 5, 2.1.5 units. in apparently healthy tigers of free range. 4.9. Total plasma protein Protein in plasma can provide information reflecting functional status of variou s organ and systems as blood is composed of approx 20 % of protein excluding haemoglobin. Ho wever, the total protein values gives the information on nutritional consequen ces or severe organ diseases as they transported the carrier of most of the constituents of the plas ma, maintains the colloid osmotic pressure, act as catalysts in biochemical reaction and play important role

in formation of fibrin polymers during clot formation (Richard, 1991). The total plasma protein in tigers was estimated 3.7-8.7 to 6.4, 1.88g /dl. The values are common ly increases in haemoconcentration and reduced in malnutrition, hepatopathy, less intake of p rotein and in neoplastic condition etc. 4.10. Plasma albumin The liver produces all the albumin and globulins while a small amount of globulins is produced by reticuloendothelial tissue (Benjamin, 1979).Liver synthetic capacity or proteinlosing nephropathy can be measured by albumin estimation in the blood plasma or serum. It also can interpret high or low calcium and magnesium level since albumin binds about one half of each of the ions (Richard, 1991). However, it appears to be a direct correlation between albumin turnover and body size because it is clinically signif icant. It is usually constituted with two third of total plasma protein and also serve as mobile amin o acids for the liver (Mc Pherson, 1991). Generally hypoalbuminism is observed in malnutrition, increased protein catabolism, nephropathy and chronic enterophathy. Shriv astav et. al. 3.5 g /dl, in free range tigers (2011) reported plasma albumin level 2.1 to 4.6, . Reduction in total albumin values is observed in malnutrition , liver diseases, stre ss, kidney dysfunction etc. 4.11. Total bilirubin Bilirubin is a breakdown product of heme about 70 percent of which i s derived from senescent red cells (Crawford et. al., 1988) however, 15 percent comes from hepatic cytoplasm and mitochondrial cytochromes and some from renal and other cytochrome s, and some from defective red blood cell broken down in the bone marrow be fore release. Shrivastav et. al. (2011) reported 0.4 to 3.2, 1.90, 1.21mg /dl, tot al bilirubin in free range tigers. The yellow color of serum or plasma is due chiefly to the p ressure of bilirubin. Increased concentration of bilirubin is commonly seen in haemolysis hepatocellul ar damage, biliary obstruction prolonged fasting reduced intake fluids etc. Tigers Blood: Haematological and Biochemical Studies 239 4.12. Creatinine Creatinine is important in muscles metabolism in that it provides stor age of high energy phosphates through synthesis of phosphocreatine (Benjamin,1979).It was estimated in tigers as 1.6 to 4.6, 2.9, 1.03 mg /dl. Serum or plasma creatinine concentration and uri nary creatine secretion are increased significantly by skeletal muscles necrosis or a trophy and defect in renal functions (Pennington, 1971) 4.13. Blood urea nitrogen

Urea is the end product of protein and amino acids and is generated in the liver through urea cycle (Woo and Cannon, 1991).Blood Urea Nitrogen is one of the important tools to know the renal function status. The values of BUN (6.5 to 48.2, 27.9 , 13.7 mg /dl) was observed in free range tigers is commonly seen in malnutrition and he patic insufficiencies, however, increased BUN is generally associated with renal disease congestive hea rt failure, shock, hypertension etc. Shrivastav et. al. (2011) observed the high rise might be also due to adlib intake of meat as the Royal Bengal Tiger can consume 35-40 kg meat of pray animal at a time (Prater, 2005). 4.14. Hepatic enzymes The serum enzymes used routinely in clinical diagnosis are synthesized in liver (Schaffner, and Schaffner, 1991). In hepatocellular or in cholestatic forms of liv er injury these hepatic enzymes are released in to the serum. The serum enzyme activities tha t are elevated in hepato cellulardamage are Alanine Aminotransferase (ALT) Aspertate Aminot ransferase (AST) Ornithine Carbamoyltransferase (OCT), Glutamic Dehydrogenase (GD) S orbitol Dehydrogenase (SDH) and arginase. The elevated serum activities that su ggest cholestasis (intra hepatic or extrahepatic) are Alkaline phosphotase (AP), Gamma gl utamyl transpeptidase (GGT) and 5 nucleotidase (5ND). The pathogenesis of the hepatic dis ease in carnivores especially in Felids are associated with viral hepatitis, pa rasitic infections or mechanical injuries (Rao and Acharjyo, 2002). The liver has great func tional reserves and signs of hepatic failure often do not develop until 70% or more of the functiona l capacity of the liver is lost (Tennant, 1997). 4.15. Alanine aminotransferase (ALT) Alanine Aminotransferase (ALT) was also termed as SGPT and used by ma ny estimations and large number are found in Hepatocytes in cats, dogs and promates (Benjamin, 1979).The ALT was estimated 21.2 to 109.0, 67.9, 27.84 IU /L in free range healthy tigers (Shrivastav et. al, 2011). 4.16. Aspertate aminotransferase (AST) Apart from liver, AST (Aspertate Aminotransferase) is also present in muscles and cardiac muscles. The higher value of AST though is not an organ specific but used as an indicator of

Blood Cell An Overview of Studies in Hematology 240 liver dysfunctions. Shrivastav, et. al. (2011) reported 14.4 to 84.0, 57.9 17.27 IU /L in the free range tigers. The haemato-biochemical profile of the Bengal tigers reported by Shriva stav et. al. (2011) was compared with the values of captive Bengal tigers (Seal et al. 1

987), and no major differences were noticed except in ALT, AST and BUN. The mean values (BUN (27.90 13.77 mg/dl), ALT (67.80 27.84 IU/L) and AST (57.9. 17.27 IU/L) in free range tigers (Table1)) are comparatively higher with the values of BUN (23.4 0.70 mg/dl), and AST (26.5 4.7 IU/L) as recorded by Seal et al. (1987).The higher values in free rang e tigers might be associated with beasts of prey, its variety and intake of flesh in natural ha bits and habitat while zoo tigers are locally dependent on monitored diet in captivity. Comprehensive information on haemato-biochemical parameters of free range tigers would be helpful for health monitoring and assessment of health status and prognosis of Bengal Tigers (Panthera tigris tigris) during treatment. Author details A.B. Shrivastav and K.P. Singh Centre for Wildlife Forensic and Health, M.P. Pashu Chikitsa Vigyan Vishwavidyalaya, Jabalpur, India Acknowledgement The Authors are highly thankful to Dr. H. S. Pabla, PCCF and Dr. Su has Kumar APCCF (Wildlife) Govt. of M.P. for their interest and constant inspiration t o support wildlife activities organized by the Centre for Wildlife Forensic and Health, M PPCVV, Jabalpur482001, India. 5. References [1] Benjamin, M.M. (1979) Outline of Veterinary Clinical Pathology, 3 rd Edn the state University Press Ames, Iowa, USA., -108-109 pp. [2] Chandranaik, B.M. Billarey,S.D. Das D; Renukaprasad, C and Krishnap pa G (2006) Studies on haematological values in Tigers (Panthera tigris) Zoos Print Journal , 21(7) 2321. [3] Crawford, J. M.; Hauser, S.C and Gollan, J.L. (1988) Formation of hepatic metabolism and transport of bile pigment.A status report semin. Liver Disease 8:105. [4] Currier, M.J. P. and Russell K.R. 1982. Haematology and Blood Che mistry of the mountain Lion (Felis concolor) Journal of Wildlife Diseases, 18:99. [5] Davidsohn L. and and Henry, J.B. (1969) Clinical Diagnosis by Lab oratory Methods Saunders, Philadelphia, Pennsylvania. Tigers Blood: Haematological and Biochemical Studies 241 [6] Douglas,A and Nelson,M.D.(1991)Basic Examination of Blood, Haematopoi esis Erythrocytic and Leukocytic Disorders. In Clinical Diagnosis and Managem ent by Laboratory Methods 18 th Edn. HBJ International Edition W. B.Saunders. [7] Fowler, ME (1986) Hematological data for some exotic species of Falidae: zo o and wild animal medicine, 2

nd Edn. Saunders, London, p 840. [8] Gopal, R. (1993) Fundamentals of Wildlife Management 2 nd Edn JH publication Allahabad. [9] Galli, S. J. et. al. (1984).Basophils and Mast Cells: morphologic insights into their biology, secretary pattern and functions, Prog. Allergy, 34:1. [10] Gallin et. al.(1982) Human neutrophils specific granules deficiencies : a model to assess the role of neutrophils specific granules in the evolution of inflamma tory response, Blood, 59:1317 pp. [11] Haden, R. L. Qualitative changes in neutrophilic leukocytes, Amer. Journal of Clinical Pathology, 5: 354-1935. [12] Harvey, J.W (1997). The Erythrocyte: Physiology, Metabolism and Bio chemical Disorders In Clinical Biochemistry of Domestic Animals Ed. Kaneko et. al. 5 th Edn Harcourt Brace Academic Press, Asia Pp 157-203. [13] Jain NC (1986) Materials and Methods for the study of the blood. Veterinary hematology, 3rd Edn. Lea & Fibiger, Philadelphia. [14] Jain, N.C. and Kno, C.S (1975). Erythrocyte Sedimentation rate in dog cat. Comparison of two methods and influence of Packed Cell Volume, Temperature and s torage of blood. Journal of Small Animal Practices 16:671. [15] Karanth K.U and Gopal, R. (2005) An Ecology based policy framework for huma n tiger coexistence in India. In People and Wildlife: Conict or co-existence 373-387. Wo odruffe, R. Thirgood, S. and Rabinowitz, A. Edn Cambridge, Cambridge University Press. [16] Mc Pherson, R. A. (1991) Specific Proteins: In Clinical Diagnosis and Management by Laboratory Methods 18 th Edn. HBJ International Edition W. B.Saunders pp 215. [17] Meyers-Wallen V.N et al. (1984) Hematologic Values in Healthy Neon atal, Weanling and Juvenile Kittens American Journal of Veterinary Research, 45: 1322. [18] Prater S. M. (2005) Indian wild animals, 7 th Edn Bombay natural history society, Bombay -37-45 pp. [19] Pennington,R.J.(1971).Biochemical aspects of muscles disease. Adv.clin.Chem .14:409. [20] Peters, T. (1977) Serum albumin: Recent progress in the understand ing of its structure and biosynthesis.Clinical Chemistry., 23:5. [21] Prasse K.W. et. al. (1973) Blood Neutrophilic Granulocyte kinetics in cats American Journal of Veterinary Research, 34:1021. [22] Rao A. T., and Acharyjo, L.N (2002) Disease of Wild Felids, Repr oprint Publisher, Bhubaneswar

[23] Richard A.M. (1991) Specific Proteins: In Clinical Diagnosis and M anagement by Laboratory Methods 18 th Edn. HBJ International Edition W. B. Saunders pp 215. [24] Schaffner, J.A. and Schaffner F (1991) Assessment of the Status o f the Liver In Clinical Diagnosis and Management by Laboratory Methods 18 th Edn. HBJ International Edition W. B. Saunders pp 229. Blood Cell An Overview of Studies in Hematology 242 [25] Seal US, Armstrong DL, Simmons LG (1987) Yohimbine hydrochloride r eversal of ketamine hydrochloride and xylazine hydrochloride immobilization of Benga l tigers and effects on haematology and serum chemistries. Journal of Wildlife Diseases 23(2):296300. [26] Shah, H.L. (1987) An integrated approach to the study of Zoonosis , Journal of Veterinary Parasitology, 1(1&2):7-12. [27] Shrivastav, A. B. (2001) Wildlife health: A new discipline: Essent ial for Tiger Conservation Programme, Intas Polivet 2: (2) 134-136. [28] Shrivastav, A. B. and Sharma, R. K.(2000) A Manual of Wildlife H ealth and Management in Protected Areas, College of of Veterinary Science and An imal Husbandry. [29] Shrivastav A .B. and Sharma R .K (2005). Health Management of Ti ger: A new discipline: Journal of Polyvet 2: 4-16. [30] Shrivastav, A. B. Singh K. P, Mittal, S. K. and Malik P.K. ( 2 011) Hematology and Biochemical Studies in Tigers, European Journal of Wildlife Research. [31] Singh, K. P. (2004) Serum Biochemistry on Prognosis of animal dis eases, In Training Manual for Field Veterinarians Published by JNKVV, 36-36. [32] Taketa F, et.al. (1967). Studies on cat haemoglobin and hybrids w ith Human Haemoglobin A. Biochemistry 6: 3809. [33] Tennant B.C. (1997) Hepatic Function In Clinical Biochemistry of Domestic A nimals Ed. Kaneko et. al. 5 th Edn Harcourt Brace Academic Press, Asia - 327. [34] WII (1985) A Guide for the Chemical Restraint of Wild animals, T echnical Report II, Wild Life Institute of India. [35] Woo, J and Cannon, D. C. (1991) Metabolic intermediates and inorganic ions. In Clinical Diagnosis and Management by Laboratory Methods 18 th Edn.HBJ International Edition W. B. Saunders pp141. Chapter 13

 2012 Druyan, licensee InTech. This is an open access chapter distributed under t he terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop erly cited. Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Blood Cells S. Druyan Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48307 1. Introduction Reduced oxygen availability to the tissues (hypoxia) poses numerous cha llenges to animal life. Hypoxia occurs as a result of diminished partial pressure of oxygen, such as occurs with increasing altitude, or reduced oxygen percentage in the air capillarie s of the lung. The oxygen partial pressure drops by approximately 7 mm Hg, i.e, approxima tely 2.5% in the case of atmospheric oxygen, for each 1,000 m increase in altitude, an d thereby reduces the amount of oxygen available to the hemoglobin in red blood cells as b lood passes through the lung. The hypoxia tolerance of birds has been suggested to be greater than that of mam mals. Early studies found that lowland house sparrows (Passer domesticus) in a win d tunnel at a simulated altitude of 6100 m behaved normally and flew for short periods [1]. Su ch findings support the anatomical and physiological evidence that the O2 transport pathway of birds has several unique characteristics that help support energetic activity and aerobic metabolism during hypoxia. The O2 cascade from inspired air to the tissue mitochondria includes several con vective and diffusive steps at which physiological adjustments can preserve the rate of O2 f lux in spite of hypoxia, thereby ensuring an uninterrupted supply of O2 to the energyproducing machinery of the cells [2]. These steps include ventilatory convection, diffusion across the bloodgas interface, circulatory convection, diffusion across the bloodtissu e interface (including myoglobin-facilitated diffusion), and O2 utilization by the tissue mi tochondria. Breathing (ventilation) is stimulated when a decline in arterial PO2 i s sensed by chemoreceptors in the carotid bodies. However, this hypoxic ventilatory response increases respiratory CO2 loss, causing a secondary hypocapnia (low partial press ure of CO2 in the Blood Cell An Overview of Studies in Hematology 244 blood) and alkalosis (high pH) in the blood [3]. Hypocapnia reflexiv

ely inhibits breathing and causes an acidbase disturbance. It has been suggested that birds h ave a higher tolerance of hypocapnia than mammals [4], possibly because of an ability to rapi dly restore blood pH in the face of CO2 challenges [5]. The significance of this tolerance i s that it would enable birds to ventilate more deeply before depletion of CO2 in the blood impairs normal function, and thereby to enhance O2 transport to the gas-exchange surf ace. It seems that every step in the O2 transport pathway can be influential, and that the relative benefit of each step changes with the level of O2 availability. The acclimatization response to hypoxia generally involves increases in hematocrit (Hct) and in hemoglobin (Hb) concentration, but this adaptive erythropoietic response is complicated [6-9]. It is reasonable to expect that an increased Hct c ould confer a physiological advantage under hypoxia, by enhancing O2-carrying capacity, but experimental results do not support this [10,11]. A moderately increase d Hct enhances arterial O2 content and therefore increases aerobic capacity [12-14], but the hi ghest attainable Hct is not necessarily associated with the highest possible aerobic power output [15,16]. This is because the associated increase in blood viscosity increases the pe ripheral vascular resistance, and this might compromise cardiac output (Q), thereby reduc ing the O2 consumption rate (VO2) [17,18]. Another mechanism that can sustain/enhance O2 transport under hypoxia is alterat ion in the O2-binding properties of Hb in the blood. These alterations could be mediated by changes in the intrinsic HbO2 affinity, changes in the sensitivity of Hb to allos teric cofactors that modulate HbO2 affinity, and/or changes in the concentration of allosteri c cofactors within the erythrocytes [19-22]. Numerous high-altitude birds, such as the bar-headed goose, the Andean goose [23], and the Tibetan chicken (Gallus gallus) [24], possess Hb with an increased O2 affinity. This can dramatically increase O2 delivery and pulmonary O2 loading in hypoxia by increasing the saturation of Hb and, consequently, the O2 content of the blood at a given O2 partial pressure. Thus it can greatly improve the O2 transport pathway [25]. Contrary to the hematological changes that are typically associated with the acc limatization response to hypoxia, genetically based changes in Hb structure that in crease intrinsic O2 affinity or that suppress sensitivity to allosteric cofactors are more important to hypoxia tolerance in naturally high-altitude birds [21,22,26], because in lowlan d birds an increased HbO2 affinity may hinder O2 unloading in the tissue capillaries.

Although these distinctive characteristics of birds should enhance hypox ia tolerance by improving the overall capacity for O2 transport, being avian is not i n itself sufficient for coping with hypoxia. Domesticated meat-type chickens (broilers) exhibit high O2 requirements because of their very fast growth and, consequently, they may have a reduced blood O2 level, i.e., hypoxemia [27-31] resulting from vigorous digesti on and metabolism which have high O2 requirements. When O2 demand increases, heart rate and cardia c output increase, thereby increasing the flow of blood through the lung and the pressure required to force blood through the arterioles and capillaries of the lung. The i ncreased flow rate and Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 245 increased transit time may not allow the red blood cells to pick up a full load of O2, so that hemoglobin O2 saturation is not complete, which causes hypoxemia [32]. Hypoxia/hypoxemia directly stimulates the endothelial and smooth muscle cells in pulmonary blood vessels, causing vasoconstriction throughout the lungs and an in crease in pulmonary blood pressure that can persist for a long time at high al titude [33,34]. This global vasoconstriction impairs O2 diffusion because it can divert blood flow aw ay from the gas-exchange surface to pulmonary shunt vessels [35], and the resultant pulmonary hypertension can cause fluid leakage into the air spaces, which, in turn, causes a thickening of the O2 diffusion barrier [36,37]. Hypoxic pulmonary hypertension can also overburden the right ventricle of the heart and can contribute to pathophysiological condit ions, such as chronic mountain sickness or ascites in broilers [9,38]. Ascites in fast-growing broilers: The commercial broiler of today represents the culmination of dramatic changes over the past 60 years. These changes were caused by genetic selection processes that foc used mainly on production traits [39,40]; it has been reported that 85-90% of the changes in commercial broilers were directly related to genetic aspects [39-42]. Commercial b roilers of 1991 were compared with the Athens-Canadian Random Bred Control Population, which represents the commercial broilers of 1957 [39,40]. Average daily weight gain of the 1957 and 1991 broilers were 10 and 31 g/d, respectively, from hatch to 3 weeks of age, and 19 and 68 g/d, respectively, from 3 to 6 weeks. The higher growth rate (GR) is driv en by a higher feed intake per unit time and higher metabolic rate and, consequently, a h igher demand for O2, from the embryonic stage onward [43-45]. However, it appears that the increase in growth

rate occurred without concomitant development in the efficiency of the cardiovas cular and the respiratory systems [41,46]. Thus, the increase in metabolic rate, coupled with exposure to environ mental conditions such as temperature, lighting and ventilation, and nutritional factors such as feed form or content, all seem to promote the development of ascites [47]. The pri mary cause of the ascites syndrome, however, is believed to be hypoxia/hypoxemia [48,49], when the bird s demand for O2 exceeds its cardiopulmonary capacity and causes pulmonary hypertension [50], which results in development of the ascites syndrome (AS) [51-53]. The etiology of the syndrome was well documented previously [52,54,55], and is characterized phenotypically by increased pulmonary hypertension, right-ve ntricle hypertrophy, fluid accumulation in the pericardium and abdominal cavity, increased hematocrit that results from increased red blood cell production (eryth ropoiesis), and a decline in arterial blood O2 saturation [41,52,56,57]. An international survey in commercial broiler flocks showed that AS af fected 4.7% of broilers worldwide [58]. Likewise, it was found that over 25% of over all broiler loss in the United Kingdom was a result of AS [59]. It is, therefore, apparent t hat this syndrome is a serious economic concern in the broiler industry. As the syndrome appe ars mainly at ages greater than 4 weeks, even 1% of mortality from AS causes significant economic losses, Blood Cell An Overview of Studies in Hematology 246 because it occurs toward the end of the growing period [58] and, the refore, affects heavy birds which have absorbed a considerable investment of labor and feed [60,61]. Two management approaches have been applied in order to minimize the actual AS morta lity in commercial flocks: (1) increasing the broiler house temperature by mean s of heating and insulation, which are costly; and (2) reducing the actual growth rate and, therefore, the metabolic rate and demand for oxygen, by providing fewer hours of light so as to reduce the quantity of feed consumed, and using low-energy mash feeds to reduce intake of dietary energy [47,62]. Thus, while the genetic potential for rapid growth of commercial broilers has been continuously improved by breeding companies [41], its full express ion is not allowed at the farm level, specifically to avoid morbidity and mortality of A S-susceptible birds. Consequently production costs are increased because of the longer perio d of rearing to marketing body weight. There are two alternative hypotheses regarding the association between GR of

contemporary broilers and their susceptibility or resistance to AS. Man y studies showed that AS does not develop in slow-growing chickens, egg-type Leghorns [see, e.g., 63,64], or slow-growing broilers [see, e.g., 65,66]. It has been suggested that high GR is the direct cause of AS, because of the consequent high demand for oxygen by tissues a nd organs of these birds. According to this hypothesis, alleles or genotypes that increase GR of broilers also increase their tendency to develop AS. Such a situation should be man ifested in a symmetrical genetic correlation between GR and AS: genetic differences in GR whether between lines or families, or between individuals within lines should be associated with corresponding differences in %AS. Symmetrically, individuals that develop AS, or families with higher %AS, should have a genetic potential for a higher GR tha n their counterparts that remain healthy under the same rearing conditions. The second hypothesis asserts that broilers do not have to be the fastest growin g birds in a flock in order to develop AS, but simply need to have their weight-g ain rate exceed the growth rate of their pulmonary vascular capacity [67-71]. According to this hypo thesis, there should be high-GR broilers that do not develop AS despite their high O2 demand, because they are genetically resistant. Similarly, there should be broilers wit h genetically low GR that, nevertheless, are susceptible to AS, although they require specia l environmental conditions to express this susceptibility. The hypotheses regarding an inherent association between AS and the genetic pote ntial for high GR were tested by examining contemporary commercial broilers in 2002 and 20 06, and an experimental low-GR slow-growing line [71]. All the lines were test ed under the same experimental protocol, that allowed measurement of GR under standard br ooding conditions (SBCs) up to d 19, and then efficiently distinguished between AS-susc eptible and AS-resistant individuals, the latter being those that remained healthy under the same highchallenge, ascites-inducing conditions (AICs) conditions based on exposu re to low ambient temperatures while receiving different forms of diet [72]. Asci tes syndrome incidence was 31 and 47% in the 2002 and 2006 birds respectively, and 32% in the 1986 slowgrowing line (Table 1). Most broilers that remained healthy under the high-challenge AICs exhibited the same early GR and BW as those that later developed AS. These resul ts, and the Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 247 relatively high incidence of AS in the slow-growing line, indicated that there i

s very little, if any, direct genetic association between AS and genetic differences in potential GR, which suggests that AS-resistant broilers can be selected for higher GR and remain healthy, even under AICs Age (d) 2002 experiment 2006 experiment 1986 broiler line 2002 broiler line 1986 broiler line 2002 broiler line Cumulative Mortality n 1 (N = 91) % n 1 (N = 42) % n 1 (N = 78) % n 1 (N = 97) % 28 0 0.0 1 2.4 0 0 a 2 2.1 a 35 5 5.5 2 4.8 3 3.8 b 10 10.3 a 42 10 11.0 b 9 21.4 a 12 15.4 b 26 26.8 a 54 2 22 24.2 -- -- 15 19.2 -- -Morbidity 3 42 or 54 7 7.6 4 9.5 11 14.1 20 20.6 Total AS incidence 29 31.8 13 31.0 26 33.3 46 47.4 a, b Mortality or morbidity percentages per line within rows (ages), within experim ental year, without common superscript differ significantly ( 2 test, P < 0.05) 1 n = number of birds with AS; N = total number of birds in the line.

2 The birds from the 1986 broiler line were kept under AIC through 54 d of age. 3 Birds that survived to the end of trial (Day 54 for the 1986 broiler line; Day 42 for the 2002 and 2006 broiler lines) but were diagnosed with AS. Table 1. Cumulative mortality and morbidity due to the ascites syndrome (AS) at various ages in broiler lines of the years 1986, 2002 and 2006, all reared together under high-c hallenge ascites-inducing conditions (AICs) from Day 19 to end of trial (According to [71]). These results, supported by several previous studies [68,70-78], suggest that there is no "true" genetic correlation between the potential GR of broilers and th eir propensity to develop AS. It seems that AS is not caused by the increased O2 requirement of a fast growth rate, but by an impairment of the O2 supply needed to sustain the fast growth ra te. Thus, a better solution would be to select against AS susceptibility, because if all broilers were resistant to AS, management-induced reduction of growth rate would no longer be needed. Breeding against AS susceptibility should aim at identifying and elimina ting all the AS-susceptible individuals in the selected population and selecting for high GR among the AS-resistant ones. The questions raised by the last hypothesis concern what might cause broilers to be susceptible to ascites, and whether it is related to physiological dis orders of the cardiovascular system. This chapter will introduce readers to the physiological Ascites Syndrome and th e complexity of the problems that highly productive broiler chickens face in coping wi th high-oxygen-demand conditions such as cold stress and high altitude. It will focus on: a. the ascites syndrome its causes and etiology in broiler chickens; b. cardiovascular functioning and responsiveness in ascitic broilers; and c. genetic and physiological aspects of coping with the sy ndrome. Blood Cell An Overview of Studies in Hematology 248 2. The Ascites syndrome: Its causes and etiology in broiler chickens Ascites physiology and etiology: The AS involves accumulation of fluids in the abdominal cavity [79], which prompted the common name of water belly to describe the syndrome; it occurs when br oilers fail to supply sufficient oxygen to support their metabolic demands [80]. In the late 19 70s AS was observed only at high altitudes [81], but since then it has been fou nd also at low altitudes [82], mainly in broilers reared at low ambient temperatures and/or fed pelleted feed with high energy content. The general pathogenesis of AS has been well documented [52,54,83,84].

