Free ASCP MLS Exam Practice Questions Mock Test: Part 40 – Hemoglobinopathies & Thalassemia

The dithionite solubility test (Sickle Dex test) is the standard, rapid, and inexpensive screening test specifically for the presence of HbS. It works on the principle that sodium dithionite reduces oxygen, causing deoxygenation. Deoxygenated HbS is insoluble and forms a turbid solution, while normal hemoglobin (HbA) remains soluble and the solution clears.

Why the others are incorrect:

• a) Alkali denaturation test: This is a quantitative test for HbF, not HbS.

• c) Heat instability test: This is a test for unstable hemoglobins that precipitate when heated.

• d) Isoelectric focusing: This is a highly sensitive diagnostic method for identifying hemoglobin variants, not a simple, cost-effective screening test. It is more complex and expensive than the solubility test.

HbH is a pathologic hemoglobin composed of four beta (β) chains (β₄). It occurs in conditions like alpha-thalassemia where there is a severe deficiency of alpha-globin chains. The excess beta chains form unstable tetramers.

It is known as a “fast” hemoglobin because it migrates more rapidly toward the anode than HbA on alkaline hemoglobin electrophoresis due to its different net charge.

Why the others are incorrect:

• a) HbA: Is a tetramer of two alpha and two beta chains (α₂β₂). It is the normal adult hemoglobin and has a standard migration pattern.

• b) HbF: Is a tetramer of two alpha and two gamma chains (α₂γ₂). It migrates in a different position than HbA but is not classified as a “fast” hemoglobin like HbH.

• c) Hb Bart’s: Is a tetramer of four gamma (γ) chains (γ₄). It is the counterpart to HbH but is present in the newborn period in alpha-thalassemia.

In heterozygous delta-beta thalassemia, there is a deletion that involves both the delta (δ) and beta (β) globin genes.

• This means the production of both δ-chains (for HbA₂) and β-chains (for HbA) is impaired.

• As a result, HbA is slightly decreased and HbA₂ is normal (not elevated, unlike in beta-thalassemia trait).

• To compensate, there is a moderate increase in HbF production (typically 5-20%), as the gamma (γ)-globin genes are unaffected.

Why the others are incorrect:

• b) Elevated HbA2, normal HbF: This is the classic finding for beta-thalassemia trait, where only the beta-globin gene is affected.

• c) Elevated HbA2, elevated HbF: This pattern is not typical for delta-beta thalassemia, as the delta gene is also deleted.

• d) Absent HbA, elevated HbF: This is the pattern for homozygous beta-thalassemia major, not the heterozygous delta-beta form.

HbE is one of the most common hemoglobinopathies worldwide and is highly prevalent in Southeast Asian populations.

On alkaline gel electrophoresis, HbE has the same migration pattern as HbC and HbA₂. This is a key diagnostic point.

Why the others are incorrect:

• a) HbD: This variant is more common in populations of the Indian subcontinent and migrates with HbS, not HbC.

• c) HbO: This is a rare variant and does not have the specific Southeast Asian association or the HbC-migration pattern as a defining characteristic.

• d) HbG: This name refers to several rare variants and is not the common Hb variant in Southeast Asia.

Hydrops fetalis, resulting from the deletion of all four alpha-globin genes, is incompatible with extrauterine life. Affected fetuses develop severe anemia, heart failure, and massive tissue edema (hydrops), typically leading to death in utero or shortly after birth. The absence of alpha chains prevents the production of any functional fetal (HbF) or adult (HbA) hemoglobin.

Why the others are incorrect:

• a) Silent carrier (1 gene deletion): Asymptomatic.

• b) Trait (2 gene deletions): Causes mild microcytic anemia but is compatible with normal life.

• c) HbH disease (3 gene deletions): Causes a moderate to severe hemolytic anemia but is compatible with life, though it may require transfusions.

In beta-thalassemia minor (trait), there is reduced synthesis of beta-globin chains. This causes a compensatory increase in the production of delta (δ)-globin chains, leading to an elevated level of HbA₂ (α₂δ₂) on hemoglobin electrophoresis. A level above 3.5% is a key diagnostic feature.

Why the others are incorrect:

• a) A decreased red blood cell count: This is incorrect. Beta-thalassemia minor characteristically has a normal or even elevated red blood cell count, despite the microcytosis.

• b) A high MCV: This is incorrect. The MCV is low (microcytosis) due to the reduced hemoglobin content in each red cell.

• d) Decreased iron stores: This is incorrect. Iron stores are typically normal in beta-thalassemia minor. It is important to distinguish it from iron deficiency anemia, which also causes microcytosis but has low iron stores.

Hemoglobin H (HbH) disease occurs when three of the four alpha-globin genes are deleted or dysfunctional. This severe alpha-globin deficiency leads to an excess of beta-globin chains, which form unstable beta-chain tetramers (β₄) known as Hemoglobin H.

Why the others are incorrect:

• b) Alpha-chain tetramers: These do not form; excess alpha chains are unstable and precipitate but don’t form functional tetramers.

