Evaluation of Anemia, Leukopenia, and Thrombocytopenia

Iron Deficiency

Iron deficiency occurs when iron utilization or loss exceeds iron absorption and results in depletion of body stores. Early in iron deficiency, iron stores are decreased, but the red cells are morphologically unaffected. Serum ferritin (normally 12 ng/mL to 300 ng/mL) is in equilibrium with tissue stores and serves as an indirect measure of storage iron in uncomplicated cases. However, ferritin is an acute phase protein, and patients with chronic inflammation or liver disease may have elevated values even in the presence of iron deficiency. After iron stores are depleted, serum iron drops and the iron transport protein, transferrin, increases, so that the total iron-binding capacity is increased. The red cells become microcytic and normochromic, and finally microcytic and hypochromic ( Fig. 11-3 ). Transferrin saturation (serum iron/total iron binding capacity) of less than 15% is virtually diagnostic of iron deficiency. Iron homeostasis, including iron uptake from the intestine and release from stores, is regulated by the liver-secreted protein hepcidin. Serum iron concentration has diurnal variation and should be measured in the morning, when it is at its highest level. The sensitivity and specificity of the CBC, transferrin saturation, and ferritin values are usually sufficient to make the diagnosis of iron deficiency without the need to perform a bone marrow study. In addition, serum-soluble transferrin receptor (sTfR) levels, which are elevated in iron deficiency but usually unaffected by inflammation, and the sTfR-ferritin index (sTfR/log ferritin) may be helpful in interpreting iron status in patients with inflammatory disease. Bioactive forms of serum hepcidin are also being investigated as markers of iron status and erythropoietin resistance in states of inflammation. In ambiguous cases, such as patients with elevated acute phase proteins or hepatic disorders, a bone marrow evaluation for iron assessment is indicated. Bone marrow iron stores and sideroblast iron should be evaluated on an aspirate smear ( Fig. 11-4, A, B ), because iron is chelated by acidic decalcifying agents and is generally underestimated in clot or trephine biopsy sections. In normal bone marrow, one or two small siderotic granules are normally identifiable in at least 10% of the normoblasts (see Fig. 11-4, A, B ). In iron deficiency, the Prussian blue reaction demonstrates loss of reticuloendothelial marrow stores and iron incorporation into normoblasts (see Fig. 11-4, C, D ; see Table 11-2 ).The absence of iron stores differentiates iron deficiency from advanced anemia of chronic disease, which may mimic an iron deficiency state. However, some authors suggest the evaluation of multiple marrow spicules before declaring the marrow as iron deficient because the iron may be irregularly distributed. If recent parenteral iron or RBC transfusion has been given to an iron-deficient individual, these findings may be misleading because the bone marrow iron stores may appear adequate. Bone marrow morphology is otherwise non-specific in iron deficiency. In severe anemia, the erythroid precursors may appear smaller, with only a narrow rim of cytoplasm. Rare individuals have iron-refractory iron-deficiency anemia that is congenital and due to defects in the TMPRSS6 gene. Some cases are due to gene mutations involving the transferrin gene or iron transport genes (DMT1, GLRX); other individuals may have cellular iron export abnormalities. As iron is not normally excreted by the body, except in menstrual periods, the etiology for blood loss should be carefully sought. The leading cause of iron-deficiency anemia in adults is occult bleeding from the gastrointestinal tract. Exclusion of Helicobacter pylori infection as a cause of unexplained iron-deficiency anemia is also important because eradication of the organisms leads to amelioration of the anemia.

