Erythrocyte morphology and hemoglobin

Erythrocytes, or red blood cells (RBCs), are the most common blood cells. Normal RBCs have a biconcave shape and contain hemoglobin but no nucleus or organelles. Dysmorphic RBCs (e.g., sickle cells, target cells) have an altered form and are often a sign of an underlying condition. Hemoglobin (Hb) is composed of heme and globin subunits and is responsible for transporting oxygen and carbon dioxide throughout the body. Hb can undergo conformational changes (e.g, depending on its oxygenated state), which influence how it binds and releases O₂ and CO₂. Deficient Hb (anemia), genetic Hb variants (e.g., HbS, HbC), and certain substances (carbon monoxide, nitrates that form methemoglobin, cyanide) affect the affinity for and ability to transport O₂, resulting in decreased tissue oxygenation.

Normal morphology

• Biconcave shape: large surface area-to-volume ratio for rapid gas exchange
• Content
  • No nucleus or organelles
  • Contain hemoglobin

Dysmorphic RBCs

	Morphology	Associated conditions
Dacryocytes (teardrop cells, teardrop erythrocytes)	Teardrop-shaped	Extramedullary hematopoiesis Myelofibrosis Thalassemia Splenomegaly
Sickle cells (drepanocytes)	Sickle-shaped	Sickle cell anemia
Schistocytes (fragmentocytes)	Fragmented	Microangiopathic hemolytic anemia (e.g., HUS, DIC, TTP) Mechanical damage: artificial cardiac valves, extracorporeal circulation
Macrocytes (megalocytes)	Large, spherical	Megaloblastic anemia (folate deficiency, vitamin B12 deficiency)
Spherocytes	Small, spherical, no central pallor	Hemolytic anemias Hereditary spherocytosis Autoimmune hemolytic anemia ABO incompatibility in infants Hemolytic transfusion reaction
Elliptocytes (ovalocytes)	Oval or elliptical	Hereditary elliptocytosis: caused by mutations in genes encoding RBC membrane proteins (e.g., spectrin, protein 4.1); usually asymptomatic May also be seen in thalassemia, myelofibrosis, and iron deficiency anemia.
Echinocytes (burr cells)	Smooth, rounded, and evenly spaced cytoplasmic projections	Uremia Liver disease Pyruvate kinase deficiency Oxidative or colloid osmotic stress in already dysmorphic erythrocytes (e.g., spherocytes) Artifacts
Target cells (codocytes)	Bullseye appearance	Hemoglobinopathies Thalassemia Hemoglobin C and S Liver disease Post-splenectomy LCAT deficiency Artifacts
Acanthocytes (spur cells)	Thorny projections	In blood Hemolytic anemia in hepatic disorders (e.g. alcoholic cirrhosis) Myelodysplastic syndromes Following splenectomy Neurodegenerative disorders Hereditary abetalipoproteinemia (Bassen-Kornzweig syndrome) In urine Glomerular damage (e.g., glomerulonephritis)
Stomatocytes	Slit-like central pallor most often caused by changes in membrane permeability	Hepatic disorders Hemolytic anemias Hereditary stomatocytosis
Degmacytes (bite cells)	One or more semicircular portions removed ("bitten off") from the cell margin	Glucose-6-phosphate dehydrogenase deficiency

RBCs with abnormal content (inclusion bodies)

	Content	Appearance	Cause
Heinz bodies	Denaturated hemoglobin	Small Red or pink	Glucose-6-phosphate dehydrogenase (G6PD) deficiency Thalassemia
Pappenheimer bodies	Iron	Small, blue or purple granules	Sideroblastic anemia Hemolytic anemia Sickle cell disease
Basophilic stippling	RNA	Small, basophilic granules throughout cytoplasm	Nonspecific finding that appears in a variety of disorders with impaired cell division Lead intoxication Sideroblastic anemia Thalassemia Severe anemias of varying etiology
Ringed sideroblasts	Iron	Perinuclear ring within mitochondria of erythroblasts: seen in bone marrow with Prussian blue stain	Sideroblastic anemia
Howell-Jolly bodies	DNA (nuclear remnants)	A typically small, basophilic spot, located toward the periphery of the cell	Asplenia

The lifespan of RBCs is about 120 days. After this time, macrophages in the reticuloendothelial system of the bone and the spleen phagocytose RBCs. They are then broken down and their parts recycled.

