90_01

Iron Deficiency and Other Hypoproliferative Anemias: Introduction

Anemias associated with normocytic and normochromic red cells and an inappropriately low reticulocyte response (reticulocyte index <2.5) are hypoproliferative anemias. This category includes early iron deficiency (before hypochromic microcytic red cells develop), acute and chronic inflammation (including many malignancies), renal disease, hypometabolic states such as protein malnutrition and endocrine deficiencies, and anemias from marrow damage. Marrow damage states are discussed in Chap. 94. Hypoproliferative anemias are the most common anemias and anemia associated with acute and chronic inflammation is the most common of these. The anemia of acute and chronic inflammation, like iron deficiency, is related in part to abnormal iron metabolism. The anemias associated with renal disease, inflammation, cancer, and hypometabolic states are characterized by an abnormal erythropoietin response to anemia.

Iron Metabolism

Iron is a critical element in the function of all cells, although the amount of iron required by individual tissues varies during development. At the same time, the body must protect itself from free iron, which is highly toxic in that it participates in chemical reactions that generate free radicals such as singlet O2 or OH–. Consequently, elaborate mechanisms have evolved that allow iron to be made available for critical physiologic functions while at the same time conserving this element and handling it in such a way that toxicity is avoided.

The major role of iron in mammals is to carry O2 as part of the heme protein that, in turn, is part of hemoglobin. O2 is also bound by a heme protein in muscle, myoglobin. Iron is a critical element in iron-containing enzymes, including the cytochrome system in mitochondria; iron distribution in the body is shown in Table 90-1. Without iron, cells lose their capacity for electron transport and energy metabolism; in erythroid cells hemoglobin synthesis is impaired, resulting in anemia and reduced O2 delivery to tissue.

The Iron Cycle in Humans

Figure 90-1 outlines the major pathways of internal iron exchange in humans. Iron absorbed from the diet or released from stores circulates in the plasma bound to transferrin, the iron transport protein. Transferrin is a bilobed glycoprotein with two iron binding sites. Transferrin that carries iron exists in two forms—monoferric (one iron atom) or diferric (two iron atoms). The turnover (half-clearance time) of transferrin-bound iron is very rapid—typically 60 to 90 min. Because the overwhelming majority of iron transported by transferrin is delivered to the erythroid marrow, the clearance time of transferrin-bound iron from the circulation is affected most by the plasma iron level and the activity of the erythroid marrow. When erythropoiesis is markedly stimulated, the pool of erythroid cells requiring iron increases and the clearance time of iron from the circulation decreases. The half-clearance time of iron in the presence of iron deficiency is as short as 10 to 15 min; this value reflects the limits of iron delivery as a function of the cardiac output going to the bone marrow. With suppression of the erythroid marrow, the plasma iron level typically is increased and the half-clearance time is prolonged to as much as several hours. Normally, the iron bound to transferrin turns over 10 to 20 times per day. Assuming a normal plasma iron level of 80 to 100 g/dL, the amount of iron passing through the transferrin pool is 20 to 24 mg/d.


Figure 90-1 Internal iron exchange. Normally about 80% of iron passing through the plasma transferrin pool is recycled from broken-down red cells. Absorption of about 1 mg/d is required from the diet in men, 1.4 mg/d in women to maintain homeostasis. As long as transferrin saturation is maintained between 20 to 60% and erythropoiesis is not increased, iron stores are not required. However, in the event of blood loss, dietary iron deficiency, or inadequate iron absorption, up to 40 mg/d of iron can be mobilized from stores. RE, reticuloendothelial.


The iron-transferrin complex circulates in the plasma until the iron-carrying transferrin interacts with specific transferrin receptors on the surface of marrow erythroid cells. Diferric transferrin has the highest affinity for transferrin receptors; apotransferrin (transferrin not carrying iron) has very little affinity. While transferrin receptors are found on cells in many tissues within the body—and all cells at some time during development will display transferrin receptors—the cell having the greatest number of receptors (300,000 to 400,000/cell) is the developing erythroblast.

Once the iron-bearing transferrin interacts with its receptor, the iron-transferrin-receptor complex is internalized via clathrin-coated pits and transported to an acidic endosome, where the iron is released at the low pH. The iron is then made available for heme synthesis while the transferrin-receptor complex is recycled to the surface of the cell, where the bulk of the transferrin is released back into the circulation and the transferrin receptor reanchors into the cell membrane. At this point a certain amount of the transferrin receptor protein may be released into circulation. Within the erythroid cell, iron that is in excess of the amount needed for hemoglobin synthesis binds to a storage protein, apoferritin, forming ferritin. This mechanism of iron exchange also takes place in other cells of the body expressing transferrin receptors, especially liver parenchymal cells where the iron can be incorporated into heme-containing enzymes or stored. The iron incorporated into hemoglobin subsequently enters the circulation as new red cells are released from the bone marrow. The iron is then part of the red cell mass and will not become available for reutilization until the red cell dies.

In a normal individual, the average red cell life span is 120 days. Thus, 0.8 to 1.0% of red cells turn over each day. At the end of its life span, the red cell is recognized as senescent by the cells of the reticuloendothelial (RE) system, and the cell undergoes phagocytosis. Once within the RE cell, the hemoglobin from the ingested red cell is broken down, the globin and other proteins are returned to the amino acid pool, and the iron is shuttled back to the surface of the RE cell, where it is presented to circulating transferrin. The "harvesting" of iron from senescent red cells is both efficient and rapid, with newly recycled iron appearing in the circulation within 10 min of ingestion of the red cell. It is the efficient and highly conserved recycling of iron from senescent red cells that supports steady state (and even mildly accelerated) erythropoiesis.

