Hematopoiesis in Adults and Fetal Development

Haematopoiesis Overview

• Haematopoiesis is the production of blood cells.
• It's a continuous process to replace old blood cells.
• Pronounced "heh-ma-tuh-poy-EE-sus".
• Erythrocytes (red blood cells): ~120 days
• Thrombocytes (platelets): 10 to 12 days
• Leukocytes (white blood cells): few days to over a year
• Begins before birth and continues throughout life.

Haematopoiesis Areas to Cover

• Ontogeny and development of blood-forming tissues
• Functions of the bone marrow
• Maturation sequence of normal blood cells and influencing factors
• Structure of normal blood cells and precursors
• Production, structure, and function of platelets
• Basic haematological investigations

Haematopoiesis Importance

• In average adults, 200 to 400 billion blood cells are destroyed and replaced daily.
• Maintains continuous blood production.
• Gives rise to multipotential haematopoietic stem cells (HSCs) - also called haemo-cytoblasts.

Fetal Development Phases

• Mesoblastic Phase (Yolk Sac):
  • Occurs early (~19th day) after fertilization in embryonic life
  • Primitive erythroblasts are formed in the mesenchyme of the yolk sac.
  • Provides oxygen to the fetus.
  • Angioblasts surround the mesoderm, becoming blood vessels.
  • Takes place first in the yolk sac, then aorta-gonad-mesonephros (AGM).
• Hepatic Phase (Liver and Spleen):
  • Begins at 4 to 5 weeks of gestation.
  • Primarily in the liver.
  • Produces clusters of developing erythroid, granulocyte, monocyte, megakaryocyte, and lymphoid cells (blood islands).
  • Spleen and lymph nodes take a limited role as secondary lymphoid organs in the mid-fetal life.
• Medullary Phase (Bone Marrow):
  • Begins around the 5th month.
  • HSCs and mesenchymal stem cells migrate to the bone marrow (BM).
  • Myeloid production becomes very active in the bone marrow.
  • Myeloid 3:1 erythroid ratio.
  • Detectable amounts of growth and stimulating factors (EPO, G-CSF, GM-CSF, Hf, Ha) are present.
  • Production in the liver diminishes.

Adult Haematopoiesis

• Bone marrow, liver, spleen, lymph nodes, and thymus are involved in blood cell proliferation and maturation.
• Bone Marrow: Erythroid, myeloid, megakaryocytic, lymphoid cell development in early stages.
• Primary Lymphoid Tissue (Bone Marrow and Thymus): T and B cells develop into immunocompetent cells.
• Secondary Lymphoid Tissue (Spleen and Lymph Nodes): Immunocompetent cells divide further, becoming effector and memory cells.

Bone Marrow

• One of the body's organs within cortical bone cavities (honeycomb-like trabecular bone).
• The primary site of haematopoiesis after birth and throughout adult life.
• Differentiates into myeloid and lymphoid lineages under growth factor influence.
• Supplies mature functional blood cells to the circulation.
• Two major components:
  • Red Marrow: Haemopoeitically active
  • Yellow Marrow: Haemopoeitically inactive (primarily adipocytes - fat cells).

Retrogression

• During infancy and early childhood, bone marrow (BM) is primarily red marrow (RM).
• Between 5 and 7 years, adipocytes increase and dominate the long bones.
• RM is eventually restricted to flat bones (sternum, vertebrae, pelvis, ribs, skull, proximal long bones).
• Areas with replaced RM contain a mixture of adipocytes, undifferentiated mesenchymal cells, and macrophages.
• Inactive yellow marrow (YM) can convert back to active RM when needed (e.g., due to blood loss).

Red Marrow

• Composed of extravascular cords containing developing blood cell lineages, stem progenitor cells, and macrophages, separated by sinusoids (endothelial and adventitial cells-located between trabeculae of spongy bone).
• Haematopoietic cells develop within defined niches within the cords.

Hematopoietic Cells in Red Marrow

• Normoblasts (developing red blood cells) cluster near vascular sinuses, some around iron-laden macrophages.
• Megakaryocytes are found near vascular sinus walls for platelet release.
• Immature myeloid cells (e.g., granulocytes) are located deeper within cords, migrating towards sinuses during maturation.