• c) Gamma-chain tetramers: These form Hemoglobin Bart’s (γ₄), which is present in newborns with alpha-thalassemia, not HbH disease in adults.

• d) Delta-chain tetramers: These are not clinically significant and do not form a recognized hemoglobin variant.

Hb Chesapeake is a well-known example of a high-oxygen-affinity hemoglobin variant. It results from a mutation in the alpha-globin chain that decreases the stability of the deoxygenated (Tense or T) state, favoring the oxygenated (Relaxed or R) state. This “left-shifts” the oxygen dissociation curve, meaning hemoglobin holds onto oxygen more tightly and releases less to the tissues.

Why the others are incorrect:

• a) HbS: Has decreased oxygen affinity.

• b) Hb Kansas: Is a classic example of a low-oxygen-affinity variant.

• d) Hb Bibba: Is an unstable hemoglobin variant; its oxygen affinity is not its defining characteristic.

The sickle solubility test (also called the dithionite solubility test) is a rapid, qualitative screening test used specifically to detect the presence of Hemoglobin S (HbS). It works by reducing oxygen tension, which causes HbS to polymerize and form a turbid solution if present.

Why the others are incorrect:

• a) HbF: Is detected by alkali denaturation test or acid elution (Kleihauer-Betke) test.

• b) HbA₂: Is quantified by hemoglobin electrophoresis or chromatography.

• d) HbC: Does not cause a positive solubility test; it is identified by its electrophoretic mobility.

The definitive test for detecting HbH inclusions is the brilliant cresyl blue supravital stain. This dye causes oxidative denaturation and precipitation of the unstable HbH (β₄ tetramers), resulting in the characteristic multiple, small, evenly distributed “golf ball” inclusions within red blood cells.

Why the others are incorrect:

• a) Wright’s stain: This is a routine stain for peripheral blood smears but cannot demonstrate HbH inclusions.

• c) Prussian blue stain: This is used to detect iron stores (hemosiderin) in bone marrow macrophages, not hemoglobin inclusions.

• d) PAS stain: This is used to detect glycogen and carbohydrates, helpful in diagnosing conditions like acute lymphoblastic leukemia, not hemoglobinopathies.

Hemoglobin Bart’s is a pathologic hemoglobin composed of four gamma (γ) globin chains (γ₄). It is formed in alpha-thalassemia when there is a severe deficiency of alpha (α) chains. In fetuses and newborns, the excess gamma chains that cannot form HbF (α₂γ₂) instead form these unstable tetramers.

Why the others are incorrect:

• a) Alpha₂Delta₂: This is the structure of HbA₂.

• c) Beta₄: This is the structure of HbH.

• d) Alpha₂Gamma₂: This is the structure of HbF.

Hemoglobin E disease is characterized by microcytic, hypochromic red blood cells. This occurs because the HbE mutation (Glu26Lys in the beta-globin chain) also creates a cryptic splice site, leading to reduced production of normal beta-globin mRNA. This results in a thalassemic phenotype, explaining the microcytosis and hypochromia, even in the heterozygous state (HbE trait).

Why the others are incorrect:

• a) Macrocytic, normochromic: This is seen in megaloblastic anemias (e.g., B12/folate deficiency), not hemoglobinopathies.

• b) Normocytic, normochromic: This is typical for simple heterozygous states like HbS or HbC trait, but not HbE trait.

• d) Microcytic, normochromic: While microcytosis is correct, the cells in HbE are typically hypochromic due to the thalassemia-like effect.

In sickle cell trait (HbAS), the individual has one normal beta-globin gene and one sickle beta-globin gene. The normal gene produces enough beta-A chains to form significant amounts of HbA.

The typical hemoglobin electrophoresis pattern shows:

• HbA: ~60%

• HbS: ~40% (usually ranging from 35-45%)

• Normal levels of HbF and HbA₂

This proportion of HbS is below the threshold needed to cause spontaneous sickling under most physiological conditions, which is why sickle cell trait is generally asymptomatic.

Why the others are incorrect:

• a) >90%: This is seen in sickle cell disease (HbSS).

• b) 70-80%: This is not a typical percentage for a common sickle cell syndrome.

• d) <5%: This is far too low; a level this low would not be classified as sickle cell trait.

HbH is an unstable hemoglobin composed of four beta chains (β4) that precipitates within red blood cells. Incubating RBCs with brilliant cresyl blue (a redox dye) causes oxidative denaturation and precipitation of HbH, resulting in a characteristic pattern of multiple, small, evenly distributed inclusions throughout the cell. This is known as the “HbH inclusion test.”

Why the others are incorrect:

• a) Prussian blue stain: This is used to stain iron (hemosiderin) in bone marrow macrophages, not hemoglobin inclusions.

• c) New methylene blue: While also a supravital stain used to demonstrate reticulocytes, it is not the standard or most specific dye used to demonstrate HbH inclusions. Brilliant cresyl blue is the classic and preferred reagent for this test.

