Hematology 425 Erythrocyte Production & Destruction Russ Morrison September 22, 2006 12/2/2018

General Discussion of Erythropoiesis RBC one of the first elements observed under a microscope – 1723 Ability of the RBC to carry O2 discovered in 1865 Bone marrow as the major source of RBC production proposed in 1886 Work continued with microscopic examination of marrow and nomenclature of marrow cells through the 1970s 12/2/2018

General Discussion of Erythropoiesis During the 1990s to present, molecular biology is defining the relationship between the morphology and physiology of erythrocytes 12/2/2018

The Erythron Term erythron describes unified functional tissue made up of RBCs in all stages in all areas of the body Includes normoblasts and developing cells in the marrow Circulating mature RBCs in the PB Vascular spaces within specific organs 12/2/2018

RBC Feedback Loop O2 Balance in health 12/2/2018

Erythropoietin RBC feedback loop proposed in 1878 Early 20th century, substance to control RBC production in response to hypoxia proposed – substance referred to as erythropoiesis stimulating factor, or erythropoietin Erythropoietin demonstrated in 1953 12/2/2018

Erythropoietin Biochemically a thermostable, nondialyzable glycoprotein hormone with MW between 20K and 30K Consists of a carbohydrate piece believed to convey specificity in target cell receptor sites Terminal sialic acid unit necessary for biologic activity in vivo 12/2/2018

Erythropoietin Produced by a peritubular interstitial cell located in the kidney When stimulated by tissue hypoxia is capable of increasing RBCs through many mechanisms Binds to membrane surface receptors of erythroid precursors Stimulates synthesis of RNA and cAMP 12/2/2018

Erythropoietin once EPO is bound to specific membrane receptors, stimulates CFU-E and controls the production of RBCs by Regulating the 3 division-reduction stages of normoblastic production Controlling the rate of production of RBCs by shortening the time element of either division or maturation, or both 12/2/2018

Erythropoietin Increasing the rate of the pentose phosphate shunt Assisting in the egression of mature RBCs through gaps of endothelium into the sinusoids via impact on walls of bone marrow sinuses Stimulating early release of shift reticulocytes Increasing rate of Hgb synthesis by transferring iron from transferrin to developing erythroid precursors 12/2/2018

Erythropoietin Other influences on production of EPO Testosterone stimulates erythropoiesis which explains gender and age-related Hgb normal reference ranges Presence or absence of specific hormones will increase or decrease production of EPO EPO may be produced by certain cysts or tumors causing polycythemia 12/2/2018

Erythropoietin May be quantitatively measured Reference range of 10-20 mU/mL Increased EPO found in urine of patients with anemia, except that seen in renal disease In RBC aplasia there is no response to EPO which indicates a possible EPO inhibitor 12/2/2018

Apoptosis Programmed RBC death also relies on binding of specific receptors to the membrane of erythroid precursors 2 receptors of apoptosis Fas located on the membrane of all erythroid progenitors The Fas ligand, FasL is acquired in later phases of erythroid differentiation and appears on the membrane of polychromatophilic and orthochromatic normoblasts. It is absent on early RBC precursors. 12/2/2018

Process of Apoptosis Low levels of EPO allow FasL erythroid precursors to cross-link with Fas-marked erythroid precursors, initiating apoptosis The interaction of Fas with FasL promotes the physiologic deletion of potentially unneeded, potentially harmful RBCs Impaired Fas-iinduced apoptosis results in pathologic RBC accumulation 12/2/2018

Process of Apoptosis High levels of EPO are required for protection of immature erythroblasts from FasL present on mature erythroblasts Apoptosis, unlike necrosis, is not associated with inflammation 12/2/2018

Morphology of Apoptotic Cells During the sequential process of apoptosis, the phollowing prophologic changes are seen Condensation of the nucleus with basophilic staining of chromatin Nucelolar disintegration Shrinkage of cell volume with associated increase in cell density and compaction of cytoplasmic organelles while mitochondria remain normal Apoptotic cell contents remain membrane-bound 12/2/2018

Apoptosis, good news and bad news Unregulated apoptosis causes severe anemia It is all a balance between production of FasL, increased or decreased production of EPO and nonexpression of FasL, the interaction of Fas and FasL, effect of cytokines and inflammatory factors. Although we are talking about RBC apoptosis, apoptosis is involved in controlling production/destruction of all human cells 12/2/2018

