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Chapter 12. Blood

Blood: Introduction
Blood is a specialized connective tissue in which cells are suspended in fluid extracellular material called plasma. Propelled mainly by
rhythmic contractions of the heart, about five liters 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 ECM surrounding blood vessels, plasma proteins react with one
another to produce a clot, which includes formed elements and a yellowish 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, citrate) can be separated by centrifugation into
layers that reflect its heterogeneity (Figure 121). Erythrocytes make up the bottom layer and their volume, normally about 45% of the total
blood volume in healthy adults, is called the hematocrit.

Figure 121.

Composition of whole blood.

A tube of blood after centrifugation (center) has about 43% 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 average
concentrations of erythrocytes, platelets and leukocytes in normal blood are included here, along with the percentage each type of leukocyte represent in the buffy coat.
A cubic millimeter of blood is equivalent to a microliter ( L).


The yellowish translucent, slightly viscous supernatant comprising 55% at the top half of the centrifugation tube is the plasma. A thin layer
between the plasma and the hematocrit, about 1% of the volume, is white or grayish in color and consists of leukocytes and platelets, both
less dense than erythrocytes.
Blood is a distributing vehicle, transporting O2 (Figure 122), CO2, metabolites, hormones, and other substances to cells throughout the

body. O2 is bound mainly to hemoglobin in erythrocytes, 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 and metabolic residues are collected from all cells and
removed from the blood by the excretory organs. Hormone distribution in blood permits the exchange of chemical messages between distant
organs for normal cellular function. Blood further participates in heat distribution, the regulation of body temperature, and the maintenance of
acid-base and osmotic balance.

Figure 122.

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 takes place between blood
and tissues.

Leukocytes have diversified 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, 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 810% of its volume. Plasma
proteins account for approximately 7% of the dissolved components, with the remainder including nutrients, nitrogenous waste products,
hormones, and many 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.
The major plasma proteins include the following:
Albumin, the most abundant plasma protein, is made in the liver and serves primarily in maintaining the osmotic pressure of the
blood.

blood.
- and -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.
-globulins, which are immunoglobulins (antibodies) secreted by lymphocytes in many locations.
Complement proteins, a system of factors important in inflammation and destruction of microorganisms.
Fibrinogen, the largest plasma protein (340 kD), also made in the liver, which during clotting polymerizes as insoluble, crosslinked fibers which block blood loss from small vessels.

Blood Cells
Blood cells are generally studied in smears or films prepared by spreading a drop of blood in a thin layer on a microscope slide (Figure 123).
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 special mixtures of acidic (eosin) and basic (methylene blue) dyes. These mixtures 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. Some of these special stains, such as Giemsa and Wright stain,
are named for hematologists who introduced their own modifications into the original mixture.

Figure 123.

Preparing a blood smear.

Erythrocytes
Erythrocytes (red blood cells) are terminally differentiated, lack nuclei, and are packed with the O2-carrying protein hemoglobin. Under
normal conditions, these corpuscles never leave the circulatory system.

Like most mammalian red blood cells, human erythrocytes suspended in an isotonic medium are flexible biconcave disks (Figure 124). They
are approximately 7.5 m in diameter, 2.6 m thick at the rim, and only 0.75 m thick in the center. This biconcave shape provides a large
surface-to-volume ratio and facilitates gas exchange. The normal concentration of erythrocytes in blood is approximately 3.95.5 million per
microliter in women and 4.16 million per microliter in men.

Figure 124.

Normal human erythrocytes.

(a): Colorized SEM of normal erythrocytes with each side concave. X3000. (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 which 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
aggregates called rouleaux. X250. H&E.