Advential Cells

• Form a discontinuous layer on the outer side of the bone marrow sinus.
• Basement membrane lies next to these cells.
• Endothelial cells form the inner layer of the sinus.
• Advential cells (reticular cells) have extending processes, forming a supportive mesh for developing blood cells.
• They contract to allow mature blood cells to pass into the bloodstream, interacting with endothelial layer.

Haematopoietic Microenvironment

• Plays a critical role in stem cell differentiation and proliferation.
• Provides a semi-fluid matrix for haematopoietic cells' anchorage, self-renewal, proliferation, and differentiation.
• Stromal cells (endothelial cells, adipocytes, macrophages, osteoblasts, osteoclasts, fibroblasts) support and regulate stem cell survival and differentiation.

Types of Stromal Cells

• Endothelial Cells: Form a continuous inner layer, controlling particle flow, and producing cytokines.
• Adipocytes: Large cells with a single fat vacuole, influencing erythropoiesis and maintaining bone integrity by secreting steroids.
• Fibroblasts (reticular cells): Produce reticular fibers supporting vascular sinuses and developing hematopoietic cells.
• Osteoblasts: Bone-forming cells.
• Osteoclasts: Bone-resorbing cells.
• Macrophages: Involved in phagocytosis and cytokine secretion, regulating haematopoiesis and located throughout the marrow space.

Liver and Spleen

• Liver: Primary site for blood cell production during fetal development, until bone marrow takes over. Also plays a role in protein synthesis for blood clotting and eliminating old/damaged blood cells.
• Spleen: Produces red blood cells (RBCs) in the fetus. Acts as a filter in adults, removing old/damaged RBCs and recycling iron. A role in the immune response is played through antibody production and white blood cell storage.

Lymph and Thymus

• Lymphatic System: Produces lymphocytes (WBCs) critical for the immune response, removing toxins and waste, transporting lymph (contains infection-fighting WBCs).
• Thymus: Essential for the maturation of T-lymphocytes (T-cells), crucial for the adaptive immune response. Secretes hormones (e.g., thymosin) promoting T-cell development and maintaining the immune system.

Haematopoietic Stem Cells (HSCs)

• Actively dividing cells capable of self-renewal and differentiation into any blood cell type.
• Give rise to haematopoietic progenitor cells (HPCs) - committed to a single blood cell lineage, not capable of self-renewal.
• Self-renewal involves maintaining multipotency & tissue regenerative potential through cell cycle entry & division, with at least one progeny remaining undifferentiated.
• Differentiate into progenitor cells (e.g., common myeloid & lymphoid progenitors).

Morphologic Features of Blood Cells During Maturation

• Overview of changes during development:
  • Decrease in cell size
  • Reduction in nucleus-to-cytoplasm ratio
  • Fluctuations in nucleus size and shape
  • Chromatin condensation
  • Loss of nucleoli
  • Cytoplasmic changes (e.g., reduction in basophilia, increase in granularity).

Regulation of Haematopoiesis

• Regulated by haematopoietic growth factors (cytokines).
• Cytokines are soluble proteins eliciting biological effects at low concentrations.
• Influence blood cell production, differentiation, trafficking, and apoptosis.
• Positive influences (e.g., KIT ligand, FLT3 ligand, GM-CSF, IL-1, IL-3, IL-6, IL-11)
• Negative influences (e.g., transforming growth factor-β, TNF-α, interferons, lymphokines, monokines, chemokines)

Colony-Stimulating Factors (CSFs)

• Cytokines that stimulate WBC production, differentiation, and function.
• Subtypes including granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and granulocyte-macrophage-CSF (GM-CSF).
• Crucial for stem cell proliferation and differentiation into various blood cell lineages (e.g., erythrocytes, granulocytes, and macrophages).

Interleukins (ILs)

• Cytokines playing a significant role in the immune system (regulating growth, differentiation, and activation of haematopoietic and immune cells).
• Key types include: IL-3 (multipotent haematopoietic stem cell growth & differentiation), IL-6 (stimulating immune responses & haematopoiesis), IL-7 (T and B lymphocyte development), IL-11 (megakaryocyte growth, precursors to platelets).

