HKMU SCI8003SEF Haematology & Transfusion Science I

Hemolytic anemia
Hemolytic anemias are defined as those anemias which result form an increase in the rate of red
cell destruction. In hemolytic disorders, red cells are destroyed prematurely, usually in a random
fashion. If the red blood cell life span is only moderately shortened, the patient will usually have
little, if any, anemia because the bone marrow is capable of increasing the rate of new red blood
cell production.

Red cell destruction usually occurs after a mean lifespan of 120 days when the cells are
removed extravasculary by the macrophages of the reticuloendothelial (RE) system, especially in
the marrow but also in the liver and spleen. The breakdown of red cells liberates iron for
recirculation via plasma transferrin to marrow erythroblasts, and protoporphyrin which is broken
down to bilirubin. This circulates to the liver where it is conjugated to glucuronides which are
excreted into the gut via bile and converted to stercobilinogen and stercobilin (excreted in feces).
Stercobilinogen and stercobilin are partly reabsorbed and excreted in urine as urobilinogen and
urobilin. A small fraction of protoporphyrin is converted to carbon monoxide (CO) and excreted
via the lung. Globin chains are broken down to amino acids which are reutilized for general
protein synthesis in the body.
Haptoglobins are proteins present in normal plasma capable of binding hemoglobin. The
hemoglobin-haptoglobin complex is removed from plasma by the RE system.

There are two general sites in which hemolysis may take place.

Intravascular hemolysis

Red blood cells are destroyed directly within the circulatory system i.e. breakdown of red cells
within blood vessels which plays little or no part in normal red cell destruction.

Extravascular hemolysis

Is more common than intravascular hemolysis and involves the destruction of red blood cells
within mononuclearphagocytic cells, often in the spleen.

1. Classification of HA

• Extrinsic: From a source outside the red cell; disorders extrinsic to the RBC are usually
acquired. The RBC may get damaged by chemicals, mechanical or physical agents or
immune destruction. Hemolysis can occur either intravascularly or extravascularly

• Intrinsic: Due to defects of the RBC itself and are usually inherited. The abnormality can
be in the membrane, cell enzymes, or the hemoglobin molecule. The cells that show
intrinsic defects are hemolyzed extravascularly because the RBCs are not severely
damaged.
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2. Laboratory findings

The major criteria for the laboratory diagnosis of hemolytic anemia are:

- Reticulocytosis
- Serum level of unconjugated/conjugated bilirubin.
- Serum level of lactic dehydrogenase (LDH) is elevated.
- Serum haptoglobin level is decreased.
- The peripheral blood smear often but not invariably shows morphologic changes in the
red blood cells compatible with hemolysis e.g. many spherocytes suggest hereditary
spherocytosis or immunohemolytic anemia and sickle cells suggest one of the sickle cell
syndromes.

Reticulocytosis is a constant feature of all hemolytic anemias. Reticulocytosis reflects the

increased activity of the bone marrow as it attempts to maintain erythrocyte mass in the
peripheral blood. If the bone marrow is able to increase erythropoiesis in order to
compensate for the decreased erythrocyte life span, the anemia does not develop. This is
referred to as compensated hemolytic disease. At any time compensated hemolytic disease
can develop into an anemia if RBC destruction exceeds beyond compensatory capacity of the
bone marrow or the bone marrow suddenly stops making RBCs.

The bone marrow shows erythroid hyperplasia of the bone marrow and the M:E ratio is
decreased.

The peripheral blood shows a normochromic, normocytic blood picture with polychromasia
and the presence of nRBCs.

Test used to evaluate heme catabolism are abnormal. Some of these tests include
haptoglobin and conjugated and unconjugated bilirubin.

3. Intrinsic HA

3.1 Membrane Defects

This is a congenital hemolytic anemia, some of which present at birth, and others later in life,
while still others may remain silent unless a physiologic stress is superimposed. Hemolytic
anemia can result from abnormalities in constituent membrane proteins or lipids, both of
which can alter the membrane’s stability, shape, deformability, and or permeability. These
abnormal RBCs are susceptible to entrapment by the splenic cords. Most of the hemolysis
associated with membrane abnormalities is extravascular, occurring in the splenic cords.

