216 part iii • the anemias
a b
■ Figure 12-10 (a) Blood film from a patient with anemia of chronic disease. Hb 96 g/L; MCV
76 fL; MCH 24 pg; MCHC 30.6 g/dL. The erythrocytes appear microcytic (peripheral blood;
Wright-Giemsa stain; 1000* magnification). (b) Bone marrow from same patient in (a) stained
with Prussian blue. Note the macrophages with abundant blue staining iron (bone marrow;
Prussian blue stain; 1000* magnification).
Bone Marrow of its pathophysiologic relationship to these anemias through a block
The bone marrow usually shows an increased M:E ratio because of a in heme synthesis.
decrease in erythrocyte precursors. The proportion of younger eryth-
roblasts increases. Poor hemoglobin production is apparent, especially Sideroblastic Anemias
in the polychromatophilic erythroblasts. The proportion of sidero- Mutations that affect the first enzymatic step in heme synthesis, the
blasts decreases to <30%; however, the macrophages appear to have formation of ALA, result in sideroblastic anemia (Chapter 6). Muta-
increased amounts of hemosiderin in the form of coarse iron aggre- tions in the subsequent steps of heme synthesis result in metabolic
gates (Figure 12-10b). This finding helps to distinguish ACD from disorders called porphyrias. Sideroblastic anemia (SA) is the result of
IDA. In IDA, macrophage iron and sideroblasts are absent. diverse clinical and biochemical manifestations that reflect multiple
Bone marrow examination is usually not necessary for distin- underlying hereditary, congenital, or acquired pathogenic mecha-
guishing ACD from IDA. An algorithm using the MCV, serum ferritin, nisms. However, all types are characterized by (1) an increase in total
and iron saturation can correctly differentiate and classify most cases body iron, (2) the presence of ring sideroblasts in the bone marrow,
of ACD and IDA. 50 and (3) hypochromic anemia.
therapy Classification
Anemia can be alleviated by successful treatment of the underlying The classification of SA is arbitrary at best, and many different
disease. Anemia is usually mild and nonprogressive; thus, transfusion schemes of classification exist. The one in Table 12-11 ★ is among
is rarely warranted except in older patients with vascular disease and the most descriptive and separates those that are inherited and those
circulatory insufficiency. that are acquired.
The most common form of hereditary SA is due to a defec-
tive X-linked recessive gene. Although carrier females often show
aneMiaS aSSOCiateD with a dimorphic population of both morphologically abnormal and
aBnOrMal heMe SyntheSiS
These anemias are associated with defects in enzymes of the heme ★ taBle 12-11 Classification of Sideroblastic Anemia
biosynthetic pathway leading to abnormal heme synthesis. Iron incor-
poration into the protoporphyrin ring to form heme can be blocked. hereditary acquired
In contrast to IDA, the positive iron balance in these anemias can • Sex linked • Idiopathic refractory sideroblastic
lead to an increase in iron stores predominantly in the spleen, liver, • Autosomal recessive anemia (IRSA) or refractory anemia
and bone marrow. Serum ferritin levels > 250 mcg/L in the male and with ring sideroblasts (RARS)
>200 mcg/L in the female indicate increased iron stores. • Secondary to drugs, toxins, lead
The conditions discussed in this section include sideroblastic • Associated with malignancy
anemia and the porphyrias. Lead poisoning also is included because
M12_MCKE6011_03_SE_C12.indd 216 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 217
normal erythrocytes, they rarely have anemia. Affected males dem- Significant ineffective erythropoiesis is characterized by bone
onstrate the typical SA findings. In rare instances, both sexes are marrow erythroid hyperplasia. This increased erythropoietic activity
equally affected, implying the presence of another hereditary form results in increased iron absorption in the gut. Iron overload can be
that is transmitted in an autosomal recessive manner. In the heredi- significant and can lead to complications such as cardiac failure and
tary forms, anemia can become apparent in infancy but most com- diabetes.
monly appears in young adulthood. Occasionally, symptoms do not Other hereditary forms of SA have been described but are less
occur until age 60. common than the sex-linked type. 65
The acquired forms of sideroblastic anemia are more common
than the hereditary forms. The acquired forms are classified accord- acquired Sideroblastic anemia
Acquired SA can be categorized as refractory anemia with ring sid-
ing to whether the basis of the anemia is unknown (idiopathic) or is eroblasts (RARS) and those secondary to drugs or toxins. RARS is an
secondary to an underlying disease or toxin (secondary type). The acquired stem cell disorder and is discussed in the chapter on myelo-
idiopathic form, refractory anemia with ring sideroblasts (RARS), dysplastic syndromes (Chapter 25).
can affect either sex in adult life. It is included in a group of acquired SA secondary to drugs or toxins is the result of the interference
stem cell disorders called myelodysplastic syndromes (Chapter 25) of the drugs or toxins with the activity of heme enzymes. Lead and
that have a tendency to terminate in acute leukemia. The acquired alcohol are the most common causes of this form of SA.
secondary type SAs are associated with malignancy, drugs, or other
toxic substances. In this type, once the underlying disorder is effec- Lead Poisoning. Lead poisoning (plumbism) has been recognized
tively treated or the toxin removed, the anemia abates. for centuries. In children, it generally results from ingestion of flaked
lead-based paint. Lead was removed from paint sold after 1978. Chil-
Pathophysiology dren from lower socioeconomic backgrounds are at increased risk;
Studies of patients with SA have shown disturbances of the enzymes those between 1 and 3 years of age are at greatest risk. Clinically, lead
regulating heme synthesis. Ring sideroblasts are specific findings for toxicity in children is associated with hyperactivity, low IQ, concentra-
these heme enzyme abnormalities. Ring sideroblasts are formed from tion disorders, hearing loss, and impaired growth and development.
an accumulation of nonferritin iron in the mitochondria that encircle In adults, lead poisoning is primarily the result of inhalation of lead
the erythroblast nucleus. The mitochondria eventually rupture as they or lead compounds from industrial processes. It is the most common
66
become iron laden. When stained with Prussian blue, the iron appears disease of toxic environmental origin in the United States.
as blue punctate deposits circling the nucleus (ring sideroblasts). Iron Many states require laboratories and physicians to report elevated
within the erythroblasts is normally deposited diffusely throughout blood lead levels to the state health department, which can report to
the cytoplasm in siderosomes. the Centers for Disease Control and Prevention (CDC). Although
average blood lead levels have dropped by 80% since the late 1970s,
hereditary Sideroblastic anemia about 7,000 adults had blood lead levels of >25 mcg/dL in 2009
67
The most common form of hereditary SA is sex linked and due to an (40 states reporting). In 2010, hundreds of thousands of children
abnormal d@aminolevulinic acid synthase enzyme (ALAS). under age 6 were reported to have blood lead levels >5 mcg/dL, the
There are two different forms of ALAS, the nonerythroid or level at which the CDC recommends public health actions be initiated
68
hepatic form (ALAS1) and the erythroid form (ALAS2). Nonery- (46 states reporting). (In 2012, the CDC set a new acceptable blood
throid ALAS1 is coded for by a gene (chromosomal region 3p21) lead level at for children [see Web Table 12-3]).
expressed in all tissues. The erythroid form, ALAS2, is coded for by a Lead serves no physiological purpose. Although lead poisoning
gene on the X chromosome (Xp21-q21). ALAS2 is the first and rate- consistently shortens the erythrocyte life span, the anemia accom-
limiting enzyme in heme synthesis. panying plumbism is not primarily the result of hemolysis but of a
Sex-linked SA (XLSA) is due to an abnormal ALAS2 gene that is marked abnormality in heme synthesis. Once ingested, lead passes
the result of heterogeneous mutations in the catalytic domain of the through the blood to the bone marrow where it accumulates in the
62
63
protein. More than 22 different mutations have been described. mitochondria of erythroblasts and inhibits cellular enzymes involved
The mutations are located in exons 5–11 with most being single-base in heme synthesis. The heme enzymes most sensitive to lead inhibition
mutations affecting the site at which the enzyme binds the cofactor are d@aminolevulinic acid dehydrase (d@ALA@D) and ferrocheletase
pyridoxal 5′@phosphate. (heme synthase) (Chapter 6). Other enzymes can be affected at higher
Evidence indicates that the activity of ALAS2 depends on the lead concentrations. Thus, the synthesis of heme is disturbed at the
presence of pyridoxal phosphate. This cofactor binds to the enzyme conversion of d@ALA to porphobilinogen (catalyzed by d@ALA@D);
and is crucial for its stability, maintenance of a conformation optimal urine excretion of d@ALA increases as a result. Incorporation of iron
for substrate binding and product release, and its catalytic activity. In into protoporphyrin to form heme (which uses the enzyme ferrochela-
17 of the ALAS2 mutations, a partial to complete clinical response to tase) is also disrupted. The effect of lead on ferrochelatase is competi-
pharmacologic doses of pyridoxine occurs. Excess pyridoxine possibly tive inhibition with iron; iron accumulates in the cell, and EP (in the
enhances the abnormal ALAS enzyme activity by stabilizing it after form of ZPP) is strikingly increased.
62
synthesis. In the pyridoxine-refractory sex-linked SA, the mutation Studies have revealed that microcytic, hypochromic anemia is
appears to affect the processing of ALAS2 precursor mRNA (which not characteristic of elevated lead levels in most children. Evidence
terminates ALAS2 translation prematurely) or abolishes its enzymatic suggests that the presence of a microcytic anemia in plumbism
function. 63,64 is most likely due to complications of ID or to the coexistence of
M12_MCKE6011_03_SE_C12.indd 217 01/08/14 9:14 pm
218 part iii • the anemias
α-thalassemia trait. 69–71 One study found that 33% of African Ameri- interfering with absorption, storage, and release. Serum iron levels
can children with lead poisoning and microcytosis had α-thalassemia are increased during drinking episodes but return to normal after
trait. 69 cessation.
Coexistent ID and lead poisoning put children at a higher risk for Associated with Malignancy. Ring sideroblasts can be found in dis-
developing even more serious complications because children absorb eases other than sideroblastic anemia, including hematologic malig-
larger portions of lead in iron-deficient states and the competitive nancies (e.g., leukemia, malignant histiocytosis, multiple myeloma,
inhibition of ferrochelatase by lead is even greater in the absence of lymphoma) (Web Table 12-5 ). Some investigators believe that the
iron. Thus, it is critical to make a diagnosis of ID when it coexists with presence of ring sideroblasts in these disorders suggests that the malig-
lead poisoning. nancy can result from an abnormal clone of pluripotential stem cells
Although the ZPP measurement has been utilized to screen that affects the erythroid as well as other cell lineages. Occasionally
for lead in the past, it is no longer recommended as an appropri- ring sideroblasts appear in the bone marrow following treatment of
ate screening tool because ZPP does not rise until lead levels reach malignant disease (e.g., multiple myeloma, Hodgkin’s disease). The
72
approximately 20 mcg/dL. Lead levels can impair neuropsychologic appearance of ring sideroblasts after treatment is considered a poor
development. Screening generally should be done by direct lead mea- prognostic sign because these cases almost always terminate in acute
surements. ZPP also cannot be used to differentiate IDA and thal- leukemia.
assemia in the presence of coexistent lead poisoning because of the
increase in erythrocyte protoporphyrin caused by lead.
Clinical Features
Alcoholism. Anemia is a common finding (up to 62%) in hospital- In patients with acquired sideroblastic anemias secondary to drugs
31
ized chronic alcoholics. Megaloblastic and sideroblastic anemia or malignancy, the manifestations of the underlying disorder domi-
occur in the majority, but anemia in this population has many other nate. Patients with hereditary sideroblastic anemia or RARS, however,
73
causes including ACD, IDA, acute blood loss, and chronic hemolysis generally show primary signs and symptoms of anemia. In hereditary
(Table 12-12 ★). Less than one-half have an isolated cause for the sideroblastic anemias, most patients also exhibit signs associated with
anemia (Web Table 12-4). Studies show that sideroblastic anemia is iron overload including hepatomegaly, splenomegaly, and diabetes. In
particularly common among alcoholics with a poor diet and can be the latter stages of the disease, cardiac function can be affected.
associated with a concomitant decrease in folic acid and megaloblasto-
sis (Chapter 15). The presence of a dimorphic erythrocyte population laboratory Features
and siderocytes in the peripheral blood are clues to a diagnosis of SA. Refer to Table 12-13 ★ for laboratory findings in sideroblastic anemia.
Alcohol is believed to interfere with some enzymes of hemoglobin Peripheral Blood
synthesis including inhibition of the synthesis of pyridoxal phosphate The anemia is usually moderate to severe. A dimorphic picture of
and of activity of uroporphyrinogen decarboxylase and ferrochelatase normochromic and hypochromic cells is characteristically seen in
but enhancement of activity of d@ALAS. Alcoholics with a poor diet inherited and acquired secondary forms of SA (Figure 12-11 ■).
can also have an inadequate intake of pyridoxine. Alcohol is directly Dual populations of macrocytes and microcytes or normocytes can
toxic to hematologic cells as evidenced by the frequent presence of be found. Hypochromic macrocytes are especially prevalent in RARS,
vacuoles in bone marrow precursor cells, thrombocytopenia, and whereas hypochromic microcytes are more common in the heredi-
granulocytopenia. tary form of SA. Macrocytes are also common in SA associated with
Interpreting laboratory tests in alcoholism is difficult because alcoholism.
the alcohol has effects on multiple parameters. Even in the absence If a dimorphic erythrocyte population is present, the MCV,
of megaloblastosis and anemia, the cells are frequently macrocytic MCH, and MCHC can be normal because these parameters represent
(MCV 100–110 fL). Alcohol has a direct effect on folate metabolism,
an average of all erythrocytes, thus emphasizing the need for careful
examination of CBC parameters and the blood smear. The RDW and
erythrocyte histogram/cytogram are useful in detecting these dual
★ taBle 12-12 Mechanisms of Anemia in Chronic populations. The RDW is increased, and the RBC histogram/cyto-
Alcoholism
gram shows two peaks representing the dual population.
• Megaloblastosis associated with folate deficiency
• Iron-deficiency anemia from chronic or acute blood loss
• Sideroblastic anemia from toxic effects of alcohol on enzymes ★ taBle 12-13 Laboratory Findings in Sideroblastic
needed for heme synthesis Anemia
• Hemolytic anemia
Chronic hemolysis due to splenic sequestration • Dual population of hypochromic and normochromic erythrocytes
Spur cell anemia occurring in the setting of severe liver dis- • Pappenheimer bodies in erythrocytes
ease, splenomegaly, and jaundice • normal or increased platelets
Transient hemolytic anemia associated with portal hypertension • Increased serum iron, serum ferritin, and percentage of saturation
and acute congestive splenomegaly • Ring sideroblasts in the bone marrow
M12_MCKE6011_03_SE_C12.indd 218 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 219
MCV, peripheral blood smear evaluation,
serum ferritin
Hypersegmented Serum ferritin
PMNs <20 ng/mL
or >3% Normal
macroovalocytes or increased IDA TIBC
or MCV > 110fL sTfR increased
Serum iron sTfR
TIBC increased
and
Serum iron serum iron
normal/
TIBC increased decreased
normal/low
■ Figure 12-11 Blood film from a patient with and sTfR
sideroblastic anemia. Two populations of erythrocytes normal
are present: hypochromic and normochromic. Anisocytosis Probable folic acid Further
is present with microcytes, macrocytes, and normocytes. deficiency evaluation
This is the dimorphic blood picture typical of sideroblastic
anemia. Note also the numerous inclusions (Pappenheimer ■ Figure 12-13 An algorithm for diagnosis of anemia in
bodies) (peripheral blood; Wright-Giemsa stain; 1000* alcoholics.
magnification).
Other laboratory test results can be abnormal. Even though the
bone marrow is usually hyperplastic, the reticulocyte production index
Other abnormalities of erythrocytes are often seen. Poikilocyto- is 6 2, indicating that the anemia has an ineffective erythropoietic
sis and target cells can be present. Erythrocytes can contain Pappen- component. Other indications of ineffective erythropoiesis include a
heimer bodies (iron deposits) (Chapter 37). When Pappenheimer slightly increased serum bilirubin, (usually 6 2.0 mg/dL), decreased
bodies are present, reticulocyte counts must be performed carefully haptoglobin, and increased lactate dehydrogenase (LD). Iron studies
because both RNA and Pappenheimer bodies take up supravital show increased serum iron, normal or decreased TIBC with increased
stains. However, Pappenheimer bodies stain with both Romanowsky saturation levels (sometimes reaching 100%), and increased serum
and Prussian blue stains, whereas reticulated RNA does not stain with ferritin. Leukocyte and platelet counts are usually normal but can be
either. Nucleated erythrocytes are rarely present. decreased. Thrombocytosis is found in about one-third of patients.
Basophilic stippling can be seen in any of the SAs. However, In alcoholics, the direct and indirect effects of alcohol complicate
coarse punctate basophilic stippling, resulting from aggregated ribo- the interpretation of laboratory tests used to diagnose anemia, includ-
somes and degenerating mitochrondria, is a particularly characteristic ing MCV, serum iron, TIBC, serum ferritin, and red cell and serum
feature of lead poisoning (Figure 12-12 ■). The punctate stippling folate (Figure 12-13 ■).
occurs in the reticulocyte and is found in developing erythroblasts. It
has been shown that the granules in these stippled erythroblasts con-
tain free ionized iron and the hemoglobin production in the cells is CaSe StuDy (continued from page 215)
grossly deficient. Many stippled cells in the peripheral blood in lead
poisoning are actually siderocytes. 8. Do the iron studies in Jose (serum iron 18 mcg/dL,
TIBC 425 mcg/dL) suggest SA?
Bone Marrow
Bone marrow changes include erythroid hyperplasia often accom-
panied by various degrees of megaloblastosis. The megaloblastosis is
sometimes responsive to folate, which indicates the presence of a com-
plicating folate deficiency. Erythroblasts appear poorly hemoglobinized
with scanty, irregular cytoplasm. Macrophages contain increased
amounts of storage iron. Ring sideroblasts constitute more than 40% of
the erythroblasts (Figure 12-14 ■). Ring sideroblasts are erythroblasts
with iron granules that encircle one-third or more of the nucleus. The
siderotic granules are larger than those found in normal sideroblasts. In
■ Figure 12-12 Peripheral blood smear from a patient hereditary SA, the abnormal granules occur primarily in the later stages
with lead poisoning. Note the heavy basophilic stippling of erythroblast development (e.g., polychromatophilic and orthochro-
in the erythrocyte (peripheral blood; Wright-Giemsa stain; matic erythroblast stages). In the idiopathic and secondary forms, the
1000* magnification). abnormal granules occur beginning with the earlier erythroblast stages.
M12_MCKE6011_03_SE_C12.indd 219 01/08/14 9:14 pm
220 part iii • the anemias
Ring sideroblasts must be present for an SA diagnosis; however, Secondary SA resulting from a disease, toxin, or drug may be cor-
it is important to recognize that other disease entities can have ring rected by successful treatment of the disease or by elimination of the
sideroblasts present without being SA. toxin/drug.
Molecular Studies
If hereditary sex-linked SA is suspected based on erythrocyte
morphology and iron studies, the patient should be tested for the heMOChrOMatOSiS
presence of ALAS2 gene mutations. This can be done by study- Hemochromatosis describes the clinical disorder that results in
63
ing genomic DNA or cDNA (from RNA) from reticulocytes parenchymal tissue damage from progressive iron overload. Hemo-
(Chapter 42). If a mutation is found, other family members should chromatosis typically is not associated with anemia but is included in
be tested because even if anemia is not present, there is a risk of this chapter because iron studies are markedly abnormal in patients
iron overload. Molecular studies also allow a distinction between with this disorder. Hemochromatosis should be considered when
hereditary SA and RARS. symptoms are present, when there is a family history of the disorder,
or when iron studies suggest iron overload. Iron overload is said to
exist when serum ferritin is 7200 mcg/L in premenopausal women
CaSe StuDy (continued from page 219) or 7300 mcg/L in men and postmenopausal women. A persistent
transferrin saturation of Ú45% is often recommended as the value
75
9. Do Jose’s laboratory test results and clinical history that indicates further investigation is necessary. In hemochromato-
indicate that a bone marrow examination is necessary? sis, excess iron deposits are stored not only in macrophages but also
in hepatocytes, cardiac cells, endocrine cells, and other parenchymal
tissue cells and interfere with the normal function of these cells. This
situation has potentially fatal consequences, especially in relation to
therapy cardiac and hepatic dysfunction.
Pyridoxine therapy is generally tried on patients with hereditary SA; Hemochromatosis is classified as hereditary or secondary. The
650% experience a return to normal hemoglobin levels with therapy, hereditary form is caused by genetic mutations. The secondary type
although some have a partial response. In some patients, iron over- is associated with other hematologic diseases and a variety of other
load decreases responsiveness to pyridoxine, but when the iron load is conditions (Table 12-14 ★).
74
reduced by phlebotomy, hemoglobin concentration increases. Folic
acid is administered to those with megaloblastic features. Although
the anemia can be treated, iron overload is the primary complica- ★ taBle 12-14 Causes of Iron Overload
tion of SA. Sometimes the risk of hemochromatosis is lessened by the hereditary
removal of excess body iron through phlebotomy or chelation ther-
apy. Iron overload is monitored by iron studies (percent saturation of • Classical hemochromatosis: HFE–associated mutations (type I)
transferrin, serum ferritin). Female carriers should also be monitored • Juvenile hemochromatosis (type 2)
for iron overload. Some patients live for many years tolerating their • Hemojuvelin (HJV) mutations (type 2A)
anemia well; others die because of complications of iron overload, • Hepcidin (HAMP) mutations (type 2B)
infections, or bone marrow failure. • Transferrin-receptor 2 deficiency (type 3)
• Ferroportin deficiency (type 4)
• DMT1 mutations
• Congenital atransferrinemia
• Aceruloplasminemia
Secondary
• Anemias with ineffective erythropoiesis (e.g., sickle cell anemia,
thalassemia, refractory anemias with erythroid hypercellular bone
marrow, X-linked sideroblastic anemia)
• Chronic transfusions
• Chronic liver disease
• Viral hepatitis
• Dietary iron overload
• Overload from injections or ingestion of iron supplements
• Alcoholism
• Insulin-resistance-associated hepatic iron overload
• African dietary iron overload
• Porphyria cutanea tarda
■ Figure 12-14 Ring sideroblasts (bone marrow; • Medicinal iron ingestion
Prussian blue stain; 1000* magnification).
M12_MCKE6011_03_SE_C12.indd 220 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 221
Hereditary Hemochromatosis mutation is cys282 S tyrosine: C282Y. Less common are the muta-
tions his63 S asp: H63D; and ser65 S cys: S65C. Clinical signs of
etiology and Pathophysiology
Hereditary hemochromatosis (HH) is characterized by increased iron the disease can be found in homozygous C282Y and compound het-
erozygosity for C282Y and H63D. The mechanism of the way muta-
absorption in the gut and progressive iron overload. It is a genetic dis- tions in the HFE protein affect iron metabolism is unclear but can
order with a prevalence of 1 in 200–250 persons. One in ten Cauca- involve interactions of HFE with other proteins in addition to TfR
sians in the United States is a carrier. It is the most common genetic (see earlier discussion of “Systemic Iron Balance, HFE”). Finding
abnormality in those with a European ancestry. Most of the heredi- decreased hepatic expression of hepcidin in HFE-related HH sug-
tary forms of iron overload involve a deficiency of hepcidin either as gests that the mutation affects upstream regulation of hepcidin. About
a result of a mutation involving the hepcidin gene (HAMP) or muta- 3–4 mg of iron a day is absorbed from the GI tract as compared with
tions of genes whose products regulate hepcidin expression (HFE, the usual 1–2 mg, resulting in an accumulation of about 0.5–1.0 gm/
TfR2, HJV, DMT1, transferrin). The result is an increase in absorption year. The capacity of cells to store iron by complexing with apoferritin
of iron in the intestine and/or uncontrolled release of iron from mac- is exceeded, and “free” intracellular iron accumulates. This free iron
rophages and duodenal enterocytes into the plasma iron pool. Iron facilitates the buildup of reactive oxygen species that cause cell injury
in excess of that needed for erythropoiesis and myoglobin produc- and cell death and leads to organ failure. Most of the excess iron is
tion is deposited in hepatocytes and parenchymal tissue cells, result- deposited in the liver. Mutations of the TfR2 gene result in a disease
ing in an increase in total body iron stores. Most mutations in genes that is clinically indistinguishable from HFE-associated HH.
that affect the regulation of iron metabolism are recessive disorders Other less common forms of HH are due to mutations in other
(Table 12-15 ★). genes involved in iron sensing and regulation, but all appear to result
The most common form of HH in populations of European ori-
76
gin is due to mutations in the HFE gene, identified in 1996. Muta- in deregulation of hepcidin expression.
Mutations in the ferroportin gene can decrease the amount of
tions result in an autosomal recessive disorder of low penetrance with functional ferroportin on the cell surface, blocking iron export and
adult onset. Clinical disease is more common in males than females. resulting in retention and accumulation of iron in macrophages. This
The HFE gene is mapped to chromosome 6p21,3. The most common
★ taBle 12-15 Summary of Acquired and Hereditary Iron Overload Disorders
Disorder Defect/Mutation Fe absorption Fe Deposition Site(s) Plasma iron Ferritin
acquired
Iron-loading anemias Erythropoietic drive Increased Macrophage Increased Increased
(ineffective erythropoi- and possibly GDF-15
esis; e.g., thalassemia, suppress hepcidin
XLSA) synthesis
Anemia of chronic dis- IL-6 increases hepcidin, Decreased Macrophage Decreased normal to increased
ease (ACD) which binds ferropor-
tin and traps iron in
macrophage
Transfusion induced Gain of 1 mg iron/mL normal Parenchymal tissue Increased Increased
erythrocytes transfused
hereditary
Hereditary hemochro- HFE gene, 6p21; TfR2 Increased Parenchymal tissue Increased Increased
matosis (autosomal gene, 7q22
recessive)
Juvenile hemochro- HJV gene 1q21; HAMP Increased Parenchymal tissue Increased Increased
matosis (autosomal (hepcidin) gene,
recessive) 19q13; HFE and TfR2
(TfR2) genes
Hereditary
hemochromatosis
(autosomal dominant)
(a) Ferroportin associ- SLC40A1 (Ferroportin) normal or low but Predominantly deposi- normal but increases Increased
ated with impaired iron gene, 2q32 defective export of iron tion in macrophages with progression of
export from macrophages disease
(b) Ferroportin associ- SLC40A1 gene, 2q32 normal but increased Parenchymal tissue: Increased Increased
ated with hepcidin export of iron from heart, liver, pancreas,
resistance duodenal enterocytes other organs
M12_MCKE6011_03_SE_C12.indd 221 01/08/14 9:14 pm
222 part iii • the anemias
type of mutation produces only minor clinical manifestations. Alter- in chronic liver disease. Serum iron studies are abnormal in 40–50% of
natively, mutations can cause resistance of ferroportin to internaliza- patients with chronic viral hepatitis, alcoholic liver disease, and non-
tion and degradation induced by hepcidin. The result is loss of control alcoholic steatohepatitis. Alcohol disrupts normal iron metabolism
of iron export from enterocytes and macrophages, which leads to iron and results in excess iron deposition in the liver in about one-third
overload in parenchymal cells of the liver and other organs. Ferropor- of alcoholics. 81
tin mutations are inherited as dominant disorders. Aceruloplasmin-
emia affects expression of ferroportin on the cell surface leading to Treatment
ferroportin degradation. Phlebotomy is usually the treatment for HH. Each unit of blood
removes about 250 mg of iron from the body. It is suggested that fol-
Clinical Findings lowing initial venesection therapy to deplete iron stores, iron status be
Clinical penetrance of the most common form of HH, HFE-mutation, monitored annually by serum ferritin levels. 82
is incomplete with only 1 in 5,000 affected individuals having clini- Treatment of secondary hemochromatosis sometimes includes
cal symptoms. About 81% of symptomatic patients have the C282Y/ iron chelation (e.g., deferoxamine) to bind iron and enable urinary
C282Y genotype. Asymptomatic patients who have the C282Y/C282Y excretion. Only a very small amount of iron is available for chelation
genotype or the H63D/C282Y genotype should have their serum iron (from the labile pool) at any given time; thus, continuous infusion of
and ferritin levels monitored because they are at risk for developing the chelator over a long period of time is required to remove this iron.
iron storage disease. Clinical findings include chronic fatigue, arthral- Chelation therapy is the primary form of treatment for transfusion-
77
gia, infertility, impotence, cardiac disease, diabetes, and/or cirrhosis. induced hemochromatosis.
