Anemia

hematology-oncology
ANEMIA: PRODUCTION DEFECTS

Stavrula Otis, MD, and Elizabeth A. Price, MD, MPH*
Definition
Red blood cell (RBC) production defects cause anemia
that is marked by a low absolute reticulocyte count. Examination of the peripheral blood count and the bone
marrow aids in classifying these disorders. The marrow
characteristically shows one of the following:
1. A normal ratio of myeloid cells to erythroid cells (M:E
ratio), normal overall cellularity, and a normal pattern of
erythroid maturation
2. Virtual absence of normal bone marrow elements caused
by aplasia (absence of marrow cells) or by replacement
of normal marrow elements by fibrosis, solid tumors,
granulomas, or leukemia
3. Erythroid hyperplasia with increased cellularity. Because
of defects of erythroid maturation, there is ineffective
erythropoiesis or intramedullary hemolysis. Erythroid
precursors die in the marrow, and few cells reach the
periphery.
Production Defects Associated with Apparently Normal
Bone Marrow
anemia of inflammation
Definition and Epidemiology

The anemia of inflammation (AI; also known as the
anemia of chronic disease) is one of the most prevalent types
of anemia, second only to iron deficiency anemia (IDA).1
It is a hypoproliferative anemia defined by a low serum
or plasma iron concentration in the presence of adequate
reticuloendothelial iron stores, in the presence of concomitant inflammatory disease.
Etiology
AI is driven by inflammatory cytokines and is classically
associated with infection, malignancy, and autoimmune disease, although it may also be seen in the context of chronic
liver disease, chronic kidney disease (CKD), congestive heart
failure, and diabetes.1,2
Pathogenesis
In the setting of inflammation, there are multiple mechanisms that contribute to anemia, the most important of
which is the hepcidin-mediated disruption of iron flow into
the plasma compartment. Hepcidin is a 25amino acid
peptide hormone that binds to ferroportin,3,4 the major iron
exporter found on the basolateral surfaces of enterocytes,
macrophages, and hepatocytes. Ferroportin is a multipass
* The authors and editors gratefully acknowledge the contributions of the previous author, Stanley L. Schrier, MD, FACP, to
the development and writing of this chapter.
Financial disclosure information is located at the end of this chapter
before the references.
2014 Decker Intellectual Properties Inc
transmembrane protein responsible for the delivery of

dietary iron from the gut into the circulation, as well as the
release into the circulation of recycled iron from macrophages and stored iron from hepatocytes. When hepcidin
binds to ferroportin, the complex is internalized, phosphorylated, and degraded.5 In the absence of ferroportin, iron is
unable to move across cell membranes into the plasma,
where it binds to its carrier protein, transferrin. This leads to
decreased plasma iron levels (hypoferremia) and a decrease
in available iron for erythroid precursors in the bone
marrow. Like other hormones, hepcidin is feedback regulated by the substance it controls: iron. Hyperferremia leads
to increased hepcidin levels, whereas hypoferremia leads
to decreased hepcidin levels.6 In addition to plasma iron,
several other extracellular signals regulate hepcidin production, including the inflammatory cytokine interleukin-6 (IL6) and lipopolysaccharide (LPS), which upregulate hepcidin,
as well as erythropoietin (EPO), erythropoiesis, hypoxia,
and anemia, which downregulate hepcidin.6
There are several other mechanisms by which inflammatory mediators contribute to AI apart from the regulation of
hepcidin. Various inflammatory cytokines have been shown
to suppress both EPO activity and erythropoiesis by direct
and indirect means. IL-1 and tumor necrosis factora (TNFa) directly suppress EPO production in vitro by inhibiting
EPO gene expression.7 TNF-a and interferon gamma can
induce EPO resistance in vitro, in part by downregulating
EPO receptors on erythroid progenitor cells, an effect that
can be overcome with higher levels of EPO.8 TNF-a and
interferon gamma also directly inhibit erythropoiesis by
inducing nitric oxide synthase. The corresponding increase
in intracellular nitric oxide results in a dose-dependent
increase in apoptosis of bone marrow progenitors.9 An
increase in apoptosis of erythroid bone marrow cells has
been seen in anemic patients with rheumatoid arthritis
compared with healthy controls, with normalization of
levels following anti-TNF treatment.10 An inflammatory
milieu is also noxious to circulating RBCs, as demonstrated
by the shortened survival of RBCs from normal healthy
controls when transfused into patients with inflammation
and, conversely, the normal survival of RBCs from patients
with chronic disease when transfused into normal healthy
controls.11
Diagnosis
AI is usually mild and asymptomatic, but more severe
cases may present with the usual symptoms of anemia
(e.g., fatigue, dyspnea on exertion, headache, dizziness). The
physical examination may vary depending on the underlying disease process driving the anemia. AI is characterized
by a mild to moderate drop in hematocrit, although 20% of
patients have a more severe anemia, with hematocrit values
less than 25%.2 When determining whether a patient has AI,
RBC and iron indices are the first parameters to consider. In
AI, the hemoglobin and hematocrit are mildly to moderately
decreased, and erythrocytes are typically normochromic
Scientific American Medicine
DOI 10.2310/7900.1044
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anemia: production defects 2

200 ng/mL were also iron deficient.13 In this setting, a useful
measure of iron deficiency is the soluble transferrin receptor
(sTfR). The transferrin receptor (TfR) is a transmembrane
protein responsible for binding serum transferrin and
importing iron into the cell. The TfR is found in particularly
high concentrations on erythroid progenitors and is upregulated by iron deficiency and increased erythropoiesis (e.g.,
hemolysis, exogenous EPO), but not by inflammation. The
sTfR is a truncated form of the tissue receptor, and its serum
levels reflect total body TfR concentration.14 The sTfR index,
defined as sTfR divided by the log of ferritin (sTfR/log
ferritin), is a simultaneous measure of the functional iron
compartment and the stored iron compartment and therefore provides a more accurate estimate of total body iron
than either metric alone.15,16 An sTfR/log ferritin less than
1.5 indicates the presence of iron deficiency in patients with
concomitant AI. In a recent study, the use of ferritin, sTfR,
and sTfR index improved the detection of IDA in patients
with concurrent AI from 41 (using ferritin alone) to 92%
(using all three).17 The use of the sTfR and sTfR/log ferritin
ratio is made somewhat more difficult by the fact that
multiple assays with variable reference ranges are currently
in clinical use.
Because of the central role it plays in iron homeostasis,
there has been a concerted effort in recent years to establish
a reliable method for measuring serum hepcidin levels.18,19
The results of one study comparing serum hepcidin and IL6 levels in patients with AI, IDA, and AI + IDA suggest that
erythroid demand for iron is a more powerful regulator of
hepcidin expression than inflammation.20 If that is the case,
and normocytic, although with more severe anemia, the

cells can become hypochromic and microcytic. Serum iron is
usually low, transferrin is decreased or low normal, and
transferrin saturation is usually normal or low due to the
concomitant decline of serum iron and transferrin concentrations [see Table 1]. Serum ferritin, on the other hand, is
elevated in AI for two reasons: (1) iron stores are increased,
and (2) ferritin behaves like an acute-phase reactant. In
contrast, IDA is typically characterized by an elevated
serum transferrin, low transferrin saturation, and low serum
ferritin.
The patient with concomitant AI and IDA is encountered
frequently and may pose a diagnostic challenge to the
clinician. The gold standard is generally felt to be evaluation
of a bone marrow aspirate for iron content, but this is an
invasive procedure and rarely done for this indication. The
distinction between isolated AI and AI with concurrent IDA
(AI + IDA) is important to make because of the difference
in patient management. Patients with AI + IDA will likely
benefit from iron supplementation, whereas patients with
AI alone will not only not benefit from iron replacement but
could also be at risk for iron overload with supplemental
iron. Due to the lack of specificity of serum iron, transferrin,
and ferritin in distinguishing AI from IDA or AI + IDA,
other diagnostic measures have been proposed. In general,
a serum ferritin less than 60 ng/mL in a patient with chronic
disease is suggestive of concomitant iron deficiency, and a
trial of intravenous iron may be appropriate.12
One study, however, showed that up to one third of
patients with AI and a serum ferritin between 100 and
Table 1 Differential Diagnosis of Hypochromic Anemias

Anemia of Chronic
Inflammation (ACI)
Iron Deficiency
Anemia (IDA)
Smear
Usually normochromic, normocytic

but can be mildly
hypochromic,
microcytic
Varies with the

degree of the
anemia
Serum iron level
Low
Low
Iron-binding capacity
Low/normal
High
Low/normal
Normal
Normal
Percent saturation
516
016
Low
Normal (2040)
6090
Serum ferritin level
Normal or high
Low
Low/normal/or
slightly high
Normal
High
Soluble transferrin
receptor (sTfR)
Normal
High
Usually normal but

can be increased
if the iron
deficiency is
severe
Variable
Variable
sTfR/log ferritin
Low
High
Normal or high
Normal or high
Marrow iron in
reticuloendothelial
cells
++ to +++
++ to +++
++++
Marrow iron in
sideroblasts
++ to +++
++++ with ringed

sideroblasts
Marrow erythroid
precursors
Normal
Generally normal;
cytoplasm may
be scanty
Normal
Usually mild
erythroid
hyperplasia
Intense erythroid
hyperplasia with
dyserythropoiesis
Laboratory Variable
+: degree of iron present, ++: moderate, +++: high, ++++: very high.

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Thalassemia
Trait
Sideroblastic Anemias
Varies with disease

and the extent of
anemia; usually
normal
Hypochromia,
target cells,
microcytes,
basophilic
stippling
Red blood cells can be

micro-, normo-, or
macrocytic; in female
carriers, a dimorphic
population can be seen
Low
Normal
High
ACI and IDA
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serum hepcidin may be an ideal marker of iron deficiency in
patients with background AI. To date, hepcidin immunoassays appear to hold the most promise of becoming the
method of choice for quantifying serum hepcidin concentration because of their low limit of detection, relatively
low cost, and high throughput21; however, serum hepcidin
assays are not readily available for clinical use at this time.
Differential Diagnosis
The differential diagnosis of anemia of inflammation
includes other types of hypoproliferative anemias, including
iron deficiency, vitamin B12/folate deficiency, copper
deficiency, and anemia of renal disease. Drug- or radiationinduced myelosuppression and infiltration of the bone
marrow by infection or malignancy should also be
considered in the differential diagnosis.
Management
Identifying and treating the underlying disease are paramount in managing AI as AI may be the presenting feature
of a serious underlying disorder. In most cases, the anemia
itself is mild and asymptomatic and does not require
treatment. In more severe cases, pharmacologic doses of
erythpoiesis-stimulating agents (ESAs) can override the
defect in EPO production. It is useful to check a baseline
(pretreatment) plasma EPO level because a response to ESAs
is unlikely in patients whose endogenous levels are above
500 mU/mL. The recommended dosing for ESAs is as
follows: for EPO, 150 units/kg three times a week or 10,000
units three times a week or 40,000 units weekly; for darbepoetin (longer-acting formulation), 2.25 g/kg weekly or
200 g every 2 weeks or 500 g every 3 weeks.11 The target
hemoglobin is 10 to 12 g/dL. If the hemoglobin level does
not rise after 12 weeks, ESAs should be discontinued. To
respond optimally, the patient must have adequate available
iron stores. If concomitant iron deficiency is suspected or
confirmed, iron therapy may be corrective.22 In patients with
AI, intravenous iron formulations may be preferable given
the possibility of elevated hepcidin levels limiting oral iron
absorption.23
Although responses to ESAs have been reported in
patients with cancer,24 growing evidence suggests that the
use of ESAs in patients with active cancer may be harmful.
Several clinical trials of patients with different types of
cancers have shown an increased risk of venous thromboembolism, as well as worse overall survival in patients
treated with ESAs.2527 The American Society of Hematology
and the American Society of Clinical Oncology have published a joint clinical practice guideline for the use of erythropoietic therapy in adult patients with cancer, with the
recommendation that careful evaluation of risk and benefits
be assessed prior to using erythropoietic therapy in patients
undergoing myelosuppressive chemotherapy who have a
hemoglobin less than 10 g/dL and further cautioning against
the use of erythropoietic therapy under other oncologic
circumstances.28
Complications
The complications of anemia of inflammation are primarily related to the underlying disorder rather than the anemia
itself, which is usually mild. In cases of severe anemia,

