L6 - Erythrocyte Structure and Function II

Why is RBC Deformability Essential for Normal Function?

• RBCs must bend to pass through narrow capillaries and return to their biconcave shape in larger vessels

• Deformation may also facilitate oxygen unloading through connections between the cytoskeleton and hemoglobin

What are the Main Components of the RBC Membrane and Their Functions?

1. Cytoskeleton: Provides shape, elasticity, rigidity, and deformability

2. Lipid bilayer: Acts as a permeability barrier, retains hemoglobin, and is relatively noncompressible

3. Membrane anchor proteins: Connect the lipid bilayer to the cytoskeleton

4. Cholesterol: Contributes to membrane stiffness and stability

What is the Role of Spectrin in RBC Structure?

Spectrin forms an elastic cytoskeletal lattice beneath the lipid bilayer

• Membrane anchor points connect this lattice to the bilayer, allowing RBCs to deform and recover their normal shape

What is the Difference between Physiologic and Pathologic RBC Deformation?

• Physiologic: Elastic, reversible deformation supported by the cytoskeletal lattice

• Pathologic: Persistent or irreversible shape changes caused by membrane or cytoskeletal injury, reducing flexibility

How does ATP Maintain RBC Membrane Function?

ATP generated through Embden–Meyerhof glycolysis supports:

• Membrane deformation

• Cation pumps

• Ionic gradients

• Cell volume regulation

ATP depletion decreases RBC deformability.

How do Ionic Gradients Differ between Most Mammalian RBCs and Dog/Cat RBCs?

• Most mammals: RBC interiors are relatively potassium-rich

• Dogs and cats: Relatively sodium-rich RBC pattern

  • ATP-dependent pumps help maintain these ionic gradients

  • Delayed blood sample processing can alter measured electrolyte concentrations

How can Plasma Lipid Abnormalities Affect RBC Morphology?

RBC membrane lipids are in equilibrium with plasma lipids

• Abnormal cholesterol or lipid concentrations can change membrane composition and produce abnormal RBC shapes, particularly acanthocytes

What are the Major Causes and Consequences of RBC Membrane Abnormalities?

Causes:

• Structural defects involving membrane proteins or lipid

• Metabolic defects involving ATP or calcium regulation

• Oxidative injury, intracellular organisms, or abnormal hemoglobin aggregates

Consequences:

• Decreased deformability

• Abnormal RBC shapes (poikilocytosis)

• Capillary obstruction or fragmentation

• Premature RBC destruction (hemolysis)

Why is Intravascular Hemolysis Potentially Harmful?

Intravascular hemolysis releases free hemoglobin into circulation

• Free hemoglobin can cause secondary tissue damage, particularly to the kidneys

What are the Normal RBC Shapes across Different Species?

• Most mammals: Biconcave, discoid RBCs

  • Dogs: Prominent central pallor

  • Cats and horses: Less obvious central pallor on routine smears

• Camelids: Normally oval or elliptical RBCs

• Birds and reptiles: Oval, nucleated RBCs.

What is Poikilocytosis, and Why is It Diagnostically Important?

Poikilocytosis refers to abnormal RBC shapes

• Specific morphologies can suggest underlying membrane damage, metabolic abnormalities, mechanical injury, or disease

What is Splenic Conditioning, and How does It Remove Abnormal RBCs?

• Healthy, deformable RBCs pass through narrow splenic red-pulp passages and return to circulation

  • Rigid or damaged RBCs become trapped and are phagocytosed by splenic macrophage

  • The spleen can also remove RBC inclusions, including nuclear remnants

• Excessive abnormal RBC production may overwhelm splenic filtration, allowing abnormal cells to appear on blood smears

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Echinocytes

Morphology: RBCs with multiple short, pointed surface projections

Causes:

• Artifacts: Underfilled EDTA tubes or rapid smear drying (crenation)

• Pathologic: Electrolyte abnormalities, uremia, and rattlesnake envenomation

Mechanism: Osmotic disturbances or membrane damage alter RBC shape

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Acanthocytes

Morphology: RBCs with irregularly distributed projections of varying lengths, often rounded or blunt

Causes:

• Hepatic disease and lipid abnormalities

• RBC fragmentation

• Neoplasia, including hemangiosarcoma, lymphoma, and osteosarcoma

Mechanism: Altered membrane lipid composition, particularly excess cholesterol in the outer bilayer leaflet, produces irregular projections

• Acanthocytes may be physiologic in pigs, calves, and rabbits

What is the Difference between Echinocytes and Acanthocytes?