Thalassemias

Thalassemias are a common cause of hypochromic microcytic anemia in which adult hemoglobin (HbA [α ₂ β ₂ ]) synthesis is quantitatively affected by decreased alpha or beta globin chain synthesis ( Table 11-3 ; see Fig. 11-2 ). Thalassemia is common in Mediterranean regions, tropical Africa, and Asia; β-thalassemia is also seen in the Middle East and India. It is caused by nearly 200 different mutations that affect one or both of the beta globin chain genes on chromosome 11. These are primarily point mutations affecting transcription, splicing, or translocation of beta globin messenger RNA. The diagnosis is best made by high-performance liquid chromatography or hemoglobin electrophoresis. In its most benign form (β-thalassemia minor), only one of the two genes is mutated, causing either decreased (β ⁺ ) or absent (β ⁰ ) beta globin protein synthesis by the affected allele. The normally produced alpha chains have insufficient beta chains with which to pair, and the excess combine with delta chains to produce HbA ₂ (α ₂ δ ₂ ). A mild elevation in HbF (α ₂ γ ₂ ) is also found in about one third of patients. If both beta chain genes are mutated, scant (β ⁺ β ⁺ or β ⁰ β ⁺ ) or no beta chains (β ⁰ β ⁰ ) are made, causing a serious childhood anemia, β-thalassemia major (Cooley's anemia). This is usually diagnosed in the first year of life as hemoglobin switches from HbF to HbA. It is associated with severe microcytic anemia, marked hemolysis, marked erythroid hyperplasia, hepatosplenomegaly, and failure to thrive ( Fig. 11-5, A, B ). Leukopenia and thrombocytopenia may occur with progressive splenomegaly. Patients who are not transfusion dependent or are diagnosed later in life are classified clinically as having β-thalassemia intermedia. Methyl violet highlights insoluble alpha chain inclusion bodies in the red cells, resembling Heinz bodies. They also occur in hemoglobinized erythroid precursors in the marrow and result in ineffective erythropoiesis. Erythropoiesis is also affected by red cell membrane abnormalities in the developing cells (abnormal ratio of spectrin to band 3 and abnormal band 4.1) that promote increased intramedullary death of the red cell precursors. In addition to erythroid hyperplasia, the bone marrow may demonstrate erythrophagocytosis and increased hemosiderin as a result of excessive absorption of dietary iron, secondary to decreased hepcidin levels.

The α-thalassemias are primarily caused by deletions of one, two, or three of the four alpha chain (αα/αα) genes located on chromosome 16. The number of deleted loci determines disease severity. A silent carrier has only one deleted gene (−α/αα), and the blood picture is normal. When two genes are involved (−α/−α in African populations, or − −/αα in Asian populations), a mild hypochromic microcytic anemia is generally present (α-thalassemia trait) ( Fig. 11-6, A ). Only one functioning alpha chain (− −/−α) results in HbH (β ₄ ) disease (see Fig. 11-6, B ). Neonates with this disorder have excess unpaired gamma globin chains that form tetramers, called hemoglobin Barts (γ ₄ ). They also produce small amounts of fetal hemoglobin until beta chain synthesis develops and replaces gamma chain synthesis. Adults and older children form beta chain tetramers (HbH) with normal amounts of fetal hemoglobin. HbH disease is most common in Asian populations, who present with variable symptoms and usually a moderate hypochromic microcytic anemia with reticulocytosis, although severe anemia similar to β-thalassemia major may be seen. RBCs have HbH inclusions that can be seen best with brilliant cresyl blue or new methylene blue stains. Unlike the globin precipitates of β-thalassemia major, these beta globin inclusions are not seen in bone marrow erythroid precursors. The remaining bone marrow findings are similar. Deletion of all four alpha chain genes (hydrops fetalis) is incompatible with life (see Fig. 11-6, C ). With the absence of alpha chain production, only hemoglobin Barts is formed. This abnormal hemoglobin has very high oxygen affinity and deprives fetal tissues of needed oxygen. An acquired form of α-thalassemia may develop in elderly patients with myelodysplastic syndrome (MDS) and an associated ATRX gene mutation that downregulates alpha chains.

A third form of thalassemia, termed δβ-thalassemia, is caused by deletions of large segments of DNA on chromosome 11, including both delta and beta genes. Heterozygotes present as thalassemia minor with microcytosis and no anemia. Their HbF (α ₂ γ ₂ ) levels are typically elevated (5% to 15%), and HbA ₂ (α ₂ δ ₂ ) is normal or low. Patients homozygous for the delta-beta mutation have 100% HbF and clinical findings similar to those of thalassemia intermedia. Finally, rare patients have a thalassemic picture due to structurally abnormal hemoglobins, such as hemoglobins Constant Spring, Lepore, and HbE.