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RBC Cell Membrane

The cell membrane serves as a semipermeable outer boundary of the cell. It allows the
passage of nutrients, ions, and information between the cytoplasm and the exterior. It also
serves as the location of surface markers for cell identity. The cell membrane is made of
phospholipid bilayer. The membrane must be flexible, and resilient. It achieves this by being
a fluid structure of globular proteins floating in lipids. The lipids comprise phospholipids and
cholesterol arranged in two layers. The phosphate end of the phospholipid and the hydroxyl
radical of cholesterol are oriented towards the inner and outer surface of the cell membrane.
The fatty acid portion of the phospholipid are directed towards the center of the cell
membrane. Within the cytoskeleton, actin, spectrin act to stabilize the membrane

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Membrane defects can be divided into two categories:

- skeletal protein abnormalities and

- lipid composition abnormalities.

The skeletal protein abnormalities can be further broken down into either vertical or
horizontal defects.

i. Vertical interactions attach integral proteins and lipids of the membrane to the
skeletal lattice on the cytoplasmic side of the cell. These interactions help to stabilize
the cell. Defects in these interactions cause the lipid bilayer to separate from the
skeletal lattice, which causes a decrease in the surface area-to-volume ratio, forming a
spherocyte. Eventually that spherocyte will be hemolyzed.
ii. Horizontal interactions provide mechanical support for the membrane. Defects in
these interactions lead to the disruption of the skeletal lattice and the membrane
becomes destabilized, forming cell fragmentation with poikilocytosis as an end result.

The Lipid composition abnormalities have an excess amount of cholesterol that accumulates
in the outer bilayer. As the spleen attempts to fix this abnormal cell, the acanthocyte is
formed.

3.1.1 Hereditary spherocytosis (HS)

HS is an autosomal dominant disorder that may become symptomatic shortly after birth, or
may not be detected until later life. Most patients with HS have spherocytes, splenomegaly,
and jaundice.

Laboratory findings

- Patients with HS have laboratory evidence of hemolysis, including anemia,

reticulocytosis, increased serum LDH level, decreased or absent haptoglobin, and often
mild hyperbilirubinemia.
- Large numbers of spherocytes, polychromasia, anisocytosis and poikilocytosis.
- Mild anemia; MCH, MCV usually normal; MCHC >36%; RDW Increased

Diagnostic Tests for HS

i. Osmotic fragility - increase

- Cells are incubated in decreasing concentrations of NaCl. Spherocytes lyse sooner than
normal red cells. Osmotic fragility test measures the RBCs resistance to hemolysis by
osmotic stress, which depends on the volume of the cell, the surface area, and its
membrane function. Because of the decreased surface to volume ratio, spherocytes can
not expand. Therefore they hemolyze.

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ii. Autohemolysis test

- Red cells are incubated at 37̊ C for 48 hours. Degree of hemolysis is increased when
spherocytes are present.
iii. Red cell membrane studies
- Membrane proteins are analyzed using gel electrophoresis.

3.1.2 Hereditary elliptocytosis (HE)

HE is an autosomal dominant disorder characterized by excessive numbers of elliptical cells. It

may also occur in thalassemia, iron deficiency sickle cell disease, and megaloblastic anemia.
These disorders, however, are accompanied by other characteristic morphologic changes as well.
Most patients are asymptomatic and anemia is absent. The hemolysis is mild and well
compensated by the bone marrow. In 10 to 15 percent of patients with HE, erythrocyte
destruction is substantially increased, leading to all the signs and symptoms of a true hemolytic
anemia.

3.2 Enzyme Deficiency

Intrinsic defects such as enzyme deficiencies can lead to hemolytic anemia. The enzyme
deficiency compromises the integrity of the cell membrane or the hemoglobin which results in
hemolysis of the red cell. The most common enzyme deficiencies are those within the hexose
monophosphate shunt and the glycolytic pathway. Although the amount of enzymes within the
rbc is limited, those involved in processes that protect the cell from oxidant damage and provide
the cell with energy are essential for cell survival. The mature RBC depends entirely on
anaerobic glucose metabolism for its energy needs, so any defect of an enzyme can hinder the
cell’s integrity.