MEDICAL APPLICATION
A decreased number of erythrocytes in the blood is usually associated with anemia. An increased number of erythrocytes (erythrocytosis, or
polycythemia) may be a physiologic adaptation found, for example, in individuals who live at high altitudes, where O2 tension is low.
Polycythemia (Gr. polys, many + kytos, cell + haima, blood), usually an increase in hematocrit, is often associated with diseases of varying
degrees of severity and increases blood viscosity; when severe, it can impair circulation through the capillaries.
Abnormal erythrocytes with diameters greater than 9 m are called macrocytes, and those with diameters less than 6 m are called
microcytes. The presence of a high percentage of erythrocytes with great variations in size is called anisocytosis (Gr. aniso, uneven, +
kytos).
Erythrocytes are normally quite flexible, which permits them to adapt to the irregular bends and small diameters of capillaries. Observations in
vivo show that when traversing the angles of capillary bifurcations, erythrocytes with normal adult hemoglobin (HbA) are easily deformed and
frequently assume a cuplike shape.
The plasmalemma of the erythrocyte, 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 these proteins include antigenic sites that
form the basis for blood typing. Several peripheral proteins are associated with the inner surface of the membrane, including spectrin, which
forms a lattice reinforcing the membrane, and ankyrin, which anchors the lattice to band 3 proteins. This meshwork permits the membrane
and cell flexibility required for passage through capillaries and important for the normal low viscosity of blood.
Erythrocyte cytoplasm 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 gas-transporting capability of hemoglobin. The combination of hemoglobin with carbon monoxide (CO) is
irreversible, however, reducing the cells' capacity to transport O2.

MEDICAL APPLICATION
Inherited alterations in hemoglobin molecules are responsible for several pathologic conditions, an example of which is sickle cell disease.
This inherited disorder is caused by a mutation of one nucleotide (a point mutation) in the gene for the chain of hemoglobin. The triplet GAA
(for glutamic acid) is changed to GUA, which specifies valine. The consequences of this substitution of a single amino acid are profound.
When the altered hemoglobin (called HbS) is deoxygenated in venous capillaries, it polymerizes and forms rigid aggregates that give the
erythrocyte a characteristic sickle shape (Figure 125). The sickled erythrocyte is inflexible and fragile and has a shortened life span leading
to anemia. It increases the blood viscosity and can damage the wall of blood vessels, promoting blood coagulation. Blood flow through the
capillaries is retarded or even stopped, leading to severe O2 shortage (anoxia) in tissues.
Anemia is a pathologic condition characterized by blood concentrations of hemoglobin below normal values. Although anemias are usually
associated with a decreased number of erythrocytes, it is also possible for the number of cells to be normal but for each cell to contain a
reduced amount of hemoglobin (hypochromic anemia). Anemia may be caused by loss of blood (hemorrhage); insufficient production of
erythrocytes by the bone marrow; production of erythrocytes with insufficient hemoglobin, usually related to iron deficiency in the diet; or
accelerated destruction of blood cells.

Figure 125.

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.

Erythrocyte differentiation (presented in Chapter 13) includes loss of the nucleus and all organelles shortly before the cells are released by
bone marrow into the circulation. Lacking mitochondria, mature 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 erythrocytes displaying such changes are removed from the circulation, mainly by macrophages of the
spleen, liver, and bone marrow.

Leukocytes
Leukocytes (white blood cells) migrate to the tissues where they become functional and perform various activities (Figure 126). According to
the type of cytoplasmic granules and the shape of their nuclei, leukocytes are divided into two groups: polymorphonuclear granulocytes and
mononuclear agranulocytes. Both types are spherical while suspended in blood plasma, but become amoeboid and motile after leaving the
blood vessels and invading the tissues. Their estimated sizes mentioned below refer to observations in blood smears in which the cells are
spread and appear slightly larger than they are in the circulation.

Figure 126.

Five types of human leukocytes.

Neutrophils, eosinophils, and basophils have granules that stain specifically with certain dyes and are called granulocytes. Lymphocytes and monocytes are considered
agranulocytes, even though they may show azurophilic granules (lysosomes), which are also present in other leukocytes.

