Chapter 13

Hemoglobinopathies
Learning objectives:
1. Describe the common denominator in hemoglobinopathies & name the
3 major categories of classification of hemoglobin defects.
2. Describe the etiology of sickle cell disease (SCD), discuss its
epidemiology & describe its clinical signs and symptoms
3. Outline laboratory findings that are typical of SCD and briefly describe
the various approaches used for SCD diagnosis.
4. Compare the conditions of - and -thalassemia and outline the
laboratory findings in the various forms of thalassemia.
5. Describe the conditions of sickle -thalassemia, sickle-C (SC), and
sickle cell trait.
6. Describe the general characteristics of hemoglobin (Hb) C disease, Hb
SC disease, Hb D disease, Hb E disease, Hb H disease,
methemoglobinemia & unstable hemoglobins.

Introduction to Hemoglobinopathies
A group of genetically determined abnormalities of the structure or
synthesis of the globin chain; the heme group is normal
Most common in people of African, Mediterranean, or Southeast
Asian origin
Majority of hemoglobinopathies result from -globin chain
abnormalities.
They frequently associate with chronic hemolytic anemia and other
complications
Globin chain abnormalities are either qualitative defects (structural
abnormalities) or a quantitative defect of the globin chain synthesis
Qualitative abnormal hemoglobin molecules result from genetic
mutation involving amino acid deletions or substitutions in the
globin protein chain; most common disorder of this type is
sickle cell anemia.
Quantitative globin disorders result from genetic defects that
lead to reduced synthesis of globin chains. This type of
quantitative disorders is known as thalassemias.

Prevalence of SCD by race

or ethnic group in the US
Race or ethnic
group
White
Black
Hispanic, total
Hispanic, eastern
states
Hispanic, western
states
Asian
Native American

Average
prevalence per
100,000 live births
1.72
289
5.28
89.8
3.14
7.61
36.2

Prevalence of SCD
worldwide

Sickle Cell Disease (SCD)

SCD is the most common type of hemoglobinopathy around
the world; about 7% of world population carry the mutation.
Greatest prevalence is in Africa (central Africa); but also
common in the Middle East, Mediterranean, India & Nepal.
Geographic areas with the highest frequency of sickle cell
gene are also areas where infection with Plasmodium
falciparum is common suggesting that individuals with
HbS trait are resistant to malarial infections (WHY ???)
Sickle cell disease is mostly inherited as an autosomal
recessive trait with over-dominance (= heterozygotes have
a selective advantage over homozygotes).
HbS heterozygotes are carriers of the defect with little or no clinical
consequences
HbSS homozygotes suffer from sickle cell disease significant
clinical consequences

Mode of inheritance of SCD / SC trait

Other forms of SCD:

Compound heterozygous states in which the person has only
one copy of the mutation that causes HbS and one copy of
another abnormal hemoglobin allele could also lead to SCD.
Such forms include:
Sickle / hemoglobin C disease (HbSC)
Sickle / beta-plus-thalassaemia (HbS/+)
Sickle / beta-zero-thalassaemia (HbS/0)

Etiology of SCD
HbS is the hemoglobin that is
produced when valine
(hydrophobic) substitutes
glutamic acid (negatively
charged) at the sixth position in
the chain
This substitution is on the surface
of the molecule a change of net
charge
Changes electrophoretic mobility
of the molecule
In the deoxygenated form,
solubility of HbS is markedly
reduced, producing a tendency
for deoxyhemoglobin S
molecules to polymerize into
rigid aggregates

Following polymerization, the

cell assumes a crescent or sickle
shape

Hemoglobin S polymer formation

Normal
Sickle Cell Disease

Electron micrograph of
Hemoglobin S polymers

Etiology (cont)
The sickling process is enhanced by:
Hypoxia
Acidosis
Extreme temperature (high or low)
Hypertonicity of microenvironment
Concentration of HbS within erythrocyte itself
(MCHC)
Presence of other intracellular hemoglobin
variants (the proportion of HbS to HbA &
HbF) presence of HbA or HbF tends to
dilute (minimize) the sickling process

Sickle Cell Disease

Etiology (Cont)

When sickled cells attempt to travel through small vessels,

they get stuck vessels become obstructed.
This initiates a pattern of blood not flowing properly to
tissue and creating a lack of oxygen.
Lack of oxygen (hypoxia) causes more sickling and more
deprivation of oxygen to tissue.
This process can produce intense pain.
When sickled cells receive oxygen, they return to their
normal shape
Repeated cycles of sickling & unsickling lead to red cell
damage, resulting in hemolysis & anemia
Additionally, sickled cells have high tendency to adhere to
vascular endothelial cells of small vessels, leading to
vaso-occlusion painful crises ischemic injury