complications may include severe fatigue, acute coronary
syndrome, congestive heart failure, arrhythmias, and severe
dyspnea.
Prognosis
The prognosis for individual patients with AI varies
depending on the underlying disease process causing the
anemia and the severity of the anemia.
anemia in renal disease
Epidemiology
More than 10% of the US population has CKD, with higher
rates in those with diabetes and hypertension.29 Over 90% of
those with severe renal disease will have anemia without
intervention, whereas 45% of those with milder forms of
renal insufficiency have anemia.30
Etiology/Pathogenesis
The predominant cause of anemia in renal disease is a
deficiency of EPO production by the diseased kidneys.
Inflammatory markers are also frequently elevated, suggesting a component of AI.31 Anorexia and poor iron intake, frequent blood sampling, and loss of erythrocytes during hemodialysis may produce iron deficiency. Folic acid
deficiency, hypersplenism, and secondary hyperparathyroidism with marrow fibrosis29 may also promote anemia.
Anemia in hemodialysis patients can be caused by aluminum toxicity as well. This anemia was initially identified
in patients who had so-called dialysis dementia. Very high
plasma aluminum levels probably result from aluminum
contamination of the dialysis fluid or gastrointestinal
absorption of the aluminum gels taken to bind dietary
phosphates. In vitro experiments have shown that aluminum inhibits the growth of the erythroid precursors colonyforming uniterythroid (CFU-E) and burst-forming unit
erythroid (BFU-E).32
Diagnosis and Differential Diagnosis

There are no findings that specifically confirm the diagnosis of anemia due to renal disease. Details should be sought
related to other possible causes of hypoproliferative anemia,
and the blood smear should be examined for findings
that suggest other causes of anemia. The presence of Heinz
bodies suggests that oxidative hemolysis has occurred,
perhaps caused by oxidants in hemodialysis fluid.
Management and Complications

ESAs have to date been the standard treatment for
patients with CKD and moderate to severe anemia. EPO
therapy can eliminate the transfusion requirement for
patients on hemodialysis and in patients with progressive
renal disease who do not yet require hemodialysis. Such
treatment significantly improves their quality of life.33
However, recent data have raised concern about the use of
ESAs and an increased risk of hypertension,34 cardiovascular
events, and death.35 In one of the largest trials to date in
patients with type 2 diabetes and CKD, 4,038 patients were
randomized to treatment with darbepoetin alfa to achieve a
hemoglobin level of 13 g/dL, or treatment with placebo,
with rescue darbepoetin alfa when the hemoglobin level

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was less than 9 g/dL (administered in 46% of placebo
patients).36 During treatment, the median hemoglobin level
was 12.5 g/dL in the darbepoetin alfa group and 10.6 g/dL
in the placebo group. There was a significant increase in the
risk of both venous and arterial thromboembolic events and,
in particular, a significant increase in the incidence of stroke
in the darbepoetin alfa group compared with the placebo
group. Recent Food and Drug Administration (FDA) guidelines indicate that no clinical trial to date has identified a
hemoglobin target, ESA dose, or dosing strategy that does
not increase the risks of serious adverse cardiovascular
events in patients with CKD and that the lowest ESA dose
sufficient to reduce the need for RBC transfusions should be
used.37 The FDA recommends the reduction or interruption
of ESA dosing if the hemoglobin level exceeds 10 g/dL in
patients with CKD not on dialysis and the reduction or
interruption of ESA dosing if the hemoglobin level exceeds
11 g/dL in patients on dialysis. Hemoglobin levels should
be monitored at least weekly when adjusting or initiating
therapy, until values are stable, and then at least monthly.
In addition, the risks and benefits of ESA therapy should
be discussed with each patient prior to initiating therapy,
and the ESA medication guide should be provided to
each patient. Side effects should be reported to the FDA
MedWatch program. Iron status must also be monitored
with ESA treatment, and parenteral iron supplementation
may improve the response to ESA therapy.38
anemia secondary to other conditions
Alcohol Abuse
Excessive alcohol ingestioneither acute or chronichas
profound hematologic effects.39 Ingestion of about 80 g of
alcohol (one bottle of wine, six pints of beer, or one third
of a bottle of whiskey) daily may produce macrocytosis,40
stomatocytosis,41 thrombocytopenia, vacuolization of proerythroblasts, ringed sideroblasts,41 a sharp drop in serum
folic acid levels, and a rise in serum iron levels; it may also
impair the reticulocyte response to administered folic acid
in a patient known to be folic acid deficient. Acute alcohol
ingestion itself does not produce a megaloblastic anemia.39
It has been postulated that alcohol-induced hematologic
toxicity is mediated through acetaldehyde, the major
metabolite of ethanol, which is far more toxic and reactive
than ethanol. The mechanism for these alcohol-induced
abnormalities may be the formation of antibodies against
acetaldehyde-hemoglobin adducts.41 Megaloblasts, macroovalocytes, and hypersegmented polymorphonuclear neutrophils (PMNs) usually appear when concomitant folic acid
deficiency is present. Chronic alcohol abuse often results
in concomitant folic acid or iron deficiency, severe liver
disease, gastrointestinal bleeding, hypersplenism, and the
anemia of chronic inflammation.
Starvation
Starvation resulting from anorexia nervosa or protein
deficiency can cause anemia and even pancytopenia. Hemolysis may also be present [see Figure 1]. The bone marrow
biopsy is hypocellular, with a characteristic gelatinous background material consisting of acid mucopolysaccharides.
The anemia can occur despite normal folic acid and cobalamin (vitamin B12) levels and can be corrected with proper
nutrition.

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Hypothyroidism
Hypothyroidism
impairs
erythrocyte
production.
Although hypothyroidism can cause a macrocytic anemia,
the clinician should additionally evaluate for other causes of
macrocytic anemia, including the presence of folic acid or
cobalamin deficiency.
Panhypopituitarism
The mild anemia that is associated with severe panhypopituitarism can be corrected by replacement of adrenal,
thyroid, and gonadal hormones; the enhancing effect of
androgens on the action of EPO is well known.
Aging
The hemoglobin levels, RBC indices, and leukocyte and
platelet counts of healthy older people are similar to those
of younger adults; this finding was confirmed in a study of
patients who were 84 years of age or older.42 Thus, a workup
is required when anemia occurs in such older patients.
Approximately 10% of community-dwelling older adults
have anemia,43 most commonly defined by World Health
Organization criteria as a hemoglobin less than 12 g/dL in
women and less than 13 g/dL in men.44 Although typically
mild (i.e., hemoglobin value > 10 g/dL),43 anemia in this
population is associated with poor outcomes, including
disability,45 decreased functional status,46 and increased
mortality. The etiology of the anemia is often apparent and
frequently treatable, as in the case of IDA.43 However, in 30
to 40% of older adults with anemia, the etiology is not found,
despite intensive clinical investigation.47,48 This entity is
variably labeled unexplained anemia of the elderly (UAE),
senile anemia, or anemia of unknown etiology. The pathophysiology of UAE remains obscure; however, several
observations are surprisingly uniform in describing this
entity, suggesting a common underlying impairment. The
anemia in UAE is hypoproliferative, and EPO levels are
relatively low for the degree of anemia.48,49 However, in
contrast to what is seen in anemia of CKD, inflammatory
markers are not markedly elevated.49 Importantly, African
Americans may have physiologically lower hemoglobin values by approximately 1 g/dL, in comparison with whites,44,45
a difference that is not fully explained by the presence of
iron deficiency or a-thalassemia.45 In addition, older African
Americans with comparable degrees of anemia may not
have the associated morbidity and mortality seen in older
whites. Whether initiating therapy with erythropoietic
agents in patients with UAE improves functional outcomes
or mortality risk is unclear.50 In one randomized, doubleblind, placebo-controlled, crossover study in older, predominantly female African-American women with UAE or AI,
treatment with epoetin alfa and the resultant increase in
hemoglobin led to improved fatigue and quality of life,
with return to baseline 16 weeks after drug withdrawal.
Importantly, the target hemoglobin in the study was 13.0 to
13.9 g/dL, a target that is higher than current guidelines
recommend for other disease states37 and that may be associated with increased adverse events.36 Thus, further study is
needed before such treatment can be considered standard of
care.
heme-onc
Figure 1 The peripheral smear changes seen in severe liver disease or starvation (a) include distinct variation in the size and shape of red blood
cells; both sharply spiculed cells (spur cells) and scalloped erythrocytes are prominent. The leukoerythroblastic blood smear (b) indicates marrow
replacement with extramedullary hematopoiesis. It is characterized by variation in the size and shape of red blood cells, by the presence of nucleated red blood cells in the peripheral blood, by giant platelets, and by immaturity in the myeloid series. In folic acid or cobalamin deficiency (c),
the smear is characterized by variation in erythrocyte size and by distinct macrocytosis. Occasionally, fish-tailed erythrocytes are present, along
with hypersegmented neutrophils.
Production Defects Associated with Marrow Aplasia or

Replacement
The combination of anemia and neutropenia or
thrombocytopenia or the combination of all three of these
abnormalities (i.e., pancytopenia) usually indicates that the
hematopoietic marrow is damaged. If the marrow cavity is
infiltrated but pluripotent stem cells are intact, extramedullary hematopoiesis will often develop in the organs of fetal
hematopoiesis (i.e., spleen, liver, and distal bones).
Pancytopenia can be caused by both acquired and
congenital causes. The finding of combined cytopenias or
of immature cells in the blood (myelocytes, metamyelocytes,
and erythroblasts)that is, a leukoerythroblastic blood
smearsuggests extramedullary hematopoiesis [see
Figure 1]. These findings are an indication for bone
marrow aspiration and biopsy.
acquired aplastic anemia
Definition
Pancytopenia (i.e., anemia, neutropenia, and thrombocytopenia) and aplastic marrow on biopsy examination [see
Figure 2] establish a working diagnosis of aplastic anemia.
The biopsy specimen must not be taken from a marrow site
that has been irradiated. It is essential to determine the
severity of aplastic anemia. Severe aplastic anemia (SAA) is
defined by (1) marrow of less than 25% normal cellularity or
marrow of less than 50% normal cellularity in which fewer
than 30% of the cells are hematopoietic and (2) two of three
abnormal peripheral blood values (absolute reticulocyte
count < 20,000/L, absolute neutrophil count [ANC] < 500/L,

or platelet level < 20,000/L).51 Some investigators prefer
to identify a cohort of patients with very severe aplastic
anemia as those who had an ANC less than 200/L.50
Epidemiology.
The incidence of acquired aplastic anemia is two cases per
million in Western countries and two- to threefold higher in
Asia.52,53 Peak incidence occurs in children and young adults
and in patients over 60 years of age.54
Etiology/Pathogenesis
Aplastic anemia has a number of causes [see Table 2],
although in many cases, the exact cause cannot be determined. Ionizing irradiation and chemotherapeutic drugs
used in the management of malignant and immunologic
disorders have the capacity to destroy hematopoietic stem
cells. With careful dosing and scheduling, recovery is
expected. Certain drugs, such as chloramphenicol, produce
marrow aplasia that is not dose dependent. Gold therapy
and the inhalation of organic solvent vapors (e.g., benzene
or glue) can also cause fatal marrow failure. The rare connective tissue disorder eosinophilic fasciitis is also associated
with aplastic anemia. In 2 to 10% of hepatitis patients, severe
aplasia occurs 2 to 3 months after a seemingly typical case
of acute disease, usually in young men. Often the hepatitis
has no obvious cause, and tests for hepatitis A, B, and C are
negative.55 There is also a high incidence of aplastic anemia
after liver transplantation in patients with severe non-A,
non-B hepatitis.56 Aplastic anemia is also, albeit rarely, seen

