Microcytic Hypochromic Anemia & Thalassemia

Department of Laboratory Medicine
Bangabandhu Sheikh Mujib Medical University

Shahbag, Dhaka.
Presentation On
Microcytic Hypochromic
Anemias & Thalassemias.
Dr Dipalok Mukherjee
Resident, Phase A.
Points To Ponder
 The Red Cells  Iron Deficiency Anemia
 Hemoglobin  Nutritional & Metabolic
 The hemoglobin- Aspects of Iron
Dissociation Curve  Anemia of Chronic Disorders
 Role of Erythropoietin
 Sideroblastic Anemias
 Normal Values & Hematinics
 Genetic Disorders of
 Red Cell Indices Hemoglobin Synthesis
 Anemias: General
 The Thalassemias
Consideration
 Microcytic Hypochromic  Diagnostic Algorithm
Anemias
© Dr Dipalok Mukherjee
The Red Cells
 Red cells are the most numerous of all blood cells
 Specialized for and C carriage
 They have a 4-month (120 days) lifespan
 Mature cells lack nuclei, and other cytoplasmic structures;
hence, cannot undergo mitosis.
 Biconcave discs with a diameter of 7-8µm in large blood
vessels with an average diameter of 7.2µm
 Shape changes to parachute-like conformation in
capillaries.
The Red Cells Photomicrograph
Fig: (A) Mature red cells in a peripheral blood film, (B) Scanning electron
micrograph of red cells (x2500).
Red Cell Metabolism
The Embden–Meyerhof
pathway
 Glucose enters the red cell
from plasma by facilitated
transfer.
 Metabolized to lactate.
 Each molecule of glucose
produces two molecules of
ATP and thus two high-
energy phosphate bonds
are generated.
 Also generates NADH
which is required by
methemoglobin reductase
to reduce functionally dead
methemoglobin containing
Fig: The Embden–Meyerhof glycolytic pathway. ferric iron.
The Luebering–Rapoport shunt regulates the
concentration of 2,3-DPG in the red cell.
Red Cell Membrane
The membrane skeleton is formed by structural proteins that include α and β spectrin,
ankyrin, protein 4.1, and actin. These proteins form a horizontal lattice on the internal
side of the red cell membrane and are important in maintaining the biconcave shape.
Fig: The structure of the red

cell membrane. Some of the
penetrating and integral
proteins carry carbohydrate
antigens; other antigens are
attached directly to the lipid
layer.
Hemoglobin
 Hemoglobin is a specialized protein
for O and CO carriage.
 Each molecule of normal adult
hemoglobin A (Hb A) consists of four
polypeptide chains, 𝛂𝟐 𝛃𝟐 , each with
its own heme group.
 Heme synthesis occurs largely in the
mitochondria.
 The structure of heme contains a 𝐅𝐞𝟐
ion at its center, each connected to
globin chains in a pocket, forming a
Fig: The structure of heme.
hemoglobin molecule.
Hemoglobin Synthesis
Hemoglobin synthesis in the
developing red cell
The mitochondria are the main

sites of protoporphyrin synthesis,
iron (Fe) is supplied from
circulating transferrin and globin
chains are synthesized on
ribosomes.
*δ-ALA, δ-aminolaevulinic acid; CoA,
coenzyme A.
The Hemoglobin Dissociation
Curve
Fig: The oxygenated and deoxygenated

hemoglobin molecule. α, β, globin chains
of normal adult hemoglobin (Hb A).
Fig: The hemoglobin O dissociation curve.

2,3-DPG, 2,3-diphosphoglycerate.
Role of Erythropoietin
 Erythropoietin is a heavily glycosylated polypeptide
hormone.
 Essential for the regulation of erythropoiesis.
 90% is produced in the peritubular interstitial cells of

the kidneys; and 10% in the liver.
 Erythropoietin stimulates erythropoiesis by increasing

the number of progenitor cells committed to
erythropoiesis.
Erythropoietin Production
 Erythropoietin stimulates
erythropoiesis and so
increases delivery.
 Hypoxia induces hypoxia
inducible factors (HIFs) α
and β, which stimulate
erythropoietin production.
 Von-Hippel–Lindau (VHL)
protein breaks down HIFs.
 Mutations in VHL, PHD2 or
HIF-1α underlie congenital
polycythemeia.
Normal Values for
Blood Cells & Hematinics
Reticulocyte count (x10 /L) (per cent)
Adults and children 50-100 (0.5-2.5%)
Full term/cord blood 120-400 (2-6%)
*Lower limit 1.5 × 109/L in some ethnic groups, e.g. in

