Acquired and Congenital Hemolytic Anemia

Suzie A. Noronha, MD*

*Division of Pediatric Hematology/Oncology, University of Rochester, Golisano Children’s Hospital, Rochester, NY.

Educational Gaps
1. Pediatricians and other general practitioners may not be aware of
the significance of central nervous system disease in children who
have sickle cell disease, particularly the more subtle silent
infarct.
2. Pediatricians frequently fail to order a reticulocyte count or detect
splenomegaly on physical examination before referring a patient with
anemia. These findings are critical to diagnosing hemolytic anemia.

Objectives After completing this article, the reader should be able to:

1. Recognize clinical features of hemolysis, including reticulocytosis and

splenomegaly.
2. List the different types of acquired autoimmune hemolytic anemias
that can manifest throughout childhood.
3. Understand the role of transfusion in the management of neurologic
disease in patients who have sickle cell disease.
4. Review the spectrum of disease of a- and b-thalassemias.
5. Recognize clinical findings associated with hereditary spherocytosis.
6. Determine when to suspect glucose-6-phosphate dehydrogenase
deficiency and how to counsel families on triggers to avoid.

INTRODUCTION

Hemolytic anemia (HA) affects a substantial proportion of the pediatric popu-

lation globally. Many children are hospitalized every year due to sequelae of
this heterogeneous disease. Clinicians should be facile in recognizing its
presentation.

PATHOPHYSIOLOGY: EXTRAVASCULAR VERSUS INTRAVASCULAR

HEMOLYSIS
AUTHOR DISCLOSURE Dr Noronha has HA may be defined as increased destruction of red blood cells (RBCs). RBCs are
disclosed no financial relationships relevant to
cleared from the circulation via extravascular or intravascular mechanisms
this article. This commentary does not contain
a discussion of an unapproved/investigative (Figure). HA can be caused by congenital or acquired RBC abnormalities
use of a commercial product/device. (Table 1).

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 Figure. Mechanism of extravascular versus
intravascular hemolysis. RBC¼red blood cell.
Courtesy of Jessica Shand, MD.

Extravascular hemolysis is mediated by the reticuloen- within the macrophage in extravascular hemolysis, during
dothelial system (RES) of the spleen and liver. Most HAs, intravascular hemolysis, circulating free hemoglobin is
such as warm autoimmune hemolytic anemia (AIHA), bound irreversibly to the plasma haptoglobin and cleared
sickle cell disease (SCD), and hereditary spherocytosis by the liver. If free hemoglobin exceeds the binding capacity
(HS), are characterized by extravascular hemolysis. The of haptoglobin, hemoglobinemia occurs. Unbound hemo-
hallmark of extravascular hemolysis is phagocytosis of globin dimers are reabsorbed by the proximal renal tubule
erythrocytes by splenic macrophages or hepatic Kupffer until the absorptive capacity is exceeded. Free hemoglobin is
cells, followed by sequestration and removal. Heme, re- subsequently excreted in the urine, which appears dark.
leased from free hemoglobin in the phagocytosed cells, is
converted to biliverdin within the phagocyte. Biliverdin is
CLINICAL FINDINGS AND DIFFERENTIAL DIAGNOSIS
subsequently converted to bilirubin.
Intravascular hemolysis is defined as damage incurred Children may present with acute or insidious onset of pallor,
by the RBC membrane directly within the vasculature due fatigue, and lightheadness as a consequence of anemia.
to shear stress, toxins, or complement-mediated lysis. Ex- New-onset or recurrent jaundice may result from unconju-
amples include mechanical valve-induced hemolysis, Shiga gated hyperbilirubinemia. Parents may describe dark urine,
toxin-associated hemolytic-uremic syndrome, and cold which is due to hemoglobinuria from intravascular hemo-
agglutinin disease. Whereas hemoglobin clearance occurs lysis. Acrocyanosis may occur, tachycardia and/or a flow

TABLE 1. Classification of Common Hemolytic Anemias

ORIGIN DISORDER MECHANISM OF DESTRUCTION

Acquired Hemolytic disease of newborn Extravascular

Warm AIHA Extravascular
Mechanical valve HA Intravascular
Hemolytic-uremic syndrome Intravascular
Cold agglutinin disease Intravascular
Malaria Intravascular/extravascular
Congenital Sickle cell disease Extravascular
Thalassemia Extravascular
Hereditary spherocytosis Extravascular
G6PD deficiency Intravascular/extravascular

AIHA¼autoimmune hemolytic anemia, G6PD¼glucose-6-phosphate dehydrogenase, HA¼hemolytic anemia

