ANEMIAS OF ABNORMAL GLOBIN DEVELOPMENT

THE THALASSEMIAS
 In thalassemia:
- either may deletion or may change in globin chain (pero MORE ON GENE DELETION)
- always has Target cells (in pictures) present in thalassemia
- specifically in chromosome 16 is the problem (either may deletion nga, or change)
- they are autosomal recessive (meaning it takes two genes to be seen)

The THALESSEMIAS Target cells are the predominant cells in thalassemia What is Hgb H disease?
- it is part of thalassemia
2 types of thalassemia:
that are common
> alpha
• The thalassemias are a diverse group of genetic disorders
> beta characterized by a primary, QUANTITATIVE reduction in globin
chain synthesis for hemoglobin. The thalassemias are probably the
most common single gene disorders in the world population.
• Thalassemias are very common in the shores of the Mediterranean
and Africa through Middle East, the Indian subcontinent, Myanmar
(Burma), and Southeast Asia (including the Philippines).
• The geographic distribution follows that of the malaria parasite
Plasmodium falciparum.
• The thalassemias are usually classified according to the particular
globin chain whose synthesis is suppressed. The types are:
Ur globin chain has • α-thalassemia
-Baby/ Neonatal - 2 alpha, 2 gamma
-Adult - alpha & beta • β -thalassemia and in that 4 globin chains, has heme inside
• γ -thalassemia Hemoglobin has 4 globin chains:
- alpha
• δ-thalassemia - beta
- delta
• δβ-thalassemia - gamma
• γδβ-thalassemia Hgb F is composed of
 The THALESSEMIAS
• The genetics of thalassemia is centralized to the structures of
CHROMOSOME 11 and 16
• Follows Mendelian principles
• α and β thalassemia are the most common
• α thalassemia is caused by a GENE DELETION
• β thalassemia is caused by POINT MUTATIONS in the DNA code
How target cells occur?
- analogy: deflated balloon, pag
2 defective genes = alpha thalassemia minor tinanggal hangin don, it deflates
3 defective genes = Hgb disease (excess in beta chain; has tetramer) and creates crenation outside,
Thalassemia, characterized by: and then it scrunches and
- low MCV shrinks the cells
- low MCH
- low MCHC When that happen, Target cells
occurs
GENERAL CONSIDERATIONS IN LABORATORY TESTING FOR
THALASSEMIAS

• HEMATOLOGY
• Microcytic hypochromic anemia
• WBC and platelets are not involved - but WBC and platelets are not involved, because the problem is in the gene formation of mRNA in RBCs

• SPECIAL HEMATOLOGY TESTS

• Bone marrow: Hyperplastic red cell precursors
• Hemoglobin electrophoresis on cellulose acetate at alkaline pH – varied results
• Citrate agar electrophoresis (acid pH)
• Purposes:
• Hb F, which is OFTEN ELEVATED IN THALASSEMIA is effectively separated from HbA
• Hemoglobin variants may migrate differently in this medium, allowing them to be
identified tentatively
• The quantities of HbA2 and Hb F and the presence of Hb H inclusions in the rbc’s can be
diagnostic
• Acid elution testfor Hb F: differentiates thalassemia from Hereditary Persistence of Fetal
Hemoglobin (HPFH)
GENERAL CONSIDERATIONS IN LABORATORY TESTING FOR
THALASSEMIAS