Hyperpigmentation of the skin is also found.
laboratory Findings CheCkpoint 12-5
Screening tests for hemochromatosis usually include percent satura- What is the risk of population genetic screening for HH?
tion of transferrin and serum ferritin. The criterion for a diagnosis of What is the benefit of population genetic screening for
hemochromatosis is usually Ú45% transferrin saturation. Saturation HH?
often approaches 100%. Adding serum ferritin analysis increases spec-
ificity for HH; iron overload is suggested when the serum ferritin is
elevated. Although an increase in transferrin saturation is sensitive for
hemochromatosis, DNA testing to identify the mutated gene is neces- POrPhyriaS
sary for a definitive diagnosis. Biopsy of the liver and/or bone marrow Heme is an iron-chelated porphyrin ring. Its biosynthesis occurs in
usually is not needed. Liver enzymes are often elevated. Anemia is not most cells, but the major sites of synthesis are erythroid cells of the
characteristic except in aceruloplasminemia (when a normocytic, bone marrow and hepatocytes. Thus, the activity of the enzymes cata-
normochromic anemia is present) and in DMT1 mutations (when a lyzing porphyrin metabolism is highest in these cells.
severe microcytic, hypochromic anemia is present). 78 The porphyrias represent a group of inherited disorders char-
Screening for HH is controversial. Some recommend popula- acterized by a block in porphyrin synthesis. Abnormal porphyrin
tion screening with transferrin saturation testing and suggest that metabolism is due to a defect in one or more of the enzymes in the
laboratories include transferrin saturation in their routine lab pan- heme synthesis pathway. As a result of these enzyme deficiencies, the
79
els. Genetic screening is still costly for population screening, and porphyrin precursors behind the enzyme defect accumulate in tissues,
unresolved ethical issues including patient privacy, counseling, and and large amounts are excreted in the urine and/or feces. These excess
insurance concerns exist. With the recognition of the low penetrance porphyrin deposits cause most of the symptoms and clinical findings
of clinical disease (61%), interest in large-scale screening has largely associated with porphyria. The most common findings include photo-
disappeared. It is recommended, however, that both symptomatic and sensitivity, abdominal pain, and neuropathy (motor dysfunction, sen-
asymptomatic first-degree relatives of those with HH be tested with sory loss, mental disturbances). However, usually adequate production
genetic screening because transferrin saturation will not detect car- of heme for hemoglobin synthesis occurs.
80
riers. The goal of screening and identifying the disease early is to Although rare, the porphyrias have received wide recognition
allow treatment with phlebotomy before the onset of clinical disease. and stimulated interest because the disease affected the royal fami-
lies of England and Scotland, especially those descended from Mary,
Secondary Hemochromatosis Queen of Scots. Historians have described George III as suffering
Secondary hemochromatosis is associated with a number of condi- from a mental and physical disorder thought to have been porphyria.
tions including anemias that have an ineffective erythropoiesis com- Princess Charlotte, the granddaughter of George III and heir to the
ponent and increased iron absorption. These include b-thalassemia, throne, died in childbirth; her death was attributed to an acute por-
congenital dyserythropoietic anemia, and X-linked sideroblastic ane- phyria attack at the time of delivery.
mia. An increase in GDF-15 associated with ineffective erythropoiesis Two forms of porphyria (erythropoietic and hepatic) are
could be responsible for suppressing hepcidin synthesis, leading to an described, depending on the primary site (bone marrow or liver) of
increase in iron absorption in these conditions. Iron overload often defective porphyrin metabolism (Table 12-16 ★). Only the erythro-
develops in patients who have transfusion-dependent anemias such poietic porphyrias affect the erythrocytes. Therefore, these porphyrias
as sickle cell disease and thalassemia. Iron overload can also be found are discussed here.
M12_MCKE6011_03_SE_C12.indd 222 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 223
★ taBle 12-16 Classification and Characteristics of the Porphyrias
Metabolites in excess
Mode of
Porphyria inheritance enzymatic Defect urine Feces erythroid Cells tissue Source
erythropoietic
Congenital Autosomal Uroporphyrinogen III Uroporphyrin I, Coproporphyrin I, Uroporphyrin I, Erythropoietic
erythropoietic recessive synthase Coproporphyrin I Uroporphyrin I Coproporphyrin I
porphyria (CEP)
Erythropoietic Autosomal Ferrocheletase normal Protoporphyrin Protoporphyrin Erythropoietic
protoporphyria (EPP) dominant (heme synthase) and occasionally
hepatic
hepatic and
erythropoietic
Hepatoerythropoietic Recessive Uroporphyrinogen Uroporphyrin Uroporphyrin ZPP Hepatic and
porphyria (HEP) decarboxylase erythropoietic
hepatic
Acute intermittent Autosomal Porphobilinogen @-ALA, Porphobilinogen Sometimes normal Hepatic
porphyria (AIP) dominant deaminase Coproporphyrin
and Protoporphyrin
Hereditary Autosomal Coproporphyrinogen Coproporphyrin III, Coproporphyrin III normal Hepatic
coproporphyria (HCP) dominant oxidase Uroporphyrin (in attack)
Variegate porphyria Autosomal Protoporphyrinogen Porphobilinogen, Protoporphyrin normal Hepatic
(PV) dominant oxidase @-ALA, Uroporphyrin, Coproporphyrin
Coproporphyrin
Porphyria cutanea Autosomal Uroporphyrinogen Uroporphyrin I, Protoporphyrin normal Hepatic
tarda (PCT) dominant decarboxylase Uroporphyrin III, Coproporphyrin
Coproporphyrin (slight)
ALA dehydratase Autosomal ALA dehydratase @-ALA, Coproporphyrin III — normal Hepatic
deficiency recessive
porphyria (ADP)
Pathophysiology coproporphyrin I (CoproI). A defect in uroporphyrinogen III syn-
The erythropoietic porphyrias result from an abnormality of the thase results from point mutations in the gene, shifting the porphobi-
linogen into the functionless UroI isomer (Figure 12-15 ■). However,
enzymes in the heme biosynthetic pathway within the erythroblasts enough UroIII is produced to generate adequate amounts of heme.
of the bone marrow. At least two types exist: congenital erythropoietic This suggests that a deficiency of uroporphyrinogen III synthetase
porphyria (CEP) and erythropoietic protoporphyria (EPP). They are is not the only abnormality. Another possibility for the excessive
classified according to the particular enzyme defect and the excessive amounts of UroI isomer is hyperactivity of uroporphyrinogen I syn-
porphyrin intermediates produced. Although CEP is associated with thase. Validation of either of these hypotheses requires purification,
hemolytic anemia, anemia in EPP is rare. characterization, and accurate measurement of these enzymes.
Porphyrins are functionless products produced by the irrevers-
The excess porphyrins are deposited in body tissues and excreted
ible oxidation of type I and type III porphyrinogens. Porphyrinogens in urine and feces. Intense fluorescence with ultraviolet light can verify
of the type III series are the precursors of heme, whereas the type I their presence. The cause of the hemolytic anemia that accompanies
isomers do not produce any useful metabolites and cannot be used CEP is unclear but is thought to be associated with the excessive por-
in heme synthesis (Chapter 6). In normal heme synthesis, both type phyrin deposits within erythrocytes. The finding that normal erythro-
I and III isomers are formed in a 1:10,000 ratio. Normally, most por- cytes infused into CEP patients have a normal life span supports this.
phyrinogens are readily converted to heme, and very small amounts The erythrocytes in CEP can be subject to photohemolysis as they
of the porphyrin intermediates are formed. If for some reason exces- pass through the dermal capillaries exposed to UV light. The erythro-
sive amounts of porphyrinogens are produced, the corresponding cytes show increased photohemolysis in vitro, but whether this occurs
oxidized porphyrin compounds also increase. Porphyrins are tetra- in vivo is uncertain.
pyrroles and resonating compounds (contain alternating single and
double bonds); erythrocytes that contain these substances show red
fluorescence with UV light. erythropoietic Protoporphyria (ePP)
EPP is characterized by an overproduction of protoporphyrin, the
Congenital erythropoietic Porphyria (CeP) immediate precursor of heme, and is associated with a defect in fer-
CEP (Gunther’s disease) is characterized by the presence of exces- rochelatase (Figure 12-10). Adequate amounts of heme are produced,
sive amounts of type I porphyrins: uroporphyrin I (UroI) and however, and no anemia is present. Excess protoporphyrin IX builds
M12_MCKE6011_03_SE_C12.indd 223 01/08/14 9:14 pm
224 part iii • the anemias
Glycine + Succinyl coenzyme A
ALA synthetase
d-Aminolevulinic acid
ALA dehydrase
Porphobilinogen
(uroporphyrinogen I Acute intermittant porphyria
synthase) (uroporphyrinogen I synthase)
+
Congenital erythropoietic porphyria
Uroporphyrin I Uroporphyrinogen I
(uroporphyrinogen III synthase)
Porphyria cutanea tarda
(uroporphyrinogen decarboxylase) Uroporphyrinogen III Uroporphyrin III
Coproporphyrin I Coproporphyrinogen I (uroporphyrinogen decarboxylase)
Coproporphyrinogen III Coproporphyrin III
Hereditary coproporphyria
(coproporphyrinogen oxidase)
Protoporphyrinogen III
Variegate porphyria
(protoporphyrinogen oxidase)
Protoporphyrin IX
Erythropoietic protoporphyria
(ferrochelatase)
Heme
■ Figure 12-15 The formation of heme from glycine and succinyl-coenzyme A involves the
production of porphyrinogen intermediates. Normally, only very small amounts of the series I iso-
mers are formed. However, in a group of congenital disorders called porphyrias, there is a block
in porphyrin metabolism due to a defect in one or more of the enzymes involved in porphyrin
metabolism, and there is a build-up of porphyrin intermediates depending on which enzyme is
involved. In congenital erythropoietic porphyria, there is an abnormality in conversion of porphobi-
linogen to uroporphyrinogen III, and large amounts of the functionless series I isomers are formed.
These isomers are oxidized to porphyrin and accumulate in the tissues. Another form of porphyria,
erythropoietic protoporphyria, is characterized by excessive production of protoporphyrin due to
an abnormality of the ferrocheletase enzyme. Other porphyrias noted in this figure are classified as
hepatic because the site of abnormal metabolism is the liver.
up in the cell, can leak into skin dermal capillaries, and can be found The first signs of CEP occur in infancy. The urine is colored
in the skin, liver, blood, and feces. Due to its insolubility in water, pro- pink to reddish brown, depending on the amount of uroporphyrin
toporphyrin is not present in the urine. This porphyria is thought to excreted. This is usually first noted as a pink stain on the infant’s diaper.
be of both erythropoietic and hepatic origin. 83 The excess porphyrins in the skin create an extreme photosensitiv-
ity to sunlight. Vesicular or bullous eruptions appear on uncovered
areas shortly after exposure to sunlight. The lesions heal slowly and
Clinical Features can become infected. Repeated eruptions and skin injury cause scar-
CEP is a rare autosomal recessive disease with ∙130 cases reported. ring and can lead to severe mutilation of the face, ears, and hands. The
EPP is inherited as an autosomal dominant trait, but recessive inheri- excess porphyrin stains the teeth a brown discoloration. Under UV
84
tance is possible. About 300 cases of EPP have been reported, but light, the teeth fluoresce bright red. Hypertrichosis affects the entire
the actual rate of occurrence is probably masked due to the subtlety of body but is especially present in exposed areas. The hair can be blond
the clinical signs and the absence of colored porphyrins in the urine. and downy or dark and coarse. Splenomegaly is a consistent finding
M12_MCKE6011_03_SE_C12.indd 224 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 225
and is usually progressive with the disease. A mild to severe hemolytic contain large amounts of protoporphyrin; protoporphyrin is not
anemia is present with erythrocyte life span decreased to as little as found in the urine. The protoporphyrin in erythrocytes is free (FEP),
18 days. Patients with CEP do not exhibit the abdominal pain or neu- not bound to zinc as in IDA and lead poisoning. The FEP is higher
rologic and psychotic signs associated with some hepatic porphyrias. than in other disorders associated with an increase in erythrocyte pro-
Clinical signs of EPP are more subtle, and its course is relatively toporphyrin levels. This block in heme synthesis, which occurs in the
mild in comparison with CEP. Photosensitivity is not severe, and scar- reaction just prior to insertion of iron into the porphyrin ring, would
ring is usually absent. Sunlight exposure leads to erythema and urti- be expected to cause an accumulation of iron within the erythroblasts.
caria. Protoporphyrin accumulates in the erythrocytes, which causes The fact that this iron buildup does not occur has not been explained.
them to fluoresce intensely, but there is no hemolytic anemia. Occa-
sionally hepatic damage occurs. Prognosis and Therapy
Laboratory Features Individuals with CEP do not usually survive beyond the fifth decade of
life. Attempts to decrease the excess porphyrins have been unsuccess-
Congenital erythropoietic Porphyria ful, but the quality of life for CEP patients has improved by minimiz-
The peripheral blood in CEP exhibits a mild to severe normocytic ing the scarring and mutilation with effective dermatologic treatment.
anemia with anisocytosis and poikilocytosis. The blood smear reveals Avoiding exposure to sunlight is critical. Splenectomy has sometimes
significant polychromatophilia and nucleated erythrocytes. The resulted in a decrease of porphyrin production and helped ameliorate
erythrocytes fluoresce with UV light. the hemolytic anemia. Evidence for long-term success with splenec-
The bone marrow in CEP shows erythroid hyperplasia. A tomy, however, is questionable. Blood transfusion in conjunction with
large portion of the erythroblasts demonstrates intense fluores- administration of chelators to reduce iron overload suppresses eryth-
cence with UV light. The fluorescence is localized principally in ropoiesis and decreases or eliminates symptoms. Bone marrow trans-
the nuclei. The fact that not all erythroblasts fluoresce suggests that plantation is suggested in severe phenotypes because the predominant
two populations of erythrocytes exist, one of which is normal. site of porphyrin production is the bone marrow.
Serum iron and storage iron are usually normal. Haptoglobin is Treatment of EPP is to protect the skin from sunlight and mini-
absent, and unconjugated bilirubin as well as urinary and fecal urobi- mize the toxic effects of protoporphyrin on the liver. In most patients
linogen are increased. with EPP, high doses of @-carotene improve tolerance to sunlight. Blood
Large amounts of uroporphyrin I and coproporphyrin I are transfusions and hematin can be utilized to suppress erythropoiesis.
excreted in the urine and feces. These isomers are also found in the Splenectomy can be helpful if hemolysis and splenomegaly are promi-
plasma and in erythrocytes. nent. Cholestyramine can promote excretion of liver protoporphyrin. 85
Genes of the heme biosynthetic pathway have been cloned, and
erythropoietic Protoporphyria (ePP) mutations associated with porphyrias have been identified. Gene therapy
82
The blood and bone marrow in EPP usually reveal no abnormalities may be an option for porphyrias in the future. This type of therapy
on routine examination; however, under UV light, the cytoplasm of involves inserting a functional gene into specific hematopoietic or hepatic
erythroblasts fluoresces intensely. The erythrocytes, plasma, and feces stem cells of the patient, restoring normal heme synthesis pathways.
Summary
hemoglobin synthesis requires adequate production of heme Abnormal porphyrin metabolism results in SA and porphyr-
and globin. inadequate amounts of either can result in ane- ias. SA is due to defective porphyrin synthesis and a block in
mia—usually microcytic, hypochromic anemia. Defects in heme the insertion of iron into the porphyrin ring to form heme. The
synthesis could be due to either faulty iron or porphyrin metab- erythrocyte population in SA characteristically contains cells
olism (Table 12-17 ★ ). that are normochromic and hypochromic (dual population).
Many proteins, including hepcidin, ferroportin1, hephaes- The bone marrow has ring sideroblasts. Hereditary SA is due to
tin, DMT1, DCytB, HFE, TfR, GDF-15, HIF-2, and transferrin, defective ALAS2, the enzyme in the first step of heme synthesis.
play a role in iron homeostasis. The anemias with a faulty iron SA can also occur because of the effects of drugs or toxins on
metabolic component include IDA and ACD. IDA is due to inad- enzymes involved in heme synthesis.
equate amounts of iron for heme synthesis; iD usually occurs Iron studies are helpful in differentiating these disorders.
because of blood loss or a nutritional deficiency of iron. ACD Serum iron is decreased in IDA and ACD; it is normal to increased
has several pathophysiologic mechanisms related to cytokines in SA. The serum transferrin is increased and saturation decreased
produced as a result of inflammation or infection. The major if total body iron is decreased, whereas if storage iron is normal
mechanism is a block in reutilization of macrophage iron. The or increased, the serum transferrin is normal or decreased with
erythrocytes are normocytic, normochromic, but in long-stand- normal or increased saturation. Serum ferritin is a reliable indicator
ing anemia, they can be microcytic, hypochromic. of iron stores except in the presence of inflammation or infection
M12_MCKE6011_03_SE_C12.indd 225 01/08/14 9:14 pm
226 part iii • the anemias
when it can be falsely increased. Serum ferritin is decreased in ID associated with iron metabolism can also cause HH. These
and normal or increased in SA, ACD, and hemochromatosis. The mutations are believed to affect the synthesis or function of
serum transferrin receptor assay is useful in differentiating ID and hepcidin. Serum iron studies reflect the excessive iron over-
ACD. Levels are increased in ID and normal in ACD. load with increased serum ferritin and a very high saturation
Hemochromatosis is a disorder characterized by total of transferrin.
body iron excess. Anemia is not a characteristic of this dis- Porphyria is a heterogeneous group of hereditary disorders
order, but it is included in this chapter to help the reader due to a block in porphyrin synthesis. The defect involves one
compare and differentiate iron study results with those associ- of the critical enzymes in the porphyrin metabolic pathway. Two
ated with defective heme synthesis. Hemochromatosis can be forms, CEP and EPP, have an erythropoietic component. Eryth-
caused by a genetic defect or chronic transfusions. The most rocytes have very high levels of free erythrocyte protoporphyrin,
common hereditary form of hemochromatosis (HH) is due to and excess porphyrins are deposited in tissues and excreted in
a mutation of the HFE gene. Mutations in several other genes feces/urine.
★ taBle 12-17 Summary of Conditions Associated with Abnormal Heme Synthesis
and/or Iron Metabolism/Utilization
Condition etiology CBC iron Studies Other
Iron-deficiency anemia inadequate iron due to Microcytic, hypochromic, Decreased serum iron, Increased ZPP (do
deficient dietary intake; anisocytosis, poikilocytosis serum ferritin, and percent not use to evaluate if
decreased absorption; saturation of transferrin; possibility of concur-
increased loss increased TIBC and sTfR rent lead poisoning);
decreased reticu-
locyte hemoglobin
(CHr, MCHr, RET-He);
increased sTfR-F index
Anemia of chronic disease Impaired release of iron normocytic, normochromic; Decreased serum iron and Increased ZPP,
from macrophages due to in long-standing cases can low to normal TIBC; low/ decreased sTfR-F index
increased hepcidin induced be microcytic, hypochromic normal transferrin satura-
by cytokines; inhibition tion; increased or normal
of EPO production and serum ferritin; sTfR normal
impaired erythropoiesis;
shortened erythrocyte
survival
Sideroblastic anemia Defect in enzymes needed Dual population of nor- Increased serum iron and Bone marrow shows
for heme synthesis; can be mochromic and hypo- transferrin saturation; nor- ring sideroblasts;
hereditary or acquired (sec- chromic erythrocytes and mal or decreased TIBC; peripheral blood eryth-
ondary to drugs/toxins) macrocytic, microcytic or increased serum ferritin rocytes can contain
macrocytic, normocytic Pappenheimer bodies
erythrocytes
Hemochromatosis (no Hereditary: genetic muta- no anemia or character- Increased serum iron; trans- Molecular testing
anemia) tions resulting in deficiency istic peripheral blood cell ferrin saturation (Ú45%), to identify mutated
of ferroportin or hepcidin; morphology except in and increased serum ferritin gene is suggested if
acquired: transfusion, iron DMT1 and ferroportin -1 transferrin saturation is
injections, chronic liver mutations more than 45%
disease
Porphyrias (anemia not Block in porphyrin synthesis CEP: normocytic hemolytic Storage iron and serum iron CEP: increased uro-
characteristic) due to defect in enzymes anemia; anisocytosis and normal porphyrin I and cop-
for heme synthesis; CEP poikilocytosis; polychro- roporphyrin I excreted
and EPP affect erythrocytes matophilia; nucleated RBCS in urine and feces,
EPP: no abnormalities plasma, and erythro-
cytes; series III isomers
also increased
CEP, EPP: normoblasts
demonstrate intense flu-
orescence with UV light
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Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 227
Review Questions
Level i 8. Which of the following is most often associated with micro-
cytic, hypochromic anemia? (Objective 5)
1. What is the iron transport protein called? (Objective 3)
A. lead poisoning
A. ferritin
B. transferrin B. ID
C. hemosiderin C. sideroblastic anemia
D. albumin D. anemia of chronic disease
2. The term sideropenic is most closely associated with which 9. Which of the following best describes hemosiderosis?
of the following anemias? (Objective 1) (Objective 7)
A. iron deficiency A. decrease in serum iron
B. sideroblastic B. increase in serum iron
C. lead poisoning C. increase in macrophage iron
D. anemia of chronic disease D. increase in total body iron
3. Microcytic, hypochromic erythrocytes are most character- 10. A patient with anemia of chronic disease would be expected
istic of which of the following anemias? (Objective 5) to have which set of laboratory test results? (Objective 5)
A. megaloblastic A. MCV decreased, serum iron increased, serum ferritin
B. lead poisoning increased, TIBC and percent saturation increased
C. iron deficiency B. MCV normal, serum iron increased, serum ferritin
decreased, TIBC and percent saturation decreased
D. anemia of chronic disease
C. MCV normal, serum iron decreased, serum ferritin
4. Which of the following individuals is most likely to require increased, TIBC and percent saturation decreased
an increased intake of iron? (Objective 4)
D. MCV decreased, serum iron decreased, serum ferritin
A. adult male
decreased, TIBC and percent saturation decreased
B. menopausal female
C. mother of three preschool children Level ii
D. 75-year-old male
Use this history for questions 1–4.
5. The basic defect in sideroblastic anemia is: (Objective 6) A 75-year-old male experiencing mental confusion and fatigue
A. inadequate iron intake was seen by his physician. Laboratory tests were ordered:
12
B. inadequate absorption of iron in the gut RBC 3.3 * 10 /L
Hb 9.3 g/dL
C. cytokine inhibition of erythropoiesis
Hct 0.29 L/L
D. defect in enzymes regulating heme synthesis PLT 168 * 10 /L
9
9
6. Anemia(s) characterized by defective heme synthesis WBC 4.0 * 10 /L
includes: (Objective 6) Differential
Segmented neutrophils 60%
A. hemochromatosis
Band neutrophils 9%
B. megaloblastic anemia Lymphocytes 25%
C. thalassemia Monocytes 3%
Eosinophils 3%
D. sideroblastic anemia
7. The most common cause of ID in middle-aged men is: RBC morphology: Anisocytosis with microcytic, hypochromic
(Objective 6) RBCs, and normocytic, normochromic RBCs present
Laboratory data for anemia workup:
A. inadequate iron in the diet
B. cancer Reticulocyte count 1.0%
Serum iron 274 mcg/dL
C. prescription drugs
Total iron-binding capacity (TIBC) 285 mcg/dL
D. chronic bleeding
M12_MCKE6011_03_SE_C12.indd 227 01/08/14 9:14 pm
228 part iii • the anemias
1. Which anemia of defective heme synthesis is associated 7. What laboratory test is best for screening for iron defi-
with this type of red cell morphology? (Objective 8) ciency in a population of 1- to 3-year-old children who
have a high incidence of elevated blood lead levels?
A. sideroblastic anemia
(Objective 14)
B. anemia of chronic disease
A. hematocrit
C. iron-deficiency anemia
B. serum ferritin
D. erythropoietic porphyria
C. serum iron
2. Which laboratory result(s) is (are) most useful in distinguish-
ing this patient’s anemia from IDA? (Objective 9) D. ZPP
A. bone marrow 8. A 2-year-old child was tested for blood lead level. The
result was 25 mcg/dL. He also had microcytic, hypochromic
B. mean cell volume anemia. The child’s parents were questioned, and it was
determined that the source of lead was a painted crib in his
C. hemoglobin
day care center. The child was enrolled in another day care
D. iron studies center. Follow-up testing revealed that the blood lead level
was within normal limits but the microcytic, hypochromic
3. From the results of these laboratory studies, how would anemia was still present. Which follow-up test(s) would you
you describe the patient’s red blood cells? (Objective 8) recommend to help identify the etiology of this anemia?
A. microcytic, hypochromic (Objectives 9,14,15)
B. dual population of microcytes and normocytes A. serum iron, serum ferritin, TIBC, % saturation
C. macrocytic, normochromic B. hemoglobin electrophoresis
D. normocytic, normochromic C. molecular diagnostic testing for sideroblastic anemia
4. A bone marrow examination was performed and sections D. molecular diagnostic testing for hemosiderosis
were stained with Prussian blue. Numerous sideroblasts 9. A health maintenance organization (HMO) has a contract
were present with a large number of ring sideroblasts. for laboratory testing services with your laboratory. The
What is the most probable cause of this anemia? (Objec- HMO has decided to screen its members for hereditary
tives 6, 8, 10) hemochromatosis. Which laboratory test will you recom-
A. poor diet mend for this screening? (Objective 18)
B. chronic blood loss A. serum iron
B. % transferrin saturation
C. abnormality of ALAS2
C. serum ferritin
D. increased iron requirement
D. molecular test for mutated HFE gene
5. A hematocrit is not recommended to screen for iron defi-
ciency in children because: (Objectives 1, 2, 8, 9) 10. In regard to question 9, what test should you recommend
be done reflexively on patients with an abnormal screening
A. it is not sensitive enough to pick up anemia in children
test? (Objective 18)
B. iron deficiency can be present without the presence of A. molecular test for HFE gene
anemia
B. serum iron
C. high levels of lead will affect the hematocrit accuracy
C. serum ferritin
D. serum ferritin is more cost effective
D. % saturation
6. If a child with lead poisoning also had a significant
microcytic, hypochromic anemia, what complicating
pathology/pathologies should be considered? (Objec-
tive 14)
A. iron deficiency
B. thalassemia
C. iron deficiency and thalassemia
D. thalassemia and sideroblastic anemia
M12_MCKE6011_03_SE_C12.indd 228 01/08/14 9:14 pm
Chapter 12 • anemias of DisorDereD iron metabolism anD heme synthesis 229
11. Which protein regulates iron absorption in the gut and 12. What effect would a high degree of ineffective erythropoi-
transport of iron from cells to the plasma? (Objective 3) esis have on iron metabolism? (Objective 4)
A. DcytB A. decreased absorption of iron in the intestine
B. DMT1 B. decreased transport of iron across the basolateral
membrane of enterocytes
C. hepcidin
C. decreased hepcidin synthesis
D. hephaestin
D. increased serum transferrin saturation
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. Find additional information to help
organize information and figures to help understand concepts.
References
1. andrews nC. pathology of iron metabolism. in: hoffman r, benz Jr. eJ, 18. Kawasaki n, morimoto K, tanimoto t et al. Control of hemoglobin synthesis
th
Shattil SJ et al., eds. Hematology: Basic Principles and Practice. 5 Ed. in erythroid differentiating K562 cells. I. Role of iron in erythroid cell heme
new york: Churchill livingstone; 2009 (available at www.mdconsult synthesis. Arch Biochem Biophys. 1996;328:289–94.
.com/books/page.do?eid=44-u1.0-B978-0-443-06715-0..X5001-8&isbn= 19. Rouault TA, Klausner RD. Iron-sulfur clusters as biosensors of oxidants and
978-0-443-06715-0&uniqid=350057742-2\#4-u1.0-b978-0-443-06715- iron. Trends Biochem Sci. 1996;21:174–77.
0..X5001-8--TOP; accessed August 6, 2012). 20. muhlenhoff U, Gerber J, richhardt n et al. Components involved in assem-
2. roy Cn, andrews nC. anemia of inflammation: the hepcidin link. Cur Opi bly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p.
