1. Hematopoeisis, Bone marrow, Erythropoiesis, RBC structure and function
Faisal Klufah
M.S.H.S, MLS(ASCP)

2. Objectives Define hematopoiesis Describe the origin of hematopoieisis
Define erythropoiesis
List proper cell maturation of the erythrocytic series
Identify three areas of RBC metabolism crucial for normal erythrocyte survival and function
Describe RBC membrane biochemical structure and the consequences of structural membrane defects
RBC metabolic pathways

3. Hematopoeisis

4. Tissue homeostasis
Maintenance of an adequate number of cells through these functions:
Proliferation
Differentiation
Death (apoptosis)

5. Cell cycle
G1 S G2 M
S phase - DNA synthesis
M phase – mitosis

6. Apoptosis vs Necrosis
Necrosis: cell death by lethal chemical, biological or physical events
Apoptosis : programmed cell death regulated by genetic material of cell
physical (e.g. radiation), chemical (e.g. carcinogens or chemotherapeutic drugs) or biological (e.g. bacteria, viruses or even insufficient oxygen) 
Increased in AIDS, neurodegenerative disorders, MDS, aplastic anemia
Decreased in cancer, autoimmune disorders, viral infections

7. Hematopoiesis Blood cell formation – production and development
Occurs bone marrow, liver, spleen, lymph nodes, thymus
Bone marrow – sole site of effective hematopoiesis in normal adults
6 billion cells/kg of body weight per day
2.5 billion red cells
2.5 billion platelets
1.0 billion white cells
Rate adjusted to need, vary from nearly zero to many times the normal
Constant turnover of cells

8. Definition of HEMATOPOIESIS
Development of different cell lineages in blood
Differentiation
Appearance of different properties in cells
Commitment
Cells derived from common precursors take separate routes
Maturation occurs from commitment to fully developed cell

9. Ontogeny of Hematopoeisis
Yolk sac > fetal liver/spleen > BM
Three developmental periods
Mesoblastic
Hepatic
Myeloid

10. Mesoblastic Blood islands of yolk sac Primarily RBC production
Embryonic hemoglobin produced
Mesoblastic:The middle germinal layer of an early embryo, consisting of undifferentiated cells destined to become the mesoderm

11. Hepatic At 6 weeks cell production in liver Fetal hemoglobin produced
Spleen, thymus, lymph nodes also active production

12. Myeloid At 5th month Bone Marrow becomes site of cell production
Liver & spleen now Extramedullary
Hemoglobin A (22)

13. Hematopoietic precursor cells
Stem cells
Progenitor cells
Maturing cells

15. Stem Cells Very small group of cells
Multipotential cells that give rise to all lineages of blood cells
High self-renewal ability
Not morphologically distinguishable
Identified by flow cytometry with marker CD34
Supporting research

17. Progenitor Cells Committed cells to differentiation into cell lines
Described as colony-forming units (CFU)
CFU-GEMM
CFU-GM
CFU-Meg
Population amplified by proliferation

18. Maturing cells Majority of precursor cells
Recognizable morphologic characteristics
Nomenclature unique for each cell line

19. Cytokines & Growth Factors
Govern precursor cell survival, self-renewal, proliferation, differentiation
Growth factor control
Interleukins numbered according to discovery
Growth factors promote cell survival by suppressing apoptosis
Growth factors promote proliferation

20. Lineage specific cytokines
Erythropoiesis
BFU-E
CFU-E dependent on EPO
Granulopoiesis and Monopoiesis
CFU-GM supported by IL-3
Megakaryocytopoiesis
CFU-Meg induced by IL-11 and TPO
Lymphopoiesis
Multiple GF in development of T & B cells

21. Bone Marrow

22. Bone marrow Blood forming tissue located between trabeculae
Bone marrow/ medullary hematopoiesis
Major hematopoietic organ
Blood forming tissue located between trabeculae
Bone marrow stroma is supporting tissue for hematopoietic cells
Red marrow/yellow marrow

23. Thymus Lymphopoietic organ in upper mediastinum
Cortex densely packed with small lymphocytes
Primary purpose
Compartment for maturation of T lymphocytes
Precursor T cells leave the bone marrow and enter the thymus

24. Spleen Upper left quadrant of abdomen Richly supplied with blood
Functions include
culling; filtering and destruction of old or damaged RBCs
Pitting: pluck our particles from RBCs
immune defense
storage: hold 1/3 of platelets

25. Lymphatic system Lymph nodes and lymphatic vessels
Nodes remove foreign particles from lymph
Functions
Immune defense
B cell production in germinal centers