Echinocytes:

• Short, pointed, more uniformly distributed projections

• Associated with electrolyte disturbances, uremia, and artifacts

Acanthocytes:

• Irregular, often blunt, unevenly distributed projections

• Associated with lipid abnormalities, liver disease, and neoplasia

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Target Cells (Codocytes)

Morphology: RBCs with a target-like central appearance

Associations:

• Regenerative anemia

• Iron deficiency anemia

• Hepatobiliary disease

Mechanism: Changes in the relationship between membrane surface area and cell volume, including reduced hemoglobin content in iron deficiency

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Schistocytes

Morphology: Mechanically fragmented RBCs

Associations:

• Thrombi

• Disseminated intravascular coagulation (DIC)

• Some vascular tumors

Mechanism: RBCs are torn by fibrin strands or abnormal blood flow within vessels

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Keratocytes

Morphology: RBCs with horn-like projections or blister-type changes

Associations:

• Thrombi

• Disseminated intravascular coagulation (DIC)

• Vascular tumors

Mechanism: Mechanical injury damages part of the RBC membrane without completely dividing the cell, producing a horned appearance

What is the Difference between Schistocytes and Keratocytes?

Schistocytes: RBC fragments resulting from mechanical shearing

Keratocytes: RBCs with horn-like projections resulting from partial membrane damage

Both suggest mechanical RBC injury, often associated with fibrin strands, thrombi, or abnormal vascular flow

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Spherocytes

Morphology: Small, rounded, dense RBCs with diminished central pallor

Mechanism:

1. Antibodies bind the RBC membrane

2. Macrophages partially phagocytose antibody-coated membrane

3. The remaining membrane reseals around the cytosol, producing a spherical RBC

What is the Clinical Significance of Spherocytes?

• Numerous spherocytes, particularly in dogs, strongly suggest immune-mediated hemolytic anemia (IMHA)

• A few spherocytes alongside schistocytes and acanthocytes may indicate RBC fragmentation

• Spherocytes must be interpreted with additional clinical and laboratory evidence

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Heinz bodies

Morphology: Aggregates of oxidized globin attached to the inner RBC membrane

Mechanism:They form when oxidative damage causes hemoglobin proteins to denature and aggregate

• These aggregates reduce RBC deformability and may contribute to hemolysis

Why are Cats Particularly Susceptible to Heinz Body Formation?

Feline hemoglobin contains numerous sulfhydryl (SH) groups that are sensitive to oxidation

• Up to approximately 5% of feline RBCs may contain Heinz bodies under normal conditions

What are Common Causes of Heinz Body Formation, and How are They Identified?

Causes:

• Oxidant exposures such as acetaminophen, mothballs, and skunk musk

• Endogenous oxidants from accumulated metabolic products

Identification:

• Routine stain: Pale red RBC protrusions

• New methylene blue: Makes Heinz bodies easier to identify

Name the Abnormal RBC Morphology and What Causes Their Formation?

ANSWER → Eccentrocytes

Morphology: RBCs with hemoglobin displaced toward one side

Causes: Eccentrocytes indicate oxidative injury and may occur with or without Heinz bodies

Mechanism: Oxidative membrane damage causes portions of the membrane to adhere to each other, displacing hemoglobin

What are the Main RBC Antioxidant Pathways and Their Functions?

• Embden–Meyerhof glycolysis: Produces ATP and NADH

• Methemoglobin reductase system: Restores oxidized heme iron (Fe³⁺) to functional Fe²⁺

• Pentose phosphate pathway (PPP): Produces NADPH to maintain reduced glutathione

• Glutathione system: Protects hemoglobin and membrane proteins from oxidative damage

How does the Glutathione Antioxidant System Protect RBCs?

1. Reduced glutathione (GSH) helps detoxify hydrogen peroxide (H₂O₂) into water

2. GSH becomes oxidized glutathione (GSSG)

3. Glutathione reductase converts GSSG back into GSH

4. This regeneration requires NADPH from the Pentose phosphate pathway

   • The lecture also emphasizes selenium-dependent antioxidant protection.

What is the Difference between Heinz Bodies and Howell–Jolly Bodies?

• Heinz bodies: Oxidized hemoglobin aggregates, typically pale red on routine smears and better visualized with new methylene blue

• Howell–Jolly bodies: Small nuclear remnants that stain blue-purple on routine smears

  • Howell–Jolly bodies may increase with regenerative anemia, splenic dysfunction, or splenectomy

What is RBC Agglutination, and What is Its Clinical Significance?

Agglutination occurs when antibodies cross-link RBC surfaces, causing cells to clump together

• It is an important hallmark of IMHA and may also occur following incompatible blood transfusions

What is RBC Senescence, and What Causes Aging RBCs to Be Removed?

Senescence is the progressive aging of RBCs as they approach the end of their species-specific lifespan

Changes include:

• ATP depletion and metabolic failure

• Reduced antioxidant protection and gradual hemoglobin oxidation

• Alterations in membrane proteins recognized by senescence antibodies

• Decreased deformability

• Aging RBCs become trapped in the spleen and are removed by macrophages

What happens to Hemoglobin after Senescent RBCs are Phagocytosed?

Hemoglobin is separated into:

1. Globin: Degraded into reusable amino acids

2. Heme: Broken down to recover iron and degrade the potentially oxidative heme ring

• Iron may be stored as ferritin or hemosiderin for future use.

