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C H A P T E R COMPOSITION OF PLASMA 12 237 Blood SUMMARY OF KEY POINTS...

C H A P T E R COMPOSITION OF PLASMA 12 237 Blood SUMMARY OF KEY POINTS 250 BLOOD CELLS 239 ASSESS YOUR KNOWLEDGE 252 Erythrocytes 239 Leukocytes 241 Platelets 247 B lood is a specialized connective tissue consisting of cells and fluid extracellular material called plasma. Propelled mainly by rhythmic contractions of the heart, about 5 L of blood in an average adult moves unidirec- tionally within the closed circulatory system. The so-called gut, while metabolic residues are collected from cells through- out the body and removed from the blood by the excretory organs. Hormone distribution in blood permits the exchange of chemical messages between distant organs regulating nor- mal organ function. Blood also participates in heat distribu- formed elements circulating in the plasma are erythrocytes tion, the regulation of body temperature, and the maintenance (red blood cells), leukocytes (white blood cells [WBCs]), and of acid-base and osmotic balance. platelets. Leukocytes have diverse functions and are one of the When blood leaves the circulatory system, either in a body’s chief defenses against infection. These cells are gen- test tube or in the extracellular matrix (ECM) surrounding erally spherical and inactive while suspended in circulating blood vessels, plasma proteins react with one another to pro- blood, but when called to sites of infection or inflammation, duce a clot, which includes formed elements and a pale yel- they cross the wall of venules, become motile and migrate into low liquid called serum. Serum contains growth factors and the tissues, and display their defensive capabilities. other proteins released from platelets during clot formation, which confer biological properties very different from those of plasma. Collected blood in which clotting is prevented by the ››COMPOSITION OF PLASMA addition of anticoagulants (eg, heparin or citrate) can be sepa- Plasma is an aqueous solution, pH 7.4, containing sub- rated by centrifugation into layers that reflect its heterogeneity stances of low or high molecular weight that make up (Figure 12–1). Erythrocytes comprise the sedimented mate- 7% of its volume. As summarized in Table 12–1, the dis- rial, and their volume, normally about 44% of the total blood solved components are mostly plasma proteins, but they volume in healthy adults, is called the hematocrit. also include nutrients, respiratory gases, nitrogenous The straw-colored, translucent, slightly viscous superna- waste products, hormones, and inorganic ions collec- tant comprising 55% at the top half of the centrifugation tube tively called electrolytes. Through the capillary walls, is the plasma. A thin gray-white layer called the buffy coat the low-molecular-weight components of plasma are in between the plasma and the hematocrit, about 1% of the vol- equilibrium with the interstitial fluid of the tissues. The ume, consists of leukocytes and platelets, both less dense than composition of plasma is usually an indicator of the mean erythrocytes. composition of the extracellular fluids in tissues. Blood is a distributing vehicle, transporting O2, CO2, The major plasma proteins include the following: metabolites, hormones, and other substances to cells through- Albumin, the most abundant plasma protein, is made out the body. Most O2 is bound to hemoglobin in erythrocytes in the liver and serves primarily to maintain the osmotic and is much more abundant in arterial than venous blood pressure of the blood. (Figure 12–2), while CO2 is carried in solution as CO2 or Globulins (α- and β-globulins), made by the liver and HCO3–, in addition to being hemoglobin-bound. Nutrients are other cells, include transferrin and other transport fac- distributed from their sites of synthesis or absorption in the tors; fibronectin; prothrombin and other coagulation 237  238 CHAPTER 12 Blood FIGURE 12–1 Composition of whole blood. Plasma (55% of whole blood) Buffy coat ( 2% dissolved in plasma, cellular respiration; carbon Symptoms of anemia include lethargy, shortness of breath, 98% bound to hemoglobin dioxide is a waste product fatigue, skin pallor, and heart palpitations. Sickle cell anemia within erythrocytes; and produced by cells during this carbon dioxide: ~7% process is caused by a homozygous mutation causing an amino acid dissolved in plasma, ~27% substitution in hemoglobin, which renders the mature RBCs bound to hemoglobin within deformed and slightly rigid (Figure 12–5) and can lead to erythrocytes, ~66% converted capillary blockage. to HCO3−) An increased concentration of erythrocytes in blood Wastes (breakdown products Waste products serve no (erythrocytosis or polycythemia) may be a physiologic of metabolism) (eg, lactic acid, function in the blood plasma; adaptation found, for example, in individuals who live at creatinine, urea, bilirubin, they are merely being high altitudes, where O2 tension is low. Elevated hematocrit ammonia) transported to the liver and kidneys where they can be increases blood viscosity, putting strain on the heart, and, if removed from the blood severe, can impair circulation through the capillaries. 12_Mescher_ch12_p237-253.indd 239 26/04/18 11:22 am  240 CHAPTER 12 Blood FIGURE 12–3 Preparing a blood smear. Lymphocyte Erythrocytes Neutrophil Withdraw blood Stain LM 640x Monocytes Platelets 1 Prick finger and collect 2 Place a drop of blood 3a Using a second slide, pull the 4 When viewed under the microscope, a small amount of blood on a slide. drop of blood across the first blood smear reveals the components using a micropipette. slide’s surface, leaving a thin of the formed elements. layer of blood on the slide. 