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Junqueira's Basic Histology, 14e >

Chapter 12: Blood

INTRODUCTION
Blood 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 unidirectionally within the closed circulatory system. The so-called
formed elements circulating in the plasma are erythrocytes (red blood cells), leukocytes (white blood cells), and platelets.

When blood leaves the circulatory system, either in a test tube or in the extracellular matrix (ECM) surrounding blood vessels, plasma proteins
react with one another to produce a clot, which includes formed elements and a pale yellow liquid called serum. Serum contains growth factors
and 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 addition of anticoagulants (eg, heparin or citrate) can be separated by centrifugation into
layers that reflect its heterogeneity (Figure 12–1). Erythrocytes comprise the sedimented material and their volume, normally about 44% of the
total blood volume in healthy adults, is called the hematocrit.

FIGURE 12–1

Composition of whole blood.

A tube of blood after centrifugation (center) has nearly half of its volume represented by erythrocytes in the bottom half of the tube, a volume
called the hematocrit. Between the sedimented erythrocytes and the supernatant light-colored plasma is a thin layer of leukocytes and platelets
called the buffy coat. The concentration ranges of erythrocytes, platelets, and leukocytes in normal blood are included here, along with the
differential count or percent range for each type of leukocyte represented in the buffy coat. A cubic millimeter of blood is equivalent to a
microliter (µL). (Complete blood count [CBC] values in this chapter are those used by the US National Board of Medical Examiners.)

The straw-colored, translucent, slightly viscous supernatant comprising 55% at the top half of the centrifugation tube is the plasma. A thin gray-

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white layer called the buffy coat between the plasma and the hematocrit, about 1% of the volume, consists of leukocytes and platelets, both less
dense than erythrocytes.

Blood is a distributing vehicle, transporting O2, CO2, metabolites, hormones, and other substances to cells throughout the body. Most O2 is
bound to hemoglobin in erythrocytes and is much more abundant in arterial than venous blood (Figure 12–2), while CO2 is carried in solution as
CO2 or HCO3−, in addition to being hemoglobin-bound. Nutrients are distributed from their sites of synthesis or absorption in the gut, while
metabolic residues are collected from cells throughout 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 normal organ function. Blood also participates in heat
distribution, the regulation of body temperature, and the maintenance of acid-base and osmotic balance.

FIGURE 12–2

Blood O2 content in each type of blood vessel.

The amount of O2 in blood (the O2 pressure) is highest in arteries and lung capillaries and decreases in tissue capillaries, where exchange of O2
and CO2 occurs between blood and tissues.

Leukocytes have diverse functions and are one of the body’s chief defenses against infection. These cells are generally spherical and inactive
while suspended in circulating blood, but, when called to sites of infection or inflammation, they cross the wall of venules, become motile and
migrate into the tissues, and display their defensive capabilities.

COMPOSITION OF PLASMA
Plasma is an aqueous solution, pH 7.4, containing substances of low or high molecular weight that make up 7% of its volume. As summarized in
Table 12–1, the dissolved components are mostly plasma proteins, but they also include nutrients, respiratory gases, nitrogenous waste products,
hormones, and inorganic ions collectively called electrolytes. Through the capillary walls, the low-molecular-weight components of plasma are
in equilibrium with the interstitial fluid of the tissues. The composition of plasma is usually an indicator of the mean composition of the
extracellular fluids in tissues.

TABLE 12–1 The composition of blood plasma.

Plasma Component (Percentage of Plasma) Functions
Is the solvent in which formed elements are suspended
Water (~92% of plasma)
and proteins and solutes are dissolved
Plasma proteins (~7% of plasma) All proteins serve to buffer against pH changes
Exerts osmotic force to retain fluid within the
microvasculature
Albumin (~58% of plasma proteins) Contributes to blood’s viscosity

Binds and transports some fatty acids, electrolytes,

hormones and drugs

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α-Globulins transport lipids and some metal ions

β-Globulins transport iron ions and lipids in bloodstream

Globulins (~37% of plasma proteins)
γ-Globulins are antibodies with various immune
functions
Participates in blood coagulation (clotting); precursor of
Fibrinogen (~4% of plasma proteins)
fibrin
Consists of enzymes, proenzymes, hormones, and the
Regulatory proteins (>1% of plasma proteins)
complement system
Other Solutes (~1% of Blood Plasma)
Help establish and maintain membrane potentials,
Electrolytes (eg, sodium, potassium, calcium, chloride, iron, bicarbonate, and
maintain pH balance, and regulate osmosis (control of the
hydrogen)
percentages of water and salt in the blood)
Energy source; precursor for synthesizing other
Nutrients (eg, amino acids, glucose, cholesterol, vitamins, fatty acids)
molecules
Respiratory gases (eg, oxygen: > 2% dissolved in plasma, 98% bound to hemoglobin Oxygen is needed for aerobic cellular respiration; carbon
within erythrocytes; and carbon dioxide: ~7% dissolved in plasma, ~27% bound to dioxide is a waste product produced by cells during this
hemoglobin within erythrocytes, ~66% converted to HCO3−) process
Waste products serve no function in the blood plasma;
Wastes (breakdown products of metabolism) (eg, lactic acid, creatinine, urea,
they are merely being transported to the liver and kidneys
bilirubin, ammonia)
where they can be removed from the blood

The major plasma proteins include the following:

Albumin, the most abundant plasma protein, is made in the liver and serves primarily to maintain the osmotic pressure of the blood.

