BLOOD PHYSIOLOGY

1. BLOOD PHYSIOLOGY

2. What will we discuss in this chapter? (Outline) Outline I. Blood composing II. Physical and chemical characteristics of blood III. Blood Cells 1. Hemopoietic process and hemopoietic stem cells 2. Hemopoietic microenvironment 3. Erythrocyte Physiology 4. Leukocyte Physiology 5. Platelet or Thrombocyte Physiology IV. Physiological Hemostasis 1. Endocrine functions of vessel endothelial cells 2. Physiological Characteristics of Platelet 3. Blood Coagulation 4. Fibrinolysis V. Blood Group 1. RBC Agglutination 2. ABO blood group system 3. Rh blood group system 4. Relation between blood volume and clinic 5. Principle of Transfusion and Cross-match test

3. Blood and Internal Environmental Homeostasis • Blood is that part of extracellular fluid within the cardiovascular system • Blood forming During animals’ evolution, extracellular fluid was gradually shaped from the age-old time with ocean which was mainly salty solution. At last, extracellular fluid was differentiated into plasma and interstitial fluid and blood came from plasma and cells. • The role of blood in internal environmental homeostasis Blood, the most active component in extracellular fluid, display functions as follows: (1) transportation; (2) pH value buffer; (3) temperature or thermal maintenance; (4) immunity and defence

4. I. Blood composing • Blood composing: plasma + blood cells • Hematocrit: blood cells occupies the percentage of total blood volume. normal value male: 40-50% female: 37-48% newborn: 55%

5. Blood component (summing-up)

6. Terminology and normal value

7. Chemical component of plasma • Water: > 90% • Small molecule: 2%, it is electrolytes, nutriment, metabolic products, hormone, enzyme,etc. • Protein: 60-80 g/L, plasma protein include albumin (40-50 g/L), globulin (20-30 g/L,α1-, α2, β-, γ- ) and fibrinogen. Most of albumin and globulin made from liver. A/G and clinic. • Function of plasma protein: (1) transportation, (2) nutrition, (3) forming colloid osmotic pressure, (4) coagulation and anticoagulation, (5) pH value buffer, (6) immunity (globulin)

8. H2O 90 - 91% Interstitial fluid Intracellular fluid Plasma Protein (Unit：mmol/L) Chemical component of plasma

9. II. Physical and chemical characteristics of blood • Specific gravity: total blood (1.050-1.060) more influenced by red blood cells; plasma (1.025-1.030) more influenced by plasma protein; RBC (1.090-1.092) more influenced by Hb. • Viscosity: Blood relative viscosity (4~5) mainly depends on the numbers of red blood cells. Plasma relative viscosity (1.6~2.4) is mainly involved in plasma protein • Plasma osmotic pressure is 300 mmol/L or 770kPa (1) Crystal osmotic pressure results from NaCl and modulates water distribution between inside and outside of cells. (2) Colloid osmotic pressure results from albumin and regulates water distribution between inside and outside of capillary. • Plasma pH value is about 7.35~7.45, and usually buffer systems are NaHCO3/H2CO3 (20:1), protein salt/protein, Na2HPO4/ NaH2PO4, Hb salt/Hb, HbO salt/ HbO2, K2HPO4/ KH2PO4, KHCO3/H2CO3, etc [lungs and kidney mainly regulate Plasma pH value ].

10. Osmosis and Osmotic Pressure • Osmosis is the movement of water down its concentration gradient. • Osmosis is determined by the number of impermeable molecules. • Osmotic pressure is the force drawing water down its concentration gradient.

11. Osmosis and Osmotic Pressure A B ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ Water ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ [Water] > [Water] [Salt] < [Salt] Osmotic Pressure < Osmotic Pressure Osmosis is the movement of water from a high concentration to a low concentration. In this illustration, two compartments (A and B) are separated by a semipermeable membrane (broken vertical line). The water concentration in compartment A is greater than the concentration in compartment B because of the presence of salt (X) in B. Therefore, water will move down its concentration gradient from A to B. The force needed to prevent this water movement is called osmotic pressure.