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Figure 2 Shown are (a) a biopsy of normal bone marrow and (b) a biopsy of bone marrow from a patient with aplastic anemia showing almost
complete aplasia.
in pregnancy and may spontaneously improve following

delivery.57
Several lines of evidence support the possibility that
immune disorders can lead to aplasia. Marrow aplasia
occurs in graft versus host disease (GVHD).58 Immunosuppressive preconditioning improves the chances of successful
transplantation of syngeneic marrow into patients with
aplastic anemia,59 and immunosuppressive therapy has
been used successfully to treat idiopathic aplastic anemia.52
The blood of some patients with aplastic anemia appears to
contain suppressor T cells that suppress the growth of the
committed progenitor cells known as colony-forming unit
granulocyte-macrophage (CFU-GM). The suppressor T cells
Table 2
Causes of Aplastic Anemia
Irradiation drugs
Agents whose use regularly causes myelosuppression
Alkylating agents: melphalan, cyclophosphamide,
chlorambucil, busulfan
Antimetabolites: azathioprine, 6-mercaptopurine,
hydroxyurea, methotrexate
Other antitumor agents: daunorubicin, doxorubicin,
carmustine, lomustine, amsacrine
Agents whose use occasionally causes myelosuppression
Chloramphenicol, gold compounds, arsenic, sulfonamides,
mephenytoin, trimethadione, phenylbutazone,
quinacrine, indomethacin, diclofenac, felbamate
Toxins
Benzene, glue vapors
Infections
Non-A, non-B, non-C hepatitis; infectious mononucleosis;
parvovirus infection (attacks erythroid precursors); HIV
Malignant diseases
Hairy-cell leukemia, acute lymphocytic leukemia, acute
myeloid leukemia (rarely), myelodysplastic syndromes
Clonal disorders
Paroxysmal nocturnal hemoglobinuria
Immune-mediated aplasia
Eosinophilic fasciitis
Inherited disorders
Fanconi anemia
Pregnancy

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may act by producing interferon gamma.52 Patients with

aplastic anemia have been found to have decreased regulatory T cells, as seen in other autoimmune disorders,60
which could lead to deficient regulation of cytotoxic T cells.
The result of these complex immune mechanisms involving
suppressor T cells is a profound decrease in primitive
hematopoietic cells as measured by both the long-term
cultureinitiating cell assay and the ability to form secondary colonies from the colonies surviving 5 weeks of marrow
culture.61
Patients with inherited mutations in genes that repair
or protect telomeres appear to be at increased risk for the
development of aplastic anemia. Telomeres are nucleoprotein complexes important in the protection, replication,
and stabilization of linear eukaryotic genomes.62 In human
somatic cells, telomeres consist of tandem repeats that are
gradually lost with cellular aging; when critical shortening
is reached, permanent growth arrest and death follow.
The telomere repair machinery includes an RNA template,
encoded by TERC, and the reverse transcriptase telomerase,
encoded by TERT. Telomeres are short in one third of
patients with apparently acquired aplastic anemia,63 and
mutations have been identified in TERT64 and TERC.65
The majority of patients with aplastic anemia will harbor
paroxysmal nocturnal hemoglobinuria (PNH) [search
this book for information on PNH] clones, when tested
with very sensitive assays.52,66 Aplasia can also be part of a
prodrome to hairy-cell leukemia [search this book for
information on chronic lymphoid leukemias and plasma cell disorders], acute lymphoblastic leukemia [search this book
for information on acute lymphoid leukemia], or, in rare cases,
acute myeloid leukemia, or it can develop in the course
of myelodysplasia [search this book for information on
myelodysplastic syndrome or acute myeloid leukemia].
Diagnosis
Presenting symptoms are entirely related to the degree
of marrow failure and resultant cytopenias. The patient
with aplastic anemia may seek medical attention because of
heme-onc
fatigue and shortness of breath. Accompanying thrombocytopenia may cause petechiae, oral blood blisters, gingival
bleeding, and hematuria depending on the level of the platelet count. By far the greatest problem associated with aplastic anemia is the recurrent bacterial infections caused by the
profound neutropenia. Sepsis, pneumonia, and urinary tract
infections are common among patients with aplastic anemia.
Invasive fungal infections may cause death, especially in
patients with severe neutropenia.
The diagnosis of aplastic anemia requires a marrow aspirate and biopsy [see Figure 2], as well as a thorough history
of drug exposures, infections, and especially symptoms
suggesting viral illnesses and serologic test results for
hepatitis, infectious mononucleosis, HIV, and parvovirus
[see Figure 3]. Measurement of RBC and white blood cell
glycosylphosphatidylinositol-linked proteins is helpful in
the diagnosis of PNH.
It is also important to determine the severity of aplastic
anemia [see Acquired Aplastic Anemia, Definition, above].
Severe cases are associated with a very low rate of spontaneous remission and a mortality of 70% within 1 year. In
contrast, 80% of patients who have milder forms of aplastic
anemia survive for 1 year.67
The differential diagnosis of pancytopenia includes
hematologic malignancies, systemic lupus erythematosus,
and congestive splenomegaly. In these diseases, however,
the marrow is not aplastic but rather shows hyperplasia of
the involved cell lines. Other conditions that cause pancytopenia include diseases that lead to marrow replacement,
including solid tumors and infection, and hypoplastic
myelodysplastic syndrome (MDS).
A careful search must be made for signs and symptoms of
inherited bone marrow failure syndromes such as Fanconi
anemia and dyskeratosis congenita, which may present with
pancytopenia, particularly in the younger adult. The most
common physical findings seen in Fanconi anemia include
short stature, thumb abnormalities, microcephaly, eye
abnormalities, hyper- or hypopigmented skin lesions, developmental delay, and gonadal abnormalities (in males).68 The
Figure 3 Giant pronormoblast, evident on this marrow smear,

strongly suggests a diagnosis of parvovirus infection.

diagnosis can be confirmed by culturing peripheral blood
lymphocytes with the clastogenic agents diepoxybutane and
mitomycin C and assessing for increased chromosomal
breakage. Patients with dyskeratosis congenita may present
with the classic triad of nail dystrophy, lacey reticular skin
pigementation, and oral leukoplakia; other findings include
early gray hair or eyebrows and developmental delay.68 The
diagnosis is made by establishing the presence of a known
mutation or by the presence of short telomeres.69 Detection
is important not only for treatment of marrow aplasia, as
standard transplant regimens are overly toxic in patients
with Fanconi anemia, but also in monitoring for other
disease manifestations, including the development of solid
tumors.
Management/Complications/Prognosis
Treatment of mild aplastic anemia Treatment of milder
forms of aplastic anemia involves removing the offending
agent and providing supportive therapy, primarily transfusion therapy, anticipating that the remaining pluripotent
stem cells will repopulate the marrow.
Supportive therapy Thrombocytopenia is often a major
problem associated with aplastic anemia. It should be managed by platelet transfusion as needed to control or prevent
bleeding. Usually, a threshold of 10,000 platelets/L is used
for transfusion, but conservative treatment is best, and as
few transfusions as possible are given. Extensive platelet
replacement may result in allosensitization to platelets and
may complicate future allogeneic bone marrow transplantation. RBC transfusions are given as required to control the
symptoms and signs of anemia
Granulocyte colony-stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF)
have been given to patients to raise the ANC and help combat infection. They are usually ineffective when used alone
because of the severe deficiency in precursor cells, which
are the target for the actions of G-CSF and GM-CSF.70 It is
generally preferable to proceed to definitive treatment:
immunosuppressive therapy or, preferably, allogeneic bone
marrow transplantation in a younger patient, if a matched
sibling donor is available [search this book for information on hematopoietic stem cell transplantation (HSCT)].
Definitive therapy Transplantation from a matched
sibling is a very effective regimen for patients with aplastic
anemia.71 The use of peripheral blood transplantations may
reduce survival compared with marrow transplantations
owing to increased GVHD.72 The results with mismatched or
unrelated matched donors are somewhat worse, although
the outcomes were significantly improved with alternative
donor transplantations performed after 1997 versus those
performed earlier.73 Patients with aplastic anemia who are
without sibling donors are often given a trial of immunosuppressive therapy before transplantation.71
The current standard immunosuppressive regimen for
aplastic anemia is the combined use of antithymocyte globulin (ATG) and cyclosporine.71 The combination of ATG and
cyclosporine results in an approximately 70% response
rate by 4 months, with most responses occurring by
6 months.74 Younger age and better baseline reticulocyte
and lymphocyte counts correlate with improved response
to immunosuppressive therapy.75 The 5-year survival rate

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with immunosuppressive therapy is approximately 75%.71
To date, there has been no benefit to adding additional
immunosuppressive medication to this standard regimen.76,77
Caution should be used in treating older patients as those
over 60 years old have an increased risk of infection, bleeding, serious cardiac events, and early death with the use of
ATG.78
Horse ATG, given as a dose of 40 mg/kg/day for 4 days,79
appears to be more effective as first-line therapy than rabbit
ATG when used to treat aplastic anemia. The toxic side
effect of ATG is serum sickness, which can usually be controlled with corticosteroids. Prednisone (60 to 100 mg/day)
is given orally in divided doses, or methylprednisolone
(40 mg) is added to the infusion bottle, and the dose can
be increased to 1 mg/kg/day. Corticosteroid therapy is
adjusted to control serum sickness, but it can usually be
tapered after 2 weeks and stopped after 30 days. Because
ATG can lower platelet counts, platelet transfusions are
given as needed to maintain the platelet count at more than
20,000/L.
Cyclosporine (10 to 12 mg/kg/day) is given orally in
two divided doses, with the aim of achieving blood trough
levels 200 to 400 ng/mL.79 The cyclosporine is continued
for at least 6 months. Cyclosporine can cause hypertension,
renal toxicity, hypomagnesemia, hypophosphatemia, vitiligo,
tremors, hypertrichosis, susceptibility to Pneumocystis jiroveci
pneumonia (PCP), and gingival hyperplasia.74 Monthly
aerosolized pentamidine is given as PCP prophylaxis.80
In contrast to patients who undergo allogeneic bone
marrow transplantation, patients who respond to immunosuppressive therapy are not actually cured. Many of these
patients continue to have moderate cytopenia81; 20 to 36%
experience relapses of aplastic anemia,80 although prolonging cyclosporine therapy beyond 6 months may reduce the
risk of relapse.82,83 Patients remain at risk for the development of clonal disorders, such as PNH, MDS, and acute
leukemia. In one study with 11 years of follow-up, the
projected relapse rate was 38%, and clonal or malignant
diseases developed in 25% of patients.74 Patients are also at
increased risk for the development of solid tumors after
treatment of aplastic anemia, but the risk appears to be
similar for patients treated with immunosuppressive therapy as for those undergoing allogeneic bone marrow transplantation.84 More than 50% of patients who have relapses
of aplastic anemia after initially responding to immunosuppressive therapy may respond to a second course of therapy.85 For unresponsive patients, a trial of rabbit ATG may
work. The rabbit ATG (3.5 mg/kg/day diluted in saline and
infused over 6 to 8 hours for 5 consecutive days)86 is given,
along with cyclosporine (5 mg/kg/day p.o. on days 1
through 180 and then tapered) and G-CSF (5 g/kg/day on
days 1 through 90).85 A third course of ATG is likely to be
beneficial only in those who have previously responded.87
Initial reports suggest that the use of the anti-CD52
monoclonal antibody alemtuzumab as a single agent may
have efficacy comparable to rabbit ATG in patients who
have relapsed following or are refractory to horse ATG
based therapy.88 Interestingly, alemtuzumab had very poor
efficacy as first-line treatment.88
There is concern that the use of G-CSF in patients with
aplastic anemia may increase the risk of development of