Globulins (α- and β-globulins), made by liver and other cells, include transferrin and other transport factors; fibronectin; prothrombin and
other coagulation factors; lipoproteins and other proteins entering blood from tissues.

Immunoglobulins (antibodies or γ-globulins) secreted by plasma cells in many locations.

Fibrinogen, the largest plasma protein (340 kD), also made in the liver, which, during clotting, polymerizes as insoluble, cross-linked
fibers of fibrin that block blood loss from small vessels.

Complement proteins, which comprise a defensive system important in inflammation and destruction of microorganisms.

BLOOD CELLS
Blood cells can be studied histologically in smears prepared by spreading a drop of blood in a thin layer on a microscope slide (Figure 12–3). In
such films the cells are clearly visible and distinct from one another, facilitating observation of their nuclei and cytoplasmic characteristics.
Blood smears are routinely stained with mixtures of acidic (eosin) and basic (methylene blue) dyes. These mixtures may also contain dyes called
azures that are more useful in staining cytoplasmic granules containing charged proteins and proteoglycans. Azurophilic granules produce
metachromasia in stained leukocytes like that seen with mast cells in connective tissue. Some of these special stains, such as Giemsa and Wright
stain, are named after hematologists who introduced their own modifications into the original mixtures.

FIGURE 12–3

Preparing a blood smear.

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Erythrocytes

Erythrocytes (red blood cells or RBCs) are terminally differentiated structures lacking nuclei and completely filled with the O2-carrying protein
hemoglobin. RBCs are the only blood cells whose function does not require them to leave the vasculature.

MEDICAL APPLICATION

Anemia is the condition of having a concentration of erythrocytes below the normal range. With fewer RBCs per milliliter of blood, tissues are
unable to receive adequate O2. Symptoms of anemia include lethargy, shortness of breath, fatigue, skin pallor, and heart palpitations. Anemia
may result from insufficient red cell production, due, for example, to iron deficiency, or from blood loss with a stomach ulcer or excessive
menses.

An increased concentration of erythrocytes in blood (erythrocytosis, or polycythemia) may be a physiologic adaptation found, for example, in
individuals who live at high altitudes, where O2 tension is low. Elevated hematocrit increases blood viscosity, putting strain on the heart, and, if
severe, can impair circulation through the capillaries.

Human erythrocytes suspended in an isotonic medium are flexible biconcave discs (Figure 12–4). They are approximately 7.5 µm in diameter,
2.6-µm thick at the rim, but only 0.75-µm thick in the center. Because of their uniform dimensions and their presence in most tissue sections,
RBCs can often be used by histologists as an internal standard to estimate the size of other nearby cells or structures.

FIGURE 12–4

Normal human erythrocytes.

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(a) Colorized SEM micrograph of normal erythrocytes with each side concave. (X1800)

(b) Diagram of an erythrocyte giving the cell’s dimensions. The biconcave shape gives the cells a very high surface-to-volume ratio and places
most hemoglobin within a short distance from the cell surface, both qualities that provide maximally efficient O2 transport. Erythrocytes are also
quite flexible and can easily bend to pass through small capillaries.

(c) In small vessels red blood cells also often stack up in loose aggregates called rouleaux. The standard size of RBCs allows one to estimate that
the vessel seen is approximately 15 mm in diameter. (X250; H&E)

The biconcave shape provides a large surface-to-volume ratio and facilitates gas exchange. The normal concentration of erythrocytes in blood is
approximately 3.9-5.5 million per microliter (µL, or mm3) in women and 4.1-6.0 million/µL in men.

Erythrocytes are normally quite flexible, which permits them to bend and adapt to the small diameters and irregular turns of capillaries.
Observations in vivo show that at the angles of capillary bifurcations, erythrocytes with normal adult hemoglobin frequently assume a cuplike
shape. In larger blood vessels RBCs may adhere to one another loosely in stacks called rouleaux (Figure 12–4c).

The erythrocyte plasmalemma, because of its ready availability, is the best-known membrane of any cell. It consists of about 40% lipid, 10%
carbohydrate, and 50% protein. Most of the latter are integral membrane proteins (see Chapter 2), including ion channels, the anion transporter
called band 3 protein, and glycophorin A. The glycosylated extracellular domains of the latter proteins include antigenic sites that form the
basis for the ABO blood typing system. Several peripheral proteins are associated with the inner surface of the membrane, including spectrin,
dimers of which form a lattice bound to underlying actin filaments, and ankyrin, which anchors the spectrin lattice to the glycophorins and band
3 proteins. This submembranous meshwork stabilizes the membrane, maintains the cell shape, and provides the cell elasticity required for
passage through capillaries.

Erythrocyte cytoplasm lacks all organelles but is densely filled with hemoglobin, the tetrameric O2-carrying protein that accounts for the cells’
uniform acidophilia. When combined with O2 or CO2, hemoglobin forms oxyhemoglobin or carbaminohemoglobin, respectively. The
reversibility of these combinations is the basis for the protein’s gas-transporting capacity.

Erythrocytes undergo terminal differentiation (discussed in Chapter 13) which includes loss of the nucleus and organelles shortly before the cells
are released by bone marrow into the circulation. Lacking mitochondria, erythrocytes rely on anaerobic glycolysis for their minimal energy
needs. Lacking nuclei, they cannot replace defective proteins.

