Sickle Cell Disease (Hb SS)

Homozygous Hb S disease is a serious chronic hemolytic anemia, first manifested in early childhood and often fatal before 30 years of age. With modern medical care, many patients live longer, but the median age of death in the United States is still only in the 40s. Hb S is found mostly in the black population; 1 of every 600 black persons in the United States has sickle cell anemia (Steinberg, 1999).

In Hb S (β6 glu→val), the glutamic acid in the sixth position on the β-chain is replaced by valine. Hb S is freely soluble when fully oxygenated; when O₂ is removed, Hb S polymerizes, with formation of tactoids (fluid crystals) that are rigid and deform the cell into the shape that gave the disease its name. A strong interaction between the side chain of β6 valine and the hydrophobic pocket of β85 phenylalanine and 88 leucine of another Hb S molecule is probably the basis of polymer formation. In homozygous Hb S disease, sickling occurs at O₂ tensions that are physiological in the peripheral microcirculation. The rigidity of the red cells is responsible for the hemolysis, as well as for most of the complications. These irreversibly sickled cells result from membrane reorganization during repeated episodes of sickling and unsickling, in addition to cell dehydration that markedly reduces cellular deformability. Sickle cells contain high calcium levels, which stimulate potassium and water loss (Gardos effect) and exaggerate cell dehydration (Weatherall & Clegg, 1999). The hemolytic component is mostly extracellular and is caused by clustering of band 3 of the cell membrane, with the consequence of increased IgG binding and recognition by macrophages. Integrins (α4β1) on the sickle cell surface attach to fibronectin and adhesion molecules expressed on endothelial cells; this is enhanced by inflammatory cytokines, von Willebrand factor, and platelet activation, and together these interactions cause vaso-occlusion.

Diagnosis.

The anemia is normochromic and normocytic; polychromasia is increased, and nucleated red blood cells are present. Sickle cells are almost always found in the stained smear (Fig. 32-22). Target cells are numerous, and Howell-Jolly and Pappenheimer bodies are regularly seen in older children and adults as a result of asplenia. The microhematocrit as an estimate of degree of anemia is unreliable because of excessive plasma trapping. Neutrophilia and thrombocytosis are usual. The marrow shows erythroid hyperplasia.

No Hb A is found if the patient has not been transfused recently; more than 80% of the hemoglobin is Hb S, 1% to 20% Hb F, and 2% to 4.5% Hb A₂ (Wrightstone & Huisman, 1974). The fetal hemoglobin is distributed unevenly among the red cells. Hb S and several D and G hemoglobins have the same electrophoretic mobility at alkaline pH, but of these, only Hb S gives a positive sickling test.

Hemoglobin SC Disease

The frequency of Hb SC disease is almost the same as that of Hb SS disease in African Americans. It causes a mild hemolytic anemia. Crises are less frequent and less painful than in sickle cell anemia. Onset is usually in childhood, but the disease might be undetected until later in life. The life expectancy is only modestly shortened. The body habitus is normal or stocky in contrast to the asthenic features in sickle cell anemia. Splenomegaly might be the only finding on physical examination. Fatigue, dyspnea on effort, frequent upper respiratory infections, attacks of mild jaundice, and arthralgias are seen. Constant hip and low back pain may be present with aseptic necrosis of the head of the femur. Hematuria from renal medullary infarction and splenic infarcts have been described. In pregnancy, crises are more frequent and there is an increase in thrombotic tendency, which can cause massive thromboembolism and sudden death following childbirth. A higher incidence of retinopathy is seen in Hb SC disease than in SS disease.

Anemia varies from moderate to very mild and is normochromic-normocytic. Anisocytosis and poikilocytosis are mild to severe, and target cells are numerous—up to 85% of the erythrocytes. Plump and angulated sickled cells are often present on the film. The sickling test is positive. Hb S is 50% and Hb C is slightly less. Hb F is usually under 2%. The proportions of Hbs are the same in patients with coexistent α-thalassemia (Steinberg et al, 1983).

Hb S/β-Thalassemia

This is the third most common sickling disorder after Hb SS and Hb SC in African Americans, and the most common one in people from the Mediterranean. It usually runs a milder course in black people (usually S/β⁺-thalassemia) but causes a severe sickling disorder with manifestations similar to those of sickle cell anemia in people of Italian, Turkish, or Greek descent.

In Hb S/β⁰-thalassemia, Hb A is absent; Hb S is 75% to 90%, Hb F is 5% to 20%, and Hb A₂ is 4% to 6%. This disorder clinically and hematologically resembles sickle cell disease, except for the spleen, which remains enlarged after childhood and into adult life. The main difference is that in Hb S/β⁰-thalassemia, the MCV and MCH are decreased, and the Hb A₂ might be significantly increased. Family study is often necessary for a clear distinction (Wrightstone & Huisman, 1974). On the blood smear, pronounced microcytosis, variable hypochromia, and many target cells are present. As a consequence of reduced cellular hemoglobin, Hb S polymers are formed more slowly, and fewer sickled cells are present on the smear.

In Hb S/β⁺-thalassemia, Hb A is 15% to 30%; Hb S is over 50%, Hb F is 1% to 20%, and Hb A₂ is 4% to 6%. Although these individuals clinically may resemble those with sickle trait (Hb AS), in S/β⁺-thalassemia, the amount of Hb S always exceeds Hb A, while in sickle cell trait Hb A always exceeds Hb S.

Hb SS/α-Thalassemia

Thirty to forty percent of patients with sickle cell anemia are heterozygous for α⁺-thalassemia, and 2% to 3% are homozygous (Higgs et al, 1982). The MCV (83 fL in heterozygotes and 72 fL in homozygotes) and MCH are decreased. The Hb level is higher and the reticulocyte count is significantly lower as compared with sickle cell anemia. On the smear, sickled cells are uncommon, similarly to Hb S/β⁰-thalassemia. Although the anemia is less severe, the vaso-occlusive disease is not, and some studies even show increased morbidity and mortality. As described earlier, δ-chains successfully compete with positively charged S-chains for a limited number of α-chains, and Hb A₂ levels increase. Hematologically, this cannot be separated from Hb S/β⁰-thalassemia (Table 32-5). Whenever microcytosis and increased Hb A₂ are present in sickle cell anemia, family or molecular studies are done to differentiate these entities.

Hemoglobin C is prevalent in West Africans and in about 2% to 3% of black people. The heterozygous state (Hb AC) is asymptomatic, without anemia, with normal or minimally reduced MCV and normal red cell life span. The MCHC might be slightly elevated. Target cells are present on blood film. Hb C makes up about 40% of the total hemoglobin. When significant microcytosis is present, it is usually caused by coexistent α-thalassemia, and Hb C is reduced to 38%, 32%, and 24% in patients with three, two, and one α-gene, respectively (Huisman, 1977).

Hb C Disease.