Hemat. 2005;12(2):107–11. EMBO J. 2003;22:4815–25.
3. Guschin H, Mackenzie B, Berger UV et al. Cloning and characteriza- 21. Killip S, Bennett JM, Chambers MD. Iron deficiency anemia. Am Fam Physi-
tion of a mammalian proton-coupled metal-iron transporter. Nature. cian. 2007;75:611–18.
1997;388:482–88. 22. Ferri C, Procianoy RS, Silveira RC. Prevalence and risk factors for iron-
4. Qiu A, Jansen M, Sakaris A et al. Identification of an intestinal folate trans- deficiency anemia in very-low-birth-weight preterm infants at 1 year of cor-
porter and the molecular basis for hereditary folate malabsorption. Cell. rected age. J Trop Pediatr. 2014;60(1):53–60.
2006;127:917–28. 23. Oski FA. Iron deficiency in infancy and childhood. NEJM. 1993;329(3):190–93.
5. Anderson GJ, Frazer DM, McLaren GD. Iron absorption and metabolism. 24. Domellof M. Iron and other micro-nutrient deficiencies in low-birth weight
Curr Opin Gastroenterol. 2009;23:129–35. infants. Nestle Nutrition Institute Workshop Series. 2013;74:197–206.
6. Gavin MW, McCarthy DM, Garry PJ. Evidence that iron stores regulate iron 25. Clark SB. Iron deficiency anemia: diagnosis and management. Curr Opin
absorption: a setpoint theory. Am J Clin Nutr 1994;59:1376–80. Gastroentero. 2009;25:122–28.
7. hentze mW, muckenthaler mU, andrews nC. balancing acts: molecular 26. Brumberg, JJ. Chlorotic girls, 1870–1920: a historical perspective on female
control of mammalian iron metabolism. Cell. 2004;117:285–97. adolescence. Child Dev. 1982;53:1468–77.
8. Frazer DM, Wilkins SJ, Becker EM et al. A rapid decrease in the expression 27. Joosten e, Ghesquiere b, linthoudt h et al. Upper and lower gastrointes-
of DMT1 and DCytb but not Ireg1 or hephaestin explains the mucosal block tinal evaluation of elderly inpatients who are iron deficient. Amer J Med.
phenomenon of iron absorption. Gut. 2003;52(3):340–46. 1999;107(1):24–29.
9. nicolas G, Chauvet C, Viatte l et al. the gene encoding the iron regula- 28. napolitano m, Dolce a, Celenza G et al. iron-dependent erythropoiesis
tory peptide hepcidin is regulated by anaemia, hypoxia, and inflamma- in women with excessive menstrual blood losses and women with normal
tion. J Clin Invest. 2002;110:1037–44. menses. Ann Hematol. 2014;993(4):557–63.
10. tanno t, bhanu nV, oneal pa et al. high levels of GDf-15 in thalassemia 29. Major A, Mathez-Loic F, Rohling R. The effect of intravenous iron on the
suppress expression of the iron regulatory protein hepcidin. Nat Med. reticulocyte response to recombinant human erythropoietin. Br J Haematol.
2007;13(9):1096–101. 1997;98(2):292–94.
11. Haase VH. Regulation of erythropoiesis by hypoxia-inducible factors. Blood 30. fernandez-rodriquez am, Guindeo-Casasus mC, molero-labarta t et al.
Rev. 2013;27:41–53. Diagnosis of iron deficiency in chronic renal disease. Amer J Kidney Dis.
12. Schmidt PJ, Toran PT, Giannetti AM et al. The transferrin receptor modu- 1999;34(3):508–13.
lates Hfe-dependent regulation of hepcidin. Cell Met. 2008;7(3):205-14. 31. Thompson WG, Meola T, Lipkin M Jr et al. Red cell distribution width, mean
doi:10.1016/j.cmet.2007.11.016 corpuscular volume, and transferrin saturation in the diagnosis of iron defi-
13. Goswami t, andrews nC. hereditary hemochromatosis protein, hfe inter- ciency. Arch Intern Med. 1988;148:2128–30.
action with transferrin receptor2, suggests a molecular mechanism for 32. Braugnara C, Zurakowski D, Dicanzio J et al. Reticulocyte hemoglobin con-
mammalian iron sensing. J. Biol. Chem. 2006;281:28494–98. tent to diagnose iron deficiency in children. JAMA. 1999;281:2247–48.
14. Ganz t, nemeth e. hepcidin and iron homeostasis. Biochim Biophys Acta. 33. Aarna PV, Austin RD, Mathew P. Pica. Indian J Dent Res. 2012;23(3):426-27.
2012;1823:1434–43. 34. Geissler PW, Shulman CE, Prince RJ et al. Geophage, iron status, and anae-
15. Kawabata H, Fleming RE, Rui D et al. Expression of hepcidin is down regu- mia among pregnant women on the coast of Kenya. Transac Royal Soc Trop
lated in TfR2 mutant mice manifesting a phenotype of hereditary hemo- Med Hyg. 1998;92(5):549–53.
chromatosis. Blood. 2004;105:378–81. 35. Idjradinata P , Pollitt E. Reversal of developmental delays in iron-deficient
16. Malyszko J. Hemojuvelin: the hepcidin story continues. Kidney Blood Press anaemic infants treated with iron. Lancet. 1993;341:1–4.
Res. 2009;32(2):71–76. 36. Roncagliolo M, Garrido M, Walter T et al. Evidence of altered central
17. Klausner rD, rouault ta, harford Jb. regulating the fate of mrna: the nervous system development in infants with iron deficiency anemia at 6
control of cellular iron metabolism. Cell. 1993;72:19–28. months: delayed maturation of auditory brainstem responses. Am J Clin
Nutri. 1998;68(3):683–90.
M12_MCKE6011_03_SE_C12.indd 229 01/08/14 9:14 pm
230 part iii • the anemias
37. Cullen p, soffker J, hopfl m, bremer C et al. hypochromic red cells and 63. May A, Bishop DF. The molecular biology and pyridoxine responsiveness of
reticulocyte hemoglobin content as markers of iron-deficient-erythropoiesis x-linked sideroblastic anemia. Haematologica. 1998;83:56–70.
in patients undergoing chronic haemodialysis. Nephrol Dial Transplant. 64. furuyama K, fujita h, nagi t et al. pyridoxine refractory x-linked sideroblas-
1999;14(3):659–65. tic anemia caused by a point mutation in the erythroid 5-aminolevulinate
38. Vreugdenhil G, Baltus CA, van Eijk HG et al. Anaemia of chronic disease: synthase gene. Blood. 1997;90 (2):822–30.
diagnostic significance of erythrocyte and serological parameters in iron 65. Koc S, Harris JW. Sideroblastic anemias: variations on imprecision in diag-
deficient rheumatoid arthritis patients. Br J Rheumatol. 1990;29:105–10. nostic criteria; proposal for an extended classification of sideroblastic ane-
39. Van Vranken M. Evaulation of microcytosis. Amer Fam Physician. mias. Am J Hematol. 1998;57:1–6.
2010;82(9):1117-22. 66. Landrigan PJ, Todd AC. Lead poisoning. West J Med. 1994;161:153–59.
40. Kis AM, Carnes M. Detecting iron deficiency in anemic patients with con- 67. CDC. Adult blood lead level epidemiology and surveillance –United States
comitant medical problems. J Gen Intern Med. 1998;13(7):455–61. 2008-09. MMWR. July 1, 2011;60(25):841–45 (available at www.cdc.gov/
41. Van den broek nr, letsky ea, White sa et al. iron status in pregnant mmwr/preview/mmwrhtml/mm6025a2.htm; accessed August 6, 2012).
women: Which measurements are valid? Br J Haematol. 1998;103(3):817–24. 68. CDC. CDC response to Advisory Committee on childhood lead poison-
42. McKay R, Scott BB. Iron deficiency anemia: How far to investigate? Gut. ing prevention recommendations in “low level lead exposure harms
1993;34:1427–28. children: a renewal call of primary prevention.” 2012;61(20) (available
43. Wong SS, Qutishat AS, Lange J et al. Detection of iron-deficiency ane- at www.cdc.gov/nceh/lead/ACCLPP/CDC_Response_lead_exposure_recs.pdf;
mia in hospitalized patients by zinc protoporphyrin. Clin Chim Acta. accessed September 6, 2013).
1996;244(1):91–101. 69. Bhambhani K, Aronow R. Lead poisoning and thalassemia trait or iron defi-
44. Rettmer Rl, Carlson TH, Origenes ML et al. Zinc protoporphyrin/heme ratio ciency: the value of the red blood cell distribution width. Am J Dis Child.
for diagnosis of preanemic iron deficiency. Pediatrics. 1999;104(3):e37. 1990;144:1231–33.
45. Labbe RF, Dewanji A. Iron assessment tests: transferrin receptor vis-à-vis 70. Carraccio CL, Bergman GE, Daley BP. Combined iron deficiency and lead
zinc protoporphyrin. Clin Biochem. 37(3):165-74.2004. poisoning in children: effect on FEP levels. Clin Pediatr. 1987;26:644–47.
46. Harthoorn-Lasthuizen EJ, Lindemans J, Langenhuipen MM. Combined use 71. Clark M, Royal J, Seeler, R. Interaction of iron deficiency and lead and the
of erythrocyte zinc protoporphyrin and mean corpuscular volume in dif- hematologic findings in children with severe lead poisoning. Pediatrics.
ferentiation of thalassemia from iron deficiency anemia. Europ J Haematol. 1988;81:247–54.
1998;60(4):245–51. 72. Rainey PM. Lead: a heavy burden for small children. Clin Chem News.
47. Junca J, Flores A, Roy C et al. Red cell distribution width, free erythrocyte 1992;18(6):37.
protoporphyrin, and England-Fraser index in the differential diagnosis of 73. Savage D, Lindenbaum J. Anemia in alcoholics. Medicine. 1986;5:322–39.
microcytosis due to iron deficiency or beta-thalassemia trait: a study of 74. Cotter PD, May A, Li L et al. Four new mutations in the erythroid-specific
200 cases of microcytic anemia. Hematol Pathol. 1991;5:33–36. 5-aminoluvulinate synthase (ALSA2) gene causing x-linked sideroblastic
48. Suominen P , Punnonen K, Ragamaki A et al. Serum transferrin receptor and anemia: increased pyridoxine responsiveness after removal of iron overload
transferrin receptor-ferritin index identify healthy subjects with subclinical by phlebotomy and coinheritance of hereditary hemochromatosis. Blood.
iron deficits. Blood. 1998;92(8):2934–39. 1999;93(5):1757–69.
49. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and 75. Brittenham, GM. Disorders of iron metabolism: iron deficiency
its ratio to serum ferritin in the diagnosis of iron deficiency. Blood. and iron overload. In: Hoffman R, Benz EJ Jr, Shattil SJ et al., eds.
th
1997;89(3):1052–57. Hematology: Basic Principles and Practice. 5 ed. new york: Churchill
50. Mulherin D, Skelly M, Saunders A et al. The diagnosis of iron deficiency in Livingstone; 2009 (available at www.mdconsult.com/books/page
patients with rheumatoid arthritis and anemia: an algorithm using simple .do?eid=4-u1.0-B978-0-443-06715-0..X5001-8&isbn=978-0-443-06715-
laboratory measures. J Rheum. 1996;23(2):237–40. 0&uniqid=350057742-2#4-u1.0-b978-0-443-06715-0..X5001-8--top;
51. ahluwalia n. Diagnostic utility of serum transferrin receptors measurement accessed September 6, 2013).
in assessing iron status. Nutri Rev. 1998;56(5 Pt1):133–41. 76. feder Jn, Gnirke a, thomas W. a novel class-i1 like gene is mutated
52. Case G. Maintaining iron balance with total-dose infusion of intravenous in patients with hereditary hemochromatosis. Nature Genet.
iron dextran. Anna J. 1998;25(1):65–68. 1996;13:399–408.
53. Adamson J. Erythropoietin, iron metabolism, and red blood cell production. 77. Kasvosve I. Effect of ferroportin polymorphism on iron homeostasis and
Sem Hematol. 1996;33:5–9. infections. Clin Chim Acta. 2013;416;20–25.
54. Chang CC, Kass L. Clinical significance of immature reticulocyte frac- 78. H(+)@coupled divalent metal-io transporter-1: functional properties, physi-
tion determined by automated reticulocyte counting. Am J Clin Pathol. ological roles and therapeutics. Curr Top Membr. 2012;70:169-214.
1997;108(1):69–73. 79. Cogswell ME, Burke W, McDonnell SM et al. Screening for hemochromato-
55. Dallman PR, Reeves JD, Driggers DA et al. Diagnosis of iron deficiency: the sis: a public health perspective. Am J Prev Med. 1999;16(2):134–40.
limitations of laboratory tests in predicting response to iron treatment in 80. Olynyk JK. Hereditary haemochromatosis: diagnosis and management in
1-year-old infants. J Pediatr. 1981;99(3):376–81. the gene era. Liver. 1999;19:73–80.
56. Krantz SB. Pathogenesis and treatment of the anemia of chronic disease. 81. Fletcher LM, Halliday JW, Powell LW. Interrelationships of alcohol and iron
Am J Med Sci. 1994;307:353–59. in liver disease with particular reference to the iron-binding proteins, fer-
57. Means RT. Clinical application of recombinant erythropoietin in the anemia ritin, and transferrin. J Gastroentero Hepat. 1999;14:202–14.
of chronic disease. Hematol Oncol Clin No Amer. 1994;8(5):933–44. 82. Adams PC, Kertesz AE, Valberg LS. Rate of iron reaccumulation following
58. Dicato M, Plawny L, Diederich M. Anemia in cancer. Ann Oncology. iron depletion in hereditary hemochromatosis: implications for venesection
2010;21(supplement 7):vii167-72. doi:10.1093/annonc/mdq284. therapy. J Clin Gastroenterol. 1993;16(3):207–10.
59. baer an, Dessypris en, Goldwasser e et al. blunted erythropoietin 83. DeVerneuil H, Ged C, Bouleihfar S et al.Porphyrias: animal models
response to anemia in rheumatoid arthritis. Br J Haematol. 1987;66:559–64. and prospects for cellular and gene therapy. J Bioenerg Biomembr.
60. Means RT Jr, Krantz SB. Progress in understanding the pathogenesis of the 1995;27(2):239–48.
anemia of chronic disease. Blood. 1992;80:1639–47. 84. Mascaro JM. Porphyrias in children. Pediatr Dermatol. 1992;9:371–72.
61. Jain s, narayan s, Jagdish s et al. evaluation of serum transferrin receptor 85. Fuller SJ, Wiley JS. Heme biosynthesis and its disorders. In: Hoffman
and sTfR indices in diagnosing and differentiating iron deficiency anemia R, Benz EJ, Silberstein LE et al., eds. Hematology: Basic Principles and
from anemia of chronic disease. Indian J Pediatr. 2010;77(2):179-83. Practice, 6th ed. new york: elsevier; 2013:457–72 (available at https:www-
62. Bottomley SS, May BK, Cox TC et al. Molecular defects of erythroid clinicalkey-com; accessed June 18, 2014).
5-aminolevulinate synthase in s-linked sideroblastic anemia. J Bioenerg Bio-
membr. 1995;27(2):161–68.
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13 Hemoglobinopathies:
Qualitative Defects
Rebecca J. Laudicina, Phd
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Define hemoglobinopathy. Objectives—Level I and Level II 231
2. Explain the basis of defects resulting in the production of variant
hemoglobins. Key Terms 232
3. Explain the basis of the hemoglobin electrophoresis method in Background Basics 232
identifying variant hemoglobins.
4. Describe the epidemiology of sickle cell anemia (SCA) and other Case Study 232
hemoglobinopathies. Overview 232
5. Identify the globin chain defects causing SCA, hemoglobin C (HbC)
disease, and hemoglobin E (HbE) disease. Introduction 232
6. Associate laboratory analyses with their use in detecting and identifying Structural Hemoglobin Variants 233
hemoglobinopathies.
7. Recognize and identify abnormal laboratory test results, including Sickle Cell Anemia 236
peripheral blood findings and screening and confirmatory tests, Other Sickling Disorders 241
typically associated with homozygous and heterozygous conditions
involving sickle hemoglobin (HbS), HbC, HbD, and HbE and compound Hemoglobin C Disease 241
heterozygous conditions involving HbS and other variant hemoglobins.
8. List major clinical findings typically associated with the Hemoglobin S/C Disease 242
hemoglobinopathies listed in Objective 7. Hemoglobin D 243
Objectives—Level II Hemoglobin E 243
At the end of this unit of study, the student should be able to: Unstable Hemoglobin Variants 244
1. Compare the synthesis and concentration of variant hemoglobins in Hemoglobin Variants with Altered
homozygous and heterozygous conditions. Oxygen Affinity 245
2. Compare the prevalence of hemoglobins S, C, D, and E.
3. Compare and contrast the pathophysiology of common hemoglobin Summary 247
variants in terms of altered solubility, function, and stability. Review Questions 247
4. Analyze the structure of the hemoglobin molecule in sickle cell anemia
(SCA) and relate it to the pathophysiology of the disease. Companion Resources 249
5. Contrast clinical findings in persons who are homozygous and References 249
heterozygous for hemoglobins S, C, D, and E and in those who have
combined heterozygosities for these variant hemoglobins.
(continued)
M13_MCKE6011_03_SE_C13.indd 231 01/08/14 12:43 pm
232 SECTION III • ThE ANEmIAS
9. Design a laboratory-testing algorithm for optimizing tests
Objectives—Level II (continued)
used in detecting and identifying variant hemoglobins.
6. Identify and explain the basis of current therapies for 10. Evaluate laboratory test results and medical history of a
SCA. clinical case for a patient with a hemoglobinopathy and
7. Evaluate and interpret mobility patterns obtained on suggest a possible diagnosis.
cellulose acetate and citrate agar gel hemoglobin 11. Explain the physiologic abnormality resulting in unstable
electrophoresis when structurally abnormal hemoglobins, methemoglobinemia, and hemoglobin
hemoglobins are present. variants with increased or decreased oxygen affinity.
8. Select, evaluate, and interpret tests used in detecting and 12. Interpret laboratory findings associated with the disorders
identifying abnormal hemoglobins. in Objective 11.
Key Terms
Aplastic crisis Hemoglobin electrophoresis Ischemic Thalassemia
Autosplenectomy Hemoglobinopathy methemoglobinemia Vaso-occlusive crisis
Compound heterozygote Irreversibly sickled cell (ISC) Sequestration crisis
Background Basics
The information in this chapter builds on the concepts learned • Name and describe basic laboratory procedures used to screen
in previous chapters. To maximize your learning experience, you for and assess anemia. (Chapters 10, 11, 37)
should review these concepts before starting this unit of study: • Recognize abnormal values and results for basic hematologic
procedures. (Chapters 10, 11, 37)
Level i • Describe the classification systems of anemias. (Chapter 11)
• Describe erythrocyte metabolism and erythrocyte destruction.
(Chapter 5) Level ii
• Describe the structure and function of hemoglobin; list variant • Explain the genetic control of globin chain synthesis. (Chapter 6)
hemoglobins. (Chapter 6) • Describe the ontogeny of hemoglobin types. (Chapter 6)
OveRview
case study
When hemoglobin’s molecular structure is altered, the molecule’s
We will refer to this case throughout the chapter. function, stability, and/or solubility can change, often resulting in
anemia and other clinical consequences. Laboratory screening tests
Shane, a 16-year-old African American male with a
previously diagnosed hemoglobinopathy, was admitted for hemoglobin variants are based on the altered characteristics of
to the hospital complaining of severe pain in his knees and the hemoglobin molecule. If screening tests are abnormal, reflex tests
back. Two of his four siblings have the same disorder. He are required to confirm the presence of a variant hemoglobin. The
has been admitted to the hospital on numerous occasions laboratory’s role is not limited to detecting variant hemoglobins but
throughout his life for complications of his disease. extends to monitoring a patient’s condition over the course of an
Physical examination reveals a thin male in acute distress, illness and during treatment. This chapter discusses the most com-
complaining of severe pain. A head, eyes, ears, nose, and mon variant hemoglobins including epidemiology, pathophysiology,
throat (HEENT) exam is positive for corkscrew vessels of clinical findings, laboratory findings, and treatment. Emphasis is on
the schlerae, schleral icterus, and small, ill-defined, mobile the correlation of clinical history and symptoms with laboratory
(shotty) cervical lymph nodes. Abdominal exam revealed tests and interpretation of test results.
no splenomegaly, hepatomegaly, tenderness, or masses.
Vital signs included temperature 37.8°C, blood pressure
95/70, and pulse 82. Blood was drawn for laboratory tests, intROductiOn
and a chest radiograph and mRI of the head were ordered. Clinical diseases that result from a genetically determined
Consider whether the patient’s current condition is abnormality of the structure or synthesis of the hemoglobin
likely to be related to his previous diagnosis and what molecule are called hemoglobinopathies. The abnormality is
the laboratory’s role is at this time. associated with the globin chains; the heme portion of the molecule
is normal. The globin abnormality can be either a qualitative defect
M13_MCKE6011_03_SE_C13.indd 232 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 233
in the globin chain (structural abnormality) or a quantitative defect disease, however, remained a mystery until Linus Pauling in 1949
in globin synthesis. discovered the altered electrophoretic mobility of the hemoglobin
4
Qualitatively abnormal hemoglobin molecules arise from genetic in patients with sickle cell disease. The molecule’s altered electrical
mutations in the coding region of a globin gene, resulting in amino charge was ascribed to a molecular abnormality of the globin chain.
acid deletions or substitutions in the globin protein chain. These More than 1100 abnormal hemoglobins have since been
5
mutations cause structural variation in one of the globin chain classes identified. A molecular database of abnormal hemoglobins can
(structural hemoglobin variants). The nomenclature of these disorders be accessed at http://globin.cse.psu.edu/globin/hbvar/menu.
is discussed in the later section “Nomenclature.” The most common html. The number of identified b@chain mutations exceeds the
clinical disorder of this type of mutation is sickle cell anemia. number identified for a@, d@, or g@chains (Table 13-1 ★). Part of the
The quantitative globin disorders result from various genetic explanation for this distribution likely resides in the genetics and phe-
defects that reduce synthesis of structurally normal globin chains. notypic expression of the globin gene loci. Hemoglobin F (g@chain)
The quantitative disorders are known collectively as thalassemias. is expressed to a significant extent only during fetal development;
Clinical disorders are also associated with combination defects therefore, variant F hemoglobins are unlikely to be detectable after
involving both structural defects and quantitative deficiencies 3–6 months of age. Mutations affecting the g@chain locus with the
of hemoglobin. Compound heterozygotes possess two different potential to produce a significant clinical abnormality would likely
abnormal alleles of a gene coding for globin chains. An example is cause fetal death. At birth and in the first few months of life, as b@chain
thalassemia in combination with sickle cell (Chapter 14). synthesis becomes predominant, residual abnormal HbF is masked by
As a result of the globin chain defects, hemoglobinopathies can the increasing concentration of HbA in children carrying mutations
be associated with a chronic hemolytic anemia or can be asymptom- of the g-chain, producing milder clinical presentations.
atic. Clinical expression of the hemoglobinopathy varies, depending Because HbA (d@chain) is usually a minor component of the
2
on the class of globin chain involved (a, b, d, or g), the severity of total hemoglobin in adults, HbA variants are also unlikely to cause
2
hemolysis, and the compensatory production of other normal globin clinical complications and are discovered “ accidentally” during labora-
chains. Some of the hemoglobinopathies produce no clinical signs tory evaluation for other purposes.
or symptoms of disease and are identified only through population Two copies of the a@chain gene are found on each of the number
studies specifically designed to reveal “silent” carriers. As discovery 16 chromosomes, resulting in a total of four genes. Thus, a mutation of
of silent carriers increases, the incidence of these genetic disorders is a single a@locus resulting in a variant a@chain@containing hemoglobin
proving to be much higher than originally thought. produces only a small amount of the abnormal hemoglobin and is less
Hemoglobinopathies are believed to be the most common lethal likely to cause clinically significant disease.
1
hereditary diseases in humans. More than 330,000 infants worldwide The b@gene, which is found in a single copy on each chromosome
are born each year with inherited hemoglobin disorders such as sickle 11, is most likely to be associated with a clinical phenotype when
2
cell and thalassemia. Many affected children live in low-income mutated because the b@globin chain is a component of HbA, the
countries, and many die before age 5. major adult hemoglobin.
Hemoglobinopathies are found worldwide but occur
most commonly in African blacks and ethnic groups from the Identification of Hemoglobin Variants
Mediterranean basin and Southeast Asia. The geographic locations Most structural hemoglobin variants result from a single amino acid
where the quantitative and qualitative hemoglobin disorders are substitution or deletion in the globin polypeptide chain. The majority
found frequently overlap; thus, it is not uncommon for individuals to of structural variants result in no clinical or hematologic abnormality
have both a structural hemoglobin variant and a form of thalassemia. and have been discovered only by population studies or family stud-
This could partly explain the extreme variation in clinical findings ies. Mutations result in clinical manifestations only when the solubility,
associated with hemoglobinopathies. stability, or function (oxygen affinity) of the hemoglobin molecule is
This chapter discusses the structural hemoglobin variants and altered. These phenotypic variants produce both clinical and hemato-
Chapter 14 discusses the thalassemias and combination disorders. logic abnormalities of varying severity, depending on the nature and site
of the mutation. Laboratory tests designed to detect and identify hemo-
globin variants are based on the molecule’s altered structure or function.
stRuctuRaL HemOgLObin vaRiants
The largest group of hemoglobinopathies results from an inherited ★ tabLe 13-1 Structural Variants of Globin Genes
structural change in one of the globin chains; however, rate of synthesis
of the abnormal chain usually is not significantly impaired. Any of the globin Polypeptide gene number of variants
globin chain classes, a, b, d, or g, can be affected. a1@ and/or a2@globin gene(s) 424
Sickle cell anemia (SCA), the most common structural b@globin gene 571
hemoglobin variant, was reported by James Herrick of Chicago in d@globin gene 69
3
1910. He described the typical crescent-shaped sickled erythrocytes Ag@globin gene 38
in a young black student from the West Indies. Following this initial gg@globin gene 58
report, additional cases of the disease were described, and the clinical
pattern of SCA was established. The pathophysiologic aspects of the from globin.cse.psu.edu/globin/hbvar/menu.html (accessed march 14, 2014).
M13_MCKE6011_03_SE_C13.indd 233 01/08/14 12:43 pm
234 SECTION III • ThE ANEmIAS
Methods of Analysis Hemoglobin Electrophoresis Controls
Hemoglobin carries an electrical charge resulting from the presence Cellulose
+
+
-
of ionized carboxyl (COO ) and protonated (H ) amino (NH ) acetate,
3
groups. The type (net positive, net negative) and strength of the pH 8.4
charge depend on both the amino acid sequence of globin chains in
the hemoglobin molecule and the pH of the surrounding medium. Origin A 2 S F A Barts
C
H
D
Many amino acid substitutions alter the molecule’s electrophoretic E G
charge, enabling the detection of a structural hemoglobin vari- O Lepore
ant by hemoglobin electrophoresis. Some substitutions can
cause identical changes in the net charge of the molecule; thus, two Citrate
different mutant hemoglobins can have identical electrophoretic agar gel,
pH 6.2
mobility. Other substitutions do not alter the charge of the globin
chain, and the variant hemoglobin migrates identical to the “normal” F A Origin S C
globin chain. By varying the medium and pH of the procedure, many D
E, A , Lepore
clinically significant hemoglobins can be detected and identified. G 2
Methods for performing hemoglobin electrophoresis, examples of O
electrophoretic patterns, and more complete discussions of the tests Patient
that follow are included in Chapter 37.
The most common clinically symptomatic hemoglobinopathies Cellulose
acetate,
involve abnormalities of the b@globin chain, resulting in a decrease pH 8.4
or absence of HbA (a b ) and sometimes an increase in HbF (a g )
2 2
2 2
and/or HbA (a d ). A band representing the mutant hemoglobin ■ FiguRe 13-1 Electrophoretic patterns of
2 2
2
may also be present. Typically, an elevation in HbF and/or HbA is hemoglobins. The top and middle patterns are
2
the clue to the presence of a hemoglobinopathy, although HbF can controls for electrophoresis by cellulose acetate at
be elevated in other hematologic disorders as well. HbF concentra- pH 8.4 and citrate agar gel at pH 6.2 showing the
tions 710% can be measured by electrophoresis and densitometry. mobility patterns for normal and abnormal hemo-
Smaller but significant increases in HbF can be measured more accu- globins. Note that some hemoglobins that move
rately by alkali denaturation and other methods. HbF distribution together on cellulose acetate (pH 8.4) (e.g., HbS, D,
G) can be separated on citrate agar gel (pH 6.2). The
among the erythrocyte population is evaluated by acid elution tests. bottom pattern shows an electrophoretic pattern
These tests are based on the fact that HbF is more resistant to alkali of a patient with normal hemoglobin using cellulose
and acid treatment than other hemoglobins. acetate at pH 8.4.