Human erythrocytes normally survive in the circulation for about 120 days. By this time defects in the membrane’s cytoskeletal lattice or ion
transport systems begin to produce swelling or other shape abnormalities, as well as changes in the cells’ surface oligosaccharide complexes.
Senescent or worn-out RBCs displaying such changes are recognized and removed from circulation, mainly by macrophages of the spleen, liver,
and bone marrow.

Leukocytes

Leukocytes (white blood cells or WBCs) leave the blood and migrate to the tissues where they become functional and perform various activities
related to immunity. Leukocytes are divided into two major groups, granulocytes and agranulocytes, based on the density of their cytoplasmic
granules (Table 12–2). All are rather spherical while suspended in blood plasma, but they become amoeboid and motile after leaving the blood

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vessels and invading the tissues. Their estimated sizes mentioned here refer to observations in blood smears in which the cells are spread and
appear slightly larger than they are in the circulation.

TABLE 12–2 Leukocytes: Numbers, structural features, and major functions.

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aColor with routine blood smear stains. There are typically 4500-11,000 total leukocytes/µL of blood in adults, higher in infants and young
children.

bThe 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.

Granulocytes possess two major types of abundant cytoplasmic granules: lysosomes (often called azurophilic granules in blood cells) and
specific granules that bind neutral, basic, or acidic stains and have specific functions.

Granulocytes also have polymorphic nuclei with two or more distinct (almost separated) lobes and include the neutrophils, eosinophils, and
basophils (Figure 12–1 and Table 12–2). All granulocytes are also terminally differentiated cells with a life span of only a few days. Their Golgi
complexes and rough ER are poorly developed, and with few mitochondria they depend largely on glycolysis for their energy needs. Most
granulocytes undergo apoptosis in the connective tissue and billions of neutrophils alone die each day in adults. The resulting cellular debris is
removed by macrophages and, like all apoptotic cell death, does not itself elicit an inflammatory response.

Agranulocytes lack specific granules, but do contain some azurophilic granules (lysosomes). The nucleus is spherical or indented but not
lobulated. This group includes the lymphocytes and monocytes (Figure 12–1 and Table 12–2). The differential count (percentage of all
leukocytes) for each type of leukocyte is also presented in Table 12–2.

FIGURE 12–5

Sickle cell erythrocyte.

A single nucleotide substitute in the hemoglobin gene produces a version of the protein that polymerizes to form rigid aggregates, leading to
greatly misshapen cells with reduced flexibility. In individuals homozygous for the mutated HbS gene, this can lead to greater blood viscosity,
and poor microvascular circulation, both features of sickle cell disease. (X6500)

All leukocytes are key players in the constant defense against invading microorganisms and in the repair of injured tissues, specifically leaving
the microvasculature in injured or infected tissues. At such sites factors termed cytokines are released from various sources and these trigger
loosening of intercellular junctions in the endothelial cells of local postcapillary venules (Figure 12–6). Simultaneously the cell adhesion protein
P-selectin appears on the endothelial cells’ luminal surfaces following exocytosis from cytoplasmic Weibel-Palade bodies. The surfaces of
neutrophils and other leukocytes display glycosylated ligands for P-selectin, and their interactions cause cells flowing through the affected
venules to slow down, like rolling tennis balls arriving at a patch of velcro. Other cytokines stimulate the now slowly rolling leukocytes to
express integrins and other adhesion factors that produce firm attachment to the endothelium (see Figure 11–21d). In a process called diapedesis
(Gr. dia, through + pedesis, to leap), the leukocytes send 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. The attraction of neutrophils to bacteria involves
chemical mediators in a process of chemotaxis, which causes leukocytes to rapidly accumulate where their defensive actions are specifically

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needed.

FIGURE 12–6

Diagram of events involving leukocytes in a postcapillary venule at sites of inflammation.

Locations in connective tissue with injuries or infection require the rapid immigration of various leukocytes to initiate cellular events for tissue
repair and removal of the invading microorganisms. The cytokines and cell binding proteins target various leukocytes and are best known for
neutrophils. The major initial events of neutrophil migration during inflammation are summarized here:

1. Local macrophages activated by bacteria or tissue damage release proinflammatory cytokines such as interleukin-1 (IL-1) or tumor
necrosis factor-α (TNF-α) that signal endothelial cells of nearby postcapillary venules to rapidly insert glycoprotein selectins on the
luminal cell surfaces.

2. Passing neutrophils with appropriate cell surface glycoproteins bind the selectins, which causes such cells to adhere loosely to the
endothelium and “roll” slowly along its surface.

3. Exposure to these and other cytokines causes expression of new integrins on the rolling leukocytes and expression of the integrin ligand
ICAM-1 (intercellular adhesion molecule-1) on the endothelial cells. Junctional complexes between the endothelial cells are selectively
downregulated, loosening these cells.

4. Integrins and their ligands provide firm endothelial adhesion of neutrophils to the endothelium, allowing the leukocytes to receive further
stimulation from the local cytokines.

5. Neutrophils become motile, probe the endothelium with pseudopodia, and, being attracted by other local injury-related factors called
chemokines, finally migrate by diapedesis between the loosened cells of the venule. Rapid transendothelial migration of neutrophils is
facilitated by the cells’ elongated and segmented nuclei. All leukocytes first become functional in the ECM after emerging from the
circulation by this process.