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murmur may be appreciated on physical examination, and Direct Antiglobulin Testing
splenomegaly may be observed due to sequestration of RBCs. Once hemolysis is identified, further management is based
Adenopathy or hepatosplenomegaly should prompt on whether the hemolysis is antibody-mediated. The direct
investigation for malignancy or lymphoproliferative disor- Coombs or antiglobulin test (DAT) is the primary method to
ders. Lymphomas, in particular, can manifest with immune detect in vivo coating of patient erythrocytes by autoanti-
cytopenias, including AIHA and immune thrombocytope- bodies. In the DAT, a nonspecific antihuman globulin is
nia. In these cases, additional evaluation for hyperuricemia added to the patient’s RBCs. If this antibody recognizes
and examination of a peripheral blood smear by a hema- immunoglobulin bound to the RBC surface, as in AIHA, it
tologist is recommended. This type of paraneoplastic auto- binds or crosslinks other bound antibodies and agglutinates
immunity may be accompanied by constitutional symptoms the antibody-bound RBCs. Subsequently adding antihuman
suggestive of malignancy (eg, fevers, night sweats, weight antibodies for complement or immunoglobulin (Ig)G iden-
loss, and fatigue). tifies the type of immunoglobulin bound to the RBC sur-
In contrast to malignancy-associated immune hemoly- face. Binding of anticomplement antibodies usually implies
sis, children are typically healthy before the onset of isolated bound IgM. The specific antibody binding patterns can
AIHA, although they may have experienced nonspecific viral help to differentiate between warm-reactive (IgG) and cold-
symptoms or fever within several weeks of diagnosis. If the reactive (IgM) AIHA.
hemolytic anemia is congenital, the stigmata of chronic hemo-
lysis may be noted, such as pigmented gallstones and re- Indirect Antiglobulin Testing (Indirect Coombs)
lated sequelae, due to excess production of bilirubin. Patient serum (rather than RBCs) is incubated with healthy
donor RBCs. If RBC autoantibodies in the patient’s serum
LABORATORY FINDINGS bind to the donor RBCs, agglutination occurs, indicating the
presence of circulating antibody. This may be performed if
Reticulocytosis is an important distinguishing feature of
AIHA is suspected but the DAT result is negative.
hemolysis and usually exceeds 2%, with the absolute retic-
ulocyte count greater than 100  103/mL (100  109/L).
However, transient reticulocytopenia can occur in up to 33% NEONATAL ALLOIMMUNE HEMOLYTIC DISEASE
of patients, due to in vivo hemolysis of reticulocytes, nutri-
Maternal antibodies to incompatible fetal RBC antigens,
tional deficiencies, concurrent parvovirus infection, toxin
such as Rhesus (Rh)D, A, or B, can cause hemolytic disease
exposure, or underlying marrow dysfunction. (1)(2)
in utero. In the postnatal period, infants may exhibit mild
anemia to hydrops fetalis. Before the introduction of anti-D
Peripheral Smear Findings
Ig prophylaxis and intrauterine transfusions, Rh disease
Microspherocytes are characteristic of AIHA due to mem-
of the newborn was the predominant cause of neonatal
brane changes that occur when immunoglobulins are bound
AIHA, which is associated with 50% mortality and often
to the RBC surface. Fragment forms, such as schistocytes or
lifelong morbidity. Rh disease remains a significant global
helmet cells, may result from toxin- or shear stress-mediated
burden. Today the most common cause of neonatal AIHA
hemolysis. Polychromasia, related to the increase in circu-
in Western countries is ABO incompatibility, ie, infants
lating reticulocytes, may also be reported.
with A or B antigen born to group O mothers with high-
Chemistry Panel titer IgG antibodies. These infants may demonstrate iso-
Unconjugated bilirubin, lactate dehydrogenase, and aspar- lated unconjugated hyperbilirubinemia rather than hyper-
tate aminotransferase values may be elevated. The latter two bilirubinemia and anemia, which is more typical of Rh
intracellular enzymes are released into the plasma with cell disease. Hyperbilirubinemia may be exacerbated by coexis-
destruction. As the plasma carrier for free hemoglobin, tent glucose-6-phosphate dehydrogenase (G6PD) deficiency
haptoglobin is often decreased. However, this is not a useful or Gilbert syndrome. In addition to ABO incompatibility,
marker in infants younger than age 3 months. prenatal alloimmunization to minor RBC antigens such as
Kell, Fy, Jk, C, and E is also rising in relative frequency and
Hemoglobin Electrophoresis may lead to severe disease.
This test identifies sickle cell and b-thalassemia variants. Onset of jaundice within the first postnatal day or pro-
Blood for hemoglobin electrophoresis must be collected longed or severe hyperbilirubinemia should prompt investi-
before transfusion because it reflects the donor hemoglobin gation of hemolytic disease. Infants with alloimmunization
profile if performed within 3 months posttransfusion. usually are DAT-positive. If the DAT result is negative, the

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 laboratory performs an indirect antiglobulin test with the nonspecific fever or viral symptoms. At diagnosis, patients
infant’s serum. If agglutination occurs, maternal antibody is present with jaundice, splenomegaly, and laboratory findings
present in the serum. If the indirect antiglobulin test result consistent with HA. If DAT is negative for anti-IgG but positive
is negative, a search for nonimmune or congenital causes of for anticomplement, further testing for cold agglutinins and
HA is warranted. paroxysmal cold hemoglobinuria (PCH) should be pursued.
Intensive phototherapy is often sufficient to address Cold agglutinin disease is caused by IgM autoantibodies,
ABO-associated hemolytic disease, although exchange which bind below 37°C (98.6°F) and are maximally reactive
transfusion may be necessary. The use of intravenous at 4°C (39.2°F). IgM autoantibodies trigger complement
immunoglobulin to prevent exchange transfusion is con- deposition in vitro, resulting in agglutination with anti-
troversial, as demonstrated in a recent meta-analysis. (3) complement. Patients may display acrocyanosis when cold,
After discharge from the nursery, late-onset anemia may which results from autoagglutination of RBCs in the skin
ensue 1 to 3 weeks after birth. Anemia results from con- capillaries, causing localized stasis. Although bedside
tinued immune-mediated destruction of RBCs and RBC autoagglutination of the blood can be observed in the test
progenitors as well as antibody-associated suppression tube as the sample cools, this may be a result of clinically
of erythropoiesis. Neonates should be closely followed after insignificant cold autoantibodies. Further testing includes
discharge to determine the need for transfusion. initial screening at room temperature (20°C [68°F]), which
should induce agglutination in the presence of cold agglu-
tinin disease. Subsequently, agglutination of the RBCs in
CHILDHOOD AUTOIMMUNE HEMOLYTIC ANEMIA
saline and albumin is observed in a staged manner from 0°
Beyond the neonatal period, AIHA is rare in children, with to 30°C (32° to 86°F). If agglutination occurs at 30°C (86°F),
an annual incidence of 0.2 per million individuals younger pathogenicity is inferred.
than age 20 years. A recent French national cohort study PCH is a rare, self-limited AIHA caused by the Donath-
suggests the incidence may be as much as 10 to 20 times Landsteiner (DL) antibody. Recurrence is unusual. Once
higher. (2) AIHA may be classified as primary or secondary. commonly associated with syphilis, it is now seen predom-
inantly in children, often preceded by an upper respiratory
Primary Autoimmune Hemolytic Anemia tract infection. The DL antibody is a cold-reactive IgG, which
Primary AIHA, for which a cause is not identified, accounts is considered biphasic because of the 2-step nature of its
for 30% to 40% of pediatric cases. Primary AIHA is further in vitro characteristics. The DL test involves incubation of
categorized by thermal reactivity or the temperature at the patient’s blood at 4°C (39.2°F) for 1 hour to allow
which the RBC autoantibody is most reactive and causes maximal binding of the IgG, followed by a second incuba-
agglutination. Agglutination at 37°C (98.6°F) constitutes tion at 37°C (98.6°F) to activate complement and induce
warm AIHA, while agglutination below 30°C (86°F) is hemolysis. The DL test is not universally available and may
defined as cold AIHA (Table 2). become negative after a few days into the clinical course
Sixty percent of adult and pediatric patients with AIHA of the condition, making diagnosis of PCH a challenging
are diagnosed with warm agglutinins, which are almost proposition. (4)
always IgG but sometimes involve complement. Most of DAT-negative AIHA may be present, particularly if cold
these patients were previously healthy but may have had antibodies are involved. Antibody may not be tightly bound

