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  Chapter 13 - Red Blood Cell and Bleeding Disorders

Jon C. Aster MD, PhD


   •   Chapter 13 - Red Blood Cell and Bleeding Disorders
                 • Normal
                         – Normal Development of Blood Cells
                               » ORIGIN AND DIFFERENTIATION OF
                                    HEMATOPOIETIC CELLS
                                       • Anatomy of Bone Marrow.
                                       • Morphology.
                 • Pathology
                         – Anemias
                               » ANEMIAS OF BLOOD LOSS
                                       • Acute Blood Loss
                                       • Chronic Blood Loss
                               » HEMOLYTIC ANEMIAS
                                       • Morphology.
                                       • Hereditary Spherocytosis (HS)
                                              • Molecular Pathology.
                                              • Morphology.
                                              • Clinical Course.
                                       • Hemolytic Disease Due to Red Cell Enzyme
                                          Defects: Glucose-6-Phosphate
                                          Dehydrogenase Deficiency
                                       • Sickle Cell Disease
                                              • Pathogenesis.
                                              • Morphology.
                                              • Clinical Course.
                                       • Thalassemia Syndromes
                                       • β-Thalassemias
                                              • Molecular Pathogenesis.
                                              • Clinical Syndromes.
                                       • Thalassemia Major.
                                       • Morphology.
                                       • Thalassemia Minor.
                                       • α-Thalassemias
                                       • Clinical Syndromes.
                                       • Silent Carrier State.
                                       • α-Thalassemia Trait.
                                       • Hemoglobin H Disease.
                                       • Hydrops Fetalis.
              •   Paroxysmal Nocturnal Hemoglobinuria
              •   Immunohemolytic Anemia
              •   Warm Antibody Immunohemolytic Anemia.
              •   Cold Agglutinin Immunohemolytic Anemia.
              •   Cold Hemolysin Hemolytic Anemia.
              •   Hemolytic Anemia Resulting from Trauma
                  to Red Cells
       » ANEMIAS OF DIMINISHED ERYTHROPOIESIS
              • Megaloblastic Anemias
                      • Morphology.
              • Anemias of Vitamin B12 Deficiency:
                  Pernicious Anemia
                      • Normal Vitamin B12 Metabolism.
                      • Etiology of Vitamin B12 Deficiency.
                      • Biochemical Functions of Vitamin
                         B12 .
                      • Incidence.
                      • Pathogenesis.
                      • Morphology.
                      • Clinical Course.
              • Anemia of Folate Deficiency
                      • Etiology.
              • Iron Deficiency Anemia
                      • Iron Metabolism.
                      • Etiology.
                      • Morphology.
              • Anemia of Chronic Disease
              • Aplastic Anemia
                      • Etiology.
                      • Pathogenesis.
                      • Morphology.
                      • Clinical Course.
              • Pure Red Cell Aplasia
              • Other Forms of Marrow Failure
–   Polycythemia
–   Bleeding Disorders: Hemorrhagic Diatheses
       » BLEEDING DISORDERS CAUSED BY VESSEL
           WALL ABNORMALITIES
       » BLEEDING RELATED TO REDUCED
           PLATELET NUMBER: THROMBOCYTOPENIA
              • Immune Thrombocytopenic Purpura (ITP)
              • Pathogenesis.
              • Morphology.
              • Clinical Features.
              • Acute Immune Thrombocytopenic Purpura
                                           •Drug-Induced Thrombocytopenia: Heparin-
                                            Induced Thrombocytopenia
                                          • HIV-Associated Thrombocytopenia
                                          • Thrombotic Microangiopathies: Thrombotic
                                            Thrombocytopenic Purpura (TTP) and
                                            Hemolytic-Uremic Syndrome (HUS)
                                   »   BLEEDING DISORDERS RELATED TO
                                       DEFECTIVE PLATELET FUNCTIONS
                                   »   HEMORRHAGIC DIATHESES RELATED TO
                                       ABNORMALITIES IN CLOTTING FACTORS
                                          • Deficiencies of Factor VIII-vWF Complex
                                          • Von Willebrand Disease
                                          • Hemophilia A (Factor VIII Deficiency)
                                          • Hemophilia B (Christmas Disease, Factor IX
                                            Deficiency)
                                   »   DISSEMINATED INTRAVASCULAR
                                       COAGULATION (DIC)
                                          • Etiology and Pathogenesis.
                                          • Morphology.
                                          • Clinical Course.



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The organs and tissues involved in hematopoiesis have been traditionally divided into
myeloid tissue, which includes the bone marrow and the cells derived from it (e.g.,
erythrocytes, platelets, granulocytes, and monocytes), and lymphoid tissue, consisting of
thymus, lymph nodes, and spleen. This subdivision is artificial with respect to both the
normal physiology of hematopoietic cells and the diseases affecting them. For example,
although bone marrow is not where most mature lymphoid cells are found, it is the source
of lymphoid stem cells. Similarly, myeloid leukemias, neoplastic disorders of myeloid
stem cells, originate in the bone marrow but secondarily involve the spleen and (to a
lesser degree) lymph nodes. Some red cell disorders (hemolytic anemias) result from the
formation of autoantibodies, signifying a primary disorder of lymphocytes. Thus, it is not
possible to draw neat lines between diseases involving the myeloid and lymphoid tissues.
Recognizing this difficulty, we somewhat arbitrarily divide diseases of the hematopoietic
tissues into two chapters. In the first, we consider diseases of red cells and those affecting
hemostasis. In the second, we discuss white cell diseases and disorders affecting
primarily the spleen and thymus.

Normal
A complete discussion of normal hematopoiesis is beyond our scope, but certain features
are helpful to an understanding of the diseases of blood.
Normal Development of Blood Cells

Blood cells first appear during the third week of fetal embryonic development in the yolk
sac, but these cells are generated from a primitive stem cell population restricted to the
production of myeloid cells. The origin of definitive hematopoietic stem cells that give
rise to lymphoid and myeloid cells is still unsettled. Most studies suggest they arise in the
mesoderm of the intraembryonic aorta/gonad/mesonephros (AGM) region,[1] but evidence
also exists for an origin within a small subset of yolk sac-derived cells. By the third
month of embryogenesis, stem cells derived from the AGM and/or yolk sac migrate to
the liver, which is the chief site of blood cell formation until shortly before birth.
Beginning in the fourth month of development, stem cells migrate to the bone marrow to
commence hematopoiesis at this site. By birth, marrow throughout the skeleton is
hematopoietically active and virtually the sole source of blood cells. In fullterm infants,
hepatic hematopoiesis dwindles to a trickle, persisting only in widely scattered small foci
that become inactive soon after birth. Up to the age of puberty, marrow throughout the
skeleton remains red and hematopoietically active. By age 18 only the vertebrae, ribs,
sternum, skull, pelvis, and proximal epiphyseal regions of the humerus and femur retain
red marrow, the remaining marrow becoming yellow, fatty, and inactive. Thus, in adults,
only about half of the marrow space is active in hematopoiesis.

Several features of this normal sequence should be emphasized. By birth, the bone
marrow is virtually the sole source of all forms of blood cells, including lymphocyte
precursors. In the premature infant, foci of hematopoiesis are frequently evident in the
liver and, rarely, in the spleen, lymph nodes, or thymus. Significant postembryonic
extramedullary hematopoiesis is abnormal in the full-term infant. With an increased
demand for blood cells in the adult, the fatty marrow can transform to red, active marrow.
For example, in the face of red cell deficiency (anemia), the marrow can increase red cell
production (erythropoiesis) as much as eight-fold. If the marrow stem cells and
microenvironment are normal and the necessary nutrients are available (e.g., adequate
amounts of iron, protein, requisite vitamins), premature loss of red cells (as occurs in
hemolytic disorders) produces anemia only when marrow compensatory mechanisms are
outstripped. Under these circumstances, extramedullary hematopoiesis can reappear
within the spleen, liver, and even lymph nodes.
ORIGIN AND DIFFERENTIATION OF HEMATOPOIETIC CELLS


The formed elements of blood—red cells, granulocytes, monocytes, platelets, and
lymphocytes—have a common origin from pluripotent hematopoietic stem cells sitting at
the apex of a complex hierarchy of progenitors ( Fig. 13-1 ). Most of the work supporting
this scheme comes from studies conducted in mice, but it is believed hematopoiesis in
man proceeds in a highly analogous fashion. The pluripotent stem cell gives rise to two
types of multipotent progenitors, the common lymphoid and the common myeloid stem
cell. The common lymphoid stem cell in turn gives rise to precursors of T cells (pro-T
cells), B cells (pro-B cells), and natural killer cells.[2] The details of lymphoid
differentiation are not discussed here, but it is worth pointing out that morphologic
distinctions among lymphoid cells at various stages of differentiation are subtle at best.
As a result, monoclonal antibodies recognizing differentiation-stage-specific antigens are
used widely to define normal lymphocyte subsets ( Chapter 14 ). From the common
myeloid stem cell arise at least three types of committed stem cells capable of
differentiating along the erythroid/megakaryocytic, eosinophilic, and granulocyte-
macrophage pathways.[3] In functional assays the committed stem cells are called colony-
forming units (CFU), because each can give rise to colonies of differentiated progeny in
vitro (see Fig. 13-1 ). From the various committed stem cells are derived intermediate
stages and ultimately the morphologically recognizable precursors of the differentiated
cells, such as proerythroblasts, myeloblasts, megakaryoblasts, monoblasts, and
eosinophiloblasts, which in turn give rise to mature progeny.

The specific characteristics of rare cells lying high up in the hierarchy shown in Figure
13-1 are still debated. What are agreed upon are certain overarching themes that apply to
hematopoiesis. Since mature blood elements are terminally differentiated cells with finite
life spans, their numbers must be replenished constantly. It follows that stem cells must
not only differentiate, but also self-renew, a critical property of stem cells. Pluripotent
stem cells have the greatest capacity for self-renewal, but normally most are not in cell
cycle. As commitment to particular lines of differentiation proceeds, self-renewal
becomes limited, but a greater fraction of committed cells divide actively. For example,
few common myeloid stem cells are normally in cell cycle, but up to 50% of CFU-GM



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Figure 13-1 Differentiation of hematopoietic cells. SCF, stem cell factor; Flt3L, Flt3 ligand; GM-CSF,
granulocyte-macrophage colonys-timulating factor; M-CSF, macrophage colony-stimulating factor; G-
CSF, granulocyte colony-stimulating factor. (Modified from Wyngaarden JB, et al [eds]: Cecil Textbook of
Medicine, 19th ed. Philadelphia, WB Saunders, 1992, p. 820.)
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(the precursors of granulocytes and macrophages) are actively dividing. This suggests
that pools of differentiated cells are replenished mainly by lineage-restricted stem cells.
Although the earliest morphologically recognizable precursors (e.g., myeloblasts or
proerythroblasts) also actively proliferate, they cannot self-renew, and eventually all of
their progeny differentiate and "die." By definition, then, they do not have the properties
of stem cells.

Many disorders of the marrow, including marrow failure (aplastic anemias) and
hematopoietic neoplasms (e.g., leukemias) are caused by stem cell dysfunction, and there
is thus great interest in the physiologic mechanisms regulating the proliferation and
differentiation of progenitor cells. These processes involve soluble factors and
hematopoietic cell-stromal cell interactions in the bone marrow. Among the
hematopoietic growth factors, some, such as stem cell factor (also called c-KIT ligand)
and FLT3-ligand, act on very early stem cells. Others, such as the granulocyte-
macrophage colony-stimulating factor (GM-CSF), act on CFU-GM. Some recombinant
factors are currently being used to stimulate hematopoiesis, including erythropoietin,
GM-CSF, G-CSF, and thrombopoietin.

Bone marrow-derived stem cells have a number of surprising properties. Although
residing mainly in the marrow, a subset circulates normally in the peripheral blood.
Hence, hematopoiesis occurs in the marrow because its specialized environment fosters
stem cell homing, survival, and differentiation, not because stem cells are restricted to
this site. The homing of stem cells, which involves surface adhesion molecules, makes it
possible to perform bone marrow transplantation by simply infusing donor stem cells into
the peripheral blood. More remarkably, circulating marrow-derived stem cells can "seed"
other tissues and develop into nonhematopoietic cells as well. The best characterized and
most widely accepted of these alternative "fates" is the differentiation of marrow stem
cells into endothelial cell precursors (hemangioblasts) [4] , which in turn give rise to
endothelial cells. This capacity is not surprising, given the close functional relationship of
blood elements and the cardiovascular system. In fact, many genes involved in the
development of hematopoietic cells also participate in the development of blood vessels
and endothelial cells. More controversial studies suggest that bone marrow-derived stem
cells can also differentiate into hepatocytes, bile duct cells, myocardium, skeletal muscle,
endothelial cells, glia, and even neurons directly.[5] [6] Other explanations for these results,
such as fusion of marrow stem cells to these other mature cell types or contamination of
hematopoietic stem cells with other types of stem cells,[4] [7] still need to be excluded (
Chapter 3 ). Nonetheless, it is hoped that marrow-derived stem cells will have sufficient
plasticity to permit their use in a variety of stem cell-based therapies.
Anatomy of Bone Marrow.


The bone marrow provides a unique microenvironment for the orderly proliferation,
differentiation, and release of blood cells. Under the electron microscope, the marrow
cavity is a vast network of thin-walled sinusoids lined by a single layer of endothelial
cells underlaid by a discontinuous layer of basement membrane and adventitial cells.
Within the interstitium lie clusters of hematopoietic cells and fat cells. Differentiated
blood cells enter sinusoids by transcellular migration through the endothelial cells. The
normal marrow is organized anatomically in subtle but important ways. For example,
normal megakaryocytes lie next to sinusoids and extend cytoplasmic processes that bud
off into the bloodstream to produce platelets. Similarly, normal immature granulocytic
myeloid forms are concentrated next to bone trabeculae, while mature granulocytes are
located more centrally. Diseases that distort the marrow architecture, such as deposits of
metastatic cancer or granulomatous disease, disturb normal function. In such instances,
an abnormal release of immature precursors into the peripheral blood can occur that is
referred to as "leukoerythroblastosis."
Morphology.


Although the morphology of hematopoietic cells within the bone marrow is best studied
in smears of marrow aspirates, additional complementary information is obtained from
bone marrow biopsy specimens. For example, a reasonable estimate of marrow activity is
obtained by examining the ratio of fat cells to hematopoietic elements in bone marrow
biopsy samples. In normal adults, this ratio is about 1:1, but with marrow hypoplasia
(e.g., aplastic anemia), the proportion of fat cells is greatly increased; conversely, fat cells
may disappear completely in diseases characterized by increased hematopoiesis (e.g.,
hemolytic anemias). Also, certain disorders (such as metastatic cancers and
granulomatous diseases) induce local marrow fibrosis, rendering the lesional cells
"inaspirable"; here too, a biopsy is the examination of choice. The limitation of biopsies
is that tissue fixation and decalcification alter the appearance of marrow cells, making
them less recognizable than in air-dried aspirate smears. However, it is not always
possible to differentiate the various "blast" forms morphologically, even in aspirate
smears. Often, tentative identification is based on "the company they keep." Thus, a
primitive cell found within a focus of maturing granulocytes is likely a myeloblast.
Pluripotent and multipotent stem cells are morphologically inconspicuous lymphocyte-
like cells constituting less than 0.1% of the marrow cellularity. Stem cells are identified
and purified away from other cell types using antibodies against discriminating markers
(e.g., CD34).

The relative proportion of hematopoietic precursors is almost always deranged in
diseases of the blood and bone marrow, which normally contains about 65% granulocytes
and their precursors; 25% erythroid precursors; and 10% lymphocytes and monocytes
and their precursors. Thus, the normal myeloid to erythroid ratio is 2 to 3:1. Prevalent cell
types in the myeloid compartment include myelocytes, metamyelocytes, and
granulocytes. In the erythroid compartment, the most common forms are
polychromatophilic and orthochromic normoblasts.
Pathology
Anemias

The function of red cells is to transport oxygen to peripheral tissues. Reduced oxygen-
carrying capacity of blood usually results from a deficiency of red cells, or anemia,
defined as a reduction below normal limits of the total circulating



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red cell mass. Measurement of red cell mass is not easy, however, and in routine practice
anemia is defined as a reduction below normal in the volume of packed red cells, as
measured by the hematocrit, or a reduction in the hemoglobin concentration of the blood.
On occasion, fluid retention can expand plasma volume and dehydration can contract
plasma volume, creating spurious abnormalities in these values.

There are innumerable classifications of anemia. An acceptable one based on underlying
mechanisms is presented in Table 13-1 . A second useful approach classifies anemia
according to alterations in red cell morphology, which often correlates with the cause of
red cell deficiency. Morphologic characteristics providing etiologic clues include red cell
size

     TABLE 13-1 -- Classification of Anemia According to Underlying Mechanism
Blood Loss
Acute: trauma
Chronic: lesions of gastrointestinal tract, gynecologic disturbances
Increased Rate of Destruction (Hemolytic Anemias)
Intrinsic (intracorpuscular) abnormalities of red cells
  Hereditary
Red cell membrane disorders
Disorders of membrane cytoskeleton:         spherocytosis, elliptocytosis
Disorders of lipid synthesis:      selective increase in membrane lecithin
Red cell enzyme deficiencies
Glycolytic enzymes: pyruvate kinase         deficiency, hexokinase deficiency
Enzymes of hexose monophosphate            shunt: G6PD, glutathione synthetase
Disorders of hemoglobin       synthesis
Deficient globin synthesis:       thalassemia syndromes
Structurally abnormal globin        synthesis (hemoglobinopathies): sickle cell anemia,
unstable hemoglobins
  Acquired
Membrane defect: paroxysmal nocturnal          hemoglobinuria
Extrinsic (extracorpuscular) abnormalities
Antibody mediated
Isohemagglutinins: transfusion          reactions, erythroblastosis fetalis
Autoantibodies: idiopathic (primary),   drug-associated, systemic lupus
erythematosus, malignant neoplasms, mycoplasmal infection
Mechanical trauma to red      cells
Microangiopathic hemolytic anemias:            thrombotic thrombocytopenic purpura,
disseminated intravascular coagulation
Cardiac traumatic hemolytic           anemia
Infections: malaria, hookworm
Chemical injury: lead    poisoning
Sequestration in mononuclear phagocyte         system: hypersplenism
Impaired Red Cell Production
Disturbance of proliferation and differentiation of stem cells: aplastic anemia, pure red
cell aplasia, anemia of renal failure, anemia of endocrine disorders
Disturbance of proliferation and maturation of erythroblasts
Defective DNA synthesis: deficiency or         impaired use of vitamin B12 and folic acid
(megaloblastic anemias)
Defective hemoglobin      synthesis
Deficient heme synthesis: iron         deficiency
Deficient globin synthesis:       thalassemias
Unknown or multiple mechanisms: sideroblastic anemia, anemia of chronic infections,
myelophthisic anemias due to marrow infiltrations


(normocytic, microcytic, or macrocytic); degree of hemoglobinization, reflected in the
color of red cells (normochromic or hypochromic); and other special features, such as
shape. These red cell indices are often judged qualitatively by physicians, but precise
quantitation is done in clinical laboratories using special instrumentation. The most useful
red cell indices are as follows:

       • Mean cell volume: the average volume of a red blood cell, expressed in
       femtoliters (cubic micrometers)
          • Mean cell hemoglobin: the average content (mass) of hemoglobin per red blood
          cell, expressed in picograms
          • Mean cell hemoglobin concentration: the average concentration of hemoglobin
          in a given volume of packed red blood cells, expressed in grams per deciliter
          • Red blood cell distribution width: the coefficient of variation of red blood cell
          volume

Adult reference ranges for red cell indices are shown in Table 13-2 .

Whatever its cause, anemia leads to certain clinical features when sufficiently severe.
Patients appear pale. Weakness, malaise, and easy fatigability are common complaints.
The lowered oxygen content of the circulating blood leads to dyspnea on mild exertion.
The nails can become brittle, lose their usual convexity, and assume a concave spoon
shape (koilonychia). Anoxia can cause fatty change in the liver, myocardium, and kidney.
If fatty changes in the myocardium are sufficiently severe, cardiac failure can develop
and compound the respiratory difficulty caused by reduced oxygen transport. On
occasion, the myocardial hypoxia manifests as angina pectoris, particularly when
complicated by preexisting coronary artery disease. With acute blood loss and shock,
oliguria and anuria can develop due to renal hypoperfusion. Central nervous system
hypoxia can cause headache, dimness of vision, and faintness.
ANEMIAS OF BLOOD LOSS

Acute Blood Loss


The clinical and morphologic reactions to blood loss depend on the rate of hemorrhage
and whether the bleeding
                                                                                          *
                   TABLE 13-2 -- Adult Reference Ranges for Red Blood Cells
Measurement (units)                                                     Men                   Women
Hemoglobin (gm/dL)                                                   13.6–17.2                12.0–15.0
Hematocrit (%)                                                         39–49                   33–43
Red cell count (106 /µL)                                              4.3–5.9                  3.5–5.0
Reticulocyte count (%)                                                           0.5–1.5
Mean cell volume (µm3 )                                                           82–96
Mean corpuscular hemoglobin (pg)                                                  27–33
Mean corpuscular hemoglobin concentration                                         33–37
(gm/dL)
RBC distribution width                                                          11.5–14.5
RBC, red blood cell.

*Reference ranges vary among laboratories. The reference ranges for the laboratory providing the result
should always be used in interpreting the test result.
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is external or internal. The effects of acute blood loss are mainly due to the loss of
intravascular volume, which can lead to cardiovascular collapse, shock, and death. If the
patient survives, the blood volume is rapidly restored by shift of water from the
interstitial fluid compartment. The resulting hemodilution lowers the hematocrit.
Reduction in the oxygenation of renal juxtaglomerular cells triggers increased production
of erythropoietin, which stimulates the proliferation of committed erythroid stem cells
(CFU-E) in the marrow. It takes about 5 days for the progeny of these CFU-Es to fully
differentiate, an event marked by the appearance of increased numbers of newly released
red cells (reticulocytes) in the peripheral blood. The iron in hemoglobin is recaptured if
red cells are lost internally, as into the peritoneal cavity, but external bleeding leads to
iron loss and possible iron deficiency, which can hamper restoration of normal red cell
counts.

The earliest change in the peripheral blood immediately after acute blood loss is
leukocytosis, due to the mobilization of granulocytes from marginal pools. Initially, red
cells appear normal in size and color (normocytic, normochromic). However, as marrow
production increases, there is a striking increase in the reticulocyte count, reaching 10%
to 15% after 7 days. Reticulocytes are recognizable as polychromatophilic macrocytes in
the usual blood smear. Early recovery from blood loss is often accompanied by
thrombocytosis, which is caused by increased platelet production.
Chronic Blood Loss


Chronic blood loss induces anemia only when the rate of loss exceeds the regenerative
capacity of the marrow or when iron reserves are depleted. Iron deficiency anemia, which
has identical features regardless of underlying cause (e.g., bleeding, malnutrition,
malabsorption states), will be discussed later.
HEMOLYTIC ANEMIAS


Hemolytic anemias share the following features:

          • A shortened red cell life span (normal = 120 days); that is, premature
          destruction of red cells
          • Elevated erythropoietin levels and increased erythropoiesis in the marrow and
          other sites, to compensate for the loss of red cells
          • Accumulation of the products of hemoglobin catabolism, due to an increased
          rate of red cell destruction
The physiologic destruction of senescent red cells takes place within the mononuclear
phagocytic cells of the spleen. In the great majority of hemolytic anemias, the premature
destruction of red cells also occurs within the mononuclear phagocyte system
(extravascular hemolysis), which undergoes a form of work-related hyperplasia marked
by splenomegaly. Much less commonly, lysis of red cells within the vascular
compartment (intravascular hemolysis) predominates.

Intravascular hemolysis of red cells is caused by mechanical injury, complement fixation,
infection by intracellular parasites such as falciparum malaria ( Chapter 8 ), or exogenous
toxic factors. Mechanical injury caused by defective cardiac valves, thrombi within the
microcirculation, or repetitive physical trauma (marathon running, bongo drum beating)
can physically lyse red cells. Complement fixation can occur on antibody-coated cells
during transfusion of mismatched blood. Toxic injury is exemplified by clostridial sepsis,
which releases toxins that attack the red cell membrane.

