Red Blood Cell/Hemoglobin Disorders

Clinical Features

The hematologic manifestation of Cbl and folate deficiency is megaloblastic anemia. The onset of anemia is gradual, and the severity at presentation is extremely variable. Some patients exhibit profound macrocytic anemia with Hb levels as low as 2 g/dL, whereas other patients may have a more subtle manifestation or present with symptoms other than anemia. Due to defective DNA synthesis, ineffective erythropoiesis or intramedullary hemolysis can lead to jaundice, markedly increased lactate dehydrogenase, and increased indirect bilirubin. The ineffective hematopoiesis and hemolysis may be so pronounced that haptoglobin is reduced and hemosiderinuria occurs. The mean corpuscular volume (MCV) is characteristically high, with clinically overt megaloblastic anemia, but may also become elevated before the development of detectable anemia. However, the MCV may be in the normal range if there is concurrent iron deficiency anemia or thalassemia. The reticulocyte count is low. Leukopenia and thrombocytopenia are often present because impairment of DNA synthesis affects all proliferating cells.

The effect of Cbl deficiency on epithelial cells may result in the classic “beefy red smooth tongue” (atrophic glossitis). More important, Cbl deficiency is often associated with a peripheral neuropathy and demyelinization of the dorsal and lateral columns of the spinal cord (i.e., subacute combined degeneration), leading to paresthesias, loss of position and vibration sense, spastic ataxia, and extensor plantar reflexes. Cognitive impairment may be seen; psychosis is a rare complication. Neurologic complications of Cbl deficiency may be only partially reversible, making early recognition of this disorder essential.

Pathologic Features

In clinically overt megaloblastic anemia, the peripheral blood film demonstrates marked variability in red cell size (anisocytosis), with large, oval red cells (macroovalocytes), teardrop cells, fragments, microcytes, and other nonspecific abnormally shaped red cells (poikilocytes; Fig. 1.2 ). Polychromasia is characteristically absent. With increasing severity, there is increased red cell fragmentation. Howell-Jolly bodies, nucleated RBCs with megaloblastic morphology, and basophilic stippling are often observed ( Fig. 1.3 ). Hypersegmented neutrophils with six or more nuclear lobes, in combination with macrocytic anemia, are highly specific for megaloblastic anemia ( Fig. 1.4 ).

Bone marrow aspirate smears typically demonstrate erythroid hyperplasia and a shift to immaturity, as well as characteristic cytologic changes in all three myeloid lineages. Maturing cells are larger than normal, with asynchronous nuclear and cytoplasmic maturation. The chromatin of the erythroid precursors appears more granular than during normal maturation, and the most mature erythroid precursors display a prominent fenestrated nuclear appearance that contrasts with the expected complete condensation of chromatin at this stage ( Fig. 1.5 ). Erythroid precursors with abnormally shaped, multiple, budding, or fragmented nuclei are often seen ( Fig. 1.6 ), resembling those seen in myelodysplastic syndromes. Giant metamyelocytes and bands with abnormally open chromatin are also characteristic ( Figs. 1.5 and 1.7 ), whereas larger than normal megakaryocytes with unattached nuclear lobes or hyperlobated nuclei reflect similar abnormalities in megakaryocytic maturation. Because of the pronounced ineffective hematopoiesis, markedly increased iron stores are seen with a Prussian blue iron stain unless there is concurrent iron deficiency. Bone marrow core biopsy specimens demonstrate a markedly hypercellular marrow with expanded and left-shifted trilineage hematopoiesis ( Fig. 1.8 ). In particular, islands of immature erythroid precursors with finely stippled chromatin and distinct, irregular, eosinophilic nucleoli are often prominent ( Fig. 1.9 ).

Ancillary Studies

Serum Cbl is fairly sensitive and specific for clinically overt Cbl deficiency. Causes of falsely low serum Cbl include pregnancy, use of oral contraceptives, anticonvulsant administration, human immunodeficiency virus infection, and folate deficiency. Mixed Cbl/folate deficiency complicates the interpretation of the laboratory workup of megaloblastic anemia ( Table 1.1 ). Falsely normal levels of Cbl are rare with clinically overt deficiency but may be observed in association with myeloproliferative disorders, liver disease, intestinal bacterial overgrowth, and congenital TC II deficiency. Detection of antibodies to intrinsic factor helps to determine the cause of a Cbl deficiency and to confirm a diagnosis of pernicious anemia.