• d) Crystal violet: This is a general histological stain, not used for demonstrating HbH inclusions.

Alpha-thalassemia is most commonly caused by deletions of one or more of the four alpha-globin genes on chromosome 16. The severity of the disease depends on the number of functional genes deleted:

• 1 gene deleted: Silent carrier (asymptomatic)

• 2 genes deleted: Alpha-thalassemia trait (mild anemia)

• 3 genes deleted: HbH disease (moderate to severe anemia)

• 4 genes deleted: Hydrops fetalis (lethal in utero or at birth)

Why the others are incorrect:

• a) Mutations in beta-globin gene: This causes beta-thalassemia, not alpha-thalassemia.

• c) Overproduction of HbF: This is a compensatory mechanism in beta-thalassemia, not the cause of alpha-thalassemia.

• d) Decreased synthesis of delta chains: This would affect HbA₂ levels but does not cause alpha-thalassemia.

Bone marrow or stem cell transplantation is currently the only potential curative treatment for β-thalassemia major. It replaces the defective hematopoietic stem cells with healthy ones from a compatible donor, allowing for normal hemoglobin production.

Why the other options are incorrect:

• a) Repeated iron supplements: This would be harmful, as thalassemia major patients already have iron overload from increased intestinal absorption and transfusions.

• c) Splenectomy alone: This may reduce transfusion requirements in some cases but is not curative and does not address the underlying genetic defect.

• d) Vitamin B12 injections: These are used to treat megaloblastic anemia, not thalassemia.

Beta-thalassemia major (also known as Cooley’s anemia) is characterized by a severe quantitative defect in beta-globin chain synthesis. The key laboratory findings include:

• Markedly elevated HbF: Due to compensatory gamma-chain synthesis, with HbF levels often >90%.

• Microcytosis and hypochromia: Due to defective hemoglobin production.

• Very low or absent HbA: Reflecting the severe deficiency of beta-chains.

• Profound anemia: With hemoglobin levels often below 7 g/dL.

• Erythroid hyperplasia: Leading to characteristic skeletal changes on X-ray.

Why the others are incorrect:

• a) Normal RBC count with macrocytosis: This is incorrect – beta-thalassemia major shows microcytosis, not macrocytosis, and typically has decreased RBC count.

• c) Decreased RBC count with normocytosis: This is incorrect – the RBCs are microcytic, not normocytic.

• d) Marked polychromasia with spherocytes: This describes hemolytic anemias like hereditary spherocytosis or autoimmune hemolytic anemia, not beta-thalassemia major.

In homozygous beta-thalassemia (also known as beta-thalassemia major), there is a severe reduction or complete absence of beta-globin chain production.

• This means very little to no HbA (α₂β₂) can be formed.

• To compensate, the body continues to produce gamma (γ)-globin chains from fetal life, leading to a marked increase in HbF (α₂γ₂).

• Hemoglobin electrophoresis in a patient with thalassemia major typically shows a predominance of HbF (often >90%), a complete absence or severe reduction of HbA, and a variable amount of HbA₂.

Why the others are incorrect:

• a) Predominantly HbA with elevated HbA2: This is the classic pattern for beta-thalassemia trait (minor), not the homozygous state.

• b) Predominantly HbS: This pattern is seen in sickle cell disease (HbSS), not beta-thalassemia.

• d) Normal pattern with microcytosis: The electrophoretic pattern is not normal in homozygous beta-thalassemia; it is dramatically altered.

β-thalassemia major is characterized by a severe, transfusion-dependent anemia that typically becomes apparent in the first year of life. The profound deficiency of β-globin chains leads to inadequate hemoglobin production, massive ineffective erythropoiesis, and hemolytic anemia, making regular blood transfusions essential for survival.

Why the others are incorrect:

• a) Mild anemia, asymptomatic: This describes β-thalassemia minor (trait).

• c) Petechiae and mucosal bleeding: These are features of thrombocytopenia or platelet function disorders, not thalassemia.

• d) Polycythemia: This is the opposite of the profound anemia seen in thalassemia major.

The hallmark laboratory finding in β-thalassemia major is a significant increase in HbF, which typically constitutes the majority of the hemoglobin (often >90%), along with an elevated HbA₂. This occurs due to the severe deficiency or absence of β-globin chains, leading to compensatory increases in gamma (γ) and delta (δ) chain production.

Why the others are incorrect:

• a) Increased HbA: This is incorrect because HbA (α₂β₂) is severely reduced or absent in β-thalassemia major due to the lack of β-globin chains.

• c) Increased HbC: This is incorrect; HbC is a variant hemoglobin not associated with β-thalassemia.

• d) Increased HbE: This is incorrect; HbE is a separate variant hemoglobin that can interact with β-thalassemia but is not a feature of β-thalassemia major itself.

• There is a failure to switch from γ-globin (fetal) to β-globin (adult) after birth.

• This means β-globin expression is reduced or absent, while γ-globin continues to be produced.