3b After the blood dries, apply a stain briefly and rinse. Place a coverslip on top. Human erythrocytes suspended in an isotonic medium The biconcave shape provides a large surface-to-volume are flexible biconcave discs (Figure 12–4). They are approxi- ratio and facilitates gas exchange. The normal concentration mately 7.5 μm in diameter, 2.6-μm thick at the rim, but only of erythrocytes in blood is approximately 3.9-5.5 million per 0.75-μm thick in the center. Because of their uniform dimen- microliter (μL, or mm3) in women and 4.1-6.0 million/μL sions and their presence in most tissue sections, RBCs can in men. often be used by histologists as an internal standard to esti- Erythrocytes are normally quite flexible, which permits mate the size of other nearby cells or structures. them to bend and adapt to the small diameters and irregular FIGURE 12–4 Normal human erythrocytes. Sectional view ~.75 µm ~2.6 µm b ~7.5 µm a c Rouleaux Erythrocytes (a) Colorized SEM micrograph of normal erythrocytes with each transport. Erythrocytes are also quite flexible and can easily bend side concave. (X1800) to pass through small capillaries. (b) Diagram of an erythrocyte giving the cell’s dimensions. The (c) In small vessels red blood cells also often stack up in loose biconcave shape gives the cells a very high surface-to-volume aggregates called rouleaux. The standard size of RBCs allows ratio and places most hemoglobin within a short distance from one to estimate that the vessel seen is approximately 15 mm in the cell surface, both qualities that provide maximally efficient O2 diameter. (X250; H&E) 12_Mescher_ch12_p237-253.indd 240 26/04/18 11:22 am  Blood Cells 241 turns of capillaries. Observations in vivo show that at the Granulocytes possess two major types of abundant angles of capillary bifurcations, erythrocytes with normal cytoplasmic granules: lysosomes (often called azurophilic C H A P T E R adult hemoglobin frequently assume a cuplike shape. In larger granules in blood cells) and specific granules that bind blood vessels RBCs may adhere to one another loosely in neutral, basic, or acidic stains and have specific functions. stacks called rouleaux (Figure 12–4c). Granulocytes also have polymorphic nuclei with two The erythrocyte plasmalemma, because of its ready or more distinct (almost separated) lobes and include the availability, is the best-known membrane of any cell. It con- neutrophils, eosinophils, and basophils (Figure 12–1 and sists of about 40% lipid, 10% carbohydrate, and 50% protein. Table 12–2). All granulocytes are also terminally differentiated 1 2 Most of the latter are integral membrane proteins (see cells with a life span of only a few days. Their Golgi complexes Chapter 2), including ion channels, the anion transporter and rough ER are poorly developed, and with few mitochon- called band 3 protein, and glycophorin A. The glycosyl- dria they depend largely on glycolysis for their energy needs. Blood Blood Cells ated extracellular domains of the latter proteins include Most granulocytes undergo apoptosis in the connective tissue antigenic sites that form the basis for the ABO blood typ- and billions of neutrophils alone die each day in adults. The ing system. Several peripheral proteins are associated with resulting cellular debris is removed by macrophages and, like the inner surface of the membrane, including spectrin, all apoptotic cell death, does not itself elicit an inflammatory dimers of which form a lattice bound to underlying actin response. filaments, and ankyrin, which anchors the spectrin lattice Agranulocytes lack specific granules, but do con- to the glycophorins and band 3 proteins. This submembra- tain some azurophilic granules (lysosomes). The nucleus nous meshwork stabilizes the membrane, maintains the cell is spherical or indented but not lobulated. This group shape, and provides the cell elasticity required for passage includes the lymphocytes and monocytes (Figure 12–1 through capillaries. and Table 12–2). The differential count (percentage of all Erythrocyte cytoplasm lacks all organelles but is densely leukocytes) for each type of leukocyte is also presented in filled with hemoglobin, the tetrameric O2-carrying protein Table 12–2. that accounts for the cells’ uniform acidophilia. When com- All leukocytes are key players in the constant defense bined with O2 or CO2, hemoglobin forms oxyhemoglobin or against invading microorganisms and in the repair of carbaminohemoglobin, respectively. The reversibility of these injured tissues, specifically leaving the microvasculature combinations is the basis for the protein’s gas-transporting in injured or infected tissues. At such sites factors termed capacity. cytokines are released from various sources and these trig- Erythrocytes undergo terminal differentiation (discussed ger loosening of intercellular junctions in the endothelial in Chapter 13) which includes loss of the nucleus and organ- cells of local postcapillary venules (Figure 12–6). Simultane- elles shortly before the cells are released by bone marrow into ously the cell adhesion protein P-selectin appears on the the circulation. Lacking mitochondria, erythrocytes rely on endothelial cells’ luminal surfaces following exocytosis from anaerobic glycolysis for their minimal energy needs. Lacking cytoplasmic Weibel-Palade bodies. The surfaces of neutro- nuclei, they cannot replace defective proteins. phils and other leukocytes display glycosylated ligands for Human erythrocytes normally survive in the circulation P-selectin, and their interactions cause cells flowing through for about 120 days. By this time defects in the membrane’s the affected venules to slow down, like rolling tennis balls cytoskeletal lattice or ion transport systems begin to produce arriving at a patch of velcro. Other cytokines stimulate the swelling or other shape abnormalities, as well as changes in now slowly rolling leukocytes to