1394330130hs and g6pd.pdf

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 can’t 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