Rapid growth requires a high resting metabolic rate, which requires adequate O2 sup ply and utilization. The broiler chicken probably has more genetic potential for growth tha n it has potential to provide O2 to sustain that growth, and in some broilers the demand for O2 might exceed the cardiopulmonary capacity to supply sufficient O2, ultimately leading to an O2 deficit [85]. The heart responds by increasing its output of (deoxygenated) blood to the lungs for oxygenation. This increased blood flow causes an increase in the blood pressure required to push the blood through the capillaries in the lung which, in turn, c auses pulmonary hypertension. This increase in work load results in an enhanced pressu re load on the right ventricular muscle wall, to which the muscle cells respond by adding parallel sarcomeres, causing thickening (hypertrophy) of the right ventricular wall. The mus cular right ventricular wall increases the pressure in the pulmonary arteries, arte rioles and capillaries of the lung. This process continues, causing additional hypertrophy. Me anwhile, the right atrio-ventricular valve thickens and starts to leak, partly because the thicker valve is now less effective and partly because of the increasing back pressure from the pulmo nary arteries and right ventricular chamber [86]. The leaking valve aggravates the e xcess pressure problem by admitting excess volume, the right ventricle dilates, and t he wall-muscle cells lengthen by producing longitudinally arranged sarcomeres. The increased blood volume raises the pressure overload until valve de ficiency occurs, causing a drop in cardiac output and pulmonary hypertension, but marke d pressure increases in the right atrium, sinus venosus, vena cava and portal ve in. This increased pressure in the sinusoids of the liver causes leakage of plasma from the liver i nto the hepatoperitoneal spaces, i.e., ascites. The leaking valve and increased venou s pressure result in hypoxemia and tissue hypoxia, and the kidney responds by producing erythropoieti n in an attempt to increase the blood s O2 carrying capacity by intensively pr oducing more red blood cells. Domestication had introduced several other insufficiencies into the card iovascular system; among them is a thicker respiratory membrane than that in oth er birds, i.e., broilers have a thicker respiratory membrane than Leghorn-type laying fowl. This leads to: a. lower efficiency of O2 transfer through the respiratory membran e; and b. lower hemoglobin oxygenation capability [62]. Research focusing on hemoglobin O2 satur ationin meat-type chickens indicated that fast-growing broilers have lower satur

ation than slowAscites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 249 growing broilers [30,87]. These results suggested that some meat-type c hickens were not fully oxygenating their hemoglobin, even at low altitude. This might have been t he result of increased blood flow rate through the lung capillary bed, which would reduce the time available for hemoglobin to be oxygenated in the lung interface [32,88 ], or to presence of immature red blood cells in the system [89]. In order to overcome th is situation an increase in erythropoiesis takes place. However, such an increase, if not coupled to plasma volume expansion would increase blood viscosity, followed by inc reased bloodflow resistance [90]. The back pressure in the veins causes venous congestion, d ilation and prominent vessels [50]. The lack of O2 in the heart muscle results in hypoxic da mage and, finally, right-ventricular hypertrophy. As cardiac output is reduced and tissue hypoxia becomes worse, the left ventricle loses muscle mass, the wall thins ( because of hypoxia and disuse atrophy), the valves thicken and the chamber enlarges. Hear t muscle damage is caused by the excess workload and by the tissue hypoxia associated with circulatory failure, not by the tissue hypoxia that increases cardiac output and triggers pulmonary hypertension. Environmental causes of ascites syndrome: Altitude: The partial pressure of oxygen becomes lower with increasing altitude. The ability of chickens to oxygenate their hemoglobin fully as the erythrocytes pa ss through the lung depends on the transit time in the lung, hemoglobin- O2 affinity, the thickness of the air hemoglobin barrier and, especially, the partial pressure of O2 in the air [62]. The effects of high altitude or hypoxia on ascites and heart disorders in broilers were reporte d as early as the 1950s and 1960s [91-97]. Those reports indicated that birds raised at high a ltitudes died because of right ventricular hypertrophy, congested and edematous lungs, and accumulation of fluid in the abdominal cavity. Significant microscopic damage to the heart, lungs and kidneys was also found in birds reared at high altitude [9 5,97], as well as in 1week-old broilers raised at high altitude [98] and in birds exposed to simulated high altitude [99-101]. Because AS was first noticed in birds raised at high altitude, the use of natura l or simulated high-altitude conditions was one of the first experimental protocols to be used [see, e.g., 47,97]. The hypobaric chamber has been shown to be an effective too

l for simulating high altitude and consistently inducing AS [102-106]; it simulates high alti tude conditions by generating a partial vacuum, thereby reducing the partial pressure of O2. Anthony and Balog [106], by simulating an altitude of 2,900 m above sea level, successfully induced 66% AS in a commercial sire line. In six lines of commercial broilers that were rear ed in the same hypobaric chamber, 47% of the birds developed AS [107]. When birds are exposed to low atmospheric O2 levels pulmonary blood vessels cons trict and pulmonary vascular resistance increases [108]. This immediate increase i n pulmonary arterial pressure can, over time, cause right ventricular hypertrophy and eventu ally result in the ascites syndrome [81,89,109-111]. Additionally, hypoxemia leads to a n increase in hematocrit, which, in turn, increases blood viscosity and results in i ncreased resistance to blood flow through the pulmonary blood vessels [90,112-116]. Blood Cell An Overview of Studies in Hematology 250 Low temperature: Temperature is the most-studied environmental cause o f ascites [see, e.g., 117-125]. In endothermic animals (mammals and birds) body tempera ture (Tb) is the most physiologically protected parameter of the body; therefore, the th ermoregulatory system in these animals operates at a very high gain, in order to hold Tb within a relatively narrow range, despite moderate to extreme changes in environmental conditions [1 26]. The ability to maintain a stable Tb springs from the mechanisms that cont rol heat production and heat loss; mechanisms that changed in the course of evolution, to enable end othermia to replace ectothermia [127,128]. Birds mostly respond to acute or chronic cold exposure by increasing their metabolic rate and oxygen requirement [129,130]. It wa s reported that a drop in environmental temperature from 20 to 2 C almost doubled the oxygen require ment of White Leghorn hens [131], and in another study there was a 32.7% increase in oxygen requirement in response to low temperatures [132]. Low temperatures were found to increase ascites by increasing both met abolic O2 requirements and pulmonary hypertension [122,133]. This increase in pulm onary arterial pressure was attributed to a cold-induced increase in cardiac output, rather than to hypoxemic pulmonary vasoconstriction [134]. As a result, low ambient te mperature has been widely used to induce AS in broilers [60,66,73,115,122,134-140]. Various pr otocols were developed, ranging from exposure to constant low temperatures [60,73,122 ,135,136,140], through gradual stepping down of ambient temperature [66,122,137,139], t o episodic

protocols under which the birds were exposed to natural fluctuations o f winter temperatures [115,138]. The efficacy of a cold-exposure protocol depen ds upon its timing, duration and magnitude, as well as husbandry and the birds genetic t endency to develop AS. The effect of the timing of a cold-stress application on ascites deve lopment in broilers indicates that exposure to low temperatures during brooding has a long -lasting effect on ascites susceptibility [62,120,125,137,141,142]. The consensus appears to be that cold stress during the first two weeks of life affects the birds metabolic rate for several weeks, and increases their susceptibility to ascites [62,120,125,137,141,142]. A novel AIC protocol for AS [72] involved rearing the tested birds in individual cages from 19 d of age, so that they could not escape the challenge of the environmental conditions, which comprised fan-in duced air movement at about 2 m/s and moderately low ambient temperatures (18 t o 20C). The effects of the environmental conditions were augmented by early use of high-energy pelleted feed to enhance rapid growth and by lighting for 23 h/d. Under this com bination of conditions, %AS among the broilers was 44% much higher than those re ported for coldstressed broilers on litter, and similar to or slightly lower than th at among broilers challenged by hypobaric chamber. The birds that developed ascites as a result of exposure to low temp eratures exhibited the same pathological symptoms as those that developed it under low O2 pa rtial pressure symptoms including increased hematocrit, hemoglobin, heart weight, and r ightventricle:total-ventricle ratios [70-72,122,124,143-147]. Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 251 3. Cardiovascular functioning and responsiveness in ascitic broilers Blood O2 transport, erythropoiesis and ascites The blood system provides the main systemic response to environmental changes and metabolic demands, either through the cardiovascular system or through alteration in O2carrying capacity. Reduced O2 availability in the blood (hypoxemia), reduces the O2 parti al pressure (PO2) of the arterial blood (PaO2). In such a situation the blood system must maintain an adequate delivery of O2 to the peripheral tissues, while maintaining an adequat e PO2 at the vascular supply source,, in order to permit O2 diffusion to the tissue mitochondria. Oxygen delivery can be enhanced by increasing the total cardiac output (Q) and by increasing the blood O2 capacitance coefficient ( O2). The latter parameter is de fined as the

ratio (CaO2 CvO2)/(PaO2 PvO2), where CaO2 CvO2 is the arterialvenous difference i n O2 concentration and PaO2 PvO2 is the arterialvenous difference in PO2. With regard to maintaining an adequate PO2 at the vascular supply source, the lo wer critical PO2 can be expressed as PvO2 = PaO2 [ O2 (Q/VO2)] 1, in which VO2 is the rate o f O2 consumption by the tissues and the product O2 (Q/VO2) is the specifi c blood O2 conductance [148,149]. Because PaO2 is determined by ventilation and O2 equilibr ation at the bloodgas interface, this equation shows that an increase in specific bl ood-O2 conductance minimizes the decline in PvO2 under hypoxia, thereby maintaining an ad equate pressure head for O2 diffusion to the tissue mitochondria [2]. Under severe hypoxia, an increased blood-O2 affinity will tend to maxi mize O2. The resultant increase in the specific blood O2 conductance helps meet cha llenges of both delivery and supply: it minimizes the expected PO2 decrement in the tissue capil laries while preserving a constant CaO2 CvO2 difference. Likewise, an increased hem oglobin concentration increases CaO2, thereby increasing blood O2 conductance if PaO2, Q and VO2 all remain constant. With excessive polycythemia, however, potential adv antages of an increased Hb concentration for O2-carrying capacity might be more than offset by a corresponding reduction in Q. Several significant alterations to the blood system in AS broilers wer e well documented: increased red blood cell numbers, through increased erythropoietin production [9 6,100,150153]; elevation of hematocrit values and blood viscosity [54,72,154], a nd central venous blood congestion [50,155]. These findings raised the question of the association between the plasma and the fluid that accumulated in the abdominal cavity, and whether the i ncrease in hematocrit resulted from a decline in plasma volume caused by plasma leakage out of the blood vessels, or from increased erythropoiesis that occurred as a compensatory reaction to the lack of oxygen in the tissue. In ascitic broilers the composition of the abdominal cavity fluid was fairly similar to that of the plasma, with regard to osmol ality, and total protein and albumin concentrations, which suggests a deficiency in the selective permeab ility of the blood vessels [89]. These findings resemble those in cirrhotic human p atients with ascites Blood Cell An Overview of Studies in Hematology 252 [156-158]. The escape of plasma fluid out of the blood vessels was probably due to increased pulmonary hypertension and central venous congestion symptoms found both in huma ns [158] and in broilers [56]. As in the case of human ascitic patients [159], AS b

roilers exhibited conservation of plasma volume similar to that of the healthy ones. However, the PCV in the AS broilers increased significantly, by up to 80%, as a result of a significant increase in the number of erythrocytes, which also contributed to a significant elevati on in blood volume. Thus, enhanced erythropoiesis, and not plasma volume reduction, was found to be involved in the hemodynamics of the ascitic broilers [89]. This finding could also account for the blood congestion and the increased blood viscosity [90] that contribute to the enhanced cardiac workload [103,134], blood pressure [103], and blood-flow resista nce [111] in AS chickens. In AS birds, the high PCV, on the one hand, and the significant dec line in blood oxygen saturation, on the other hand [30,57,66] raised the possibility of an impairment of blood O2carrying capacity. Increased erythrocyte rigidity appears to be another important factor in AS [54,62,113]: the nucleated erythrocytes will normally curl or fold to pass through lung capillaries [160], but hypoxemia and high hemoglobin concentrations decr ease the deformability of erythrocytes [62]. Further calculations of hemoglobin content per 1,000 red blood cells revealed a significant reduction in the AS broilers compar ed with that in the healthy and control broilers [57,89]. These results suggest the possibi lity of inefficient enhancement of the erythropoiesis process. Ascites-induction conditions elicited enhanced erythropoiesis, which resul ted in an increased proportion of immature erythrocytes in the bloodstream. However, where as in the healthy broilers only a moderate proportion (7.2%) of immature erythrocytes was observed, in the AS ones, immature erythrocytes contributed up to 23.5% to the total eryth rocyte count [89]. The significant increase in immature erythrocytes, coupled with the signif icant decline in hemoglobin content, might provide the explanation for the decline of O2 satur ation in the blood of AS broilers [30,57,72,134]. The differences between healthy and AS chickens in their production of erythrocytes in general, and of immature erythrocytes in particular, suggest that eryth ropoiesis regulation in the ascitic birds is defective. The heart The avian heart is different from that of mammals in that the right atrio-ventri cular valve is composed of a muscle loop made up mainly of muscle fibers from the right ventricle wall. The anatomy of this valve makes birds very susceptible to valve insuff iciency [52,161,162]: when the right ventricle responds to an increased workload it becomes hypertroph ic and the

valve hypertrophies along with the ventricle [161]. This thickening of the valve interferes with its effectiveness and may lead to rapidly developing valve failur e and ascites [161]. Although litter oiling did not reduce the average ascites score, litte r oiling improved air quality significantly in the pens and also improved heart morphology by reducing the right ventricle area from 0.44 to 0.36 cm 2 in ad libitum birds [163, 164]. Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 253 Alterations in the electrocardiogram (ECG) are seen in conjunction with AS. Of most importance has been the finding that increased S-wave amplitude in sta ndard limb lead II indicates increased susceptibility to AS [111]. However, there were no ECG readings indicative of primary pulmonary hypertension in most birds that developed ascite s [165]. A slower heart rate (bradycardia) [55,116], as well as reduction in the pulse rate had been found in birds developing AS [55] and in acutely cold-exposed birds [116]. Heart rate on days 1 and 7 was found to be significantly higher in the AS-suscep tible (AS-S) genetic broiler line than in the AS-resistant (AS-R) broiler line, with only the lowest quartile of individual heart rates in the AS-S line overlapping the highest qu artile in the AS-R line [57]. These results were in agreement with those of Druyan et al. [1 66], who found that generation S3 chicks from their AS-S line had a significantly higher heart rate on day of hatch than that of generation S3 chicks from the AS-R line. It was reported [167] that heart rate began to increase shortly after hatch, and reached a peak close to 4 wk of age; thereafter, it declined slowly [168]. The AS-S selected line exhibited increased heart rate only between d 1 and d 7, with a decline thereafter toward d 17, while the birds were kept unde r standard brooding condition [57]. Mild hypoxia was found to elicit an increase in heart rate [169,170], which suggests that the AS-S birds in that study experienced O2 shortage already at the time of hatch, even when kept under optimal conditions. A higher mean partial pressur e of CO2 in broilers venous blood (a marker for lung ventilation rate) on d 11 was found to be associated with increased ascites susceptibility [171,172]. Those results indicate that ASsusceptible birds suffer O2 shortage at an early age. However, it also suggests that as long as the susceptible birds are under SBC, higher heart rate can compensate for a mild hypoxemia, and no other physiological parameter would be affected. Effect on heart and blood vessels Birds with ascites induced by either low ventilation or cold temperatu

res exhibited hypertrophy of the medial layer of arteriols, which was probably a re sponse to primary pulmonary hypertension [173]. In low, ventilation-induced ascites, the b roilers had significant inflammation or osseous-nodule formation in the lungs [174,1 75], whereas in cold-stress-induced ascites, birds showed no inflammation [173]. Wideman et al. [50] suggested that increases in pulmonary vascular resistance initiate incre ases in venous pressure by challenging the capacity of the right ventricle to thrust all the re turning venous blood through the lungs. An acute reversal of systemic hypoxemia was reported to have no effect on pulmonary hypertension a finding that discounted the influen ce of hypoxic pulmonary vasoconstriction [176]. It was hypothesized that this reversal of systemic hypoxemia increased total peripheral resistance and normalized arterial pressure and cardiac output, but could not decrease pulmonary hypertension because o f the overwhelming influence of sustained pulmonary vascular resistance [176]. Development of techniques to measure changes in pulmonary arterial pressure and change s in wedge pressure helped to clarify that changes in pulmonary arterial pressure contribute to the mismatch between pulmonary vascular capacity and cardiac output, and th at pulmonary hypertension is initiated as a consequence of excessive pulmonary arter ial or arteriolar resistance [177,178]. The difference between individual broilers susceptibility to ascites may Blood Cell An Overview of Studies in Hematology 254 be related to an innate or acquired variability in their pulmonary vascular resp onsiveness to vasoactive mediators [179]. 4. Genetic and physiological aspects of coping with the syndrome The genetic control of susceptibility to AS Recent reports [71,72,107] indicate that about 50% of the broilers in commercial stocks develop AS under experimental protocols of high-challenge AIC. The term high chal lenge is used for AICs that apparently induce AS in all AS-susceptible individuals, wh ereas lowchallenge AICs induce lower rates of AS, probably only in the AS-S in dividuals whose higher growth rates necessitate higher O2 demands. The rates of AS reported in r ecent years are similar to those found under high-challenge AIC in the 1990s [68, 73]. In recent years, however, actual AS mortality in commercial flocks has been significantl y reduced or even completely eliminated by management practices that reduce feed intake a nd growth rate and, consequently, reduce the physiological O2 demand [47,62]. The prob lem with this

approach is that it compromises the efficiency of broiler production. A better solution would be to select against AS susceptibility: once all the broilers were resistant to AS, a managed reduction in growth rate would no longer be needed. H owever, breeding is feasible only if there is an inherent susceptibility to AS and if ef fective selection against it can be applied. Several studies have found the tendency of broilers to develop AS to be under genetic control, with estimates of heritability ranging from 0.1 to 0.7 [72-74 ,180,181]. Significant heritability of 0.5 to 0.6 has also been found for the ratio of rig ht ventricle weight to total ventricle weight (RV:TV) a postmortem indicator for AS development and severity [7274,182]. These data indicate the feasibility of selecting against susceptibility to AS, but only if all the genetically susceptible birds are identified at the phenotypic level. Mortality or morbidity caused by AS provides the ultimate identification of AS-S individuals. However, actual development of AS in susceptible birds depends on envi ronmental conditions that lead to hypoxemia, either by reducing O2 supply or in creasing the O2 demand [62]. It was found that a hypobaric chamber with a reduced O2 partial pressure, equivalent to that at 2,900 m above sea level, successfully induced 66% AS in a commercial sire line, suggesting full exposure of genetic variation in AS suscept ibility [103]. Surgical inactivation of one lung induced AS in all or most of the susceptibl e individuals [32,68,183,184]. The AIC protocol for broilers housed in individual cag es, where the tested broilers could not avoid the environmental conditions that were based on movemen t of cool air driven by a fan, combined with high-energy pelleted feed and 23 h of light per day, resulted in about 50% AS among commercial broilers [70-72], suggesting that all or, at least, most of the susceptible broilers developed AS. The successful induction of AS by means of any of these approaches suggests that breeding for AS resistance can be achieved by keeping all selection candidates under high-challenge AIC and awaiting mortality of all susceptible individuals. However, thi s direct-selection Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 255 approach has not been used by breeding companies, because it would fo rce them to compromise the selection for more important traits, such as growth rat e and meat yield, which are not fully expressed under AIC. Indirect selection against susceptibility to AS, cardiovascular indicators: Many studies focused on identifying reliable diagnostic indicators for AS in broilers.

Hematocrit (HCT) is a marker for high rate of erythropoiesis in ascit ic birds, therefore it is always significantly higher in AS broilers than in their healthy count erparts reared under the same conditions [30,54,60,115,124,125,139,154]. HCT values from broilers age d 35 and 44 d were used to screen one sire line and two dam lines for AS susceptibility [154 ]: they were used to select individuals that were considered the most (> 36%) and least (< 29%) AS susceptible, and the males and females with the highest and lowest H CT values, from the two dam lines, were selected and classified as high hematocrit (H) an d low hematocrit (L) groups. These individuals were then reared under broiler breeder managem ent conditions. Males and females within each group were mated, to create offspring that were HH , HM-no definition for HM, LM, and LL. The progeny underwent screening for hematocrit on days 6, 42, and 49, and from d 33 onward birds were subjected to cold stres s. Differences in HCT values were seen at d 6: the HH chicks had significantly higher values than all other groups. On d 49 HCT values of the HH birds were significantly higher than t hose of the LL birds. Cold stress increased AS mortality in all combinations, but the HH bi rds had significantly higher AS mortality then the LL birds, which suggests that HCT value is heritable. It was also suggested that HCT screening and selection based on HCT values could be eff ective in developing resistant populations of broilers. However, later studies rev ealed that the variation in HCT was a secondary manifestation of developing AS, therefore it co uld not be used as an early indicator of AS sensitivity under normal conditions [57,72]. He art rate (HR), measured by pulse oximetry or by encephalography, was found to be low er in broilers suffering from AS than in healthy ones [111,163,185]. At 35 days of age, HR in feedrestricted broilers was significantly higher than that in fast-growing broilers, and the HR of broilers suffering from congestive heart failure, which is associated with hypox emia and AS, was significantly lower than that of feed-restricted, slow-growing broilers and healthy fastgrowing broilers [64]. Broilers with AS were found to have a signific antly lower SaO2 than their healthy counterparts at the age of 6 weeks (62.1 and 86.0%, respectively) [30]. Broilers with AS induced by a pulmonary artery clamp had a significantly lower SaO2 and higher right-ventricle:total-ventricle weight ratio (hypertrophy of the right ve ntricle RV:TV) than those of healthy, non-AS broilers [32]. Therefore, low SaO2 was sugges ted to be a reliable genetic early indicator for AS susceptibility [186]. In recent years, some breed ing companies

have selected against broilers with low SaO2, as measured in selection candidate s at 5 wk of age [187]. However, because of the low %AS in these unstressed flocks, high SaO2 levels are expected in susceptible individuals that do not develop AS; also, low heritabili ty (0.15) was reported for SaO2 at 5 wk of age in commercial breeding lines [187]. Because of this low heritability and only moderate genetic correlation with actual manifesta tion of AS, the effectiveness of 5-wk SaO2 as an indicator for selection against AS s usceptibility must be limited. All the cited findings suggest that there is a genetic component for AS mortality and Blood Cell An Overview of Studies in Hematology 256 also for several parameters (e.g., RV:TV and HCT) that have been foun d to be associated with development of AS; however, the exact biochemical and physiologica l precursor factors related to the genetic propensity to develop AS are still not known. It is often difficult to determine whether a particular change is primary in natur e, and therefore determinative, or is a subsequent secondary manifestation in the develo pment of AS. If parameters to specifically predict AS susceptibility or resistance are sought, it is of paramount importance that the primary changes be determined and evaluat ed. Moreover, in order to assess their significance as criteria for selection, it i s necessary to estimate the heritability of these parameters, and their genetic correlation with co nsequent AS development under AIC. In order to conduct advanced physiological and genomic research on AS, and to find the primary cause of AS, identification of all AS-susceptible individuals i s crucial. This identification depends solely on mortality or morbidity under AIC. Under low- or mediumchallenge AIC, relatively slow-growing broilers or those that can bette r withstand cold stress, have a relatively lower demand for oxygen and, therefore, do not develop AS. Incorrect identification of AS-susceptible chicks as AS-resistant leads to biased findings regarding the true genetic association between the measured traits and the genetic difference in broilers susceptibility to AS. To effectively select against AS susceptibility without interfering with the nor mal expression of other selected traits, one has to identify the genes responsible for the prim ary cause of AS or measure their phenotypic expression. There is evidence that the pri mary cause of AS is manifested in the prenatal or very early postnatal phases, when the cardiovascul ar system is being developed and is starting to function [188-190]. Measurements of such a

manifestation, especially at the embryonic stage, necessitate sacrificing the investigated individuals, rendering it impossible to later determine, under AIC, if these ind ividuals were susceptible or resistant to AS. Therefore, to conduct advanced physiolo gical and genomic research on AS, one needs a pair of selected lines in which all the individuals are either AS-S or AS-R. Comparisons of tissues or functions of individuals from the divergent lines can help to identify the primary cause of AS and thereby to provide an effective indicator for selection against susceptibility. Resource populations derived from cross es between such divergent lines might facilitate genomic research aimed at identifying the genes involved in susceptibility or resistance to AS. Direct selection against susceptibility to AS Successful selection against AS susceptibility was conducted in a fully pedigreed elite commercial broiler breeder line [68,184]. Only males and females that did not develop AS following AS-inducing surgery, i.e., unilateral pulmonary artery occlusio n, were used for reproduction. After two cycles of such selection, %AS among males that were exposed to low temperatures (14 C) from 17 to 49 d of age was reduced to 4%, fr om 31% in the base population and 15% after one cycle. That study demonstrated the feasib ility of selection based on mortality of AS-susceptible individuals under a protocol of h igh-challenge AIC. Divergent selection for AS mortality was conducted by Anthony et al. [78]: the AS was Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 257 induced in a hypobaric chamber where oxygen content was reduced to the level equ ivalent to 2,900 m above sea level. After 10 generations of divergent sire-fa mily selection, %AS increased to about 90% in the AS-susceptible line and decreased to ab out 20% in the ASresistant line, thus reaching a divergence of about 70% [78]. Similarl y successful divergent selection was applied by Druyan et al. [70]: the 1st selection cycle was based on progeny testing for AS mortality under low-challenge AIC, and two further cycl es of full-pedigree progeny testing were conducted under a high-challenge AIC protocol [70, 72]. Two divergent lines were established: AS-susceptible (AS-S) and AS-resistant (AS-R), with, respectively, 95 and 5% AS incidence, i.e., a divergence of 90%, when reared tog ether under the same high-challenge AIC [70]. Genomic selection against susceptibility to AS The very rapid genetic divergence between the selected lines, along wit h pedigree analysis of %ASF within the AS-S- and AS-R-selected lines implies that a single or a few