Anemia of Chronic Disease

Anemia of chronic disease (ACD), also known as anemia of inflammation , is second only to iron-deficiency anemia in frequency. It is observed in patients with infectious, inflammatory, traumatic, or neoplastic disorders and is thus common among hospitalized patients. ACD is characterized by a low serum iron concentration in the face of normal or increased iron stores. It results from cytokine-induced hepcidin production that causes a mild shortening of RBC life span, impaired mobilization of iron from reticuloendothelial stores to erythroid precursors, lower than expected erythropoietin production, and an inadequate response of erythroid precursors to erythropoietin. The relative contribution of each of these findings may vary according to the underlying disease. The recently discovered liver peptide hormone hepcidin is a major regulator of iron homeostasis and plays a central role in the pathogenesis of ACD. The expression of hepcidin is upregulated by interleukin (IL)-6 (which in turn can be increased by IL-1) and inhibited by tumor necrosis factor-α. Hepcidin blocks the activity of ferroportin, a transport protein that facilitates basolateral movement of iron from the intestinal apical cells, as well as from histiocytes. Thus, an increase in hepcidin in inflammation leads to hypoferremia and impaired delivery of iron to erythroid precursors (see Fig. 11-2 ). Hepcidin antagonists are currently being explored as promising therapies for ACD. Serum ferritin increases in proportion to the increase in hepcidin. Suppression of erythropoiesis in ACD may be reversible by exogenous erythropoietin (or derivatives), but responses depend on the type of cytokines involved in stimulating hepcidin production. For example, erythropoietin treatment is less effective for correcting the anemia of malignancy than for treating the anemia of chronic renal failure. The beneficial or detrimental effects of providing supplemental iron also appear to depend on the underlying condition.

The normochromic normocytic form of ACD is generally mild to moderate and is far more prevalent than the hypochromic microcytic type, which is usually seen with progression and exacerbation of the underlying disease ( Fig. 11-7, A ). Iron studies are helpful to exclude iron deficiency. However, ferritin can be difficult to interpret because it is an acute phase protein; a ferritin level greater than 60 µg/L should be considered indicative of adequate iron stores. The bone marrow is usually normocellular, with normal or slightly decreased numbers of erythroid precursors (see Fig. 11-7, B ). Bone marrow examination is helpful primarily in assessing the iron status when iron studies are indeterminate. A Prussian blue stain of the aspirate shows increased accumulation of reticuloendothelial iron stores in histiocytes and decreased sideroblast iron ( Fig. 11-8 ; see Table 11-2 ). This staining pattern excludes iron deficiency; chronic blood loss also becomes a less likely cause.

Sideroblastic Anemias

Sideroblastic anemias are a heterogeneous group of disorders that are unified pathologically by abnormal accumulation of mitochondrial iron and impaired heme synthesis. The blood film often shows a striking dimorphic RBC picture with varying numbers of hypochromic and normochromic RBCs ( Figs. 11-9 and 11-10 ). The constellation of blood findings (see Table 11-1 ) merits a bone marrow examination for definitive diagnosis. The bone marrow exhibits erythroid hyperplasia with normoblastic to megaloblastic maturation ( Figs. 11-11 and 11-12 ). Occasional dysplastic changes are seen, especially in the acquired clonal disorders. In some congenital disorders, such as Pearson marrow-pancreas syndrome, large coalescent vacuoles may be found in the cytoplasm of bone marrow precursors ( Fig. 11-13 ). The diagnostic feature is the presence of increased iron stores and ring sideroblasts, in which five or more large, siderotic granules are found in a perinuclear ring around one third or more of the nucleus ( Fig. 11-14 ). Sideroblastic iron granules are often more numerous and larger than normal. When examined by electron microscopy, large electron-dense deposits are found within mitochondria. Ineffective hematopoiesis is the primary cause of the anemia. Although the mechanisms responsible for sideroblastic anemia are not fully understood, the adverse effects of excess iron on mitochondrial heme synthesis and pyridoxine metabolism play a large role.