12. Tonicity • The tonicity of a solution refers to the effect of the solution on cell volume. • A hypertonic extracellular solution is one in which the water concentration is less outside the cell than inside; water leaves the cell; cell volume decreases. • An isotonic extracellular solution is one in which the water concentration is the same inside and outside the cell; no water movement; cell volume does not change. • A hypotonic solution is one in which the water concentration is greater outside than inside the cell; water enters the cell; cell volume increases. • An isosmotic solution may not be an isotonic solution if the particles are permeable to the cell membrane.

13. III.Blood Cells Blood cells are erythrocyte (red blood cell, RBC), leukocyte (white blood cell, WBC) and thrombocyte (platelet, P).

14. Blood Cells • The forming processes of erythrocyte (red blood cell, RBC), leukocyte (white blood cell, WBC) and thrombocyte (platelet, P) originating from hematopoietic stem cells are hemopoiesis. • Transfer of blood cells forming place: yolk sac hemopoiesis(earlyembryo period) →liver and spleen (second embryo month) → marrow↑and liver, spleen↓ (after fourth embryo month) → marrow (fetus birth time) and liver, spleen as complementary role. During adulthood (after 18), red marrow (flat bones, e.g. vertebra,ilium, sternum, rib, skull and long bone ending) rather than yellow marrow has hematopoietic functions.

15. 1. Hemopoietic process and hemopoietic stem cells Hemopoietic process Stage one: Hemopoietic stem cells self renewal, steady numbers, active differentiation. Stage two: committed progenitors directionaldifferentiation (CFU-GEMM, CFU-E, CFU- GM, CFU-MK, CFU-TB). [CFU: colony- forming unit Stage three: precursors morphologic occurrence of various original blood cells.

16. Hemopoietic stem cells Basic characteristics • Self renewal in high degree, constant from young to old age. • Multi- directional differentiation • Large potential proliferation, Hemopoietic stem cells produce about 1×1011 blood cells releasing to blood for use. • Surface sign According to CFU (colony forming unit), using fluorescence-activated cell sorting (FACS), its main surface sign is CD34+CD38-Lin-and CD34-CD38-Lin-. Note CD: cluster of differentiation of antigen on the white blood cells; Lin: systemic specific antigen on the hemopoietic cells.

17. 2.Hemopoietic microenvironment • Hemopoietic microenvironment: It includes stromal cell secreting extracellular matrix (ECM), multihemopoietic regulating factor, hemopoietic nerves and blood vessels. • Stromal cells in the marrow come from fibrocyte, reticulocyte, endothelial cell, ectoblast cell, monocyte, engulfing cell, osteoblast and osteoclast. • Stromal cells supply two material: one is soluble hemopoietic growth factor, another is membrane-combined adhesive molecule. • Extracellular stroma synthesized and secreted by marrow stromal cell filling cellular interstice contains big molecules, such as collagen (typeI, II, III, IV), glycoprotein (fibronectin, laminin, hemopoieticnectin ) and protein amylose (sulfate cartilagetin, sulfate heparin, hyaluronic acid and sulfate dermatin, etc). • Hemopoietic cells must adhere to stromal cell and is in the hemopoietic microenvironment for survival.

18. Hemopoietic process

19. Hemopoietic process

20. Hemopoietic process

21. 3.Erythrocyte Physiology Shape and number of red blood cells (RBC) • Shape of RBC: like biconcave disc Its diameter is about 7~8 µm, peripheral thickness about 2.5 µm, central thickness about 1 µm and cubage about 90 µm3.

22. biconcave disc like Reason for shape of RBC

23. Erythrocyte Physiology Number of RBC: It is most numbers in the blood. Normal value about RBC Male adult, 4.5~5.5×1012/L; average, 5.0×1012/L Female adult, 3.8~4.6× 1012/L; average, 4.2×1012/L Newborn, ≥ 6.0×1012/L Protein within RBC is hemoglobin (Hb). Hb in male adult, 120~160 g/L; Hb in female adult, 110~150 g/L; Hb in newborn (within 5 days), ≥ 200 g/L Pregnant female, numbers of RBC and Hb are relatively less (because of more plasma). Dweller lived in plateau, numbers of RBC and Hb are relatively more (because of compensation for anoxia).