major genes were responsible for the difference in %AS between the lines [70]. It was conclu ded that one or more genes was/were involved in the response to a two-cycle select ion against AS susceptibility [68]. Single-gene inheritance was also suggested after a complex segregation analysis of data on oxygen saturation of the hemoglobin in arterial blood (SaO2) [188], a trait known to be closely related to the AS [30,72]. Data on SaO2 from 12 ,000 males in fully pedigreed populations of a male line that had been closed for 30 to 40 generations were available for that study. The results suggested that a single dialleli c dominant locus was responsible for 90% of the genetic variation in SaO2, with high levels of SaO2 i ndicating AS resistance and low levels indicating AS susceptibility. Data from testcrosses between fully divergent AS-S and AS-R lines suggested a model of complementary inter action between the dominant alleles of two unlinked major genes [77]. If, indeed, only a few genes are involved in genetic control of susc eptibility to AS, and in light of the current rapid development and application of genomic tools, the AS genes seem likely to be detected and mapped in the near future. Once mapped, with the help of current and future genomic methodologies, the causative SNPs (or closely linked ones, used as markers) in these genes will be identified. High-throughput genomic ass ays may soon facilitate efficient genotyping of these marker SNPs, and their routine utilization in commercial breeding programs. With availability of such markers, high-chal lenge AIC will not be needed to effectively select against susceptibility to AS, because breede rs will be able to easily detect and cull individual birds, within the elite lines, that carry the alleles for AS susceptibility. All major broiler-breeding companies have been heavily i nvolved in R&D efforts aimed at achieving this goal. 5. Overall conclusions Broilers, being highly productive birds, have difficulties in maintainin g a dynamic steadystate balance between higher metabolic rate, on the one hand, and, on the other hand, the consequently higher demand for O2 a demand that might exceed the car diovascular Blood Cell An Overview of Studies in Hematology 258 system s capacity to satisfy the O2 needs. This non-steady-state situat ion leads to the development of the physiological syndrome ascites. Following exposure to AIC of birds from various backgrounds, birds tha t manifested AS were found to differ significantly from their healthy counterparts, in traits that were measured after initiation of the various AIC protocols, e.g., RV:TV ra

tio, hematocrit, erythrocyte counts, SaO2, heart rate, weight gain (WG). These differences are co nsistent with findings of numerous reports; they represent changes in secondary manif estations of AS and, therefore, could be useful in diagnosis of birds that are develo ping AS, but not in prediction of AS susceptibility. Only Druyans lines that were divergently selected for AS were found to differ sig nificantly in heart rate during the first week of life, when reared under standard brooding conditions (SBCs). Heart rate was significantly higher in the AS-S line than the AS-R line, but before the manifestation of the syndrome no such differences were found between the sick an d healthy birds from commercial flocks that were kept under SBCs. Therefore, it appears t hat higher heart rate cannot be used as a general indicator to identify AS-susceptible broi ler chicks. It is expected that the problem of AS will be solved by genetic eradication of t he alleles for AS susceptibility. However, manifestation of AS by genetically susceptib le individuals depends on environmental conditions as well as genetic variation in growth rate. Therefore genomic information is required for effective integration of selection against AS susceptibility into breeding programs of commercial broiler stocks. Author details S. Druyan Department of Poultry and Aquaculture Science, ARO, the Volcani Center, Bet-Daga n, Israel 6. References [1] Tucker VA (1968) Respiratory physiology of house sparrows in relation to hi gh altitude flight. J. Exp. Biol. 48: 55-66. [2] Storz JF, Scott GR, Cheviron ZA (2010) Phenotypic plasticity and genetic adaptation to high-altitude hypoxia in vertebrates. J. Exp. Biol. 213: 4125-4136. [3] Scott GR, Milsom WK (2009) Control of Breathing in Birds: Implica tions for High Altitude Flight. In: Glass ML, Wood SC, editors. Cardio-Respiratory Con trol in Vertebrates: Comparative and Evolutionary Aspects. Berlin: Springer-Verlag . pp. 429448 [4] Scheid P (1990) Avian Respiratory System and Gas Exchange. In: Su tton JR, Coates G, Remmers JE, editors. Hypoxia: the Adaptations. Toronto: B.C. Decker. pp. 4-7. [5] Dodd GAA, Scott GR, Milsom WK (2007) Ventilatory roll off during sustained hypercapnia is gender specific in Pekin ducks. Respir. Physiol. Neurobiol. 156: 47-60. [6] Bullard RW (1972) Vertebrates at Altitude. In: Yousef MK, Horvath SM, Bullard RW, editors. Physiological Adaptations. New York: Academic Press. pp. 209-225. Ascites Syndrome in Broiler Chickens A Physiological Syndrome Affected by Red Bl ood Cells 259

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ood Cells 269 [175] Maxwell MH, Dick LA, Anderson IA, Mitchell MA (1989) Ectopic ca rtilagious and osseous lung nodules induced in the young broiler by inadequate ventil ation. Avian Pathol. 18: 113-124. [176] Wideman RF Jr, Fedde MR, Tackett CD, Weigle GE (2000) Cardio-pulmonary fun ction in preascitic (hypoxemic) or normal broilers inhaling ambient air or 1 00% oxygen. Poult. Sci. 79: 415-425. [177] Forman MF, Wideman RF Jr (2000) Measurements of pulmonary arteri al pressure in anesthetized male broilers at two to seven weeks of age. Poult. Sci. 79: 1645- 1 649. [178] Chapman ME, Wideman RF Jr (2001) Pulmonary wedge pressures confirm pulmona ry hypertension in broilers is initiated by an excessive pulmonary arteria l (precapillary) resistance. Poult. Sci. 80: 468-473. [179] Wideman RF Jr, Erf GF, Chapman ME (2001) Intravenous endotoxin triggers pulmonary vasoconstriction and pulmonary hypertension in broiler chickens. Poult . Sci. 80: 647-655. [180] de Greef KH, Janss LLG, Vereijken ALJ, Pit R, Gerritsen CLM (20 01) Disease-induced variability of genetic correlations: Ascites in broilers as a case stu dy. J. Anim. Sci. 79: 17231733. [181] Moghadam HK, McMillan I, Chambers JR, Julian RJ (2001) Estimatio n of genetic parameters for ascites syndrome in broiler chickens. Poult. Sci. 80: 844-848. [182] Pakdel A, Van Arendonk JA, Vereijken AL, Bovenhuis H (2002) Dir ect maternal genetic effect for ascites related traits in broilers. Poult. Sci. 81: 12731279. [183] Wideman RF Jr, Kirby YK, Owen RL, French H (1997) Chronic unilateral occlu sion of an extrapulmonary primary bronchus induces pulmonary hypertension syndrom e (ascites) in male and female broilers. Poult. Sci. 76: 400-404. [184] Wideman RF Jr, French H (1999) Broiler breeder survivors of chr onic unilateral pulmonary artery occlusion produce progeny resistant to pulmonary hypert ension syndrome (ascites) induced by cool temperatures. Poult. Sci. 78: 404-411. [185] Kirby YK, McNew RW, Kirby JD, Wideman RF Jr (1997) Evaluation of logistic versus linear regression models for predicting pulmonary hypertension syndrome (ascites) using cold exposure or pulmonary artery clamp models in broilers. Poul t. Sci. 76: 392399. [186] Druyan S, Cahaner A, Bellaiche M, Rosner A, Shlosberg A (1999). Genetics of blood oxygenation, heart rate, and ECG waveforms, and their association with ascites in broilers. In: Proc. 1st European Poultry Genetics Symposium, Mariensee, Germany.. p. 112.

[187] Navarro P, Visscher, PM, Chatziplis D, Koerhuis AN, Haley CS (2 006) Segregation analysis of blood oxygen saturation in broilers suggests a major gene influence on ascites. Br. Poult. Sci. 47: 671684. [188] Dewil E, Buys N, Albers GAA, Decuypere E (1996) Different chara cteristics in chick embryos of two broiler lines differing in susceptibility to ascites. Br. Poult. Sci. 37: 10031013. [189] De Smit L, Tona K, Bruggeman V, Onagbesan O, Hassanzadeh M, Ar ckens L, Decuypere E (2005) Comparison of three lines of broilers differing in ascites Blood Cell An Overview of Studies in Hematology 270 susceptibility or growth rate. 2. Egg weight loss, gas pressures, embr yonic heat production, and physiological hormone levels. Poult. Sci. 84: 14461452. [190] Tona K, Kemps B, Bruggeman V, Bamelis F, De Smit L, Onagbesan O, De Baerdemaeker J, Decuypere E (2005) Comparison of three lines of broile r breeders differing in ascites susceptibility or growth rate. 1. Relationship bet ween acoustic resonance data and embryonic or hatching parameters. Poult. Sci. 84: 14391445. Chapter 14

2012 Yamashita, licensee InTech. This is an open access chapter distributed unde r the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body Kikuji Yamashita Additional information is available at the end of the chapter http://dx.doi.org/10.5772/36005 1. Introduction 1.1. A far infrared ray (FIR) energy The power of the energy radiated from any material is dependent on t he temperature. For example, the sun with the temperature of 6,000C at the surface and ~1 5,000,000C at the depths radiates the radio-magnetic ray with from the weak to the strong energy. The radiomagnetic ray with strong energy is absorbed before the arrives at the surface of the earth. But, if a human bathe in the ultraviolet ray even if it arrives onl y about 0.6% of whole ultraviolet ray radiated by the sun, the surface of skin is burned a nd strongly damaged. If the cell is poured the large quantities of ultraviolet ray, the DNA of nuclear is injured to induce a liver spot, freckles and aging of skin. Still more, it is thought that the immunity is

inhibited and a cataract and a skin cancer can be induced by the ul traviolet ray. It was thought that the sunlight arrived at the surface of earth was finally composed of the ultraviolet ray (UVA, UVB) of 6%, the visible ray of 52% and the infrared ray of 42%. It was well known that the energy of these lights bring a lots of the good effects for living bodies. The other sides, the materials on the earth we are living radiated the narrow ra nge of energy from 5 to 50m in FIR under 30C by getting the energy of sunlight. In other words, thought the radiating energy on the earth creating a life was only FIR of 5~50m by getting the energy of visual ray and FIR, the earth was filled with the energy of only FIR of narrow range. It was means that the energy of FIR may make contribut ion to the birth of a life. The National Aeronautics and Space Administration (NASA) report ed which ray indispensable for the maintenance of the life was FIR of 4~20m as the artificial sun in the space station. Therefore, it comes to a conclusion that the FIR is the raising ray for the living body (Fig.1). Recently, there have been many studies of the ef fects of FIR on health and food preservation. The available evidence indicates that whole-body FIR irra diation has Blood Cell An Overview of Studies in Hematology 272 biological effects [1-7]. Whole-body FIR irradiation is believed to improve huma n health and sleep by enhancing blood circulation in the skin [1, 2]. However, the effects of FIR on cells are not clearly understood. These thinking let me realize that there are a lot of natural materials radiating strongly the energy of FIR as a charcoal, stone, soil and tree. So, these natural materials were being used for our studies on the energy of FIR. Figure 1. The classes of the electromagnetic wave by the wave length m 2. The characteristics of rhyolite In order to study strictly the effects of the energy of FIR in 4~20m on the livi ng body, the radiating machine of the energy of FIR should be developed. In order to develop it, the selection of a good FIR radiator were started at first. The most noticeable effe ct of FIR energy radiation was the activation of water molecules. The change of the we ight of the ultrapure water on the FIR ceramics containing the five natural stones, 4 chemical product s of mineral oxide and charcoal was exactly measured with time at 37C. Though it was made clea r that some natural stones activated the evaporation of pure water, the rhyolite especi ally strongly activated. Then, the rhyolite (MATERA Inc. Toon, Ehime, Japan) mined a t Toon, Ehime in Shikoku Island was selected as the FIR radiation ceramics for the dev elopment of the

radiating machine of the energy of FIR (Fig.2). The characteristic of rhyolite w as the volcanic rock containing 70% over silicate dioxide with the flowing patterns fo rmed by the phenocryst of magma (Fig.2). Though the components are the quartz, fel dspar and biotite, the rhyolite is similar the granite. When the FIR energy radiated fro m the rhyolite was measured by Fourier transformed infrared spectrophotometer, it was made clear that the rhyolite was radiated 90% over the ideal black body at whole range of 5~20m (Fig. 3). It was proved that the rhyolite was the excellent radiator of FIR energy of 5~20m. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 273 Figure 2. The photograph of the rhyolite Figure 3. The radiation energy of the far infrared ray from the radiator The powder of rhyolite showed the antibacterial action to the Staphylo coccus aureus ATCC6538P at 1/5012. The adhesive sheet with the 15% powder of rhyoli te showed at 1/1995. Still more, the paint containg the 10 % powder of rhyolite s howed the antibacterial action to the Methicillin-Resistant Staphylococcus Aureus MRSAIID 1677 at 1/5012 . Black body Ryolite Wavelength () D i s p e r s i o n r a d i a t i o n ( W / m 2 s t r

 ) Blood Cell An Overview of Studies in Hematology 274 The values of antibacterial action of the materials containing rhyolite are resp ectively 3.7, 3.3 and 3.2. Though the values of antibacterial action over 2.0 was estimated as the antibacterial substance, the rhyolite was regarded to be the fairly mighty the antibacterial s ubstance. Still more, the antibacterial action of rhyolite to the colon bacillus was verified in neutral condition at our laboratory also (Fig.4, Table 1). These results sugge sted that the antibacterial action of rhyolite did not depend on the change of pH. This graph showed the effects of rhyolite on the oxidation-reduction potential of water (Fig.5). Item The water treated with the powder of rhyolite The water treated with the stone of rhyolite Control Biochemical oxygen demand mg/1 2.1 2.4 2.4 Chemical Oxygen Demand mg/1 3.4 3.9 3.9 Suspended solids 12 10 8 Dissolved Oxygen 9 8.9 9.1 Escherichia coli group number MPN/100ml 0 2200 14000 Conductivity 18.6 19.5 19 Table 1. The chemical analysis of the rhyolite treated water Figure 4. The effect of the rhyolite on Escherichia coli; Escherichia coli (DH50 ) were cultured for 16 hours in the sterilized pure water, the water containing the rhyolite and the wa ter containing 0.1% rhyolite powder. The proliferation of the Escherichia coli inhibited at 15.6% i n the water containing the rhyolite and 69.9% in the water containing 0.1% rhyolite powder. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 275 Figure 5. The change of the oxidation-reduction potential affected by the rhyoli te. The oxidationreduction potential (The longitudinal axis shows ORP, mv) decreased depending on the contents of the rhyolite(The transverse axis shows %) after 0.5-48h. Though the oxidation-reduction potential of tap water is about 550, it decreased with time to be about 280 after 48 hours. When the powder of rhyolite was put into tap water at 0.1%, 1%, 5%, the oxidation-reduction potential of tap water decreased respectiv

ely up to 480, 340 and 220. Then, it decreased with time up to about 130 afte r 24 hours. It was thought that the high oxidation-reduction potential of the tap water c aused by the residual chlorine. The reason why the oxidation-reduction potential of the tap decreased with time might be that the hypochlorous acid in tap water evaporated as the gas of chlorine with time. The oxidation-reduction potential decreased in the dependent upon the density of the rhyolite powder. It was thought that the reason w as by the elution of any reducing agent from rhyolite. The small amounts of any elements w ith oxidative reaction were left in the ultrapure water. Therefore, the small amounts of rhyol ite powder could reduce the most of oxidizers in the ultrapure water. The mighty oxidative powers of the residual chlorine attack the cell membrane of a microorganism and a virus to degenerate the inner protein and show the germicidal and disinfectant effects. It was thought that the reducer generally do not show a mighty antibacterial effect. Why the rhyolite shows the mighty antibacterial effects? Then, the 500g of rhyolite powd er was put into 1l of ultrapure water. After keeping 18hours, the supernatant of the water was collected and filtrated with the filtering paper. Still more, the filt rate was centrifuged at O x i d a t i o n r e d u c t i o n p o t e n t i a l

( O R P ) m v Ultrapure water Tap water Concentration of rhyolite %

Blood Cell An Overview of Studies in Hematology 276 3000rpm for 10min. The supernatant of 600ml dried at 200C for 5 hours up to get 1 3.4mg of white precipitates. The precipitates were analyzed by the x ray di ffraction and the electron-micro analyzer. The results showed that the precipitates mainly consist of calcium and silicon (Fig.6). As the rhyolite contained 70% over silica te, the results suggested that the ceramics structure consist of silicate oxide should disintegrate. Then, the silicate radical molecule containing an additional electron could b e formed. It was thought that the radical molecules are so active that the bacterium e l.al was contacted to collapse the cell membrane with an antibacterial action. Though the me chanism to form the silicate radical molecule was not clear, the antibacterial effects of the rhyolite containing the 70% over silica were the experimental facts. Figure 6. The analysis by the x ray diffraction of the components in the solutio n 3. The development of the FIR energy radiator In order to clarify the effects of the FIR energy radiation on the living body a t the rebel of a cell and animal, the FIR incubator for cell culture and animal raisin g apparatus was developed utilizing the valid ceramics as the rhyolite and the purebla ck that the resemble effects as the ideal black body are well known. The CO2 incubator must be exactly controlled the temperature inside of the incubator at 37 0.5C. Therefore, though the water jacket of about 40litter is equipped for the s uppression The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 277 of heat loss, the equipment of incubator becomes solid and heavy. Then, 0.5% CO2 gas need be supply into the incubator to activate the culture cell breathing. Still more, the gas of 0.5% CO2 in air must be supplied though the water layer in order to keep the humidity near 100%. So, we remodel the existing CO2 incubator. The heating system w as changed to FIR panel heater for keeping the temperature in the incubator at 37 0.5C. Th e five face at up,

down, both side and behind in the incubator except the front glass w ere coated with the valid ceramics as the rhyolite and the pureblack close to black body for raising the efficiency of FIR radiation. The all shelves were also coated with the same cer amics. Still more, the water jacket was change to the simple insulation, because the FIR ene rgy directly and instantly heats the surface of objects even if the air and space lie between the surface of incubator and the objects. As a result, the light and compact CO2 in cubator that can continuously radiate the FIR energy for 24hours was developed with a great succe ss (Fig.7) [8]. So, the strict comparative experiments on the effect of the FIR energy radiation on the culture cells become possible utilizing the same type of CO2 incubator without remodeling as control. Figure 7. The FIR CO2 incubator Then, the FIR animal raising apparatus with same FIR radiating system of the FIR CO2 incubator to control the temperature inside 20-40C at will and break d own any smell chemical products by photo-degradation with the light catalyst of TiO2 could be also developed (Fig.8). The FIR animal raising apparatus has the two chambers which t he upper chamber is coated with the valid ceramics as the rhyolite and the lo wer chamber is no coating [9]. So, the strict comparative experiments on the effect of FIR energy radiation on animals become also possible by using the FIR animal raising apparatus. Blood Cell An Overview of Studies in Hematology 278 Figure 8. The FIR animal raising apparatus 4. The effects of FIR energy radiation on water 4.1. The volatility and fluidity of water Water is indispensable for human life. As water is generally present surrounding us, the characteristics of water is hard to be considered as a strange matter . But, the truth that a boiling point of water is 100C is peculiar comparing with the other e lements with the similar molecular weight. For examples, though a sulfur is next of oxygen in the periodic table of the elements, the boiling point of hydrogen sulfate (H2S) is -60.7C. But, as the molecular weight of oxygen is smaller than that of sulfur, the boilin g point must be smaller than -60.7C. It is supposed that the boiling point of water should be the oretically at about -80C. Then, the coagulating point of water is appropriate theoretically at about 110C. Well, why the boiling and coagulating points are respectively at 100C and 0C? The reason is by the formation of cluster through the pulling against eac h other among the

water molecules by a hydrogen bond et al. The imaginary molecular weight inferre d from the boiling and coagulating points is about 100 by which 5, 6 water molecules gathered. Then, a water molecule has as much as four hydrogen bonds. Moreover, it was thought that the shapes of the cluster shows a straight chain, from a square to eleven-cornered shape, and the average shape is the pentagon. Moreover, it was thought that the cluster of water is not stable for long time, but it was formed and broken at the cycles of 10 -12 seconds. But, this condition of cluster is only applied to the very pure water w ithout any The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 279 ion and impurity. The real water actually contained various impurities. Even if the very pure water is kept in air, the condition of water changes moment by moment by melting of a gas and molecules from air. Then, in order to estimate the pre sent conditions of energy in the water, the change of the weight of water is exactly m easured for the indicator of the volatility. When the difference of volatility between the tap w ater and the deionized water are compared, the deionized water is clearly volatilize d more quickly than the tap water (Fig.9). Figure 9. The change of the weight of the deionized water and the tap water The high volatility means that a lot of water molecules spring out of the surfac e of water. Therefore, it is supposed that the energy of the water molecules is high and its cluster is small. This graph shows the change of weight of the deionized water radiated FIR energy through the grass layer by keeping in the FIR incubator for 12 hours and the untreated deionized water (Fig.10). Though the deionized water more quickly los ses the weight than the tap water, the deionized water radiated FIR energy losses mu ch bigger weight than the deionized water. In other words, it was made clear that the deionized water radiated FIR energy has high energy in the molecules to evaporate much easy. The results mean that the cluster of the water can become small by the FIR energy radiation. If that is the case, why the cluster formed by the water molecules changes by a ccepted the FIR energy? The water molecule consisting of two [O-H] bonds opening with 104has three types of the vibration energies. One is the deformation vibration of 1594cm -1 and the others are the symmetrical and asymmetrical expansion and contraction e

nergy of each 3656 cm -1 and 3756 cm -1 . When the energy were convert to the wave length, the deformation vibration is 6.27m and the expansion and contraction energy are 2.74m and 2.66m [10]. If the FIR energy of 5~20m radiated the water molecule s, the deformation vibration of the water molecule was activated by the trans mission of the energy through the resonance effect because the energy of deformation vibration was coincident with the radiating energy. Consequently, it was thought that the kine tic energy Blood Cell An Overview of Studies in Hematology 280 of the water molecule increased to rush out of a cluster. The cluster in the act ivated water by FIR energy radiation ought to raise the volatility. On the contrary, t he strength of FIR energy radiation by any materials can be estimated by measuring the c hange of the volatility. Then, when the boiling points of water was measured, that of FIR radiated water was 97.4C and that of control water was 98.9C. The boiling point of water decreased clearly by FIR energy radiation. These results also suggested that the water molecules are at the condition to rush out easy from the surface of water. Still more, when the viscosity of water was measured, it was clarified that the viscos ity clearly decreased by the FIR energy radiation (Fig.11). These results also suggest that the water become to flow easy by the same change of the cluster in water. It is thought that the wat er radiated FIR energy increases the volatility and fluidity at the same time. It is a very important finding. For example, it is thought that the blood and lymph flow in the human body become smoothly and activates by radiation of the FIR energy. It is only natural that the collection of lymph fluid into a lymph vessel rides the swelling in the body. Th en, a tear, sweat and digestive juices become similarly to flow easy also. It is suggested that the activation of the secretion of a tear stimulate the para-sympathetic n erve system. Still more, the activation of the secretion of a sweat stimulates the vario us metabolisms in whole body. The activation of the secretion of the saliva and digesti ve juices stimulates the digestive function to make the body vigorously. Figure 10. The change of the deionized water weight affected by the rhyolite as the FIR radiator

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 281 Figure 11. The change of the viscosity of water by the rhyolite as the FIR radia tor 4.2. The solubility of water It was found that the water radiated FIR energy by the rhyolite beco me to dissolve variable products. The plastic culture dish adhered proteins as bovine serum albumin (BSA) were prepared for the estimation of the solubility of water. The FIR energ y radiated ultrapure water by the rhyolite and non-treated same water poured into the dish, and was collected for measurement of the UV absorbance for protein. The results sh owed that the FIR energy radiated ultrapure water could dissolve much more BSA than the non-tr eated same water (Fig.12). Not only protein but also sugar and sodium hydroxide were c hecked the solubility. The results showed that the FIR energy radiated ultrap ure water could dissolve much more them. When the rhyolite powder was inserted into a fish tank at about 0.5%, the water in the tank became clear just after 4 days. T hese results were considered by which the deposited protein and other products in the t ank dissolved in water to raise the degree of transparency by the FIR energy radiation from the r hyolite. It was confirmed that the fishes and shell fishes become live longer than usual and keep the freshness in the tank with the rhyolite. For the application of these effects of the FIR energy radiation on water, the development of the contact lens cleaner was tried. The force of cleaning in the FIR energy radiated ultrapure water were estimated to t he contact lens attached the BSA on the surface. The results suggested that the force of cleaning of water was surely raised by FIR energy radiation from rhyolite. But, as the force of FIR was defeated to a detergent, the development of the contact lens cleaner was given up. However, it was expected that the effects of the FIR energy radiation from rhyolite, on which the force of solubilizing in water was activated, will be applied in vario us fields in future. ** Blood Cell An Overview of Studies in Hematology 282 Figure 12. The solubility of BSA in the FIR energy radiated 5. Experiment 5.1. The effects of the FIR energy radiation on an animal 5.1.1. The effect of the FIR energy radiation on the change animal When the rats were raised by using the FIR animal the increase of the body weight of rats radiated FIR energy became gentler than water of body weight of an raising equipment, that of control (Fig

.13). Though the same experiments on the body weight of rats repeated five times, the results showed the same tendency that the FIR energy radiation inhibited the increase of the body weights. In some laboratories, the rats and the mice eat foods and drink water a s much as wish. As a result, the rats and the mice naturally grow fat within a year. Therefore, it sh ould be thought that the FIR energy radiation activates the exercise activity and keep s away the switchover to the obesity conditions. Figure 13. The change of the body weight affected by FIR. The growth of the rats was inhibited by the rhyolite as the FIR radiator. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 283 5.1.2. The effect of the FIR energy radiation on the change of blood flow of an human The Rhyolite powder with a peak diameter of about 10m was printed to the blanket cloths made in Japan at 15%, like polka dots of about 2 cm (MATERA Inc. T oon, Ehime, Japan). The Rhyolite containing blanket and the control blanket were applied t o the healthy volunteers for application of FIR energy of 20 min at room temperatur e 22C and room humidity 50%, after acclimation of the condition in the room for 10 min. The velocity and the quantity of blood were continuously measured by the laser Doppler blood perfusion monitor and imagers in the same way for 20 min. The order of the application bet ween the FIR and the control blankets was often changed at the condition of d ouble blind. The velocity and the quantity of blood flow were accelerated 32.2% (Fig.14 ) and 19.1% (Fig.15) by the application of the Rhyolite containing cloths. Figure 14. The change of the velocity of blood by the FIR blanket. This graph sh owed the velocity of the blood after 60 min of using the rhyolite containing blanket. The velocity of th e blood increased 32.2% in the rhyolite containing blanket compared with the control blanket. Figure 15. The change of the quantity of blood by the FIR blanket. This graph sh owed the quantity of the blood after 60 min of using the rhyolite containing blanket. The quantity o f the blood increased19.1% in the rhyolite containing blanket compared with the control blan ket. Blood Cell An Overview of Studies in Hematology 284 5.1.3. The effect of the FIR energy radiation on the change of blood catecholami ne The amount of adrenaline, noradrenaline and dopamine in blood of the 40 mice kee ping in the FIR and control animal raising apparatus for 40 days were measured at the bl ood center.

