Haematopoiesis, Haemoglobin Structure, and Blood Cell Abnormalities Study Guide

Learning Aims and Outcomes

• Theoretical Grounding Framework:
  • Normal haematopoiesis including Erythropoiesis, Thrombopoiesis, and Leukopoiesis.
  • Haemoglobin function and variants.
  • Introduction to basic red cell pathology.
• Learning Outcomes:
  • Demonstrate the basic cellular process of haematopoiesis.
  • Identify light microphotographic images of all cell lines.
  • Understand hormonal controls for generating and regulating blood cells.
  • Recognize light microphotographs of elementary red cell pathologies.
  • Develop a professional vocabulary in haematologica.

General Classification and Origins of Blood Cells

• Origin: All blood cells originate in the bone marrow.
• Lineage Classification:
  • Myeloid Lineage: Includes Neutrophils, Monocytes, Platelets, and Eosinophils.
  • Lymphoid Lineage: Includes Lymphocytes.
• Sites of Haematopoiesis:
  • Myeloid Tissue: Occurs in bone marrow. Produces Erythrocytes, Platelets, Granulocytes, and Monocytes.
  • Lymphoid Tissue: Occurs in the Thymus, Lymph nodes, and Spleen.

Specific Cell Morphology and Function

• Neutrophils:
  • Morphology: Purple hue with granular appearance; nucleus has $4-6$ lobes.
  • Role: Innate immunity; effective against bacteria and debris.
  • Detection: FBC (Full Blood Count), Blood Film, Flow Cytometry.
• Monocytes:
  • Morphology: Lighter purple hue, mildly granular, very large mononuclear cell containing vacuoles.
  • Role: Innate immunity; differentiate into tissue macrophages; effective against bacteria and debris.
  • Detection: FBC, Blood Film, Flow Cytometry.
• Eosinophils:
  • Morphology: Striking red hue; very granular; nucleus is bi-lobed or tri-lobed.
  • Role: Innate immunity; involved in allergic reactions and response to parasites.
  • Detection: FBC, Blood Film, Flow Cytometry.
• Basophils:
  • Morphology: Striking blue hue; very granular; the nucleus is often almost occluded by the density of the granules.
  • Role: Largely unknown.
  • Detection: FBC, Blood Film, Flow Cytometry.
• Erythrocytes:
  • Morphology: Anuclear bi-concave disc; distinctive pink/red hue.
  • Role: Transport of oxygen to tissues.
  • Detection: FBC, Blood Film, Flow Cytometry.

Developmental Timeline and Niche Biology

• Ontogeny of Haematopoiesis:
  • $3/52$ (3 weeks): Formation of blood islands in the yolk sac.
  • $6/52$ (6 weeks): Liver becomes the primary haematopoietic organ.
  • $6/52$ to 8th month: Spleen contributes to production.
  • $12-14/52$ (12-14 weeks): Bone marrow becomes involved; source of blood cells for the remainder of life.
• The Haematopoietic Niche:
  • Occurs in a suitable microenvironment called a "niche" provided by a stromal matrix.
  • Vascular Niche: Lined by endothelium.
  • Endosteal Niche: Lined by osteoblasts.
  • Involves binding of extracellular glycoproteins and other compounds.
• Cellular Proliferation: A single stem cell can give rise to over $10^6$ mature cells after multiple divisions. As cells mature, they lose the capacity for self-renewal.

The Stem Cell Hierarchy and Surface Markers

• Stem Cell Characteristics:
  • CD34 and c-kit expression.
  • Pale staining with rhodamine and Hoechst dyes.
• Lineage-Specific Markers:
  • B-cell: $CD19, CD20, (CD22), CD79a$.
  • T-cell: $CD3, CD2, CD5, CD4/CD8$.
  • NK-cell: $CD16, CD57, CD56$.
• Comparative HSC Markers:
  • Human HSC: $Lin^- CD90^+ CD38^- CD34^+$
  • Mouse HSC: $Lin^- Thy1.1^{low} c-Kit^+ Sca1^+ flk2^- CD34^- Slamf1^+$
• Progenitors:
  • CLP: Common Lymphoid Progenitor.
  • CMP: Common Myeloid Progenitor.
  • MEP: Megakaryocyte-Erythroid Progenitor.
  • GMP: Granulocyte-Macrophage Progenitor.