Middle East and black-skinned people.
**Normal ranges differ between different laboratories.
PCV, packed cell volume.
Red Cell Indices:
MCV & MCH
Mean corpuscular volume (MCV) is derived from packed cell volume (PCV) and total
number of red cells in that particular volume.
 Expressed in femtoliter (fL).
 It helps in morphological classification of anemias.
MCV= PCV/number of red cells.
Adult 80-95
At birth 106
At one year 70-80
10-12 years 77-91
Mean corpuscular hemoglobin (MCH) describes mean amount of Hb per red blood
cells and expressed in picograms (pg). A
 Subnormal MCH occurs in microcytosis with or without low Hb concentration as
in thalassemia minor or iron deficiency.
MCH= Hb conc. (gm/L)/number of RBC/L
Adult MCH: 27-34pg.
Red Cell Indices:
MCHC & RDW
Mean corpuscular hemoglobin concentration (MCHC) is the measure of Hb concentration
(gm/dL) in a volume of red cells (PCV) and expressed in gm/dL.
MCHC = Hb conc. (gm/dL)/PCV
 A subnormal MCHC is indicative of defective Hb synthesis as in thalassemia or iron

deficiency.
 Elevated values indicate cellular dehydration eg. spherocytosis.
Adult MCHC: 31-35 gm/dL.
Red cell distribution width (RDW) provides an assessment of variation in cell volume
which corresponds to the microscopic analysis of the degree of anisocytosis.
 RDW-CV (percent of coefficient of variation) has been found to be more useful in

nutritional anemias like iron deficiency and megaloblastic anemias.
Adult RDW-CV: 12.8±1.2 percent.
Anemias: General Consideration
Anemia is defined as a reduction in the hemoglobin concentration of the
blood below normal for age and sex.
Global incidence
 The World Health Organization (WHO) defines anemia in adults as a
hemoglobin less than 130g/L in males and 120g/L in females.
 On this basis, anemia was estimated in 2010 to occur in about 33% of
the global population.
 Prevalence was greater in females than males at all ages most
frequent in children less than 5 years old.
 Anemia was most frequent in South Asia; and Central, West and East
Sub-Saharan Africa.
 The main causes are iron deficiency (life-long poor diet combined
with menstruation, repeated pregnancies, hookworm,
schistosomiasis), the anemia of chronic disorders, sickle cell
diseases, thalassemia, and malaria.
Classification of Anemias
Anemias can be classified as:
 Morphologically depending on the red cells indices.
 Etiological classification.
Morphological classification Etiological classification
 Microcytic, hypochromic  Blood loss (hemorrhagic)
 Normocytic, normochromic  Impaired red cell production

(dyserythropoietic)
 Macrocytic  Excessively rapid red cell
destruction (hemolytic).
Morphological
Classification of Anemias
 The most useful classification is that based on red cell indices.
 Suggests nature of the primary defect.
 May also indicate an underlying abnormality before overt anemia has
developed.
Microcytic, hypochromic Normocytic, Macrocytic
normochromic
MCV <80fL MCV 80-95fL MCV >95fL
MCH <27pg MCH ≥27pg Megaloblastic: vitamin 𝐁𝟏𝟐
or folate deficiency.
Iron deficiency anemia, Many hemolytic anemias, Non-megaloblastic:
thalassemia, anemia of anemia of chronic diseases alcohol, liver disease,
chronic diseases (some (some cases), after acute myelodysplasia, aplastic
cases), sideroblastic anemia. hemorrhage. anemia.
Lead poisoning. Mixed deficiencies
Bone marrow failure (eg.
post chemotherapy,
infiltration by carcinoma).
Basic Pathophysiological
Categories of Anemias
 Blood loss
 Impaired red cell production
a) Inadequate supply of nutrients essential for erythropoiesis: iron
deficiency, vitamin 𝟏𝟐 or folate deficiency, combined deficiency, protein-energy
malnutrition, vitamin C and other less common deficiencies.
b) Depression of erythropoietic activity
c) Anemia associated with chronic disorders such as infection, connective
tissue disorders, disseminated malignancies, inflammatory disorders.
d) Anemia associated with renal failure
e) Aplastic anemia
f) Anemia due to replacement of normal bone marrow by: leukemia,
lymphoma, myeloproliferative disorders, myeloma, myelodysplastic disorder.
g) Anemia due to inherited disorders such as thalassemia.
 Excessive red cell destruction
a) Due to intrinsic defects in red cells (intra corpuscular defects)
b) Due to extrinsic effects on red cells (extra corpuscular defects).
Clinical Features of Anemias
The major adaptations to anemia occur in the
 cardiovascular system with increased stroke volume and tachycardia
 the hemoglobin dissociation curve.
The presence or absence of clinical features can be considered under
four major headings.
a) Speed of onset
b) Severity
c) Age
d) The hemoglobin dissociation curve.
Anemias: Clinical Manifestations
Symptoms
Common Fatigue, tiredness, effort intolerance, effort dyspnea,
palpitations.
Less common Faintness, giddiness, pounding in the ears, effort angina*.
Signs
General
Common Pallor
Less common High cardiac output state, congestive cardiac failure*.
Specific
Koilonychia Iron deficiency anemia
Jaundice hemolytic, megaloblastic anemias,
Leg ulcers Sickle cell anemia
Bone deformities Thalassemia major.
*More likely in the elderly patients.
Anemias: Physical Signs
(a) (b) (c)
Fig: Pallor of the conjunctival mucosa (a) and of the nail bed (b) in two patients
with severe anemia (hemoglobin 60g/L), (c) Retinal hemorrhages in a patient
with severe anemia (hemoglobin 25g/L) caused by severe hemorrhage.
Microcytic Hypochromic Anemias
(Pathophysiological Classification)
Disorders of Iron metabolism
 Iron deficiency anemia
 Anemia of chronic disorders
 Atransferrinemia
 Familial microcytic hypochromic anemias
Disorders of globin synthesis
 Thalassemia
 Hb-E Trait and Hb-E disease
 Hb - C disease
Disorders of porphyrin and heme synthesis
 Defective δ-aminolevulinic acid (ALA) synthesis
 Vit B12 deficiency
 Defective ALA synthetase activity
 Defective vit 𝟏𝟐 metabolism
 Deficiency of heme synthesis
 Lead intoxication.
Microcytic Hypochromic Anemias:
Differentials
 Iron deficiency anemia
 The thalassemias
 Anemia of chronic
disorders
Fig: The causes of a hypochromic microcytic anemia.
 Sideroblastic anemias. These include iron deficiency or of iron release from
macrophages to serum (anemia of chronic
inflammation or malignancy), failure of
protoporphyrin synthesis (sideroblastic anemia) or
of globin synthesis (α- or β-thalassemia). Lead also
inhibits heme and globin synthesis.
Iron Deficiency Anemia (IDA)
 Iron is one of the most common elements in the Earth’s crust.
 Yet iron deficiency is the most common cause of anemia, affecting about 500
million people worldwide.
 It is particularly frequent in low-income populations, such as in sub-Saharan