Granulocytes possess two types of granules: the specific granules that bind neutral, basic, or acidic stains and have specific functions and
the azurophilic granules, which are specialized lysosomes, stain darkly, and are present at some level in all leukocytes. When the cells
phagocytose microorganisms, several azurophilic granule proteins act collectively to kill and then digest them. The bactericidal proteins
include myeloperoxidase, which generates hypochlorite and other reactive agents toxic to bacteria; cationic polypeptides called defensins
which bind and produce holes in cell membranes of microorganisms; and lysozyme, which dissolves bacterial cell wall components. The
major protein components of specific and azurophilic granules are listed in Table 121.

Table 121. Granule composition in human granulocytes.

Cell Type

Specific Granules

Azurophilic Granules

Neutrophil

Alkaline phosphatase

Acid phosphatase

Collagenase

Lactoferrin

Lysozyme

-Galactosidase

Several nonenzymatic antibacterial basic proteins

-Glucuronidase

-Mannosidase
Arylsulfatase

Cathepsin

5'-Nucleotidase

Elastase

Collagenase

Myeloperoxidase

Lysozyme

Defensins

Eosinophil

Acid phosphatase

Arylsulfatase

-Glucuronidase

Cathepsin

Phospholipase

RNAase

Eosinophilic peroxidase

Major basic protein

Basophil

Eosinophilic chemotactic factor

Heparin

Histamine

Peroxidase

Granulocytes have polymorphic nuclei with two or more lobes and include the neutrophils, eosinophils, and basophils (Figures 121 and
126). All granulocytes are terminally differentiated cells with a life span of only a few days. Their Golgi complexes and rough ER are poorly
developed. They have few mitochondria and depend largely on glycolysis for their low energy needs, containing glycogen that allows them to
function in tissue with little O2, such as inflamed areas. Granulocytes normally die by apoptosis in the connective tissue and billions of
neutrophils alone die by apoptosis each day in the adult human. The resulting cellular debris is removed by macrophages and like all
apoptotic cell death does not elicit an inflammatory response.

Agranulocytes do not have specific granules, but they do contain azurophilic granules (lysosomes). The nucleus is round or indented. This
group includes lymphocytes and monocytes (Figures 121 and 126). The differential count of all types of leukocytes is presented in Table
122.

Table 122. Number and percentage of blood corpuscles (blood count).

Corpuscle Type

Approximate Number per L

Approximate Percentage

Erythrocyte

Female:

3.9 5.5 x 106/ L

Male:

4.1 6 x 106/ L

Reticulocyte

1% of the erythrocyte count

Leukocyte

600010,000

Neutrophil

5000

60-70%

Eosinophil

150

2-4%

Eosinophil

150

2-4%

Basophil

30

0.5%

Lymphocyte

2400

28%

Monocyte

350

5%

Platelet

300,000

All leukocytes are key players in the defense against invading microorganisms, and in the repair of injured tissues. How they leave the
circulation and become active at the specific sites where needed has been especially well-studied for neutrophils, the most abundant
leukocyte specially adapted for bacteria removal. At sites of injury or infection, various substances termed cytokines are released that trigger
loosening of intercellular junctions in the endothelial cells of local postcapillary venules and the rapid appearance of P-selectin on their
luminal surfaces from Weibel-Palade bodies. Neutrophils and other leukocytes have on their surfaces ligands for P-selectin and interaction
between these proteins causes cells flowing through the 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 (Figure 1120d). In a process called diapedesis (Gr. dia, through, + pedesis, to leap) the leukocytes quickly send extensions
into the new intercellular openings, migrate out of the venules into surrounding tissue spaces, and head directly for the bacterial cells. The
attraction of neutrophils to bacteria involves chemical mediators in a process of chemotaxis, which causes leukocytes to rapidly concentrate
where their defensive actions are specifically needed.
The number of leukocytes in the blood varies according to age, sex, and physiologic conditions. In healthy adults, there are roughly 6000
10,000 leukocytes per microliter of blood (Table 122).

