Hemoglobinopathies

What is a hemoglobinopathy?

A disease involving the HGB molecule, caused by genetic mutations affecting globin synthesis

What are the two main ways hemoglobinopathies affect hemoglobin synthesis?

Qualitatively and Quantitatively

What is Qualitatively hemoglobinopathies

It involve a structural defect due to an altered amino acid sequence but maintain a normal synthesis rate.

What is Quantitatively hemoglobinopathies

It result in decreased hemoglobin production but do not alter the amino acid sequence.

How are hemoglobinopathies classified?

• Structural defects (qualitative hemoglobinopathies): Hb S, Hb C

• Thalassemias (quantitative hemoglobinopathies)

Which chromosome contains the α-like globin genes, and which specific genes are they?

Chromosome 16 with Alpha and Zeta

Which chromosome contains the β-like globin genes, and which specific genes are they?

Chromosome 11 with Beta, Gamma, Delta, and Elipson

What is the basic composition of a human hemoglobin molecule?

4 globin chains and two alpha like chains and 2 beta like chains

Which globin genes are activated during the first 3 months of embryonic life, and what hemoglobin is produced?

Gower I Zeta and Epsilon

What changes in globin chain synthesis occur shortly after the first 3 months of embryonic development?

Gower II alpha 2 and epsilon 2 and Portland zeta 2 and gamma 2

What happens to globin chain synthesis later in fetal development?

HGB F Alpha 2 and Gamma 2

How does hemoglobin composition change during the first 6 months after birth?

HGB A Alpha 2 and Beta 2

What are the typical percentages of hemoglobins found in normal adults?

1. Hb A: 95%

2. HB A2: 3.5%

3. Hb F 1-2%

How does the sickle cell mutation affect red blood cells?

A single amino acid sub causes HGB to polymerize, forming long HGB crystals. These crystals stretch the RBC membrane, producing the crescent moon sickle shape

What happens in homozygous β-hemoglobinopathies?

Both B genes are mutated leading to the absence of normal Hb A for example Hb SS and CC

How do heterozygous β-hemoglobinopathies differ from homozygous forms?

One B gene is normal producing both normal Hb A and variant hemoglobin’s. This is present in lower amounts, leading to mild or no symptoms like: Hb AS and HB AC

How did Fishleder and Hoffman classify structural hemoglobins?

1. Hemolytic Anemia- associated hemoglobin Hb S

2. Methemoglobinemia associated hemoglobin Hb M

3. Hemoglobin’s with altered oxygen affinity (increase or decrease)

4. Clinical or functionally insignificant hemoglobin’s (no effect)

Which hemoglobinopathy is the most frequent and severe?

Hb S with sickle cell is the most common and severe structural hemoglobinopathy.

What advantage does sickle cell trait provide?

Resistance to Plasmodium falciparum malaria

How is sickle cell inherited?

As an autosomal codominant inheritance pattern

What genetic combinations result in sickle cell disease (SCD)?

• Hb SS (Sickle Cell Anemia) → Homozygous (S gene from both parents).

• Hb SC Disease → Compound heterozygous (S gene from one parent, C gene from the other).

• Hb S–β-thalassemia → Compound heterozygous (S gene from one parent, β-thalassemia gene from the other).

What genetic mutation causes Hb S?

When Glutamic acid is replaced by Valine at position 6 on the beta globin chain

How does the Hb S mutation affect hemoglobin charge?

• Glutamic acid has a net charge of -1.

• Valine has a net charge of 0.

• This substitution results in a +1 charge change, which alters the electrophoretic mobility of the hemoglobin molecule.

How does the amino acid substitution of sickle cell affect hemoglobin interactions?

The change from Glu to Val affects HGB solubility and molecular interactions, leading to polymerization of Hb S under low oxygen conditions, which contribute to erythrocyte sickling and disease patholgy

At what oxygen saturation levels does sickling occur in homozygotes

When O2 saturation drops to less than 85%

How does the formation of sickle cells affect blood flow?

It increases blood viscosity, which slows blood flow making it difficult got blood to circulate, particularly in small vessels

At what oxygen saturation levels does sickling occur in heterozygotes?

When O2 saturation drops to less than 40%

How does reduced oxygen tension, low pH, and increased 2,3-bisphosphoglycerate (2,3-BPG) contribute to sickling?

They reduce HGB oxygen affinity and expose HB S to hypoxia, making RBC more prone to sickling and polymerization

What is the end result of the sickling process?

It leads to the blocking of capillaries and arterioles by sickled RBC’s. leading to infraction or tissue damage in the surrounding areas due to lack of blood flow

What are reversible sickle cells?

They are Hb S RBC that change shape in response to oxygen tension

How do reversible sickle cells behave when fully oxygenated?