TABLE 2. Classification and Features of Autoimmune Hemolytic Anemia

THERMAL REACTIVITY CLINICAL SIGNS, SYMPTOMS DAT FINDINGS TREATMENT OPTIONS

Warm Jaundice þIgG, -C3 First-line: corticosteroids

Splenomegaly Second-line: rituximab, splenectomy
Cold Acrocyanosis -IgG, þC3 Avoidance of cold
Hemoglobinuria
Biphasic Hemoglobinuria, self-limited -IgG, þC3 Supportive care

C¼complement, DAT¼direct antiglobulin test, Ig¼immunoglobulin

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to RBCs and may be eliminated with the eluate in vitro. DAT- after recovery, the DAT may remain positive for years or
negative AIHA may also involve IgA or natural killer (NK) indefinitely. Recurrence is more likely if an underlying
cells, which are not routinely screened for by the standard autoimmune disease or immunodeficiency exists. If the
DAT. (4) More detailed immunologic evaluation of the RBCs patient does not tolerate reduction of corticosteroids or if
using flow cytometry, gel card diagnostics, or more sensitive the agents are ineffective, second-line treatment is indi-
Coombs reagents may be available through select reference cated. Both rituximab and splenectomy have been used but
laboratories. have not been compared in clinical trials.
Splenectomy has been used as second-line therapy for
Secondary Autoimmune Hemolytic Anemia refractory warm AIHA since the 1950s. There is no role for
A triggering cause was identified in 63% of cases of AIHA in splenectomy in cold agglutinin disease because hemolysis
the French national cohort study, characterizing the condi- in those cases is intravascular. There are surprisingly few
tion as secondary. A defined infection was diagnosed in 22% data on the efficacy of splenectomy in children with refrac-
of patients, and almost 50% of these individuals ultimately tory warm AIHA. Several large case series of adult patients
were diagnosed with an immune disorder. Associated or- undergoing splenectomy for benign hematologic diseases
ganisms included Epstein-Barr virus, cytomegalovirus, show that it is relatively safe, particularly if the spleen can be
Mycoplasma, pneumococcus, and parvovirus. (2) removed laparoscopically. Patients have an increased risk of
Although less common than in adults, pediatric autoim- thrombosis, especially affecting the portal or mesenteric
mune cytopenias can be associated with malignancies such veins, perhaps exacerbated by postsplenectomy thrombocy-
as Hodgkin lymphoma. AIHA can be seen with autoim- tosis. However, the risk of thrombotic events may be more
mune lymphoproliferative syndrome (ALPS), common var- a function of the underlying hematologic disorder; thalas-
iable immunodeficiency, systemic lupus erythematosus, semia intermedia and major confer hypercoagulability. (5)
and after solid organ and allogeneic stem cell transplan- The most feared complication is postsplenectomy sepsis
tation. Disordered immune regulation through multi- due to encapsulated organisms. Splenectomy should be
factorial mechanisms such as altered regulatory T-cell delayed in children younger than age 5 years because the
function, abnormal complement activity, and abnormal risk of sepsis is greatest in this age group. Vaccination
apoptosis predisposes these individuals to autoimmunity. against encapsulated organisms, including the pneumococcal
An abrupt decline in hemoglobin values after initiation conjugate series and Haemophilus influenzae type b (Hib)
of certain medications should prompt consideration of drug- series should be completed by 15 months of age. Pneumococcal
associated immune hemolytic anemia (DAIHA). The in- polysaccharide and meningococcal polysaccharide can be
cidence of DAIHA is estimated at 1 per 1 million pediatric administered after age 2 years and should be provided at
and adult patients, but this is likely an underestimate. Rec- least 2 weeks before splenectomy. Postsplenectomy, the
ognizing this potentially severe complication allows discon- patient should continue to receive antibiotic prophylaxis,
tinuation of the drug and resolution of hemolysis. One usually penicillin, for at least 5 years or through age 18
proposed mechanism asserts that certain drugs bind co- years. The family must be educated about fever as an
valently to RBC antigens, stimulating hapten-dependent indicator of bacterial sepsis.
antibodies that activate macrophages and Fc-mediated Rituximab, a chimeric monoclonal antibody specific to
extravascular destruction. Another mechanism involves the B-lymphocyte CD20 antigen, may supplant splenectomy
direct stimulation of RBC autoantibody production. Cefo- as an alternative for corticosteroid-refractory AIHA. Rituximab
tetan, ceftriaxone, piperacillin, fludarabine, and diclofenac efficiently eliminates B lymphocytes and has been used to
have been implicated. Specialized reference laboratories treat other autoimmune diseases. The typical regimen is
can perform drug-independent and -dependent assays to 375 mg/m2 weekly for 3 to 4 weeks. In a case series, 87% of
facilitate diagnosis. patients had a sustained response at 13 months. Three of the
15 patients suffered recurrence but responded to a second
course of rituximab. (6) The aggregate results of other small
MANAGEMENT OF AUTOIMMUNE HEMOLYTIC ANEMIA
studies support a durable response to rituximab with few
Corticosteroids (prednisone 1 mg/kg per day) are first-line adverse effects. Infusion reactions include fever, hypoten-
therapy for warm AIHA and are associated with an 80% sion, respiratory distress, and rash; these complications
response rate. Improvement usually occurs within 24 to 72 respond to infusion rate reduction and antihistamine
hours of initiation. Once anemia is corrected, corticoste- use. Premedication with antipyretics, antihistamines,
roids are weaned over several months to avoid relapse. Even and corticosteroids usually prevents such reactions.