The number of leukocytes in the blood varies according to age, sex, and physiologic conditions. Healthy adults have 4500-11,000 leukocytes per
microliter of blood.

Neutrophils (Polymorphonuclear Leukocytes)

Mature neutrophils constitute 50%-70% of circulating leukocytes, a figure that includes slightly immature forms released to the circulation.
Neutrophils are 12-15 µm in diameter in blood smears, with nuclei having two to five lobes linked by thin nuclear extensions (Table 12–2; Figure

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12–7). In females, the inactive X chromosome may appear as a drumstick-like appendage on one of the lobes of the nucleus (Figure 12–7c)
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 ECM substrates such as collagen.

FIGURE 12–7

Neutrophils.

(a) In blood smears neutrophils can be identified by their multilobulated nuclei, with lobules held together by very thin strands. With this feature,
the cells are often called polymorphonuclear leukocytes, PMNs, or just polymorphs. The cells are dynamic and the nuclear shape changes
frequently. (X1500; Giemsa)

(b) Neutrophils typically have diameters ranging from 12 to 15 µm, approximately twice that of the surrounding erythrocytes. The cytoplasmic
granules are relatively sparse and have heterogeneous staining properties, although generally pale and not obscuring the nucleus. (X1500;
Giemsa)

(c) Micrograph showing a neutrophil from a female in which the condensed X chromosome appears as a drumstick appendage to a nuclear lobe
(arrow). (X1500; Wright)

Neutrophils are usually the first leukocytes to arrive at sites of infection where they actively pursue bacterial cells using chemotaxis and remove
the invaders or their debris by phagocytosis.

The cytoplasmic granules of neutrophils provide the cells’ functional activities and are of two main types (Figure 12–8). Azurophilic primary
granules or lysosomes are large, dense vesicles with a major role in both killing and degrading engulfed microorganisms. They contain proteases
and antibacterial proteins, including the following:

Myeloperoxidase (MPO), which generates hypochlorite and other agents toxic to bacteria

Lysozyme, which degrades components of bacterial cell walls

Defensins, small cysteine-rich proteins that bind and disrupt the cell membranes of many types of bacteria and other microorganisms.

FIGURE 12–8

Neutrophil ultrastructure.

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A TEM of a sectioned human neutrophil reveals the two types of cytoplasmic granules: the small, pale, more variably stained specific granules
(S) and the larger, electron-dense azurophilic granules (A).

 Specific granules undergo exocytosis during and after diapedesis, releasing many factors with various activities, including enzymes to digest
ECM components and bactericidal factors. Azurophilic granules are modified lysosomes with components to kill engulfed bacteria.

 The nucleus (N) is lobulated and the central Golgi apparatus (G) is small. Rough ER and mitochondria are not abundant, because this cell
utilizes glycolysis and is in the terminal stage of its differentiation. (X25,000)

MEDICAL APPLICATION

Several kinds of neutrophil defects, often genetic in origin, can affect function of these cells, for example, by decreasing 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
typically experience more frequent and more persistent bacterial infections, although macrophages and other leukocytes may substitute for
certain neutrophil functions.

Specific secondary granules are smaller and less dense, stain faintly pink, and have diverse functions, including secretion 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 that begins the process of restoring the
normal tissue microenvironment. They release many polypeptide chemokines that attract other leukocytes and cytokines that direct activities of
these and local cells of the tissue. Important lipid mediators of inflammation are also released from neutrophils.

Neutrophils contain glycogen, which is broken down into glucose to yield energy via the glycolytic pathway. The citric acid cycle is less
important, as might be expected in view of the paucity of mitochondria in these cells. The ability of neutrophils to survive in an anaerobic
environment is highly advantageous, because they can kill bacteria and help clean up debris in poorly oxygenated regions, for example, damaged
or necrotic tissue lacking normal microvasculature.

Neutrophils are short-lived cells with a half-life of 6-8 hours in blood and a life span of 1-4 days in connective tissues before dying by apoptosis.

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MEDICAL APPLICATION

Neutrophils look for bacteria to engulf by pseudopodia and internalize them in vacuoles called phagosomes. Immediately thereafter, specific
granules fuse with and discharge their contents into the phagosomes which are then acidified by proton pumps. Azurophilic granules then
discharge their enzymes into this acidified vesicle, killing and digesting the engulfed microorganisms.

During phagocytosis, a burst of O2 consumption leads to the formation of superoxide anions (O2−) and hydrogen peroxide (H2O2). O2− is a
short-lived, highly reactive free radical that, together with MPO and halide ions, forms a powerful microbial killing system inside the
neutrophils. Besides the activity of lysozyme cleaving cell wall peptidoglycans to kill certain bacteria, the protein lactoferrin avidly binds iron, a
crucial element in bacterial nutrition whose lack of availability then causes bacteria to die. A combination of these mechanisms will kill most
microorganisms, which are then digested by lysosomal enzymes. Apoptotic neutrophils, bacteria, semidigested material, and tissue-fluid form a
viscous, usually yellow collection of fluid called pus.

Several neutrophil hereditary dysfunctions have been described. In one of them, actin does not polymerize normally, reducing neutrophil
motility. With a NADPH oxidase deficiency, there is a failure to produce H2O2 and hypochlorite, reducing the cells’ microbial killing power.
Children with such dysfunctions can experience more persistent bacterial infections.

