Case 3 + 4: Alexandria Vardalos + Ms. LW

Hb: Types

HbA

HbA2

HbF

HbS

HbA

Adult Hb

Subunits: α₂β₂

• 2 alpha chains

• 2 beta chains

• Contain heme groups

O2 Binding Affinity: High

• 4 O2 molecules

Major HbA variant

• 96-98% of total Hb

HbA2

Adult Hb2

Subunits: α₂δ₂

• 2 alpha chains

• 2 delta chains

O2 Binding Affinity: Higher than HbA

• Minor HbA variant (2-3%)

HbF

Fetal Hb

Subunits: α₂γ₂

• 2 alpha chains

• 2 gamma chains

O2 Binding Affinity: Higher than HbA

HbS

Sickle Hb

Cause sickle cell disease

Subunits: α^A₂β^S₂

• 2 normal alpha chains

• 2 abnormal beta subunits

O2 Binding Affinity: Lower than HbA

• De-O2 form = Sickled rod shape = RBC distortion = RBC stick together

• Occlude blood vessels

Hb Electrophoresis

Quantify Hb types in blood using electrical current to separate (by charge)

Diagnose sickle cell anemia and thalassemia

Hb Electrophoresis: Procedure

1. Hemolyze blood sample

2. Add sample to gel electrophoresis buffer + apply electric field

3. Hb types separate by charge

• Neg Hb → Anode

4. Stain gel to visualize

• Greatest → Least Migration: HbA > HbF > HbS > HbA2 and Hb C

Erythropoiesis: Fetus

Mesoblastic Period: 2 weeks - 2 months

• Yolk Sac: Produce embryonic Hb

  • No survival into adult life

  • No O2 delivery

Hepatic Period: 2-7 months

• Liver: Produce HbF and RBC

• Spleen, Thymus, Lymph Nodes: Produce RBC and lymphocytes

Before Birth: 7-9 months

• Bone Marrow: Produce RBC, HbF, HbA

Erythropoiesis: Adult

After Birth: < 6 months

• Bone Marrow:

  • Decrease HbF production (decrease gamma chains)

  • Increase HbA production (increase beta chains)

Infant: 6+ months

• Bone Marrow: HbA > HbA2 > HbF

Adult:

• Intramedullary Hematopoiesis: Bone marrow (most)

• Extramedullary Hematopoiesis: Outside bone marrow

  • Liver and spleen = Hepatosplenomegaly

  • When needed: Bone marrow infiltration, tumour

Hb Destruction

1. RBC circulate for 120 days

• Biochemical and structural changes = Mark for clearance

2. RBCs phagocytosed by macrophages

• In Cells: Liver, spleen, bone marrow

• In Spleen: Filtration (older RBC undergo hemolysis, healthy RBC survive)

3. Hb degraded in macrophages

• Breakdown globin chains → Amino acids

• Breakdown heme moiety → Fe, biliverdin, CO

4. Fe bind transferrin → Transport to…

• Bone Marrow: Produce new RBCs

• Liver: Storage as ferritin

5. Biliverdin converted to bilirubin (yellow pigment)

• Converted to bile in liver → Excreted into intestines → Colour stool brown

6. CO exhaled from lungs

Bilirubin: Types

Direct: Measured conjugated bilirubin

• Water-soluble

• Increased = Hepatic dysfunction

Indirect: Unmeasured unconjugated bilirubin

• Lipid-soluble

• Increased = RBC breakdown/improper degradation

• Calculated from total bilirubin

Total: Measured

• Direct + indirect bilirubin

• Jaundice: High bilirubin in blood

O2-Hb Dissociation Curve

O2 binding to Hb as function of PO2

Sigmoidal Shape: Positive cooperativity

• O2 binding increases affinity of heme to bind additional O2

P50: PO2 of 50% Hb saturation

O2-Hb Dissociation Curve: In Lungs

PaO2 = 100 mmHg

100% saturation

• High Hb affinity for O2

• Tight binding to maximize O2 loading

Can tolerate O2 drops until 60 mmHg

O2-Hb Dissociation Curve: In Tissues

PVO2 = 40 mmHg

75 % saturation

• Decreased Hb affinity for O2

• Loose binding to increase O2 unloading

O2-Hb Dissociation Curve: Right Shift

Decreased Hb affinity for O2 = Increased unloading

• Decrease pH

• Increase temp

• Increase 2,3-biphosphoglycerate (BPG)