It was shown that the amount of the dopamine in blood catecholamine significantly decreased, and the adrenaline and the noradrenaline in the blood catec holamine tend to decrease by radiated the FIR energy (Fig.16). These results suggested that the FIR energy radiation decreased the amount of the blood catecholamine in mice to inhibit the sympathetic nerve and activate the parasympathetic nerve. A mamma as a human bod y has the autonomous nervous system with the motor nervous and sensory nervo us system. The autonomous nervous system distributes all internal organs to work in o rder to sustain a persons life independent of his mind. Figure 16. The change of the concentration of catecholamine in the blood affecte d by the FIR energy radiation. The concentration of the adrenaline, noradrenaline and dopamine in th e FIR group were significantly lower 14.4% There are the sympathetic nerves and the parasympathetic nerves in the autonomous nervous system which distribute every internal organ at the same times to control the function of each internal organ in spite of his mind. When the sympa thetic nerves excited, the heart beat violently, the blood pressure increase, the breath become hard an d the stress build up in the body. On the contrary, when the parasympathetic nerves excited, the feeling become confortable as at home to become feeling an appetite, sleep an d sexual desire. It is thought that the rats and the mice radiate FIR energy become loss of the stress in the cage and lives the active and healthy life. In short, FIR energy radiation may avoid the stress and obesity. * Dopamine Noradrenalin Adrenalin The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 285 5.1.4. The effect of the FIR energy radiation on the biological macromolecules The effect of FIR energy radiation on the biological macromolecules is being mad e clear. The effects of FIR energy radiation on the activity of the enzyme can be estimated by using the FIR incubator developed by us. An enzyme is the special protein which has the various amino acids forming the three-dimensional structure with the various bonds to ge t a special role to a chemical reaction. In case that the activities of the 19 kinds of the enzymes appear in radiating FIR energy, it was made clear that the activity of some enzymes were accelerated or restrained. These results suggested that the efficiency of the enzyme activity changed because the various bond conforming the three-dimensional struct ure of the enzyme was affected by the FIR energy. In the enzymes measured by us

, the results suggested that the activities of esterase, esterase lipase, lipase, leu cin allylamidase, betaglucuronidase were accelerated and that of alkaline-phosphatase, acid-pho sphatase, naphtol-AS-BI-phospho hydrase were restrained. Especially, esterase, ester ase lipase and lipase are the enzymes to decompose a fat. Though the experiments were prelimina ry study, it was made clear that the FIR energy radiation changed the three-dimensional st ructure of the various enzymes to accelerate or restrain the activities. Still mo re, it was possible from the results that the increase of body weight restrained because some decompositi on enzyme of fat were restrained by FIR energy radiation. These results suggested tha t the FIR energy radiation has a diet effect. . Figure 17. The change of activity of lipase by FIR energy radiation. The activit y in the FIR radiation group was higher 40.5% than the control group at 530nm. A b s o r b a n c e Blood Cell An Overview of Studies in Hematology 286 5.2. The effects of FIR energy radiation skin 5.2.1. The effect of FIR energy radiation on skin repair The wound of circle with 8mm diameter was artificially made at the skin of rats keeping the FIR and the control animal raising apparatus. Then, the area of the wound was measured each several days. And the surface condition was observed. The results showed th at though the surface of the wound in the FIR radiation group wet and soft, that of the co ntrol group was covered the hard scab (Fig.18). Figure 18. The surface of wound of skin affected by the FIR energy radiation. Co ntrol : (A) FIR energy radiation : (B) The repair area of FIR radiation group increased each 30.54%, 28.27% and 14.22% after 5, 6 and 7days compared with control group (Fig.19). These results showed that the FI R energy radiation from out of body accelerated the wound healing at the surfa ce of rat skin. Still more, the repair area of wound increased each 44.39% and 24.29% after 3 and 5 days by

application of the ointment containing the 10% rhyolite as the FIR en ergy radiating ceramics. In other words, it was clarified that the FIR energy radiation promote d the wound hearing of skin, even if the radiation of FIR energy were by far from body or by contact with skin. If the blanket and sheet with FIR energy radiation effects were developed, the bedsore of the bedridden old persons or the sick persons in bed for a long time would be suppressed and restored to regenerate the skin and ease the pain of the patients. Now, thou gh the rise in B A The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 287 a fee for medical treatment becomes a social problem, if the bedsore could be su ppressed to shorten the term of the admission to a hospital, the fee for the medical treatme nt would be reduced in the future. Still more, if the space of the residence was constructed with the FIR energy radiating building material as the rhyolite, the metabolism of skin would be promoted to hinder the aging of skin. Then, the FIR energy radiating ceramics as rhyolite may be tied together with a development of the creams and cosmetics to hinder th e aging of skin. Figure 19. The repair area of wound hearing. The speed of repair was accelerated affected by the FIR energy radiation. 5.2.2. Effect of the FIR energy radiation on a atopic dermatitis The mouse naturally induced a crisis of the inflammation of the skin as the atop ic dermatitis with the application of a picric acid as the material raising the in flammation was imported from the United States. Then, the effects of the FIR energy radiation on the ind uction of the inflammation of the skin were analyzed by using the mouse. No an inflammation of skin in the mouse radiated FIR energy was detected after the application of a picric aci d, though the atopic dermatitis of control mouse in the same condition were all ind uced without the FIR energy radiation (Fig.20). It was made clear that the area of the inflammation o f skin and the number of the mast cells at the inflammation of skin as an atopic d ermatitis clearly decreased by the FIR energy radiation (Fig.21). These results suggested that the FIR energy radiation was effective fo r the prevention and the medical treatment to the inflammation of the skin as an atopic dermatitis. I n the future, it is thought that the FIR ceramics as the rhyolite are tied together with the d evelopment of the medicine or the cure for the inflammation of the skin as an atopic dermatiti s.

Blood Cell An Overview of Studies in Hematology 288 Figure 20. The change of the inflammation of the skin as the atopic dermatitis. The induction of the inflammation of the skin on all mice was suppressed by the FIR energy radiation Figure 21. The area of the inflammation of skin and the number of the mast cells at the inflammation. Though the area of the inflammation in control group was 1.96cm 2 , no inflammation was detected in the FIR energy radiation group (A). The number of must cells was smaller 26.9% in th e FIR energy radiation group than the control group (B). B A

The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 289 5.3. The effects of the FIR energy radiation on bone 5.3.1. The effect of the FIR energy radiation on the cultured osteoblast-like ce lls The culturing osteoblast-like MC3T3-E1 cell (Riken Cell Banc, Tsukuba, Japan) were separated in the two groups in the FIR and the control CO2 incubator. After 1, 3 , 5, 7, 10 and 14days, the cell number of living cells in both groups were measured by a hemato cytometer and observed by phase contrast microscope CK40 (Olympus, Tokyo, Japan). Still more, the gene expression of the 4 days culture cells in both groups was analy zed by the cluster analysis the microarray hybridization method (http://www.godatabase.org). The proliferation of the osteoblast-like MC3T3-E1 cells in FIR group s ignificantly inhibited each 19.08% and 12.24% after 3day and 10days culture compared with control group . But, it was considered that the formation of the calcified nodules in the FIR group was more than the control group by the observation of the culture cells stained with von kossa and with a phase-contrast microscope (Fig.22). Figure 22. The effects of the FIR energy radiation on the bone nodules formed by MC3T3-E1 cells. The cultured MC3T3-E1 cells in FIR group (A) and control group (B) was observed by t he phase contrast microscope CK40 (Olympus, Tokyo, Japan). Some samples after 4weeks culture were stained with Von Kossa method and observed by the same method. FIR group (C) Control group (D) Ba r: 100m The results showed that the number and the area of the calcified nod ules of the FIR group were 148.8 and 158.8 m 2 , though no calcified nodules was formed yet in control group after 2 weeks culture. Still more, that of FIR group increased each 2.92tim es and 4.91times compared with the control group after 4weeks (Fig.23). Consequently, the

formation of the calcified nodules was clearly promoted by the FIR energy radiation. On the other hand, it was made clear by the results of cluster analysis after 4 day culture that the g enes expression of Interferon activated gene 205, 203 and Interferon -indu i le protein 27 in the cell to cell signal field were reinforced and the gene expression of Interleukin 1 family member 6, Interleukin 17D, 17 receptor C, 17B, and Interleukin 3 related with i nflammation were oppositely dropped (Table 2). It was also made clear that the genes expression of Platelet-derived growth fact or (PDGF) D polypeptide, Transforming growth factor (TGF) induced and TGF inducible early growth response 1 in the cell proliferation field were reinforced, and the gene expression of PDGF B polypeptide and Fibroblast growth factor (FGF) 2, 21, 8 were oppositely dropped (Table 2). It was clarified by these data that the cell differentiati on as the function of osteoblast estimated by the formation of calcified nodules were promote d by FIR energy Blood Cell An Overview of Studies in Hematology 290 radiation, though the proliferation was inhibited by it. In short, the FIR energy radiation shorts the term of cell proliferation and advances the timing of cell differentiation on the osteoblast. Finally, it was considered that the FIR energy radiation c ontrol these genes expression to inhibit the cell proliferation and promote the cell differ entiation of osteoblast [8, 9]. It was made clear by these facts that the differentiation of cultured os teoblast-like cell as an osteoblast is promoted by the radiation of FIR energy. Cell-cell signaling Gene Name Acc. No. Fold change Interferon activated gene 205 Interleukin 15 receptor, alpha chain Interferon alpha-inducible protein 27 Mitogen activated protein 3K 7 Interferon activated gene 203 NM_172648 NM_008358 AK010014 BC006665 NM_008328 1.597 1.422 1.388 1.365 1.328 Interleukin 1 family, member 6 Interleukin 17D

Heparin-binding EGF-like growth factor Interleukin 3 Interleukin 17 receptor Interleukin 17B NM_019450 NM_145837 NM_010415 NM_010556 NM_134159 NM_019508 -1.348 -1.393 -1.395 -1.471 -1.481 -1.631 Growth Gene Name Acc. No. Fold change Platelet-derived growth factor, D polypeptide Transforming growth factor, beta induced TGFb Inducible early growth response 1 Mitogen activated protein kinase kinase kinase 7 Growth differentiation factor 15 AK003359 NM_009369 NM_013692 BC006665 NM_011819 1.555 1.505 1.383 1.365 1.322 Fibroblast growth factor 8 Fibroblast growth factor 21 Epidermal growth factor receptor related gene Fibroblast growth factor 2 Heparin-binding EGF-like growth factor Early growth response 3 Growth factor receptor binding protein2-ap 1(Gab1) Platelet-derived growth factor, B polypeptide D12483 NM_020013 M99623 NM_008006 NM_010415 NM_018781 NM_021356 NM_011057 -1.323 -1.332 -1.337 -1.342 -1.395 -1.565

-1.667 -2.304 Table 2. The cluster analysis of the gene expression in MC3T3-E1 cells affected by FIR energy radiation by the microarray hybridigestion method These genes were reinforced and repress ed at 1.3 over by the FIR energy radiation in the cluster of the Cell-to signaling and the growth. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 291 Figure 23. The effects of the FIR energy radiation on the number and the area of the bone nodules formed by MC3T3-E1 cells. Each five samples stained with Von Kossa method after 1, 2, 3 and 4 weeks in both groups were measured the nodules (A) and the number of the area (B) by B io-system microscope BX-51. The measurement was analyzed by the average of five field of visions of each samples. Statistical analysis was carried out by t-test. *P<0.01 5.3.1.1. The effects of FIR energy radiation on the titanium implant Pure titanium post (RTP post titanium #Ti, Dentech, Tokyo) were treated with the snowtech O solution (Nissan Chemical Industries, Tokyo) for 48 hours and immers ed in MEM medium (Minimum Essential Medium Eagle, Sigma, St. Louis, MO, USA) for 5 days. Control group was only immersed in MEM medium at same condition. The 20 S.D. rat s of 6 weeks old were implanted the titanium post into the central position of the femu r. Left side was control group and Right side was the snowtech O solution treated group. These rats were separated two groups which the FIR energy radiating group kept i n the FIR animal raising apparatus and the control group was kept in the normal animal raising apparatus. The femur with the titanium post was extracted and measured the inten sity between the titanium and the bone by the tension test with the small omnipotent testing machine (Autograph, Shimadzu, Kyoto) after 1, 2 and 4 weeks raising. The same experiments were repeated twice. Though the data were easy affected by the damage of the surgical operation and the extent of the inflammation, the identical tendency was shown. The tensio n intensity of the implanted titanium post with the snowtech O solution in the F IR energy radiating group significantly reinforced 2.68 times as much as the same implant in the con trol group after 2 weeks (Fig.24). ** Blood Cell An Overview of Studies in Hematology 292 Figure 24. The effect of the FIR radiation on the strength of the bone bonding t o titanium. The bone bonding to titanium was clearly promoted by the FIR energy radiation. It was been thinking that FIR energy was too weak to permeate deep into the tissue of the

living body. But, if the FIR energy radiation affects the water molec ules in the blood as mentioned previously, the blood of the whole body was affected by FIR energy even if the deep position. The possibility is supported by the experimental results which th e new bone formation inside of the femur was activated by the FIR energy radiation at the s urface of the skin from outside of body. It is suggested that the deformational vibration of the water molecules in the b lood affected by the FIR energy radiation was activated by the transmission of the energy through the resonance effect because the energy of deformation vibration was coinci dent with the radiating energy. It was proved that the blood containing the activate d water molecules become quickly flow into the bone to activate the new bone formation. 5.3.1.2. The effects of the feed containing the FIR ceramics on the growth of bo ne The powder of the rhyolite mined at the vein of ore located on the Median Tectonic Line around Ehime prefecture was utilized for this experiment. The compositi on of the ingredient is SiO2 66.8%, Al2O3 13.52%, Fe2O3 6.98%, K2O 4.19%, Na2O 3.98%, CaO 2.55%. The 60 rats of SD strain at 4 weeks old were separated the three g roups of 0.01% and 0.1%FIR treated and control. The each group was raised every 2 or 3 days with th e standard feed containing 0.01% and 0.1% rhyolite and nothing for the each group in the me asurement the amount of the eating feed. After 115days, all rats were killed by the over a nesthesia and the femur and the tibia were measured at many parts of width and the length. By the results of experiments, the amount of the eating feed of 0.01 % FIR treated group increased up to 6.3%, that of 0. 1% FIR treated group decreased on the contrary compared with the control group. The width of the right femur at distal, cent ral and postal position significantly grew each 3.2%, 21.4% and 12.3% and that of the right tibia at cen tral position The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 293 and the left femur at the distal position grew 1.3% also. The width of the right femur at central and distal position significantly grew each 3.7% and 6.1%, and that of t he right tibia at central position and the left tibia at the distal position grew 1 1.8% also. Then, on the length of the long bone, the left tibia significantly grew 2.4% alone. The speci al affected parts were shown at Fig.25. It was clarified that the width of the femur and tibia qui ckly grew as shown in Fig.25. Figure 25. The effect of the eating the feed containing rhyolite on the growth o f the bone. The measurement parts of the femur and the tibia were shown (A). The growth of the f

emur and the tibia of these three groups of 0.01% and 0.1%FIR treated and control were compared after 115days. Right : (B), Left : (C) It was clarified that the width of the femur and tibia quickly grew i n the 0.01% FIR group. It was made clear by these results that the bone formation was promo ted by the inside radiation from the digestive organs by eating the feed containing the FIR energy radiating ceramics as same as the outside radiation of the FIR energy. These r esults suggest that the FIR energy radiation from the digestive organs arrives easy the deep posited bone of the living body. Still more, it was thought that the growth of length wa s a little inhibited and that of width was promoted. In other words, the FIR energy radiation does not pr omote the intrachondral ossification but the intra-membranous ossification that the osteob last directly differentiates from the undifferentiated cells. As the effects of the bone affected more sensitive than the cartilage with the blood capillary, it was thought that the b one formation by the osteoblast is promoted by the FIR energy radiation. The result s also prove that the Blood Cell An Overview of Studies in Hematology 294 FIR energy radiation affected the living body though the blood contain ing many water molecules. It was clarified that the FIR energy radiation promotes the bone formation though the energy transmission to water molecules. 5.4. The Effects of the FIR energy radiation on the cancer 5.4.1. The Effects of the FIR energy radiation on the culture cancer cells In order to clarify the effects of the FIR energy radiation on the culture cancer cells, A431 human epithelial vulva carcinoma cells, Sa3 human gingival squamous car cinoma cells, HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were cultured for the analysis of t he effects on the proliferation, the shape of cells and the induction of apoptosis. A431, A549, and MCF7 cells were cultured in Dulbeccos modified Eagles m edium/Hams F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagles basal medium (Sigma). All culture medium was supplemented with 1 0% heatinactivated fetal bovine serum. The cells (510 4 ) were plated and measured on days 8 using 0.2% trypan blue and the hemocytometer. Incorporation of 5-bromo-2-deoxyuridine ( BrdU) was used to determine the amount of DNA synthesis. Cells were observe d with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained w ith hematoxylin and eosin. Still more, apoptotic cells were identified using an Apo-Br dU in situ DNA

Fragmentation Assay Kit (Bio Vision, Mountain View, CA, USA). Although the proliferation of A549, HSC3, and Sa3 cells was significantl y suppressed from day 6 of culture (59.0%, 75.4%, and 76.2%, respectively) up to at least day 10, FIR irradiation had little effect on the growth of A431 or MCF7 cells. Measurement o f BrdU incorporation on day 4 of culture also showed a significant suppression of growth by FIR irradiation in A549, HSC3, and Sa3 cells but not in A431 or MCF7 cells. Observation of the morp hology by the phase contrast microscopy revealed that the cytoplasm and nucleus was enlarged in A549 cells. Some of the HSC3 cells also showed hypertrophy of the cytoplasm and nucleus and others tended to show atrophy. Finally, some of the Sa3 cells sh owed hypertrophy of the cytoplasm (to refer JEMAA 2010, 2, 382-394, Fig.1) [10] To determine whether the inhibition of proliferation by FIR energy radiation was associated with apoptosis or necrosis, cancer cells were subjected to FIR irradia tion of 48 h and analyzed on day 4 by staining with annexin V-FITC and PI and by TUN EL (TdT-mediated dUTP-biotin nick end labeling) (to refer JEMAA, 2010, 2, 382-394, Fig. 3)[10]. These results indicated that FIR did not induce apoptosis in A431, HSC3, or Sa3 ce lls. A few of the Sa3 cells showed significant necrosis. These results indicate that the inhibition of proliferation by FIR in the cancer cells, particularly in HSC3 and Sa3 cells, was not due to apoptosis. Still more, in order to analyze all expressing genes induced by FIR energy radiation, the control and the FIR-irradiated samples after four days culture was monitored u sing a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) with elimination of low s ignal feature of background signal + 2.6 SD, and P value < 0.01. Genes were further classified for process and function according to their The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 295 Table 3. The analysis of the extracted genes induced by the FIR energy radiatio n. The 10 most promoted genes and the 10 most inhibited genes in these changed genes showed Blood Cell An Overview of Studies in Hematology 296 By the results that the significant changed genes of the 5 cancer ce lls by the FIR energy radiation were search, the cells inhibited the proliferation and showed that the many genes related the proliferation changed. The number of the inhibited g enes by FIR energy radiation is more than that of the promoted gene except for t he HSC3 cells. The 10 most promoted genes and the 10 most inhibited genes in these chan ged genes were shown at Table 3. There is no common gene in the expressed gene of 5 cells. Thes

e genes contained mainly the transcriptional factor and the control factor of the transcriptional factor. 5.4.2. The control system in the cancer cells for the FIR energy radiation At the process of the analysis of the gene expression induced by the FIR energy radiation, it was made clear that the gene expression of HSP (Heat Shock Protein) 70 was reinf orced by the FIR energy radiation. Therefore, the hyper expression cells and th e knockdown cells of HSP70 were formed to analyze the effect on the proliferation. In order to directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70 A and HSC3-HSP70A cells, respectively). Control cells were transfected wit h empty pcDNA3.1 (A431-Neo and HSC3-Neo). In our initial experiments, we found that the exposure of HSC3 and Sa3 cells but not A431 cells to the FIR energy radiation causes G2/M arrest and induces partial hypertrophy to the necrosis (data not shown) [12]. To determine whether the increased expression of HSP70A confers protection against the FIR energy radiation, the cell survival was examined in FIR-irradiated A431 -HSP70, A431Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found th at over expression of HSP70A increased the cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiate d) A431-HSP70A cells was similar. The survival rate after 6 days of FIR irradiation was signifi cantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cell s. The BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells. Although the BrdU incorporati on of FIRirradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated H SC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A ce lls (to refer Med.Oncol 2008, 25, 229-237, Fig.3)[9]. HSP70 appears to be present in a variety of normal cell types and i ts expression may be induced by several stressors, such as hyperthermia, cardiac ischemia, i nfection, UV radiation, endotoxin, and nitric oxide to suppress or denature any for eign protein and restore an injured protein from lethal effects [13]. HSP70 seems parti cularly important for cancer cells. In human breast cancer, the expression of HSP70 correlates with in creased cell proliferation, poor differentiation, lymph node metastases, and poor the

rapeutic outcome [14]. In vivo animal studies and clinical trials have revealed that hyperthermia may serve as a powerful tool in the treatment of prostate cancer [15-20]; at the cellular lev el, hyperthermic The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 297 stress induces HSPs. Moreover, chemotherapeutic agents such as cisplatin, adriam ycin, and bleomycin, as well as r i tion induce HSPs. HSP70 participates in cytop rotection and is associated with cellular resistance to lethal external effects [18-21]. However, in the present study, HSP70 was never induced by FIR. These results suggested that F IR has anti-tumor activity without inducing HSP70 as an anti-stress factor. This characte ristic indicates that FIR may be suitable for medical treatment. We next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A/C siRNA effecti vely decreased HSP70A and HSP70C mRNA (Fig. 4A) and protein levels in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it sup pressed BrdU incorporation in cells irradiated with FIR. Similarly, the HSP70A/C siR NA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation a lso significantly suppressed BrdU incorporation in HSC3 cells transfected with the negati ve control siRNA. These results indicate that a decrease in HSP70 protein mediate s the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells (to refer Med.Oncol 2008, 25, 229-237, Fig.4)[9]. 5.4.3. The effects of the FIR energy radiation on the implanted cancer cell to m ice For experiments on body weight change, 10-week-old male skid mice (CB1 7/Icr-Prkdc) and 6- to 8-week-old male nude mice (Crlj:CD1-Foxn1) purchased from Ch arles River Japan (Yokohama, Japan) were housed with temperature control and a 12h light-dark cycle. Male skid mice were weighed every 5 days, beginning at 10 wee ks of age. FIR treatment by our developed animal raiser (FIR group), n=7 and control (control group), n=8. The log-phase cancer cells cultured in 10% FBS and DMEM/HamF-12 medium (Sigma, St. Louis, USA) resuspended at a cell density of 110 7 ml in PBS containing 500 g/ml of MatrigelTM basement membrane matrix (Becton Dickinson Labware, Two Oak Park, Bedford, MA). The tumor cells (110

6 ) were suspended in 0.1 ml of PBS containing 50 g of MatrigelTM and were slowly hypodermically injected in the back using a 25-gauge needle. After injecting tumor cells, mice were separated two groups: FIR group and control group. During the experiments, the long and short diameters of the resulting tumor were measured in five-day intervals, and tumor volume was calculated by the followin g formula: tumor volume (mm3) =1/2 (long diameter) (short diameter) 2. For purpose of the ascertaining tumor organization change by the FIR energy radi ation, the excised tumor was observed after stained with hematoxylin and eosin to detect tumor organization change after 30 days. Then we carried out immunohistochemical analyses to detect matrix metal loproteinases (MMP) members MMP-1, MMP-9, MMP-10 and MMP-13 [9]. The increase in tumor volume Blood Cell An Overview of Studies in Hematology 298 after implantation was significantly suppressed by whole-body FIR starti ng from day 15, compared to the control group (to refer J Table1). The cDNA microarra y analysis of tumor samples of control and FIR-treated showed that MMP-1, MMP-9, MMP-10, and MMP-13 in the MMP family were significantly down-regulated in the FIR group compared with control group (to refer ITEL 2005, 6(6), 597-601, Table1) [9]. The expression profiles obtained from cDNA microarray analyses as analyzed by quantitative real-time RT-PCR s howed the control group to have highly significant overexpression of the genes f or MMP-1, MMP-9, MMP-10, and MMP-13 compared to the FIR group (to refer ITEL 2005, 6( 6), 597-601, Fig.4) [9]. These results suggest that suppression of tumor volume increase by in vivo FIR radiation was due to inhibition of MMPs by FIR radiation. In other words, FIR suppresses invasion and metastasis of tumor cells by inhibiting the expression of MMP-1, 9, 10 and 1 3. As shown in (to refer ITEL 2005, 6(6), 597-601, Fig 5) [9], extensive portions o f tumor tissue in the FIR group was encapsulated and necrotized in intra tumor divis ion. On the other hand, in the control group tumor cells showed active proliferation and invasion into the surrounding muscular tissue. In addition, evaluation of the immunohistoc hemical expression of MMP-1, MMP-9, MMP-10 and MMP-13 using tumor tissues of control and FIR groups on day 30 after implantation showed MMP-1 and MMP-9 to be positively expr essed in the tumor stroma of the control group, while MMP-10 and MMP-13 we re strongly positive in tumor parenchyma (the part positively dyed at Cytokeratin10 ) (to refer ITEL

2005, 6(6), 597-601, Fig. 6) [9]. However, these MMPs in the FIR group were not significantly expressed. The result of immunohistological detection for these MMPs was concord ant with the results of cDNA microarray and QRT-PCR. That is, FIR radiation se emed to suppress invasion of tumor cells by inhibiting the expression of MMP-1, MMP-9, MMP-10, an d MMP13. The increased expression and activity of MMPs is associated with tumor invasion, metastasis, and angiogenesis [22]. The role of MMP-1 in tumor invasion and metas tasis has only recently been determined [23]. MMP-1 has been shown to cleave en tactin, thus contributing to the degradation of basement membranes and hence potentially cont ributing to the transitioning across epithelial barriers by tumor cells [24]. I mmunohistochemical detection of MMP-1 expression is also associated with increased invasive potenti al and poor prognosis in colon and esophageal cancers [25, 26]. The first barrier to tumor i nvasion is the basement membrane, and because one of its principle constituents is ty pe I collagen, the gelatinases (MMP-2 and MMP-9) are thought to play important roles in its degrada tion [27]. MMP-10 (stromelysin-2) is known to degrade various components of the e xtracellular matrix, and though it has been reported that MMP-10 (stromelysin-2) ex pression by lymphoma cells accelerates the growth of thymic lymphoma [28], the rol e of MMP-10 in other types of tumor growth is relatively unknown. MMP-13 (collagenase-3) is exp ressed in breast carcinoma and in articular cartilage of arthritic patients [29]. In addit ion, MMP-13 has high collagenolytic and gelatinolytic activity, and MMP-13 may be of i mportance in The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 299 degradative processes involved in breast cancer progression [29]. In th e present study, it was suggested that MMP-1, MMP-9, MMP-10, and MMP-13 are important for the invasion and metastasis of epithelial cancer of the human vulva A431 cell line in vivo and that the activities of MMP-1, MMP-9, MMP-10, and MMP-13 are selectively inhibite d by FIR irradiation. In conclusion, FIR irradiation can suppress tumor growth and invasion of epithelial cancer of the human vulva tumor by inhibiting the expression of MMP-1, MMP-9 , MMP-10, and MMP-13 without critical side effects. Inhibition of MMP expression was considered to be one of the mechanisms by which multiplication of A431 tumor cells was suppressed in FIR irradiation. 6. Conclusion