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MDS or acute leukemia.89 However, in a recent European
Group for Blood and Marrow Transplantation (EBMT) study
randomizing patients on immunosuppressive treatment to
G-CSF or no G-CSF, the addition of G-CSF did not increase
the risk of secondary neoplasms or a new PNH clone.90
Longer follow-up may be needed to more fully exclude this
possibility. Although the addition of G-CSF led to fewer
infectious episodes (24% versus 36%), there was no improvement in response rates or overall survival in the G-CSF
arm.
An intriguing report concerns 67 patients with SAA who
were treated with high-dose intravenous cyclophosphamide
(50 mg/kg/day) for 4 consecutive days without stem cell
rescue.91 The median time to neutrophil recovery of 0.5
109/L was 60 days. In the treatment-naive group, the
response rate was 71%, and in the previously treated group,
the response rate was 48%. The actuarial probability of
survival at 10 years for the treatment-naive patients was
88%, with a median follow-up of 58 months. However, a
trial comparing high-dose cyclophosphamide with ATG
closed early because of excessive cyclophosphamideinduced morbidity and mortality.92 Therefore, the exact
role that high-dose cyclophosphamide should play in the
treatment of aplastic anemia requires further study.
Daclizumab is a humanized monoclonal antibody that
binds to the IL-2 receptor and blocks production of IL-2 by
activated T cells. Nineteen of 45 (42%) patients with moderate aplastic anemia responded within 3 months of treatment
with daclizumab, with another two patients responding to
a second course of treatment.93 In this study, pretreatment
transfusion independence was a positive predictor of
response.
Treatment of SAA The choice of first-line therapy
for patients with SAA is influenced by age and disease
severity. Allogeneic bone marrow transplantation should be
performed in patients younger than 20 years if a matched
sibling donor is available. Although there are risks, including chronic GVHD and organ dysfunction caused by the
conditioning program,59 75 to 80% of patients may be cured54;
the incidence of later clonal disorders is very low.94 Patients
younger than 20 years who do not have a matched sibling
donor should consider transplantation from a matched
unrelated donor. Allogeneic transplantation from a matched
unrelated donor initially produced a 2-year survival rate of
only 29% because of severe GVHD.70 However, the outcome
of unrelated donor transplants for aplastic anemia has
improved markedly over earlier efforts, likely due to
improved donor/recipient HLA matching and better
conditioning regimens and supportive care.71,95,96 There is
controversy regarding the upper age limit for which bone
marrow transplant should be first-line therapy in patients
with aplastic anemia.54 In general, patients between 20 and
40 years of age who are in excellent health and have a
fully matched sibling donor are also referred for first-line
treatment with bone marrow transplantation, with 5-year
survival rates of 70 to 80%.54 Patients older than 40 years are
treated with immunosuppressive therapy, although select
older patients may do well in the transplant setting if a
suitably matched related donor is available.54
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acquired pure red cell aplasia
Definition and Epidemiology

In adults, pure red cell aplasia (PRCA) is a rare acquired
syndrome characterized by a severe normocytic anemia,
profound reticulocytopenia (often 0%), and the absence
of RBC precursors in an otherwise normal bone marrow.
The hematocrit is usually less than 20% and the absolute
reticulocyte count less than 10,000/L, whereas the platelet
count, white blood cell count, and differential are normal,
a reflection of ongoing normal granulopoiesis and megakaryopoiesis in the bone marrow.
Etiology/Genetics
PRCA can present as a primary hematologic disorder in
the absence of underlying disease or occur secondary
to lymphoproliferative disorders, autoimmune disease,
parvovirus, thymoma and other solid tumors, treatment
with recombinant human EPO and other drugs, ABOincompatible HSCT, and pregnancy.97
Pathogenesis
Most cases of PRCA appear to be immunologically
mediated, either by humoral mechanisms or cell-mediated
mechanisms, depending on the underlying cause. Antibodydependent PRCA has been demonstrated in patients with
primary idiopathic PRCA,98101 as well as patients treated
with ESAs102,103 and ABO-mismatched HSCT.104 Although
cases of PRCA arising from autoantibodies against endogenous EPO have been described,105 most cases of EPO-related
PRCA occur in patients treated with ESAs. The antibodies
that form in response to exogenous EPO inhibit the growth
of erythroid progenitor colonies in vitro and cross-react with
endogenous EPO and all currently available ESAs.102,103
There was a sharp peak in the incidence of ESA-related
PRCA in 2002 that was attributed to leachates from uncoated rubber plungers in syringes,106 which have since been
replaced with Teflon stoppers, although sporadic cases
are still seen.107 PRCA arising from ABO-mismatched HSCT
results from preexisting isoantibodies in the host that
react with incompatible blood antigens on donor erythroid
progenitors; these isoantibodies may also be produced by
long-lived plasma cells derived from the host.104
For patients with PRCA in whom an IgG inhibitor cannot
be demonstrated, lymphocyte-mediated inhibition of erythropoiesis is thought to be the major mechanism of pathogenesis, with the notable exceptions of parvovirus-induced
PRCA and PRCA that occurs as a prodrome to MDS (discussed below). The association of PRCA with large granular
lymphocyte (LGL) leukemia is increasingly being recognized. In fact, more recent studies show that LGL leukemia
is the disorder most commonly associated with PRCA.108
This may shed light on the pathogenesis of PRCA associated
with other lymphoproliferative disorders, such as lymphomas and leukemias, as well as PRCA associated with solid
tumors, thymoma, and autoimmune diseases, including collagen vascular diseases, as clonal and nonclonal expansions
of LGLs have been associated with all of the aforementioned
disorders.109
LGL leukemia is defined as a clonal expansion of T cell
LGLs or natural killer (NK) cell LGLs. There are several

possible mechanisms by which LGLs may be activated
to destroy erythroid precursors; these mechanisms are
characterized by an inappropriate imbalance between LGL
activation and inhibition and rely on the fact that maturing
erythroid cells express diminishing levels of major histocompatibility complex (MHC) class 1 molecules, which are
essential for establishing immune tolerance, in part by binding to killer cell inhibitory receptors (KIRs) on T and NK
cells. The activation signals triggering LGL cytolysis may
include (1) binding of T cell receptor (TCR)-ab to an RBC
peptide displayed by an MHC class I molecule; (2) MHCunrestricted binding of TCR-cd to a ligand expressed on the
surface of RBC precursors; or (3) binding of erythroblastspecific autoantibodies to CD16 (the receptor for the Fc
portion of the IgG antibody) on NK-LGLs.97 Although other
myeloid cells may express the same peptide antigens or
ligands as the erythroid precursors, they are protected from
LGL-mediated cytolysis by the KIRs-MHC1 inhibitory
signal. In contrast, the activation signal goes unchecked in
erythroblasts due to low levels of MHC class 1 molecules,
resulting in LGL-mediated destruction of erythroid
progenitors.97
Parvovirus-induced PRCA appears to be a consequence of
direct toxic effects of the virus on the erythroblast. Parvovirus has limited tissue tropism and uses the blood group P
antigen as a primary receptor for virus entry into human
erythroid progenitors.110 Viral replication culminates in lysis
of RBC precursors and can cause a transient aplastic crisis in
patients with underlying severe hemolytic disorders such as
sickle cell anemia or hereditary spherocytosis. PRCA may
also present as a prodrome to the MDS or acute myeloid
leukemia. In a series of 360 cases of MDS diagnosed in a
single institution, six (1.6%) were found to have MDS with
erythroid hypoplasia/aplasia.111
Diagnosis
The patient with PRCA presents with symptoms characteristic of anemianamely, weakness, fatigue, and shortness of breath. WBCs and platelets are normal in number,
morphology, and functionality. A very low reticulocyte
counteither a relative reticulocyte value of less than
0.2% or a very low absolute reticulocyte count of less than
10,000/Lshould prompt the physician to order a bone
marrow aspirate. Typically, the transferrin saturation is
elevated as unused iron is shuttled out of the marrow. In
a patient with PRCA, a bone marrow aspirate and biopsy
typically show normal myelopoiesis, lymphopoiesis, and
megakaryocytopoiesis; erythropoiesis is virtually absent. In
the absence of any apparent cause of PRCA, four conditions
must be considered: idiopathic PRCA, thymoma, hypoplastic MDSs, and LGL leukemia. The workup to diagnose
PRCA usually includes computed tomography of the chest
to evaluate the possibility of thymoma, immunophenotypic
analysis, and T cell receptor clonality studies of circulating
blood or marrow lymphocytes to identify LGL proliferation,
marrow cytogenetics to evaluate the possibility of MDS, and
tests for parvovirus.112 A diagnostic hallmark of parvovirus
infection is the appearance of giant pronormoblasts in the
marrow [see Figure 3]. The distinction between PRCA associated with the MDSs and acute myeloid leukemia may
be difficult to determine at the time of diagnosis unless a
typical myelodysplastic cytogenetic abnormality is detected
during a bone marrow examination.

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If antibody-mediated PRCA is suspected, the patient
should be tested for neutralizing anti-EPO antibodies.113
Another clue to the diagnosis of antibody-mediated PRCA
is a low serum EPO level,103 in contrast to the high serum
EPO levels typically seen in other forms of PRCA.
PRCA is self-evident on the bone marrow biopsy, and the
differential diagnosis should include all possible causal
agents, such as parvovirus, thymoma, and lymphoproliferative disorders. Aplastic anemia should also be considered if
there are cytopenias in other cell lines.
Management
Initial management of PRCA should focus on supporting
the patient with RBC transfusions as needed for symptomatic
anemia and cessation of any causal or potentially exacerbating agents, such as ESAs and other drugs. No additional
therapy is required in those forms of PRCA that are usually
self-limited, such as in the case of ABO-incompatible bone
marrow transplantation.97,114 For cases of PRCA in which
there is a clearly identifiable cause, therapy should be
directed accordingly. For example, in cases of thymomarelated PRCA, thymectomy can lead to remission in 30 to 40%
of cases, although additional treatment may be required.115,116
In cases of parvovirus-induced PRCA, treatment with intravenous immune globulin (IVIg) (0.4 g/kg/day 5 days) can
accelerate recovery of erythropoiesis.112 For AIDS patients
with parvovirus infection and PRCA, IVIg may have to be
continued on a long-term basis.117 In cases of PRCA associated with ESAs, the ESA should be stopped immediately
and immunosuppressive therapy should be initiated. In one
study, 57% of patients who received immunosuppression
recovered, compared with only 2% of those who did not
receive immunosuppressive therapy.118 It is safe to rechallenge patients with ESAs if the neutralizing antibody is no
longer detectable,118 although subsequent relapse has been
reported.119 Peginesatide (formerly Hematide, Affymax
Inc., Palo Alto, CA) is a synthetic EPO agonist with no
structural similarities to human EPO that does not appear
to cross-react with EPO and was used successfully in an
anti-EPO antibody-mediated PRCA rat model.120 Peginesatide was successful in improving hemoglobin and leading to
transfusion independence in 13 of 14 patients with antibodymediated PRCA.121
Primary idiopathic PRCA and secondary PRCA that
fails to respond to therapy directed at the underlying disease should be treated with immunosuppressive therapy
based on the presumption that the disorder is immune
mediated. Remissions have been reported using corticosteroids, cyclophosphamide, cyclosporine, ATG, splenectomy,
plasmapheresis, rituximab, and alemtuzumab.97 In very
refractory cases, allogeneic bone marrow transplantation
can be effective.122 Historically, corticosteroids have been the
most commonly used first-line agent. Oral prednisone is
given at a dose of 1 mg/kg/day until remission is induced
(hematocrit > 35%) and then tapered very slowly over a 3- to
4-month period.112 Although remission is achieved in 30 to
62% of patients within 2 to 4 weeks, relapse is common and
occurs most often during the taper.123 Long-term maintenance therapy with steroids is undesirable due to the side