express integrins and other the cells’ surface oligosaccharide complexes. Senescent or adhesion factors that produce firm attachment to the endo- worn-out RBCs displaying such changes are recognized and thelium (see Figure 11–21d). In a process called diapedesis removed from circulation, mainly by macrophages of the (Gr. dia, through + pedesis, to leap), the leukocytes send spleen, liver, and bone marrow. extensions through the openings between the endothelial cells, migrate out of the venules into the surrounding tissue space, and head directly for the site of injury or invasion. Leukocytes The attraction of neutrophils to bacteria involves chemical Leukocytes (WBCs) leave the blood and migrate to the tissues mediators in a process of chemotaxis, which causes leuko- where they become functional and perform various activities cytes to rapidly accumulate where their defensive actions are related to immunity. Leukocytes are divided into two major specifically needed. groups, granulocytes and agranulocytes, based on the The number of leukocytes in the blood varies according density of their cytoplasmic granules (Table 12–2). All are to age, sex, and physiologic conditions. Healthy adults have rather spherical while suspended in blood plasma, but they 4500-11,000 leukocytes per microliter of blood. become amoeboid and motile after leaving the blood vessels and invading the tissues. Their estimated sizes mentioned here refer to observations in blood smears in which the cells Neutrophils (Polymorphonuclear Leukocytes) are spread and appear slightly larger than they are in the Mature neutrophils constitute 50%-70% of circulating leuko- circulation. cytes, a figure that includes slightly immature forms released to 12_Mescher_ch12_p237-253.indd 241 26/04/18 11:22 am  242 CHAPTER 12 Blood      TABLE 12–2 Leukocytes: Numbers, structural features, and major functions. Eosinophil Neutrophil Basophil Granulocytes Agranulocytes Lymphocyte Monocyte Differential Type Nucleus Specific Granulesa Countb (%) Life Span Major Functions Granulocytes Neutrophils 3-5 lobes Faint/light pink 50-70 1-4 d Kill and phagocytose bacteria Eosinophils Bilobed Red/dark pink 1-4 1-2 wk Kill helminthic and other parasites; modulate local inflammation Basophils Bilobed or S-shaped Dark blue/purple 0.5-1 Several months Modulate inflammation, release histamine during allergy Agranulocytes Lymphocytes Rather spherical (none) 20-40 Hours to many years Effector and regulatory cells for adaptive immunity Monocytes Indented or C-shaped (none) 2-8 Hours to years Precursors of macrophages and other mononuclear phagocytic cells a Color with routine blood smear stains. There are typically 4500-11,000 total leukocytes/µL of blood in adults, higher in infants and young children. b The percentage ranges given for each type of leukocyte are those used by the US National Board of Medical Examiners. The value for neutrophils includes 3%-5% circulating, immature band forms. All micrographs X1600. 12_Mescher_ch12_p237-253.indd 242 26/04/18 11:22 am  Blood Cells 243 FIGURE 12–5 Sickle cell erythrocyte. › ›› MEDICAL APPLICATION C H A P T E R Several kinds of neutrophil defects, often genetic in origin, can affect function of these cells, for example, by decreas- ing adhesion to the wall of venules, by causing the absence of specific granules, or with deficits in certain factors of the azurophilic granules. Individuals with such disorders typi- cally experience more frequent and more persistent bacterial 1 2 infections, although macrophages and other leukocytes may substitute for certain neutrophil functions. Blood Blood Cells Specific secondary granules are smaller and less dense, stain faintly pink, and have diverse functions, including secre- tion of various ECM-degrading enzymes such as collagenases, delivery of additional bactericidal proteins to phagolysosomes, and insertion of new cell membrane components. Activated neutrophils at infected or injured sites also have important roles in the inflammatory response, which begins the process of restoring the normal tissue microenvironment. They release many polypeptide chemokines that attract other leukocytes and cytokines, which direct activities of these and A single nucleotide substitute in the hemoglobin gene produces local cells of the tissue. Important lipid mediators of inflam- a version of the protein that polymerizes to form rigid aggre- gates, leading to greatly misshapen cells with reduced flexibility. mation are also released from neutrophils. In individuals homozygous for the mutated HbS gene, this can Neutrophils contain glycogen, which is broken down into lead to greater blood viscosity and poor microvascular circula- glucose to yield energy via the glycolytic pathway. The citric tion, both features of sickle cell disease. (X6500) acid cycle is less important, as might be expected in view of the paucity of mitochondria in these cells. The ability of neu- trophils to survive in an anaerobic environment is highly advantageous, because they can kill bacteria and help clean up the circulation. Neutrophils are 12-15 μm in diameter in blood debris in poorly oxygenated regions, for example, damaged or smears, with nuclei having two to five lobes linked by thin necrotic tissue lacking normal microvasculature. nuclear extensions (see Table 12–2; Figure 12–7). In females, Neutrophils are short-lived cells with a half-life of the inactive X chromosome may appear as a drumstick-like 6-8 hours in blood and a life span of 1-4 days in connective appendage on one of the lobes of the nucleus (Figure 12–7c) tissues before dying by apoptosis. although this characteristic is not always seen. Neutrophils are inactive and spherical while circulating but become amoeboid and highly active during diapedesis and upon adhering to › ›› MEDICAL APPLICATION ECM substrates such