• As a result, adults with HPFH have persistently high levels of HbF (α₂γ₂) instead of the usual HbA (α₂β₂).

🔹 Quick check on the options:

• Alpha → unaffected (needed for both HbA and HbF).

• Beta → expression lost → HbA absent. ✅

• Gamma → persists (not lost).

• Delta → minor component (HbA₂), not the key issue here.

Hydrops fetalis is the most severe form of alpha-thalassemia. It is caused by the deletion of all four alpha-globin genes.

• With no functional alpha-globin chains, a fetus cannot produce any normal fetal hemoglobin (HbF, α₂γ₂) or adult hemoglobins (HbA, α₂β₂).

• Instead, abnormal hemoglobins like Hemoglobin Bart’s (γ₄) and Hemoglobin H (β₄) are formed, which have extremely high oxygen affinity and cannot effectively deliver oxygen to tissues.

• This results in severe, life-threatening anemia and heart failure (hydrops) in utero or shortly after birth.

Why the others are incorrect:

• a) 1 gene deleted: Alpha-thalassemia silent carrier (asymptomatic).

• b) 2 genes deleted: Alpha-thalassemia trait (mild microcytic anemia).

• c) 3 genes deleted: HbH disease (moderate to severe hemolytic anemia).

Target cells (codocytes) are characterized by a bull’s-eye appearance and are commonly associated with:

1. Thalassemia: Due to the reduced hemoglobin content and increased red cell surface area.

2. Liver Disease: Due to the accumulation of excess cholesterol and phospholipids in the red cell membrane, increasing its surface area.

Why the others are incorrect:

• b) Megaloblastic anemia: Typically shows macro-ovalocytes, not target cells.

• c) Iron deficiency anemia: May show target cells, but they are more characteristic of thalassemia.

• d) Aplastic anemia: Shows a general paucity of all cell lines without specific morphological changes like target cells.

In HbH disease, the peripheral blood smear typically shows:

1. Target cells due to the excess red cell membrane surface area relative to hemoglobin content.

2. “Golf ball” inclusions which are multiple, small, evenly distributed precipitates of HbH that appear when red cells are stained with brilliant cresyl blue. These are the hallmark finding for HbH disease.

Why the others are incorrect:

• a) Spherocytes: These are characteristic of hereditary spherocytosis and immune hemolytic anemias.

• c) Howell–Jolly bodies: These nuclear remnants are seen in asplenic states, not specifically in HbH disease.

• d) Bite cells: These are associated with oxidative hemolysis (e.g., G6PD deficiency), not HbH disease.

Vaso-occlusive crises (also called painful crises) are the hallmark clinical presentation of sickle cell disease. They occur when sickled red blood cells block small blood vessels, causing tissue ischemia, infarction, and severe pain. These crises most commonly affect the bones, chest, abdomen, and joints.

Why the others are incorrect:

• a) Hemarthrosis: This refers to blood in a joint space, which is not characteristic of sickle cell disease.

• c) Petechiae: These are small bleeding spots associated with low platelet counts, not a feature of sickle cell disease.

• d) Splenomegaly only: While splenomegaly can occur in young children with sickle cell disease, adults typically develop autosplenectomy (shrunken, non-functional spleen) due to repeated infarctions.

Hemoglobin C disease is characterized by the formation of intraerythrocytic crystals, often described as rod-shaped or hexagonal, especially after splenectomy. This occurs because the HbC variant (β6 Glu→Lys) reduces the solubility of hemoglobin, promoting its crystallization under certain conditions.

Why the others are incorrect:

• a) HbS: Characterized by sickle-shaped cells, not crystals, due to polymerization of deoxygenated hemoglobin.

• c) HbSC: This compound heterozygote can show features of both disorders, including some sickle cells and rare “HbC crystals,” but the classic, prominent rod-shaped crystals are a hallmark of homozygous HbC disease.

• d) HbD: Does not typically form crystals.

Hemoglobin H disease occurs when three out of four α-globin genes are deleted.

• 1 gene deletion → Silent carrier (no symptoms)

• 2 gene deletions → α-thalassemia trait (mild anemia)

• 3 gene deletions → Hemoglobin H disease (moderate to severe anemia, HbH forms = β4 tetramers)

• 4 gene deletions → Hydrops fetalis (incompatible with life, Hb Bart’s = γ4 tetramers)

• HbE trait is a classic cause of microcytosis without anemia (or with a very mild anemia).

• HbE is a β-globin chain variant (Glu26Lys). This mutation also affects the splicing of β-globin mRNA, leading to reduced production of normal β-globin chains. This results in a mild thalassemia-like phenotype, explaining the microcytosis.

• On hemoglobin electrophoresis, HbE co-migrates with HbA₂ and HbC at an alkaline pH.

• The findings—normal hemoglobin level, microcytic RBCs, and 30% of a variant that migrates like HbA₂—are highly characteristic of HbE trait.

Why the others are incorrect:

• a) HbC trait: Typically causes normocytic cells, not microcytic.

