1. Structure and Function of Erythropoietic Tissue
The RBCs

2. Erythropoiesis (RBC production)
Mature   erythrocytes are derived from committed erythroid proginator cells through a series of mitotic divisions and maturation  phases.
Erythropoietin, a humoral agent produced
mainly by  the kidneys stimulates erythropoiesis  by  acting  on committed stem cells to induce proliferation and differentiation of erythrocytes in the bone marrow.  

3. Erythropoiesis
Tissue  hypoxia (lack of oxygen) is the main stimulus for erythropoietin production.
Nucleated red cell precursors in the bone marrow are collectively called normoblasts or erythroblasts.
RBCs that have matured to the non-nucleated stage gain entry to the peripheral blood.
Once the cells have lost their nuclei, they are called erythrocytes.

4. Erythropoiesis Young erythrocytes that contain
residual  RNA  are  called reticulocytes.
Bone marrow normoblast proliferation and maturation occurs in  an orderly and well defined sequence.
The  process  involves  a gradual decrease in  cell  size, condensation and eventual expulsion of the nucleus, and an increase in hemoglobin production.  

5. Basic blood cell maturation
Nearly all hematopoietic cells mature in the manner shown below. For RBCs the nucleus is eventually extruded and the cytoplasm increase correlates with hemoglobin increase.

6. Erythropoiesis
For red cell production to be efficient , 85% or more of the erythroid activity must have a balanced incorporation of heme and globin to form hemoglobin.
The immature, nucleated RBC must have  an adequate supply of iron‚ as well as normal production of porphyrin and globin polypeptide chains‚ for adequate synthesis of hemoglobin.
Folic  acid  and vitamin B12‚ are also needed in adequate amounts to maintain proliferation and  differentiation.
Defects  may occur at any stage of development and these defects will lead to the death of the cell.

7. Erythropoiesis
Normally 1-15% of the RBCs die during  maturation.
Ineffective erythropoiesis occurs when there is  a  failure  to deliver the appropriate number of erythrocytes to the peripheral blood.  
Normoblasts normally spend 4-7 days proliferating and maturing in the bone marrow.
The stages of maturation from the most immature to the most mature are:

8. Pronormoblast or rubriblast

9. Pronormoblast (A)

10. Basophilic normoblast or prorubicyte

11. Basophilic normoblast

12. Polychromatophilic normoblast or rubicyte

13. Polychromatophilic normoblasts

14. Orthochromic normoblast or metarubicyte

15. Orthochromic normoblast

16. Reticulocyte or polychromatophilic erythrocyte

17. Reticulocyte

18. Mature erythrocyte

19. Erythropoiesis

20. Erythropoiesis
Reticulocytes are released from the bone marrow into the peripheral blood where they mature into erythrocytes , usually within 24 hours.
It is rare to see more than 1% reticulocytes in the peripheral smear from an adult, but common in healthy newborns.
They can be visualized more easily by staining with new methylene blue which allows for visualization of the remnants of the ribosomes on the endoplasmic reticulum.

21. Erythropoiesis
Mature RBCs have a lifespan of days and senescent RBCs are removed by the spleen.
3 areas of RBC structure/metabolism are crucial for normal erythrocyte maturation, survival and function:
The RBC membrane
Hemoglobin structure and function
Cellular energetics

22. Erythropoiesis
Defects or problems associated with any of these will result in impaired RBC survival.
The RBC must be flexible in order to squeeze through the capillaries of the spleen.
Flexibility is a property of the membrane and the fluidity of the cell’s content.
Any decrease in flexibility results in a decrease in RBC deformability and a decrease in RBC survival in passage through the spleen.

23. The RBC membrane
The RBC membrane is a semi-permeable lipid bilayer supported by a protein cytoskeleton (contains both integral and peripheral proteins).
Since the mature cells lack enzymes and cellular organelles necessary to synthesize new lipid or protein, extensive damage cannot be repaired and the cell will be culled in the spleen.

24. The RBC membrane The constituents of the RBC membrane include:
Phospholipids- exchange between phospholipids in the membrane and the plasma may occur.
Since the fatty acid content of the diet and the plasma are correlated, changes in the diet may have an effect on the fatty acid composition of the phospholipids in the RBC membrane which can adversely effect the flexibility of the RBC and may result in an RBC with a decreased survival time.

25. The RBC membrane
Cholesterol- membrane cholesterol exists in free equilibrium with plasma cholesterol.
Therefore, an increase in free plasma cholesterol results in an accumulation of cholesterol in the RBC membrane.
RBCs with increased cholesterol appear distorted and the increased cholesterol results in the formation of target cells, and acanthocytes.
An increase in the cholesterol to phospholipid ratio results in a cell membrane that is less deformable and therefore, the RBC has a decreased survival time.