Sideroblastic anemias can be classified as congenital or acquired ( Box 11-1 ). The congenital sideroblastic anemias (CSA) are most common in children, presenting soon after birth to later in childhood. They most often affect males and show an X-linked pattern of inheritance. The most common form of X-linked sideroblastic anemia is due to a mutation in the gene that encodes δ-aminolevulinic acid (ALA) synthetase, an enzyme important in the early steps of heme synthesis (see Fig. 11-2 ). The mutation affects the enzyme's affinity for its cofactor, pyridoxal-5′-phosphate. Some patients with this abnormality may respond to pyridoxine; in others, the mutation decreases the stability of the enzyme, and they are resistant to pyridoxine therapy. Another form, X-linked sideroblastic anemia with ataxia, is due to a mutation in the gene that encodes the transporter protein ABCB7. Autosomal recessive forms include sideroblastic anemias due to defects in heme synthesis or iron-sulfur biogenesis. CSAs due to abnormal mitochondrial protein synthesis have been identified. Perhaps the best example is Pearson marrow-pancreas syndrome (see Fig. 11-13 ), which occurs sporadically and is characterized by lactic acidosis, exocrine pancreatic insufficiency, sideroblastic anemia, and large deletions or duplications in mtDNA. Another CSA, mitochondrial myopathy with lactic acidosis and ring sideroblasts (MLASA), is due to defective mitochondrial protein expression. The primary or idiopathic acquired forms of sideroblastic anemia include clonal disorders that fall in the spectrum of MDS, and they are discussed in Chapter 45 . Point mutations in mitochondrial DNA (mtDNA) have been reported in patients with primary acquired sideroblastic anemia, but their significance in the pathophysiology of the disease process is not yet clear.

The secondary and less common forms of acquired sideroblastic anemia are the result of drugs and exposure to toxins, many of which have been characterized. For example, the drug isoniazid inhibits pyridoxine metabolism; lead inhibits δ-ALA dehydratase and heme synthetase; and alcohol produces a direct toxic effect on erythroid precursors (found in 30% of hospitalized alcoholics). The anemia can be reversed by administration of pyridoxal phosphate and discontinuation of the offending drug. Copper deficiency anemia, often secondary to zinc overload, is discussed in more detail in the neutropenia section of this chapter; the red cells may be microcytic, normocytic, or macrocytic.

Pure Red Cell Aplasia

Pure red cell aplasia is an isolated failure of erythropoiesis that results in anemia with reticulocytopenia and normal neutrophil and platelet counts. The marrow shows absent or diminished erythroid precursors, often with a left shift in erythroid maturation ( Fig. 11-15 ). The anemia may be acute and transient or chronic, depending on the cause ( Box 11-2 ). The congenital form, Diamond-Blackfan syndrome, is described under the macrocytic anemias. The acquired forms of pure red cell aplasia more frequently present with normochromic normocytic anemia. Parvovirus B19 is the most common identifiable cause of red cell aplasia in children and immunocompromised adults. The virus selectively invades and replicates in erythroid progenitor cells, causing direct cytotoxic effects with interruption of erythrocyte production. In children, it is associated with erythema infectiosum (fifth disease), a transient, asymptomatic drop in hemoglobin of about 1 g/dL, with recovery in 10 to 19 days. Children with a hemolytic disorder that shortens the RBC life span, such as red cell enzyme deficiencies, membrane abnormalities, hemoglobinopathies, or malaria infection, often have a more profound anemia and “aplastic crisis” ( Fig. 11-16 ). Parvovirus B19 may persist in immunocompromised individuals who fail to produce neutralizing antibodies to eradicate the virus. Infection manifests as a chronic instead of acute pure red cell aplasia unless patients are treated with intravenous immunoglobulin therapy. Bone marrow findings depend on the timing of the evaluation. Initial RBC depletion may be followed by a wave of early progenitors without maturation. Giant pronormoblasts with intranuclear viral inclusions are transient but may be occasionally identified, particularly in immunocompromised individuals. Viral-associated suppression of myelopoiesis and megakaryopoiesis occurs with rare cases of marrow necrosis. Serum polymerase chain reaction studies for parvovirus B19 DNA, elevated IgM antibody titers, and immunohistochemistry or in situ hybridization for parvovirus on marrow biopsy sections are diagnostic.