Sickle cell Disease

Normal RBCs
flexible, disc
shaped
move easily
throughout blood
vessels
lasts four months
in bloodstream
Abnormal RBCs
stiff, curved shape
resembling a sickle
(crescent moon shaped)
clogs blood vessels
last 10-20 days in
bloodstream which can
lead to anemia

Clinical signs and Symptoms

Sickling leads to damage & defects in various body organs & tissues
enlarged heart, progressive loss of pulmonary & renal function, strokes,
arthritis, liver damage, skeletal damage leads to crises of different
forms: vaso-occlusive crisis, aplastic crisis, sequestration crisis &
hemolytic crisis; most episodes of crises last for 5-7 days.
Symptoms usually appear after the age of 6 months
Symptoms of the disease include:
Severe hemolytic anemia
Vaso-occlusion symptoms: develops between 12 months- 6 years.
Hand-Foot syndrome (resulting from vaso-occlusion crises)
Infection (Streptococcus pneumonia and Haemophilus influenzae) is
the major cause of death among children below age of 5 years.
Leg ulcers
Aplastic crises due to viral infection
Lagging growth & development
Bone and joint destruction result from repeated ischemia and
infarctions.
Pulmonary complications
Strokes

Laboratory Findings
Decreased hemoglobin (10-5 g/dL)
Decreased hematocrit, red cell count, and
increased WBC count.
Blood film: Anisopoikilocytosis, hypochromia,
target cells, microcytes, polychromasia, red cell
fragments, and sickled red cells.
Reticulocytosis
Increased unconjugated Bilirubin
Decreased haptoglobin & hemopexin

Laboratory Investigations of SCD

1. Solubility test

Principle: in the deoxygenated form, HbS becomes

insoluble & precipitates causes the solution to be turbid.

Procedure:

Few drops of whole blood in a test tube are mixed with a

solution containing ) hemolyzing agent (saponin) & a
reducing agent (sodium dithionite).
The tube is held in front of a white card with narrow black
lines and turbidity is read in comparison of a negative and
positive controls.
If the black lines cannot be seen the solution is turbid & the
test is positive for HbS.
The test gives positive results for HbAS, HbSS, & HbSC
It does not differentiate between heterozygous and
homozygous forms.
Hence, a positive test is further confirmed by electrophoresis.

Solubility Test
Red Cells
+
Saponin
+
Sodium Dithionite

Positive

Negative

2. Sickling test of Whole blood

The sickling phenomenon can also be

demonstrated by making a thin wet
film of whole blood:
a small drop of blood is added to a
slide
mix with a reducing agent like
sodium metabisulfite or sodium
dithionite & covered with a cover
slip
Cover slip is sealed at the edges
observation of sickled cells under
the microscope indicate a positive
result for HbS
But test does not differentiate
between heterozygous and
homozygous states
Hence, Hb electrophoresis should
follow

3. Hemoglobin Electrophoresis

Cellulose Acetate Electrophoresis at Alkaline pH

At alkaline pH (pH=8.6), hemoglobins will be
negatively charged Migrate from cathode (negative
pole) to anode (positive pole)
Hemoglobin variants with highest negative charge will
move the fastest
At alkaline pH, hemoglobins D & G migrate with HbS
while hemoglobins C & E migrate with HbA2.
Therefore, electrophoresis at acidic pH should be done
to differentiate these hemoglobins from each others.

Hemoglobin Electrophoresis
pH 8.6

Cathode (-)

Anode (+)

HbAA
HbAS
HbSS
PA A2
C
E

S F
D
G

Hemoglobin Electrophoresis
pH 8.6
Cathode (-)
AA

Anode (+)

AS
SS
SC
AC
CC
A2
C
E

S
D
G

Hemoglobin Electrophoresis (Cont)

Citrate Agar Electrophoresis (Acidic pH, 6.2)

Citrate agar electrophoresis separates

hemoglobin fractions that migrate together on
cellulose acetate.
These fractions are hemoglobins S, D, G,
C, E & A2 as shown in diagram below

Citrate Agar Electrophoresis (Acidic pH, 6.2)

Cathode

Anode
AA

AS

SS

SC

AC

AE?