as collagen. Neutrophils look for bacteria to engulf by pseudopodia and Neutrophils are usually the first leukocytes to arrive at internalize them in vacuoles called phagosomes. Immedi- sites of infection where they actively pursue bacterial cells ately thereafter, specific granules fuse with and discharge using chemotaxis and remove the invaders or their debris by their contents into the phagosomes that are then acidified phagocytosis. by proton pumps. Azurophilic granules then discharge their The cytoplasmic granules of neutrophils provide the cells’ enzymes into this acidified vesicle, killing and digesting the functional activities and are of two main types (Figure 12–8). engulfed microorganisms. Azurophilic primary granules or lysosomes are large, During phagocytosis, a burst of O2 consumption leads dense vesicles with a major role in both killing and degrading to the formation of superoxide anions (O2–) and hydrogen engulfed microorganisms. They contain proteases and anti- peroxide (H2O2). O2– is a short-lived, highly reactive free radi- bacterial proteins, including the following: cal that, together with MPO and halide ions, forms a powerful microbial killing system inside the neutrophils. Besides the Myeloperoxidase (MPO), which generates hypochlo- activity of lysozyme cleaving cell wall peptidoglycans to kill rite and other agents toxic to bacteria certain bacteria, the protein lactoferrin avidly binds iron, a Lysozyme, which degrades components of bacterial cell crucial element in bacterial nutrition whose lack of availability walls then causes bacteria to die. A combination of these mecha- Defensins, small cysteine-rich proteins that bind and nisms will kill most microorganisms, which are then digested disrupt the cell membranes of many types of bacteria by lysosomal enzymes. Apoptotic neutrophils, bacteria, and other microorganisms. 12_Mescher_ch12_p237-253.indd 243 26/04/18 11:22 am  244 CHAPTER 12 Blood FIGURE 12–6 Diagram of events involving leukocytes in a postcapillary venule at sites of inflammation. Endothelial cells Selectin ligands Integrins Neutrophil 3 Lumen of venule 2 Selectins 4 Cytokines (IL-1 & TNF-α) 5 1 Integrin receptors (ICAM-1) Interstitial space in connective tissue Activated macrophage Locations in connective tissue with injuries or infection require 3. Exposure to these and other cytokines causes expression of new the rapid immigration of various leukocytes to initiate cellular integrins on the rolling leukocytes and expression of the inte- events for tissue repair and removal of the invading microorgan- grin ligand ICAM-1 (intercellular adhesion molecule-1) on the isms. The cytokines and cell binding proteins target various leuko- endothelial cells. Junctional complexes between the endothelial cytes and are best known for neutrophils. The major initial events cells are selectively downregulated, loosening these cells. of neutrophil migration during inflammation are summarized 4. Integrins and their ligands provide firm endothelial adhesion here: of neutrophils to the endothelium, allowing the leukocytes to 1. Local macrophages activated by bacteria or tissue damage receive further stimulation from the local cytokines. release proinflammatory cytokines such as interleukin-1 (IL-1) 5. Neutrophils become motile, probe the endothelium with pseu- or tumor necrosis factor-α (TNF-α) that signal endothelial cells of dopodia, and, being attracted by other local injury-related fac- nearby postcapillary venules to rapidly insert glycoprotein selec- tors called chemokines, finally migrate by diapedesis between tins on the luminal cell surfaces. the loosened cells of the venule. Rapid transendothelial migra- 2. Passing neutrophils with appropriate cell surface glycoproteins tion of neutrophils is facilitated by the cells’ elongated and seg- bind the selectins, which causes such cells to adhere loosely to mented nuclei. All leukocytes first become functional in the ECM the endothelium and “roll” slowly along its surface. after emerging from the circulation by this process. main identifying characteristic is the abundance of large, aci- semidigested material, and tissue-fluid form a viscous, usually dophilic specific granules typically staining pink or red. yellow collection of fluid called pus. Ultrastructurally the eosinophilic-specific granules Several neutrophil hereditary dysfunctions have been are seen to be oval in shape, with flattened crystalloid cores described. In one of them, actin does not polymerize nor- (Figure 12–9c) containing major basic proteins (MBP), an mally, reducing neutrophil motility. With an NADPH oxidase arginine-rich factor that accounts for the granule’s acidophilia deficiency, there is a failure to produce H2O2 and hypochlo- and constitutes up to 50% of the total granule protein. MBPs, rite, reducing the cells’ microbial killing power. Children with along with eosinophilic peroxidase, other enzymes and tox- such dysfunctions can experience more persistent bacterial ins, act to kill parasitic worms or helminths. Eosinophils also infections. modulate inflammatory responses by releasing chemokines, cytokines, and lipid mediators, with an important role in the inflammatory response triggered by allergies. The number of circulating eosinophils increases during helminthic infections Eosinophils and allergic reactions. These leukocytes also remove antigen- Eosinophils are far less numerous than neutrophils, consti- antibody complexes from interstitial fluid by phagocytosis. tuting only 1%-4% of leukocytes. In blood smears, this cell is Eosinophils are particularly abundant in connective tis- about the same size as a neutrophil or slightly larger, but with sue of the intestinal lining and at sites of chronic inflamma- a characteristic bilobed nucleus (Table 12–2; Figure 12–9). The tion, such as lung tissues of asthma patients. 