24. Physiological Characteristics and Functions of RBC Characteristics of RBC • Permeability: semipermeable membrane, gas and urea freely passing through, negative ions easily in or out of RBC, and positive ions not. There are Na-K ATPase as pump on the membrane of RBC and low-temperature-stored plasma easily has high kalium. Why? • Plasticity and metamorphose: Plasticity and metamorphose depend on: 1) surface area-cubage ratio, 2) viscosity of Hb, 3) membrane elasticity and viscosity.

25. Physiological Characteristics and Functions of RBC Characteristics of RBC • Suspension stability: it cab be described by erythrocyte sedimentation rate (ESR) which is RBC descending distance per hour and suspension stability is inverse proportion to ESR. Normal value of ESR: male, 0~15 mm/h; female, 0~20 mm/h. ESR and clinic: some diseases bring about rouleaux formation (mainly involved in plasma component, e.g. globulin, fibrinogen, cholesterol) and speed up ESR.

26. Physiological Characteristics and Functions of RBC Characteristics of RBC • Osmotic fragility: Changes in RBC put into lower osmotic salty solution. Osmotic fragility of aged RBC is large and easily results in rupture (hemolysis and ghost cell). Isosmotic solution, e.g. 0.85% NaCl, 1.4%NaHCO3, 5% glucose, etc. Isotonic solution, e.g. 0.85% NaCl Isosmotic solution does not equal to isotonic solution. Isosmotic solution, isotonic solution and clinic

27. Physiological Characteristics and Functions of RBC Functions of RBC • RBC can be used for transportation of O2 and CO2 in the blood. • RBC can be served as pH buffer.

28. Erythropoiesis • Hemopoietic material for erythropoiesis: iron (Fe++) and protein, [reason for anemia] • Influencing factors of RBC maturity: Vitamin B12 and folic acid (DNA metabolism), [clinic relation] • Process of erythropoiesis: Hemopoietic stem cells→multi systemic hemopoietic progenitor cells→RBC-committed progenitor cells (BFU-E→CFU-E)→original RBC→ earlier infantile RBC→medium-term infantile RBC→terminal infantile RBC→reticular RBC→mature RBC→blood for circulation. This process requires 6~7 days. [mitosis several times] [apoptosis]

29. Place for Erythropoiesis Main place for Erythropoiesis is bone marrow. Aother place is liver.

30. Regulation of Erythropoiesis • 0.8% of total RBCs has self renewal, that is to say, 160×106 RBC production every minute. • Burst forming unit-erythroid, BUF-E, important to earlier erythropoiesis, depends on stimulation of burst promoting activity, BPA outside body. BPA made by leucocyte is a glycoprotein whose molecular weight is about 25000~40000 • Colony forming unit-erythroid, CFU-E, important to terminal erythropoiesis, depends on erythropoietin, EPO which is also a glycoprotein, molecular weight, 34000, plasma concentration 10 pmol/L, half life 5 hours, increasing release when anoxia.

31. Regulation of Erythropoiesis

32. Life and breakage of RBC • Life-span: 120 days, about 4 months, each RBC circulates 27 km averagely in vessels, short life-span for aged RBC • Breakage: places are liver, spleen and lymphatic node, and after breakage, Hb released from RBC immediately combine with plasma α2-globulin (Hb touched protein) which is taken in by liver for iron reuse. • Hb, very toxic if it get into blood, normally, it can be metabolized into bile pigment in liver. • Clinic relation.

33. 4.Leukocyte PhysiologyClassification and numbers of Leukocyte • Number of Leukocyte (white blood cells, WBC): • (4.0~10)×109/L • Classification: It is granulocyte (neutrophil, • eosinophil, basophil), monocyte and lymphocyte.

34. Classification and numbers of Leukocyte TABLE. Classification and normal value of Leukocyte Absolute Value (×109/L) Percentage (%) Total numbers of leukocytes 4.0~10.0 Neutrophil (bacilliform nucleus) 0.04~0.5 1~5 Neutrophil (foliiform nucleus) 2.0~7.0 50~70 Eosinophil 0.02~0.5 0.5~5 Basophil 0.0~0.1 0~1 Monocyte 0.12~0.8 3~8 Lymphocyte 0.8~4.0 20~40 For Clinic Use

35. Physiological Changes in Numbers of Leukocyte • Newborn: Number is higher, 15×109/L, after birth 3 or 4 days to 3 months, being about 10×109/L, mainly, neutrophil, 70%; secondarily, lymphocyte. • Circadian changes: Number of WBC is more in the afternoon than in the morning. • Food taking, ache and mood excitation: Number of WBC is remarkably higher. • Heavy exercise and laboring: Increasing numbers, about 35×109/L, return to original level after action stop. • Terminal pregnancy of female: Numbers changes in 12~17×109/L, and during parturition, 34×109/L, and after parturition 2~5 days, number return to original level.