They circulate as normal biconcave discs

What happens to reversible sickle cells during deoxygenation?

HGB polymerization occurs, increasing viscosity and causing the RBC to change into sickle shapes

How do reversible sickle cells contribute to vasoocclusion?

They can travel into microvasculature when oxygenates, but as they deoxygenate they become distorted and viscous potentially causing blockage.

What are irreversible sickle cells?

Sickle cells that do not change shape regardless of oxygen tension of hemoglobin polymerization

How do irreversible sickle cells appear on a peripheral blood film?

They appear as elongated sickle cells (stay as sickle shape) with pointed ends on a peripheral blood film

How does the spleen react to irreversible sickle cells?

It recognizes them as abnormal and removes them from circulation

Why are irreversible sickle cells prevented from causing vasoocclusion?

Because they are removed from circulation by the spleen, irreversible sickle cells do not enter the microcirculation, preventing vasoocclusion

When do individuals with SCD typically start showing symptoms, and why?

After 7 months of life, due to mutated B chains being produced, replacing normal gamma chains. This causes Hb S to increase and Hb F to decrease

What complications can arise due to the increase in Hb S levels in SCD?

Progressive hemolytic anemia and splenomegaly

What are the different types of crises that can occur in individuals with SCD?

• Vasoocclusive or "painful" crisis

• Aplastic crisis

• Megaloblastic crisis

• Sequestration crisis

• Chronic hemolytic crisis

What is the hallmark of sickle cell disease (SCD)?

Vasoocclusive crisis

What factors can trigger a vasoocclusive crisis in SCD?

1. Acidosis

2. Hypoxia

3. Dehydration

4. Infection and Fever

5. Exposure to extreme cold

Where do painful episodes most often manifest in individuals with SCD during a vasoocclusive crisis?

1. Bones

2. Lungs

3. Liver

4. Spleen

5. Penis

6. Eyes

7. CNS

8. Urinary Tract

What percentage of SCD patients develop cutaneous manifestations such as ulcers or sores on the lower leg?

About 8%

What often triggers a sickle cell crisis

An acute upper respiratory infection or GI infection

What are common symptoms of a sickle cell crisis

1. Weakness

2. Anorexia

3. Fever

4. Vomiting

5. Pain in joints, back, and abdomen

What are the effects of chronic sickle cell

1. Growth retardation

2. Delayed sexual maturation

3. Decreased fertility

4. Priapism

What are the effects of repeated splenic infarcts in sickle cell disease?

It can lead to scarring, resulting in diminished splenic tissue and abnormal functions. This can lead to autosplenectomy

What is splenic sequestration in sickle cell disease

It is the sudden trapping of blood in the spleen, leading to a rapid decline in HGB levels

What is frequently the first symptom in sickle cell disease in young children

Dactylitis or vaso-occusive pain in hands and feet

How does splenic sequestration affect sickle cell patients' spleens and hemoglobin levels?

It causes rapid splenic enlargement and results in lower HBG levels

What is autosplenectomy, and how is it identified?

It refers to the gradual loss of splenic function in SCD, and is often identified by the presence of howell jolly and pappenheimer bodies in RBC on the PB film

How does pulmonary infarction affect sickle cell disease patients?

It occurs when sickling in the microvasculature of the lungs causes acute chest syndrome

What are the key characteristics of acute chest syndrome in sickle cell disease?

Fever, chest pain, and the presence of pulmonary infiltrates on chest radiographs

What is the leading cause of death among adults with sickle cell disease?

Acute chest syndrome

What are megaloblastic episodes in sickle cell disease, and how are they caused?

It happens from sudden arrest of erythropoiesis due to folate depletion

What are aplastic episodes in sickle cell disease, and what causes them?

They are life threatening hematologic complications in SCD, most commonly caused by viral infections like Parvovirus. They are characterized by bone marrow failure and the absence of retic’s to compensate for the decrease in RBC survival

How is the anemia in sickle cell disease classified morphologically?

As chronic hemolytic anemia, morphologically as normocytic and normochromic

What other cells and abnormalities can be observed in the peripheral blood film of a patient with sickle cell disease?

1. Poikilocytosis

2. Anisocytosis

3. Target cells

4. Nucleated RBC

5. Spherocytes

6. Basophilic stippling

7. Pappenheimer bodies

8. Howell-Jolly bodies

What is the hallmark of sickle cell disease in a peripheral blood film?

Sickle cells and Target cells

How does the RBC distribution width (RDW) appear in sickle cell disease?

It increases

How is the mean cell volume (MCV) affected in sickle cell disease despite an elevated reticulocyte count?

Normal

What does a decreased reticulocyte count in sickle cell disease suggest?

Aplastic crisis

What are the common findings in the leukocyte count of patients with sickle cell disease?