Reminder of Microenvironment of the Bone Marrow Must be conducive to blood cell production Increased CO2 due to sluggish circulation of the sinusoids – promotes Hgb production and formation of priomordial blood cells Wetted sticky surfaces – promotes formation of a cellular bed essential to differentiation and development of varied cell lines 12/2/2018

Reminder of Microenvironment of the Bone Marrow 3. Locale and structure of the marrow – macrophages bring iron to red cell precursors to be used in the synthesis of Hgb 12/2/2018

Normoblastic Maturation A steady, continuous process of replication and maturation reflecting the mechanism of the RBC feedback loop Pluripotential stem cells supply blasts committed to erythropoiesis This cell line undergoes three divisions (figure 7-2) Maximum number of mature RBCs generated by 1 multi-potential stem cell stimulated by EPO is 16 12/2/2018

Normoblastic Maturation In cases of severe anemia EPO can increase the mitotic potential of the polychromatic normoblast by a factor of 2, producing a mzximum of 32 mature RBCs per stem cell 12/2/2018

Nomenclature of RBC Maturation Normoblastic Rubriblastic Erythroblastic Pronormoblast Rubriblast Proerythroblast Basophilic normoblast Prorubricyte Basophilic erythroblast Polychromatic normoblast Rubricyte Polychromic erythroblast Orthochromic normoblast Metarubricyte Orthochromic erythroblast Reticulocyte Erythrocyte 12/2/2018

Criteria of ID of RBC Precursors All dependent on a good working stain, normally Wright’s or Wright-Giemsa Table 7-2 summarizes microscopic criteria for identification/classification Cell size Nuclear-to-cytoplasmic ratio Nucleoli (presence of) Stain characteristics (to a lesser extent) 12/2/2018

The Maturation Sequence - Pronormoblast Nucleus round to oval, containing one or two nucleoli Chromatin contains fine clumps Cytoplasm intensely blue Golgi complex may be visible next to the nucleus Demonstrate small tufts of irregular cytoplasm along the periphery of the membrane Undergoes mitosis giving rise to two daughter pronormoblasts which mature into basophilic normoblasts 12/2/2018

The Maturation Sequence – Basophilic Normoblast Chromatin of nucleus has begun to condense, staining reaction is a deep purple-red Cytoplasm is a deeper, richer blue than the blast Hgb synthesis is taking place, but increased RNA masks the Hgb pigmentation Undergoes mitosis, giving rise to 2 daughter cells that mature into 2 polychromatic normoblasts 12/2/2018

The Maturation Sequence – Polychromatic Normoblast Chromatin of nucleus becomes variable with condensation of chromatin allowing cell size to decrease dramatically Color of cytoplasm is murky gray-blue reflecting increasing Hgb pigmentation and decreasing RNA Polychromatic normoblast divides, producing two daughter cells that mature into orthochromic normoblasts In severe anemia, 2 divisions take place at this stage Last stage in which the cell undergoes mitosis 12/2/2018

The Maturation Sequence – Orthochromic Normoblast Nucleus appears pyknotic and is incapable of DNA synthesis. The nucleus is ejected from the cell at this stage. Cytoplasm reflects Hgb production and is pink-orange Residual cytoplasmic organelles contribute bluish cast to the cell 12/2/2018

The Maturation Sequence - Reticulocyte No nucleus present Full complement of Hgb, but retains bluish cast until organelles are absorbed The “shift” reticulocyte moves through the wall of the sinusoids and enters the peripheral blood stream becoming a reticulocyte Supravital stain will show residual RNA Cell is an indicator of bone marrow activity (increased when RBC production increases) 12/2/2018

The Maturation Sequence - Erythrocyte Mature PB erythrocyte is biconcave disc Surface-to-volume ratio optimizes gaseous exchange Main function is delivery of O2 to all tissues Has selective permeability and deformability Life span of 120 days Travels 300 miles during life span Interior of RBC contains 90% Hgb and 10% H2O 12/2/2018

The RBC Membrane Main physiologic functions are Maintain cell shape deformability for osmotic balance between plasma and the cell cytoplasm Acts as a supporting skeletal system for surface antigens Helps in the transportation of essential cellular ions and gases 12/2/2018

The RBC Membrane RBC membrane allows water and anions to freely enter the cell and pumps out excess intracellular sodium and potassium and calcium This active cation pump maintains balance of intracellular and extracellular sodium and potassium and calcium Allows the RBC to maintain normal bi-concave structure and shape Increased cation permeability is a result of sickle cell disease, when the SC deoxygenates there is increased cation permeability 12/2/2018