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 Increased susceptibility to infection and viral reactivation Expression of sickle hemoglobin results from a point
are theoretical concerns but are infrequent and usually seen mutation in the b-globin gene. Deoxygenation causes ab-
in patients undergoing stem cell transplant. (7) Rituximab normal polymerization of sickle hemoglobin, transforming
has been used with success in some patients who have the erythrocyte into the characteristic crescent or sickle
cytopenias associated with underlying immune disorders, shape. Vaso-occlusion by these poorly deformable RBCs is
(8)(9) although patients with AIHA associated with ALPS the hallmark of the disease, but there is extensive literature
did not respond to rituximab. (10) describing the molecular and cellular changes that contribute
Most cases of cold agglutinin disease result in chronic to SCD pathophysiology. (12)
mild HA. Patients are advised to avoid the cold. Immuno- The spleen is also subject to multifactorial injury, invari-
suppressive therapy, such as corticosteroids, cyclophospha- ably leading to splenic dysfunction early in life and ulti-
mide, chlorambucil, fludarabine, and rituximab, has been mately autoinfarction. Before autoinfarction, the spleen
used without significant efficacy or durability of response. can sometimes sequester blood cells. Splenic sequestration
(11) tends to occur in early childhood in those who have SS
Regardless of classification, if a patient ultimately diag- disease and later in patients with milder disease. Seques-
nosed with AIHA presents with severe anemia that may tration may vary from self-limited, with a mild drop in
cause cardiovascular compromise (hemoglobin <5 g/dL [50 hemoglobin and splenomegaly, to severe, in which the
g/L]) or severe anemia with reticulocytopenia, transfusion is volume of sequestered blood is life-threatening and patients
necessary. Communication with the blood bank about the present with cardiovascular collapse. Parents are taught to
clinical scenario is imperative because autoantibodies of- palpate their children’s spleens because they may be the first
ten obscure the RBC phenotype and make crossmatching to detect this complication. Small-aliquot transfusions may
difficult. If the child has not been transfused before, it be considered for severe anemia. Recurrent severe splenic
is reasonable to proceed with transfusion despite positive sequestration may warrant splenectomy in select cases.
crossmatching tests because alloimmunization is rare. If the However, no robust data indicate survival benefit or
patient has received a previous transfusion, the blood bank decrease in morbidity with splenectomy.
performs specialized testing to clarify the presence of Early impairment in splenic function underlies patients’
alloantibodies. vulnerability to infection with encapsulated organisms
such as Hib and Streptococcus pneumoniae. Before advances
in prophylaxis, children younger than age 3 years with SS
CONGENITAL HEMOLYTIC ANEMIA
disease frequently contracted pneumococcal bacteremia
Inherited molecular defects that affect the stability of the with significant mortality. The advent of universal newborn
RBC, including its shape (SCD), hemoglobin content (thal- screening, prompt initiation of penicillin prophylaxis in
assemia), membrane stability (spherocytosis), or metabolic infancy, and introduction of Hib and pneumococcal vacci-
stability (G6PD deficiency), can cause hemolysis. In this nations have dramatically decreased rates of sepsis and
section, we review the pathophysiology, clinical presenta- death in infants with SCD. Breakthrough pneumococcal
tion, and management recommendations for these congen- disease still occurs, (13) so fevers in those who have SCD
ital hemolytic anemias. must be promptly evaluated. Clinicians should order, at a
minimum, a complete blood cell (CBC) count, reticulocyte
count, and blood culture and administer parenteral antibi-
HEMOGLOBINOPATHIES
otics with effective antipneumococcal activity in the setting
Qualitative Defects: Sickle Cell Disease of fever.
SCD occurs in 1 in 300 to 400 African American births in Although survival has improved for children who have
the United States. The spectrum of SCD encompasses SCD, quality of life is frequently compromised by pain and
multiple sickling variants, which are diagnosed on newborn multiorgan dysfunction. Recurrent ischemic injury and
screen. The most common and most severe type is homo- chronic inflammation can eventually lead to widespread
zygous SS disease, in which both parents contribute the irreversible organ damage in patients with the most severe
sickle hemoglobin mutation. The remaining genotypes are disease (Table 3). (14) The greatest progress has been made
compound heterozygotes. Coinheritance of hemoglobins S in understanding neurologic disease in SCD.
and C as well as hemoglobin S and b-thalassemia occur to a The Cooperative Study of Sickle Cell Disease, the largest
lesser degree. In people of Asian descent, coinheritance of natural history study of SCD in the United States, reported
hemoglobins S and E is rising in incidence. that stroke occurred in 10% of those who had SS disease by