Eosinophils

Eosinophils are far less numerous than neutrophils, constituting only 1%-4% of leukocytes. In blood smears, this cell is about the same size as a
neutrophil or slightly larger, but with a characteristic bilobed nucleus (Table 12–2; Figure 12–9). The main identifying characteristic is the
abundance of large, acidophilic specific granules typically staining pink or red.

FIGURE 12–9

Eosinophils.

Eosinophils are about the same size as neutrophils but have bilobed nuclei and more abundant coarse cytoplasmic granules. The cytoplasm is
often filled with brightly eosinophilic specific granules, but it also includes some azurophilic granules. (a) Micrograph shows an eosinophil (E)
next to a neutrophil (N) and a small lymphocyte (L). (X1500; Wright)

(b) Even with granules filling the cytoplasm, the two nuclear lobes of eosinophils are usually clear. (X1500; Giemsa)

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(c) Ultrastructurally a sectioned eosinophil clearly shows the unique specific eosinophilic granules (EG), as oval structures with disc-shaped
electron-dense, crystalline cores. These granules, along with a few lysosomes and mitochondria (M), fill the cytoplasm around the bilobed
nucleus (N). (X20,000)

Ultrastructurally the eosinophilic specific granules are seen to be oval in shape, with flattened crystalloid cores (Figure 12–9c) containing major
basic proteins (MBP), an arginine-rich factor that accounts for the granule’s acidophilia and constitutes up to 50% of the total granule protein.
MBPs, along with eosinophilic peroxidase, other enzymes and toxins, act to kill parasitic worms or helminths. Eosinophils also 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 and allergic reactions. These leukocytes also remove
antigen-antibody complexes from interstitial fluid by phagocytosis.

Eosinophils are particularly abundant in connective tissue of the intestinal lining and at sites of chronic inflammation, such as lung tissues of
asthma patients.

MEDICAL APPLICATION

An increase in the number of eosinophils in blood (eosinophilia) is associated with allergic reactions and helminthic infections. In patients with
such conditions, eosinophils are found in the connective tissues underlying epithelia of the bronchi, gastrointestinal tract, uterus, and vagina, and
surrounding any parasitic worms present. In addition, these cells produce substances that modulate inflammation by inactivating the leukotrienes
and histamine produced by other cells. Corticosteroids (hormones from the adrenal cortex) produce a rapid decrease in the number of blood
eosinophils, probably by interfering with their release from the bone marrow into the bloodstream.

Basophils

Basophils are also 12-15 µm in diameter but make up less than 1% of circulating leukocytes and are therefore difficult to find in normal blood
smears. The nucleus is divided into two irregular lobes, but the large specific granules overlying the nucleus usually obscure its shape.

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 (Table 12–2; Figure 12–10). The strong basophilia of the granules is due to the
presence of heparin and other sulfated GAGs. Basophilic specific granules also contain much histamine and various other mediators of
inflammation, including platelet activating factor, eosinophil chemotactic factor, and the enzyme phospholipase A that catalyzes an initial step in
producing lipid-derived proinflammatory factors called leukotrienes.

FIGURE 12–10

Basophils.

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(a-c) Basophils are also approximately the same size as neutrophils and eosinophils, but they have large, strongly basophilic specific granules
that usually obstruct the appearance of the nucleus which usually has two large irregular lobes. (a and b: X1500, Wright; c: X1500, Giemsa)

(d) A TEM of a sectioned basophil reveals the single bilobed nucleus (N) and the large, electron-dense specific basophilic granules (B).
Basophils exert many activities modulating the immune response and inflammation and have many functional similarities with mast cells, which
are normal, longer-term residents of connective tissue. (X25,000)

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 metachromatic granules containing heparin and histamine, have surface receptors for immunoglobulin E (IgE),
and secrete their granular components in response to certain antigens and allergens.

MEDICAL APPLICATION

In some individuals a second exposure to a strong allergen, such as that delivered in a bee sting, may produce an intense, adverse systemic
response. Basophils and mast cells may rapidly degranulate, producing vasodilation in many organs, a sudden drop in blood pressure, and other
effects comprising a potentially lethal condition called anaphylaxis or anaphylactic shock.

Basophils and mast cells also are central to immediate 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 combines with the receptor-bound IgE molecules, causing them to cross-link and
aggregate on the cell surfaces and triggering rapid exocytosis of the cytoplasmic granules. Release of the inflammatory mediators in this manner
can result in bronchial asthma, cutaneous hives, rhinitis, conjunctivitis, or allergic gastroenteritis.

Lymphocytes

By far the most numerous type of agranulocyte in normal blood smears, lymphocytes constitute a family of leukocytes with spherical nuclei
(Table 12–2; Figure 12–11). Lymphocytes are typically the smallest leukocytes and constitute approximately a third of these cells. Although they
are morphologically similar, mature lymphocytes can be subdivided into functional groups by distinctive surface molecules (called “cluster of
differentiation” or CD markers) that can be distinguished using antibodies with immunocytochemistry or flow cytometry. Major classes include
B lymphocytes, helper and cytotoxic T lymphocytes (CD4+ and CD8+, respectively), and natural killer (NK) cells. These and other types of

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lymphocytes have diverse roles in immune defenses against invading microorganisms and certain parasites or abnormal cells. T lymphocytes,
unlike B cells and all other circulating leukocytes, differentiate outside the bone marrow in the thymus. Functions and formation of lymphocytes
are discussed with the immune system in Chapter 14.