The sudden onset of pure red cell aplasia is often associated with a history of a recent respiratory or gastrointestinal viral infection or the use of drugs administered for infectious or inflammatory conditions. Box 11-2 provides a partial list of drugs that may be responsible, with resolution of the aplasia typically occurring with drug cessation. The rare formation of anti-erythropoietin antibodies secondary to erythropoietin treatment, particularly in patients with renal failure, is more of a problem. Red cell aplasia persists despite stopping erythropoietin treatment, and immunosuppressive therapy is required. Transient erythroblastopenia of childhood is a common finding in children undergoing bone marrow examination for anemia. The cause of this acute, transient disorder remains elusive. Most chronic, acquired pure red cell aplasias have an autoimmune basis, with impairment or suppression of erythropoiesis by humoral or cellular immune mechanisms. Classic causes include thymoma, hematologic malignancies, and systemic autoimmune disorders. Despite the clearly established association between red cell aplasia and thymoma, less than 10% of individuals with aplasia are found to have thymomas on radiographic evaluation. Clonal proliferations of T cells or altered Th1/Th2 ratios have been implicated in many cases of chronic pure red cell aplasia. In addition, a significant proportion of idiopathic cases are likely secondary to the frequently underdiagnosed T-cell large granular lymphocyte (LGL) leukemia. Antibody-mediated processes may affect cells directly or indirectly through complement-mediated processes. Alternatively, erythropoietin may be targeted, as previously described. In refractory patients without a clear underlying cause and normal cytogenetic studies, pure red cell aplasia may be the initial presentation for MDS. Patients with MDS may have aberrant expression of antigens on their erythroid precursors, such as CD71, which may be useful in distinguishing MDS from other causes of persistent anemia.

Aplastic Anemia

Aplastic anemia usually presents with pancytopenia and is discussed under bone marrow failure syndromes.

Myelophthisic Anemias

Myelophthisic anemias are caused by replacement of normal marrow cells by tumor, granuloma, histiocytes in storage disease, or fibrosis and usually exhibit bicytopenia or pancytopenia. Although the anemia is typically normochromic and normocytic, red cell fragmentation, spherocytes, and teardrop forms are frequently encountered. Normoblasts and left-shifted granulocyte precursors produce a “leukoerythroblastic” blood picture in most cases associated with metastatic tumor or fibrosis ( Fig. 11-17, A, B, and C ). Bone marrow evaluation is essential to identify the underlying disorder.

Anemia of Chronic Renal Failure

Anemia of chronic renal failure often has a multifactorial cause, including the effect of certain still ill-defined plasma factors. However, a primary cause is erythropoietin underproduction by the damaged kidneys (see Chapter 12 ).

Posthemorrhagic Anemia

Posthemorrhagic anemia due to recent blood loss is normochromic and normocytic and is accompanied by a reticulocytosis that first manifests 3 to 5 days after blood loss. By 7 to 10 days, the reticulocytes may be so numerous that they increase the MCV up to 100 to 110 fL. Shortly after the hemorrhage, the first notable change in the blood is thrombocytosis, followed by demargination of neutrophils from the release of adrenergic hormones. Finally, the hemoglobin falls as extravascular fluids enter the vascular space.

Hemolytic Anemias

Hemolytic anemias are usually normochromic normocytic anemias in which an elevated reticulocyte count reflects compensation for increased RBC destruction. The process may be episodic or persistent. Hemolysis is caused by four basic abnormalities: intrinsic red cell defects, plasma factors, disruption of the cells by mechanical or thermal damage, and infectious agents ( Table 11-4 ; see Fig. 11-1 ). Patients with hemolytic anemia often have similar clinical and laboratory findings: normochromic normocytic anemia, reticulocytosis, shortened red cell life span, elevated erythropoietin level, increased indirect bilirubin, increased lactate dehydrogenase, markedly decreased haptoglobin, and jaundice. Those with extravascular hemolysis also develop splenomegaly and gallstones. Bone marrow evaluation invariably shows erythroid hyperplasia, even in patients with only mild compensated hemolysis. Circulating red cells with characteristic shape changes (i.e., sickle cells or spherocytes) are helpful in the diagnosis, whereas the erythroid precursors in the marrow usually have an unremarkable appearance. Identifying or confirming the cause of a hemolytic anemia relies on the patient's history (including the family history) and on definitive laboratory studies, as summarized in Table 11-4 .