Whatever the mechanism, intravascular hemolysis is manifested by (1) hemoglobinemia,
(2) hemoglobinuria, (3) jaundice, and (4) hemosiderinuria. Free hemoglobin in plasma is
promptly bound by an α2 -globulin (haptoglobin), producing a complex that is rapidly
cleared by the mononuclear phagocyte system, thus preventing excretion into the urine.
Decreased serum haptoglobin is characteristic of intravascular hemolysis. When the
haptoglobin is depleted, free hemoglobin is prone to oxidation to methemoglobin, which
is brown in color. The renal proximal tubular cells reabsorb and catabolize much of the
filtered hemoglobin and methemoglobin, but some passes out with the urine, imparting a
red-brown color. Iron released from hemoglobin can accumulate within tubular cells,
giving rise to renal hemosiderosis. Concomitantly, heme groups derived from the
complexes are catabolized to bilirubin within the mononuclear phagocyte system, leading
to jaundice. In hemolytic anemias, the serum bilirubin is unconjugated and the level of
hyperbilirubinemia depends on the functional capacity of the liver and the rate of
hemolysis. When the liver is normal, jaundice is rarely severe. Excessive bilirubin
excreted by the liver into the gastrointestinal tract leads to increased formation and fecal
excretion of urobilin ( Chapter 18 ).

Extravascular hemolysis takes place whenever red cells are rendered "foreign" or become
less deformable. Since extreme alterations in shape are required for red cells to navigate
the splenic sinusoids successfully, reduced deformability makes the passage difficult and
leads to sequestration within the cords, followed by phagocytosis ( Fig. 13-2 ). This is an
important pathogenetic mechanism of extravascular hemolysis in a variety of hemolytic
anemias. With extravascular hemolysis,
Figure 13-2 Schematic of splenic sinus (electron micrograph). A red cell is in the process of squeezing
from the red pulp cords into the sinus lumen. Note the degree of deformability required for red cells to pass
through the wall of the sinus.




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hemoglobinemia and hemoglobinuria are not observed, and its principal features are
anemia and jaundice. However, some hemoglobin inevitably escapes from phagocytes,
leading to decreases in plasma haptoglobin. The morphologic changes are identical to
those in intravascular hemolysis, except that "work" hyperplasia of the mononuclear
phagocyte system often leads to splenomegaly.
Morphology.


Certain morphologic changes are common in hemolytic anemias, regardless of cause or
type. Anemia and lowered tissue oxygen tension stimulate increased production of
erythropoietin, which leads to the appearance of increased numbers of erythroid
precursors (normoblasts) in the marrow ( Fig. 13-3 ). If the anemia is severe,
extramedullary hematopoiesis can appear in the liver, spleen, and lymph nodes. The
accelerated erythropoiesis leads to a prominent reticulocytosis in the peripheral blood.
Elevated biliary excretion of bilirubin promotes the formation of pigment gallstones
(cholelithiasis). If chronic, phagocytosis of red cells leads to hemosiderosis, usually
confined to the mononuclear phagocyte system.

The hemolytic anemias are classified in a variety of ways. One has already been
mentioned, namely, division into intravascular and extravascular hemolytic disorders.
However, since disorders with predominantly intravascular hemolysis are quite
uncommon, this classification is not entirely satisfactory. A second pathogenetic
classification is based on whether the underlying cause of red cell destruction is extrinsic
(extracorpuscular mechanism) or intrinsic to the red cell (intracorpuscular defect). These
anemias can also be divided into hereditary and acquired disorders. In general, hereditary
disorders are due to intrinsic defects and the acquired disorders to extrinsic factors such
as autoantibodies. Each of the classifications has value. Here we follow the intrinsic-
extrinsic outline given in Table 13-1 , limiting our discussion to the more common
entities.




Figure 13-3 Marrow smear from a patient with hemolytic anemia. The marrow reveals greatly increased
numbers of maturing erythroid progenitors (normoblasts). (Courtesy of Dr. Steven Kroft, Department of
Pathology, University of Texas Southwestern Medical School, Dallas, TX.)


Hereditary Spherocytosis (HS)


This inherited disorder is caused by intrinsic defects in the red cell membrane that render
red cells spheroid, less deformable, and vulnerable to splenic sequestration and
destruction. The prevalence of HS is highest in northern Europe, where rates of 1 in 5000
are reported. An autosomal dominant inheritance pattern is seen in three fourths of cases.
The remaining patients have a more severe autosomal recessive form of the disease.
Molecular Pathology.


The remarkable elasticity and durability of the normal red cell are attributable to the
physicochemical properties of its specialized membrane skeleton ( Fig. 13-4 ), which lies
closely apposed to the internal surface of the plasma membrane. Its chief protein
component, spectrin, consists of two polypeptide chains, α and β, which form intertwined
(helical) flexible heterodimers. The "head" regions of spectrin dimers self-associate to
form tetramers, while the "tails" associate with actin oligomers. Each actin oligomer can
bind multiple spectrin tetramers, thus creating a 2-dimensional spectrin-actin skeleton
that is connected to the cell membrane by two distinct interactions. The first, involving
the proteins ankyrin and band 4.2, binds spectrin to the transmembrane ion transporter,
band 3. The second, involving protein 4.1, binds the "tail" of spectrin to another
transmembrane protein, glycophorin A.

HS is caused by diverse mutations affecting ankyrin, band 3, spectrin, or band 4.2, the
proteins involved in the first of these two tethering interactions, [8] presumably because
this complex is particularly important in stabilizing the lipid bilayer. The most common
cause of autosomal dominant HS is mutation of red cell ankyrin.[9] Another 20% of
autosomal dominant HS cases are caused by mutations in band 3.[10] The remaining cases
are associated mostly with mutations in α-spectrin, β-spectrin, or band 4.2. Regardless of
the molecular defect, reduced membrane stability leads to loss of membrane fragments
during exposure to shear stresses in the circulation (see Fig. 13-4 ). The loss of
membrane relative to cytoplasm "forces" the cells to assume the smallest possible
diameter for a given volume, namely, a sphere.

Although there is much to learn about the molecular defects in HS, the travails of the
spherocytic red cells are fairly well defined ( Fig. 13-5 ). In the life of the "portly,"
inflexible spherocyte, the spleen is the villain. Red cells must undergo extreme
deformation to leave the cords of Billroth and enter the sinusoids. Because of their
spheroidal shape and reduced membrane plasticity, spherocytes attempting to squeeze out
of the cords are like an "obese man attempting to bend at the waist."[11] As spherocytes are
trapped in the spleen, the already sluggish circulation of the cords stagnates further,
producing a progressively more hostile environment. Lactic acid accumulates and the pH
falls, inhibiting glycolysis. The inability to generate adenosine triphosphate impairs the
ability of red cells to extrude sodium, adding an element of osmotic injury. Stagnation in
the cords also promotes contact with plentiful macrophages, which phagocytose the
hapless spherocytes. The cardinal role of the spleen in the premature demise of the
spherocytes is proved by the invariably beneficial effect of splenectomy. The spherocytes
persist, but the anemia is corrected.



                                            626
Figure 13-4 Schematic representation of the red cell membrane cytoskeleton and alterations leading to
spherocytosis and hemolysis. Mutations weakening interactions involving α-spectrin, β-spectrin, ankyrin,
band 4.2, or band 3 all cause the normal biconcave red cell to lose membrane fragments and adopt a
spherical shape. Such spherocytic cells are less deformable than normal and therefore become trapped in
the splenic cords, where they are phagocytosed by macrophages.


Morphology.


The most outstanding morphologic finding in this disease are spherocytes, apparent on
smears as abnormally small, dark-staining (hyperchromic) red cells lacking the normal
central zone of pallor ( Fig. 13-6 ). Spherocytosis, although distinctive, is not
pathognomonic, as it is also seen in autoimmune hemolytic anemias. Present also are
changes associated with all hemolytic anemias, including reticulocytosis, marrow
hyperplasia due to increased
Figure 13-5 Model of the pathophysiology of hereditary spherocytosis. (Adapted from Wyngaarden JB, et
al [eds]: Cecil Textbook of Medicine, 19th ed. Philadelphia, WB Saunders, 1992, p. 859.)



erythropoiesis, hemosiderosis, and mild jaundice. Cholelithiasis (pigment stones) occurs
in 40% to 50% of the affected adults. Other alterations are fairly distinctive. Moderate
splenic enlargement is characteristic (500 to 1000 gm); in few other hemolytic anemias is
the spleen enlarged as much or as often. It results from congestion of the cords of Billroth
and "work hyperplasia" due to markedly increased erythrophagocytosis.


                                                 627
Figure 13-6 Hereditary spherocytosis (peripheral smear). Note the anisocytosis and several dark-appearing
spherocytes with no central pallor. Howell-Jolly bodies (small dark nuclear remnants) are also present in
red cells of this asplenic patient. (Courtesy of Dr. Robert W. McKenna, Department of Pathology,
University of Texas Southwestern Medical School, Dallas, TX.)


Clinical Course.


The characteristic clinical features are anemia, splenomegaly, and jaundice. The severity
of the disease varies greatly from one patient to another. In a minority, HS presents at
birth with marked jaundice, requiring exchange transfusion. In 20% to 30% of patients,
the disease is virtually asymptomatic because the decreased red cell survival is readily
compensated for by increased erythropoiesis. In most, however, the compensatory
changes are outpaced, producing a chronic hemolytic anemia, usually of mild to moderate
severity. The generally stable clinical course is sometimes punctuated by an aplastic
crisis, usually triggered by an acute parvovirus infection. Parvovirus infects and kills red
cell progenitors, causing red cell production to cease until an effective immune response
commences, generally in 1 to 2 weeks. Because the lifespan of red cells in HS is
shortened to 10 to 20 days, cessation of erythropoiesis for even short time periods leads
to sudden worsening of the anemia accompanied by reticulocytopenia. Transfusions may
be necessary to support the patient until the immune response clears the infection.
Hemolytic crises are produced by intercurrent events leading to increased splenic
destruction of red cells (e.g., infectious mononucleosis); these are clinically less
significant than aplastic crises. Gallstones, found in many patients, can also produce
symptoms. Diagnosis of HS is based on family history, hematologic findings, and several
pieces of laboratory evidence. In two thirds of the patients, the red cells are abnormally
sensitive to osmotic lysis when incubated in solutions of hypotonic salt, which induce the
influx of water into spherocytes with little margin for expansion. Spherocytes retain most
of the cytoplasm they were "born" with and lose sodium and water during conditioning in
the circulation, leading to an increased mean cell hemoglobin concentration in most
patients. As mentioned earlier, splenectomy is often beneficial.
Hemolytic Disease Due to Red Cell Enzyme Defects: Glucose-6-Phosphate Dehydrogenase Deficiency


The red cell is vulnerable to injury by exogenous and endogenous oxidants.
Abnormalities in the hexose monophosphate shunt or glutathione metabolism resulting
from deficient or impaired enzyme function reduce the ability of red cells to protect
themselves against oxidative injuries, leading to hemolytic disease. The most important
of these enzyme derangements is the hereditary deficiency of glucose-6-phosphate
dehydrogenase (G6PD) activity. As noted in Figure 13-7 , G6PD reduces NADP to
NADPH while oxidizing glucose-6-phosphate. NADPH then provides reducing
equivalents needed for conversion of oxidized glutathione to reduced glutathione, which
protects against oxidant injury by catalyzing the breakdown of compounds such as H2 O2
.

Several hundred G6PD genetic variants are known, but most are harmless. Only two
variants, designated G6PD A- and G6PD Mediterranean, cause most clinically significant
hemolytic anemias.[12] G6PD A- is present in about 10% of American blacks; G6PD
Mediterranean, as the name implies, is prevalent in the Middle East. The high frequency
of these variants in each population is believed to stem from a protective effect against
Plasmodium falciparum malaria.[13]

G6PD variants associated with hemolysis destabilize the enzyme. Compared to the most
common normal variant, G6PD B, the half-life of G6PD A- is moderately reduced,
whereas that of G6PD Mediterranean is more markedly abnormal. Elucidation of the
crystal structure of G6PD has revealed that both disease-associated mutations result in
misfolding of the protein, making it more susceptible to proteolytic degradation.[14]
Because mature red cells do not synthesize new proteins, G6PD A- or G6PD
Mediterranean enzyme activities fall quickly as red cells age to levels inadequate to
protect against oxidant stress.

G6PD deficiency is a recessive X-linked trait, placing males at highest risk for
symptomatic disease. G6PD deficiency manifests in several distinct clinical patterns.
Most common




Figure 13-7 Role of glucose-6-phosphate dehydrogenase (G6PD) in defense against oxidant injury. The
disposal of H2 O2 , a potential oxidant, is dependent on the adequacy of reduced glutathione (GSH), which
is generated by the action of NADPH. The synthesis of NADPH is dependent on the activity of G6PD.
GSSG, oxidized glutathione.
                                             628


is hemolysis after exposure to oxidant stress. This can occur due to ingestion of certain
drugs or foods, or (more commonly) exposure to oxidant free radicals generated by
leukocytes in the course of infections. The oxidant drugs implicated are numerous,
including antimalarials (e.g., primaquine and chloroquine), sulfonamides, nitrofurantoins,
and others. Some drugs cause hemolysis only in those with the more severe
Mediterranean variant. Many infections can trigger hemolysis; viral hepatitis, pneumonia,
and typhoid fever are among those most likely to do so. Hemolysis can also occur after
ingestion of fava beans (favism), which generate oxidants when metabolized. Favism is
endemic in the Mediterranean, Middle East, and parts of Africa where consumption of
fava beans is prevalent. Uncommonly, G6PD deficiency presents as neonatal jaundice or
a chronic low-grade hemolytic anemia in the absence of infection or known
environmental triggers.

G6PD deficiency causes episodic intravascular and extravascular hemolysis, which seems
to involve the following sequence. When G6PD-deficient red cells are exposed to high
levels of oxidants, there is oxidation of reactive sulfhydryl groups on globin chains,
which become denatured and form membrane-bound precipitates known as Heinz bodies.
These are seen within red cells stained with crystal violet as dark inclusions ( Fig. 13-8 ).
Heinz bodies can damage the membrane sufficiently to cause intravascular hemolysis.
Less severe membrane damage results in decreased red cell deformability. As inclusion-
bearing red cells pass through the splenic cords, macrophages pluck out the Heinz bodies.
Due to membrane damage, some of these partially devoured cells retain an abnormal
shape, appearing to have a bite of cytoplasm removed ("bite cells") (see Fig. 13-8 ). Other
less severely damaged cells revert to a spherocytic shape due to loss of membrane surface
area. Both bite cells and spherocytes are highly prone to trapping in splenic cords and
rapid removal via erythrophagocytosis.

Acute intravascular hemolysis marked by anemia, hemoglobinemia, and hemoglobinuria
usually begins 2 to 3 days following
Figure 13-8 G6PD deficiency: effects of oxidant drug exposure (peripheral blood smear). Inset, Red cells
with precipitates of denatured globin (Heinz bodies) revealed by supravital staining. As the splenic
macrophages pluck out these inclusions, "bite cells" like the one in this smear are produced. (Courtesy of
Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School,
Dallas, TX.)



exposure of G6PD-deficient individuals to oxidants. The hemolysis tends to be greater in
individuals with highly unstable G6PD Mediterranean. Since only older red cells are at
risk for lysis, the episode is self-limited, as hemolysis stops when only the younger red
cells remain (even if administration of an offending drug continues). The recovery phase
is heralded by reticulocytosis. Since hemolytic episodes related to G6PD deficiency occur
intermittently, most features of chronic hemolytic anemias (e.g., splenomegaly,
cholelithiasis) are absent.
Sickle Cell Disease


Sickle cell disease is an important hereditary hemoglobinopathy, a type of disease
characterized by production of defective hemoglobins. Hemoglobin, as you recall, is a
tetrameric protein composed of two like pairs of globin chains, each with its own heme
group. Normal adult red cells contain mainly HbA (α2 β2 ) along with small amounts of
HbA2 (α2 δ2 ) and fetal hemoglobin (α2 γ2 ). The clinically significant hemoglobinopathies
result from mutations in the β-globin gene. Sickle cell anemia is caused by a point
mutation at the sixth position of the β-globin chain leading to the substitution of a valine
residue for a glutamic acid residue. The abnormal physiochemical properties of the
resulting sickle hemoglobin (HbS) are responsible for sickle cell disease. Several hundred
other abnormal hemoglobins have been identified containing point mutations or deletions
in one of the globin chains.

About 8% of black Americans are heterozygous for HbS. If an individual is homozygous
for the sickle mutation, almost all the hemoglobin in the red cell is HbS (α2 βs 2 ). In
heterozygotes, only about 40% of the hemoglobin is HbS, the remainder being normal
hemoglobins. Where malaria is endemic in Africa, as many as 30% of the native
population are heterozygous. This high frequency is likely related to protection against
falciparum malaria afforded by HbS, particularly in infants.[15]
Pathogenesis.


When deoxygenated, HbS molecules undergo aggregation and polymerization. Initially,
the red cell cytosol converts from a freely flowing liquid to a viscous gel as HbS
aggregates form. With continued deoxygenation, aggregated HbS molecules assemble
into long needle-like fibers within red cells, producing a distorted sickle or holly-leaf
shape ( Fig. 13-9 ).

Sickling of red cells is initially a reversible phenomenon; with oxygenation, HbS
depolymerizes and the cell shape normalizes. However, with repeated episodes of
sickling, membrane damage occurs and cells become irreversibly sickled, retaining their
abnormal shape even when fully oxygenated. The precipitation of HbS fibers also causes
oxidant damage, not only in irreversibly sickled cells but also in normal-appearing cells.
With membrane injury, red cells become loaded with calcium, which is normally
excluded rigorously. Calcium ions activate a potassium ion channel, leading to the efflux
of potassium and water, intracellular dehydration, and an increase in the mean cell
hemoglobin concentration. [16] In addition, lesions produced by repeated episodes of
deoxygenation render sickle red cells abnormally sticky.[17] These membrane changes are
important in the pathogenesis of microvascular occlusions, described later.

A number of factors affect the rate and degree of sickling.



                                                    629




Figure 13-9 Sickle cell anemia (peripheral blood smear). A, Low magnification show sickle cells,
anisocytosis, and poikilocytosis. B, Higher magnification shows an irreversibly sickled cell in the center.
(Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical
School, Dallas, TX.)



        • Perhaps most important is the amount of HbS and its interaction with the other
        hemoglobin chains in the cell. In heterozygotes, approximately 40% of the
        hemoglobin is HbS, the rest being HbA, which interacts only weakly with HbS
        when deoxygenated. Both the relatively low concentration of HbS and the
        presence of interfering HbA act to prevent efficient HbS aggregation and
        polymerization, and thus red cells in heterozygous individuals do not sickle
        except under conditions of severe hypoxia. Such individuals have sickle cell trait,
        an asymptomatic carrier state. In contrast, homozygous HbS individuals have full-
        blown sickle cell anemia. Hemoglobins other than the normal HbA also influence
        the aggregation and polymerization of HbS and thus the severity of sickle cell
        anemia profoundly. Fetal hemoglobin (HbF) inhibits the polymerization of HbS,
        and hence newborns do not manifest the disease until they are 5 to 6 months of
        age, when the amount of HbF in the cells falls close to adult levels. Another
        hemoglobin modifying the effect of HbS is HbC, which has a point mutation in
        the β-globin chain leading to substitution of lysine for glutamate at position 6.
       HbC has a greater tendency to form aggregates with deoxygenated HbS than
       HbA. As a result, individuals with HbS and HbC have a symptomatic sickling
       disorder (designated HbSC disease) that is generally milder than sickle cell
       anemia. About 2% to 3% of American blacks are asymptomatic HbC/HbA
       heterozygotes, and about 1 in 1250 has HbSC disease.
       • The rate of HbS polymerization is strongly dependent upon the hemoglobin
       concentration per cell, that is, the mean corpuscular hemoglobin concentration
       (MCHC). Higher HbS concentrations increase the probability that aggregation
       and polymerization will occur during any given period of deoxygenation. Thus,
       intracellular dehydration, which increases the MCHC, facilitates sickling and
       vascular occlusion (see later). Conversely, conditions that decrease the MCHC
       reduce disease severity. This is most clearly illustrated when homozygous sickle
       cell anemia co-exists with α-thalassemia. These patients have milder disease
       because thalassemia reduces globin synthesis and limits the total hemoglobin
       concentration per cell.
       • A decrease in pH reduces the oxygen affinity of hemoglobin, thereby increasing
       the fraction of deoxygenated HbS at any given oxygen tension and augmenting
       the tendency for sickling.
       • The length of time red cells are exposed to low oxygen tension is an important
       variable. Normal transit times for red cells passing through capillaries are not
       sufficient for significant aggregation of deoxygenated HbS to occur. Hence,
       sickling of red cells is confined to microvascular beds where blood flow is
       sluggish. This is normally the case in the spleen and the bone marrow, which are
       prominently affected by sickle cell disease. Two factors play particularly
       important pathogenic roles in occlusive episodes involving other vascular beds:
       inflammation and increased red cell adhesion. As you will recall, the exodus of
       blood from inflamed tissues is slowed, due to the adhesion of leukocytes and red
       cells to activated endothelium and the transudation of fluid through leaky vessels.
       As a result, inflamed vascular beds have longer red cell transit times and are
       prone to induce clinically significant sickling. For reasons that are unclear, sickle
       red cells also express adhesion molecules at increased levels on their surfaces. In
       fact, adhesion of sickle red cells to cultured endothelial cells in vitro correlates
       with clinical severity, presumably because "stickiness" influences transit time in
       vivo.[17]

The clinical manifestations of sickle cell disease are dominated by chronic hemolysis and
ischemic tissue damage resulting from occlusion of small blood vessels ( Fig. 13-10 ).

Irreversibly sickled cells have rigid, nondeformable cell membranes that lead to difficulty
in negotiating the splenic sinusoids, sequestration, and rapid phagocytosis. Some
intravascular hemolysis can also occur because of the increased mechanical fragility of
severely damaged cells. The red cell survival correlates with the percentage of
irreversibly sickled cells in the circulation, supporting the concept that the hemolytic
anemia results primarily from premature removal of irreversibly sickled cells.
The pathogenesis of microvascular occlusions, a clinically important component of sickle
cell anemia, is less certain. There is no correlation between the number of irreversibly
sickled cells and the frequency or severity of ischemic episodes, suggesting that
reversibly sickled cells initiate microvascular occlusion. As mentioned above, reversibly
sickled cells express higher than normal levels of adhesion



                                                  630




Figure 13-10 Pathophysiology of sickle cell anemia.



molecules and appear abnormally sticky in certain assays. In vivo, it is hypothesized that
increased adhesiveness makes reversibly sickled red cells more likely to arrest during
transit through the microvasculature, particularly in areas of sluggish flow. This tendency
is likely enhanced by inflammation, which up-regulates the expression of adhesion
molecules on endothelial cells ( Chapter 2 ), making adherence of granulocytes,
monocytes, and reversibly sickled cells more likely. An important role for pro-
inflammatory granulocytes is supported by observations showing that leukocytosis
correlates with disease severity.[18] The arrest of sickle red cells within hypoxic, inflamed
vascular beds results in an extended exposure to a low oxygen tension, causing sickling
and vascular obstruction. Indeed it appears that sickled cells, themselves, can induce
adhesion molecules on the endothelium.[19] Once this process starts, it is easy to envision
how a vicious cycle of sickling, obstruction, hypoxia, and more sickling ensures.[20] In
recent years increasing attention is being focused on the role of nitric oxide (NO) in
microvascular occlusions. It is thought that in patients with sickle cell anemia, plasma
hemoglobin (released from lysed RBC) binds to and inactivates NO. Recall that NO is a
potent vasodilator and inhibits platelet aggregation. Such reduction in bioavailable NO
predisposes to increased vascular tone (narrowing) and platelet aggregation. These
findings provide rationale for NO therapy in sickle cell disease.[21]
Morphology.


