THE THALASSEMIAS

A group of disorders, each of which results from an inherited

abnormality of globin chains production.
Hemoglobin electrophoresis in normal adults:

HbA = 22 = 97%
HbA2 = 22 = 2%
HbF = 22 = 1%

At birth HbF is more then 90% and gradually, in the first year of
life, is replaced with Hb A (adult-type);

Globin chains:

are encoded by two genes on chromosome 16

, , are encoded by genes on chromosome 11

COMMON FEATURES
microcytic, hypochromic anemia - due to impaired
hemoglobin synthesis;
peripheral blood smear: anisocytosis (small cells),
target cells,
signs of hemolysis: LDH1,2, indirect-reacting
serum bilirubin;
hemolytic jaundice, splenomegaly
elevated levels of serum iron, elevated transferrin
saturation
hemoglobin electrophoresis confirms the
diagnosis

1. ALPHA () THALASSEMIA = QUANTITATIVE

IMPAIRED SYNTHESIS OF GLOBIN CHAINS
The inability to form globin chains leads to synthesis of
two abnormal types of hemoglobin:
Hb Barts = 4 and Hb H = 4.
Hb 4 and 4 are soluble and do not precipitate in the
marrow. However, they are unstable and precipitate in
older red cells. Hence, the anemia of thalassemia is
hemolytic rather than dyserythropoietic.
In addition Hb H and Barts have a higher affinity for
oxygen and it is more difficult to release the oxygen in the
tissues - in fact they are useless as oxygen carriers.
In thalassemia one to four genes may be affected (by
deletions or mutations) leading to clinical syndromes of
variable severity

a) HYDROPS FETALIS WITH HB

BART'S (HB BARTS = HB 4)
Absence of all four of the genes on chromosome 16
Clinical picture: premature, pale, and bloated infant
who, if not stillborn, has significant cardiorespiratory
distress at birth.
Death usually occurs within 1 hour of birth and results
from severe hypoxia, a consequence of the high oxygen
affinity of Hb Bart's.
The hemoglobin concentration varies from 4 to 10 g/dl.
The peripheral blood smear: anisopoikilocytosis, severe
hypochromia, and nucleated red blood cells.
Hb electrophoresis: Hb Bart's, Hb F and Hb A are
absent

b) HEMOGLOBIN H DISEASE (HB H =

4)
Deletion of three of the four -globin genes
clinical picture is that of thalassemia intermedia: affected
infants appear well at birth but develop anemia and
splenomegaly by 1 year of age; thereafter, jaundice and
hepatosplenomegaly are prominent.
Approximately one third of these patients have skeletal
changes associated with an expanded erythron.
Transfusion therapy is unnecessary, except during
intercurrent illness.
Moderate microcytic hypochromic anemia: Hb= 7 to 10 g/dl
Reticulocytes: 5 and 10%.
Hb electrophoresis : at birth Hb Barts =20-40%, replaced
gradually during the first months by Hb H = 5-40%

c) ALPHA () THALASSEMIA MINOR

Deletion of two of the four -globin genes

It is asymptomatic and usually is detected during
routine hematologic examination
Mild hypochromic, microcytic anemia Hb = 10 to 11 g/dl
Hb electrophoresis: in the newborn period detection of
Hb Bart's, Hb H usually undetectable in adults

d) SILENT CARRIER STATE

Deletion of a single globin gene
No clinical or laboratory tests changes:
In very rare cases at birth Hb Barts 1-2%
Gene mapping is the only reliable means for precise
identification

2. BETA () THALASSEMIA = QUANTITATIVE

IMPAIRED SYNTHESIS OF GLOBIN CHAINS

The pregnancy is normal and the newborns are well at

birth (the synthesis of HbF - 22 is normal).
Anemia usually develops during the first few months of
life due to total or partial impaired synthesis of chains.
Hemoglobin electrophoresis confirms the diagnosis
showing decreased level of Hb A and increased level of
Hb A2 and Hb F.
Hb 4 is very unstable and precipitate in the marrow .
Red cell precursors that contain Hb 4 are recognized
by the local macrophage and are phagocytized resulting
in ineffective erythropoiesis.
Therefore Hb electrophoresis will not reveal Hb 4.