Results of laboratory tests provide essential information when a
hemoglobinopathy is suspected. The hemoglobin concentration from Other traditional tests for abnormal hemoglobins are based on
the CBC indicates whether anemia is present. RDW and erythrocyte altered physical properties of the structural variants. These include
indices should be evaluated to help distinguish hemoglobinopathies solubility tests, heat precipitation tests, and tests for Heinz bodies. The
from thalassemia, which usually causes a microcytic, hypochromic uses of these methods are included in the following discussion of the
anemia. A review of the blood smear can reveal specific poikilocytes, corresponding specific structural variants. Procedures are included
such as sickle cells. in Chapter 37.
An important next step is to detect hemoglobin variants and Techniques are also available to identify the specific molecular
10
6
quantify hemoglobins A and F. Several options, ranging from defect of hemoglobin disorders. These techniques are discussed in
2
traditional electrophoresis on solid media to newer methods, are avail- Chapter 42. The polymerase chain reaction (PCR) has been incorporated
able; many laboratories use a combination of tests. Figure 13-1 ■ shows into diagnostic procedures for identifying point mutations because it
the electrophoretic mobility of normal and abnormal hemoglobins enhances sensitivity and reduces the amount of DNA and time required
using cellulose acetate (pH 8.4) and citrate agar gel (pH 6.2). Some for analysis. Prenatal diagnosis can be carried out in the first trimester of
laboratories are using advanced testing methods such as isoelectric pregnancy using DNA obtained from chorionic villus sampling. In cases
focusing (IEF), high-performance liquid chromatography (HPLC), or of hemoglobinopathies caused by known common mutations, such as
7
capillary electrophoresis (CE). These laboratory methods are suitable sickle cell anemia, the fetal DNA can be analyzed directly. If prenatal
for testing large numbers of individuals for hemoglobinopathies and diagnosis is desired when the exact mutation is not known, the parents’
offer improved resolution and identification of certain hemoglobin DNA could be analyzed first (e.g., restriction fragment length polymor-
8
variants over results obtained with alkaline electrophoresis. HPLC has phism [RFLP]) to help identify the presence of a mutation (Chapter 42).
the advantage over electrophoresis of quantifying low concentrations
7
of HbA and HbF. All states in the United States mandate newborn CheCkpoint 13-1
2
9
screening programs for sickle cell disease, often using HPLC or mass
spectrometry methods that are appropriate for high-volume testing. Why is it not possible for all structural hemoglobin variants
Unfortunately, these methods may be unavailable for testing residents to be identified by hemoglobin electrophoresis?
of developing and low-income countries.
M13_MCKE6011_03_SE_C13.indd 234 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 235
of disease, the abnormal hemoglobin usually constitutes 90–95% of
case study (continued from page 232) the total hemoglobin. This is explained by the effect of the abnormal
globin chain on the formation of the hemoglobin tetramers. The
1. Identify a laboratory test needed to determine Shane’s normal a@chain has a net positive charge; the normal b@chain (b )
A
hemoglobinopathy.
has a negative charge, and ab dimers form initially through positive-
negative electrostatic interactions. b@chain mutants with a lesser
A
A
negative charge than b form ab dimers more slowly than do b
Nomenclature (and, conversely mutations that increased the negative charge of the
S
C
b@globin chain would form ab dimers more rapidly). Both b and b
The first abnormal hemoglobin discovered was called
hemoglobin S (HbS) because it was associated with crescent (sickle)- mutations cause a net reduction of the b@chain negative charge and
C
S
A
shaped erythrocytes (S for sickle). Subsequently, other hemoglobin form ab or ab dimers more slowly than ab dimers. As a result,
variants were discovered and were given successive letters of the heterozygotes have ∙60% HbA and ∙35940% HbS or HbC. If HbS
alphabet according to electrophoretic mobility beginning with the or HbC constitutes 750% of the total hemoglobin in a heterozygote,
letter C. The letter A was already being used to describe the normal the patient could have inherited two different abnormal hemoglobin
adult hemoglobin, HbA. The letter B was not used to avoid confusion genes (compound heterozygote) or a form of thalassemia in
with the ABO blood group system. The letter F had been designated combination with the structural hemoglobin variant (Chapter 14).
to describe fetal hemoglobin, HbF. The letter M was given to those
hemoglobins that tended to form methemoglobin (HbM). CheCkpoint 13-2
As more variants were discovered, it was recognized that the
alphabetical system was not sufficient and a different nomenclature What does the term silent carrier mean when referring to
system was needed. Thus, subsequent hemoglobins were given a hemoglobinopathy?
common names according to the geographic area in which they
were discovered (e.g., Hb Ft. Worth). It also became apparent that
some variants with the same letter designation (same electrophoretic
mobility) had different structural variations. If the hemoglobin has case study
the electrophoretic mobility of a previously lettered hemoglobin,
that letter is used in addition to the geographic area (e.g., HbG Results of hemoglobin electrophoresis were 90% HbS,
Honolulu). 9% hbf, and 1% HbA 2 .
A standardized hemoglobin nomenclature has been recommended
for use. All variants should be given a scientific designation as well as 2. What is the abnormal hemoglobin causing Shane’s
a common name. The scientific designation includes the following: disease?
(1) the mutated globin chain, (2) the position of the affected amino acid, 3. Is Shane heterozygous or homozygous for the disorder?
(3) the helical position of the mutation, and (4) the amino acid substitu- 4. What is this disorder called?
tion. (If the mutation affects amino acids between helices, the number of
the amino acid and the letters of the two bracketing helices are used.) For
example, HbS is designated b6 (A3) Glu S Val. The mutation is in the
b@chain affecting the amino acid in the sixth position of the completed Pathophysiology
polypeptide located in the A3 helix position. The amino acid valine is The structural hemoglobin variants cause symptoms if the amino
substituted for glutamic acid. Hemoglobins with amino acid deletions acid substitution occurs at a critical site within the molecule. Muta-
include the word missing after the amino acid and helix designation (e.g., tions that cause clinical signs of disease affect the solubility, function
b56959 [D7–E3] missing). The advantage of the helical designation is (oxygen-affinity), and/or stability of the hemoglobin molecule.
that amino acid substitutions in the same helix can lead to similar func-
tional and structural alterations of the hemoglobin molecule, allowing a altered solubility
better understanding of the clinical manifestations of each. If a nonpolar amino acid is substituted for a polar residue near the
Not all globin chain mutations cause symptoms of disease; thus, molecule’s surface, the solubility of the hemoglobin molecule can be
many go undetected. Only those that cause clinical symptoms are likely affected. Hemoglobin S and hemoglobin C are examples of this type of
to be brought to a physician’s attention. If an individual is homozygous substitution. In the deoxygenated state, the HbS molecule polymerizes
for the gene coding for a structural b@globin mutant, no HbA is pro- into insoluble, rigid aggregates. The majority of surface substitutions,
duced, and the term disease or anemia is used to describe the specific however, do not affect the tertiary structure, heme function, or subunit
disorder (e.g., sickle cell anemia). If one of the genes coding for the interactions and are therefore innocuous.
b@chain is normal and the other b@gene codes for a structural variant,
both HbA and the abnormal hemoglobin are produced, and the word altered Function
trait is used to describe the heterozygous disorder (e.g., sickle cell trait). Some amino acid substitutions can affect the oxygen affinity of
With the most common b@chain hemoglobin variants (HbS, hemoglobin by stabilizing heme iron in the ferric state, producing
HbC), the abnormal hemoglobin usually accounts for 650% of the methemoglobin, which cannot combine with oxygen (Chapter 6).
total hemoglobin in the trait form, whereas in the homozygous state Hemoglobins M and Chesapeake are examples. Mutations within the
M13_MCKE6011_03_SE_C13.indd 235 01/08/14 12:43 pm
236 SECTION III • ThE ANEmIAS
subunit interface, a b , can affect the allosteric properties of the molecule, frequency. Children with sickle cell trait are infected with the malar-
1 2
leading to increased or decreased oxygen affinity. Considerable move- ial parasite, but the parasite counts remain low. It has been suggested
ment occurs at the a b contact region on oxygenation, which triggers that resistance to malaria occurs because parasitized cells sickle more
1 2
these allosteric interactions (Chapter 6). High oxygen-affinity hemo- readily, leading to sequestration and phagocytosis of the infected cell
globin variants produce congenital erythrocytosis whereas decreased by the spleen. Other as yet undefined factors could also contribute
12
oxygen-affinity variants produce pseudoanemia and cyanosis. to reduced malarial susceptibility in individuals with HbS. Epide-
miological data suggest there could be similar selective advantages
11
altered stability to HbE and HbC. Molecular evidence indicates that the identical
Amino acid substitutions that reduce the stability of the hemoglobin sickle mutation arose independently in these geographic areas at least
tetramer result in unstable hemoglobins. The mutations usually five times.
disrupt hydrogen bonding or hydrophobic interactions that retain
the heme component within the heme-binding pocket of the globin Pathophysiology
chain or that hold the tetramer together. The result is a weakening
of the binding of heme to globin and detachment of the heme or Sickle cell anemia is caused by a mutation (GAG S GTG conversion)
the disruption of the integrity of hemoglobin’s tetrameric structure. in the HBB gene (hemoglobin b gene). This mutation results in the sub-
Consequently, hemoglobin denatures, aggregates, and precipitates stitution of nonpolar valine for polar glutamic acid at the sixth amino
as Heinz bodies. Clinically, the unstable hemoglobin variants are acid position in the A3 helix of the bchain:b6 (A3) Glu S Val (see
sometimes known as congenital Heinz body hemolytic anemias. the previous “Nomenclature” section for explanation)—and produces
In addition to altering the molecule’s stability, disruption of normal the mutant hemoglobin, HbS. The amino acid substitution is on the
conformation also can affect the molecule’s function. surface of the molecule, producing a net decrease in negative charge;
hence, it changes the molecule’s electrophoretic mobility. The solubility
of HbS in the deoxygenated state is markedly reduced, producing a
CheCkpoint 13-3 tendency for deoxyhemoglobin S molecules to polymerize into rigid
aggregates. The cells may assume a crescent shape, depending on the
The mutation in HbJ-Capetown, a92, Arg S gln, stabilizes extent of polymerization of the HbS molecules. Polymerization is
hemoglobin in the R state (Chapter 6). What functional reversible on reoxygenation.
effect does this have on the hemoglobin molecule?
Polymerization is time dependent. A time delay occurs between
deoxygenation and the formation of a significant amount of HbS
polymers. The development of significant polymerization and red
sickLe ceLL anemia cell distortion takes ∙294 minutes. The delay time for polymeriza-
Worldwide, sickle cell anemia is the most common symptomatic tion is important in considering the overall clinical consequences of
hemoglobinopathy with greatest prevalence in tropical Africa HbS. Even though most red cells contain some sickle hemoglobin
(Table 13-2 ★). Gene frequency in equatorial Africa can exceed 20%. polymer at the oxygen concentration in venous blood, the majority
The sickle cell gene is also common in areas around the Mediterranean, of the cells do not sickle during their journey through the circulation
the Middle East, India, Nepal, and in geographic regions in which because they reach the lungs and are reoxygenated before significant
there has been migration from endemic areas, such as North, Central, polymerization and cell distortion occur. The length of delay depends
11
and South America. Sickle cell anemia occurs in 0.3–1.3%, and sickle highly on temperature, pH, ionic strength, and oxygen tension in the
cell trait occurs in 8–10% of African Americans. cell’s environment. Hypoxia, acidosis, hypertonicity, and temperatures
It is interesting to note that geographic areas with the highest higher than 37°C promote deoxygenation and the formation of HbS
frequency of sickle cell genes are also areas where infection with polymers. The spleen, kidney, retina, and bone marrow provide a suf-
Plasmodium falciparum is common. This correlation strongly ficiently hypoxic, acidotic, and hypertonic microenvironment to pro-
suggests that HbS in heterozygotes confers a selective advantage mote HbS polymerization and sickling.
against fatal malarial infections, resulting in an increase in the gene
★ tabLe 13-2 Summary of the Most Common Hemoglobin Variants
Hb Peripheral blood, Homozygotes Hb Present on electrophoresis mutation Lys geographic distribution
HbS Normocytic, normochromic anemia; homozygous: hbS, f, A 2 b6(A3) glu S Val Tropical Africa and mediterranean
reticulocytosis; poikilocytosis with heterozygous: hbA, S, f, A 2 areas, middle East, India, Nepal
sickled and boat-shaped cells
HbE microcytic, hypochromic anemia; homozygous: hbE, f, A 2 b26(B8) glu S Lys Burma, Thailand, Cambodia,
target cells malaysia, Indonesia
heterozygous: hbA, E, f, A 2
HbC Normocytic, normochromic anemia homozygous: hbC, f, A 2 b6(A3) glu S Lys West Africa
with reticulocytosis; poikilocytosis
heterozygous: hbA, C, f, A 2
with folded, irregularly contracted
cells; target cells
M13_MCKE6011_03_SE_C13.indd 236 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 237
Sickling (delay time) also depends on intracellular hemoglobin Oxygen affinity of Hbs
composition (the proportion of HbA, HbS, HbA , and HbF present) The oxygen affinity of HbS differs from that of HbA, resulting in
2
as well as total hemoglobin concentration (MCHC). Non-S hemo- important physiologic changes in vivo. HbS has decreased oxygen
globins increase the delay time for polymerization, presumably by affinity, and the 2,3-BPG level of homozygotes is increased. This
interfering with the HbS polymerization process. Altering erythro- decreased oxygen affinity, depicted by a shift to the right in the
cyte contents by increasing the concentration of HbF forms the basis oxygen dissociation curve, facilitates the release of more oxygen to
of an important treatment for SCA. The delay time is also inversely the tissues. However, this phenomenon increases the concentration of
related to the total hemoglobin concentration. The more concentrated deoxyhemoglobin S, promoting the formation of sickle cells.
the hemoglobin solution is within the cell (the higher the MCHC),
the shorter is the delay time and the greater is the potential for HbS Red blood cell (Rbc) destruction
aggregates to form. Using this concept, attempts are made to treat the The primary cause of anemia in sickle cell anemia is extravascular
disease by hydrating the cells, which would decrease the MCHC and hemolysis. Erythrocyte survival depends on intracellular HbF
prevent sickling. concentration and degree of membrane damage. Changes in
16
Polymerization of deoxyhemoglobin S begins when the oxygen the erythrocyte membrane resulting in increased fragility coupled
saturation of hemoglobin falls below 85% and is generally complete with Heinz body formation from denatured HbS lead to increased
at about 38% oxygen saturation. The HbS aggregates cause the membrane shedding (vesiculation), a decreased cell surface area, and
erythrocyte to become rigid and less deformable. With repeated the removal of the cell by the mononuclear phagocyte system. The life
17
cycles of deoxygenation, polymerization, and sickling, disruption of span of circulating HbS erythrocytes can decrease to as few as 14 days.
cation homeostasis occurs, resulting in an increase in intracellular The sluggish blood flow and the hypoglycemic, hypoxic environment of
++
+
Ca and loss of K and water from the cell. This cellular dehydration the spleen promotes HbS polymerization and sickling, further slowing
increases the MCHC, predisposing the cell to sickling on subsequent blood circulation in the splenic cords and enhancing phagocytosis of
deoxygenation. In addition, the increased MCHC is associated with an erythrocytes containing HbS. Eventually, however, the spleen loses its
increase in cytoplasmic viscosity and a decrease in cell deformability. functional capacity as repeated ischemic crises (see “Vaso-Occlusive
HbS is also prone to oxidation. Oxidation of membrane proteins and Crisis”) lead to splenic tissue necrosis and atrophy. With splenic atro-
lipids weakens critical skeletal associations. Repeated sickling tends phy, other cells of the mononuclear phagocyte system in the liver and
to decouple the lipid bilayer from the membrane skeleton, and loss bone marrow take over the destruction of these abnormal cells.
of membrane phospholipid asymmetry occurs. The aggregates also
damage the erythrocyte membrane directly, and with the weakened Clinical Findings
cytoskeleton, the cell’s fragility increases. The first clinical signs of sickle cell anemia appear at about 6 months of
age when the concentration of HbS predominates over HbF. Clinical
irreversibly sickled cells manifestations result from chronic hemolytic anemia, vaso-occlusion
The sickled erythrocyte can return to a normal biconcave shape upon of the microvasculature, overwhelming infections, and acute splenic
reoxygenation of the hemoglobin; however, as described above, with sequestration.
repeated cycles of sickling, the erythrocyte membrane undergoes
changes that cause it to become leaky and rigid. After repeated sickling anemia
episodes, the cells become irreversibly sickled cells (ISCs), which A moderate to severe chronic anemia as the result of extravascular
are locked in a sickle shape whether oxygenated or deoxygenated (i.e., hemolysis is characteristic of the disease. Gallstones, a complication
regardless of polymerization state of the hemoglobin molecules). This of any chronic hemolytic disorder, are commonly found due to
is due to abnormal interactions of red cell skeletal proteins, most likely cholestasis and increased bilirubin turnover. Folate deficiency due to
++
caused by oxidative damage and increased Ca concentrations. The increased erythrocyte turnover can further exacerbate the anemia,
ISC is the result of permanent alterations of the submembrane skel- producing megaloblastosis (Chapter 15). Over time, iron overload
13
etal lattice. From 5–50% of the circulating erythrocytes in sickle cell and resultant complications, can develop.
anemia are ISCs. This varies from person to person but is relatively Hemodynamic changes occur in an attempt to compensate for
constant for a given individual. The ISCs have a very high MCHC and the tissue oxygen deficit; as a result, symptoms of cardiac overload
a low MCV. They are ovoid or boat shaped with a smooth outline and including cardiac hypertrophy, cardiac enlargement, and eventually
lack the spicules characteristic of deoxygenated sickled cells. congestive heart failure are frequent complications of the disease.
The ISCs are removed by mononuclear phagocytes in the The hyperplastic bone marrow, secondary to chronic hemolysis,
spleen, liver, or bone marrow. ISCs account for most, if not all, of is accompanied by bone changes, such as thinning of cortices and a
the sickle forms on a peripheral blood smear (most reversible sickle “hair-on-end” appearance in x-rays of the skull. Hyperplasia results
cells regain a discocyte shape when the blood is exposed to air while from a futile attempt by the marrow to compensate for premature
making the slide). The ISCs can also initiate or increase the severity erythrocyte destruction. Conversely, aplastic crises can accompany
of vaso-occlusive crises because of impaired cell deformability and or follow viral, bacterial, and mycoplasmal infections. This temporary
increased cell adherence to vascular endothelium. The mechanism for cessation of erythropoiesis in the face of chronic hemolysis leads to
this interaction between sickled cells and the endothelium is unclear an acute worsening of the anemia. The aplasia can last from a few
but could be related to changes in the surface properties of sickled cells days to a week and because of the significantly decreased red cell life
and endothelial cells as well as plasma factors. 14,15 span, can induce a catastrophic fall in the hemoglobin concentration.
M13_MCKE6011_03_SE_C13.indd 237 01/08/14 12:43 pm
238 SECTION III • ThE ANEmIAS
Increasing evidence suggests that many cases of aplasia occur as a Recurrent occlusive episodes can lead to infarctions of tissue
result of infection with human parvovirus B19. Parvovirus also causes of the genitourinary tract, liver, bone, lung, and spleen. The chronic
a cessation of erythropoiesis in normal individuals, but with a normal organ damage is accompanied by organ dysfunction. Although
RBC life span, normal blood cell production in these individuals is splenomegaly is present in early childhood, repeated splenic infarc-
restored before any clinically significant changes in erythrocyte tions eventually result in splenic fibrosis and calcifications (usually
concentration take place. by age 4 or 5). This organ damage, secondary to infarction, is known
as autosplenectomy. As a result, splenomegaly is rare in adults
vaso-Occlusive crisis with this disease. Aseptic necrosis of the head of the femur is com-
HbS cells are poorly deformable. Sickled cells have difficulty mon. Dactylitis, a painful symmetrical swelling of the hands and feet
squeezing through small capillaries, and consequently, the rigid (hand-foot syndrome) caused by infarction of the metacarpals and
cells tend to aggregate in the microvasculature, increasing vascular metatarsals, is often the first sign of the disease in infants. Recurrent
stasis. Erythrocytes behind the blockage release their oxygen to the priapism is a characteristic, painful complication that occasionally
surrounding hypoxic tissue, deoxygenate, polymerize, and sickle, requires surgical intervention.
increasing the plug’s size (Figure 13-2 ■). Erythrocytes from nearby The slow flow of blood in occlusive areas can lead to
capillaries are forced to give up more oxygen than they normally thrombosis. Thrombosis of the cerebral arteries resulting in stroke
would to feed the oxygen-deprived tissue around the blockage. These is common. Magnetic resonance imaging shows evidence of sub-
cells then form rigid aggregates of deoxyhemoglobin S, expand- clinical cerebral infarction in 20–30% of children with sickle cell
ing the blockaded region. If severe, lack of oxygen can cause local anemia. If the arterioles of the eye are affected, blindness can
tissue necrosis. Vaso-occlusion occurs more often in tissues prone to occur. Chronic leg ulcers, found also in other hemolytic anemias,
vascular stasis (spleen, marrow, retina, kidney). can occur at any age. The ulcers appear without any known injury.
The blockage of the microvasculature by rigid sickled cells These painful sores do not readily respond to treatment and can
accounts for the majority of the clinical signs of sickle cell anemia. take months to heal.
The occlusions do not occur continuously but sporadically, causing Placental infarctions in pregnant women with sickle cell disease
acute signs of distress. These episodes are called vaso- occlusive can be a hazard to the fetus. Maternal anemia often becomes more
crises and are the most frequent causes of hospitalization. The crises severe during pregnancy. In addition, other clinical findings can
can be triggered by infection, decreased atmospheric oxygen pressure, be exacerbated during pregnancy, endangering the life of both the
dehydration, or slow blood flow, but frequently they occur without mother and the fetus.
any known cause. The occlusions are accompanied by pain, low-grade
fever, organ dysfunction, and tissue necrosis. The episodes generally bacterial infection
last for 4–5 days and subside spontaneously. Overwhelming bacterial infection is a common cause of death in
young patients, and fever is treated as a medical emergency. The risk of
septicemia from encapsulated microorganisms, such as Streptococcus
pneumoniae and Haemophilus influenzae, is extremely high in chil-
Normal RBC dren with SCA who have not received vaccines against these organisms.
Normal flexible RBC
Bacterial pneumonia is the most common infection, but meningitis is
also prevalent. The reasons for this increased susceptibility to infec-
Normal
tion are not fully understood but could be related to functional asple-
18
nia, impaired opsonization, and abnormal complement activation.
An RBC that has The spleen is particularly important for host defense in the young.
RBC with HbS undergone repeated Significant impairment of in vivo neutrophil adherence to vascular
sickling episodes endothelium in HbS disease also occurs. This could prevent neutro-
becomes stiff. 18
Nonflexible RBC is phils from rapidly relocating to areas of inflammation. Prophylactic
caught up in the micro- penicillin is given to children with sickle cell anemia beginning at age
vasculature three months to reduce morbidity and mortality from infection, but
O 2
Sickled RBCs compliance with therapy may not be optimal. The pneumococcal,
RBCs with HbS behind meningococcal, and H. influenzae type B vaccines protect patients
the nonflexible RBC from infections with S. pneumoniae, Neisseria meningitidis, and H.
give up their oxygen to
the surrounding influenzae, reducing the incidence of infection. Children with sickle
hypoxic tissue causing cell anemia are also routinely immunized for hepatitis B and seasonal
them to sickle influenza.
O 2
As more RBCs with
HbS give up their CheCkpoint 13-4
oxygen a blockade is
formed
O 2 Why do newborns with sickle cell anemia not experience
■ FiguRe 13-2 An illustration showing the way sickle episodes of vaso-occlusive crisis?
cells block a vessel and precipitate a vaso-occlusive crisis.
M13_MCKE6011_03_SE_C13.indd 238 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 239
acute splenic sequestration counts than do normal individuals, especially children 610 years of
21
In young children sudden splenic pooling of sickled erythrocytes age. Platelet counts are also frequently higher than normal. After the
(sequestration crisis) can cause a massive decrease in erythrocyte age of 40, the hemoglobin concentration, reticulocyte count, leukocyte
mass within a few hours. Thrombocytopenia can also occur. Hypo- count, and platelet count decrease. 22
volemia and shock follow. At one time, splenic sequestration was the The blood smear shows variable anisocytosis with polychro-
leading cause of death in infants with sickle cell anemia. Early diagno- matophilic macrocytes and variable poikilocytosis with the presence
sis, instruction of parents in detecting an enlarging spleen, and rapid of sickled cells and target cells. Nucleated erythrocytes can usually be
intervention with transfusion have decreased morbidity and mortality found. The RDW is increased. During and following a hemolytic crisis,
23
associated with splenic sequestration. the RDW increases linearly with increases in reticulocytes. If the
patient is not experiencing a crisis, sickled cells may not be present.
acute chest syndrome In older children and adults, signs of splenic hypofunction
This illness resembling pneumonia is the most common cause of are apparent on the peripheral blood smear with the presence of
death in children with sickle cell disease and the second most common basophilic stippling, Howell-Jolly bodies, siderocytes, and poikilocytes.
19
cause of hospitalization. Clinical findings include cough, fever, chest
pain, dyspnea, chills, wheezing, and pulmonary infiltrates. Hemoglo-
20
bin concentration and oxygen saturation decrease. The etiology of case study
acute chest syndrome is not clear. In children, an infectious agent can
often be identified. Other possible causes include pulmonary edema Admission laboratory data on Shane included:
from overhydration, fat embolism from infarcted bone marrow, and
9
hypoventilation due to pain from rib infarcts or from narcotic analge- WBC 16.4 * 10 /L
12
sics used to combat pain. The long-term effects of recurrent episodes RBC 2.5 * 10 /L
of acute chest syndrome are unknown. Hb 78 g/L
Hct 0.24 L/L
9
iron Overload PLT 467 * 10 /L
Excessive iron storage (hemosiderosis) is observed as a complication Differential: Segs 76%, bands 10%, lymphs 9%, monos
for some patients due to the use of transfusion therapy, erythropoiesis- 3%, eos 1%, basos 1%; RbC morphology: Sickle cells
induced increase in iron absorption, and an overall increase in lon- 3+, target cells 1+, ovalocytes 1+, polychromasia, 3
gevity for patients with SCA. Hepatic fibrosis and cirrhosis related to NRBC/100 WBCs, Howell-Jolly bodies
hemosiderosis can occur. The serum ferritin assay is useful for moni-
toring storage iron levels in patients with SCA and can indicate the 8. Which of Shane’s hematologic test results are
consistent with a diagnosis of sickle cell anemia?
need for iron chelation therapy.
9. What does the presence of polychromatophilic
erythrocytes signify?
case study (continued from page 235) 10. Why is the absolute neutrophil count elevated?
11. What is the significance of ovalocytes on the blood
The chest radiograph showed consolidation in the left smear?
lower lobe, indicating that Shane has pneumonia.
12. What is the significance of Howell-Jolly bodies on the
5. What physiological conditions does Shane have that smear?
could lead to sickling of his erythrocytes?
6. What is the cause of Shane’s pain and acute distress?
7. Why might Shane be more susceptible to pneumonia
than an individual without sickle cell disease?
Laboratory Findings
Peripheral blood
A normocytic, normochromic anemia is characteristic of sickle cell
anemia; however, with marked reticulocytosis, the anemia can appear
macrocytic (Figure 13-3 ■). Reticulocytosis from 10–20% is typical.
