ASH Hematology Review Series

Diagnostic Testing for Hemoglobinopathies

Alice Ma, MD, FACP
Disclosures
• No relevant disclosures
Overview

• Thalassemias
• Thalassemia-like hemoglobinopathies
• Other hemoglobinopathies
Normal Hemoglobin
• Hemoglobin is a
heterotetramer
• 2 alpha chains
• 2 beta or beta-like chains
• Each globin chain has a
heme moiety, bound to
iron
The globin gene loci
Chr 16—The alpha globin gene locus

5’ ζ α1 α2 3’

Chr 11—The beta globin gene locus

5’ ε γ1 γ2 δ β 3’
Globin genes combine to form different hemoglobin species

Chr 16

5’ ζ α α 3’

Chr 11

5’ ε γ γ δ β 3’

α
γ

Polypeptide chains α2γ2

Hemoglobins HbF
Globin genes combine to form different hemoglobin species

Chr 16

5’ ζ α α 3’

Chr 11

5’ ε γ γ δ β 3’

α
δ

Polypeptide chains
α2δ2
Hemoglobins HbA2
Globin genes combine to form different hemoglobin species

Chr 16

5’ ζ α α 3’

Chr 11

5’ ε γ γ δ β 3’

α
β

Polypeptide chains α2β2

Hemoglobins HbA
Globin genes combine to form different hemoglobin species

Chr 16

5’ ζ α α 3’

Chr 11

5’ ε γ γ δ β 3’

α
γ δ β

Polypeptide chains α2γ2 α2δ2 α2β2

Hemoglobins HbF HbA2 HbA

Normal Hemoglobin Percentages in Adults
HbA 95-98%
HbA2 2-3%
HbF 0.8-2%
 Thalassemia
• Definition: genetically heterogeneous
conditions resulting from imbalance
between the amounts of alpha and beta
globin chains that are synthesized.
• These arise from mutations which
partially or completely inactivate
production of either alpha or beta
globin chains.
– In general, we tend to have full Worldwide distribution of alpha and beta
thalassemia
deletions of alpha genes due to crossing - corresponds to distribution of falciparum
over errors during meiosis malaria
– And point mutations in the beta genes
Thalassemia Classification

• Severity of clinical manifestations

– Thalassemia minor (very mild symptoms, if any)
• Symptoms result from hypoproliferation—not hemolysis
– Thalassemia major (severe symptoms, pts are transfusion dependent)
• Hemolysis is prominent
– Thalassemia intermedia (between minor and major—pts not necessarily
transfusion dependent)
• The hemoglobin phenotype
– e.g. Hemoglobin Barts, Hemoglobin H
• The genotype
The beta thalassemias

• Distribution – most common in southern Europe. Also seen in SE Asia,

Africa, Middle East
• If a mutation causes absent beta globin synthesis, it is β0 thalassemia.
• The β+ thalassemia mutations have decreased (but still present) beta
globin synthesis.
• These mutations can be heterozygous, homozygous, or compounded
with other mutations of the other β globin gene.
Transfusion-dependent beta thalassemias

β Thalassemia major (Cooley’s Anemia) (b0/b0)

β Thalassemia intermedia (b+/b0 orb+/b+)
– Clinically, severe anemia develops b/w 2 and 12 mo. Infants are well at birth, since HbF and HbA2 predominate, and
less HbA is needed
• Typically seen in those of Mediterranean descent
• Diagnosis by hemoglobin electrophoresis showing α 4 tetramers and absence (or relative
deficiency) of normal hemoglobin A
• Symptoms include
– Anemia
– Splenomegaly
– Bony deformities
– Those due to iron overload (bronze skin, liver failure, heart failure, endocrine failure)
Thalassemia major--pathophysiology
• Deficiency of beta globin chains leads to relative excess of alpha chains—thus precipitate out
as α 4 tetramers
α 4 tetramers are unstable and leads to:
– Ineffective erythropoiesis (aka intramedullary hemolysis—hemolysis of red cell
precursors inside the marrow)
– Destruction of produced RBCs
• Anemia leads to increased epo production, leading to more ineffective erythropoiesis
• Splenomegaly develops, which worsens anemia due to sequestration
• Marrow expansion leads to skeletal deformities with “frontal bossing”, thinning bone cortex,
fractures
• Iron overload develops due to:
– Transfusions
– Iron hyper-absorption from gut
Beta thalassemia minor (trait)
• This is usually a heterozygous state—where one allele is normal and one allele is usually
β+
• Clinically asymptomatic since there are enough beta chains to pair with the alpha—so
not so many α 4 tetramers
• Lab findings
– Microcytic (MCV usually around 70)
– May or may not be anemic, if anemia is present, it is very mild
– RBC count will typically be very elevated
– RDW is normal, since ALL of the cells are microcytic and hypochromic
• Diagnosis by hemoglobin electrophoresis in adults
– Proportion of delta chains is increased, so Hgb A2 percentage will rise
– Typically see Hb A2 levels 4-8%
– We do not see this increase in HbA2 in newborns, since newborn hemoglobin species are
predominantly HbF
Going back to the globin gene loci. . .