Folate status may be assessed by either serum or red cell folate assays. Serum folate may occasionally be misleading due to fluctuations related to short-term dietary changes. Hemolysis (in vivo and in vitro) will cause falsely elevated serum folate, whereas alcohol intake can cause transient low levels despite adequate stores. Red cell folate levels better reflect body folate stores and are less prone to short-term fluctuation. Importantly, Cbl deficiency can cause reduced red cell folate levels, but this is usually associated with normal or increased serum levels (see Table 1.1 ).

Measurements of serum homocysteine and methylmalonic acid are also useful in the assessment of megaloblastic anemia. Homocysteine accumulates in both Cbl and folate deficiency because of interruption of the biochemical pathway illustrated in Fig. 1.1 . Unlike folate deficiency, Cbl deficiency is associated with an increase in serum methylmalonic acid because of interruption of the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. A normal methylmalonic acid level generally rules out Cbl deficiency; however, both metabolites may be mildly elevated with renal insufficiency. Elevated serum homocysteine may also be seen with vitamin B ₆ deficiency, vitamin B ₂ (riboflavin) deficiency, hypothyroidism, some drugs, and genetic mutations affecting metabolism of homocysteine.

Differential Diagnosis

The major differential diagnoses of megaloblastic anemia owing to folate or Cbl deficiency are megaloblastic changes related to drug therapy and myelodysplastic syndromes. A variety of agents that interfere with DNA synthesis can cause megaloblastic changes in maturing marrow elements, including methotrexate, zidovudine, hydroxyurea, and azathioprine. Megaloblastic morphology due to drugs tends to be confined to the erythroid compartment, although this may not be the case with long-term administration. In general, an accurate drug history and biochemical tests for folate and Cbl deficiency will distinguish this cause of megaloblastosis.

Megaloblastic anemia may be confused with myelodysplastic syndromes because of the potential for dyserythropoiesis in megaloblastic anemia or the potential for megaloblastic changes in myelodysplastic syndromes. However, when megaloblastic changes are seen in the erythroid elements of myelodysplastic syndromes, they are generally accompanied by a spectrum of other abnormalities that are not a feature of megaloblastic anemia, such as neutrophil hyposegmentation or hypogranularity, hypogranular platelets, hypolobated megakaryocytes, or increased blasts. Furthermore, giant bands and metamyelocytes as seen in megaloblastic anemia are usually not a notable feature of myelodysplastic syndromes. As a general rule, a very high MCV (more than 130 fL) would favor a megaloblastic anemia due to Cbl or folate deficiency versus other causes.

In addition to other causes of megaloblastosis, nonmegaloblastic macrocytosis can also enter into the differential diagnosis of megaloblastic anemia. These diagnoses include reticulocytosis, liver disease, alcohol abuse, drug effects, and hypothyroidism. Reticulocytosis is easily discerned either with a reticulocyte count or with prominent polychromasia on blood smear examination. Other causes of nonmegaloblastic macrocytosis do not produce the degree of anisopoikilocytosis, oval macrocytes, or hypersegmented neutrophils seen in megaloblastic anemia. Liver disease, in particular, is characterized by a uniform population of round macrocytic cells and target cells.

Prognosis and Therapy

Standard therapy for Cbl deficiency, when the cause is not dietary deficiency, has traditionally been intramuscular injection of vitamin B ₁₂ ; however, oral therapy is effective for some patients. To prevent adverse neurologic consequences because of concurrent or overlooked Cbl deficiency, Cbl status must be established before folate therapy is initiated. This may be difficult to do in practice because folate deficiency can cause low serum Cbl for reasons that are not well understood. If there is any doubt, Cbl supplementation should be administered along with folate therapy. Standard therapy for folate deficiency is oral supplementation. Parenteral administration of folate may be needed in cases caused by intractable malabsorption.

Clinical Features

Most signs and symptoms are those seen in any form of anemia, such as fatigue, dyspnea, and pallor of skin and mucous membranes. Because the anemia develops over a protracted period and compensatory mechanisms have ample time to develop, IDA is surprisingly well tolerated, even when extremely severe. Pica, koilonychia (i.e., spoon-shaped nails), and esophageal webs are rare but have a strong association with IDA. Angular cheilitis and atrophic glossitis can also be seen but are not specific. The anemia ranges from mild to profound, with Hb levels as low as 2 g/dL in the most severe cases. IDA typically is a microcytic and hypochromic anemia, reflected as a decreased MCV and mean corpuscular Hb concentration (MCHC), respectively. However, the MCV may be normal in early iron deficiency; the degree of microcytosis roughly correlates with the degree of anemia. Likewise, the MCHC may be normal in early iron deficiency when only a minority of cells is hypochromic.