Since each milliliter of red cells contains 1 mg of elemental iron, the amount of iron needed to replace those red cells lost through senescence amounts to 16 to 20 mg/d (assuming an adult with a red cell mass of 2 L). Any additional iron required for daily red cell production comes from the diet. Normally, an adult male will need to absorb at least 1 mg of elemental iron daily to meet needs, while females in the childbearing years will need to absorb an average of 1.4 mg/d. However, to achieve a maximum proliferative erythroid marrow response to anemia, additional iron must be available. With markedly stimulated erythropoiesis, demands for iron are increased by as much as six- to eightfold. With hemolytic anemias, the rate of red cell destruction is increased, but the iron recovered from the red cells is efficiently reutilized for hemoglobin synthesis. In contrast, with blood loss anemia the rate of red cell production is limited by the amount of iron that can be mobilized from ferritin and hemosiderin stores. Typically, the rate of mobilization under these circumstances will not support red cell production more than 2.5 to 3 times normal. If the delivery of iron to the stimulated marrow is suboptimal, the marrow's proliferative response is blunted and normal hemoglobin synthesis is impaired. The result is a hypoproliferative marrow accompanied by microcytic, hypochromic anemia.

While blood loss or hemolysis places a demand for iron to be supplied to the erythroid marrow, other conditions such as inflammation interfere with iron release from stores and can result in a rapid decrease in the serum iron (see below).

Nutritional Iron Balance

The balance of iron metabolism in the organism is tightly controlled and designed to conserve iron for reutilization. There is no excretory pathway for iron, and the only mechanisms by which iron is lost from the body are blood loss (via gastrointestinal bleeding, menses, or other forms of bleeding) and the loss of epidermal cells from the skin and gut. Normally, the only route by which iron comes into the body is via absorption from food (dietary iron intake) or from medicinal iron taken orally. Iron may also enter the body through red cell transfusions or injection of iron complexes. The margin between the amount of iron available for absorption and the requirement for iron in growing infants and the adult female is narrow. The narrowness of this margin accounts for the great prevalence of iron deficiency worldwide—currently estimated at one-half billion people.

External iron exchange—the amount of iron required from the diet to replace losses—averages about 10% of body iron content a year in men and 15% in women of childbearing age, equivalent to 1.0 and 1.4 mg of elemental iron daily, respectively. Dietary iron content is closely related to total caloric intake (approximately 6 mg of elemental iron per 1000 calories). Iron bioavailability is affected by the nature of the foodstuff, with heme iron (e.g., red meat) being most readily absorbed. In the United States, the average iron intake in an adult male is 15 mg/d with 6% absorption; for the average female, the daily intake is 11 mg/d with 12% absorption. An individual with iron deficiency can increase iron absorption to about 20% of the iron present in a meat-containing diet but only 5 to 10% of the iron in a vegetarian diet. As a result, nearly one-third of the female population in the United States has virtually no iron stores. Vegetarians are at an additional disadvantage because certain foodstuffs that include phytates and phosphates reduce iron absorption by about 50%. When ionizable iron salts are given together with food, the amount of iron absorbed is reduced. This is particularly true with iron in the ferric state. When the percentage of iron absorbed from individual food items is compared with the percentage for an equivalent amount of ferrous salt, iron in vegetables is only about one-twentieth as available, egg iron one-eighth, liver iron one-half, and heme iron one-half to two-thirds. Therefore, liver and heme iron are absorbed nearly as well as iron salt added to food, while the iron in vegetables and eggs is much less available.

Infants, children, and adolescents may be unable to maintain normal iron balance because of the demands of body growth and lower dietary intake of iron. In pregnancy during the last two trimesters, daily iron requirements increase to 5 to 6 mg. That is the reason why iron supplements are strongly recommended for pregnant women in developed countries. Enthusiasm for supplementing foods such as bread and cereals with iron has waned in the face of concerns that the very prevalent hemochromatosis gene would result in an unacceptable risk of iron overload.

Iron absorption takes place largely in the proximal small intestine and is a carefully regulated process. For absorption, iron must be taken up by the luminal cell. That process is facilitated by the acidic contents of the stomach, which maintains the iron in solution. At the brush border of the absorptive cell, the ferric iron is converted to the ferrous form by a ferrireductase. Transport across the membrane is accomplished by divalent metal transporter 1 (DMT 1, also known as Nramp 2 or DCT 1). DMT 1 is a general cation transporter. Once iron is inside the gut cell, the iron may be stored as ferritin or transported through the cell to be released at the basolateral surface to plasma transferrin. It is likely that another transporter acts here in concert with hephaestin, another ferroxidase. Hephaestin is similar to ceruloplasmin, the copper-carrying protein.

Iron absorption is influenced by a number of physiologic states. Erythroid hyperplasia, for example, stimulates iron absorption, even in the face of normal or increased iron stores. Patients with anemias associated with high levels of ineffective erythropoiesis absorb excess amounts of dietary iron. Over time, this may lead to iron overload and tissue damage. In iron deficiency, iron is much more efficiently absorbed from a given diet; the contrary is true in the presence of iron overload. This is possibly mediated through signals that become fixed before the jejunal crypt cell migrates up the villus to become an absorptive cell. The normal individual can reduce iron absorption in situations of excessive intake or medicinal iron intake; however, while the percentage of iron absorbed goes down, the absolute amount goes up. This accounts for the acute iron toxicity occasionally seen when children ingest large numbers of iron tablets. Under these circumstances, the amount of iron absorbed exceeds the transferrin binding capacity of the plasma, resulting in free iron that affects critical organs such as cardiac muscle cells.

Iron Deficiency Anemia

Stages of Iron Deficiency

Iron deficiency anemia is the condition in which there is anemia and clear evidence of iron deficiency. However, iron deficiency occurs in steps (Fig. 90-2). These can be divided into three stages. The first stage is negative iron balance, in which the demands for (or losses of) iron exceed the body's ability to absorb iron from the diet. This stage can result from a number of physiologic mechanisms including blood loss, pregnancy (in which the demands for red cell production by the fetus outstrip the mother's ability to provide iron), rapid growth spurts in the adolescent, or inadequate dietary iron intake. Most commonly, the growth needs of the fetus or rapidly growing child exceed the individual's ability to absorb the iron necessary for hemoglobin synthesis from the diet. Blood loss in excess of 10 to 20 mL of red cells per day is greater than the amount of iron that the gut can absorb from a normal diet. Under these circumstances the iron deficit must be made up by mobilization of iron from RE storage sites. During this period measurements of iron stores—such as the serum ferritin level or the appearance of stainable iron on bone marrow aspirations—will decrease. As long as iron stores are present and can be mobilized, the serum iron, total iron-binding capacity (TIBC), and red cell protoporphyrin levels remain within normal limits. At this stage, red cell morphology and indices are normal.