26. Erythropoiesis

27. Erythron
Total population of erythrocytes and precursors in peripheral blood and bone marrow
RBC production
RBC release
RBC destruction
Primary signal regulating RBC production is oxygen tension
 tissue oxygenation due to anemia or pulmonary insufficiency

28. Erythropoiesis
Stimulated by Erythropoietin (EPO), a glycoprotein hormone produced in the kidney
EPO accelerates commitment of pluripotent stem cell to CFU-E and erythroid development

29. Maturation characteristics
Cells accumulate hemoglobin
Lose their protein-synthesizing apparatus
Nuclear chromatin pattern changes
cells become smaller
Nucleus to cytoplasm ratio decreases

30. Sequence of RBCs Maturation
Pronormoblast
Basophilic normoblast
Polychromatic normoblast
Orthochromic normoblast
Reticulocyte
Erythrocyte

31. All stages of erythropoeisis

32. RBC structure and function

33. RBC Membrane Composition
Proteins
integral: Extend from outer surface to inner
peripheral: cytoplasmic surface beneath lipid bilayer
Trilaminar structure
outer hydrophilic
central hydrophobic
inner hydrophilic
Hydrophilic substances are water loving.

34. Schematic of RBC membrane

35. RBC Membrane Lipids 95% of lipid content Remaining 5%
Unesterified Cholesterol
Phospholipid bilayer
Remaining 5%
Glycolipids
Antigenic properties of the membrane
Free fatty acids
Unesterified Cholesterol
Affects surface area; passive cation permeability
Phospholipid bilayer
Mobility contributes to membrane fluidity

36. Membrane Proteins:Integral
Glycophorin A,B,C
Carry RBC antigens and give the RBC it’s negative charge
Band 3
Functions as anion exchange protein

37. Membrane Proteins: Peripheral
Peripheral (form membrane “skeleton”)
Contribute to cell shape,membrane stability, deformability and gives it the viscoelastic properties

38. RBC Deformability
Flexibility of the RBC to squeeze through capillaries
Increased conc of hgb or decreased fluidity = decreased deformability.
Accumulation of membrane calcium result in rigid, shrunken cells & reduced deformability

39. RBC Permeability Freely permeable to H2O, Cl-,
Cation pump regulates balance of Na+and K+

40. RBC Metabolism
Limited because of absence of nucleus, mitochondria, and other organelles
Pathways described contribute energy to maintain :
high intracellular K+, low intracellular Na+, very low intracellular Ca++
Hemoglobin in reduced form
Membrane integrity and deformability

41. Pathways:
1- Embden-Meyerhof Pathway 2- Hexose Monophosphate Shunt 3-Methemoglobin reductase 4-Rapoport- Luebering Shunt

42. Pathways: Embden-Meyerhof Pathway
90-95% of rbc glucose consumption
Glucose enters cell by diffusion and metabolized to lactate
net gain of two moles of ATP/mole of glucose
Key enzymes: pyruvate kinase, phosphofructokinase
Key role:ATP necessary for RBC shape, flexibility and membrane integrity

43. Gylcolysis in RBCs

44. Hexose Monophosphate Shunt
produces reduced NADPH and reduced glutathione(GSH)
Functionally dependent on G6PD
GSH protects cell from permanent oxidant damage
Key enzymes:glutathione reductase, G6PD
Key role:maintain reduced GSH and reduced NADP
Nicotinamide adenine dinucleotide phosphate,
NADPH provides the reducing equivalents for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of ROS (reactive oxygen species), allowing the regeneration of GSH (reduced glutathione)

45. Diagram of HMS

46. Methemoglobin reductase
Pathway that maintains heme iron in reduced ferrous (Fe2+)
Hgb in ferric state is methemoglobin(Fe3+)
Key enzyme: methemoglobin reductase
Key role: prevent hypoxia

47. Rapoport- Luebering Shunt
causes accumulation of 2,3 DPG thus regulating oxygen delivery to the tissues.
Key enzyme:DPG-synthetase
Key role: affects oxygen affinity of hemoglobin

48. Erythrocyte Destruction
RBC begins to undergo senescence
Reticuloendothelial System (RES) daily removes 1% of old RBCs via macrophages
As RBC ages, glycolytic enzymes decrease activity resulting in less energy and less deformability

49. Extravascular Hemolysis
Occurs in RES macrophages
90% of RBC destruction
iron returned to erythroid precursors
globin amino acids returned to AA pool
heme protoporphyrin ring disassembled.
Balances RBC number with production and use

50. Intravascular Hemolysis
5-10% rbc destruction(within blood vessel)
Free hemoglobin in the blood
Iron bound to transferrin
Released Hgb complexed to haptoglobin therefore decreased haptoglobin in the plasma