Homozygous Hb C disease is a mild hemolytic anemia with splenomegaly that is often asymptomatic but occasionally results in jaundice and abdominal discomfort. Life expectancy is normal. In the United States, 0.02% of black people have Hb C disease (Schneider et al, 1976). Reticulocytosis is low for the degree of anemia. The anemia is largely a consequence of low O₂ affinity of Hb C (Bunn & Forget, 1986) and should not be treated. The MCV is usually decreased, and the MCHC is normal or increased. Numerous target cells with an admixture of microspherocytes and minimal polychromasia are seen in the blood. Osmotic fragility is biphasic, with both increased and decreased fragility, but is not used in the diagnosis today. Hexagonal or rod-shaped crystals may be seen in erythrocytes in the stained smear, especially after splenectomy or after slow drying of the smear (Fig. 32-23). As opposed to Hb S, crystals of Hb C tend to melt at low partial pressure of oxygen (pO₂) and do not cause vaso-occlusive disease. The red blood cells are dehydrated (causing the increased MCHC), owing to loss of cations and water as a result of interaction of Hb C with the red cell membrane. As a consequence, the cells are more rigid and less deformable than normal, increasing their likelihood of being trapped and destroyed in the spleen. Hb F is elevated at 2% to 4%, and Hb A₂, when measured by HPLC, might be slightly increased; the remainder of hemoglobin is Hb C.

This is the most common α-chain variant in black people (Schneider et al, 1976). Almost invariably, the linked α-gene is deleted (−α^G). Simple heterozygotes (−α^G/αα) have 30% Hb G and normal red cell indices, but double heterozygotes with α-gene deletion on the other chromosome as well (−α^G/−α) have 45% Hb G and a thalassemia trait phenotype. As with other α-chain variants, a minor hemoglobin representing a combination of α^G with δ is present close to Hb A₂ and helps to differentiate Hb G Philadelphia from β-chain variant G and D hemoglobins (sometimes called G₂ hemoglobin).

Hemoglobins Associated with High Oxygen Affinity and Polycythemia

Eighty-six abnormal Hbs with high O₂ affinity that are associated with familial erythrocytosis are known today (Globin Gene Server, 2015). Some are listed in Table 32-3. The O₂ dissociation curve is shifted to the left. The p₅₀, the pO₂ at which Hb is 50% saturated, is decreased. Under physiologic conditions, the normal p₅₀ of whole blood is 26 mm Hg; in this disorder, it has ranged from 5 to 23 mm Hg. Because the Hb has a high affinity for O₂, it cannot unload the oxygen and the tissues are relatively hypoxic, resulting in increased EPO production and polycythemia. These disorders are autosomal dominant; only heterozygotes have been described. The Hb concentration has ranged from 15 to 23.8 g/dL. Measurement of O₂ affinity is required to establish the diagnosis (Bunn & Forget, 1986). Because the amino acid substitution is inside the molecule, often the abnormal hemoglobin is indistinguishable from Hb A on electrophoresis or HPLC.

Hemoglobins Associated with Low Oxygen Affinity

There are far fewer variants with decreased O₂ affinity (see Table 32-3; Bunn & Forget, 1986). The O₂ dissociation curve is shifted to the right (increased p₅₀). These individuals have mild “anemia,” as they can unload more O₂ to the tissues and they simply do not need that much Hb. A handful of variants with markedly decreased affinity are associated with cyanosis. In these, O₂ uptake is impaired in the lung and the level of deoxyhemoglobin is more than 5 g/dL, causing cyanosis. The skin and mucous membranes have a slate gray color. There is no anemia. Many of the unstable Hbs also have decreased O₂ affinity; however, the hemolytic state dominates the clinical picture.

M Hemoglobins: Pseudocyanosis

Nine abnormal Hbs are associated with clinical methemoglobinemia and cyanosis that do not respond to methylthioninium chloride (methylene blue) (Globin Gene Server, 2015; Bunn, 1994). The color is similar to cyanosis—a brownish color, caused by methemoglobin. The common feature is that all have an amino acid substitution at or near the heme group, so that methemoglobin is unusually stable, and reduction to ferrous heme and hence reversible binding of O₂ are prevented. Methemoglobin constitutes no more than 3% of the total Hb in normal humans.

Cyanosis from birth is seen in Hb M disease with α-chain abnormalities, or in fetal Hb M (Hb FM Osaka). In the latter, cyanosis will disappear after the γ-chains have been replaced by β-chains by 6 months of age. Cyanosis does not appear until nearly 6 months of age in Hb M variants with β-chain abnormalities, for the same reason (Bunn, 1994). Of course, the cyanosis is not associated with enzyme abnormalities in the red cell, toxic drugs, or cyanotic heart disease—conditions that must be considered in the differential diagnosis. Patients usually have no other symptoms.

All Hb M disorders thus far discovered have been reported in heterozygotes, probably because homozygosity is lethal. Some types of Hb M do not separate from Hb A on alkaline electrophoresis. If the hemolysate is first converted to methemoglobin, the Hb M will migrate differently from normal methemoglobin at pH 7.1. The absorption spectra of the eluted Hb M, which may be distinctive, can be compared with those of normal methemoglobin (Bunn, 1994).

Unstable Hemoglobins

More than 100 Hbs, mostly β-chain variants have been described in which the Hb stable tetrameric structure is not maintained and Hgb precipitates within the red cell as Heinz bodies (Bunn, 1998). Some are listed in Table 32-3. Rare unstable hemoglobins such as Hb F Poole are γ-chain variants. The precipitated Hb attaches to the cell membrane making the cells inflexible. Heinz bodies are removed by the spleen that further damages the cells and causes shortened survival. The O₂ affinity is usually abnormal but may be increased or decreased. Some of these unstable Hbs cause “congenital Heinz body hemolytic anemias.”

All patients have been heterozygous. Clinical features have shown considerable variation, from severe hemolytic anemia in the first year of life (e.g., Hb Hammersmith, Hb Bristol) to a very mild chronic hemolytic anemia (e.g., Hb Louisville, Hb Hasharon) that may be exacerbated by drugs (e.g., Hb Zurich). A few unstable Hbs have been discovered incidentally in clinically normal individuals (e.g., Hb Tacoma, Hb Sogn).

Jaundice and splenomegaly are common, as in other hemolytic anemias. More distinctive in some cases is the excretion of darkly pigmented urine (only during hemolytic crises in mild variants). Cyanosis is present in some patients and is due to methemoglobinuria and sulfhemoglobinemia or to low O₂ affinity.

The anemia is normocytic and normochromic to hypochromic, the latter because of the removal of precipitated Hb from aging red cells by macrophages of the spleen and other reticuloendothelial organs. Prominent basophilic stippling, probably related to excessive clumping of ribosomes, is a common feature. Occasional “bite cells” may be seen. Patients with relatively high Hb concentrations in the steady state usually have Hb variants with a high O₂ affinity and an unexpectedly high reticulocyte count (e.g., Hb Köln, Hb Gun Hill). On the other hand, patients with rather low Hb concentrations may be relatively asymptomatic if their Hb has a low O₂ affinity; their reticulocyte counts are unexpectedly low for the Hb concentration (e.g., Hb Hammersmith). Heinz bodies are rarely seen in circulating red cells before splenectomy, although sometimes they may be generated by incubating the red cells with brilliant cresyl blue or new methylene blue. After splenectomy, Heinz bodies are readily demonstrable in a large proportion of cells; the blood film shows irregularly contracted cells and basophilic stippling that may be pronounced.

In splenectomized patients, the Heinz bodies may interfere with Hb determinations and with electronic platelet and white blood cell counts. Before the absorbance of the hemolysate is measured, it should be centrifuged to remove the Heinz bodies. Platelet and leukocyte counts should be performed by visual methods. Hb electrophoresis is normal in about one fourth of patients. Hb A₂ may be elevated in β-chain variants because of the loss of abnormal Hb from the cells; this phenotype may resemble thalassemia intermedia. Hb F may be increased to a level of 10% to 15%. Key laboratory determinations include heat instability and isopropanol precipitation tests.