Figure 90-2 Laboratory studies in the evolution of iron deficiency. Measurements of marrow iron stores, serum ferritin, and total iron-binding capacity (TIBC) are sensitive to early iron-store depletion. Iron-deficient erythropoiesis is recognized from additional abnormalities in the serum iron (SI), percent transferrin saturation, the pattern of marrow sideroblasts, and the red cell protoporphyrin level. Patients with iron deficiency anemia demonstrate all the same abnormalities plus hypochromic microcytic anemia.


When iron stores become depleted, the serum iron begins to fall. Gradually, the TIBC increases, as do red cell protoporphyrin levels. By definition, marrow iron stores are absent when the serum ferritin level is <15 g/L. As long as the serum iron remains within the normal range, hemoglobin synthesis is unaffected despite the dwindling iron stores. Once the transferrin saturation falls to 15 to 20%, hemoglobin synthesis becomes impaired. This is a period of iron-deficient erythropoiesis. Careful evaluation of the peripheral blood smear reveals the first appearance of microcytic cells, and if the laboratory technology is available, one finds hypochromic reticulocytes in circulation. Gradually, the hemoglobin and hematocrit begin to fall, reflecting iron deficiency anemia. The transferrin saturation at this point is 10 to 15%.

When moderate anemia is present (hemoglobin 10–13 g/dL), the bone marrow remains hypoproliferative. With more severe anemia (hemoglobin 7–8 g/dL), hypochromia and microcytosis become more prominent, misshapen red cells (poikilocytes) appear on the blood smear as cigar- or pencil-shaped forms and target cells, and the erythroid marrow becomes increasingly ineffective. Consequently, with severe prolonged iron deficiency anemia, erythroid hyperplasia of the marrow develops rather than hypoproliferation.

Causes of Iron Deficiency

Conditions that increase demand for iron, increase iron loss, or decrease iron intake or absorption can produce iron deficiency (Table 90-2).

Clinical Presentation of Iron Deficiency

Certain clinical conditions carry an increased likelihood of iron deficiency. Pregnancy, adolescence, periods of rapid growth, and an intermittent history of blood loss of any kind should alert the clinician to possible iron deficiency. A cardinal rule is that the appearance of iron deficiency in an adult male means gastrointestinal blood loss until proven otherwise. Signs related to iron deficiency depend on the severity and chronicity of the anemia in addition to the usual signs of anemia—fatigue, pallor, and reduced exercise capacity. Cheilosis (fissures at the corners of the mouth) and koilonychia (spooning of the fingernails) are signs of advanced tissue iron deficiency. The diagnosis of iron deficiency is typically based on laboratory results.

Laboratory Iron Studies

Serum Iron and Total Iron-Binding Capacity

The serum iron level represents the amount of circulating iron bound to transferrin. The TIBC is an indirect measure of the circulating transferrin. The normal range for the serum iron is 50 to 150 g/dL; the normal range for TIBC is 300 to 360 g/dL. Transferrin saturation, which is normally 25 to 50%, is obtained by the following formula: serum iron x 100 ÷ TIBC. Iron deficiency states are associated with saturation levels below 18%. In evaluating the serum iron, the clinician should be aware that there is a diurnal variation in the value. A transferrin saturation of >50% indicates that a disproportionate amount of the iron bound to transferrin is being delivered to nonerythroid tissues. If this condition persists for an extended time, tissue iron overload may occur.

Serum Ferritin

Free iron is toxic to cells, and the body has established an elaborate set of protective mechanisms to bind iron in various tissue compartments. Within cells, iron is stored complexed to protein as ferritin or hemosiderin. Apoferritin binds to free ferrous iron and stores it in the ferric state. As ferritin accumulates within cells of the RE system, protein aggregates are formed as hemosiderin. Iron in ferritin or hemosiderin can be extracted for release by the RE cells although hemosiderin is less readily available. Under steady state conditions, the serum ferritin level correlates with total body iron stores; thus, the serum ferritin level is the most convenient laboratory test to estimate iron stores. The normal value for ferritin varies according to the age and gender of the individual (Fig. 90-3). Adult males have serum ferritin values averaging about 100 g/L, while adult females have levels averaging 30 g/L. As iron stores are depleted, the serum ferritin falls to <15 g/L. Such levels are virtually always diagnostic of absent body iron stores.


Figure 90-3 Serum ferritin levels as a function of sex and age. Iron store depletion and iron deficiency are accompanied by a fall in serum ferritin level below 20 g/L.


Evaluation of Bone Marrow Iron Stores

Although RE cell iron stores can also be estimated from the iron stain of a bone marrow aspirate or biopsy, the measurement of serum ferritin has largely supplanted bone marrow aspirates for determination of storage iron (Table 90-3). The serum ferritin level is a better indicator of iron overload than the marrow iron stain. However, in addition to storage iron the marrow iron stain provides information about the effective delivery of iron to developing erythroblasts. Normally, 20 to 40% of developing erythroblasts—called sideroblasts—will have visible ferritin granules in their cytoplasm. This represents iron in excess of that needed for hemoglobin synthesis. In states in which release of iron from storage sites is blocked, RE iron will be detectable, and there will be few or no sideroblasts. In the myelodysplastic syndromes, mitochondrial dysfunction occurs, and accumulation of iron in mitochondria appears in a necklace fashion around the nucleus of the erythroblast. Such cells are referred to as ringed sideroblasts.

Red Cell Protoporphyrin Levels

Protoporphyrin is an intermediate in the pathway to heme synthesis. Under conditions in which heme synthesis is impaired, protoporphyrin accumulates within the red cell. This can reflect an inadequate iron supply to erythroid precursors to support hemoglobin synthesis. Normal values are less than 30 g/dL of red cells. In iron deficiency, values in excess of 100 g/dL are seen. The most common causes of increased red cell protoporphyrin levels are absolute or relative iron deficiency and lead poisoning.

Serum Levels of Transferrin Receptor Protein

Because erythroid cells have the highest numbers of transferrin receptors on their surface of any cell in the body, and because transferrin receptor protein (TRP) is released by cells into the circulation, serum levels of TRP reflect the total erythroid marrow mass. Another condition in which TRP levels are elevated is absolute iron deficiency. Normal values are 4 to 9 g/L determined by immunoassay. This laboratory test is becoming increasingly available and has been proposed to measure the serial expansion of the erythroid marrow in response to recombinant erythropoietin therapy.

