Chapter 4 Blood and Hematopoiesis Guanhua Huo September ， 2013

1. Chapter 4 Blood and Hematopoiesis Guanhua Huo September，2013

2. Blood • Blood (about 5.5 L in a man) consists of the cells and fluid. Blood is made up of two parts: formed elements, or blood cells, and plasma, the liquid in which the formed elements are suspended. The formed elements are erythrocytes (red blood cells), platelets, and leukocytes (white blood cells). • If blood is removed from the circulatory system, it will clot. This clot contains formed elements and a clear yellow liquid called serum, which separates from the coagulum. • Blood that is collected and kept from coagulating by the addition of anticoagulants (eg, heparin, citrate) separates, when centrifuged, into layers that reflect its heterogeneity. The hematocrit is an estimate of the volume of packed erythrocytes per unit volume of blood. The normal value is 40–50% in men and 35–45% in women.

3. Plasm (I.M.) : 55% The formed elements, blood cells： erythrocytes leucocytes blood platelets Plasm Platlets& Leucocytes buffy coat Erythrocyte *using anticoagulants

4. serum Blood clots components same as that of plasma but fibrinogen Serum 1. Plasma 90% water albumin 3.5-5.0g/dl 10% solutes :fibrinogen • plasma pr. enzymes (7%) globular pro. α,β, γ: Ig. etc. • organic mollecules: • Lipid • hormones • Vitamins • inorganic salts • metabolites

5. BLOOD CELLS • blood smear stains • wiwh Wright or Giemsa Erythrocytes:4 ～ 6×1012/L 4.2～5.5×1012/L(M) 3.5～5.0×1012/L(W) *Hb:120～150g/L(M)；105～135g/L(W) Leukocytes:4～10×109/L neutrophils 50～70% Granulocytes eosinophils 0.5～3 % basophils 0～1% Agranulocytes lymphocytes 20～30% monocytes 3～8% Platelets: 100～300×109/L

6. Erythrocyte • biconcave discs—maximize surface-to-volume ratio， 7～8μm，flaxible • no nuclei，no organelles • rich in Hb：105～150g/L • blood group (ABO; Rh…): • dependent on the agglutinogen on the membrane • Life span:120 days Central pallor The normal concentration of erythrocytes in blood is approximately 3.9–5.5 million per microliter in women and 4.1–6 million per microliter in men.

7. Pathology • Anemia：R.B.C < 3×1012/L or Hb< 100g/L megaloblastic anemia(巨细胞贫血) : >9 μm iron deficiency anemia: (缺铁性贫血) <6 μm • Osmotic stress abnormal: hypotonic(低滲): RBC swelling ＆hemolysis (remain ghost血影)； hypertonic (高滲): RBC collapsing • An increased number of erythrocytes (erythrocytosis, or polycythemia) may be a physiological adaptation. It is found, for example, in people who live at high altitudes, where O2 tension is low. Polycythemia , which is often associated with diseases of varying degrees of severity, increases blood viscosity; when severe, it can impair circulation of blood through the capillaries. Polycythemia might be better characterized as an increased hematocrit, ie, an increased volume occupied by erythrocytes. • Erythrocytes with diameters greater than 9 m are called macrocytes, and those with diameters less than 6 m are called microcytes. The presence of a high percentage of erythrocytes with great variations in size is called anisocytosis .

8. Reticulocytes • 0.5～1.5% • containing RER residual, ribosomes & Mit. (stained by brilliant cresyl blue) • increase in anemia • Indicator of hematopoietic capacity of the bone marrow

9. Erythrocytes recently released by the bone marrow into the bloodstream often contain residual ribosomal RNA, which, in the presence of some dyes (eg, brilliant cresyl blue), can be precipitated and stained. Under these conditions, the younger erythrocytes, called reticulocytes, may have a few granules or a netlike structure in their cytoplasm. • Reticulocytes normally constitute about 1% of the total number of circulating erythrocytes; this is the rate at which erythrocytes are replaced daily by the bone marrow. Increased numbers of reticulocytes indicate a demand for increased O2–carrying capacity, which may be caused by factors such as hemorrhage or a recent ascent to high altitude.

