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Hemolytic anemias

Hereditary spherocytosis
G6PD deficiency

Erythrocyte membrane
15 major proteins
Integral
Cross lipid bilayer
Glycophorins, Rh proteins, band 3, ATP-ases

Peripheral
Inside the lipid bilayer (Membrane skeleton)
Membrane skeleton provides support to lipid bilayer
Spectrin is major protein of membrane skeleton
Alpha and beta chains
These chains intertwine to form heterodimers

Ankyrin binds spectrin to Band 3

Erythrocyte membrane

Erythrocyte membrane defects


Spherocytes
Hereditary spherocytosis
Autoimmune hemolysis

Elliptocytes
Hereditary elliptocytosis

Poikilocytes
Hereditary elliptocytosis

Stomatocytes
Vinca alkaloids, Alcohol

Target cells
Iron deficinecy,
Thalassemias
Hepatic dysfunction

Acanthocytes
Abetalipoprotienemia

Echinocytes
Uremia
Vitamin K deficiency

Normal RBCs

Hereditary spherocytosis
Erythrocyte membrane disorder characterized by

Loss of vertical interaction of erythrocyte membrane


RBCs assume a spherical shape
Spherical RBCs are sequestered by intact spleen
Intact spleen enlarges, and destroys RBCs (Hemolysis)
This leads to anemia, jaundice, splenomegaly
Peripheral smear shows spherical RBCs
Spherical RBCs are fragile (increased osmotic fragility)
RBC life span is about 10-20 days

Mostly autosomal dominant (75%)

Normal

membrane

cytoskeleton
Hereditary spherocytosis

Hereditary spherocytosis: Pathogenesis


Molecularly heterogenous disorder
Membrane defects

Spectrin deficiency
Ankyrin deficiency
Band 3 deficiency
Combined deficiency

Erythrocyte abnormalities
Increased Na and K flux across membrane
RBCs are dehydrated, and less deformable
This makes RBCs more fragile

Hereditary spherocytosis: Pathogenesis


Splenic sequestration
Spherocytes are unable to move through splenic
sinusoids due to impaired deformability
These abnormal cells get entrapped and then
lysed
If spleen is removed, hemolysis does not take
place.

Pathogenesis of HS
Spectrin,ankyrin,band 3 defect
Decreased spectrin incorporation into
membrane/band 3 deficiency
Destabilisation of lipid bilayer
Microspherocytosis-Decreased RBC deformability
Stagnation in splenic cords and contact with
macrophages
Phagocytosis of spherocytes/Conditioning of
spherocytes
Further loss of membrane surface area

Pathogenesis of HS

ATP depletion
Increased glycolysis
Decreased 2,3 dpg,Ph falls
Passive cation leak
Increased Na/K Pump activity
Water leak and cell dehydration

Clinical features
Pallor
Icterus
Splenomegaly

Lab findings
Peripheral Smear :
Spherocytes
Reticulocytosis
Nucleated RBCs
Bone marrow :
Erythroid hyperplasia
Serum Iron Increased
Decreased MCV,Increased MCHC
Hemosiderosis
Unconjugated bilirubin increased
Osmotic fragility test - hemolysis

Spherocytes
Formed by partial phagocytosis
Decreased deformability
Denser, smaller (in appearance!), round RBC
without central pallor

100

% of hemolysis

75
50
25

80 70 60 50 40

30

% of NaCl

20

10

Molecular studies
Quantification of major proteins using PAGE
Mutation screening/Direct DNA sequencing

Complications
Worsening anemia
Aplastic crisis
Exhaustion of folate reserves

Choilelithiasis
Bilirubin pigment stones

Splenectomy is mainstay of treatment

Erythrocyte metabolism
Glucose is the main substrate of red cells
Two pathways
Glycolytic/Energy producing pathway
HMP Shunt/Protective pathway

Major products of glycolytic pathway: ATP, 2-3


DPG NADH
Major product of HMP pathway: NADPH

Classification of enzyme disorders


Disorders of HMP Shunt and glutathione
metabolism
G6PD def
Glutathione reductase def
Glutathione peroxidase def

Glycolytic enzyme abnormalities


Abnormalities of purine and pyrimidine
metabolism
Pyrimidine 5 Nucleotidase deficiency
ADA excess
Adenylate kinase (

Glucose-6-Phosphate Dehydrogenase
Deficiency
Basic defect
Inability of red cells to protect themselves
against oxidative injuries
Leading to hemolytic disease
Abnormalities in the hexose monophosphate
shunt or glutathione metabolism

Glucose-6-Phosphate Dehydrogenase
Deficiency
Variants
G6PD B Normal variant
G6PD A- 10% of African Americans
G6PD Mediterranean-clinically significant
hemolytic anemias

Protective effect against Plasmodium


falciparum malaria
X- Linked recessive
Males at highest risk

G6PD variants
Class I (Severe deficiency,chronic hemolytic
anemia
Class II (Severe def, intermittent hemolysis)
Class III (Moderate def, intermittent
hemolysis)
Class IV (No def)
Class V (Increased activity)

Activity of G6PD decrease with aging red cells


Normal half life of enzyme is 62 days
Normal old red cells have sufficient NADPH
G6PD Variants associated with hemolysis have
much shorter half life

Glucose-6-Phosphate Dehydrogenase
Deficiency
Clinical patterns1. Foods- fava beans (favism),
2. Medications - ***antimalarials (e.g.,
primaquine and chloroquine), sulfonamides,
nitrofurantoins,
3. Infections- viral hepatitis, pneumonia, and
typhoid fever
Hemolysis causes both intravascular and
Extravascular lysis
after exposure to oxidant stress

Glucose-6-Phosphate Dehydrogenase
Deficiency
Why do the red cells die?

They cant reduce peroxides

Peroxides attack hemoglobin bonds


Heme breaks away from globin
Globin denatures and sticks to RBC membrane, forming
Heinz body which may lead to intra-vascular hemolysis

Remaining cells are trapped in splenic cords


Macrophages bite out Heinz bodies, leaving cell fragile
and deformed may lead to extravascular hemolysis

Clinical and hematologic features


Acute hemolytic anemia after oxidant stress
pallour,jaundice,dark urine,abdominal pain
Bite cells and blister cells on peripheral smear
Heinz bodies on supravital stains
Features related to chronic hemolysis
(splenomegaly,cholelithiasis absent)
Congenital Nonspherocytic Hemolytic anemia

Glucose-6-phosphate dehydrogenase deficiency

Bite cells and Heinz Bodies

Lab diagnosis
Spectrophotometrically measure NADPH
generation (hemolysate+NADP+G6P)
Flourescent spot test
Methemoglobin reduction test using
methylene blue as hydrogen acceptor