Differential Diagnosis

Other than iron deficiency, only three conditions need to be considered in the differential diagnosis of a hypochromic microcytic anemia (Table 90-4). The first is inherited defects in globin chain synthesis: the thalassemias. These are differentiated from iron deficiency most readily by serum iron values, since it is characteristic to have normal or increased serum iron levels and transferrin saturation with the thalassemias.

The second condition is chronic inflammatory disease with inadequate iron supply to the erythroid marrow. The distinction between true iron deficiency anemia and the anemia associated with chronic inflammatory states is among the most common diagnostic problems encountered by clinicians (see below). Usually the anemia of chronic disease is normocytic and normochromic. Again, the iron values usually make the differential diagnosis clear, as the ferritin level is normal or increased and the TIBC is typically below normal.

Finally, the myelodysplastic syndromes represent the third and most rare condition. Occasionally, patients with myelodysplasia have impaired hemoglobin synthesis with mitochondrial dysfunction resulting in impaired iron incorporation into heme. The iron values again reveal normal stores and more than an adequate supply to the marrow, despite the microcytosis and hypochromia.

Treatment

The severity and cause of iron deficiency anemia will determine the appropriate approach to treatment. As an example, symptomatic elderly patients with severe iron deficiency anemia and cardiovascular instability may require red cell transfusions. Younger individuals who have compensated for their anemia can be treated more conservatively with iron replacement. The foremost issue for the latter patient is the precise identification of the cause of the iron deficiency.

For the majority of cases of iron deficiency (pregnant women, growing children and adolescents, patients with infrequent episodes of bleeding, and those with inadequate dietary intake of iron), oral iron therapy will suffice. For patients with unusual blood loss or malabsorption, specific diagnostic tests and appropriate therapy take priority. Once the diagnosis of iron deficiency anemia and its cause is made, and a therapeutic approach is charted, there are three major approaches.

Red Cell Transfusion

Transfusion therapy is reserved for those individuals who have symptoms of anemia, cardiovascular instability, and continued and excessive blood loss from whatever source, and those who require immediate intervention. The management of these patients is less related to the iron deficiency than it is to the consequences of the severe anemia. Not only do transfusions correct the anemia acutely, but the transfused red cells provide a source of iron for reutilization, assuming they are not lost through continued bleeding. Transfusion therapy will stabilize the patient while other options are reviewed.

Oral Iron Therapy

In the patient with established iron deficiency anemia who is asymptomatic, treatment with oral iron is usually adequate. Multiple preparations are available ranging from simple iron salts to complex iron compounds designed for sustained release throughout the small intestine (Table 90-5). While the various preparations contain different amounts of iron, they are generally all absorbed well and are effective in treatment. Some come with other compounds designed to enhance iron absorption, such as ascorbic acid. It is not clear whether the benefits of such compounds justify their costs. Typically, for iron replacement therapy up to 300 mg of elemental iron per day is given, usually as three or four iron tablets (each containing 50 to 65 mg elemental iron) given over the course of the day. Ideally, oral iron preparations should be taken on an empty stomach, since foods may inhibit iron absorption. Some patients with gastric disease or prior gastric surgery require special treatment with iron solutions, since the retention capacity of the stomach may be reduced. The retention capacity is necessary for dissolving the shell of the iron tablet before the release of iron. A dose of 200 to 300 mg of elemental iron per day should result in the absorption of iron up to 50 mg/d. This supports a red cell production level of two to three times normal in an individual with a normally functioning marrow and appropriate erythropoietin stimulus. However, as the hemoglobin level rises, erythropoietin stimulation decreases, and the amount of iron absorbed is reduced. The goal of therapy in individuals with iron deficiency anemia is not only to repair the anemia, but also to provide stores of at least 0.5 to 1.0 g of iron. Sustained treatment for a period of 6 to 12 months after correction of the anemia will be necessary to achieve this.

Of the complications of oral iron therapy, gastrointestinal distress is the most prominent and is seen in 15 to 20% of patients. For these patients, abdominal pain, nausea, vomiting, or constipation often lead to noncompliance. Although small doses of iron or iron preparations with delayed release may help somewhat, the gastrointestinal side effects are a major impediment to the effective treatment of a number of patients.

The response to iron therapy varies, depending on the erythropoietin stimulus and the rate of absorption. Typically, the reticulocyte count should begin to increase within 4 to 7 days after initiation of therapy and peak at 11/2 weeks. The absence of a response may be due to poor adsorption, noncompliance (which is common), or a confounding diagnosis. If iron deficiency persists, it may be necessary to switch to parenteral iron therapy.

Parenteral Iron Therapy

Intravenous iron can be given to patients who are unable to tolerate oral iron, whose needs are relatively acute, or who need iron on an ongoing basis, usually due to persistent gastrointestinal blood loss. Parenteral iron use has been rising rapidly in the last several years with the recognition that recombinant erythropoietin therapy induces a large demand for iron—a demand that frequently cannot be met through the physiologic release of iron from RE sources. Concern has been raised about the safety of parenteral iron—particularly iron dextran. The serious adverse reaction rate to intravenous iron dextran is 0.7%. Fortunately, newer iron complexes are available in the United States that have a much lower rate of adverse effects. The most recently approved preparations are intravenous sodium ferric gluconate (Ferrlecit) and iron sucrose (Venofer).

