• Erythron: highly specialized tissue responsible for O₂ transport
• Bone marrow: site of RBC production
• RBC’s: "containers" for Hgb, lack nucleus and mitochondria
• Hgb: protein ideally suited for O₂ transport
• RBC membrane: provides strength and flexibility
• RBC metabolism: provides energy and protects RBC/Hgb from environmental insults

• Production of RBCs:
• (1) Fetal Life – Liver, Spleen, Bone Marrow (BM)
• (2) Child/Adult – BM. (Child-lots of the skeleton is involved, Adult-mostly the axial skeleton is involved).
• (3) Extreme hematologic Stress – Liver and Spleen can revert to making RBC ("Extramedullary Hematopoiesis"). Ex: kid with severe anemia due to thalassemia with hepatosplenomegaly.
• BM Environment: Well suited to support cellular proliferation and maturation. A fine reticular meshwork supports cellular elements as vascular sinuses course through the marrow cavity allowing for the inflow of plasma nutrients but retaining developing cells until they are mature. RBCs mature around a central macrophage.
• RBC Development: Stem cell Þ multipotent stem cell Þ BFU-E Þ CFU-E Þ erythroblast Þ Þ RBC
• RBC Lifespan: From earliest recognizable erythroblast to a mature RBC it takes 3-4 days, reticulocyte (nucleus has been extruded, but some RNA is left over) for about 1 day, and mature RBC lives 120 days.
• What happens: During cell differentiation cell size decreases, the nucleus contracts and is eventually extruded, protein synthetic ability declines and Hgb synthesis increases giving rise to a progressive pink appearance of cells. The reticulocyte is the immediate precursor of the mature RBC and, within 24 hours of release into the peripheral circulation, evolves into the mature RBC. Normally reticulocytes account for about 1% of the circulating RBC mass. In anemias associated with blood loss or premature red cell destruction, the reticulocyte count increases (an appropriate BM response to anemia. Anemias associated with BM failure (i.e. aplastic anemia) are associated with an inadequate reticulocyte response.
• Nutritional factors:
• Fe – deficiency leads to marked limitation of erythropoesis.
• B₁₂/Folate – both must be present for normal methionine and thymidylate synthesis which are key for normal DNA synthesis, deficiency leads to disturbed RBC maturation and ineffective erythropoesis
• Hemoglobin synthesis: Three components of hemoglobin: (1) globin, (2) protoporphyrin, (3) iron (protoporphyrin and iron combine to form Heme). Iron enters the developing RBC and ultimately enters the mitochondria to support heme synthesis. In the mitochondria the first step of heme synthesis takes place as glycine and succinyl CoA combine to form delta aminolevulinic acid. Synthesis shifts into the cytoplasm but ultimately returns to the mitochondria for final steps in the formation of protoporphyrin IX and, eventually heme, as protoporphyrin and iron combine.
• "Sideroblastic anemia" = Congenital absence of enzymes along the path of proto-porphyrin synthesis may lead to severe impairment of heme synthesis.
• Globin chain synthesis: Each globin chain is a polypeptide of approximately 150 amino acids in length. The sequential expression and activation of globin genes gives rise to the various Hgb's:
• Hgb A = 2 alpha chains and 2 beta chains
• Hgb A₂ = 2 alpha chains and 2 delta chains
• Hgb F = 2 alpha chains and 2 gamma chains
• Changes of Hgb throughout life:
• Birth – most Hgb present is of the fetal variety
• 4-6 months – gradual decline in synthesis of fetal Hgb with a corresponding increase in Hgb A
• 6-8 months – approximately 97% of Hgb is Hgb A, 2% is Hgb A-2 and 1% is Hgb F
• Þ Clinical correlate: beta chain Hgb disorders such as sickle cell disease do not clinically manifest until 4-6 months of age since fetal Hgb predominates during early infancy
• Mature RBC = biconcave disk, no nucleus, can’t reproduce, can’t produce energy (no mitoch), very little cytoplasm, diameter = 8 m m, width = 2 m m, volume = 90 femtoliters
• Three Constituents of RBCs: RBC membrane + internal metabolic apparatus (ie a bit of cytoplasm) + hemoglobin
• RBC membrane: Has lipid bilayer membrane with proteins in it. Underneath it is a cytoskeleton of proteins allowing rubbery elasticity with main protein being Spectrin. Ankyrin anchors cytoskeleton to membrane. Na/K ATPase channel is abundant on membrane (ATP from pentose phosphate shunt – that’s why G6PD deficient people have problems).
• Membrane antigen structure – there are over 300 RBC membrane Ags. They are Polysaccharides. (A, B, Rh, Duffy, etc).