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effects of myopathy, hyperglycemia, infection, osteoporosis,
and compression fractures. Cyclosporine is emerging as
a potentially better first-line treatment option.124,125 The
response rates are excellent, with 65 to 87% of patients
achieving remission within 2 to 12 weeks.123 The reported
dosages range from 2 to 7 mg/kg, with total dosages of
200 to 300 mg/day most commonly used. Maintenance
cyclosporine is more effective in sustaining remission than
maintenance corticosteroids, but relapse rates are high when
therapy is discontinued altogether.123 Cyclophosphamide
has been also effective in inducing remissions in PRCA and
is often used in patients with associated LGL leukemia.108,126
Usually low doses (50 to 100 mg/day p.o.) for 3 to 6 months
suffice to produce remission. As with corticosteroids,
however, the long-term toxicity profile is unacceptable,
particularly the increased risk of MDS, bladder cancer, and
gonadal toxicity.
Induction of remission in patients with acquired PRCA is,
for the most part, easy to achieve using any one (or a combination) of the aforementioned therapies. Sustaining the
remission is more of a challenge as the disease has a
tendency to relapse. In light of that, the long-term toxicity
profile must be considered when choosing first- and secondline medications. There are few reports to date on the
efficacy of biologic therapies such as ATG, rituximab, and
alemtuzumab, and very little is known about their longterm efficacy.97,127,128 Of the therapies most frequently used,
cyclosporine is as effective, if not more so, than other agents
and has the most acceptable toxicity profile. That being said,
the minimum required dose to maintain remission should
be used, and the patient should be closely monitored for
nephrotoxicity and hypertension.
Complications
The complications of PRCA are those of severe anemia
and include profound fatigue, weakness, dyspnea on
exertion, and acute coronary syndrome. A leading cause
of morbidity and mortality in this patient population is
infection, a treatment-related complication.
Prognosis
Acquired PRCA is usually a chronic condition, with
relapses occurring anywhere from 3 months to 8 years after
treatment-induced remission.125,129 Overall survival varies
depending on the underlying cause and can range from
4 years in patients with secondary PRCA to over 10 years in
patients with idiopathic PRCA.130 Some types of secondary
PRCA, such as pregnancy-related and thymoma-related
PRCA, have a much better overall survival.131,132
Production Defects with Marrow Erythroid Hyperplasia
and Ineffective Erythropoiesis
definition
Anemia with a low reticulocyte count may occur despite
intense marrow erythroid hyperplasia. This paradoxical
situation is the hallmark of ineffective erythropoiesis or
intramedullary hemolysis. Generalized erythroid impairment may be present, or specific subpopulations of erythroid
precursors may be involved. Some of these subpopulations
heme-onc
escape death in the marrow, but their progeny are so
severely damaged that they are rapidly removed from the
circulation, thus giving the picture of peripheral hemolysis.
Other signs of ineffective erythropoiesis include jaundice, a
very high serum lactate dehydrogenase level, and 75 to 90%
saturation of serum iron-binding capacity. The classic ferrokinetic picture shows rapid plasma iron clearance, which
indicates intense erythroid precursor activity. The delivery
of labeled RBCs to the peripheral circulation, however, is
dramatically reduced, which suggests that the precursors
are being destroyed by intramedullary hemolysis.
The differential diagnosis includes megaloblastic anemias,
sideroblastic anemias (SAs), thalassemia [search this
book for information on hemoglobinopathies and hemolytic
anemias], MDSs [search this book for information on MDS
and acute myeloid leukemia], and agnogenic myeloid metaplasia [search this book for information on myelofibrosis and
myeloproliferative neoplasms].
megaloblastic anemias
Etiology
Megaloblastic anemias are caused by cobalamin or folic
acid deficiency, by drugs that interfere with the synthesis of
DNA or with the absorption or metabolism of cobalamin,
and by genetic disorders that interfere with DNA metabolism or with the absorption or distribution of cobalamin.
Pathophysiology
Megaloblastic erythropoiesis is characterized by defective
DNA synthesis and arrest at the G2 phase, with impaired
maturation and a buildup of cells that do not synthesize
DNA and that contain anomalous DNA. This condition
leads to asynchronous maturation between the nucleus and
the cytoplasm.133 RNA production and protein synthesis
continue; thus, larger cells, or megaloblasts, are produced.
Ineffective erythropoiesis results, and there is disagreement
about the presence of increased apoptosis.134,135 It is presumed that similar defects in DNA synthesis characterize
the mucosal abnormalities of the stomach and tongue. In
the granulocytic line, the presence of giant metamyelocytes
represents ineffective granulopoiesis.133
Role of folic acid and cobalamin The interactions
between folic acid and cobalamin are critical in the metabolism of single carbon units, mainly methylene and formyl
analogues, which have a key role in the synthesis of DNA
and purines [see Figure 4a].136 There are two major coenzymes
of cobalamin, adenosylcobalamin and methylcobalamin.
Adenosylcobalamin is the coenzyme for methylmalonyl
coenzyme A mutase, which catalyzes a step in the catabolism of propionic acid [see Figure 4b]. Methylcobalamin
is the coenzyme for methionine synthase, which functions
as a methyltransferase in the reaction that converts 5methyltetrahydrofolate (CH3-THF1) to tetrahydrofolate
(THF1) [see Figure 4a].135 Cobalamin and folic acid [see
Figure 5] combine in the methionine synthase reaction [see
Figure 4a], in which the methyl group of CH3-THF1 is transferred to cobalamin to form methylcobalamin. Methylcobalamin then transfers its methyl group to homocysteine to

form methionine. The monoglutamated THF1 that is formed
by this reaction is polyglutamated by the enzyme folylpolyglutamate synthase, and a methylene group is added to it by
the serine-glycine methyltransferase to form 5,10-methylene
THFn. 5,10-Methylene THFn provides its methylene to convert deoxyuridylate to thymidylate, a key step in DNA synthesis. 5,10-Methylene THFn can also be directly converted
to CH3-THF1 by the enzyme 5,10-methylenetetrahydrofolate
reductase, thereby making its methyl group available.
Formyl THFn (also called leucovorin, folinic acid, or
citrovorum factor) has an important role in purine synthesis
and DNA metabolism. It can be generated by oxidation of
5,10-methylene THFn or directly from THFn by the enzyme
formyl THF synthase, with methionine providing the
formate group [see Figure 4a].136 When cobalamin is deficient,
CH3-THF1 cannot transfer its methyl group to cobalamin;
therefore, THF1 is not free to be polyglutamated by folylpolyglutamate synthase [see Figure 4a]. The polyglutamated
form is required for synthesis of either 5,10-methylene THFn
or formyl THFn; thus, DNA synthesis and purine synthesis
are blocked. This hypothesis, the methylfolate trap hypothesis, is supported by the finding of increased levels of
CH3-THF1 in the plasma of cobalamin-deficient patients. An
alternative explanation is the formate starvation hypothesis,
wherein cobalamin deficiency impairs methionine generation, which therefore cannot provide the methyl groups
needed by the enzyme formyl THFn synthase to produce
formyl THFn.
Other aspects of folic acid and cobalamin metabolism
Neither folic acid nor cobalamin is produced by humans
in adequate amounts; both must be absorbed from food.
Cobalamin, in particular, is derived from microbial sources
and is ingested in the form of meat or eggs.
Most of the dietary folic acid is in the polyglutamate form
and is absorbed at the intestinal mucosa. Absorption of
radioactively labeled folic acid approaches 80% of a 200 g
dose.133,136 The serum folic acid level appears to be maintained by folic acid absorbed from food. Enterohepatic
circulation of folic acid has been observed in which folic
acid passing into the bile and small intestine is quantitatively
reabsorbed. In an animal model, ethanol administration
blocks the entry of folic acid into the bile. This effect could
account, in part, for the sharp fall in the serum folic acid
level seen 8 hours after alcohol consumption. A similar
fall in serum folic acid level follows phenytoin ingestion.
The daily requirement of cobalamin is about 1 g; however,
the recommended daily intake is 2.4 g for adults to ensure
the availability of that 1 g. A typical Western diet that
is rich in animal products usually provides about 5 to
15 g.137
R proteins are a class of cobalamin-binding glycoproteins
found in saliva and gastric juice; they are produced by granulocytes and other tissues. Intrinsic factor (IF) is a 45 kDa
glycoprotein, secreted by gastric parietal cells, that is highly
specific for unaltered cobalamin. After ingestion, cobalamin
enters the stomach bound to animal proteins. Cobalamin is
released from animal proteins by pepsin and hydrochloric
acid. Free cobalamin is then bound to R proteins. The R
proteincobalamin complex does not bind to ileal receptors

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heme-onc
a
Adenosyltransferase
Methionine Synthase
Homocysteine
Methionine
S-Adenosylmethionine
Cobalamin
Methylcobalamin
CH3-THF1
THF1
Folylpolyglutamate Synthase
Serine-Glycine
Methyltransferase
5,10-Methylene THFn
THFn
Formyl THF Synthase
Oxidation
Formyl THFn
Purine Synthesis
5,10-Methylenetetrahydrofolate Reductase
Deoxyuridylate
Thymidylate
CH3-THFn
Methylmalonyl-CoA
Mutase
b
Propionyl-CoA
Methylmalonyl-CoA
Adenosylcobalamin
Succinyl-CoA
Figure 4 (a) Intracellular interdependent cofactor activity of cobalamin and folic acid is essential in DNA synthesis and metabolism.61
(b) Adenosylcobalamin is a cofactor in the synthesis of succinylcoenzyme A from methylmalonylcoenzyme A.61 CoA = coenzyme A; THF1 =
tetrahydrofolate.
Figure 5 Folic acid functions as a coenzyme in single-carbon transfer reactions. It is not physiologically active until it is reduced at
positions 5, 6, 7, and 8 to tetrahydrofolate. Single-carbon groups (R)
such as methyl analogues and formate are added at either position 5
or position 10, or they may bridge positions 5 to 10, as shown. Several
glutamates may be attached in sequence (R1), which convert the
monoglutamate to the polyglutamate form. Enzymes of the intestinal
mucosa split polyglutamates back to monoglutamate, whereas liver
enzymes add glutamate to tetrahydrofolate or to other reduced folic
acids.