Bone Marrow Examination/Procedure

• Involves an invasive procedure for haematologist (specialist).
• Preferred site for collection is posterior iliac crest in adults.
• Indications for examination include unexplained anemia, abnormal/irregular blood components, cytopenia, disorders diagnosis/staging of malignant disorders, and treatment monitoring, suspected bone marrow metastases.
• Should be evaluated within 24 hours of peripheral blood counts for accurate context.

Bone Marrow Collection

• Aspirates (flakes).
• Trephine biopsy using appropriate instruments (e.g., needles, cannulas).
• Collection materials & techniques are detailed to maintain sterility and prevent contamination.

Dry Tap

• Occurs when no bone marrow is obtained during the procedure due to various underlying issues (e.g., marrow fibrosis, increased adipose tissue, neoplastic infiltration).
• Can also be due to technical issues in the aspiration procedure.

Bone Marrow Processing

• Thick and thin smears prepared using specific stains (May-Grunwald-Giemsa (MGG), Wright, Prussian Blue (Perl's) for iron).
• Additional methods include cytochemistry, flow cytometry, molecular study, cytogenetic study in culture bottles for histological staining of trephine biopsies, and FISH (fluorescence in situ hybridization) studies of particle clots.

Peripheral Blood

• Adult blood volume: approximately 4-6 liters (~8% of total body weight).
• Composition: 55% plasma & 45% cells (erythrocytes, leukocytes, thrombocytes - platelets).
• Leukocytes (white blood cells): neutrophils, lymphocytes, monocytes, eosinophils, basophils - types based on granular and nuclear characteristics.

RBC (Erythrocyte)

• Structure: Anucleate (no nucleus), biconcave shape for maximum oxygen/carbon dioxide surface area. Filled with haemoglobin (protein that binds to oxygen and carbon dioxide).
• Production: Erythropoiesis in red bone marrow from stem cells (process with multiple stages).
• Life Cycle: ~100-120 days. Recycled by macrophages in the spleen, liver, and bone marrow.
• Function: Transport oxygen from lungs to tissues and carbon dioxide from tissues to lungs.

WBC (Leukocyte)

• Structure: Granulocytes (e.g., neutrophils, eosinophils, basophils) have granules, agranulocytes (e.g., lymphocytes, monocytes) lack granules, varying nucleus shapes and sizes.
• Production: In bone marrow from haematopoietic stem cells, differentiating into various types based on their specific roles in immune function.
• Life Cycle: Lifespan varies depending on the WBC type (e.g., neutrophils a few hours to days).
• Function: Crucial for the body's immune system (e.g., protecting against infections, removing dead cells).

Neutrophil

• Structure: Multi-lobed nucleus, cytoplasmic granules with antimicrobial substances.
• Production: In bone marrow, maturing before entering bloodstream.
• Life Cycle: Short lifespan (<24 hours in circulation).
• Function: First responders to bacterial infections (phagocytosis, eliminating pathogens).

Eosinophil

• Structure: Bi-lobed nucleus, cytoplasmic granules staining red with eosin.
• Production: In bone marrow, mature in tissues.
• Life Cycle: Circulate in blood (8-12 hours), residing in tissues for several days.
• Function: Combat parasitic infections, involved in allergic reactions/Asthma (by releasing toxic granules).

Basophil

• Structure: Large, cytoplasmic granules staining blue with basic dyes.
• Production: Produced in bone marrow, released into the bloodstream.
• Life Cycle: Short lifespan (hours to days).
• Function: Release histamine and heparin during inflammatory and allergic reactions, participating in immune responses to parasites.

Lymphocyte

• Structure: Large, round nucleus with minimal cytoplasm (agranulocyte).
• Production: Bone marrow, maturing in lymphoid organs (thymus & spleen) into B and T lymphocytes.
• Life Cycle: Varying lifespan (weeks to years) based on cell type.
• Function: Crucial for adaptive immunity (B cells produce antibodies while T cells destroy infected cells/regulate immune responses).

Monocyte

• Structure: Largest white blood cells with a kidney-shaped nucleus.
• Production: In bone marrow, circulate in the blood before migrating to tissues.
• Life Cycle: Circulate in blood (1-3 days) prior to differentiating into macrophages or dendritic cells in tissues.
• Function: Phagocytosis (ingesting pathogens and dead cells), immune regulation, and tissue repair.

