1. Hematology 425 Erythrocyte Production & Destruction
Russ Morrison
September 22, 2006
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2. 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
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3. General Discussion of Erythropoiesis
During the 1990s to present, molecular biology is defining the relationship between the morphology and physiology of erythrocytes
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4. 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
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5. RBC Feedback Loop O2
Balance in health
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6. 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
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7. 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
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8. 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
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9. 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
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10. 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
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11. 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
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12. Erythropoietin May be quantitatively measured
Reference range of 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
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13. 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.
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14. 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
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15. 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
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16. 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
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17. 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
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18. 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
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19. 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
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20. 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
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21. 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
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22. 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
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23. 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)
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24. 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
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25. 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
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26. 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
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27. 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
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28. 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)
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29. 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
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30. 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
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31. 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
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32. 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
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33. 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
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34. 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
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35. 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
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36. 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
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37. 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.
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38. 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.
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39. 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
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40. 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.
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41. 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
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42. 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
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43. 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
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