Week 8 Part 1 - Hematopoiesis

Hematopoesis:

• Formation and development of blood cells

• The proliferation, differentiation and maturation

  of blood cells

• Primarily takes place in the bone marrow

• Only mature cells are released into peripheral

  blood

Formed elements of the blood go through developmental stages:

• As cells mature, they are able to move through the sinusoids of the marrow because of decreased overall cell size, decreased nuclear cytoplasmic ratio, and increased flexibility and mobility.

• Normal peripheral blood cells include lymphocytes, basophils, eosinophils, segmented neutrophils, monocytes, and band neutrophils.

• Each cell type has a normal life span and function.

• Normally, only mature cells are seen in the peripheral blood circulation

  • Immature cells may appear in the peripheral blood in certain disease states, called a shift to the left

Characteristics of Hematopoesis:

• Replacement of circulating cells

• Proliferation of precursor cells that retain mitotic

  capability

• Governed by multiple cytokines

• Specialized microenvironment

Differentiation:

• Process that generates the diverse cell populations

• Appearance of different properties in cells which were

  initially equivalent

Commitment:

• When two cells derived from the same precursor take

  a separate route of development

• They commit to that path

Maturation:

• The entire process from commitment to when the cell

  has all of its characteristics

Two primary characteristics of hematopoesis:

1. The variety of distinct blood cell types produced

2. The relatively brief life span of the individual cells

Circulating cells are:

• Mature

• Incapable of mitosis

  • Exception: lymphocytes

• Limited life span or terminally differentiated

  • Must be replaced by less differentiated, mitotically active precursor cells

Stages of Hematopoesis:

• Divided into three phases:

  • Mesoblastic, Hepatic, Myeloid

• Sites of hematopoiesis by age

Mesoblastic stage:

• Begins in early embryonic development

• Starts with yolk sac, as early as 19 days after

  fertilization

• Confined to erythropoiesis from blood islands

• Remains active for 8-12 weeks

• Embryonic Hemoglobin: Gower 1, Gower 2, and

  Portland

  • (do not carry oxygen well)

  • Differs from fetal and adult hematopoiesis because it occurs intravascularly – (within developing blood vessels)

Hepatic Stage:

• Begins at 5-7 weeks gestational age

• Reaches peak by third month.

• Third month of fetal development liver becomes active along with kidney, spleen, and lymph nodes

• Thymus (first fully developed fetal organ) becomes major site for T cell lymphocyte production

• Kidney and Spleen produce B cell lymphocytes

  • Keep in mind cells differentiate in the lymph nodes, but aren’t produced there.

• Granulocytes and lymphocytes are at a minimum

• Hemoglobin production: A, A2 and F

• Continues to about the fifth month

Myeloid Stage:

• Occurs in bone marrow (called medullary) because it occurs in medulla

• Begins in 4th and 5th month of fetal development

• 6th month the bone marrow becomes the primary

  site for hematopoiesis and continues throughout

  life

• Measurable levels of EPO, G-CSF, GM-CSF,

  HgbF, HgbA can be detected

• Cells at various stages of maturation (for all cell

  lineages) can be seen

Bone Marrow:

• One of the largest organs in the body

• Composed of yellow and red marrow

• Intricate supply of nutrients and blood vessels

• Consists of vessels, nerves, differentiated and undifferentiated hematopoietic cells, reticuloendothelial cells

Bone Marrow Structure consists of:

• Vascular Compartment

• Hematopoietic Compartment

Vascular Compartment:

• Consists primarily of venous vessels referred to as sinuses

Hematopoietic Compartment:

• Hematopoietic stem cells residing in this compartment

  proliferate and differentiate into the lymphocytic,

  granulocytic, monocytic, megakaryocytic and

  erythroid lineages

• All cells go into sinus before going to peripheral

  blood

• Megakaryocytes – cytoplasm pinches off to

  become platelets

Bone Marrow Activity:

• During first few years of life the marrow of all

  bones is red and cellular

• By age 18 the red marrow is found in the vertebra, ribs, sternum, skull, and pelvis

• Past the age of 40, marrow in the sternum, ribs, pelvis, and vertebra are composed of equal amounts of red and yellow marrow

Medullary Hematopoiesis:

• Hematopoiesis that takes place in the bone marrow

Extra-Medullary Hematopoiesis:

• When there is an increased demand for hematopoiesis it may (again) begin in the spleen and liver (as a last resort kind of)

Bone Marrow Procedure:

• Iliac crest of pelvis site preferred site for bone marrow analysis

• 200 cell differential is performed (new recommendation is 500 cell differential)

Bone Marrow Biopsy or Core:

• To identify placement of cells to see where each cell belongs and the cellularity

• Touch preparation used to study cellular details.