The hemoglobin ranges from 6–10 g/dL (60–100 g/L) and the hema-
tocrit from 18–30% (0.18–0.30 L/L). A calculated hematocrit from an
electronic cell counter is more reliable than a centrifuged microhema-
tocrit because excessive plasma trapped by sickled cells in centrifuged ■ FiguRe 13-3 Hemoglobin S disease (sickle cell
specimens falsely elevates the manual hematocrit. anemia). Note abnormal boat-shaped, sickled, and
The Cooperative Study of Sickle Cell Disease revealed that ovoid erythrocytes (peripheral blood; Wright-Giemsa
individuals homozygous for HbS have higher steady-state leukocyte stain; 1000* magnification).
M13_MCKE6011_03_SE_C13.indd 239 01/08/14 12:43 pm
240 SECTION III • ThE ANEmIAS
bone marrow Other diagnostic tests
Bone marrow aspiration shows erythroid hyperplasia, reflecting the IEF in agar gels, CE, and HPLC can be used to identify abnormal
attempt of the bone marrow to compensate for chronic hemolysis. hemoglobins such as HbS. Preferred methods for prenatal screening
Erythrocyte production increases to 4–5 times normal. If the patient and diagnosis use DNA-based analysis (polymerase chain reaction)
is deficient in folic acid, megaloblastosis can be seen. Iron stores are to detect point mutations in globin gene sequences. DNA testing pro-
most often increased but can be diminished if hematuria is excessive. vides a genotype diagnosis and eliminates the need for later neonatal
Bone marrow examination is not usually performed because it yields testing when the phenotype results can be inconclusive. The molecular
no definitive diagnostic information. techniques are discussed in Chapter 42.
Hemoglobin electrophoresis Other Laboratory Findings
The presence of HbS is often confirmed by hemoglobin electrophoresis, Other laboratory findings are less specific. The hemolytic nature of the
although other methods for its detection can be used. Electrophoresis disease causes indirect bilirubin to increase, haptoglobin to decrease,
on cellulose acetate at pH 8.4 shows 80–95% HbS (Table 13-3 ★). and uric acid and serum lactic dehydrogenase (LD) to increase.
HbF ranges from 5–20%. High levels of HbF (25–35%) can indicate Serum ferritin is typically increased. These tests offer no diagnostic
compound heterozygosity for HbS and hereditary persistence of fetal information on sickle cell anemia but can be performed to evaluate
hemoglobin (Chapter 14). HbA is normal. Newborns have 60–80% complicating conditions.
2
HbF with the remainder HbS. In infants 63 months of age with small
amounts of HbS, electrophoresis on citrate agar gel at pH 6.2 permits
more reliable separation of HbF from both HbA and HbS. Citrate agar
gel electrophoresis is also useful in separating HbD and HbG from case study (continued from page 239)
HbS. Both of these nonsickling hemoglobins migrate with HbS on
alkaline electrophoresis with paper or starch gel but migrate with HbA The LD level was reported as 1260 U/L (reference interval
on agar gel electrophoresis at acid pH. 75–200 U/L).
13. What is the significance of Shane’s elevated LD?
solubility test
The solubility test is a rapid test for detecting HbS in the heterozygous
or homozygous state. In severe anemia, the amount of HbS can be
too low to be accurately detected, and the procedure may need to be Therapy
altered. This test should not be used as a screening test for newborns
because of high levels of HbF and the low concentration of HbS in this Disease management and treatment for patients with SCA is extensive
age group. Unstable hemoglobins can give a false positive test if many and multifaceted, and frequent monitoring of patient status using
Heinz bodies are present. Other rare hemoglobin variants (e.g., HbC laboratory assays is needed. Treatment includes both immediate
Harlem, HbI) can also give positive tests. False positive tests can occur and prophylactic or long-term approaches. Immediate transfusion
with elevated plasma proteins and lipids. therapy may be ordered for complications of SCA, including
stroke, vaso-occlusion in other organs, splenic sequestration, acute
sickling test chest syndrome, aplastic crises, and others. Transfusion with units
Another confirmatory test that is performed less often is the sodium from normal donors dilutes the amount of HbS present, restores
metabisulfite slide test for sickling. This test is positive in both sickle oxygenation of tissues, and temporarily suppresses erythropoiesis of
cell anemia and sickle cell trait. new cells containing HbS. Simple transfusion or exchange transfusion
may be used, often with units that are screened for HbS and fully (or
24
partially) antigen matched. Preoperative transfusion is helpful in
★ tabLe 13-3 Hemoglobin Electrophoresis Results preventing the complications of anesthesia-induced sickling. Long-
in Clinical Conditions term transfusion therapy can be useful in preventing complications
Hemoglobin (%) of sickle cell anemia but is somewhat controversial due to potential
side effects and lack of definitive treatment guidelines. Recent data
condition a a2 F s c have shown that transfusion therapy is useful in prevention of stroke
25
Normal adult 97 3 61 0 0 and other complications in children with SCA. Complications of
Normal neonate 20–25 61 75–80 0 0 chronic transfusion therapy include transmission of blood-borne
*SCD (SS) 0 N 5–20 80–95 0 diseases, hyperviscosity, alloimmunization, expense, inconvenience,
S trait (SA) 50–65 N N 35–45 0 and iron overload.
C disease 0 N 67 0 790 Various pharmacologic agents have been used to reduce
C trait (CA) 60–70 N N 0 30–40 intracellular sickling by increasing the level of HbF. Of these, only
SC disease 0 ? ? 750 650 hydroxyurea (HU) has FDA approval for preventing vaso-occlusive
crises in patients with sickle cell anemia. HU is widely used and
*SCD = sickle cell disease
elevates HbF in most HbS-containing erythrocytes by activating
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ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 241
genes controlling g@globin chain production. Children and adults because, statistically, one of four children born to parents each of
respond well to HU, experiencing fewer vaso-occlusive crises and whom has the trait will have sickle cell anemia and two of four will
hospitalizations. HU is an antineoplastic and antimetabolite drug have the trait.
with significant side effects of neutropenia, reticulocytopenia, Complications of splenic infarction and renal papillary necro-
and thrombocytopenia. Monitoring cell counts biweekly is sis have occasionally been reported in affected individuals subjected
recommended. HU is also a potential teratogen, so contraceptive to extreme and prolonged hypoxia such as flying at high altitude in
precautions are recommended. HU does not seem to produce unpressurized aircraft or following general anesthesia.
serious irreversible toxicity and is not associated with long-term Hematologic parameters, including hemoglobin concentration,
26
adverse effects on growth or development in children. Long-term are normal. No sickled cells or other abnormalities are observed
risks of malignancy are unknown. Other pharmacologic agents for on smears. Sickling can be induced with the sodium metabisulfite
treating SCA are being investigated. test, however, and the solubility test is also positive. Hemoglobin
Additional aspects of treatment and patient management of SCA electrophoresis results show 50–65% HbA, 35–45% HbS, normal
include prophylactic penicillin, folic acid, immunizations, hydration, HbF, and normal or slightly increased HbA . If HbA constitutes
2
and analgesics for pain. 650% of the total hemoglobin in sickle cell trait, the patient is
Hematopoietic stem cell transplantation (SCT) affords the probably heterozygous for another hemoglobinopathy such as
potential for the cure of sickle cell disease. Results of multicenter case thalassemia.
series show disease-free survival rates ranging from 80–85% with
the best outcomes obtained when children received stem cells from OtHeR sickLing disORdeRs
HLA-identical siblings. Risk of complications, including graft versus Sickle cell disease/sickle cell disorder (SCD) are terms used to
host disease and neurological problems, is high for patients with describe clinical conditions in which erythrocyte sickling occurs due
sickle cell disease undergoing SCT, especially when nonmyeloabla- to the presence of HbS. Compound heterozygous conditions (geno-
27
tive approaches or cells from unrelated donors are used. Candidates types with two different hemoglobin mutations, heterozygous for
for SCT include children with severe complications of SCA who have each) may be considered SCD with variable clinical consequences.
matched sibling donors. Furthermore, SCT is extremely expensive Some but not all of the clinical problems typical of sickle cell anemia
and can be beyond the financial means of many patients. Gene apply to other SCD. Hydroxyurea and transfusion therapies could be
therapy, in which normal genes are inserted into a patient’s defective needed, depending on disease severity. The combination of b with
S
stem cells and returned to the patient, also holds a promise of cure but some forms of b thalassemia can be quite severe, requiring patient
is not available at this time. Progress in finding a stable gene vector management and therapies comparable to those of sickle cell anemia.
capable of expressing therapeutic levels of normal b@globin chains Hemoglobin electrophoresis and other methods including IEF and
over time has been slow. 28,29
HPLC are needed to confirm the diagnosis of a combined disorder
involving HbS. See “Hemoglobin S/C Disease” and Chapter 14 for
additional information on combined disorders.
CheCkpoint 13-5
Outline the treatment options for a patient with HbS
disease, pneumonia, and vaso-occlusive crisis. Discuss CheCkpoint 13-6
how each would affect the patient’s clinical condition.
A child’s parents both have sickle cell trait. The physi-
cian orders a hemoglobin electrophoresis on the child.
Results of electrophoresis on cellulose acetate at pH 8.4
Sickle Cell Trait show 65% hbS, 30% hbA, 3% hbf, 2% HbA 2 . Explain
S
Sickle cell trait is the heterozygous b state, and typically the patient these results, and suggest further testing that could help
S
A S
has one normal b@gene and one b @gene (b b ). Sickle cell trait is in diagnosis.
not as severe a disorder as sickle cell anemia because the presence
of HbA or other non-S hemoglobins interferes with the process of
HbS polymerization, preventing sickling under most physiologic
conditions. However, sickle cell trait cells can sickle under very low HemOgLObin c disease
oxygen tension (∙15 mmHg). Although some hemoglobins other Hemoglobin C, the second hemoglobinopathy to be recognized, is the
11
than HbS (especially HbC, HbD, HbE) can be incorporated into third most prevalent hemoglobin variant worldwide. The first cases
the polymer of deoxyhemoglobin S, the presence of molecules of of HbC were discovered in the combined heterozygous state with HbS;
these hemoglobins intermixed with the molecules of HbS creates this is not surprising because both hemoglobinopathies are prevalent
a weakened structure and decreases the degree of polymerization. in the same geographic area. Hemoglobin C is found predominantly
Because HbA constitutes 750% of the total hemoglobin, sickle cell in West African blacks in whom the incidence of the trait can reach
trait rarely results in clinical symptoms and physical examinations 17–28% of the population. From 2–3% of African Americans carry the
are normal. However, it is important to diagnose sickle cell trait trait, and 0.02% have the disease.
M13_MCKE6011_03_SE_C13.indd 241 01/08/14 12:43 pm
242 SECTION III • ThE ANEmIAS
then homozygous HbS but more severe than homozygous HbC.
Hb S/C disease is considered a sickling disorder. The concentration
of hemoglobin in individual erythrocytes (MCHC) is increased,
and the concentration of HbS is more than in sickle cell trait (the
C
percentage of HbS is higher than that of HbC because the b @globin
S
has a less negative charge than the b @globin; thus, the cell does not
S
C
form ab @dimers as readily as ab @dimers). The presence of HbC
makes the HbS/C cells more prone to sickling than cells that contain
HbA/HbS because HbC molecules participate in the polymerization
process with HbS molecules more easily than do HbA molecules.
Increased erythrocyte rigidity is noted at oxygen tensions 650 mm
Hg. In addition, cells containing HbS/C have formation of aggregates
30
of intracellular HbC crystals. Thus, in HbS/C disease, both sickling
■ FiguRe 13-4 Hemoglobin C disease. Note the cell in and crystal formation contribute to the pathophysiology of the
the top center with a HbC crystal and target cells (periph- disease.
eral blood; Wright-Giemsa stain; 1000* magnification).
The clinical signs and symptoms of the disease are similar to those
of mild sickle cell anemia, and treatment can be indicated. Because of
the poorly deformable red cells, patients can develop vaso-occlusive
Hemoglobin C is produced when lysine is substituted for glutamic
acid at the sixth position (A3) in the b@chain [b6 (A3) Glu S Lys]. crises leading to the complications associated with this pathology. A
The mutation in the HBB gene is in the same position as the HbS notable difference from sickle cell anemia, however, is that in HbS/C
mutation. Because the substitution is a nonpolar amino acid for disease, splenomegaly is prominent.
a polar amino acid as with HbS, hemoglobin solubility decreases. Mild to moderate normocytic, normochromic anemia is
Intraerythrocytic crystals of oxygenated HbC can be found in the present. The hematocrit is usually >25% (0.25 L/L), and the hemo-
red cells, especially in splenectomized individuals. HbF inhibits the globin concentration is between 10 and 14 g/dL (100 and 140 g/L).
formation of crystals. Crystal formation is enhanced when cells are The higher hemoglobin concentration does not necessarily mean
dehydrated or in hypertonic solutions. Erythrocytes with crystals that hemolysis is less severe than in sickle cell anemia; it could be
become rigid and are trapped and destroyed in the spleen, thereby that the higher oxygen affinity of HbS/C cells results in higher
reducing erythrocyte life span to 30–55 days. erythropoietin levels. Peripheral blood smears reveal a large num-
C C
Hemoglobin C disease (b b ) is usually asymptomatic, but ber of target cells (up to 85%), folded cells, and boat-shaped cells but
patients occasionally experience joint and abdominal pain. In contrast rarely sickled forms ( Figure 13-5 ■). Typical HbC crystals are rarely
to sickle cell anemia, the spleen is most often enlarged. Variable found. Some erythrocytes contain a single eccentrically located,
hemolysis results in a mild to moderate anemia. densely stained, round mass of hemoglobin that makes part of the
The hemoglobin ranges from 8–10 g/dL (80–120 g/L) and the cell appear empty. These cells have been referred to as billiard-ball
30
hematocrit from 25–35% (0.25–0.35 L/L). The anemia is accompanied cells. Anisocytosis and poikilocytosis range from mild to severe.
by a slight to moderate increase in reticulocytes. The stained blood Small, dense, misshapen cells, some with crystals of various shapes
31
smear contains small cells that appear to be folded and irregularly jutting out at angles, have been referred to as HbSC poikilocytes.
contracted as well as many target cells (Figure 13-4 ■). Intracellular Hemoglobin electrophoresis shows a higher concentration of HbS
hemoglobin crystals can be found if the smear has been dried slowly.
Microspherocytes are occasionally present.
On cellulose acetate at an alkaline pH, HbC migrates with
HbA , HbE, and HbO-Arab. HbC can be separated from these other
2
hemoglobins by agar gel electrophoresis at an acid pH (Figure 13-1).
For individuals with HbC disease, electrophoresis on citrate agar gel
at acid pH demonstrates 790% HbC with a slight increase in HbF
(not 77%).
A
C
Hemoglobin C trait (b /b ) is asymptomatic. No hematologic
abnormalities are produced except that target cells are noted on
blood smears. Mild hypochromia can be present. About 60–70% of
the hemoglobin is HbA and 30–40% is HbC.
HemOgLObin s/c disease ■ FiguRe 13-5 Hemoglobin S/C disease. Notice the
elongated cells, the cell with hemoglobin contracted to
In hemoglobin S/C disease, both b@chains are structurally one side of the cell (billiard ball cell; arrow), and boat-
S
abnormal. One b@gene codes for b @chains, and the other gene shaped cells. The small contracted cells are typical of
S C
C
codes for b @chains (b b ); thus, HbA is absent. The combined those seen in hemoglobin C disease (peripheral blood;
heterozygous state for HbS and HbC results in a disease less severe Wright-Giemsa stain; 1000* magnification).
M13_MCKE6011_03_SE_C13.indd 242 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 243
than HbC. HbF can be increased up to 7%. No HbA is found due to CheCkpoint 13-8
the absence of normal b@chains.
A 13-year-old black female had a routine physical. Her
CBC was normal, but the differential revealed many
CheCkpoint 13-7 target cells. Hemoglobin electrophoresis revealed a
band that migrated like HbS on cellulose acetate at pH
What is the functional abnormality of HbC and HbS?
Why do these two abnormal hemoglobins have the same 8.4. Her hemoglobin solubility test was negative. Explain
the results and suggest a follow-up test to determine a
altered functions?
diagnosis.
HemOgLObin d HemOgLObin e
Hemoglobin D (HbD) is the result of several molecular variants
of which the identical variants HbD Punjab and HbD Los Angeles Hemoglobin E is the second most prevalent hemoglobinopathy
11
(b121[GH4] Glu S Gln) are the most common. HbD migrates with worldwide. It is most often encountered in individuals from south-
HbS on hemoglobin electrophoresis at alkaline pH; however, the HbD east Asia. The trait has reached frequencies of almost 50% in areas of
molecules do not sickle and have normal solubility properties. HbD Thailand. It is estimated that 15–30% of immigrants from southeast
variants are found in various ethnic groups including Indians and Asia living in North America have HbE with the highest frequencies
African Americans. An uncommon a@chain variant, HbG Philadel- occurring in those from Cambodia and Laos. Although found mainly
phia, is also included in this group due to its similar electrophoretic in Asians, the HbE trait can also occur in blacks.
properties. The rarely observed HbD homozygous state is associated Hemoglobin E is the result of a substitution of lysine for glutamic
with a mild hemolytic anemia and the presence of target cells, but the acid in the b@chain (b26[B8] Glu S Lys). The hemoglobin is slightly
heterozygous state is asymptomatic. In homozygous HbD, electropho- unstable when subjected to oxidant stress. Also, the nucleotide substitu-
resis on cellulose acetate at pH 8.4 demonstrates about 95% HbD with tion creates a potential new splicing sequence so that some of the mRNA
the same electrophoretic mobility as HbS. Electrophoresis on citrate may be improperly processed. As a result, synthesis of the abnormal
agar at pH 6.0 allows separation of HbS and HbD. At acid pH, HbD b@chain is decreased, and HbE trait and disease have some thalassemia-
migrates with HbA. like characteristics including an increased ratio of a:non@a@chain syn-
Although rare, the combined heterozygous state of HbD thesis and a@chain excess (Chapter 14). The oxygen dissociation curve
and HbS exists. HbD molecules can interact with HbS molecules, is shifted to the right, indicating that HbE has decreased oxygen affinity.
producing aggregates of deoxyhemoglobin (Figure 13-6 ■). This Homozygous HbE is characterized by the presence of a
produces a relatively mild form of sickle cell anemia. HbD is also mild, asymptomatic, microcytic hypochromic anemia (similar to
found in combination with b@thalassemia, resulting in a more seri- thalassemia) with decreased erythrocyte survival (Figure 13-7 ■).
ous clinical condition than when HbD is combined with a normal Target cells are prominently observed on smears. Electrophoresis
2
b allele. Compound heterozygotes can be recognized by results of demonstrates mostly HbE (90% or more) with the remainder HbA
2
electrophoresis and erythrocyte indices. and HbF. On alkaline electrophoresis, HbE migrates with HbA , HbC,
and HbO-Arab. On agar gel at an acid pH, HbE migrates with HbA.
HbE trait is asymptomatic, and hematologic parameters are
normal except for slight microcytosis. Hemoglobin electrophoresis at
■ FiguRe 13-6 A blood film from a patient with hemo-
globin D/S. Homozygous HbD does not usually cause
anemia, but when combined with HbS, it potentiates ■ FiguRe 13-7 A blood film from a patient with homozy-
the aggregating of deoxyhemoglobin and sickling of gous hemoglobin E (HbEE). Note the microcytosis (com-
erythrocytes, producing a mild sickle cell anemia. Notice pare red cell size to that of the nucleus of the lymphocyte)
the boat-shaped cells and target cells (peripheral blood; and prominent target cells (peripheral blood; Wright-
Wright-Giemsa stain; 1000* magnification). Giemsa stain; 1000* magnification).
M13_MCKE6011_03_SE_C13.indd 243 01/08/14 12:43 pm
244 SECTION III • ThE ANEmIAS
alkaline pH shows about 35–45% HbE. The remainder is HbA with 5. Replacement of a hydrophobic residue with a more hydrophilic
normal HbA and HbF. amino acid in the hydrophobic pocket, disrupting the conforma-
2
Combined heterozygosity for HbE and forms of thalassemia tion of the hydrophobic heme cleft
are commonly observed in some population groups. The degree of Unstable hemoglobin denatures and precipitates as Heinz bodies,
clinical severity is variable and depends on specific genotype. See which attach to the inner surface of the membrane, thereby decreasing
Chapter 14.
cell deformability. The inclusions are pitted by macrophages in the spleen,
leaving the cell with less hemoglobin and decreased membrane. This
process leads to rigid cells and their premature destruction in the spleen.
CheCkpoint 13-9
In addition to altering the molecule’s stability, disruption of the
The red cell morphology in HbE disease and b@thalassemia normal conformation also can affect the molecule’s function. Many
are similar: microcytic, hypochromic anemia with target of the unstable hemoglobins also have altered oxygen affinity. If the
cells. What laboratory test(s) could differentiate these two amino acid substitution is strategically located to affect the oxygen-
conditions? binding site, oxygen affinity can be increased or decreased. In addition,
some unstable hemoglobin variants have a tendency to spontaneously
oxidize to methemoglobin.
unstabLe HemOgLObin vaRiants Clinical Findings
Unstable hemoglobins can result from structurally abnormal globin Congenital hemolytic anemia indicative of an unstable hemoglobin
chains, and 7140 unstable variants have been described. The disorder requires further investigation to establish a definitive
abnormal chains contain amino acid mutations at critical internal diagnosis. Family history is extremely important in defining the heredi-
32
portions of the chains, which affect the molecular stability. The tary nature of the disease. Patient history can also provide information
disorders are characterized by denaturation and precipitation of about the nature of events such as infection or drug administration
the abnormal hemoglobin in the form of Heinz bodies, causing cell that precipitate acute hemolytic episodes. The severity of the disorder
rigidity, membrane damage, and subsequent erythrocyte hemoly- can range from asymptomatic to a chronic severe hemolytic anemia.
sis. Although hemoglobin denaturation and hemolysis can occur The severity of the anemia depends on the degree of instability
spontaneously, symptoms associated with acute hemolysis usually of the hemoglobin tetramer and the change, if any, in oxygen affinity.
occur after drug administration, infection, or other events that change For example, Hb Köln is an unstable hemoglobin (b@chain mutation)
the hemoglobin molecule’s normal environment. Clinical conditions that also demonstrates increased oxygen affinity. Hemoglobins with
associated with unstable variants are known as unstable hemoglobin increased oxygen affinity (hemoglobin-oxygen dissociation curve is
disorders or congenital Heinz body hemolytic anemias. shifted left) release less oxygen to the tissues, which results in relatively
higher levels of tissue hypoxia and of erythropoietin as well as higher
Pathophysiology hematocrits than expected relative to the severity of hemolysis.
Most affected individuals with unstable hemoglobins are heterozygous. Conversely, unstable hemoglobins with decreased oxygen affinity (e.g.,
Most unstable hemoglobins are inherited as autosomal- dominant Hb Hammersmith) have a hemoglobin-oxygen dissociation curve
disorders, but a large number arise from spontaneous mutations that is shifted right, increasing the oxygen delivery to the tissues and
with no evidence of hemoglobin instability in parents or other family allowing patients to function at a lower hemoglobin concentration.
members. When the oxygen affinity of an unstable hemoglobin is decreased, the
Unstable hemoglobin variants can result from a variety of reticulocyte count is not increased as much as would be expected for
globin chain amino acid substitutions or deletions that disrupt the degree of anemia relative to an uncomplicated hemolytic anemia.
the stability of the globin subunit or hemoglobin tetramer, or the Most clinical findings occur as the result of increased erythro-
binding of heme to globin. Such mutations resulting in unstable cyte hemolysis. Jaundice and splenomegaly are common when there
hemoglobin variants include: is chronic extravascular hemolysis. Cyanosis can result from the for-
mation of sulfhemoglobin and methemoglobin that accompanies
1. Mutation of a globin amino acid that is involved in contact with hemoglobin denaturation. Weakness and jaundice frequently follow
the heme group or that results in a tendency to dissociate heme the administration of oxidant drugs, which increase the hemoglobin’s
from the abnormal globin chains instability. Acute hemolysis can also be accompanied by the excretion
2. Replacement of nonpolar by polar amino acids at the interior of of dark urine due to the presence of dipyrroles in the urine.
the molecule resulting in a distortion of the folding of the globin
chain Laboratory Findings
3. Deletion or insertion of additional amino acids, particularly in the Peripheral blood
helical regions of the molecule, creating instability The anemia of congenital hemolytic anemia involving unstable
4. Mutations of amino acids at intersubunit contacts (especially the hemoglobin variants is usually normocytic and normochromic. Occa-
b contact points), creating instability and a tendency to disso- sionally, MCV and MCH are slightly decreased because of the removal
a 1 1
ciate into monomers of Heinz bodies from erythrocytes and loss of hemoglobin in the spleen.
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ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 245
The reticulocyte count is typically increased. The blood smear can show state. Other substitutions that affect oxygen affinity include those near
basophilic stippling, pitted cells (bite cells), and small contracted cells. the a b contacts, at the C terminal end of the b@chain, and near the
1 2
If splenic function is efficient, Heinz bodies are not detectable in 2,3-BPG binding site. These are critical sites involved in the allosteric
peripheral blood cells. Heinz bodies can be found in the peripheral properties of hemoglobin and/or in the physiologic regulation of hemo-
blood following splenectomy; however, this finding is not specific globin affinity for oxygen. Either the a@ or b@chain can be affected, but
for unstable hemoglobin disorders. Heinz bodies can also be seen most identified substitutions are associated with the b@chain.
in erythrocyte enzyme abnormalities that permit oxidation of Increased oxygen affinity results in congenital erythrocytosis, a
hemoglobin, in thalassemia, and after administration of oxidant drugs compensatory mechanism for the reduced release of oxygen to the
in normal individuals. Heinz bodies of unstable hemoglobins can be tissues. Hemoglobinopathies causing permanent methemoglobin
generated in vitro by incubation of the erythrocytes with brilliant formation are associated with pseudocyanosis, whereas cyanosis
cresyl blue or other redox agents. These intracellular inclusions cannot characterizes hemoglobins with decreased oxygen affinity.
be observed on smears stained with Wright’s stain.
Hemoglobin Variants with
Other Laboratory Findings Increased Oxygen Affinity
Many unstable hemoglobins identified to date have the same charge High-affinity hemoglobins are inherited as autosomal dominant traits.
and electrophoretic mobility as normal hemoglobin. Only about 45% All such hemoglobins discovered have been in the heterozygous state.
can be identified by electrophoresis. Hemoglobin A and HbF are The variants can result from amino acid substitutions that involve the
2
sometimes increased, which could suggest the presence of an abnormal a b contacts. Mutations alter the quaternary structure of hemoglo-
1 2
hemoglobin when it is not detected by its electrophoretic pattern. bin to favor the R (oxy) state by either stabilizing this conformation
The presence of an unstable hemoglobin (i.e., one that precipitates
more readily than normal hemoglobin) may be demonstrated by or destabilizing the T (deoxy) state. Other substitutions affect the
C-terminal end of the b@chain, which is important in maintaining the
screening tests, including the heat denaturation test or isopropanol stability of the T form. A few substitutions affect the 2,3-BPG binding
precipitation test. When a hemoglobin solution is heated to 50°C sites, which alter oxygen affinity when bound to hemoglobin.
for 1 hour, an unstable hemoglobin variant will show precipitation. The high-affinity hemoglobins bind oxygen more readily than
Normal hemoglobins will not precipitate. An unstable hemoglobin normal and retain more oxygen at lower PO levels, which results
2
is also precipitated within 20 minutes in the isopropanol (17% by in a shift to the left of the oxygen dissociation curve. The P of the
50
volume) precipitation test. Normal hemoglobin remains in solution hemoglobin is decreased to 12–18 mm Hg, meaning that less oxygen
30–40 minutes. More definitive identification of unstable hemoglobin is released to tissues at the tissue PO of ∙26 mm Hg. The resulting
2
variants can be made by using isoelectric focusing, HPLC analysis of tissue hypoxia stimulates erythropoietin release and, subsequently,
globin chains, or genetic testing for globin gene abnormalities.
formation of a compensatory increased erythrocyte mass. In addition
to the primary effect of increasing oxygen affinity, secondary effects of
CheCkpoint 13-10 the mutations, including hemoglobin instability, reduced Bohr effect,
and reduced cooperativity of the oxygen binding, can also occur. 32
a. Explain why patients with an unstable hemoglobin Erythrocyte counts and hematocrit levels are increased, and hemo-
variant usually experience acute hemolysis only after globin levels are increased to about 20 g/dL (200 g/L). Other hemato-
administration of certain drugs or with infections. logic parameters are normal. About half of the hemoglobin variants have
b. A patient is suspected of having a congenital Heinz an altered electrophoretic mobility, enabling diagnosis by starch gel or
body hemolytic anemia but hemoglobin electrophoresis cellulose acetate electrophoresis. Diagnosis is established by measuring
is normal. Why is it necessary to perform additional oxygen affinity (determining the P of the patient’s hemoglobin).