The anatomic alterations are caused by chronic hemolysis, increased formation of
bilirubin, and small vessel stasis and thrombosis. The consequences of the increased red
cell destruction and anemia have been detailed in the general discussion of all hemolytic
anemias. The bone marrow is hyperplastic because of a compensatory hyperplasia of
erythroid progenitors. Expansion of the marrow leads to bone resorption and secondary
new bone formation, resulting in prominent cheekbones and changes in the skull that
resemble a crew-cut in roentgenograms. Extramedullary hematopoiesis can also appear.

In children, during the early phase of the disease, the spleen is commonly enlarged up to
500 gm. On histologic examination, there is marked congestion of the red pulp, due
mainly to the trapping of sickled red cells in the splenic cords and sinuses ( Fig. 13-11 ).
This erythrostasis in the spleen leads to marked tissue hypoxia, thrombosis, infarction,
and fibrosis. Continued scarring causes progressive shrinkage of the spleen so that by
adolescence or early adulthood only a small nubbin of fibrous tissue is left; this process is
called autosplenectomy ( Fig. 13-12 ). Infarction secondary to vascular occlusions and
anoxia can occur in many other tissues as well, including the bones, brain, kidney, liver,
retina, and pulmonary vessels, the latter sometimes producing cor pulmonale. Vascular
stagnation in subcutaneous tissues often leads to leg ulcers in adult patients; this
complication is rare in children. As in other hemolytic anemias, increased breakdown of
hemoglobin can cause pigment gallstones, and all patients develop hyperbilirubinemia
during periods of active hemolysis.



                                             631
Figure 13-11 A, Spleen in sickle cell anemia (low power). Red pulp cords and sinusoids are markedly
congested; between the congested areas, pale areas of fibrosis resulting from ischemic damage are evident.
B, Under high power, splenic sinusoids are dilated and filled with sickled red cells. (Courtesy of Dr. Darren
Wirthwein, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.)


Clinical Course.


From the description of the disease to this point, it is evident that patients are beset with
problems stemming from (1) severe anemia, (2) vaso-occlusive complications, and (3)
chronic hyperbilirubinemia. [22] Increased susceptibility to infection with encapsulated
organisms, another threat, has at least two causes. First, splenic function is severely
impaired, in children because of congestion and poor blood flow, and in adults because of
infarction and autosplenectomy. Secondly, defects in the alternative complement pathway
impair opsonization of encapsulated bacteria such as pneumococci and Haemophilus
influenzae. Septicemia and meningitis caused by these two organisms are the most
common causes of death in children with sickle cell anemia.

Chronic hemolysis induces moderately severe anemia (hematocrit values between 18%
and 30%) associated with striking reticulocytosis and hyperbilirubinemia. Irreversibly
sickled cells, ranging in frequency from 5% to 15%, are seen in peripheral smears.

The protracted course is frequently exacerbated by a variety of "crises." Vaso-occlusive
crises, also called pain crises, represent episodes of hypoxic injury and infarction
associated with




Figure 13-12 Splenic remnant in sickle cell anemia. (Courtesy of Drs. Dennis Burns and Darren
Wirthwein, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.)



severe pain in the affected region. Although infection, dehydration, and acidosis (all of
which favor sickling) sometimes act as triggers, in most instances no predisposing causes
are identified. The most commonly involved sites are the bones, lungs, liver, brain,
spleen, and penis. In children, painful bone crises are extremely common and often
difficult to distinguish from acute osteomyelitis. They frequently manifest as the hand-
foot syndrome, a dactylitis of the bones of the hands or feet or both.[23] Particularly
dangerous are vaso-occlusive crises involving the lungs, which typically present with
fever, cough, chest pain, and a pulmonary infiltrate. Also known as acute chest syndrome,
these are sometimes initiated by a simple lung infection. [24] Due to inflammation, blood
flow becomes sluggish and "spleenlike," leading to sickling and vaso-occlusion within
pulmonary vascular beds. This further compromises pulmonary function, creating a
potentially fatal cycle of worsening pulmonary and systemic hypoxemia, sickling, and
vaso-occlusion. Other organs affected by vaso-occlusive crises include the central
nervous system, where hypoxia can provoke seizures or strokes, and the cutaneous tissues
of the leg, leading to the appearance of ulcers.

Although pain crises are the most common cause of patient morbidity and mortality,
several other acute events complicate the course of sickle cell disease. Sequestration
crises occur in children with intact spleens. Massive sequestration of sickled red cells
leads to rapid splenic enlargement, hypovolemia, and sometimes shock. Both
sequestration crises and the acute chest syndrome may require treatment with exchange
transfusions if the patient is to survive. In aplastic crises, there is a transient cessation of
bone marrow erythropoiesis due to an acute infection of erythroid progenitor cells by
parvovirus B19. Reticulocytes disappear from the peripheral blood, causing a sudden and
rapid worsening of anemia.

In addition to these dramatic crises, chronic tissue hypoxia also takes a subtle yet
important toll. Chronic hypoxia is responsible for a generalized impairment of growth
and development as well as organ damage affecting spleen, heart, kidneys, and lungs.
Damage to the renal medulla leads to hyposthenuria (inability to concentrate urine),
which causes an increased propensity for dehydration and its attendant



                                               632


risks. It must be emphasized that there is great variation in the clinical manifestations of
sickle cell anemia. Some individuals are crippled by repeated vaso-occlusive crises,
whereas others have only mild symptoms. The basis for this wide range in disease
expression is not understood, but variation in unidentified modifying genes seems likely.

The diagnosis is suggested by clinical findings and the presence of irreversibly sickled
cells in peripheral blood smears and is confirmed by various tests for sickle hemoglobin.
In general, these involve mixing a blood sample with an oxygen-consuming reagent, such
as metabisulfite, which induces sickling of red cells if HbS is present. Hemoglobin
electrophoresis is also used to demonstrate the presence of HbS and exclude other sickle
syndromes, such as HbSC disease. Prenatal diagnosis is possible based on analysis of
fetal DNA obtained by amniocentesis or chorionic biopsy. The outlook for patients with
sickle cell anemia has improved considerably as a result of better supportive care.
Approximately 90% of patients survive to age 20, and close to 50% survive beyond the
fifth decade. A major recent advance in sickle cell anemia is treatment with the cancer
therapeutic drug hydroxyurea, which has several beneficial effects.[25] Through uncertain
mechanisms, hydroxyurea causes a significant increase in the concentration of HbF in red
cells, which (as we have discussed) interferes with the polymerization of HbS. However,
the therapeutic response to hydroxyurea often precedes the rise in HbF levels, implying
that other mechanisms are also important, several of which have been proposed. Firstly,
hydroxyurea acts as an anti-inflammatory agent by inhibiting the production of white
cells, which may reduce inflammation-related red cell stasis and sickling. Secondly,
hydroxyurea increases the mean red cell volume and thereby decreases the concentration
of HbS. Thirdly, hydroxyurea can be oxidized by heme groups to produce No. It is
hypothesized all of these actions contribute to ability of hydroxyurea to reduce pain crises
in children and adults.[26] [27]
Thalassemia Syndromes


The thalassemia syndromes are a heterogeneous group of inherited disorders caused by
genetic lesions leading to decreased synthesis of either the α- or β-globin chain of HbA
(α2 β2 ). β-Thalassemia is caused by deficient synthesis of the β chain, whereas α-
thalassemia is caused by deficient synthesis of the α chain. The hematologic
consequences of diminished synthesis of one globin chain stem not only from low
intracellular




Figure 13-13 Diagrammatic representation of the β-globin gene. Arrows denote sites where point
mutations giving rise to thalassemia have been identified.



hemoglobin (hypochromia), but also from a relative excess of the unimpaired chain. For
example, in β-thalassemia, excess free α chains aggregate into insoluble inclusions within
red cells and their precursors, leading to premature destruction of maturing erythroblasts
in the marrow (ineffective erythropoiesis) and lysis of mature red cells in the spleen
(hemolysis).
β-Thalassemias


The abnormality common to all β-thalassemias is diminished synthesis of structurally
normal β-globin chains, coupled with unimpaired synthesis of α chains. The clinical
severity of the anemia varies due to heterogeneity in the causative mutations. We begin
our discussion with the molecular lesions in β-thalassemia, and then relate the clinical
variants to specific underlying molecular defects.
Molecular Pathogenesis.
Adult hemoglobin (HbA) is a tetramer composed of two α chains and two β chains
encoded by a pair of functional α-globin genes on chromosome 16 and a single β-globin
gene on chromosome 11. β-Thalassemia syndromes are classified into two categories: (1)
β0 -thalassemia, associated with total absence of β-globin chains in the homozygous state,
and (2) β+ -thalassemia, characterized by reduced (but detectable) β-globin synthesis in
the homozygous state. Sequencing of β-thalassemia genes has revealed approximately
100 different causative mutations. [28] Most are point mutations; unlike α-thalassemia,
gene deletions are uncommon in β-thalassemia. Details of these mutations and their
effects on β-globin synthesis are found in specialized texts. A few illustrative examples
are cited ( Fig. 13-13 ).

       • Promoter region mutations. Point mutations within promoter sequences prevent
       RNA polymerase from binding normally, reducing transcription by 75% to 80%.
       Some normal β-globin is synthesized, producing β+ -thalassemia.
       • Chain terminator mutations. Two types of mutations cause premature
       termination of mRNA translation. One creates a new stop codon within an exon;
       the second consists of small insertions or deletions that shift the mRNA reading
       frames and introduce downstream stop codons that terminate protein synthesis
       (frameshift mutations; see Chapter 5 ). In both cases, synthesis of functional β-
       globin is prevented by premature chain termination, leading to β0 -thalassemia.
       • Splicing mutations. Mutations leading to aberrant splicing are the most common
       cause of β-thalassemia. Most affect


                                               633


       introns; others are located within exons. Some of these mutations alter normal
       splice junctions, such that normal splicing does not occur at all. Unspliced mRNA
       is degraded within the nucleus, and β0 -thalassemia results. Other mutations
       occurring in introns create new "ectopic" splice sites at abnormal locations—
       within an intron, for example. Because normal splice sites remain, both normal
       and abnormal splicing occurs, giving rise to normal and abnormal β-globin
       mRNA. These mutations cause β+ -thalassemia.

Impaired β-globin synthesis results in anemia by two mechanisms ( Fig. 13-14 ). The
deficit in HbA synthesis produces "under-hemoglobinized," hypochromic, microcytic red
cells
Figure 13-14 Pathogenesis of β-thalassemia major. Note that aggregates of unpaired α-globin chains are
not visible in routinely stained blood smears. Blood transfusions are a double-edged sword, correcting the
anemia and thereby reducing the stimulus for marrow expansion, but also adding to systemic iron overload.



with subnormal oxygen transport capacity. A more important factor is diminished
survival of red cells and their precursors, resulting from the imbalance in α- and β-chain
synthesis. Free α chains precipitate within the normoblasts, forming insoluble inclusions.
These inclusions cause a variety of untoward effects, but cell membrane damage is the
proximal cause of most red cell pathology. Many developing normoblasts in the marrow
succumb to these membrane lesions, undergoing apoptosis. In severe β-thalassemia, it is
estimated that 70% to 85% of normoblasts suffer this fate, leading to ineffective
erythropoiesis. [28] The inclusion-bearing red cells derived from precursors escaping
intramedullary death are prone to splenic sequestration and destruction due to cell
membrane damage and decreased deformability.
                                              634


In severe β-thalassemia, marked anemia produced by ineffective erythropoiesis and
hemolysis leads to several additional problems. Erythropoietin secretion in the setting of
severe uncompensated anemia leads to massive erythroid hyperplasia in the marrow and
sites of extramedullary hematopoiesis. The expanding mass of erythropoietic marrow
invades the bony cortex, impairs bone growth, and produces other skeletal abnormalities,
described later. Extramedullary hematopoiesis involves the liver, spleen, and lymph
nodes, and in extreme cases produces extraosseous masses in the thorax, abdomen, and
pelvis. The metabolically active erythroid progenitors steal nutrients from other tissues
that are already oxygen starved, causing severe cachexia in untreated patients. Another
disastrous complication seen in severe β-thalassemia (as well as in other causes of
ineffective erythropoiesis) is excessive absorption of dietary iron. This, coupled with the
iron accumulation due to the repeated blood transfusions required by these patients, leads
to a state of severe iron overload. Secondary injury to parenchymal organs, particularly
the iron-laden liver, often follows and sometimes induces secondary hemochromatosis (
Chapter 18 ).
Clinical Syndromes.


Clinical classification of β-thalassemias is based on the severity of the anemia, which in
turn depends on the type of genetic defect (β+ or β0 ) and the gene dosage (homozygous or
heterozygous).[29] In general, individuals homozygous for β-thalassemia genes (β+ /β+ or β0
/β0 ) have a severe, transfusion-dependent anemia called β-thalassemia major.
Heterozygotes with one β-thalassemia gene and one normal gene (β+ /β or β0 /β) usually
have a mild microcytic anemia that causes no symptoms. This condition is referred to as
β-thalassemia minor or β-thalassemia trait. A third clinical variant of intermediate
severity is called β-thalassemia intermedia. β-thalassemia intermedia is genetically
heterogeneous. This category includes milder variants of β+ /β+ or β+ /β0 -thalassemia and
unusually severe variants of heterozygous β-thalassemia (β0 /? or β+ /?). Ironically, the
presence of an α-thalassemia gene defect often decreases the severity of β-thalassemia
major, since the imbalance in α- and β-chain synthesis is lessened; this combination can
also result in a clinical phenotype resembling β-thalassemia intermedia. The clinical and
morphologic features of thalassemia intermedia are not described separately but can be
surmised from

               TABLE 13-3 -- Clinical and Genetic Classification of Thalassemias
Clinical                                                                   Molecular
Nomenclature             Genotype                   Disease                Genetics
β-Thalassemias
Thalassemia           Homozygous β0 - Severe; requires blood           Rare gene deletions
major                 thalassemia (β0 /β0 transfusions                 in β0 /β0
                      )                                                Defects in
              TABLE 13-3 -- Clinical and Genetic Classification of Thalassemias
Clinical                                                                      Molecular
Nomenclature              Genotype                    Disease                 Genetics
                     Homozygous β+ -                                      transcription,
                     thalassemia (β+ /β+                                  processing, or
                     )                                                    translation of β-
                                                                          globin mRNA
Thalassemia          β0 /β                 Severe, but does not require
intermedia                                 regular blood transfusions
                     β+ /β+
Thalassemia          β0 /β                 Asymptomatic with mild or
minor                                      absent anemia; red cell
                                           abnormalities seen
                     β+ /β
α-Thalassemias
Hydrops fetails -/- -/-                    Lethal in utero without
                                           transfusions
HbH disease          -/- -/α               Severe; resembles β-
                                           thalassemia intermedia
α-Thalassemia        -/- α/α (Asian)       Asymptomatic, like β-          Mainly gene
trait                                      thalassemia minor              deletions
                     -/α -/α (black
                     African)
Silent carrier       -/α α/α               Asymptomatic; no red cell
                                           abnormality


the following discussions of thalassemia major and minor ( Table 13-3 ).
Thalassemia Major.


β-Thalassemia is most common in Mediterranean countries and parts of Africa and
Southeast Asia. In the United States, the incidence is highest in immigrants from these
areas. As indicated in Table 13-3 , the genotype of affected patients can be β+ /β+ , β0 /β0 ,
or β0 /β+ . With all these genotypes, the anemia manifests 6 to 9 months after birth, as
hemoglobin synthesis switches from HbF to HbA. In untransfused patients, hemoglobin
levels range between 3 and 6 gm/dL. The peripheral blood smear shows severe red cell
morphologic abnormalities, including marked anisocytosis and poikilocytosis (variation
in size and shape, respectively), microcytosis (small size), and hypochromia (poor
hemoglobinization). Target cells (so called because hemoglobin collects in the center of
the cells), basophilic stippling, and fragmented red cells are also common. Inclusions of
aggregated α chains are efficiently removed by the spleen and not easily found in
peripheral blood smears. The reticulocyte count is elevated, but because of ineffective
erythropoiesis is lower than expected for the severity of anemia. Variable numbers of
poorly hemoglobinized normoblasts are seen in the peripheral blood due to "stress"
erythropoiesis and abnormal release of progenitors from sites of extramedullary
hematopoiesis. The red cells can completely lack HbA (β0 /β0 genotype) or contain small
amounts (β+ /β+ or β0 /β+ genotypes). HbF is markedly increased and indeed constitutes
the major red cell hemoglobin. HbA2 levels may be normal, low, or high.
Morphology.


The major morphologic alterations, in addition to those found in all hemolytic anemias,
involve the bone marrow and spleen. In the untransfused patient, there is striking
expansion of hematopoietically active marrow, particularly in facial bones. This erodes
existing cortical bone and induces new bone formation, giving rise to a "crew-cut"
appearance on X-rays ( Fig. 13-15 ). Both mononuclear phagocytic cell hyperplasia and
extramedullary hematopoiesis contribute to enlargement of the spleen, which can weigh
up to 1500 gm.



                                                  635




Figure 13-15 Thalassemia: x-ray film of the skull showing new bone formation on the outer table,
producing perpendicular radiations resembling a crew-cut. (Courtesy of Dr. Jack Reynolds, Department of
Radiology, University of Texas Southwestern Medical School, Dallas, TX.)



Hemosiderosis and secondary hemochromatosis, the two manifestations of iron overload
( Chapter 18 ), occur in almost all patients due to numerous blood transfusions and
increased absorption of dietary iron. Iron deposition often causes damage to several
organs, most notably the heart, liver, and pancreas ( Chapter 18 ).
The clinical course of β-thalassemia major is brief unless blood transfusions are given.
Untreated children suffer from growth retardation and die at an early age from the
profound effects of anemia. Blood transfusions not only improve the anemia but also
suppress secondary features related to excessive erythropoiesis. In those who survive
long enough, the cheekbones and other bony prominences are enlarged and distorted.
Hepatosplenomegaly due to extramedullary hematopoiesis is usually present. Cardiac
disease resulting from progressive iron overload and secondary hemochromatosis (
Chapter 18 ) is an important cause of death, particularly in heavily transfused patients.
Administration of iron chelators can forestall or prevent this complication. With
transfusions and iron chelation, survival into the third decade is possible, but the overall
outlook remains guarded. Bone marrow transplantation from an HLA-identical sibling is
currently the only therapy offering a cure. Prenatal diagnosis is possible by molecular
analysis of DNA.
Thalassemia Minor.


Thalassemia minor is much more common than thalassemia major and understandably
affects the same ethnic groups. Most patients are heterozygous carriers of a β+ or β0 gene.
Thalassemia trait may offer resistance against falciparum malaria, accounting for its
prevalence in parts of the world where malaria is endemic. These patients are usually
asymptomatic, and anemia is mild if present. The peripheral blood smear typically shows
some red cell abnormalities, including hypochromia, microcytosis, basophilic stippling,
and target cells. Mild erythroid hyperplasia is seen in the bone marrow. Hemoglobin
electrophoresis characteristically reveals an increase in HbA2 to 4% to 8% of the total
hemoglobin (normal, 2.5% ± 0.3%). HbF levels can be normal or slightly increased.
Recognition of β-thalassemia trait is important on two counts: (1) differentiation from the
hypochromic microcytic anemia of iron deficiency and (2) genetic counseling. Iron
deficiency can usually be excluded as the cause of a microcytic anemia through
measurement of serum iron, total iron-binding capacity, and serum ferritin (see Iron
Deficiency Anemia). Hemoglobin electrophoresis is a very helpful confirmatory test for
β-thalassemia trait, particularly in individuals (such as women of childbearing age) at risk
for both thalassemia trait and iron deficiency.
α-Thalassemias


The α-thalassemia disorders are characterized by reduced or absent synthesis of α-globin
chains. There are normally four α-globin genes. The severity of α-thalassemia varies
greatly depending on the number of α-globin genes affected. As in β-thalassemias, the
anemia stems both from lack of adequate hemoglobin and the effects of excess unpaired
non-α chains (β, γ, δ). However, the situation is complicated somewhat by synthesis of
different non-α chains at varying times of development. Thus, in the newborn with α-
thalassemia, excess unpaired γ-globin forms γ4 -tetramers known as hemoglobin Barts,
whereas in adults excess β-globin chains form β4 tetramers known as HbH. Since free β
and γ chains are more soluble than free α chains and form fairly stable homotetramers,
hemolysis and ineffective erythropoiesis are less severe than in β-thalassemias. A variety
of molecular lesions result in α-thalassemia, but the most common cause of reduced α-
chain synthesis is deletion of α-globin genes.[29]
Clinical Syndromes.


Clinical syndromes are determined and classified by the number and position of deleted
α-globin genes. Each of the four α-globin genes, which occur in two linked pairs on each
copy of chromosome 16, normally contributes approximately 25% of the α-globin chains.
α-Thalassemia syndromes stem from combinations of deletions that remove one to four
α-globin gene copies. Not surprisingly, the severity of the clinical syndrome is
proportional to the number of missing α-globin genes. Categories of α-thalassemias are
given in Table 13-3 , which lists clinical terms, along with their genetic equivalents and
salient clinical features.
Silent Carrier State.


This occurs if a single α-globin gene is deleted. It is associated with a barely detectable
reduction in α-globin chain synthesis that is insufficient to result in anemia. These
individuals are completely asymptomatic.
α-Thalassemia Trait.


This is caused by deletion of two α-globin genes. The two involved genes can be from the
same chromosome (α/α -/-), or one α-globin gene can be deleted from each of the two
chromosomes (α/- α/-) (see Table 13-3 ). The former genotype is more common in Asian
populations, the latter in regions of Africa. Both genotypes produce similar quantitative
deficiencies of α-globin chains and are clinically identical, but only matings involving
individuals with the α/α -/- genotype are at risk for producing offspring with severe α-
thalassemia (HbH disease or hydrops fetalis). As is evident from Table 13-3 , in black
African populations, α-thalassemia trait is associated with an α/- α/- genotype, and
mating of two such individuals cannot result in progeny with HbH disease or hydrops
fetalis.



                                             636


The clinical picture in α-thalassemia trait is identical to that described for β-thalassemia
minor, that is, small red cells (microcytosis), minimal or no anemia, and no abnormal
physical signs.
Hemoglobin H Disease.


This is caused by deletion of three α-globin genes. As already discussed, HbH disease is
seen most commonly in Asian populations and rarely in those of African origin. With
only one normal α-globin gene, the synthesis of α chains is markedly reduced and
tetramers of excess β-globin, called HbH, form. HbH has extremely high affinity for
oxygen and therefore is not useful for oxygen exchange, leading to tissue hypoxia
disproportionate to the level of hemoglobin. Additionally, HbH is prone to oxidation,
leading to the formation of intracellular inclusions that can be demonstrated by staining
with vital dyes. The instability of HbH is a major cause of anemia, as precipitates of
oxidized HbH form in older red cells, which are then removed by splenic macrophages.
This produces a moderately severe anemia resembling β-thalassemia intermedia.
Hydrops Fetalis.


This most severe form of α-thalassemia is caused by deletion of all four α-globin genes.
In the fetus, excess γ-globin chains form tetramers (hemoglobin Barts) with such a high
affinity for oxygen that they deliver almost no oxygen to tissues. Survival in early
development is due to the expression of δ chains, an embryonic globin that pairs with γ
chains to form a functional Hb tetramer (δ2 γ2 ). Signs of fetal distress usually become
evident by the third trimester of pregnancy. In the past, severe tissue anoxia invariably
led to intrauterine fetal death; with intrauterine transfusion, many such infants can be
saved. The fetus shows severe pallor, generalized edema, and massive
hepatosplenomegaly similar to that seen in erythroblastosis fetalis ( Chapter 10 ).
Paroxysmal Nocturnal Hemoglobinuria


Despite its rarity, paroxysmal nocturnal hemoglobinuria (PNH) has fascinated
hematologists because it is the only hemolytic anemia caused by an acquired intrinsic
defect in the cell membrane. Before we describe its molecular basis, it is instructive to
recall that proteins are anchored into the lipid bilayer in two ways. Most have a
hydrophobic sequence that spans the cell membrane ( Fig. 13-16 ); these are called
transmembrane proteins. The remainder are attached to the cell membrane by covalent
linkage to a specialized phospholipid called glycosylphosphatidylinositol (GPI). PNH
results from acquired mutations in phosphatidylinositol glycan A (PIGA), which is
essential for the synthesis of the GPI anchor. [30] Because PIGA is X-linked and subject to
lyonization (see Chapter 5 ), only the active PIGA gene needs to be mutated to produce a
functional deficiency. The causative somatic mutations occur in pluripotent stem cells;
hence, all its clonal progeny (red cells, white cells, and platelets) are deficient in proteins
attached to the cell membrane via GPI. Several GPI-linked proteins inactivate
complement. Their absence in PNH renders blood cells unusually sensitive to lysis by
complement. Not all blood cells are affected in PNH patients, indicating that the mutant
clone exists side by side with progeny of normal stem cells.