• Embden-Meyerhof Pathway - glycolysis from glucose to lactate, net 2 ATPs produced and used to support membrane ion pumps. When deficiencies of the E-M path exist, RBC survival is reduced, leading to hemolysis. Examples of deficiencies: pyruvate kinase deficiency, glucose phosphate isomerase deficiency (both of which lead to chronic hemolytic anemia), as well as blood bank stored blood with decrease cellular ATP leading to shorter survival time in transfused patients.
• Methemoglobin Reductase Pathway - prevents iron of Hgb from being oxidized, makes NADH which is reducing power. Hgb iron must be in reduced state (Fe⁺²) in order to transport O ₂. The environment is constantly generating oxidant stress and therefore a tendency to oxidize iron to Fe³⁺. The methemoglobin reductase pathway counteracts this by reducing iron to the +2 state. The E-M path generates NADH which is, ultimately, "reducing power". Methemoglobin reductase reduces iron as NADH is converted to NAD. Patients with methemoglobin reductase deficiency have a substantial quantity of methemoglobin (Hgb with iron in the oxidated state) associated with reduced O ₂ carrying capacity.
• Luebering-Rapaport Pathway - modifies affinity of binding of Hgb and O₂, makes 2,3-DPG which shifts saturation curve to the right. This pathway is an off-shute of the E-M pathway leading to generation of 2-3 DPG. 2-3 DPG is an important regulator of Hgb-O₂ release (increased 2-3 DPG giving rise to increased O₂ release). An increased rate of glycolysis leads to an increase in intracellular 2-3 DPG concentration. When venous blood is increasingly deoxygenated, the rate of glycolysis increases leading to increased 2-3 DPG production and increased O₂ release to the tissues. This is an appropriate response to ensure adequate O₂ delivery.
• Hexose-Monophosphate Shunt (Phosphoglucoate Pathway) - This pathway couples oxidative metabolism with NADP and glutathione reductase to provide anti-oxidant substrate which ultimately combats the effects of oxygen stresses (environmental, medications). If the shunt is defective (as is the case in patients with G6PD deficiency) oxidative insults lead to oxidation of globin chains and denaturation of Hgb leading to precipitates (Heinz bodies) in the RBC, membrane damage and, ultimately, cell death.

Hemoglobin function – Cooperative binding of O₂ (ie when binds one O₂ binding of further O₂ becomes easier).

• P₅₀ = PO₂ at which Hgb is 50% saturated
• Right shift (O2 released easily) of the curve is caused by: ß O₂ affinity, ß pH, Ý 2,3-DPG, Ý pCO₂, Ý temp
• Left shift (O2 bound tightly) of the curve is caused by: Ý O₂ affinity, Ý pH, ß 2,3-DPG, ß pCO₂, ß temp
• Kidneys are sensors (Juxtatubular cells) for O₂ delivery Þ Ý EPO
• Note: too much/little Hgb content must be interpreted based on the altitude where the person is located.
• As patients become progressively anemic, EPO Ý

RBC life cycle – RBCs are subjected to much stress, live for about 120 days, spleen removes dying RBCs.

• Reticuloendothelial cells participate in the destruction of senescent RBC's. The spleen is a well suited site of RBC destruction given that cells must course through 2-3 micron apertures in the walls of splenic sinusoids, which is an ultimate test of cell pliability. Rigid cells are entrapped and phagocytosed. Intra-erythrocyte inclusions are removed during splenic circulation.
• Destruction of RBCs happens within reticuloendothelial cells – NOT in the circulation. Globin and heme get recycled, porphyrin is degraded to bilirubin which is conjugated by the liver and excreted in the gut. Rate limiting step is conjugation. Indirect (unconjugated) bilirubin is result if this doesn’t happen.
• Normally ~10% RBCs lyse while in circulation Þ Hgb gets released into circulation and rapidly disassociates into alpha and beta dimers which are bound by haptoglobin. The Hgb/haptoglobin complex is transported to the liver. If haptoglobin is depleted, free Hgb circulates and is filtered by the kidney. Free Hgb is either reabsorbed by renal tubular cells or excreted as free Hgb in the urine.