Mesenchymal Stem Cells (MSC) and Stroma

• MSC Surface Markers: $CD34^-, CD45^-, BMPR, CD44, CD54, CD105, c-Kit, Sca 1, Stro 1, Thy 1$.
• MSC Differentiation Pathways:
  • Adipocyte: markers ALBP, FATP.
  • Osteoblast/Osteocyte: Hydroxyapatite, Osteocalcin, E11/gp38, DMP1, Phex.
  • Chondrocyte: markers Collagen 2, Sulfated Proteoglycan.
  • Myocyte (Skeletal/Cardiac/Smooth): Myog, Pax 7, Myo D, MR4, Cripto, GATA4, Myosin light chain, SM-MHC, VE-cadherin.
  • Fibroblast/Tenocyte: $CD146, FSP1$.

Growth Factors and Lineage Regulation

• Role of Growth Factors: Growth factors stimulate proliferation, direct differentiation, promote maturation, suppress apoptosis, and enhance the function of mature cells.
• Key Regulatory Factors:
  • Erythropoiesis: Erythropoietin ($EPO$), $GM-CSF$.
  • Thrombopoiesis: Thrombopoietin ($TPO$), $IL-11$.
  • Neutropoiesis: $G-CSF, GM-CSF, IL-3$.
  • Monopoiesis: $M-CSF, GM-CSF, IL-3$.
  • Eosinopoiesis: $IL-5, IL-3, GM-CSF$.
  • Basopoiesis/Mastpoiesis: $SCF, IL-3$.
  • Lymphopoiesis (B-cells): Early stage involves $IL-7, SCF$. Later stages involve $IL-4, IL-6$. Final proliferation/secretion involves $IL-6, GM-CSF$.
  • Lymphopoiesis (T-cells): $IL-2, IL-7$. CD4/CD8 differentiation involves $Ag, TCR/CD3, CD28$.

Erythropoiesis and Granulocyte Maturation Stages

• Erythropoiesis Stages:
  1. Hemocytoblast (Stem Cell)
  2. Proerythroblast (Committed Cell)
  3. Phase 1: Early erythroblast (Ribosome synthesis)
  4. Phase 2: Late erythroblast/Normoblast (Hemoglobin accumulation)
  5. Phase 3: Reticulocyte (Ejection of nucleus)
  6. Mature Erythrocyte
• Granulocyte Maturation Proportions:
  • Myeloblasts: $≈ 2\%$
  • Promyelocytes: $≈ 5\%$
  • Myelocytes: $5-20\%$
  • Metamyelocytes: $≈ 22\%$

B-cell and T-cell Maturation Pathways

• B-cell Maturation:
  • Early B Precursor: $CD34^+, TdT^+, CD19, DR, CD10$.
  • Pre-B: $CD19, DR, CD10, Cμ$ (cytoplasmic mu chain), $TdT^+$.
  • B Cell: Surface Immunoglobulin ($SIg$), $CD19, CD20, CD21, CD22$.
  • Plasma Cell: Antibody secretion.
  • Genetic Events: Ig heavy chain rearrangement followed by light chain rearrangement.
• T-cell Maturation:
  • Early Thymocytes: $CD34^+, TdT^+, CD7, CD2, CD5$.
  • Common Thymocytes: $CD1, CD2, CD4, CD8, CD5, CD7, TdT^+$.
  • Mature T Cells: Helper-inducer ($CD4^+$) or Cytotoxic ($CD8^+$). Both express the $CD3$/TCR complex.

Red Blood Cell Structure and Membrane

• Membrane Dimensions: Lipid bilayer is approximately $5\,nm$ thick.
• Lipid Composition by Weight (RBC Plasma Membrane):
  • Cholesterol: $23\%$
  • Phosphatidylethanolamine: $18\%$
  • Phosphatidylserine: $7\%$
  • Phosphatidylcholine: $17\%$
  • Sphingomyelin: $18\%$
  • Glycolipids: $3\%$
  • Others: $13\%$
• Membrane Interactions:
  • Vertical Interactions: Link the lipid bilayer to the cytoskeleton via proteins like Band 3, Ankyrin, and Protein 4.2.
  • Horizontal Interactions: Maintain the integrity of the cytoskeleton. Involve alpha-spectrin, beta-spectrin, Actin, and Tropomyosin.
  • Proteins: Glycophorin A, B, and C.