Three GPI-linked proteins that regulate complement activity—decay-accelerating factor,
or CD55; membrane inhibitor of reactive lysis, or CD59; and a C8 binding protein—are
deficient in PNH. Of these, CD59 is the most
Figure 13-16 Two kinds of membrane proteins: transmembrane and glycosyl phosphatidyl inositol (GPI)-
linked. The latter are anchored to cell membranes through a covalent attachment to a glycosyl phospatidyl
inositol moiety. In PNH, GPI cannot be synthesized, leading to a global deficiency of GPI-linked
membrane proteins.



important. It is a potent inhibitor of C3 convertase, and thereby prevents spontaneous
activation of the alternative complement pathway in vivo. These defects are not limited to
red blood cells, as deficient platelets and granulocytes are also more sensitive to lysis by
complement.

The intravascular hemolysis is actually paroxysmal and nocturnal in only 25% of cases.
Chronic hemolysis without dramatic hemoglobinuria is more common. During the
disease course, hemosiderinuria eventually leads to iron deficiency. An inconstant but
severe clinical manifestation is episodic venous thrombosis, often involving the hepatic,
portal, or cerebral veins; this thrombosis is fatal in 50% of cases. Dysfunction of platelets
due to the absence of certain GPI-linked proteins contributes to the prothrombotic state.
PNH patients are also at increased risk for developing acute myelogenous leukemia. The
basis for this association is unclear.

Remarkably, it is now appreciated that normal individuals harbor small numbers of bone
marrow cells with PIGA mutations identical to those causing PNH.[30] It is assumed that
these cells increase in numbers (thus producing clinically evident PNH) only in very rare
instances where they have a selective advantage. Consistent with this view, PNH often
arises in the setting of primary bone marrow failure (aplastic anemia), which can be
caused by immune-mediated destruction or suppression of marrow stem cells. Based on
this scenario, immunosuppression is being evaluated as a therapeutic approach, as it may
permit reconstitution of the marrow with the offspring of residual normal stem cells.
Immunohemolytic Anemia


Hemolytic anemias in this category are caused by extracorpuscular mechanisms.
Although these disorders are commonly referred to as autoimmune hemolytic anemias,
the designation immunohemolytic anemias is preferred because in some instances the
immune reaction is initiated by drug ingestion. [31] The immunohemolytic disorders are
classified in various ways, most centered on the characteristics of the responsible
antibody ( Table 13-4 ).

The diagnosis of immunohemolytic anemias requires the detection of antibodies and/or
complement on patient red cells. This is done using the direct Coombs antiglobulin test.
In



                                              637




               TABLE 13-4 -- Classification of Immunohemolytic Anemias
Warm Antibody Type
The antibody is of the IgG type, does not usually fix complement, and is active at 37°C.
Primary (idiopathic)
Secondary
Lymphomas and leukemias
Other neoplastic diseases
Autoimmune disorder (particularly       systemic lupus erythematosus)
Drugs
Cold Agglutinin Type
The antibodies are IgM and most active in vitro at 0° to 4°C.
Antibodies dissociate at 30°C or above; agglutination of cells by IgM and complement
fixation occurs only in peripheral cool parts of the body (e.g., fingers, ears, and toes).
Acute (mycoplasmal infection, infectious mononucleosis)
Chronic
Idiopathic
Associated with lymphoma
Cold Hemolysins (Paroxysmal Cold Hemoglobinuria)
IgG antibodies bind red cells at low temperature, fix complement, and cause hemolysis
when the temperature is raised above 30°C.

this test, patient red cells are mixed with heterologous antisera specific for human
immunoglobulins or complement. If either is present, red cells are cross-linked by
multivalent antibodies, causing clumping or agglutination. The indirect Coombs
antiglobulin test, in which patient serum is tested for its ability to agglutinate defined test
red cells, can then be used to characterize the target of the autoantibody. The temperature
dependence of this reaction also helps to define the type of antibody responsible.
Quantitative immunologic tests to measure such antibodies directly are also available.
Warm Antibody Immunohemolytic Anemia.


This is the most common form (48% to 70%) of immune hemolytic anemia. About 50%
of cases are idiopathic (primary); the remainder arise secondarily in the setting of a
predisposing condition (see Table 13-4 ) or drug exposure. Most causative antibodies are
of the immunoglobulin G (IgG) class; only sometimes are IgA antibodies culpable. Most
red cell destruction in this form of hemolytic disease is extravascular. IgG-coated red
cells bind Fc receptors on monocytes and splenic macrophages, which results in loss of
red cell membrane during "partial" phagocytosis. As in hereditary spherocytosis, the loss
of cell membrane converts the red cells to spherocytes, which are sequestered and
removed in the spleen, the major site of red cell destruction in this disorder. Thus,
moderate splenomegaly is characteristic of this form of anemia.

As with other forms of autoimmunity, the cause of autoantibody formation is largely
unknown. In many cases, the antibodies are directed against the Rh blood group antigens.
The mechanisms of drug-induced hemolysis are better understood. Two predominant
immunologic mechanisms have been implicated.[31]

         • Hapten model. The drugs—exemplified by penicillin and cephalosporins—act
         as haptens by binding to the red cell membrane. Antibodies directed against the
         cell-bound drug result in the destructive sequence cited before. This form of
         hemolytic anemia is usually caused by large intravenous doses of the antibiotic
         and occurs 1 to 2 weeks after onset of therapy. Sometimes the antibodies bind
         only to the offending drug, as in penicillin-induced hemolytic anemia. In other
         cases, such as quinidine-induced hemolysis, the antibodies recognize a complex
         of the drug and a membrane protein. In drug-induced hemolytic anemias, the
         destruction of red cells can occur intravascularly after fixation of complement or
         extravascularly in the mononuclear phagocyte system.
         • Autoantibody model. These drugs, of which the antihypertensive agent α-
         methyldopa is the prototype, in some manner initiate the production of antibodies
         directed against intrinsic red cell antigens, in particular the Rh blood group
         antigens. Approximately 10% of patients taking α-methyldopa develop
         autoantibodies, as assessed by the direct Coombs test. However, only 1%
         develops clinically significant hemolysis.
Cold Agglutinin Immunohemolytic Anemia.


This form of immunohemolytic anemia is caused by so-called cold agglutinins, IgM
antibodies that bind and agglutinate red cells avidly at low temperatures (0° to 4°C).[32] It
is less common than warm antibody immunohemolytic anemia, accounting for 16% to
32% of cases of immunohemolytic anemia. Such antibodies appear acutely during the
recovery phase of certain infectious disorders, such as mycoplasma pneumonia and
infectious mononucleosis. In these settings, the disorder is self-limited and rarely induces
clinical manifestations of hemolysis. Other infectious agents associated with this form of
anemia include cytomegalovirus, influenza virus, and human immunodeficiency virus
(HIV). Chronic cold agglutinin immunohemolytic anemias occur in association with
certain lymphoid neoplasms or as an idiopathic condition. Clinical symptoms result from
binding of IgM to red cells at sites such as exposed fingers, toes, and ears where the
temperature is below 30°C. IgM binding agglutinates red cells and rapidly fixes
complement on their surface. As the blood recirculates and warms, IgM is rapidly
released, usually before complement-mediated hemolysis can occur. However, the
transient interaction with IgM is sufficient to deposit sublytic quantities of C3b, an
excellent opsonin, leading to rapid removal of affected red cells by mononuclear
phagocytes in the liver and spleen. The hemolysis is of variable severity. Vascular
obstruction caused by red cell agglutinates results in pallor, cyanosis of the body parts
exposed to cold temperatures, and Raynaud phenomenon ( Chapter 11 ).
Cold Hemolysin Hemolytic Anemia.


Cold hemolysins are autoantibodies responsible for an unusual entity known as
paroxysmal cold hemoglobinuria, characterized by acute intermittent massive
intravascular hemolysis, frequently with hemoglobinuria, after exposure to cold
temperatures. This is the least common form of immunohemolytic anemia. Lysis is
clearly complement dependent. The autoantibodies are IgGs that bind to the P blood
group antigen on the red cell surface at low temperatures. Complement-mediated
intravascular lysis does not occur until the cells recirculate to warm central regions, as the
enzymes of the complement cascade function more efficiently at 37°C. The antibody,
also known as the Donath-Landsteiner antibody, was first recognized in association with
syphilis. Today, most cases of paroxysmal cold hemoglobinuria follow infections such as
mycoplasma pneumonia,



                                                      638


measles, mumps, and ill-defined viral and "flu" syndromes. The mechanisms responsible
for production of such autoantibodies in these settings are unknown.
Hemolytic Anemia Resulting from Trauma to Red Cells


Red blood cells can be disrupted by physical trauma in a variety of circumstances. Of
these, hemolytic anemias caused by cardiac valve prostheses, or narrowing or
obstruction of the microvasculature, are most important clinically. Severe traumatic
hemolytic anemia is more frequently associated with artificial mechanical valves than
bioprosthetic porcine valves. Hemolysis in both instances stems from shear stresses
produced by turbulent blood flow and abnormal pressure gradients. Microangiopathic
hemolytic anemia, on the other hand, occurs when red cells are forced to squeeze through
abnormally narrowed small vessels. Narrowing is most often caused by fibrin deposition
in association with disseminated intravascular coagulation (discussed later in this
chapter). Other causes of microangiopathic hemolytic anemia include malignant
hypertension, systemic lupus erythematosus, thrombotic thrombocytopenic purpura
(TTP), hemolytic-uremic syndrome (HUS), and disseminated cancer, most of which are
discussed elsewhere in this book. The common feature among all these disorders is a
microvascular lesion that causes mechanical injury to circulating red cells. This damage
is evident in peripheral blood smears in the form of red cell fragments (schistocytes),
"burr cells," "helmet cells," and "triangle cells" ( Fig. 13-17 ). Except for TTP and HUS,
hemolysis is not a major clinical problem in most instances.




Figure 13-17 Microangiopathic hemolytic anemia. A peripheral blood smear from a patient with
hemolytic-uremic syndrome shows several fragmented red cells. (Courtesy of Dr. Robert W. McKenna,
Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.)


ANEMIAS OF DIMINISHED ERYTHROPOIESIS


Anemias often result from deficiencies of vital nutrients necessary for red cell formation.
Included in this group are the anemias of vitamin B12 and folate deficiency, characterized
by defective DNA synthesis (megaloblastic anemias), and iron deficiency anemias, in
which heme synthesis is impaired. Other causes of decreased erythropoiesis include
anemia of chronic disease, anemia of renal failure, and "marrow stem cell failure," which
embraces such conditions as aplastic anemia and pure red cell aplasia.
Megaloblastic Anemias


The following discussion attempts first to characterize the major features of these
anemias and then to discuss the two principal types of megaloblastic anemia: (1)
pernicious anemia, the major form of vitamin B12 deficiency anemia, and (2) folate
deficiency anemia.
The megaloblastic anemias constitute a diverse group of entities, having in common
impaired DNA synthesis and distinctive morphologic changes in the blood and bone
marrow. As the name implies, erythroid precursors and red cells are abnormally large due
to defective cell maturation and division. The precise basis for these changes is not fully
understood.

Some of the metabolic roles of vitamin B12 and folate are considered later, but for now it
suffices that vitamin B12 and folic acid are coenzymes required for synthesis of
thymidine, one of the four bases found in DNA. A deficiency of these vitamins or
impairment in their metabolism results in defective nuclear maturation due to deranged or
inadequate DNA synthesis, with an attendant delay or block in cell division. The
synthesis of RNA and protein is relatively unaffected, however, so cytoplasmic
maturation proceeds in advance of nuclear maturation, a situation described as
nuclear/cytoplasmic asynchrony.
Morphology.


Certain morphologic features are common to all forms of megaloblastic anemia. A
peripheral blood examination usually reveals pancytopenia, as all myeloid lineages are
affected. There is marked variation in the size and shape of red cells (anisocytosis), which
nonetheless are normochromic. Many red cells are macrocytic and oval
(macroovalocytes), with mean corpuscular (cell) volumes above 100 fl (normal, 82 to
98). Because they are thicker than normal and well-hemoglobinized, most macrocytes
lack the central pallor of normal red cells and can even appear "hyperchromic," but the
MCHC is not elevated. The reticulocyte count is low, and nucleated red cells
occasionally appear in the circulating blood with severe anemia. Neutrophils are also
larger than normal (macropolymorphonuclear) and hypersegmented; that is, they
have five to six or more nuclear lobules ( Fig. 13-18 ). The marrow is usually markedly
hypercellular due to increased numbers of all types of myeloid precursors, which may
completely replace the fatty marrow. Megaloblastic change is detected in all stages of red
cell development. The most primitive cells (promegaloblasts) are large, with a deeply
basophilic cytoplasm, prominent



                                            639
Figure 13-18 Megaloblastic anemia. A peripheral blood smear shows a hypersegmented neutrophil with a
six-lobed nucleus. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas
Southwestern Medical School, Dallas, TX.)



nucleoli, and a distinctive fine nuclear chromatin pattern ( Fig. 13-19 , cell A). As these
cells differentiate and begin to accumulate hemoglobin, the nucleus retains its finely
distributed chromatin and thus fails to undergo the chromatin clumping typical of the
normoblast. For example, orthochromatic megaloblasts have a large amount of pink,
well-hemoglobinized cytoplasm, but the nucleus, instead of becoming pyknotic, remains
relatively large and immature. Because DNA synthesis is impaired in all proliferating
cells, granulocytic precursors also display nuclear-cytoplasmic asynchrony in the form of
giant metamyelocytes and band forms. Megakaryocytes, too, can be abnormally large and
have bizarre, multilobate nuclei.

The marrow hyperplasia usually seen in megaloblastic anemias is a response to increased
levels of growth factors such as erythropoietin. However, due to the derangement in
DNA synthesis, most myeloid precursors undergo apoptosis in the marrow (another
example of ineffective hematopoiesis), leading to pancytopenia. The anemia is further
exacerbated by increased hemolytic destruction of red cells in the periphery. The basis for
hemolysis is not entirely clear; both an acquired intracorpuscular defect and a poorly
characterized plasma factor have been suggested to contribute. As in other states
associated with ineffective erythropoiesis, if the deficiency persists enhanced uptake of
iron in the gut can lead to anatomic signs of mild to moderate iron overload after several
years ( Chapter 18 ).
Anemias of Vitamin B12 Deficiency: Pernicious Anemia


The major causes of megaloblastic anemia are listed in Table 13-5 . As mentioned at the
outset, pernicious anemia is an important cause of vitamin B12 deficiency. Pernicious
anemia is a specific form of megaloblastic anemia caused by atrophic gastritis and an
attendant failure of intrinsic factor production that leads to vitamin B12 deficiency.
Figure 13-19 Megaloblastic anemia (bone marrow aspirate). A to C, Megaloblasts in various stages of
differentiation. Note that the orthochromatic megaloblast (B) is hemoglobinized (as revealed by
cytoplasmic color), but in contrast to normal orthochromatic normoblasts, the nucleus is not pyknotic. The
granulocytic precursors are also large and have abnormally "immature" chromatin. (Courtesy of Dr. Jose
Hernandez, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.)



We first review vitamin B12 metabolism, as this helps to place pernicious anemia in
perspective relative to the other causes of vitamin B12 deficiency anemia.
Normal Vitamin B12 Metabolism.


Vitamin B12 is a complex organometallic compound known as cobalamin. Under normal
circumstances, humans are totally dependent on dietary animal products for their vitamin
B12 requirement. Microorganisms are the ultimate origin of cobalamin in the food chain.
Plants and vegetables contain little cobalamin save that contributed by microbial
contamination; strictly vegetarian or macrobiotic diets, then, do not provide adequate
amounts of this essential nutrient. The daily requirement is 2 to 3 mg. A balanced diet
contains significantly larger amounts and normally results in accumulation of vitamin B12
in sufficient quantities to last for several years.

Absorption of vitamin B12 requires intrinsic factor, which is secreted by the parietal cells
of the fundic mucosa ( Fig. 13-20 ). First, vitamin B12 is freed from binding proteins in
food through the action of pepsin in the stomach. Free vitamin B12 then binds to salivary
proteins called cobalophilins, or R-binders. In the duodenum, cobalophilin-vitamin B12
complexes are broken down by the action of pancreatic proteases, and released vitamin
B12 then associates with intrinsic factor. This complex is transported to the ileum, where it
is endocytosed by ileal enterocytes that express intrinsic factor-specific receptors on their
surfaces. Within ileal cells, vitamin B12 associates with a major carrier protein,
transcobalamin II, and is secreted into the plasma. Transcobalamin II delivers vitamin B12
to the liver and other cells of the body, particularly rapidly proliferating cells in the bone



                                              640




                       TABLE 13-5 -- Causes of Megaloblastic Anemia
Vitamin B12 Deficiency
Decreased intake
Inadequate diet,     vegetarianism
Impaired absorption
Intrinsic factor deficiency
Pernicious anemia
Gastrectomy
Malabsorption states
Diffuse intestinal disease, e.g.,    lymphoma, systemic sclerosis
Ileal resection, ileitis
Competitive parasitic uptake
Fish tapeworm infestation
Bacterial overgrowth in blind loops and       diverticula of bowel
Increased requirement
Pregnancy, hyperthyroidism,          disseminated cancer
Folic Acid Deficiency
Decreased intake
Inadequate diet—alcoholism,         infancy
Impaired absorption
Malabsorption states
Intrinsic intestinal disease
Anticonvulsants, oral      contraceptives
Increased loss
Hemodialysis
Increased requirement
Pregnancy, infancy, disseminated       cancer, markedly increased hematopoiesis
Impaired use
Folic acid antagonists
Unresponsive to Vitamin B12 or Folic Acid Therapy
Metabolic inhibitors of DNA synthesis and/or folate metabolism, e.g., methotrexate
Modified from Beck WS: Megaloblastic anemias. In Wyngaarden JB, Smith LH (eds):
Cecil Textbook of Medicine, 18th ed. Philadelphia, WB Saunders, 1988, p. 900.

marrow and mucosal lining of the gastrointestinal tract. In addition to the intrinsic-factor
dependent pathway, evidence also supports the existence of an alternative mechanism
that is not dependent on availability of intrinsic factor or intact terminal ileum. The
mechanism involved is not entirely clear but up to 1% of a large oral dose can be
absorbed by this pathway, thus making it feasible to treat pernicious anemia by oral
vitamin B12 therapy.[33]
Etiology of Vitamin B12 Deficiency.


With this background, we can consider the various causes of vitamin B12 deficiency (see
Table 13-5 ). Inadequate diet is obvious but must be present for many years to deplete
reserves. The absorption of vitamin B12 can be impaired by disruption of any one of the
steps outlined earlier. With achlorhydria and loss of pepsin secretion (which occurs in
some elderly individuals), vitamin B12 is not readily released from proteins in food. With
gastrectomy and pernicious anemia, intrinsic factor is not available for transport to the
ileum. With loss of exocrine pancreatic function, vitamin B12 cannot be released from R-
binder-vitamin B12 complexes. Ileal resection or diffuse ileal disease can remove or
damage the site of intrinsic factor-vitamin B12 complex absorption. Tapeworm infestation,
by competing for the nutrient, can induce a deficiency state. Under some circumstances,
for example, pregnancy, hyperthyroidism, disseminated cancer, and chronic infections,
Figure 13-20 Schematic illustration of vitamin B12 absorption.




                                                    641


the demand for vitamin B12 can be so great as to produce a relative deficiency, even with
normal absorption.
Biochemical Functions of Vitamin B12 .


Two reactions in humans are known to require vitamin B12 . Methylcobalamin is an
essential cofactor for methionine synthase, an enzyme involved in the conversion of
homocysteine to methionine ( Fig. 13-21 ). In the process, methylcobalamin yields a
methyl group and is regenerated from N5 -methyltetrahydrofolic acid (N5 -methyl FH4 ),
the principal form of folic acid in plasma. In the same reaction, N5 -methyl FH4 is
converted to tetrahydrofolic acid (FH4 ). FH4 is crucial, since it is required (through its
derivative N5,10 -methylene FH4 ) for conversion of deoxyuridine monophosphate to
deoxythymidine monophosphate, an immediate precursor of DNA. It has been postulated
that the fundamental cause of impaired DNA synthesis in vitamin B12 deficiency is the
reduced availability of FH4 , most of which is "trapped" as N5 -methyl FH4 .[34] In addition,
the deficit in FH4 can be exacerbated by an "internal" folate deficiency caused by a failure
to synthesize metabolically active polyglutamylated forms.[35] This may stem from a
requirement for vitamin B12 in synthesis of methionine, which contributes a carbon group
needed in the metabolic reactions that create folate polyglutamates (see Fig. 13-21 ).
Whatever the mechanism of internal folate deficiency, lack of folate is the proximate
cause of anemia in vitamin B12 deficiency, as the anemia inevitably improves with
administration of folic acid.

The neurologic complications associated with vitamin B12 deficiency are an enigma,[36] as
treatment with folate fails to improve neurologic deficits. In addition to the
transmethylation reaction discussed previously, the second reaction depending on
cobalamin is the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A,
which requires adenosylcobalamin as a prosthetic group on the enzyme methylmalonyl-
coenzyme A mutase. A deficiency of vitamin B12 thus leads to increased plasma and urine
levels of methylmalonic acid. Interruption of the succinyl pathway and consequent
Figure 13-21 Relationship of N5 -methyl FH4 , methionine synthase, and thymidylate synthetase. In
cobalamin deficiency, folate is sequestered as N5 -methyl FH4 . This ultimately deprives thymidylate
synthetase of its folate coenzyme (N5,10 -methylene FH4 ), thereby impairing DNA synthesis.



build-up of methylmalonate and propionate (a precursor) could lead to the formation and
incorporation of abnormal fatty acids into neuronal lipids. It has been suggested that this
biochemical abnormality predisposes to myelin breakdown and thereby produces the
neurologic complications of vitamin B12 deficiency ( Chapter 28 ). However, rare
individuals with hereditary deficiencies of methylmalonyl-coenzyme A mutase, while
having complications related to methylmalonyl acidemia, do not suffer from the
neurologic abnormalities seen in vitamin B12 deficiency,[37] casting doubt on this
explanation.

With this overview of vitamin B12 metabolism, we can now turn our attention to
pernicious anemia.
Incidence.


Although somewhat more prevalent in Scandinavian and "English-speaking" populations,
pernicious anemia occurs in all racial groups, including, in the United States, blacks and
Hispanics. A disease of older age, it is generally diagnosed in the fifth to eighth decades
of life. A genetic predisposition is strongly suspected, but no definable genetic pattern of
transmission has been discerned. As described below, these patients probably have a
tendency to form antibodies against multiple self-antigens.
Pathogenesis.
Pernicious anemia is believed to result from immunologically mediated, possibly
autoimmune, destruction of gastric mucosa. The resultant chronic atrophic gastritis is
marked by a loss of parietal cells, a prominent infiltrate of lymphocytes and plasma cells,
and megaloblastic changes in mucosal cells similar to those found in erythroid
precursors. A number of immunologic reactions are associated with these morphologic
changes. Three types of antibodies are present in many but not all patients with
pernicious anemia. About 75% of patients have a type I antibody that blocks binding of
vitamin B12 to intrinsic factor. Type I antibodies are found in both plasma and gastric
juice. Type II antibodies prevent binding of the intrinsic factor-vitamin B12 complex to its
ileal receptor. These immunoglobulins are also found in a large proportion of patients
with pernicious anemia. The third type of antibodies, present in 85% to 90% of patients,
recognize the α and β subunits of the gastric proton pump,[38] which is normally localized
to the microvilli of the canalicular system of the gastric parietal cell. Type III antibodies
are not specific for pernicious anemia or other autoimmune diseases, as they are found in
up to 50% of elderly patients with idiopathic chronic gastritis not associated with
pernicious anemia. As discussed next, they most likely result from gastric injury, rather
than cause it.