Pathologic Features

The characteristic findings on the peripheral blood film are small (microcytic), under-hemoglobinized (hypochromic) RBCs with variability in size (anisocytosis) and abnormal shapes (poikilocytosis; Fig. 1.10 ). Elliptocytes are prominent poikilocytes in IDA; often they are long and narrow (pencil cells). Target cells, teardrop cells, very small hypochromic microcytes, and various nonspecific poikilocytes are also common. Occasional red cell fragments may be observed, but they are not prominent. Thrombocytosis is common, but platelet counts may also be normal or reduced. Variations from the typical findings occur when there has been recent partial repletion of iron or when additional factors, such as Cbl or folate deficiency, are also contributing to anemia.

The bone marrow in IDA may demonstrate erythroid hyperplasia, but this is generally of only a mild degree. Erythroid precursors may have scant cytoplasm with frayed cytoplasmic borders.

Ancillary Studies

Serum ferritin measurement is the most useful single laboratory test for iron deficiency. Serum ferritin below the lower limit of the reference range (about 12 mg/dL) is essentially diagnostic of iron deficiency. However, ferritin is an acute-phase reactant, and normal levels may be seen when iron deficiency coexists with infection, inflammation, or malignancy. When this occurs, the ferritin level will usually be low-normal; therefore low-normal values in an individual with an active inflammatory process support a diagnosis of iron deficiency in the appropriate setting. Ferritin levels may even be higher in patients with acute liver injury, despite iron deficiency. Measurement of serum iron alone is not useful because of diurnal fluctuations of serum iron levels and the rapid changes that may occur with dietary intake, inflammation, and blood loss. Transferrin, measured either directly or indirectly as the total iron-binding capacity, is typically increased in IDA, but this response can be blunted in the presence of hypoproteinemia. The transferrin saturation (the ratio of serum iron to total iron-binding capacity) is typically less than 15% in iron deficiency, but saturation levels in this range may also be seen in anemia of chronic disease (ACD).

Increased serum soluble transferrin receptor (sTfR) is a sensitive marker of iron deficiency, reflecting increased expression of this receptor on RBC precursors as a response to depleted tissue iron. This parameter is elevated during iron-deficient erythropoiesis before the development of overt anemia. Furthermore, inflammation does not raise sTfR levels substantially. However, conditions with an increased mass of RBC precursors such as hemolytic anemias and ineffective erythropoiesis (e.g., megaloblastic anemia) will manifest elevated sTfR; therefore this finding is not specific for iron deficiency.

When biochemical tests are equivocal, the absence of stainable iron on a Prussian blue–stained marrow aspirate smear with adequate spicules is considered to be the gold standard for the diagnosis of IDA. An iron stain performed on a bone marrow core biopsy may also be helpful, but iron can be leached out during decalcification of the bone marrow biopsy and result in the false interpretation of absent iron stores.

In IDA, hepcidin level is generally suppressed. Hepcidin blocks iron absorption from the diet and the iron release from macrophages and liver cells. Although measurement of serum hepcidin is helpful, development of an accurate clinical assay is challenging.

Differential Diagnosis

Other common causes of hypochromic, microcytic anemia are thalassemia and ACD. Sideroblastic anemia with microcytosis is rarer. The hematologic and biochemical tests that are useful to differentiate the major causes of microcytic anemia are listed in Table 1.2 . In addition, mild anemia associated with thalassemia trait or a thalassemia-like hemoglobinopathy (e.g., Hb E) characteristically exhibits a red blood cell count greater than 5 × 10 ¹² cells/L, which is unusual for IDA. Hb electrophoresis would help establish the diagnosis of most β-thalassemias and thalassemia-like hemoglobinopathies. Patients with ACD are also at risk of developing IDA. Serum hepcidin can help identify concomitant IDA in this situation because levels are lower compared to pure ACD. When the interpretation of biochemical studies for iron status is particularly difficult, monitoring the response of Hb or the reticulocyte count to a trial of iron supplementation may be sufficient to confirm iron deficiency. An iron stain performed on a bone marrow aspirate smear can also help to resolve uncertainty about iron status.

Prognosis and Therapy

Iron supplementation with oral iron salts, most commonly ferrous sulfate, is standard therapy for uncomplicated IDA. Response to iron supplementation is usually rapid if the iron deficiency is the result of inadequate iron intake. If an appropriate response is not observed or when occult blood loss is more likely, additional evaluation to determine the cause of IDA should be considered. Parenteral iron may be needed for patients with uncontrolled blood loss, intolerance to oral iron, or malabsorption.

Because of the physiologic attempts to compensate for severe chronic anemia that has gradually developed, a rapid blood transfusion can result in hypervolemia and cardiac dilatation. Therefore red cell transfusions should be reserved for patients with severe anemia causing cardiac compromise.