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age 20 years. Ischemic stroke accounted for most events. hemoglobin S-b-null-thalassemia). Once identified, affected
(15) The risk of recurrence, if SCD is untreated, ranges from patients are started on monthly RBC transfusions.
47% to 66%, (16)(17) but this risk is substantially decreased Cessation of transfusions, even after TCD findings nor-
with chronic transfusions. malize, was associated with reversion to abnormal TCD
The cause of stroke in SCD is multifactorial and remains velocities as well as increased rate of stroke in the STOP
incompletely understood. Abnormal adherence of the sick- 2 study. (22) The current standard of care is to continue
led RBCs to the vascular endothelium, recurrent endothe- chronic transfusions indefinitely with concomitant iron
lial injury, a hypercoagulable state, nitric oxide deficiency, chelation.
altered vasomotor tone, and poor cerebrovascular reserve in Silent cerebral infarcts (SCIs) are emerging as a risk
the setting of chronic anemia all likely contribute to the factor for neurocognitive deficit in those who have SCD.
pathogenesis of stroke. (18) Childhood prevalence increases with age and is estimated
Transcranial Doppler (TCD) ultrasonography is a com- to be 12% to 37%, by age 14. (23) SCIs are more common
mon, noninvasive radiologic technique for assessing the among those who have SS disease but are also detected in
patency of intracranial vessels. Use of TCD ultrasonography other sickle variants. Defined as ischemic cerebral lesions
is predicated on the fact that flow velocity is inversely on magnetic resonance imaging (MRI) without correspond-
proportional to arterial diameter, with high flow velocity ing neurologic deficits, SCIs have been associated with
suggesting arterial stenosis. Elevated cerebral arterial flow lower reading, math, and IQ scores as well as a higher risk
velocity (>200 cm/second) on TCD ultrasonography is for overt strokes. Schools should be informed of these
highly predictive of stroke in those who have SCD. (19) findings, so that affected students may receive academic
(20) The landmark randomized, controlled Stroke Preven- support. From a medical standpoint, recent results from the
tion Trial in Sickle Cell Anemia (STOP) study showed that Silent Cerebral Infarct Multi-Center Clinical Trial indicated
in patients with abnormal TCD ultrasonography findings, that chronic transfusions could halt progression of SCIs.
chronic transfusion to reduce sickle hemoglobin to less than (24) However, it is unclear yet how this will affect practice,
30% was effective as primary prevention of stroke. (21) given the short duration of the study (3 years) and the lack of
Annual TCD ultrasonography imaging, starting at age 2 uniform guidelines for MRI screening in this population.
years, is now a critical component of health care mainte-
nance for those who have severe SCD (ie, hemoglobin SS, Quantitative Defects: The Thalassemias
The thalassemias are among the most common genetic
disorders worldwide. Almost 5% of the global population
carries a globin mutation related to a- or b-thalassemia. The
TABLE 3. Clinical Manifestations of Sickle Cell thalassemias represent a spectrum of disorders of decreased
Disease or absent globin chain production due to hundreds of point
mutations and less commonly, deletions. In addition to
ORGAN SYSTEM CLINICAL MANIFESTATIONS
ineffective erythropoiesis, the resulting excess of unpaired
Neurologic Overt strokes, silent infarcts, globins produces insoluble tetramers that inflict oxidant
neurocognitive deficit
injury on RBC membrane lipids and proteins.
Retina Sickle retinopathy Hemoglobin E is a b-globin variant common in many
Musculoskeletal Acute and chronic pain, avascular necrosis Asian populations around the world. It produces a thalassemia-
Cardiac Cardiomegaly, pulmonary hypertension like picture, so it will be reviewed here.
a-Thalassemia. Understanding a-thalassemia can be
Pulmonary Acute chest syndrome, chronic lung disease,
nighttime hypoxia aided by familiarity with developmental hemoglobin expres-
Renal Hyposthenuria, renal papillary necrosis, sickle
sion. In healthy individuals, 2 a-globin gene loci on each
nephropathy chromosome 16 are expressed. The 4 genes express 2 a-
Spleen Splenic sequestration, functional asplenia, globin molecules, which partner with 2 g-globin molecules
hypersplenism to form fetal hemoglobin in utero and for several months
Liver/gallbladder Jaundice, cholelithiasis, sickle hepatopathy after birth. Hemoglobin production undergoes a progres-
sive developmental switch from 6 to 12 months of age,
Coagulation Hypercoagulability
which leads to preferential expression of b-globin rather
Genitourinary Nocturnal enuresis, delayed puberty,
priapism
than g-globin. The b-globin molecules then partner with the
2 a-globin molecules to form normal adult hemoglobin.