Despite the presence of these autoantibodies, it is not established that they are the
primary cause of gastric changes. It is believed that an autoreactive T-cell response
initiates gastric mucosal injury, triggering the formation of autoantibodies, which may
exacerbate epithelial injury. When the mass of intrinsic factor-secreting cells falls below
a threshold (and reserves of stored vitamin B12 are depleted), anemia develops. In an
animal model of autoimmune gastritis mediated by CD4+ T cells, a pattern of
autoantibodies resembling that seen in pernicious anemia develops, thus supporting the
primacy of T-cell autoimmunity.[38] An autoimmune basis is also supported by the
association of pernicious anemia with other autoimmune disorders, particularly
autoimmune thyroiditis and adrenalitis. Conversely, patients who present with other
autoimmune diseases are predisposed to develop antibodies against intrinsic factor.



                                             642

Morphology.


The major specific changes in pernicious anemia are found in the bone marrow,
alimentary tract, and central nervous system. The changes in the bone marrow and blood
are similar to those described earlier for all megaloblastic anemias.

In the alimentary system, abnormalities are regularly found in the tongue and stomach.
The tongue is shiny, glazed, and "beefy" (atrophic glossitis). The changes in the stomach
are those of diffuse chronic gastritis ( Chapter 17 ). The most characteristic histologic
alteration is the atrophy of the fundic glands, affecting both chief cells and parietal cells,
the latter being virtually absent. The glandular lining epithelium is replaced by mucus-
secreting goblet cells that resemble those lining the large intestine, a form of metaplasia
referred to as intestinalization. Some of the cells as well as their nuclei may increase to
double the normal size, a form of "megaloblastic" change exactly analogous to that seen
in the marrow. As will be seen, patients with pernicious anemia have a higher incidence
of gastric cancer. The gastric atrophic and metaplastic changes are due to autoimmunity
and not vitamin B12 deficiency; hence, parenteral administration of vitamin B12 corrects
the bone marrow changes, but gastric atrophy and achlorhydria persist.

Central nervous system lesions are found in approximately three fourths of all cases of
fulminant pernicious anemia, but in some instances neuronal involvement is seen in the
absence of overt megaloblastic anemia. The principal alterations involve the spinal
cord, where there is degeneration of myelin in the dorsal and lateral tracts,
sometimes followed by loss of axons. These changes give rise to spastic paraparesis,
sensory ataxia, and severe paresthesias in the lower limbs. Less frequently, degenerative
changes occur in the ganglia of the posterior roots and in peripheral nerves ( Chapter 28 ).
Because both sensory and motor pathways are involved, the term "subacute combined
degeneration" or "combined system disease" is sometimes used to describe the neurologic
changes associated with vitamin B12 deficiency.
Clinical Course.


Pernicious anemia is insidious in onset, so the anemia is often quite severe by the time
the patient seeks medical attention. The course is progressive unless halted by therapy.

Diagnostic features include (1) a moderate to severe megaloblastic anemia, (2)
leukopenia with hypersegmented granulocytes, (3) mild to moderate thrombocytopenia,
(4) mild jaundice due to ineffective erythropoiesis and peripheral hemolysis of red cells,
(5) neurologic changes related to involvement of the posterolateral spinal tracts, (6)
achlorhydria even after histamine stimulation, (7) inability to absorb an oral dose of
cobalamin (assessed by urinary excretion of radiolabeled cyanocobalamin given orally,
called the Schilling test), (8) low serum levels of vitamin B12 , (9) elevated levels of
homocysteine and methyl malonic acid in the serum (this is more sensitive than serum
levels of vitamin B12 ) (10) a striking reticulocytic response and improvement in
hematocrit levels beginning about 5 days after parenteral administration of vitamin B12 .
Serum antibodies to intrinsic factor are highly specific for pernicious anemia. Their
presence attests to the cause of vitamin B12 deficiency, rather than the presence or
absence of cobalamin deficiency.

As mentioned, serum homocysteine and methylmalonic acid levels are raised in patients
with deficiency of vitamin B12 , and high levels of homocysteine are a risk factor for
atherosclerosis and thrombosis. It is still not definite whether such an elevation in patients
with folate or vitamin B12 deficiency increases the risk of vascular disease. The cytologic
aberrations in the gastric mucosa are associated with an increased risk of gastric cancer (
Chapter 17 ). With parenteral or high dose oral vitamin B12 , the anemia can be cured and
the peripheral neurologic changes reversed, or at least halted in their progression, but the
changes in the gastric mucosa are unaffected. Overall longevity can be restored virtually
to normal.
Anemia of Folate Deficiency
A deficiency of folic acid, more properly pteroylmonoglutamic acid, results in a
megaloblastic anemia having the same characteristics as that caused by vitamin B12
deficiency. However, the neurologic changes seen in vitamin B12 deficiency do not occur.
Folic acid (or more specifically, tetrahydrofolate [FH4 ] derivatives) acts as an
intermediate in the transfer of one-carbon units such as formyl and methyl groups to
various compounds ( Fig. 13-22 ). In the process, FH4 also serves as an acceptor of one-
carbon fragments from compounds such as serine and formiminoglutamic acid (FIGlu).
The FH4 derivatives so generated in turn donate the acquired one-carbon fragments in
reactions synthesizing various metabolites. FH4 , then, can be viewed as the biologic
"middleman" in a series of swaps involving one-carbon moieties. The most important
metabolic processes dependent on such one-carbon transfers are (1) purine synthesis; (2)
conversion of homocysteine to methionine, a reaction also requiring vitamin B12 ; and (3)
deoxythmidylate monophosphate synthesis. In the first two reactions, FH4 is regenerated
from its one-carbon carrier derivatives and is available to accept another one-carbon
fragment and re-enter the donor pool. In the synthesis of thymidylate, a dihydrofolate is
produced that must be reduced by dihydrofolate reductase for reentry into the FH4 pool.
The reductase step is significant, since this enzyme is susceptible to inhibition by various
drugs. Among the biologically active molecules whose synthesis is dependent on folates,
thymidylate is perhaps the most important. As discussed earlier in relation to pernicious
anemia, deoxythymidylate monophosphate is required for DNA synthesis. It should be
apparent from our




Figure 13-22 Role of folate derivatives in the transfer of one-carbon fragments for synthesis of biologic
macromolecules. FH4 , tetrahydrofolic acid; FH2 , dihydrofolic acid; FIGlu, formiminoglutamate; dTMP,
deoxythymidylate monophosphate. *Synthesis of methionine also requires vitamin B 12 .




                                                    643


discussion that suppressed synthesis of DNA, the common denominator of folic acid and
vitamin B12 deficiency, is the immediate cause of megaloblastosis.
Etiology.


Humans are entirely dependent on dietary sources for their folic acid requirement, which
is 50 to 200 mg daily. Most normal diets contain ample amounts. The richest sources are
green vegetables such as lettuce, spinach, asparagus, and broccoli. Certain fruits (e.g.,
lemons, bananas, melons) and animal proteins (e.g., liver) contain lesser amounts. The
folic acid in these foods is largely in the form of folylpolyglutamates. Despite their
abundance in raw foods, polyglutamates (depending on the specific form) are sensitive to
heat; boiling, steaming, or frying of foods for 5 to 10 minutes destroys up to 95% of the
folate content. Intestinal conjugases split the polyglutamates into monoglutamates that
are readily absorbed in the proximal jejunum. During intestinal absorption, they are
modified to 5-methyltetrahydrofolate, the normal transport form of folate. The body's
reserves of folate are relatively modest, and a deficiency can arise with months of a
negative balance. There are three major causes of folic acid deficiency: (1) decreased
intake, (2) increased requirements, and (3) impaired use ( Table 13-5 ).

Decreased intake can result from either a nutritionally inadequate diet or impairment of
intestinal absorption. A normal daily diet contains folate in excess of the minimal daily
adult requirement. Inadequate dietary intakes are almost invariably associated with
grossly deficient diets, particularly those lacking vitamins such as the "B group." Such
dietary inadequacies are most frequently encountered in chronic alcoholics, the indigent,
and the very elderly. In alcoholics with cirrhosis, other mechanisms of folate deficiency
such as trapping of folate within the liver, excessive urinary loss, and disordered folate
metabolism have also been implicated. Under these circumstances, the megaloblastic
anemia is often accompanied by general malnutrition and manifestations of other
avitaminoses, including cheilosis, glossitis, and dermatitis. Malabsorption syndromes
such as nontropical and tropical sprue can lead to inadequate absorption of this nutrient,
as can diffuse infiltrative diseases of the small intestine (e.g., lymphoma). In addition,
certain drugs, particularly the anticonvulsant phenytoin and oral contraceptives, interfere
with absorption.

Despite adequate intake of folic acid, a relative deficiency can be encountered in states of
increased requirement, such as pregnancy, infancy, hematologic derangements associated
with hyperactive hematopoiesis (hemolytic anemias), and disseminated cancer. In all
these circumstances, the demands of active DNA synthesis render normal intake
inadequate.

Folic acid antagonists, such as methotrexate, inhibit dihydrofolate reductase and lead to a
deficiency of tetrahydrofolate. With inhibition of folate metabolism, all rapidly growing
cells are affected, thus leading to ulcerative lesions within the gastrointestinal tract as
well as megaloblastic anemia. Many other chemotherapeutic drugs damage DNA or
inhibit DNA synthesis through other mechanisms; these can also cause megaloblastic
changes in rapidly dividing cells. Owing to their growth inhibitory actions,
antimetabolites are used in cancer therapy.

As mentioned at the outset, megaloblastic anemia resulting from a deficiency of folic acid
is identical to that encountered in vitamin B12 deficiency. Thus, the diagnosis of folate
deficiency can be made only by demonstration of decreased folate levels in the serum or
red cells. As in vitamin B12 deficiency, serum homocysteine levels are increased.

Although prompt hematologic response heralded by reticulocytosis follows the
administration of folic acid, it should be cautioned that the hematologic symptoms of a
vitamin B12 deficiency anemia also respond to folate therapy. However, folate does not
prevent (and may even exacerbate) the progression of the neurologic deficits typical of
the vitamin B12 deficiency states. It is thus essential to exclude vitamin B12 deficiency in
megaloblastic anemia before initiating therapy with folate.
Iron Deficiency Anemia


Deficiency of iron is probably the most common nutritional disorder in the world.
Although the prevalence of iron deficiency anemia is higher in developing countries, this
form of anemia is also common in the United States, particularly in toddlers, adolescent
girls, and women of childbearing age.[39] The factors underlying the iron deficiency differ
somewhat in various population groups and can be best considered in the context of
normal iron metabolism.
Iron Metabolism.


As might be expected given the very high prevalence of iron deficiency in human
populations, evolutionary pressures have yielded iron metabolism pathways that are
strongly biased toward the retention of iron. There is no regulated pathway for iron
excretion, which is limited to the 1 to 2 mg per day lost by shedding of mucosal and skin
epithelial cells. Iron balance, therefore, is maintained largely by regulating the
absorption of dietary iron. The normal daily Western diet contains approximately 10 to
20 mg of iron, most in the form of heme contained in animal products, with the remainder
being inorganic iron in vegetables. About 20% of heme iron (in contrast to 1% to 2% of
nonheme iron) is absorbable, so the average Western diet contains sufficient iron to
balance fixed daily losses. The total body iron content is normally about 2 gm in women
and up to 6 gm in men. As indicated in Table 13-6 , it is divided into functional and
storage compartments. Approximately 80% of the functional iron is found in hemoglobin;
myoglobin and iron-containing enzymes such as catalase and the cytochromes contain the
rest. The storage pool represented by hemosiderin and ferritin contains approximately
15% to 20% of total body iron. Healthy young females have substantially smaller stores
of iron than do males, primarily due to blood loss during menstruation. This precarious
iron balance is easily tipped into deficiency by excessive losses or increased demands
associated with menstruation and pregnancy, respectively.


                   TABLE 13-6 -- Iron Distribution in Healthy Young Adults (mg)
Pool                                                         Men              Women
Total                                                            3450                   2450
Functional
             TABLE 13-6 -- Iron Distribution in Healthy Young Adults (mg)
Pool                                                         Men              Women
Hemoglobin                                                      2100                    1750
Myoglobin                                                          300                    250
Enzymes                                                             50                     50
Storage
Ferritin, hemosiderin                                           1000                      400



                                             644


Free iron is highly toxic, and the pool of storage iron is tightly bound to either ferritin or
hemosiderin.[40] Ferritin is a protein-iron complex found in all tissues but particularly in
liver, spleen, bone marrow, and skeletal muscles. In the liver, most ferritin is stored
within the parenchymal cells; in other tissues, such as spleen and bone marrow, it is
mainly in the mononuclear phagocytic cells. Hepatocytic iron is derived from plasma
transferrin, whereas storage iron in the mononuclear phagocytic cells (Kupffer cells) is
derived from the breakdown of red cells ( Fig. 13-23 ). Intracellular ferritin is located in
both the cytosol and lysosomes, in which partially degraded protein shells of ferritin
aggregate into hemosiderin granules. With a hematoxylin and eosin stain, hemosiderin
appears in cells as golden yellow granules. The iron in hemosiderin is chemically reactive
and turns blue-black when exposed to potassium ferrocyanide, which is the basis for the
Prussian blue stain. With normal iron stores, only trace amounts of hemosiderin are found
in the body, principally in mononuclear phagocytic cells in the bone marrow, spleen, and
liver. In iron-overloaded cells, most iron is stored in hemosiderin.

Very small amounts of ferritin normally circulate in the plasma. Since plasma ferritin is
derived largely from the storage pool of body iron, its levels correlate well with body iron
stores. In iron deficiency, serum ferritin is always below 12 µg/L, whereas in iron
overload, high values approaching 5000 µg/L can be seen. Of physiologic importance,
the storage iron pool can be readily mobilized if iron requirements increase, as may occur
after loss of blood.

Iron is transported in plasma by an iron-binding glycoprotein called transferrin (see Fig.
13-23 ), which is synthesized in the liver. In normal individuals, transferrin is about 33%
saturated with iron, yielding serum iron levels that average 120 µg/dL in men and 100
µg/dL in women. Thus, the total
Figure 13-23 The internal iron cycle. Plasma iron bound to transferrin is transported to the marrow, where
it is transferred to developing red cells and incorporated into hemoglobin. Mature red blood cells are
released into the circulation and, after 120 days, are ingested by macrophages in the reticuloendothelial
system (RES). Here iron is extracted from hemoglobin and returned to the plasma, completing the cycle.
(From Wyngaarden JB, et al [eds]: Cecil Textbook of Medicine, 19th ed. Philadelphia, WB Saunders,
1992, p. 841.)



iron-binding capacity of serum is in the range of 300 to 350 µg/dL. The major function of
plasma transferrin is to deliver iron to cells, including erythroid precursors, where iron is
required for hemoglobin synthesis. Immature red cells possess high-affinity receptors for
transferrin, and iron is transported into erythroblasts by receptor-mediated endocytosis.

The absorption of iron and its regulation have become better understood over the last
several years.[40] Most iron is absorbed in the duodenum, where uptake of heme and
nonheme iron occurs through two distinct pathways ( Fig. 13-24 ). Nonheme iron
traverses the apical and basolateral membranes of villus enterocytes through the action of
two distinct transporters. After reduction to ferrous (Fe++ ) iron by a membrane-associated
cytochrome B, divalent metal transporter 1 (DMT1) first moves nonheme iron across the
apical membrane. At least two proteins are then required for the basolateral transfer of
iron to transferrin in the plasma: ferroportin, a transporter, and hephaestin, an iron
oxidase. Both DMT1 and ferroportin are widely distributed in the body, suggesting their
involvement in iron transport in other tissues as well. For example, DMT1 also appears to
be important in uptake of iron into erythroblasts. Dietary heme iron is absorbed through a
different transporter that is not yet well characterized.

The efficacy of enterocyte uptake of heme and nonheme iron differs dramatically.
Approximately 25% of the heme iron derived from hemoglobin, myoglobin, and other
animal proteins is absorbed. The absorption of nonheme iron is more variable, being
influenced by substances in the diet that inhibit (phytates, tannates, and phosphates) or
enhance (ascorbic and amino acids) uptake, but frequently less than 5% of that
consumed. After absorption, both heme and nonheme iron appear to enter a common pool
in the mucosal cell. Normally, a fraction of the iron that enters the cell is rapidly
delivered to plasma transferrin. Most, however, is deposited as ferritin, some to be
transferred more slowly to plasma transferrin, and some to be lost with exfoliation of
mucosal cells. The extent to which the mucosal iron is distributed along these various
pathways depends on the body's iron requirements. When the body is replete with iron,
formation of ferritin within the mucosal cells is maximal, whereas transport into plasma
is enhanced in iron deficiency.

Since body losses of iron are limited, iron balance is maintained by regulating absorptive
intake. The mechanisms that regulate the absorption of available iron into the mucosal
cell are still incompletely understood.[41] Important clues come from observations
demonstrating that the rate and level of absorption are dependent on total body iron
content and erythropoietic activity, or more specifically the iron needs of the erythroid
precursors. As body stores rise, the absorption of iron falls, and vice versa. With
ineffective erythropoiesis, as may occur in β-thalassemias, iron absorption is increased
despite an excess of stored body iron. Some signal must be delivered to the mucosal cell,
modifying its uptake and transfer of iron. An excellent candidate for a negative "iron
metabolism regulatory hormone" is hepcidin, a small, liver-derived plasma peptide.
Hepcidin inhibits iron uptake in the duodenum and iron release from macrophages.[42] The
concentration of hepcidin falls as iron stores become depleted, and hepcidin knockout
mice develop hemochromatosis. Conversely, over-expression of hepcidin in transgenic
mice causes an iron deficiency



                                            645
Figure 13-24 Diagrammatic representation of iron absorption. Mucosal uptake of heme and nonheme iron
is depicted. When the storage sites of the body are replete with iron and erythropoietic activity is normal,
most of the absorbed iron is lost into the gut by shedding of the epithelial cells. Conversely, when body iron
needs increase or when erythropoiesis is stimulated, a greater fraction of the absorbed iron is transferred
into plasma transferrin, with a concomitant decrease in iron loss through mucosal ferritin.



anemia. These findings suggest the existence of a hepcidin receptor on duodenal
enterocytes. The HFE gene, which encodes an HLA-like transmembrane protein, is also
clearly involved in regulation of iron absorption. [41] As discussed in Chapter 18 , mutation
in this gene leads to excessive absorption of dietary iron and eventual hemochromatosis,
a disease characterized by systemic iron overload.
Etiology.


To maintain a normal iron balance, approximately 1 mg of iron must be absorbed from
the diet every day. Because only 10% to 15% of ingested iron is absorbed, the daily iron
requirement is 7 to 10 mg for adult men and 7 to 20 mg for adult women. Since the
average daily dietary intake of iron in the Western world is about 15 to 20 mg, most men
ingest more than adequate iron, whereas many women consume marginally adequate
amounts of iron.

The bioavailability of dietary iron is as important as the overall content. Heme iron is
much more absorbable than inorganic iron. The absorption of the latter is influenced by
other dietary contents. Ascorbic acid, citric acid, amino acids, and sugars in the diet
enhance absorption of inorganic iron, but tannates (as in tea), carbonates, oxalates, and
phosphates inhibit its absorption.

An iron deficiency can result from (1) dietary lack, (2) impaired absorption, (3)
increased requirement, or (4) chronic blood loss.

Dietary lack is a rare cause of iron deficiency in industrialized countries having abundant
food supplies (including meat) where about two thirds of the dietary iron is in the readily
assimilable heme form. The situation is different in developing countries, where food is
less abundant and diets are predominantly vegetarian, containing poorly absorbable
inorganic iron. Despite the availability of iron, dietary inadequacy still occurs in
privileged societies in these groups:

            • Infants are at high risk because milk diets contain very small amounts of iron.
            Human breast milk, for example, provides only about 0.3 mg/L of iron. Cow's
            milk contains about twice as much iron as human breast milk, but the iron in
            cow's milk has poor bioavailability.
            • Children, especially during the early years of life, have increased dietary iron
            needs to accommodate growth, development, and the accompanying expansion of
            blood volume.
            • The impoverished, at any age, can have suboptimal diets for socioeconomic
            reasons.
       • The elderly often have restricted diets with little meat because of limited income
       or poor dentition.

Impaired absorption is found in sprue, other causes of intestinal steatorrhea, and chronic
diarrhea. Gastrectomy impairs iron absorption by decreasing hydrochloric acid and transit
time through the duodenum. Specific items in the diet, as is evident from the preceding
discussion, can also affect absorption.

Increased requirement is an important potential cause of iron deficiency. Growing infants
and children, adolescents, and premenopausal (particularly pregnant) women have a
much greater requirement for iron than do nonmenstruating adults. Particularly at risk are
economically deprived women having multiple, frequent pregnancies.

Chronic blood loss is the most common cause of iron deficiency in the Western world. If
bleeding occurs into tissues or cavities of the body, the heme iron can be totally
recovered and recycled. However, external hemorrhage, as can occur from the
gastrointestinal tract (e.g., peptic ulcers, hemorrhagic gastritis, gastric carcinoma, colonic
carcinoma, hemorrhoids, or hookworm or pinworm disease), the urinary tract (e.g., renal,
pelvic, or bladder tumors), or the genital tract (e.g., menorrhagia, uterine cancer),
depletes iron reserves.

When all the potential causes of an iron deficiency are taken into consideration,
deficiency in adult men and postmenopausal women in the Western world must be
attributed to gastrointestinal blood loss until proven otherwise. To prematurely ascribe
iron deficiency in such individuals to any other cause is to run the risk of missing an
occult gastrointestinal cancer or other bleeding lesion.

Whatever its basis, iron deficiency induces a hypochromic microcytic anemia.
Simultaneously, depletion of essential iron-containing enzymes in cells throughout the
body can cause other changes, including koilonychia, alopecia, atrophic changes in the
tongue and gastric mucosa, and intestinal malabsorption. These changes are seen with
severe and long-standing iron deficiency. Uncommonly, esophageal webs appear to
complete the triad of major findings in the Plummer-Vinson syndrome: (1) microcytic
hypochromic anemia, (2) atrophic glossitis, and (3) esophageal webs ( Chapter 17 ).



                                              646


At the outset of chronic blood loss or other states of negative iron balance, reserves in the
form of ferritin and hemosiderin may be adequate to maintain normal hemoglobin and
hematocrit levels as well as normal serum iron and transferrin saturation. Progressive
depletion of these reserves first lowers serum iron and transferrin saturation levels,
without producing anemia. In this early stage, there is increased erythroid activity in the
bone marrow. Anemia only appears when iron stores are completely depleted,
accompanied by low serum iron, serum ferritin, and transferrin saturation.
Morphology.


The bone marrow reveals a mild to moderate increase in erythroid progenitors
(normoblasts). A diagnostically significant finding is the disappearance of stainable iron
from mononuclear phagocytic cells in the bone marrow, which is assessed by performing
Prussian blue stains on aspirated or sectioned bone marrow. In the peripheral blood
smear, red cells are small (microcytic) and pale (hypochromic). Normal well-
hemoglobinized red cells have a zone of central pallor measuring about one third of the
cell diameter. In established iron deficiency, the zone of pallor is enlarged; hemoglobin
may be seen only in a narrow peripheral rim ( Fig. 13-25 ). Poikilocytosis in the form of
small, elongated red cells (pencil cells) is also characteristic.