Homozygous β-Thalassemia (Thalassemia Major; Cooley's Anemia)

In the absence (β⁰) or a marked decrease (β⁺) of β-chain production, there is an excess of α-chains. Aggregates of α-chains are unstable and precipitate in the normoblast or erythrocyte and damage the cells. Excess α-chains and their degradation products—heme, hemin, and iron, which serve as foci for the generation of reactive O₂ species—result in the partial oxidation of band 4.1 and a reduced spectrin/band 3 ratio in red blood cell precursors. Precipitates and cells are removed, causing ineffective erythropoiesis and severe hemolytic anemia. Furthermore, clustering of band 3 in the membrane may be followed by opsonization with autologous IgG and complement and removal by macrophages (Weatherall & Clegg, 1999).

Clinical findings include jaundice and splenomegaly, which become evident early in childhood. Prominent frontal bones, cheekbones, and jaws impart a mongoloid appearance. These changes and X-ray findings of a thinned cortex of the long and flat bones and thickening of the skull with osteoporosis (“hair-on-end” appearance) reflect extreme bone marrow hyperplasia. Growth is stunted, and puberty is delayed. Most patients require regular transfusions and develop problems caused by iron loading. Iron overload commonly develops, and the major cause of death is cardiac failure due to myocardial siderosis by the end of the third decade.

Unlike most hemolytic diseases, the anemia is hypochromic and microcytic. Extreme poikilocytosis with bizarre shapes, target cells, ovalocytosis, Cabot rings, Howell-Jolly bodies, nuclear fragments, siderocytes, anisochromia, anisocytosis, and often extreme normoblastosis are present (Fig. 32-26). Poikilocytosis is more striking in patients with intact spleen, while nucleated red blood cells are more numerous after splenectomy. Nucleated red blood cells have hypochromic cytoplasm and, especially after splenectomy, aggregates of densely staining Hb, which probably represent precipitated α-chains. Incubation of the blood with methyl violet stains these precipitates in both red cells and erythroblasts. The reticulocyte count is less elevated than expected for the degree of anemia because of destruction of erythroid precursors in the marrow. Osmotic resistance of the red cells, serum iron, and indirect-reacting bilirubin are increased.

In the marrow, marked erythroid hyperplasia is present. Many late normoblasts show inclusion bodies, as in the blood. Gaucher-like cells are present. Storage iron and sideroblasts are increased.

In β⁰-thalassemia, Hb A is absent, Hb F is as high as 98%, and Hb A₂ is about 2%. In β⁺-thalassemias (Mediterranean), Hb F is 60% to 95%, with Hb A present. Although Hb A₂ may or may not be increased, the ratio of A₂ to A is always increased. In black people with β⁺-thalassemia, the clinical features are less severe (thalassemia intermedia), and transfusion is usually unnecessary; Hb F is 20% to 40%, Hb A₂ is 2% to 5%, and the rest is Hb A (see Table 32-7).

Heterozygous β-Thalassemia (β-Thalassemia Trait; Thalassemia Minor; Cooley's Trait)

This is caused by the β⁰-thalassemia gene with absent, or the β⁺-thalassemia gene with reduced, β-globin chain synthesis. There are usually no symptoms or abnormal physical signs. The only clinical presentation might be a refractory anemia of pregnancy (Weatherall & Clegg, 2001).

Most β-thalassemia heterozygotes have a mild anemia but, occasionally, Hct and Hb might be normal. Those of African origin have higher Hb levels than people from the Mediterranean region, reflecting their milder genotype. Characteristically, the RBC is elevated (5 to 7 M/µL), the MCH is low (usually less than 22 pg), and the MCV is low (between 55 and 70 fL). The MCHC is sometimes low but often normal. The reticulocyte count is twice the normal value. On stained films, the cells have a moderate degree of microcytosis, hypochromia, and poikilocytosis; target cells and basophilic stippling are often, but not always, present (Fig. 32-27). In the marrow, there is mild erythroid hyperplasia with ragged cytoplasmic borders—a sign of defective hemoglobinization.

Hb A₂ is elevated in the 3.5% to 7% range, and Hb F is slightly elevated (1% to 3%) in about half of cases. In the few cases where Hb F exceeds 4%, it is likely that a gene for HPFH is also present. The relatively rare deletional forms tend to have higher levels of Hb F (up to 9%), and in a few families, in which the deletion included the promoter region, Hb F was found to be unusually high (up to 14%) (Weatherall & Clegg, 2001). In infants, a slower than normal decline in Hb F level is observed, and the adult steady-state level is not reached until adolescence. This is a particularly important consideration in double heterozygosity for β-thalassemia and Hb S, when Hb F level is used to predict prognosis.

β-Thalassemia Trait with Normal Hb A₂

In a few cases, both Hb A₂ and F are normal. These are difficult to distinguish from α-thalassemia trait, and only molecular studies might be definitive. The bulk of these cases results from the coinheritance of δ-thalassemia (Weatherall, 1994), either in trans or in cis, to the β-thalassemia gene. In other patients with normal Hb pattern, only minimal hematologic changes are noted (the “silent” β-thalassemia gene), and only a more severe β-thalassemia syndrome in a family member suggests the presence of a very mild β-thalassemia mutation.

Iron studies are little different from normal (Weatherall & Clegg, 2001), although iron deficiency often complicates thalassemia trait in childhood and during pregnancy. In severe iron deficiency, the level of Hb A₂ may fall into the normal range, thus obscuring the diagnosis of β-thalassemia trait, but this is unusual. Most often, although the level of A₂ falls, it remains elevated above the normal range (Weatherall & Clegg, 2001). Nevertheless, when iron deficiency is present, repeat measurement of Hb A₂ is recommended after replenishment of iron.

The differential diagnosis between iron deficiency and β- or α-thalassemia trait can be difficult. Increased RBCs in the presence of decreased MCV is the hallmark of thalassemia trait. An MCV/RBC ratio less than 13 suggests thalassemia trait; a ratio greater than 13 is more consistent with iron deficiency, but this and other formulas are not conclusive enough for diagnosis.

δβ⁰-Thalassemia

This is sometimes called F-thalassemia, a helpful name, as you have to think of it when there are thalassemic indices and significantly increased levels of Hb F. β- and δ-chains are not produced; this is nearly, although not completely, compensated for by increased output of γ-chains. The heterozygous state is similar to a mild β-thalassemia trait, except that Hb A₂ is not increased or even slightly reduced (mean level is 2.4%), and Hb F is significantly increased (5.4% to 20%). In the homozygous state, hemoglobin consists of only Hb F.

Clinically, (δβ)⁰-thalassemia behaves as a mild form of β-thalassemia. In the heterozygous state, the Hb is normal or slightly reduced, the MCH is between 21 and 26 pg, and the MCV is 65 to 79 fL. There are no clinical symptoms. Homozygotes have a mild form of thalassemia intermedia with a Hb level of 10 to 13 g/dL, mildly thalassemic red cell indices, and only minimal hepatosplenomegaly. It is most common in the Mediterranean population. The mild phenotype is the result of increased production of γ-chains, which compensate to some degree for the lack of β-chains.

The molecular defect is a long deletion involving the β- and δ- and often also the Aγ-gene. Twenty-one different deletions have been described (Globin Gene Server, 2015), but the hematologic findings are essentially the same. When the Aγ-gene is also deleted, the accurate nomenclature is (Aγδβ)⁰-thalassemia; homozygotes have a somewhat more severe phenotype.