50
tests? Individuals with these hemoglobin variants are asymptomatic. A
ruddy complexion is occasionally apparent as a result of the erythro-
cytosis. The importance of identifying the presence of a high-affinity
Therapy hemoglobin is for the differential diagnosis and exclusion of other
causes of erythrocytosis and the avoidance of unnecessary and expen-
A wide variation in the clinical presentation of patients with unstable sive diagnostic and therapeutic interventions.
hemoglobin variants exists, and many do not require therapy. Sple-
nectomy can be performed if hemolysis is severe. Patients are advised
to avoid oxidizing drugs, which can precipitate a hemolytic episode. Hemoglobin Variants with
Decreased Oxygen Affinity
HemOgLObin vaRiants witH Low oxygen-affinity hemoglobins result from mutations that stabilize
or favor the deoxygenated (T) conformation of the hemoglobin mol-
aLteRed Oxygen aFFinity ecule or destabilize the oxygenated (R) form. These mutations impair
Amino acid substitutions in the globin chains near the heme pocket can oxygen binding or reduce heme–heme subunit interactions (coop-
affect the hemoglobin’s ability to carry oxygen by preventing heme from erativity). Low oxygen-affinity hemoglobins are often associated with
binding to the globin chain or by stabilizing iron in the oxidized ferric mutations involving the a b contact points, which are involved in
1 2
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246 SECTION III • ThE ANEmIAS
intramolecular movement as hemoglobin goes from the oxy (R) to not occur until about the sixth month after birth when HbA (a b )
2 2
the deoxy (T) state. The low-affinity variants result in a right-shifted becomes the major hemoglobin.
oxygen dissociation curve. The presence of methemoglobin imparts a brownish color
Most low oxygen-affinity hemoglobins possess enough oxygen to the blood. Except for this abnormal color, no other hematologic
affinity to become fully saturated in the lungs but at the capillary PO abnormality is present because methemoglobin levels are rarely higher
2
in the peripheral tissues, they deliver higher than normal amounts of than 30%. Patients with methemoglobinemia appear to be cyanotic,
oxygen (i.e., become more unsaturated). Two physiologic effects result: but unlike truly cyanotic people, arterial blood PO levels are usually
2
normal (i.e., pseudocyanosis).
• Because oxygen delivery is so efficient, oxygen requirements of Hemoglobin M does not always separate from HbA at an
the tissues can be met by lower than normal hemoglobin con- alkaline pH. Hemoglobin electrophoresis on agar gel at pH
centration, producing a pseudoanemia (hemoglobin lower than 7.1 of a blood sample containing HbM reveals a brown band
“normal”).
(HbM) running anodal to a red band (HbA). The pattern can
• The amount of deoxygenated hemoglobin in the capillaries and appear sharper with IEF. Oxidizing the erythrocyte hemoly-
veins is increased, resulting in cyanosis. However, no adverse clini- sate with ferricyanide before electrophoresis reveals a sharp
cal effect is associated with this cyanosis. Patients are asymptomatic separation of congenital HbM and methemoglobin formed from
and require no treatment. HbA. Methemoglobin formed from HbA by oxidation of iron
Diagnosis requires determining the P (the PO at which has no change in molecular charge and therefore has the same
2
50
hemoglobin is 50% saturated). HPLC can also be useful in identifying electrophoretic mobility as HbA.
hemoglobin variants with low oxygen affinity. Although HbM can be detected by spectral abnormalities of the
hemoglobin (absorption peaks at ∙630 and 502 nm), methemoglobin
Methemoglobinemias formed in NADH-diaphorase deficiency has a normal absorption
spectrum. In addition, methemoglobin formed as the result of
Methemoglobin is hemoglobin with iron oxidized to the ferric state, NADH-diaphorase deficiency can be readily reduced by incubation
which cannot carry oxygen. Methemoglobinemia is a clinical con- of blood with methylene blue whereas methemoglobin caused by a
dition that occurs when methemoglobin encompasses 71% of the structural hemoglobin variant such as HbM does not reduce with the
hemoglobin (Chapter 6). This condition can occur as a result of either methylene blue. To confirm NADH-diaphorase deficiency, a quanti-
acquired or inherited defects in the methemoglobin reductase system tative assay of the enzyme activity is necessary. Refer to Table 13-4 ★
or as a result of the presence of a structurally abnormal globin chain. for a summary of differentiation of types of methemoglobinemia (see
In the latter case, the defect results in increased formation of methe- also Chapter 6, Table 6-6).
moglobin by rendering the molecule relatively resistant to reduction No treatment for the HbM structural variants exists because
by methemoglobin reductase. the abnormal hemoglobin resists reduction. However, most patients
This structural variant of hemoglobin is called hemoglobin M are asymptomatic and require no management. Most patients have
(HbM). Nine variants of it have been described; all variants have methemoglobin levels below the level at which symptoms of oxygen
been found only in the heterozygous state. Most HbM variants are deprivation occur.
produced by a tyrosine substitution for the proximal or distal his-
tidine in the heme pocket of the a@ or b@chains. Tyrosine forms a
covalent link with heme iron, stabilizing the iron in the ferric state. CheCkpoint 13-11
The stabilized, oxidized iron is relatively resistant to reduction by the
methemoglobin reductase pathway. If the substitution occurs in the Why should red cell enzyme assays and hemoglobin
a@chain, cyanosis (or actually pseudocyanosis) is present from birth electrophoresis both be performed on a patient with
because the a@chain is a component of HbF (a g ), the major hemo- congenital cyanosis?
2 2
globin at birth. If the substitution occurs in the b@chain, cyanosis does
★ tabLe 13-4 Causes and Characteristics of Methemoglobinemia
Reduction with
type cause electrophoretic Pattern nadH-diaphorase activity methylene blue
Acquired Hb oxidized at a rate exceeding Normal Normal Yes
capacity of methemoglobin reductase
system
Congenital
Recessive Defect in methemoglobin reductase Normal Decreased Yes
system
Dominant Structural abnormality of hemoglobin (hbm) hbm variant Normal No
M13_MCKE6011_03_SE_C13.indd 246 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 247
Summary
The hemoglobinopathies are a group of chronic hemolytic rigid aggregates and reduced erythrocyte deformability. The
anemias caused by qualitative defects in the globin chains of rigid cells aggregate in the microvasculature, resulting in tissue
hemoglobin. Quantitative defects in globin chain synthesis hypoxia. In HbC disease, the erythrocytes become trapped and
(thalassemias) are discussed in Chapter 14. destroyed in the spleen when intracellular crystals of HbC form.
Qualitative defects are due to genetic mutations that cause generally, these variants produce a normocytic, normochromic
a structural change in the globin chain. Although the mutation anemia with anisocytosis and poikilocytosis characteristic for the
can affect any of the globin chain classes, most clinically specific hemoglobinopathy. The blood smear in HbS disease can
significant mutations affect the b@chain. The mutation can show sickled forms and target cells. HbC disease has numerous
affect the hemoglobin molecule’s solubility, stability, or function target cells and irregularly contracted cells, and the erythrocytes
(oxygen affinity). Hemoglobin electrophoresis can be used to occasionally contain HbC crystals. In the United States, both
detect those mutants in which amino acid mutations cause a HbS and HbC are found in highest frequency among African
change in the hemoglobin molecule’s electrophoretic mobility. Americans. Hemoglobin E is especially prevalent in people
However, not all variants can be detected and identified by elec- from southeast Asia. HbE disease is characterized by a mild,
trophoresis, and other methods such as hplC, IEf , or CE can asymptomatic microcytic anemia.
be used. Other tests detect alterations in hemoglobin solubility Unstable hemoglobin variants produce congenital Heinz
and/or stability or oxygen affinity. molecular techniques are body hemolytic anemia. The hemoglobin denatures in the form
available to help definitively diagnose hemoglobinopathies but of Heinz bodies, decreasing cell deformability and resulting in
are not always necessary for routine diagnostic purposes. membrane damage and premature destruction. Hemoglobin
In the United States, the most common structural variants are variants with increased or decreased oxygen affinity can result
HbS and HbC. Both are characterized by decreased hemoglobin in erythrocytosis or cyanosis, respectively. Hemoglobin variants
solubility and can be detected electrophoretically. Hemoglo- resulting in methemoglobin formation are associated with
bin S has decreased solubility when deoxygenated, forming pseudocyanosis.
Review Questions
Level i 3. Which part of the standard CBC provides the most use-
ful screening information when differentiating between a
1. A patient has been previously diagnosed as being patient diagnosis of sickle cell anemia versus thalassemia?
heterozygous for hemoglobin D. What is an important (Objective 6)
identifying characteristic of this hemoglobin variant?
(Objective 7) A. hemoglobin concentration alone
A. It migrates with HbS on hemoglobin electrophoresis at B. hemoglobin and hematocrit together
alkaline pH.
C. erythrocyte indices
B. Sickled RBCs are typically observed on smears.
D. total WBC count and differential
C. Hemoglobin electrophoresis at alkaline pH is needed
for its separation from another common variant. 4. Which homozygous hemoglobinopathy is most consistent
with the following laboratory test results? (Objective 7)
D. It causes severe hemolytic anemia with many target
cells in heterozygotes. Hemoglobin level mildly decreased, MCV decreased,
target cells and hypochromia observed on smear, a large
2. Which of the following laboratory assays are useful in band in the A 2 region on alkaline electrophoresis
recognizing and confirming a diagnosis for patients with
hemoglobinopathies? (Objective 6) A. hemoglobin C
1. Hemoglobin electrophoresis B. hemoglobin D
2. RBC indices C. hemoglobin E
3. RBC morphology
4. Hemoglobin level D. hemoglobin S
5. Chemistry assays including bilirubin, ferritin, and others
A. 1 and 5 only
B. 2 and 4 only
C. 1, 3, and 4 only
D. all of the above
M13_MCKE6011_03_SE_C13.indd 247 01/08/14 12:43 pm
248 SECTION III • ThE ANEmIAS
5. Which of the following is a clinical condition whose associ- 10. A hemoglobin electrophoresis showed 49% HbS, 42%
ated laboratory findings are seen in children with sickle cell HbC, 6% HbF, and 3% HbA 2 . These results are consistent
anemia? (Objectives 7, 8) with a diagnosis of: (Objective 7)
A. vaso-occlusive crisis denoted by the presence of many A. sickle cell anemia
sickled RBCs on smears
B. sickle cell trait
B. erythrocyte aplasia denoted by a rapid increase in
hemoglobin C. hemoglobin S/C disease
D. hemoglobin C trait
C. bacterial infection denoted by the appearance of
Howell-Jolly bodies on smears
Level ii
D. thrombosis denoted by an increase in WBCs and
abnormal differential 1. In which U.S. population group would you expect to
observe the lowest prevalence of clinically significant
6. Hemoglobinopathies are clinical diseases that result from hemoglobinopathies? (Objective 2)
genetically determined abnormalities of the hemoglobin
molecule and include those involving: (Objective 1) A. African Americans
1. heme structure B. Caucasian Americans
2. decreased heme synthesis
3. globin chain structure C. Immigrants from southeast Asia
4. reduced globin chain synthesis D. Immigrants from the mediterranean basin
A. 1 and 3 only 2. An African American 2-year-old is seen in an outpatient
clinic with signs and symptoms consistent with a severe
B. 1 and 2 only
hemoglobinopathy. Based on this presentation, this child
C. 3 and 4 only is most likely homozygous for which hemoglobin variant?
(Objective 5)
D. all of the above
A. HbC
7. Amino acid substitutions on globin chains often alter
the hemoglobin molecule’s charge and mobility. This is B. HbD
the principle of which test for identifying hemoglobins?
(Objective 3) C. HbE
D. HbS
A. HbA 2 quantitation
3. A patient with a long history of anemia is referred to
B. hbf quantitation
a hematologist for diagnosis. This patient recently was
C. hemoglobin electrophoresis treated with a drug for an unrelated condition, but the
therapy was discontinued following an acute episode
D. solubility test of severe anemia. Other family members also appear
8. Tests that quantitate HbF and HbA 2 are useful in detecting to suffer from a similar condition. The hematologist
hemoglobinopathies because: (Objective 6) suspects the patient has an unstable hemoglobin vari-
ant and asks for test suggestions. What laboratory
A. they are the only valid tests available for this results would best support the preliminary diagnosis?
purpose (Objective 10)
B. they are routinely performed in most laboratories A. Heinz bodies observed upon staining, increased
C. hbf and HbA 2 are typically decreased in reticulocytes, bite cells on peripheral blood smear
hemoglobinopathies B. decreased reticulocytes, decreased indices, target
D. hbf and HbA 2 are typically increased in cells on smears
hemoglobinopathies C. Howell-Jolly bodies and sickle cells observed on
9. The presence of many sickled erythrocytes on a peripheral smears
blood smear is most likely to be found in a person who is: D. increased WBC concentration with abnormal
(Objective 7) differential
A. heterozygous for HbC
B. homozygous for HbC
C. heterozygous for HbS
D. homozygous for HbS
M13_MCKE6011_03_SE_C13.indd 248 01/08/14 12:43 pm
ChApTER 13 • hEmOglObINOpAThIES: QuAlITATIvE DEfECTS 249
4. What is an advanced testing method suitable for detecting 7. HbS is an example of a hemoglobin with a globin chain
and identifying hemoglobin variants that you might select mutation that alters hemoglobin: (Objective 3)
for your high-volume laboratory in a large, urban medical
center? (Objective 9) A. function
B. solubility
A. hemoglobin electrophoresis performed at alkaline pH
only C. stability
B. hemoglobin solubility test D. oxygen binding
C. high-performance liquid chromatography 8. Cells containing large amounts of HbS sickle when which
of the following conditions occur? (Objective 4)
D. hemoglobin electrophoresis performed at acid pH
only A. high oxygen tension and acidosis
5. An asymptomatic patient presents with persistent erythro- B. hypoxia and alkalosis
cytosis. The hematologist has ruled out polycythemia vera
and other causes. What is an additional explanation for the C. temperatures 637°C and alkalosis
erythrocytosis, and what laboratory assay might be useful D. temperatures 737°C and hypoxia
in this case? (Objective 11)
9. A hemoglobin electrophoresis on cellulose acetate at pH
A. an unstable hemoglobin variant identified by 8.5 is performed on a 2-year-old African American patient
performing a Heinz body stain with severe anemia. An abnormal band appears halfway
between the HbA and HbA 2 positions on the strip. The
B. a high-affinity hemoglobin variant identified by
isoelectric focusing HbF level is 10%, and the HbA 2 level is normal. No HbA is
present. What is the most likely identity of the abnormal
C. a crystalizable hemoglobin variant demonstrated by hemoglobin? (Objective 7)
observing hemoglobin C crystals on a smear
A. HbC
D. a quantitative decrease in globin chain produc-
tion indicated by observing an increased level of B. HbS
hbf C. HbD
6. Rank the following hemoglobinopathies on the basis of quan- D. HbE
tity of HbS starting with the lowest amount. (Objective 1) 10. The presence of HbE disease in an adult is best confirmed
1. adult with sickle cell disease using which routine laboratory test? (Objective 8)
2. adult with sickle cell trait
3. neonate with sickle cell disease A. hemoglobin electrophoresis
A. 3, 2, 1 B. CBC and peripheral blood smear
B. 2, 3, 1 C. solubility test
C. 1, 2, 3 D. PCR for molecular defect
D. 2, 1, 3
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. find additional information to help
organize information and figures to help understand concepts.
References
1. Weatherall DJ, Clegg Jb. genetic disorders of hemoglobin. Semin 6. Clarke gm, higgins TN. laboratory investigation of hemoglobinopathies
Hematol. 1999;36(4)(suppl 7):24–37. and thalassemias: Review and update. Clin Chem. 2000;46:1284–90.
2. modell b, Darlison m. global epidemiology of haemoglobin disorders and 7. Abou-Diwan C, Young AN, molinaro RJ. hemoglobinopathies and clinical
derived service indicators. Bull World Health Organ. 2008;86:480–487. laboratory testing. Med Lab Obs. 2009 41:8–18.
3. Herrick JB. Peculiar elongated and sickle-shaped red blood corpuscles in a 8. houtovsky A, hadzi-Nesic J, Nardi mA. hplC retention time as a diag-
case of severe anemia. Trans Assoc Am Physicians. 1910;25:553–61. nostic tool for hemoglobin variants and hemoglobinopathies: a study
4. Pauling L, Itano HA, Singer SJ et al. Sickle cell anemia: A molecular disease. of 60,000 samples in a clinical diagnostics laboratory. Clin Chem.
Science. 1949;110:543–48. 2004:50:1736–47.
5. huisman Th, Carver mf, baysal E et al. A database of human hemoglobin 9. u.S. preventive Services Task force. Screening for sickle cell disease
variants and thalassemias. http://globin.cse.psu.edu/globin/hbvar/menu in newborns: u.S. preventive Services Task force recommenda-
.html (accessed march, 1414). tion statement. Rockville (mD): Agency for healthcare Research and
M13_MCKE6011_03_SE_C13.indd 249 01/08/14 12:43 pm
250 SECTION III • ThE ANEmIAS
Quality (AhRQ); 2007 (available at www.guideline.gov; accessed 21. West mS, Wethers D, Smith J et al. laboratory profile of sickle cell disease:
march 14, 2014). a cross-sectional analysis. The cooperative study of sickle cell disease.
10. Old Jm. Screening and genetic diagnosis of haemoglobin disorder. Blood J Clin Epidemiol. 1992;45:893–909.
Rev. 2003;17:43–53. 22. morris J, Dunn D, beckford m et al. The haematology of homozygous sickle
11. flint J, harding Rm, boyce AJ et al. The population genetics of the cell disease after the age of 40 years. Br J Haematol. 1991;77:382–85.
haemoglobinopathies. Baillieres Clin Haematol. 1998;11:1–51. 23. el Sayed hl, Tawfik Zm. Red cell profile in normal and sickle cell diseased
12. Bunyaratvej A, Butthep P, Sae-Ung N et al. Reduced deformability of children. J Egypt Soc Parasitol. 1994;24:147–54.
thalassemic erythrocytes and erythrocytes with abnormal hemoglobins 24. Quirolo K. How do I transfuse patients with sickle cell disease? Transfusion.
and relation with susceptibility to Plasmodium falciparum invasion. Blood. 2010;50:1881–86.
1992;79:2460–63. 25. miller ST, Wright E, Abboud m. Impact of chronic transfusion on incidence
13. Liu SC, Derick LH, Palek J. Dependence of the permanent deformation of pain and acute chest syndrome during the Stroke Prevention Trial (STOP)
of red blood cell membranes on spectrin dimer-tetramer equilibrium: in sickle-cell anemia. J Pediatr. 2001;139:785–89.
implication for permanent membrane deformation of irreversibly sickled 26. Trompeter S, Roberts I. haemoglobin f modulation in childhood sickle cell
cells. Blood. 1993;81:522–28. disease. Br J Haematology. 2008;144:308–16.
14. Ballas SK, Smith ED. Red blood cell changes during the evolution of the 27. Thompson AA. Advances in the management of sickle cell disease. Pediatr
sickle cell painful crisis. Blood. 1992;79:2154–63. Blood Cancer. 2005;46:533–39.
15. mackie lh, hochmuth Rm. The influence of oxygen tension, temperature, 28. persons DA, Nienhuis AW. gene therapy for the hemoglobin disorders:
and hemoglobin concentration on the rheologic properties of sickle past, present, and future. Proc Natl Acad Sci USA. 2000;97(10):5022–24.
erythrocytes. Blood. 1990;76:1256–61. 29. Walters mC. Stem cell therapy for sickle cells disease: transplantation and
16. Steinberg mh. pathophysiology of sickle cell disease. Baillieres Clin gene therapy. Hematology 2005. Washington DC: American Society for
Haematol. 1998;11:163–84. Hematology. 2005:66–73
17. liu SC, Yi SJ, mehta JR et al. Red cell membrane remodeling in sickle cell 30. lawrence C, fabry mE, Nagel Rl. The unique red cell heterogeneity of SC
anemia. J Clin Invest. 1996;97:29–36. disease: crystal formation, dense reticulocytes, and unusual morphology.
18. boghossian Sh, Nash g, Dormandy J et al. Abnormal neutrophil Blood. 1991;78:2104–12.
adhesion in sickle cell anaemia and crisis: relationship to blood rheology. 31. bain bJ. blood film features of sickle cell–haemoglobin C disease.
Br J Haematol. 1991;78:437–41. Br J Haematol. 1993;83:516–18.
19. Ballas SK. Sickle cell disease: Clinical management. Baillieres Clin Haematol. 32. Steinberg mh. hemoglobins with altered oxygen affinity, unstable hemo-
1998;11:185–14. globins, m-hemoglobins, and dyshemoglobinemias. In: greer Jp , foester J,
th
20. Quinn CT, buchanan gR. The acute chest syndrome of sickle cell disease. Rodgers gm, et al., eds. Wintrobe’s Clinical Hematology, 12 ed. Philadel-
J Pediatr. 1999;135:416–22. phia; Wolters Kluwer health/lippincott Williams & Wilkins; 2009.
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14 Thalassemia
Tim R. Randolph, phd
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Define thalassemia. Objectives—Level I and Level II 251
2. Differentiate thalassemias from hemoglobinopathies based on defini-
tion and basic pathophysiology. Key Terms 252
3. Describe the typical peripheral blood morphology associated with Background Basics 252
thalassemia.
4. Compare and contrast the etiology of a@ and b@thalassemia. Case Study 252
5. For each of the four genotypes of a@thalassemia, describe the: Overview 252
a. Number of affected alleles
b. Individuals affected Introduction 252
c. Basic pathophysiology
d. Symptoms a-Thalassemia 257
e. Laboratory results including blood cell morphology and hemoglobin b-Thalassemia 261
electrophoresis
6. For each of the six genotypes of b@thalassemia describe the: Other Thalassemias and Thalassemia-Like
a. Individuals affected Conditions 267
b. Basic pathophysiology Differential Diagnosis of Thalassemia 271
c. Symptoms
d. Laboratory results including blood cell morphology and hemoglobin Summary 272
electrophoresis
Review Questions 272
Objectives—Level II Companion Resources 274
At the end of this unit of study, the student should be able to: References 274
1. List and describe five primary genetic defects found in thalassemias.
2. Compare and contrast a@ and b@thalassemia.
3. Correlate the outcomes in hemoglobin synthesis resulting from the five
genetic defects in thalassemia.
(continued)
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252 SECTION III • ThE ANEmIAS
7. Compare and contrast other thalassemia and thalassemia-
Objectives—Level II (continued) like conditions including:
4. For all four genotypes of a@thalassemia: a. db@thalassemia
a. Correlate all three nomenclature systems: genotype, b. gdb@thalassemia
genotype description, and phenotype. c. hemoglobin Constant Spring
b. Explain advanced pathophysiology. d. hereditary persistence of fetal hemoglobin (hPFh)
c. Describe treatment and prognosis. e. hemoglobin Lepore
5. For all four phenotypes of b@thalassemia: f. Thalassemia/hemoglobinopathy combination
a. List expected genotypes. disorders
b. Explain advanced pathophysiology. 8. Differentiate iron deficiency-anemia and hPFh from thal-
c. Describe treatment and prognosis. assemia based on results of laboratory tests and clinical
6. Correlate clinical severities of both a@ and b@thalassemia findings.
with their respective genotypes.
Key Terms
Allele Double heterozygous Genotype Ineffective erythropoiesis
Compression syndrome Extramedullary erythropoiesis haplotype P 50 value
Crossover Functional hyposplenism heterozygous Phenotype
Diploid Gene cluster homozygous Zygosity
Background Basics
The information in this chapter builds on concepts presented in • Describe the basic structure and function of hemoglobin and
previous chapters. To maximize your learning experience, you identify the globin chain composition of normal hemoglobin
should review the following concepts before beginning this unit types. (Chapter 6)
of study: • Describe the extravascular destruction of erythrocytes and deg-
radation of hemoglobin. (Chapters 5, 6)
level i
• Describe the pathophysiology of hemoglobinopathies. level ii
(Chapter 13) • Summarize the synthesis and molecular structure of hemoglobin
• Describe the morphologic and functional classification of ane- and correlate alterations in structure with function; describe globin
mias and the associated lab tests. (Chapter 11) chain synthesis in utero and throughout life. (Chapters 3, 6)
• Interpret routine laboratory tests, such as CBC and differential, • Interpret hemoglobin migration patterns on electrophoresis,
and apply both normal and abnormal results in the diagnosis of acid elution of fetal hemoglobins, hemoglobin solubility tests,
anemias. (Chapters 10, 11, 37) and iron panels to distinguish iron metabolism disorders and
hemoglobinopathies from thalassemias. (Chapters 12, 13, 37)
and laboratory findings. Subsequently, the following are discussed for
case sTudy each type: pathophysiology, clinical findings, laboratory results, treat-
ment, and prognosis. Other thalassemia-like conditions are described
We will refer to this case throughout the chapter.
and compared and contrasted with thalassemia. The chapter con-
John is a 4-year-old boy who frequently complains of cludes by describing the laboratory differential diagnosis of types of
weakness, fatigue, and dyspnea. The family moved to thalassemia and other disorders that have similar peripheral blood
the United States from Greece before the child’s birth. morphology.
Both parents experienced fatigue from time to time but
never consulted a physician. Consider the types of ane-
mia most often found at this age and the laboratory tests inTRoducTion
that could help establish a diagnosis.
What is the significance, if any, of knowing the par- Thalassemia constitutes a family of inherited disorders in which
ents’ background and medical history? mutations in one or more of the globin genes of hemoglobin cause
decreased or absent synthesis of the corresponding globin chains.
More than 400 unique mutations have been described among this
1
diverse group of disorders. Consequences of the mutation depend
oveRview on the particular chain affected and the amount of globin chain
This chapter discusses a group of hereditary anemias collectively produced. Limited availability of globin chains results in a reduc-
called thalassemia. It begins with a general description of thalassemia, tion in the assembly of hemoglobin. Patients with mild genetic
including the genetic defects and types, pathophysiology, and clinical defects are generally asymptomatic. Patients with more severe
M14_MCKE6011_03_SE_C14.indd 252 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 253
defects present with symptoms that result from one or more of large screening and education programs to detect carriers. This has
the following: decreased production of normal hemoglobin, syn- drastically reduced the number of individuals born with both homo-
thesis of abnormal hemoglobins, unbalanced synthesis of a@ and zygous and heterozygous forms of the disease.
non-a@globin chains and ineffective erythropoiesis. Symptoms
include anemia, hepatosplenomegaly, infections, gallstones, and Thalassemia Versus Hemoglobinopathy
bone deformities that alter facial features and result in pathologic
fractures. The system of categorizing thalassemias and hemoglobinopathies
The thalassemias are classified according to the affected glo- differs among hematologists. Some use hemoglobinopathy as a dis-
bin chain. Clinically, the most important thalassemias involve the ease category to encompass both structural variants of hemoglobin
a@ and b@chains because they are components of hemoglobin A, (e.g., sickle cell anemia) and thalassemias. Others categorize only the
which makes up 97% of normal adult hemoglobin. a@Thalassemia structural variants as hemoglobinopathies and describe thalassemias
results from decreased or absent production of a@globin chains, as a separate disease entity. In this text, we refer to the two diseases as
and b@thalassemia is caused by decreased or absent production of separate entities.
b@globin chains. Reduction in the synthesis of the d@chain, a com- In the preceding chapter, hemoglobinopathies were defined as
ponent of hemoglobin A , can occur but is not associated with ane- qualitative defects in the structure of globin chains resulting in pro-
2
mia. Severe impairment of z@, P@, or g@globin synthesis is usually duction of abnormal hemoglobin molecules. Thalassemias, on the
lethal in utero. other hand, are typically quantitative disorders of hemoglobin syn-
Thomas Cooley offered the first clinical description of thesis that produce reduced amounts of normal hemoglobin.