• CHEMISTRY
• Ferritin: Most important test to differentiate Iron deficiency from thalassemia
• Transferrin saturation >60%
• Serum Iron >160ug/dL
• Note: Transferrin and serum iron are useful in transfusion dependent
thalassemias in which iron overload is important especially in thalassemia major
and intermedia.
Some important points to consider:
• Testing in infancy
• At birth, the normal red cells are macrocytic but the MCV
subsequently begins to fall
• Hb F is NOT DIAGNOSTIC of b thalassemia in infants, since it is
normally elevated in infants
• Interference of blood transfusion
• Knowledge of transfusion status is important for correct
interpretation of electrophoresis results
• Abnormal hemoglobin fraction may be masked on hemoglobin
electrophoresis as long as transfused cells are still circulating
Some important points to consider:
• Effects of concurrent iron deficiency on test results
• Concurrent iron deficiency anemia in thalassemia is known to lower
concentration of Hb A2 and Hb H as well as serum iron and ferritin
which may mask the diagnosis of heterozygous b thalassemia and Hb
H disease, respectively
• Hb H inclusions may be more difficult to detect in erythrocytes. If a
subject is iron deficient, iron stores SHOULD BE FIRST DEPLETED, then
diagnostic tests for thalassemia may be performed.
α-thalassemia
• Nomenclature
• α thalassemia-1 or 1° thalassemia - a DOUBLE gene deletion (- - )
• α thalassemia-2 – a SINGLE gene deletion (- a)
• Normally, a total of 4 a genes is inherited, two from each parent
• These 4 genes produce a genes in a quantity equal to that b genes,
resulting in b/a chain ratio of 1:1
• Most forms of a thalassemia result from a DECREASE IN THE NUMBER OF a
CHAINS, because of a DELETION in one, two, three, or four genes
• Without a chains, excess g and b chains accumulate and forms tetramers:
g4 (Hb Bart’s) and b4 (Hb H)
• Severity of the disease ranges from lethal (when all 4 a genes are deleted)
to no clinical abnormalities (when only one gene is deleted)
Types
BART’S HYDROPS FETALIS
• Caused by a deletion of all 4 a globin chains (- -/- -)
that results in the production of Hemoglobin
Bart’s (g4)
• Both parents of affected individuals have
heterozygous a° thalassemia ( - -/aa)
• Almost exclusively seen in Southeast Asians
• Disorder is lethal and infants are usually stillborn
or die within a few hours of birth
• Survival into the 3rd trimester of fetal life is
attributed to the presence of Hb Portland (z2g2)
which effectively delivers oxygen to the tissues
• Hb Bart’s has INCREASED AFFINITY to oxygen and
thus will not effectively release oxygen to tissues
• Infants who are liveborn are underweight,
edematous, demonstrate ascites, and have
distended abdomens with marked
hepatosplenomegaly.
Types
HbH DISEASE
• Deletion of 3 or 4 a globin genes (- -/- α)
• Non deletional forms exists (ααT/ααT and ααT/- -)
• Widespread in Southeast Asia, parts of the Middle East, and Mediterraneam
island populations (Greece, Italy, Cyprus)
• The disorder results from the decreased synthesis of α chains with resultant
formation of unstable Hb H (β4)
Types Composed of:

Hb H – CONSTANT SPRING DISEASE (Hb CS)

2 normal B chain
1 normal A chain
1 abnormal A chain

• Hb CS consists of 2 normal b chains, one normal a chain and one abnormal a

chain that has 172 amino acids as opposed to normal 141.
• Occurs frequently in Orientals (SE Asia)
 Types
α THALASSEMIA MINOR

• Has two genotypic forms:

• Heterozygous α° (- -/αα)
• Homozygous α+ (-α/-α)
• Both forms are commion in SE Asians, Chinese and Filipinos
• Homozygous α+ thalassemia is common in American blacks and in the
Mediterranean
• Heterozygous α° is rare in blacks
Types
THE SILENT CARRIER
• Also known as Heterozygous a+ thalassemia
• Associated with one a gene deletion (-α/αα)
• Common in SE Asians, Chinese and Filipinos
β THALASSEMIA
• An unbalanced globin chain synthesis due to the LACK OF, OR THE
REDUCED PRODUCTION OF b CHAINS that causes an excess of a
chains which are very unstable.
• Fates:
• If the imbalance is minor, unpaired a chains are simply removed by
proteolysis during erythroid maturation
• If the degradation is overwhelmed by massive imbalance, excess free a chains
precipitate, causing severe erythrocyte dysfunction
• Most occur in Europe, Middle East and North Africa, China, India, and
Central Africa
 Types
β THALASSEMIA MAJOR
• There are 4 genotypes associated with β thalassemia, three of
which use the nomenclature:
• β° (no globin chain production)
• β+ (decreased b globin chain production)
• Four genotypes of Beta Thalassemia
• β°/β° -- both
MAJOR
deletion

• β+/β+ (Mediterranean form) -- both decrease

INTERMEDIA
• β°/β+ -- one deleted, one decrease
MINOR
• δβ Lepore/δβ Lepore (referred as Hb Lepore)
Hb Lepore is - beta and
delta fuse/ nagdikit
• Hb Lepore is composed of normal a chains and two abnormal a
chains formed by fusion of N-terminal end of the d chain and the C-
terminal of the b chain
• Hb Lepore consists of 3 types:
• Hb Lepore-Baltimore
• Hb Lepore-Boston (most common)
• Hb Lepore-Hollandia
Types
β THALASSEMIA MAJOR
• Manifests with severe anemia within the 1st
year of life with a difficult clinical course:
• Bone marrow expansion resulting from
excessive ineffective erythropoiesis that
causes marked skeletal deformities with
frontal bossing, cheek and jaw protrusion (or
“Chipmunk facies”, distortion of the ribs and
vertebrae, and pathologic fractures of long
bones
• Hepatosplenomegaly
• Cardiomegaly
• Gallstones
• Chronic leg ulcers
• Hypersplenism
• Growth and sexual retardation
• Intercurrent infections
Types
β THALASSEMIA INTERMEDIA ALL HOMOZYGOUS