The clinical manifestations of the anemia are nonspecific and were detailed earlier. The
dominating signs and symptoms




Figure 13-25 Hypochromic microcytic anemia of iron deficiency (peripheral blood smear). Note the small
red cells containing a narrow rim of peripheral hemoglobin. Scattered fully hemoglobinized cells, present
due to recent blood transfusion, stand in contrast. (Courtesy of Dr. Robert W. McKenna, Department of
Pathology, University of Texas Southwestern Medical School, Dallas, TX.)



frequently relate to the underlying cause of the anemia, for example, gastrointestinal or
gynecologic disease, malnutrition, pregnancy, and malabsorption.

The diagnosis of iron deficiency anemia ultimately rests on laboratory studies. Both the
hemoglobin and hematocrit are depressed, usually to moderate levels, in association with
hypochromia, microcytosis, and some poikilocytosis. The serum iron and ferritin are
low, and the total plasma iron-binding capacity (reflecting transferrin concentration) is
high. Low serum iron with increased iron-binding capacity results in a reduction of
transferrin saturation levels to below 15%. Reduced iron stores inhibit hepcidin synthesis
and its serum levels fall. As mentioned earlier, transferrin receptor, expressed on the
surface of many cells, is required for the transport of iron into cells. The level of soluble
transferrin receptors, which are mostly derived from erythroid progenitors in the marrow,
is elevated in iron deficiency due to a mild expansion of erythroid progenitors and an
increased rate of transferrin receptor shedding. Reduced heme synthesis leads to elevation
of free erythrocyte protoporphyrin. An alert clinician investigating unexplained iron
deficiency anemia will occasionally discover an occult bleed or cancer and thereby save a
life.
Anemia of Chronic Disease


Impaired red cell production associated with chronic diseases is perhaps the most
common cause of anemia among hospitalized patients in the United States. It is
associated with reduced erythroid proliferation and impaired iron utilization and can
therefore mimic iron deficiency.[43] The chronic illnesses associated with this form of
anemia can be grouped into three categories:

          • Chronic microbial infections, such as osteomyelitis, bacterial endocarditis, and
          lung abscess
          • Chronic immune disorders, such as rheumatoid arthritis and regional enteritis
          • Neoplasms, such as Hodgkin lymphoma and carcinomas of the lung and breast

The common features characterizing anemia in these diverse clinical settings are low
serum iron and reduced total iron-binding capacity in association with abundant stored
iron in the mononuclear phagocytic cells. This combination suggests some impediment in
the transfer of iron from the storage pool to the erythroid precursors. In addition, marrow
erythroid progenitors do not proliferate adequately because erythropoietin levels are
inappropriately low for the degree of anemia. The reduction in renal erythropoietin
generation is caused by the action of interleukin-1, tumor necrosis factor (TNF), and
interferon-γ, secretion of which is triggered by the underlying chronic inflammatory or
neoplastic disease.[44] These cytokines also stimulate hepcidin synthesis in the liver, which
in turn inhibits the release of iron from the storage pool.[42] The anemia is usually mild,
and the dominant symptoms are those of the underlying disease. The red blood cells can
be normocytic and normochromic or hypochromic and microcytic as in anemia of iron
deficiency. The presence of increased storage iron in the marrow macrophages, a high
serum ferritin level, and reduced total iron-binding capacity readily rule out iron
deficiency as the cause of anemia. Only successful treatment of the underlying condition
reliably corrects the anemia. However, many patients benefit from administration of
erythropoietin.



                                               647
Aplastic Anemia


Aplastic anemia is a somewhat misleading term applied to a syndrome of marrow failure
associated with pancytopenia (anemia, neutropenia, and thrombocytopenia). The marrow
failure stems from suppression or disappearance of multipotent myeloid stem cells.
Etiology.


The major circumstances under which aplastic anemia can appear are listed in Table 13-7
.

Most cases of aplastic anemia of "known" etiology follow exposure to chemicals and
drugs. With some agents, marrow damage is predictable, dose related and, in most
instances, reversible when exposure to the offending agent is stopped. Among the best-
known dose-dependent myelotoxins are benzene, chloramphenicol, alkylating agents, and
antimetabolites (e.g., 6-mercaptopurine, vincristine, and busulfan). In other instances,
however, pancytopenia appears as an apparent idiosyncratic reaction to very small doses
of known myelotoxins (e.g., chloramphenicol) or nonmyelotoxic drugs such as
phenylbutazone, methylphenylethylhydantoin, streptomycin, and chlorpromazine. In such
idiosyncratic reactions, the aplasia can be severe, irreversible, and fatal.

Whole-body irradiation is another insult that can destroy hematopoietic stem cells in a
dose-dependent fashion. Persons who receive therapeutic irradiation or are exposed to
radiation in nuclear accidents (e.g., Chernobyl) are at risk.

Aplastic anemia can appear after a variety of viral infections, most commonly viral
hepatitis of the non-A, non-B, non-C, and non-G types. Why certain individuals develop
this complication is not understood, but it is not related to the severity of infection.

Fanconi anemia is a rare autosomal recessive disorder caused by defects in a component
of a multiprotein complex

                    TABLE 13-7 -- Major Causes of Aplastic Anemia
Acquired
Idiopathic
Primary stem cell defect
Immune mediated
Chemical agents
Dose related
Alkylating agents
Antimetabolites
Benzene
Chloramphenicol
Inorganic arsenicals
Idiosyncratic
Chloramphenicol
Phenylbutazone
Organic arsenicals
Methylphenylethylhydantoin
Streptomycin
Chlorpromazine
Insecticides (e.g., DDT,        parathion)
Physical agents (e.g., whole-body irradiation)
Viral infections
Hepatitis (unknown virus)
Cytomegalovirus infections
Epstein-Barr virus infections
Herpes varicella-zoster
Miscellaneous
Infrequently, many other drugs and      chemicals
Inherited
Fanconi anemia


required for DNA repair[45] ( Chapter 7 ). Marrow hypofunction in Fanconi anemia
becomes evident early in life and is accompanied by multiple congenital anomalies, such
as hypoplasia of the kidney and spleen and hypoplastic anomalies of bone, often
involving the thumbs or radii.

Despite all these possible causes, no provocative factor can be identified in fully 65% of
the cases, which are lumped into the idiopathic category.
Pathogenesis.


The pathogenesis of aplastic anemia is not fully understood. Indeed, it is unlikely that a
single mechanism underlies all cases. Two major etiologies have been invoked: an
immunologically mediated suppression and an intrinsic abnormality of stem cells ( Fig.
13-26 ).
Recent studies suggest that aplastic anemia results most commonly from suppression of
stem cell function by activated T cells.[46] It is postulated that stem cells are first
antigenically altered by exposure to drugs, infectious agents, or other unidentified
environmental insults. This evokes a cellular immune response, during which activated T
cells produce cytokines such as interferon-γ and TNF that prevent normal stem cell
growth and development. This scenario is supported by several observations.
Immunosuppressive therapy with antithymocyte globulin combined with drugs such as
cyclosporine produces responses in 60% to 70% of patients, and successful bone marrow
transplantation requires "conditioning" with high doses of myelotoxic drugs or radiation.
In both instances, it is hypothesized these therapies work by suppressing or killing
autoreactive T-cell clones. The target antigens for T-cell attack are not well defined. In
some instances GPI-linked proteins may be the targets of sensitized T cells,




Figure 13-26 Pathophysiology of aplastic anemia. Damaged stem cells can produce progeny expressing
neo-antigens that evoke an autoimmune reaction, or give rise to a clonal population with reduced
proliferative capacity. Either pathway could lead to marrow aplasia.
                                            648


possibly explaining why aplastic anemia sometimes evolves to paroxysmal nocturnal
hemoglobinuria.

Alternatively, the notion that aplastic anemia results from a fundamental stem cell
abnormality is supported by the presence of karyotypic aberrations in many cases and
occasional transformation of aplasia to myeloid neoplasms, typically myelodysplasia or
acute myelogenous leukemia.[46] Some marrow insult presumably causes genetic damage
that limits the proliferative and differentiative capacity of stem cells. If the damage is
extensive enough, aplastic anemia results. These two mechanisms are not mutually
exclusive, as genetically altered stem cells might also express "neo-antigens" that could
serve as targets for T-cell attack.
Morphology.


The markedly hypocellular bone marrow is largely devoid of hematopoietic cells; often
only fat cells, fibrous stroma, and scattered or clustered foci of lymphocytes and plasma
cells remain. A marrow aspirate often yields little material (a "dry tap"). Hence, marrow
aplasia is best appreciated in a bone marrow biopsy ( Fig. 13-27 ). A number of
additional pathologic changes commonly accompanying marrow failure are related to
granulocytopenia and thrombocytopenia, such as mucocutaneous bacterial infections and
abnormal bleeding, respectively. The toxic drug or agent sometimes injures other tissues
as well. Benzene, for example, can cause fatty changes in the liver and kidneys. If the
anemia necessitates multiple transfusions, systemic hemosiderosis can appear.
Clinical Course.


Aplastic anemia can occur at any age and in either sex. The onset is usually insidious.
Initial manifestations vary somewhat, depending on which cell line is predominantly
affected. Anemia can cause progressive weakness, pallor, and dyspnea. Petechiae and
ecchymoses can herald thrombocytopenia. Granulocytopenia can manifest as frequent
and persistent minor infections or the sudden onset of chills, fever, and prostration.
Splenomegaly is characteristically absent; if present, the diagnosis of aplastic anemia
should be seriously questioned. The red cells are typically normocytic and
normochromic, although slight macrocytosis is occasionally present. Reticulocytopenia is
the rule.
Figure 13-27 Aplastic anemia (bone marrow biopsy). Markedly hypocellular marrow contains mainly fat
cells. A, Low power. B, High power. (Courtesy of Dr. Steven Kroft, Department of Pathology, University of
Texas Southwestern Medical School, Dallas, TX.)



The diagnosis rests on examination of bone marrow biopsy and peripheral blood. It is
important to distinguish aplastic anemia from other causes of pancytopenia, such as
"aleukemic" leukemia and myelodysplastic syndromes ( Chapter 14 ), that can present
with identical clinical manifestations. In aplastic anemia, the marrow is hypocellular (and
usually markedly so), whereas myeloid neoplasms are associated with hypercellular
marrow filled with abnormal myeloid progenitors. The prognosis of aplastic aplasia is
unpredictable. As mentioned earlier, withdrawal of toxic drugs can lead to recovery in
some cases. Spontaneous remission in idiopathic cases is unfortunately uncommon. In
younger patients, allogeneic bone marrow transplantation offers a hope for cure. Older
patients or those without suitable donors often respond well to immunosuppressive
therapies (antithymocyte globulin and cyclosporine).
Pure Red Cell Aplasia


Pure red cell aplasia is a rare form of marrow failure characterized by a marked
hypoplasia of marrow erythroid elements in the setting of normal granulopoiesis and
thrombopoiesis.[47] Pure red cell aplasia can be primary, without any associated disease, or
arise secondarily to neoplasms, particularly thymic tumors (thymomas) and large
granular lymphocytic leukemia ( Chapter 14 ), drug exposures, or autoimmune disorders.
The association with thymoma raises the question of some thymus-related immunologic
mechanism; indeed, in about half the patients, resection of the thymic tumor is followed
by hematologic improvement. In all likelihood, the primary form is also related to
autoimmunity against erythroid precursors, and immunosuppressive therapy can be
beneficial in such patients. Plasmapheresis has also been used with some success in
refractory cases.
Other Forms of Marrow Failure


Space-occupying lesions that destroy significant amounts of bone marrow or disturb the
marrow architecture depress its productive capacity. This form of marrow failure is
referred to as myelophthisic anemia. As you might anticipate, all formed blood elements
are affected. Characteristically, immature erythroid and myeloid progenitors appear in the
peripheral blood



                                             649


(leukoerythroblastosis). Infiltrative diseases of the marrow typically cause reactive
fibrosis and distortion of the marrow architecture; presumably, this disturbs the normal
mechanisms that restrict the release of immature erythroid and myeloid cells into the
peripheral blood. The most common cause of myelophthisic anemia is metastatic cancer,
most often carcinomas arising in the breast, lung, and prostate. However, any tumor or
infiltrative process (e.g., granulomatous disease) involving the marrow can produce
identical findings. Myelophthisic anemia is also observed in myelofibrosis and other
myeloproliferative disorders ( Chapter 14 ), all of which often cause marrow fibrosis.

Diffuse liver disease, whether toxic, infectious, or cirrhotic, is associated with an anemia
attributed to hypofunction of the marrow. Concomitant folate deficiency and iron
deficiency due to gastrointestinal blood loss (varices, hemorrhoids) can also contribute to
the anemia. Most often, erythroid progenitors are preferentially affected; depression of
the white cell count and platelets is less common, but has been described. The anemia is
often slightly macrocytic due to lipid abnormalities associated with liver failure, which
cause red cell membranes to acquire phospholipid and cholesterol in the periphery.

Chronic renal failure, whatever its cause, is almost invariably associated with anemia
that tends to be roughly proportional to the severity of the uremia. The basis of the
anemia is multifactorial. There is evidence of an extracorpuscular defect inducing chronic
hemolysis. Some patients have an iron deficiency secondary to the bleeding tendency
often encountered in uremia. Concomitantly, there is reduced red cell production, related
to advanced destruction of the kidneys and inadequate synthesis of erythropoietin, which
appears to be the dominant cause of anemia. Not surprisingly, therefore, administration of
recombinant erythropoietin results in significant improvement of the anemia associated
with renal failure,[48] although optimal response may require aggressive concomitant iron
replacement therapy.




Polycythemia

Polycythemia, or erythrocytosis, denotes an abnormally high concentration of red cells,
usually with a corresponding increase in hemoglobin level. The increase in red cells can
be relative, when there is hemoconcentration due to decreased plasma volume, or
absolute, when there is an increase in total red cell mass. Relative polycythemia results
from any cause of dehydration, such as deprivation of water, prolonged vomiting,
diarrhea, or excessive use of diuretics. It is also associated with an obscure condition of
unknown etiology called stress polycythemia, or Gaisböck syndrome. Affected
individuals are usually hypertensive, obese, and anxious ("stressed"). Absolute
polycythemia is primary when it results from an intrinsic abnormality of the myeloid
stem cells and secondary when the red cell progenitors are responding to increased levels
of erythropoietin. Primary polycythemia (polycythemia vera) is one of several neoplasms
originating from myeloid stem cells (considered in Chapter 14 ). Another much less
common form of "primary" polycythemia results from mutations in the erythropoietin
receptor that cause hyperresponsiveness to erythropoietin. Affected individuals have
congenital polycythemia.[49] One such individual won an Olympic gold medal in cross-
country skiing, having benefited from this natural form of blood doping![50] Secondary
polycythemias can be caused by an increase in erythropoietin secretion that is
physiologically appropriate (e.g., chronic hypoxia) or inappropriate (pathologic) ( Table
13-8 ).

Bleeding Disorders: Hemorrhagic Diatheses

Excessive bleeding can result from (1) increased fragility of vessels, (2) platelet
deficiency or dysfunction, (3) derangement of coagulation, and (4) combinations of these.

Before discussing these specific bleeding disorders, it is helpful to review normal
hemostasis ( Chapter 4 ) and the common laboratory tests used in the evaluation of a
bleeding diathesis. It should be recalled from the discussion in Chapter 4 that the normal
hemostatic response involves the blood vessel wall, the platelets, and the clotting
cascade. Tests used to evaluate different aspects of hemostasis are the following:

       • Bleeding time. This measures the time taken for a standardized skin puncture to
       stop bleeding and provides an in vivo assessment of platelet response to limited
       vascular injury. The reference range depends on the actual method employed and
       varies from 2 to 9 minutes. Prolongation generally indicates a defect in platelet
       numbers or function. Bleeding time test is fraught with variability and poor
       reproducibility. Hence new instrument-based assay systems such as platelet
       function analyzer-100 (PFA-100) that provide a quantitative measure of platelet
       function under conditions of high shear stress are being evaluated as replacements
       for the bleeding time test.[51]
       • Platelet counts. These are obtained on anticoagulated blood using an electronic
       particle counter. The reference range is 150 to 300 × 103 /µL. Counts well outside
       this range need to be confirmed by a visual inspection of a peripheral blood
       smear, as clumping of platelets can cause spurious "thrombocytopenia" during
       automated counting, and high counts may be indicative of a myeloproliferative
       disorder.
       • Prothrombin time (PT). This assay tests the extrinsic and common coagulation
       pathways. The clotting of plasma after addition of an exogenous source of tissue
       thromboplastin (e.g., brain extract) and Ca2+ ions is measured in seconds. A
       prolonged PT can result from deficiency or dysfunction of factor V, factor VII,
       factor X, prothrombin, or fibrinogen.
       • Partial thromboplastin time (PTT). This assay tests the intrinsic and common
       clotting pathways. The clotting of


                                                650


       plasma after addition of kaolin, cephalin, and calcium ions is measured in
       seconds. Kaolin serves to activate the contact-dependent factor XII, and cephalin
       substitutes for platelet phospholipids. Prolongation of the PTT can be due to
       deficiency or dysfunction of factor V, VIII, IX, X, XI, or XII, prothrombin, or
       fibrinogen.

            TABLE 13-8 -- Pathophysiologic Classification of Polycythemia
Relative         Reduced plasma volume (hemoconcentration)
Absolute
Primary          Polycythemia vera, rare erythropoietin receptor mutations (low
                 erythropoietin)
Secondary        High erythropoietin
                 Appropriate: lung disease, high-altitude living, cyanotic heart disease
                 Inappropriate: erythropoietin-secreting tumors (e.g., renal cell
                 carcinoma, hepatocellular carcinoma, cerebellar hemangioblastoma)


More specialized tests are available to measure the levels of specific clotting factors,
fibrinogen, fibrin split products, the presence of circulating anticoagulants, and platelet
function. With this overview, we can turn to the various categories of bleeding disorders.
BLEEDING DISORDERS CAUSED BY VESSEL WALL ABNORMALITIES


Disorders within this category, sometimes called nonthrombocytopenic purpuras, are
relatively common but do not usually cause serious bleeding problems. Most often, they
induce small hemorrhages (petechiae and purpura) in the skin or mucous membranes,
particularly the gingivae. On occasion, however, more significant hemorrhages can occur
into joints, muscles, and subperiosteal locations or take the form of menorrhagia,
nosebleeds, gastrointestinal bleeding, or hematuria. The platelet count, bleeding time, and
results of the coagulation tests (PT, PTT) are usually normal.

The varied clinical conditions in which hemorrhages can be related to abnormalities in
the vessel wall include the following:

       • Many infections induce petechial and purpuric hemorrhages, but especially
       implicated are meningococcemia, other forms of septicemia, infective
       endocarditis, and several of the rickettsioses. The involved mechanism is
       presumably microbial damage to the microvasculature (vasculitis) or disseminated
       intravascular coagulation (DIC). Failure to recognize meningococcemia as a cause
       of petechiae and purpura can be catastrophic for the patient.
       • Drug reactions sometimes induce cutaneous petechiae and purpura without
       causing thrombocytopenia. In many instances, the vascular injury is mediated by
       drug-induced antibodies and deposition of immune complexes in the vessel walls,
       leading to hypersensitivity (leukocytoclastic) vasculitis ( Chapter 11 ).
       • Scurvy and the Ehlers-Danlos syndrome are associated with microvascular
       bleeding resulting from impaired formation of collagens needed for support of
       vessel walls. The same mechanism may account for spontaneous purpura
       commonly seen in the very elderly. The predisposition to skin hemorrhages in
       Cushing syndrome, in which the protein-wasting effects of excessive
       corticosteroid production cause loss of perivascular supporting tissue, has a
       similar etiology.
       • Henoch-Schönlein purpura is a systemic hypersensitivity disease of unknown
       cause characterized by a purpuric rash, colicky abdominal pain (presumably due
       to focal hemorrhages into the gastrointestinal tract), polyarthralgia, and acute
       glomerulonephritis ( Chapter 20 ). All these changes result from the deposition of
       circulating immune complexes within vessels throughout the body and within the
       glomerular mesangial regions.
       • Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder
       characterized by dilated, tortuous blood vessels with thin walls that bleed readily.
       Bleeding can occur anywhere in the body but is most common under the mucous
       membranes of the nose (epistaxis), tongue, mouth, and eyes and throughout the
       gastrointestinal tract.
       • Amyloid infiltration of blood vessels. Systemic amyloidosis is associated with
       perivascular deposition of amyloid and consequent weakening of blood vessel
       wall. This is most commonly observed in plasma cell dyscrasias ( Chapter 14 )
       and is manifested as mucocutaneous petechiae.

Bleeding in these conditions is rarely life threatening with the exception of some cases of
hereditary telangiectasia. Recognition of the presenting symptoms should prompt further
studies to establish a specific diagnosis.
BLEEDING RELATED TO REDUCED PLATELET NUMBER: THROMBOCYTOPENIA


Reduction in platelet number constitutes an important cause of generalized bleeding.
Normal platelet counts range from 150,000 to 300,000/µL. A count below 100,000/µL is
generally considered to constitute thrombocytopenia. However, spontaneous bleeding
does not become evident until the count falls below 20,000/µL. Platelet counts in the
range of 20,000 to 50,000/µL can aggravate post-traumatic bleeding. Bleeding resulting
from thrombocytopenia alone is associated with a prolonged bleeding time and normal
PT and PTT.

The important role of platelets in hemostasis is discussed in Chapter 4 . It hardly needs
reiteration that these cells are critical for hemostasis, as they form temporary plugs that
quickly stop bleeding and promote key reactions in the clotting cascade. Spontaneous
bleeding associated with thrombocytopenia most often involves small vessels. The
common sites of such hemorrhage are the skin and the mucous membranes of the
gastrointestinal and genitourinary tracts. Intracranial bleeding is a threat to any patient
with a markedly depressed platelet count.

The many causes of thrombocytopenia can be classified into the four major categories
listed in Table 13-9 .

       • Decreased production of platelets. This can accompany generalized diseases of
       bone marrow such as aplastic anemia and leukemias or result from diseases that
       affect the megakaryocytes somewhat selectively. In vitamin B12 or folic acid
       deficiency, there is poor development and accelerated destruction of
       megakaryocytes within the bone marrow (ineffective megakaryopoiesis) because
       DNA synthesis is impaired.
       • Decreased platelet survival. This important cause of thrombocytopenia can have
       an immunologic or nonimmunologic etiology. In the immune conditions, platelet
       destruction is caused by circulating antiplatelet antibodies or, less often, immune
       complexes. The antiplatelet antibodies can be directed against a self-antigen on
       the platelets (autoantibodies) or against platelet antigens that differ among
       different individuals (alloantibodies). Common antigenic targets of both
       autoantibodies and alloantibodies are the platelet membrane glycoprotein
       complexes IIb-IIIa and Ib-IX. Autoimmune thrombocytopenias include idiopathic
       thrombocytopenic purpura, certain drug-induced thrombocytopenias, and HIV-
       associated thrombocytopenias. All of these are discussed later. Alloimmune
       thrombocytopenias arise when an individual is exposed to platelets


                                                  651


       of another person, as may occur after blood transfusion or during pregnancy. In
       the latter case, neonatal or even fetal thrombocytopenia occurs by a mechanism
       analogous to erythroblastosis fetalis.[52]
       Nonimmunologic destruction of platelets may be caused by mechanical injury, in
       a manner analogous to red cell destruction in microangiopathic hemolytic anemia.
       The underlying conditions are also similar, including prosthetic heart valves and
       diffuse narrowing of the microvessels (e.g., malignant hypertension).
       • Sequestration. Thrombocytopenia, usually moderate in severity, may develop in
       any patient with marked splenomegaly, a condition sometimes referred to as
       hypersplenism ( Chapter 14 ). The spleen normally sequesters 30% to 40% of the
       body's platelets, which remain in equilibrium with the circulating pool. When
       necessary, hypersplenic thrombocytopenia can be ameliorated by splenectomy.
       • Dilutional. Massive transfusions can produce a dilutional thrombocytopenia.
       Blood stored for longer than 24 hours contains virtually no viable platelets; thus,
       plasma volume and red cell mass are reconstituted by transfusion, but the number
       of circulating platelets is relatively reduced.
                            TABLE 13-9 -- Causes of Thrombocytopenia
Decreased production of platelets
Generalized diseases of bone marrow
Aplastic anemia: congenital and             acquired (see Table 13-7 )
Marrow infiltration: leukemia,             disseminated cancer
Selective impairment of platelet production
Drug-induced: alcohol, thiazides,            cytotoxic drugs
Infections: measles, human              immunodeficiency virus (HIV)
Ineffective megakaryopoiesis
Megaloblastic anemia
Myelodysplastic syndromes
Decreased platelet survival
Immunologic destruction
Autoimmune: idiopathic thrombocytopenic                 purpura, systemic lupus erythematosus
Isoimmune: post-transfusion and              neonatal
Drug-associated: quinidine, heparin,            sulfa compounds
Infections: infectious mononucleosis,              HIV infection, cytomegalovirus
Nonimmunologic destruction
Disseminated intravascular              coagulation
Thrombotic thrombocytopenic                purpura
Giant hemangiomas
Microangiopathic hemolytic               anemias
Sequestration
Hypersplenism
Dilutional


Immune Thrombocytopenic Purpura (ITP)


ITP can occur in the setting of a variety of conditions and exposures (secondary ITP) or
in the absence of any known risk factors (primary or idiopathic ITP). There are two
clinical subtypes of primary ITP, acute and chronic; both are autoimmune disorders in
which platelet destruction results from the formation of antiplatelet autoantibodies. We
first discuss the more common chronic form of primary ITP; acute ITP, a self-limited
disease of children, is discussed later.
Immunologically mediated destruction of platelets (immune thrombocytopenia) occurs in
many different settings, including systemic lupus erythematosus, acquired
immunodeficiency syndrome (AIDS), after viral infections, and as a complication of drug
therapy. These secondary forms of immune thrombocytopenia can sometimes mimic the
idiopathic autoimmune variety, and hence the diagnosis of this disorder should be made
only after exclusion of other known causes of thrombocytopenia. Particularly important
in this regard is systemic lupus erythematosus, a multisystem autoimmune disease (
Chapter 6 ) that can present with thrombocytopenia.
Pathogenesis.