2
thalassemia in Detroit in 1925. At that time, thalassemia was The different clinical presentations of hemoglobinopathies
thought to be a rare disorder restricted to the Mediterranean and thalassemia are a direct result of the differences in the
ethnicities. Dr. Cooley’s work broadened our understanding of the types of mutations encountered in these disease states. Most
nationalities that potentially could be affected by thalassemia and hemoglobinopathies result predominantly from a point mutation
suggested that the disease was hemolytic in nature. By 1960, it was within a globin gene that is translated into a globin chain
apparent that the thalassemias composed a heterogeneous group containing a single amino acid substitution. Hemoglobin types
of genetic disorders. With the advent of molecular biology, many containing a point mutation in the globin may be produced at
groups of researchers have widely studied thalassemias. Methods normal levels but are structurally abnormal. They can be unstable
developed in the last 25 years have enabled researchers to mea- or have abnormal function. In contrast, thalassemias result from
sure the quantity of globin chains synthesized and identify specific both deletional and nondeletional mutations in globin genes that
genetic mutations. reduce or eliminate the synthesis of the corresponding globin
Thalassemia is now recognized as one of the most common chain. This results in the assembly of inadequate amounts of nor-
genetic disorders affecting the world’s population. Approximately mal hemoglobin and a reduced oxygen-carrying capacity of the
3
1–5% of people are thought to be carriers of b@thalassemia. It is esti- blood. Unlike hemoglobinopathies, the amino acid sequence of the
mated that between 100,000 and 200,000 individuals worldwide are chain, if produced, is usually normal, and the chain is assembled
born each year with severe forms of thalassemia, and approximately into the appropriate hemoglobin, albeit in reduced amounts. In
3
60,000 of those have b@thalassemia. In North America, about 20% some of the less common thalassemias, the globin chains can be
of immigrants from Southeast Asia and 6–11% of African Americans lengthened or truncated (Table 14-1 ★).
have detectable a@thalassemia. Many more are silent carriers. About
6% of individuals with Mediterranean ancestry, 5% of Southeast
Asians, and 0.8% of African Americans have b@thalassemia along with CheCkpoint 14-1
people from the Middle East and India. 4
Thalassemia is a major health problem in countries where these Differentiate the etiology of thalassemias and hemoglo-
disorders are prevalent. Prevention is seen as an essential part in the binopathies.
management of the problem. Thus, many of these countries now have
★ TaBle 14-1 Comparison of Hemoglobinopathies and Thalassemias
erythrocyte hb solubility Reticulocyte
disease RBc count indices morphology abnormal hb Test ancestry count
hemoglobinopathy T Normocytic, Target cells, sickle cells hbS, hbC, + in hbS, hb African, mediterranean, c c
normochromic (in hbS), hbC crystals hbE, etc. Bart’s, and middle Eastern, South-
(in hbC), others hbC harlem east Asian
Thalassemia cCompared with microcytic, Target cells, basophilic hbh (b ), hb Negative mediterranean, South- c
4
4
what is expected hypochromic stippling Bart’s (g ) east Asian, African
for the hb level
T = slight decrease; c = slight increase; c c = moderate increase; hb = hemoglobin, RBC = red blood cell; + = positive
M14_MCKE6011_03_SE_C14.indd 253 01/08/14 12:50 pm
254 SECTION III • ThE ANEmIAS
Genetic Defects in Thalassemia three remaining globin chains (a, b, and d) along with the g@chains
are considered normal adult globin chains and combine to form
Nearly all thalassemic mutations fall into one of five categories of
2 2
2
2 2
genetic lesions: gene deletion, promoter mutation, nonsense muta- hemoglobin A (a b ), hemoglobin A (a d ), and hemoglobin F
2 2
tion, mutated termination (stop) codon, and splice site mutation (a g ), respectively. Approximately 97% of normal adult hemoglobin
(Table 14-2 ★). Regardless of the type of mutation encountered, the is HbA; thus, a deficiency of either a@ or b@chains affects hemoglobin
results are the same: the globin chain encoded by the mutated globin A assembly, reducing HbA concentration and affecting the blood’s
gene is absent, reduced in concentration, or occasionally somewhat oxygen-carrying capacity.
longer or shorter than normal. Two major types of classical thalassemia, a@thalassemia and
If all globin genes of a single type of globin chain are affected, the b@thalassemia, have been described. When synthesis of the a@chain
corresponding hemoglobin is absent. Deleted genes are most com- is impaired, the disease is a@thalassemia. When synthesis of the
mon in a@thalassemia. Reduction in globin chain production from b@chain is affected, the disease is b@thalassemia. There have been
nondeletional mutations is more common in the b@thalassemias. The reports of d@thalassemia, but its occurrence is rare and it is not clini-
degree of reduction in globin chain production is a direct reflection cally significant because the d@chain is a component of the minor
2
of the type of mutation encountered and parallels the severity of the hemoglobin HbA , which comprises only 2.5% of total hemo-
clinical disorder (Table 14-2). globin. Combinations of gene deletions such as db@thalassemia or
gdb@thalassemia occur but are rare. The synthesis of all the indicated
chains is reduced.
CheCkpoint 14-2 Occasionally synthesis of a structural globin variant decreases
globin chain synthesis, producing the clinical picture of thalassemia
What are the most common genetic mutations associated (thalassemic hemoglobinopathy). These structural variants include
with a@thalassemia? those hemoglobins with abnormally long or short globin chains (e.g.,
hemoglobin Constant Spring) as well as variants with a point mutation
(e.g., hemoglobin E). Hemoglobin Lepore is a hemoglobin variant in
which the non@a@globin chains are not only structurally abnormal
Types of Thalassemia but also ineffectively synthesized. Because of their clinical similarity
Because six different normal globin genes exist (a, b, g, d, P, z), at to thalassemias, these particular structural variants are discussed in
least six versions of thalassemia are possible. In addition, deletions this section.
can occur to entire gene clusters, concurrently affecting more than A variant of b@thalassemia known as hereditary persistence of
one globin chain. Of the six normal globin genes, P and z (P, z) are fetal hemoglobin (HPFH) is characterized by continued production
normally synthesized only early in utero, and g is produced in high of increased amounts of HbF throughout life. This disorder is
amounts from approximately the third trimester of pregnancy until characterized by a failure in the switch of g@chain production to
birth. Shortly before birth, g@chain synthesis begins to decrease but b@chain production after birth. In homozygotes, 100% of circulating
can still be detected in low amounts in adult life in some people. The hemoglobin is HbF.
★ TaBle 14-2 Five Common Genetic Defects in Thalassemia
mutation Type Thalassemia encountered effect on Gene effect on Globin chain
Deletion (large) Predominantly a@thalassemia, some Loss of gene Absence of production
b@thalassemia
Promoter Predominantly b@thalassemia Impaired transcription Reduced or absent production
Nonsense Predominantly b@thalassemia In frame substitution Amino acid change
Frame shift Amino acid changes distal to shift
Longer or shorter globin chains
Stop codon Predominantly b@thalassemia Convert stop codon to amino acid codon Slightly lengthened globin chain (retained)
Significantly lengthened globin chain
(degraded)
Splice site Predominantly b@thalassemia Create new splice sites Slightly shortened globin chain (retained)
Significantly shortened globin chain
(degraded)
Loss of splice sites Slightly lengthened globin chain (retained)
Significantly lengthened globin chain
(degraded)
Unaltered globin chain
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ChAPTEr 14 • ThALASSEmIA 255
Pathophysiology transporter of oxygen. In the fetus, when a@chains are decreased,
excess g@chains can combine to form hemoglobin molecules with four
Normally, equal amounts of a@ and b@chains are synthesized by the
4
maturing erythrocyte, resulting in a b@chain to a@chain ratio of 1:1. g@chains, hemoglobin Bart’s (Hb Bart’s, g ). This hemoglobin also has
In a@ and b@thalassemia, synthesis of one of these chains is decreased a very high oxygen affinity.
or absent, resulting in an excess of the other chain. If the a@chain is
affected, there is an excess of b@chains, and if the b@chain is affected, Clinical Findings
there is an excess of a@chains. This unbalanced synthesis of chains Clinical findings are related to anemia, chronic hemolysis, and
contributes substantially to the pathophysiology in thalassemia and ineffective erythropoiesis (Table 14-3 ★). The combination of
produces several effects, all of which contribute to anemia: (1) a reduced HbA synthesis, ineffective erythropoiesis, and hemolysis
decrease in total erythrocyte hemoglobin production, (2) ineffective results in anemia. The severity of anemia varies widely, depending
erythropoiesis, and (3) chronic hemolysis. on the specific genetic mutation and number of genes affected.
The exact nature of the contribution of unbalanced chain pro- Hypoxia from anemia is exacerbated in some cases by the presence
duction depends on which globin chain accumulates in excess and of abnormal hemoglobins that have a high oxygen affinity (HbH
on the amount of that excess. There is no compensatory downward and Hb Bart’s). These hemoglobins do not release oxygen readily
adjustment of production of one globin chain when its partner globin to the tissues.
subunit is not synthesized normally. Although excess b@ and g@chains Chronic hemolysis has several adverse effects. Splenomegaly is
can combine as tetramers (b4, g4) to form abnormal hemoglobins, frequently present because the spleen is a major site of extravascu-
a@chains do not. Excess a@chains are highly insoluble and precipi- lar hemolysis. Occasionally, the spleen can become overburdened
tate within the cell. The precipitates (Fessas bodies) bind to the cell by the process of erythrocyte destruction resulting in functional
membrane, causing membrane damage (which leads to apoptosis) and hyposplenism. In this case, the spleen’s function as a secondary
decreased erythrocyte deformability. Macrophages may destroy the lymphoid tissue is compromised, leading to an increase in infec-
precipitate-filled developing erythroblasts in the bone marrow, result- tions (Chapter 7). Chronic hemolysis can also result in the for-
ing in a large degree of ineffective erythropoiesis. Cells that survive mation of gallstones formed from the large amounts of bilirubin
the marrow environment and enter the circulation also contain pre- excreted by the liver. The chronic demand for erythrocytes also has
cipitates that are subsequently pitted and/or removed by the spleen, adverse effects. The bone marrow responds by increasing eryth-
causing chronic extravascular hemolysis. ropoiesis, resulting in erythroid hyperplasia, and in some of the
Excess b@chains can combine to form hemoglobin molecules more severe thalassemias, bone marrow expansion and thinning of
containing four b@chains, hemoglobin H (HbH, b ). This hemoglo- calcified bone. Consequently, patients develop skeletal abnormali-
4
bin has a high oxygen affinity and is also unstable. Thus, it is a poor ties and pathologic fractures. The increased iron demand needed
★ TaBle 14-3 Clinical and Laboratory Findings Associated with Thalassemia
clinical Finding pathophysiology laboratory Finding
Anemia/hypoxia Decreased hemoglobin production/erythropoiesis T/N RBC count, T hemoglobin, T hematocrit
Ineffective erythropoiesis microcytic/hypochromic rBCs
Presence of high-affinity hemoglobins (hbh and hb Bart’s) T mCV, T mCh, T mChC
Increased extravascular hemolysis c Reticulocyte count
Anisocytosis and poikilocytosis
Target cells, basophilic stippling, nucleated RBCs
Bm erythroid hyperplasia
c RDW
Abnormal hemoglobin electrophoresis
Splenomegaly/hemolysis Splenic removal of abnormal erythrocytes c Bilirubin
Ineffective erythropoiesis T haptoglobin
Gallstones Increased intravascular and extravascular hemolysis c Bilirubin
Skeletal abnormalities Expansion of bone marrow Bm erythroid hyperplasia
Pathologic fractures Thinning of calcified bone
Iron toxicity Iron overload
multiple transfusions c Prussian blue staining in Bm
Increased iron absorption c Serum iron/ferritin and T TIBC
Bm = bone marrow; mCh = mean corpuscular hemoglobin; mChC = mean corpuscular hemoglobin concentration; mCV = mean corpuscular vol-
ume; RBC = red blood cell; RDW = red cell distribution width; TIBC = total iron-binding capacity
M14_MCKE6011_03_SE_C14.indd 255 01/08/14 12:50 pm
256 SECTION III • ThE ANEmIAS
to support the erythropoietic activity stimulates the increased present even in cases without anemia. Basophilic stippling and
absorption of iron, more than the amount required for erythro- nucleated erythrocytes can be present. Anisocytosis and poikilo-
poiesis (Chapter 12). This additional iron is not effectively incor- cytosis are common. Precipitates of excess chains or unstable
porated into hemoglobin, so it accumulates in macrophages in hemoglobin can be visualized with supravital stains. Reticulocytes
the bone marrow, liver, and spleen. As this process continues, iron and bilirubin are usually increased due to the chronic hemolysis,
eventually accumulates in parenchymal cells of various organs and whereas haptoglobin can be decreased, depending on the degree of
adversely affects organ function. Iron toxicity commonly affects intravascular hemolysis.
such organs as the liver, pituitary, heart, and bone, resulting in Hemoglobin electrophoresis is always indicated if thalassemia
dysfunctions such as cirrhosis, hypogonadism and growth fail- is suspected. HbA is usually decreased. HbF and HbA are increased
2
ure, arrhythmias and cardiomyopathies, and pathologic fractures, in b@thalassemia but decreased in a@thalassemia. Hemoglobin
5
respectively. The additional iron introduced through transfusion Bart’s and HbH can also be present in some of the a@thalassemia
therapy exacerbates iron overload. Ineffective erythropoiesis in the syndromes.
bone marrow can be accompanied by extramedullary erythro- Bone marrow studies are not necessary for diagnosis but when
poiesis in the liver and spleen. Extramedullary erythropoiesis can performed show marked erythroid hyperplasia. Erythroblasts appear
produce masses large enough to cause compression syndromes abnormal with very little cytoplasm, uneven cytoplasmic membranes,
(Table 14-3). and striking basophilic stippling. Prussian blue stain reveals an abun-
Pregnant women with thalassemia have physiological demands dance of iron and occasionally a few ringed sideroblasts. Phagocytic
that impact the developing fetus to a greater extent than the mother. “foam” cells similar to Gaucher cells have been reported in the more
A developing fetus can experience diminished growth, premature severe forms of the disease. This morphology results from partially
birth, and even intrauterine death if the mother’s oxygen concen- digested red cell membrane lipids associated with intense ineffective
tration falls below 70 mm Hg. Pregnant women who present with a erythropoiesis.
hemoglobin level of between 9 and 10 g/dL (90–100 g/L) around the In areas of high prevalence of thalassemia and in certain
time of delivery are often given a blood transfusion to improve oxygen populations, screening programs to detect thalassemia carriers have
delivery. To avoid iron overload caused by the combination of transfu- been developed. Thus, assays that are uncomplicated, time efficient,
sion therapy and increased iron absorption, pregnant women can be and accurate need to be used. A variety of PCR-based nucleic acid
given deferoxamine (an iron chelator used to help eliminate excess assays can be performed to identify mutations in thalassemia,
iron) during and after the transfusion. 6 including allele-specific oligonucleotide hybridization (ASO), dot
blot and reverse dot blot assays, amplification refractory mutation
9
system (ARMS), and direct sequencing (Chapter 43). Given the
CheCkpoint 14-3 large number of mutations present in the human globin genes,
a small panel of probes for mutations found in a specific ethnic
Why do a@ and b@thalassemia result in more clinically group are initially screened for together with the wild-type allele.
severe disease than other types of thalassemia? Using this approach, identification of the mutation(s) is achieved
790% of the time. In cases where the mutation(s) are not iden-
tified, a second round of multiplex screening can be performed
using a panel of more rare mutations; this is successful in most of
Laboratory Findings the remaining 10% of cases. Gene sequencing can be performed to
10
Peripheral blood findings provide clues to the disease (Table 14-3). identify mutations that evade both rounds of screening. The dot
Thalassemias are characterized by microcytic, hypochromic anemia blot methodology is used to identify a specific mutation when one
with a decrease in MCV, MCH, and usually MCHC. The erythrocyte has been identified in a specific kindred as opposed to screening for
count is often normal or slightly decreased, but increased relative unknown mutation(s). 11
to the hemoglobin and hematocrit levels. The Mentzer Index can With the development of capillary sequencing techniques, PCR
be helpful in differentiating thalassemia from iron-deficiency amplified target genes can be sequenced and compared with wild-
anemia (a common cause of microcytic hypochromic anemia) type globin DNA to detect mutations. Until recently, direct sequenc-
7
( Chapter 12). The index is calculated by dividing the MCV (fL) ing was expensive and labor intensive, so hybridization methods for
12
by the RBC count (*10 /L). If the result is 613, diagnosis favors nucleic acid analysis were favored. Direct sequencing is now a com-
thalassemia whereas if the result is 713, diagnosis favors iron mon method of mutation identification.
deficiency. The RDW can be increased or within the reference inter- Nucleic acid-based methods are being developed for prenatal
8
val. In iron deficiency, the RDW is most often increased (715). In diagnosis of thalassemias and hemoglobinopathies. Extracellular fetal
practice, the CBC, Mentzer Index, and RDW should not be used as DNA is present in the maternal circulation. If the father is a known
the only parameters to differentiate the two pathologies. In micro- heterozygote for a particular thalassemia mutation that the mother
cytic hypochromic anemia, iron studies (e.g., serum ferritin) should does not have, detection of this allele in the maternal circulation con-
be performed routinely. Target cells and microcytosis usually are firms inheritance of the paternal gene by the child. 12
M14_MCKE6011_03_SE_C14.indd 256 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 257
A@Thalassemia affected individuals
General Considerations a@thalassemia is found primarily in people of Mediterranean, Asian, and
African ancestry. In particular, it is commonly seen in blacks, Indians,
a@Thalassemia is a group of four disorders characterized by decreased Chinese, and Middle Eastern people, with blacks usually expressing a
synthesis of a@chains. Although each is discussed separately, several milder version of the disease. The reason patients of African descent tend
features common to each type are presented here. to present with a milder version of a@thalassemia is because the deletion
in this ethnic group usually involves the lower-producing a @gene.
etiology 1
In the human genome, two a@genes are located on each of the two #16 Genotypes
chromosomes, totaling four a@genes in the diploid state (Figure 14-1 ■). Three nomenclature systems—genotypic, genotypic description, and
Mutations can affect one or more of the a@genes, resulting in four phenotypic—have been developed to classify the a@thalassemias into
discrete clinical severities. A patient in whom all four a@genes are deleted five discrete categories. The addition of the normal genotype pro-
produces no a@chains, a condition referred to as hydrops fetalis. When duces a total of six possibilities. The genotypic system designates
three of the four a@genes are deleted, the disorder is known as hemo- deleted genes as (-) and unaffected genes as (a). The genotypic
globin H (HbH) disease. The deletion of two a@genes is known as description system combines the zygosity state, homozygous or
a@thalassemia minor, and the deletion of a single a@gene is known as heterozygous, with either a gene symbol (a or a ) or a nominal
+
0
silent carrier. Though less common, nondeletional mutations and muta- descriptor (a@thal@1 or a@thal@2) to designate the number of deleted
tions that produce unstable a@chains are also found in a@thalassemia. a@genes on each chromosome. Both a@thal@1 and a indicate the
0
The outcome is usually the same as that of a deletion mutation, a reduc- deletion of both a@genes on the same chromosome (cis deletion)
tion in a@chains and in the corresponding a@containing hemoglobins. (-, -). a@thal@2 and a refer to one deleted and one unaffected
+
a@gene on a given chromosome (-, a). The phenotypic system
affected alleles describes four clinical types, hydrops fetalis, hemoglobin H disease,
The amount of a@chains synthesized is somewhat proportional to the a@thalassemia minor, and silent carrier with the a@thalassemia
number of affected alleles. However, sometimes erythrocytes produce minor type exhibiting two clinical severities (Table 14-4 ★).
13
higher concentrations of a@chains than the number of affected alleles About 15–20% of patients have a nondeletional mutation of the
would predict. There are two main reasons for this phenomenon. First, a@thalassemia gene, designated as a , that functions to reduce but
T
the two a@genes on each of the #16 chromosomes are designated as not eliminate a@chain production from that gene.
a and a , with the a @gene positioned upstream (5 ) of the a @gene
2
2
1
1
(Figure 14-1). The a @gene produces two to three times the amount of 0 0
2
13
mRNA as the a @gene. Therefore, a deletion of the a @gene would A@Thalassemia Major (A /A or
1
2
reduce a@chain production to a greater degree than would a deletion A@thal@1/A@thal@1; Hydrops Fetalis)
of the a @gene. Second, the erythropoietic system has an internal The most severe form of a@thalassemia, a@thalassemia major, involves
1
mechanism designed to stimulate increased production of a@chains the deletion of all four a@genes (- -/- -). Both parents of the thal-
from the unaffected genes to compensate for deletions, thus lessening assemia patient must have a@thalassemia to have a child with hydrops
the net reduction of a@chains. fetalis because both a@genes on each parental chromosome inherited
by the child are deleted (a@thal@1; -, -). a@Thalassemia major is
z cz ca 2 ca 1 a 2 a 1 found almost exclusively in Asians because that is the major ethnic
Maternal 5' 3' group carrying the a@thal@1 allele.
allele
1 2 pathophysiology
z cz ca 2 ca 1 a 2 a 1 Because all four a@genes are deleted in hydrops fetalis, no physiologi-
Paternal 5' 3' cally useful hemoglobins can be synthesized beyond the embryonic
allele state. Therefore, this disorder is incompatible with life, and infants
3 4 are either stillborn or die within hours of birth. In the absence of
z = zeta cz = psi zeta ca = psi alpha 1 a@chains, erythrocytes assemble hemoglobin using the g@, d@, and
1
a = alpha 2 ca = psi alpha 2 a = alpha 1 b@chains available. Therefore, abnormal hemoglobin tetramers
2
1
2
involving g@chains (Hb Bart’s, g ) are produced. Hb Bart’s has a very
■ FiGuRe 14-1 A short section of chromosome 4
16 showing the 5 to 3 orientation of three functional high oxygen affinity and no Bohr effect (Chapter 6). Therefore, this
genes z, a 2 , and a 1 along with three pseudogenes cz, ca 2 , hemoglobin cannot supply tissues with sufficient oxygen to sustain
and ca 1 . Pseudogenes are the result of partial gene dupli- life, and the developing infant usually dies of hypoxia and congestive
cations but are not expressed. There are two functional heart failure in utero. Hemoglobin Portland, although normally absent
a@genes on each chromosome; the a 2 @gene expresses 2–3 following the first trimester, continues to be synthesized until birth in
0
0
times as much protein product as the a 1 @gene. a /a thalassemia because it does not contain a@chains.
M14_MCKE6011_03_SE_C14.indd 257 01/08/14 12:50 pm
258 SECTION III • ThE ANEmIAS
★ TaBle 14-4 Characteristics of a@Thalassemia
hemoglobins
Genotype Genotypic description phenotype hematologic Findings severity present
(- -/- -) homozygous a@thal@1 hydrops fetalis marked anemia Fatal hb Bart’s (80–90%)
0
a /a 0 microcytic/hypochromic rBCs hb Portland (10–20%)
c c c anisopoikilocytosis
c NRBC
(- -/-a) heterozygous hemoglobin h disease moderate to marked anemia Chronic, moderately Birth = hb Bart’s;
0 + severe hemolytic
a /a a@thalassemia@1/ microcytic/hypochromic rBCs Adult = hbh
anemia
a@thalassemia@2 Target cells
Basophilic stippling
Poikilocytosis
(- -/aa) heterozygous a@thalassemia@minor Slight anemia mild to moderate Birth = hb Bart’s;
0
a /a a@thalassemia@1 microcytic/hypochromic rBCs Adult = normal
Target cells
Basophilic stippling
Poikilocytosis
(-a/-a) homozygous a@thalassemia@minor Slight anemia mild Birth = hb Bart’s;
+
a /a + a@thalassemia@2 microcytic/hypochromic rBCs Adult = normal
Target cells
Basophilic stippling
Poikilocytosis
(-a/aa) heterozygous Silent carrier Normocytic or slightly microcytic Normal Normal
+
a /a a@thalassemia@2 RBCs
(aa/aa) Normal None Normal Normal Normal
c c c = marked increase; c = slight increase
clinical Findings C G
Infants who survive until birth exhibit significant physical abnormali- O D
ties upon routine exam. The babies are underweight and edematous E S
with a distended abdomen. The liver and often the spleen are enlarged CA CS A2 Lepore F A Portland Barts H
due to extramedullary hematopoiesis. There is massive bone marrow Hydrops
hyperplasia. Hemolysis in the fetus is severe and there is extensive fetalis
deposition of hemosiderin. Hb H
(neonate)
Hb H
laboratory Results (adult)
Laboratory results confirm the clinical observation of severe anemia Hb H/
with hemoglobin values ranging from 3–10 g/dL (30–100 g/L) and CS
erythrocytes that are markedly microcytic and hypochromic. Hemo- a-Thal
globin electrophoresis on cellulose acetate or agarose at alkaline pH minor (neonates
shows 80–90% Hb Bart’s and 10–20% Hb Portland; HbH is sometimes Silent carrier only)
also detectable. HbA, HbA , and HbF are absent due to the lack of
2
a@chain production (Figure 14-2 ■). CA = Carbonic anhydrase
CS = Constant Spring
■ FiGuRe 14-2 Hemoglobin electrophoresis on cellulose
acetate or agarose at pH 8.4 is helpful in distinguishing
the type of thalassemia and in differentiating thalas-
CheCkpoint 14-4 semias from hemoglobinopathies. In a@thalassemias,
there is a reduction in a@containing hemoglobins (HbA,
Which of the three normal adult hemoglobins would be HbA 2 , and HbF) proportional to the number of deleted
affected in hydrops fetalis? a@genes and in the more severe cases, the emergence of
non@a@containing hemoglobins (HbH and Hb Bart’s).
M14_MCKE6011_03_SE_C14.indd 258 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 259
0
+
Hemoglobin H Disease (A /A Hemoglobin H can also occur as an acquired defect in eryth-
or A@thal@1/A@thal@2) roleukemia and other myeloproliferative neoplasms. However, the
clinical manifestations and hematological abnormalities of these
HbH disease, a symptomatic but nonfatal type of a@thalassemia, was acquired disorders make it possible to distinguish them from HbH
the first type to be described in 1956. It occurs when three of the four disease. Acquired HbH is probably due to a defect that prohibits the
a@genes are deleted (-,-/-, a). African Americans seldom pres- transcription of the a@gene.
ent with HbH disease because they rarely express a deletion of two
a@genes on the same chromosome. 14
This disorder usually results when two parents, one with het-
erozygous a@thal@1 (- -/aa) and the other with the heterozygous CheCkpoint 14-5
13
a@thal@2 (-a/aa) genotype, bear children. All children from a patient Compare oxygen-binding characteristics of hbh relative
with HbH disease will have a type of a@thalassemia, the severity of which to hbA and myoglobin.
depends on the allele inherited and the other parent’s genotype.
pathophysiology
The dramatic reduction in a@chain synthesis (25–30% of normal) clinical Findings
results in a decrease in the assembly of HbA, HbA , and HbF. In addi- Symptoms are related to anemia and chronic hemolysis. Hemoglobin
2
tion, a decrease in a@chains creates a relative excess of b@chains, which H disease shows a wide variation from mild to severe anemia, which
assemble to form b@chain tetramers called HbH. g@Chains also are worsens during pregnancy, in infectious states, and during adminis-
produced in excess of a@chains, especially at birth, and combine to tration of oxidant drugs. Splenomegaly and, less often, hepatomegaly
form g@chain tetramers or Hb Bart’s. are present. Less than half of affected patients exhibit skeletal changes
HbH is thermolabile, unstable, and tends to precipitate inside similar to those found in b@thalassemia major, and there is relatively
erythrocytes triggering chronic hemolytic anemia. Its oxygen affinity little ineffective erythropoiesis.
is 10 times that of HbA, reducing oxygen delivery to the tissues. Its
high oxygen affinity is attributed to the lack of heme–heme interac- laboratory Results
tion and absence of the Bohr effect (Chapter 6). This increased oxygen Hemoglobin H disease is characterized by a microcytic, hypochromic
affinity is reflected in the lower P value of HbH relative to HbA and anemia with hemoglobin levels usually ranging from 8–10 g/dL. Retic-
50
myoglobin (Figure 14-3 ■). ulocytes are moderately increased to 5–10%, and nucleated red blood
cells are observed on the peripheral blood smear (Figure 14-4a ■).