Parenteral iron is used in two ways: one is to administer the total dose of iron required to correct the hemoglobin deficit and provide the patient with at least 500 mg of iron stores; the second is to give repeated small doses of parenteral iron over a protracted period. The latter approach is common in dialysis centers, where it is not unusual for 100 mg of elemental iron to be given weekly for 10 weeks to augment the response to recombinant erythropoietin therapy. The amount of iron needed by an individual patient is calculated by the following formula:



In administering intravenous iron dextran, anaphylaxis is a concern. Anaphylaxis is almost never seen with the newer preparations. The factors that have correlated with a serious anaphylactic-like reaction include a history of multiple allergies or a prior allergic reaction to dextran (in the case of iron dextran). Generalized symptoms appearing several days after the infusion of a large dose of iron can include arthralgias, skin rash, and low-grade fever. This may be dose-related, but it does not preclude the further use of parenteral iron in the patient. To date, patients with sensitivity to iron dextran have been safely treated with iron gluconate. If a large dose of iron dextran is to be given (>100 mg) the iron preparation should be diluted in 5% dextrose in water or 0.9% NaCl solution. The iron solution can then be infused over a 60- to 90-min period (for larger doses) or at a rate convenient for the attending nurse or physician. While a test dose (25 mg) of parenteral iron is recommended, in reality a slow infusion of a larger dose of parenteral iron solution will afford the same kind of early warning as a separately injected test dose. Early in the infusion of iron, if chest pain, wheezing, a fall in blood pressure, or other systemic manifestations occur, the infusion of iron—whether as a large solution or a test dose—should be interrupted immediately.

Other Hypoproliferative Anemias

In addition to mild to moderate iron deficiency anemia, the hypoproliferative anemias can be divided into four categories: (1) chronic inflammation/infection; (2) renal disease; (3) endocrine and nutritional deficiencies (hypometabolic states); and (4) marrow damage (Chap. 94). With chronic inflammation, renal disease, or hypometabolism, endogenous erythropoietin production is inadequate for the degree of anemia observed. For the anemia of chronic inflammation (anemia of chronic disease), the erythroid marrow also responds inadequately to stimulation, due in part to defects in iron reutilization. As a result of the lack of adequate erythropoietin stimulation, an examination of the peripheral blood smear will disclose only an occasional polychromatophilic ("shift") reticulocyte. In the cases of iron deficiency or marrow damage, appropriate elevations in endogenous erythropoietin levels are typically found, and shift reticulocytes will be present on the blood smear.

Anemia of Acute and Chronic Inflammation/Infection (the Anemia of Chronic Disease)

The anemia of chronic disease—which encompasses inflammation, infection, tissue injury, and conditions associated with the release of proinflammatory cytokines (such as cancer)—is one of the most common forms of anemia seen clinically and is probably the most important in the differential diagnosis of iron deficiency, since many of the features of the anemia are brought about by inadequate iron delivery to the marrow, despite the presence of normal or increased iron stores. This is reflected by a low serum iron, increased red cell protoporphyrin, a hypoproliferative marrow, transferrin saturation in the range of 15 to 20%, and a normal or increased serum ferritin. The serum ferritin values are often the most distinguishing feature between true iron deficiency anemia and the iron-deficient erythropoiesis associated with inflammation. Typically, serum ferritin values increase threefold over basal levels in the face of inflammation. All of these changes are due to the effects of inflammatory cytokines and hepcidin, the storage iron regulator, acting at several levels of erythropoiesis (Fig. 90-4). Interleukin 1 (IL-1) directly decreases erythropoietin production in response to anemia. IL-1, acting through accessory cell release of interferon (IFN-), suppresses the response of the erythroid marrow to erythropoietin—an effect that can be overcome by increased erythropoietin administration in vitro and in vivo. In addition, tumor necrosis factor (TNF), acting through the release of IFN- by marrow stromal cells, also suppresses the response to erythropoietin. Hepcidin, made by the liver, is increased in inflammation and acts to suppress iron absorption and iron release from storage sites. The overall result is a chronic hypoproliferative anemia with classic changes in iron metabolism. The anemia is further compounded by a mild to moderate shortening in red cell survival.


Figure 90-4 Suppression of erythropoiesis by inflammatory cytokines. Through the release of tumor necrosis factor (TNF) and interferon (IFN-) neoplasms and bacterial infections suppress erythropoietin (EPO) production, the release of iron from reticuloendothelial stores, and the proliferation of erythroid progenitors [erythroid burst-forming units and erythroid colony-forming units (BFU/CFU-E)]. The mediators in patients with vasculitis and rheumatoid arthritis include interleukin 1 (IL-1) and IFN-. The red arrows indicate sites of inflammatory cytokine inhibitory effects.


With chronic inflammation/infection, the primary disease will determine the severity and characteristics of the anemia. For instance, many patients with cancer also have anemia that is typically normocytic and normochromic. In contrast, patients with long-standing active rheumatoid arthritis or chronic infections such as tuberculosis will have a microcytic, hypochromic anemia. In both cases, the bone marrow is hypoproliferative, but the differences in red cell indices reflect differences in the availability of iron for hemoglobin synthesis. Occasionally, conditions associated with chronic inflammation are also associated with chronic blood loss. Under these circumstances, a bone marrow aspirate stained for iron may be necessary to rule out absolute iron deficiency. However, the administration of iron in this case will correct the iron deficiency component of the anemia and leave the inflammatory component unaffected.

The anemia associated with acute infection or inflammation is typically mild but becomes more pronounced over time. Acute infection can produce a fall in hemoglobin levels of 2 to 3 g/dL within 1 or 2 days; this is largely related to the hemolysis of red cells near the end of their natural life span. The fever and cytokines released exert a selective pressure against cells with more limited capacity to maintain the red cell membrane. In most individuals the mild anemia is reasonably well tolerated, and symptoms, if present, are associated with the underlying disease. Occasionally, in patients with preexisting cardiac disease, moderate anemia (hemoglobin 10–11 g/dL) may be associated with angina, exercise intolerance, and shortness of breath. The red cell indices vary from normocytic, normochromic to microcytic, hypochromic. The serum iron values tend to correlate with the red cell indices. The erythropoietic profile that distinguishes the anemia of inflammation from the other causes of hypoproliferative anemias is shown in Table 90-6.

Anemia of Renal Disease

Chronic renal failure is usually associated with a moderate to severe hypoproliferative anemia; the level of the anemia correlates with the severity of the renal failure. Red cells are typically normocytic and normochromic. Reticulocytes are decreased. The anemia is due to a failure to produce adequate amounts of erythropoietin and a reduction in red cell survival. In certain forms of acute renal failure, the correlation between the anemia and renal function is weaker. Patients with the hemolytic-uremic syndrome increase erythropoiesis in response to the hemolysis, despite renal failure requiring dialysis. Polycystic renal disease also shows a smaller degree of erythropoietin deficiency for a given level of renal failure. By contrast, patients with diabetes have more severe erythropoietin deficiency for a given level of renal failure.