Hereditary Spherocytosis

Hereditary spherocytosis (HS) is a common cause of non-immune hemolytic anemia due to abnormalities in the RBC transmembrane proteins. The defect leads to local discohesion of the membrane skeleton from the lipid bilayer, which creates a microvesicle with subsequent loss of membrane and formation of a spherocyte. Spherocytosis is the hallmark of HS and should be suspected if the red cell indices include a normal or low MCV and the MCHC after warming remains 36 g/dL or greater ( Fig. 11-18 ). The less deformable spherocytes are selectively trapped in the spleen and are vulnerable to further surface membrane loss and destruction. Genetic defects vary among different racial groups, with heterogeneous molecular abnormalities that are often family specific. Gene mutations typically shift the normal reading frames or introduce premature stop codons that result in mutant alleles that fail to produce protein. The specific gene involved (i.e., the molecular phenotype) may not strictly relate to the biochemical phenotype (i.e., the abnormal protein produced). For example, an ankyrin gene defect may manifest as spectrin protein deficiency. It is usually the spectrin content of the red cell that best correlates with the degree of anemia, percentage of circulating spherocytes, reticulocyte count, and increased osmotic fragility.

Clinically, anemia is the presenting complaint in nearly half of patients, although disease severity varies widely among individuals. Mild compensated hemolysis is observed in about 20% of individuals, with the majority of affected people (60%) having moderate hemolysis with a hemoglobin of 8 to 11 g/dL and reticulocyte percentage generally higher than 8%. At birth, HS patients usually have a normal hemoglobin value that may sharply and transiently decrease during the first 20 days of life to a level that requires blood transfusions. The more asymptomatic forms of HS may not be identified until a hemolytic crisis develops during childhood, often triggered by a viral infection. Less commonly, an aplastic crisis develops secondary to parvovirus B19 infection (see Fig. 11-16 ). Although a family history of HS is often elicited in individuals suspected of having HS, the most severe forms of the disease are recessive and associated with α-spectrin and some ankyrin defects. Sporadic mutations are particularly common in the autosomal recessive forms of HS. Several diagnostic methods for HS are available, including osmotic fragility studies, glycerol lysis tests, cryohemolysis test, and osmotic gradient ektacytometry, yet each test may have considerable false positive and false negatives. Flow cytometric analysis of eosin-5′-maleimide (EMA)–labeled red blood cells exhibits the greatest disease specificity for HS at 98; however, a subset of cases of HS may still be unrecognized with EMA analysis alone. Although EMA binds specifically with band 3 protein, membrane protein abnormalities in HS other than band 3 deficiency affect binding and therefore the fluorescent intensity of the dye measured by flow cytometry. Coupled with peripheral blood smear review and depending on availability of testing, the recommended laboratory tests include flow cytometry for EMA binding and cryohemolysis test. Splenectomy has been the primary mode of therapy.

Hereditary Elliptocytosis and Hereditary Pyropoikilocytosis

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) were originally described as distinct entities, but recent molecular studies have established that HPP is a subset of HE (see Tables 11-4 and 11-5 ). They are caused by defects in the horizontal protein interactions that hold the membrane skeleton together. The abnormality that best correlates with disease severity is a failure of spectrin homodimers to self-associate into heterodimers, the basic building blocks of the membrane skeleton. Differences in the clinical severity of HE cannot always be explained by a specific genetic defect. The most prevalent form of the disease is a single gene defect (heterozygous) that causes the red cells to elongate and form elliptocytes in circulation, without anemia or significant splenomegaly. The more severe form of the disease, HPP, is due to a combination of two defective membrane protein genes that result in marked spectrin deficiency in addition to functionally abnormal proteins. The MCV may be very low because of marked RBC fragmentation, rendering the clinical presentation atypical for a hemolytic anemia, with possible microcytic rather than normocytic RBC indices ( Fig. 11-19 ). A disorder related to HE, called Southeast Asian ovalocytosis , is found in people from Malaysia, Indonesia, the Philippines, and Papua New Guinea. Only a subset of affected individuals has hemolytic anemia, with distinctive oval stomatocytes. This variant red cell may protect individuals against cerebral malaria.

Hereditary Stomatocytosis Syndromes.