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and thus is not absorbed. In the stomach, cobalamin binds

preferentially to R proteins rather than to IF133; thus, it is the
physiologically inactive R proteincobalamin complex that
is discharged into the duodenum. In the duodenum and
small intestine, however, the pancreatic proteases degrade
the R proteins, freeing cobalamin and allowing it to bind to
IF. The IF-cobalamin complex, in the presence of Ca2+ and
at a pH level greater than 5.4, binds to the receptor for
IF-cobalamin, cubilin, expressed in the terminal portion
of the ileum, where absorption takes place [see Figure 6].138
This absorption is facilitated by the transmembrane protein
amnionless.138
In the plasma, most of the cobalamin is bound to haptocorrins, with 10 to 30% bound to transcobalamin.138 The
cobalamin-transcobalamin complex binds to a receptor
protein on cell surfaces and is internalized. Cobalamin is
primarily stored in the liver and kidney.
The elevation of cobalamin levels seen in patients with
chronic granulocytic leukemia or significant granulocytosis
is caused by increases in haptocorrin, which is produced in
granulocytes.
heme-onc

of anti-IF antibodies: one of these antibodies blocks attachment of cobalamin to IF, and the other blocks attachment of
the IF-cobalamin complex to ileal receptors.143 Clinically,
highly specific anti-IF antibodies are found in about 70% of
patients with pernicious anemia. Pernicious anemia may
also be caused by chronic atrophic gastritis, leading to loss
of IF production. The chronic atrophic gastritis in pernicious
anemia is also associated with an increased risk of intestinaltype gastric cancer and of gastric carcinoid tumors.144 Pernicious anemia occurs in association with other autoimmune
disorders. In one study, autoimmune thyroid disorders were
observed in 24% of 162 patients with pernicious anemia.145
An increase in vitamin B12 deficiency is also seen in patients
treated with the antidiabetic agent metformin,146 possibly
due to inhibition of calcium-dependent uptake of vitamin
B12 by ileal receptors.147
l
l
lI
lI
ll
ll
ll
l ll
ll
l
ll
Figure 6 Cobalamin assimilation. Dietary cobalamin (Cbl) enters

the stomach and binds to R protein. This physiologically inactive complex enters the duodenum. In the small intestine, pancreatic enzymes
and pepsin digest the R protein, and Cbl binds to intrinsic factor (IF).
The Cbl-IF complex passes through the intestine until it reaches receptors on the microvilli of mucosal cells in the distal ileum. The Cbl
is then transferred to transcobalamin II (TC-II), which circulates in
the blood until it binds to receptors on cells in the body and is
internalized.
megaloblastic anemia caused by cobalamin

deficiency
Epidemiology
The overall prevalence of vitamin B12 deficiency is
unknown; however, the incidence appears to rise with age.139
Importantly, although 10 to 20% of elderly patients will
have low cobalamin levels, less than 10% of these will have
clinical evidence of deficiency.140 Vitamin B12 deficiency is
common following gastric bypass procedures.141
Pathophysiology
The etiology of cobalamin deficiency can be categorized
as due to dietary deficiency, pernicious anemia (the lack of
IF), food-cobalamin malabsorption (the inability to release
cobalamin from food and make it available to gastric IF),
small bowel disease, pancreatic insufficiency (although it
is debated whether pancreatic insufficiency can truly
cause cobalamin deficiency), and rare congenital disorders.
Whereas food-cobalamin malabsorption is common among
patients with subclinical deficiency, those with clinical manifestations of cobalamin deficiency are more likely to have
pernicious anemia.142 Cobalamin deficiency in pernicious
anemia is thought to result from an autoimmune gastritis
and an autoimmune attack on gastric IF. There are two types
Diagnosis
Clinical manifestations and physical examination In
addition to macrocytic and megaloblastic anemia, the
patient with cobalamin deficiency may present with weakness, lethargy, jaundice, and dementia, as well as atrophy of
the lingual papillae and glossitis. Neuropathy is the presenting feature in about 12% of patients with cobalamin (vitamin
B12) deficiency without concomitant anemia.148 Patients with
severe cobalamin deficiency initially complain of paresthesia. The sense of touch and temperature sensitivity may be
minimally impaired. Memory impairment and depression
may be prominent. The disease may progress, involving the
dorsal columns, causing ataxia and weakness. The physical
examination reveals a broad-based gait, Romberg sign,
slowed reflexes, and a loss of a sense of position and a
feeling of vibration (especially when tested with a 256 Hz
tuning fork). If the disorder is not detected and treated, the
lateral columns become involved, resulting in weakness,
spasticity, inability to walk, sustained clonus, hyperreflexia,
and Babinski sign. Because the peripheral nerves, as well
as the dorsal and lateral columns, are involved, these
neurologic manifestations are sometimes termed subacute
combined degeneration or subacute combined system
disease.
Cobalamin deficiency appears to be the cause of various
neuropsychiatric disorders, with symptoms such as paresthesia, ataxia, limb weakness, gait disturbance, memory
defects, hallucinations, and personality and mood changes.148
These symptoms, however, cannot be easily accounted for
by the type of spinal cord lesions that occur in patients with
cobalamin deficiency. Investigators have tried to determine
whether a defect in methionine synthesis or an abnormality
in propionic acid metabolism accounts for the neuropathy
associated with cobalamin deficiency [see Figure 4b], but
the exact mechanism remains obscure. Accruing evidence
supports the impairment of methionine synthase as the
cause of the neuropathy.148 A study in which various
metabolites of cobalamin were measured discovered that
only high levels of plasma cysteine were predictive of
neurologic dysfunction.140
Laboratory tests The evaluation of suspected cobalamin
deficiency generally proceeds in two stages: documenting
the presence of the vitamin deficiency and determining its

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cause (e.g., pernicious anemia, malabsorption, dietary lack).
The diagnosis can often be established by (1) measurement
of the serum cobalamin concentration, (2) evaluation of
specific metabolites, and (3) use of the Schilling test to
establish malabsorption of cobalamin. Unfortunately,
the Schilling test is no longer available in most parts of the
United States, although there is interest in resuscitating it as
an important test in distinguishing between malabsorptive
and nonmalabsorptive etiologies of cobalamin deficiency.149
Macrocytosis (mean corpuscular volume [MCV] greater
than 100 fL) is a hallmark of cobalamin deficiency, but it
may be masked by concurrent disorders, such as iron
deficiency. If macrocytosis is not apparent on examination of
the peripheral smear, it is easily detected when RBC counts
are made with an electronic particle counter. The peripheral
smear shows macro-ovalocytes, fish-tailed RBCs, hypersegmented neutrophils, and, occasionally, nucleated RBCs [see
Figure 2]. The finding that a single PMN has six lobes or that
5% of PMNs have five lobes constitutes strong evidence
of megaloblastic anemia. In severe cases, granulocytopenia
and thrombocytopenia are present. If severe iron deficiency
is concurrent with macrocytosis, the full morphologic
expression of megaloblastosis is blocked, although the giant
metamyelocytes in the marrow and hypersegmented PMNs
in the peripheral blood will still be present.
Plasma cobalamin levels and RBC folic acid levels should
be measured if the MCV is greater than 100 fL. If performed,
a bone marrow aspirate and biopsy typically reveal
enormous megaloblastic erythroid hyperplasia with giant
metamyelocytes.133 The hypercellularity detected on a bone
marrow examination can be so dramatic and megaloblasts
so immature that clinicians still sometimes make the erroneous diagnosis of leukemia.150 Although the diagnosis may
be clear in symptomatic patients with low vitamin B12
levels, patients who are in the low-normal range (i.e., 200 to
350 pg/mL) may have true deficiency.14 Elevated methylmalonic acid levels in these cases can provide further confirmation of deficiency, although levels may also be elevated if the
patient has concomitant renal insufficiency.146
The standard approach to determining the cause of
proven cobalamin deficiency has traditionally relied on the
Schilling test. The Schilling test measures the absorption of
cobalamin labeled with cobalt 57 (57Co). After 1 g of radioactively labeled cobalamin is given orally, 1,000 g of unlabeled cobalamin is given parenterally. The parenteral dose
saturates transcobalamin and haptocorrin, so a significant
portion of the absorbed material is flushed and excreted in
the urine. If the amount of 57Co-labeled cobalamin measured
in an accurately collected 24-hour urine sample is less than
10% of the dose that was administered orally, cobalamin
absorption is poor.
Malabsorption of cobalamin can be demonstrated by
means of a food Schilling test, which is not available clinically. This test is performed with eggs from chickens that
have been injected with radioactive cobalamin151 and indicates whether there is insufficient acid-pepsin to split the
cobalamin-enzyme complex and release free cobalamin to be
bound by IF. If pernicious anemia is strongly suspected in a
patient whose Schilling test result is apparently normal and
whose plasma cobalamin is not diagnostically low, other
steps should be taken to confirm the diagnosis, including

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examination of the blood cell morphology, measurement of
the anti-IF antibody, or performance of a therapeutic trial
with parenterally administered cobalamin. Measurement
of the serum levels of homocysteine and methylmalonic acid
is increasingly being used because both are elevated as a
consequence of cobalamin deficiency [see Figure 4].
If the initial Schilling test demonstrates reduced excretion
of cobalamin, a second phase of the test may be conducted,
aimed at correcting cobalamin absorption caused by pernicious anemia. In this phase of the test, supplementary
oral IF is administered and will normalize the cobalamin
absorption unless the supplementary IF is not fully active,
the patient secretes antibodies to IF, or the patient is taking
drugs that interfere with cobalamin absorption. In no case,
however, will supplementary IF be effective in patients with
intestinal malabsorption. It is important to recognize that
prolonged cobalamin deficiency impairs intestinal epithelial
cells and thus impairs absorption. Therefore, the second
stage of the test should be performed only after several
weeks of cobalamin replacement therapy. If the result of
the second stage of the Schilling test is abnormally low, this
suggests the presence of intestinal malabsorption, such as
may occur in sprue, pancreatic insufficiency, or blind loop
syndromes.
An alternative to use of the Schilling test is to test for
anti-IF antibodies, with a sensitivity of 50 to 84% and a high
specificity.152 Antigastric parietal cell antibodies are less
sensitive and specific.
Concurrent a-thalassemia may minimize the macrocytosis
of pernicious anemia.153 This possibility should be considered particularly in patients of African descent, among
whom there is a high incidence of a-thalassemia (about
30%). Anemia of chronic inflammation or anemia resulting
from blood loss and iron deficiency can also reduce the
degree of macrocytosis but will not affect the hypersegmentation of neutrophils. In one study, iron deficiency was
discovered in 20% of 121 patients with pernicious anemia154;
in another study, 19% of patients with pernicious anemia
were not anemic, and 33% did not have macrocytosis.155
Falsely low serum cobalamin levels occur during pregnancy and in folic acid deficiency states.156 In the past, a
decline in the serum cobalamin level was usually not
considered important unless the value was very low (i.e.,
< 150 pg/mL). It has become clear, however, that patients
with serum cobalamin levels as high as 250 pg/mL and
perhaps higher may have cobalamin deficiency.151,155,157
Fortunately, the finding of macro-ovalocytes or hypersegmented PMNs on the peripheral smear remains a sensitive
indicator for the presence of cobalamin deficiency.
Determining the underlying cause After the presence
of macrocytosis and a reduced cobalamin level have been
identified, the cause of these conditions must be determined.
It is important to remember that macrocytosis can be caused
by conditions other than pernicious anemia, including folic
acid deficiency, liver disease, alcohol abuse, reticulocytosis,
and ingestion of drugs such as antimetabolites, alkylating
agents, and zidovudine.153,158 Cobalamin deficiency can be
caused by inadequate absorption resulting from gastric
abnormalities (e.g., pernicious anemia, gastritis), small
bowel disease (e.g., tropical sprue, Crohn disease), and
pancreatic insufficiency [see Table 3].
heme-onc
Table 3 Causes of Cobalamin Deficiency