• c) HbO trait: This is rare and does not typically present with this specific electrophoretic pattern or microcytosis as a hallmark feature.

• d) HbD trait: Does not cause microcytosis and migrates with HbS, not HbA₂, on alkaline electrophoresis.

Thalassemia is fundamentally defined by a quantitative reduction in the synthesis of one or more of the globin chains (alpha or beta) that make up hemoglobin. This imbalance leads to inadequate hemoglobin production, precipitation of excess unpaired chains, and destruction of red blood cells (ineffective erythropoiesis and hemolysis).

Why the others are incorrect:

• a) Abnormal heme synthesis: This is the defect in sideroblastic anemias, not thalassemia.

• c) Structural instability of hemoglobin: This describes unstable hemoglobin variants (e.g., Hb Köln), which are qualitative, not quantitative, defects.

• d) Iron incorporation defect: This is also characteristic of sideroblastic anemia, where iron cannot be properly incorporated into the heme ring.

The major risk of repeated transfusions in thalassemia major is iron overload. Each unit of transfused blood contains approximately 200-250 mg of iron, which the human body cannot actively excrete. This excess iron accumulates in tissues (liver, heart, endocrine organs), leading to hemosiderosis and organ damage (cardiomyopathy, liver cirrhosis, endocrine failure).

Why the others are incorrect:

• a) Hemolysis: This is not a typical complication of transfusion in thalassemia when properly matched blood is used.

• c) Pancytopenia: This is not caused by transfusions; it may occur due to other complications like parvovirus B19 infection.

• d) Megaloblastosis: This is caused by folate or B12 deficiency, not transfusions.

Splenic sequestration crisis is a life-threatening complication most commonly seen in sickle cell disease (particularly in young children before auto-splenectomy occurs). It involves sickled red blood cells becoming trapped in the spleen, causing rapid splenic enlargement, a dramatic drop in hemoglobin, and hypovolemic shock.

Why the others are incorrect:

• a) HbC trait: This is asymptomatic and does not cause splenic sequestration.

• c) Beta-thalassemia trait: This causes mild microcytic anemia without acute splenic complications.

• d) HbE disease: While it can cause mild splenomegaly, it does not typically cause acute sequestration crises like sickle cell disease.

Thalassemia trait (especially β-thalassemia trait and α-thalassemia trait) is the most common hemoglobinopathy worldwide, particularly prevalent in Mediterranean, Middle Eastern, South Asian, and Southeast Asian populations.

👉 Explanation:

• HbS disease (Sickle cell disease): Common in Africa and parts of India, but less globally widespread than thalassemia.

• HbC disease: Seen mostly in West Africa, but not very common worldwide.

• HbE trait: Very common in Southeast Asia, but still less common overall than thalassemia trait.

Target cells are the most characteristic peripheral blood finding in hemoglobin C disease. The presence of the less soluble HbC leads to red cell membrane damage and loss of potassium and water, resulting in a dehydrated cell with a high surface-area-to-volume ratio. This causes the red cell to assume a target appearance on the blood smear.

Why the others are incorrect:

• a) Macrocytes: This is incorrect. Hemoglobin C disease is associated with normocytic or mildly microcytic cells, not macrocytes.

• b) Spherocytes: This is incorrect. Spherocytes are the hallmark of hereditary spherocytosis and some immune hemolytic anemias, not hemoglobin C disease.

• d) Rouleaux formation: This is incorrect. Rouleaux formation is characteristic of conditions with high levels of circulating proteins, such as multiple myeloma (due to paraproteins), not hemoglobin C disease.

Hemoglobin S (HbS) results from a single point mutation in the beta-globin gene. This mutation causes the substitution of valine for glutamic acid at the sixth position of the beta-globin chain.

• This change replaces a hydrophilic (charged) amino acid with a hydrophobic one.

• This allows deoxygenated HbS to polymerize, forming long fibers that distort the red cell into a sickle shape.

Why the others are incorrect:

• b) Valine → Glutamic acid: This is the reverse of the correct substitution.

• c) Lysine → Glutamic acid at position 26: This describes the mutation for Hemoglobin E.

• d) Glutamic acid → Lysine at position 26: This also describes the mutation for Hemoglobin E.

Hb Bart’s (γ₄) is elevated in newborns with alpha-thalassemia. The severity correlates with the number of alpha-globin gene deletions:

• Hydrops fetalis (4 gene deletions): Hb Bart’s is the predominant hemoglobin (80-100%)

• HbH disease (3 gene deletions): Hb Bart’s levels are typically 20-40%

• Alpha-thalassemia trait (2 gene deletions): Hb Bart’s levels are usually 3-10%

This occurs because alpha-chain deficiency in the fetus leads to excess gamma chains that form Hb Bart’s tetramers.

Why the others are incorrect:

• a) Beta-thalassemia minor: Shows elevated HbA₂ and/or HbF, not Hb Bart’s.

• c) HbC trait: Shows HbC on electrophoresis, not Hb Bart’s.

