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Haemolytic anaemias
Ahmad Sh. Silmi
Msc Haematology, FIBMS
Haemolytic anaemias (HAs)
• HAs are defined as those anaemias which
result from an increase in the rate of red
cell destruction.
• any condition which leads to a reduction
in the mean lifespan of the red cell is a
haemolytic disorder.
2
• Because of erythropoietic hyperplasia and
anatomical extension of bone marrow, red
cell destruction may be increased several
fold before the patient become anaemic --compensated haemolytic anaemia.
• The normal adult marrow, after full
expansion, is able to produce red cells at
6-8 times the normal rate.
• Therefore HA may not be seen until the
red cell lifespan is less than 30 days.
3
Classification of HA
• The HA can be classified in several different
ways:
1- Site of haemolysis:
• Extravascular haemolytic disorders macrophages of the RES
• Intravascular haemolytic disorders- within the
circulatory system
• In many of the cases there is a combination of
both extra and intravascular haemolysis.
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Extravascular haemolysis
• Red cell destruction
usually occurs in the
cell of the RES.
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Intravascular haemolysis
• Destruction of red
cells occur inside the
blood vessels.
6
Classification of HA
2- Site of defect:
• Intrinsic defect (intracorpuscular)structural or functional defect within the
red cell.
• Extrinsic defect (extracorpuscular)- an
abnormality in the red cell environment.
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Classification of HA
3- Inherited or acquired:
• Inherited HA are usually caused by intrinsic
defect.
• While acquired HA are caused by an extrinsic
defect.
• However there are some exceptions:
Paroxysmal nocturnal haemoglobinuria (PNH)
which is an acquired intrinsic defect, and severe
hereditaryG6PD enz deficiency which requires
the presence of an extrinsic trigger such as the
antimalarial drug for the intrinsic defect to
8 manifest.
Inherited & acquired HA
Hereditary HA
• Membrane defects e.g
hereditary spherocytosis
• Metabolic defect e.g
G6PD deficiency.
• Haemoglobin defects e.g
sickle cell disease.
9
Acquired HA
– Immune
-Autoimmune eg AIHA
-Alloimmune e.g HDN,
HTR
– Red cell fragmentation
syndromes
– March
haemoglobinaemia
– Infections
– Chemical and physical
agents.
– PNH
Inherited Haemolytic Disorders
•
•
•
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Disorders of globin synthesis and / or
structure (thalassaemia and
haemoglobinopathies and these are
previously described).
Primary membrane disorders.
Enzyme disorders.
Primary membrane disorders
• Primary disorders of the red cell
membrane are associated with alteration
of cell shape. Many of these disorders are
classified according to the shape of the
abnormal cells. This scheme is by far the
most common employed, and so will be
used.
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Hereditary Spherocytosis
• Hereditary spherocytosis (HS), also known as congenital
haemolytic anaemia and congenital haemolytic jaundice,
is the most common of the inherited primary red cell
membrane abnormalities.
• Incidence rate: at least 1 in 5000 in North European
populations.
• Inheritance: autosomal (non-sex linked) dominant,
although a less common autosomal recessive variant
exists. To date, no homozygous for the autosomal
dominant have been described, suggesting that this
state is incompatible with life.
12
Pathophysiology
• The defective red cells in HS have an increased flux of
Na+ ions into the cell, because of weakened membrane
structure, leading to greatly increased activity of the
cation pump and necessitating an increased rate of
glycolysis.
• Normally, the osmotic balance of the red cell is
maintained with sufficient glucose and ATP to expel
sodium at a rate equal to its influx. However, spherocyte
consume glucose at a higher rate. When the amount of
glucose is low, as in splenic cords, there is an increased
rate of destruction of red cells. The water content of red
cells is increases, and as a result, swelling and
haemolysis of the red blood cells occur.
13
Cause
• The exact nature of the red cell defect is
unknown.
• A variety of cytoskeletal defects have been
described.
• Evidence suggests the primary defect involve
the structure of spectrin, a skeletal protein in the
red cell membrane.
• A quantitative decreases in spectrin that,
correlates with the degree of spherocytosis and
the severity of the disorder.
14
Symptoms
•
•
•
•
•
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Congenital haemolytic anaemia with a
variable degree of spherocytosis.
Increased red cell osmotic fragility.
Episodic jaundice.
Variable splenomegally.
Cholelithiasis (pigment gallstones// less
common).
Laboratory findings
•
•
•
•
•
•
•
•
•
•
•
•
•
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Moderate anaemia.
Peripheral blood shows variable spherocytic cell varies from few
to many.
Decrease RBC's diameter and increase MCHC.
Increase reticulocyte count up to 20%.
Few NRBC's present.