What is the Pathway of Heme Degradation to Bilirubin?

1. Heme oxygenase converts heme into biliverdin

2. Biliverdin reductase converts biliverdin into bilirubin

3. Unconjugated (indirect) bilirubin binds albumin and travels through plasma to the liver

4. Hepatocytes conjugate bilirubin with glucuronide

5. Conjugated (direct) bilirubin enters bile and travels to the intestine

What is the Difference between Unconjugated and Conjugated Bilirubin?

• Unconjugated (indirect): Produced from heme degradation, poorly water-soluble, and transported bound to albumin

• Conjugated (direct): Processed by hepatocytes through glucuronidation, becoming more water-soluble for biliary excretion

What Happens to Bilirubin after It Enters the Intestine?

Intestinal bacteria process bilirubin into downstream products, including urobilinogen

• These products contribute to:

  • Stercobilin: Fecal pigment

  • Urobilin: Urinary pigment

How can Hemolysis Affect Bilirubin Metabolism?

Increased RBC destruction produces more heme for degradation, increasing bilirubin production

• If bilirubin production exceeds the body's ability to process and eliminate it, bilirubin may accumulate and contribute to icterus

What are the Major Stages Where Bilirubin Metabolism can Be Disrupted?

These disturbances can cause abnormal bilirubin accumulation or excretion

1. Prehepatic: Increased RBC destruction and bilirubin production

2. Hepatic: Impaired bilirubin uptake, conjugation, or handling by the liver

3. Posthepatic: Impaired biliary excretion or downstream elimination

Table 1. RBC Membrane Components & Functions

Component	Main Function
Cytoskeleton (spectrin)	Maintains RBC shape, elasticity, and deformability
Lipid bilayer	Permeability barrier; retains hemoglobin
Membrane anchor proteins	Connect lipid bilayer to cytoskeleton
Cholesterol	Contributes to membrane stiffness and stability
ATP (glycolysis)	Supports deformation, ion pumps, and cell volume regulation
Cation pumps	Maintain ionic gradients and osmotic balance

Key concept: RBC deformability depends on the interaction between the cytoskeleton, lipid bilayer, and ATP-dependent processes.

2. Poikilocytes — Morphology, Mechanism & Clinical Associations

RBC Type	Morphology	Mechanism / Associations
Echinocyte	Multiple short, pointed projections	Osmotic/electrolyte disturbances, uremia, envenomation, or EDTA artifact
Acanthocyte	Irregular, blunt projections	Altered membrane lipids, liver disease, fragmentation, or neoplasia
Codocyte (target cell)	Target-like central appearance	Altered membrane-to-volume relationship; iron deficiency, regenerative anemia, liver disease
Schistocyte	Fragmented RBC	Mechanical shearing from fibrin, thrombi, or DIC
Keratocyte	Horn-like projections	Partial membrane damage from mechanical fragmentation
Spherocyte	Round, dense, reduced central pallor	Membrane loss following partial macrophage phagocytosis; IMHA
Eccentrocyte	Hemoglobin displaced to one side	Oxidative injury causes membrane adhesion

Important distinctions:

• Echinocytes vs. Acanthocytes: Uniform pointed projections vs. irregular blunt projections.

• Schistocytes vs. Keratocytes: Complete fragmentation vs. partial membrane damage.

• Spherocytes: Strongly associated with IMHA when numerous, especially in dogs.

Table 3. RBC Oxidative Damage & Antioxidant Pathways

Pathway / System	Main Function	Key Molecule
Embden–Meyerhof glycolysis	Supplies energy and reducing equivalents	ATP, NADH
Methemoglobin reductase	Restores oxidized heme iron (Fe³⁺ → Fe²⁺)	NADH
Pentose phosphate pathway	Supplies reducing power for antioxidant defense	NADPH
Glutathione system	Detoxifies peroxides and protects RBC membrane/globin	GSH
Glutathione reductase	Regenerates reduced glutathione (GSSG → GSH)	NADPH

Key distinction: Methemoglobin reductase protects hemoglobin's functional iron state, whereas the glutathione system protects membrane and globin proteins against oxidative damage.

Table 4. RBC & Hemoglobin Degradation

Step	Process	Product / Fate
1	Splenic macrophages phagocytose senescent RBCs	Hemoglobin released for degradation
2	Globin breakdown	Amino acids recycled
3	Heme breakdown	Iron recovered and heme ring degraded
4	Heme oxygenase	Heme → biliverdin
5	Biliverdin reductase	Biliverdin → unconjugated bilirubin
6	Albumin transport	Unconjugated bilirubin → liver
7	Hepatic conjugation	Conjugated bilirubin
8	Biliary excretion	Conjugated bilirubin → bile → intestine
9	Intestinal bacterial metabolism	Urobilinogen and downstream pigments
10	Final elimination	Stercobilin (feces), Urobilin (urine)

Remember: Iron and globin are recycled, while the potentially oxidative heme ring must be degraded.