• d) HbE trait: Shows HbE on electrophoresis, not Hb Bart’s.

• Caused by a mutation in the stop codon of the α-globin gene → translation continues → results in an elongated α-globin chain.

• This chain is unstable, leading to decreased α-globin production (like α-thalassemia).

• Clinical effect: resembles α-thalassemia trait or HbH disease, depending on zygosity/associated deletions.

🔹 Option breakdown:

• a) Short α-chain variant →  opposite; HbCS is elongated, not shortened.

• b) Fusion hemoglobin → describes Hb Lepore (δβ fusion).

• c) Elongated α-chain variant → true for HbCS.

• d) β-chain variant → not correct; HbCS involves α-chain.

Thalassemia trait characteristically shows a normal or elevated red blood cell (RBC) count despite the presence of microcytic, hypochromic anemia. This is because the bone marrow compensates for defective hemoglobin production by increasing the number of red cells, even though they are small and pale.

Why the others are incorrect:

• b) Low RBC count and high RDW: This pattern is more typical of iron deficiency anemia.

• c) High MCV and low ferritin: Thalassemia trait has a low MCV, not high.

• d) Low iron and low transferrin saturation: These findings are characteristic of iron deficiency, not thalassemia trait (which has normal iron studies).

HbD has the same net electrical charge as HbS at an alkaline pH. This causes them to migrate to the same position on an alkaline electrophoresis gel. However, HbD does not have the same amino acid substitution (valine for glutamic acid) that causes HbS to polymerize and form sickled cells under low oxygen conditions. Therefore, it is not clinically sickling.

• HbA and HbF migrate to different, distinct positions.

• HbC migrates more slowly than HbS.

The main distinguishing feature between iron deficiency anemia (IDA) and thalassemia trait is ferritin levels:

• IDA: Low ferritin (indicating depleted iron stores)

• Thalassemia trait: Normal or elevated ferritin

This is the most reliable differentiating factor since both conditions share features like microcytosis and hypochromia.

Why the others are incorrect:

• a) Microcytosis in both: This is a shared feature, not a distinguishing one.

• c) High HbA₂ in IDA: Incorrect; HbA₂ is typically low in IDA and elevated in beta-thalassemia trait.

• d) Increased RDW in thalassemia: Incorrect; RDW is typically normal in thalassemia trait but increased in IDA.

Hemoglobin electrophoresis is the primary and most specific method for identifying common hemoglobin variants. It separates hemoglobins based on their electrical charge in a pH buffer, creating a characteristic migration pattern that allows for the identification of variants like HbS, HbC, HbE, HbD, etc.

Why the others are incorrect:

• a) Hemoglobin solubility test: This is a screening test specific only for HbS. It cannot identify other variants.

• c) Osmotic fragility test: This is used to diagnose hereditary spherocytosis, not hemoglobinopathies.

• d) Reticulocyte count: This is a non-specific test that measures bone marrow response to anemia and does not identify hemoglobin variants.

Hemoglobin Lepore is a variant hemoglobin caused by a fusion gene created from the non-homologous crossover between the delta (δ) and beta (β)-globin genes during meiosis. The resulting chromosome has a single δ-β fusion gene (Lepore), which produces a fused δ-β globin chain. The reciprocal product of this crossover is the anti-Lepore (β-δ fusion) hemoglobin.

Sickle cell anemia is caused by a specific point mutation in the beta-globin gene where a single nucleotide substitution (GAG → GTG) results in the amino acid valine replacing glutamic acid at the sixth position of the beta-globin chain. This structural change causes HbS to polymerize under low oxygen conditions, leading to sickling of red blood cells.

Why the others are incorrect:

• a) Lysine replacing glutamic acid at position 6: This describes Hemoglobin C, not HbS.

• c) Deletion of alpha-globin gene: This causes alpha-thalassemia, not sickle cell disease.

• d) Duplication of delta-globin gene: This is not a recognized cause of a major hemoglobinopathy.

HbF (fetal hemoglobin) has a higher oxygen affinity than HbA (adult hemoglobin). This is physiologically essential for efficient oxygen transfer from the maternal circulation (with HbA) to the fetus (with HbF) across the placenta. The higher affinity is due to HbF’s reduced binding to 2,3-bisphosphoglycerate (2,3-BPG) compared to HbA.

Why the others are incorrect:

• a) HbA: Has a normal, lower oxygen affinity than HbF.

• b) HbS: Has a lower oxygen affinity than HbA.

• d) HbC: Has oxygen affinity similar to or slightly lower than HbA.

Howell–Jolly bodies are nuclear remnants (fragments of DNA) that are normally pitted from red blood cells by the spleen. They are a classic finding in the blood smears of asplenic or splenectomized individuals, including those with sickle cell disease who have undergone autosplenectomy (functional asplenia) or surgical splenectomy.

Why the others are incorrect:

• a) Heinz bodies: These are precipitates of denatured hemoglobin associated with unstable hemoglobins or oxidative stress, not a specific consequence of splenectomy.