Neutrophils (Polymorphonuclear Leukocytes)

Neutrophils constitute 6070% of circulating leukocytes. They are 1215 m in diameter in blood smears, with nuclei having two to five lobes
linked by thin nuclear extensions (Figures 121, 126, and 127). In females, the inactive X chromosome may appear as a drumstick-like
appendage on one of the lobes of the nucleus (Figure 127c) although this characteristic is not obvious in every neutrophil. Neutrophils are
inactive and spherical while circulating but become actively amoeboid during diapedesis and upon adhering to solid substrates such as
collagen in the ECM.

Figure 127.

Neutrophils.
(a): In blood smears neutrophils can be identified by their multilobulated nuclei, with lobules held together by thin strands. With this feature the cells are often called
polymorphonuclear leukocytes, or just polymorphs. The cells are dynamic and the nuclear shape changes frequently. X1500. Giemsa. (b): Other identifying features of
neutrophils include overall diameter of 1215 m, approximately twice that of the surrounding erythrocytes. The cytoplasmic granules are relatively sparse and
heterogeneous in their staining properties, although generally pale and not obscuring the nucleus. X1500. Giemsa. (c): Micrograph shows a neutrophil from a female in
which the condensed X chromosome appears as a drumstick appendage to a nuclear lobe (arrow). X1500. Wright.

MEDICAL APPLICATION
Immature neutrophils that have recently entered the blood circulation have a nonsegmented nucleus in the shape of a horseshoe (band
forms). An increased number of band neutrophils in the blood indicates a higher production of neutrophils, probably in response to a bacterial
infection.
The cytoplasm of the neutrophil contains two main types of granules: the more abundant specific granules, which are very small and near
the limit of light microscope resolution (Figure 127), and azurophilic granules, which are specialized lysosomes with components to kill
ingested bacteria (Figure 128). Neutrophils are active phagocytes of bacteria and other small particles and are usually the first leukocytes to
arrive at sites of infection, where they actively pursue bacterial cells using chemotaxis.

Figure 128.

Neutrophil ultrastructure.
TEM of a sectioned human neutrophil immunostained for peroxidase reveals the two types of cytoplasmic granules: the small, pale, peroxidase-negative specific
granules and the larger, dense, peroxidase-positive azurophilic granules. Specific granules undergo exocytosis during and after diapedesis, releasing many factors
with various activities, including enzymes to digest ECM components and bacteriostatic factors. Azurophilic granules are modified lysosomes with components to kill
engulfed bacteria. The nucleus is lobulated and the central Golgi apparatus is small. Rough ER and mitochondria are not abundant, because this cell utilizes glycolysis
and is in the terminal stage of its differentiation. X27,000. (Reproduced, with permission, from Bainton D.F.: Fed. Proc. 1981;40:1443.)


Neutrophils also 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, since they can kill bacteria and help clean up debris in poorly oxygenated regions, eg, inflamed or
necrotic tissue.
Neutrophils are short-lived cells with a half-life of 67 hours in blood and a life span of 14 days in connective tissues before dying by
apoptosis.

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. By means of proton pumps in the phagosome membrane, the pH of the
vacuole is lowered to about 5.0, a favorable pH for maximal activity of lysosomal enzymes. Azurophilic granules then discharge their
enzymes into the acid environment, killing and digesting the engulfed microorganisms.
During phagocytosis, a burst of O2 consumption leads to the formation of superoxide anions and hydrogen peroxide (H2O2). O2 is a shortlived free radical formed by the gain of one electron by O2. It is a highly reactive radical that kills microorganisms ingested by neutrophils.
Together with myeloperoxidase and halide ions, it forms a powerful killing system. Other strong oxidizing agents (eg, hypochlorite) can
inactivate proteins. Lysozyme has the function of specifically cleaving a bond in the peptidoglycan that forms the cell wall of some grampositive bacteria, thus causing their death. Lactoferrin avidly binds iron; because iron is a crucial element in bacterial nutrition, lack of its
availability leads to bacterial death. The acid environment of phagocytic vacuoles can itself cause the death of certain microorganisms. 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, and the neutrophils are
sluggish. In another, there is a failure to produce , H2O2 and hypochlorite, and microbial killing power is reduced. This dysfunction results
from a deficiency of NADPH oxidase, leading to a deficient respiratory burst. Children with these dysfunctions are subject to persistent
bacterial infections. More severe infections result when neutrophil dysfunction and macrophage dysfunction occur simultaneously.