Clinical Features

The most common congenital form of SA is X-linked due to mutations in 5-aminolevulinate synthase ( ALAS2 ) and manifests predominantly in boys who have mild to moderate microcytic anemia. Girls with the same heterozygous mutations are usually asymptomatic. Autosomal-recessive SA due to mutated SLC25A38 , which encodes an erythroid mitochondrial transporter, usually presents in the first year of life with severe, transfusion-dependent microcytic anemia refractory to pyridoxine treatment.

Pathologic Features

The peripheral blood smear reveals anemia with dimorphic red blood cell morphology, including a distinct population of hypochromic red blood cells ( Fig. 1.11 ). The anemia in congenital SA is usually, but not always, microcytic, and the acquired forms are often not microcytic. Ring sideroblasts in bone marrow aspirate smears are the hallmark of SA and are due to deregulated iron metabolism and mitochondrial iron overload in the erythroid precursors ( Fig. 1.12 ). Hemosiderin-laden macrophages are increased due to ineffective erythropoiesis. A peculiar morphologic clue in bone marrow smears associated with copper deficiency is the presence of hemosiderin in plasma cells ( Fig. 1.13 ).

Ancillary Studies

Iron studies often reflect associated iron overload. A definitive diagnosis of congenital SA can be made when specific mutations are identified. Evaluation for copper deficiency includes serum copper and ceruloplasmin.

Differential Diagnosis

The differential diagnosis for SA with microcytosis includes IDA, thalassemia, and ACD. Thus iron studies and hemoglobin electrophoresis are helpful. In adults, the acquired forms of SA are more common than congenital forms.

Prognosis and Therapy

SA with mutated ALAS2 may respond to pyridoxine treatment. Transfusions with therapy to manage iron overload provides relief for patients with severe anemia. Hematopoietic stem cell transplantation has been successful in some patients with congenital SA. For acquired SA, the anemia is often reversible after the offending agents have been removed (e.g., alcohol, drugs) or treatment for copper deficiency has been implemented.

Clinical Features

The clinical spectrum is highly variable. CDA I and II are autosomal recessive, whereas CDA III is autosomal dominant. CDA II is the most common, and many patients come to attention as adults due to mild or moderate normocytic anemia associated with jaundice, splenomegaly, and/or hepatomegaly. Presentation of CDA I and III is similar to CDA II, but CDA I is usually macrocytic, comes to attention at a younger age, and can be associated with skeletal abnormalities. CDA I may also present as hydrops fetalis or as severe neonatal anemia. Cholelithiasis and rarely extramedullary hematopoiesis-related mass lesions can occur with CDA. Secondary hemochromatosis from iron overload is a major complication.

Pathologic Features

The peripheral blood film reveals various poikilocytes, basophilic stippling, and, in CDA II, occasional binucleated erythroblasts. The bone marrow shows erythroid hyperplasia and dyserythropoiesis. The nonerythroid lineages are morphologically normal. A highly specific feature for CDA I on bone marrow examination is thin chromatin bridges between paired erythroblasts. CDA II is often first recognized based on binucleated and rarely multinucleated erythroblasts in more than 10% of erythroid precursors. The binucleation is often restricted to the late-stage erythroblasts ( Fig. 1.14 ). CDA III is characterized by giant, multinucleated erythroblasts with up to 12 nuclei.

Ancillary Studies

The diagnosis of CDA is based on long-standing hemolytic anemia with characteristic bone marrow findings and lack of an appropriate reticulocyte response. In the past, laboratory tests to confirm the diagnosis included a positive HAM test for CDA II, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify hypoglycosylated membrane proteins in CDA II (e.g., band 3 is narrowed and shows faster migration), and electron microcopy for ultrastructural abnormalities distinct in CDA I, II, and III (e.g., “Swiss cheese–like” heterochromatin in CDA I and “double plasma membrane” in CDA II). When the clinical picture, peripheral blood, and bone marrow findings are compatible with a diagnosis of CDA, sequencing for known mutations can confirm the diagnosis in many cases and exclude other rare inherited anemias. A summary of some key features and mutated genes recognized so far for CDA is shown in Table 1.3 . The interested reader may consult the reference list for more information.

Differential Diagnosis

Clinically, CDA may be confused with thalassemia or hemolytic anemia. However, a relatively low reticulocyte count incommensurate to the degree of anemia and the unique bone marrow findings will lead to the correct diagnosis. CDA II is often misdiagnosed as hereditary spherocytosis (HS) due to hemolysis-related symptoms and occasional spherocytes in the peripheral blood. However, more numerous spherocytes and a greater reticulocyte response are expected for HS. The differential diagnosis of dyserythropoiesis includes many conditions such as B ₁₂ /folate deficiency, exposure to heavy metals, copper deficiency, chemotherapy, medication effect, and myelodysplastic syndromes. However, the morphologic abnormalities predominant in the classic types of CDAs are distinct and not the main feature in any of these conditions.