12_Mescher_ch12_p237-253.indd 244 26/04/18 11:22 am  Blood Cells 245 two irregular lobes, but the large specific granules overlying FIGURE 12–7 Neutrophils. the nucleus usually obscure its shape. C H A P T E R The specific granules (0.5 μm in diameter) typically stain purple with the basic dye of blood smear stains and are fewer, larger, and more irregularly shaped than the granules of other granulocytes (see Table 12–2; Figure 12–10). The strong baso- philia of the granules is due to the presence of heparin and other sulfated GAGs. Basophilic-specific granules also contain 1 2 much histamine and various other mediators of inflamma- tion, including platelet activating factor, eosinophil chemotac- tic factor, and the enzyme phospholipase A that catalyzes an Blood Blood Cells b initial step in producing lipid-derived proinflammatory fac- tors called leukotrienes. By migrating into connective tissues, basophils appear to supplement the functions of mast cells, which are described in Chapter 5. Both basophils and mast cells have metachro- matic granules containing heparin and histamine, have sur- face receptors for immunoglobulin E (IgE), and secrete a c their granular components in response to certain antigens and allergens. (a) In blood smears neutrophils can be identified by their multi- lobulated nuclei, with lobules held together by very thin strands. With this feature, the cells are often called polymorphonuclear › ›› MEDICAL APPLICATION leukocytes, PMNs, or just polymorphs. The cells are dynamic In some individuals a second exposure to a strong allergen, and the nuclear shape changes frequently. (X1500; Giemsa) such as that delivered in a bee sting, may produce an intense, (b) Neutrophils typically have diameters ranging from 12 to adverse systemic response. Basophils and mast cells may rap- 15 μm, approximately twice that of the surrounding erythro- idly degranulate, producing vasodilation in many organs, a cytes. The cytoplasmic granules are relatively sparse and have sudden drop in blood pressure, and other effects comprising heterogeneous staining properties, although generally pale and a potentially lethal condition called anaphylaxis or anaphy- not obscuring the nucleus. (X1500; Giemsa) lactic shock. (c) Micrograph showing a neutrophil from a female in which the condensed X chromosome appears as a drumstick appendage Basophils and mast cells also are central to immediate to a nuclear lobe (arrow). (X1500; Wright) or type 1 hypersensitivity. In some individuals substances such as certain pollen proteins or specific proteins in food are allergenic, that is, elicit production of specific IgE antibodies, which then bind to receptors on mast cells and immigrating basophils. Upon subsequent exposure, the allergen com- › ›› MEDICAL APPLICATION bines with the receptor-bound IgE molecules, causing them An increase in the number of eosinophils in blood (eosino- to cross-link and aggregate on the cell surfaces and trigger- philia) is associated with allergic reactions and helminthic ing rapid exocytosis of the cytoplasmic granules. Release infections. In patients with such conditions, eosinophils are of the inflammatory mediators in this manner can result in found in the connective tissues underlying epithelia of the bronchial asthma, cutaneous hives, rhinitis, conjunctivitis, bronchi, gastrointestinal tract, uterus, and vagina, and sur- or allergic gastroenteritis. rounding any parasitic worms present. In addition, these cells produce substances that modulate inflammation by inac- tivating the leukotrienes and histamine produced by other Lymphocytes cells. Corticosteroids (hormones from the adrenal cortex) By far the most numerous type of agranulocyte in normal produce a rapid decrease in the number of blood eosinophils, blood smears, lymphocytes constitute a family of leukocytes probably by interfering with their release from the bone mar- with spherical nuclei (Table 12–2; Figure 12–11). Lympho- row into the bloodstream. cytes are typically the smallest leukocytes and constitute approximately one-third of these cells. Although they are morphologically similar, mature lymphocytes can be subdi- Basophils vided into functional groups by distinctive surface molecules Basophils are also 12-15 μm in diameter but make up less (called “cluster of differentiation” or CD markers) that can be than 1% of circulating leukocytes and are therefore difficult distinguished using antibodies with immunocytochemistry or to find in normal blood smears. The nucleus is divided into flow cytometry. Major classes include B lymphocytes, helper 12_Mescher_ch12_p237-253.indd 245 26/04/18 11:22 am  246 CHAPTER 12 Blood FIGURE 12–8 Neutrophil ultrastructure. A S N G N A TEM of a sectioned human neutrophil reveals the two types of Azurophilic granules are modified lysosomes with components to cytoplasmic granules: the small, pale, more variably stained specific kill engulfed bacteria. granules (S) and the larger, electron-dense azurophilic granules (A). The nucleus (N) is lobulated and the central Golgi apparatus (G) Specific granules undergo exocytosis during and after dia- is small. Rough ER and mitochondria are not abundant, because pedesis, releasing many factors with various activities, including this cell utilizes glycolysis and is in the terminal stage of its differen- enzymes to digest ECM components and bactericidal factors. tiation. (X25,000) and cytotoxic T lymphocytes (CD4+ and CD8+, respectively), indented nuclei and more cytoplasm that is slightly baso- and natural killer (NK) cells. These and other types of lym- philic, with a few azurophilic granules, mitochondria, free phocytes have diverse roles in immune defenses against invad- polysomes, and other organelles (Figure 12–11d). ing microorganisms and certain parasites or abnormal cells. Lymphocytes