It was made clear that the motion of the water molecules was surely and physically activated by the radiation of FIR with the CO2 incubator and the ani mal raising apparatus. The analysis of the change of blood circulation conducted the conclusion that th e FIR energy radiation activated not only the motion of water molecules but also the blood ci rculation in the living body. Still more, it was made clear that the activation e ffects on the water molecules developed the activation effects of the skin regeneration and the new bone formation. Moreover, the proliferation or metastasis of the various can cer cells were inhibited by the FIR energy radiation connected with the activation of blood cir culation. It is expected that the present studies on the FIR energy radiation connect with the d evelopment of medical treatment for the regeneration of the tissue and organ as a skin and a bone, and the prevention medicine for cancer. Author details Kikuji Yamashita Department of Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan 7. References [1] Inou S, Kabaya M. Biological activities caused by far-infrared radi ation. Int J Biometeorol 1989;33(3) 145-150. [2] Honda K, Inou S. Sleep-enhancing effects of far-infrared radiation in rats. Int J Biometeorol 1988;32(2) 92-94. [3] Udagawa Y, Nagasawa H. Effects of far-infrared ray on reproduction, growth, behaviour and some physiological parameters in mice. In Vivo 2000;14(2) 321-326. Blood Cell An Overview of Studies in Hematology 300 [4] Udagawa Y, Nagasawa H,Kiyokawa S. Inhibition by whole- body hypert hermia with far-infrared rays of the growth of spontaneous mammary tumors in mice. Anticancer Res 1999;19(5B) 4125-4130. [5] Toyokawa H, Matsui Y, Uhara J, Tsuchiya H, Teshima S, Nakanishi H, Kwon A-H, Azuma Y, Nagaoka T, Ogawa T, Kamiyama Y. Promotive effects of far-infrared ray o n full-thickness skin wound healing in rats. Exp Biol Med (Maywood) 2003 ;228(6) 724729. [6] Udagawa Y, Inada K, Nagasawa H. Inhibition by Single Whole-Body Hyperthermi a with Glucose Administration of the Growth of Spontaneous Mammary Tumors in Mice. Jpn J Hyperthermic Oncol 2000;16(4)229-236. [7] Nagasawa H, Inada K, Ishigame H, Kusakawa S, Udagawa Y. Different Schedules of Whole-Body Hyperthermia with or without Glucose for the Inhibition of Mammary Tumors and Uterine Adenomyosis in SHN Mice. Bulletin of the School of Agriculture,

Meiji University 2001 43-51. [8] Yamashita K, Hosokawa H, Ishibashi J, Ishikawa N, Morimoto H, Ish ikawa T, Nagayama M, Kitamura S. Development of CO2 Incubator with Limited FarInfrared Radiation for Activation of Glucose Metabolism. ITE Letters BNTN 2005; 6(5) 53-57. [9] Hosokawa H, Yamashita K, Ishibashi J, Ishikawa N, Morimoto H, Ishikawa T, K itamura M, Nagayama M. A New Animal Raiser: Effect of Limited Infrared Radiation on Tumo r Growth of A431 Cells. ITE Letters BNTM 2005; 6(6) 597-602. [10] Egawa H, Takada K, Sasaki Y. (Eds) Far infrared ray. Ningentoreki shisha Inc Tokyo 1999; 3. [11] Ishibashi J, Yamashita K, Ishikawa T, Hosokawa H, Sumida K, Nagayama M, Kit amura S. The effects inhibiting the proliferation of cancer cells by far-inf rared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A. Me d Oncol (Northwood, London, England) 2008; 25(2 229237. [12] Yamashita K, Shine-Od Dalkhsuren, Ishikawa T, Sumida K, Ishibashi J, Hosokawa H, Ueno A, Nasu F, Kitamura S. Far-infrared ray radiation inhibits the p roliferation of A549, HSC and Sa3 cancer cells through enhancing the expression of AT F3 gene. J Electromag Anal 2010; 4(2) 382-394. [13] Wong H R, et al. Increased expression of heat shock protein-70 p rotects A549 cells against hyperoxia. Am J Physiol 1998;275(4 Pt 1) L836-41. [14] Nylandsted J, Brand K, Jaattela M. Heat shock protein 70 is required for th e survival of cancer cells. Ann N Y Acad Sci 2000; 926:122-125. [15] Kaplan I, Kapp DS, Bagshaw MA. Secondary external-beam radiotherapy and hyperthermia for local recurrence after 125-iodine implantation in adeno carcinoma of the prostate. Int J Radiat Oncol Biol Phys 1991;20(3) 551-554. [16] Kaver I, Ware JL, Koontz WW Jr.The effect of hyperthermia on hum an prostatic carcinoma cell lines: evaluation in vitro.Jr J Urol 1989;141(4) 1025-1027. The Effects of the Far-Infrared Ray (FIR) Energy Radiation on Living Body 301 [17] Paulus JA, Tucker RD, Flanagan SW, Moseley PL, Loening SA, Park JB. et al. Heat shock protein response in a prostate tumor model to interstitial thermotherap y: implications for clinical treatment. Prostate 1993;23(3) 263-270. [18] Roigas J, Wallen ES, Loening SA, Moseley PL. Effects of combined treatment of chemotherapeutics and hyperthermia on survival and the regulation of heat shock proteins in Dunning R3327 prostate carcinoma cells. Prostate 1998 ;34(3) 195202. [19] Servadio C, Leib Z. Local hyperthermia for prostate cancer. Urolog y 1991;38(4) 307-

309. [20] Yeushalmi A. Localized, non-invasive deep microwave hyperthermia for the treatment of prostatic tumors: the first 5 years. Recent Results Cance r Res 1988;107:141-146. [21] Bellmann K, Jttel M, Wissing D, Burkart V, Kolb H. Heat shock prot ein hsp70 overexpression confers resistance against nitric oxide. FEBS Lett 1996;3 91(1-2) 185188. [22] Spiliotis ET, Kinoshita M, Nelson WJ. A mitotic septin scaffold r equired for Mammalian chromosome congression and segregation. Science 2005;307(5716) 1781-1785. [23] Robinson CM, Stone AM, Shields JD, Huntley S, Paterson IC, Prime SS. Functional significance of MMP-2 and MMP-9 expression by human malignant oral ker atinocyte cell lines. Arch Oral Biol 2003;48(11) 779-786. [24] Sossey-Alaoui K, Ranalli TA, Li X, Bakin AV, Cowell JK. WAVE3 promotes cell motility and invasion through the regulation of MMP-1, MMP-3, and MMP-9 express ion. Exp Cell Res 2005;308(1) 135-145 [25] Jia Y, Zeng ZZ, Markwart SM, Rockwood KF, Ignatoski KM, Ethier S P, Livant DL. Integrin fibronectin receptors in matrix metalloproteinase-1-dependent invas ion by breast cancer and mammary epithelial cells. Cancer Res 2004;64(23) 86748681. [26] Sires UI, Griffin GL, Broekelmann TJ, Mecham RP, Murphy G, Chung AE, Welgus HG, Senior RM. Degradation of entactin by matrix metalloproteina ses. Susceptibility to matrilysin and identification of cleavage sites. J Bi ol Chem1993;268(3) 2069-2074. [27] Hall C, Nelson DM, Ye X, Baker K, DeCaprio JA, Seeholzer S, Lipinski M, Ada ms PD. HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cycli n-cdk2 substrate whose expression blocks S-phase progression. Mol Cell Biol 20 01;21(5) 1854-1865. [28] Murray GI, Duncan ME, O Neil P, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in colorectal canc er. Nat Med 1996;2(4) 461-462. Blood Cell An Overview of Studies in Hematology 302 [29] Murray GI, Duncan ME, O Neil P, McKay JA, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal can cer. J Pathol 1998;185(3) 256-261. Chapter 15

 2012 Zeh et al., licensee InTech. This is an open access chapter distributed und er the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Laboratory Reference Intervals in Africa Clement E. Zeh, Collins O. Odhiambo and Lisa A. Mills Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48250 1. Introduction Over the last decade, there has been a significant increase in the n umber of clinical trials taking place in sub-Saharan Africa in a concerted effort to identify safe and effective prevention and treatment strategies to combat the heavy burden of infe ctious diseases in this region [1-3]. This is because numerous viral, parasitic and bacterial disea ses are endemic in this region, including: 66% of the global HIV/AIDS infections, 31% of tuberculosis infections, and 86% of malaria cases [3, 4]. Routine capacity for clinical labor atory testing is also increasing in Africa. Clinical trials and clinical care in sub-Sa haran Africa require accurate laboratory reference intervals for appropriate assessment of pa tients/participants, monitoring disease progression, and reporting of possible toxicity and adverse e vents. This is particularly important in phase I and II clinical trials. Pha se I trials often enroll a small group of healthy participants in order to determine the metabolic and phar macologic actions of drugs, side effects associated with increasing doses and early eviden ce of efficacy. Phase II trials on the other hand are controlled clinical studies conducted to e valuate efficacy of drug/vaccine for a particular indication in a larger group of part icipants and to further evaluate its safety. Many HIV vaccine trials are slated for Phase IIII trials in Africa. The inception of the US President s Emergency Plan for AIDS Relief in 2004, with a m andate to treat 2 million HIV infections with anti-retroviral therapy by 2008 ha s accelerated the implementation of lymphocyte immunophenotyping in urban and rural areas in Africa as initiation of therapy is often predicated on absolute CD4 T- lymphocyt e counts. Central to any HIV vaccine and/or care and treatment program is the capability t o measure absolute CD4 counts. CD4 counts are important in the context of breakthrough infections d uring HIV vaccine trials and informing treatment. Correct diagnosis in patient ma nagement often involves accurate interpretation of results from laboratory testing [5]. Hence i t is critical for medical professionals to have access to an accurate management resource such as

reference intervals. Blood Cell An Overview of Studies in Hematology 304 Historically, clinical studies as well as routine clinical patient management in most African countries have relied on European-generated automated instrument values, US established reference intervals or the U.S. NIH division of AIDS (DAIDS) toxicity grading tables in assessing clinical parameters in study participants. The US-established reference intervals are obtained from the Massachusetts General Hospital reference values a nd serve as the standard reference interval comparison for most studies [6]. The DAIDS toxicity tables, also derived from a Caucasian population, are used for grading the severity of adult and pediatric adverse events, whether or not they are considered to be re lated to the study intervention [7]. DAIDS provides guidelines for estimating severity of adverse e vents using specific reference intervals (Table 1) as criteria for determining what is normal and among abnormal values, how to grade the severity of the abnormality. PARAMETER GRADE 1 MILD GRADE 2 MODERATE GRADE 3 SEVERE GRADE 4 POTENTIALLY LIFE-THREATENING HEMOGLOBIN 10.0 10.9 g/dL 9.0 9.9 g/dL 7.0 8.9 g/dL < 7.0 g/dL NEUTROPHILS 1.0 1.3 x 10 9 cells/L 0.75 0.999 x 10 9 cells/L 0.5 0.749 x 10 9 cells/L < 0.5 x 10 9 cells/L Adult and pediatric values for age >57 days, HIV-negative from the DAIDS toxicit y tables version 1.0, December 2004; clarification August 2009.

Table 1. Examples of DAIDS criteria of estimating severity grading based on lab oratory parameters. Reference values, in general, refer to the value or test result obtained by the observation or measurement of a particular type of quantity on an adequate number of persons (r eference sample group) selected to represent the general population. Reference v alues are usually presented as reference intervals which refer to the interval between, and including two reference limits i.e., from the lower reference limit to the upper reference lim it defined by a specific percentage (usually 95%). In certain parameters such as absolu te counts of monocytes, eosinophils and basophils, only one reference limit (decision limit), more often the upper reference limit is of biological significance hence the lowe r reference limit assumes a value of zero. Reference values go hand in hand with toxicity grading or decision li mits, which can be defined as specific levels of the analyte that correspond to mild to life threatening clinical situations. Toxicity grading is particularly useful in the decision-maki ng process of interpreting a measured value and assessing the health status of the subject bei ng tested. For this reason reference values or toxicity grading are routinely used in clinical trials at enrollment to determine eligibility, establish baseline measures, and als o during the course of the trial to monitor the participants health. Moreover, several analytes are u sed either as markers for the possible presence of a disease or as direct evidence for that disease. Reference values, especially hematological and immunologic indices, are influenc ed by such factors as genetics, dietary patterns, pregnancy, gender, age, ethnic o rigin and prior exposure to environmental pathogens. Thus, it is important to consider these factors when Laboratory Reference Intervals in Africa 305 applying reference intervals in diagnostics as well as in recruitment in clinical trials. According to the Clinical Laboratory and Standards Institute (CLSI) gui delines [8], it is recommended that laboratories establish their own reference intervals fr om the local population or validate the use of those obtained from a different set ting. Despite this, clinicians and researchers in Africa have continued to use reference v alues of European or North American populations. Our group in Kenya has recently published reference intervals based on the CLSI guidelines and are currently assisting reg ional laboratories to establish their own reference intervals[9]. In this chapter, we give a brief background on the current status of participant recruitment

in clinical trials and patient management in Africa. We will also des cribe how to select a reference population from which to derive the reference sample group. In addition, we describe various studies advocating the establishment of reference inter vals performed in different regions of the African continent including our own. These studies show differences in hematological, biochemical and immunologic parameters between various African populations but these differences are statistically insignificant. Howeve r, most hematological, biochemical and immunologic parameters considered in the A frican studies are significantly different when compared to American and European deri ved values. This chapter will also discuss the proposed partitioning of adolescent males from adults given their increased recruitment into clinical trials. Adult males have sign ificantly higher values for most hematological and biochemical parameters and we provide an ex planation why this is so. While pregnant women and infants undergo physiological pro cesses that alter their hematological and biochemical parameters, the partitioning of thes e cohorts has been slow. We discuss how pregnancy induces these changes and describe the particular parameters affected. We also highlight the dynamic changes in these pa rameters during infancy and how they differ from western-derived values. In this chapter, we als o illustrate the downside of using inappropriate reference intervals in the recruitment of pa rticipants in clinical trials and patient management. We show how the use of such values results in exclusion of clinically healthy participants from clinical trials and may lead t o inappropriate reporting of adverse events during the course of these studies. This potentially results in escalation of costs in the conduct of clinical trials. In this book chapter, we also propose the development of laboratory-derived African toxicity grades that, in addit ion to the already developed reference values, would be used for reporting adverse events in clinic al trials and for determining critical values in routine health care. 2. Use of reference intervals, consequence of misclassification and selection of a reference population 2.1. The use of reference intervals Reference intervals are useful both in the clinical and research envir onment. Medical laboratory reference intervals are primarily used for clinical purposes. They c an be used as an indicator of good health. Alternatively, reference intervals/limits can be us ed to screen for physiological or pathological conditions hence important in routine heal th assessment, Blood Cell An Overview of Studies in Hematology 306

particularly for screening of anemia, blood disorders and diseases of the immune system. Reference intervals are important for accurate interpretation of laboratory data and provide assistance to the clinician in creating a more comprehensive clinical perspective for diagnosis and management of patients [10]. Of particular importance is the use o f reference values as surrogate markers for monitoring disease progression and resp onse to antiretroviral therapy in HIV-infected individuals [11]. For example, de cisions to initiate, continue, or change antiretroviral therapy regimens are determined using CD4+ Tlymphocyte cell (CD4) counts, while drug toxicity is monitored using l iver function tests, renal function tests, and full blood counts (FBC) [12, 13]. The hemog lobin concentration is used as a marker of anemia. As part of the management of anemia, th e clinician conducts additional tests to identify a reversible etiology for anemia (eg, iro n deficiency, infection) and if present treats it appropriately. However, in the clinical envir onment, the statistical definition of reference intervals may not allow certain clinical uses. Because t hese reference intervals have been derived statistically from a healthy population, they may no t be used to rule in or rule out specific medical conditions. The statistically der ived 95% reference interval would mean that 5% of normal subjects would have abnormal la boratory values. This is erroneously interpreted that 95% of diseased individuals would fall outside the derived reference interval. It is recommended that the number of diseased indivi duals who fall outside the defined 95% reference intervals be determined through a study of the distribution of such persons with the target condition [14]. Thus, it is necessary to confirm the validity of the proposed reference intervals with clinicians using a particular test to manage patients. In the research environment, however, the aim is to define a reference populatio n that is as similar as possible to that for which a particular test will be applied with the exception of the presence of the disease. During clinical trials, reference intervals re levant to the study of interest are required to interpret normal values of standard laboratory test res ults from the target population [15]. This is particularly important during phase I/I I safety trials where healthy individuals are assessed without a control group [15-17]. Moreo ver, clinical reference intervals are necessary in order to accurately assess potenti al adverse events observed during the course of clinical trials. 2.2. Consequences of misclassification A majority of clinically healthy participants have been excluded from

several clinical trials in Africa because laboratory hematological and biochemical parameters ar e classified as abnormal [18-20]. Unnecessary exclusion of potential participants general ly results in increased cost for study recruitment to achieve the target sample size . Accurate reference intervals are required for monitoring adverse events during vaccine and drug tri als to limit misclassification that might otherwise lead to discontinuation of such trials or erroneous conclusions that the trial interventions are associated with adverse ev ents. A study documented that the expense of adverse event investigation and reporting account ed for at least one-third of the study cost, irrespective of the adverse event grade [18]. To overcome Laboratory Reference Intervals in Africa 307 these challenges, there is a need to establish accurate, locally derived referen ce intervals for the target population. Within the last decade, several studies in subSaharan Africa have attempted to establish hematological and biochemical reference intervals for use in clinical monitoring and patient management. 2.3. Selection of a reference population The selection of a reference population is as per described in the C linical Laboratory Standards Institute (CLSI, Wayne, PA, USA) guidelines [21]. The guideli nes state that reference individuals selected for the determination of reference interv als should closely resemble the patient population undergoing medical examination and shoul d be of similar age to be clinically significant [21]. The reference individuals should not be h ospital or clinic patients unless absolutely necessary. The guidelines describe two select ion methods for a reference population: a priori and a posteriori. A priori sampling method involv es selection of reference individuals based on well-defined exclusion and partition crit eria. The entire selection process takes place before any blood sample is drawn and a sufficient number of reference individuals are targeted to provide statistical validity. A p osteriori sampling method involves selection of the reference population after the analyte has been tested. The CLSI guidelines recommend a minimum of 120 individuals to allow 90% confidence l imits to be non-parametrically calculated for the reference limits [22]. Partitioning of reference intervals either by gender or age is recommended if clinically useful or physiologically w ell grounded. Even though 120 samples remains the recommended standard, an efficient laboratory, by considering the CLSI revised guideline strategies [8], can determine re ference intervals using fewer samples [23]. Alternatively, a laboratory can adopt reference intervals es

tablished from another laboratory if the values are verified using the procedures set out in th e guidelines. In our study [9] of adolescents and adults living in rural western K enya, all participants were screened by a review of medical history, a physical examination, tested for HIV and pregnancy (for females), and treated for any illnesses diagnosed. Participants w ere included if they were a permanent resident of the study area, between 13 and 34 years of age and able to provide informed consent or assent if a minor. Participants were e xcluded if they were HIV-seropositive, pregnant, exhibiting febrile symptoms or on any medica tion. Blood samples were therefore obtained from clinically healthy participants sel ected to generate hematologic and biochemical reference intervals. Data were partitioned by age (< 18 years of age as adolescents and 18 as adults) and gender; median and 95th percentile inter vals were calculated. The lower 95% reference limit was defined as the 2.5th pe rcentile while the upper limit was defined as the 97.5th percentile. A Wilcoxon rank-sum test was u sed to test for age and gender differences. We compared our data against reference interval s from the Massachusetts General Hospital (MGH-USA) [6] and the U.S. NIH Division of AIDS (DAIDS) toxicity tables [7] to determine the number of study participa nts with values outside the MGH ranges or who had any adverse event as graded by th e DAIDS criteria. However, while the CLSI guidelines recommend a description of the population fro m which reference intervals are derived, the DAIDS and Massachusetts General Ho spital reference values do not provide such information. Blood Cell An Overview of Studies in Hematology 308 3. Current status of reference values in Africa Reference intervals for clinical laboratory parameters have traditionally been o btained from European and North American populations [2]. However, differences have been reported between these values when compared to healthy African population values [16]. These include lower hemoglobin, red blood cell counts, hematocrit, mean corpu scular volume, platelets and neutrophils, and higher monocyte and eosinophil levels for African population compared to their Western counterparts [16,24-26] and Africans of European decen t [27, 28]. Moreover, variations in several indices have been reported between different Afr ican ethnic groups [26, 29-31]. These differences are postulated to occur due to factors suc h as genetics, dietary patterns, gender, age, ethnic origin and environmental pathogens which a re known to influence hematological and immunologic indices [32-35].

While the differences observed in some laboratory parameters between Af rican and Caucasian/Western populations may be attributed to nutritional difference s, genetic polymorphisms, or more intense environmental exposure to endemic pathogens, it m ust be stressed that these reference values are being derived from populationbased statistical analyses of norms among healthy persons. For example, healthy African s tend to have lower white blood cell counts than Caucasians, but there is no evidence that the y suffer any additional risk of developing severe infection or other sequelae. Als o, African American populations, with environmental exposures more like their white American counterparts, tend to have lower normal values in hematologic parameters than Caucasia n Americans, suggesting a genetic basis for these population differences. 3.1 Variation in specific laboratory parameters a. Hematologic parameters The normal values of red cell counts and indices (i.e., hemoglobin concentration , hematocrit, mean corpuscular volume, red blood cell count), white cell counts and platelet counts are known to vary with age, sex and pregnancy [9, 16, 20, 31, 36]. In addition, genetic and environmental factors can also affect the reference intervals in certai n populations [32-34, 37]. It is of particular importance that these differences in reference interva ls be considered by clinicians in different settings. i. Red blood cell (RBC) components African RBC component values were significantly lower when compared to reference intervals obtained from the Massachusetts General Hospital [6] from a North American population, and thus a significant proportion are misclassified when th e NIH DAIDS toxicity tables are applied [9, 34, 38]. Differences observed in the RBC compone nts between African and Caucasian populations may be attributed to lower dietary i ron intake, genetic polymorphisms such as thalassemia and sickle cell trait or chronic exp osure to endemic parasites including helminths, malaria and schistosomiasis. Statistically significant differences in median RBC, hemoglobin concentra tion (Hb) and hematocrit (Hct), mean corpuscular volume (MCV) and mean corpuscular he moglobin Laboratory Reference Intervals in Africa 309 (MCH) by gender have been observed in several African studies, with a dult males having higher values than adult females in East Africa [9, 16, 20, 31, 39, 40], Souther n Africa [20, 36], West Africa [41] and Central Africa [42]. These gender differences in RBC parameters as illustrated in our findings (Table 2), are consistent with previously establishe d evidence that

males have higher values than females for these parameters and is par tly attributed to the influence of the androgen hormone on erythropoiesis [43, 44] and to menstrual bl ood loss in women [16, 25, 39, 42, 45]. It has been reported that estrogens lowe r the Hb through hemodilution while testosterone increases the plasma volume but increases circul ating RBC to an even greater extent [46]. Age 13-17 years Age 18-34 years Parameter Gender n Median (95 th percentile) p-value (gender) n Median (95 th percentile) p-value (gender) p-value (age) Hemoglobin (g/dL) Female 57 12.2 (8.1 - 14.2) <.0001 83 12.1(8.0 14.2) <.0001 0.3243 Male 76 13.1 (10.6 - 15.6) 77 14.2 (11.4 - 16.9) <.0001 Hematocrit (%) Female 57 35.6 (24.8 - 43.1) <.0001 83 35.8 (23.2 - 44.3) <.0001 0.8015 Male 76 38.8 (29.3 48.1) 77 41.7 (32.6- 51.5) <.0001 RBC (x10 12 /L) Female 57 4.7 (3.3 - 5.4) 0.0001 83 4.5 (3.4 - 5.7) <.0001 0.2638 Male 76 4.9 (4.1 - 5.8) 77 5.3 (4.3 - 6.5) <.0001 PLT(x10 9 /L) Female 57 233 (134 439) 0.2958 83 220 (88 439) 0.0222 0.4034 Male 76 224 (103 386) 77 201 (102 -307) 0.0094 WBC (x10 9 /L) Female 57 5.2 (3.9-10.2) 0.6359 83 5.6 (3.3-9.7) 0.0189 0.2038 Male 76 5.6 (3.3-8.3) 77 5.3 (2.5-7.4) 0.6382 Lymphocytes (x10 9 /L) Female 57 2.2 (1.1 - 3.1) 0.9820 83 2.2 (1.3 - 3.8) 0.6901 0.9388 Male 76 2.2 (1.0 - 4.2) 77 2.2 (1.0 3.5) 0.585 Ab Neutrophils (x10 9 /L) Female 57 2.0 (1.0-6.2) 0.4991 83 2.3 (1.3-5.4) 0.0004 0.0576

