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

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

Hematopoeisis

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

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

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

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

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

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

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

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

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

Hematopoietic precursor cells Stem cells Progenitor cells Maturing cells

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

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

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

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

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

Bone Marrow

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

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

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

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

Erythropoiesis

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

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

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

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

All stages of erythropoeisis http://www.tau.ac.il/~inter05/eryt.htm

RBC structure and function

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.

Schematic of RBC membrane

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

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

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

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

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

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

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

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

Gylcolysis in RBCs http://www.vet.ed.ac.uk/clive/cal/RUMENCAL/Info/infFerm.html

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)

Diagram of HMS http://www.uq.edu.au/vdu/HexoseMonophosphateShunt.gif

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

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

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

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

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