Therapy

Treatment depends on the type of CDA and the degree of anemia. Blood transfusions with therapy to prevent iron overload may be required for patients with more severe anemia. Interferon gamma may alleviate the symptoms of CDA I. Hematopoietic stem cell transplantation can be curative.

Clinical Features

Progressive uremia leads to plasma expansion and hemodilution; therefore the Hb level immediately before hemodialysis is lower than that immediately afterward. Cardiovascular and renal attempts to compensate for anemia contribute to the development of left ventricular hypertrophy and can lead to congestive heart failure. Treatment with erythropoiesis-stimulating agents (ESAs), such as epoetin alfa or darbepoetin alfa, can improve the quality of life of these patients. However, the response to these therapies is limited if either absolute or functional iron deficiency develops. Functional iron deficiency is due to depletion of the iron pool immediately available for erythropoiesis. Mobilization of iron stores becomes rate limiting and is inadequate to meet the accelerated demand for iron during aggressive ESA therapy.

Pathologic Features

In ACKD, the peripheral blood film shows a normochromic, normocytic anemia with minimal polychromasia and minimal anisocytosis. Occasional echinocytes (burr cells) and other poikilocytes may be observed, such as acanthocytes and rare fragments. There is no increase in polychromasia.

The bone marrow is usually normocellular or slightly hypocellular; in the absence of ESA therapy, it contains normal to slightly reduced erythroid precursors. The amount of sideroblastic iron and storage iron will depend on overall iron status. Morphologic features of renal osteodystrophy may be noted on a bone marrow core biopsy specimen, especially in association with end-stage renal disease and in patients who have been treated with long-term dialysis. Renal osteodystrophy is a spectrum of abnormalities of bone turnover that variably exhibits thinning or thickening of bony trabeculae, excess osteoid without mineralization (osteomalacia), stromal fibrosis, and osteitis fibrosa cystica ( Fig. 1.15 ).

Ancillary Studies

Plasma erythropoietin concentration and the reticulocyte count are inappropriately low for the degree of anemia in ACKD. An increase in reticulocytes is a helpful indicator of a response to therapy. However, measuring plasma erythropoietin concentration is not usually recommended.

Detection of concomitant iron deficiency may be compromised by the fact that patients with CKD may have associated inflammation; therefore ferritin levels may be difficult to interpret. Even more challenging is the detection of the functional iron deficiency that may compromise the effectiveness of ESA therapy. The small numbers of hypochromic RBCs produced in this circumstance will not be detected reliably by the MCV or MCHC. High-throughput hematology analyses provide additional parameters of hypochromia, which vary based on the manufacturer but which can help identify functional iron deficiency (e.g., percentage of hypochromic red cells, reticulocyte hemoglobin content, and low hemoglobin density).

Differential Diagnosis

Nutritional deficiencies, infections, and other causes of inflammation commonly have a role in the development of anemia in patients with CKD. In addition to malnutrition, blood loss associated with hemodialysis can contribute to iron deficiency. The diagnosis of iron deficiency in the setting of CKD is challenging due to poor predictive ability of serum iron indices. Recent guidelines (Kidney Disease: Improving Global Outcomes) recommend a course of iron therapy in patients with CKD and anemia if the transferrin saturation is less than 30% and the serum ferritin level is less than 500 mg/L. Patients receiving dialysis are also at increased risk of megaloblastic anemia, because Cbl and folate are water soluble and dialyzable.

Elevated markers of inflammation, such as C-reactive protein or erythrocyte sedimentation rate, indicate that chronic infection or inflammation may be contributing factors for anemia. Patients with high levels of inflammatory cytokines are more resistant to ESA therapy.

Hypothyroidism and secondary hyperparathyroidism can also contribute to anemia and resistance to ESA therapy among patients with CKD. Similar to ACKD, anemia related to hypothyroidism manifests with an inappropriately low reticulocyte response and is usually normocytic and normochromic. Aluminum toxicity associated with dialysis is another occasional cause of anemia in patients with renal failure; when chronic, this anemia becomes hypochromic and microcytic. In addition, a sudden lack of response to erythropoietin therapy could herald pure red cell aplasia caused by the development of antierythropoietin antibodies; however, this is rare and mostly related to a formulation that had been used outside of the United States.