vary in life span according to their specific T lymphocytes, unlike B cells and all other circulating leuko- functions; some live only a few days and others survive in the cytes, differentiate outside the bone marrow in the thymus. circulating blood or other tissues for many years. Functions and formation of lymphocytes are discussed with the immune system in Chapter 14. Although generally small, circulating lymphocytes have › ›› MEDICAL APPLICATION a wider range of sizes than most leukocytes. Small, newly Given their central roles in immunity, lymphocytes are released lymphocytes have diameters similar to those of RBCs; obviously important in many diseases. Lymphomas are a medium and large lymphocytes are 9-18 μm in diameter, with group of disorders involving neoplastic proliferation of lym- the latter representing activated lymphocytes or NK cells. The phocytes or the failure of these cells to undergo apoptosis. small lymphocytes are characterized by spherical nuclei with Although often slow-growing, all lymphomas are considered highly condensed chromatin and only a thin surrounding malignant because they can very easily become widely rim of scant cytoplasm, making them easily distinguishable spread throughout the body. from granulocytes. Larger lymphocytes have larger, slightly 12_Mescher_ch12_p237-253.indd 246 26/04/18 11:22 am  Blood Cells 247 FIGURE 12–9 Eosinophils. C H A P T E R EG E 1 2 N Blood Blood Cells a L N M b c Eosinophils are about the same size as neutrophils but have (c) Ultrastructurally a sectioned eosinophil clearly shows the bilobed nuclei and more abundant coarse cytoplasmic granules. unique specific eosinophilic granules (EG), as oval structures with The cytoplasm is often filled with brightly eosinophilic specific disc-shaped electron-dense, crystalline cores. These granules, granules, but it also includes some azurophilic granules. (a) Micro- along with a few lysosomes and mitochondria (M), fill the cyto- graph shows an eosinophil (E) next to a neutrophil (N) and a small plasm around the bilobed nucleus (N). (X20,000) lymphocyte (L). (X1500; Wright) (b) Even with granules filling the cytoplasm, the two nuclear lobes of eosinophils are usually clear. (X1500; Giemsa) Monocytes rough ER are present, along with a Golgi apparatus involved in Monocytes are precursor cells of macrophages, osteoclasts, the formation of lysosomes (Figure 12–12e). microglia, and other cells of the mononuclear phago- cyte system in connective tissue of nearly all organs (see › ›› MEDICAL APPLICATION Chapter 5). Circulating monocytes have diameters of 12-15 μm Extravasation or the accumulation of immigrating monocytes and have nuclei that are large and usually distinctly indented occurs in the early phase of inflammation following tissue injury. or C-shaped (Figure 12–12). The chromatin is less condensed Acute inflammation is usually short-lived as macrophages than in lymphocytes and typically stains lighter than that of undergo apoptosis or leave the site, but chronic inflammation large lymphocytes. Cells of the mononuclear phagocyte sys- usually involves the continued recruitment of monocytes. The tem arise in developing organs from monocytes formed in the resulting continuous presence of macrophages can lead to embryonic yolk sac and are supplemented throughout life by excessive tissue damage that is typical of chronic inflammation. monocytes from the bone marrow. All monocyte-derived cells are antigen-presenting cells with important roles in immune defense as well as tissue repair. Platelets The cytoplasm of the monocyte is basophilic and contains Blood platelets (or thrombocytes) are very small non-nucleated, many small lysosomal azurophilic granules, some of which are membrane-bound cell fragments only 2-4 μm in diameter at the limit of the light microscope’s resolution. These gran- (Figure 12–13a). As described in Chapter 13, platelets origi- ules are distributed through the cytoplasm, giving it a bluish- nate by separation from the ends of cytoplasmic processes gray color in stained smears. Mitochondria and small areas of extending from giant polyploid bone marrow cells called 12_Mescher_ch12_p237-253.indd 247 26/04/18 11:22 am  248 CHAPTER 12 Blood FIGURE 12–10 Basophils. a B B N b N c d (a-c) Basophils are also approximately the same size as neutrophils (d) A TEM of a sectioned basophil reveals the single bilobed nucleus and eosinophils, but they have large, strongly basophilic specific (N) and the large, electron-dense specific basophilic granules (B). granules that usually obstruct the appearance of the nucleus Basophils exert many activities modulating the immune response and which usually has two large irregular lobes. (a and b: X1500, Wright; inflammation and have many functional similarities with mast cells, c: X1500, Giemsa) which are normal, longer-term residents of connective tissue. (X25,000) megakaryocytes. Platelets promote blood clotting and help adhesion to collagen or other substrates outside the vascular repair minor tears or leaks in the walls of small blood vessels, endothelium. preventing loss of blood from the microvasculature. Normal Besides specific granules, the central granulomere has platelet counts range from 150,000 to 400,000/μL (mm3) of a sparse population of mitochondria and glycogen parti- blood. Circulating platelets have a life span of about 10 days. cles (Figure 12–13b). Electron-dense delta granules (δG), In stained blood smears, platelets often appear in clumps. 