Africa or South-South East Asia.
 Poor food quality and parasites (e.g. hookworm or schistosomiasis), which

cause iron loss due to hemorrhage, may be present.
 Moreover, the body has limited ability to absorb iron.
 It is always secondary to underlying disorder.
 Occurs at any age but especially common in women of childbearing age.
Dietary Iron
 Iron is present in food as ferric
hydroxides and ferric–protein and
heme–protein complexes.
 Both the iron content and the
proportion of iron absorbed differ
from food to food.
 In general meat, in particular liver,
is a better source than vegetables,
eggs or dairy foods.
 The average diet contains 10–15
mg iron daily, from which only 5–
10% is normally absorbed. The
proportion can be increased to 20–
30% in iron deficiency or
pregnancy.
 Even in these situations most
dietary iron remains unabsorbed.
Nutritional & Metabolic Aspects of
Iron
Table: Distribution of body iron.
Total body iron content of the normal adult varies from 3-5gm depending on
age, sex, and weight of the individual. It is greater in male than female.
Regulation of Iron Absorption
Figure shows the regulation

of iron absorption. Dietary
ferric 𝟑
iron is reduced
to 𝟐 and its entry to the
enterocyte is through the
divalent cation binder DMT-
1. Its export into portal
plasma is controlled by
ferroportin. It is oxidized to
before binding to
transferrin in plasma. heme
is absorbed after binding to
its receptor protein.
Factors Affecting Iron Absorption
The following table enumerates factors affecting luminal
iron absorption.
Cellular Uptake of Iron
Transferrin
+
Iron (𝐅𝐞𝟑 ) Fig: Mechanisms
involved in tissue iron
uptake.
Transferrin-Iron-
Transferrin receptor
1 (TfR1) complex on
cell surface
Complex fuses 𝐅𝐞𝟑 is converted

Transferrin-TfR1
with endosome to 𝐅𝐞𝟐 by
complex recycled
and acidification Ferrireductase
to the cell surface
𝐅𝐞𝟐 -DMT-1 → 𝐅𝐞𝟐 to Divalent

Ferrioxidase → Metal Transporter
Ferritin 1 (DMT-1)
Daily Iron Cycle
Fig: Daily iron cycle. Most of the iron in the body is contained in circulating
hemoglobin and is reutilized for hemoglobin synthesis after the red cells die. Iron is
transferred from macrophages to plasma transferrin and so to bone marrow
erythroblasts. Iron absorption is normally just sufficient to make up for iron loss.
Hepcidin: The Iron Homeostasis
Regulator
 Hepcidin is a polypeptide produced by liver cells.
 It is the major hormonal regulator of iron homeostasis.
 It inhibits iron release from macrophages, intestinal epithelial cells, and

from other cells by its interaction with the transmembrane iron exporter,
ferroportin.
 It accelerates degradation of ferroportin.
 Raised hepcidin levels therefore profoundly affect iron metabolism by

reducing iron absorption, and its release from macrophages.
Factors Affecting Hepcidin Synthesis
Factors favoring synthesis Factors reducing synthesis
High transferrin saturation (iron Erythroblasts → erythroferrone