FIGURE 12–11

Lymphocytes.

Lymphocytes are agranulocytes and lack the specific granules characteristic of granulocytes. Lymphocytes circulating in blood generally range in
size from 6 to 15 µm in diameter and are sometimes classified arbitrarily as small, medium, and large.

(a) The most numerous small lymphocytes shown here are slightly larger than the neighboring erythrocytes and have only a thin rim of
cytoplasm surrounding the spherical nucleus. (X1500; Giemsa)

(b) Medium lymphocytes are distinctly larger than erythrocytes. (X1500; Wright)

(c) Large lymphocytes, much larger than erythrocytes, may represent activated cells that have returned to the circulation. (X1500; Giemsa)

(d) Ultrastructurally a medium-sized lymphocytes is seen to be mostly filled with a euchromatic nucleus (N) surrounded by cytoplasm containing
mitochondria (M), free polysomes, and a few dark lysosomes (azurophilic granules). (X22,000)

Although generally small, circulating lymphocytes have a wider range of sizes than most leukocytes. Small, newly released lymphocytes have
diameters similar to those of RBCs; medium and large lymphocytes are 9-18 µm in diameter, with the latter representing activated lymphocytes
or NK cells. The small lymphocytes are characterized by spherical nuclei with highly condensed chromatin and only a thin surrounding rim of
scant cytoplasm, making them easily distinguishable from granulocytes. Larger lymphocytes have larger, slightly indented nuclei and more
cytoplasm that is slightly basophilic, with a few azurophilic granules, mitochondria, free polysomes, and other organelles (Figure 12–11d).

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Lymphocytes vary in life span according to their specific functions; some live only a few days and others survive in the circulating blood or other
tissues for many years.

MEDICAL APPLICATION

Given their central roles in immunity, lymphocytes are obviously important in many diseases. Lymphomas are a group of disorders involving
neoplastic proliferation of lymphocytes or the failure of these cells to undergo apoptosis. Although often slow-growing, all lymphomas are
considered malignant because they can very easily become widely spread throughout the body.

Monocytes

Monocytes are agranulocytes that are precursor cells of macrophages, osteoclasts, microglia, and other cells of the mononuclear phagocyte
system in connective tissue (see Chapter 5). All monocyte-derived cells are antigen-presenting cells and have important roles in immune defense
of tissues. Circulating monocytes have diameters of 12-15 µm, but macrophages are often somewhat larger. The monocyte nucleus is large and
usually distinctly indented or C-shaped (Figure 12–12). The chromatin is less condensed than in lymphocytes and typically stains lighter than
that of large lymphocytes.

FIGURE 12–12

Monocytes.

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Monocytes are large agranulocytes with diameters from 12 to 20 µm that circulate as precursors to macrophages and other cells of the
mononuclear phagocyte system.

(a-d) Micrographs of monocytes showing their distinctive nuclei which are indented, kidney-shaped, or C-shaped. (a: X1500, Giemsa; b-d:
X1500, Wright)

(e) Ultrastructurally the cytoplasm of a monocyte shows a Golgi apparatus (G), mitochondria (M), and lysosomes or azurophilic granules (A).
Rough ER is poorly developed and there are some free polysomes (R). (X22,000)

  (Figure 12–12e, used with permission from D.F. Bainton and M.G. Farquhar, Department of Pathology, University of California at San
Francisco, CA.)

The cytoplasm of the monocyte is basophilic and contains many small lysosomal azurophilic granules, some of which are at the limit of the light
microscope’s resolution. These granules are distributed through the cytoplasm, giving it a bluish-gray color in stained smears. Mitochondria and
small areas of rough ER are present, along with a Golgi apparatus involved in the formation of lysosomes (Figure 12–12e).

MEDICAL APPLICATION

Extravasation or the accumulation of immigrating monocytes occurs in the early phase of inflammation following tissue injury. Acute
inflammation is usually short-lived as macrophages undergo apoptosis or leave the site, but chronic inflammation usually involves the continued
recruitment of monocytes. The resulting continuous presence of macrophages can lead to excessive tissue damage that is typical of chronic
inflammation.

Platelets

Blood platelets (or thrombocytes) are very small non-nucleated, membrane-bound cell fragments only 2-4 µm in diameter (Figure 12–13a). As
described in Chapter 13, platelets originate by separation from the ends of cytoplasmic processes extending from giant polyploid bone marrow
cells called megakaryocytes. Platelets promote blood clotting and help repair minor tears or leaks in the walls of small blood vessels, preventing
loss of blood from the microvasculature. Normal platelet counts range from 150,000 to 400,000/µL (mm3) of blood. Circulating platelets have a
life span of about 10 days.

FIGURE 12–13

Platelets.

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Platelets are cell fragments 2-4 µm in diameter derived from megakaryocytes of bone marrow. Their primary function is to rapidly release the
content of their granules upon contact with collagen (or other materials outside of the endothelium) to begin the process of clot formation and
reduce blood loss from the vasculature.