Globin metabolism

Globin chains are released and converted into amino acids.

Hemoglobin metabolism

Process: heme (red) → biliverdin (green pigment) → bilirubin (yellow pigment)

1. Heme is converted to biliverdin by heme oxygenase.
   • Requires NADPH + H⁺
   • Carbon monoxide (CO) is released.
   • Iron is released, oxidized, and taken up by transferrin to be recycled.
2. Biliverdin is converted to bilirubin by biliverdin reductase.
   • Requires NADPH + H⁺

Heme breakdown is responsible for the color changes in hematomas!

Bilirubin metabolism

1. Unconjugated bilirubin (insoluble in water) is released into the blood by macrophages → binds to albumin and reaches the liver
2. Unconjugated bilirubin is converted into bilirubin via the enzyme UDP-glucuronosyltransferase in the liver.
   • Bilirubin is conjugated with glucuronic acid → bilirubin diglucuronide = conjugated bilirubin (water soluble)
   • Most conjugated bilirubin is excreted into the GI tract via bile, while some is released into the blood.
3. Conjugated bilirubin excreted in bile is broken down by GI bacteria into urobilinogen
   • Most urobilinogen is converted to stercobilin → excreted in feces (brown color).
   • Part of urobilinogen oxidates to urobilin → excreted in the urine (yellow color).

2,3- bisphosphoglycerate shunt

• 2,3-bisphosphoglycerate mutase is vital for the formation of 1,3-bisphosphoglycerate (intermediate in glycolysis) → 2,3-BPG
  • 2,3-BPG mutase is unique to erythrocytes and placental cells.
• 2,3-BPG binds to hemoglobin → conformational change → oxygen is released into local tissues
• 2,3-BPG binds with greater affinity to deoxygenated hemoglobin than oxygenated hemoglobin.

Energy production

• Mature RBCs are unable to generate energy via the Krebs cycle because they lack mitochondria.
• Important biochemical pathways available in RBCs:
  • Glycolysis
  • Pentose-phosphate pathway
  • Glutathione reduction

References:^[1][2]

Overview

Hb is a circulating globular protein composed of a heme moiety with a central iron ion and four subunits of globin.

• The main function of Hb is to take up O₂ from the lungs and deliver it to tissues.
• It can undergo conformational changes (e.g., depending on its state of oxygenation), which influence how it binds and releases O₂ and CO₂.
• Deficient or defective Hb can ultimately affect the transport of O₂ (see Hb variants for details).
• For more information about disorders of Hb, see the learning card on anemia.

Heme synthesis

• Heme is synthesized from protoporphyrin, a porphyrin.
• Porphyrins: a group of intermediates in the heme synthesis pathway that is composed of four subunit rings
  • Includes porphobilinogen and uroporphyrinogen
  • Binding of a central ferrous iron (Fe²⁺) forms a porphyrin complex (i.e., heme).
• The steps of heme synthesis occur both in the cytoplasm and the mitochondria (the first and final steps occur in mitochondria).

Location	Reaction	Enzyme	Clinical significance
Mitochondria	Glycine + succinyl CoA → δ-aminolevulinic acid (ALA)	ALA synthase Activated by CYP450 inducers , ethanol, ↓ heme Inhibited by glucose, ↑ heme Cofactor: pyridoxal phosphate (vitamin B6)	Sideroblastic anemia due to: X-linked sideroblastic anemia: hereditary deficiency of ALA synthase Alcohol use Vitamin B6 deficiency
Cytoplasm	Two molecules of δ-ALA combine → porphobilinogen	ALA dehydratase	Sideroblastic anemia due to lead poisoning (inhibits ALA dehydratase)
	Porphobilinogen → hydroxymethylbilane	Porphobilinogen deaminase	Enzyme deficiency causes acute intermittent porphyria.
	Hydroxymethylbilane → Uroporphyrinogen III	Uroporphyrinogen-III synthase	-
	Uroporphyrinogen III → coproporphyrinogen III	Uroporphyrinogen decarboxylase	Enzyme deficiency causes porphyria cutanea tarda.
	Coproporphyrinogen III (in cytoplasm) → protoporphyrinogen III (in mitochondria)	Coproporphyrinogen III oxidase	-
Mitochondria	Protoporphyrinogen III → protoporphyrin IX	Protoporphyrin oxidase	-
	Protoporphyrin binds iron (Fe2+) → heme	Ferrochelatase	Sideroblastic anemia due to: Lead poisoning (inhibits ferrochelatase) Basophilic stippling: lead inhibits the breakdown of rRNA in RBCs → cells accumulate undegraded rRNA → small, basophilic granules on microscopic examination of RBCs Drugs Isoniazid Chloramphenicol Linezolid Cycloserine Copper deficiency