overload)
Inflammation → IL6 Hypoxia
Erythropoietin
Matriptase (iron deficiency)
Low transferrin saturation
Iron Excretion & Requirements
Iron is lost through the following:
 Desquamated epithelial cells from the gastrointestinal tract in stool
 Excretion in the urine and sweat
 Loss of hair and nails.
Table: Estimated daily iron requirements. Units are mg/day.
Iron Deficiency Anemia: Causes
Chronic blood loss Increased demands
a) Uterine  Prematurity
b) Gastrointestinal  Growth
 Chronic upper GI bleeding: bleeding  Pregnancy
peptic ulcer, esophageal varices,
aspirin (or other non-steroidal anti-  Erythropoietin therapy
inflammatory drugs) ingestion. Malabsorption
 Malignancies: Carcinoma of the  Gluten-induced enteropathy,
stomach, caecum, colon or rectum.
 Gastrectomy
 Parasites: hookworm, schistosomiasis.
 Autoimmune gastritis
 Chronic inflammatory conditions:
angiodysplasia, inflammatory bowel Poor diet
disease, piles, diverticulosis. A major factor in many developing
c) Rarely: hematuria, hemoglobinuria, countries.
pulmonary hemosiderosis, self-inflicted
blood loss.
Clinical Features
 When iron deficiency is developing, the reticuloendothelial stores (hemosiderin and
ferritin) become completely depleted before anemia occurs.
 As the condition develops, the patient may show the general symptoms
and signs of anemia.
 A painless glossitis, angular stomatitis, brittle, ridged or spoon nails (koilonychia),

and an unusual dietary cravings (pica). The cause of the epithelial cell changes may
be related to reduction of iron-containing enzymes.
 Retina: hemorrhages and papilledema in severe cases.
 A syndrome of dysphagia and glossitis as a result of pharyngeal webs (Paterson-Kelly

or Plummer-Vinson Syndrome). It usually occurs in middle aged or elderly women
with chronic iron deficiency.
 Neonatal iron deficiency is associated with cognitive and behavioral abnormalities.
 While in children it can cause irritability, poor cognitive function, and a decline in
psychomotor development.
Physical Findings
Fig: Iron deficiency anemia. (a) Koilonychia: typical ‘spoon’ nails. (b)
Angular cheilosis: fissuring and ulceration of the corner of the mouth.
Iron Deficiency Anemia: Diagnosis
The diagnosis of iron deficiency anemia can be

confirmed by
 Lab investigations relating to the blood cells and

hematinics
 Investigation of the cause of iron deficiency.
Laboratory Findings
Investigations Findings
MCV/MCH Reduced in relation to severity of anemia.
Peripheral blood film Anisocytosis, poikilocytosis: hypochromic,

microscopic cells with pencil-shaped
poikilocytes, occasional target cells.
Serum iron Reduced
TIBC Raised
Serum ferritin Reduced
Bone marrow iron stores Absent
Erythroblast iron Absent
Hb electrophoresis Normal.
Peripheral Blood Film: IDA
Fig: The peripheral blood film in severe iron deficiency anemia. The cells are
microcytic and hypochromic (blue arrowhead) with good number of pencil-
shaped poikilocytes (red arrowhead).
Investigation of the Cause of
Iron Deficiency
Cause Investigations
Menorrhagia Test for coagulation eg. PT, aPTT; test for von-
Willebrand disease.
Gastrointestinal blood loss Occult blood test
Stool routine microscopy for ova
Urea breath test for Helicobacter infection
Appropriate upper and lower GI endoscopy, and/or
radiology.
Autoimmune gastritis Parietal cell antibodies, serum gastrin levels
Gluten induced enteropathy Transglutaminase antibodies, duodenal biopsy
Celiac axis angiogram Angiodysplasia
Hematuria Urinary routine microscopy
Chest x-rays Pulmonary hemosiderosis.
Iron Deficiency Anemia: Treatment
 The underlying cause is treated as far as Table: Failure of response to oral iron.
possible.
 Iron is given to correct the anemia and
replenish iron stores.
 Oral iron therapy should be given for long
enough both to correct the anemia and to
replenish body iron stores, which usually
means for at least 6 months.
 Best oral preparation is ferrous sulphate.
Other preparations maybe ferrous
fumerate, ferrous gluconate.
 The hematological response to parenteral
iron is no faster than to adequate dosage
of oral iron, but the stores are
replenished faster.
 Parenteral iron should not be given in 1st
trimester of pregnancy.
Anemia of Chronic Disorders (ACDs)
One of the most common anemias; associated with many chronic inflammatory
and malignant diseases.
With iron deficiency it accounts for about two-thirds of the world’s anemias.
The characteristic features are:
 Normochromic, normocytic or mildly hypochromic indices and red cell

morphology; MCV rarely <75fL.
 Mild and non-progressive anemia (hemoglobin rarely <90g/L) - the severity

being related to the severity of the underlying disease.
 Both serum iron and TIBC are reduced.
 The serum ferritin is normal or raised.
 Bone marrow storage (reticuloendothelial) iron is normal, but erythroblast