Eosinophils
Eosinophils are far less numerous than neutrophils, constituting only 24% of leukocytes in normal blood. In blood smears, this cell is about
the same size as a neutrophil, but with a characteristic bilobed nucleus (Figures 121, 126, and 129). The main identifying characteristic is
the abundance of large, red specific granules (about 200 per cell) that are stained by eosin.

Figure 129.

Eosinophils.
Eosinophils are about the same size as neutrophils but have bilobed nuclei and abundant coarse cytoplasmic granules. The cytoplasm is often filled with brightly
eosinophilic specific granules, but also includes some azurophilic granules. (a): Micrograph shows an eosinophil next to a neutrophil for comparison with its nucleus and
granules. X1500. Wright. (b): Even with granules filling the cytoplasm, the two nuclear lobes of eosinophils are usually clear. X1500. Giemsa. (c): TEM of a sectioned
eosinophil clearly shows the unique specific granules, as oval structures with disk-shaped electron-dense crystalline cores (EG). These along with lysosomes and a few
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 many having a flattened crystalline core (Figure 129)
containing major basic protein, an arginine-rich factor accounting for the granule's intense acidophilia. This protein constitutes 50% of the
total granule protein. The major basic protein, along with eosinophilic peroxidase, other enzymes and toxins, have cytotoxic effects on
parasites such as helminthic worms and protozoa. Eosinophils also phagocytose antigen-antibody complexes and modulate inflammatory
responses in many ways. They are an important source of the factors mediating allergic reactions and asthma.

MEDICAL APPLICATION

An increase in the number of eosinophils in blood (eosinophilia) is associated with allergic reactions and helminthic (parasitic) infections. In
tissues, eosinophils are found in the connective tissues underlying epithelia of the bronchi, gastrointestinal tract, uterus, and vagina, and
surrounding 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 about 1215 m in diameter, but make up less than 1% of blood leukocytes and are therefore difficult to find in smears of
normal blood. The nucleus is divided into two or more irregular lobes, but the large specific granules overlying the nucleus usually obscure its
shape.
The azurophilic specific granules (0.5 m in diameter) stain dark blue or metachromatically with the basic dye of blood smear stains and are
fewer and more irregular in size and shape than the granules of the other granulocytes (Figures 121, 126, and 1210). The metachromasia
is due to the presence of heparin and other sulfated glycosaminoglycans (GAGs) in the granules. Basophilic specific granules also contain
much histamine and various mediators of inflammation, including platelet activating factor, eosinophil chemotactic factor, and phospholipase
A which produces low molecular weight factors called leukotrienes.

Figure 1210.

Basophils.
(a, b, c): Basophils are approximately the same size as neutrophils and eosinophils, but have large, strongly basophilic specific granules which usually obstruct the
appearance of the nucleus having two or three irregular lobes. a and b: X1500, Wright; c: X1500, Giemsa. (d): TEM of a sectioned basophil reveals the lobulated
nucleus (N), appearing as three separated portions, the large specific basophilic granules (B), mitochondria (M), and Golgi complex (G). Basophils exert many activities
modulating the immune response and inflammation and share many functions with mast cells, which are normal, longer term residents of connective tissue. X16,000.
(Figure 1210d reproduced, with permission, from Terry R.W. et al: Lab. Invest 1969;21:65.)

By migrating into connective tissues, basophils may supplement the functions of mast cells, with which they share a common progenitor cell
origin. Both basophils and mast cells have metachromatic granules containing heparin and histamine, have IgE bound to surface receptors,
and secrete their granular components in response to certain antigens (see Chapter 5).

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.
In the dermatologic disease called cutaneous basophil hypersensitivity, basophils are the major cell type at the site of inflammation.