δβ⁺-Thalassemia: Lepore Hemoglobins

In the Lepore hemoglobins, an abnormal δβ-fusion chain is produced, a result of chromosome crossing-over and fusion of genetic material at the δβ-genes. No normal δ- or β-chain synthesis is directed from the affected chromosome. Because Hb F production is only slightly increased and the composite δβ-chain is synthesized at a very slow rate, a severe thalassemic phenotype is produced. Hematologic abnormalities are similar to those seen in β⁰-thalassemia. Hb Lepore migrates slightly faster than Hb S on alkaline electrophoresis and usually constitutes about 10% of total Hb in the heterozygotes; Hb A₂ averages 2%, and Hb F is 2% to 3% (Efremov et al, 1978). In homozygotes, Hb Lepore is 10% to 15%, and the rest is Hb F. Different Hb Lepores have been described, depending on the point of fusion, but they behave similarly. This is a much more severe thalassemia gene than the (δβ)⁰ form and causes transfusion-dependent thalassemia major in the homozygous state.

In a similar mechanism, anti-Lepore hemoglobins have βδ-fusion gene. Several of these hemoglobins have been described, including P-Nilotic.

Deletional Pancellular HPFH

In the six deletional forms, the δβ-gene complex is deleted. The black and Ghanaian forms are the most common, but Indian, Italian, and Southeast Asian forms are also well documented. Homozygotes have slightly microcytic, hypochromic red cells, but no anemia. Hb F is 100%; no Hb A or Hb A₂ is present. The Hct can even be high—a result of high O₂ affinity of Hb F. In the heterozygote, no hematologic abnormalities are found. Hb F is 15% to 30%, and Hb A₂ is decreased at 1% to 2.1%.

Hb Kenya

This is a Hb analogous to the Lepore Hbs, which is associated with an HPFH phenotype. It contains an ^Aγβ-fusion gene. In the heterozygote, Hb Kenya is around 10%, F is 7%, and A₂ is reduced.

Nondeletional Pancellular HPFH

A mutation in the promoter region of one of the γ-genes results in increased synthesis of Hb F. Output from the δ- and β-genes in cis is reduced. Levels of Hb F range from 3% to 31% in the different forms, Hb A₂ is invariably low, and the red cell indices are close to normal. There are two particulars to keep in mind. First, β-chain synthesis is decreased, but not absent from the affected chromosome, and compound heterozygotes of this form of HPFH and Hb S on the other chromosome do have Hb A (≈30%). The hemoglobin composition is similar to that seen in Hb S/β+-thalassemia, except that Hb A₂ is reduced. Second, it is not rare to find it together with an α-thalassemia gene, which is highly prevalent in African Americans.

Heterocellular or Swiss-type HPFH

Heterocellular or Swiss-type HPFH has less Hb F, ranging from 2% to 5%. The distribution of Hb F is uneven (heterocellular): Both F cells and erythrocytes completely lacking Hb F are present. It was first described among healthy Swiss army recruits but is present in other populations and is quite common. It is caused by polymorphic variations at three well-known and other lesser known loci that regulate adult Hb F production (Thein & Menzel, 2009). In normals or in carriers of β-thalassemia or Hb S, there is only a minimal increase in Hb F. When inherited with Hb SS or homozygous β-thalassemia, however, the result could be an unusually high level of Hb F and milder disease.

α-Thalassemia Syndromes

Four α-thalassemia syndromes result from the combination of these genotypes (Bunn & Forget, 1986), which roughly correspond to the loss of 4, 3, 2, or 1 genes from the normal complement (αα/αα) (Table 32-8). In the following discussion, nondeletion forms are not always depicted separately, for the sake of simplicity.

Hemoglobin H Disease (−α/− −).

Three of the four α-genes are absent. A chronic hemolytic anemia occurs with the clinical picture of thalassemia intermedia in a minority of cases, although the severity varies, and most patients do well. Hb H disease is very common in Southeast Asia but is also seen in the Mediterranean and the Middle East; it is very rare, however, in black people, as α⁰-thalassemia is uncommon in this group. Splenomegaly and sometimes hepatomegaly are present. Hb values average 3 g/dL less than in age- and sex-matched controls. Transfusion is rarely needed. The anemia may become more severe during pregnancy, but the Hb rarely falls below 7 g/dL. The MCV (60 to 70 fL) and MCH (17 to 21 pg) are decreased (Higgs et al, 1989), and RBC is increased (6 to 6.2 M/µL). The blood film shows hypochromia, basophilic stippling, and anisopoikilocytosis with target cells (Fig. 32-28). Reticulocytes range from 4% to 5%.

Hemoglobin electrophoresis shows a rapidly migrating band of Hb H (β4), accounting for 1% to 40% (average 9%) of the hemoglobin, and the slightly less rapidly migrating Hb Bart's in half of the cases. Hb H can precipitate and be lost from the hemolysate in vitro by careless handling or prolonged storage. In old hemolysates, Hb H appears as a series of bands on electrophoresis. The percentage of Hb Bart's is 2% to 40% at birth; it gradually falls thereafter, averaging 4.8%, but the level in adults is variable. As in other α-thalassemia syndromes, more Hb Bart's is present at birth than Hb H in adult life. Hb A₂ is diminished.

Hemoglobin H preparation.

Vital staining of the blood with an oxidizing dye such as brilliant cresyl blue induces inclusion bodies (Hb H precipitates) in many of the red blood cells. During incubation of two parts of blood in one part of 1% brilliant cresyl blue stain, the unstable Hb H (β₄) gradually precipitates as multiple small pale blue inclusions uniformly distributed on the red cell membrane (Fig. 32-29) (Jones et al, 1981). Hb H inclusions must be distinguished from (1) the granules and reticular networks of reticulocytes, which are darker blue in color and (2) preformed Heinz bodies, which are larger, also darker blue, and are often attached to the membrane. After 20 minutes of incubation at room temperature, Hb H inclusions are present in at least half of the red cells in Hb H disease, and in rare red cells in α-thalassemia trait. The larger, single Heinz bodies may be found after splenectomy in Hb H disease.

Heinz Bodies

When a globin chain denatures, it forms precipitates that are known as Heinz bodies (Dacie & Lewis, 1991). These precipitates cannot be detected in Romanowsky's-stained, air-dried blood films, but after vital staining with methyl violet or crystal violet, Heinz bodies stain deep purple. They vary from 1 to 4 µm in diameter and often attach to the red cell membrane. They also stain, but less intensely, as pale blue inclusions in reticulocyte stains (e.g., new methylene blue) (Fig. 32-31).

The presence of Heinz bodies in freshly drawn blood indicates that (1) an oxidizing drug or chemical (e.g., phenylhydrazine, chlorate, naphthalene, dapsone) has been ingested in sufficient amount to overwhelm the normal protective mechanisms of the red cell and denature Hb; (2) a drug such as primaquine has been ingested by an individual with G6PD deficiency (or another defect resulting in a deficiency of reduced glutathione), so that Hb is not protected from oxidative denaturation; or (3) the subject has an unstable Hb.

Ascorbate Cyanide Test

When blood is incubated with a solution of sodium cyanide and sodium ascorbate, hydrogen peroxide is generated from the coupled oxidation of ascorbate and oxyhemoglobin (Dacie & Lewis, 1991). Cyanide inhibits catalase, hydrogen peroxide is available to oxidize Hb, and the brown color of methemoglobin is discernible. This occurs more rapidly in G6PD-deficient cells than in normal cells.

The ascorbate cyanide test is not specific, in that abnormalities in the glutathione synthesis or maintenance pathways can produce positive results.