a) THALASSEMIA MAJOR = ANEMIA COOLEY

Clinical picture develops gradually after the first few

months
increased hematopoiesis leads to bone marrow
expansion, impaired growth & thinning of cortical:
frontal bossing, long bone fractures,
hepatosplenomegaly, jaundice
secondary hemosiderosis: liver cirrhosis
(hepatosplenomegaly), myocarditis, arrhythmia,
congestive heart failure, growth retardation,
hypogonadotropic hypogonadism

HAIR-ON-END APPEARANCE OF SKULL PRODUCED BY

TRABECULAR STRIATIONS RADIATING OUTWARD FROM THE
INNER TABLE

LABORATORY TESTS
Severe hypochromic, microcytic anemia (Hb = 2-6
g/dl, MCV, MCHC)
reticulocytosis = 5-15% - lower than expected
peripheral blood smear: anisocytosis (small cells),
target cells
Hb electrophoresis:

Hb

A absent or
Hb F= 40-90%
Hb A = 24-10%

b) THALASSEMIA INTERMEDIA
Signs and symptoms of thalassemia intermedia are
comparable to those of thalassemia major but are of
lesser magnitude
Hb = 6-9 g/dl, MCV, MCHC (hypochromic /
microcytic)
reticulocytosis = 3-10%
peripheral blood smear: anisocytosis (small cells),
target cells,
Hb electrophoresis:
Hb

A=
HbF = 10%
HbA2 = 3-7%

c)

THALASSEMIA MINOR

Usually

is asymptomatic; detected during routine hematologic

examination
Hb = 10-12 g/dl, MCV, MCHC (hypochromic / microcytic)
Reticulocytosis = 2-4%
Peripheral blood smear: anisocytosis (small cells), target
cells
Hb electrophoresis: HbA , HbF ,HbA2

thalassemia minima also called the "silent

thalassemia" - virtually no clinical or laboratory changes

EXTRACORPUSCULAR HEMOLYTIC
ANEMIAS

1.
2.
3.
4.
5.
6.

Autoimmune hemolytic anemias

Drug-related immune hemolytic anemias
Alloimmune hemolytic disease of the newborn
Microangiopathic and macroangiopathic
hemolytic anemias
Hemolytic anemia due to infections with
microorganisms malaria
Hypersplenism

1. AUTOIMMUNE HEMOLYTIC
ANEMIAS (AHA)
Autoimmune hemolytic anemias (AHA) occur when a
patient produces pathologic autoantibodies that
attach to and destroy red blood cells, causing anemia.
AHA are classified according to the characteristic
temperature activity of the antibodies:

Warm-active

antibodies have their greatest affinity at

37C. (80% of all AHA)
Cold-active antibodies display increasing affinity for the
RBCs as the temperature approaches 0C. (20%)

a) AUTOIMMUNE HEMOLYTIC ANEMIAS

CAUSED BY WARM-ACTIVE ANTIBODIES
Warm-active antibodies are typically of the IgG variety,
they have the highest affinity at 37C (the major share of
autoantibodies is commonly bound to the patient's
circulating erythrocytes at this temperature)
50% of warm-antibody AHA have autoantibodies specific
for epitopes on Rh proteins. The autoantibodies of such
patients do not react with Rh negative RBC.
Autoantibodies with such specificity have been designated
as Rh related.
50% of warm-antibody AHA have IgG autoantibodies that
are fully reactive with Rh null RBC. The exact specificity of
the autoantibodies for many of these patients is undefined.