10. CO2+HbO2 HbCO2+O2 Function of RBC Binding & transporting O2and CO2 In lungs In organs & tissues

11. The erythrocyte is quite flexible, a property that permits it to adapt to the irregular shapes and small diameters of capillaries. Observations in vivo show that when traversing the angles of capillary bifurcations, erythrocytes containing normal adult hemoglobin (HbA) are easily deformed and frequently assume a cuplike shape. • Erythrocytes are surrounded by a plasmalemma; because of its ready availability. It consists of about 40% lipid (eg, phospholipids, cholesterol, glycolipids), 50% protein, and 10% carbohydrate. About half the proteins span the lipid bilayer and are known as integral membrane proteins.

12. Several peripheral proteins are associated with the inner surface of the erythrocyte membrane. The peripheral proteins seem to serve as a membrane skeleton that determines the shape of the erythrocyte. One protein associated with the inner surface of the erythrocyte membrane is the cytoskeletal spectrin, which links several membrane components with other cytoskeletal elements, forming a meshwork that reinforces the erythrocyte membrane. This meshwork also permits the flexibility of the membrane necessary for the large changes in shape that occur when the erythrocyte passes through capillaries. Because erythrocytes are not rigid, the viscosity of blood normally remains low. • In their interiors, erythrocytes contain a 33% solution of hemoglobin, the O2-carrying protein that accounts for their acidophilia. In addition, there are enzymes of the glycolytic and hexose-monophosphate-shunt pathways of glucose metabolism.

13. The image is a cryo electron micrograph of unstained frozen-hydrated membrane skeletons. Spectrin tetramers (indicated by arrow heads) cross-link actin junctional complexes (indicated by arrows) to form an extensive 2-dimensional network.

16. Leukocytes, WBC • Spherical, nucleate & colorless • Contain specific or nonspecific granules • Amoeboid movement • Phagocytosis except for lymphocytes • Involved in defense against foreign materials

17. Leukocytes: 4～10×109/L neutrophil 50 ～75% Granulocyte eosinophil 1～4% basophil 0～1% Agranulocyte lymphocyte 20 ～30% monocyte 3 ～8%

18. Neutrophils, Polymorphonuclear Leukocytes • 60~70%; 10～12μm; • Nucleus: polymorphous, 2～5 lobes; band neutrophils-Immature • Specific Granules: 80%, fine (0.3～0.8μm ) neutrophilic (salmon pink) • Azurophilic granules: 20%, larger, reddish-purple

19. Immature neutrophils that have recently entered the blood circulation have a nonsegmented nucleus in the shape of a horseshoe (band forms). An increased number of band neutrophils in the blood indicates a higher production of neutrophils, probably in response to a bacterial infection, or to a treatment for aplastic anemia, left shift of nucleus. Conversely, an increased number of neutrophils with 4~5 lobes, right shift of nucleus, indicates hematopoietic dysfunction of bone marrow. • Neutrophils with more than five lobes are called hypersegmented and are typically old cells. Although the maturation of the neutrophil parallels the increase in the number of nuclear lobes under normal conditions, in some pathological conditions, young cells appear with five or more lobes.

20. E.M. • Specific Granules: medium-dense, containing: • Alkaline phosphatase • Phagocytins • Azurophilic Granules: lysosomes, containing: • Acid phosphatase; • peroxidase • Arylsulfatase; • Myeloperoxidase; • Lysozyme • Cationic antibacterial proteins The cytoplasm contains two types of granules: the small, pale, peroxidase-negative specific granules and the larger, dense, peroxidase-positive azurophilic granules. The nucleus is lobulated, and the Golgi complex is small. RER and mitochondria are not abundant, because this cell is in the terminal stage of its differentiation. x27,000.

21. Function • Mobility(chemotaxis): Attracted by polymorpho- nuclear chemotactic factor (released by macrophages) • Phagocytose & destroy bacteria: the number of leukocytes↑ acute percentage of neutrophils↑bacteria infection ** cell mark: peroxidase and CD15 They are active phagocytes of bacteria and other small particles. Neutrophils are inactive and spherical while circulating but show an active ameboid movement upon adhering to a solid substrate, such as collagen in the extracellular matrix.