Lymphocytes
Lymphocytes constitute a family of leukocytes with spherical nuclei (Figures 121 and 126). They can be subdivided into functional groups
according to distinctive surface molecules (markers) that can best be distinguished immunocytochemically, notably T lymphocytes, B
lymphocytes, and natural killer (NK) cells. Lymphocytes have diverse functional roles related to immune defense against invading
microorganisms, foreign or abnormal antigens, and cancer cells. Additional information on the different types of lymphocytes and the
functional characteristics in immune responses is presented in Chapter 14.

Most lymphocytes in the blood are small with diameters of 68 m; medium and large lymphocytes range in size from 9 to 18 m in diameter.
Some larger lymphocytes may be cells that have been activated by specific antigens. The small lymphocytes that predominate in the blood
are characterized by spherical nuclei, sometimes indented, and condensed, very basophilic chromatin, making them easily distinguishable
from granulocytes.
The cytoplasm of the small lymphocyte is scanty, and in blood smears it appears as only a thin rim around the nucleus. It is slightly basophilic
and may contain a few azurophilic granules, along with a few mitochondria and a small Golgi apparatus; it contains free polyribosomes
(Figure 1211).

Figure 1211.

Lymphocytes.
Lymphocytes are agranulocytes and lack the specific granules characteristic of granulocytes. Lymphocytes circulating in blood range in size from 6 to 15 m 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 often 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), with a nucleolus (Nu), surrounded by cytoplasm containing
mitochondria (M), free polysomes, and a few lysosomes (azurophilic granules). X22,000.

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. They are the only type of leukocytes that, following diapedesis, can return from the tissues back to the blood.

Monocytes
Monocytes are bone marrowderived agranulocytes with diameters varying from 12 to 20 m. The nucleus is large, off-center, and may be
oval, kidney-shaped, or distinctly U-shaped (Figure 1212). The chromatin is less condensed than in lymphocytes and stains lighter than that
of large lymphocytes.

Figure 1212.

Monocytes.
Monocytes are large agranulocytes with diameters from 12 to 20 m that circulate as precursors to macrophages and other cells of the mononuclear phagocyte system.
(a, b, c, d): Micrographs of monocytes that show their eccentric nuclei indented, kidney-shaped, or U-shaped. a: X1500, Giemsa; bd: X1500, Wright. (e): TEM of 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 ribosomes (R). X22,000. (Figure 12-12e, with permission, from D.F. Bainton and M.G. Farquhar, Department of Pathology, University of California at San
Francisco.).

The cytoplasm of the monocyte is basophilic and contains very small azurophilic granules (lysosomes), 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. In the
electron microscope, nucleoli may be seen in the nucleus, and a small quantity of rough ER, free polyribosomes, and many small
mitochondria are observed. A Golgi apparatus involved in the formation of lysosomes is present and many microvilli and pinocytotic vesicles
are found at the cell surface (Figure 1212).
Circulating monocytes are precursor cells of the mononuclear phagocyte system (see Chapter 5). After crossing the walls of postcapillary
venules, monocytes differentiate into macrophages in connective tissues, microglia in the CNS, osteoclasts in bone, etc.

Platelets
Blood platelets (thrombocytes) are nonnucleated, disklike cell fragments 24 m in diameter. Platelets originate by fragmentation at the ends
of cytoplasmic processes extending from giant polyploid cells called megakaryocytes in the bone marrow (Chapter 13). Platelets promote
blood clotting and help repair minor tears or leaks in the walls of blood vessels, preventing loss of blood. Normal platelet counts range from

200,000 to 400,000 per microliter of blood. Platelets have a life span of about 10 days.
In stained blood smears, platelets often appear in clumps. Each platelet has a lightly stained peripheral zone, the hyalomere, and a central
zone containing darker-staining granules, called the granulomere (Figure 1213).

Figure 1213.