Chr 16

5’ ζ α α 3’

Chr 11

5’ ε γ γ δ β 3’

α
γ δ β

Polypeptide chains α2γ2 α2δ2 α2β2

Hemoglobins HbF HbA2 HbA

 in H H

aem1s

-{ - .'American Society of Hematology

The alpha thalassemias
• Seen mostly in SE Asia and W Africa
• Let’s do this by genotype
αα/α-: one alpha gene deletion
– Typically silent.
– May have minimal microcytosis.
– Anemia not present.
– Hg electrophoresis normal
αα/−− and − α / − α : Two gene deletions
– Mildly anemic (Hg 10-11),
– Microcytosis with MCV around 70.
– Hgb electrophoresis normal in adults
– Newborns will make Hemoglobin Barts (γ4), so the newborn screen (electrophoresis) will be abnormal
– Make dx by molecular techniques in adults.
– Africans and African-Americans have α − / α − in 5-7% of population (deletion intrans)
– Asians tend to have α α / − − (deletion in cis)
Why is the hemoglobin electrophoresis normal in alpha trait?

• With the beta thalassemias, the proportions change as the

beta chains change, so the electrophoresis percentages
change
• With alpha thalassemia trait—the proportions of the beta
chains don’t change, so the proportions of HbA to HbA2 to
HbF don’t change—so the electrophoresis percentages are
normal
A deli analogy for you

• Think of gamma chains as tuna, and delta chains as turkey and

beta chains as roast beef. Alpha chains are bread
• Decreasing the amount of bread doesn’t change the
proportion of filling—just the number of sandwiches you can
make
The alpha thalassemias

• a-/--: Hemoglobin H disease

– Hemoglobin H = b4 tetramers.
– Hgb H is unstable and precipitates as the RBC ages, forming Heinz bodies, which
causes bite cells and a hemolytic anemia.
– Variable presentation
• Most are like β-thal intermedia
• Some are barely affected and some are severely affected
– Most have splenomegaly
– Hgb b/w 7-11, but may be as low as 3-4
– Low MCV and MCH, high RDW
The alpha thalassemias

• --/--: Hydrops fetalis

– No alpha chains form
– Predominant chain synthesized is γ4tetramers = Hemoglobin Barts
– Intrauterine death, followed by stillbirth at 25-40 weeks
– Can be treated in utero by exchange transfusions or stem cell
transplantation
Hemoglobin S
• Sixth aa in b chain is changed from
glutamate (charged) to valine (hydrophobic)
• Different electrophoretic mobility
• Deoxygenated HbS is 50x less soluble than
deoxy HbA
– Polymerizes and forms long fibers
– Rate of polymerization is logarithmically related
to intracellular concentration of HbS
– Heterozygous cells do not usually sickle in vivo
 Hemoglobin C

• Sixth aa in b chain is changed

from glutamate to lysine.
• Leads to increased cellular
dehydration and crystal
formation
• Found in individuals of West
African descent Target Cells (red arrows) and a Hemoglobin C crystal
(yellow arrow) in homozygous CC disease
Sickle Cell Disease

• Associated with 3 genotypes: SS, SC, and S b-thalassemia.

• SS disease = Sickle cell anemia
• SC disease
– The presence of HbC leads to more intracellular dehydration, worsening
sickling
• Sickle b thalassemia
– S-β0 thalassemia – indistinguishable from SS
– S-β+ thalassemia – milder disease, since some normal beta chains are
produced
 Sickle Cell Trait
• Hematologic parameters are normal (ie—no microcytosis)
• In general, should not have crises or splenic infarctions unless severely hypoxic
• May have renal manifestations
– Isosthenuria
– Microscopic hematuria
• Athletes and screening
– Military recruits and athletes more prone to heat stroke and rhabdomyolysis
during training under unfavorable conditions
– 30X increase in sudden death during boot camp
• Goes away with frequent breaks, good hydration
Hemoglobin C Disease

• Hemoglobin C Trait
– 30-40% HbC
– Not anemic
– Microcytic, increased MCHC
• Hemoglobin C disease
– Mild hemolysis
– Microcytosis and target cells, increased MCHC
– Splenomegaly
Q

• What is the disease?

