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Hemoglobinopathies

Hemoglobinopathies (Qualitative)

Hereditary Disorders Abnormal Hgb Structure > 300 Types Abnormal Hgb (Hgb S Disorders - Most Prevalent Molecular abnormalities in hemoglobinopathies

Substitution of one amino acid for another (most common) Substitution of more than one amino acid Deletion of one or more amino acids Fusions of hemoglobin chains Extension of an amino acid chain

Possible Consequences of a Hemoglobinopathy


No detectable effect Instability of Hgb molecule (Unstable Hgb) Increase or a decrease in O2 affinity Inability to maintain iron in its reduced state (methemoglobinemia) Dec solubility of hgb molecule

Hemoglobin M Disease

Substitution at either proximal or distal histidine loci (his tyr) iron-phenolate complex Fe+++ state is incapable of binding O2 cyanosis Methemoglobinemia

Unstable Hemoglobinopathies

Mostly mutation in heme pocket region


H2O gains access to hydrophobic region Result: heme instability, denaturation & release of heme from its binding site

Heinz Bodies evidence of an unstable hgb mutant

Hemoglobinopathy Altering Oxygen Affinity

Increased O2 Affinity: Stabilization of Oxyconformation = inc O2 affinity Confirmed by left shift O2 Saturation Curve Typically exhibit erythrocytosis

Decreased O2 Affinity: Stabilization of Deoxyconformation = dec O2 affinity Confirmed by right shift O2 Saturation Curve Typically somewhat anemic

HEMOGLOBINOPATHIES

Hb E (GLULYSINE 26TH BETA)


AGAR GEL E AT ACID Ph UNSTABLE IN OXIDANT EXPOSURE Very low MCV (55-65 fL)

Few

to many codocytes

Hb M
OR B- CHAIN MUTATION IN HIS TYR RESIDUE OF HEME POCKET Autooxidation of iron to Fe3+) methemoglobin (+) SCHUMMS TEST
A

Hgb C-Harlem (C-Georgetown) double sub at B chain


6th valine for glutamic acid 73rd aspartic acid for asparagine (Korle Bu mutation) of beta & alpha chain variants that migrate in an alkaline pH at the same electrophoretic position as HgB S

Hgb D & G:
Group

Hgb O-Arab:
121st

B chain lysine for glutamic acid Mild hemolytic anemia; many codocytes The only HgB to move just slightly away from the point of application toward the cathode on citrate agar at an acidic pH

Clinically Significant Variant Hemoglobins


Hemoglobinopathy Hemoglobin S Hemoglobin C Hemoglobin E Hemoglobin Constant Spring Hemoglobin H a2bS2 a2bC2 a2bE2 Alpha Normal Normal Normal Beta Defective Defective Defective Normal Normal Hemolytic anemia, splenomegaly, target cells are characteristic Benign, common in SE Asia Long alpha chain Paucity of alpha chains Screening test: Heat instability test Comments

HCSpr Defective b4 Absent

Hemoglobin Barts
Hemoglobin M

g4

Absent
Normal

Normal
Normal

Not compatible with life


A group of abnormal hemoglobins in which a single amino acid substitution favors the formation of methemoglobin.

HEREDITARY PERSISTENCE OF Hb F

PARTIAL/ TOTAL SUPPRESSION OF BETA & DELTA CHAINS


Lab Dx:
ALKALI

DENATURATION TEST KLEIHAUER- BETKE (ACID DENATURATION TEST)

KLEIHAUER- BETKE (ACID DENATURATION / Elution TEST)


Principle: Detects fetal-maternal hemorrhage Blood smear + acid buffer (adult Hgb loses Hgb into the buffer, only the stroma (cell membrane) is left Fetal RBCs unaffected & retain Hgb

Fetal cells: bright pink Adult cells: "ghost cells


Cord blood is used as the positive control

Hemoglobinopathy S

AR Chronic hemolytic anemia Pallor, Jaundice, Dark urine, HSM Types:


Homozygous

(Disease

or Anemia) Heterozygous (Trait) SC, SD, STHALASSEMIA

Homozygous: sickling begins when O2 saturation < 85% Heterozygous: sickling begins when O2 saturation < 40%

Hemoglobin S Disorders

Discovered 1904 man from Grenada Point Mutation of B Globin Gene (146 aa of B globin chain) Incidence:

8% in American Blacks 30% in Some African

Confers resistance to P. falciparum (Hgb S Trait)

Complications

Hemolytic Crisis Hyperhemolytic crisis Thrombotic crisis


Heart infarction CNS hemiplegia Mesentery abd. pain

Sequestration crisis: Destruction of RBC in liver & spleen shock HSM Aplastic crisis: Aplasia of BM

Hand-Foot Syndrome: children < 3 y/o w/ sickle cell anemia exhibit painful swelling in the hands and feet

4 triggers of sickle cell crisis


Acidosis Hypoxia Infection Fever

Sickle Cell Anemia Lab Findings:

BM: erythroid hyperplasia PBS:


Marked poikilocytosis & anisocytosis Drepanocytes NRBCs Basophilic stippling Pappenheimer bodies Howell-jolly bodies Moderate to marked polychromasia

Retic: 10-15% High plt count & moderate neutrophilia Low ESR Low osmotic fragility test

HEMOGLOBINOPATHY S LAB DX:

PBS: SICKLE, BRONZE ELLIPTOCYTES; Normocytic Normochromic Reticulocytic count BM exam: erythroid hyperplasia

HEMOGLOBINOPATHY S LAB DX:

SOLUBILITY TESTS (Na DITHIONATE Test)

Sickle Cell screening test Hgb <7g/dL Use of 10 x 75mm tube, instead of 12 x 75 Infants <6 months of age

FALSE NEGATIVE in:


FALSE POSITIVE in:

Lipemia Very high WBC

Sickle Cell Disorders Diagnosis:

(+) SICKLING PHENOMENON (Na METABISULFIDE TEST) Sickledex - In Vitro Sickling After Adding Reducing Agt
Normal RBCs Sickled RBCs

Sickle Cell Disorders Diagnosis:

Hgb Electrophoresis: ID Hgb S (Hb S- INC/ Hb F- 1- 10% / Hb A2- N)


Sickle

Cell confirmation test

DNA Analysis Prenatal Testing

Treatment

During attacks:

Rest in bed Hydration Sedation Antibiotics Transfusion

Between attacks:

Transfusion Iron chelating agents Splenectomy Hydroxyurea Butyrate Increases proportion of HgB F in adults

Reactivate Fetal Hemoglobin Production using Hydroxyurea Chemical inhibition of Hb S polymerization Increase in intracellular hydration Altering RBC/Endothelial cell interactions BMT Gene therapy

Prognosis: Improve with age

HEMOGLOBINOPATHY C: Hb C (GLULYSINE 6TH BETA)

HGB C-A TRAIT:


ASYMPTOMATIC

Heterozygous HgB AC:


HgB

C (30%) HgB A (60%)

HEMOGLOBINOPATHIES

HGB C-C DISEASE:


NN anemia Marked increased in codocytes (target cells) Tetragonal crystals of HgB C on blood smear Marked increase in retics Very little to no HgB A HgB C (90%) HgB F (7%) HgB A2 (3%)

Homozygous HgB CC:


HgB SC lab findings:

NN anemia HgB: 11 - 13 g/dL Retic: 3 - 5% PBS: Few sickle cells, codocytes, & intraerythrocytic crystalline structures
Moderate hemolytic anemia Moderate splenomegaly Sodium dithionite test (+)

WWW Sites of Interest

Joint Center for Sickle Cell and Thalassemic Disorders: http://wwwrics.bwh.harvard.edu/sickle/ (Overview of sickle cell disease, thalassemia and iron kinetics) The Sickle Cell Information Center, Emory University: http://www.emory.edu:80/PEDS/SICKLE/ (Includes PowerPoint presentations on sickle cell disease)