HEMOPOIESIS & ERYTHROPOIESIS D.Rezazadeh Department of

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HEMOPOIESIS ERYTHROPOIESIS

• D.Rezazadeh
• Department of Medical Laboratory Science
• Kermanshah,Faculty of Paramedical

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HEMOPOIESIS INTRO

• Hemo Referring to blood cells
• Poiesis The development or production of
• The word Hemopoiesis refers to the production
  development of all the blood cells
• Erythrocytes Erythropoiesis
• Leucocytes Leucopoiesis
• Thrombocytes Thrombopoiesis.
• Begins in the fetal liver spleen, continues in
  the bone marrow till young adulthood beyond!

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SITES OF HEMOPOIESIS

• Appendicular skeleton
• Bones of the Upper Lower limbs
• In Adults active hemopoietic marrow is found only
  in
• The axial skeleton
• The proximal ends of the appendicular skeleton.

• Active Hemopoietic marrow is found, in children
  throughout the
• Axial skeleton
• Cranium
• Ribs.
• Sternum
• Vertebrae
• Pelvis

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STEM CELL THEORY

• The dazzling array of all the blood cells are
  produced by the bone marrow.
• They all come from a single class of primitive
  mother cells called as
• PLURIPOTENT STEM CELLS.
• These cells give rise to blood cells of
• Myeloid series Cells arising mainly from the
  bone marrow.
• Lymphoid series cells arising from lymphoid
  tissues.

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CLONAL HEMOPOIESIS
PLURIPOTENT STEM CELL
MULTIPLICATION
COMMITTMENT
COMMITTED STEM CELL
STEM CELL
MULTIPLICATION
COMMITTED STEM CELL
PROGENITOR CELL
CFU COLONY FORMING UNIT
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CLONAL HEMOPOIESIS (Contd)
COLONY FORMING UNIT (CFU)
INTERMEDIATE BLAST CELLS
MORPHOLOGICALLY RECOGNIZABLE
MATURE BLOOD CELLS END CELLS FINITE LIFE SPAN
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STEM CELLS

• These cells have extensive proliferative capacity
  and also the
• Ability to give rise to new stem cells (Self
  Renewal)
• Ability to differentiate into any blood cells
  lines (Pluripotency)
• They grow and develop in the bone marrow.
• The bone marrow spleen form a supporting
  system, called the
• hemopoietic microenvironment

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Control of Hemopoiesis
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STEM CELLS Types

• Pluripotent Stem cells
• Has a diameter of 18 23 µ.
• Giving rise to both Myeloid and Lymphoid series
  of cells
• Capable of extensive self-renewal.
• Myeloid Stem cells Generate myeloid cells
• Erythrocytes
• Granulocytes PMNs, Eosinophils Basophils.
• Thrombocytes.
• monocytes
• Lymphoid Stem cells Giving rise only to
• Lymphocytes T type mainly.

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PROGENITOR CELLS

• Committed stem cells lose their capacity for
  self-renewal.
• They become irreversibly committed.
• These cells are termed as Progenitor cells
• They are regulated by certain hormones or
  substances so that they can
• Proliferate
• Undergo Maturation

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CELL LINE REGULATORS
CELL LINE REGULATOR
ERYTHROID BFU-E CFU E BURST PROMOTING ACTIVITY ERYTHROPOIETIN
GRANULOCYTE-MONOCYTE CFU-GM COLONY STIMULATING FACTOR(CSF)
MEGAKARYOCYTES CFU-M MEGAKARYOCYTIC STIMULATING ACTIVITY
MEGAKARYOCYTES THROMBOPOIETIN
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BLOOD CELLS DEVELOPMENT
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ERYTHROPOIESIS
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ERYTHROPOIESIS SITES/PHASES

• INTRAUTERINE LIFE
• INTRAVASCULAR PHASE Under 3rd month of Intra
  Uterine Life.
• HEPATIC PHASE 3rd to 5th month IUL
• Liver Spleen
• MYELOID PHASE From 5th month of IUL onwards.