• c) Pappenheimer bodies: These are iron-containing granules seen in sideroblastic anemias and other conditions of iron overload.

• d) Basophilic stippling: This represents aggregated ribosomes and is seen in lead poisoning, thalassemias, and other dyserythropoietic states.

The standard cyanmethemoglobin method for hemoglobin measurement converts most hemoglobin derivatives to cyanmethemoglobin, which is then measured photometrically.

• Sulfhemoglobin is a stable, green-pigmented derivative that does not convert to cyanmethemoglobin and is therefore not detected by this method, leading to a falsely low reading.

Why the others are incorrect:

• a) Carboxyhemoglobin: Is slowly converted to cyanmethemoglobin and is measured, though inaccuracies can occur with high levels.

• b) Methemoglobin: Is readily converted to cyanmethemoglobin and is measured.

• d) Deoxyhemoglobin: Is converted to cyanmethemoglobin and is measured.

HbE disease is one of the most common hemoglobinopathies globally and is highly prevalent in Southeast Asia, particularly in countries like Thailand, Cambodia, Laos, and Vietnam. The gene frequency in some regions can be as high as 30-50%.

Why the others are incorrect:

• a) Middle East: This region has higher prevalence of beta-thalassemia and HbS, not HbE.

• b) Africa: This continent has high prevalence of HbS and HbC, not HbE.

• d) South America: Has lower prevalence of HbE compared to Southeast Asia.

The combination of a mildly elevated HbF (typically 5-15%) with a normal HbA2 level is the classic electrophoretic finding for heterozygous delta-beta thalassemia.

• The deletion of both the delta (δ) and beta (β) genes explains this pattern:

  • Loss of the δ-gene prevents an elevated HbA₂.

  • Loss of the β-gene causes a mild thalassemic picture and a compensatory increase in HbF.

Why the others are incorrect:

• a) Alpha thalassemia minor: Typically shows a normal HbF and a normal HbA₂ on electrophoresis. The diagnosis is often one of exclusion.

• b) Beta thalassemia minor: Characteristically shows an elevated HbA₂ (>3.5%), not a normal level. HbF may be slightly elevated, but the high HbA₂ is the key finding.

• d) Hereditary Persistence of Fetal Hemoglobin (HPFH): In the heterozygous state, HPFH shows higher levels of HbF (typically 15-35%) that are evenly distributed among red cells (pancellular), unlike the lower, unevenly distributed HbF seen in delta-beta thalassemia.

Hb Lepore is a variant hemoglobin caused by a fusion gene created from the non-homologous crossover between the delta (δ) and beta (β)-globin genes during meiosis. The resulting chromosome has a single δ-β fusion gene (Lepore), which produces a fused δ-β globin chain. The reciprocal product of this crossover is the anti-Lepore (β-δ fusion) hemoglobin.

Why the others are incorrect:

• b) Deletion of alpha genes: This causes alpha-thalassemia.

• c) Deletion of beta genes: This causes beta-thalassemia.

• d) Point mutation in gamma gene: This would affect HbF, not create Hb Lepore.

The hemoglobin solubility test (e.g., Sickledex) depends on detecting a specific amount of insoluble HbS to cause turbidity. If the hematocrit is severely decreased (as in severe anemia), the concentration of HbS in the test sample may be too low to produce a positive (turbid) result, leading to a false negative.

Why the others are incorrect:

• a) A very high reticulocyte count: This does not typically cause a false negative.

• c) Using a specimen older than 2 hours: While old specimens can give unreliable results, the test is generally reliable on blood samples that are several days old if stored properly.

• d) A high blood glucose level: Hyperglycemia does not interfere with the solubility test’s ability to detect HbS.

Hemoglobin electrophoresis is the most important test for evaluating the severity of thalassemia because it quantifies the different hemoglobin types:

• In beta-thalassemia major: Shows predominantly HbF with absent/reduced HbA

• In beta-thalassemia intermedia: Shows elevated HbF with some HbA present

• In beta-thalassemia minor: Shows elevated HbA₂ (>3.5%)

This differentiation directly correlates with clinical severity and guides management decisions.

Why the others are incorrect:

• a) Serum ferritin: Measures iron stores, important for monitoring transfusion overload but not for initial severity assessment.

• c) Reticulocyte count: Indicates bone marrow response but doesn’t differentiate thalassemia types.

• d) Peripheral smear only: Shows microcytosis and hypochromia but cannot quantify hemoglobin variants or determine severity.

Hemoglobin S polymerizes under low oxygen tension (deoxygenated conditions). When hemoglobin releases oxygen in the tissues, the deoxygenated form of HbS undergoes a conformational change that exposes a hydrophobic region where valine at position 6 interacts with complementary sites on other hemoglobin molecules, leading to the formation of long, rigid polymers that distort the red cell into a sickle shape.

Why the others are incorrect:

• a) Alkaline pH: Polymerization is actually inhibited at alkaline pH.

