Intravascular hemolysis results from the rupture
or lysis of red blood cells within the circulation, i.e.
the red cells are lysing in vivo. When the membrane of
erythrocytes rupture, they release their hemoglobin into the
hemoglobin (which is a tetramer) breaks down into hemoglobin
dimers in plasma. Haptoglobin (an α-2 globulin produced
in the liver) binds the liberated free hemoglobin dimers. However,
haptoglobin is readily saturated (this occurs at around a hemoglobin
concentration of 150 mg/dL). If intravascular hemolysis continues,
the hemoglobin dimers are in excess in plasma and are filtered
readily through the glomerulus (because they are < 20 kd
in size). This will cause a hemoglobinuria (see image below
on right) and a positive reaction for heme protein on the dipstick
(with no erythrocytes evident in the urine sediment). Because
hemoglobin concentrations >20 mg/dL will cause visible discoloration
of plasma (light pink to dark red, depending on how much hemoglobin
is present), hemoglobinemia is often visible with intravascular
hemolysis. The hemolytic
index provided on Cornell University's chemistry panel is
often quite high in patients with intravascular hemolysis (i.e.
> 200 units). The image on the left shows severe hemolysis
(red discolored supernatant plasma of blood centrifuged in a
microhematocrit tube) in a dog with an immune-mediated hemolytic
anemia (with intravascular hemolysis). The hemolytic index in
such a patient would be > 500 units.
The hemoglobin dimers that remain in circulation are oxidized
to methemoglobin, which disassociates into a free heme and globin
chains. The oxidized free heme (met-heme) binds to hemopexin
(a β-globulin, Hpx) and the met-heme and hemopexin complex
(met-heme/Hpx) is taken up by a receptor on hepatocytes and
macrophages within the spleen, liver and bone marrow (only hepatocyte
uptake is illustrated in the image above). Similarly, the hemoglobin/haptoglobin
complex is taken up by hepatocytes and macrophages (to a lesser
extent). Within these cells, the hemoglobin disassociates into
heme and globin chains. The globins are broken down to amino
acids, which are then used for protein synthesis. The heme is
oxidized by heme oxygenase forming biliverdin and releasing
iron. The iron can be transferred to apotransferrin (the iron
transport protein) in plasma or can be stored within cells as
ferritin (i.e. the iron is bound to the storage protein, apoferritin).
The remaining porphyrin ring (biliverdin) is degraded to unconjugated
bilirubin by biliverdin reductase. If the hemoglobin/haptoglobin
complex is internalized by macrophages, the unconjugated bilirubin
is released into the plasma, where it binds to albumin (to render
it water-soluble) and is taken up by hepatocytes through the
haptoglobin receptor. Thus, with intravascular hemolysis, increases
in bilirubin are usually due to unconjugated bilirubin (indirect)
and are likely of macrophage (rather than hepatocyte) origin.
Note that it is unusual for intravascular hemolysis to occur
alone, i.e. it is usually accompanied by extravascular hemolysis.
This extravascular hemolysis is the likely source of most of
the unconjugated bilirubin that is produced by macrophages in
a hemolytic anemia. Because haptoglobin is consumed during intravascular
hemolysis, serum values of this protein usually decline with
intravascular hemolytic anemias or when hemoglobin is liberated
into plasma by artifactual lysis of red cells in vitro (e.g.
freezing of red cells, old samples - see below). Haptoglobin
is a positive acute phase reactant and values will increase
as part of the acute phase response (an evolutionary conserved
innate response to inflammation, injury or infection). In fact,
an increase in haptoglobin is one of the main reasons for the
high α-2 peak seen in acute phase responses in serum electrophoresis.
Corticosteroids will also increase serum values of haptoglobin
The process of intravascular hemolysis with resulting hemoglobinemia,
hemoglobinuria and bilirubinemia is illustrated in the image
Since heme oxygenase is also present in renal
tubular cells, the renal epithelium is capable of converting
hemoglobin to bilirubin. However, this only occurs when there
is intravascular hemolysis with hemoglobinuria (i.e. the renal
epithelium does not take up unconjugated bilirubin or hemoglobin
from blood!). The renal epithelium absorbs the filtered hemoglobin
from the urine, converting it to unconjugated bilirubin and
then conjugating it for excretion into the urine (see image
below). This may be responsible for some of the bilirubinuria
seen in animals with intravascular hemolysis, however in most
of these animals, there is concurrent cholestasis that is responsible
for the bilirubinuria (which is conjugated).
Note that red cells can also lyse or rupture in vitro (either
in the blood collection tube or during collection). When this
occurs, the hemolysis is considered an artifact and does
not indicate the animal has a hemolytic anemia.
- Artifactual hemolysis: Poor
venipuncture technique, prolonged blood storage, exposure
to temperature extremes (hot or cold enough to freeze the
cells), and certain anticoagulants (fluoride-oxalate) will
cause artifactual red cell lysis. Red cells are also more
fragile in lipemic samples and tend to lyse more readily in
these samples, even if the blood is stored or handled correctly.
This artifactual red cell lysis can mimic intravascular hemolysis
and it can be very difficult to tell them apart (particularly
in the laboratory where all we see is the sample and not the
patient). However, if the animal is anemic and has hemoglobinuria,
true intravascular hemolysis, i.e. a pathological hemolytic
anemia, is likely.
Intravascular hemolysis ("true" in vivo hemolysis)
results in a hemolytic anemia in affected animals, but is far
far less common than hemolytic anemias due to extravascular
hemolysis in animals.
There are several causes of intravascular hemolysis:
- Immune-mediated hemolytic anemia:
Complement fixation by IgG or IgM causes assembly of the membrane
attack complex (MAC, C6-C9) on red cell membranes in vivo
which lyses the cells. A variant of an immune-mediated
hemolytic anemia is an acute hemolytic transfusion reaction
where transfusion of incompatible blood into an animal will
cause acute intravascular hemolysis when antibodies bind to
the transfused "foreign" red blood cells and activate
the complement cascade.
infection in a reindeer with intravascular hemolytic
anemia. Single (arrowhead) and paired (arrow) pyriform
organisms are seen in several RBCs.
- Erythroparasites: Babesia
species replicate inside erythrocytes and rupture the
cells when they exit to continue their life cycle. This results
in an intravascular hemolysis. Indeed, Babesia bovis infections
are often called "red-water" disease due to the
- Other organisms: Bacteria,
such as Clostridium hemolyticum and Leptospira,
can cause lysis of red cells in vivo. There have been
reports of bee stings, spider bites and snake venoms causing
a hemolytic anemia due to intravascular hemolysis (due to
phospholipases in the venom).
- Oxidant injury: Oxidant
injury (e.g. copper poisoning in sheep or dogs, red maple
toxicity in horses, zinc toxicity in dogs) can result in an
hemolytic anemia due to ingestion of wilted red maple
leaves in a horse. Eccentrocytes (black arrow) and pyknocytes
(black arrowheads) indicate oxidant injury, whereas
ghost cells (red arrow) indicate intravascular hemolysis.
- Metabolic conditions:
Acute liver disease in horses can result in intravascular
hemolysis. Because phosphate is essential for ATP production
and maintenance of the integrity of red cell membranes, intravascular
hemolysis can occur with severe hypophosphatemia (e.g. phosphate
-depleted dogs and cats with diabetes mellitus that are treated
with insulin, post-partum hypophosphatemia in dairy cows).
- Inherited red cell defects:
Dogs with phosphofructokinase deficiency can suffer from bouts
of intravascular hemolysis with exercise, due to alkaline
fragility of their red blood cells.