Moderate increase in WBC

What is a common finding in the platelet count of sickle cell disease patients?

Elevated

What bone marrow changes are typically observed in sickle cell disease?

Erythroid hyperplasia

What is the sodium metabisulfite test for detecting Hb S insolubility?

It involves inducing sickle cell formation on a glass slide by mixing a drop of blood with a drop of 2% sodium metabisulfite (a reducing agent) on the slide. The mixture was sealed under a coverslip, and the resultant sickle formation was identified microscopically

How does the sodium metabisulfite test for sickle cell disease work?

It reduces the HGB inside the RBC cells to its deoxygenated form, which induces polymerization and leads to sickle cell formation

What is the most common screening test for Hb S?

The tube HGB solubility test

How does the Tube hemoglobin solubility test work?

It works by capitalizing on the decreased solubility of deoxygenated Hb S in solution, which produces turbidity

What substances are used in the Tube hemoglobin solubility test?

Blood is added to a buffered salt solution containing a reduced agent like sodium dithionite and a detergent based lysing agent, saponin

What role does saponin play in the hemoglobin solubility test?

It dissolves the membrane lipids of the blood cells, causing the release of HGB

What is the role of dithionite in the hemoglobin solubility test?

It reduces the iron in HGB from the ferrous to the ferric oxidation state, which prevents oxygen binding and converts the HGB to the deoxygenated form

What happens when deoxygenated Hb S is present in the solution?

It polymerizes in the solution, which renders it turbid

What happens to the solution if nonsickling hemoglobins are present instead?

The solution remains clear

What can cause false-positive results in the hemoglobin solubility test for Hb S?

Hyperlipidemia

What can cause false-negative results in the hemoglobin solubility test for Hb S?

Low RBC and Low HCT

Which other hemoglobins can give a positive result on the hemoglobin solubility test?

1. Georgetown

2. Providence

3. Memphis

What is the basis of alkaline hemoglobin electrophoresis?

It is based on the separation of HGB molecules in an electric field, primarily due to differences in total molecular charge. HGB molecules assume a negative charge and migrate towards the anode (positive pole)

Why might hemoglobins that exhibit an abnormal electrophoretic pattern at an alkaline pH be subjected to electrophoresis at an acid pH?

HGB with similar charges may show the same pattern at an alkaline pH. Electrophoresis at an acid pH helps to clarify separate these HGB by altering their charge

How does Hb S behave during alkaline electrophoresis?

It migrates with Hb D and Hb G and Lepore

How does Hb S behave during acid electrophoresis?

It separates from Hb D and Hb G and Lepore

How does Hb C behave during alkaline electrophoresis?

It migrates with Hb E and Hb O

How does Hb C behave during acid electrophoresis?

It separates from Hb E and Hb O

What is the mainstay of therapy for sickle cell disease (SCD)?

Supportive care

What new therapies are evolving for sickle cell disease (SCD)?

1. Neonatal screening

2. Bone marrow transplant

3. Treatment with hydroxyurea and butyrate in adults

What do hydroxyurea and butyrate do

They induce production of HB F

How are painful episodes managed in sickle cell disease?

Hydration, oxygen therapy, or effective pain therapy

Why is bone marrow transplantation considered high risk for sickle cell patients

Due to drug toxicity

Under what conditions can individuals with sickle cell trait experience adverse events?

1. Severe hypoxia

2. Severe respiratory infections

3. Unpressurised flight at high altitudes

4. Anesthesia

What is the only consistent abnormality found in patients with sickle cell trait?

They are unable to concentrate urine

Which material is used for the alkaline electrophoresis for SCD, and at what pH

Cellulose Acetate at 8.4

Which material is used for the acid electrophoresis for SCD, and at what pH

Citrate Agar at 6.0-6.2

In what population is Hb C found most commonly

African American

What is the structural change in the hemoglobin molecule that defines Hemoglobin C?

It is defined by a sub of Lysine for Glutamic acid at position 6 of the B chain

How does Hemoglobin C polymerize and what is the resulting effect on red blood cells?

Hb C polymerizes under low oxygen tension and forms short, thick crystals.

How do the polymers of Hemoglobin C affect red blood cells differently than Hemoglobin S?

The short, thick crystals of Hb C do not alter RBC shape to the same extent, which leads to less splenic sequestration and hemolysis

Do individuals with Hemoglobin C experience vasoocclusive crises like those with Hemoglobin S?

No

What is the typical anemia seen in homozygous Hemoglobin C (Hb C) disease?

Mild to moderate, normochromic and normocytic anemia. With occasional microcytosis and mild hypochromia.

What is commonly observed in the peripheral blood of individuals with Hemoglobin C disease?

1. Increase Target cells

2. Slight-Mod increase in Retic

3. Nucleated RBC