(a) In a blood smear, platelets (arrows) are often found as aggregates. Individually they show a lightly stained hyalomere region surrounding a
more darkly stained central granulomere containing membrane-enclosed granules. (X1500; Wright)

(b) Ultrastructurally a platelet shows a system of microtubules and actin filaments near the periphery, called the marginal bundle (MB), which
is formed as the platelet pinches off from megakaryocyte (Chapter 13), and helps maintain its shape. An open canalicular system (OCS) of
invaginating membrane vesicles continuous with the plasmalemma facilitates rapid degranulation upon activation and Ca2+ release. The central
granulomere region contains small dense delta granules (δG), larger and more numerous alpha granules (αG), and glycogen (G). (X40,000)

 (Figure 12–13b, used with permission from Dr M. J. G. Harrison, Middlesex Hospital and University College London, UK.)

In stained blood smears, platelets often appear in clumps. Each individual platelet is generally discoid, with a very lightly stained peripheral
zone, the hyalomere, and a darker-staining central zone rich in granules, called the granulomere. A sparse glycocalyx surrounding the platelet
plasmalemma is involved in adhesion and activation during blood coagulation.

Ultrastructural analysis (Figure 12–13b) reveals a peripheral marginal bundle of microtubules and microfilaments, which helps to maintain the
platelet’s shape. Also in the hyalomere are two systems of membrane channels. An open canalicular system of vesicles is connected to
invaginations of the plasma membrane, which may facilitate platelets’ uptake of factors from plasma. A much less prominent set of irregular
tubular vesicles comprising the dense tubular system is derived from the ER and stores Ca2+ ions. Together, these two membranous systems
facilitate the extremely rapid exocytosis of proteins from platelets (degranulation) upon adhesion to collagen or other substrates outside the

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vascular endothelium.

Besides specific granules, the central granulomere has a sparse population of mitochondria and glycogen particles (Figure 12–13b). Electron-
dense delta granules (δG), 250-300 nm in diameter, contain ADP, ATP, and serotonin (5-hydroxytryptamine) taken up from plasma. Alpha
granules (αG) are larger (300-500 nm in diameter) and contain platelet-derived growth factor (PDGF), platelet factor 4, and several other
platelet-specific proteins. Most of the stained granules seen in platelets with the light microscope are alpha granules.

The role of platelets in controlling blood loss (hemorrhage) and in wound healing can be summarized as follows:

Primary aggregation: Disruptions in the microvascular endothelium, which are very common, allow the platelet glycocalyx to adhere to
collagen in the vascular basal lamina or wall. Thus, a platelet plug is formed as a first step to stop bleeding (Figure 12–14).

Secondary aggregation: Platelets in the plug release a specific adhesive glycoprotein and ADP, which induce further platelet aggregation
and increase the size of the platelet plug.

Blood coagulation: During platelet aggregation, fibrinogen from plasma, von Willebrand factor and other proteins released from the
damaged endothelium, and platelet factor 4 from platelet granules promote the sequential interaction (cascade) of plasma proteins, giving
rise to a fibrin polymer that forms a three-dimensional network of fibers trapping red blood cells and more platelets to form a blood clot,
or thrombus (Figure 12–14). Platelet factor 4 is a chemokine for monocytes, neutrophils, and fibroblasts and proliferation of the
fibroblasts is stimulated by PDGF.

Clot retraction: The clot that initially bulges into the blood vessel lumen contracts slightly due to the activity of platelet-derived actin and
myosin.

Clot removal: Protected by the clot, the endothelium and surrounding tunic are restored by new tissue, and the clot is then removed,
mainly dissolved by the proteolytic enzyme plasmin, which is formed continuously through the local action of plasminogen activators
from the endothelium on plasminogen from plasma.

FIGURE 12–14

Platelet aggregation, degranulation, and fibrin clot formation.

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Minor trauma to vessels of the microvasculature is a routine occurrence in active individuals and quickly results in a fibrin clot, shown here by
SEM (a). Upon contact with collagen in the vascular basement membrane, platelets (P) aggregate, swell, and release factors that trigger
formation of a fibrin meshwork (F) that traps erythrocytes (E) and more degranulating platelets. Platelets in various states of degranulation are
shown. Such a clot grows until blood loss from the vasculature stops. After repair of the vessel wall, fibrin clots are removed by proteolysis due
primarily to locally generated plasmin, a nonspecific protease. (X4100)

(b) Platelets aggregate at the onset of clot formation. This TEM section shows platelets in a platelet plug adhering to collagen (C). Upon
adhering to collagen, platelets are activated and their granules undergo exocytosis into the open canalicular system, which facilitates extremely
rapid release of factors involved in blood coagulation. When their contents are completely released, the swollen degranulated platelets (arrows)
remain as part of the aggregate until the clot is removed. Several other key proteins for blood coagulation are released locally from adjacent
endothelial cell processes (EP) and from the plasma. Part of an erythrocyte (E) is seen at the right. (X7500)

MEDICAL APPLICATION

Aspirin and other nonsteroidal anti-inflammatory agents have an inhibitory effect on platelet function and blood coagulation because they block
the local prostaglandin synthesis that is needed for platelet aggregation, contraction, and exocytosis at sites of injury. Bleeding disorders result
from abnormally slow blood clotting. One such disease directly related to a defect in the platelets is a rare autosomal recessive glycoprotein Ib
deficiency, involving a factor on the platelet surface needed to bind subendothelial collagen and begin the cascade of events leading to clot
formation.

SUMMARY OF KEY POINTS

Blood

The liquid portion of circulating blood is plasma, while the cells and platelets comprise the formed elements; upon clotting, some
proteins are removed from plasma and others are released from platelets, forming a new liquid termed serum.