Normal WBC & platelet, except during haemolysis, where
there is leukocytosis and thrombocytosis.
Osmotic Fragilty is increased.
Increase bilirubin.
Increase urine and stool urobilinogen.
Decrease haptoglobin and may be undetectable.
Direct combs' is negative.
Bone marrow is hypercellular where 25-60% is erythroid.
Treatment
Splenectomy, which leads to:
•
•
•
•
•
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Increase RBC's lifespan.
Increase Hb, Hct, and RBC's.
Decrease bilirubin.
Decrease reticulocyte.
Osmotic fragility will continue increased.
Hereditary Elliptocytosis
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Hereditary Elliptocytosis
• Hereditary
Elliptocytosis
disorders
are
characterized by the presence of a large
proportion of oval or elliptical red cells.
• The frequency of HE is difficult to estimate
because most forms are clinically silent but may
be as high as 1 in 1000.
• The condition typically is transmitted as an
autosomal dominant characteristic.
• In contrast to HS, homozygous form is well
recognized as a severe transfusion-dependent
haemolytic anaemia.
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Cause
• A wide range of cytoskeletal defects has
been described in association with HE.
• The most common cytoskeletal defects
are structural abnormalities of domain 1 of
α spectrin, the region responsible for
dimer-dimer self-association in
cytoskeleton. Resulting in free
unconnected dimers.
20
Pathophysiology
• Most workers currently believe that,
because of the weakened forces holding
the cytoskeleton together in HE red cells;
repeated
passage
through
the
microvasculature resulting in red cell
fragmentation, poikilocytosis and microelliptocytosis.
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Symptoms
• Approximately 90% of the individuals
showing elliptocytosis have no clinical
symptoms other than the presence of
elliptical red blood cells. The remaining
patients display a haemolytic anaemia and
splenomegally.
22
• Laboratory findings :The characteristic
feature is increased osmotic fragility test.
• Treatment: Splenectomy typically
provides a functional cure
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Hereditary Enzyme Deficiency
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The pathways of glucose metabolism
G6PD function
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Glucose 6-Phosphate Dehydrogenase
Functions
• Regenerates NADPH, allowing regeneration of
glutathione
• Protects against oxidative stress
• Lack of G6PD leads to hemolysis during
oxidative stress
– Infection
– Medications
– Fava beans
• Oxidative stress leads to Heinz body formation,
 extravascular hemolysis
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Glucose-6-phosphate dehdrogenase
deficiency
The reduction of oxidized glutathione by NADPH
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Geographical distribution
• Deficiency of G-6-PD has been reported in most populations of the
world but is most commonly seen in Western and Central Africa, the
Mediterranean region, the Middle East and SE Asia.
• Normally active G6PD has been designated type Gd B.
• It is the most common form of the enzyme in all populations and
exists in 99% of Whites. Among whites, G6PD Mediterranean is the
most common variant, although the overall prevalence is low.
• Another variety of the G6PD enzyme that is commonly found in
Africans also has normal activity but differs from the Gd B by a
single amino acid substitution that alter its electrophoretic mobility.
This variant is designated as Gd A. The Gd A variant is found in
about 20% of American black men.
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Glucose 6-Phosphate Dehydrogenase
G6PD Activity (%)
Different Isozymes
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
RBC Age (Days)
Normal (GdB)
Mediterranean (Gd Med)
30
Black Variant (GdA-)
120
Mode of Inheritance
• The gene, which encodes G-6-PD, is located on the tip
of the q arm of the X chromosome, close to the factor
VIII gene.
• The disorder is fully expressed in men (hemizygote) who
inherit the mutant gene.
• In women, full expression of the disorder occurs only
when two mutant genes (homozygous) are inherited.
• The heterozygous woman has two populations of red
blood cells, one population with normal enzyme activity
and the other with deficient enzyme activity.
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Pathophysiology
Oxidants cause two kinds of damage to red
cells:
•
•
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Damage to haemoglobin
Damage to the membrane.
Tests which could be done in the diagnosis of
glucose-6-phosphate dehydrogenase deficiency





elevated bilirubin levels
low serum haptoglobin
haemoglobinuria
elevated absolute reticulocyte count
low red blood cell count and
haemoglobinuria
 Heinz bodies present on examination of
the peripheral blood smear using stains
 methylene blue test
 methemoglobin reduction test
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Acquired Haemolytic Disorders
The acquired haemolytic disorders can be subclassified into five groups, according to the
nature of the defect:
• Haemolysis secondary to immune mechanisms.
• Haemolysis secondary to the action of chemicals,
drugs or toxins.
• Haemolysis secondary to infection.
• Haemolysis secondary to physical damage.