Fluorescent Spot Test

Whole blood is added to a mixture of glucose-6-phosphate (G6P), NADP, saponin, and buffer, and a spot of this mixture is placed on filter paper and is observed for fluorescence with ultraviolet light. If G6PD is present, NADP is converted to NADPH. Because phosphogluconate dehydrogenase is present in most hemolysates, further NADP is converted to NADPH (see Fig. 32-30). NADPH fluoresces, but NADP does not. The normal control sample fluoresces brightly, and lack of fluorescence indicates G6PD deficiency. By reoxidizing any small amounts of NADPH formed, oxidized glutathione (GSSG) enhances the ability of the test to detect mild G6PD deficiency. This is the recommended screening test for G6PD deficiency (Beutler et al, 1979).

Pyruvate Kinase (PK) Deficiency

The most common red cell enzyme deficiency involving the Embden-Meyerhof glycolytic pathway, PK deficiency results in mild to moderately severe nonspherocytic chronic hemolytic anemia with splenomegaly. The prevalence of PK deficiency is estimated to be 1 : 20,000 in the general white population (Zanella et al, 2005). The anemia may be detected in infancy, or not until adult life in milder cases. Neonatal jaundice occurs in more than half of cases, which may require exchange transfusion. Patients tend to tolerate the anemia rather well because of high levels of 2,3-DPG, which occur as a result of the block in glycolysis. The blood film may show no notable red cell abnormalities until after splenectomy, when echinocytes, irregularly contracted cells, and crenated red cells may be prominent. Reticulocyte counts are elevated and increase further after splenectomy.

PK isoenzymes are produced by two separate genes: one on chromosome 15, encoding the M (muscle) isoforms, and the other on chromosome 1, encoding the L (liver) and R (red cell) isoforms. The complexity of PK isoforms is further complicated by the fact that functional enzyme is a tetramer. Inheritance is autosomal recessive, but this is probably true only in consanguineous families. PK mutants are numerous and are not detected in phenotypically normal heterozygotes that have one half the normal PK activity. Most individuals with PK-deficient hemolytic anemias are therefore probably double heterozygotes for two mutant genes (Valentine, 1979). Acquired PK deficiency occurs occasionally in myelodysplastic disorders and leukemias (Valentine, 1979; Miwa, 1981).

The autohemolysis test gives variable results. Some patients show only a mild increase in autohemolysis that is partially prevented by glucose (type I), and others have a greater increase that is not prevented by glucose (type II). Heinz bodies are not found. The diagnosis is made by a specific screening test or enzyme assay.

The reticulocyte count in unsplenectomized patients is usually increased, although not proportional to the degree of anemia, because younger PK-deficient erythrocytes are selectively sequestered in the spleen (Zanella et al, 2005). Splenectomy is indicated in cases requiring transfusions. After splenectomy, the Hb concentration usually increases by 1 to 2 g/dL and the reticulocytes increase sharply, although hemolysis persists (Miwa, 1981).

Other Glycolytic Enzyme Deficiencies

Other enzyme deficiencies in the Embden-Meyerhof pathway are rarer (Beutler, 1994; van Solinge & van Wijk, 2010). When severe, they produce hemolytic anemia, with two exceptions: (1) LDH deficiency has no clinical manifestations; and (2) deficiencies of 2,3-DPG mutase and 2,3-DPG phosphatase activities occur together and result in erythrocytosis as a result of lack of 2,3-DPG, shifting the O₂ dissociation curve of Hb to the left, and relative tissue hypoxia.

Other enzyme deficiencies in the hexose monophosphate shunt are rare. They include the two enzymes involved in glutathione synthesis: γ-glutamyl cysteine synthase and glutathione synthetase. As in G6PD deficiency, hemolysis increases with oxidant drug exposure or infection.

No good evidence is available, however, for the causation of chronic hemolytic anemia by 6-phosphogluconate deficiency, catalase, enolase, glutathione reductase (GR) deficiency, or glutathione peroxidase (GP_x) deficiency (Beutler, 1994; van Solinge & van Wijk, 2010). GR contains flavine-adenine dinucleotide and is often partially deficient because of dietary riboflavin deficiency. GP_x is one-half normal in about 30% of the Jewish population as a result of homozygosity for a gene with low GP_x activity; in addition, GP_x activity is dependent on selenium intake in the diet. In neither case is there an association with a hematologic disorder.

Pyrimidine-5′-Nucleotidase Deficiency

When RNA is degraded in the reticulocyte, pyrimidine nucleotides must be dephosphorylated by pyrimidine-5′-nucleotidase (PN) to cross the red cell membrane (Paglia & Valentine, 1981). Autosomal recessive PN deficiency results in accumulation of pyrimidines, and the impaired degradation of RNA results in pronounced basophilic stippling in red cells on the blood film. This is probably one of the more common enzyme deficiencies responsible for nonspherocytic hereditary hemolytic anemia, once G6PD and PK deficiencies are excluded (Rees et al, 2003).

The disorder is characterized by mild to moderate chronic hemolysis, jaundice, reticulocytosis (≈10%), marked basophilic stippling (about 5% of the red cells), and splenomegaly without notable improvement after splenectomy. However, no cases of transfusion dependency associated with PN deficiency have been reported (Rees et al, 2003). A screening test compares the ultraviolet absorption of deproteinized extracts of red cells at 260 nm with that at 280 nm. In PN deficiency, the major absorption peak is shifted from the normal 260 nm to 280 nm, which is the maximum for the pyrimidines uridine diphosphate and cytidine diphosphate. Confirmation requires an assay showing decreased nucleotidase activity (Beutler, 1984).

Acquired PN deficiency occurs in lead poisoning and is probably responsible for the basophilic stippling in that condition. It has been also reported in β-thalassemia trait, acute leukemias, chronic myeloid leukemia, chronic lymphocytic leukemia, and lymphomas (Rees et al, 2003).

Traumatic Hemolysis

Hemolytic anemia characterized by striking morphologic abnormalities of the red cells, which include fragments (schistocytes) and irregularly contracted cells (triangular cells, helmet cells), has been attributed to physical trauma to the red cells. The International Council for Standardization in Haematology (ICSH) recommends the classification of schistocytes into four categories (crescentic/triangular, keratocytes, helmet cells, and microspherocytes), and it also recommends the use of 1% threshold to designate increased schistocytes (Zini et al, 2012). The basis of the hemolytic process is probably damage to the red cells in their contact with loose fibrin meshworks (intravascular coagulation) or with pathologic vascular lesions. Fragmentation of the cells results with or without intravascular lysis. Two general categories are recognized in this group of disorders, aptly termed red cell fragmentation syndrome: macroangiopathic and microangiopathic.