26. Acanthocytes

27. Target cells

28. The RBC membrane
RBC membrane proteins- 10 major and over 200 minor proteins are asymmetrically organized in the RBC membrane.
Integral proteins- many carry RBC antigens and act as receptors or are transport proteins. Glycophorins are the major integral membrane proteins in the RBC.
Located in the membrane are proteins that function as cationic pumps.
The RBC maintains its volume and water homeostasis by controlling the intracellular concentrations of Na+ and K+ via these cationic pumps which require ATP.
ATP is also required in the Ca++ pump system that prevents excessive intracellular build-up of Ca++.
In ATP depleted cells there is an intracellular build-up of Na+ and Ca++ and a loss of K+ and water. This leads to dehydrated, rigid cells that are culled by the spleen.  

29. The RBC membrane
Any abnormality that increases membrane permeability or alters cationic transport may lead to decreased RBC survival.
The major peripheral protein is spectrin and it binds with other peripheral proteins such as actin to form a skeleton of microfilaments on the inner surface of the membrane. This strengthens the membrane and gives it its elastic properties.
For spectrin to participate in this interaction, it must be phosphorylated by a protein kinase that requires ATP.
Thus, a decrease in ATP leads to decreased phosphorylation of spectrin.
Unphosphorylated spectrin can no longer bind to actin to give the membrane its elastic properties.
This then leads to a loss in membrane deformability and a decreased RBC survival time.

30. RBC membrane structure

31. Hemoglobin Structure and Function
Hemoglobin occupies 33% of the RBC volume and % of the dry weight.
65% of the hemoglobin synthesis occurs in the nucleated stages of RBC maturation and 35% during the reticulocyte stage.
Normal hemoglobin consists of 4 heme groups, which contain a protoporphyrin ring plus iron, and globin, which is a tetramer of 2 pairs of polypeptide chains.

32. Structure of hemoglobin

33. Hemoglobin Structure and Function
Normal hemoglobin production is dependent upon 3 processes: Adequate iron delivery and supply, adequate synthesis of protoporphyrins and adequate globin synthesis.
Iron delivery and supply:
Iron is delivered to the RBC precursor by transferrin. It goes to the mitochondria where it is inserted into protoporphyrin to form heme.
Synthesis of protoporphyrin:
Begins in the mitochondria where glycine + succinyl CoA  delta aminolevulenic acid ( ALA). This is the rate limiting step.
In the cytoplasm 2  ALA prophobilinogen (PBG)

34. Hemoglobin Structure and Function
4 prophobilinogen (PBG) uroporphyrinogen I and III (UPG I and III). Only type III is used. Type I represents a dead-end pathway. PBG deaminase and UPG cosynthase are both required for UPG III synthesis. UPG I synthesis requires only PBG deaminase. In the absence of UPG cosynthase large amounts of UPG I accumulate in the RBCs , bone marrow, and urine causing a condition called congenital erythropoietic porphyria (more on this later).
 Decarboxylation of UPG III  coproporphyrinogen III (CPG III). This moves to the mitochondria.
In the mitochondria CPG III  protoporphyrin IX
Fe is added to form ferroprotoporphyrin IX= HEME

35. SUMMARY OF HEME SYNTHESIS

36. HEME SYNTHESIS

37. Structure of heme

38. HEMOGLOBIN SYNTHESIS

39. Hemoglobin Structure and Function
Since porphyrinogens are readily oxidized to form porphyrins, excess formation of porphyrins can occur if any of the normal enzymatic steps in heme synthesis is blocked. Metabolic disorders in which this occurs are called porphyrias. There are 2 categories of porphyrias: inherited and acquired
Inherited
Erythropoietic porphyria - results from excessive production of porphyrins in the bone marrow.
Hepatic porphyria - results from excessive production of porphyrins in the liver.
Acquired
Lead intoxication - interferes with protoporphyrin synthesis
Chronic alcoholic liver disease

40. HEMOGLOBIN STRUCTURE AND FUNCTION
Globin Synthesis
In the yolk sac, the embryonic hemoglobins epsilon and zeta are produced.
In the fetus and the adult, 4 types of hemoglobin chains may be formed: alpha ( α), beta (β ), gamma ( γ), and delta ( δ).
Normal hemoglobin's contain 4 globin chains.
Hemoglobin (hgb) F= α2 γ2 and is the predominant hgb formed during liver and bone marrow erythropoiesis in the fetus. A normal, full term baby has 50-85% hgbF.
Near the end of the first year of life, normal adult hgb levels are reached. All adult normal hgbs are formed as tetramers containing 2 α chains + 2 non-α chains. Normal adult RBCs contain:

41. Hemoglobin Structure and Function
92-95% hgb A=α2β2
 3-5% hgb Ac= glycosylated α2β2
 2-3% hgb A2= α2δ2
 1-2% hgb F (fetal hgb)= α2γ2
Each globin chain links with heme to form hgb= 4 globin + 4 heme.
The precise order of the amino acids is critical for hgb structure and function.
An adequate amount of globin synthesis is also important. A decreased production in 1 chain results in thalassemia (discussed later).