Male 76 1.9 (0.8-5.0) 77 2.0 (0.8-3.9) 0.6575 CD4: Absolute Female 58 934 (465- 1553) 0.4074 83 866 (440-1602) 0.0141 0.509 Male 76 874 (367-1571) 77 811 (462-1306) 0.0209 CD8: Absolute Female 58 506 (195-1068) 0.4506 83 472 (262 - 1167) 0.8706 0.9213 Male 76 468 (195-988) 77 468 (201-1104) 0.4194 CD4/CD8 ratio Female 58 1.8 (0.9-3.2) 0.9215 83 1.8 (0.8-3.0) 0.0728 0.4879 Male 76 1.8 (0.8-2.8) 77 1.6 (0.8-2.8) 0.0543 Table 2. Test of difference in hematologic and immunologic parameters between g ender and agegroups from healthy 13-34 year olds in a rural western Kenya cohort (2003-2005). Blood Cell An Overview of Studies in Hematology 310 Age-related differences in the RBC component have also been observed a mong male participants, with adults (18 years) having higher levels of Hb, Hct and RBC com pared to male adolescents (13-17 years) as shown here (Table 2) [9]. This age variation i s similar to that reported in a study of Caucasian adolescents [34]. This difference could be attr ibuted to higher levels of androgen hormones among older males. This explanation is further stren gthened by the absence of age-related hematological difference among female participants. I t has also been postulated that an increase in the size and mass of muscle fibers as occurs in males is associated with an increase in the number of circulating red blood cells [47]. There are limited data comparing reference intervals for hematologic values amon g African children compared to Caucasian children and also few studies on releva nt local reference values for African infants. However, these studies, similar to the adu lt studies, have highlighted differences in RBC components compared to values obtained f rom Caucasian children [48]. The lower RBC parameters, as in the adolescent and adu lt groups, may be attributed to impaired hematopoiesis as a result of lower dietary iron intake, c hronic blood loss due to hookworm infestation or chronic malaria infection [25]. Endemic sick le cell trait (HbS) and -t l ssemi may also play an important role [49]. ii. Platelets In general, lower platelet counts are more common in African than in Western pop ulations. While the lower platelet counts in African populations are consistent in several African studies [24, 25, 30, 31, 40], its etiology is unknown. Possibilities such as dietary, environmental and genetic factors have been proposed [24, 30, 31]. Nev ertheless, the significant difference in the lower limit of the reference interval be tween African and

Caucasian populations warrants consideration when interpreting platelet counts i n patients or during clinical trial recruitment in African populations. Among Africans males, significant age-related differences have been obse rved in platelet counts with adults having higher platelet counts compared to adolescents [9]. Th is variation is observed as a progressive increase with age from adolescence to yo ung adulthood. In comparison, there is little age variation in platelet counts among females. Howe ver, females have higher platelet counts than males both in adolescence [9] and adulthood [9, 16]. These gender differences in platelet counts have been attributed to hormonal influences [50]. Platelet count have been observed to falls at the onset of menstruation while pe ak values are obtained in mid-cycle indicating that hormonal influences and/or menstrual blood loss may be involved [27]. While platelet levels remain stable during pregnancy, a decrea se has been reported immediately after delivery, likely due to consumption during s eparation and delivery of the placenta. iii. White Blood Cell (WBC) components A high proportion of participants in the African studies have WBC counts below t he lower range of the Massachusetts General Hospital US population-derived values [9, 20]. This phenomenon is consistent with a number of studies that have reported lower WBC c ounts in Laboratory Reference Intervals in Africa 311 African populations and those of African ancestry, including African Am ericans, than in Caucasian populations [24, 30, 51-53]. Because the reference interval f or WBC counts is significantly different from that of Caucasian populations, it is advisable to u se appropriate ethnic group intervals when interpreting blood counts [31]. Gender differences in the WBC counts exist in both African and Caucasian populat ions with females having higher values than males [9, 20, 54]. Age-related diffe rence in WBC counts has been reported in several African studies [9, 25, 33]. Adolescents have higher WBC counts compared to adults as shown in our study (Table 2) [9]. 1. Neutrophils Within the U.S., lower neutrophil counts are more common among blacks compared to Caucasians [28]. Thus, it is not unsurprising to observe a higher proportion of African study participants (22.5-35%) having neutrophil counts below the lower range of Massachusetts General Hospitals population-derived reference interval [9]. It is estimate d that about 25% to 50% of Africans have benign ethnic neutropenia, maintaining consistently low ab solute neutrophil counts with no evidence of increased susceptibility to infection or o ther adverse

events [28]. Possibile explanations for the lower neutrophil count incl ude diet, genetic or environmental influences [53, 55]. In general, there are significant differences in neutrophil counts betw een male and female adults, with the females having higher neutrophil counts than males. T his increase in neutrophil counts observed in women may be related to estrogen since a decrease in counts has been reported after menopause [39]. Oral contraceptives have also been implicated in neutrophilia [56]. Additionally, several studies in southern Africa have documented high rates of n eutropenia in infants of women receiving Prevention of Mother to Child Transmissi on interventions [57-591]. Evaluation of neutropenia in infants receiving antiretroviral prophylaxis or treatment (directly or indirectly through maternal exposure in utero or through breastfeeding) remains a challenge. Neutropenia is a known side effect of zidovu dine [60] and trimethoprim/sulfamethoxazole, which is often prescribed for preventi on of opportunistic infections in HIV-infected and/or HIV-exposed infants/childr en. This problem is further compounded by the paucity of normative data for hematologic v alues in African infants. 2. Basophils and eosinophils Basophil and eosinophil counts in African populations are significantly elevated in both genders when compared to the US-based reference intervals [9, 20]. Thi s may be due to a high prevalence of parasitic infections in the environment including sc histosomiasis, helminthic infections, perennial malaria and exposure to a broader rang e of environmental antigens [25, 39]. However, the eosinophil counts do not vary significantly by g ender or by age, as assessed between adolescent and adult African participants [9, 34]. Blood Cell An Overview of Studies in Hematology 312 3. Monocytes Generally, no ethnic or age differences are observed between Caucasian and African populations [46]. Monocyte counts in Eastern and Southern Africa are comparable to the US derived valuesand thus there is no need for separate reference intervals [9, 20] . In the African studies, no differences are observed in absolute monocy te counts between adolescents and adults or by gender [9, 20]. Previous studies from Ea stern and Southern African populations indicate an increase in monocyte counts in males c ompared with females but the difference is not significant [3, 24, 26, 29, 30]. 4. Lymphocytes Among healthy, HIV-uninfected persons, there are no significant differences in l ymphocyte

counts between Caucasian and African populations but females generally have higher lymphocyte counts than males [54]. This is corroborated by studies wit hin Africa that indicated higher CD4 cell percentage and absolute CD4 counts in female s compared to males [9, 26, 61]. However, geographical variation exists in lymphocyte counts with some p opulations in Southern African showing significantly lower reference values than other parts of Africa [62]. In assessing age-related variability, younger age is associated w ith higher CD4 cell counts and a higher CD4:CD8 ratio. However, the differences are not significantl y except for CD4 cell counts between male adolescent and male adults [9]. These ag e and geographical variations need to be considered when interpreting lymphocyte counts. b. Clinical chemistry parameters Most African studies [9, 15, 16, 20] report reference intervals for most paramet ers (creatinine, direct bilirubin, amylase and albumin) that are in agreement with refe rence intervals published in the United States [6]. However, certain parameters such a s Creatine Kinase (CK) and Lactose Dehydrogenase (LDH) have upper intervals that are sub stantially higher than those published in the Massachusetts General Hospital intervals [1 6, 20]. Other parameters with a similar trend include total bilirubin (T-bil) and bl ood urea nitrogen (BUN). The upper range for T-bil is about twice as high as that of the US-derived upper reference limit while the lower range for BUN is a about a third of the US-derived lower reference limit [9, 15, 20]. The etiology of high T-bil in the Afric an population may arise from a number of factors including RBC hemolysis caused by malaria infection or sickle cell disease, malnutrition or physical exertion. Moreover, the presence of s imilar trends among other African populations is suggestive of a common environmental or genetic fac tor. Our findings indicated gender and age variations in blood chemistry an alytes of liver and renal function among African adolescents and adults. Male adolescents a nd adults had higher values for alanine aminotransferase (ALT), aspartate aminotransfer ase (AST), T-bil and creatinine than females adolescents and adults (Table 3). These gender diffe rences were significantly greater for T-bil and creatinine in both adolescents and adults while for AST, the difference was significant only among the adolescents. However, these differ ences were Laboratory Reference Intervals in Africa 313 not clinically significant. There were no gender differences in BUN and glucose levels for all age groups and no significant differences in T-bil, AST, ALT and gluco

se between the two age groups for both males and females. However adult men and women had higher v alues for creatinine and BUN compared to adolescent males and females, respectively. Age 13-17 years Age 18-34 years Parameter Gender n Median (95 th percentile) n Median (95 th percentile) p-value (gender) p-value (age) AST/SGOT (/L) Female 62 22.6 (12.0 43.1) 0.0102 82 22.2 (13.5 - 48.5) 0.0822 0.5905 Male 77 26.9 (17.0 59.2) 77 26.7 (12.5-69.3) 0.9147 ALT/SGPT (/L) Female 62 17.4 (4.2-65.3) 0.6289 82 18.9 (10.7-61.3) 0.2247 0.1305 Male 77 20.5 (4.9-42.4) 77 22.4 (12.0-80.6) 0.0901 Total Bilirubin (mol/L) Female 62 9.7 (3.7-38.5) 0.0331 82 11.5 (5.8-36.1) 0.0368 0.7132 Male 77 13.9 (5.7 62.6) 77 13.8 (5.3 - 50.7) 0.6662 Creatinine (mol/L) Female 62 64.5 (48.0-87.6) 0.0229 82 70.7 (52.4-96.8) <.0001 0.0013 Male 77 66.3 (49.6-103.7) 77 83.1(54.2-137.8) <.0001 Table 3. Test of difference in clinical chemistry parameters between gender and age-groups from healthy 13-34 year olds in a rural western Kenya cohort (2003-2005). 4. Should establishing separate normal ranges for African adolescents and pregnant women be considered? A number of studies similar to our published data [9], have reported age-related variation between male adolescents as compared to adults for Hb, Hct and RBC l evels [25, 34, 45]. This observation is physiologically grounded on hormonal influence and as per the CLSI guidelines, partitioning reference intervals by age (or other subgroup considerations) may be appropriate. While these observations may not be of any medical si gnificance, it should be taken into consideration whenever clinical trials target this popula tion. To satisfy the statistical requirement for partitioning, there is need for further research on reference values among adolescents, as their participation in clinical trials increases. Other than the RBC components mentioned above, no significant age differences ha ve been observed in other laboratory parameters measured among males or in any parameters measured among females except for creatinine and BUN. Thus, for such

parameters for which no differences are reported, adult values can be used in clinic al trials involving adolescents. With the advent of antiretroviral therapy for HIV and other interventi ons to improve maternal and child health, pregnant women and infants have become the focus of many health programs. However, few data exist regarding these important popu lations, despite increased clinical trials aimed at reducing mother-to-child HIV transmis sion. Although pregnancy-induced changes occur in hematological values including Hb, Hc t and RBC count, very few laboratories provide specific reference ranges for pregnant wome n [63, 64]. Blood Cell An Overview of Studies in Hematology 314 In pregnancy, blood volume increases resulting in hemodilution. While t he red cell mass increases during pregnancy, the plasma volume increases more resulting in a relative anemia. This leads to a lower Hb level, Hct and RBC. Hb is known to vary with ge stational age with the highest values within the first and last trimesters and lowest duri ng the second trimester. Similarly, the Hct and RBC decreases with gestational age. A stable higher upper reference limit for WBC count during pregnancy has been reported [65, 66]. WBC count is known to peak at delivery, thus limiting the use of this parameter as a marker f or infection during delivery. This increase in WBC count results primarily from an increase i n neutrophil counts and a slight increase in lymphocyte counts. Currently, there exists no A frican study designed to establish reference intervals during pregnancy and most laboratory i nformation systems report reference values based on samples obtained from nonpregna nt women which may not be useful for clinical decisions during pregnancy. Thus, there is an increased risk of overlooking important physiologic alterations resulting from pathologica l conditions and of misinterpreting normal changes as pathological events [64]. It is therefo re important to develop reference intervals for women during pregnancy and the postpartum per iod for use in patient monitoring and management. 5. A case for African/ Region specific toxicity tables? Under a research-based approach, applying the US Massachusetts General Hospital derived reference intervals to our reference population from western Kenya duri ng screening for a clinical trial (Table 4), over 58% of the volunteers would have been excluded from the trial despite having laboratory results consistent with the general population from which they were derived. This erroneous screening out of otherwise healthy volunte ers would have

important implications on study costs, work load and time, as more vo lunteers would be need to be screened in order to meet the required target [15]. Similarly, applying the DAIDS toxicity tables to our population, some of our calculated reference intervals fall between the normal, and grade 12 toxicity grad ing in the DAIDS system (Table 4). Using the clinic based approach, 40% of our otherwi se healthy study participants would have erroneously been considered to have at least one laborat ory-based grade 14 toxicity adverse event. The lower range for Hb, neutrophil counts, as we ll as the upper range for eosinophil counts and bilirubin would be considered as grade 2 adverse events, for example. Even though studies have documented these findings, this i nformation is not widely known and as a result, DAIDS has issued only 1 set o f standard toxicity tables without considering racial or ethnic differences [57]. Thus, dur ing international clinical trials, these tables are used as guidelines in the conduct o f such trials. This may result in a situation where the results of a clinical trial cannot b e generalized to the population in question since a majority of otherwise healthy participan ts are screened out. Moreover, given that the investigational product is intended for use w ithin the same population being sampled, this may complicate post-market analysis or a pplication of the product for the general population. Unfortunately, there are no compara ble tables from Africa on which such clinical decisions can be based. It is therefore important that African countries carry out large studies in different regions of Africa for such parameters to establish African toxicity tables. Laboratory Reference Intervals in Africa 315 MGH USA reference intervals (25th percentile)[6] Division of AIDS (DAIDS) toxicity grading out of range Comparison Grade 1 Grade 2 Grade 3 Grade 4 Parameter n 95 th percentile n % n % n % n % n % Hemoglobin Males (g/dl) 140 13.5-17.5 65 46 2 1.3 0 0 2 1.3 0 0 Hemoglobin Females (g/dl) 153 12-16 61 40 11 7.9 8 5.7 14 10 0 0 Hct (females) (%) 140 36-46 74 53 Hct (males) (%) 153 41-53 88 58

RBC (males) (10 12 cells/L) 140 4.5-5.9 29 19 RBC (females) (10 12 cells/L) 153 4.0-5.2 32 23 MCV (fL) 293 80-100 157 54 Platelets (10 9 cells/L) 293 150-350 53 18 6 2 6 2 0 0 0 0 WBC (10 9 cells/L) 293 4.5-11.0 66 23 2 0.7 0 0 0 0 0 0 Lymphocyte count (10 9 cells/L) 293 1.0-4.8 6 2 0 0 0 0 0 0 0 0 Neutrophil count (10 9 cells/L) 293 1.8-7.7 110 38 25 8.5 9 3.1 1 0.3 0 0 Eosinophil (10 9 cells/L) 293 0-0.5 130 44 60 20.5 12 4.1 0 0 0 0 Basophil count (10 9 cells/L) 293 0-0.2 5 2 Monocyte count (10 9 cells/L) 293 0-0.8 0 0 ALT (SGPT) (U/ L) 293 0-35 30 10 12 4.1 1 0.3 0 0 0 0 AST (SGOT) (U/ L) 293 0-35 40 13 9 3.1 3 1 0 0 0 0 Total Bilirubin (mol/L) 293 5.1-17.0 90 30 37 12.7 27 9.2 4 1.4 1 0.3 Creatinine (mol/L) 293 0-133 4 1 4 1.4 0 0 0 0 0 0 Glucose mmol/L 293 4.2-6.4 210 71 BUN (mmol/L) 293 3.6-7.1 246 84 *CD4 (Cells/ l) 293 404-1612 6 2 3 1 1 0.3 0 0 0 0 *CD8 (Cells/ l) 293 220-1129 13 4 *Reference ranges provided by Becton-Dickinson with the MultiTEST IMK Kit Reagen t package (12/2000;23-3602-02) DAIDS- Division of AIDS tables for grading the severity of adult and pediatric a dverse events [26] - MGHMassachusetts General Hospital weekly case records [25] Table 4. Frequency of adverse events and out of range values comparing western Kenyan cohort to DAIDS and North American derived MGH values 6. Conclusion While it is desirable to generate reference intervals for different populations, the procedure remains a challenge due to the prohibitive cost involved in performing these stu dies and the limitation in identifying suitable healthy reference individuals. Thus, the CLSI recommendation that all diagnostic laboratories should determine and mai ntain their own reference interval for each laboratory parameter is impractical. The revised CLS

I guidelines Blood Cell An Overview of Studies in Hematology 316 have recommended that if it is not possible to establish detailed ref erence studies, then validation of published reference intervals can be performed using methodology t ailored for the population served by the laboratory. As few as 20 specimens can be used to validate reference values within each laboratory by performing a formal outlier test. Given the number of clinical trials and persons receiving clinical ser vices is expected to increase substantially in sub-Saharan Africa, there is a need for the establishm ent of locally derived clinical laboratory reference values to ensure appropriate general healt h assessment, treatment monitoring, and efficient implementation of clinical trials. Even more important is the need for the establishment of toxicity grading tables for applicat ion in clinical care among Africans based on the documented differences between laboratory r eference values from African populations and Caucasians or Western populations of mixed ethnic o rigin. Author details Clement E. Zeh, Collins O. Odhiambo and Lisa A. Mills U.S. Centers for Disease Control and Prevention (CDC-Kenya), Kisumu, Kenya, Centre for Global Health Research, Kenya Medical Research Institute/U.S. CDC Res earch and Public Health, Kisumu, Kenya Acknowledgement The authors would like to acknowledge Kayla Laserson and Laurie Kamimoto and Pro fessor Barbara Bains for their thorough review of this chapter. The authors would also like to appreciate vital contributions made by KEMRI/CDC Kisumu, HIV-Research La boratory. This chapter is published with the approval of the Director of KEMRI. Disclaimer: The findings and conclusions in this article are those of the author s and do not necessarily represent the views of the U.S. Centers for Disease Control and Prev ention. Use of trade names is for identification purposes only and does not const itute endorsement by the U.S. Centers for Disease Control and Prevention or the Department of Health and Human Services. 7. References [1] Esparza J, O.S., HIV vaccines: a global perspective. Current Molecular Medi cine, 2003. 3: p. 183193. [2] Jaoko W, N.F., Anzala O, Manyonyi GO, Birungi J, Nanvubya A, Bas hir F, Bhatt K, Ogutu H, Wakasiaka S, Matu L, Waruingi W, Odada J, Oyaro M, Indangasi J, NdinyaAchola J, Konde C, Mugisha E, Fast P, Schmidt C, Gilmour J, Tarragon a T, Smith C, Barin B, Dally L, Johnson B, Muluubya A, Nielsen L, Hayes P, Boaz M , Hughes P, Hanke T, McMichael A, Bwayo J, Kaleebu P, Safety and immunogenicity o f recombinant

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2012 Mamak and Aytekin, licensee InTech. This is an open access chapter distribu ted under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by /3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the ori ginal work is properly cited. Principles of Blood Transfusion Nuri Mamak and smail Aytekin Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48332 1. Introduction The aim of this chapter is to present a revised overview of small a nd large animal transfusion medicine based on a review of the veterinary literature. B lood transfusion has become more performable in small and large animal practice. By donor selection and the availability of blood component substitutes, usage of the blood products improve d. The use of blood component therapy safely needed knowledge of blood groups, antibody pre valence and the impact of blood groups on veterinary transfusion medicine. Ani mal blood transfusions antibodies against blood group antigens also play a role. In addition knowledge of the means to decrease the risk of adverse reactions by using proper donors and screening assays that simplify detection of serological incompatibility is i mportant. The clinical significance of blood group antigens in veterinary medicine is generall y in the areas of transfusion reactions and neonatal isoerythrolysis (NI). This chapter includes an update on canine and feline, horse, donkey, cattle, sheep, gaot, pig, llama and alpaca blood groups and known blood incompatibilities, donor selection and blood collection, storage of blood components, available equine blood products and indications for transfus ion, whole blood (WB) and blood product transfusion in ruminants and camelids, blood co mponent and blood substitute therapy, administration, and adverse reactions in small and large animal blood transfusion. 2. Blood types in dogs and cats

Blood types are classified according to specific antigens on the surfa ce of erythrocytes. Platelets, leukocytes, and body tissues and fluids may also consists o f erytrocyte antigens. [1]. In immunogenicity and clinical significance these antigens can differ. They can serve as markers of disease in some cases and taking part in recognition of s elf. The clinical significance of blood group antigens is generally noted in transfusion reactions and neonatal isoerythrolysis (NI) in veterinary medicine [2]. These antigens can characterist ically trigger a reaction caused by circulating anti-erythrocyte antibodies in the opposi te host or donor. Blood Cell An Overview of Studies in Hematology 322 These antibodies can occur naturally. Also they can be induced by a previous transfusion. Interaction leads to the destruction by hemolysis of red blood cells (RBCs). Thi s is one of the severe and potentially life-threatening situation. [3]. The dog erytrocyte antigen types or blood types are categorized by th e DEA (Dog Erythrocyte Antigen) system. DEA 1.1, 1.2, and 1.3 are termed A system. There ar e also DEA 3, DEA 4, DEA 5, DEA 6, DEA 7 and DEA 8. [2]. In the United States the incidence of DEA 1.1 is approximately 45% and DEA 1.2 is 20% [4]. DEA 1.3 is common in German she pherd dogs and has been reported only in Australia [5]. Frequency of DEA 1.1 in Kangal Dog was found as 61.1% in Turkey [6]. In Croatia where the closest data stud ied the rate was 66.7% [7]. The rate was also 56.9% in Portugal [8] and 55% in Japan [9]. Approximately 60 % of the canine population is in DEAs 1.1 and 1.2 group. DEA 1.1 is the strongest antigen in the dog. Two membrane proteins of 50 and 200 kD has been identified by a monoclonal antib ody to DEA 1.1 using immunoprecipitation techniques. [10]. Presenting in a sin gle band DEA 1.2 has been found to be an 85-kD protein [11]. DEA 1.1 is the most antigenic group in respect to transfusion medicine. Little i s investigated about DEA 3, 4, 5 and 7 in comparison to DEA 1.1. In literature, the frequency of DEA 3 is lower in comparison to DEA 1.1 blood type. In the United States it is determined that approximately 6% of the general dog population is DEA 3 positive [12]. This rate is reported as 13% in Brazil [13]. In Turkey, DEA 3 is most found blood type in the Kangal D og [6]. In the canine blood groups DEA 4 is the most common type. In USA, it is indicated t hat overall 98% of the general dog population have DEA 4 blood [12]. In Brazil, all dogs blood type were positive for DEA 4 [13]. The molecular weight of DEA 4 present in a single band has

been found to be 32 to 40 kD using immunoprecipitation techniques [11]. In the United States typing sera can be commercially obtained only for DEA 1.1, 1.2, 3, 4, 5, and 7 [4]. In Brazil a report studied on German shepherd dogs determ ined that 14% of the dogs were positive for DEA 5 and 8% were positive for DEA 7 [13]. The frequency of DEA 5 and 7 positive dogs was 55.5% and 71.7% respectively in Turkey [6]. Also, DEA 7 may cause an antibody response in dogs that lack it. A system of nomenclature about antigen Tr has described. The Tr antigen system is a 3-phenotype, 6-genotype system [ 14]. The molecular weight of DEA 7 present in 3 distinct bands has been found to be 53, 58, and 63 kD by using immunoprecipitation techniques [11]. An exact definition of a canine universal donor is not agreed among veterinary t ransfusion experts. Well excepted description of the universal donor is that a dog negative for DEA 1.1, 1.2, DEA 3, DEA 5, DEA 7, and positive for DEA 4. It is difficult to find DEA 4 negative dog because 98% of all dogs are positive for DEA 4. Thus there is a very little chan ce to influence donor selection. If the dog is DEA 7 positive, some other experts do not exclude it from the donor pool [15]. In most populations the incidence of DEA 4 blood ty pe is more than 98% [16]. Because of this in transfusion medicine these dogs are the best candidate for being a donor. If other donors are known to be compatible with the recipient they can also be utilized [17]. DEA 3, 5 and 7 negative dogs have naturally occurring antibodies to DEA 3, 5 and 7 positive red cells. However during the first transfusion these blood groups do not Principles of Blood Transfusion 323 possess a major transfusion reaction [4]. In Turkey, the most common blood types were DEA 1.1, 4 and 7. Because all Kangal dogs have DEA 4 positivity it does not seem to be important in respect to transfusion medicine. The prevalence and antige nic properties of DEA 1.1 and 7 are significantly important. If unmatched transfusion is performed in Turkish Kangal dogs they can constitute acute hemolytic transfusion reactions [ 6]. Dogs with DEA 1.1 or 1.2 are called group A positive. Adversely, dogs do not have DEA 1.1 or 1 .2 are called group A negative [1]. A blood group system described as N-acetylneuraminic acid and N-glycolylneuramin ic acid present on gangliosides (hematosides) of the RBC membrane in Japan [18 ]. It is referred as the D system. This system is consist of two antigens, D1 and D2, with phenotypes , D1, D2, and D1D2. The D1 and D2 antigens are codominanat factors. Anti-D1 is

identical to antiDEA3. The importance of this system in transfusion medicine pointed out by trans fusion of D2 type blood into a D1 type patient, or of D1 type blood into a D2 type patient consistently cause severe acute transfusion reactions [19, 20]. RBCs of some dogs designated as type C at titre sup to 128 are aglutinated rather than lectin extracted from se eds of Clerodendron tricotomum. Type C is completely negative for other dogs. C system wa s compared to the DEA system and determined to be different [10, 19, 21]. Specific IgG alloantibodies in previously sensitized Dalmatian dog by blood transfusion is described a s the Dal blood type. The frequency is not known. Typing sera for this antigen also is commercially not available [2, 22, 23]. Three blood types are described in the feline AB blood group system and mik grou p system. In cats a new blood group defined as Mik. It is named after the alloantibody ide ntified in the first blood donor cat, Mike. In three cats that had not previously received tran sfusions Mik antibodies were detected. They are defined as a cause of incompatibili ties between donor and recipient blood that are not related to the AB blood group system [24]. The phenotypes type A, type B, and type AB are occured. A null phenotype is not exist. The most common blood type is Type A. Type B is less common. Type AB is rare [2, 25] . Type B is indicated in Australia (26.3%), and Greece (20.3%) ([26] , [27] ). In large studies of both pedigree and non-pedigree cats in the USA distribution of type AB cats is demons trated to be rare (0.14%) ([28] ). Type AB were 0.4% in Australia (([26]). In Scotland the incidence of AB cats is 4.4% ([29] ). Type B is indicated in Australia (26.3%), and Greece (20.3%) ([26, 27]. In large studies of both pedigree and non-pedigree cats in the USA distribution of type AB cats is demons trated to be rare (0.14%) [28]. Type AB were 0.4% in Australia [26]. In Scotla nd the incidence of AB cats is 4.4% [29]. In Turkey, 60 % of Van cats and 46.4 % of Angora cats are type B [30]. And 220 (73.1%) nonpedigree domestic cats had type A blood, 74 (24.6%) had type B an d seven (2.3%) had type AB [31] in Turkey. Except type AB group, cats have naturally occurring allo antibodies. It is known that cats have naturally occurring alloantibodies (isoantib odies) against the blood type they are lacking. Because of this to prevent blood incompa tibility reactions in cats feline blood typing is important in clinical practice. Blood type incompatibility can Blood Cell An Overview of Studies in Hematology