• The role of the RBC in the transport of O₂ from the lungs to the tissues is central. A variety of integrated physiologic components contributes to active O₂ supply to tissues including pulmonary function, and hemodynamic factors (C.O., regional blood flow, blood volume, viscosity). All are capable of physiologic responses to hypoxic stress.
• Regulation of the Erythron: In the basal ideal state, blood enters the tissue at a PO₂ of 95 and exits at a PO₂ of 40. Therefore, 25% of O₂ transported by Hgb is release.
• O₂ delivery may be altered by: (1) Hgb-O₂ affinity and (2) increase in the number of RBC's.
• Pulmonary function/hemodynamic factors: The lungs and heart manifest a physiologic response in the face of decreased O₂ carrying capacity associated with anemia. Alterations in regional blood flow as well as a marked increase in cardiac output can compensate for 50% fall in O₂ carrying capacity in the anemic patient. As a result, patients with significant anemia frequently have tachycardia and an increased cardiac ejection fraction.

• Measurements:
• In-vitro culture techniques – evaluation of CFU-E and BUF-E
• Cellularity of BM and Erythroid-Myeloid Ratio (normally 1:3) – helpful index of marrow erythroid proliferative capacity.
• Reticulocyte index – best assessment of effective RBC production. Normally, 1% of circulating RBC's are reticulocytes. In the anemic patient, the percentage of reticulocytes is less meaningful since the total number of RBC's may be markedly reduced. In addition, reticulocytes are released earlier from the BM under anemic stress and are therefore present in the peripheral circulation longer. Correction factors may be applied to the reticulocyte count to derive the so-called "reticulocyte index" which is the best single determinant of BM adequacy in the face of anemia.
• Serum Indirect Bilirubin Level and LDH – Hgb catabolism is best measured by these; both Ý during Ý RBC destruction.
• Radioactive Tagged RBCs – RBC survival studies.
• Three patterns of abnormal erythropoiesis which define the mechanisms responsible for anemia:
• (1) patients with hemolytic anemia's have an adequate reticulocyte index, an active erythroid BM, and hyperbilirubinemia;
• (2) patients with hypoproliferative erythropoiesis have an inadequate reticulocyte index, suboptimal marrow erythroid activity and decreased levels of bilirubin;
• (3) patients with ineffective erythropoiesis (intra marrow RBC destruction) have an inadequate reticulocyte index, an active erythroid marrow and elevated bilirubin levels.
• Patterns of abnormal erythropoiesis:

• In the basal state, the normal adult male has:
• Hgb concentration of 15 grams/dl
• hematocrit of approximately 45%
• RBC mass of 2,000 cc's
• red cell turnover of approximately 1% per day
• reticulocyte production index is about 1.0
• bilirubin and LDH level are normal
• peripheral blood smear demonstrates normal morphology and no evidence of polychromasia.
• Under hypoxic/anemic stimulus the erythroid response is governed by 3 factors:
• Time - An initial reticulocyte response occurs within 2-3 days but a full response requires 4-7 days.
• Magnitude of the response - which is related to the degree of anemia
• iron supply - since inadequate iron can blunt an appropriate response
• Þ In the case of an acute fall in Hgb individuals are capable of increasing basal RBC production by four fold. Patients with chronic hemolysis may increase basal production by a factor of 8 related to expansion of BM into peripheral sites and extramedullary hematopoiesis
• Mechanisms of anemia:
• Acute blood loss with hemodilution
• Decreased production (ex: renal failure, aplastic anemia)
• Ineffective erythropoiesis (ex: B-12 or folate deficiency)
• Increased destruction (ex: acute hemolysis due to autoimmune hemolytic anemia, chronic hemolysis due to sickle cell anemia).

• The red blood cell, in essence, may be thought of as a "container" for Hgb. Hgb is responsible for the critical function of O₂ transport and delivery from the lungs to tissues throughout the body for support of aerobic metabolism. The RBC membrane and metabolic machinery support the role of Hgb in its O₂ transport/delivery function. Ultimately, appropriate tissue O₂ supply is dependent upon the integrated psychologic functions of the erythron, lungs, heart and vasculature, kidneys, and cellular and humoral regulators.