The RBC Membrane Composition consists of 2 interrelated parts Outer bilayer of lipids with integral proteins embedded in it - serves as a barrier separating interior contents of RBC from the external environment (blood plasma) Underlying protein membrane skeleton - responsible for shape, structure and deformability of the RBC - also contains pumps and channels for ion and other substance movement between RBC interior and blood plasma 12/2/2018

The RBC Membrane Proteins in the RBC membrane act as receptors, RBC antigens and enzymes Glycolipids embedded in the external half of the membrane have carbohydrate pieces that extend into the aqueous phase of the blood. These glycolipids carry A, B, H and P blood group antigens. The Rh polypeptides span the lipid bilayer of the membrane and are necessary for normal RBC membrane integrity 12/2/2018

The RBC Membrane The lipid membrane bilayer is attached to a protein skeleton Membrane proteins are classified as integral or peripheral Integral proteins penetrate the lipid bilayer and can interact with the hydrophobic lipid area Integral proteinsgive the RBC its negative charge and carry membrane receptors and RBC antigens Peripheral proteins interact with protein or lipids at the membrane surface but do not penetrate the bilayer 12/2/2018

The RBC Membrane Peripheral proteins form a membrane skeleton This cytoskeleton modulates cell shape and deformability Abnormalities in these proteins are related to morphologic disorders, spherocytosis, elliptocytosis that may have clinical consequences The strength of the skeleton is derived from spectrin 12/2/2018

Erythrocyte Destruction As the cell ages and dies or undergoes senescence a series of changes occur A sequence of activities is initiated by decreased generation of ATP by the nonoxidative glycolytic pathway Decreased cholesterol and phospholipids cause a loss of selective permeability increasing Na+ and losing K+, resulting in decreased surface-to-volume ratio RBC become spheroidal in shape IgG accumulates on RBC surface Methemoglobin accumulates Metabolic activities gradually shut down 12/2/2018

Extravascular Hemolysis Vulnerable RBCs circulate through the spleen and are phagocytized by sensitive macrophages. The RBC is degraded by digestive enzymatic activity of the macrophage Hgb molecules are disassembled, iron is bound to transferrin and transported to the hepatocytes for storage Amino acids are transferred to body amino acid pools Components of protoporphyrin are chemically separated, carbon is exhaled as CO2, the tetrapyrrole ring is convereted to biliverdin, conveyed to the liver and conjugated to bilirubin glucuronide. 12/2/2018

Extravascular Hemolysis Bilirubin glucuronide enters the intestine via the bile where it is excreted as urobilinogen. This routine destruction of senescent RBCs takes place in lymphoid tissue and accounts for 90% of RBC degradation. This process of extravascular hemolysis balances RBC number with production and physiological need. 12/2/2018

Extravascular Hemolysis Anemias associated with extravascular hemolysis include: Inherited RBC defects -membrane abnormalities, enzyme deficiencies, hemoglobinopathies, thalassemias Acquired RBC deficiencies -megaloblastic anemias, vitamin E deficinecy in newborns Immunohemolytic anemias -autoimmune, drug-induced 12/2/2018

Intravascular Hemolysis 10% or RBC hemolysis occurs within the lumen of the blood vessels (intravascular hemolysis) The RBC is lysed and its contents enter the blood stream where transport proteins present in the plasma, ind, remove and convey the substances to designated sites for storage or disassembly. 12/2/2018

Intravascular Hemolysis Findings indicating intravascular hemolysis Depleted serum haptoglobin and hemopexin Formation of methemoglobin and methemalbumin Increased indirect bilirubin Increased iron stores Increased LDH, isoenzymes 1 and 2 Increased reticulocyte count Hyperplastic bone marrow 12/2/2018

Intravascular Hemolysis Anemias associated with Intravascular Hemolysis Complement activation on the RBC membrane -PNH, PCH, AI hemolytic anemias, specific alloimmune hemolytic anemias Physical or mechanical trauma to the RBC membrane -microangiopathic hemolytic anemia, osmotic trauma, heart and great vessel abnormalities, prosthetic valves, DIC 12/2/2018

Intravascular Hemolysis Anemias associated with Intravascular Hemolysis Toxic microenvironment -bacterial infections (toxins), plasmodium falciparum infection, venoms, drug poisoning, acute drug reaction in glucose-6-phosphate dehydrogenase 12/2/2018