iron is reduced.
Causes of ACDs
Chronic inflammatory diseases & conditions
 Infections e.g. pulmonary abscess, tuberculosis, osteomyelitis,
pneumonia, bacterial endocarditis)
 Non-infectious e.g. rheumatoid arthritis, systemic lupus erythematosus,
and other connective tissue diseases, sarcoidosis, inflammatory bowel
disease.
 Congestive heart failure
 Chronic pulmonary disease
 Chronic renal disease
 Obesity
Malignant diseases
Carcinoma, lymphoma, sarcoma.
Pathogenesis of ACDs
Chronic Fig: Flow chart
inflammation, depicting the
malignancy pathogenesis of
anemia of chronic
disorders.
↑ Cytokines eg.
IL6, IL1 ↑ Cytokines eg. IL6,
IL1, TNF, direct
inhibition by IFN-γ
↑ Hepcidin
↓ Erythropoietin
↓ Release of 𝐅𝐞𝟑
from
macrophages. ANEMIA (ACDs)
Peripheral Blood Film: ACDs
Fig: Despite a reduction in the total number of red cells, the RBC indices are
normal, and the cells have the typical area of central pallor without
widespread variation in size and shape (American Society of Hematology).
Sideroblastic Anemias
 This is a refractory anemia defined by the presence of many pathological ring
sideroblasts in the bone marrow.
 These are abnormal erythroblasts containing numerous iron granules
arranged in a ring or collar around the nucleus.
 There is also usually erythroid hyperplasia with ineffective erythropoiesis.
 Sideroblastic anemia is diagnosed when ≥15% of marrow erythroblasts are
ring sideroblasts.
 They can be found at lower numbers in a variety of other hematological
conditions.
 Sideroblastic anemia is classified into different types and the common link is a
defect in heme synthesis.
Classifications of
Hereditary
 X chromosome-linked ALA-S* mutation
 Rarely with spinocerebellar degeneration and ataxia (usually occurs in males,
transmitted by females; also occurs rarely in females).
 Other rare autosomal types involving mitochondrial proteins or thiamine
phosphorylation.
Acquired
 Primary
Myelodysplasia (refractory anemia with ring sideroblasts)
 Secondary (reversible)
a) Drugs, e.g. anti-tuberculous eg. Isoniazid; alcohol, lead poisoning.
b) Other benign conditions e.g. hemolytic anemia, megaloblastic anemia,
rheumatoid arthritis.
*ALA-S, δ-aminolaevulinic acid synthase.
Siderocytes & Sideroblasts
Siderocytes
Red cells containing granules of non-heme iron which give a positive Prussian
blue reaction. Siderocytes are not normally found in red cells in the peripheral
blood, but small numbers are seen following splenectomy.
Sideroblasts
Sideroblasts are erythroblasts with Prussian blue positive iron granules in their
cytoplasm.
Three types of sideroblasts are defined on the basis of the number, size, and
distribution of the siderotic granules:
 The first type is seen in normal bone marrow.
 The second type is abnormal and may be found in conditions with
dyserythropoiesis or hemolysis.
 The third type found where synthesis is disturbed, e.g. the primary and
secondary sideroblastic anemias.
Laboratory Findings
MCV/MCH  Usually low in congenital types

 Raised in acquired types.
Peripheral blood film  Markedly hypochromic, microcytic in
congenital type.
 Dimorphic in acquired.
Serum iron Raised
TIBC Normal
Serum ferritin Raised
Bone marrow iron stores Present
Erythroblast iron Ring forms
Hb electrophoresis Normal.
PBF & Bone Marrow in
Fig: Pappenheimer bodies in peripheral blood Fig: Ring sideroblasts with a perinuclear ring
following splenectomy. of iron granules in sideroblastic anemia.
Genetic Disorders of Hemoglobin
Synthesis
Genetic disorders of hemoglobins may be classified into two broad groups:
 The hemoglobinopathies are defective Hb due to abnormal globin moiety
formation.
The common abnormal hemoglobins are
Hemoglobin Structural formula
Hb-S α β
→
Hb-C α β
→
Hb-E α β
→
Hb-D Punjab α β
→
glu, glutamic acid; val, valine; lys, lysine; gln,

glutamine.
The thalassemias are normal hemoglobins with absent or decreased synthesis of

one or more types of globin polypeptide chains.
Geographical Distribution
Fig: The geographical distribution of the thalassemias, and the

more common, inherited, structural hemoglobin abnormalities.
Normal Hemoglobins In Humans
 Hb is a conjugated protein of molecular weight of 64,000 consisting of two polypeptide
chains.
 Each polypeptide chains is attached to a heme moiety.
 Human Hb exists in a number of types depending on the globin moiety.
 The heme, however, is identical in all types.
Table: Normal variants of human hemoglobins.

Stages Hb Structural formula Percentage
Adult Hb A 𝛂𝟐 𝛃𝟐 ~97%
Hb A 𝛂 𝟐 𝛅𝟐 1.5-3.2
Fetal Hb F 𝛂𝟐 𝛌𝟐 70-90 (at term)