Thal Major

S-Thal

|
C

A
A2
G
D
E

Treatment of Sickle Cell Disease

There is no cure for SCD but symptoms can be treated.

Crises accompanied by extreme pain, common problem, is
treated with pain relievers.
Special precautions are taken before any type of surgery;
for major surgery some patients receive transfusions to boost
[Hb]. Blood transfusions may also be used to treat/prevent
anemia, spleen enlargement, and recurring strokes.
Infants diagnosed with the disease receive daily doses of
penicillin to prevent infections.
Adults with SCD now take hydroxyurea, an anticancer
drug that causes the body to produce RBCs resistant to
sickling average life expectancy in the US of SCD patients
increased from 42 years in males & 48 years in females to >50
yrs.

Thalassemia
Basics of Hb structure & synthesis

Basics of Hb synthesis
Adult Hb is made of 2 gene loci:
globin locus on chromosome 11 and
the locus on chromosome 16
locus contains 2 copies of the same
gene aligned one after the other
total genes/cell = 4); each
contributes ~25% of the total
globin chains made in the cell.
The 2 globin genes are active
during fetal growth and produce HbF.
Very early on during embryonic
development, 2 (1 & 2) make
chains instead of globin chains)
Adult gene, becomes active after
birth.
Each of the four globin genes
contribute to the synthesis of the
HbA protein.

RBC Hb composition under normal conditions

Adult
Fetal

Hemoglobin

Structural formula

Hb-A

2 2 97%

Hb-A2

2 2 1.5-3.5%

Hb-F

2 2 0.5-1%

Hb-Barts

Embryonic Hb-Gower 1

2 2

Hb-Gower 2

2 2

Hb-Portland

2 2

Hemoglobinopathies
Thalassemia
Thalassemias result due to absence or reduced synthesis of
or chain protein.
Inherited as autosomal recessive.
Globin genes are located at chromosomes 11 (beta chain) and
16 (the alpha chain).
Only one gene per chromosome, (two per diploid cell),
specifies the -chain.
Two genes on each homologous chromosome (4/diploid
cell), specify the inheritance of the -globin chain.
Deficiency of the -chain lead to alpha thalassemia
deficiency of the beta chain lead to beta thalassemia.

Classification & Terminology

Beta Thalassemia
Normal
Minor (heterozy.)
Intermedia
Major (homozy.)

/
/0
/+
0/+
0/0
+/+

Keep in mind that + means that there is some synthesis

within a wide range (from very little to almost normal)

-Thalassemias

1. -thalassemia minor: the heterozygous, characterized by:

mild anemia with microcytosis & abnormal erythrocytes
morphology
splenomegaly
2. Thalassemia intermedia: anemia is moderate with presence
of HbA in addition to HbF
3. -thalassemia major (Cooleys anemia): the homozygous
form, characterized by:
severe anemia
transfusion dependence
organ damage secondary to iron overload
extramedullary erythropoiesis (bone damage)

-Thalassemia minor
Lab Findings:
Beta Thalassemia minor could be mistaken by
mild iron deficiency anemia on peripheral blood
film.
Characterized by increased HbA2 (diagnostic
test) and decreased MCV.
Normal range for HbA2 is 1.5-3.5%, but in thalassemia
minor it is 3.5-8.0%

-Thalassemia Major
Symptoms appear several months after birth following the switch
from to chain synthesis
Pathophysiology: Decreased synthesis of chain leads to excess
of -chain excess free chains are unstable and precipitate
within the cell causing membrane damage contributes to
destruction of RBCs and development of anemia
Lab Findings:
Decreased Hb, Hct & RBC count
Significantly reduced MCV, MCH, and MCHC
Anisocytosis, poikilocytosis, hypochromia, target cells,
polychromasia, and nRBCs.
Increased RDW, reticulocytes, bilirubin, serum iron & serum
ferritin.
Electrophoresis reveals increased HbF & decreased/absent HbA

-Thalassemia

1.

Major cause of -thalassemia is deletion of one or more of

the genes coding for -chain on chromosome 16.
Can be classified into four types according to number of
gene deletion:
Silent carrier (one gene is inactive) three remaining
genes can synthesize adequate amount of producing
normal amount of Hb no anemia

2. -thalassemia trait (two genes are inactive) imbalance of

- and -chain synthesis creates an excess in chains.
These excess chains may aggregate forming a tetramer
(4) known as HbH. HbH is an unstable hemoglobin that
precipitates on cell membrane. Affected individuals are
clinically normal but frequently have minimal anemia and
reduced MCV & MCH; RBC count is usually increased,
typically exceeding 5.5 1012/L.