Sideroblastic anemia is due to ineffective heme synthesis, which may be congenital (X-linked defect in the δ-ALA synthase gene) or acquired (e.g., vitamin B₆ deficiency, or lead poisoning leading to sequential inhibition of δ-ALA dehydratase and ferrochelatase).

Globin

• Globin is an integral part of the Hb molecule.
• Tetramer consisting of four individual subunits that bind heme
  • Composed of amino acids that fold to form 8 alpha helices
• There are 6 types of globin chains.
  • β, δ, γ, ε are encoded on chromosome 11.
  • α and ζ are encoded on chromosome 16.
• The combination of subunits in the Hb molecule determines the type of Hb (e.g., embryonic, fetal, newborn, or adult Hb).
• Mutations in genes encoding globin results in Hb variants.

Hemoglobin patterns

For genetic variants of hemoglobin patterns, see hemoglobin variants below.

			Chromosome 16
			HBA1 gene	HBA2 gene	HBZ1 gene	HBZ2 gene
			α globin		ζ globin
Chromosome 11	HBB gene	β globin	HbA1 (the main adult hemoglobin): ααββ Adult: > 95–98% Newborn: ∼ 15% HbA1c: glycosylated Hb (seen in diabetes mellitus) Due to nonenzymatic glycosylation		Portland 2 (ζζββ): embryonic Hb
	HBD gene	δ globin	HbA2: ααδδ Adult: ∼ 2–3% Newborn: ∼ 0.5%		No Hb
	HBG1 gene	γ globin	HbF (fetal hemoglobin): ααγγ Adult: ∼ 0.8–2% Newborn: ∼ 80% Synthesized in Yolk sac (weeks 3–8) Liver (week 6–birth) Spleen (weeks 10–28) Bone marrow (> week 18) Has ↓ binding of 2,3-bisphosphoglycerate (2,3-BPG) → ↑ affinity for O2 → ↑ O2 extraction from the maternal circulation (via the placenta)		Portland 1 (ζζγγ): embryonic Hb
	HBG2 gene
	HBE gene	ε globin	Gower 2 (ααεε): embryonic		Gower 1 (ζζεε): embryonic Hb

References:^[3][4]

Overview

• O₂, CO₂, and protons all bind Hb and influence one another's affinity to Hb, which is important for gas exchange.
  • Hb releases CO₂ more easily with increasing pO₂ (Haldane effect).
  • Hb binds CO₂ more easily if the pO₂ is low.
  • Hb releases O₂ easier with increasing H⁺(Bohr effect).
• CO₂ is mainly carried in three forms in the body:
  • Plasma bicarbonate, HCO₃^- (70%)
  • Bound to Hb (carbamino-Hb, HbCO₂) at the N-terminus of globin (does not compete with O₂ at heme)
  • Dissolved in blood (5%)

Oxygenation and deoxygenation of Hb

• Oxyhemoglobin: hemoglobin with oxygen bound to its heme component (oxygenated) → bright red blood
• Deoxyhemoglobin: hemoglobin with no oxygen bound to its heme component (deoxygenated) → dark red blood; blue to purple appearance of tissue during hypoxia → "cyanosis"

Bicarbonate buffer system

• RBCs carry carbonic anhydrase, which converts HCO₃^- and H⁺ to H₂O and CO₂ in the following steps: HCO₃⁻ + H⁺ ⇄ H₂CO₃ ⇄ H₂O + CO₂
• Ultimately, excess H⁺ during acidic states is eliminated through conversion to CO₂, which can be exhaled.
• During basic states, the bicarbonate buffer system can reverse so that CO₂ is converted to HCO₃⁻ + H⁺.
  • Chloride shift: Excess intracellular HCO₃⁻ produced this way is released into the plasma in exchange for Cl^-.
• This phenomenon makes HCO₃^- the most important buffer in the body. HCO₃^- accounts for 50% of the blood buffer capacity.
• For more details on the buffering mechanisms of the body, see compensation in acid-base disorders.