22. B. Eosinophilis • 10～15μm; • Nu.: 2 ～5 lobes • Abundant large (0.5～1.5μm ) and elongated refractile specific granules that are stained by eosin -lysosomes • A few redish-purple azurophilic granules

23. E.M. • specific granules: Lysosome :ovoid; a crystalline core (internum) that lies parallel to the long axis of the granule. The less dense material surrounding the internum is known as the externum, or matrix. • Containing: acid phosphatase, peroxidase, histaminase major basic protein arylsulfatase eosinophilic peroxidase Typical eosinophilic granules are clearly seen. Each granule has a disk-shaped electron-dense crystalline core that appears surrounded by a matrix enveloped by a unit membrane. EG, eosinophil granule; N, nucleus; M, mitochondria. x20,000.

24. Function • Chemotaxis: attracted by chemotactic factor from the mast cells • Phagocytize: antigen–antibody complexes • Releasearylsulfatase and histaminase:modulate inflammation by inactivating the leukotrienes and histamine produced by other cells-- reduces allergic reaction • Antiparasite • major basic protein : • It is increased in patients infected with allergic reaction or parasite • An increase in the number of eosinophils in blood (eosinophilia) is associated with allergic reactions and helminthic (parasitic) infections. In tissues, eosinophils are found in the connective tissues underlying epithelia of the bronchi, gastrointestinal tract, uterus, and vagina, and surrounding the parasitic worms.

25. Basophils, basophilic granulocytes • 10 ～ 12μm • Nucleus: • Irregular or S-shaped • Specific granules:large, variable-sized, basophilic (redish-violet to dark blue), obscure nucleus, distribute unevenly & metachromasia Fewer redish-purple azurophilic granules

26. E.M.: Specific granules, containing • Eosinophilic chemotactic factor • Heparin • Histamine • Peroxidase • Leukotriene • Founction: anticoagulate responsible to allergic reaction Electron micrograph of a rabbit basophil. The lobulated nucleus (N) appears as three separated portions. Note the basophilic granule (B), mitochondria (M), and Golgi complex (G). x16,000.

27. D. Monocytes • 12～20μm, • bluish-gray color Nu.: oval, kidney- or horseshoe-shaped with delicate net-like Chromatin, eccentrically • Cytoplasm: abundant, faint blue-gray, basophilic and frequently contains very fine azurophilic granules (lysosomes)

28. E.M : RER, polyribosomes, small mitochondria, Golgi complex microvilli and pinocytotic vesicles-surface, free ribosomes azurophilic granules: lysosomes peroxidase, acid phosphatase, non-specific esterase Function: • amoeboid movement & obvious chemotaxis • Phagocytosis (mono- nuclear phagocytic system) • differentiate into macrophage **mark: CD14 Golgi complex (G), mitochondria (M), and azurophilic granules (A). RER is poorly developed. Some free ribosomes (R). x22,000.

29. Lymphocytes • Lymphocytes can be classified into several groups according to distinctive surface molecules (markers), which can be distinguished by immunocytochemical methods. They also have diverse functional roles, all related to immune reactions in defending against invading microorganisms, foreign macromolecules, and cancer cells.

30. 3 types: small 6～8μm; medium-sized 9～12μm; large 13～20μm • Small lymphocyte(90%): LM: Round or ovoid; Nu.:spherical with an indentation, condensed chromatin, appears as coarse clumps intensely stained-purplish blue to black Cytoplasm:scanty, appearsas a thin rim around the nucleus, slightly basophilic, assuming a light blue color. A few azurophilic granules, no specific granules

31. Electron micrograph of a human blood lymphocyte. This cell has little rough endoplasmic reticulum but a moderate quantity of free polyribosomes. Note the nucleus (N), the nucleolus (Nu), and the mitochondria (M). Reduced from x22,000.

