21:RBC Deformability and Intrinsic Disorders

• Intrinsic disorders causing hemolytic anemia involve abnormalities in the RBC membrane, metabolic enzymes, or hemoglobin, and are mostly hereditary.
• RBC shape maintenance relies on the plasma membrane, a lipid bilayer with embedded proteins connected to an underlying protein cytoskeleton.

RBC Deformability Factors

• Biconcave, discoid geometry
• Membrane elasticity
• Cytoplasmic viscosity

Geometry

• It's vital for RBC deformability
• Maintained by transmembrane and cytoskeletal protein interactions

Vertical Interactions

• Proteins like ankyrin and actin anchor the lipid bilayer to the spectrin cytoskeleton, critical for structural integrity.
• Defects can destabilize the membrane, leading to shape abnormalities like spherocytosis.

Horizontal Interactions

• Spectrin dimers associate laterally to form tetramers.
• This process creating a flexible lattice for membrane stability under stress

Elasticity

• Spectrin-cytoskeleton interactions allows the membrane to stretch and return to its shape
• Spectrin unfolding and refolding contributes to RBC elasticity.

Cytoplasmic Viscosity

• Dependent on hemoglobin concentration and proper cell volume
• Regulated by ion channels and pumps
• Changes affect deformability, potentially leading to premature destruction.

Hemolytic Anemia: Vertical Defects

• Disrupting the lipid bilayer-cytoskeleton anchor, as in hereditary spherocytosis (HS). This results in detachment of the lipid membrane, forming vesicles
• Decreases surface area-to-volume ratio, leading to non-deformable spherocytes
• These cells are trapped and destroyed in the spleen, causing extravascular hemolysis.

Hemolytic Anemia: Horizontal Defects

• Mutations in proteins like spectrin or protein 4.1 weaken interactions, causing cytoskeleton fragility
• In hereditary elliptocytosis (HE), RBCs become elliptical under mechanical stress. Severe cases cause fragmentation and reduced deformability.
• Damaged RBCs are sequestered in the spleen, leading to hemolysis.

Structural Support

• Vertical: Ankyrin + protein 4.1

• Horizontal: Spectrin α and β heterodimers with actin and protein 4.1

• Impaired RBC deformability results in premature destruction, causing hemolytic anemia

• Symptoms depend on severity

• Can result in spherocytes, elliptocytes, increased bilirubin, and a positive osmotic fragility test

Hereditary Spherocytosis (HS)

• Spherical RBCs with reduced surface area-to-volume ratio
• Defects in vertical membrane protein interactions cause loss of the lipid bilayer
• This results in spherical, less deformable RBCs that are destroyed in the spleen
• Common protein mutations in ankyrin, band 3, spectrin (commonly β-spectrin), or protein 4.2.

Hereditary Elliptocytosis (HE)

• Elliptical (cigar-shaped) RBCs
• Defects in horizontal membrane protein interactions weaken the spectrin-actin lattice, leading to elliptical cell shapes
• Common protein mutations in spectrin α or β chains and protein 4.1
• The most common mutation is in SPTA1, coding for α-spectrin

Hereditary Ovalocytosis (HO)

• Oval-shaped RBCs (ovalocytes), often with increased rigidity
• Mutations in membrane proteins contribute to altered RBC deformability and membrane rigidity
• Commonly associated with mutations in band 3 (such as Southeast Asian Ovalocytosis)
• HO often causes less severe hemolysis compared to HS and HE

Comparison

• HS affects vertical interactions (resulting in spherocytes), while HE affects horizontal interactions (resulting in elliptocytes)
• HO less commonly associated with severe hemolysis
• Often overlaps in features with HS and HE, but stands out due to band 3 mutations and increased membrane rigidity

RBC Morphology: Hereditary Spherocytosis

• Spherocytes are the hallmark
• Small, round, lack central pallor, with a reduced surface area-to-volume ratio
• No RBC agglutination