• c) High temperature: While fever can trigger sickling crises, this is due to increased metabolic demand and hypoxia, not direct temperature-induced polymerization.

• d) Normal oxygen conditions: HbS remains soluble and does not polymerize under normal oxygenated conditions.

Thalassemia is caused by mutations in the genes controlling α-globin or β-globin chain production.

• In α-thalassemia, there’s reduced or absent synthesis of α-globin chains.

• In β-thalassemia, there’s reduced or absent synthesis of β-globin chains.

The HbS trait (sickle cell trait) provides significant resistance to severe Plasmodium falciparum malaria. The mechanism involves several factors: the parasite’s difficulty metabolizing hemoglobin S, impaired cytoadherence of infected red cells, and enhanced removal of infected cells by the spleen. This heterozygote advantage is why the sickle cell gene remains prevalent in malaria-endemic regions.

Why the others are incorrect:

• a) HbC: Also provides some protection against malaria, but the evidence is strongest for HbS.

• c) HbE: May offer slight protection but not to the same extent as HbS.

• d) HbD: Is not associated with malaria resistance.

The hemoglobin solubility test (e.g., Sickledex) is based on the unique property of deoxygenated HbS to polymerize and form a turbid solution. When a reducing agent like sodium dithionite is added to lyzed blood, it deoxygenates the hemoglobin. If HbS is present, it becomes insoluble, causing the solution to appear turbid. A clear solution indicates a negative result (no HbS present).

Why the others are incorrect:

• a) HbA, b) HbF, and d) HbA2: These hemoglobins remain soluble under the test conditions and will not cause turbidity.

The Kleihauer-Betke test is a cytological (acid elution) test used to quantify fetal hemoglobin (HbF) by identifying red blood cells that contain it. When a blood smear is exposed to an acidic buffer, HbA is eluted from the cells, but HbF is resistant to elution. Cells containing HbF stain dark pink, while adult red cells appear as “ghost cells.”

Primary Clinical Uses:

1. To detect and quantify fetomaternal hemorrhage (the presence of fetal cells in the maternal circulation).

2. To confirm the pancellular (even) distribution of HbF in Hereditary Persistence of Fetal Hemoglobin (HPFH).

Why the others are incorrect:

• a) HbA₂: Quantified by hemoglobin electrophoresis or chromatography.

• b) HbS: Detected by solubility test or electrophoresis.

• d) HbH: Detected by brilliant cresyl blue inclusion test.

Sickle cell trait (HbAS) is typically a benign, asymptomatic carrier state. Individuals have one normal beta-globin gene and one sickle beta-globin gene, resulting in a hemoglobin composition of approximately 60% HbA and 40% HbS. The presence of sufficient normal HbA prevents the widespread polymerization and sickling that causes the severe complications of sickle cell disease (HbSS).

Why the others are incorrect:

• b) Associated with severe anemia: This is a feature of sickle cell disease (HbSS), not the trait.

• c) Lethal in infancy: This is incorrect; sickle cell trait has a normal life expectancy.

• d) Associated with microcytosis only: Sickle cell trait does not cause microcytosis. This finding is characteristic of thalassemias.

Beta-thalassemia trait characteristically presents with microcytic, hypochromic red blood cells but has normal iron studies (normal serum iron, normal ferritin). This distinguishes it from iron deficiency anemia, which also causes microcytosis and hypochromia but shows low iron stores.

Why the others are incorrect:

• a) Iron deficiency anemia: Shows microcytic, hypochromic RBCs but with low iron stores.

• c) Sideroblastic anemia: Can show microcytosis but typically has elevated iron stores and ringed sideroblasts in the bone marrow.

• d) Anemia of chronic disease: Is typically normocytic, normochromic, though it can sometimes be microcytic.

HbSC disease typically presents with milder symptoms compared to homozygous HbSS disease. Patients with HbSC generally have:

• Higher hemoglobin levels (8-12 g/dL vs 6-9 g/dL in HbSS)

• Fewer vaso-occlusive crises

• Less severe hemolytic anemia

• Later onset of complications

However, HbSC disease is still a significant sickling disorder and can cause serious complications like proliferative retinopathy, avascular necrosis, and renal disease, which may be even more prevalent than in HbSS.

Why the others are incorrect:

• b) Identical severity as HbSS: Incorrect; HbSC is clinically milder overall.

• c) Asymptomatic condition: Incorrect; HbSC causes symptomatic disease.

• d) Severe transfusion dependence: Incorrect; most HbSC patients are not chronically transfusion-dependent.

Hb Köln is a classic example of an unstable hemoglobin variant. The amino acid substitution (Val→Met at position 98 of the beta chain) disrupts the hemoglobin molecule’s stability, causing it to precipitate and form Heinz bodies within red blood cells. This leads to chronic hemolytic anemia.

Why the others are incorrect:

• a) HbA: This is the normal, stable adult hemoglobin.

• b) HbF: This is the normal, stable fetal hemoglobin.

• d) HbA₂: This is the normal, stable minor adult hemoglobin.