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Regulation of Erythropoiesis

• CFU E
• Proerythroblasts
• Mature Erythrocytes
• Tissue Oxygenation
• Factors decreasing
• Hypovolemia
• Anemia
• Poor blood flow
• Pulmonary Disease

Stimulates
An example of a Negative feed back mechanism
ERYTHROPOIETIN
Decreases
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Signaling Pathways
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FACTORS REGULATINGERYTHROPOIESIS

• SINGLE MOST IMPORTANT REGULATOR TISSUE
  OXYGENATION
• BURST PROMOTING ACTIVITY
• ERYTHROPOIETIN
• IRON
• VITAMINS
• Vitamin B12
• Folic Acid
• MISCELLANEOUS

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NAME CELLULAR SOURCE CELL TYPES PRODUCED IN INCREASED NUMBERS
Erythropoietin (EPO) Kidney cells, Kupffer cells rbc
G-CSF Monocytes, fibroblasts, endothelial cells n
M-CSF Monocytes, fibroblasts, endothelial cells m
GM-GSF T cells, monocytes, fibroblasts, endothelial cells n, m, e, meg, rbc
IL-1 Macrophages, endothelial cells, fibroblasts n, m, e, b, meg, rbc
IL-3 T cells n, m, e, b, meg, rbc
IL-4 T cells b
IL-5 T cells e
IL-6 Macrophages, endothelial cells, fibroblasts n, m, e, b, meg, rbc
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ERYTHROPOIETIN

• A hormone produced by the Kidney.
• A circulating Glycoprotein
• Nowadays available as Synthetic Epoietin
• Acts mainly on CFU E.
• Increases the number of
• Nucleated precursors in the marrow.
• Reticulocytes Mature Erythrocytes in the blood.

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HAEMATOPOIETIC GROWTH FACTORS

• Thrombopoietin
• Stimulated survival and proliferation of
  haemopoietic stem and megakaryocyte
  colony-forming cells
• Stimulated formation of megakaryocytes,
  including megakaryocyte maturation and release of
  platelet into the blood

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Colony-Stimulating Factors

• GM-CSF
• Its ability to stimulate colonies of neutrophils
  and macrophagess
• Acts on many cell types including early
  multipotential stem cells, megakaryocyte,
  eosinophil, erythroid and mature neutrophils and
  macrophages
• Stimulates of cytotoxic and phagocyte activity
  against bacteria, yeast, and parasites
• May also raise the platelet count and possibly
  the reticulocyte level

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Colony-Stimulating Factors

• G-CSF
• Stimulate predominantly neutrophil colony
  formation

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Interleukin-1

• Defined as a macrophage product inducing IL-2
  receptor expression on T lymphocytes
• Activation of cells involved in
• early stages of hematopoiesis
• The inflammatory response
• Wound healing
• Activates lymphocytes, neutrophils and
  macrophages and NK cells
• Acts as endogenous pyrogens
• Induces proliferation of fibroblasts, epithelial
  and endothelial cells

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Interleukin-2

• Is T-cell growth factor
• Promotes the proliferation of T-lymphocytes,
  B-lymphocytes and monocytes
• Promotes cytotoxic function by stimulating the
  proliferation and activity of NK cells
• May inhibit both granulocyte-macrophage colony
  formation and erythropoiesis

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Interleukin-5

• Knows as eosinophil differentiation factor
• Support the proliferation, maturation and
  function of eosinophils

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Interleukin-6

• Has a wide effects in hemopoiesis
• Interacts synergistically with other growth
  factors to stimulate myeloid proliferation
• Is a major factor in the immune response and
  inflammation and act as acute phase protein
• Stimulates granulocyte-macrophage and
  megakaryocyte colony formation

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Other cytokines

• Flt-3 ligand acts on relatively primitive
  progenitor stem cells showing synergies with
  G-CSF, GM-CSF, M-CSF, IL-3 and SCF