36. WBC Diapedisis BloodVessel Chemotaxis Metamorphose Physiological Characteristics and Functions of WBC Terminology • Diapedisis: Metamorphosed WBCs pass through vessel wall getting into interstitial fluid. • Chemotaxis: It is a process that WBCs shift to some chemical material (metabolic production, antigen-antibody complex, bacteria, toxin, etc). • Phagocytosis: It is a process that WBCs enclose and engulf exotic or extraneous material, and use intracellular enzyme digesting them.

37. Physiological Characteristics and Functions of WBC • Neutrophil • Another name, polymorphonuclear, PMN, 6~8 h in the vessels, diapedisis, chemotaxis and phagocytosis (using its hydrolyzed enzyme) • Function: It plays a very important role in nonspecific cellular immunity system which is against pathogenic microorganism, such as bacteria, virus, parasite, etc. • Clinic relation: Number of neutrophil greatly increase occurring in acute inflammation and earlier time of chronic inflammation. number decrease of neutrophil will result in poor resistibility and easily suffering from infection.

38. Physiological Characteristics and Functions of WBC • Eosinophil • Circadian changes: Its number is lower in the morning • and higher at night. • Function: • 1. It limits and modulates the effects of basophil on fast • allergic reaction. • 2. It is involved in immune reaction against worm with • opsonization. • Clinic relation: Its number increase when person suffers • from parasite infection or allergic reaction.

39. Physiological Characteristics and Functions of WBC • Basophil • Circulatory time: 12 hours • Basogranules contain heparin, histamine, chemotactic • factors and chronic reactive material for allergic reaction. • Function: It is also involved in allergic reaction. • 1. Heparin serves as lipase cobase and speeds up fatty • decomposition. • 2. Histamine and chronic reactive material increase • permeability of capillary and contract bronchia smooth • muscle, and result in allergic reaction such as measles, • asthma. • 3. Eosinophil chemotactic factor A released by basophil • can attract eosinophil collection and modify eosinophil • function.

40. Physiological Characteristicsand Functions of WBC • Monocyte • Its body is large, diameter about 15~30 µm withoutgranule • Function: • 1. It contains many nonspecific lipase and displays the • powerful phagocytosis. • 2. As soon as monocytes get into tissue from blood , it change • name called macrophage activating monocyte- macrophage • system to release many cytokins, such as colony stimulating • factor (CSF), IL-1, IL-3, IL-6, TNFα, INF-α,β ,etc. • 3. Cytokins induced by monocyte may modulate other cells • growth. • 4. Monocyte- macrophage system plays a very important role in • specific immune responsive induction and regulation.

41. Physiological Characteristicsand Functions of WBC • Lymphocyte • Classification: It can be separated into T- Lymphocyte and • B- Lymphocyte. • Function: • 1. Lymphocytes serve as a nuclear role in immune • responsive reaction. • 2. T- Lymphocytes involved in cellular immunity. • 3. B- Lymphocytes involved in humoral immunity. • Clinic relation: Numbers increase of lymphocytes occur in • chronic inflammation and late time of infection.

42. Leukopoiesis, Regulation and Breakage • Birth place: bone marrow, originating from hemopoietic stem cells, and leukopoiesis process is similar to RBC. • Leukopoiesis, differentiation and growth are influenced by hemopoietic growth factor, HGF which are glycoprotein secreted by lymphocyte, monocyte- macrophage, fibrous cell and endothelial cell. • Colony stimulating factor, CSF, such as GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3) also influence Leukopoiesis. • Life span: several hours to 3 or 4 days. • Leukocyte breakage: site are liver, spleen and lymphatic node. • Pus or purulence forming

43. 5.Platelet or Thrombocyte Physiology • Shape: Biconvex disk like, diameter about 2~4 µm, average cubage 8 µm3. • Complicated structure: under the electronic microscope, there are α-granule, dense body, lysin peroxide enzyme, opening tubular system, dense tubular system, canaliculus,etc. • Dense body: It contains ADP, ATP, 5-HT, Ca2+, epinephrine,etc. • Source: Platelet comes from megakaryocyte fractionlet release • in the marrow.