RBC Morphology: Immune-Associated Hemolytic Anemia

• Spherocytes can appear, especially in warm autoimmune hemolytic anemia (AIHA), due to antibody-mediated cell membrane damage
• RBC agglutination may be seen, particularly in cold agglutinin disease, due to cold-reacting antibodies

Laboratory Findings: Hereditary Spherocytosis

• Osmotic Fragility Test: Increased, reflecting reduced surface area-to-volume ratio
• Direct Antiglobulin Test (DAT): Negative
• Reticulocytosis (increased reticulocyte count)
• Elevated mean corpuscular hemoglobin concentration (MCHC) due to cell dehydration
• Peripheral smear shows isolated spherocytes without agglutination

Laboratory Findings: Immune-Associated Hemolytic Anemia

• Direct Antiglobulin Test (DAT): Positive, indicating antibodies or complement on the RBC surface
• Reticulocytosis (variable)
• Peripheral smear: Spherocytes (in warm AIHA) or agglutinated RBCs (in cold agglutinin disease)
• Indirect hyperbilirubinemia and increased lactate dehydrogenase (LDH), reflecting hemolysis

Key Differences

• Defect is intrinsic to the RBC membrane in HS (usually due to vertical protein interaction defects), while IAHA involves extrinsic immune-mediated destruction
• DAT results are negative in HS but positive in IAHA
• RBC agglutination is seen only in specific types of IAHA (e.g., cold agglutinin disease)

Osmotic Fragility Test

• RBCs are exposed to increasingly hypotonic solutions, which cause the cells to swell and eventually lyse
• Spherocytes have increased fragility and lyse at higher concentrations of saline than normal RBCs due to their reduced surface area-to-volume ratio
• Increased fragility indicates membrane abnormalities consistent with HS
• Reticulocytosis or recent transfusions can interfere with results
• Lacks specificity; other hemolytic conditions (e.g., immune hemolysis) can show increased osmotic fragility
• Requires fresh blood and precise lab handling
• Cannot differentiate between hereditary and acquired spherocytosis

Eosin-5'-Maleimide (EMA) Binding Test

• EMA is a fluorescent dye that binds to transmembrane proteins (e.g., band 3, Rh, RhAg, CD47). The fluorescence intensity (mean fluorescence intensity, MFI) is measured via flow cytometry.
• Reduced protein interactions in HS lead to lower MFI compared to normal controls
• Lower MFI confirms HS with high sensitivity (93–97%) and specificity (94–99%)
• Can distinguish HS from immune-mediated hemolysis, which usually shows normal MFI
• Requires a small sample
• Can be completed in about 3hrs with samples stored up to 7 days
• Gating eliminates interference from transfused or fragmented RBCs
• Positive results can also occur in conditions like congenital dyserythropoietic anemia type II, Southeast Asian ovalocytosis, or hereditary pyropoikilocytosis (HPP)
• Not universally available
• EMA binding test is more sensitive, specific, and practical for confirming HS

Overhydrated Hereditary Stomatocytosis (OHS)

• Defective membrane cation permeability leads to sodium influx exceeding potassium efflux, causing water retention
• Moderate to severe hemolytic anemia
• 5–50% stomatocytes on smear; decreased MCHC, increased MCV

Dehydrated Hereditary Stomatocytosis (DHS)

• Autosomal dominant mutation causes potassium leakage without sodium compensation, leading to water loss
• Mild to moderate anemia, jaundice, splenomegaly
• Reticulocytosis; increased MCHC; presence of stomatocytes, target cells, echinocytes

Acquired Stomatocytosis

• Often due to drying artifacts

Acanthocytosis: Hereditary Forms

• Disorders under neuroacanthocytosis, such as abetalipoproteinemia, McLeod syndrome, and chorea acanthocytosis

Acanthocytosis: Acquired Spur Cell Anemia

• Defective lipid distribution in RBC membranes due to excess free cholesterol, often seen in severe liver disease
• Acanthocytes (spur cells) RBC Morphology