250-300 nm in diameter, contain ADP, ATP, and serotonin Each individual platelet is generally discoid, with a very lightly (5-hydroxytryptamine) taken up from plasma. Alpha granules stained peripheral zone, the hyalomere, and a darker-staining (αG) are larger (300-500 nm in diameter) and contain platelet- central zone rich in granules, called the granulomere. A derived growth factor (PDGF), platelet factor 4, and several sparse glycocalyx surrounding the platelet plasmalemma is other platelet-specific proteins. Most of the stained granules involved in adhesion and activation during blood coagulation. seen in platelets with the light microscope are alpha granules. Ultrastructural analysis (Figure 12–13b) reveals a The role of platelets in controlling blood loss (hemor- peripheral marginal bundle of microtubules and microfila- rhage) and in wound healing can be summarized as follows: ments, which helps to maintain the platelet’s shape. Also in the hyalomere are two systems of membrane channels. An Primary aggregation: Disruptions in the microvas- open canalicular system of vesicles is connected to invagi- cular endothelium, which are very common, allow the nations of the plasma membrane, which may facilitate plate- platelet glycocalyx to adhere to collagen in the vascular lets’ uptake of factors from plasma. A much less prominent basal lamina or wall. Thus, a platelet plug is formed as set of irregular tubular vesicles comprising the dense tubular a first step to stop bleeding (Figure 12–14). system is derived from the ER and stores Ca2+ ions. Together, Secondary aggregation: Platelets in the plug release a spe- these two membranous systems facilitate the extremely rapid cific adhesive glycoprotein and ADP, which induce further exocytosis of proteins from platelets (degranulation) upon platelet aggregation and increase the size of the platelet plug. 12_Mescher_ch12_p237-253.indd 248 26/04/18 11:22 am  Blood Cells 249 FIGURE 12–11 Lymphocytes. C H A P T E R 1 2 M Blood Blood Cells a M N M b c d Lymphocytes are agranulocytes and lack the specific granules (b) Medium lymphocytes are distinctly larger than erythrocytes. characteristic of granulocytes. Lymphocytes circulating in blood (X1500; Wright) generally range in size from 6 to 15 μm in diameter and are some- (c) Large lymphocytes, much larger than erythrocytes, may represent times classified arbitrarily as small, medium, and large. activated cells that have returned to the circulation. (X1500; Giemsa) (a) The most numerous small lymphocytes shown here are (d) Ultrastructurally a medium-sized lymphocytes is seen to be slightly larger than the neighboring erythrocytes and have only a mostly filled with a euchromatic nucleus (N) surrounded by cyto- thin rim of cytoplasm surrounding the spherical nucleus. (X1500; plasm containing mitochondria (M), free polysomes, and a few Giemsa) dark lysosomes (azurophilic granules). (X22,000) Blood coagulation: During platelet aggregation, enzyme plasmin, which is formed continuously through fibrinogen from plasma, von Willebrand factor the local action of plasminogen activators from the and other proteins released from the damaged endo- endothelium on plasminogen from plasma. thelium, and platelet factor 4 from platelet granules promote the sequential interaction (cascade) of plasma proteins, giving rise to a fibrin polymer that forms a › ›› MEDICAL APPLICATION three-dimensional network of fibers trapping RBCs Aspirin and other nonsteroidal anti-inflammatory agents and more platelets to form a blood clot, or thrombus have an inhibitory effect on platelet function and blood (Figure 12–14). Platelet factor 4 is a chemokine for coagulation because they block the local prostaglandin monocytes, neutrophils, and fibroblasts, and prolifera- synthesis, which is needed for platelet aggregation, contrac- tion of the fibroblasts is stimulated by PDGF. tion, and exocytosis at sites of injury. Bleeding disorders Clot retraction: The clot initially bulges into the blood result from abnormally slow blood clotting. One such disease vessel lumen, but soon contracts slightly due to the activ- directly related to a defect in the platelets is a rare autosomal ity of platelet-derived actin and myosin. recessive glycoprotein Ib deficiency, involving a factor on Clot removal: Protected by the clot, the endothelium the platelet surface needed to bind subendothelial collagen and surrounding tunic are restored by new tissue, and the and begin the cascade of events leading to clot formation. clot is then removed, mainly dissolved by the proteolytic 12_Mescher_ch12_p237-253.indd 249 26/04/18 11:22 am  250 CHAPTER 12 Blood FIGURE 12–12 Monocytes. A R R M M a M A G b c M A L d e Monocytes are large agranulocytes with diameters from 12 to apparatus (G), mitochondria (M), and lysosomes or azurophilic 20 μm that circulate as precursors to macrophages and other cells granules (A). Rough ER is poorly developed and there are some of the mononuclear phagocyte system. free polysomes (R). (X22,000) (a-d) Micrographs of monocytes showing their distinctive nuclei (Figure 12-12e, used with permission from D. F. Bainton and which are indented, kidney-shaped, or C-shaped. (a: X1500, M. G. Farquhar, Department of Pathology, University of California, Giemsa; b-d: X1500, Wright) San Francisco, CA.) (e) Ultrastructurally the cytoplasm of a monocyte shows a Golgi Blood SUMMARY OF KEY POINTS The liquid portion of circulating blood is plasma, while the cells transport, and release of O2, and with a normal life span of about and platelets comprise the formed elements; upon clotting, some 120 days. proteins are removed from plasma and others are released from WBCs or leukocytes are broadly grouped as granulocytes platelets, forming a new liquid termed serum. (neutrophils, eosinophils, basophils) or agranulocytes (lympho- Important protein components of plasma include albumin, cytes, monocytes). diverse α- and β-globulins, proteins of the complement system, All leukocytes become active outside the circulation, specifically and fibrinogen, all of which are secreted within the liver, as well as leaving the microvasculature in a process involving cytokines, selec- the immunoglobulins. tive adhesion, changes in the endothelium, and transendothelial RBCs or erythrocytes, which make up the hematocrit por- migration or diapedesis. tion (~45%) of a blood sample, are enucleated, biconcave discs All granulocytes have specialized lysosomes called azurophilic 7.5 μm in diameter, filled with hemoglobin for the uptake, granules and smaller specific granules with proteins for various cell-specific functions. 