Chronic ITP is caused by the formation of autoantibodies against platelet membrane
glycoproteins, most often IIb-IIIa or Ib-IX.[53] Antibodies reactive with these membrane
glycoproteins can be demonstrated in the plasma as well as bound to the platelet surface
(platelet-associated immunoglobulins) in approximately 80% of patients. In the
overwhelming majority of cases, the antiplatelet antibodies are of the IgG class.

The mechanism of platelet destruction is similar to that seen in autoimmune hemolytic
anemias. Opsonized platelets are rendered susceptible to phagocytosis by the cells of the
mononuclear phagocyte system. About 75% to 80% of patients are remarkably improved
after splenectomy, indicating that the spleen is the major site of removal of sensitized
platelets. Since it is also an important site of autoantibody synthesis, the beneficial effects
of splenectomy may in part derive from removal of the source of autoantibodies.
Although destruction of sensitized platelets is the major mechanism responsible for
thrombocytopenia, there is some evidence that megakaryocytes may be damaged by
autoantibodies, leading to impairment of platelet production. In most cases, however,
megakaryocyte injury is not significant enough to deplete their numbers.
Morphology.


The principal morphologic lesions of thrombocytopenic purpura are found in the spleen
and bone marrow but they are not diagnostic. Secondary changes related to the bleeding
diathesis may be found in any tissue or structure in the body.

The spleen is normal in size. On histologic examination, there is congestion of the
sinusoids and hyperactivity and enlargement of the splenic follicles, manifested by the
formation of prominent germinal centers. In many instances, scattered megakaryocytes
are found within the sinuses and sinusoidal walls. This may represent a very mild form of
extramedullary hematopoiesis driven by elevated levels of thrombopoietin. These splenic
findings are not sufficiently distinctive to be considered diagnostic.

Bone marrow reveals a modestly increased number of megakaryocytes. Some are
apparently immature, with large, nonlobulated, single nuclei. These findings are not
specific for autoimmune thrombocytopenic purpura but merely reflect accelerated
thrombopoiesis, being found in most forms of thrombocytopenia resulting from increased
platelet destruction. The importance of bone marrow examination is to rule out
thrombocytopenias resulting from bone marrow failure. A decrease in the number of
megakaryocytes argues against the diagnosis of ITP. The secondary changes relate to the
hemorrhages that are dispersed throughout the body.



                                            652

Clinical Features.


Chronic ITP occurs most commonly in adult women younger than age 40 years. The
female-to-male ratio is 3:1. This disorder is often insidious in onset and is characterized
by bleeding into the skin and mucosal surfaces. Cutaneous bleeding is seen in the form of
pinpoint hemorrhages (petechiae), especially prominent in the dependent areas where the
capillary pressure is higher. Petechiae can become confluent, giving rise to ecchymoses.
Often there is a history of easy bruising, nosebleeds, bleeding from the gums, and
hemorrhages into soft tissues from relatively minor trauma. The disease may manifest
first with melena, hematuria, or excessive menstrual flow. Subarachnoid hemorrhage and
intracerebral hemorrhage are serious consequences of thrombocytopenic purpura but,
fortunately, are rare in treated patients. Splenomegaly and lymphadenopathy are
uncommon in primary ITP, and their presence should lead one to consider other possible
diagnoses.

The clinical signs and symptoms associated with ITP are not specific for this condition
but rather reflective of thrombocytopenia. Destruction of platelets as the cause of
thrombocytopenia is supported by the findings of a low platelet count and normal or
increased megakaryocytes in the bone marrow. Accelerated thrombopoiesis often leads to
the formation of abnormally large platelets (megathrombocytes), detected easily in a
blood smear. The bleeding time is prolonged, but PT and PTT are normal. Tests for
platelet autoantibodies are not widely available. Therefore, a diagnosis of ITP should be
made only after other causes of platelet deficiencies, such as those listed in Table 13-9 ,
have been ruled out.

Almost all patients respond to immunosuppressive doses of glucocorticoids, but many
eventually relapse and come to splenectomy. Most maintain safe platelet counts
postsplenectomy and require no further therapy. A significant minority, however, have
refractory forms of ITP that can be very difficult to treat. Various immunosuppressive
approaches may be effective in such patients.
Acute Immune Thrombocytopenic Purpura


Like chronic ITP, this condition is caused by antiplatelet autoantibodies, but its clinical
features and course are distinct. Acute ITP is a disease of childhood occurring with equal
frequency in both sexes. The onset of thrombocytopenia is abrupt and is preceded in
many cases by a viral illness. The usual interval between the infection and onset of
purpura is 2 weeks. Unlike the adult chronic form of ITP, the childhood disease is self-
limited, usually resolving spontaneously within 6 months. Steroid therapy is indicated
only if thrombocytopenia is severe. Approximately 20% of the children, usually those
without a viral prodrome, have persistent low platelet counts beyond 6 months and appear
to have chronic ITP similar in most respects to the adult disease.
Drug-Induced Thrombocytopenia: Heparin-Induced Thrombocytopenia


Like hemolytic anemia, thrombocytopenia can result from immunologically mediated
destruction of platelets after drug ingestion.[54] The drugs most commonly involved are
quinine, quinidine, sulfonamide antibiotics, and heparin. Heparin-induced
thrombocytopenia (HIT) is of particular importance because this anticoagulant is used
widely and failure to make a correct diagnosis can have severe consequences.
Thrombocytopenia occurs in approximately 5% of patients receiving heparin. Most
develop so-called type I thrombocytopenia, which occurs rapidly after onset of therapy, is
modest in severity and clinically insignificant, and may resolve despite continuation of
heparin therapy. It most likely results from a direct platelet-aggregating effect of heparin.

Type II thrombocytopenia is more severe. It occurs 5 to 14 days after commencement of
therapy (or sometimes sooner if the patient has been previously sensitized to heparin) and
can, paradoxically, lead to life-threatening venous and arterial thrombosis.[54] HIT is
caused by an immune reaction directed against a complex of heparin and platelet factor 4,
a normal component of platelet granules that binds tightly to heparin. It appears that
heparin binding modifies the conformation of platelet factor 4, making it susceptible to
immune recognition. [55] Binding of antibody to platelet factor 4 produces immune
complexes that activate platelets, promoting thrombosis even in the setting of marked
thrombocytopenia. The mechanism of platelet activation is not understood. Unless
therapy is immediately discontinued, clots within large arteries may lead to vascular
insufficiency and limb loss, and emboli from deep venous thrombosis can cause fatal
pulmonary thromboembolism.
HIV-Associated Thrombocytopenia


Thrombocytopenia is perhaps the most common hematologic manifestation of HIV
infection. Both impaired platelet production and increased destruction are responsible.
CD4, the receptor for HIV on T cells, has also been demonstrated on megakaryocytes,
making it possible for these cells to be infected by HIV.[56] Infected megakaryocytes are
prone to apoptosis and are impaired in terms of platelet production. HIV infection also
causes hyperplasia and dysregulation of B cells, which predispose to the development of
immune-mediated thrombocytopenia. Antibodies directed against platelet membrane
glycoprotein IIb-III complexes are detected in some patients' sera. These autoantibodies,
which sometimes cross-react with HIV-associated gp120, are believed to act as opsonins,
thus promoting the phagocytosis of platelets by splenic phagocytes. Some studies also
implicate nonspecific deposition of immune complexes on platelets as a factor in their
premature destruction by the mononuclear phagocyte system.
Thrombotic Microangiopathies: Thrombotic Thrombocytopenic Purpura (TTP) and Hemolytic-Uremic Syndrome (HUS)


The term thrombotic microangiopathy encompasses a spectrum of clinical syndromes
that includes TTP and HUS. TTP, as originally defined, is associated with the pentad of
fever, thrombocytopenia, microangiopathic hemolytic anemia, transient neurologic
deficits, and renal failure. HUS is also associated with microangiopathic hemolytic
anemia and thrombocytopenia but is distinguished from TTP by the absence of
neurologic symptoms, the prominence of acute renal failure, and frequent affliction of
children. Recent studies, however, have tended to blur these clinical distinctions. Many
adult patients with "TTP" lack one or more of the five criteria, and some patients with
"HUS" have fever and neurologic dysfunction. The common fundamental feature in both
of these conditions is widespread formation of hyaline thrombi, comprised primarily of
platelet aggregates, in the



                                              653


microcirculation. Consumption of platelets leads to thrombocytopenia, and the
intravascular thrombi provide a likely mechanism for the microangiopathic hemolytic
anemia and widespread organ dysfunction. It is believed the varied clinical
manifestations of TTP and HUS are related to differing proclivities for thrombus
formation in specific microvascular beds.

For many years, the pathogenesis of TTP was enigmatic, although treatment with plasma
exchange (initiated in the early 1970s) changed an almost uniformly fatal condition into
one that is successfully treated in more than 80% of cases. Recently, the underlying cause
of many, but not all, cases of TTP has been elucidated. In brief, symptomatic patients are
often deficient in an enzyme called ADAMTS 13. This enzyme is designated "vWF
metalloprotease" and it normally degrades very high molecular weight multimers of von
Willebrand factor (vWF).[57] (ADAMTS 13 is unrelated to the other tissue
metalloproteases that cleave extracellular matrix.) In the absence of this enzyme, very
high molecular weight multimers of vWF accumulate in plasma and, under some
circumstances, promote platelet microaggregate formation throughout the
microcirculation, leading to the symptoms of TTP. Superimposition of endothelial cell
injury (caused by some other condition) may further predispose a patient to
microaggregate formation, thus initiating or exacerbating clinically evident TTP.

The deficiency of ADAMTS 13 may be inherited or acquired.[58] In many patients an
antibody that inhibits vWF metalloprotease is detected.[57] Much less commonly the
patients have inherited an inactivating mutation in the gene encoding this enzyme.
Despite these advances, it is clear that factors other than vWF metalloprotease deficiency
must be involved in triggering full-blown TTP, because symptoms are episodic even in
those with hereditary deficiency of vWF metalloprotease. It is important to consider the
possibility of TTP in any patient presenting with thrombocytopenia and microangiopathic
hemolytic anemia, as any delay in diagnosis and treatment can be fatal. Plasma exchange
can be life saving by providing the missing enzyme.

In contrast to TTP, most patients with HUS have normal levels of vWF metalloprotease,
indicating that HUS usually has a different pathogenesis. [59] One important cause of HUS
in children and the elderly is infectious gastroenteritis caused by E. coli strain 0157:H7.[60]
This strain elaborates a Shiga-like toxin that is absorbed from the inflamed
gastrointestinal mucosa. It binds to and damages endothelial cells in the glomerulus and
elsewhere, thus initiating platelet activation and aggregation. Affected children present
with bloody diarrhea, and a few days later HUS makes its appearance. With appropriate
supportive care, affected children often recover completely, but irreversible renal damage
and death can occur in more severe cases. HUS can also be seen in adults following
exposures that damage endothelial cells (e.g., certain drugs, radiation therapy). The
prognosis of adults with HUS is guarded, as it is most often seen in the setting of other
chronic, life-threatening conditions.

While DIC and thrombotic microangiopathies share features such as microvascular
occlusion and microangiopathic hemolytic anemia, they are pathogenetically distinct. In
TTP and HUS (unlike DIC), activation of the coagulation cascade is not of primary
importance, and hence results of laboratory tests of coagulation, such as PT and PTT, are
usually normal.
BLEEDING DISORDERS RELATED TO DEFECTIVE PLATELET FUNCTIONS


Qualitative defects of platelet function can be congenital or acquired. Several congenital
disorders characterized by prolonged bleeding time and normal platelet count have been
described. A brief discussion of these rare diseases is warranted by the fact that they
provide excellent models for investigating the molecular mechanisms of platelet
function.[61]

Congenital disorders of platelet function can be classified into three groups on the basis
of the specific functional abnormality: (1) defects of adhesion, (2) defects of aggregation,
and (3) disorders of platelet secretion (release reaction).

       • Bleeding resulting from defective adhesion of platelets to subendothelial matrix
       is best illustrated by the autosomal recessive disorder Bernard-Soulier syndrome,
       which is caused by an inherited deficiency of the platelet membrane glycoprotein
       complex Ib-IX. This glycoprotein is a receptor for vWF and is essential for
       normal platelet adhesion to subendothelial matrix ( Chapter 4 ).
       • Bleeding due to defective platelet aggregation is exemplified by Glanzmann's
       thrombasthenia, which is also transmitted as an autosomal recessive trait.
       Thrombasthenic platelets fail to aggregate in response to adenosine diphosphate
       (ADP), collagen, epinephrine, or thrombin owing to deficiency or dysfunction of
       glycoprotein IIb-IIIa, a protein complex that participates in the formation of
       "bridges" between platelets by binding fibrinogen and vWF.
       • Disorders of platelet secretion are characterized by normal initial aggregation
       with collagen or ADP, but subsequent responses, such as secretion of
       thromboxanes and release of granule-bound ADP, are impaired. The underlying
       biochemical defects of these so-called storage pool disorders are varied, complex,
       and beyond the scope of our discussion.

Among the acquired defects of platelet function, two are clinically significant.[62] The first
is ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, which
significantly prolongs the bleeding time. Aspirin is a potent, irreversible inhibitor of the
enzyme cyclooxygenase, which is required for the synthesis of thromboxane A2 and
prostaglandins ( Chapter 2 ). These mediators play important roles in platelet aggregation
and subsequent release reactions ( Chapter 4 ). The antiplatelet effects of aspirin form the
basis for its use in the prophylaxis of thrombosis ( Chapter 12 ). Uremia ( Chapter 20 ) is
the second condition exemplifying an acquired defect in platelet function. Although the
pathogenesis of bleeding in uremia is complex and not fully understood, several
abnormalities of platelet function are found.
HEMORRHAGIC DIATHESES RELATED TO ABNORMALITIES IN CLOTTING FACTORS


A deficiency of every clotting factor has been reported to be the cause of a bleeding
disorder, with the exception of factor XII deficiency, which does not cause bleeding. The
bleeding in factor deficiencies differs from platelet deficiencies in that spontaneous
petechiae or purpura are uncommon. Rather, the bleeding is manifested by large post-
traumatic ecchymoses or hematomas, or prolonged bleeding after a laceration or any
form of surgical procedure. Bleeding into the gastrointestinal and urinary tracts, and
particularly into weight-bearing joints, is common. Typical stories include the patient
who continues to



                                             654


ooze for days after a tooth extraction or who develops a hemarthrosis after relatively
trivial stress on a knee joint. The course of history may have been changed by a
hereditary coagulation defect present in the intermarried royal families of Great Britain
and other parts of Europe. Clotting abnormalities can also be acquired in many different
conditions.

Acquired disorders are usually characterized by multiple clotting abnormalities. Vitamin
K deficiency ( Chapter 9 ) results in impaired synthesis of factors II, VII, IX, and X and
protein C. Since the liver makes virtually all the clotting factors, severe parenchymal
liver disease can be associated with a hemorrhagic diathesis. Disseminated intravascular
coagulation produces a deficiency of multiple coagulation factors.

Hereditary deficiencies have been identified for each of the clotting factors. Deficiencies
of factor VIII (hemophilia A) and of factor IX (Christmas disease, or hemophilia B) are
transmitted as sex-linked recessive disorders. Most others follow autosomal patterns of
transmission. These hereditary disorders typically involve a single clotting factor.
Deficiencies of Factor VIII-vWF Complex


Hemophilia A and von Willebrand disease, two of the most common inherited disorders
of bleeding, are caused by qualitative or quantitative defects involving the factor VIII-
vWF complex. Before we can discuss these disorders, it is essential to review the
structure and function of these proteins.[63] [64]
Plasma factor VIII-vWF is a complex made up of two separate proteins (factor VIII and
vWF) that can be characterized according to functional, biochemical, and immunologic
criteria. Factor VIII procoagulant protein, or factor VIII ( Fig. 13-28 ;




Figure 13-28 Structure and function of factor VIII-von Willebrand factor (vWF) complex. Factor VIII is
synthesized in the liver and kidney, and vWF is made in endothelial cells and megakaryocytes. The two
associate to form a complex in the circulation. vWF is also present in the subendothelial matrix of normal
blood vessels and the alpha granules of platelets. Following endothelial injury, exposure of subendothelial
vWF causes adhesion of platelets, primarily via glycoprotein lb platelet receptor. Circulating vWF and
vWF released from the alpha granules of activated platelets can bind exposed subendothelial matrix, further
contributing to platelet adhesion and activation. Activated platelets form hemostatic aggregates; fibrinogen
(and possibly vWF) participate in aggregation through bridging interactions with the platelet receptor
GpIIb/III. Factor VIII takes part in the coagulation cascade as a cofactor in the activation of factor X on the
surface of activated platelets.



also see Chapter 4 ), is an intrinsic pathway component required for activation of factor
X. Deficiency of factor VIII gives rise to hemophilia A. Circulating factor VIII is
noncovalently associated with very large vWF multimers containing up to 100 subunits;
the molecular mass of individual multimers can exceed 20 × 106 daltons. vWF also
interacts with several other proteins involved in hemostasis, including collagen, heparin,
and platelet membrane glycoproteins (Ib-IX and IIb-IIIa). Glycoprotein Ib-IX serves as
the major receptor for vWF. The most important function of vWF in vivo is to promote
the adhesion of platelets to subendothelial matrix, which is accomplished in two ways.
Some vWF secreted by endothelial cells is normally deposited in the subendothelial
matrix, where it promotes platelet adhesion should the endothelial lining be disrupted
(see Fig. 13-28 ). Endothelial cells and platelets also release vWF into the circulation, and
upon vascular injury this second pool of vWF is adsorbed to exposed subendothelial
matrix and further augments platelet adhesion. vWF multimers can also promote platelet
aggregation by binding to activated GpIIb/IIIa receptors; this activity may be of particular
importance under conditions of high shear stress (such as occurs in small vessels). That
vWF is crucial to the normal process of hemostasis ( Chapter 4 ) is supported by the
occurrence of a bleeding diathesis known as von Willebrand disease when there is
deficiency of this factor.

vWF multimers also serve as a carrier for factor VIII and are important for its stability.
The half-life of factor VIII in the circulation is 12 hours if vWF levels are normal but
only 2.4 hours if it is deficient or abnormal (as in patients with von Willebrand disease).

vWF can be assayed by immunologic techniques or by the so-called ristocetin
agglutination test. This assay, which can be



                                             655


performed with formalin-fixed platelets, measures the ability of ristocetin (developed as
an antibiotic) to promote the interaction between vWF and platelet membrane
glycoprotein Ib. Multivalent ristocetin-dependent binding of vWF creates interplatelet
"bridges," leading to the formation of platelet clumps (agglutination), an event easily
measured in a device called an aggregometer. Thus, the degree of ristocetin-dependent
platelet agglutination caused by the addition of patient plasma provides a bioassay for
vWF.

The two components of the factor VIII-vWF complex are encoded by separate genes and
synthesized in different cells. vWF is produced by endothelial cells and megakaryocytes
and can be demonstrated in platelet α-granules. Endothelial cells are the major source of
subendothelial and plasma vWF. Factor VIII is made in several tissues; sinusoidal
endothelial cells and Kupffer cells in the liver and glomerular and tubular epithelial cells
in the kidney appear to be particularly important sites of synthesis. To summarize, the
two components of factor VIII-vWF complex, synthesized separately, come together and
circulate in the plasma as a unit that serves to promote clotting as well as platelet-vessel
wall interactions necessary to ensure hemostasis. With this background, we can discuss
the diseases resulting from deficiencies of factor VIII-vWF complex.
Von Willebrand Disease


With an estimated frequency of 1%, von Willebrand disease is believed to be one of the
most common inherited disorders of bleeding in humans. Clinically, it is characterized by
spontaneous bleeding from mucous membranes, excessive bleeding from wounds,
menorrhagia, and a prolonged bleeding time in the presence of a normal platelet count. In
most cases, it is transmitted as an autosomal dominant disorder, but several rare
autosomal recessive variants have been identified.[65]

More than 20 variants of von Willebrand disease have been described, which can be
grouped into two major categories:

          • Type 1 and type 3 von Willebrand disease are associated with a reduced
          quantity of circulating vWF. Type 1, an autosomal dominant disorder, accounts
          for approximately 70% of all cases and is relatively mild. Reduced penetrance and
          variable expressivity characterize this type, and hence clinical manifestations are
          varied. Type 3 (an autosomal recessive disorder) is associated with extremely low
          levels of functional vWF, and the clinical manifestations are correspondingly
          severe. Because a severe deficiency of vWF has a marked affect on the stability of
          factor VIII, some of the bleeding characteristics resemble those seen in
          hemophilia. The nature of the mutations in the vast majority of patients with type
          1 disease is poorly defined. In some cases missense mutations have been found. In
          others, it is suspected that both alleles are affected by distinct mutations
          (compound heterozygotes) producing an apparent dominant inheritance. Type 3
          disease is associated with deletions or frameshift mutations.[66]
          • Type 2 von Willebrand disease is characterized by qualitative defects in vWF;
          there are several subtypes, of which type 2A is the most common. It is inherited
          as an autosomal dominant disorder. Because of missense mutations, the vWF
          formed is abnormal, leading to defective multimer assembly. Large and
          intermediate multimers, representing the most active forms of vWF, are missing
          from plasma. Type 2 von Willebrand disease accounts for 25% of all cases and is
          associated with mild to moderate bleeding.

Patients with von Willebrand disease have prolonged bleeding time despite a normal
platelet count. The plasma level of active vWF, measured as the ristocetin cofactor
activity, is reduced. Because vWF stabilizes factor VIII by binding to it, a deficiency of
vWF gives rise to a secondary decrease in factor VIII levels. This may be reflected by a
prolongation of the PTT in von Willebrand disease types 1 and 3.

