Priniciple of Electrophersis

• Hemolysate is prepared • Globin is run on a gel at acid and alkaline ph Millicent Sutton MD October 28, 2005 • Mobility base on charge

NORMAL

• Consist of 2 alpha chains and 2 non alpha chains •Hb A = α2β2 •Hb F= α 2γ2 •Hb A2 = α2δ2

Hemoglobin Variants Abnormal Hb variant

Altered the conformational dynamic of • Hemolysate run on HPLC column (cation) globin: globin interaction • Molecule is split into heme and globin • Globins are dissociated Altered oxygen affinity but not denatured • Globins eluted based on mobility (charge Altered the rheology and physiology of the difference) red cell • As Hb elutes it pass thru a photometer generate a chromatogram

1 Genetics of

• Autosomal recessive inheritance • 8-10% African American (AA) carry the Hb S gene • Gene arose as evolutionary selection for resistance to falciparum malaria • Approximately 1in 600 AA are affected with SCD • The offspring of two carriers has 1:4 chance being affected

New Born Screening Hb S

• New York State one • Hb S tends to aggregates (valine) of 43 states that test • Deoxygenated Hb S in solution forms gel for hemoglobin variant as part of causes elongation of the cells and newborn screening resulting in a distorted red cell •NYS Newborn • Sickling is also influenced by amount and screening for sickle type of Hb in the cell hemoglobin began in • 2,3 DPG 1976

Molecular Genetics

• SSD is due to the substitution of thymine for adenine in the glutamic acid DNA codon (GAG ↑GTG) resulting in valine substitutiion at the β6 position •HbS= α2β2 (6glu↑val)

2 Clinical Laboratory SCD Role of Leukocytes

• Chronic hemolytic • Suggestion by clinical observation • Steady state Hb 5-11gm/dl -Severity of disease • Normochromic anemia -Increased leukocytes associated with • Reticulocytosis increased mortality • Normal MCHC -Increased silent infarcts • Decrease amount of erythropoeitin relative • Neutrophils binds sickle cell in vitro to degree of anemia

Natural History of SCD

Table 1. Sickle Hemoglobinopathies: Neonatal Screening and Diagnostic Test Results.

Neonatal screening Hematologic studies by 2 years results • FIRST DECADE • SECOND MCV2 3 Hb A2 Hb F Hb F (%) (%) distribution • Infection/Sepsis DECADE

Disorder • Cerebral infarct • Chronic organ SS FS N or I <3.6 <25 Heterocellular SC FSC N or D NA <15 NA •ACS damage S beta-thal FSA or FS 7 N or D >3.6 <25 NA S betao-thal FS D >3.6 <25 Heterocellular • Arthropathy • Renal disease S delta/beta--thal FS D <2.5 <25 Heterocellular S-HPFH FS N or D <2.5 <25 Pancellular • Cholelithiasis • Leg ulcers Hb = hemoglobin, thal = , N = normal, I = increased, D= decreased • Priapism • Neurologic disease • Poor growth • Sudden death /Development

New Model of Vaso-occlusion Functional asplenia

• Unlike normal red • The red pulp of the spleen is hypoxic and cells sickle cells and acidotic and favors sickling . adhere to the endothelium cells in • Sickle cells lack the deformability vitro necessary for passage through the splenic • Membrane damage sinus gaps. induces oxygen • Sluggish flow, increased viscosity and low radical generation O2 and pH lead to chronic congestion of • Increased expression the red pulp and results in multiple micro- of adhesion infarcts and hemorrhages. molecules

3 Splenic Sequestration Transcranial Doppler (TCD) in • Occurs when there is rapid enlargement SCD of the spleen accompanied by an acute fall in hemoglobin. • A measure of cerebral blood flood velocity • The mechanism for the triggering of • Abnormal TCD predicts increased risk of these episodes are not clear but be stroke in SCD. accompanied by viral illnesses • Velocity in excess of 200cm/sec is • 10-20% of children with sickle cell associated with increased risk of stroke. disease between the ages of 6 mons to 3 years will have at least one episode.

Stroke in Sickle Cell Disease: Prevention of Stroke in SCD • STOP I (Stroke Prevention) Study :A • Affects as many as 15-25% of patients randomized clinical trial of patients with with SS disease (rare in Hgb SC) abnormal TCD, compared chronic • Hemorrhage or thrombosis transfusion to reduce Hb S level ≤ 30% • Peak age for cerebral thrombosis and to a non transfusion arm. infarction is 7-10 years • There were 92% fewer strokes in the • Subarachnoid hemorrhage more transfusion arm compared to the non common in the second decade. transfused arm.

Patho-physiology of Stroke Prevention of Stroke in SCD in Sickle Cell Disease: • Intimal and medial hyperplasia and STOP II proliferation secondary to damaged • Patients with abnormal TCD who received endothelial from sickle cells. transfusion therapy for 36 months • Narrowing or complete occlusion of the randomized to continue or discontinue Tx cerebral vessels; anterior and and/or after 36 months. middle cerebral arteries. • Study was halted early because of • Multi-vessel involvement is common even increase stroke in discontinued Tx arm. in patients will unilateral findings.