Assessment of iron status provides information to distinguish the anemia of renal disease from the other forms of hypoproliferative anemia (Table 90-6) and to guide management. Patients with the anemia of renal disease usually present with normal serum iron, TIBC, and ferritin levels. However, those maintained on chronic hemodialysis may develop iron deficiency from blood loss through the dialysis procedure. Iron must be replenished in these patients to ensure an adequate response to erythropoietin therapy (see below).

Anemia in Hypometabolic States

Patients who are starving, particularly for protein, and those with a variety of endocrine disorders that produce lower metabolic rates may develop a mild to moderate hypoproliferative anemia. The release of erythropoietin from the kidney is sensitive to the need for O2, not just O2 levels. Thus, erythropoietin production is triggered at lower levels of O2 tension in disease states (such as hypothyroidism and starvation) where metabolic activity, and thus O2 demand, is decreased.

Endocrine Deficiency States

The difference in the levels of hemoglobin between men and women is related to the effects of androgen and estrogen on erythropoiesis. Testosterone and anabolic steroids augment erythropoiesis; castration and estrogen administration to males decrease erythropoiesis. Patients who are hypothyroid or have deficits in pituitary hormones also may develop a mild anemia. Pathogenesis may be complicated by other nutritional deficiencies as iron and folic acid absorption can be affected by these disorders. Usually, correction of the hormone deficiency reverses the anemia.

Anemia may be more severe in Addison's disease, depending on the level of thyroid and androgen hormone dysfunction; however, anemia may be masked by decreases in plasma volume. Once such patients are given cortisol and volume replacement, the hemoglobin level may fall rapidly. Mild anemia complicating hyperparathyroidism may be due to decreased erythropoietin production as a consequence of the renal effects of hypercalcemia or to impaired proliferation of erythroid progenitors.

Protein Starvation

Decreased dietary intake of protein may lead to mild to moderate hypoproliferative anemia; this form of anemia may be prevalent in the elderly. The anemia can be more severe in patients with a greater degree of starvation. In marasmus, where patients are both protein- and calorie-deficient, the release of erythropoietin is impaired in proportion to the reduction in metabolic rate; however, the degree of anemia may be masked by volume depletion and becomes apparent after refeeding. Deficiencies in other nutrients (iron, folate) may also complicate the clinical picture but may not be apparent at diagnosis. Changes in the erythrocyte indices on refeeding should prompt evaluation of iron, folate, and B12 status.

Anemia in Liver Disease

A mild hypoproliferative anemia may develop in patients with chronic liver disease from nearly any cause. The peripheral blood smear may show spur cells and stomatocytes from the accumulation of excess cholesterol in the membrane from a deficiency of lecithin cholesterol acyltransferase. Red cell survival is shortened, and the production of erythropoietin is inadequate to compensate. In alcoholic liver disease, nutritional deficiencies can add complexity to the management. Folate deficiency from inadequate intake, as well as iron deficiency from blood loss and inadequate intake, can alter the red cell indices.

Treatment

Many patients with hypoproliferative anemias experience recovery of normal hemoglobin levels when the underlying disease is appropriately treated. For those in whom such reversals are not possible—such as patients with end-stage renal failure, cancer, and chronic inflammatory diseases—symptomatic anemia requires treatment. The two major forms of treatment are transfusions and erythropoietin.

Transfusions

Thresholds for transfusion should be altered based on the patient's symptoms. In general, patients without serious underlying cardiovascular or pulmonary disease can tolerate hemoglobin levels above 8 g/dL and do not require intervention until the hemoglobin falls below that level. Patients with more physiologic compromise may need to have their hemoglobin levels kept above 11 g/dL. A typical unit of packed red cells increases the hemoglobin level by 1 g/dL. Transfusions are associated with certain infectious risks (Chap. 99), and chronic transfusions can produce iron overload.

Erythropoietin

Erythropoietin is particularly useful in anemias in which endogenous erythropoietin levels are inappropriately low, such as the hypoproliferative anemias. Iron status must be evaluated and iron repleted to obtain optimal effects from erythropoietin. In patients with chronic renal failure, the usual dose of erythropoietin is 50 to 150 U/kg three times a week subcutaneously. Hemoglobin levels of 10 to 12 g/dL are usually reached within 4 to 6 weeks if iron levels are adequate; 90% of these patients respond. Once a target hemoglobin level is reached, the erythropoietin dose can be decreased. A fall in hemoglobin level occurring in the face of erythropoietin therapy usually signifies the development of an infection or iron depletion. Aluminum toxicity and hyperparathyroidism can also compromise the erythropoietin response. When an infection intervenes, it is best to interrupt the erythropoietin therapy and rely on transfusion to correct the anemia until the infection is adequately treated. The dose needed to correct the anemia in patients with cancer is higher, up to 300 U/kg three times a week, and only about 60% of patients respond.

Hemoglobinopathies: Introduction

Hemoglobin is critical for normal oxygen delivery to tissues; it is also present in erythrocytes in such high concentrations that it can alter red cell shape, deformability, and viscosity. Hemoglobinopathies are disorders affecting the structure, function, or production of hemoglobin. These conditions are usually inherited and range in severity from asymptomatic laboratory abnormalities to death in utero. Different forms may present as hemolytic anemia, erythrocytosis, cyanosis, or vasoocclusive stigmata.

Properties of the Human Hemoglobins

Hemoglobin Structure

Different hemoglobins are produced during embryonic, fetal, and adult life (Fig. 91-1). Each consists of a tetramer of globin polypeptide chains: a pair of -like chains 141 amino acids long and a pair of -like chains 146 amino acids long. The major adult hemoglobin, HbA, has the structure 22. HbF (22) predominates during most of gestation, and HbA2 (22) is a minor adult hemoglobin. Embryonic hemoglobins need not be considered here.


Figure 91-1 The globin genes. The -like genes (,) are encoded on chromosome 16; the -like genes (,,,) are encoded on chromosome 11. The and genes encode embryonic globins.


