Hemolytic anemia

Basic concept

Bone marrow
Constantly producing red blood cells

Stem cell » lymphoid + myeloid

Myeloid »
- granulocytes/ monocytes
- erythroblastic/ megakaryocytic
- basophilic

E/ Mg » eryhthroblates »»»»reticulocytes » RBCs

Within 120 days, RBCs will be destroyed, because they don't have nucleus so they
can't make new proteins as their proteins get denaturated.

RBC
In the inner side there is a network of proteins interconnected unconnected with
membrane proteins making the cytoskeleton of the RBC, most important of them
are spectrin and encirine.
Role of cytoskeleton : hold the membrane with the content of RBC.

RBC is provided by extra membrane to increase the surface of gas exchanges while
it has less substance
=» biconcave shape
Example of a slim woman woth a large dress.

In circulation, RBCs have to go through narrow places like cords of Billroth in

spleen, they have to twist to pass through them, the flexibility of the cytoskeleton
proteins makes this possible

cytoskeleton
- increase surface for gas exchange
- flexibility

As RBC gets older, it's proteins get denatured, it becomes stiff (denatured
cytoskeleton), less flexible,it gets stuck in narrow points, sometimes it looses a part
of its membrane. As parts of the membrane are lost, the remaining parts fuse
together:
- substance RBC content is the same
- membrane is less
The biconcave shape is transformed to spherical.
In the other hand, the loss of Na+ channels leads to the accumulation of Na within
the RBCs and their swelling.

Spleen
physiological apparatus to check RBCs flexibility

in spleen : arteriols » narrower cords of Billroth

Flexible RBCs can pass through the splits between between the endothelial cells of
cords of Billroth while older RBCs get stuck there and they are taken by local
macrophages which degrade them, leading to the formation of bilirubin.

Hemoglobin is highly toxic this why it is kept inside RBCs.

Pathologic Hemolysis is the premature rapid destruction of rbc's

Normally, a n rBC is destroyed Within 120 days.

In accelerated hemolysis the destruction occurs before 120 days,
Example within 20 days, the hemolysis here is 6 times more rapid.

Excessive hemolysis > decreased hemoglobin > decreased oxygen supply to the
kidneys > erythropoietin released by juxtaglomerular apparatus.
Endothelial cells of peritubular capillaries, and connective tissu cells are very
sensitive to oxygen amounts.
less O2 » endoth c of PTcapillaries » genes activation » EPO » Receptors of EPO
on pro erythroblasts and erythroblasts » stop of apoptotic process
EPO the message of life
proEB and erythroB have a suicadal tendancy, if left alone in a petry dish with all
life requirement they undergo apoptosis unless EPO is provided.
when they are alive they proliferate.
EPO doesn't allow erythroid precursors to undergo apoptosis
In the presence of EPO the amount of red bone marrow expands and becomes more
cellular : erythroid hyperplasia
Bone marrow is trying to compensate the RBC loss
If bone marrow is healthy and it's provided with all the nutrients required, it can
increase its production of red blood cells 8 to 10 times.
If the bone marrow increase production of RBC he's able to compensate excessive
hemolysis which means Hemoglobin levels will be maintained, in this case we say
there is compensated hemolytic disease, there is no anemia since hemoglobin
levels are maintained.

10X vs 6X ( 20 days)

10X vs 12X (10 days life)

when the compensatory mechanisms of bone marrow are dépassés Hb levels start
decreasing progressively » hemolytic anemia

Compensatoey mechanisms dépassés :

- hemolysis rate is very excessive
- BM can t compensate (iron deficiency, folate deficiency, BM disease) even if
hemolysis increase by 3 times only can t be compensated
We will be looking for evidence of
1- excessive hemolysis
2- excessive erythropoiesis
3- extra or intravascular hemolysis
4- acute or chronic hemolysis

A- excessive Hemolysis

1) Jaundice with
unconjugated hyperbilirubinemia
- if the liver is healthy, hyperbilirubinemia will be moderate since hepatocytes will
increase their conjugation activity to eliminate excessive bilirubin
Palor due to anemia
Jaundice+palor = lemon jello color
2) increased urobilinogen in urine, but urine color is normal since urobilinogen is
not a strong pigment (urine color turns dark when it contains bilirubin in the case
of conjugated hyperbilirubinemia)
Acholiuric jaundice
3) increase stercobiinogen » dark stool
4) increased LDH
5) decreased haptoglobin
- extravascular hemolysis » slight decrease
- intravascular hemolysis » very important decrease almost absent

B- Increased erythropoiesis
1) reticulocytosis
2) macrocytosis
3) polychromia
4) bone marrow biopsy: Hypercellularity

C- extra or intravascular
Extravascular : - splenomegaly
ExtraVascular hemolysis » macrophages have to work more » they undergo
workloads hyperplasia » splenomegaly
Intravascular
- important decrease of haptoglubinemia
- hemoglobinemia
- methemoglobinemia
- hemoglobinuria
- acute tubular necrosis

D- acute or chronic
In a chronic hemolysis
- gallbladder bilirubin stones
- hemosiderinuria : Proximal convoluted tubule cells finds a way to handle the
chronically taken small amounts of hemoglobin by expressing the genes of
apoferritine » ferritin » hemosiderin.
PCT cells loaded with hemosiderin when should into urine and can be stained by
prussian Blue.