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 a-Thalassemias demonstrate a remarkably close correla- The inheritance of 2 severe b-globin mutations in a
tion between genotype and phenotype. Deletion of 1 gene is homozygous or compound heterozygous pattern causes
designated silent carrier because it is clinically asymptom- b-thalassemia major. Irritability, lethargy, and failure to
atic. Two-gene deletion represents a-thalassemia minor or thrive develop in the second 6 months after birth, as fetal
trait. Affected individuals may exhibit mild microcytic ane- hemoglobin expression declines and anemia develops. If
mia that does not impair growth or development. Com- the disease remains unrecognized, the stigmata of ineffec-
pound heterozygosity with a-thalassemia trait and sickle cell tive erythropoiesis emerge, including frontal bossing as the
anemia can ameliorate the severity of SCD. marrow compartment expands to compensate, hepatosple-
Hemoglobin H disease, or 3-gene deletion, can be diag- nomegaly and paravertebral pseudotumors due to extrame-
nosed at birth or later in childhood. At birth, 3-gene deletion dullary hematopoiesis, and growth failure due to chronic
leads to decreased a-globin production as well as insoluble anemia.
tetramers of g-globin, which is identified as hemoglobin Severe microcytic anemia in an older infant who has
Barts on newborn screen. After the developmental switch, normal iron stores and poor growth should prompt inves-
insoluble tetramers of b-globin are identified as hemoglo- tigation for b-thalassemia major. Hemoglobin electropho-
bin H on hemoglobin electrophoresis. The oxidant injury resis assists in diagnosis. The child should be referred to
produced by hemoglobin H results in chronic hemolysis. pediatric hematology for initiation of chronic transfu-
Patients may require RBC transfusions during their sec- sions, which suppress endogenous erythropoiesis. Trans-
ond decade of life. Despite only episodic need for trans- fusions improve oxygen-carrying capacity, growth, and
fusions, patients with hemoglobin H disease are at risk cardiac status. Patients are transfused every 2 to 4 weeks
for iron overload due to increased gastrointestinal iron to maintain a pretransfusion hemoglobin of 9 to 10 g/dL
absorption. (90-100 g/L).
Four-gene deletion has historically been incompatible Because each unit of blood introduces 200 to 250 mg of
with life due to the inability to express a-globin and, thus, elemental iron, most of which cannot be actively excreted,
fetal hemoglobin. Profound tissue hypoxia and consequent iron overload inevitably develops. The RES sequesters
congestive heart failure develop in utero, leading to anasarca excess iron. When the capacity of the RES is exceeded, iron
and a hydropic state. However, intrauterine transfusions is deposited in organ parenchyma, causing toxicity. Without
have allowed some patients to survive the perinatal period, effective chelation, patients are at risk for liver dysfunction,
albeit with the sequelae of congenital anomalies and devel- cardiac failure, hypogonadism, and other endocrinopathies.
opmental delay. They require chronic transfusions after (25)
birth and eventually bone marrow transplantation. The gold standard measurement of iron overload is
b-Thalassemia. In contrast to a-globin, b-globin is ex- via liver biopsy. Most institutions use ferritin as a surrogate
pressed by a single gene locus on chromosome 11. More marker for iron stores, with MRI of the liver and heart as an
than 200 point mutations and, rarely, deletions can cause accurate, noninvasive method of confirmation. Most chil-
b-thalassemia, with variable severity. Inheritance of 1 muta- dren are started on deferasirox or deferoxamine at age 2
tion leads to b-thalassemia trait or minor, a benign condition years. These agents bind iron and facilitate its urinary and
characterized by mild microcytic anemia. Clinicians may biliary excretion.
encounter b-thalassemia trait when evaluating a child who If a matched sibling donor is available, some children
has mild microcytic anemia unresponsive to iron supple- with b-thalassemia may be candidates for curative hemato-
mentation. Elevated hemoglobin A2 on hemoglobin elec- poietic stem cell transplant. Emerging therapies include
trophoresis and normal results of iron studies confirm the induction of hemoglobin F expression and gene therapy.
diagnosis. In the interim, lifelong chronic transfusion remains the
b-Thalassemia intermedia results from inheritance of mainstay of therapy for b-thalassemia.
2 b-globin mutations, 1 of which yields a mild phenotype. Hemoglobin E. The frequency of the hemoglobin E allele
Affected patients are not transfusion-dependent but may be ranges from 15% to 60% on the Indian subcontinent and
at long-term risk for high-output cardiac failure and pul- in Southeast Asia. The product of a splice site mutation in
monary hypertension from persistent tissue hypoxia. Thus, the b-globin gene, hemoglobin E mRNA is transcribed at
chronic transfusion may be warranted. Patients with thal- reduced levels and is translated into an abnormal b-globin
assemia intermedia may also develop iron overload due to molecule. The reduced expression of this abnormal b-globin
inappropriately brisk gastrointestinal iron absorption, and yields clinical findings equivalent to b-thalassemia trait in
they benefit from chelation. patients with homozygous E disease or hemoglobin E trait.

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 b-Thalassemia variants are common in these geographic most often are the osmotic fragility test and eosin-5-
regions, so coinheritance of b-thalassemia and hemoglobin maleimide (EMA) flow cytometry. Osmotic fragility testing
E occurs frequently. The clinical spectrum of hemoglobin takes advantage of the fact that HS erythrocytes lyse more
E/b-thalassemia ranges from moderate anemia to severe readily in hypotonic solutions than normal erythrocytes.
transfusion-dependent anemia, similar to b-thalassemia However, because the sensitivity and specificity of osmotic
major. These diagnoses may be suspected when a patient fragility testing are not optimal, EMA flow cytometry is
of Asian ancestry presents with microcytic anemia and increasingly being used. EMA is a fluorescent dye that binds
normal iron stores. Hemoglobin electrophoresis can eluci- to band 3 and indirectly to other RBC membrane proteins.
date these variants. Hemoglobin E/b-thalassemia of mod- The degree of fluorescence can be used to identify reduc-
erate severity may result in iron overload through increased tions in band 3, spectrin, or ankyrin. This testing method
iron absorption, and affected patients benefit from chela- has a sensitivity and specificity of greater than 90%.
tion. Patients with severe hemoglobin E/b-thalassemia Most patients who have HS exhibit mild hemolytic
should be referred to a hematologist early in life to start anemia, but moderate and severe disease exists, as defined
chronic transfusion therapy. by the degree of anemia and compensation for hemolysis.
Patients with the most severe HS almost always have auto-
somal recessive disease and are transfusion-dependent.
MEMBRANOPATHIES: HEREDITARY SPHEROCYTOSIS
Individuals with mild disease have well-compensated hemo-
The RBC cytoskeleton interacts with the outer lipid bilayer lysis and no evidence of anemia.
through horizontal spectrin molecules and vertical proteins Management depends on disease severity. Patients
4.1 and 4.2 as well as band 3 and ankyrin molecules. Mutations with mild HS are asymptomatic. Patients with moderate-
in 1 or more of these proteins are responsible for HS, which to-severe HS benefit from folate supplementation. Chole-
demonstrates autosomal dominant inheritance in 75% of cystectomy may be required for symptomatic cholelithiasis.
cases. The remaining 25% result from spontaneous mutation Splenectomy was formerly a standard recommendation for
or autosomal recessive inheritance. Defects of any of these moderate-to-severe HS; short-term benefits of splenectomy
proteins lead to membrane instability and RBC destruction via include improved hemoglobin, reduction in transfusion
extravascular hemolysis. This is the most common cause of number, and decreased cholelithiasis. Postsplenectomy
inherited anemia in individuals of northern European ances- sepsis due to encapsulated organisms is a substantial risk
try but has been observed in all races and ethnicities. for patients, even after appropriate immunization. In addi-
HS may present at any age but is usually diagnosed in tion, a growing body of literature describes long-term risks
childhood. Children may present with unexplained anemia, of vascular disease after splenectomy, including thrombosis,
reticulocytosis, jaundice, and/or splenomegaly. Aplasia due pulmonary hypertension, and atherosclerosis. Although no
to parvovirus B19 infection may be the initial presentation for randomized, controlled trials have been conducted, the gen-
HS, even in adulthood. The CBC count may reveal elevated red eral consensus still supports splenectomy for patients with
cell hemoglobin concentration (mean corpuscular hemoglo- severe HS in whom growth failure, skeletal abnormalities,
bin concentration ‡34.5 g/dL [345 g/L]) due to relative cellular and exercise intolerance are typical. The decision regarding
dehydration. RBCs may be normocytic or mildly microcytic. splenectomy in moderate HS is made on a case-by-case basis.
HS should be considered in a neonate who has early Patients should be vaccinated against encapsulated organ-
jaundice requiring phototherapy or exchange transfusion. isms at least 2 weeks before splenectomy.
The degree of hyperbilirubinemia does not predict future
severity of disease. Some neonates develop severe anemia
ENZYMOPATHIES: GLUCOSE-6-PHOSPHATE
within the first few postnatal weeks that necessitates RBC
DEHYDROGENASE DEFICIENCY
transfusion, but they become transfusion-independent during
their first year after birth. A subset of patients suffers anemia G6PD is critical for RBC defense against oxidative injury
in utero and requires transfusion at birth; these patients due to its role in the replenishment of nicotinamide adenine
typically exhibit severe disease throughout their lives. (26) dinucleotide phosphate hydrogen via the pentose phosphate
The diagnosis of HS is straightforward in the child who pathway. Deficiency of this enzyme predisposes individuals
has a positive family history and nonimmune spherocytic to the development of intravascular HA when their eryth-
hemolytic anemia. Further testing should be pursued for rocytes are under oxidative stress.
individuals who have evidence of chronic nonimmune Worldwide, G6PD deficiency has a prevalence of 400
hemolytic anemia but no family history. The studies used million cases, making it the most common RBC enzymopathy.