Each globin chain enfolds a single heme moiety, consisting of a protoporphyrin IX ring complexed with a single iron atom in the ferrous state (Fe2+), positioned in a manner optimal for reversible binding of oxygen. Each heme moiety can bind a single oxygen molecule; every molecule of hemoglobin can thus transport up to four oxygen molecules.

The amino acid sequences of the various globins are highly homologous to one another. Each has a highly helical secondary structure. Their globular tertiary structures can cause the exterior surfaces to be rich in polar (hydrophilic) amino acids that enhance solubility and the interior to be lined with nonpolar groups, forming a hydrophobic pocket into which heme is inserted. The tetrameric quaternary structure of HbA contains two dimers. Numerous tight interactions (i.e., 11 contacts) hold the and chains together. The complete tetramer is held together by interfaces (i.e., 12 contacts) between the -like chain of one dimer and the non- chain of the other dimer.

The hemoglobin tetramer is highly soluble but individual globin chains are insoluble. Unpaired globin precipitates, forming inclusions that damage the cell. Normal globin chain synthesis is balanced so that each newly synthesized or non- globin chain will have an available partner with which to pair to form hemoglobin.

Solubility and reversible oxygen binding are the key properties deranged in hemoglobinopathies. Both depend most on the hydrophilic surface amino acids, the hydrophobic amino acids lining the heme pocket, a key histidine in the F helix, and the amino acids forming the 11 and 12 contact points. Mutations in these strategic regions tend to be the ones that alter clinical behavior.

Function of Hemoglobin

To support oxygen transport, hemoglobin must bind O2 efficiently at the partial pressure of oxygen (PO2) of the alveolus, retain it, and release it to tissues at the PO2 of tissue capillary beds. Oxygen acquisition and delivery over a relatively narrow range of oxygen tensions depend on a property inherent in the tetrameric arrangement of heme and globin subunits within the hemoglobin molecule called cooperativity or heme-heme interaction.

At low oxygen tensions, the hemoglobin tetramer is fully deoxygenated (Fig. 91-2). Oxygen binding begins slowly as O2 tension rises. However, as soon as some oxygen has been bound by the tetramer, an abrupt increase occurs in the slope of the curve. Thus, hemoglobin molecules that have bound some oxygen develop a higher oxygen affinity, greatly accelerating their ability to combine with more oxygen. This S-shaped oxygen equilibrium curve (Fig. 91-2), along which substantial amounts of oxygen loading and unloading can occur over a narrow range of oxygen tensions, is physiologically more useful than the high-affinity hyperbolic curve of individual monomers.


Figure 91-2 Hemoglobin-oxygen dissociation curve. The hemoglobin tetramer can bind up to four molecules of oxygen in the iron-containing sites of the heme molecules. As oxygen is bound, 2,3-BPG and CO2 are expelled. Salt bridges are broken, and each of the globin molecules changes its conformation to facilitate oxygen binding. Oxygen release to the tissues is the reverse process, salt bridges being formed and 2,3-BPG and CO2 bound. Deoxyhemoglobin does not bind oxygen efficiently until the cell returns to conditions of higher pH, the most important modulator of O2 affinity (Bohr effect). When acid is produced in the tissues, the dissociation curve shifts to the right, facilitating oxygen release and CO2 binding. Alkalosis has the opposite effect, reducing oxygen delivery.


Oxygen affinity is modulated by several factors. The Bohr effect arises from the stabilizing action of protons on deoxyhemoglobin, which binds protons more readily than oxyhemoglobin because it is a weaker acid (Fig. 91-2). Thus, hemoglobin has a lower oxygen affinity at low pH, facilitating delivery to tissues. The major small molecule that alters oxygen affinity in humans is 2,3-bisphosphoglycerate (2,3-BPG, formerly 2,3-DPG), which lowers oxygen affinity when bound to hemoglobin. HbA has a reasonably high affinity for 2,3-BPG. HbF does not bind 2,3-BPG, so it tends to have a higher oxygen affinity in vivo. Hemoglobin also binds nitric oxide reversibly; this interaction may influence vascular tone, but its physiologic relevance remains unclear.

To understand hemoglobinopathies, it is important to understand that proper oxygen transport depends on the tetrameric structure of the proteins, the proper arrangement of the charged amino acids, and interaction with low-molecular-weight substances such as protons or 2,3-BPG.

Developmental Biology of Human Hemoglobins

Red cells first appearing at about 6 weeks after conception contain the embyronic hemoglobins Hb Portland (22), Hb Gower I (22), and Hb Gower II (22). At 10 to 11 weeks, fetal hemoglobin (HbF; 22) becomes predominant. The switch to nearly exclusive synthesis of adult hemoglobin (HbA; 22) occurs at about 38 weeks (Fig. 91-1). Fetuses and newborns therefore require -globin but not -globin for normal gestation. Small amounts of HbF are produced during postnatal life. A few red cell clones called F cells are progeny of a small pool of immature committed erythroid precursors (BFU-e) that retain the ability to produce HbF. Profound erythroid stress, such as that seen in severe hemolytic anemias, after bone marrow transplant, or during chemotherapy, causes more of the F-potent BFU-e to be recruited. HbF levels thus tend to rise in some patients with sickle cell anemia or thalassemia. This phenomenon is also important because it probably explains the ability of hydroxyurea to increase levels of HbF in adults. Fetal globin genes can also be activated partially after birth by agents such as butyrate that inhibit histone deacetylase and modify the structure of chromatin.

Genetics and Biosynthesis of Human Hemoglobin

The human hemoglobins are encoded in two tightly linked gene clusters; the -like globin genes are clustered on chromosome 16, and the -like genes on chromosome 11 (Fig. 91-1). The -like cluster consists of two -globin genes and a single copy of the gene. The non- gene cluster consists of a single gene, the G and A fetal globin genes, and the adult and genes.

