2. Hematopoiesis

Basic Morphology and Concepts

  • Study of blood and related disorders

  • Average blood volume:

    • Women: 4-5 L

    • Men: 5-6 L

  • Blood constitutes approximately 8% of body weight

  • Blood pH range: 7.35-7.45

  • Blood composition:

    • 55% plasma

    • 45% formed elements

    • 44% Red Blood Cells (RBC)

    • 1% White Blood Cells (WBC) and platelets

Plasma Composition

  • Plasma is composed of 91.5% water and 8.5% solutes:

    • Solutes:

    • Albumin: 55%

    • Globulins: 38%

    • Fibrinogen: 7%

    • Miscellaneous components (including electrolytes, hormones, non-protein nitrogen compounds, and gases)

Cell Morphology from Normal Blood Smear

  • Erythrocytes:

    • Evaluated under conditions where cells are evenly distributed without overlap

    • Structure: Biconcave disc, size 7-8 µm, volume of 90 femtoliters

    • Central region creates an area of central pallor

    • Uniform shape and size with no nucleus or inclusions

Platelets

  • Evaluated in the same areas as RBCs

  • Size: 1-4 µm, varying in shape

  • Normal observation shows an average of 7-15 platelets per oil immersion field

  • To estimate, count in 10 fields

  • Appearance: Reddish-purple granules with a small amount of bluish cytoplasm

  • Function: Contain molecules needed for hemostasis, including adhering, aggregating, and serving as surfaces for coagulation reactions

Leukocytes

  • Different types of leukocytes include:

    • Neutrophils (segmented and band forms)

    • Eosinophils

    • Basophils

    • Lymphocytes

    • Monocytes

  • Any presence of immature cells is considered abnormal

Types of Neutrophils

  • Segmented Neutrophil:

    • Comprise 50-70% in adults and older children

    • Typically has 3 lobes

    • Chromatin appears heavily clumped, coarse, or pyknotic

    • Cytoplasm is light pink with secondary granules in pink or neutral shades

    • Generally double the size of RBC

    • Functions in inflammation and phagocytosis

  • Band Neutrophil (Non-segmented):

    • Resembles a horseshoe or sausage shape (C or S shaped)

    • Clumped chromatin with pyknotic masses at each end

    • Secondary granules are small and exhibit various shades of pink

    • Distinguishing features from segmented neutrophils require various criteria; when in doubt, assess maturity for clarity

Eosinophils

  • Constitute 0-4% of leukocytes

  • Characterized by round refractile granules with eosin affinity, showing orange/reddish-orange coloration

  • Slightly larger than neutrophils, typically bi-lobed with condensed chromatin

  • Exhibit diurnal variation with increased levels observed at night

Basophils

  • Comprise 0-2%

  • Large, abundant violet-blue (purple-black) granules exhibiting affinity for blue dyes

  • Granules are water-soluble, which may result in loss of visibility if poorly fixed

  • Similar to eosinophils, they show diurnal variation

Lymphocytes

  • Represent 20-44% of leukocytes in adults

  • Measure 7-10 µm with size variability based on slide thickness

  • The edges of large lymphocytes may appear indented by neighboring RBCs (known as holly-leaf appearance)\n

  • Cytoplasm is blue and clear with more intense peripheral blue, generally lacking granular presence; if present, red granules may be visible

  • N:C ratio is typically between 4:1 to 2:1

  • Clumped chromatin and possible nucleoli visible in some cells under light microscopy

Monocytes

  • Represent 2-9%

  • Measure 12-18 µm with a nucleus-to-cytoplasm (N:C) ratio of 1:1 to 2:1

  • Cytoplasm appears dull blue-gray with fine granules contributing to a “ground glass” effect

  • Nuclear morphology includes kidney bean shapes or lobulation

Hematopoiesis

  • Defined as:

    • The process of blood cell production and development of blood cell varieties

  • It originates from the progeny of hematopoietic stem cells

  • The system is responsible for maintaining the populations of RBCs, WBCs, and platelets

    • RBCs: Transport oxygen and carbon dioxide

    • Granulocytes: Fight infections

    • Lymphocytes: Immune functions

    • Platelets: Maintain hemostasis to halt bleeding

Self Replication and Proliferation

  • The bone marrow microenvironment harbors stem cells, characterized as multipotential and able to differentiate into diverse cell types based on received stimuli

  • Mature cells in peripheral blood arise through growth factors (cytokines) such as:

    • Colony Stimulating Factors (CSF)

    • Interleukins

  • Recombinant technology allows for cytokine production for therapeutics:

    • Interleukins

    • EPO (Erythropoietin)

    • Granulocyte Colony-Stimulating Factor (G-CSF)

    • Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

Production Rates of Blood Cells

  • Bone marrow can produce:

    • 3 billion RBCs

    • 1.5 billion WBCs

    • 2.5 billion platelets
      per day per body weight

  • Functions of bone marrow structure:

    1. Constant supply of mature cells to peripheral circulation

    2. Ability to increase production based on hematologic conditions

    3. Compensate for decreased production by recruiting non-bone marrow sites like the liver and spleen

Stages of Hematopoiesis

  • Hemopoiesis: Formation of blood cells from hematopoietic stem cells

  • Differentiation Pathways:

    • Myeloid Progenitor Cells

    • Generate all types of granulocytes and platelets

    • Lymphoid Progenitor Cells

    • Generate T cells, B cells, and natural killer cells

Key Hematopoietic Sites

  • Key components include:

    • Bone marrow (primary site)