Hemoglobin electrophoresis of affected neonates shows about
25% Hb Bart’s with decreased levels of HbA, HbA , and HbF. Shortly
100 2
before birth, b@chains begin to replace g@chains, and HbH eventu-
ally replaces Hb Bart’s. Hemoglobin H, a fast-migrating hemoglobin
HbH Myoglobin Hb A
at alkaline pH, constitutes 2–40% of the hemoglobin in adults with
75 P 50 is the partial pressure of HbH disease. HbA is decreased to about 1.5%, but HbF is normal.
2
oxygen at which the hemoglobin A trace of Hb Bart’s can be demonstrated in approximately 10% of
% Hb saturation 50 measure of the binding affinity Other laboratory tests are available to assess patients with HbH dis-
affected adults with remaining hemoglobin being HbA (Figure 14-2).
tested is 50% saturated. It is a
ease. Hemoglobin H inclusions are easily found upon incubation of
of the hemoglobin. A higher P 50
indicates a lower binding affinity
blood with brilliant cresyl blue (Figure 14-4b). These inclusions tend
and a greater ability to release
to cover the inside of the plasma membrane, giving the appearance
oxygen to tissues.
of a golf ball.
25 HbH – P 50 = 6 mm Hg
Myoglobin – P 50 = 12 mm Hg Treatment and prognosis
HbA – P 50 = 26 mm Hg
Treatment for patients with HbH disease is variable but in severe
26 cases can involve long-term transfusion therapy and splenectomy.
0 Regular transfusions minimize the stunting of growth and the other
0 6 12 25 50 75 100
Partial pressure of oxygen (mm Hg) consequences of chronic severe anemia. Iron chelation therapy with
deferoxamine avoids iron overload and the effects of iron toxicity.
■ FiGuRe 14-3 The Hb dissociation curve illustrates the Deferaserox (licensed in the United States in 2006) and deferipone
relative binding affinities of HbA, HbH, and myoglobin (not yet licensed in the United States) are oral chelators that serve
using the P 50 value. The monomeric myoglobin molecule
lacks heme–heme interactions, causing it to bind oxygen as an alternative to deferoxamine but have significant dose-related
4
tightly, decreasing the P 50 value relative to HbA. The P 50 sequelae including neutropenia, arthropathy, and agranulocytosis.
value is even lower for HbH, indicating an even stronger The increased and ineffective erythropoiesis places high demands on
affinity for oxygen estimated to be 10 times more than the the bone marrow requiring folate supplementation to avoid a defi-
5
oxygen affinity of HbA. ciency. Early treatment is necessary to prevent the typical clinical
M14_MCKE6011_03_SE_C14.indd 259 01/08/14 12:50 pm
260 SECTION III • ThE ANEmIAS
a b
■ FiGuRe 14-4 (a) This peripheral blood smear is from a patient with HbH disease. Note the
microcytic, hypochromic anemia with target cells (Wright-Giemsa stain; 1000* magnification).
(b) Peripheral blood from patient in Figure 14-4a after incubation with brilliant cresyl blue.
Notice the cells that have dimples and look like golf balls. These are the cells with precipi-
tated HbH. (Brilliant cresyl blue stain; 1000* magnification).
manifestations of thalassemia. With supportive care and behavioral decreases to undetectable levels and hemoglobin electrophoresis
interventions, patients with HbH disease experience a normal life becomes normal. The only persistent hematological abnormality
expectancy. For patients in whom transfusion and chelation therapy thereafter is a mild microcytic, hypochromic anemia.
are not efficacious, a bone marrow transplant could be indicated. 5 In adult patients, hemoglobin levels are above 10 g/dL
12
(100 g/L), and the erythrocyte count is above 5 * 10 /L. The
A@Thalassemia Minor (A@thal@2/A@thal@2 peripheral blood film usually demonstrates significant microcyto-
0
[a /a ], or A@thal@1/normal [A /A]) sis with an MCV of 60–70 fL with few target cells (Figure 14-5 ■).
Occasional cells can exhibit HbH inclusions after incubation with
The a@thalassemia trait (homozygous a@thal@2 or a@thal@1 trait)
occurs when two of the four a@genes, either on the same (cis) or brilliant cresyl blue.
opposite (trans) chromosomes, are missing. The condition is found In some cases, a@thalassemia may be masked by iron-deficiency
15
in all geographic locations. In African Americans, the homozygous anemia. Persistence of microcytes following successful treatment of
a@thal@2 form is the most common presentation. In individuals of iron deficiency is suggestive of thalassemia, but further investigation
Southeast Asian or Mediterranean descent, genetic testing has identi- is needed for conclusive diagnosis.
fied at least nine haplotypes (groups of alleles of different genes on
the same chromosome that are closely linked and usually inherited Treatment and prognosis
together), representing different mutations, all of which result in the These patients are usually asymptomatic, have a normal life span, and
deletion of both a@genes on the same chromosome. 16 do not require medical intervention for their thalassemia.
pathophysiology
Although a measurable decrease in the production of a@containing
hemoglobins occurs, the unaffected a@globin genes are able to direct
synthesis of a@globin chains to a greater than normal degree and
therefore partially compensate for the deleted genes. Only minor
changes occur in the erythrocyte count, indices, hemoglobin electro-
phoresis patterns, and red cell morphology.
clinical Findings
Patients with a@thalassemia trait are asymptomatic with a mild ane-
mia and are often diagnosed incidentally or when being evaluated for
family studies. This mild phenotype is the reason that this form is
called thalassemia minor.
■ FiGuRe 14-5 This is a peripheral blood smear from a
patient with a@thalassemia trait. The hemoglobin is 15 g/
laboratory Results dL, RBC count 6.4 * 10 /L, MCV 69.4 fL. The high RBC
12
The most demonstrable laboratory abnormalities are observed in the count with microcytosis is typical of thalassemia minor
newborn. The presence of 5–6% Hb Bart’s in neonates can be helpful or trait (peripheral blood; Wright-Giemsa stain; 1000*
17
in diagnosing this condition. Three months after birth, Hb Bart’s magnification).
M14_MCKE6011_03_SE_C14.indd 260 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 261
+
Silent Carrier (A@thal@2/normal; A /A) Genetics
The silent carrier version of a@thalassemia (a@thal@2 trait) is missing Whereas a total of four a@globin genes results in four major geno-
only one of four functioning a@genes. More than 25% of African types of a@thalassemia, there are only two b@globin genes, one located
Americans have been shown to express a deletion of one a@gene. 18–20 on each chromosome 11 (Figure 14-6 ■). If the prominent type of
mutation found in b@thalassemia were also deletional, one would
expect two severities, the severe homozygote and the mild heterozy-
pathogenesis/clinical Findings/laboratory Results
In the silent carrier state, the three remaining a@genes direct the syn- gote. However, in b@thalassemia, most mutations are nondeletional,
resulting in a near continuum of clinical severities. Two classifica-
thesis of an adequate number of a@chains for normal hemoglobin tion systems are currently used for this diverse group of diseases. The
synthesis. This carrier state is asymptomatic and benign, but adults genotypic system classifies b@thalassemia patients into six genotypes
16
often present with a borderline normal MCV of around 78–80 fL. based on zygosity and the degree of alteration of the b@genes, and the
In affected infants, 1–2% Hb Bart’s may be found at birth but cannot phenotypic system divides patients into four categories based on the
be detected after three months of age. The only definitive diagnostic severity of clinical symptoms.
test for thalassemias in adults with one or two gene deletions is globin In the genotypic system, all b@gene mutations are categorized
gene analysis.
into two groups based on the impact of the mutation on b@globin
0
+
production. The two gene varieties are termed b and b . The
Treatment and prognosis b @gene mutation causes a partial block in b@chain synthesis, and the
+
Patients with the silent carrier phenotype require no treatment and b @gene mutation results in a complete absence of b@chain synthe-
0
have a normal life span. sis from that allele. In addition, a minimally affected b@allele called
SC
silent carrier (b ) has been identified. It is found only in the most
benign version of b@thalassemia. In the heterozygous state, hemato-
logical features are normal; it is clinically benign. Diagnosis can be
case sTudy (continued from page 252) made only by finding a slight imbalance of a@/non-a@chain synthe-
9
+
SC
0
sis. When the three gene designations, b , b , and b are combined
The parents took John, the 4-year-old Greek patient, with the normal allele (b), and the two possible zygosity patterns
to a pediatrician for a checkup. A CBC ordered had (homozygous and heterozygous) are considered, eight possible geno-
the following results: types (b /b , b /b , b /b ,/b /b, b/b , b/b, b/b , b /b ) emerge
+
o
+
SC
SC
SC
+
o
o
o
+
(Table 14-5 ★).
cBc differential
9
WBC 11.4 * 10 /L Segs 55% b@thalassemia is the result of several different types of molecu-
12
RBC 1.7 * 10 /L Bands 1% lar defects. More than 200 mutations resulting in partial to complete
hb 8.3 g/dL Lymphs 36% absence of b@gene expression have been described, but only 20
1,14
hct 0.24L/L mono 7% mutations account for 80% of the diagnosed b@thalassemias.
mCV 69 fL Eos 1% b@thalassemia is rarely due to deletion of the structural gene as is
mCh 21 pg moderate poikilocytosis, poly- the case in the a@thalassemias. Most defects in b@thalassemia are
mChC 29.2 g/dL chromasia, and many target point mutations in regions of the DNA that control b@gene expres-
9
Plt 172 * 10 /L cells; few teardrop cells sion. These types of mutations can affect gene expression ranging
from minor reductions in b@globin production to complete absence
1. Based on the indices, classify the anemia morph ologically. of synthesis. Mutations can affect any step in the pathway of glo-
2. Name the dominant poikilocyte observed in this bin gene expression, including gene transcription, RNA processing,
peripheral blood smear. mRNA translation, and post-translational integrity of the protein. 21,22
3. Name three disorders that frequently present with
the same poikilocyte that dominates in this peripheral e G g A g cb d b
blood smear. Maternal 5' 3'
allele
4. List two additional lab tests that would help to confirm 1
the diagnosis and predict the results of each.
e G g A g cb d b
Paternal 5' 3'
allele
2
B@Thalassemia e = epsilon G = G gamma A g = A gamma
g
General Considerations cb = psi beta d = delta b = beta
■ FiGuRe 14-6 Chromosome 11 is the location of four
As with a@thalassemias, some features of b@thalassemias are common types of globin genes (P, g, d, b). The 5 to 3 orienta-
to all forms of the disease. The genetics of b@thalassemia and the indi- tion of the genes is depicted. There is a gene for P, d,
viduals affected with the disorder are presented before the discussion and b and two g@genes. A b@pseudogene (cb) has been
of its various forms. identified but does not express protein product.
M14_MCKE6011_03_SE_C14.indd 261 01/08/14 12:50 pm
262 SECTION III • ThE ANEmIAS
★ TaBle 14-5 Characteristics of b@Thalassemia
Genotype Zygosity phenotype RBc count RBc morphology hb electrophoresis severity
0
b /b 0 homozygous major Relative c c c c Target cells No A, c A 2 , c c F Severe
0
b /b + Double heterozygous major Relative c c c c Target cells T T A, c A 2 , c c F Severe
Intermedia c c Target cells T A, c A 2 , c c F moderate
+
b /b + homozygous major Relative c c c c Target cells T T A, c A 2 , c c F Severe
Intermedia c c Target cells T A, c A 2 , c c F moderate
0
b /b heterozygous Intermedia Relative c c c Target cells T A, c A 2 , c F moderate
minor c Target cells T A, c A 2 , c F mild
+
b /b heterozygous minor Relative c c Target cells T A, c A 2 , c F mild
SC
b /b SC homozygous mild Intermedia Relative c cTarget cells T A, c A 2 , c F mild
SC
b /b heterozygous minima Normal { Target cells Normal Normal
b/b homozygous Normal Normal Normal Normal Normal
c = slight increase; c c = moderate increase; c c c = marked increase; +/- = occasionally seen; T = slight decrease; T T = moderate decrease;
SC
A = hbA, A 2 = hbA 2 ; F = hbF; b = silent carrier
Within a given population, a few genetic lesions account for most of or absence of b@chain synthesis. As can be seen in Table 14-5, two
+ +
0 +
the b@thalassemia mutations. For instance, in Greece, five mutations of these three genotypes (b b , b b ) can also present as the
account for 87% of the gene defects. 22 milder b@thalassemia intermedia phenotype that will be discussed
The phenotypic classification recognizes four groups of patients subsequently.
categorized by the severity of symptoms, medical interventions,
and prognoses. The four groups, listed in order from most severe pathophysiology
to least severe, are b@thalassemia major, b@thalassemia intermedia,
b@thalassemia minor, and b@thalassemia minima (Table 14-5). The dramatic reduction or absence of b@chain synthesis affects the
The phenotypic classification does not accurately reflect the production of HbA. The symptoms that result from b@thalassemia
genetic description of the disease. However, a disadvantage to the major begin to manifest in infants approximately six months after
genotypic system is that patients with identical genotype designa- birth. Other non@b@containing hemoglobins, HbA and HbF, are
2
tions can express b@thalassemia that is phenotypically diverse. For increased in partial compensation for the decreased HbA levels.
+
+
instance, a severe form of b /b @thalassemia (Mediterranean form) The pathophysiologic mechanisms that result from a lack
is characterized by an increase in HbF (50–90%) and a normal or only of b@chain production can be classified into four categories
+
+
slightly elevated HbA , whereas a milder form of b /b @thalassemia (Figure 14-7 ■):
2
(Black form) has 20–40% HbF with normal or elevated HbA , and the • reduced HbA
2
remainder HbA. For this reason, some clinicians prefer the pheno- • compensatory production of other hemoglobins
typic system that more closely parallels symptoms and better predicts
clinical interventions necessary for appropriate management of the • ineffective erythropoiesis with hemolysis
patient. • erythroid hyperplasia
A dramatic reduction in HbA compromises the blood’s oxygen-
affected individuals
0
The most severe mutation (b ) is found more frequently in the Medi- carrying capacity. Other non@b@containing hemoglobins, HbF and
HbA , are increased. HbF has a higher affinity for oxygen than HbA
2
terranean regions—specifically in northern Italy, Greece, Algeria, (Chapter 6). The result is to exacerbate the already compromised oxy-
+
Saudi Arabia—and Southeast Asia. Two severities of the b mutation gen delivery to tissues.
tend to originate in different ethnic populations. The more severe ver- In b@thalassemia major reduced synthesis of b@chains results
sion is observed in the Mediterranean region, the Middle East, the in an excess of free a@chains and a b@to@a chain ratio of 60.25.
Indian subcontinent, and Southeast Asia; the milder version is local- The excess free a@chains cannot form hemoglobin tetramers, so
ized to patients of African descent. 15
they precipitate within the cell, damaging the cell membrane, and
23
+
+
0
0
0
+
B@Thalassemia Major (B /B , B /B , B /B ) leading to chronic hemolysis. The accumulating a@chains con-
tain free iron and hemichromes that generate reactive oxygen spe-
expected Genotypes cies (ROS). The ROS damage hemoglobin as well as the membrane
b@Thalassemia major, also referred to as Cooley’s anemia, is caused proteins and lipids, decreasing membrane stability. 24,25 Oxidation
0 +
+ +
0
0
by a homozygous (b /b , b b ) or double heterozygous (b b ) of membrane Band 3 produces clustering of the proteins, creating
26
inheritance of abnormal b@genes resulting in marked reduction new antigens on the cell surface that bind IgG and complement.
M14_MCKE6011_03_SE_C14.indd 262 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 263
Mutated b–gene (s)
Normal a, g, d genes
Excess a–chains Normal synthesis of a–chains Hb A
Reduced synthesis of b–chains
Enhanced synthesis of g–, d–chains
Precipitation of
a–chains Hb F, Hb A 2
Oxygen affinity Reduced oxygen delivery
Damaged RBC membranes & apoptosis ANEMIA Iron
P.B. circulation Bone marrow: Phagocytosis EPO Toxicity
Ineffective erythropoiesis B.M. expansion
Splenic Hepatosplenomegaly Fractures
hemolysis Mongoloid
facies
■ FiGuRe 14-7 In b@thalassemia major, the decreased synthesis of
b@chains reduces the production of HbA and increases the production of
non@b@chain containing hemoglobins (HbA 2 and HbF). Excess a@chains form
insoluble precipitates inside erythrocytes, damaging the membranes and
reducing RBC lifespan through splenic sequestration and ineffective eryth-
ropoiesis. All of these factors contribute to a reduced oxygen delivery to
the tissues resulting in anemia and hypoxia. The compensatory erythroid
hyperplasia in the bone marrow expands the marrow cavity, resulting in
pathologic fractures and mongoloid facial features.
Many IgG- and complement-sensitized erythrocytes in the bone clinical Findings
marrow are destroyed by binding to marrow macrophages via Fc Early symptoms of b@thalassemia first observed in infants include
receptors, resulting in phagocytosis and a large degree of hemolysis. irritability, pallor, and a failure to thrive and gain weight, beginning at
The majority of the hemolysis occurs within the bone marrow about 6 months of age. Diarrhea, fever, and an enlarged abdomen are
primarily at the polychromatophilic erythroblast stage of erythroid also common findings. If therapy does not begin during early child-
27
development, resulting in ineffective erythropoiesis. The ineffective hood, the clinical picture of thalassemia develops within a few years.
erythropoiesis is due to activation of apoptotic mechanisms by the Severe anemia is the clinical condition responsible for many prob-
precipitated a@globin chains and damaged cellular components. 28,29 If lems experienced by these children. The anemia places a tremendous
the patient has inherited a@thalassemia with b@thalassemia, the symp- burden on the cardiovascular system as it attempts to maintain tissue
toms associated with hemolysis may be reduced because the relative perfusion. Constant high output of blood usually results in cardiac fail-
excess of a@chains is reduced by the co-inherited a@thalassemia. ure in the first decade of life and is the major cause of death in untreated
The combination of reduced HbA, increased HbF, ineffective children. Growth is retarded, and a bronze pigmentation of the skin is
erythropoiesis, and chronic hemolysis results in significant anemia. notable. Chronic hemolysis often produces gallstones, gout, and icterus.
The body attempts to compensate by stimulating erythropoiesis. The Bone changes accompany the hyperplastic marrow. Marrow
resulting erythroid hyperplasia causes bone marrow expansion and cavities enlarge in every bone, expanding the bone and producing
thinning of calcified bone. Increased erythropoietic activity also stim- characteristic bossing of the skull, facial deformities, and “hair-on-
ulates the absorption of more iron in the gut, leading to iron toxicity. end” appearance of the skull on x-ray (Figure 14-8 ■). The thinning
Ineffective erythropoiesis in the bone marrow can be accompanied by cortical bone in long bones can lead to pathologic fractures.
extramedullary hematopoiesis in the liver and spleen, often producing Extramedullary hematopoiesis can be found in the liver and
hepatosplenomegaly (Figure 14-7). spleen and occasionally elsewhere in the body. The spleen can become
massively enlarged and congested with abnormal erythrocytes.
Other clinical findings are associated with the body’s attempt to
CheCkpoint 14-6 increase erythrocyte production. Features that suggest an increased
metabolic rate include fever, lethargy, weakened musculature,
Why are the symptoms of b@thalassemia major delayed decreased body fat, and decreased appetite. Infection is a common
until approximately the sixth month of life? cause of death. Folic acid deficiency can develop as a consequence of
increased utilization by the hyperplastic marrow.
M14_MCKE6011_03_SE_C14.indd 263 01/08/14 12:50 pm
264 SECTION III • ThE ANEmIAS
■ FiGuRe 14-9 Peripheral blood smear from a patient
with b@thalassemia major showing marked anisopoikilocy-
tosis. Target cells, schistocytes, teardrops, and ovalocytes
are the major poikilocytes observed. An NRBC is also
present (Wright-Giemsa stain; 1000* magnification).
+
+
0
+
The other genotypes, b /b and b /b , show some HbA, but the
majority of the hemoglobin is HbF with normal to increased HbA 2 33
(Figure 14-10 ■). The increased HbF in thalassemia is thought to be
due to the expansion of a subpopulation of erythrocytes that have
the ability to synthesize g@chains. The distribution of HbF among
erythrocytes is heterogeneous.
Definitive diagnosis of b@thalassemia can be made by demon-
■ FiGuRe 14-8 Increased erythropoiesis in the bone stration of a b@to@a@chain ratio of 60.25. Molecular techniques dem-
marrow of patients with b@thalassemia major expands onstrating specific genetic mutations can also define the presence of
the marrow cavity producing the typical “hair-on-end”
appearance as seen on this radiograph of the skull of a thalassemia (Chapter 42).
boy with b@thalassemia.
Treatment
Most children with b@thalassemia major participate in a regular trans-
laboratory Results fusion program, which prolongs life into at least the second or third
The hemoglobin level can be as low as 2 or 3 g/dL (20–30 g/L) in the decade and allows normal development and growth patterns. Initial
more severe forms of the disease. The anemia is markedly microcytic treatment protocols were mainly palliative and designed to maintain
30
and hypochromic with an MCV of 667 fL and a markedly reduced
MCH and MCHC. The peripheral blood smear shows marked aniso-
C
cytosis and poikilocytosis (Figure 14-9 ■). Precipitates of a@chains O G
D
can be visualized with methyl violet stain. Variable basophilic stippling E S
and polychromasia are noted. Reticulocytes are not increased to the CA CS A2 Lepore F A Portland Bart's H
degree expected for the severity of the anemia because of the high b-thal
degree of ineffective erythropoiesis. Nucleated erythrocytes are almost major
31
always found, and the RDW can be normal or increased. Second- b-thal
ary leukopenia and thrombocytopenia can be produced because these major (med)
components also become trapped in the enlarged spleen. Chronic b-thal
hemolysis is reflected by increased unconjugated bilirubin. Urine can intermediate
appear dark brown from the presence of dipyrroles. b-thal
Bone marrow studies are not usually necessary for diagnosis but minor
when performed show marked erythroid hyperplasia with an M:E b-thal
ratio of 1:10 or less. minima
Hemoglobin electrophoresis performed on cord blood CA = Carbonic anhydrase
samples provides evidence of deficient b@chain production at birth. CS = Constant Spring
Although normal cord blood contains about 20% HbA, cord blood med = Mediterranean
21
from infants with b@thalassemia major has 62% HbA. In adults, ■ FiGuRe 14-10 Hemoglobin electrophoresis pat-
hemoglobin electrophoresis shows variable results, depending on tern from patients with b@thalassemia shows a
the thalassemia alleles inherited. Absence of HbA, 90% HbF, and low, reduction in b@containing HbA and an increase in
32
0
0
normal, or increased HbA is characteristic of b /b @thalassemia. non@b@containing HbA 2 and HbF.
2
M14_MCKE6011_03_SE_C14.indd 264 01/08/14 12:50 pm
ChAPTEr 14 • ThALASSEmIA 265
the hemoglobin level of approximately 7–8 g/dL (70–80 g/L), but evi- expression of Oct4, Sox2, Klf4, and c-Myc, transcription factors essen-
dence of erythroid expansion and increased iron absorption persisted. tial for maintaining pluripotency in early embryos and embryonic
Clinical evidence suggests that the more aggressive hypertransfusion stem cells. 62,63 When grown in culture, these cells could be induced
programs (maintaining a hemoglobin level of 9–10.5 g/dL) offer the to differentiate into multipotent hematopoietic stem cells and then
34
highest quality of life without significant sequelae. The objectives autotransplanted.
of hypertransfusion programs are to minimize the anemia, reduce Although significant obstacles must be overcome, correction of
excess iron absorption, and suppress ineffective erythropoiesis. The the molecular defect with gene therapy is thought to be achievable in
large doses of iron received with these transfusions, however, lead to the future. 64
tissue damage from iron overload similar to that seen in hereditary
hemochromatosis. Iron-chelating agents such as deferoxamine and prognosis
deferasirox are given to decrease the deposition of iron in the tis- Untreated patients generally expire during the first or second decade
sues. 35,36 Splenectomy can be performed in an attempt to decrease of life. Patients enrolled in a hypertransfusion program with chelation
hemolysis and prolong red cell survival. However, splenectomy is therapy can extend their life expectancy by at least a decade. Usually
65
usually reserved for patients older than 5 years of age who receive in the second decade of life, endocrine disorders (e.g., diabetes) and
7200 mLs of packed red blood cells (PRBCs)/kg/yr, have leukope- hepatic and cardiac disturbances develop from excessive deposits of
nia, thrombocytopenia, splenomegaly pain, or iron overload with iron in these tissues if chelation therapy has not been successful.
35
chelation therapy. Risks associated with splenectomy include sepsis,
thrombosis, and possibly pulmonary hypertension. 37,38 0 +
Bone marrow transplants (BMT) have been attempted in an B@Thalassemia Minor (B /B or B /B)
effort to provide the individual with stem cells capable of produc- expected Genotypes
ing normal erythrocytes. Although BMT is an option for thalassemia b@Thalassemia minor results from the heterozygous inheritance of
39
+
0
patients with a suitable donor, this technology is not widely used. either a b @ or b @gene with one normal b@gene (Table 14-5). No
BMT can be replaced with stem cell transplants (SCT) that yield major clinical difference seems to exist in the expression between the
40
610% mortality, minimal morbidity, and less infertility issues. Clini- two thalassemia genes in the heterozygous state. About 1% of African
cal efficacy is controversial with the highest risk factor being a lack of Americans are heterozygous for b@thalassemia.
engraftment. Event-free survival is 670% for BMT and between 80
and 90% for SCT in patients with b@thalassemia who receive HLA- pathophysiology
identical, related BMT. 40–44 The success rate is lower for adults with The normal b@gene directs synthesis of sufficient amounts of
45
advanced disease. It is difficult to find HLA-matched related donors, b@chains to synthesize enough HbA for nearly normal oxygen deliv-
and success is much lower (ranging from 20–76%) using matched, ery and erythrocyte survival. In the case of a heterozygous b patient,
+
unrelated donors. 46–49 Haploidentical mother-to-child transplanta- the thalassemic gene will also contribute to b@chain production.
45
tion can be an option for children without a matched donor. Cord
blood transplants are being investigated and show promise. 50–52 With
a typical life expectancy of around 50 years when patients follow rec- clinical Findings
ommended transfusion and chelation protocols, supportive therapy The heterozygote appears to be asymptomatic except in periods of
must be considered over curative therapy unless the patient is young, stress that can occur during pregnancy and with infections. Under
fit, and considered for transplant prior to developing thalassemia- such conditions, a moderate microcytic anemia can develop. Con-
related sequalae. 4 comitant folate deficiency can produce a macrocytic anemia.
Drugs to treat leukemia are being used to induce the re-
expression of latent g@genes that would combine with the excess laboratory Results
a@chains and produce HbF. 53–55 The two major risk factors for The anemia in b@thalassemia minor is mild with hemoglobin values
this treatment approach include failure of g@chain stimulation and in the range of 9–14 g/dL with the mean value for women being
induction of malignancy from the antileukemic drugs. 56,57 10.9 g/dL and for men 12.9 g/dL. 66–68 The erythrocyte count is
12
There is continuing interest in gene therapy approaches in which increased (75 * 10 /L) for what is expected at the given hemoglobin
autologous stem cells are harvested and the b@gene complex trans- concentration. The condition is usually discovered incidentally during
fected using viral vectors and transplanted back into the host. How- testing for unrelated symptoms or during family study workups.
ever, several complications prevent widespread use. Human globin The erythrocytes are microcytic (MCV = 55-70 fL) and hypo-
genes are large and complicated, making the finding of adequate viral chromic (MCHC = 29-33 g/dL) or sometimes normochromic
vectors difficult. In clinical trials of gene therapy for severe immuno- with an MCH that is usually 622 pg (Figure 14-10). The degree of
deficiency diseases, some patients have developed leukemias when the microcytosis, as indicated by the MCV, is directly related to the sever-
30
transfected genes insert next to or within critical hematopoiesis con- ity of the anemia. Although the anemia is mild, the peripheral blood
trol genes. 58–60 The first thalassemia patient to receive gene therapy smear shows variable anisocytosis and poikilocytosis with target cells
was reported in 2007. 61 and basophilic stippling. Nucleated red blood cells are not usually
The use of induced pluripotent stem cells (iPS) shows promise for found, but anemic patients can have a slightly elevated reticulocyte
hematopoietic and genetic disorders in the future. Somatic skin fibro- count. Bone marrow shows slight erythroid hyperplasia and erythro-
blasts can be harvested and reprogrammed into iPSs by inducing the blasts poorly filled with hemoglobin.
M14_MCKE6011_03_SE_C14.indd 265 01/08/14 12:50 pm
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