• (Sample applied to several coverslips)

• Takes a whole piece of bone marrow

Bone Marrow Aspirate:

• To identify morphologically, cells are not in natural architecture

• Gives poor estimation of marrow cellularity

• Takes fluid from the bone marrow

Bone Marrow Differential Count:

• Birth: Predominance of granulocytes (first line of defense)

• One Month: Switch to lymphocytes

• Adult: Granulocytes and Erythrocytes predominate

Stem Cell Cycle:

• Bone Marrow is capable of producing massive

  amounts of cells daily

• Per kilogram of body weight

• The determining factor controlling the rate of production is physiologic need.

• Stem cell ratio in Bone Marrow

• 1:1000 (1 stem cell per 1000 nucleated RBC)

Origin of Marrow Cells:

• Bone Marrow contains pluripotent stem cells

• These cells have ability to differentiate and proliferate with self renewal.

• Influenced by growth factors and cytokines

Stem cell division:

• One reverts to stem cell

• Other becomes a colony forming unit (CFU)

• CFU stimulated to proceed in any direction

• Growth factors or cytokines regulate

  • proliferation

  • differentiation

  • maturation of hematopoietic precursor cells

Cell Cycle":

• G0 Resting Stage

• G1 RNA and Protein Synthesis

• S DNA Synthesis

• G2 Pre-mitotic

• M Mitosis

• Apoptosis: programmed cell death

  * In leukemia – no apoptosis

Growth factors & Cytokines:

• Often interchanged terms

Growth Factors:

• Proteins that bind to receptors on the cell surface of cells and result in proliferation or differentiation of the affected

  cells.

Cytokines:

• often compared with growth factors, affect primarily the cells of the immune system and orchestrate immune responses.

• Soluble protein, cellular products, that influence the

  function or activity of other cells.

• Have important functions in hematopoiesis and other

  cellular activities (especially immunology)

Cytokines include:

• Colony stimulating factors

• Interferons

• Interleukins

• Lymphokines

• Monokines

• Chemokines

• Cytokine Influence

How are cytokines & growth factors produced:

• By stromal cells in hematopoietic microenvironment

  • Exception is erythropoietin (EPO)

    • Produced mainly in the kidney

    • EPO specifically targets synthesis of RBC

• Most are not lineage specific

  • Each has multiple functions acting on more than one cell type (pleiotropy)

• Hematopoietic Growth Factors

  • SCF also called kit ligand

G-CSF:

• (Granulocyte colony stimulating factor)

• Produced by monocytes/macrophages, placenta, endothelial cells and stimulates development of the granulocytic lineage

GM-CSF:

• (Granulocyte Macrophage CSF)

• Produced by T lymphocytes, stromal cells, and macrophages and is an essential cytokine for development of granulocytic and monocytic lineages

Eythropoietin:

• Glycoprotein produced by kidney for the stimulation of red cell production

Thrombopoeitin:

• Glycoprotein produced by kidney, stromal cells, and

  hepatocytes to stimulate platelet production

KIT Ligand:

• (stem cell factor or SCF)

• Tyrosine-protein kinase expressed on HSC (Hematopoietic Stem Cell) , gets down regulated during differentiation, stimulates proliferation.