Hemolytic-Uremic Syndrome

HUS is classified into diarrhea-associated (D+) or typical form, and diarrhea-negative (D−) or atypical form. D+ HUS constitutes approximately 95% of cases in children and is less common in adults (Zheng & Sadler, 2008). D+ HUS occurs most commonly in infants younger than 2 years of age, but it affects all ages. It is caused by one of several serotypes of Escherichia coli that produce a verotoxin (named for its toxicity against African green monkey kidney [vero] cells). The most common is E. coli O157:H7, which produces Shiga toxins 1 and 2. This microorganism is one of the cattle gut commensals. E. coli O104:H4 has been associated with epidemic infection in Europe (Rosove, 2014). The annual incidence of infection with E. coli O157 is approximately 8 per 100,000 people in North America. The percentage of cases with bloody diarrhea that progress to HUS is 3% to 7% for sporadic cases and 20% to 30% for some outbreaks (Zheng & Sadler, 2008). HUS usually occurs 4 to 6 days after the onset of diarrhea. Shiga toxins cause bloody diarrhea, then enter the circulation and travel in the plasma on the surface of platelets or monocytes (Moake, 2002). Recent studies suggest that these toxins become attached to glomerular capillary endothelium and other renal epithelial cells. Shiga toxins, together with locally secreted cytokines, cause the release of unusually large multimers of von Willebrand factor (vWF), which promote platelet aggregation in renal vasculature (Moake, 2002). Endothelial cell damage is the hallmark of D+ HUS in children, and endothelial swelling may be severe enough to occlude the capillary lumen. The thrombi of D+ HUS are composed predominantly of fibrin with few platelets and little vWF (Zheng & Sadler, 2008). There is growing evidence that shiga toxin also activates complement component in the renal circulation, which may contribute to cell damage and thrombogenesis (Rosove, 2014). Hemolytic anemia with schistocytes (due to interaction of red cells with microvascular thrombotic lesions), variable thrombocytopenia, and uremia are the cardinal features. Death formerly occurred in almost half of cases; the renal pathology has included acute glomerulonephritis and thrombotic and necrotic vascular lesions associated with patchy, bilateral renal cortical necrosis. However, with advanced supportive therapy, including transfusions and dialysis, some investigators have reported mortality reduced to 5% to 15%. Nonetheless, minor to major renal impairment has been reported in approximately 50% of patients. Five to ten percent of patients with HUS have D− HUS, with no antecedent diarrhea of Shiga-toxin–producing organisms, and some of these patients have familial history of the disease (Zheng & Sadler, 2008). First presentations are more common in children, including neonates, although some cases present in adulthood (Rosove, 2014). Infections and pregnancy may trigger hemolytic crises. Approximately 50% of patients with D− HUS have mutations in one of the complement regulatory proteins: factor H, factor I, membrane cofactor protein (MCP), or factor B. These patients have a much higher mortality rate (Moake, 2002). Approximately 5% of D− HUS patients have antibodies against factor H, raising the possibility of using plasma exchange or immunosuppressants for therapeutic strategy (Zheng & Sadler, 2008).

Thrombotic Thrombocytopenic Purpura (TTP)

TTP may occur at any age but has a peak incidence in the third decade; it occurs more often in females than in males. The annual incidence is 3.7 cases per 100,000 with the acquired form being more common, with an incidence of 0.2 to 1 per 100,000 compared to the much less common familial form, which has an incidence of 0.05 to 0.4 per 100,000 (Rosove, 2014). The triad of clinical manifestations present in most patients includes hemolytic anemia, thrombocytopenia, and neurologic symptoms; in addition, fever and renal diseases are often present. This constitutes the classic pentad. Pathologically, microvascular occlusive lesions with hyaline thrombi and endothelial proliferation are widespread throughout the body. Until 1997, the origin was unknown. Different theories included endothelial injury by oxidant stress, antiendothelial antibodies, anti-CD36 (glycoprotein IV located on microvasculature) antibodies, platelet aggregating factors, and release of large vWF multimers into the plasma. In 1996, a protease that cleaves vWF subunits was identified in normal plasma. In 1997 and the following years, several reports indicated deficient vWF-cleaving protease in patients with TTP due to low enzyme levels (in familial cases) or to the presence of IgG enzyme inhibitor (in nonfamilial cases). These observations were confirmed by additional studies, which showed normal levels of vWF-cleaving protease in HUS (Moake, 2004). The enzyme has been further characterized as number 13 in a family of 19 distinct ADAMTS-type enzymes identified to date (a disintegrin and metalloprotease with eight thrmobospondin-1-like domains). Therefore, the vWF-cleaving metalloprotease is now referred to as ADAMTS13 (Moake, 2004). In the absence of vWF-cleaving protease, ultra-large vWF multimers secreted by endothelial cells would not be processed normally. These highly adhesive multimers may induce platelet clumping in the microcirculation under shear stress, resulting in the clinical manifestation of TTP. The disease is acute and until the 1980s was fatal in well over half of cases. With the therapy of plasma exchange by plasmapheresis and plasma transfusions (in addition to platelet inhibitors), the remission rate has improved, and long-term survival now approaches 90% (Moake, 2004). TTP can now be classified into three main categories: familial, idiopathic, and secondary. The familial form (Upshaw-Schulman syndrome) results from a mutation in the ADAMTS13 gene and is associated with a very low activity of the enzyme. More than 50 mutations have been described to date. Approximately 50% of patients with familial TTP experience their first episode during childhood, and exacerbations may occur in association with minor infections (Zheng & Sadler, 2008). The idiopathic form is caused by antibodies to ADAMTS13. The secondary form may occur along the course of other conditions, such as malignancies, organ transplantation, chemotherapy, infection, and certain medications, such as cyclosporine, quinine, ticlopidine, and clopidogrel. The mechanism of TTP under these circumstances is unknown, although direct endothelial cytotoxicity and drug-induced autoimmunity are possible mechanisms (Zheng & Sadler, 2008).

TMA after organ tranplantation is a well-recognized entity occurring as a complication of renal, lung, and allogeneic hematopoietic stem cell transplant. In this setting, medications, such as sirolimus and cyclosporin, infections, and graft-versus-host disease are important factors (Rosove, 2014).

Preeclampsia/Eclampsia

Preeclampsia and eclampsia are microangiopathic disorders occurring with pregnancy and sharing some features of HUS or TTP. Approximately 4% to 39% of patients with severe preeclampsia develop HELLP syndrome: hemolysis, elevated liver enzymes and low platelet count. The syndrome becomes manifest usually during the third trimester, although some patients may develop it during the postpartum period. Most patients recover within a few days after delivery. In a small subset of patients, severe persistent multisystem disease requires plasma exchange (McMinn & George, 2001). ADAMSTS13 has been reported to be low in 10% to 20% of cases (Rosove, 2014)

Autoimmune Hemolytic Anemia

The autoimmune hemolytic anemias (AIHAs) are due to an altered immune response, resulting in the production of antibody against the host's own erythrocytes, with subsequent hemolysis. The incidence of AIHA is estimated at 10 to 30 cases per 1 million population (Gehrs & Friedberg, 2002). The AIHAs can be classified according to serologic or clinical characteristics (Table 32-10). Some AIHAs are mediated by antibodies with maximum binding affinity at 37° C, and other AIHAs are mediated by antibodies with maximum binding affinity at 4° C. In addition, AIHAs could be viewed according to their association with other disorders. In a study of 1834 patients, approximately 40% of cases of AIHA have been associated with an underlying disease, while the remainder were idiopathic (Sokol & Booker, 1992). Lymphoproliferative disorders account for approximately half of cases of both warm and cold AIHA (Gehrs & Friedberg, 2002).

Cold Agglutinin Disease

Cold agglutinin disease occurs in individuals usually over 50 years of age and in females more often than in males. In some cases, cold agglutinin disease is associated with a lymphoproliferative disorder or infection (especially with M. pneumoniae or infectious mononucleosis). Cases unassociated with an underlying disorder are listed as idiopathic. Virtually all sera from healthy individuals contain low-titer cold agglutinins, regarded as benign or harmless, and are polyclonal. Postinfectious cold agglutinins are usually polyclonal. By contrast, monoclonal cold agglutinins are generally pathogenetic and may arise from B cell lymphoma (Gehrs & Friedberg, 2002). Cold agglutinin disease represents approximately 20% of AIHA.

