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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

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

dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs
dysfunction • Acanthocytes – Abetalipoprotienemia • Echinocytes – Uremia – Vitamin K deficiency Normal RBCs

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

Normal membrane cytoskeleton

cytoskeleton

Normal membrane cytoskeleton
Normal membrane cytoskeleton

Hereditary spherocytosis

Hereditary spherocytosis
Hereditary spherocytosis
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

Lab findings • Peripheral Smear : Spherocytes Reticulocytosis Nucleated RBCs • Bone marrow : Erythroid hyperplasia

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

phagocytosis ” – Decreased deformability – Denser, smaller (in appearance!), round RBC without central pallor
100 % of hemolysis 75 50 25 80 70 60 50 40 30 20 10
100 % of hemolysis 75 50 25 80 70 60 50 40 30 20 10
100 % of hemolysis 75 50 25 80 70 60 50 40 30 20 10
100
% of hemolysis
75
50
25
80
70
60
50
40
30
20
10

% of NaCl

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

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 patterns-

1. Foods- fava beans (favism),

2. Medications - ***antimalarials (e.g.,

primaquine and chloroquine), sulfonamides,

nitrofurantoins,

3. Infections- viral hepatitis, pneumonia, and

typhoid fever

Hemolysiscauses 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

Glucose-6-phosphate dehydrogenase deficiency

Bite cells and Heinz Bodies

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