Hereditary stomatocytosis syndromes are a group of disorders of the RBC membrane characterized by a mouth-shaped central area of pallor and abnormal permeability to sodium and potassium ( Fig. 11-20 ). This rare red cell disorder is subdivided into two entities: xerocytosis or dehydrated hereditary stomatocytosis (DHSt) and overhydrated hereditary stomatocytosis (OHS). Loss of potassium leads to RBC dehydration and a mild to moderate anemia in the more frequent form, DHSt. Automated counts show an increased MCHC and normal MCV (falsely elevated on some automated counters). A misdiagnosis of atypical HSt is often made. Recently, mutations in PIEZO1 protein (encoded by FAM38A gene) have been identified in DHSt. The PIEZO protein may play a critical role in red cell cation and volume homeostasis. The second subtype, OHS, is rare and leads to a severe hemolytic phenotype. It is characterized by a 20- to 40-fold increase in cation leak, leading to hydrated red cells, large increase in MCV, and decreased MCHC. Patients with hereditary stomatocytosis have severe thrombotic complications after splenectomy; thus, avoidance of this procedure is important.

Red Blood Cell Enzyme Defects.

RBC energy requirements are met primarily through the metabolism of glucose by the Embden-Meyerhof glycolytic pathway. Alternatively, approximately 10% of glucose is metabolized by the hexose monophosphate shunt. Erythrocyte disorders due to enzyme deficiencies of the glycolytic pathway are extremely rare, and approximately 90% of these are deficiencies of pyruvate kinase caused primarily by PK-LR gene mutations on chromosome 1q21 (see Table 11-4 ). The majority are inherited as autosomal recessive traits and first detected in infancy or childhood with the clinical presentation of chronic hemolysis. The direct antiglobulin test (Coombs test), hemoglobin electrophoresis, and osmotic fragility are normal. The peripheral blood film shows normochromic normocytic RBCs without spherocytes. The remaining morphologic findings are non-specific but include reticulocytosis and erythroid hyperplasia.

Hereditary disorders of the hexose monophosphate shunt enzymes are also rare, except glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is one of the most prevalent inborn errors of metabolism. More than 400 variants of G6PD and at least 30 mutations (missense point mutations) have been described ( Table 11-6 ). It is particularly prevalent in populations from geographic areas with endemic malaria, suggesting that evolutionary polymorphisms were formed to counteract the effects of this parasite. The G6PD gene is carried on the X chromosome, and full expression of G6PD deficiency is found only in males; female carriers may have partial deficiency. Clinical manifestations of G6PD deficiency include neonatal jaundice and hereditary non-spherocytic hemolytic anemia. The most serious consequence of G6PD deficiency is neonatal jaundice leading to kernicterus, which is worsened by associated Gilbert's disease. Although a few patients have chronic hemolytic anemia, the majority have episodic anemia induced by increased oxidative stress in erythrocytes from certain foods (fava beans), a number of drugs (sulfonamides, nitrofurans, quinine derivatives, aspirin, rasburicase), and chemicals (naphthalene, toluidine blue). Erythrocytes deficient in G6PD are unable to maintain sufficient reduced glutathione for the generation of NADH, a cofactor that maintains hemoglobin integrity. The WHO has classified G6PD variants based on their degree of enzyme deficiency and severity of hemolysis: class I, less than 10% enzyme activity with severe chronic (non-spherocytic) hemolytic anemia, to class V, increased enzyme level with no hemolysis or clinical sequelae. Oxidant damage is reflected by marked anisopoikilocytosis with “bite” cells and increased polychromatophilia on the peripheral blood film ( Fig. 11-21 ). Supravital staining demonstrates denatured hemoglobin precipitates (Heinz bodies) ( Fig. 11-22 ). The bone marrow most commonly demonstrates erythroid hyperplasia.

Table 11-6

Common Glucose-6-Phosphate Dehydrogenase (G6PD) Variants

Isoform	Ethnic Group	Comments
G6PD B	All	Most common, normal variant
G6PD A	Blacks (20%)	Normal variant, no hemolysis
G6PD A−	Blacks (11%)	Group of variants with same mutation as G6PD A, but with one additional mutation Moderate hemolysis Unstable enzyme, ↑ decay
G6PD MED	Greeks, Arabs, Sicilians, Sephardic Jews	Severe hemolysis Protects against Plasmodium falciparum
G6PD CANTON	Asians	Moderate hemolysis

Hereditary pyrimidine 5′-nucleotidase deficiency is the third most common cause of chronic non-spherocytic hemolytic anemia related to red cell enzyme defects, after deficiency of PK and G6PD. The peripheral blood smear is characterized by red cells with prominent coarse basophilic stippling secondary to accumulation of precipitated pyrimidine nucleotides.