324 especially result in two fatal reactions. The first is acute haemolyti c transfusion reactions, occur particularly in cat transfused with type A blood [32]. Feline n eonatal isoerythrolysis (NI) is the second incompatibility reaction. It occurs when type A or AB kittens born to type B queens are nursing. Naturally occurring anti-A alloantibodies result in blood incompatibility reaction in the type B queens colostrum and milk [25, 30]. Cats constitute non-self antibodies in contrast to dogs. As a result of this non -self antibodies potentially fatal antibody-mediated reactions can occur towards non-self red blood cells. Nearly 20% of type A cats have anti-B antibodies. These antibodies ar e usually weak. All type B cats have strong anti-A antibodies. In contrast AB cats do no t have alloantibodies [32]. In previously unsensitized cats naturally occuring isoantibodies a re responsible for transfusion reactions. Nearly all type B cats have highly titered anti -A agglutinins and hemolysins. RBCs can be destructed rapidly in type B cats taking type A blood. In type B cats the high titres of naturally occurring anti-A antibodies cause ra pid intravascular destruction of transfused type A red blood cells [33]. This can be m ediated by IgM, complement fixation and the release of potent vasoactive compounds. As a result of this shock can develop usually due to possessed antibodies towards the transfused RBC s [3, 34]. This can cause severe transfusion reaction and death even if as little as 1 ml o f type A blood is administered to a type B-cat [2, 35]. Because of their endotheliochorial plac enta newborn kittens have no alloantibodies. Nevertheless colostral transfer of immunoglobuli n (Ig) G and a small amount of IgM occurs. Neonatal isoerythrolysis develops in cat s. It is one of the cause of the fading kitten syndrome. Kittens that are type A or AB and those tha t are born to type B queens are at risk. In affected kittens Clinical sings can range from unapparent, to severe hemolytic anemia with hemoglobinuria, icterus, and death [1, 36, 37, 38]. 3. Transfusion therapy Packed red blood cells (pRBCs) and fresh frozen plasma (FFP) are comp onents generally provided for canine transfusions. If processed at once, 1-4 each unit (450 mL) o f whole blood can be seperated into 1 unit of pRBCs and 1 unit of FFP. It is difficult to prep are components from a small volume of blood. Because of this cat blood transfusions are usually administered as fresh or stored whole blood. If patients requires spec ific components like pRBCs and FFP, in this case whole blood can be separated into them [39]. In veterinary medicine, red blood cell transfusions are used more freq uent recently. They

are the integral part of lifesaving. They are used in critically ill as advanced treatment. Situations required transfusions include life-threatening anemia from acu te hemorrhage or surgical blood loss, hemolysis from drugs or toxins, immune-mediated di seases, severe nonregenerative conditions, and neonatal isoerythrolysis [40]. Indications of red blood cell transfusions are in the treatment of an emia caused by hemorrhage, hemolysis, or ineffective erythropoiesis. Oxygen is poorly s oluble in plasma. Because of this oxygen in blood is mostly carried by hemoglobin (Hgb) . In anemic patient, RBC transfusions increase the oxygen-carrying capacity. Therefore inadequ ate delivery of oxygen to tissues with consequent tissue hypoxia are prevented or treated [41]. Principles of Blood Transfusion 325 The treatment of severe anemia caused by hemorrhage, hemolysis, ineffec tive erythropoiesis, auto-immune hemolytic anemia, or neoplasia is primary indication for blood transfusion. Lethargy and altered mentation, increased respiratory effort , pale mucous membranes and tachycardia are the clinical signs of anaemia. The body carry out a number of adaptive responses physiologically, to maintain carrying of oxygen to the tis sues [42, 43]. The solution of oxygen in plasma is weak. Because of this hemoglobin (Hgb) carries approximately whole oxygen in blood [41]. The decision to conduct a R BC transfusion is generally based on a measurement of the patient s packed cell volume (PCV), hematocrit (Hct) or Hgb concentration (Hgb) and especially on clinical evaluation of the patient [41]. Clinically animals should be evaluated individually. Generally when the hematocrit is less than 10%, the treatment of anemia is transfusion. However, animals wit h acute-onset anemia usually require transfusion before their hematocrit decreases to 15%. Thi s contrasts with the situation in animals with chronic anemia. Other indications f or transfusion are hypovolemia, thrombocytopenia, clotting factor deficiency, and hypoprotein emia [1]. Electrocardiographic signs of myocardial ischaemia are similar to those identifi ed in human patients with myocardial infarction. It can ocur with anemia [44]. The usage of administration of FFP are for the treatment of a single or multiple clotting factor deficiency, vitamin K deficiency or antagonism, surgical bleeding or wher e a massive transfusion is required [45]. Hypoalbuminaemia and coagulopathies especia lly due to liver disease are the main reported indications for FFP transfusions in cats [46]. Stored blood is more than 8 hours old. The length of storage depends on the

anticoagulant/preservative solution used. It varies from 48 hours for 3.8% sodiu m citrate (no preservative) to 4 weeks for CPD-A1 (citrate, phosphate, dextrose, and adenine). Acid citrate dextrose (ACD), citrate phosphate dextrose (CPD and CP2D), and citrate phosphatedextrose-adenine (CPDA-1) are mostly used as preservatives. The viability of RBCs is provided by the added dextrose, phosphate, and adenine. Due t o the preservative used, the storage can extend up to 3 to 5 week ([3, 41, 47]. In patients that are hypothermic or receiving large volumes of blood, refrigerated RBC products should be prewarmed to temperatures between 22C and 37C immediately befor e transfusion. In the routine practice of RBC products to normovolemic a nemic patients, refrigerated blood components do not need warming before transfusion. W arming may accelerate the deterioration of stored RBCs and may cause rapid growth of contaminating microorganisms [48]. In clinical practice advances in safety of blood transfusion is import ant in preventing transfusion-transmitted infections (TTI). The most frequent severe infect ious outcome of transfusion has been known as bacterial contamination of platelets, with resulta nt sepsis in the recipient recently. Using automated or semi-automated blood culture devices, apheresis platelets and prestorage pooled platelets are most often tested [49]. Generally, before a blood transfusion is given to animals, blood typin g and/or crossmatching of the recipent and donor should be done to avoid the likelihood of a t ransfusion reaction. Also, ineffective therapy is caused by shortened survival of transfuse d mismatched Blood Cell An Overview of Studies in Hematology 326 red cells. In order to prevent primary sensitization and risk of developing hemo lytic disease in breeding females, cross-matching and/or blood typing is important. In general veterinary practise, blood typing for canine DEA 1.1 and for feline types A and B is applie d [1]. To decrease adverse reactions one sould pay attention to blood typing and crossmatching procedures as much as monitoring. There is always risk in blood trans fusions. For this reason, they should be performed only when warranted. When taking hist ory, previous transfusion therapy should be asked and in a history of previous transfusion the rapy crossmatching is necessary [1, 50]. Depending on availability and indication for transfusion, whole-blood or blood-c omponent therapy may be administered. RBCs, white blood cells (WBCs), platelets, all the coagulation factors, albumin and immunoglobulins constitute whole blood (WB) [51].

In cats, fresh whole blood is the most common product used recently. S tored whole blood, packed red blood cells and fresh frozen plasma (FFP) are also given as transfusi ons [45]. The heavier cellular elements from the supernatant plasma are sedimented by cent rifugation of whole blood sediments. Due to separation of blood collection within 8 hours all protein activity and concentration are maintained in the plasma. The obtained supernatant usually frozen. For subsequent transfusion, it is stored as fresh frozen plasm a (FFP). In addition it can also processed to provide cryoprecipitate and cryosupernatant. It can also b e transfused immediately as fresh plasma [52, 53]. Fresh frozen plasma have to be stored froz en at -30C before used. Also it should be identified with the donor blood type, name and collection date. Samples thawed and not used sould discarded or stored in a fri dge and used within 12-24 h and should not be refrozen [43]. Recently an ultra-purified polymerised bovine haemoglobin solution is th e only commercially available alternative to red cell transfusion (Oxyglobin). It is not licensed in cats but it has been used in treatment of anaemia in cats and also in therapy of carbon monoxide poisoning [54, 55]. Hemostatic protein deficiencies lead to hemorrhagic disorders and the t reatment is done principally by plasma components [56]. In animals with von Willebrand disease (v WD) and hereditary coagulation factor deficiencies active hemorrhage is controlle d by plasma components. Plasma components are also used for preoperative prophylaxis in these diseases [53]. For preparation of plasma components sterile plastic bags are used. Af ter that they are stored and transferred as frozen in individual boxes. Products have to be stored at -20C or lower. Just before transfusion they warmed to 37C in a water bath or incubator. Preferred route of administration is the intravenous transfusion of plasma components. If attempts at vascular access have failed, intraosseous transfusion can be used in e mergency situations. When acut allergic reactions occur transfusion is stopped and antihista mines and/or shortacting steroids are given [53, 57]. Cats have antibodies to non-self blood types within the plasma. Becaus e of this only typespecific plasma should be administered to cats in contrast to dogs. U sing one of the Principles of Blood Transfusion 327 commercially available systems whole blood can be separated into FFP and packed red cells

if it is taken aseptically. The blood spun at 3800 rpm at 10 C in a refrigerated c entrifuge for 12 mins. Using a plasma extractor the plasma is extracted and stored at 20C [57]. In hypoalbuminemic dogs and cats, human serum albumin has been used f or therapeutic use [58]. 3.1. Platelet transfusion Correction of coagulation by fresh platelets are shown by in vitro co agulation studies. Freshly collected platelets correct thrombocytopenia, control associated hemorrhage, and prevent death from bleeding. Hemorrhagic diathesis are prevented by platelet rep lacement for thrombocytopenia [59]. Severe thrombocytopenia or thrombopathia result in bleeding. Platelet transfusio n is used for the control of this bleeding. In veterinary medicine platelet transfusion ha s been used rarely compared to red blood cell (RBC) and plasma transfusion. In dog s, reports related to platelet transfusion are generally associated with experimental hematopoietic stem cell transplantation. Platelet-rich blood products consist of fresh whole blood (FWB) , plateletrich plasma (PRP) and platelet concentrate (PC). They are used for ag gressive anticancer therapy and treating complex hematologic disorders. Centrifugation of wh ole blood constitute platelet-rich plasma (PRP) and centrifugation of platelet-rich plasma constitude platelet concentrates (PC). Platelet activation is induced by centrifuga tion so that the resuspension of the platelet pellet during PC preparation from dogs is difficult. The preparation efficiency of PC from dogs can be improved by addition of PGE1 in PRP before the centrifugation of PRP. Also therapeutic efficacy of the platelets are maintained. In 10-28 kg body weight dogs plateletpheresis has been used successful ly. On the canine donor thrombocytopenia and hypocalcemia are the main adverse effects of plateletpheresis [60-62]. At room temperature (RT) (20-24C), PRP and PC can be stored for 5-7 days with con tinuous or intermittent agitation. At RT FWB can be stored for up to 8 hours. The intere st in freezed (4C) storage of platelets is increasing because of the increased risk of bacteria l proliferation at RT storage. Storage of human PRP and PC are limited to 5 days because of prev ention of bacterial proliferation at room temperature [60- 63]. Platelet transfusions as with RBC and plasma components should be perf ormed with 170 m filters standard blood administration sets. Transfusion sets which can bind platelets should be exempt from latex [60]. The most common reaction to PC are febrile reactions. The frequency i s decreased by pre-

storage leukoreduction. In immunocompetent dogs receiving multiple transf usions, alloimmunization to platelet antigens occurs. Leukocyte reduction and ul traviolet B irradiation are recently accepted methods for preventing the development of platelet alloimmunization [64-66]. Blood Cell An Overview of Studies in Hematology 328 Recently platelet cryopreservation are used to provide long-term storage and immediate availability of platelet products for transfusion. When fresh platelets are unavailable cryopreserved platelets can be activated in vitro and provide therapeutic benefi t [63]. 3.2. Granulocyte transfusion Granulocyte transfusion can be used as supportive therapy. It is used in patients with lifethreatening neutropenia caused by bone marrow failure or in patients w ith neutrophil dysfunction. Granulocyte transfusions is shown to be useful in treatmen t of infections in patients after treatment with high-dose chemotherapy. It is helpful esp ecially in the chemotherapy associated with conditioning for hematopoietic stem cell transplant . By using granulocyte colony-stimulated factors higher doses of granulocytes for t ransfusion are produced. Thus recently the use of therapeutic granulocyte transfusion has been increased. The outcome of transfusion are effected by the type of infection being treated, the likelihood of recipient marrow recovery, and recipient alloimmunization [67]. In small animals therapeutic granulocyte transfusions have been used es pecially in experimental models of myelosuppression and neonatal sepsis. In clinical veterinary medicine they have been used rarely. Granulocytes can be used to iden tify the site of inflammation. Beside leukapheresis, centrifugation of FWB, with or witho ut colloidfacilitated sedimentation, may be used to isolate canine and feline bu ffy coats. Only sedimentation may also be used in the cat. At RT granulocytes are st ored immobil for 24 hours. The dose for beginning is 1 x 10 11 granulocytes/kg in a volume of 15mL/kg. It is used once to twice in a day [68-70]. 4. Donor selection To select permanent blood donors, blood typing have to be performed. Donors should be healthy young adults. They undergo routine physical check up and hematology and clinical chemistry evaluations are done. They should never taken a blood transfusion and should be free of blood parasites and other infectious diseases [1]. Nulliparous and spayed female dog and cat donors have to be chosen.

Blood have be collected via jugular venipuncture aseptically. Acepromazine interferes w ith platelet function. Because of this donors should not be sedated with it [1]. Every 3 to 4 weeks, dogs can donate between 13 and 17 ml of blood per kilogram of body weight. Features of donors sould include well nourished, supplemented with oral iron, bled less than once per month to prevent iron deficiency, greater than 25 kg, and negative for antigens for DEAs 1.1, 1.2, 3, 5, and 7. Donors should not have hea rtworm disease, babesiosis, brucellosis, ehrlichiosis, and Rocky Mountain spotted fever. Donors have appropriate neck skin that allows easy entrance to the jugular vein, have a packed cell volume that is at least 0.40 L/L, have demonstrated a good temperamen t and be in good physical condition, have no past time history of transfusion or pregna ncy, and have got sufficient levels of von Willebrand factor (vWF) [1, 3]. Principles of Blood Transfusion 329 The ideal feline blood donors should be healthy, indoor-only cats with an agreeable temperament for easy handling and restraint. Owned pet cats should be donate maximum once every 2 months [43]. The features of feline donor sould be as follows; weig h more than 4.5 kg, have a packed cell volume that is at least 0.35 L/L, have demonstrated a good temperament, and be in good physical condition [3]. Donor cats can donate betwee n 10 and 12 ml/kg. Adult healthy cats can donate 50 ml every weeks. Donors have to be typ e A. Type B donors may be demanded depending on breed prevalence and geography. Feline leukemia virus, feline immunodeficiency virus (FIV), feline infectious peritonit is, heartworm disease, and Hemobartonella sp have to be excluded in donor cats [1]. For appropriate care of donors some processes needed. These are curren t vaccinations, if there is contact with new animals every 6 mo fecal floatation, monitorization of hemogram every year, analysing clinical chemistry, screening for infectious disea ses and in the dog preventative heartworm therapy in areas where it is necessary. When bl ood collection is taken the donor s weight, temperature, and packed cell volume have to be analyse d [3, 71]. PCV or Hb are measured by taking a blood sample. Preferentially cat s with a PCV of 30 35% are used but cats with lownormal PCVs should not be used [43]. In the cat, blood can be taken by using a 19- to 20 gauge needle or butterfly in to a syringe via jugular vein venipuncture. The region over the jugular vein is clipped and prepared aseptically and sedation is administered. It is prefered to use a 1:1 combinatio

n of ketamine 100 mg/ml and midazolam 5 mg/ml. It is made up in a small syringe and given intravenously up to a maximum dose of 5 mg/kg ketamine (0.1 ml/kg of combination). Syringe consists of either ACD, CPD, or CPDA- 1 (1 mL/9 mL of blood ), or heparin (5 units/mL of blood). Before a preservative solution is used it can be pl aced in a small blood bag. To access the jugular vein a 19-21G butterfly needle is used. The blood is collected over a total of 10 15 mins. At once a maximum of 10-12 ml/kg blood can be donated. Isotonic crystalloid fluid therapy post-donation at a rate of 60 ml/h for 3 h is given to the cat [3, 43]. 5. Administration Precaution is necessary to prevent damage of the blood product and ha rm to recipient. Blood typing or crossmatching have to be carried out to provide compa tibility before RBC transfusion [41]. Transfusions of red blood cell should be administered through a filter. The filt er is arranged to remove clots and particles which are potentially harmful to the pa tient. Blood infusion sets have in-line filters. These filters trap large cells, cellular debris, and coagulated proteins. The pore size range from 170m to 260m. A filter may be used to admin ister 2-4 units of blood to a patient or for a maximum time limit of 4 hours according to human blood banking standards. High protein concentration at the filter surface and room temperature conditions promote proliferation of any contaminating microorganisms. The rate of flow slowed down by accumulated material. After 5 days or more of refriger ated storage constituted microaggregates composed of degenerating platelets, white blood cell s (WBCs), and fibrin strands in blood. They are removed by other blood filters with a pore size of 20-40 Blood Cell An Overview of Studies in Hematology 330 Jim. For transfusions of RBCs primarily microaggregate filters are desi gned. In administering small volumes of blood (<50 mL WB or <25mL pRBCs) to cats and smal l dogs a pediatric micro-aggregate blood filter (18 um pore size, priming space <lmL) i s especially helpful. Because of a progressive decrease in pore size due to increased blood f iltered larger volumes of blood administration can result in hemolysis [41]. If plasma is taken from blood preservative solutions can be put in. Blood preservative solutions are dextrose, adenine, mannitol, and the sodium chloride. The y are necessary for RBCs to carry on their energy metabolism and viability during storage [3]. Canine pRBCs

stored in a RBC preservative can be applied directly. Other pRBC prod ucts have to be diluted by putting 10mL of saline feline pRBCs or 100mL of saline to the blood b ag so that the viscosity of the donor blood decreased [41]. In the dog, if sedation is needed, butorphanol (0.1 mg/kg BW, IV) is generally used for sedation. But acepromazine should not be used because it may cause pl atelet function disturbance [72]. In the cat, ketamin may be used 2 to 4 mg/kg BW, IV for sedation. In addition to ketamin is very successful when it is used together with 0.1 to 0.2 mg/kg BW diazepam [3]. Also combinations of ketamine hydrochloride, midazolam and butorphanol tartrate, or mask administration of sevoflurane can be used [73, 74]. Generally, intravenous administration is used for RBC transfusions. In addition intraosseous administration is a perfect alternative. Peripheral veins m ay be preferred to central veins because of an increased bleeding predisposition [41]. Blood is administered through administration sets containing 0.9% saline intravenously. Contraindications include hypotonic saline, 5% dextrose in water and la ctated Ringer s solution. Cardiac arrest may be caused by injection of undiluted citra te containing anticoagulants [1]. Using a syringe driver or by hand the transfusion should begin slowly at 0.25 ml /kg/h. If no adverse affects are encountered after the first 3060 mins of administra tion the rate can be increased. Due to the urgency of the requirement for whole blood and any underlying concurrent disease the rate of administration can vary [75]. With a PCV of 20%, dogs and cats with chronic anemia can be cardiovascularly sta ble [76]. Conversely in patients with an acute onset of anemia and continuing b lood loss or hemolysis, transfusion to a higher PCV is necessary for stabilization. Generally administration of 2mL/kg of WB or lmL/ kg of pRBCs will increase the patient s P CV by 1% if there is no continuing hemorrhage or hemolysis [41]. Patient s overall condition determine the rate of blood administration. The maxi mum rate of transfusion is 10-20mL/ kg/h in normovolemic anemic patients, to avoid circulatory overload [41]. To provide blood volume again fluid therapy with crystalloids or collo ids is necessary. If the patient s total blood volume do not decrease under 20% this is usually enoug h for losses. If losses are more than 20% whole blood or packed red cell transfusion is used. Between 20% and 50% of blood volume losses are treated by crystalloids and packed RBCs [3, 7 7]. Principles of Blood Transfusion

331 Blood components like cryoprecipitate and platelet-rich plasma are used infrequently. Cryoprecipitate contains vWF, factors VIII, XIII, fibrinogen, and fibron ectin. In vWFdeficient patients cryoprecipitate is recommended particularly when surge ry is planned or patient affected by blood loss. Bleeding hemophilia A patients, or pat ients having hypo or dysfibrinogenemia are the other indications for choosing it [3, 78]. Sometimes platelet-rich plasma is used in veterinary practice. In small -sized animals it is more useful because in larger dogs it is difficult to gain enough volume and man agement of platelet count. An alternative to platelet-rich plasma are frozen platelet conce ntrates [79]. For expansion of plasma volume, different types of colloids as dextran s and hetastarch are used as alternatives to blood products. Altering hemostasis is one of the problems of dextrans and hetastarch. Oxyglobin is a hemoglobin-based oxygen carrier. It is a pproved for use in the dog in 1998. In emergency situations it is used instead of blood products when there is limited time for preparing it or performing compatibility testing [3, 8 0]. In clinical signs of anaemia and as a therapy for carbon monoxide po isoning oxyglobin is used in cats. Because it is a potent colloid (colloid osmotic pressure 43 mmHg), the main risk associated with administration is volume overload. In patients with nor movolaemic anaemia conservative administration rates are needed such as as low as 0.2-0.4 m l/kg/h and to a maximum of 1 ml/kg/h. Careful monitorization of patients with pa ying particular attention to their heart and respiratory rate is recommended [81, 82]. A recent study described the clinical outcome in dogs experiencing mas sive transfusion. Also this study documented predictable changes in electrolytes and coag ulation status. Massive transfusion is different from usual transfusions in terms of v olume and rate of blood transfusion and blood components administered. Transfusion of a v olume of whole blood or blood components has been described as massive transfusion. T he administrated blood is greater than the patient s predicted blood volume within a 2 4-hour period or arranged as replacement of half the patient s predicted blood volume in 3 hours. In a study, massive transfusion receiving dogs were investigated and in this study the mean volumes of pRBCs was 66.5mL/kg and FFP was 22.2mL/kg. As a result of this mean plasma, RBC ratio was 1:3. After transfusion clinicopathologic changes consists of electro lytes disturbances, dilutional coagulopathy, ionized hypocalcemia and hypomagnesemia and prog ressive thrombocytopenia and prolongation of prothrombin and activated partial t

hromboplastin times [41, 83]. 6. Preparations used for transfusions and blood transfusions indications The gold standard approach is that the donor and recipient are crossmatched before administration. Administration is maintained mainly intravascular with th e use of peripheral or centrally placed catheter. Also intraosseous catheters can be used to administer all blood products. It is useful in collapsed neonatal pati ents where vascular access is difficult [43, 75, 84]. Blood Cell An Overview of Studies in Hematology 332 In acute hemorrhage, anemia, decreased red cell mass, severe methaemogl obinaemia, paracetamol toxicity, chronic non-regenerative anaemia, coagulation disord ers, and thrombocytopenia fresh whole blood is used [1, 45]. The reason of anaemia in cats requiring transfusion are haemorrhage and primary immunemediated haemolytic anaemia. Hemorrhage is caused as a result of perior postoperative bleeding, trauma, gastrointestinal bleeding, abdominal neoplasia, primary immunemediated thrombocytopenia and coagulopathies [85, 86, 87]. Also in a number of i nfectious diseases anaemia is reported such as especially feline immuno-deficiency virus ( FIV) and feline leukaemia virus (FeLV) infections, and feline infectious peritoni tis [88, 89]. Other infectious diseases which cause anemia are Ehrlichia species, Bartonella species, Haemoplasmas (Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis), Anaplasma phagocytophilum, Neorickett sia risticii, Cytauxzoon felis and Rickettsia felis have additionally been associated with anaemia [43, 90]. The indication of whole blood is in a patient whom needed several blood componen ts or has acutely lost more than 50% of its total blood volume. When 50% of total blood vo lume is lost oxygen carrying capacity and oncotic activity should be recovered. In anemia, st ored whole blood is used. For anemic animals packed erythrocytes especially those with volume overload are prefered. For tissue reoxygenation the transfusion of pack ed RBCs are used. They are also useful for normovolemic, anemic patient. Before administration, to dilute any potentially damaging antibodies these erythrocytes can be washed with saline. Re frigerated whole blood should be warmed to room temperature. Before administration it sould be gently agitated to resuspend the red blood cells. Infusion rate is limited by colder blood which has a higher viscosity [3, 41, 91].