Symptoms and signs vary widely. Some individuals may complain of acrocyanosis or Raynaud's phenomenon. Others will have episodes of hemolysis, including hemoglobinuria, following exposure to cold. Temperatures of 30° C and lower are normally attained in the superficial skin vessels of those body parts exposed to cold. The thermal range of the antibody is clinically more important than the agglutination titer (Petz, 2008).

Laboratory findings usually indicate an anemia. Spherocytes and polychromatophilic erythrocytes are present to a variable degree in the blood film. There may be marked red cell agglutination, which should be differentiated from rouleaux formation (see Figs. 30-25 and 30-26). A mild leukocytosis can exist. Red cell agglutination may interfere with automated hematology counts, particularly MCHC, which tends to be high.

The cold antibody is usually an IgM with anti-I or, less frequently anti-i, specificity. Rarely do other specificities exist. In the chronic idiopathic form of cold agglutinin disease, the antibody tends to be monoclonal IgM, k with anti-I specificity (Gehrs & Friedberg, 2002). The autoantibody is also capable of fixing complement. When the titer of cold antibody is very high, the thermal range of antibody activity may extend up to 37° C. The direct antiglobulin test is positive only if the reagents contain anticomplement activity. Thus, one usually observes a positive antiglobulin reaction with broad-spectrum and non–γ-reagents, but no agglutination with only the γ-reagent. On cold exposure and antibody binding to red cells, the complement may be activated to the stage of C3b, which adheres to the red cells after entering the systemic circulation. In the hepatic circulation, macrophages carry specific receptors for C3b. However, cells usually escape hepatic destruction. Most patients experience chronic mild to moderate anemia, which they tolerate well, and patient outcome is significantly better than with warm-antibody AIHA (Petz, 2008). Some authors remain with the opinion that chronic cold agglutinin disease is a variant of Waldenström's macroglobulinemia in which the IgM M-component has cold agglutinin activity.

Paroxysmal Cold Hemoglobinuria

Paroxysmal cold hemoglobinuria is a very rare disorder that can occur in an individual of any age. However, more cases are now being reported in young children (Petz, 2008). Females are as frequently involved as males. Patients have symptoms of acute hemolysis following exposure to the cold. Chills, fever, pain in the back and legs, and hemoglobinuria are reported. The acute form may follow an acute upper respiratory illness or immunization by 1 to 2 weeks, but the chronic form is associated with congenital syphilis. Hemoglobinuria following exposure to cold is rare in the acute form.

Laboratory features consist of anemia, elevated reticulocyte count, increased concentration of indirect bilirubin, and the presence of Hb in the urine. Anemia is frequently severe and rapidly progressive.

The serum contains a cold hemolysin with biphasic activity. This antibody, first described by Donath and Landsteiner (1904), is an IgG that fixes the first components of complement (C1–C4) in the cold (4° C). As the temperature rises to 25° to 37° C, the antibody dissociates, but the remainder of the complement proteins are activated, and erythrocyte lysis results; this biphasic hemolysis is the basis for the Donath-Landsteiner autohemolysis test for the diagnosis of this condition. The direct antiglobulin test is positive with anti-C3 but is generally negative with anti-IgG unless performed at colder temperature. The specificity of the antibody is directed against the P antigen. In general, the prognosis is good.

AIHA Associated with Warm and Cold Antibodies

AIHA associated with both warm and cold autoantibodies is mediated by IgG warm antibodies and complement, as well as IgM cold hemagglutinins. Females are more likely involved than males. There appears to be an association between the combined warm and cold antibody AIHA and SLE in that 15% to 42% of patients have SLE (Shulman et al, 1985; Sokol & Hewitt, 1985). In one study (Shulman et al, 1985), 6 of 12 patients (50%) had idiopathic AIHA, and 4 of these (33%) had concomitant thrombocytopenia (Evans syndrome); all 12 patients had severe hemolysis that responded dramatically to corticosteroid therapy.

Alloimmune Hemolytic Disease of the Newborn

Alloimmune hemolytic disease of the newborn (HDN) usually occurs following the transplacental passage of maternal antifetal red cell antibodies. HDN most frequently results from incompatibility in Rh and ABO erythrocyte antigens between mother and fetus. In rare cases, some other red cell antigen may be responsible for this disorder.

In HDN due to Rh incompatibility, prior sensitization is necessary to initiate the disease process. This sensitization usually occurs during pregnancy when Rh(D)-positive fetal red cells cross the placenta and enter the circulation of a mother with Rh-negative cells. Most women have less than 1 mL of fetal blood in their circulation following delivery. However, intrapartum fetomaternal hemorrhage of more than 30 mL occurs in approximately 1% of pregnancies (Ramasethu & Luban, 2010). Maternal sensitization can also occur by a previous incompatible transfusion. Under either of these circumstances, maternal IgG antibodies are produced against the fetal cells. If a subsequent pregnancy occurs in a sensitized mother, fetal erythrocytes again reach the maternal circulation and restimulate an antibody response, resulting in transfer of anti-Rh(D) antibody across the placenta and reduced fetal red cell survival.

In the ABO system, anti-A or anti-B antibodies of the IgG class may arise spontaneously in the mother; their presence does not require prior transfusion or pregnancy. As a result, first-born children may suffer from HDN when ABO incompatibility exists. Although ABO incompatibility occurs in 15% of group O pregnancies, ABO hemolytic disease is estimated to occur in only 3% of all births (Ramasethu & Luban, 2010).

The clinical features of HDN due to Rh incompatibility vary greatly. Some newborns experience only mild jaundice. Others initially appear markedly pale and then develop jaundice. They can have prominent hepatosplenomegaly. The disease may be complicated by a bleeding diathesis, marked acid-base abnormalities, and kernicterus. In very severe cases, patients can present with hydrops fetalis.

Early examination of the blood usually reveals an increase in nucleated erythrocytes, which may include forms as immature as pronormoblasts. Although this finding gave the disease its name, erythroblastosis fetalis, erythroblastosis is not always present, especially if the examination is not performed immediately after birth.

Up to 2.0 × 10⁹ nucleated red cells per liter in term infants and up to 5.0 × 10⁹/L in premature infants are commonly seen in this disorder. Normally, nucleated red cells average 0.5 × 10⁹/L in term infants and 1.0 to 1.5 × 10⁹/L in premature infants. Blood from the umbilical vein for early examination is more reliable than peripheral (capillary) blood because the erythrocyte count and the Hb may be significantly altered between birth and ligation of the cord.

Generally, there is macrocytic anemia of varying severity and an increase in reticulocytes. Occasionally, anemia may develop suddenly on the second or third day. The leukocyte count is frequently elevated, with immature leukocytes. Normoblastic hyperplasia of the marrow is pronounced.

In severely affected infants, thrombocytopenia, depression of prothrombin complex procoagulants, or diffuse intravascular coagulation may occur.

A direct antiglobulin test on fetal erythrocytes indicates the presence of an IgG antibody. When the maternal serum and an eluate from the fetal erythrocytes are incubated separately with a panel of O cells, one can usually demonstrate the presence of antibody with Rh(D) specificity. In Rh-negative pregnant women known to be sensitized to Rh(D), the titer of anti-Rh(D) antibody is measured periodically during pregnancy to serve as a guide for performing amniocentesis.

HDN associated with ABO incompatibility is less severe than that observed with Rh incompatibility. Occasionally, the diagnosis is suggested by the presence of unexplained hyperbilirubinemia in a group A or B newborn infant from a group O mother.