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 G6PD deficiency is X-linked, affecting predominantly males, COMMON CLINICAL MANAGEMENT POINTS FOR ALL
although female carriers can exhibit decreased expression of CHRONIC HEMOLYTIC DISORDERS
G6PD due to lyonization. Hundreds of G6PD mutations are
Parvovirus B19 Aplasia
known, leading to a diverse spectrum of disease. The World
All patients with chronic hemolysis are at risk from tran-
Health Organization (WHO) developed a classification of
sient parvovirus red cell aplasia. Parvovirus B19 preferen-
mutations based on enzyme activity and severity of hemoly-
tially infects erythroid precursors that, in a patient with
sis. Class I variants express the lowest enzyme activity and
increased RBC turnover, can cause life-threatening anemia.
cause the most severe disease, resulting in chronic extravas-
Fevers in this patient population should be evaluated with
cular HA even without exposure to oxidative stress. Class II
CBC and reticulocyte counts, which will be substantially
variants, including G6PD Mediterranean, are characterized
below baseline in the presence of parvovirus infection.
by severe deficiency and intermittent hemolysis. Class III
Patients may demonstrate fatigue, lethargy, pallor, or signs
variants such as G6PD A-, which is most common in
of congestive heart failure. As viremia declines, erythropoi-
individuals of African descent, demonstrate moderate
esis recovers in 1 to 2 weeks.
enzyme deficiency and intermittent hemolysis.
G6PD deficiency is probably responsible for approxi- Gallbladder Disease
mately 33% of cases of neonatal jaundice in male infants Upper abdominal pain in patients with hemolytic disorders
and a smaller fraction of female patients. (27) Jaundice should trigger evaluation for pigmented gallstones. The prev-
usually develops in the first postnatal days. Hemolysis of alence of cholelithiasis in this population approaches 50% by
G6PD-deficient cells does not appear to be a major com- adulthood. Cholecystectomy is often recommended because
ponent of neonatal jaundice; it may instead relate to imma- recurrence is common and may be complicated by cholecystitis,
ture bilirubin metabolism. Screening for G6PD deficiency choledocholithiasis, pancreatitis, and ascending cholangitis.
should be considered in neonates with early or severe un-
conjugated hyperbilirubinemia. (28) ACKNOWLEDGEMENTS
Beyond infancy, most G6PD-deficient individuals are
asymptomatic and unaware of their diagnosis. They may The author would like to thank Drs. Jessica Shand and Gary
come to medical attention with acute HA indicated by Noronha for reviewing this manuscript.
fatigue, back pain, dark urine, and jaundice. In the medical
history, patients may identify an oxidative stressor, such as
illness, fava bean ingestion, or certain medications. Infec-
Summary
tions associated with hemolysis in these patients include
• On the basis of strong research evidence, hemolytic anemia
hepatitis A and B and cytomegalovirus. Laboratory testing affects all age groups and may be classified as acquired or
may reveal anemia, reticulocytosis, high unconjugated congenital. It is characterized by genetic and clinical
bilirubin and lactate dehydrogenase, and decreased hapto- heterogeneity, even within individual disorders. (2)(14)(26)
globin. Diagnosis is made by quantitation of enzyme activity • Recognition of hemolytic anemia is critical for proper treatment.
when patients are well. G6PD activity may be falsely ele- A thorough history, physical examination, complete blood cell
vated immediately after a hemolytic episode, when the most count, reticulocyte count, and direct antiglobulin test can help to
narrow the differential diagnosis.
G6PD-deficient RBCs are cleared from circulation. Because
• Stroke causes significant morbidity and mortality in those who
immature RBCs have greater G6PD activity than older
have sickle cell disease. On the basis of strong research evidence,
RBCs, reticulocytosis also elevates overall G6PD activity.
stroke risk can be predicted using transcranial Doppler
Enzyme activity in a neonate may be deceptively high ultrasonography. (19)(20) Both primary and secondary stroke
because the RBC population is relatively immature. prevention can be achieved with a chronic transfusion program.
Hemolytic episodes are typically self-limited, but some (21)
individuals require transfusion for severe anemia. G6PD- • On the basis of strong research evidence, chronic transfusions
deficient patients benefit from counseling regarding avoid- play an important role in the management of several congenital
ance of oxidative triggers. Several support organizations hemolytic anemias. (21)(24) The resultant iron overload must be
managed. (25)
maintain updated lists of drugs and chemicals that can induce
hemolysis, such as primaquine, sulfa drugs, nitrofurantoin,
and naphthalene. Different G6PD variants are sensitive to
different medications, so delineating a patient’s WHO clas- CME quiz and References for this article are at http://pedsinre-
sification can facilitate more individualized guidance. view.aappublications.org/content/37/6/235.