3.2.1 Glucose-6-phophate dehydrogenase (G-6-PD)

Deficiency of the enzyme glucose-6-phophate dehydrogenase (G-6-PD) is by far the most
common inherited erythrocyte enzyme deficiency, affecting more than 100 million people. The
gene for G-6-P is sexlinked. Because of the X-linkage, male patients are more severely affected
than female patients. It is a key enzyme in the hexose monophosphate pathway (HMP). As a
result of the deficiency, denatured hemoglobin precipitates in the RBC, ultimately resulting in
hemolysis. There is a correlation with the occurrence of G-6-P-D deficiency and malaria.
Individuals that have the G6PD deficiency are protected against malarial parasites. G6PD
activity is highest in young red cells, decreases as cells age.

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In a normal RBC, G6PD is necessary for maintaining adequate levels of GSH for reducing
cellular oxidants which keeps hemoglobin in a reduced functional state (ferrous) and preserves
cellular enzymes. In a RBC without G6PD, the generation of NADPH, and subsequently GSH is
impaired and hydrogen peroxide and oxygen radicals accumulate. The presence of these
substances oxidize hemoglobin to methemoglobin which precipitates in the form of a Heinz
body. Heinz bodies are intracellular precipitates of denatured hemoglobin. The Heinz body is
removed from the RBC by the macrophages in the spleen, resulting in bite and blister cells. As
increased loss of the membrane occurs, spherocytes can be seen. Heinz bodies can also be
removed from the circulation by intravascular hemolysis.

Laboratory Findings
In individuals with G6PD deficiency, anemia is absent and the peripheral blood is normal
EXCEPT during hemolytic attacks. Once the patient’s RBCs are exposed to oxidant stress, a
hemolytic attack occurs. At this point the Hb and Hct decreases, hemoglobinuria is noted,
bilirubin and LD are increased.

4. Extrinsic Anaemia
Immune Haemolytic Anaemia (IHA)
IHA are the result of the binding of antibody, complement, or antibody plus complement to red
cells. Antibodies formed against erythrocyte antigens may be either warm (active at 37oC) or
cold (active at room temperature and below). In some cases, these antibodies activate a series of
proteins, referred to collectively as complement; in others, the red cells are coated with antibody
alone. As a result of complement activation by hemolytic antibodies, intravascular red cell lysis
and release of hemoglobin may occur. Alternatively, and more frequently, immune lysis, due to
either antibody or complement, is extravascular and happens slowly within cells of the
macrophagic-phagocytic system (RES), particularly in the spleen.
Classification of IHA
IHA can be classified into three major categories:
1. autoimmune, in which the patient makes an autoantibody against his or her own red cells;
2. alloimmune, where the patient’s antibody is directed against foreign red cells; and
3. drug-induced, where a drug-dependent or related antibody is responsible for hemolysis

4.1 Autoimmune hemolytic anemia (AIHA)

AIHA is charcterized by premature RBC destruction caused by autoantibodies that bind to the
RBC surface. Normally, autoimmunity is kept in control by the T suppressor lymphocytes.
However in autoimmune hemolytic anemia, this control is lost resulting in the production of
autoantibodies against RBC self-antigens. It is believed that the autoimmune disease occur
because of a number of factors including genetic predisposition, exposure to infectious agents
that can induce antibody production and defects in the mechanism regulating immune tolerance.

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4.1.1 Warm AIHA

The majority of the warm autoantibodies are of the IgG class and they cause extravascular
hemolysis of the RBC. Most often the specificity of the antibody is directed against antigens of
the Rh system. The red cells are usually coated with IgG alone, IgG and complement or
complement alone, but a minority of cases show IgA or IgM coating alone or combined with IgG
antibody. The complement component detectable is C3d, the degraded fragment of C3. The
AIHA in SLE is typically of the IgG + complement type. Red cells coated with IgG are taken up
the RE macrophages, especially in the spleen, which have receptors for the Fc fragment. Part of
the coated membrane is lost so the cell becomes progressively more spherical to maintain the
same volume and is ultimately prematurely destroyed, usually predominantly in the spleen. Red
cells with complement coating alone or in addition to IgG are destroyed more generally in the
RE system, and not particularly in the spleen. The disease may occur at any age in either sex and
presents as a hemolytic anemia of varying severity.
Warm autoimmune hemolytic anemias shows the greatest antigen activity at temperatures above
300. About 60% of these cases are idiopathic, with the remaining percentage due to another
underlying disease, such as lymphoma, chronic lymphocytic leukemia, viral infections and
immunodeficiency syndromes.