• Sickle cell anemia

Right Shift: Decrease pH

Increase metabolic activity = Increase CO2 = Decrease pH

Increase O2 unloading to tissues = Match O2 demand

Right Shift: Increase Temp

Increase metabolic activity = Increase heat

Increase O2 unloading to tissues = Match O2 demand

Right Shift: Increase 2,3-BPG

RBC glycolysis byproduct

• Increase during hypoxemia

Bind beta chains = Decrease affinity for O2

Right Shift: Sickle Cell Anemia

Increased 2,3-BPG binding + Sickled Hb chains = Decrease affinity for O2

O2-Hb Dissociation Curve: Left Shift

Increased Hb affinity for O2 = Decreased unloading

• Increase pH

• Decrease temp

• Decrease 2,3-BPG

• HbF

Left Shift: Increase pH

Decrease metabolic activity = Decrease CO2 = Increase pH

Low O2 demand = Decrease O2 unloading to tissues

Left Shift: Decrease Temp

Decrease metabolic activity = Decrease heat

Low O2 demand = Decrease O2 unloading to tissues

Left Shift: HbF

Decreased 2,3-BPG binding to gamma chains = Increase affinity for O2

Autosomal Recessive Inheritance

Require 2 copies of nonsex alleles to express phenotype

Usually more severe than autosomal dominant conditions

Autosomal Recessive Pedigree

1 Homozygous Parent: 0% expressed, 100% carriers

1 Heterozygous + 1 Homozygous Parent: 50% expressed, 50% carriers

Heterozygous Parents: 25% expressed, 50% carriers, 25% unaffected

Thalassemia: Description

Hereditary Hb disorder from:

• Alpha: Alpha-chain mutations

• Beta: Beta-chain mutations

Thalassemia: Epidemiology

Alpha: Common in Asia and Africa

Beta: Common in Mediterranean descent

Partial resistance to malaria

Thalassemia: Etiology

Autosomal recessive gene mutations

Alpha Thalassemia: Etiology

Deletion of alleles in alpha-globin gene on chromosome 16

Severity depend on number of defective alleles

• Silent Carrier: 1 defective allele

• Alpha-Thalassemia Trait: 2 defective alleles

• Hb H Disease: 3 defective alleles

  • Increase HbH production (unstable beta chain = Aggregate in RBC)

• Hb Bart’s Disease: 4 defective alleles

  • Increase Hb Bart production (4 gamma chains)

Beta Thalassemia: Etiology

Point mutation in beta-globin locus on chromosome 11

Minor: 1 defective allele

Major (Cooley Anemia): 2 defective alleles

Thalassemia: Pathophysiology

Anemia from:

• Decreased erythropoiesis

• Increased hemolysis

Thalassemia Pathophysiology: Decreased Erythropoiesis

Beta:

• Decreased beta chain synthesis = Increase gamma/delta chains = Increase HbF (infant) → HbA2 (adult)

• Low HbA and high HbA2 = Decreased erythropoiesis

Alpha:

• Decreased alpha chain synthesis = Low alpha chain pairing with beta/gamma chains = Increase free beta/gamma chains = Increase HbH and Hb Bart

• Low HbA = Decreased erythropoiesis

Thalassemia Pathophysiology: Increased Hemolysis

Low alpha/beta chains = Compensatory overproduction of other chains = Precipitation form inclusions in RBCs = Hemolysis

Alpha Thalassemia: Clinical Presentation

Silent Carrier: No anemia

Alpha-Thalassemia Trait: No/mild anemia

Hb H Disease:

• Jaundice

• Anemia (chronic hemolytic)

• Hepatosplenomegaly

Hb Bart’s Disease: Incompatible with life

• Intrauterine ascites

• Edema

• Hepatosplenomegaly

• Cardiac + skeletal anomalies

Beta Thalassemia: Clinical Presentation

Minor: No/mild anemia

Intermedia:

• Variable anemia

• Jaundice

Major:

• Severe hemolytic anemia

• Hepatosplenomegaly

• Growth retardation

• Skeletal deformities

Thalassemia: Investigations

CBC

Hemolysis evaluation

PBS

Hb electrophoresis

*Genetic studies

Thalassemia: CBC

Microcytic hypochromic anemia

**Normal RDW

*High RBC

Mentzer Index: MCV/RBC ratio

• Thalassemia: < 13

• IDA: > 13

Thalassemia: Hemolysis Evaluation

Low haptoglobin

• Plasma glycoprotein binding free Hb for clearance

High lactate dehydrogenase

High reticulocytes

Hyperbilirubinemia

Normal Fe studies

Thalassemia: PBS

*Target cells

Dacrocytes (teardrop cells)

*Anisocytosis (diff/abnormal RBC sizes) and poikilocytosis (diff/abnormal RBC shapes)

**Inclusion bodies

**Erythroblasts

Thalassemia: Hb Electrophoresis

Beta:

• Increased HbA2 and HbF

• Less HbA

Alpha:

• Normal

• HbH/Hb Bart’s

Thalassemia: Genetic Studies

Determine diagnosis and mutations

Thalassemia: Treatment/Management

General:

• Patient education

• Genetic counseling

• Screening for relatives

Minor:

• No treatment

• *Folic acid supplements

Major/Intermedia:

• Transfusion

  • Hb < 7 g/dL

  • Target: Hb > 9-10 g/dL

• ***Splenectomy

• Allogenic HSC transplant

  • Stem cells from genetically similar donor

• Gene therapy

Thalassemia: Complications

Fe overload

Hypercoagulopathy

Sickle Cell Disease (SCD): Description

Genetic disorders causing HbS production

• HbS polymerization

• Vasoocclusion

• Hemolytic anemia

SCD: Epidemiology

Most common in African and Eastern Mediterranean descent

SCD: Etiology

Autosomal recessive point mutations in beta-globin gene on chromosome 11

Glutamic Acid → Valine: HbS

• Heterozygotes (HbAS): 1 normal allele + 1 sickle allele → Sickle cell trait (carrier)

• Homozygotes (HbSS): 2 sickle alleles → Sickle cell anemia

Glutamic Acid → Lysine: HbC

• Heterozygotes

  • HbSC: 1 HbS allele + 1 HbC allele → HbSC disease

    • More severe than sickle cell trait

    • Less severe than SCD

  • HbAC: 1 normal allele + 1 HbC allele → HbC trait (carrier)

• Homozygotes (HbCC): 2 HbC alleles → HbC disease

SCD: Pathophysiology

1. Deoxygenated HbS = Polymerization = Sickled RBCs

• Deoxygenation from low O2 tension:

  • Infections

  • Dehydration

  • Acidosis

  • Stress

2. Sickle cells = Low elasticity + Adhere to vascular endothelium = Occlude blood vessels = Tissue infarction

3. Increased RBC hemolysis = Anemia

• Increase HbF production to compensate

SCD: Clinical Presentation

Sickle cell trait

• Asymptomatic

• Hematuria (papillary necrosis)

• Nocturia

• Renal medullary carcinoma

SCD: Start after 3-6 months from decreasing HbF and increasing HbS

• Vascular occlusion

  • Dactylitis (fingers/toes inflammation)

  • Severe pain

• Increased infections

• Acute hemolytic crisis

  • Splenic sequestration: Splenic vasoocclusion (blood trapping) cause pain and hypotension

  • Aplastic crisis: Mass hemolytic anemia

• Chronic hemolytic anemia

• Pigmented kidney stones

SCD: Investigations

Neonatal screening

Confirmatory studies: < 2 months

• Hb electrophoresis

• High performance liquid chromatography

• Capillary electrophoresis

CBC

PBS

SCD: Neonatal Screening

Positive and inconclusive results → Confirmatory studies

SCD: Hb Electrophoresis

HbS and HbC findings

SCD: High Performance Liquid Chromatography

Low HbA

High HbS

High HbF

SCD: CBC

Anemia with reticulocytes

Increased neutrophils and platelets

SCD: PBS

Crescent-shaped sickle RBCs

Howel-Jolly Bodies: Basophilic DNA remnants in immature RBCs

• Indicate splenic dysfunction

SCD: Treatment/Management

Vaccines

Prophylactic penicillin

Hydroxyurea therapy

RBC transfusion

Allogenic HSC transplant

SCD Management: Vaccines

Prevent infections

SCD Management: Prophylactic Penicillin

Children < 5 years

Prevent pneumococcal infection

SCD Management: Hydroxyurea Therapy

Antineoplastic drug prevent cell proliferation

Increase erythropoiesis + HbF levels = Decrease HbS and sickle cell crises

SCD Management: RBC Transfusion

Simple Transfusion: Dilute patient blood with donor erythrocytes

• Preferred to manage acute anemia

Exchange Transfusion: Remove patient blood and replace with donor erythrocytes

• Preferred to prevent vasoocclusive events

Indications: Acute complications

Risks:

• Simple:

  • Increase Hct levels = Increase viscosity = Increase vasoosclusive event risk

  • Fe overload

• Exchange: Increase alloimmunization (immune response against foreign antigens) risk

SCD Management: Allogenic HSC Transplant

Indications:

• Sickle cell anemia (HBSS)

• Increased complications

SCD: Complications

Recurring vascular occlusion = Infarction = Organ damage + function loss

• Homozygotes: High morbidity and mortality

• Heterozygotes: Rare

*Hematuria from papillary necrosis

Stroke

**Decreased phagocytosis in spleen = Infection risk