• Miscellaneous disorders
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IMMUNE HEMOLYTIC ANEMIA
General Principles
• All require antigen-antibody reactions
• Types of reactions dependent on:
–
–
–
–
–
Class of Antibody
Number & Spacing of antigenic sites on cell
Availability of complement
Environmental Temperature
Functional status of reticuloendothelial system
• Manifestations
– Intravascular hemolysis
– Extravascular hemolysis
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IMMUNE HEMOLYTIC ANEMIA
General Principles - 2
• Antibodies combine with RBC, & either:
1.Activate complement cascade, &/or
2.Opsonize RBC for immune system
• If 1, if all of complement cascade is fixed to red
cell, intravascular cell lysis occurs
• If 2, &/or if complement is only partially fixed,
macrophages recognize Fc receptor of Ig &/or
C3b of complement & phagocytize RBC, causing
extravascular RBC destruction
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IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Direct
• Looks for immunoglobulin &/or complement
of surface of red blood cell (normally neither
found on RBC surface)
• Coombs reagent - combination of anti-human
immunoglobulin & anti-human complement
• Mixed with patient’s red cells; if
immunoglobulin or complement are on
surface, Coombs reagent will link cells
together and cause agglutination of RBCs
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Direct Antiglobulin Test
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IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Indirect
• Looks for anti-red blood cell antibodies in
the patient’s serum, using a panel of red
cells with known surface antigens
• Combine patient’s serum with cells from a
panel of RBC’s with known antigens
• Add Coombs’ reagent to this mixture
• If anti-RBC antigens are in serum,
agglutination occurs
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Indirect Antiglobulin Test
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HEMOLYTIC ANEMIA - IMMUNE
• Drug-Related Hemolysis
• Alloimmune Hemolysis
– Hemolytic Transfusion Reaction
– Hemolytic Disease of the Newborn
• Autoimmune Hemolysis
– Warm autoimmune hemolysis
– Cold autoimmune hemolysis
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IMMUNE HEMOLYSIS
Drug-Related
• Immune Complex Mechanism
– Quinidine, Quinine, Isoniazid
• “Haptenic” Immune Mechanism
– Penicillins, Cephalosporins
• True Autoimmune Mechanism
– Methyldopa, L-DOPA, Procaineamide,
Ibuprofen
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DRUG-INDUCED HEMOLYSIS
Immune Complex Mechanism
• Drug & antibody bind in the plasma
• Immune complexes either
– Activate complement in the plasma, or
– Sit on red blood cell
• Antigen-antibody complex recognized by RE
system
• Red cells lysed as “innocent bystander” of
destruction of immune complex
• REQUIRES DRUG IN SYSTEM
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DRUG-INDUCED HEMOLYSIS
Haptenic Mechanism
• Drug binds to & reacts with red cell
surface proteins
• Antibodies recognize altered protein, ±
drug, as foreign
• Antibodies bind to altered protein & initiate
process leading to hemolysis
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DRUG-INDUCED HEMOLYSIS
True Autoantibody Formation
• Certain drugs appear to cause antibodies
that react with antigens normally found on
RBC surface, and do so even in the
absence of the drug
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ALLOIMUNE HEMOLYSIS
Hemolytic Transfusion Reaction
• Caused by recognition of foreign antigens on
transfused blood cells
• Several types
– Immediate Intravascular Hemolysis (Minutes) - Due to
preformed antibodies; life-threatening
– Slow extravascular hemolysis (Days) - Usually due to
repeat exposure to a foreign antigen to which there was
a previous exposure; usually only mild symptoms
– Delayed sensitization - (Weeks) - Usually due to 1st
exposure to foreign antigen; asymptomatic
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INCOMPATIBLE RBC TRANSFUSION
Rate of Hemolysis
Surviving Cells (%)
100
80
60
40
20
0
0
1
2
3
4
5
6
Weeks Post-Transfusion
Normal
Slow Extravascular Hemolysis
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Immediate Intravascular Hemolysis
Delayed Extravascular Hemolysis
7
ALLOIMMUNE HEMOLYSIS
Testing Pre-transfusion
• ABO & Rh Type of both donor & recipient
• Antibody Screen of Donor & Recipient,
including indirect Coombs
• Major cross-match by same procedure
(recipient serum & donor red cells)
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ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn
• Due to incompatibility between mother negative
for an antigen & fetus/father positive for that
antigen. Rh incompatibility, ABO incompatibility
most common causes.