Laboratory Findings
Depending on the severity of the anemia, and the ability of the bone marrow to compensate for
that anemia will determine the extent of the anemia.

1. Positive Direct Antiglobulin Test

2. Normocytic, normochromic anaemia
3. Increase recticulocytes
4. Spherocytes
5. Presence of autoantibody
6. Increased bilirubin
7. Decreased serum haptoglobin

4.1.2 Cold agglutinin disease (CAD)

Cold agglutinin disease occurs predominantly in older individuals with peak incidence after 50
years of age. Affects men and women equally. The antibodies in this disorder show the greatest
reactivity with antigens at temperatures below 300 C. These antibodies are usually of the IgM
class and they bind complement. Cases of cold agglutinin disease can be idiopathic or secondary
to another underlying condition, such as infections with Mycoplasma pneumoniae or Infectious
mononucleosis.
Hemolytic syndromes of varying severity may occur depending on the titer of the antibody in the
serum, its affinity for red cells, its ability to bind complement, and its thermal amplitude
(whether or not it bids to red cells at 37oC). Agglutination of red cells by the antibody often
causes peripheral circulation abnormalities. The antibody may then detach from red cells when
they pass to the warmer central circulation but, if complement has been bound, the direct
antiglbobulin test remains positive-of complement only type- and the cells are liable to be
destroyed in the whole RE system, especially the liver, giving rise to a chronic hemolytic
anemia.

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4.2 Drug-Induced HA

- Drugs that attach to the RBC membrane or alter it in some way

o Methyldopa/ procainamide, penicillin, cephalosporins
- Patient produces antibodies directed against a particular drug, its metabolites or the RBC
coated with drug
- Acquired
- The drug itself does not cause the RBC injury, and not all people taking the drug are
affected

4.3 Alloimmune hemolytic anemia (AHA)

AHA occurs as the result of antibody development to an erythrocyte antigen that the individual
lacks. When an individual is exposed to erythrocytes from another person, antigens on transfused
cells may be lacking on the erythrocytes of the recipient. So the recipient’s lymphocytes
recognizes these RBCs are foreign and stimulates antibody production. The antibody coats the
foreign RBCs and shortens RBC survival. A hallmark of alloimmune hemolytic anemia is a
positive DAT.

There are two main types:

- hemolytic transfusion reactions and
- hemolytic disease of the fetus and newborn.

4.3.1 Hemolytic transfusion reactions

Acute transfusion reactions occurs within minutes to hours after the infusion of blood from
donor blood. Incorrect ABO type is the most common cause of acute transfusion reactions. For
example, if an “O” patient receives “A” rbcs- “O” patients can ONLY get “O” blood. If the
patient gets “A” rbcs, this would trigger an acute transfusion reactions with symptoms such as
fever, shaking, chills, and buring at the IV site. Intravascular hemolysis occurs due to the
complete activation of complement mediated by Ig M antibodies.
The direct Coombs test is positive due complement fixation, but may become negative within
hours to days, depending on how rapidly the group a cells are destroyed. The presence of urine
hemosiderin beginning 3 to 5 days after the transfusion attests to the recent presence of
hemoglobinemia.

Delayed transfusion reaction which occur days to weeks after the transfusion. When the
patient is re-exposed to non-ABO blood groups, such as Duffy or Kidd, antibodies will build and
the delayed reaction takes place. Symptoms are mild and non-specific. RBCs experience
extravascular hemolysis as they are coated with IgG antibodies and removed by the macrophages.
Complement is not involved.

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4.3.2 Haemolytic Disease of New-born

This hemolytic process actually begins in utero to the baby of a mother with IgG red cell
antibodies. IgG antibodies readily cross the placenta, as opposed to IgM antibodies, which
cannot. In the past, many Rh(D)-negative women became sensitized to the red cell antigen D at
the time of birth of a first Rh-positive child, because at birth it is common for a small volume of
fetal cells to enter the maternal circulation. Today, Rh sensitization is much less common and
largely preventable. Rh-positive fetuses carried by a sensitized Rh-negative mother can be
severely affected by the IgG anti-D. Some babies develop profound in utero anemia with
congestive heat failure (hydrops fetalis), leading to stillbirth

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