4 Acute Chest Syndrome Indications for Episodic Transfusion

•ACS • Pulmonary disease (ACS) is the second • Surgery most common reason for admission to the • Erythroid aplasia hospital in patients with SCD. • Splenic/ Hepatic sequestration • It accounts for 25-40% of premature • Intractable Pain deaths in SCD. • Priaprism

Indications: Chronic Transfusion

Patho-physiology of ACS: • Primary Stroke Prevention Pathogenesis • Prevention of recurrent stroke

Endothelial Injury • Intractable pain (Cytokines) • Symptomatic anemia Increased Permeability Vasospasm Platelet/WBC activation (Pulmonary Edema) Pulmonary Hypertension Thrombosis/Inflammation • Pulmonary Hypertension/Sickle Cell Chronic Lung • Recurrent Splenic Sequestration

Transfusion for SCD Exchange Transfusion

• Goals • Advantages -Improve anemia and oxygen carrying capacity Fast : allows emergent intervention -Reduce or prevent the occurrence of the Eliminates risks of increase in viscosity and complications volume -Reduce Hb S levels • Methods Decreased iron loading compared to simple transfusion Episodic Chronic • Major role Exchange Treatement/prevention of life threatening events

5 Complications of Transfusion Hydroxyurea

• Hydroxyurea (HU) is widely used in the • Transfusion reactions treatment of sickle cell disease (SCD) in the US and abroad. • Iron Overload • The Multi-center Hydroxyurea Study (MSH), a • Alloimmunization randomized clinical trials demonstrated a • Hypersplenism reduction in frequency and severity of ACS and vaso-occlusive crisis by 30% and 50% • Hyperviscosity respectively. • Transfusion transmitted infections • Clinical benefit in the HU treated patients correlated with cyto-reductive effects

Pharmacological Agents that MSH follow-up Study Stimulate Fetal Hemoglobin Production 299 of the 500 patients enrolled in the • 5-Azacytidine original MSH study •Hydroxyurea 9 year follow- up period • Butyrate • Erythropoietin • -40% decrease in mortality in patients • Combinations treated with HU for at least 3 yrs • -28% of all deaths were pulmonary

Why is Fetal Hemoglobin Beneficial PHT : Risk Factor of death in SCD in Sickle Cell Disease Gladwin et al NEJM 350:9 2004 • Hb F interferes with polymerization of Hb S in -Prevalent in 32 % adult pts with vitro. SCD • Infants with SCD do not have complications during the first 6 months of life. -Development of PHT in SCD • SCD patients from Saudi Arabia and India with high Hb F levels have milder clinical disease. associated with anemia, chronic • CSSCD showed an inverse correlation between hemolysis and either existing Hb F levels and clinical severity cardiac or renal disease - Hb F levels or HU therapy did not appear to have protective effect.

6 Summary of HU + BU Combination Therapy Beta Globin Gene Expression

• Genes arranged in the order they are 40 expressed during 35 30 development 25 Baseline • Developmental 20

Hb F HU switching of human 15 % HU+BU globin 10

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0 12 3

Patho-physiology Thalassemia Clinical Manifestation

• Disease results from • When Hb F levels fall which are normal and imbalance in the offset by increased Hb A levels synthesis of alpha and beta globin Findings: chains Extra-medullary hematopoeisis Hemolysis Hyperbilirubinemia Congestive heart failure.

Molecular Genetics of Thalassemia Types of Beta

• There are over 200+ different thalassemia Type Hb levels Clinical mutations. Features • from β0β0 Hb F 100% Tx dependent a single base substitution Thal Major β+ β0 Hb F +/- Tx 40-80% Thal intermedia β+ β+ Hb F +/- Tx Thal intermedia 30-95%

7 Complications of Disease Transfusion therapy

• Bone expansion • Short stature • 10-20 cc/kg every 4-6 weeks to maiantin • Osteoprosis/Arthopathy Hb in 9.5-11 gm/dl range. • Delayed puberty • Endocrinopathy/Hypopituitary • Suppression of the erythropoeitic drive -IDDM, Hypothyrodism, Hypo • Decrease GI iron absorption, less bone -Testicular and ovarian failure demineralization and • Cardiomyopathy • Splenectomy can reduce transfusion • Pulmonary hypertension requirements.

Therapy Iron Balance in Thalassemia

Transfusion:

Mainstay of the therapy to correct the anemia.

Suppress the ineffective erythropoiesis

Prevent excessive marrow expansion

When to transfuse ? Other Therapies for Thalassemia

• Thal intermedia Hb levels 6-9 gm/dl • Thal Major Hb levels 3-4 gm/dl • Erythropoetin • Fetal Hb Augmentation Hb Level that are insufficient and that lead • Antioxidant to cardiovascular compromise, impaired Curative: BMT/ HPSC linear growth and excessive marrow expansion.

8 Support Therapies

• Chelation Therapy Subcutaneous Desferoxamine Deferipone, L1 Defarasirox (oral chelator) • Osteoclast repelacment therapy bisphosphonates and Vitamin D

• Hormonal repalcement

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