CLASSIFICATION
Primary or idiopathic warm-antibody AHA - no
recognizable underlying disease is present.
Secondary warm-antibody AHA - as a
manifestation or complication of an underlying
disorder
Lymphocytic malignancies, particularly chronic
lymphocytic leukemia (CLL) and lymphomas
Systemic lupus erythematosus (SLE)

PATHOGENESIS
Erythrocyte autoantibodies in warm-antibody AHA are pathogenic.
In warm-antibody AHA, the patient's RBC typically are coated with IgG
autoantibodies with or without complement proteins.
Autoantibody-coated RBC are trapped by macrophages in the spleen and, to a
lesser extent, by Kupffer cells in the liver.
The macrophage has surface receptors for the Fc region of IgG and surface
receptors for opsonic fragments of C3 (C3b) and C4 (C4b).
When present together on the RBC surface, IgG and C3b appear to act
cooperatively as opsonins to enhance trapping and phagocytosis.
Interaction of a trapped RBC with splenic macrophages may result in
phagocytosis of the entire cell or a type of partial phagocytosis that results in the
formation of spherocytes (the noningested portion of the RBC assumes a
spherical shape, the shape with the lowest ratio of surface area to volume).
Spherical RBC are more rigid and less deformable than normal RBC and are
fragmented further and/or destroyed in future passages through the spleen.
Spherocytosis is a consistent and diagnostically important hallmark of AHA,
and the degree of spherocytosis correlates well with the severity of hemolysis.
Direct complement-mediated hemolysis with hemoglobinuria is unusual in warmantibody AHA, despite the fact that many warm autoantibodies fix complement.

CLINICAL FEATURES

Symptoms are usually slow and insidious in onset over

several months:
symptoms

of anemia (tachycardia, pallor and adinamia)

jaundice and modest splenomegaly

Occasionally in very severe cases, particularly those of

acute onset, patients may present with:
fever,

pallor,
jaundice, hepatosplenomegaly,
hyperpnea, tachycardia, angina or heart failure

LABORATORY TESTS
severe to mild normochromic normocytic (MCV, MHCH
normal) anemia (Hb = 5-11 g/dl)
reticulocyte count is elevated
blood smear: spherocytes (if hereditary spherocytosis is
excluded, this finding suggests an immune hemolytic
process) and schizocytes (red blood cell fragments).
Total bilirubin is increased, up to 5 mg/dl, (>85%
unconjugated bilirubin)
Urinary urobilinogen is increased regularly, Serum
haptoglobin levels are low and LDH levels are elevated.
positive direct Coombs' test
positive indirect Coombs test

DIRECT COOMBS TEST

(DIRECT ANTIGLOBULIN TEST DAT)

detects autoantibody or complement bound to the patient's RBC

INDIRECT COOMBS TEST

(INDIRECT ANTIGLOBULIN TEST IAT)

detects free autoantibody in the plasma of these patients

b) AUTOIMMUNE HEMOLYTIC ANEMIA

CAUSED BY COLD-ACTIVE ANTIBODIES

AHA caused by cold active autoantibodies are chronic

autoimmune hemolytic anemia in which the autoantibody
directly agglutinates RBCs at temperatures below body
temperature, maximally at 0 to 5C.

Fixation of complement to RBCs by cold agglutinins

occurs at higher temperatures, but below 37C.
Cold-active antibodies (cold agglutinins) are of the IgM
type, fix complement, and may lead to immediate
intravascular destruction of erythrocytes or their
removal from the circulation by the liver.
Most cold agglutinins have specificity for
oligosaccharide antigens (I or i) of the RBC.

CLASSIFICATION

Primary or idiopathic cold agglutinin mediated

AHA - no recognizable underlying disease is
present.
Secondary cold agglutinin mediated AHA - as a
manifestation of an underlying disorder
Mycoplasma pneumoniae infections or infectious
mononucleosis - EBV
malignant lymphoproliferative diseases.

PATHOGENESIS (I)
Most cold agglutinins are unable to agglutinate RBC at
temperatures above 30C .
The highest temperature at which these antibodies cause
detectable agglutination is termed the thermal amplitude. This
value may vary considerably from one patient to another.
Patients with cold agglutinins of higher thermal amplitudes have a
greater risk for cold-agglutinin disease.
The cold agglutinins bind host RBC and activate complement
(process called complement fixation)
Complement fixation by these antibodies occur optimally at 20 to
25C and even higher temperatures.
Cold agglutinins bind to RBC in superficial vessels of the
extremities, where the temperature ranges between 28 and 31C
and cause RBC to aggregate, thereby impeding RBC flow and
producing acrocyanosis