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• Acquired mutation in hematopoietic stem cells leads to a lack of GPI-anchored proteins (e.g., CD55, CD59)
• This makes RBCs susceptible to complement-mediated destruction
• Mild to severe anemia, hemoglobinuria, thrombosis (e.g., Budd-Chiari syndrome), bone marrow failure
• Variable anemia; hemoglobinuria; thrombosis; reticulocytosis (elevated MCV); pancytopenia
• Flow cytometry to detect absence of CD59 and FLAER marker
• Treated with Eculizumab (complement inhibitor), HSCT, supportive therapies

Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• X-linked defect impairs hexose monophosphate shunt, limiting the regeneration of NADPH and glutathione
• RBCs succumb to oxidative damage during stress
• Acute hemolysis (triggered by drugs, infections, or fava beans)
• Neonatal jaundice, chronic nonspherocytic hemolytic anemia
• Protection in some cases against Plasmodium falciparum
• Heinz bodies, bite cells, anisopoikilocytosis, hemoglobinuria, increased reticulocytes, indirect bilirubin elevation
• Diagnosis via enzymatic testing (qualitative and quantitative) and molecular genotyping
• Avoid testing during hemolysis due to transiently increased reticulocyte G6PD levels

Pyruvate Kinase (PK) Deficiency

• Mutation leads to decreased PK activity
• This causes ATP depletion and 2,3-BPG accumulation, ultimately reducing RBC lifespan
• Chronic hemolysis, splenomegaly, fatigue
• Burr cells, reticulocytosis, various smear abnormalities
• Testing via enzymatic assays, similar to G6PD deficiency diagnostics

Intrinsic Hemolytic Anemia: Stomatocytosis

• Possible Cause: Membrane defect causing altered cation permeability
• Clinical: Moderate to severe hemolytic anemia for OHS; mild anemia, jaundice, and splenomegaly for DHS.
• Laboratory: OHS increased MCV, decreased MCHC, and 5–50% stomatocytes on smear. DHS increased MCHC, reticulocytosis, stomatocytes, target cells, and burr cells.
• Peripheral Blood Smear: Presence of stomatocytes, echinocytes, and puddled hemoglobin in DHS.

Intrinsic Hemolytic Anemia: Acquired Spur Cell Anemia

• Possible Cause: Associated with severe liver disease leading to altered lipid composition in the RBC membrane
• Clinical: Jaundice and anemia due to increased splenic sequestration
• Laboratory: Presence of acanthocytes (spur cells) on smear
• Peripheral Blood Smear: Acanthocytes with irregularly spiked membranes caused by lipid imbalance

Intrinsic Hemolytic Anemia: Paroxysmal Nocturnal Hemoglobinuria (PNH)

• Possible Cause: Acquired mutation in GPI-anchor synthesis leading to CD55 and CD59 deficiency
• Clinical: Anemia, episodes of hemoglobinuria (dark urine), fatigue, and thrombosis
• Laboratory: Hemoglobinuria, increased reticulocytes, variable MCV, pancytopenia, and iron deficiency. Flow cytometry confirms absence of CD59 and FLAER marker
• Peripheral Blood Smear: Usually normal RBC morphology but prone to intravascular hemolysis

Intrinsic Hemolytic Anemia: Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• Possible Cause: Oxidative stress due to G6PD dysfunction causing RBC lysis
• Clinical: Symptoms triggered by oxidative stress (e.g., drugs, infection, or fava beans); jaundice, hemoglobinuria, and fatigue
• Laboratory: Bite cells, Heinz bodies on smear. Increased reticulocyte count and bilirubin
• Peripheral Blood Smear: Bite cells and anisocytosis during hemolytic episodes

Intrinsic Hemolytic Anemia: Pyruvate Kinase (PK) Deficiency

• Possible Cause: Reduced PK activity leads to ATP depletion and RBC hemolysis
• Clinical: Chronic hemolysis with splenomegaly and fatigue
• Laboratory: Reticulocytosis, elevated indirect bilirubin, decreased haptoglobin
• Peripheral Blood Smear: Burr cells observed