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ERYTHROPOIESIS SEQUENTIAL CHANGES
I II III
IV
V VI
I N T E R N B L A S T
E AR L Y N B L A S T
MA T UR E RB C
L A T E N B L A S T
R E T I C U L OC Y T E
PRONORMOB L A S T
MITOCHONDRIA BASOPHILIA HEMOGLOBIN
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ERYTHROID PROGENITOR CELLS

• BFU-E Burst Forming Unit Erythrocyte
• Give rise each to thousands of nucleated
  erythroid precursor cells, in vitro.
• Undergo some changes to become the Colony Forming
  Units-Erythrocyte (CFU-E)
• Regulator Burst Promoting Activity (BPA)

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ERYTHROID PROGENITOR CELLS

• CFU-E Colony Forming Unit- Erythrocyte
• Well differentiated erythroid progenitor cell.
• Present only in the Red Bone Marrow.
• Can form upto 64 nucleated erythroid precursor
  cells.
• Regulator Erythropoietin.
• Both these Progenitor cells cannot be
  distinguished except by in vitro culture methods.

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Normoblastic Precursors

• PROERYTHROBLAST
• Large cell 15 20 Microns in diameter.
• Cytoplasm is deep violet-blue staining
• Has no Hemoglobin.
• Large nucleus 12 Microns occupies 3/4th of the
  cell volume.
• Nucleus has fine stippled reticulum many
  nucleoli.

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Normoblastic Precursors

• EARLY NORMOBLAST
• Smaller in size.
• Shows active Mitosis.
• No nucleoli in the nucleus.
• Fine chromatin network with few condensation
  nodes found.
• Hemoglobin begins to form.
• Cytoplasm still Basophilic.

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Normoblastic Precursors

• INTERMEDIATE NORMOBLAST
• Has a diameter of 10 14 Microns.
• Shows active Mitosis.
• Increased Hemoglobin content in the cytoplasm
• Cytoplasm is Polychromatophilic.

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Normoblastic Precursors

• LATE NORMOBLAST
• Diameter is 7 10 Microns.
• Nucleus shrinks with condensed chromatin.
• Appears like a Cartwheel
• Cytoplasm has a Eosinophilic appearance.

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Normoblastic Precursors

• RETICULOCYTE
• The penultimate stage cell.
• Has a fine network of reticulum like a heavy
  wreath or as clumps of dots
• This is the remnant of the basophilic cytoplasm,
  comprising RNA.
• In the Neonates, Count is 2 6/Cu.mm.
• Falls to lt1 in the first week of life.
• Reticulocytosis is the first change seen in
  patients treated with Vit B12

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Normoblastic Precursors

• MATURE ERYTHROCYTE
• Biconcave disc.
• No nucleus.
• About One-third filled with Hemoglobin.

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VITAMINS

• B12 Cyanocobalamine Folic Acid
• Is also called Extrinsic Factor of Castle.
• Needs the Intrinsic Factor from the Gastric juice
  for absorption from Small Intestine.
• Deficiency causes Pernicious (When IF is missing)
  or Megaloblastic Anemia.
• Stimulates Erythropoiesis
• Is found in meat diary products.

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IRON

• Essential for the synthesis of Hemoglobin.
• Deficiency causes Microcytic, Hypochromic Anemia.
• The MCV, Color Index MCH are low.

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Pronormoblast
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Basophilic Normoblast
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Polychromatophilic normoblast
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Orthochromic normoblast
Graphic accessed http//www.wadsworth.org/chemheme
/heme/glass/cytopix/slide011_nrbc2.jpg, 2000.
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Reticulocyte
Basophilic stippling
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Red Blood Cell
Graphic accessed http//www-medlib.med.utah.edu/We
bPath/jpeg5/HEME002.jpg, 2000.
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HEMOPOIESIS REVIEW I

• Hemopoiesis Production of blood cells.
• Site Predominantly in the Bone Marrow.
• Both axial appendicular skeletons.
• Erythropoiesis Production of Erythrocytes.
• Leucopoiesis Production of Leucocytes.
• Thrombopoiesis Production of Thrombocytes.

