Extravascular hemolysis occurs when RBCs are phagocytized
by macrophages in the spleen, liver and bone marrow (see image
of an erythrophage to the right). Extravascular hemolysis is
the most common form of hemolytic anemia in animals.
It usually occurs alone (without intravascular hemolysis), but
will always (to some extent) accompany intravascular hemolysis.
Note that during the normal aging of red cells in the circulation,
effete red cells are destroyed by macrophages, i.e. extravascular
hemolysis is always occurring to some degree. However, this
is a physiologic process and does not result in anemia or excessive
unconjugated bilirubin production.
With extravascular hemolysis, the erythrocytes are degraded
within macrophages, so hemoglobin is not released free
into the cytoplasm. Thus, we do not see hemoglobinemia or hemoglobinuria
with extravascular hemolysis alone, unless it is accompanying
|Within macrophages, the hemoglobin is broken
down into its constituents, i.e. the heme ring (a porphyrin
ring with iron in its center) and globin chains.
- The globins are broken down to amino acids, which
are then used for protein synthesis.
- The porphyrin ring of heme is oxidized by microsomal
heme oxygenase, producing biliverdin and releasing the
iron (Fe3+). The iron can then
be exported into plasma through iron channels, where
it binds to apotransferrin (the iron transport protein)
forming transferrin or can be stored within cells as
ferritin (i.e. the iron is bound to the storage protein,
apoferritin). With time, ferritin becomes oxidized and
degrades to form hemosiderin. Hemosiderin can be visualized
within macrophages as a dusky blue-gray pigment and
can be definitively stained with Prussian blue (which
turns hemosiderin blue).
- Biliverdin is reduced by biliverdin reductase to unconjugated
bilirubin (water insoluble). The unconjugated bilirubin
is released into the plasma, where it binds to albumin
(to render it water-soluble) and is taken up by hepatocytes.
There are many causes of extravascular hemolysis. Hemolytic
anemias (intravascular or extravascular) are usually regenerative
(if sufficient time is given for the marrow to regenerate and
if there are no additional factors suppressing erythropoiesis).
hemolytic anemia (IMHA)
hemolytic anemia in a dog. Many spherocytes (smaller
RBCs which lack central pallor) can be seen in
Attachment of IgG or IgM causes fixation of complement
(to C3b) on red cell membranes. Macrophages possess receptors
for the Fc portion of IgG and IgM as well as for C3b,
thus causing red cells with attached immunoglobulin or
C3b to be phagocytized (see image below). Partial phagocytosis
of erythrocytes forms spherocytes which, in large numbers,
are pathognomonic for IMHA. Note, that spherocytes are
most readily seen in the dog, because central pallor is
usually present in canine erythrocytes. The immunoglobulin-
and complement-coated red cells can be detected in a direct
Coombs test using a Coombs reagent, which consists of
species-specific anti-Ig and/or anti-C3. Thus, a positive
Coombs test is further supportive evidence of IMHA, but
false positives and negatives do occur. IMHA can be primary
or secondary to drugs (e.g. penicillin in horses) or erythroparasites.
|Auto-reactive B cells secrete
immunoglobulins (IgG and/or IgM) that recognize a "self"
epitope on red blood cells. Strong complement-fixing antibodies
result in formation of the membrane attack complex, which
punches holes in the red cell membrane causing them to
rupture within the circulation (intravascular hemolysis).
Weaker complement-fixing antibodies only generate the
opsonin C3b, which also attaches to RBC membranes. Ig-
or C3b-bound red blood cells are destroyed by macrophages
(which contain receptors for C3b and the Fc portion of
immunoglobulins) as they traverse through organs such
as the spleen (extravascular hemolysis). Multivalent antibodies,
such as IgM, can crosslink adjacent red blood cells, resulting
- Erythroparasites: Many
erythroparasites cause a hemolytic anemia due to extravascular
hemolysis, e.g. Mycoplasma hemofelis (feline infectious
anemia), Anaplasma bovis. With many of these organisms,
there is a concurrent immune-mediated component to the anemia
(the organisms make the red cells antigenic).
- Other organisms: Bacteria,
such as Clostridium sp and Leptospira,
can cause an extravascular hemolytic anemia, as can rickettsial
and viral (e.g. equine infectious anemia) agents.
hemolytic anemia due to acetaminophen toxicity in a
cat. Large heinz bodies (arrowheads) can be seen on
- Oxidant injury: Oxidant
injury (e.g. acetaminophen toxicity in cats) can result in
extravascular hemolysis. Heinz bodies, eccentrocytes and pyknocytes
are seen with oxidant injury (although this is species dependent).
The Heinz body-containing red blood cells are removed prematurely
from the circulation by macrophages (principally in the spleen).
Inherited defects in red cell enzymes that help the red blood
cell combat oxidant injury (e.g. glucose-6-phosphate dehydrogenase
deficiency in horses) can result in an oxidant-induced hemolytic
- Fragmentation injury:
This usually occurs secondary to vascular disease (e.g. hemangiosarcoma)
or disseminated intravascular coagulation (DIC). Keratocytes,
schistocytes, and acanthocytes are observed in peripheral
blood in fragmentation anemias. A few spherocytes may be observed
in fragmentation anemias and do not indicate immune-mediated
disease in this setting. Fragmentation anemias may be non-regenerative
as cytokines associated with the primary disease often suppress
the bone marrow. Note that some degree of intravascular hemolysis
does also occur with fragmentation injury (particularly when
fibrin strands shear red cells in DIC), however the amount
of hemoglobin released into the circulation is insufficient
to cause visible hemoglobinemia or hemoglobinuria.
- Histiocytic disorders:
In these disorders, instead of red cell destruction occuring
due to the red cells being abnormal, they are destroyed because
the macrophages are stimulated by cytokines (usually liberated
from T cells, i.e. the macrophages are reactive) or are neoplastic
(e.g. histiocytic sarcoma). Macrophage variants of histiocytic
sarcoma have been identified in dogs (particularly large breeds,
like Golden Retrievers and Labradors) and can produce an extravascular
hemolytic anemia, that can mimic IMHA.
- Inherited red cell defects:
Inherited defects of red cell enzymes (e.g. pyruvate kinase
deficiency) and membranes (e.g. hereditary stomatocytosis)
can result in extravascular hemolytic anemias. These have
primarily been identified in dogs, but can also occur in cats.