44. Normal Value and Function of Platelet • Normal value: 100×109 ~ 300×109, range from 6%~10% • Normal changes: more number in the afternoon than in the morning, more in winter than in spring, more in the venous blood than capillary, after sport↑, pregnacy↑. • *Functions: 1. It maintains capillary endothelial cells smooth and integrated (repairing endothelium and providing nutrition). 2. It is involved in physiological hemostasis. • Platelet and clinic relation: decrease of platelet, abnormal immune reaction, will results in hemorrhage or bleeding, purpuric symptom.

45. Platelet Forming and Regulation • Platelet forming: Birth place is bone marrow, originating from hemopoietic stem cells, and differentiating into burst forming unit- megakaryocyte, BFU-MK, then continuously into CFU-MK, and into megakaryocyte, demarcation membrane system, DMS, into fractionlet release to the blood requiring 8~10 days. (one megakaryocyte can produce 200~7700 platelet). • Regulation: Protein, Mpl, expressed by c-mpl (oncogene) exists in CD34+ located at hemopoietic stem cells/ committed progenitors, megakaryocyte and platelet, found by Methin in 1993, and its ligand named thrombopoietin, TPO was discovered in 1994 which promoted hemopoietic stem cells differentiating into megakaryocyte as hemopoietic stem cells positive regulating factor.

46. Life- Span and Breakage of Platelet • Life-span: Averagely, 7~14 days in the blood. It can be consumed when it displays physiological functions. • Breakage: Aged platelet can be processed by phagocytosis in liver, spleen and lymphatic node.

47. IV. Physiological Hemostasis • *Definition: The process from vessel bleeding to automatic hemostasia. • *Bleeding time: The time from vessel bleeding to automatic hemostasia. Normal time is 1~3 min and it is longer when platelet decrease. • Process of hemostasis: 1. Blood vessel contraction or convulsion (induced by neuroreflex; 5-hydroxytryptamine,5-HT; thromboxane A2, TXA2; endothelin, ET ) 2. Platelet thrombosis forming (made by platelet adhesion, aggregation, release and contraction) 3. fibrin, clot forming and maintenance (made by blood coagulation activation)

48. Physiological Hemostasis

49. 1.Endocrine functions of vessel endothelial cells • Material related to hemostasis are basal membrane, collagen (III, IV), microfibril, elastin, laminin, ectonectin, fibronectin, von Willebrand factor (vWF), protein enzyme, protein enzyme inhibitor, adhesive amylose, etc. • Anticoagulative material: They are prostacyclin (PGI2), endothelium-derived relaxing factor (EDRF or nitric oxide, NO), tissue-type plasminogen activator (tPA), uPA, ADPase, ATIII, heparin sulfate, protein C, thrombomomodulin (TM), plasminogen activator (PA). • Promoting coagulative material: Tissue factor, vWF, blood clotting factor V, plasminogen activator inhibitor (PAI-1, PAI-2, ATIII), TNFα, interleukin-1 (IL-1). • Vessel constricting and relaxing modulators: endothelin-1 (ET-1), EDRF (NO), PGI2, etc.

50. Roles of Vessel Endothelial Cells in Physiological Hemostasis Roles are close related to its endocrine functions • Vessel endothelium serves as barrier between underendothelial structure (namely, collagen) and blood. As soon as collagen expose to blood, hemostasis of platelet is immediately activated to form thrombus blocking wounded vessels. • Platelet activation can releases constrictive factors (TXA2, ET-1, 5-HT, etc) making vessel convulsion, lasting about 60 sec. • Stimulated vessel endothelial cells release coagulative factors and Promoting coagulative material to realize, speed up blood coagulation. At the same time, cells also release anticoagulative factors and fibrinolysis material to modify blood coagulation.