Inadequate diet
Strict vegetarianism
Pernicious anemia (loss of IF)
Food-cobalamin malabsorption (inability to release food-bound
cobalamin and make it available to gastric IF)
Gastric achlorhydria
Gastric resection/reconstruction
Gastritis
Helicobacter pylori infection
Chronic alcoholism
Other causes of malabsorption
Small bowel disease
Ileal resection or bypass
Blind loop syndrome with abnormal gut flora
Celiac sprue
Crohn disease
Drugs that interfere with cobalamin absorption (generally after
years of use)
Neomycin
Biguanides
Colchicine
Ethanol
Aminosalicylic acid
Omeprazole
Fish tapeworm competing for cobalamin
Degradation of cobalamin coenzymes
N2O anesthesia
Rare congenital disorders
Transcobalamin II deficiency
Defective IF production
Imerslund-Grasbeck syndrome (selective cobalamin
malabsorption with proteinuria)
IF = intrinsic factor.
Gastric surgery in which the IF, pepsin, and acid-secreting

components are removed often results in cobalamin
deficiency (it occurred in 31% of patients in one study159).
Patients who have undergone gastric surgery should be
regularly screened by measurements of plasma cobalamin
or homocysteine levels and supplemented with lifelong
cobalamin therapy if the levels are low.159
Pancreatic insufficiency may result in malabsorption of
cobalamin if the damaged pancreas does not produce enough
trypsin and chymotrypsin for digesting the R protein
cobalamin complex and freeing the vitamin to form the
complex with IF [see Figure 6] [search this book for
information on diseases of the pancreas].
There are other causes of cobalamin deficiency. In vegetarians, especially vegans, profound nutritional megaloblastic anemia can develop as a result of very low cobalamin
intake. Deficiencies of folic acid and iron have also been
observed in vegans.160 A careful patient history should
indicate the possibility of inadequate dietary intake of
cobalamin. Infants of vegan mothers can become severely
cobalamin deficient, particularly when they are breast-fed.161
Cobalamin deficiency is surprisingly common in less developed countries, where people are not strict vegans. The
incidence is particularly high in pregnant women and in
preschool-age children.161 Rare causes of cobalamin deficiency
include congenital genetic defects in IF, cubilin, and amnionless.138
Management
Specific replacement should be started promptly after the
diagnosis has been made and serum samples have been
taken to determine cobalamin levels. Patients who have a
low serum cobalamin level and macrocytic anemia should
undergo a trial of parenteral cobalamin therapy. The diagnosis of cobalamin deficiency is confirmed if cobalamin
therapy produces a reticulocytosis in 4 to 6 days that is
associated with a rise in the hemoglobin level and a fall in
the MCV.
If the patient has symptoms of severe anemia, packed
RBCs can be transfused; the transfusion should be administered very slowly to avoid precipitating or aggravating
congestive heart failure. This circumstance is one of the few
in which a single-unit transfusion may be justified because
it may produce a 25% increase in oxygen-carrying capacity.
A large dose of cobalamin should be given because the
retention of parenterally administered cobalamin is poor but
variable; the vitamin is inexpensive and has no harmful side
effects. The reticulocyte response begins in 4 to 6 days, and
the granulocyte count, if low, begins to increase at the same
time. The hypersegmentation of PMNs disappears after
10 to 14 days, which suggests that in the megaloblastic
anemias, granulopoiesis is affected by cobalamin deficiency
at two different steps: (1) the lobe number of the PMNs is
determined, and (2) granulocytes mature and leave the
marrow. Weekly dosages of 1,000 g of parenteral cobalamin for 6 weeks should be followed by parenteral dosages
of 1,000 g monthly for life. The standard parenteral
preparation is cyanocobalamin. For pancreatic insufficiency,
cobalamin can be given parenterally or pancreatic enzymes
can be administered orally. Specific therapy must be designed
for patients with intestinal forms of malabsorption.
Because a small amount of cobalamin is absorbed even in
the absence of IF and because only 2.4 g/day is required,
oral cobalamin has proved adequate for replacement in
patients with pernicious anemia, freeing the patient from
monthly injections (2,000 g/day p.o. is recommended).162
megaloblastic anemia caused by folic acid
deficiency
Etiology and Epidemiology

Folic acid deficiency is most frequently caused by poor
dietary intake but may also result from inadequate absorption secondary to disease or drug administration [see
Table 4]. Ingestion of ethanol by well-nourished individuals
does not produce megaloblastosis, but in patients with
borderline folic acid stores, ethanol can lower serum folic
acid levels and block the reticulocyte response to folic acid
administration. Alcohol may block release of folic acid from
tissues to the serum.
Megaloblastic anemia occurring as a consequence of drug
administration or pregnancy is likely to be caused by folic
acid deficiency. Many of the antineoplastic and immunosuppressive agents produce megaloblastosis; these include
fluorouracil, hydroxyurea, mercaptopurine, thioguanine,
cytarabine, and azathioprine. In pregnant women, the
presence of megaloblastosis may not be initially apparent.
Because the combination of folic acid and iron deficiency is
common, full expression of megaloblastosis is often blocked,

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Table 4 Causes of Folic Acid Deficiency

Mechanism
Cause
Absolutely inadequate
intake
Alcoholism
Nutritional deficiencies
Relatively inadequate
intake (resulting from
increased folic acid
requirements)
Pregnancy
Severe hemolysis
Chronic hemodialysis or peritoneal
dialysis
Inadequate absorption
Tropical sprue
Gluten-sensitive enteropathy
(nontropical sprue)
Crohn disease
Lymphoma or amyloidosis of small
bowel
Diabetic enteropathy
Intestinal resections or diversions
Drug-induced interference with folic acid

metabolism
Action of dihydrofolate reductase

blocked by methotrexate,
trimethoprim, pyrimethamine
Reduced folate absorption and
tissue folate depletion caused by
sulfasalazine
Interference of unknown mechanism
caused by phenytoin, ethanol,
antituberculosis drugs, ? oral
contraceptives
and the patient will have a dimorphic anemia rather than

the easily identifiable macro-ovalocytosis. Hypersegmentation of PMNs persists.133
An abnormality in folate metabolism can be caused by a
chromosomal mutation, and women who are homozygous
for this defect are thought to be at higher risk for pregnancies affected by neural tube defects. One of the enzymes that
regulates homocysteine levels, 5,10-methylenetetrahydrofolate reductase, has a genetic variant, C677T. Individuals
homozygous for this variant have increased plasma homocysteine levels that are lowered by folate supplementation.
About 5 to 10% of the general population are homozygous
for this variant. Both pregnant and nonpregnant women
who are homozygous for the C677T mutation have significantly lower RBC folic acid levels.163 These women may be
susceptible to cardiovascular disease and stroke and may
bear children with neural tube defects.164 It would be advisable to know before pregnancy that a woman is homozygous for this variant, and genetic testing would be helpful if
a woman has a family history of this defect.
A number of intestinal disorders cause folic acid deficiency. These include severe pancreatic disease and small bowel
disease, including malabsorption, ileal disease, Crohn
disease, resection, and bypass [see Table 4]. When there is no
apparent cause of folate deficiency, it may be practical to
suspect an undiagnosed disease of malabsorption. In one
prospective study of patients who had laboratory-defined
folate deficiency, 10.9% were positive for celiac disease
antibodies and 4.7% had histologically confirmed celiac
disease.165
Diagnosis
The patient with folic acid deficiency has a clinical presentation that is distinct from that of the patient with cobalamin
deficiency.142 The patient may abuse alcohol or other drugs

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and have poor dietary intake of folic acid. Following mandatory fortification of grains with folic acid in the United States
in 1998, the prevalence of folic acid deficiency has decreased
dramatically.166 The hematologic manifestations of folic acid
deficiency are the same as those of cobalamin deficiency:
severe macrocytic anemia, a low absolute reticulocyte count,
and a characteristic blood smear showing macro-ovalocytes,
occasional megaloblasts, and hypersegmented neutrophils.
Patients with megaloblastic anemia who do not have glossitis, a family history of pernicious anemia, or the neurologic features described for cobalamin deficiency may have
folic acid deficiency.
Diagnosis and Differential Diagnosis

A meticulous dietary history is important because food
faddism, poor dietary intake, and alcoholism are the usual
causes of severe folic acid deficiency [see Table 4]. Cobalamin
and folic acid deficiencies frequently coexist and are not
easily distinguished. In evaluating patients for folic acid
deficiency, values for the levels of serum folic acid, serum
cobalamin, and RBC folic acid must be obtained. The RBC
folic acid level reflects tissue stores167 but may be reduced in
patients with severe cobalamin deficiency. In isolated cases,
the serum folic acid level of cobalamin-deficient patients is
usually normal or elevated. Severe, long-standing cobalamin deficiency leads to anorexia and gastrointestinal disturbances, which may cause dietary folic acid deficiency. As a
result, both serum cobalamin and folic acid levels are low,
producing a double-deficiency state.
A serum folic acid level less than 2 ng/mL is consistent
with folic acid deficiency, as is an RBC folic acid level less
than 150 ng/mL. If the test results are inconclusive or if it
is necessary to distinguish the megaloblastosis of folic acid
deficiency from that of cobalamin deficiency, measurements
of the serum methylmalonate and homocysteine levels are
helpful. If both metabolite tests are normal (i.e., methylmalonate level of 70 to 270 nmol/L and a total homocysteine
level of 5 to 14 mol/L), deficiency of both vitamins is ruled
out. If the methylmalonate level is normal but the total
homocysteine level is increased, folic acid deficiency is
likely, and investigation into the underlying cause is
appropriate.167
Management
Standard therapy for folic acid deficiency is 1 mg/day
orally. The response, manifested by reticulocytosis in 4 to
6 days, loss of megaloblastosis, and the return of normal
blood counts, confirms the diagnosis of folic acid deficiency.
Neutrophil hypersegmentation disappears only after 10 to
14 days, however.133 Patients with megaloblastosis and
severe bone marrow depression secondary to administration of drugs that block dihydrofolate reductase, such as
pyrimethamine and methotrexate, may be treated with
folinic acid. In the case of toxicity after single large doses
of methotrexate, a single equivalent dose of intramuscular
folinic acid (i.e., milligram for milligram) will suffice. For
toxicity after chronic pyrimethamine therapy, 1 to 5 mg of
folinic acid daily can be given without blocking the antimalarial effects of pyrimethamine. Megaloblastosis caused by
anticonvulsant therapy can be treated with 1 mg of folic acid
daily. Supplementation during pregnancy is advised and
heme-onc
may also be useful for patients who have severe chronic
hemolysis.
In most patients (i.e., those who do not require a large
amount of folic acid because of conditions such as hemolysis
or pregnancy), a hematologic response occurs after administration of 200 g of folic acid daily. The increased demand
of folic acid during pregnancy requires administration of
about 200 to 300 g/day.168 Furthermore, folic acid supplementation seems to prevent fetal neural tube defects.169 Such
neural tube defects may occur in the embryo or very early
in gestationeven before the pregnancy is confirmed.
Therefore, it is recommended that women of childbearing
age or those who plan to become pregnant receive about
400 g of folic acid a day. Women who are homozygous for
the C677T mutation should also take folic acid supplements.
Staple foods such as flour and cereal grains can be fortified
with folic acid. Concern has been expressed, however, that
folic acid supplementation may mask the megaloblastosis
of pernicious anemia, causing the development of severe
neuropathy rather than anemia.170
copper deficiency
Epidemiology
Although acquired copper deficiency is rare in the United
States, it may be an underrecognized cause of anemia,
neutropenia, and myelodysplasia due to its low prevalence,
overlapping morphologic features with MDS, and the low
level of clinical suspicion in the medical community.
Etiology
The ubiquitous distribution and low daily requirement
of elemental copper make acquired deficiency rare. Copper
deficiency has been reported in the setting of zinc supplementation,171,172 chronic tube feeding,173175 total parenteral
nutrition,176,177 and various malabsorptive syndromes,
including celiac disease,178 amyloidosis,179 and postgastric
bypass surgery and postgastrectomy.180185
Pathogenesis
The mechanisms by which copper deficiency causes
cytopenias are not well understood. The anemia probably
occurs as a result of decreased activity of multiple copperdependent enzymes, including hephaestin, ceruloplasmin,
and cytochrome-c oxidase, all of which are involved in iron
metabolism and transportation.186190 Neutropenia is less
commonly seen and even less well understood, but there is
speculation that copper deficiency may lead to destruction
of myeloid progenitors in the bone marrow, impaired
maturation of myeloid precursors, impaired egress of
neutrophils from the bone marrow, and increased clearance
of neutrophils from the circulation.191,192
Of interest is the physiologic relationship between copper
and zinc. Increased zinc levels lead to copper deficiency
by upregulating metallothionein, an intracellular copperbinding protein. This results in sequestration of copper in
enterocytes and the eventual loss of copper in the stool from
normal intestinal sloughing.193,194
Diagnosis
Copper deficiency should be considered in all patients
with an otherwise unexplained anemia, neutropenia, or