Bohr effect

• The O₂ affinity of Hb is inversely proportional to the CO₂ content and pH of blood.
• High CO₂ and H⁺ concentrations (from tissue metabolism) cause decreased affinity for O₂. → O₂ that is bound to Hb is released to tissue (the O₂-Hb dissociation curve is shifted to the right).
  • HbO₂ + H⁺ ⇄ H⁺Hb + O₂
  • HbO₂ + CO₂ ⇄ Hb-COO^- + H⁺ + O₂

Haldane effect

• The CO₂ affinity of Hb is inversely proportional to the oxygenation of Hb.
• When Hb is deoxygenated (typically in peripheral tissue), uptake of CO₂ is facilitated.
• When Hb is oxygenated (in high pO₂, for example, in the lungs):
  • Oxygenated Hb has a decreased affinity for CO_2. → CO₂ that is bound to Hb is released in the pulmonary arteries to diffuse into the alveoli (the O2-Hb dissociation curve is shifted to the left).
  • Hb releases bound H⁺ → ↑ H⁺ shifts equilibrium to CO₂ production (see equation above) → CO₂ is exhaled in lungs

References:^[3][4]

Overview

	CaO2	SaO2	PaO2	O2-Hb dissociation curve
Anemia (e.g., due to chronic blood loss)	↓	Normal	Normal	Normal
CO poisoning (carboxyhemoglobin) and methemoglobinemia	↓	↓	Normal	Left-shifted
Cyanide poisoning	Normal	Normal	Normal	Normal
Polycythemia	↑	Normal	Normal	Normal
High-altitude exposure	↓	Normal	↓	Right-shifted

Carbon monoxide poisoning

• Formation of carboxyhemoglobin: Hemoglobin that is removed from oxygen transportation because its oxygen binding site is occupied by carbon monoxide
• See carbon monoxide intoxication for details.

Methemoglobinemia

• Methemoglobin contains iron in its oxidized (ferric = Fe³⁺) rather than its reduced state (ferrous = Fe2⁺) and cannot bind oxygen.
• Methemoglobin levels
  • 0–3% of total hemoglobin: physiological
  • > 3%: visible cyanosis (a brownish-blue or grayish coloration of the skin and membranes)
  • > 20%: clinical symptoms of oxygen deprivation; brown blood ("chocolate-colored blood")
  • > 70%: fatal
• Causes of methemoglobinemia
  • Exposure to substances that increase methemoglobin levels (most common cause)
    • Nitrate or nitrite
    • Drugs: nitroglycerin, sulfonamides, dapsone, inhaled nitric oxide, topical anesthetics such as lidocaine, and aniline derivatives
    • Nitro and amino compounds of benzene
  • Congenital (hereditary) methemoglobinemia
    • Deficiency of methemoglobin reductase (NADH-cytochrome b5 reductase)
      • In newborns, enzyme activity of methemoglobin reductase is low → increased risk of intoxication through drinking water with increased nitrate levels
  • Glucose-6-phosphate dehydrogenase deficiency
• Diagnosis
  • Dark brown coloration of blood
  • Reduced oxygen saturation and total oxygen content with normal PaO₂ on arterial blood gas analysis despite clinical signs of cyanosis
  • Confirm with CO-oximeter.
• Treatment
  • Methylene blue: At lower concentrations, it is very effective in reducing methemoglobin to hemoglobin.
  • Reducing agents such as ascorbic acid (vitamin C) and riboflavin may help reduce methemoglobin to hemoglobin, although they are not always effective.
  • In cases of acquired methemoglobinemia: Investigate pharmaceutical causes and cease therapy with agents that may be at fault.
  • Last resort: exchange transfusion

Because MetHb artificially increases pulse oximeter readings, oxygen concentration measured via pulse oximetry remains high (> 80%) even if methemoglobin levels are very high!

References:^[5][6]

• Anemia
• Iron deficiency anemia
• Hemolytic anemia
• Vitamin B₁₂ deficiency
• Folate deficiency
• Thalassemia
• Sickle cell anemia
• Hemolytic anemia
• Hereditary spherocytosis
• Glucose-6-phosphate dehydrogenase deficiency