– HbA 60%
– HbA2 4%
– HbF 1%
– HbS 35%
Q
• What is the disease? • What is the disease?
– HbA 60% – HbA 35%
– HbA2 4% – HbA2 4%
– HbF 1% – HbF 1%
– HbS 35% – HbS 60%
Q

Sickle trait Sickle beta+ thal

• What is the disease? • What is the disease?
– HbA 60% – HbA 35%
– HbA2 4% – HbA2 4%
– HbF 1% – HbF 1%
– HbS 35% – HbS 60%
Q
• What is the disease? • What is the disease?
– HbA 35% – MCV 72, nl ferritin
– HbA2 4% – HbA 70%
– HbF 1% – HbA2 4%
– HbS 60% – HbF 1%
– HbS 25%
Q

Sickle beta+ thal Sickle trait/alpha thal trait

• What is the disease? • What is the disease?

– HbA 35% – MCV 72
– HbA2 4% – HbA 70%
– HbF 1% – HbA2 4%
– HbS 60% – HbF 1%
– HbS 25%
Q

• What is the disease? • What is the disease?

– MCV 72 nl ferritin – MCV 72
– HbA 70% – HbA 0%
– HbA2 4% – HbA2 4%
– HbF 1% – HbF 1%
– HbS 25% – HbS 47%
– Abnormal hemoglobin 48%
• Migrates with HbA2 on alkaline
electrophoresis
• Overlaps with HbA2 on HPLC
Q
Sickle trait/alpha thal trait
Sickle/HbE
• What is the disease? • What is the disease?
– MCV 72 – MCV 72
– HbA 70% – HbA 0%
– HbA2 4% – HbA2 4%
– HbF 1% – HbF 1%
– HbS 25% – HbS 47%
– Abnormal hemoglobin 48%
• Migrates with HbA2 on alkaline
electrophoresis
• Overlaps with HbA2 on HPLC
Hemoglobin migrations

Cellulose Acetate at alkaline pH Agarose at Acid pH

• A is slowest • F is slowest
• F • A comigrates with E and A2
• S • S comigrates with O-Arab
• C comigrates with E, A2 and • C is fastest
O-Arab
Hemoglobin E

• This is a beta chain mutation leading to decreased beta chain synthesis (ie it is a thalassemic
mutation)
• Very common on Indian subcontinent—gene frequency up to 60%!
• Migrates with A2 and C in Alkaline electrophoresis, and with A and A2 in Acid on agarose
• Heterozygotes (hemoglobin E trait = HbAE) are not usually anemic, but may have minimal
microcytosis and hypochromia. Hemoglobin analysis shows approximately 30 percent
HbE, 1 percent HbF, and 70 percent HbA.
• Homozygotes (hemoglobin E disease, HbEE) have minimal anemia along with hypochromia,
target cells, and prominent microcytosis. Hemoglobin analysis shows >90 percent HbE and no
Hb A, with the remainder being HbF. Relatively asymptomatic
• Migrates like HbA2 on gel electrophoresis (but we never get 30% HbA2)
• A variable and occasionally much more severe phenotype is seen when this mutation is
combined with other
alpha and/or beta globin chain mutations (ie, sickle hemoglobin or combinations of alpha
and/or beta thalassemia)
 Hemoglobin Lepore
• Hb Lepore arises from an unequal crossing over and recombination event between adjacent delta and beta
globin genes. The fused globin forms a functional and stable hemoglobin with the mobility of HbS on
alkaline electrophoresis and HbA on acid electrophoresis.
• Hb Lepore does not undergo sickling, but may be confused with HbS as it migrates to the same position as
HbS on alkaline electrophoresis.
• Because the production of the abnormal globin is under the control of the delta globin promoter, (which is
only 2 to 3 percent as active as the beta globin promoter), there is severe underproduction of this abnormal
globin chain, such that Hb Lepore represents only approximately 3 to 20 percent of total hemoglobin in the
affected subject, rather than the expected level of approximately 50 percent. Since no functional delta chain
can be produced by the abnormal chromosome, the concentration of hemoglobin A2 is decreased to
approximately 50 percent of normal. HbF levels are usually slightly increased.
• There is unbalanced production of alpha and beta-like globin chains, producing thalassemia of moderate-to-
high severity in homozygotes and a mild beta thalassemia minor-like picture in heterozygotes
• More common in individuals of Central-European descent (ie Balkans)
What’s the disease?
• Hb 12 • Hb 5-7
• MCV 70 • MCV 60s
– HbA 85% • HbF 3%
– HbA2 1% • Abnormal hemoglobin 97%
– HbF 2% • Migrates with HbS on
– Abnormal hemoglobin 15% alkaline gel
• Migrates with HbS on • Migrates with HbA on acid
alkaline gel gel
• Migrates with HbA on acid • HPLC migrates with HbA2
gel
• HPLC migrates with HbA2
Q