12_Mescher_ch12_p237-253.indd 250 26/04/18 11:22 am  Blood Cells 251 FIGURE 12–13 Platelets. C H A P T E R 1 2 δG Blood Blood Cells αG G a OCS MB b Platelets are cell fragments 2-4 μm in diameter derived from (b) Ultrastructurally a platelet shows a system of microtubules and megakaryocytes of bone marrow. Their primary function is to actin filaments near the periphery, called the marginal bundle rapidly release the content of their granules upon contact with (MB), which is formed as the platelet pinches off from megakaryo- collagen (or other materials outside of the endothelium) to begin cyte (Chapter 13), and helps maintain its shape. An open canalicu- the process of clot formation and reduce blood loss from the lar system (OCS) of invaginating membrane vesicles continuous vasculature. with the plasmalemma facilitates rapid degranulation upon acti- (a) In a blood smear, platelets (arrows) are often found as aggre- vation and Ca2+ release. The central granulomere region contains gates. Individually they show a lightly stained hyalomere region small dense delta granules (δG), larger and more numerous alpha surrounding a more darkly stained central granulomere contain- granules (αG), and glycogen (G). (X40,000) ing membrane-enclosed granules. (X1500; Wright) (Figure 12-13b, used with permission from Dr M. J. G. Harrison, Middlesex Hospital and University College London, UK.) Neutrophils, the most abundant type of leukocyte, have polymor- Lymphocytes, agranulocytes with many functions as T- and B-cell phic, multilobed nuclei, and faint pink cytoplasmic granules that subtypes in the immune system, range widely in size, depending on contain many factors for highly efficient phagolysosomal killing their activation state, and have roughly spherical nuclei with little and removal of bacteria. cytoplasm and few organelles. Eosinophils have bilobed nuclei and eosinophilic-specific gran- Monocytes are larger agranulocytes with distinctly indented or ules containing factors for destruction of helminthic parasites and C-shaped nuclei, which circulate as precursors of macrophages and for modulating inflammation. other cells of the mononuclear phagocyte system. Basophils, the rarest type of circulating leukocyte, have irregular Platelets are small (2-4 μm) cell fragments derived from megakary- bilobed nuclei and resemble mast cells with strongly basophilic ocytes in bone marrow, with a marginal bundle of actin filaments, specific granules containing factors important in allergies and alpha granules and delta granules, and an open canalicular system chronic inflammatory conditions, including histamine, heparin, of membranous vesicles; rapid degranulation on contact with col- chemokines, and various hydrolases. lagen triggers blood clotting. 12_Mescher_ch12_p237-253.indd 251 26/04/18 11:22 am  P 252 CHAPTER 12 Blood P F FIGURE 12–14 Platelet aggregation, degranulation, and fibrin clot formation. E a EP C E P P EP F C E a b EP C Minor trauma to vessels of the microvasculature is a routine occur- (b) Platelets aggregate at the onset of clot formation. This TEM rence in active individuals and quickly results in a fibrin clot, shown section shows platelets in a platelet plug adhering to collagen here by SEM (a). Upon contact with collagen in the vascular base- (C). Upon adhering to collagen, platelets are activated and their E ment membrane, platelets (P) aggregate, swell, and release factors granules undergo exocytosis into the open canalicular system, that trigger formation of a fibrin meshwork (F) that traps eryth- which facilitates extremely rapid release of factors involved in rocytes (E) and more degranulating platelets. Platelets in various blood coagulation. When their contents are completely released, states of degranulation are shown. Such a clot grows until blood the swollen degranulated platelets (arrows) remain as part of the EP loss from the vasculature stops. After repair of the vessel wall, fibrin aggregate until the clot is removed. Several other key proteins for clots are removed by proteolysis due primarily to locally generated blood coagulation are released locally from adjacent endothelial plasmin, a nonspecific protease. (X4100) cell processes (EP) and from the plasma. Part of an erythrocyte (E) is seen at the right. (X7500) C b Blood ASSESS YOUR KNOWLEDGE 1. Which biochemical component of the erythrocyte cell surface is 4. Which cell type has cytoplasmic granules that contain heparin and primarily responsible for determining blood type (eg, the A-B-O histamine? system). a. Eosinophils a. Fatty acid b. Basophils b. Carbohydrate c. Lymphocytes c. Nucleic acid d. Monocytes d. Protein e. Neutrophils e. Cholesterol 5. A differential cell count of a blood smear from a patient with a para- 2. What cell in circulating blood is the precursor to microglia and sitic infection is likely to reveal an increase in the circulating num- most antigen-presenting cells? bers of which cell type? a. Eosinophil a. Neutrophils b. Basophil b. Lymphocytes c. Lymphocyte c. Monocytes d. Monocyte d. Basophils e. Mast cell e. Eosinophils 3. What is the approximate life span of a circulating erythrocyte? 6. Which of the following blood cells differentiate outside of the bone a. 8 days marrow? b. 20 days a. Neutrophils c. 5 weeks b. Basophils d. 4 months c. Eosinophils e. 1 year d. T lymphocytes e. Megakaryocytes 12_Mescher_ch12_p237-253.indd 252 26/04/18 11:22 am