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

Hereditary spherocytosis

membrane

cytoskeleton

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

80 50 70 60 30 40 20 10

25

100

75

50

% of NaCl

% o

f hem

oly

sis

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 patterns-

1. 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

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

Why do the red cells die?

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