PATHOGENESIS (II)
In addition, the RBC-bound cold agglutinin may activate complement
via the classical pathway.
Once activated complement proteins are deposited onto the RBC
surface, it is no longer necessary for the cold agglutinin to remain
bound to the RBC for hemolysis to occur.
Complement fixation may effect RBC injury by two major mechanisms:
(1) direct lysis (requires propagation of the full C1-to-C9 sequence on
the RBC membrane) => intravascular hemolysis leading to
hemoglobinemia and hemoglobinuria
(2) opsonization (C3b,C4b)for hepatic and splenic macrophages =>
extravascular hemolysis
A RBC heavily coated with C3b may be removed from the circulation
by macrophages either in the liver or, to a lesser extent, the spleen.
The trapped RBC may be ingested entirely or released back into the
circulation as a spherocyte after losing some of its plasma
membrane.

CLINICAL FEATURES

Most patients with cold-agglutinin

hemolytic anemia have chronic hemolytic
anemia with or without jaundice.
In others, the principal feature is
episodic, acute hemolysis with
hemoglobinuria induced by chilling.
Combinations of these clinical features
may occur.
Acrocyanosis affecting the fingers, toes,
nose, and ears are associated with
sludging (agglutination) of RBC in the
cutaneous microvasculature.

LABORATORY TESTS
mild to moderate normochromic normocytic anemia (Hb =8-11 g/dl).
reticulocyte count is elevated
blood smear: spherocytosis (less marked than in typical cases of warmantibody autoimmune hemolytic anemia).

RBC

auto agglutination may be noted at room temperature and intensified by

cooling the blood to 4C

Mild hyperbilirubinemia (>85% unconjugated bilirubin)

Serum haptoglobin levels are low and LDH levels are elevated.
Hemoglobinemia, hemoglobinuria appear in acute hemolytic episodes
positive direct Coombs' test for complement,
negative direct Coombs test for autoantibodies
negative indirect Coombs test (Coombs tests are performed at 37C)
Cold agglutinins are distinguished by their ability to agglutinate
saline-suspended human RBC at low temperature, maximally at
0 to 5C (32 to 41F). This reaction is reversible by warming.

1,2

2. DRUG-RELATED IMMUNE
HEMOLYTIC ANEMIA

a)
b)
c)

Hapten or drug adsorption mechanism - penicillin very

high doses, cephalosporin, tetracycline
Ternary complex mechanism (drug- antibody-target cell
complex)- quinine, quinidine, sulfonamides, rifampicin
Autoantibody induction. alpha methyldopa, levodopa

a) HAPTEN OR DRUG ADSORPTION MECHANISM

PENICILLIN VERY HIGH DOSES,
CEPHALOSPORIN, TETRACYCLINE

Induction of antidrug antibody is thought to require

firm chemical coupling of the drug (as a hapten) to a
protein carrier.
The drug binds firmly to red cell membrane proteins in
vivo.
Antidrug antibody (usually IgG) binds to the proteinbound drug.
Red cells coated with penicillin and IgG antipenicillin
antibody are destroyed by splenic macrophages.

b) TERNARY COMPLEX MECHANISM

(DRUG-ANTIBODY-TARGET CELL COMPLEX)
QUININE, QUINIDINE, SULFONAMIDES, RIFAMPICIN
Drug binds loosely or in undetectable amounts to red cell
membrane. However, in the presence of appropriate antidrug
antibody, a stable trimolecular (ternary) complex is formed among
drug, red cell membrane protein, and antibody. In general, the
antibody-combining site (Fab) recognizes both drug and
membrane protein components but binds only weakly to either
drug or protein unless both are present in the reaction mixture
Drugs can induce immune injury not only of red cells but also of
platelets or granulocytes
The process differs in several ways from the mechanism of
hapten/drug adsorption.