32. Classification & Function Classification* function# T-Cell: 75% cellular immunity B-Cell: 10-15% humoral immunity large granule lymphocyte (K & NK cell) * based on the source, characteristics on cellular membrane, life & function # immunological defence

33. Blood platelets • Megakaryocyte’ s fragment, in clumps • LM: 2～4μm，disc-like or irregular, anucleated. Two regions: ① Granulomere: central zone containing purple-blue granules ② Hyalomere: peripheral light blue-stained transparent zone

34. Megakaryocyte With maturation of the megakaryocyte, numerous invaginations of the plasma membrane ramify throughout the cytoplasm, forming the demarcation membranes. This system defines areas of a megakaryocyte's cytoplasm that shed platelets, extruding them into the circulation.

35. E.M.: Blood platelet

36. Cell coat: abundant GAG & glycoproteins, associated to platelet adhesion; Hyalomere: a marginal bundle of microtubules, around the periphery, helping to maintain the platelet's ovoid shape. A number of electron-dense irregular tubes, the dense tubular system. Actin and myosin molecules in the hyalomere can assemble to form a contractile system- actin-like microfilaments that functions in platelet movement and aggregation. A system of channels: a. the open canalicular system: connects to invaginations of the platelet plasma membrane, facilitats the liberation of active molecules stored in platelets, increasing the surface area for platelet adhesion. b. the dense tubule system: in hyalomere; likes sER , support, collect Ca2+ and synthesize prostaglandin.

37. The granulomere: A few mitochondria, glycogen granules, various purple granules Granules: αgranules(special granules): containing blood clotting factors (12 types found so far), fibrinogen, acidic hydrolases and platelet-derived growth factor δ granules: containing Ca2+ , pyrophosphate, ADP, ATP, take up and store serotonin (5-hydroxytryptamine, 5-HT), adrenine λ-granules: lysosomes, contain only lysosomal enzymes

38. Function 1. Hemostasia:collagenous fiber in injure area platelet plug release serotonin constraction of blood vessels slow & stop bleeding. 2.Coagulation:thrombogen coagulation factor III thrombase Fibrinogen Fibrin hemocyte coagulation 3.Protect blood vessel endothelium, repairing endothelium, prevent atherosclerosis

39. 二、Hematopoiesis • Hematopoietic organ yolk sac （early embryo) →Liver / spleen (6w—5m)→bone marrow (4m and after) • Structure of bone marrow red bone marrow: hematopoietic tissue + blood sinusoid yellow bone marrow: adipose tissue + immature hemocyte

40. Red bone marrow • hematopoietic tissue: reticular connective tissue+hemocytes in various stage, macrophages, adipocytes, mesenchymal cells • blood sinusoid: discontinuous endothelia, incomplete basement membrane

41. Section of active bone marrow ( red bone marrow) showing some of its components. Five blood sinusoid capillaries containing many erythrocytes are indicated by arrowheads. Note the thinness of the blood capillary wall.

42. Hemopoietic stem cell (HSP) and Hemopoietic progenitor cell (HPC) Hemopoietic stem cell self-replication powerful proliferation potency multi-differentiation Potency Mark:CD34, CDw90 Hemopoietic progenitor cell Unipotential or bipotential Produce mature blood cells Erythrocytes granulocyte series monocyte series megakaryocyteseries * Lymphocytes derived from lymphatic organs & tissues

44. Morphologic criteria of haemopoiesis ① Stages: the original stage the infant stage the mature stage② Cell volume:↓, megakaryocytes:↑ ③ cytoplasm: ↑，ratio of Nucleus / cytoplasm:↓

45. ④ Nucleus: size↓(RBC: abesent; megakaryocytes↑） nucleoli: number & size↓, until disappear chromatin: fine & scanty → coarse & dense; 　staining: pale → deep ⑤ Specific granules: in granulocytes & Hbin erythrocytes:↑ other organelles in red cell↓until disappear ⑥ mitotic ability:↓, until disappear（but lymphocyte)

46. proerythroblast prorubricyte orthochromatic erythroblast polychromatophilic erythroblast

47. myeloblast promyelocyte Mb M,Ma,Mb: myelocyte M1： metamyelocyte Neutrophilic series