Heterozygous Hb Lepore Homozygous Hb Lepore

• Hb 12 • Hb 5-7
• MCV 70 • MCV 60s
– HbA 85% • HbF 3%
– HbA2 1% • Abnormal hemoglobin 97%
– HbF 2% • Migrates with HbS on alkaline gel
– Abnormal hemoglobin 15% • Migrates with HbA on acid gel
• Migrates with HbS on alkaline gel • HPLC migrates with HbA2
• Migrates with HbA on acid gel
• HPLC migrates with HbA2
Hemoglobin Constant Spring

• Hemoglobin Constant Spring is a structural alpha globin chain termination variant that is quite
common in Southeast Asia. The phenotype depends on whether there are other alpha chain
deletions. It shows the presence of a minor, very slowly migrating abnormal hemoglobin component
on electrophoresis. It is caused by a chain termination mutation such that it not only results in a
deficiency of normal alpha chains, but hemoglobin Constant Spring is itself toxic
• Let’s revisit HbH disease
– Deletional HbH (a-/--)
• Patient has only ONE alpha gene and makes b4 tetramers
– Non-Deletional HbH
• Patient has 2 alpha genes, but one is Hb Constant Springs, which is messed up and interferes with transcription of the
other normal alpha gene
• More severe than deletional HbH disease
Q

• What is the disease?

– Hb 12.5
– MCV 88
– HbA 0%
– HbA2 1.5%
– HbF 28.5%
– HbS 70%
Q

• HbS/HPFH
– Hb 12.5
– MCV 88
– HbA 0%
– HbA2 1.5%
– HbF 28.5%
– HbS 70%
Q

• What is the disease?

– Hb 12.5
– MCV 78
– HbA 70%
– HbA2 2.5%
– HbF 27.5%
Q

• Heterozygous HPFH
– Hb 12.5
– MCV 78
– HbA 70%
– HbA2 2.5%
– HbF 27.5%
• What is the disease? • What is the disease?
– Hb 10.5 – Hb 12.5
– MCV 108 – MCV 88
– HbA 0% – HbA 0%
– HbA2 4.5% – HbA2 1.5%
– HbF 22.5% – HbF 28.5%
– HbS 73% – HbS 70%
 Sickle Cell Anemia on Hydrea Sickle Cell Anemia with HPFH

• What is the disease? • What is the disease?

– Hb 10.5 – Hb 12.5
– MCV 108 – MCV 88
– HbA 0% – HbA 0%
– HbA2 4.5% – HbA2 1.5%
– HbF 22.5% – HbF 28.5%
– HbS 73% – HbS 70%
OK, this one’s hard

• I’ll give you a hint. It’s homozygous for the abnormality

• Hb is 6
• MCV is 50
• HbF 100%
• HbA2 0%
• HbA 0%
OK, this one’s hard

• This is delta/beta thalassemia—we’ve deleted the delta gene

AND the beta gene—cannot make anything but HbF
• Hb is 6
• MCV is 50
• HbF 100%
• HbA2 0%
• HbA 0%
Recall question
• Hb 12
• MCV 70
• Alkaline gel
– HbA 85%
– HbA2 1%
– HbF 2%
– HbS 15%
• Sickledex negative
Recall question
Heterozygous Hemoglobin Lepore—runs with HbS on alkaline gel, but doesn’t sickle
• Hb 12
• MCV 70
• Alkaline gel
– HbA 85%
– HbA2 1%
– HbF 2%
– HbS 15%
• Sickledex negative
Summary

• HbF=α2γ2, HbA2=α2δ2, HbA = α2β2

• Sickle trait 60%A, 40%S (NOT microcytic)
• Sickle β+ thalassemia, 60%S, 40%A
• Sickle α thal trait- 65%A, 35% S,microcytic
• Review Hb Lepore, HbE, Hb Constant Spring—thalassemic
hemoglobinopathies
• HbS and HbC—structural hemoglobinopathies
A personal recommendation from me