Platelets.
Platelets are cell fragments 24 m 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 typically shows a system of microtubules and actin filaments near
the periphery to help maintain its shape and an open canalicular system of vesicles continuous with the plasmalemma. The central granulomere region contains
glycogen and secretory granules of different types. X40,000. (c): TEM section shows platelets adhering to collagen (C). Upon adhesion to collagen, platelets exocytose
their granules into the canalicular system, which allows the very rapid secretion of factors involved in blood coagulation. Degranulating platelets (arrows) remain as an
aggregate until their contents are exhausted. Other proteins involved in coagulation come from the plasma and from processes of adjacent endothelial cells (EP). The
electron-dense structure on the right is part of an erythrocyte. X;7500. (Figure 12-13b, with permission, from Dr. M. J. G. Harrison, Middlesex Hospital and University
College London.).

A coat rich in GAGs and glycoproteins, 1520 nm thick, lies outside the plasmalemma and is involved in platelet adhesion. Ultrastructural
analysis (Figure 1213) reveals around the platelet periphery a marginal bundle of microtubules and microfilaments which helps to maintain
the platelet's ovoid shape. Also in the hyalomere are two systems of membrane channels. An open canalicular system of vesicles connected
to invaginations of the plasma membrane, which may facilitate platelets' uptake of factors such as fibrinogen and serotonin from plasma.
Another 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 vascular endothelium.

The central granulomere possesses a variety of membrane-bound granules and a sparse population of mitochondria and glycogen particles
(Figure 1213). Electron-dense delta granules, 250300 nm in diameter, contain adenosine diphosphate (ADP), adenosine triphosphate
(ATP), and serotonin (5-hydroxytryptamine) taken up from plasma. Alpha granules are larger (300500 nm in diameter) and contain plateletderived growth factor, platelet factor 4, and several other platelet-specific proteins. Most of the stained granules seen with the light
microscope in platelets are alpha granules. Small vesicles, 175250 nm in diameter, have been shown to contain only lysosomal enzymes
and have been termed lambda granules.
The role of platelets in controlling hemorrhage can be summarized as follows:
Primary aggregation. Disruptions in the microvascular endothelium, which are common, allow platelet aggregation to collagen
via collagen-binding protein in the platelet membrane. Thus, a platelet plug is formed as a first step to stop bleeding (Figure 12
13c).
Secondary aggregation. Platelets in the plug release an adhesive glycoprotein and ADP, both of which are potent inducers of
platelet aggregation, increasing the size of the platelet plug.
Blood coagulation. During platelet aggregation, fibrinogen from plasma, von Willebrand factor and others from damaged
endothelium, and various factors from platelets 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 1214).
Clot retraction. The clot that initially bulges into the blood vessel lumen contracts slightly because of the interaction of platelet
actin and myosin.
Clot removal. Protected by the clot, the vessel wall is restored by new tissue, and the clot is then removed, mainly by the
proteolytic enzyme plasmin, formed continuously through the local action of plasminogen activators from the endothelium on
plasminogen from plasma. Enzymes released from platelet lambda granules also contribute to clot removal.


Figure 1214.

Fibrin clot.
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 meshwork of
polymeric proteins composed largely of fibrin traps erythrocytes 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.

MEDICAL APPLICATION
Hemophilia A and B are clinically identical, differing only in the deficient factor. Both are due to sex-linked recessive inherited disorders.
Blood from hemophiliac patients does not coagulate normally: the blood clotting time is prolonged. Persons with this disease bleed severely
even after mild injuries, such as a skin cut, and may bleed to death after more severe injuries. The blood plasma of patients with hemophilia
A is deficient in clotting factor VIII or contains a defective factor VIII, one of the plasma proteins involved in fibrin generation; in hemophilia B,
the defect is in factor IX. In severe cases the blood is incoagulable. There are spontaneous hemorrhages in body cavities, such as major
joints and the urinary tract. Generally, only males are affected by hemophilia A, because the recessive gene to factor VIII is on the X
chromosome. Females may have one defective X chromosome, but the other one is usually normal. Females develop hemophilia only when
they have the abnormal gene in both X chromosomes, a rare event. However, women with a defective X chromosome may transmit the
disease to their male children.