The usage of transfusion of fresh-frozen or stored-frozen plasma (FFP) are as fo llows; lack of coagulation factors associated with hepatic insufficiency, disseminated i ntravascular coagulation (DIC), vitamin K deficiency, rodenticide toxicosis, liver in sufficiency, biliary tract obstruction, sepsis/multiple organ dysfunction syndrome, pancreatiti s, hypoalbuminemia, and DIC without associated laboratoryproven coagulopathy, malassimilation syndrome, chronic antibiotic use, a need for plasma volume expan sion, or a massive blood loss within a few hours. Other It is also used in con genital or a hereditary deficiency in coagulation factors (i.e hemophilia A, B, or von Willebr and s disease and hypoproteinemia), [1, 3, 39]. Plasma (FP or FFP) is used especially i n the emergency conditions like excessive protein loss such as enteropathy, nephropathy, exudative dermatitis or inadequate intake. It is not appropriate for using as l ong-term source of protein in these patients [3, 92]. In cats, reactions have not been reported following transfusions of FFP [46]. The collection and re-transfusion of the cats own blood is called auto transfusion. It is a useful technique in an emergency situation. It can be obtained when a nimals bleed into body cavities. It should not be used if the blood is contaminated with urine, ba cteria or bile. Blood is collected from the body cavity in a sterile manner. After t hat it re-transfused into Principles of Blood Transfusion 333 the patient through an appropriate fitler. To prevent clotting anticoag ulant like acid citrate dextrose should be included at a ratio of 1:7 [39, 43]. 7. Transfusions reactions The indication of transfusion reactions can be immunologic or nonimmuno logic. They can be immediate or delayed. Antibodies to surface antigens of transfused erythrocytes cause immune-mediated hemolytic reactions. According to surface antigens canine blood is grouped. For six of these antigens typing is available. Except DEA 4, canine uni versal donor is negative for all dog erythrocyte antigens (DEAs). Universal donors should be examined. If other donors are known to be compatible with the recipient they can be also used. Acute hypersensitivities mediated by IgE antibodies are one of the possible immunologi c reaction. The other can be leukocyte or platelet sensitivity caused by recipient antibodies to the donor s white cells or platelets. The mechanisms of nonimmunologic reac tions are various. According to the specific reaction the type and severity of clinical

signs vary [17] Adverse reaction occurs in 2 types. First one is immediate reaction and follo wing transfusion it occurs within 1 to 2 h. Second is delayed reaction and it may begin within days, months, or years later [17]. Adverse reaction varies from mild (fever) to severe (death). Transfusion reactions can be acute or delayed. In animals receiving incompatible t ransfusions, acute intravascular hemolysis with hemoglobinemia and hemoglobinuria may be se en. Acute hemolytic reaction is the most serious transfusion reaction that can b e prevented. It is an immunological reaction and it happens when circulating natural or acqui red antibodies towards donor erythrocytic antigens are given. Hemoglobinuria, vasoconstr iction, renal ischemia occur due to intravascular hemolysis. Intravascular hemolysis d etermine clinical signs. Disseminated intravascular coagulopathy (DIC) can be caused by r elease of thromboplastic substances. Secondary to the release of vasoactive substa nces, hypotension and shock can ocur. Also acute renal failure and death can develop. After transfusion a decrease in hematocrit between 2 days and 2 weeks resulted in suspici on of delayed hemolysis. As a result of extravascular hemolysis, hyperbilirubinemia and biliru binuria may occur. In dogs clinical signs are as follows: fever, tachycardia or b radycardia, hypotension, dyspnea, cyanosis, excessive salivation, tearing, urination, defecation, vomiting, collapse, opisthotonos, cardiac arrest, hemoglobinemia, and hemoglobinuria. When an acute hemolytic reaction occured transfusion sould be interrupted at once and shock should be treated. Also blood product being used sould be checked out and the steps that led to the transfusion sould be examined [1, 3, 17, 93]. To detect transfusion reactions earlier requires careful evaluation of patient s behavior, vital signs, and perfusion before, during, and after a RBC transfusion. Preand post-transfusion measurement of PCV and total solids for example instantly and at 24 hours are ne eded. Also evaluation of the plasma and urine for the presence of Hgb is done [41]. In the dog the acute hemolytic reaction is rare because in this spec ies naturally occurring anti-erythrocytic antibodies prevalence is low [3]. Alloantibodies against the c ommon canine erythrocyte antigens 1.1 and 1.2 do not exist in dogs. As a result of this generally first Blood Cell An Overview of Studies in Hematology 334 transfusion can be safely given without regard for donor blood type. Thus the re cipient can be sensitized to immunogenic antigens (i.e 1.1, 1.2, 7, and others). On first tr

ansfusion it can cause shortened survival times of the transfused cells. Subsequent pred isposition to severe transfusion reaction can develop. DEA 1.1 which is the strongest antig en in dogs, leads to the most severe transfusion reaction [1]. In the second transfusion es pecially when DEA-1 type blood is applied twice to a DEA- 1-negative dog there is more risk [3]. In cats receiving typed or crossmatched transfusions low rates of tran sfusion reactions have been indicated. Transfusions with whole blood or packed red blood cells transfus ion reactions were reported [45]. But transfusions with FFP no reactions have been reported in cats [46]. Initial or subsequent AB-mismatched transfusions in cats can cause acut e hemolytic incompatibility reactions. Erythrocytes are destroyed immediately in cats because of alloantibodies. On the contrary in dogs, delayed transfusion reactions are more often occur. A type B transfusion to type A cat causes mild signs. In this situation shortene d erythrocyte survival can occur. This causes ineffective therapy. Acute hemolytic tr ansfusion reaction with massive intravascular hemolysis with serious clinical signs occurs in type A transfusion to a type B cat. These symptoms may occur even if it is the first transfusion. Type AB or A blood can be received by type AB cats safely [1, 94]. The transfusion should be stopped immediately if a transfusion reaction is suspected. The recipient sould be monitored continually for follow up. The most severe is acute haemolytic transfusion reactions developing as a result of naturally occurring alloantibodi es [32]. Clinical signs are restlessness, vocalisation, tachypnoea, bradycardia, t achycardia, hypotension and hypertension. Pyrexia is seen frequently as a result o f reactions to donor leukocytes, platelets and plasma proteins. As a result of binding by citrate, th ere is potential for hypocalcaemia when administering large volumes of blood products. Thus, if t he patient is showing clinical signs of hypocalcaemia calcium should be measured [38, 43]. The next hour after transfusion nonhemolytic fever can ocur as adverse reactions. If contaminated blood products applied by mistake, fever may occur in an acute hemolytic reaction in association with septicemia. Vomiting or diarrhea can be s een after plasma administration. Rarely urticaria may cause trouble to patient. It can be treated with antihistamines, with or without glucocorticosteroids. If whole blood is administered with rapid administration of a large volume of blood component to normovolemic cats o r smallsized dogs hypervolemia can be observed. Hypervolemia can result in pu lmonary edema. Cough, tachypnea, dyspnea, or cyanosis can occur due to hypervolemia. Treatment can be

done by stopping the transfusion, administering diuretics (furosemide) t o reduce pulmonary edema, and providing oxygen support [3,72, 93]. The recipient should be carefully examined before the procedure. Its heart rate , respiratory rate, mucous membrane colour, capillary refill time and temperature sou ld be recorded. Also the PCV and total plasma protein should be recorded [43, 51] . Delayed adverse transfusion reactions are consist of delayed hemolytic reaction, transmission of infectious disease, and posttransfusion purpura. Posttran sfusion purpura Principles of Blood Transfusion

335 has been reported in the dog. It is characterized by the appearance of severe thrombocytopenia in the week following a second transfusion. [3, 95, 96]. Anemia, regardless of underlying cause, is troublesome for clinicians in respect to stabilising and supporting the patient. The survival rate of all reaso ns for a transfusion is 84% in the first 24 h. It is 75% for blood loss anaemia and 49.6% for ineffectiv e erythropoeisis at 10 days [43, 97]. 8. Cross-Matching blood The incompatibilities between the donors red blood cells and recipients plasma are identified by major cross-match. The incompatibilities between the donors plasma and recipients red blood cells is identified by a minor cross-match [43]. Cross-Matching usually is identified as either major or minor cross-matches. major cross-match include putting patient serum into donor cells and determin e the presence of agglutinating and/or hemolytic antibodies in the patient aganist the do nor antigens. The principle of this test is hemolytic or agglutinating reaction. In this test the reagent or antibody reacts with the RBCs. Serological discordance between a candidate donor and the patient is identified by the crossmatching. It does not determine the blood group [3]. A positive in vitro reaction is caused by the presence of antibodies. I n patients that had no antibodies at the time of transfusion, a mild reaction can be seen i n 4 to 14 days after mismatched transfusions. When blood is transfused to a patient in whic h antibodies are already present, a severe reaction occurs. This antibody can be developed by eit her naturally occurring or as a result of a previous mismatched transfusion. Further more, high concentrations of antibodies can be caused by isosensitization from tra nsplacental immunization. In dogs that have received transfusions before, a crossma tch should always be performed. A minor cross-match include putting donor serum into patient eryt hrocytes.

This step is not necessary for the donor whom previously tested negat ive for antibodies. Transfusing packed or washed erythrocytes rather than whole blood can prevent administration of antibodies in donor blood against patient erythrocytes [1]. Before transfusion the reason of analysis with these methods are to prevent acut e hemolytic reaction due to transfusion, to provide optimal lifetime of the transf used RBCs, to prevent next discordant blood transfusions and to prevent neonatal isoerythrolysis [3]. Because there are blood types that have not been described and it is not possibl e to type for Mik it is recommended that cross-matching is performed before any tran sfusion. If the recipient has received a transfusion before more than 4 days cross-mat ching should be performed [98]. 9. Principles of blood transfusion in horses Horses have eight RBC groups or systems: A, C, D, K, P, Q, U, and T. The first seven systems are recognized by the International Society of Animal Blood Gr ouping Research. Blood Cell An Overview of Studies in Hematology 336 Blood-typing antiserum is not readily available for horses. Because of this to identify suitable donors equine blood-group testing can be performed by only fe w diagnostic laboratories. Over 30 different factors have been identified within the se seven equine systems. Experimentally many more systems have been identified [99, 1 00]. Red cell antigens Ca, Aa, and Qa are play an important role in transfusion re actions and neonatal isoerythrolysis. There is no universal equine blood donor. Because of this to prevent inadvertent sensitization of brood mares against the two most common alloantigen s (Aa and Qa) involved in neonatal isoerythrolysis, the preferred donor should be negative for factors Aa, Qa, and Ca [100, 101]. Aa and Qa alloantigens are most immunogen ic, and most neonatal isoerythrolysis cases are associated with anti-Aa or Qa antibo dies. The horse is clinically relevant for blood group incompatibilities. It is the only livestock species for this situation. Blood group antibodies can laed to transfusion reactions or NI and can be found in horses either naturally or as a result of a blood group incompatible pregnancy [2]. A donkey RBC antigen that has not been found in the horse has been identified, it is unique to the donkey and the mule [1]. In horses, requirement of blood transfusion include correction of anemia arising from acute blood loss secondary to trauma, surgical complications, ruptured uterine artery, guttural

pouch mycosis, and neonatal isoerythrolysis [99, 102]. Generally, whole blood transfusions are applied to horses that have acute blood loss caused by trauma, surgery, or some other conditions like splenic rupture or uterine artery hemorrhage. The transfusion recovers blood volume and oxygen-carrying capacity i n cases of blood loss. There is no certain indicative variables for the beginning of tra nsfusion so that physical examination and clinicopathologic parameters should be used to make the transfusion decision. In cases of acute hemorrhage one sould remember that the p acked cell volume (PCV) may be normal for up to 12 hours because of the time required for fluid redistribution and the effects of splenic contraction. As the horse is rehydrated with intravenous fluids, serial monitoring of PCV and total protein (TP) can estimate the amount of blood loss. The transfusion decision is made by suspection of larg e volume blood loss, together with tachycardia, tachypnea, pale mucous membranes, lethargy, a nd decreasing TP. During an acute bleeding episode when the PCV fall under 20%, bl ood transfusion is probably required. In acute severe cases, transfusion may be required before there is a significant fall in PCV. PVC shows the need for beginning of transfusion in chro nic anemia better whereas in acute hemorrhage, with transfusions proposed for hors es with demonstration of tissue hypoxia and a PCV less than 10-12% [103, 104]. Blood is collected and stored in glass bottles containing acidcitratedext rose (ACD). The method traditionally used for collecting blood from donor horses. Glass bottles containing ACD are easy and suitable for rapid vacuum blood draw. Because of th is they are recommended for equine whole-blood collection. For equine whole blood the optima l storage method is commercial citratephosphatedextrose with adenine (CPDA-1) bags [105, 106 ]. Packed RBCs (pRBCs) are specified for normovolemic anemia (i.e neonatal isoerythrolysis, erythropoietic failure, and chronic blood loss). Markers of tissue oxyg enation, for example Principles of Blood Transfusion 337 lactate and oxygen extraction are useful in chronic or hemolytic anemi a cases. In horses, disseminated intravascular coagulation, clotting factor deficiency, hypoal buminemia, decreased colloid oncotic pressure, and failure of transfer of passive immunity (FPT) are treated by plasma [104]. Colloid is usually used in patients with a total protein less than 4 .0g/dL or serum albumin concentration less than 2.0g/dL. When there is oncotic pressure less than 14 mmH

g, clinical symptoms like ventral edema, and conditions which increase microvascular permeability like sepsis are other indications for colloid usage [104]. According to plasma obtained by plasmapheresis and centrifugation prepar ations, plasma prepared by gravity sedimentation contains greater numbers of erythrocytes and l eucocytes. The risk of a transfusion reaction can be increased by these cells. During stora ge leukocytes can degranulate and fragment and release pyrogens and proinflammatory s ubstances [107, 108, 112]. Multiple hyperimmune plasma products are avaible with bacterial or vira l specific antibodies. For the treatment of equine endotoxemia, the efficacy of E . coli (J5) and Salmonella tiyphiimiriuni hyperimmune plasma has proved to be useful in some rep orts; in contrast, there are some reports which disapprove the utility of such products. For the protection of R. equi, the use of Rhodococcus equi hyperimmune plasma has also been controversial. For treatment of specific disease additional plasma produ cts like botulism antitoxin, West Nile virus antibody, and Streptococcus equi antibody are usable. In general equine practice plasma is administered to neonates to provide protective immunog lobulins. Protective immunoglobulins are used for treatment of failure of transfe r of passive immunity or prophylaxis against Rhodococcus equi. Also, the albumin con tent of the plasma used as a colloid for circulatory volume support and in the t reatment of proteinlosing enteropathies. In horses heritable and acquired coagulopathies ca n occur. Specific coagulation factors are not available for supplementation. Also indicati ons include coagulopathies, protein-losing nephropathy and protein loss through third spacing into a body cavity (occurring with peritonitis or pleuritis) [104, 109-113]. Fresh frozen plasma must be separated and frozen within 8 hours of blood collect ion. Then it can be colder at -18 C and stored for up to 1 year. Frozen plasma is considere d as plasma separated any time after 8 hours of blood storage [112, 114, 115]. 9.1. Blood donor selection Healthy, young gelding weighing at least 500 kg is the ideal equine blood donor. Donor horses should be performed current vaccinations. To prevent from equine infectio us anemia donors should be tested each year. RBC antigens Aa and Qa are the m ost immunogenic antigens. Because of this in the ideal donor, the Aa and Qa alloanti gens should be absent. There are breed-specific blood factor frequencies. Thus a donor of the same breed as the recipient, particularly when blood typing is absent may be preferable. Horses that have

taken blood or plasma transfusions and mares that have had foals are not appropriate as Blood Cell An Overview of Studies in Hematology 338 donors. Because they have a higher risk of carrying RBC alloantibodies . Donkeys have a RBC antigen known as "donkey factor". Horses do not have this antigen . Thus donkeys or mules should not be used as donors for horses because horses can dev elop anti-donkey factor antibodies if transfusion takes place [1, 104, 116]. An immediate blood transfusion can be applied for the first time in an emergency situation with a very minor risk of serious transfusion reaction. Horses can de velop alloantibodies within 1 week of transfusion. Thus blood typing and crossmatching are recommended before a second transfusion is given. A second blood transfusion may b e given confidently without a blood crossmatch within 2-3 days of the first transfusion. Blood typing and alloantibody screening can be used for the transfusion needed patient to find the most suitable donor horse. Blood typing and antibody screening before initia l transfusion are more important for horses. Because subsequent blood transfusions are an ticipated and if sensitized to other blood group factors broodmares may produce foals w ith neonatal isoerythrolysis (NI). For detection of equine RBC antigens Ca and Aa, a rapid ag glutination method has been developed. It can be more suitable for pretransfusion testing [9 9, 103, 104]. 9.2. Collection techniques Blood is collected from the jugular vein of the donor horse. For this purpose tw o way used; direct needle cannulation or catheteri-zation. When a large volume of blood is r equired, a 10 or 12 gauge catheter is recommended. A 14 gauge catheter is also sufficient. Pla stic bags and vacum-collection glass bottles in sizes ranging from 450 mL to 2 L a re suitable for blood accumulation. Anticoagulation with 3.2% sodium citrate is enough when b lood is received for immediate transfusion. In saline-adenine-glucose-mannitol solution red blood cell concentrates stored and they can be used for transfusion for up to 3 5 days after blood accumulation. Equine blood storage condition resemble to canine and human blood storage condition. According to both in vitro tests and human parameters after 35 days of storage equine erythrocytes remain appropriate for transfusion. Fresh frozen plasma is o btained by separation of erythrocytes and plasma. Both of them can be used alone . RBC survival evaluation sould be doen in vivo [104, 117]. To allow separation of red blood cells by gravity sedimentation the b

lood is stored in a refrigerator at 5 C for 48 hours in an upright position. Then the pl asma is decanted into a sterile 3-L bag with sterile plastic connecting tubing using gravity. 3-L bags containes a constant weight of plasma (3.4 kg). The red cell fraction is thrown out. The pla sma bags are sealed, labeled with the horses name and the date of decantation. They are stored at -20 C until needed for plasma transfusion [112, 118]. 9.3. Administration In acute blood loss cases, PCV is usually impractical for estimation of volume to be transfused because it does not exactly indicate blood loss. Instead of this the volume of blood needed are predicted by estimation of blood loss and evaluation of clinical Principles of Blood Transfusion 339 parameters. Fluid shifts will replace much of the circulating volume s o between 25% and 50% of the total blood lost should be replaced by transfusion. Pay attention sou ld be give to that up to 75% of RBCs lost into a body cavity like hemoperitoneum are within 24 -72 hours autotransfused back into circulation. Thus in cases of intracavitary he morrhage lower percentages of blood volume replacement can be needed. To remove small clots and fibrin blood and plasma products should be given with an in-line filter [104, 119]. 9.4. Adverse reactions Blood should be given at a rate of approximately 0.3mL/ kg over the first 10-20 minutes for monitoring the transfusion reactions. Heart rate, body temperature, and respiratory rate sould be monitored. Additionally horses have to be monitored for signs of muscle fasciculation, piloerection, and urticaria. Urticaria, hemolysis, pruritis , edema, tachycardia, tachypnea, pyrexia, colic, changes in mentation and acute anaphylactic reactions are adverse reactions indicated in horses taking blood transfusions. The rate of a dverse reaction to WB transfusion has been reported as 16% which are mild urticarial reactio ns and worsening hemolysis. Also 1 of 44 horses (2%) exhibit a fatal anaphylactic reaction [103, 113]. Transfusion reactions may vary from mild urticarial reactions to anaphy laxis. They are divided into immunogenic and nonimmunogenic reactions. Immunogenic reactions inc lude anaphylaxis, hemolysis, fever, hives, acute lung injury, posttransfusion purpura, immunosuppression, and neonatal isoerythrolysis. Nonimmunogenic reactions include circulatory overload, bacterial contamination, citrate toxicity, coagulopa thy, hyperammonemia, and transmission of disease. In horses that have receiv

ed fresh frozen plasma serum hepatitis has been observed [52, 93, 112, 120]. In a second plasma or blood transfusion there exists risk for severe adverse reactions in dogs. Also there is a risk of development of neonatal isoerythrolysis in gravid mares. The risk is much more in whole blood transfusions [26, 33, 112]. In horses suffered from normovolemic anemia polymerized ultrapurified bo vine hemoglobin (PUBH) improves hemodynamics and oxygen transport parameters . During infusion to be informed about any adverse reactions patients should be monitored closely. Intense pruritus, tachycardia, and tachypnea can be resolved shortly af ter stopping the infusion [121]. 10. Principles of and indications for blood transfusion in ruminants and camelids Eleven blood groups have been classified in cattle. The greatest clini cal relevance is in groups B and J. The B group is extremely complex, thus closely match ed transfusions are very difficult. Newborn calves do not have the J antigen. During the first six m onths of life they generally acquire it. Cows can be sensitized to erythrocyte antigens by vac cinations of blood origin like some anaplasmosis and babesiosis vaccines. As a resu lt of this neonatal isoerythrolysis in subsequent calves occur. [1]. Blood Cell An Overview of Studies in Hematology 340 Seven blood groups have been classified in sheep. The B group in these animals i s resemble to the B group in cattle, and the R group is resemble to the J gr oup in cattle. For example, antigens are soluble and soluble antigens passively absorbed to erythro cytes. In the goat, five blood groups are identified which resemble to those of sheep [1]. Blood group AO expression is affected by 16 porcine blood groups and the S gene. Carbohydrate antigens like AO blood group antigens and minor histocompatibility antigens can be important targets for the immune response to transplanted organ s or tissues. These antigens remain an unknown and untested variable in many transplant st udies using pigs. Depending, on work performed in some Europian country pig blood groups developed and expanded largely. The source of blood typing reagents is especially fr om isoimmune sera. Most antibodies behave as agglutinins and a few as hemolysins. Interna tionally sixteen genetic systems are recognized [2, 122-124]. In two domestic South American camelids, Ilama and alpaca, our knowledge is litt le about group variation. Six blood groups factors were identified (e.g A, B, C, D, E and F) . from isoand heteroimmune sera constituted for these animals [2].

In ruminants and camelids indications for WB and plasma transfusion are similar to horses. Chronic anemia may be a more common problem in ruminants. Gastrointest inal parasites, particularly Haemonchus contains, and ectoparasites (e.g. Haematopinus sp p. and Linognathus spp.) are causes of chronic blood loss anemia, and iron-de ficiency anemia. These can affect neonatal calves [104, 121, 125]. Studies with camelids and bovines has showed that the neonatal intesti ne can only successfully absorb colostral immunoglobulins for 1224 hours postpartum. Passive transfer (FPT) is failed in 19% to 24% of neonatal camelids. A common indicat ion for plasma transfusion in neonatal calves and crias is failure of transfer of pa ssive immunity. Hyperimmune serum products are existing for subcutaneous and intramuscul ar dosing in ruminants. These are products with antibodies against E. coli, Pasturel la, Aercanobacter pyogenes, Salmonella typhimurium and Clostridium [104, 126-129]. An integral component of neonatal camelid care is IV plasma transfusion. It is u sed for the purpose of antibody supplementation and fluid resuscitation in critical illness. Neonates are immunocompetent at birth but due to initial postpartum absorption of colostrum f or passive acquisition of immunoglobulins (especially IgG) they are severely hypogammaglobu linemic [130, 131]. In cattle, the first blood transfusion should usually be safe, regardl ess of the donor. Jnegative donor is ideal. Because agglutination reactions do not develop , routine crossmatching is not useful in ruminants. First transfusions are usuall y safe to apply without a blood cross-match but crossmatching is recommended when more than 48-72 hours have passed away since the first blood transfusion. Blood donors should not have disease like bovine leukosis virus, anaplasmosis, and bovine viral diarrhea viru s [104]. Total blood volume estimated in cattle is 80 mL/kg. From the donor animal up to 20-25% of total blood volume can be removed. Usually needle cannulation or jugul ar catheterization Principles of Blood Transfusion 341 used in this situation. Blood can be collected into bottles or bags using citrat e anticoagulant (e.g CPDA-1) in equine transfusions [104]. Blood samples can be taken from the jugular vein in sheep. A 500 ml transfer bag system including a needle can use for the storage. These bags include 70 ml of CPDA-1-stabiliser. Then the blood should be put into four 150 ml transfer bags. These bags can be s tored on a

horizontal shaker. It shows the best preservation of platelet function. Also it can be used for the storage experiment consecutively [132]. Platelet count and aggregability of CPDA-1-stabilised ovine blood is kept most c ovenient at room temperature. It provides adequate haemostatic function for ex vivo experiments for one working day. In ovine blood functional loss and high percentage o f platelets within aggregates can be observed at refrigerator temperature. This should be considere d in blood transfusion in sheep [132]. 10.1. Administration and adverse reactions In order to monitor transfusion reactions blood should first be transp orted slowly. Ruminant blood type discordance result in primarily complement-mediated hemolysis. Volume overload should not be given. Also in neonates and small ruminants volume should carefully be given [104]. Intestinal absorption of antibodies declines sharply within the first 24 hours p ostpartum. For treatment of crias with failure of passive transfer (FPT) IV or intraperitoneal administration of 2040 mL/kg of camelid plasma is recommended. In compromised neonates requiring fluid resuscitation IV administration of plasma is generally preferred. It is used for the correction of FPT and colloid support. In foals during extensive plasm a volume expansion careful monitoring is needed to prevent cardiopulmonary complications. F ollowing IV plasma administration the cardiovascular and pulmonary effects of plasma volume expansion have not been specifically worked out in camelids. But in s everal species (i.e sheep and cat) plasma volume overexpansion depending on excessive IV f luid administration has been associated with reduced lung function and pulmo nary edema formation in clinical and experimental settings. In addition according to measures in presumed hypovolemic human patients administration of colloids can induc e a greater reduction in lung function than crystalloids [130, 133-137]. Measurable plasma volume expansion and a concurrent reduction in pulmonary funct ional residual capacity (FRC) is caused by IV administration of 30 mL/kg ca melid plasma to neonatal crias. In healthy neonatal crias administration of this quanti ty of plasma seems to be safe. But with underlying cardiopulmonary or systemic disease changes in lun g volume associated with plasma administration could create risks for crias (131). Adverse effects of transfusing blood stored for prolonged periods in l amps is encountered more often in patients with reduced vascular nitric oxide levels becau se of endothelial dysfunction. These patients can benefit from transfusion of fresh PRBC if available. Also

Blood Cell An Overview of Studies in Hematology 342 inhaled nitric oxide supplementation can prevent pulmonary hypertension associated with transfusion of stored PRBC [138]. In previously untransfused pigs, hemolytic transfusion reactions do not appear t o develop. But there have been two reports about adverse reactions in pigs under going liver transplants by the use of AO incompatible transfusions. Pulmonary hypert ension and decreased fibrinogen with an associated increase in fibrin degradation products occured in pigs that received AO incompatible transfusions [139]. In a study, two pigs that administered AO incompatible blood transfusions during liver transplants died bec ause of disseminated intravascular coagulation (DIC), bleeding and progressive hypotensi on [140]. 11. Conclusion Vital part of veterinary emergency and critical care medicine is trans fusion medicine. It is also therapy of some disease of patient. Blood and blood products can be obtaine d through the purchase of blood products or donors. Potentially fatal adverse transfusion reactions risk is higher in cats than in dogs. Also, adverse transfusion reactions a re very important for large animals. By using known donors and screening assays that permit detection of incompatibility of blood typing or crossmatching, the risk can be decreased in b oth species. Author details Nuri Mamak Department of Internal Medicine, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, Turkey smail Aytekin Department of Internal Medicine, Faculty of Veterinary Medicine, University of Balikesir, Turkey 12. References [1] Brown D and Vap L. Principles of Blood Transfusions and Cross-Mat ching, In: Thrall, M.A., Baker, D.C., Campbell, T.W., DeNicola, D., Fettman, M.J., Lassen, E.D., Re bar, A., and Glade, W. (eds.) Veterinary Hematology and Clinical Chemistry. US A: Blackwell Publishing; 2006. p.197-202. [2] Andrews GA and Penedo MC. Erythrocyte Antigens and Blood Groups. In: Weiss DJ and Wardrop KJ (eds.) Schalms Veterinary Hematology (Sixth Edition). USA: Blackwe ll Publishing Ltd; 2010. p.711-724. [3] Lanevschi A, Wardrop KJ. Principles of transfusion medicine in small animal s. Can Vet J 2001;42 447-454. [4] Hale AS. Canine blood groups and their importance in veterinary transfusion medicine. Vet Clin North Am (Small Anim Pract) 1995;25 1323-1332.

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