25% (at 1st month)
5% (at 6 months)
Hb Bart’s 𝛌𝟒 ---
Embryonic Gower-1 𝛇𝟐 𝛆𝟐 ---
Gower-2 𝛂𝟐 𝛆𝟐 ---
Portland 𝛇𝟐 𝛌𝟐 ---
Molecular Aspects of
Hemoglobin Synthesis
Fig: The globin gene clusters on
chromosomes 16p and 11q. In
embryonic, fetal and adult life,
different genes are activated or
suppressed. The different globin
chains are synthesized
independently and then combine
with each other to produce the
different hemoglobins.
(b) Synthesis of individual globin
chains in prenatal and postnatal
life.
The α-globin cluster contains ζ and α genes on

chromosome 16p. Whereas, the β-globin cluster
contains ε, λ, δ, and β genes on chromosome 11q.
The Thalassemias
The thalassemias are a heterogeneous group of genetic disorders that result
from a reduced rate of synthesis of α- or β-globin chains.
 β-Thalassemia is more common in the Mediterranean region,
 While α-thalassemia is more common in South and South-East Asia.
Clinically there are three main syndromes of thalassemias.
a) Transfusion dependent thalassemia major,
b) Non-transfusion-dependent thalassemia (thalassemia intermedia) with

a moderate degree of anemia due to a variety of genetic defects eg. δβ-
thalassemia, Hb-H disease, Hb E/β thalassemia.
c) Thalassemia minor, usually due to a carrier state for α- or β-

thalassemia and characterized by erythrocyte microcytosis and mild or
no anemia.
α-Thalassemias
 These are caused by α-globin gene deletions or less frequently mutations.
 The clinical severity is related to the number of the four α-globin genes missing or
inactive.
 Deficiency of α chains leads to an excess of λ chains in fetus, and forms the tetramar Hb
Bart’s (𝛌𝟒 ).
 In adults, the unstable β chains precipitate and form Hb H (𝛃𝟒 ).
Table: Classification of the α-thalassemia syndromes.

Clinical Genetic Genotype Number of genes
α-thalassemia 2 Heterozygous α αα/-α 3
α-thalassemia 1 Homozygous α -α/-α 2
Heterozygous α αα/-- 2
Hb H disease Double heterozygote -α/-- 1
Hb Bart’s Homozygous α --/-- 0
(hydrops fetalis)
β-Thalassemias
 The genetic mutations of β-thalassemias leads to a decrease rate of β chain synthesis.
 Ultimately, causes a reduction in normal Hb A amount in red cells.
Two main types are recognized.
 𝛃 thalassemia is characterized by incomplete suppression
 𝛃𝟎 thalassemia by complete absence of chain synthesis.
Table: The β-thalassemias and related syndromes: electrophoretic phenotypes
Hemoglobin (%)
Disorders
A 𝐀𝟐 F
β-thalassemia minor 90-95 3.5-7.0 1-5
δβ-thalassemia minor 80-95 1.0-3.5 5-20
β-thalassemia major
β-thal+ 10-90 1.5-4.0 10-90
β-thal0 0 1.0-2.0 98
HPFH* 60-85 1.0-2.0 15-35
*HPFH, hereditary persistence of fetal hemoglobin
α-:β-Globin Chain Synthesis Ratio
Fig: The ratio of α-:β-globin chain synthesis (y axis) depending on