• Usually occurs with 2nd or later pregnancies
• Requires maternal IgG antibodies vs. RBC
antigens in fetus
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Erythroblastosis Fetalis
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ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn - #2
• Can cause severe anemia in fetus, with
erythroblastosis and heart failure
• Hyperbilirubinemia can lead to severe brain
damage (kernicterus) if not promptly treated
• HDN due to Rh incompatibility can be almost
totally prevented by administration of anti-Rh D
to Rh negative mothers after each pregnancy
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AUTOIMMUNE HEMOLYSIS
• Due to formation of autoantibodies that
attack patient’s own RBC’s
• Type characterized by ability of
autoantibodies to fix complement & site of
RBC destruction
• Often associated with either
lymphoproliferative disease or collagen
vascular disease
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AUTOIMMUNE HEMOLYSIS
Warm Type
•
•
•
•
•
•
•
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Usually IgG antibodies
Fix complement only to level of C3, if at all
Immunoglobulin binding occurs at all temps
Fc receptors/C3b recognized by macrophages.
Hemolysis primarily extravascular
70% associated with other illnesses
Responsive to steroids/splenectomy
AUTOIMMUNE HEMOLYSIS
Cold Type
•
•
•
•
Most commonly IgM mediated
Antibodies bind best at 30º or lower
Fix entire complement cascade
Leads to formation of membrane attack
complex, which leads to RBC lysis in
vasculature
• Typically only complement found on cells
• 90% associated with other illnesses
• Poorly responsive to steroids, splenectomy;
responsive to plasmapheresis
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HEMOLYTIC ANEMIA
Summary
• Myriad causes of increased RBC
destruction
• Marrow function usually normal
• Often requires extra folic acid to maintain
hematopoiesis
• Anything that turns off the bone marrow
can result in acute, life-threatening anemia
55
Paroxysmal Nocturnal
Haemoglobinuria
Paroxysmal Nocturnal Haemoglobinuria
Sudden
At Night
Blood in
Urine
Nocturnal haemolysis was once thought
to be associated with a slight drop in
plasma pH during sleep, but this is now
known to be untrue
57
Paroxysmal Nocturnal
Haemoglobinuria
• Paroxysmal nocturnal haemoglobinuria
(PNH) is an acquired clonal disorder,
which arises following a somatic mutation
in a multipotential stem cell
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Characteristics
• RBC’s are susceptible to the lytic action of
complement.
• Chronic but episodic intravascular haemolysis.
• Occurs mainly during sleep.
• Moderately to severe panhypoplasia.
• Neutrophil dysfunction.
• Renal insufficiency.
• Dysphagia.
• Tendency to venous thrombosis.
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Classification
• Broadly, the red cells in cases of PNH can
be divided into three groups:
1- PNH I red cells are normal with regard to their
sensitivity to complement-mediated lysis.
2- PNH II red cells show a moderately increased
sensitivity.
3- PNH III red cells show a markedly increased
sensitivity.
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The size of hemolysis
• The major severity of haemolysis is the size of
the PNH III red cell population.
Where more than 50% of the circulating red cells
are PNH III cells haemolysis is severe and
relatively constant.
Typical episodic or sleep-related hamolysis is
seen when 20-50% of the circulating red cells is
a PNH III cell.
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Pathogenesis
Deficiency of several red cell membrane
proteins has been noted in PNH, at least
some of which contribute to the
pathogenesis of the haemolysis:
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Acetylcholesterase
Acetylcholesterase deficiency is relatively
constant finding in PNH red cells and was
originally thought to be implicated in the
pathogenesis. However, this is untrue
because artificially-induced inhibition to
this enzyme has no effect on red cell life
span in vitro or in vivo.
63
Decay Accelerating Factor (DAF)
• Red cell membrane integral protein which binds
to complement components C3b and C4b on the
membrane surface, thereby inhibiting the
construction of C3.
• DAF therefore protects the red cell from
complement-mediated haemolysis.
• Deficiency of DAF causes a relatively mild
increase in susceptibility to complement64 mediated haemolysis.
Membrane inhibitor of reactive lysis
(MIRL)
• This is a red cell membrane protein which
protect normal red cell from complementmediated haemolysis by inhibiting the
assembly of the complement membrane
attack complex, C5b-9.
65
Homologous Restriction Factor
(HRF)
• This is a red cell membrane integral
protein, which, in normal cells, limits the
assembly of the complement membrane
attack complex by binding C8. HRF has
been shown to be absent in PNH III.
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Clinical picture
•
•
•
•
•
67
Episodes of intravascular haemolysis.
Haemoglobinuria.
Haemorrhage.
Infection.
Thrombotic complications.
Triggering factors for haemolysis
•
•
•
•
•
68
Mostly is unknown.
Infection.
Vaccination.
Blood transfusion.
Menstrual cycle.
Causes of death
• The excess of bacterial and fungal infection
secondary to leukopenia and neutrophil
dysfunction is a common cause of death.
• The leading cause of death in PNH, however, is
venous thrombosis.
• Thrombosis is explained by the release of
thromboplastin-like substances from lysed red
cells and platelet aggregation.
69