Important regulatory sequences flank each gene. Immediately upstream are typical promoter elements needed for the assembly of the transcription initiation complex. Sequences in the 5' flanking region of the and the genes appear to be crucial for the correct developmental regulation of these genes, while elements that function like classic enhancers and silencers are in the 3' flanking regions. The locus control region (LCR) elements located far upstream appear to control the overall level of expression of each cluster. These elements achieve their regulatory effects by interacting with trans-acting transcription factors. Some of these factors are ubiquitous (e.g., Sp1 and YY1), while others are more or less limited to erythroid cells (e.g., GATA-1, NFE-2, and EKLF). The LCR controlling the globin gene cluster is modulated by a SWI/SNF-like protein called ATRX; this protein appears to influence chromatin remodeling and DNA methylation. The association of thalassemia with mental retardation and myelodysplasia in some families appears to be related to mutations in the pathway governed by ATRX. The latter also appear to modulate genes specifically expressed during erythropoiesis, such as the genes that encode the enzymes for heme biosynthesis. This is relevant since normal red blood cell (RBC) differentiation also requires the coordinated expression of the globin genes with the genes responsible for heme and iron metabolism.

Classification of Hemoglobinopathies

There are five major classes of hemoglobinopathies (Table 91-1). Structural hemoglobinopathies occur when mutations alter the amino acid sequence of a globin chain, altering the physiologic properties of the variant hemoglobins and producing the characteristic clinical abnormalities. The variant hemoglobins relevant to this chapter polymerize abnormally, as in sickle cell anemia, or exhibit altered solubility or oxygen-binding affinity. Thalassemia syndromes arise from mutations that impair production or translation of globin mRNA, leading to deficient globin chain biosynthesis. Clinical abnormalities are attributable to the inadequate supply of hemoglobin and the imbalances in the production of individual globin chains, leading to premature destruction of erythroblasts and red cells. Thalassemic hemoglobin variants combine features of thalassemia (e.g., abnormal globin biosynthesis) and of structural hemoglobinopathies (e.g., an abnormal amino acid sequence). Hereditary persistence of fetal hemoglobin (HPFH) is characterized by synthesis of high levels of fetal hemoglobin in adult life. Acquired hemoglobinopathies include modifications of the hemoglobin molecule by toxins (e.g., acquired methemoglobinemia) and abnormal hemoglobin synthesis (e.g., high levels of HbF production in preleukemia and thalassemia in myeloproliferative disorders).

Epidemiology

Hemoglobinopathies are especially common in areas in which malaria is endemic. This clustering of hemoglobinopathies is assumed to reflect a selective survival advantage for the abnormal red cells, which presumably provide a less hospitable environment during the obligate intraerythrocytic stages of the parasitic life cycle. Very young children with thalassemia are more susceptible to infection with the nonlethal Plasmodium vivax. Thalassemia might then favor a natural protection against infection with the more lethal P. falciparum.

Thalassemias are the most common genetic disorders in the world, affecting nearly 200 million people worldwide. About 15% of American blacks are silent carriers for thalassemia; -thalassemia trait (minor) occurs in 3% of American blacks and in 1 to 15% of persons of Mediterranean origin. Thalassemia has a 10 to 15% incidence in individuals from the Mediterranean and Southeast Asia and 0.8% in American blacks. The number of severe cases of thalassemia in the United States is about 1000. Sickle cell disease is the most common structural hemoglobinopathy occurring in heterozygous form in about 8% of American blacks and in homozygous form in 1 in 400. Between 2 and 3% of American blacks carry a hemoglobin C allele.

Inheritance and Ontogeny

Hemoglobinopathies are autosomal codominant traits—compound heterozygotes who inherit a different abnormal mutant allele from each parent exhibit composite features of each. For example, patients inheriting sickle thalassemia exhibit features of thalassemia and sickle cell anemia. The chain is present in HbA, HbA2, and HbF; -chain mutations thus cause abnormalities in all three. The -globin hemoglobinopathies are symptomatic in utero and after birth because normal function of the -globin gene is required throughout gestation and adult life. In contrast, infants with -globin hemoglobinopathies tend to be asymptomatic until 3 to 9 months of age, when HbA has largely replaced HbF.

Detection and Characterization of Hemoglobinopathies—General Methods

Of the many methods available for hemoglobin analysis, electrophoretic techniques are used for routine clinical purposes. Electrophoresis at pH 8.6 on cellulose acetate membranes is especially simple, inexpensive, and reliable for initial screening. Agar gel electrophoresis at pH 6.1 in citrate buffer is often used as a complementary method because each method detects different variants. Comparison of results obtained in each system usually allows unambiguous diagnosis, but some important variants are electrophoretically silent. These mutant hemoglobins can usually be characterized by more specialized techniques such as isoelectric focusing and/or high pressure liquid chromatography (HPLC).

Quantitation of the hemoglobin profile is often desirable. HbA2 is frequently elevated in -thalassemia trait and depressed in iron deficiency. HbF is elevated in HPFH, some -thalassemia syndromes, and occasional periods of erythroid stress or marrow dysplasia. For characterization of sickle cell trait, sickle thalassemia syndromes, or HbSC disease, and for monitoring the progress of exchange transfusion therapy to lower the percentage of circulating HbS, quantitation of individual hemoglobins is also required. In most laboratories, quantitation is performed only if the test is specifically ordered.

Because some variants can comigrate with HbA or HbS (sickle hemoglobin), electrophoretic assessment should always be regarded as incomplete unless functional assays for hemoglobin sickling, solubility, or oxygen affinity are also performed, as dictated by the clinical presentation. The best sickling assays involve measurement of the degree to which the hemoglobin sample becomes insoluble, or gelated, as it is deoxygenated (i.e., sickle solubility test). Unstable hemoglobins are detected by their precipitation in isopropanol or after heating to 50°C. High-O2 affinity and low-O2 affinity variants are detected by quantitating the P50, the partial pressure of oxygen at which the hemoglobin sample becomes 50% saturated with oxygen. Direct tests for the percent carboxyhemoglobin and methemoglobin, employing spectrophotometric techniques, can readily be obtained from most clinical laboratories on an urgent basis.

Complete characterization, including amino acid sequencing or gene cloning and sequencing, is available from several investigational laboratories around the world. The advent of the polymerase chain reaction (PCR), allele-specific oligonucleotide hybridization, and automated DNA sequencing has made it possible to identify globin gene mutations in a few days.

Laboratory evaluation remains an adjunct, rather than the primary diagnostic aid. Diagnosis is best established by recognition of a characteristic history, physical findings, peripheral blood smear morphology, and abnormalities of the complete blood cell count (e.g., profound microcytosis with minimal anemia in thalassemia trait).