Haemoglobin Structure and Variants

• Basic Structure: Composed of four globin chains ($α_1, α_2, β_1, β_2$) each containing a Haem group with an Iron ($Fe$) core.
• Synthesis:
  • Mitochondria: Main site of protoporphyrin synthesis.
  • Ribosomes: Site of globin chain synthesis.
  • Iron ($Fe$) is supplied by circulating transferrin.
  • Precursors include $δ$-aminolaevulinic acid ($δ–ALA$) and Coenzyme A ($CoA$).
• Sickling Haemoglobin Variants: Besides Hb S, variants include Hb SAntilles, Hb CZiquinchor, Hb CHarlem, Hb SProvidence, Hb SOman, Hb STravis, Hb SSouth End, Hb SJamaica Plain, and Hb SCameroon.

Haemoglobin Function and Oxygen Binding

• Oxyhaemoglobin: Oxygenated state; $O_2$ bound to haem.
• Deoxyhaemoglobin: Deoxygenated state; contains $2,3-DPG$ in the central cavity.
• Cooperative Binding: The oxygen binding affinity increases as more $O_2$ molecules bind to the haemoglobin (Hb, $HbO_2$, $HbO_4$, $HbO_6$, $HbO_8$).
• Gas Transport: Transports $O_2$ to tissues and returns $CO_2$ to the lungs via the following chemical equilibrium:
  • $CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons H^+ + HCO_3^-$

Oxygen-Hemoglobin Dissociation Curve

• Curve Shape: Sigmoid-shaped.
• The Bohr Effect: A reduction in the total binding capacity of Hb to oxygen due to reduced pH (shifting the curve down and right).
• Right Shift (Reduced Oxygen Affinity - Increased Release to Tissues):
  1. High $[H^+]$ (Low pH).
  2. High $PCO_2$.
  3. High Temperature.
  4. High $2,3-DPG$ (e.g., Pyruvate kinase deficiency, Hyperthyroidism, Anemia, Chronic hypoxemia).
  5. Specific hemoglobinopathies (Hb Kansas, Hb Seattle).
• Left Shift (Increased Oxygen Affinity - Reduced Release to Tissues):
  1. Low $[H^+]$ (High pH).
  2. Low $PCO_2$.
  3. Low Temperature.
  4. Low $2,3-DPG$ (e.g., Hexokinase deficiency, Hypothyroidism, Banked blood).
  5. Congenital hemoglobinopathies (Hb Rainier, Hb Hiroshima, Hb San Francisco).
  6. Carboxyhemoglobin.

Red Cell Pathology and Morphology

• General Terms:
  • Anisocytosis: Cells of unequal/various sizes.
  • Poikilocytosis: Cells of various/abnormal shapes.
• Specific Cell Abnormalities:
  • Elliptocytes: Seen in hereditary elliptocytosis, iron deficiency anaemia, myelofibrosis, megaloblastic anemia, sickle cell anaemia.
  • Spherocytes: Seen in hereditary spherocytosis, acquired haemolytic anaemia (AIHA), physical/chemical injury, and heat damage.
  • Leptocytes (Target Cells): Seen in liver disease (obstructive jaundice), post-splenectomy, thalassemia, Hb C disease, and Hb H disease.
  • Schistocytes (Cell Fragments): Indicate haemolysis; seen in megaloblastic anaemia, severe burns, traumatic haemolysis, and microangiopathic haemolytic anemia.
  • Acanthocytes (Spicule Cells): Irregular surface spicules with bulbous ends; seen in liver disease.
  • Echinocytes (Crenated/Burr Cells): Regular smooth surface undulations; seen in uremia, artefact, and hyperosmolarity.
  • Bite Cells: Result from membrane removal by splenic macrophages; seen in G6PD deficiency.
  • Dacrocytes (Teardrop Cells): Seen in thalassemia and myelofibrosis.
  • Drepanocytes (Sickle Cells): Diagnostic of sickle cell anemia.
  • Rouleaux: RBCs stacked like coins; characteristic of multiple myeloma.
• Red Cell Inclusions:
  • Howell-Jolly Body: Remnant of nuclear chromatin. Single bodies seen in megaloblastic/hemolytic anemia or post-splenectomy. Multiple bodies suggest abnormal erythropoiesis.
  • Heinz Bodies: Inclusions of denatured haemoglobin; associated with G6PD deficiency.