Classification & Terminology Alpha Thalassemia

Normal
Silent carrier
Minor

/
- /
-/-
--/
Hb H disease
--/-
Barts (hydrops fetalis) --/--

3. Hemoglobin H disease (three genes are inactive) excess

-chains aggregate forming again HbH precipitate on
cell membrane cell damage & short RBC life span.
HbH inclusions (precipitate) are detected when cells
are stained with brilliant cresyl blue.
HbH associates with chronic, moderately severe
hemolytic anemia; Hb ranges from 8-10 g/dL & all
RBCindices are decreased.
HbH migrates ahead of HbA in electrophoretic gel.
Hb electrophoresis reveals HbH 4-30% (ahead of
HbA); may also show a small amount of Hb Barts
(4).
4. Hydrops fetalis with Hb Barts (four genes are inactive)
incompatible with life. Affected fetuses die either in utero
or shortly after birth. On electrophoresis mainly Hb Barts
is present.

Other Hemoglobinopathies
Hemoglobin

Structural formula

Hb-S

2 2 6 glu val

Hb-C

2 2 6 glu lys

Hb-E

2 2 26 glu lys

Hb-D Punjab

2 2 121 glu gln

Other Hemoglobinopathies
1. Hemoglobin C disease (HbC)
Differ from HbA by the substitution of lysine instead of
glutamic acid at position 6 of the beta globin chain.
DeoxyHbC has decreased solubility and forms
intracellular crystals (cigar-shaped crystals).
Homozygous form (HbCC) results in mild chronic
hemolytic anemia with >50% target cells in blood film.
Hemoglobin C trait (HbAC) is symptomless, with target
cells and mild hypochromia.
At alkaline pH electrophoresis, HbC migrates with A2.
At acidic pH it remains at origin

2. Hemoglobin SC disease (HbSC)

Result from the inheritance of one S gene and one C
gene; milder than SCD (SS) although HbC tends to
enhance sickling. Blood film reveals target cells, folded
erythrocytes & intracellular crystals.
3. Hemoglobin D disease (Hb D)
Both homozygous and heterozygous are asymptomatic.
HbD migrates at same position as HbS & HbG at
alkaline pH but migrates with HbA at acidic pH.
4. Hemoglobin E disease (Hb E)
In some areas of Thailand, frequency of HbE trait is
almost 50%; heterozygous (HbAE) is asymptomatic
while homozygous (HbEE) is mildly anemic.

Patient with thalassemia major:

Note the prominent target cells, anisopoikilocytosis & 3
nucleated red cells (normoblasts)

Peripheral blood smear from a patient with -thalassemia

major showing marked anisopoikilocytosis: target cells,
schistocytes, teardrops, and ovalocytesn, RBCs. (WrightGiemsa stain)

Alpha thalassemia

Brilliant Cresyl Blue Stain

Incubation with
brilliant cresyl blue
stain causes HbH to
precipitate
Results in
characteristic
appearance of
multiple discrete
inclusions -golf ball
appearance of RBCs.
Inclusions smaller
than Heinz bodies
and are evenly
distributed
throughout cell.
44

Acid Elution Stain for Detection of HbF

Based on KleihauerBetke procedure.
Acidic pH will dissolve
HbA from RBCs but not
HbF because HbF is
resistant to denaturation
& remains in the cell.
Stain slide with eosin.
Normal adult cells
appear as "ghost" cells
while cells with HbF
stain varying shades of
pink.

Cells with HbF

"ghost" cells

45

Sickle Cells
Sickle cells (drepanocytes) contain a sickling HbS which
polymerizes into long rigid crystals upon exposure to decreased
oxygen or low pH sickle shape with decreased ability to pass
through small vessels & increased mechanical fragility.

Hemoglobin C Crystals

Hemoglobin H Inclusions (Golf ball-shape)

Differential Diagnosis of Microcytic, Hypochromic

Anemias
RDW

Serum Iron

TIBC

Serum Ferritin

Inc

Dec

Inc

Dec

Alpha Thal

Norm

Norm

Norm

Norm

Beta Thal

Norm

Norm

Norm

Norm

Hgb E Disease

Norm

Norm

Norm

Norm

Anemia of Chronic
Disease

Norm

Dec

Dec

Inc

Inc

Inc

Norm

Inc

Norm

Norm

Norm

Norm

Iron Deficiency

Sideroblastic
Anemia
Lead Poisoning