Hemoglobinopathies.

Hemoglobinopathies are abnormalities of hemoglobin structure due to abnormal amino acid sequences in either the alpha or beta globin chains. The most prevalent abnormal hemoglobin is HbS, produced by the substitution of glutamate for valine at the sixth position of the beta globin chain. The gene for HbS has autosomal dominant inheritance and is found in areas of the world where malaria is common. Approximately 8% to 10% of the African American population carries at least one HbS gene. Sickle cell disease occurs in individuals with homozygous sickle mutations (termed HbSS or sickle cell anemia ) or compound heterozygous mutations, most commonly sickle cell β-thalassemia or hemoglobin sickle cell (HbSC) disease. RBC sickling is induced under conditions of deoxygenation, vasoconstriction, acidosis, increased HbS concentration, and infection. The clinical symptoms of the sickle cell disorders vary greatly in severity among individuals, but they are often due to the increased tendency of sickle cells to adhere to vascular endothelium and to the ensuing vaso-occlusive complications. Cells become irreversibly sickled and are removed by the reticuloendothelial system. The hallmark of these disorders is morphologically altered red cells ( Fig. 11-23 ). In addition to the sickle cells, irregularly shaped cells, targets, spherocytes, and polychromatophilic cells may be found on the blood film. Howell-Jolly bodies are usually identified in older individuals as a result of autosplenectomy. A left-shifted neutrophilia with toxic features and thrombocytosis are common during an acute crisis. Heterozygous disorders may additionally show microcytosis (Sβ-thalassemia) and intracellular crystals (HbSC) ( Fig. 11-24 ). Patients with sickle cell anemia may also develop acute splenic sequestration, parvovirus-related red cell aplasia, and bone marrow necrosis. In addition to erythroid hyperplasia, bone marrow biopsies frequently show increased arterial fibrosis. Patients with sickle cell disease with a genotype other than HbSS (e.g., Sβ-thalassemia) appear to be at risk for bone marrow necrosis and fat embolism syndrome.

Among the numerous other known hemoglobinopathies, HbC and HbE are the next most common causes of chronic hemolysis. The HbC gene mutation is most prevalent in West Africans; the HbE gene is found primarily in Southeast Asians. Homozygous HbE is unusual in its presentation as a mild to moderate, hypochromic microcytic anemia (MCV 50 to 65 fL). HbC is recognized morphologically by the unique intracellular crystalline structures in erythrocytes on the blood film ( Fig. 11-25 ).

Immune-Mediated Hemolytic Anemia

Autoimmune Hemolytic Anemias.

Autoimmune hemolytic anemias (AIHAs) are categorized by the temperature at which the autoantibody has the greatest avidity for the target red cell antigen, and they are detected by a positive direct antiglobulin test. Warm AIHA is most common (70% of AIHAs) and is clinically significant because it occurs at body temperature. IgG antibody– or occasionally IgA antibody–coated RBCs act to bind Fc receptors on splenic macrophages and, with or without subsequent complement fixation, are removed from circulation. Partial phagocytosis of the RBC membrane produces spherocytes ( Fig. 11-26 ). Cold AIHA is due to IgM coating of red cells at low temperatures, leading to RBC agglutination and complement fixation. The antibody is most often directed at the I antigen on the red cell membrane. Some hemolysis occurs secondary to intravascular destruction of the agglutinated cells. However, if the antibody is active at temperatures approaching 37° C, complement becomes activated, and clinically significant intravascular and sometimes extravascular complement-mediated hemolysis occurs in the liver (80% of time). Smears typically show agglutinated cells unless the blood tube was previously warmed; spherocytes are less frequent ( Fig. 11-27 ). Autoantibody formation in both warm- and cold-type AIHA most likely represents a derangement of normal immune function. Approximately 50% of AIHA is idiopathic (primary) and observed in older patients. In contrast, secondary AIHA develops in patients with underlying disease, predominantly lymphoproliferative disorders but also autoimmune disorders, infections, and carcinoma. Young patients, in particular, develop a self-limited cold-type AIHA after Mycoplasma pneumoniae infection or infectious mononucleosis.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here