Platelets (Thrombocytes)

• Structure: Small, disc-shaped cell fragments without a nucleus, derived from megakaryocytes.
• Production: Thrombopoiesis in the bone marrow, megakaryocytes release platelets into the bloodstream.
• Life Cycle: ~7-10 days. Removed from circulation by the spleen (old/damaged).
• Function: Crucial role in haemostasis (blood clotting). Adhere to damaged vessels, aggregate for a plug, activate the coagulation cascade.

Complete Blood Count (CBC)

• Most commonly ordered test in haematology.
• Provides information on overall health of bone marrow, medical condition diagnosis, and treatment monitoring.
• Performed using EDTA whole blood.
• Measures: Leukocytes (WBC count & differentials), Erythrocytes (RBC count, haemoglobin, RBC indices), & Platelets (platelet count).
• Manual and automatic methods available.

SLS - Haemoglobin Method

• In haematology, a method for automatically measuring haemoglobin used on large/fully automated instruments.
• Faster conversion speeds & no poisonous substances compared to past methods.

CBC - RBC Indices (HCT, MCV, MCH, MCHC)

• HCT (haematocrit/PCV): Percentage or volume fraction of red blood cells in whole blood (using microhematocrit tubes).
• MCV (mean corpuscular volume): Average volume of red blood cells (fL). High in macrocytic anemia (e.g., vitamin B12 or folate deficiency), low in microcytic anemia (e.g., iron deficiency).
• MCH (mean corpuscular haemoglobin): Average haemoglobin content per red blood cell (pg). High in macrocytic anemia/hyperthyroidism, low in microcytic anemia/thalassemia.
• MCHC (mean corpuscular haemoglobin concentration): Average concentration of haemoglobin per red blood cell volume (g/dL). High in hereditary spherocytosis/autoimmune hemolytic anemia; low in iron deficiency anemia/chronic inflammation/infection.
• RDW (red cell distribution width): Standard deviation of red blood cell volume (%). High in anisocytosis (abnormal variation in cell size) and nutritional deficiencies (e.g., vitamin B12, iron deficiency); low in thalassemia.

Reticulocyte Count (RET)

• Measures the last immature red blood cell stage (reticulocytes) in peripheral blood.
• Stained with supravital stains (e.g. new methylene blue/brilliant cresyl blue) to observe the remnant RNA/organelles (reticulum)
• Clinically used for assessing the bone marrow's erythropoietic activity.
• Reflects the rate of red blood cell production.

Erythrocyte Sedimentation Rate (ESR)

• Measures the rate at which red blood cells settle in a blood sample over a set period (typically 1 hour).
• Useful in diagnosis like inflammatory conditions, infections, and certain diseases.
• Reflects the presence of inflammatory markers (e.g., increased ESR with increased concentration of inflammatory proteins).

Automated CBC- Based on Sysmex XN-1000

• Haematology analysis utilizing hydrodynamically focused DC detection method (for RBC & PLT counts/sizes), a flow cytometry method (using a semiconductor laser), and a SLS hemoglobin method.

Forward Scatter and Side Scatter Light

• Forward scattering (measuring the intensity of light scattered forward): measures the size & granularity of particles.
• Side scattering (evaluating intensity of scattered light at an angle): provides information about cell interior characteristics (e.g., nucleus size, granularity).

References & Further Reading

• Hoffbrand AV, Moss PAH. (2016) Hoffbrand's Essential Hematology, 7th ed.
• Turgeon ML (2018) Clinical Hematology: theory & procedures.
• Keohane EM, Otto CN, Walenga JM (2020) Rodak's Hematology.
• Harmening D (2024) Clinical hematology and fundamentals of hemostasis, 6th ed.

Other Methods/Procedures (In different sections)

• Detailed procedures, processes, and/or required materials for bone marrow examination, collections, and/or analyses.

This quiz delves into the critical processes of hematopoiesis, highlighting its significance in adults and its phases during fetal development. Questions cover various aspects, including blood cell lifespan, the role of stem cells, and the function of bone marrow.