Leukopoiesis

• Leukopoiesis refers to the process of white blood cell (leukocyte) production in the bone marrow.
• Leukopoiesis is essential for maintaining immune system health and homeostasis

Areas of Focus

• Leukopoiesis itself
• Structure and morphology of normal leukocytes
• Functions of normal leukocytes

What are Leukocytes?

• Leukocytes protect a host from infectious agents and pathogens.
• Leukocytes develop from hematopoietic stem cells in the bone marrow.
• The cells differentiate and mature.
• Leukocyte morphology is either polymorphonuclear or mononuclear.
• Leukocytes function in innate and adaptive immune responses.
• Classification can be done using Romanowsky stained blood smears under a light microscope, or by identifying surface markers like Cluster of Differentiation (CD).
• Leukocyte count varies based on sex, age, activity, time of day, and ethnicity.
• Leukocyte count also varies due to stress, disorders and BM production
• Normal adult WBC count: 4 - 10 x 109/L

Stages of Leukopoiesis

• Stem cells: Hematopoietic stem cells (HSCs) give rise to all blood cell types including leukocytes
• Differentiation: HSCs differentiate into myeloid and lymphoid progenitor cells
• Myeloid Progenitor Cells: Develops into granulocytes and monocytes
• Lymphoid Progenitor Cells: Becomes B cells, T cells and NK cells
• Maturation stages are influenced by growth factors until they become fully functional
• Once mature, leukocytes are released into the bloodstream to fight infections, and protect against foreign invaders

Sites of Leukopoiesis

• Leukopoiesis primarily occurs in the bone marrow (BM).
• It also occurs in specific bodily locations that are areas of immune response
• Bone marrow: Primary site for production/maturation of WBCs i.e. granulocytes and monocytes from HSCs
• Thymus: Crucial for T lymphocyte (T cell) maturation. Immature T cells migrate here and become functional T cells.
• Lymph Nodes are secondary lymphoid organs where lymphocytes e.g. B/T cells mature and proliferate in response to antigens.
• The spleen supports proliferation/maturation of lymphocytes, especially B cells.
• The spleen filters blood and removes old/damaged blood cells.
• Peripheral blood: Mature leukocytes circulate here to perform their immune functions.

Granulopoiesis

• Granulopoiesis is the process by which granulocytes (neutrophils, eosinophils, basophils) are produced in bone marrow.
• HSCs differentiate into myeloid progenitor cells (granulocyte precursors).
• Myeloid progenitor cells differentiate into Granulocyte-Macrophage Progenitor Cells (GMPs), which becomes granulocytes and monocytes.
• Lineage Commitment: GMPs commit to generate specialized granulocyte precursors
• Myeloblasts are the earliest recognizable granulocyte precursors.
• Promyelocytes contain primary granules (next stage).
• Myelocytes form specialized granules characteristic of neutrophils, eosinophils or basophils.
• Metamyelocytes have indented nuclei, which is a further maturation of their granules.
• Band cells are immature granulocytes with a band-shaped nucleus, particularly in neutrophil development.
• Band cells mature into fully functional granulocytes
• Neutrophils: abundant granulocyte fighting bacteria/fungi.
• Eosinophils important for combating parasites and allergic reactions.
• Basophils are important for inflammatory responses and the release of histamine release during allergic reactions.

Neutrophil Development

• Derived from granulocyte-monocyte progenitor in BM
• Cytokines GM-CSF, IL-3 and G-CSF are responsible for growth and differentiation
• Six stages: myeloblast, promyelocyte, myelocyte, metamyelocyte, band and neutrophil
• Myeloblast maturation to myelocyte = 3-6days, another 5-7 days in the maturation pool, then release to the peripheral blood
• Average lifespan is 6-10 hours in the peripheral blood. After which, migration can take days
• Storage and circulating pools exist of mature neutrophils exist in BM

Neutrophils: Myeloblast to Myelocyte

• Myeloblast
• Size: 15-20μm
• Nucleus: round to oval
• Nucleoli: 2-5
• Chromatin: Fine
• Cytoplasm: Moderate basophilia
• Granules: Absent or up to 20.
• N/C Ratio: 4:1
• Promyelocyte:
• Size: 14-24µm
• Nucleus: round and oval
• Nucleoli: 1-3 or more
• Chromatin: but slightly coarser than myeloblast
• Cytoplasm: Basophilic.
• Granules: >20 to many; their color is red to purple or burgundy, some secondary granules
• Myelocyte
• Size 12-18 µm
• Nucleus: round to oval, may be a clearing next to the nucleus indicating Golgi location
• Nucleoli: usually not visible
• Chromatin: Coarse, more prominent than promyelocyte
• Cytoplasm: Slightly basophilic to cream-colored
• Can contain Primary and Secondary Granules