the number of functioning α and β chain genes (x axis).
Thalassemias: Clinical Features
 Severe anemia becomes  Infections occur frequently.
apparent at 3–6 months after
a) Due to iron overload: Klebsiella
birth.
or Yersinia, and fungal infection.
 Failure to thrive, pallor, and a b) Following splenectomy:
swollen abdomen within the first Pneumococcal, Haemophilus,
year. and meningococcal infections.
 Enlargement of the liver and  Hepatitis
spleen. Transfusional hepatitis B, C, and HIV
 Expansion of bones caused by infections; and hemosiderosis.
intense marrow hyperplasia leads  Hepatocellular carcinoma from
to a thalassemic facies. hepatic fibrosis/cirrhosis
a) Due to iron overload
 Thalassemia major is the disease
that most frequently underlies b) Due to chronic hepatitis B or C.
transfusional siderosis.  Osteoporosis
 Endocrinopathies
 Congestive cardiac failure from  Cardiomyopathy and
plasma overload.
arrhythmias.
Hydrops Fetalis
Fig: α-Thalassemia: hydrops fetalis, the result of the deletion of all four α-globin
genes (homozygous 𝛂𝟎 thalassemia). The main hemoglobin present is Hb Barts
( 𝛌𝟒 ). The condition is incompatible with life beyond the fetal stage.
Clinical Features
Fig: Thalassemic facies The skull is Fig: Skull X-ray in β-thalassemia major without
bossed with prominent frontal and chronic transfusion support. There is a ‘hair-on-
parietal bones; the maxilla is also end’ appearance as a result of expansion of the
enlarged. Results from chronic anemia bone marrow into cortical bone.
and compensatory hyperplasia of the
marrow. This can be prevented by
transfusion.
Thalassemias:
Laboratory Diagnosis
MCV/MCH Reduced, very low for the degree of anemia.
Peripheral blood film Marked hypochromic, microcytic cells
with significant poikilocytosis, target cells, and fragmented
cells (hemolysis), polychromatic cells; nucleated red cells
are also seen in post-splenectomy state; basophilic
stippling.
Reticulocyte count Usually increased; >5%
Serum iron Normal
TIBC Normal
Serum ferritin Normal
Bone marrow iron stores Present
Bone marrow morphology Hyperplastic, micronormoblastic; in relation to the degree of
anemia.
Erythroblast iron Present
Hb electrophoresis Hb A raised in β forms.
α-Thalassemias:
Peripheral Blood Films
Fig: (a) α-Thalassemia: Hb H disease. The blood film shows marked hypochromic, microcytic
cells with target cells, and poikilocytosis. (b) α-Thalassemia: Hb H disease. Supravital
staining with brilliant cresyl blue or methylene blue reveals multiple fine, deeply stained
deposits (‘golf ball’ cells) caused by precipitation of aggregates of β-globin chains.
β-Thalassemias:
Peripheral Blood Films
Fig: β-Thalassemia major: Peripheral blood Fig: β-Thalassemia major: Peripheral blood film
film showing prominent hypochromic after splenectomy in which hypochromic cells,
microcytic cells, target cells, and an target cells, and erythroblasts are prominent.
erythroblast. Some normochromic cells are Pappenheimer and Howell–Jolly bodies are
present from a previous blood transfusion. also seen, and the platelet count is raised.
HPLC &
Hemoglobin Electrophoresis
Fig: (a) High-performance
liquid chromatography (HPLC).
The different hemoglobins elute
at different times from the
column and their
concentrations are read
automatically. In this example,
the patient is a carrier of sickle
cell disease. (b) Hemoglobin
electrophoretic patterns in
normal adult human blood and
in subjects with sickle cell (Hb S)
trait or disease, β-thalassaemia
trait, β-thalassaemia major, Hb
S/β-thalassemia or Hb S/Hb C
disease and Hb H disease.
Thalassemias:
Treatment Modalities
 Regular blood transfusions is required usually 2-3 units every 3-4 weeks.
 Iron chelation is essential. The available drugs are deferiprone,
deferoxamine, and deferasirox that have improved quality of life.
 Regular folic acid supplementation should be provided.
 Where possible, splenectomy should be deferred until over 6 years of age.
Lifelong oral penicillin is required.
 Endocrine therapy.
 Immunization against hepatitis B, C, and before splenectomy.
 Allogeneic stem cell transplantation offers the prospect of permanent
cure.
 Trials of gene therapy for treating the thalassemia major phenotype are in
progress.
β-Thalassaemia Trait (Minor)
This is a common, usually symptomless abnormality characterized like
α-thalassemia trait by
 a hypochromic, microcytic blood picture
 MCV and MCH low but high red cell count (>5.5 × 1012/L) and
 mild anemia (hemoglobin 100–120 g/L)
 usually more severe than α-thalassemia trait
 a raised level of Hb (>3.5%) confirms the diagnosis.
The diagnosis of β-thalassaemia trait allows the possibility of prenatal

counselling, which is legally mandated in some areas of the world with a
high incidence of thalassemia major, including Bangladesh.
If the partner also has β-thalassemia trait, there is a 25% risk of a child
with thalassemia major.
Non Transfusion Dependent Thalassemia
(Thalassemia Intermedia): A Clinical
Syndrome
 Thalassemia intermedia is a clinical syndrome caused by a variety of
genetic defects requiring intermittent transfusions.
 This is thalassemia of moderate severity (hemoglobin 70–100g/L)
without the need for regular transfusions.
 Although transfusion may occasionally be required, for example
during an acute illness with temporary suppression of erythropoiesis.
Few examples are
 Hb H disease
 Hb E/β Thalassemia
 β-Thalassemia with coinherited hereditary persistence of fetal
hemoglobin (HPFH)
 δβ-thalassaemia
Special Tests & Upcoming Modalities:
α-Thalassemias
 Genetic testing is currently available to establish the diagnosis and clarify the
genetic abnormalities in patients with a family history or laboratory results
suggestive of an α-thalassemia syndrome.
 Polymerase chain reaction (PCR) and restriction endonuclease testing may
be used.
 Recombinant DNA technology can be diagnostic, but is still considered
research.
 Other genetic tests include gene mapping and anti-L globin monoclonal
anti-bodies.
 Genetic techniques can be helpful in some cases in which both the patient’s
and the parents’ α-chain configurations are elucidated exactly, which can be
useful in predicting the risk that a couple’s future offspring will be affected.
Special Tests & Upcoming Modalities:
β-Thalassemias
 Prenatal diagnosis is possible through analysis of DNA obtained via chorionic
villi sampling at 8-10 weeks’ fetal gestation or by amniocentesis at 14-20
weeks’ gestation.
 In most laboratories, the DNA is amplified using PCR and then is analyzed for
the presence of the thalassemia mutation using a panel of oligonucleotide
probes corresponding to known thalassemia mutations.
 Since the genetic defects are quite variable, family genotyping usually must
be completed for diagnostic linkage (segregation) analysis.
 With the anticipated availability of large-scale mutation screening by DNA chip
technology, extensive pedigree analyses may be obviated.
 Physicians can perform fetal blood sampling for Hb chain synthesis at 18-22
weeks’ gestation, but this procedure is not as reliable as DNA analysis
sampling methods.
 Genetic therapy strategies are currently in the early stages of development.
Laboratory Diagnosis of
Microcytic Hypochromic Anemias: In
Essence
Criteria IDA ACDs Thalassemias Sideroblastic IRIDA*
anemias
MCV/MCH ↓ N or Very ↓ ↓ or ↑ ↓
mild ↓
PBF MH NN or mild MH, poiks basophilic Dimorphic MH

MH stippling
S. iron ↓ ↓ N ↑ ↓
TIBC ↑ ↓ N N ↓
S. Ferritin ↓ ↑ N ↑ ↑
Bone marrow iron Absent Present Present Present Present
Erythroblast iron Absent Absent Present Ring forms Absent
Hb Normal Normal Hb A raised in β Normal ---

electrophoresis forms
*IRIDA: iron refractory IDA due to matriptase 2, DMT-1 mutations.