Drugs

in this group exhibit only weak direct binding to blood cell

membranes.
A relatively small dose of drug is capable of triggering destruction of
blood cells.
Cellular injury appears to be mediated chiefly by complement activation
at the cell surface

c) AUTOANTIBODY INDUCTION
ALPHAMETHYLDOPA, LEVODOPA
The mechanism by which a drug can induce formation
of an autoantibody is unknown
Drug-induced antibodies can bind avidly to red cell
membrane proteins (usually Rh proteins) in the
absence of the inducing drug and are indistinguishable
from the antired cell autoantibodies of patients with
autoimmune hemolytic anemia

3. ALLOIMMUNE HEMOLYTIC DISEASE OF

THE NEWBORN
Rh hemolytic disease of the newborn
Rh hemolytic disease of the newborn occurs as a result of immunization
(during transplacental passage of fetal red cells) of the Rh negative
mother to red cell of the Rh positive fetus.
This sensitization results in the transplacental passage of maternal IgG
antibodies anti antigen D that bind to the fetal red cells, causing
hemolysis (extravascular noncomplement-mediated phagocytosis and
lysis), and as a consequence of the hemolytic process, anemia,
extramedullary hematopoiesis, and neonatal hyperbilirubinemia,
sometimes with devastating morbidity for the fetus and newborn infant.

ABO

hemolytic disease of the newborn is limited to mothers who are blood group
type O and whose babies are group A or B. Although ABO incompatibility exists
in 15 percent of O group pregnancies, ABO hemolytic disease is estimated to
occur only in about 3 percent of all births. Most anti-A and anti-B antibodies are
of the IgM type and do not cross the placenta. A small number of group O women
produce anti-A and anti-B antibodies of the IgG type that can cross the placenta.

4. MICROANGIOPATHIC AND
MACROANGIOPATHIC HEMOLYTIC ANEMIA

a)

MICROANGIOPATHIC HEMOLYTIC ANEMIA

b)

MARCH HEMOGLOBINURIA

c)

TRAUMATIC CARDIAC HEMOLYTIC ANEMIA

a) MICROANGIOPATHIC HEMOLYTIC ANEMIA

Clinical disorders characterized by the fragmentation of red
cells as they pass through the platelet-fibrin mesh present in
microthrombi which are deposited in capillaries and
arterioles.
The formation of arteriolar microthrombi can be caused by a
variety of mechanisms:

activation

of the coagulation system as occurs in disseminated

intravascular coagulation (DIC),
antineoplastic and immunosuppressive agents as well as radiation
therapy and bacterial toxins may induce endothelial cell injury
leading to the formation of microthrombi in the affected vessels.
formation of platelet aggregates induced by the release of very large
von Willebrand factor multimers as in thrombotic thrombocytopenic
purpura (TTP).

b) MARCH HEMOGLOBINURIA

In marchers and runners, traumatic hemolysis (red

cells were destroyed in the soles of the feet during
running) may cause hemoglobinuria and anemia,
Similar traumatic red cell destruction with
hemoglobinuria has been reported after beating the
head against a wall, hand-strengthening exercises in
a practitioner of karate and playing the conga
drums

c) TRAUMATIC CARDIAC HEMOLYTIC ANEMIA

The abrasive effect on red cells of arteriosclerotic or
stenotic cardiac valves is usually minimal, resulting at
most in a mild, often compensated hemolytic anemia.
However, the introduction of artificial valves was
initially associated with marked red cell destruction and
the development of an overt hemolytic anemia.
Recently, the design of the artificial valves and the use of
more compatible plastics or biologic materials have greatly
reduced their traumatic effects and minimized hemolysis.
Actually, the potential thrombogenic effect of artificial
valves far outstrips their destructive effect on red cells,
and cardiac hemolytic anemia is now an almost
nonexistent problem.

5. HEMOLYTIC ANEMIAS RESULTING

FROM DIRECT EFFECTS OF
INFECTIOUS AGENTS
Malaria - febrile disease caused by four species of
Plasmodia: vivax, falciparum, malariae and ovale
which are capable of parasitizing erythrocytes.
P. falciparum invades erythrocytes of all ages =>50%
of RBC may be affected.
Within the erythrocyte, the malarial organisms divide
asexually, the cell ruptures, and the merozoites are
released to invade other cells.