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PIR Quiz
There are two ways to access the journal CME quizzes:
1. Individual CME quizzes are available via a handy blue CME link under the article title in the Table of Contents of any issue.
2. To access all CME articles, click “Journal CME” from Gateway’s orange main menu or go directly to: http://www.aappublications.
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1. A 3-year-old boy was diagnosed with autoimmune hemolytic anemia due to a warm REQUIREMENTS: Learners
antibody. The child rapidly responded to corticosteroids within 1 week. However, any can take Pediatrics in
attempt to decrease the prednisone dose below 0.5 mg/kg per day resulted in a recurrence Review quizzes and claim
of hemolysis during the ensuing 8 weeks. His current hemoglobin is 8.5 g/dL (85 g/L). credit online only at:
Which of the following is the most appropriate next therapeutic step? http://pedsinreview.org.
A. Continue the full dose of prednisone for 4 weeks.
B. Exchange transfusion. To successfully complete
C. Packed red blood cell transfusion. 2016 Pediatrics in Review
D. Rituximab administration. articles for AMA PRA
E. Splenectomy. Category 1 CreditTM,
2. A 10-month-old girl with known homozygous sickle cell anemia (SS disease) presents to learners must
your office with a history of a temperature of 40.1°C (104.2°F) at home. Before coming to demonstrate a minimum
the office, her parents gave her acetaminophen. Upon arrival at your office 40 minutes performance level of 60%
later, her temperature is down to 38.6°C (101.5°F). The girl has nasal congestion but no or higher on this
cough and is comfortable without distress. Which of the following is the most appropriate assessment, which
next step in management? measures achievement of
A. Ensure the parents are giving the prophylactic penicillin and send her home. the educational purpose
B. Have the family watch her at home and return if there is clinical worsening. and/or objectives of this
C. Observe her in the office for 4 hours and send her home if she looks well. activity. If you score less
D. Obtain a complete blood cell count, hemoglobin, reticulocyte count, and blood than 60% on the
culture and give intravenous antibiotics. assessment, you will be
E. Obtain a complete blood cell count and blood culture and send the child home if given additional
the former is at baseline. opportunities to answer
questions until an overall
3. A 7-year-old boy with sickle cell anemia (SS) undergoes his annual transcranial Doppler
60% or greater score is
ultrasonography study and is found to have a cerebral arterial flow velocity of 205 cm/
achieved.
second. The study is repeated and the result confirmed. His physical examination yields
normal results, and he has no history suggestive of neurologic complications. He has one
full sibling who also has sickle cell disease and one half-sibling. The most appropriate next This journal-based CME
step in management is to: activity is available
A. Initiate a chronic transfusion program. through Dec. 31, 2018,
B. Order cerebral magnetic resonance imaging. however, credit will be
C. Perform a hematopoietic stem cell transplant. recorded in the year in
D. Prescribe sumatriptan. which the learner
E. Wait 6 months and repeat the transcranial Doppler ultrasonography study. completes the quiz.
4. You are examining a 4-year-old who recently emigrated from Burma for the first time in
your practice. Physical examination results are normal. A complete blood cell count is
normal except for a hemoglobin of 9.5 g/dL (95 g/L) and mean corpuscular volume of
59 mm3 (59 fL) with a normal red cell distribution width of 13.3%. Results of iron studies are
normal. Hemoglobin electrophoresis reveals an elevated hemoglobin A2 of 4.9%. Which of
the following is the most likely diagnosis?
A. a-Thalassemia trait.
B. b-Thalassemia trait.
C. Hemoglobin E trait.
D. Homozygous hemoglobin E disease.
E. Early iron deficiency anemia.
5. An 11-year-old boy was well until 48 hours ago, when he developed fever, nasal
congestion, and mild bilateral cervical adenopathy. Twenty-four hours later, he developed
fatigue, jaundice, and very dark urine. Complete blood cell count reveals hemoglobin of 6.1
g/dL (61 g/L), mean corpuscular volume of 83 mm3 (83 fL), and mean corpuscular

Vol. 37 No. 6 JUNE 2016 245

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 hemoglobin concentration of 32 g/dL (320 g/L). Reticulocyte count is more than twice
normal at 250  103/mL (250  109/L). Other results include an unconjugated bilirubin of
4.1 mg/dL (70.1 mmol/L) and conjugated bilirubin of 0.54 mg/dL (9.2 mmol/L). Lactate
dehydrogenase is elevated to 550 U/L (9.2 mkat/L). Peripheral blood smear reveals
polychromasia without spherocytes or fragmented erythrocytes. Hemoglobin
electrophoresis results are normal. Direct antiglobulin test is normal. The most likely
diagnosis is:
A. Autoimmune hemolytic anemia.
B. E b-thalassemia.
C. Hemoglobin glucose-6-phosphate dehydrogenase deficiency.
D. Hereditary spherocytosis.
E. Sickle cell (SS) disease.

246 Pediatrics in Review

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 Acquired and Congenital Hemolytic Anemia
Suzie A. Noronha
Pediatrics in Review 2016;37;235
DOI: 10.1542/pir.2015-0053

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 Acquired and Congenital Hemolytic Anemia
Suzie A. Noronha
Pediatrics in Review 2016;37;235
DOI: 10.1542/pir.2015-0053

The online version of this article, along with updated information and services, is
located on the World Wide Web at:
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Pediatrics in Review is the official journal of the American Academy of Pediatrics. A monthly
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