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HEMOPOIESIS REVIEW II

• Cell lines
• Pluripotent Stem Cell
• Progenitor Stem Cells Committed
• Proerythroblasts
• Myeloblasts
• Monoblasts
• Lymphoid cells
• Megakaryoblasts

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Thank you!
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• Table 11. Findings That May Lead to a Hematology
  Consultation
• Decreased hemoglobin concentration (anemia)
• Increased hemoglobin concentration (polycythemia)
• Elevated serum ferritin level
• Accelerated sedimentation rate
• Leukopenia or neutropenia
• Immature granulocytes or nucleated red cells in
  the blood
• Pancytopenia
• Granulocytosis neutrophilia, eosinophilia,
  basophilia, or mastocytosis
• Monocytosis
• Lymphocytosis
• Lymphadenopathy
• Splenomegaly
• Hypergammaglobulinemia monoclonal or polyclonal
• Purpura
• Thrombocytopenia
• Thrombocytosis
• Exaggerated bleeding spontaneous or trauma
  related
• Prolonged partial thromboplastin or prothrombin
  coagulation times

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RBC Membrane

• Shape - biconcave disc
• 40 lipids
• 52 protein
• 8 carbohydrate
• Primary physiologic functions
• facilitates O2-CO2 transport
• Blood buffer
• Waste disposal
• Maintain deformability
• Keeper of surface antigens

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RBC Membrane

• Proteins
• peripheral
• alpha beta spectrin
• bands 1 2
• actin
• integral (lots of sialic acid)
• glycophorin A
• band 3
• inorganic anion transport channel
• cation pump regulates Na/K at ratios 112/251

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RBC Membrane

• Lipids
• phospholipids
• lipid bilayer
• unesterified cholesterol
• facilitates selective cation permeability
• 11 equilibrium w/plasma cholesterol
• increases in membrane cholesterol target
  cells!!

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Extravascular Erythrocyte Destruction

• Normal end-of-life event
• Inherited RBC Defects
• Membrane abnormalities
• Enzyme deficiencies
• Hemoglobinopathies
• Thalassemias
• Acquired RBC Defects
• B12 Deficiency
• Immune-mediated destruction
• Drug-induced
• Autoimmune

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IntravascularErythrocyte Destruction

• Intravascular hemolysis
• Normal end-of-life event
• Complement Activation
• PNH
• PCH
• Physical/mechanical Trauma
• DIC
• Prosthetic heart valves
• Toxins
• Arsenic poisoning
• Bacterial Infections

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Lab Evidence of Hemolysis

• Extravascular
• Urine urobilinogen
• Fecal urobilin
• Yellow-colored plasma

• Intravascular
• Increased Reticulocyte Index
• Decreased haptoglobin
• Decreased hemopexin
• Presence of Methemoglobin/methalbumin
• Increased indirect bilirubin
• Increased urobilinogen
• Decreased RBC
• Increased iron stores
• Hemosiderinuria
• Ferritin

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RBC Sources of Energy

• EMP (anaerobic)
• glucose to lactate
• ATP
• Hexose-monophosphate Shunt (aerobic)
• G-6-P to 6-PG
• NADPH
• Rapoport-Luebering Shunt
• 1,3-diphosphoglycerate to 2,3- DPG
• Methemoglobin Reductase
• G-3-P to 1,3-Bisphosphoglycerate
• NADH

Graphic accessed http//evolvels.elsevier.com/sect
ion/default.asp?id1138_ccalvo7_0001, 2009.
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Thymus
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Role of Metabolic Pathways
Pathway Enzymes Role Problems
EMP Phosphofructokinase Pyruvate kinase (PK) Produce ATP RBC Shape (ion pumps) Hemolytic Anemia PK Deficiency
HMS Glutathione Reductase G6PD NADPH Production OXY-METH HB Balance Hemolytic Anemias
RLB DPG Synthetase 2,3-DPG Production HB Oxygen Affinity Hypoxia
MHBR Methemoglobin reductase Protects HB from Oxidation via NADH Hemolytic Anemia Hypoxia
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