Laboratory findings usually show a mild anemia and modest reticulocytosis. In contrast to Rh isoimmune disease, spherocytosis in ABO isoimmune disease may be prominent. However, there may be no anemia. Fetal cells are usually weakly positive with the antiglobulin reagents. Serum from the newborn and eluates from the cells should contain anti-A or anti-B antibody. In addition, maternal serum should contain high titers of anti-A or anti-B antibodies of the IgG subclass.

Drug-Induced Immune Hemolytic Anemia

Immune hemolytic anemia may occur following the administration of drugs. Most cases in the 1970s were associated with methyldopa or high-dose intravenous penicillin therapy. Recently, this picture has changed, with second- and third-generation cephalosporins being the most common causative agent and accounting for more than 80% of drug-induced immune hemolytic anemia (Garratty & Arndt, 2007). Four mechanisms appear to mediate immune hemolysis (Packman, 2010).

Megaloblastic Marrow

If the marrow is megaloblastic, with characteristic changes in both red cell and white cell precursors, the anemia in all likelihood is due to folate or cobalamin deficiency.

See earlier sections on diagnosis of cobalamin deficiency and of folate deficiency. Once the type of deficiency is defined, the cause must be determined.

Nonmegaloblastic Marrow

If the marrow is not megaloblastic, conditions that can be associated with macrocytosis should be investigated. These include liver disease; hemolytic anemias; hypothyroidism; excessive alcohol intake; aplastic anemias; and myelodysplastic syndromes. Anemias associated with these disorders, although they may be macrocytic, are more usually normocytic, and thus are considered with the normocytic anemias as well.

Optimal Marrow Response: Reticulocyte Production Index Greater Than Two

If the output of reticulocytes has exceeded two times normal, as determined by the absolute reticulocyte count, or RPI, it can be assumed that the marrow has reached an optimal response. The cause for the anemia is then acute blood loss or hemolysis. If blood loss cannot be proved, evidence that hemolysis is in fact present must be sought.

Erythroid hyperplasia of the marrow, serum bilirubin, and urine or fecal urobilinogen will indicate whether erythropoietic activity and destruction are increased. Red cell survival determination may be needed to prove hemolysis in some cases. Low serum haptoglobin and high LDH point to hemolysis, but a normal level does not exclude it. None of these measurements will specify whether hemolysis is intravascular or extravascular, but elevated plasma Hb, hemoglobinuria, and hemosiderinuria indicate intravascular hemolysis.

Once it is determined that excessive hemolysis is occurring, the type of hemolytic mechanism must be ascertained.

Inadequate Marrow Response: Reticulocyte Production Index Less Than Two

The mechanism of the anemia may be ineffective erythropoiesis. Conditions with the greatest degree of ineffective erythropoiesis appear in other categories (e.g., megaloblastic anemia, thalassemia), but most of the myelodysplastic syndromes have a hyperplastic bone marrow and impaired delivery of cells to the blood. In some of these, morphologic changes in erythroid precursors may overlap with megaloblastic anemia, but granulocytic and megakaryocytic changes usually seen in megaloblastic anemia are lacking.

A low reticulocyte count may indicate decreased production caused by inadequate stimulation of the marrow. Chronic renal disease may result in impaired production of EPO. Certain endocrinopathies, such as hypopituitarism or hypothyroidism, may result in regulation of Hb production at a lower level as a result of decreased tissue need for O₂.

A large group of normochromic anemias associated with various chronic diseases form a heterogeneous group characterized by failure of the marrow to meet the need for slightly decreased red cell survival. Some of these are ACDs associated with infection, cancer, or rheumatoid arthritis; they have the defect in iron metabolism noted previously in the Microcytic and Hypochromic Anemias section earlier in the chapter.

Inability of the marrow to respond to EPO may be due to damage to the marrow by drugs or toxic chemicals, to unknown causes, or to infiltration of the marrow by neoplastic cells or fibrous tissue.

In those conditions with low reticulocyte counts in which the marrow is not effectively producing erythrocytes, it is usually helpful to examine the bone marrow. Other studies conducted to determine the underlying disease process can then proceed according to the marrow picture, the assessment of erythrokinetics, and the clinical findings.

Renal Disorders

In other neoplasms or growths (e.g., renal cysts, hydronephrosis, ovarian carcinoma, some hepatomas), it appears that the mass impinging on the kidney induces increased renal production of EPO as a result of increased pressure or local hypoxia within the kidney. Renal artery stenosis is also associated with polycythemia. Posttransplant erythrocytosis occurs in 10% to 20% of renal transplant recipients. The prevelance is much higher in patients who receive a renal transplant without removing their original kidneys. The therapeutic effects of angiotensin-converting enzyme inhibitors in this condition suggest a role for angiotensin II in regulating erythropoiesis (Prchal, 2003).

Familial Polycythemia

The most common familial polycythemia is due to the presence of a high oxygen affinity hemoglobin, which is inherited as an autosomal dominant trait. Congenital polycythemia may be due to a defect in the hypoxia-sensing mechanism. Von Hippel–Lindau syndrome is an autosomal dominant disorder associated with mutations in the VHL gene. These mutations result in increased levels of HIF-1α and increased levels of EPO (Prchal, 2003; Gordeuk et al, 2005). Chuvash polycythemia is an autosomal recessive congenital polycythemia that is endemic in the Chuvash population of the Russian federation. It has been reported to be associated with a high mortality rate caused by thrombotic and hemorrhagic complications. VHL gene mutations have been recently described in Croatian population as well.

Congenital polycythemia may occur secondary to a defect in the EPO receptor in the presence of a normal hypoxia-sensing mechanism. Primary familial and congenital polycythemia (PFCP) is an autosomal dominant disorder that is present at birth. PFCP is associated with low serum EPO levels and in vitro hypersensitivity of erythroid progenitors to EPO. Sixteen germline mutations in the EPO receptor gene (EPOR) with resulting truncated receptors have been described in PFCP to date (Patnaik & Tefferi, 2009). As mentioned earlier, marked decrease in red cell 2,3-DPG associated with deficiency of 2,3-DPG mutase activity results in polycythemia and appears to be inherited as an autosomal recessive condition.

Hereditary Coproporphyria (HCP)

Also inherited in an autosomal dominant fashion with incomplete penetrance, HCP is less common than AIP. It is due to a deficiency of coproporphyrinogen oxidase. Clinically, HCP resembles a milder form of AIP with its neurovisceral attacks, but cutaneous manifestations are seen in roughly one third of patients (Chemmanur & Bonovsky, 2004). Fecal coproporphyrinogen III is excreted both during and between attacks, and ALA and PBG are seen in urine during an acute crisis. ALA excretion usually exceeds that of PBG, and this, coupled with the cutaneous findings (if present) helps to differentiate HCP from AIP. Prevention, including sunscreen, is central to management.

Congenital Erythropoietic Porphyria (CEP)

CEP is distinct from the other porphyrias discussed earlier in both its inheritance (it is a recessive disorder) and its severity. Patients present shortly after birth with red-pigmented urine, hemolytic anemia, and severe cutaneous photosensitivity. Erythrodontia is striking and may be a useful clue if the diagnosis has not already been made. The deficient enzyme is uroporphyrinogen III synthase with over 35 mutations identified, and ALA synthase activity is increased. Coproporphyrin I and uroporphyrin I are present in urine. The prognosis for CEP is significantly worse than for the other porphyrias, with death occurring at an early age in many cases.