6. HYPERSPLENISM
Hypersplenism occurs when the size of the spleen is
increased by tissue components or by vascular
engorgement.
This augments its filtering function, and even normal
blood cells experience a delayed transit and
temporary sequestration.
The trapped red cells are usually destroyed causing a
hemolytic anemia.
Splenectomy is called for if the hypersplenic
cytopenias are severe enough to demand intervention
or if the enlarged spleen causes pain and discomfort.

Test

Patient value

Normal values

Units

Hb

8,5

11 - 16

g/dl

Ht

25.7%

36-46 %

Red Blood Cells

2.1410

410 - 5.510/mmc

Reticulocytes

0.5%

0.5 1.5%(!)

MCV

120

80 - 100

mc(fl)

MCHC

33

32 - 36

g/dl E

White Blood Cells

3.200

4.000 10.000/mmc

Platelets

110.000

150.000-400.000/mmc

LDH

220

100 - 180

U/L

Total Bilirubin

1.7

0.3 1

mg/dl

Direct Bilirubin

0.4

0 0.2

mg/dl

Indirect Bilirubin

1.3

0 0.8

mg/dl

Serum Iron

148

50 150

g/dl

TIBC

380

300 400

g/dl

Serum Cobalamin

120

220 850

pg/dl

Serum Folate

5.7

>5.5

ng/dl

Schilling test assays : first part 1% urinary excretion ; second part 19%
urinary excretion

Test

Patient value

Normal values

Units

Hb

11 - 16

g/dl

Ht

28.5%

36-46 %

Red Blood Cells

3.910

410 - 5.510/mmc

Reticulocytes

1%

0.5 1.5%

MCV

73

80 100

mc(fl)

MCHC

28

32 36

g/dl E

White Blood Cells

5.500

4.000 10.000/mmc

Platelets

300.000

150.000-400.000/mmc

LDH

100 180

U/L

Total Bilirubin

0.7

0.3 1

mg/dl

Direct Bilirubin

0.2

0 0.2

mg/dl

Indirect Bilirubin

0.5

0 0.8

mg/dl

Serum Iron

33

50 150

g/dl

TIBC

423

300 400

g/dl

Serum Cobalamin

220 850

pg/dl

Serum Folate

>5.5

ng/dl

Test

Patient value

Normal values

Units

Hb

11 - 16

g/dl

Ht

26%

36-46 %

Red Blood Cells

3.710

410 - 5.510/mmc

Reticulocytes

7%

0.5 1.5%

MCV

70

80 100

mc(fl)

MCHC

27

32 36

g/dl E

White Blood Cells

5.200

4.000 10.000/mmc

Platelets

320.000

150.000-400.000/mmc

LDH

290

100 180

U/L

Total Bilirubin

2.7

0,3 1

mg/dl

Direct Bilirubin

0.4

0 0,2

mg/dl

Indirect Bilirubin

2.3

0 0,8

mg/dl

Serum Iron

220

50 150

g/dl

TIBC

323

300 400

g/dl

Serum Cobalamin

232

220 850

pg/dl

Serum Folate

5.6

>5.5

ng/dl

Hb electrophoresis: Hb A = 86%; HbA2 = 5%; HbF = 9%

Test

Patient value

Normal values

Units

Hb

10

11 - 16

g/dl

Ht

30%

36-46 %

Red Blood Cells

3.5610

410 - 5.510/mmc

Reticulocytes

4%

0.5 1.5%

MCV

84.2

80 - 100

mc(fl)

MCHC

33

32 - 36

g/dl E

White Blood Cells

8.200

4.000 10.000/mmc

Platelets

382.000

150.000-400.000/mmc

LDH

210

100 - 180

U/L

Total Bilirubin

2.2

0.3 1

mg/dl

Direct Bilirubin

0.3

0 0.2

mg/dl

Indirect Bilirubin

1.9

0 0.8

mg/dl

Serum Iron

131

50 - 150

g/dl

TIBC

353

300 - 400

g/dl

positive direct Coombs' test for complement,

negative direct Coombs test for autoantibodies
negative indirect Coombs test