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Important protein components of plasma include albumin, diverse α- and β-globulins, proteins of the complement system, and
fibrinogen, all of which are secreted within the liver, as well as the immunoglobulins.

Red blood cells or erythrocytes, which make up the hematocrit portion (~45%) of a blood sample, are enucleated, biconcave discs 7.5
µm in diameter, filled with hemoglobin for the uptake, transport, and release of O2, and with a normal life span of about 120 days.

White blood cells or leukocytes are broadly grouped as granulocytes (neutrophils, eosinophils, basophils) or agranulocytes
(lymphocytes, monocytes).

All leukocytes become active outside the circulation, specifically leaving the microvasculature in a process involving cytokines, selective
adhesion, changes in the endothelium, and transendothelial migration or diapedesis.

All granulocytes have specialized lysosomes called azurophilic granules and smaller specific granules with proteins for various cell-
specific functions.

Neutrophils, the most abundant type of leukocyte, have polymorphic, multilobed nuclei, and faint pink cytoplasmic granules that
contain many factors for highly efficient phagolysosomal killing and removal of bacteria.

Eosinophils have bilobed nuclei and eosinophilic specific granules containing factors for destruction of helminthic parasites and for
modulating inflammation.

Basophils, the rarest type of circulating leukocyte, have irregular bilobed nuclei and resemble mast cells with strongly basophilic specific
granules containing factors important in allergies and chronic inflammatory conditions, including histamine, heparin, chemokines, and
various hydrolases.

Lymphocytes, agranulocytes with many functions as T- and B-cell subtypes in the immune system, range widely in size, depending on
their activation state, and have roughly spherical nuclei with little cytoplasm and few organelles.

Monocytes are larger agranulocytes with distinctly indented or C-shaped nuclei that circulate as precursors of macrophages and other cells
of the mononuclear phagocyte system.

Platelets are small (2-4 µm) cell fragments derived from megakaryocytes in bone marrow, with a marginal bundle of actin filaments,
alpha granules and delta granules, and an open canalicular system of membranous vesicles; rapid degranulation on contact with
collagen triggers blood clotting.

ASSESS YOUR KNOWLEDGE

1. Which biochemical component of the erythrocyte cell surface is primarily responsible for determining blood type (eg, the A-B-O system).

 a. Fatty acid

 b. Carbohydrate

 c. Nucleic acid

 d. Protein

 e. Cholesterol

2. What cell in circulating blood is the precursor to microglia and most antigen-presenting cells?

 a. Eosinophil

 b. Basophil

 c. Lymphocyte

 d. Monocyte

 e. Mast cell

3. What is the approximate life span of a circulating erythrocyte?

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 a. 8 days

 b. 20 days

 c. 5 weeks

 d. 4 months

 e. 1 year

4. Which cell type has cytoplasmic granules that contain heparin and histamine?

 a. Eosinophils

 b. Basophils

 c. Lymphocytes

 d. Monocytes

 e. Neutrophils

5. A differential cell count of a blood smear from a patient with a parasitic infection is likely to reveal an increase in the circulating numbers of
which cell type?

 a. Neutrophils

 b. Lymphocytes

 c. Monocytes

 d. Basophils

 e. Eosinophils

6. Which of the following blood cells differentiate outside of the bone marrow?

 a. Neutrophils

 b. Basophils

 c. Eosinophils

 d. T lymphocytes

 e. Megakaryocytes

7. Examination of a normal peripheral blood smear reveals a cell more than twice the diameter of an erythrocyte with a kidney-shaped nucleus.
There cells are < 10% of the total leukocytes. Which of the following cell types is being described?

 a. Monocyte

 b. Basophil

 c. Eosinophil

 d. Neutrophil

 e. Lymphocyte

8. A 43-year-old anatomy professor is working in her garden, pruning rose bushes without gloves, when a thorn deeply penetrates her forefinger.
The next day the area has become infected. She removes the tip of the thorn, but there is still pus remaining at the wound site. Which of the
following cells function in the formation of pus?

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 a. Cells with spherical nuclei and scant cytoplasm

 b. Biconcave cells with no nuclei

 c. Cells with bilobed nuclei and many acidophilic cytoplasmic granules

 d. Very small, cell-like elements with no nuclei but many granules

 e. Cells with polymorphic, multiply lobed nuclei

9. A 35-year-old woman’s physician orders laboratory blood tests. Her fresh blood is drawn and centrifuged in the presence of heparin as an
anticoagulant to obtain a hematocrit. From top to bottom, the fractions resulting from centrifugation are which of the following?

 a. Serum, packed erythrocytes, and leukocytes

 b. Leukocytes, erythrocytes, and serum proteins

 c. Plasma, buffy coat, and packed erythrocytes

 d. Fibrinogen, platelets, buffy coat, and erythrocytes

 e. Albumin, plasma lipoproteins, and erythrocytes

10. A hematologist diagnoses a 34-year-old woman with idiopathic thromobocytic purpura (ITP). Which of the following
symptoms/characteristics would one expect in this patient?

 a. Normal blood count

 b. Hypercoagulation

 c. Decreased clotting time

 d. Abnormal bruising

 e. Light menstrual periods

ANSWERS
Answers: 1b, 2d, 3d, 4b, 5e, 6d, 7a, 8e, 9c, 10d

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