• Essential for early stage hematopoiesis

FLT3:

• Tyrosine-protein kinase expressed on HSC. Works

  synergistically with SCF and IL-3, GM-CSF to early stage hematopoiesis

IL11 (Interleukin 11):

• Produced by stromal cells and IL-1 stimulated fibroblasts to stimulate proliferation/differentiation of myeloid lineage

IL3 (Interleukin 3):

• Produced by activated T cells, eosinophils, and mast cells,

  stimulates colony formation for myeloid lineage WBC, RBC, PLT (but not lymphs)

IL7 (Interleukin 7):

• Produced by stromal cells stimulates proliferation/differentiation of lymphoid lineage

Colony Forming Units:

• Influenced by colony stimulating factors (CSF)

• CSF produced by many cells and have high specificity for target cells

• Highly active at low concentrations

• Capable of influencing multiple cell linages

• Different types of CSF such as:

  • G-CSF (Granulocytic CSF)

  • GM-CSF (Granulocytic/Monocytic)

Lymphoid Stem Cell CFU-L:

• Gives rise to two types of lymphocytes

• B cell: in the bone marrow, these transform to plasma cells

  • Participate in humoral immunity by synthesizing immunoglobulins

• T cell: migrate to thymus

  • Regulate humoral immunity through T-helper cell and T-suppressor cells subpopulations

  • Helper to suppressor – 2:1 ratio

Myeloid Stem Cell CFU-GEMM:

• Granulocytes – From myeloblast to the release

  of the mature cell is about 9 days

  • Remain in circulation 3-6 hours, later moving to tissue for 1-4 days

• Neutrophils: primary function to phagocytize

  extracellular pathogens

• Eosinophil: defense against parasites, also plays

  role in allergic reaction

• Basophil: plays role in hypersensitivity

Monocyte maturity:

• 3-5 days

  • Circulate 16-36 hours

  • Move to tissue where they transform to macrophages (or histiocytes)

• Histiocytes (macrophage) resides in tissues, aids in phagocytosis as part of innate immune response

Megakaryocytes maturity:

• 4-5 das

  • Function in hemostasis, through production of platelets

  • Platelets are small pieces of megakaryocyte cytoplasm

Erythroid Cells:

• arise from the BFU (burst forming unit)

  • Mature in about 3-5 days

  • Remain in circulation for 120 days

  • Filled with hemoglobin (oxygen carrying protein)

Myeloid:Erythroid Ratio (M:E):

• The relative proportions of the two principle

  bone marrow cells

• Compares the relative numbers of developing

  granulocytes with the relative number of

  erythroid precursors

• Normal M:E ratio in adults is 2:1 to 4:1

• Birth – 3:1 to 4.5:1

• Granulopoietic tissue occupies 2 to 4 times greater

  marrow space than the erythropoietic precursors

Hematopoietic Maturation Process:

• Overall size of cells

  • As a rule, primitive cells are large but as they mature their size decreases

  • Exceptions to the rule are megakaryocytes

Hematopoietic Maturation Process: Nuclear to cytoplasm ratio (N:C):

• N:C ratio decreases as cell matures

• Blasts contain about 4:1 ratio

• Nucleus gets smaller with maturation

• Exceptions: platelet (cytoplasm) RBC (loses nucleus) and lymph ratio aprox. 3:1

Hematopoietic Maturation Process: Chromatin Pattern:

• Nucleus is made up of DNA

• Young cells have very fine and loose chromatin

• As the cell matures the chromatin becomes dense, clumped and typically stains dark blue

Hematopoietic Maturation Process: Nuclear Shape:

• Younger cells have round to oval nucleus

• Monocyte has a folded shape

• Granulocyte nucleus is segmented

Hematopoietic Maturation Process: Nucleolus or Nucleoli:

• Made up of RNA with indicates metabolic activity and

  growth

• Stains light blue and as the cell matures they are not

  visible

• Blasts typically have between 1-5

Hematopoietic Maturation Process: Cytoplasmic RNA:

• Young cells have a large amount of RNA and typically

  stain dark blue, as the cell matures they become a

  light blue to pink

Hematopoietic Maturation Process: Golgi Zone:

• Immature cells demonstrate a clear zone adjacent to

  the nucleus

• Golgi apparatus is responsible for transporting,

  modifying, and packaging proteins

• When visible, often indicates a younger cell

Hematopoietic Maturation Process: Granulation:

• Progresses from no granules to non-specific, to specific granules

• Granulocytes are noted for their distinctive granules

• Primary are big and burgundy color

• Secondary are more common and are small

• Reference Ranges for Red Cell Parameters

• Reference Ranges for WBC Parameters

• Erythropoiesis

• Asynchronous Erythropoiesis