Algorithm to Diagnose A Case of
Microcytic Hypochromic Anemia
Microcytic hypochromic
RBCs, ↓ MCV, ↓ MCH
Decreased Hemoglobin Mildly decreased/near normal
Reticulocyte count Increased Normal RDW;

↑ RBC count
Smear shows abnormal Consider

Decreased
RBC morphology/nRBC Thalassemia trait
Consider homozygous
Serum iron studies Thal/ Hb-pathies Hb electrophoresis/HPLC
confirmation
↓ S. Fe, ↑ S. Fe,
↓ S. Fe, ↑TIBC,
normal/↓ TIBC, normal TIBC,
↓ Ferritin
normal/↑Ferritin ↑ Ferritin
Consider ACD: Consider Sideroblastic

IDA
review. anemia: BM w/ iron stain

Microcytic Hypochromic Anemia &amp; Thalassemia

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Department of Laboratory Medicine

Bangabandhu Sheikh Mujib Medical University

Fig: The structure of the red

The mitochondria are the main

Fig: The oxygenated and deoxygenated

Fig: The hemoglobin O dissociation curve.

 Essential for the regulation of erythropoiesis.

 90% is produced in the peritubular interstitial cells of

 Erythropoietin stimulates erythropoiesis by increasing

*Lower limit 1.5 × 109/L in some ethnic groups, e.g. in

MCHC = Hb conc. (gm/dL)/PCV

 A subnormal MCHC is indicative of defective Hb synthesis as in thalassemia or iron

 Elevated values indicate cellular dehydration eg. spherocytosis.

Adult MCHC: 31-35 gm/dL.

 RDW-CV (percent of coefficient of variation) has been found to be more useful in

Adult RDW-CV: 12.8±1.2 percent.

 Normocytic, normochromic  Impaired red cell production

*More likely in the elderly patients.

(a) (b) (c)

 Iron deficiency anemia

 It is particularly frequent in low-income populations, such as in sub-Saharan

 Poor food quality and parasites (e.g. hookworm or schistosomiasis), which

 Moreover, the body has limited ability to absorb iron.

 It is always secondary to underlying disorder.

 Occurs at any age but especially common in women of childbearing age.

Figure shows the regulation

Complex fuses 𝐅𝐞𝟑 is converted

𝐅𝐞𝟐 -DMT-1 → 𝐅𝐞𝟐 to Divalent

 It is the major hormonal regulator of iron homeostasis.

 It inhibits iron release from macrophages, intestinal epithelial cells, and

 It accelerates degradation of ferroportin.

 Raised hepcidin levels therefore profoundly affect iron metabolism by

High transferrin saturation (iron Erythroblasts → erythroferrone

Inflammation → IL6 Hypoxia

Matriptase (iron deficiency)

Low transferrin saturation

Table: Estimated daily iron requirements. Units are mg/day.

 A painless glossitis, angular stomatitis, brittle, ridged or spoon nails (koilonychia),

 Retina: hemorrhages and papilledema in severe cases.

 A syndrome of dysphagia and glossitis as a result of pharyngeal webs (Paterson-Kelly

 Neonatal iron deficiency is associated with cognitive and behavioral abnormalities.

The diagnosis of iron deficiency anemia can be

 Lab investigations relating to the blood cells and

 Investigation of the cause of iron deficiency.

MCV/MCH Reduced in relation to severity of anemia.

Peripheral blood film Anisocytosis, poikilocytosis: hypochromic,

Serum iron Reduced

Serum ferritin Reduced

Bone marrow iron stores Absent

Erythroblast iron Absent

The characteristic features are:

 Normochromic, normocytic or mildly hypochromic indices and red cell

 Mild and non-progressive anemia (hemoglobin rarely <90g/L) - the severity

 Both serum iron and TIBC are reduced.

 The serum ferritin is normal or raised.

 Bone marrow storage (reticuloendothelial) iron is normal, but erythroblast

*ALA-S, δ-aminolaevulinic acid synthase.

MCV/MCH  Usually low in congenital types

glu, glutamic acid; val, valine; lys, lysine; gln,

The thalassemias are normal hemoglobins with absent or decreased synthesis of

Fig: The geographical distribution of the thalassemias, and the

Table: Normal variants of human hemoglobins.

Fetal Hb F 𝛂𝟐 𝛌𝟐 70-90 (at term)

Microcytic Hypochromic Anemia & Thalassemia

Microcytic Hypochromic Anemia & Thalassemia