Explanation of some points:

LDH is released when there is injury too many tissues, including RBC

Haptoglobin
Hemoglobin is very toxic this is why it is maintained within the RBC, if
hemoglobin is released into the circulation it is rapidly taken by haptoglobin which
is produced by liver to prevent free hemoglobinemia which is toxic.
In extravascular hemolysis, hemoglobin is further degraded into iron bilirubin
some of it only can leack to the circulation, while in intravascular hemolysis,
hemoglobin is directly released into circulation, bound to haptoglobin is will lead
to importance decrease in haptoglobin levels

Reticulocytes are bigger than mature RBCs

In reticulocytes, hemoglobin still being synthesized, there are still some mRNA and
ribosomes working. Ender super violet light, these will stain blue.

Retic means Network which refers to the network of mRNA and ribosomes that
stains blue

Rapidly released in a hurry fresh RBCs have increased size

Increased number of reticulocytes » macrocytosis MCV increased

In the circulation

Reticulocytes » blue

Mature RBCs » red

multiple color shades » polychromia

Intravascular hemolysis
Release of hemoglobin tetramers » dimers
The dimers can filter into urinary space :
- some of them are released into urine hemoglobinuria
- some are taken by proximal convoluted tubule cells, which once loaded with iron
will undergo acute tubular necrosis, acutely.

The Chronic uptake of mild amounts of hemoglobin by PCT cells give them the
ability to handle this iron by expressing the genes of apoferritin to store it as
hemosiderin.
Hemosiderinuria

Methemoglobinemia due to the oxidation of hemoglobin within the circulation

Hemolytic anemias classification

Hemolytic anemia, bring me to a breakdown of RBCs

- intrinsic or intracorpuscular
- extrinsic or extracorpuscular

A. Intra Corpuscular
#Inherited
1- Membrane defects
- heriditary spherocytosis
- hereditary elliptocytosis
2- enzymes defect
- glycolysis pathway Hexokinase and pyruvate kinase
- HMP pathway: G6PD and glutathione synthetase
3- hemoglobin defects
Globin synthesis defects
quantitative thalassemias
Qualitative hemoglobinopathies
#Acquired : Paroxysmal nocturnal hemoglobinuria

B. Extra corpuscular
1- immune mediated
ISO antibodies
- ABO incompatibility
- RH incompatibility
auto antibodies
- idiopathic
- infections
- malignancies
- drugs
2- Non immune
Mechanical causes
- cardiac mechanical valves
- DIC disseminated intravascular coagulation
- TTP thrombotic thrombocytopenic purpura
- March hemoglobinuria
Infections Malaria
Chemicals
Splenic sequestration

RBC is made of less cytoplasm an extra amount of membrane

Like a thin woman with an extra large dress.
In the inner part of the membrane there is a horizontal Network proteins which are
held with the membrane by vertical proteins.
These proteins make the cytoskeleton of RBC.

Membrane : Lipid bilayer

Horizontal network of cytoskeletal proteins
Vertical proteins bind the two

The horizontal network is very flexible allowing the RBC to twist as it passes
through narrow points
The vertical Network keeps the membrane held with the horizontal Network (the
large dress with elastics)
An inherited defect lead into mutated vertical proteins leads to hereditary
spherocytosis, the spherocytes will get stuck easily in narrow points and we'll be
prematurely destroyed.
If the defect is within the horizontal proteins it will lead to hereditary
elliptocytosis.

Spherocytosis can be due to anything that's damaged is the membrane of RBCs

Spherocytosis can be due to :
- hereditary spherocytosis
- IgG anti RBC, once coated by Ab, the RBC is captured by macrophages, but it
runs away leaving behind it the piece of membrane bound with Ab.
- venom with phospholipase activity
- clostridium infection releasing toxins with forceful lipase activity

In the RBC there are two important metabolic pathways

- Glycolysis pathway
- Hexose Monophosphate pathway
Which allows the production of NAD PH which gets oxydated to allow the
redaction of Glutathion.
The reduced form of glutathione prevents the oxidation of RBC proteins the RBC
is under oxidative stress.
HMP pathway is important to recycle reduced form of glutathione.
G6PD is an important enzyme in this pathways.
Glutathione synthetase is another enzyme required to make glutathione.

Paroxysmal nocturnal hemoglobinuria is an acquired cause of intrinsic hemolysis.

The nomenclature does not represent the characteristics of the disease which is
chronic, daytime also, hemoglobinuria is not alone (+WBC, +Plt)
RBCs, wBCs and the platelets, while circulating in the blood, they may pass
through harmful areas where complement system is activated, the compliment
May harm the membrane leading to hemolysis this is why these cells are coated
with special proteins which are complements cutters preventing the harmful effects
of complement.
In PNH, these proteins are defective.

Immune-mediated hemolysis
auto antibodies
Either by molecular mimicry or alteration of self antigens.

DIC
For some reason there is excessive activation of coagulation Cascade leading to
fibrin formation, fibrin within the blood vessels I e intravascular act like a knife to
RBC

TTP platelets within the blood vessels acting like a cutter for RBCs that try tobpass
through them

Splenic sequestration
When there is splenomegaly, RBCs have to go through increase number of cords of
Billroth there will be excessive destruction.

Notes taken by

Meddah Chahrazad Loubna