To summarize, patients with von Willebrand disease have a compound defect involving
platelet function and the coagulation pathway. Even within families in which a single
defective allele is segregating, there is often wide variability in the clinical expression of
von Willebrand disease. This appears to be due to additional genetic factors that influence
circulating levels of vWF, which vary greatly in normal populations.[67] However, except
in the most severely affected type 3 patients, adverse complications of factor VIII
deficiency, such as bleeding into the joints, are uncommon.
Hemophilia A (Factor VIII Deficiency)


Hemophilia A is the most common hereditary disease associated with serious bleeding.
It is caused by a reduction in the amount or activity of factor VIII. This protein serves as
a cofactor for factor IX in the activation of factor X in the coagulation cascade ( Chapter
4 ). Hemophilia A is inherited as an X-linked recessive trait, and thus occurs in males and
in homozygous females. However, excessive bleeding has been described in
heterozygous females, presumably due to extremely unfavorable lyonization (inactivation
of the normal X chromosome in most of the cells). Approximately 30% of patients have
no family history; their disease is presumably caused by new mutations.

Hemophilia A exhibits a wide range of clinical severity that correlates well with the level
of factor VIII activity. Those with less than 1% of normal activity develop severe disease;
levels between 2% and 5% of normal are associated with moderate disease; and patients
with 6% to 50% of activity develop mild disease. The variable degrees of factor VIII
deficiency are largely explained by heterogeneity in the causative mutations.[68] As with β-
thalassemias, several genetic lesions (deletions, nonsense mutations that create stop
codons, splicing errors) have been documented. Most severe deficiencies result from an
unusual inversion involving the X chromosome that completely abolishes the synthesis of
factor VIII. Less commonly, severe hemophilia A is associated with point mutations in
factor VIII that impair the function of the protein. In such cases, levels of factor VIII
appear normal by immunoassay. Mutations permitting some active factor VIII to be
synthesized are associated with mild to moderate disease. The disease in such patients
may be modified by other genetic factors that influence factor VIII expression levels,
which vary widely in normal individuals.[69]

In all symptomatic cases, there is a tendency toward easy bruising and massive
hemorrhage after trauma or operative procedures. In addition, "spontaneous"
hemorrhages frequently occur in regions of the body normally subject to trauma,
particularly the joints, where they are known as hemarthroses. Recurrent bleeding into
the joints leads to progressive



                                              656


deformities that can be crippling. Petechiae are characteristically absent.

Patients with hemophilia A typically have a normal bleeding time, platelet count, and PT,
and a prolonged PTT. These tests point to an abnormality of the intrinsic coagulation
pathway. Factor VIII-specific assays are required for diagnosis.

Given that one arm of the coagulation cascade, the extrinsic pathway, is intact in
hemophilia A, it seems reasonable to ask, why do these patients bleed? Obviously, test
tube assays of coagulation (discussed briefly below under DIC) are imperfect surrogates
for what occurs in vivo, and it must be that in the face of factor VIII deficiency, fibrin
deposition is inadequate to achieve hemostasis reliably. It is beyond our scope to discuss
this issue in detail, but recent studies suggest the following.[70] First, it appears that the
chief role of the extrinsic pathway in hemostasis is to produce a limited initial burst of
thrombin activation upon tissue injury. This is reinforced and amplified by a critical
feedback loop whereby thrombin activates factors XI and IX of the intrinsic pathway. In
addition, high levels of thrombin are required to activate TAFI (thrombin activatable
fibrinolysis inhibitor), a factor that augments fibrin deposition by inhibiting fibrinolysis.
Thus, both inadequate coagulation (fibrinogenesis) and inappropriate clot removal
(fibrinolysis) contribute to the bleeding diathesis in hemophilia. The precise explanation
for the tendency of hemophiliacs to bleed at particular sites (joints, muscles, and the
central nervous system) remains uncertain.

Treatment of hemophilia A involves infusion of recombinant factor VIII. Approximately
15% of patients with low or absent factor VIII develop antibodies that bind to and inhibit
factor VIII. Inhibitors are most likely to develop in patients with severe factor VIII
deficiency (possibly because the protein is perceived as foreign, having never been "seen"
before by the immune system) and represent very difficult therapeutic challenges. There
are other hazards of replacement therapy as well, the most serious of which has been the
risk of transmission of viral diseases. Until the mid-1980s, before routine screening of
blood for HIV antibodies was instituted, thousands of hemophiliacs received plasma-
derived factor VIII concentrates containing HIV, and many developed AIDS ( Chapter 6
). With the availability of recombinant factor VIII, the risk of HIV transmission has been
eliminated, but tragically too late for an entire generation of hemophiliacs. Efforts to
develop somatic gene therapy for hemophilia are also under way.
Hemophilia B (Christmas Disease, Factor IX Deficiency)


Severe factor IX deficiency produces a disorder clinically indistinguishable from factor
VIII deficiency (hemophilia A). This should not be surprising, given that factor VIII and
IX function together to activate factor X. A wide spectrum of mutations involving the
factor IX gene are found in hemophilia B.[68] Like hemophilia A, it is inherited as an X-
linked recessive trait and shows variable clinical severity. In about 14% of these patients,
factor IX is present but nonfunctional. As with hemophilia A, the PTT is prolonged and
the PT is normal, as is the bleeding time. Identification of Christmas disease (named after
the first patient with this condition and not the holiday) is possible only by assay of the
factor levels. Recombinant factor IX is used for treatment.
DISSEMINATED INTRAVASCULAR COAGULATION (DIC)


DIC is an acute, subacute, or chronic thrombohemorrhagic disorder occurring as a
secondary complication in a variety of diseases. It is characterized by activation of the
coagulation sequence that leads to the formation of microthrombi throughout the
microcirculation of the body, often in a quixotically uneven distribution. Sometimes the
coagulopathy is localized to a specific organ or tissue. As a consequence of the
thrombotic diathesis, there is consumption of platelets, fibrin, and coagulation factors
and, secondarily, activation of fibrinolytic mechanisms. Thus, DIC can present with signs
and symptoms relating to tissue hypoxia and infarction caused by the myriad
microthrombi or as a hemorrhagic disorder related to depletion of the elements required
for hemostasis (hence, the term "consumption coagulopathy" is sometimes used to
describe DIC). Activation of the fibrinolytic mechanism aggravates the hemorrhagic
diathesis.
Etiology and Pathogenesis.


At the outset, it must be emphasized that DIC is not a primary disease. It is a
coagulopathy that occurs in the course of a variety of clinical conditions. In discussing
the general mechanisms underlying DIC, it is useful to briefly review the normal process
of blood coagulation and clot removal. Clotting can be initiated by either of two
pathways: (1) the extrinsic pathway, which is triggered by the release of tissue factor
("tissue thromboplastin"), and (2) the intrinsic pathway, which involves the activation of
factor XII by surface contact with collagen or other negatively charged substances. Both
pathways, through a series of intermediate steps, result in the generation of thrombin,
which in turn converts fibrinogen to fibrin. Once activated at the site of injury, thrombin
further augments local fibrin deposition through feedback activation of the intrinsic
pathway and inhibition of fibrinolysis. Remarkably, as excess thrombin is swept away in
the blood from sites of tissue injury it is converted to an anticoagulant. Upon binding a
surface protein called thrombomodulin on intact endothelial cells, thrombin becomes
capable of activating protein C, an inhibitor of the pro-coagulant factors V and VIII.
Other important clot-inhibiting factors include the activation of fibrinolysis by plasmin
and the clearance of activated clotting factors by the mononuclear phagocyte system and
the liver. These and additional checks and balances normally ensure that just enough
clotting occurs at the right place and time.

From this brief review, it should be clear that DIC could result from pathologic activation
of the extrinsic and/or intrinsic pathways of coagulation or impairment of clot-inhibiting
influences. Since the latter rarely constitute primary mechanisms of DIC, we focus our
attention on the abnormal initiation of clotting. [71]

Two major mechanisms trigger DIC: (1) release of tissue factor or thromboplastic
substances into the circulation and (2) widespread injury to the endothelial cells. Tissue
thromboplastic substances can be derived from a variety of sources, such as the placenta
in obstetric complications ( Table 13-10 ) and the granules of leukemic cells in acute
promyelocytic leukemia. Mucus released from certain adenocarcinomas can also act as a
thromboplastic substance by directly activating factor X, independent of factor VII. In
gram-negative sepsis (an important cause of DIC), bacterial endotoxins cause activated
monocytes to release interleukin-1 and TNF, both of which



                                            657




     TABLE 13-10 -- Major Disorders Associated with Disseminated Intravascular
                                  Coagulation
Obstetric Complications
Abruptio placentae
Retained dead fetus
Septic abortion
Amniotic fluid embolism
Toxemia
Infections
Gram-negative sepsis
Meningococcemia
Rocky Mountain spotted fever
Histoplasmosis
Aspergillosis
Malaria
Neoplasms
Carcinomas of pancreas, prostate, lung, and stomach
Acute promyelocytic leukemia
Massive Tissue Injury
Traumatic
Burns
Extensive surgery
Miscellaneous
Acute intravascular hemolysis, snakebite, giant hemangioma, shock, heat stroke,
vasculitis, aortic aneurysm, liver disease

increase the expression of tissue factor on endothelial cell membranes and simultaneously
decrease the expression of thrombomodulin.[72] The net result is a shift in balance toward
procoagulation.

Endothelial injury, the other major trigger, can initiate DIC by causing release of tissue
factor, promoting platelet aggregation, and activating the intrinsic coagulation pathway.
TNF is an extremely important mediator of endothelial cell inflammation and injury in
septic shock. In addition to the effects previously mentioned, TNF up-regulates the
expression of adhesion molecules on endothelial cells and thus favors adhesion of
leukocytes, which in turn damage endothelial cells by releasing oxygen-derived free
radicals and preformed proteases.[72] Even subtle endothelial injury can unleash
procoagulant activity by enhancing membrane expression of tissue factor. Widespread
endothelial injury may be produced by deposition of antigen-antibody complexes (e.g.,
systemic lupus erythematosus), temperature extremes (e.g., heat stroke, burns), or
microorganisms (e.g., meningococci, rickettsiae).

Several disorders associated with DIC are listed in Table 13-10 . Of these, DIC is most
likely to follow obstetric complications, malignant neoplasia, sepsis, and major trauma.
The initiating factors in these conditions are often multiple and interrelated. For example,
particularly in infections caused by gram-negative bacteria, released endotoxins can
activate both the intrinsic and extrinsic pathways by producing endothelial cell injury and
release of thromboplastins from inflammatory cells; furthermore, endotoxins inhibit the
anticoagulant activity of protein C by suppressing thrombomodulin expression on
endothelium. Endothelial cell damage can also be produced directly by meningococci,
rickettsiae, and viruses. Antigen-antibody complexes formed during the infection can
activate the classical complement pathway, and complement fragments can secondarily
activate both platelets and granulocytes. Endotoxins as well as other bacterial products
are also capable of directly activating factor XII. In massive trauma, extensive surgery,
and severe burns, the major mechanism of DIC is believed to be the release of tissue
thromboplastins. In obstetric conditions, thromboplastins derived from the placenta, dead
retained fetus, or amniotic fluid may enter the circulation. However, hypoxia, acidosis,
and shock, which often coexist with the surgical and obstetric conditions, also cause
widespread endothelial injury. Supervening infection can complicate the problems
further. Among cancers, acute promyelocytic leukemia and carcinomas of the lung,
pancreas, colon, and stomach are most frequently associated with DIC. These tumors
release of a variety of thromboplastic substances, including tissue factors, proteolytic
enzymes, mucin, and other undefined tumor products.

The consequences of DIC are twofold. First, there is widespread deposition of fibrin
within the microcirculation. This can lead to ischemia of the more severely affected or
more vulnerable organs and to a hemolytic anemia resulting from fragmentation of red
cells as they squeeze through the narrowed microvasculature (microangiopathic
hemolytic anemia). Second, a hemorrhagic diathesis can dominate the clinical picture.
This results from consumption of platelets and clotting factors as well as activation of
plasminogen. Plasmin can not only cleave fibrin, but also digest factors V and VIII,
thereby reducing their concentration further. In addition, fibrinolysis leads to the
formation of fibrin degradation products, which inhibit platelet aggregation and fibrin
polymerization and have antithrombin activity. All these influences lead to the hemostatic
failure seen in DIC ( Fig. 13-29 ).
Morphology.


In general, thrombi are found in the following sites in decreasing order of frequency:
brain, heart, lungs, kidneys, adrenals, spleen, and liver. However, no tissue is spared, and
thrombi are occasionally found in only one or several organs without affecting others. In
giant hemangiomas, for example, thrombi are localized to the neoplasm, where they are
believed to form due to local stasis and recurrent trauma to fragile blood vessels. The
affected kidneys can reveal small thrombi in the glomeruli that may evoke only reactive
swelling of endothelial cells or, in severe cases, microinfarcts or even bilateral renal
cortical necrosis. Numerous fibrin thrombi may be found in alveolar capillaries,
sometimes associated with pulmonary edema and fibrin exudation, creating "hyaline
membranes" reminiscent of acute respiratory distress syndrome ( Chapter 15 ). In the
central nervous system, fibrin thrombi can cause microinfarcts, occasionally complicated
by simultaneous hemorrhage. Such changes are the basis for the bizarre neurologic signs
and symptoms sometimes observed in DIC. The manifestations of DIC in the endocrine
glands are of considerable interest. In meningococcemia, fibrin thrombi within the
microcirculation of the adrenal cortex are the likely basis for the massive adrenal
hemorrhages seen in Waterhouse-Friderichsen syndrome ( Chapter 24 ). Similarly,
Sheehan postpartum pituitary necrosis ( Chapter 24 ) is a form of DIC complicating labor
and delivery. In toxemia of pregnancy ( Chapter 22 ), the placenta exhibits widespread
microthrombi, providing a plausible
                                                  658




Figure 13-29 Pathophysiology of disseminated intravascular coagulation.



explanation for the premature atrophy of the cytotrophoblast and syncytiotrophoblast
encountered in this condition.

The bleeding manifestations of DIC are not dissimilar to those encountered in the
hereditary and acquired disorders affecting the hemostatic mechanisms discussed earlier.
Clinical Course.


The onset can be fulminant, as in endotoxic shock or amniotic fluid embolism, or
insidious and chronic, as in cases of carcinomatosis or retention of a dead fetus. Overall,
about 50% of individuals with DIC are obstetric patients having complications of
pregnancy. In this setting, the disorder tends to be reversible with delivery of the fetus.
About 33% of the patients have carcinomatosis. The remaining cases are associated with
the various entities previously listed.

It is almost impossible to detail all the potential clinical presentations, but a few common
patterns are worthy of description. These include microangiopathic hemolytic anemia;
dyspnea, cyanosis, and respiratory failure; convulsions and coma; oliguria and acute renal
failure; and sudden or progressive circulatory failure and shock. In general, acute DIC,
associated with obstetric complications or major trauma, for example, is dominated by a
bleeding diathesis, whereas chronic DIC, such as occurs in cancer patients, tends to
present initially with thrombotic complications. Accurate clinical observation and
laboratory studies are necessary for the diagnosis. It is usually necessary to monitor
fibrinogen, platelets, PT, PTT, and fibrin degradation products.
The prognosis is highly variable and depends, to a considerable extent, on the underlying
disorder. The management of these cases requires meticulous maneuvering between the
Scylla of thrombosis and the Charybdis of bleeding diathesis. Administration of
anticoagulants or procoagulants has been advocated in specific settings, but not without
controversy. The only definitive treatment is to remove or treat the inciting cause
whenever possible.

References

1. Dzierzak    E: Embryonic beginnings of definitive hematopoietic stem cells. Ann NY Acad Sci 872:256,
1999.

2. Kondo M, et al: Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell
91:661, 1997.

3. AkashiK, et al: A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature
404:193, 2000.

      SH, Zon LL: Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nature
4. Orkin
Immunol 3:323–328, 2002.

5. Clarke   D, Frisen J: Differentiation potential of adult stem cells. Curr Opin Genet Dev 11:575, 2001.

6. Vogel    G: Stem cell policy. Can adult stem cells suffice? Science 292:1820, 2001.

7. Wang X, et al: Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422:897–
901, 2003.

8. Delaunay    J: Molecular basis of red cell membrane disorders. Acta Haematol 108:210, 2002.

9. EberSW, et al: Ankyrin-1 mutations are a major cause of dominant and recessive hereditary
spherocytosis. Nat Genet 13:214, 1996.

10. JarolimP, et al: Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band
3 deficiency. Blood 88:4366, 1996.

11. Jandl   J, et al: Red cell filtration and the pathogenesis of certain hemolytic anemias. Blood 18:33, 1961.

12. MehtaA, Mason PJ, Vulliamy TJ: Glucose-6-phosphate dehydrogenase deficiency. Bailliers Best Pract
Res Clin Hematol 13:21, 2000.

13. Tishkoff  SA, et al: Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of
alleles that confer malarial resistance. Science 293:455, 2001.

14. Gomez-GallegoF, et al: Structural defects underlying protein dysfunction in human glucose-6-
phosphate dehydrogenase A(-) deficiency. J Biol Chem 275:9256, 2000.

        M, et al: Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet
15. Aidoo
359:1311, 2002.

16. Brugnara    C, et al: Erythrocyte-active agents and treatment of sickle cell disease. Semin Hematol 38:324,
2001.

17. Hebbel     RP: Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest 100:S83, 1997.




                                                        659




         ST, et al: Prediction of adverse outcomes in children with sickle cell disease. N Engl J Med
18. Miller
342:83, 2000.

19. Zachlederora M, Jarolim, P: Gene expression profile of microvascular endothelial cells after stimuli
implicated in pathogenesis of vaso-occlusion. Blood Cell Molecules Dis 30:71, 2003.

20. Frenette   PS: Sickle cell vaso-occlusion: multistep and multicellular paradigm. Curr Opin Hematol 9:101,
2002.

21. Liao    JC: Blood feud: keeping hemoglobin from nixing NO. Nature Med 8:1350, 2002.

22. Ballas   SK: Sickle cell disease: current clinical management. Semin Hematol 38:307, 2001.

23. Smith    J: Bone disorders in sickle cell disease. Hematol Oncol Clin North Am 10:1345, 1996.

24. Platt   OS: The acute chest syndrome of sickle cell disease. N Engl J Med 342:1904, 2000.

25. Bunn    HF: Pathogenesis and treatment of sickle cell disease. N Engl J Med 337:762, 1997.

26. DaviesSC, Gilmore A: The role of hydroxyurea in the management of sickle cell disease. Blood Rev
17:99, 2003.

          A, et al: Five years of experience with hydroxyurea in children and young adults with sickle cell
27. Ferster
disease. Blood 97:3628, 2001.

28. Olivieri   NF: The β-thalassemias. N Engl J Med 341:99, 1999.

       D, Rachmilewitz E: Pathophysiology of α- and β-thalassemia: therapeutic implications. Semin
29. Rund
Hematol 38:343, 2001.

30. Rosse    WF: New insights into paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol 8:61, 2001.

31. WrightMS: Drug-induced hemolytic anemias: increasing complications to therapeutic interventions.
Clin Lab Sci 12:115, 1999.

32. Gehrs    BC, Friedberg RC: Autoimmune hemolytic anemia. Am J Hematol 69:258, 2002.

33. Oh   RC, Brown DL: Vitamin B12 deficiency. Am Fam Physican 67:979, 2003.

34. Hoffbrand AV, Jackson BF: Correction of the DNA synthesis defect in vitamin B12 deficiency by
tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic
anaemia in vitamin B12 deficiency. Br J Haematol 83:643, 1993.

35. Chanarin    I, et al: Cobalamin and folate: recent developments. J Clin Pathol 45:277, 1992.

36. Wickramasinghe SN: The wide spectrum and unresolved issues of megaloblastic anemia. Semin
Hematol 36:3, 1999.

         MI, et al: Varying neurological phenotypes among mut o and mut- patients with
37. Shevell
methylmalonylCoA mutase deficiency. Am J Med Genet 45:619, 1993.

38. Toh   BH, et al: Pernicious anemia. N Engl J Med 337:1441, 1997.

39. Looker    AC, et al: Prevalence of iron deficiency in the United States. JAMA 277:973, 1997.

40. Andrews,    NC: A genetic view of iron homeostasis. Semin Hematol 39:227, 2002.

41. FlemingRE, Sly WS: Mechanisms of iron accumulation in hereditary hemochromatosis. Annu Rev
Physiol 64:663, 2002.

42. GanzT: Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood
102:783, 2003.

43. MeansRT, Jr: Erythropoietin in the treatment of anemia in chronic infectious, inflammatory, and
malignant diseases. Curr Opin Hematol 2:210, 1995.

44. Spivak    J: Iron and anemia of chronic disease. Oncology (Huntingt) 16 (Suppl 10):25, 2002.

45. Dokal    I: Inherited aplastic anemia. Hematol J 4:3, 2003.

46. Young    NS: Acquired aplastic anemia. Ann Intern Med 136:534, 2002.

47. Erslev   AJ, Soltan A: Pure red-cell aplasia: a review. Blood Rev 10:20, 1996.

48. Eschbach    JW: Current concepts of anemia management in chronic renal failure. Semin Nephrol 20:320,
2000.

49. Gregg    XT, Prchal JT: Erythropoietin receptor mutations and human disease. Semin Hematol 34:70,
1997.

50. Longmore     GD: Erythropoietin receptor mutations and Olympic glory. Nat Genet 4:108, 1993.

51. Rand    ML, Leung R, Packham MA: Platelet function assays. Trans Apher Sci 28:307, 2003.

52. Bussel   JB: Alloimmune thrombocytopenia in the fetus and newborn. Semin Thromb Hemost 27:245,
2001.

53. Cines   DB, Blanchette VS: Immune thrombocytopenic purpura. N Engl J Med 346:995, 2002.

54. Aster   RH: Drug-induced immune thrombocytopenia: an overview of pathogenesis. Semin Hematol 36:2,
1999.

55. Visentin   GP, et al: Heparin is not required for detection of antibodies associated with heparin-induced
thrombocytopenia/thrombosis. J Lab Clin Med 138:22, 2001.

56. Scaradavou    A: HIV-related thrombocytopenia. Blood Rev 16:73, 2002.

       HM: Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura. Arterioscler Thromb
57. Tsai
Vasc Biol 23:388, 2003.

58. LevyGG, et al: Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic
purpura. Nature 413:488, 2001.

59. MoakeJL: Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab
Med 126:1430, 2002.

60. Zoja   C, et al: The role of the endothelium in hemolytic uremic syndrome. J Nephrol 14(Suppl 4):S58,
2001.

      AK: Congenital disorders of platelet function: disorders of signal transduction and secretion. Am J
61. Rao
Med Sci 316:69, 1998.

62. BickRL: Platelet function defects associated with hemorrhage or thrombosis. Med Clin North Am
78:577, 1994.

          PJ, et al: The life cycle of coagulation factor VIII in view of its structure and function. Blood
63. Lenting
92:3983, 1998.

64. RuggeriZM: Structure of von Willebrand factor and its function in platelet adhesion and thrombus
formation. Best Pract Res Clin Haematol 14:257, 2001.

65. Sadler   JE, et al: Impact, diagnosis and treatment of von Willebrand disease. Thromb Haemost 84:160,
2000.

66. Castman  G, et al: von Willebrand disease in year 2003: towards the complete identification of gene
defects for correct diagnosis and treatment. Hematologica 88:94, 2003.

67. LevyG, Ginsburg D: Getting at the variable expressivity of von Willebrand disease. Thromb Haemost
86:144, 2001.

68. Bowen    DJ: Haemophilia A and haemophilia B: molecular insights. Mol Pathol 55:1, 2002.

69. LensenR, et al: High factor VIII levels contribute to the thrombotic risk in families with factor V
Leiden. Br J Haematol 114:380, 2001.

70. Mosnier LO, et al: The defective down-regulation of fibrinolysis in haemophilia A can be restored by
increasing the TAFI plasma concentration. Thromb Haemost 86:1035, 2001.

      RL: Disseminated intravascular coagulation: a review of etiology, pathophysiology, diagnosis, and
71. Bick
management: guidelines for care. Clin Appl Thromb Hemost 8:1, 2002.

72. Esmon    CT, et al: Inflammation, sepsis, and coagulation. Haematologica 84:254, 1999.

				
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