• Describes certain β thalassemias which are clinically milder than

thalassemia major but more severe than thalassemia minor kinda in the middle
• Appears that the genes are less severely affected and that impairment of β
chain synthesis is less than that usually seen in thalassemia major
Types:
• Homozygous β+ thalassemia (β+/β+) Mild Black Form
• Less impairment of β chain synthesis than the homozygous β+ thalassemia
• Homozygous δβ thalassemia (δβ°/δβ°)
• Caused by deletion on the δ and β structural genes on chromosome 11
• More efficient synthesis of γ chains; the γ chain bind with α chains to form Hb
F, which constitutes 100% of hemoglobins in patients)
• HbA and Hb A2 cannot be synthesized since no δ or β chains are produced
• Doubly heterozygous δβ thalassemia varieties
• Only Hb F and Hb Lepore are seen in Hgb electrophoresis
Types
β THALASSEMIA MINOR ALL HETEROZYGOUS

• An asymptomatic β thalassemia disorder in which there is

little or no associated anemia, although peripheral blood
erythrocyte morphology is significantly abnormal
• Three syndromes categorized as thalassemia minor, which are
generally indistinguishable:
• Heterozygous β° (β°/β) Thalassemia (β Thalassemia Trait or High Hb A2
Thalassemia)
• Heterozygous δβ Thalassemia ([δβ]°/β)
• Heterozygous Hb Lepore ([δβ]Lepore/β)
 Types
β THALASSEMIA MINIMA (SILENT β THALASSEMIA TRAIT)

• Describes a form of β thalassemia in which no clinical or laboratory abnormality

is usually detected
• Accidentally diagnosed in family studies
HEREDITARY PERSISTENCE OF FETAL
HEMOGLOBIN (HPFH)

• Describes a heterogenous group of inherited disorders characterized

by increased levels of Hb F in a dults in the absence of usual clinical
and hematologic features of thalassemia
• Characterized either by deletion or inactivation of the b and d
structural gene complexes
• Sometimes classified as a Thalassemia Minor
• 2 categories:
• Pancellular HPFH – all cells contain increased levels of HbF as determined by
the acid elution test
• Heterocellular HPFH – only a subpopulation of cells contain HbF
THALASSEMIAS WITH Hb VARIANTS
• β chain variants (Hb S, C, and E) can sometimes coexist with β
thalassemia
• If affected gene are different (i.e. α thalassemia + Hb S), the β chain
variant is lower than Hb A.
γδβ, γ, and δ THALASSEMIAS
• Not common

Not Common Thalassemias:

- Gamma, Delta, Beta

- Gamma
- Delta
Differentiation of Thalassemia from IDA
TEST THALASSEMIA IRON DEFICIENCY
MINOR
RBC Count (X1012/L) >5.5 <5.5
Random Distribution Normal to slightly 🡩 🡩
Width (RDW)
Ferritin Normal to slightly 🡩 🡩
Serum Iron Normal 🡩
Total Iron Binding Normal 🡩
Capacity (TIBC)
Transferrin Saturation Normal 🡩
(%)
Zinc protoporphyrin/ Normal 🡩
heme ratio (ZnPP/H)
Tests useful in the diagnosis of thalassemia

• Hemoglobin electrophoresis
• Quantitation of Hb F
• Quantitation of Hb A2 by microchromatography
• Brilliant Cresyl Blue (BCB) Test for Hb H Inclusions
• Principle: Cells containing an unstable hemoglobin, such as Hb H, normally
are removed promptly from the circulation by the RES. When relatively
young rbc’s containing sufficient Hb H to form inclusions are released in
the circulation, they are diluted in the population of cells with nearly
balanced globin chain synthesis and normal life span.
• When blood sample is centrifuged, the red cells taken just below the buffy
coat layer consists of primarily young and buoyant cells. These young cells
are the most capable of forming typical Hb H inclusions if a thalassemia is
present. These are stained by BCB and viewed microscopically.
Tests useful in the diagnosis of thalassemia

• Acid Elution Slide Test for Hb F

• Principle: All Hemoglobins with the exception of Hb F are eluted from the red
cell by citric acid phosphate buffer at acid pH (3.3-3.5). Blood films are
stained after elution with Ehrlich acid hematoxylin and counterstained by
erythrosine. Slides are examined microscopically.
• Cells containing HbF will be stained BRIGHT PINK TO RED whereas normal
adult cells appear as “ghost cells” (only the outer membranes are visible.