myelodysplastic bone marrow, especially in the setting of
peripheral neuropathy. The anemia due to copper deficiency
has been described as microcytic, normocytic, and macrocytic.195 Neutropenia and, less commonly, thrombocytopenia
may also be present.196199 The bone marrow may be hypo- or
hypercellular and frequently reveals cytoplasmic vacuolization of myeloid and erythroid precursors.195,196,198 Dysplasia
and ringed sideroblasts have also been described.200 Recent
studies suggest that hematogone hyperplasia, detected both
morphologically and by flow cytometry, may be common in
copper deficiency and may serve as an important diagnostic
clue in distinguishing nonclonal reactive conditions, such as
copper deficiency, from MDS, which is characterized by a
decrease in (or absence of) hematogones.179,201
To establish the diagnosis of copper deficiency, hypocupremia must be demonstrated. Serum copper levels are
subject to fluctuations and may be relatively insensitive
to detecting milder forms of copper deficiency.202 Serum
ceruloplasmin, on the other hand, is an excellent surrogate
marker of copper status and is a better way to detect copper
deficiency.203
The differential diagnosis of copper deficiency is broad
and must include other hematologic and neurologic disorders, such as MDS, SA, vitamin B12/folate deficiency, central
nervous system infection, multiple sclerosis, lupus, multiple
myeloma, leukemia, and other malignancies.
Management
Copper repletion is the mainstay of treatment and can
be achieved rapidly using oral preparations such as copper
gluconate,201 copper chloride,195,200 or copper sulfate.195,200
Intravenous copper may also be administered.195,201 A complete hematologic response is usually seen within 3 to 4
weeks. Copper repletion halts neurologic deterioration,
but neurologic improvement is limited.171,197,200,204,205 Cessation of zinc is necessary when treating copper deficiency due
to excess zinc consumption.
Complications
Complications of copper deficiency can include symptomatic anemia and infection due to severe neutropenia. Neurologic complications can be similar to the myeloneuropathy
observed with cobalamin deficiency and may be profound
and irreversible.171,197,200,204,205
Prognosis
If recognized in a timely fashion and treated appropriately, the prognosis of copper deficiency is excellent, with
complete resolution of hematologic abnormalities and variable resolution of neurologic symptoms. If copper deficiency
goes unrecognized, not only can the neurologic and hematologic consequences be serious, but patients may also be
exposed to highly toxic treatments such as chemotherapy
and/or HSCT (for presumed MDS) and be subject to the
morbidity and mortality associated with such treatments.
sideroblastic anemias
Definition/Epidemiology
The SAs are a heterogeneous group of inherited and
acquired disorders that have three common features: anemia

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(of varying degrees), the presence of ringed sideroblasts in
the bone marrow, and ineffective erythropoiesis.206 Ringed
sideroblasts are found exclusively in pathologic disorders
and are defined by the International Working Group on
Morphology of Myelodysplastic Syndrome (IWGM-MDS)
as erythroblasts that contain a minimum of five siderotic
granules covering at least one third of the circumference of
the nucleus.207 Irrespective of cause, the SAs are rare.
Etiology, Genetics, and Pathophysiology

When stained with Prussian blue, normal erythroblasts
contain a few blue granules scattered within the cytoplasm
called siderosomes, endosomes filled with excess iron not
used for heme synthesis. The iron in these siderosomes is
stored in cytosolic ferritin, whose subunits are encoded by
FTH1 and FTL genes. The iron in ringed sideroblasts, on
the other hand, is stored in mitochondrial ferritin, which is
encoded by the FTMT gene.208 On Prussian blue stain,
ringed sideroblasts are identified as erythroblasts with a
perinuclear ring of blue granules.
Abnormalities of heme synthesis are probably the most
frequent cause of the hereditary SAs.209 In these conditions,
iron enters erythroblasts normally but is not incorporated
into heme, resulting in an accumulation of iron in the cristae
of mitochondria.210 The most common form of congenital SA
is the X-linked form due to a mutation in the d-aminolevulinate synthase gene (ALAS2), a mitochondrially located
enzyme that catalyzes the first step in the heme synthetic
pathway.209,210 Impairment of ALAS2 profoundly affects
heme synthesis. The second most common form of hereditary SA (the most common autosomal recessive form) arises
from a mutation in the SLC25A38 gene, which encodes an
erythroid-specific mitochondrial carrier protein important
for the biosynthesis of heme.211 Several other mutations have
been identified in the pathogenesis of congenital SA, including mutations in the GLRX5, ABCB7, PUS1, YARS2, and
SLC19A2 genes, as well as major deletions in mitochondrial
DNA.212
Acquired forms of SA are classified as clonal and nonclonal. The most common forms of clonal SA are refractory
anemia with ringed sideroblasts (RARS) and refractory anemia with ringed sideroblasts and marked thrombocytosis
(RARS-T).213 According to the World Health Organization
Classification of Tumours of Haematopoietic and Lymphoid
Tissues published in 2008,214 the former is characterized by
isolated anemia, erythroid dysplasia (only), less than 5%
blasts, and more than 15% ringed sideroblasts in the bone
marrow. RARS-T is a myelodysplastic/myeloproliferative
overlap syndrome characterized by anemia with ringed
sideroblasts (> 15%) and marked thrombocytosis.213 RARS-T
has been associated with Jak-2 mutations and thrombopoietin receptor (MPL) mutations, both of which are commonly
found in myeloproliferative disorders.213 The vast majority
of patients with RARS have no cytogenetic abnormalities,
and none of the genes that are typically associated with
congenital SA have been implicated in acquired clonal SA.
Although acquired nonclonal SA can be seen with alcohol,
folic acid deficiency, and drugs (e.g., isoniazid and chloramphenicol), other causes of secondary SA remain largely
unknown.

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Diagnosis
SA must be included in the differential diagnosis of a
hypoproliferative (i.e., low reticulocyte count), hypochromic, microcytic anemia. Suspicion for SA increases in the
setting of a normal or elevated serum ferritin, elevated
serum iron-binding capacity (transferrin becomes saturated
due to ineffective erythropoiesis), elevated lactate dehydrogenase (due to ineffective erythropoiesis), and a normal
hemoglobin electrophoretic profile. In addition to profound
hypochromia and microcytosis, the peripheral blood smear
may show distorted RBCs, basophilic stippling, and anisocytosis, sometimes with a dimorphic population of RBCs.
Occasionally, Pappenheimer bodies (deposits of iron that
stain with Prussian blue) are present in mature RBCs. Ringed
sideroblasts must be seen on the bone marrow aspirate to
make the diagnosis of SA [see Figure 7].
If SA is confirmed on bone marrow aspirate (> 15% ringed
sideroblasts), the disorder must be further classified as
hereditary, acquired clonal, or acquired nonclonal. Patients
with hereditary forms of SA generally present early in life,
although, occasionally, patients with X-linked sideroblastic
anemia (XLSA) may present later in life due to variable
phenotypic expression.215 The diagnosis of XLSA is usually
made in male patients presenting in the first two decades
of life who demonstrate the ALAS2 mutation. In contrast to
patients with XLSA, patients homozygous for the SLC25A38
mutation become transfusion dependent very early in life,
and the more aggressive clinical course is suggestive of the
diagnosis, but demonstration of the SLC25A38 mutation
is required for a definitive diagnosis.211 The presence of
dysplasia and/or myeloproliferation is suggestive of a clonal disorder and should prompt cytogenetic studies and/or
molecular studies for Jak-2 and/or MPL in the appropriate
setting.213 If a hereditary form of SA is unlikely or ruled out,
and there are no abnormalities on the bone marrow to
suggest a clonal process, then the diagnosis of secondary SA
is made, and a careful history must be obtained to identify
possible causative agents (e.g., alcohol, medications).
The differential diagnosis of SAs includes the other forms
of hypoproliferative anemias covered in this chapter.
Figure 7 Prussian blue stain shows ringed sideroblasts in the bone

marrow of a patient who has idiopathic sideroblastic anemia.
heme-onc
Prognosis and Management

Treatment strategy and prognosis depend on whether SA
is hereditary, acquired clonal, or acquired nonclonal. It is
important to recognize reversible forms of SA (e.g., those
caused by alcoholism, folic acid deficiency, and drugs) and
to discontinue any potentially offending agents. Hereditary
forms are treated differently depending on the underlying
genetic abnormality. For example, the course of XLSA is
relatively benign, and in most cases, the anemia responds
to pyridoxine (200 to 600 mg/day).210 Occasionally, phlebotomy is implemented to prevent iron overload (even in
patients who do not require transfusions). Patients homozygous for SLC25A38 mutation, on the other hand, do not
respond to pyridoxine and become transfusion dependent
early in life, requiring iron chelation therapy to prevent iron
overload.211 Their clinical course is similar to thalassemia
major. As such, they should be considered for allogeneic
bone marrow transplantation at a young age as this is the
only curative therapy for this disease.216
Acquired clonal disorders, namely RARS and RARS-T, are
treated like other myelodysplastic and myelodysplastic/
myeloproliferative overlap syndromes and are discussed
in detail elsewhere [search this book for information on
myelodysplastic syndrome and myeloproliferative neoplasm].
The authors have no commercial relationships with manufacturers
of products or providers of services discussed in this chapter.
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Acknowledgement
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Tom Moore

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hematology-oncology

ANEMIA: PRODUCTION DEFECTS

Definition and Epidemiology

2014 Decker Intellectual Properties Inc

transmembrane protein responsible for the delivery of

anemia: production defects 2

and normocytic, although with more severe anemia, the

Table 1 Differential Diagnosis of Hypochromic Anemias

Usually normochromic, normocytic

Varies with the

Serum iron level

Serum ferritin level

Usually normal but

++++ with ringed

Scientific American Medicine

Varies with disease

Red blood cells can be

ACI and IDA

anemia: production defects 3

Diagnosis and Differential Diagnosis

Management and Complications

Scientific American Medicine

Scientific American Medicine

anemia: production defects 4

anemia: production defects 5

Production Defects Associated with Marrow Aplasia or

count < 20,000/L, absolute neutrophil count [ANC] < 500/L,

Scientific American Medicine

anemia: production defects 6

in pregnancy and may spontaneously improve following

Causes of Aplastic Anemia

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may act by producing interferon gamma.52 Patients with

Figure 3 Giant pronormoblast, evident on this marrow smear,

anemia: production defects 7

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Scientific American Medicine

anemia: production defects 8

Definition and Epidemiology

anemia: production defects 9

Scientific American Medicine

Scientific American Medicine

anemia: production defects 10

anemia: production defects 11

Scientific American Medicine

anemia: production defects 12

Formyl THF Synthase

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and thus is not absorbed. In the stomach, cobalamin binds

anemia: production defects 13

Figure 6 Cobalamin assimilation. Dietary cobalamin (Cbl) enters

megaloblastic anemia caused by cobalamin

Scientific American Medicine

Scientific American Medicine

anemia: production defects 14

Table 3 Causes of Cobalamin Deficiency

Gastric surgery in which the IF, pepsin, and acid-secreting

anemia: production defects 15

Etiology and Epidemiology

Scientific American Medicine

anemia: production defects 16

Table 4 Causes of Folic Acid Deficiency

Drug-induced interference with folic acid

Action of dihydrofolate reductase