    • Liver

    • Spleen

    • Lymph nodes

    • Thymus

  • These sites facilitate blood cell development and adjust production in response to various stimuli (e.g., infection, bleeding, hypoxia)

Timeline of Hematopoiesis in Fetus and Adults

  • Embryonic Life:

    • First weeks: Mesoderm of yolk sac produces nucleated erythroid cells

    • 2 months: Production declines, migrating to liver (until 7 months) and hematopoiesis occurs in spleen

  • Post-Birth Transition:

    • At birth: Liver and spleen cease production; the bone marrow takes over

  • Aging:

    • 4 years: Hematopoiesis decreases

    • 18-20 years: Hematopoiesis shifts to axial skeleton and specific flat bones (sternum, ribs, pelvis, vertebrae, skull)

Extramedullary Hematopoiesis

  • Occurs under conditions of increased demand or stress

  • Refers to hematopoiesis outside bone marrow (primarily in liver and spleen)

  • Can lead to hepatosplenomegaly due to organ strain

Functions of the Spleen

  • Important in red blood cell activities: filtration, production, and cellular immunity

  • Location: Left side of the body under the rib cage; fist-shaped organ weighing about 8 oz

  • Structure consists of:

    • Red pulp: Primarily involved in red blood cell filtration

    • White pulp: Involved in lymphocyte processing

    • Marginal zone: Stores white blood cells and platelets

  • Spleen Functions:

    1. Reservoir/storage for platelets and granulocytes

    2. Inspection mechanism for red blood cells and platelets, identifying abnormal cells

    3. Larger role in immunity and opsonizing encapsulated organisms

Hematopoietic System Pools

  • Various pools noted include:

    • Stem cell pool (undifferentiated cells)

    • Bone marrow pool with proliferating and maturing cells

    • Peripheral blood containing functional and storage cells

    • Marginating storage pool: Neutrophils lining blood vessel walls

Proliferation in Response to Demand

  • Erythropoietin (EPO) stimulates erythroid precursors to proliferate in cases of anemia or hypoxia

  • Resulting increase in polychromatophilic cells (reticulocytes) indicates ongoing production

Erythropoietin (EPO)

  • EPO is a hormone cytokine produced by the kidneys and acts as a targeted erythroid growth factor

  • It promotes red blood cell production via receptors on young red blood cell precursors (pronormoblasts)

  • Regulation:

    • Daily secretion of EPO increases in response to tissue hypoxia in anemia, accelerating RBC production

    • Complete maturation usually takes 5 days, but in accelerated erythropoiesis can decrease to 3-4 days

  • A recombinant form of EPO is available for therapeutic uses

Mature RBC Characteristics

  • Mature RBCs are characterized by:

    • Anucleate state, lacks mitochondria and ribosomes

    • Exhibit pliability, flexibility, and deformability

Erythropoiesis

  • Definition: The process of producing erythrocytes primarily in the bone marrow

  • Stimulated by erythropoietin, produced in the kidneys

  • Maturation involves:

    • Reduction of cell volume and N:C ratio

    • Chromatin condensation and decrease in nucleoli formation

    • Downregulation of RNA and mitochondrial content

    • Incresed hemoglobin synthesis

Myelopoiesis (Granulocytopoiesis)

  • Production of neutrophils, eosinophils, and basophils

  • After release, half circulate in blood, while others remain in the marginating pool on blood vessel walls (liver, lungs, spleen)

  • Once in tissue, they do not migrate back to circulation or bone marrow

Morphological Changes in Myelopoiesis

  • Changes during differentiation include:

    • Nuclear volume reduction

    • Chromatin condensation

    • Changes in nuclear shapes

    • Appearance/disappearance of primary and secondary granules

    • Changes in cytoplasmic color and cell size

Stages of Neutrophil Maturation

  • Maturation sequence:

    1. Myeloblast

    2. Promyelocyte

    3. Neutrophilic Myelocyte

    4. Neutrophilic Metamyelocyte

    5. Neutrophilic Band

    6. Segmented Neutrophil (Polymorphonuclear Neutrophil)

Tissue Neutrophils

  • Generally non-phagocytic

  • Occasionally found in bone marrow, relevant in conditions like chronic myelocytic leukemia

Tissue Eosinophils/Basophils

  • Tissue basophils (mast cells) participate in allergic reactions

  • Contain histamine and heparin relevant in responses

Monopoiesis

  • The process leading to the production of monocytes, macrophages, and their precursors (monoblasts, promonocytes)

Macrophage Characteristics

  • Monocytes migrate to tissues and differentiate into macrophages, which do not generally re-enter circulation

  • Macrophages can also be termed histiocytes

Lymphopoiesis

  • Lymphoid progenitor cells differentiate into T, B cells, or null cells, indistinguishable morphologically

  • Primary lymphoid organs facilitate proliferation and maturation into functional immune cells; secondary organs interact with antigen-presenting cells, phagocytes, etc. during immune response

Plasma Cells

  • Final stage of B lymphocyte development

  • Cytoplasm embodying deep blue color with perinuclear zones appearing pale

  • Eccentric nucleus, producing proteinaceous materials appearing as round globules (Russell Bodies)

Megakaryocytopoiesis

  • Involvement of the largest hematopoietic cells in producing platelets through fragmentation

  • Megakaryocyte maturation leads to membrane rupture and thrombopoiesis

Bone Derived Cells

  • Osteoblasts: Found in marrow from young children and are responsible for bone formation, calcification, and maintenance

  • Osteoclasts: Multinucleated cells active in bone phagocytosis, often confused with megakaryocytes