Neutrophils: Metamyleocyte to Segmented Neutrophils

• Metamyelocyte
• Size: 10-15 μm
• Nucleus is indented, but it is less than 50% of the width it would be if it were a hypothetical round nucleus
• Chromatin: moderately clumped
• Cytoplasm transitions from pink to colorless.
  • Many secondary, few primary, granules present
• N/C Ratio: 1.5:1
• Band Cell
• Size: 10-15 μm
• Nucleus: Is more than 50% indented
• Chromatin must be visible in constriction
• Cytoplasm: pale pink to blue with primary and secondary granules
• Segmented Neutrophil
• Nuclear lobes: segmented and connected by thin filaments with clumped chromatin
• Cytoplasm is abundant and predominates
• Primary granules are rare; secondary Abundant

Neutrophil Functions

• Phagocytosis: Neutrophils engulf and digest pathogens like bacteria/fungi.
• Granules filled with enzymes and antimicrobial proteins are released to kill and digest microbes.
• A web-like structure forms called Neutrophil Extracellular Traps (NETs), in order to trap and kill pathogens.
• They migrate quickly in a process called Chemotaxis, to areas of infection or inflammation via chemical signals (chemokines).
• Release signal molecules, and cause Inflammatory Response to help recruit other immune sites, and amplify the inflammatory response.
• Interaction with other immune system cells like macrophages and lymphocytes, enhance immune response.

Neutrophil Granules

• Primary granules (azurophilic, non-specific): Promyelocyte
• Myeloperoxidase (MPO), lysozyme, hydrolases, bacterial permeability-increasing protein (BPI)
• Secondary granules (specific) and myelocyte/metamyelocyte forms
• Contain lactoferrin, lysozyme, collagenase
• Tertiary granules form in metamyelocyte and band forms -Contain lysozyme, collagenase
• Secretory granules occur in band and segmented neutrophils
• Alkaline phosphatase

Eosinophil Development

• Derived from eosinophil-basophil progenitor
• GM-CSF, IL-3 and IL-5 are cytokines that facilitate differentiation/growth of eosinophils, whereas IL-5 specifically is critical for growth/survival
• Eosinophil myelocyte is the first maturation phase as eosinophil under light microscope by Romanowsky stain
• The half-life of an eosinophil is ~18 hours in circulation, and 2-5 days in tissues

Eosinophils: Myelocyte to Mature Eosinophiles

• Myelocyte
• Size 12-18 µm
• Nucleus: round and oval, possible slight flattening
• Nucleoli: usually not visible
• Chromatin: Coarse, more condensed than promyelocyte
• Cytoplasm: Colorless to pink
• Few to moderate primary
• Variable number of eosinophil specific granules (pale orange with a refractile appearance
• Metamyelocytes
• Size 10-15 µm, indented nucleus
• Smaller % of the indented nucleus
• Few primary granules
• Many eosinophil specific granules.
• Eosinophilic band
• Chromatin is condensed, coarse, and has threadlike features
• Granules exhibit refractile or pale color
• Cytoplasm is more transparent
• Few primary granules,
• Abundant eosinophilic specific granules
• Eosinophils
• Size is 12-17µm
• Nucleus is 2-5 lobes connected by thin filaments without visible chromatin
• Are rare primary granules
• Have abundant orange granules

Eosinophil Functions

• Defends against parasitic worms (helminths): toxic granules releases enzymes/proteins like major basic protein (MBP) and eosinophil cationic protein (ECP that can damage/kill parasites
• Body's response to allergens: Release inflammatory mediators e.g. histamine and leukotrienes (allergy response)
• Release cytokines/chemokines (modulate inflammatory/immune response
• Tissue repair by releasing growth factors
• Antigen-presenting cells (APCs):, process, and presenting antigens to T-Cells in order to help activate adaptive immune

Basophil Development

• Basophils derive from eosinophil-basophil progenitors and need cytokines like IL-3, IL-5, and GM-CSF for their development.
• Transforming growth factor-Beta suppresses eosinophil differentiation and increases basophil differentiation.
• Basophils and mast cells share similar morphology and function.
• Basophils mature only in the bone marrow and circulate in blood.
• Mast precursor cells mature in blood
• A basophil's average lifespan in circulation is approximately 4 days.

Mature Basophil Characteristics

• Size: 10-14 μm
• Nucleus: Usually two lobes connected by thin filaments without visible chromatin
• Nucleoi: Not Visible
• Chromatin: Coarse, clumped
• Cytoplasm: Lavender to colorless
• Granules: deep purple to black, unevenly distributed, and water soluble

Basophil Functions

• Basophils release histamine from the response to allergens
• Promotes inflammatory responses (e.g. leukotrienes)
• Similar to eosinophils, basophils are useful in defense against parasites
• Coordinate an effective immune response
• Prevent clots that could restrict blood flow (release heparin)

Monopoiesis

• Monopoiesis refers to monocyte the process by which monocytes are developed in the bone marrow (BM)
• Monocytes derive from granulocyte-monocyte progenitors
• Cytokines GM-CSF, IL-3, and M-CSF are responsible for their growth and differentiation
• Average lifespan in PB is 20-40hrs before specific function maturation occurs
• Circulating and marginal pools exist in blood

Monocyte Characteristics

• Size: 12-18 μm
• Nucleus: Round to oval, can be irregular
• Nucleoli: 1-2 -may or not be visible
• Chromatin: Fine
• Cytoplasm Light blue to grey.
• Very few granules.
• N/C ratio: 4:1
• Promonocyte
• Irregularly nucleated; has brainlike convolutions
• Chromatin is Fine to lacy.
• N/C ratio is between 2 and 3:1
• Monocytes
• Nucleus is variable, but horseshoe orkidney shaped is the most prevalent
• Abundant cytoplasm with pseudopods and fine granules

Monocyte Functions

• Help to clear injuries and eliminate diseased tissue
• Migrate from the bloodstream to the tissue to become macrophages (longer-lived and reside in tissue) or dendritic cells. -Cytokine production signaling immune responses
• Inflammatory or Immune Response: travel to injury sites to coordinate immune response through inflammation
• help to activate and control the adaptive immune response by Antigen presentation to T cells.

Lymphopoiesis

• Lymphopoiesis (or lymphocytopoiesis) is the process by which lymphocytes are developed in BM from common lymphoid progenitor
• Able to produce antibodies (antigenic/immunological specificity)
• Assist defense against other foreign invasion
• Includes the NK, T, B lymphocyte types
• Humoral immunity and cell-mediated immunity are two types of response
• Development occurs across primary Lymphoids (Bone Marrow, thymus)
• The bone marrow and Thymus are primary where the spleen and etc, (lymph nodes, lymphoid tissues of alimentary and respiratory tracts are secondary

Lymphocytes

• T cells
• Origin: Thymus -Tissue distribution: Parafollicular areas of cortex in nodes, periarteriolar in spleen
• Receptors: TCR for ag
• Function: CMI against intracellular organisms
• Characteristic surface markers: CD1 through CD8
• Genes rearranged: TCR α, β, γ, δ
• B cells
• Origin: Bone Marrow -Tissue distribution: Germinal centres of lymph nodes, spleen, gut, tracts
• Receptors: BCR (immunuglobin) ag
• Function: Humoral immunity
• Characteristic surface markers: CD9,10,19,20,22, and HLA Class 1+2
• Genes rearranged: IgH, Igk, Iga

Lymphoblast to mature small Lymph Characteristics

• Lymphoblast

• Size: 10-20 μm
• Nucleus: Nucleoli: ≥1
• Cytoplasm Size: Scant, varies by basophicity and granules
• N/C ratio: 7:1 to 4:1

• Prolymphocyte
• Size : 9-18 μm
• Prominent nucleous
• Smaller nucleus, N/C 3-4: 1 ratio
• Small Lymphocyte
• Size 7-18μm
• Condensed, blocky, and smaller round nucleus
• Scant to moderate sky blue, can express granules but mostly no

Large Lymphocyte and Plasma cell

• Large granular lymphocytes are cytotoxic T or can cause infections
• Plasma cells Size: 8-20 μm
• Nucleoi: none
• Coarse chromatin and is deeply basophilic
• N/C RATIO: 2:1- 1:1

Lymphocytes

• Produce antibodies against antigen
• NK patrol for viruses without antigen presenting