42. GLOBIN SYNTHESIS

43. Assembly of hemoglobin

44. Hemoglobin Structure and Function
Hemoglobin formation is regulated by several mechanisms:
The regulation of globin chain synthesis. The rate of globin synthesis is directly related to the rate of heme synthesis because heme stimulates globin synthesis by inactivating an inhibitor of globin translation.
Negative feedback of heme. High concentrations of heme prevent the mitochondrial import of the first enzyme in heme synthesis,  ALA synthase ( ALAS).
The concentration of iron. An iron responsive element-binding protein (IRE-BP) binds to mRNA iron response elements (IRE) to affect the translation of the mRNA for  ALAS, ferritin (discussed later), and transferrin receptors (discussed later).

45. Hemoglobin Structure and Function
The affinity of IRE-BP for IRE is determined by the amount of cellular iron.
When iron levels are low, there is a high binding affinity which acts to inhibit the translation of  ALAS mRNA resulting in a decrease in heme synthesis.
When iron levels are sufficient, the binding affinity is low, thus allowing translation of  ALAS mRNA and stimulation of heme synthesis.

46. How iron levels affect heme synthesis

47. Hemoglobin Structure and Function
If either heme or globin synthesis is impaired, iron accumulates in the RBC.
An RBC with accumlated iron is then called a siderocyte and the iron can be visualized using a Prussian blue stain.
When protoporphyrin synthesis is impaired, mitochondria become encrusted with iron. This is visible as a ring around the nucleus of the RBC precursor when stained with Prussian blue.
A precursor cell with a ring of iron around the nucleus is called a ringed sideroblast.

48. RINGED SIDEROBLASTS AND SIDEROCYTE

49. Ringed sideroblast

50. Hemoglobin Structure and Function
Hemoglobin function
The primary function of hgb is gas transport. The hgb molecule is capable of a considerable amount of allosteric movement as it loads and unloads O2. This is due to the multichain structure of the molecule.
In unloading of O2, the space between the chains widens and 2,3 diphosphoglycerate (DPG) binds. This is the T (tense) form of hgb and it is called deoxyhgb. It has a lower affinity for O2, so O2 unloads from the hbg.
When hgb loads O2 and becomes oxyhgb, the chains are pulled together, expelling 2,3 DPG. This is the R (relaxed) form of hgb. It has a higher affinity for O2, so O2 binds to or loads onto the hgb.  

51. Oxy versus deoxy hemoglobin

52. Hemoglobin Structure and Function
Binding and dissociation of O2 are not directly proportional to the O2 concentration. Note the hgb-O2 dissociation curve below:

53. Hemoglobin Structure and Function
The sigmoid shape of the curve shows that a significant amount of O2 delivery will occur with a small drop in O2 tension.
O2 affinity of hgb is expressed as the partial (P) O2 pressure (in mm Hg) at which hgb is 50% saturated with O2.
Increased O2 affinity means that hgb does not readily give up its O2.
Decreased O2 affinity means that hgb releases the O2 more readily.
Normally the partial O2 pressure in the lungs is 100 mm. and the hgb is 100 saturated with O2. In tissues the partial pressure is 40mm. and the hgb is 75% saturated with O2. Therefore 25% of the O2 is delivered to the tissues

54. Hemoglobin Structure and Function
In hypoxia there is a compensatory shift to the right in the dissociation curve. This is mediated by an increase in 2,3 DPG and results in decreased hgb affinity for O2 and increased O2 delivery to the tissues. Therefore the RBCs are more efficient in O2 delivery.
A patient suffering from anemia due to blood loss may compensate by shifting the O2 dissociation curve to the right.
A shift to the right also occurs in acidosis and when the body temperature is increased.

55. RIGHT SHIFT IN O2 DISSOCIATION CURVE

56. Hemoglobin Structure and Function
A shift to the left in the O2 dissociation curve results in decreased O2 delivery to the tissues. This occurs:
In alkalosis
When there are increased quantities of abnormal hemoglobins such as methgb and carboxyhgb
When there is an increase in hgb F which has a higher affinity for O2 than does hgb A or
When a patient has received multiple transfusions with 2,3 DPG depleted blood.

57. LEFT SHIFT IN O2 DISSOCIATION CURVE

58. COMPARISON OF AN O2 DISSOCIATION CURVE AT NORMAL PH AND WITH ACIDOSIS OR ALKALOSIS

59. Hemoglobin Structure and Function
Inherited abnormalities in hgb may result in either type of shift and can have profound effects on the RBCs ability to provide the tissues with O2. Acquired abnormal hgbs of clinical importance are those that have been altered post- translationally to produce hgbs that are unable to transport or deliver O2 and they include:
Carboxyhgb - CO replaces O2 and binds 200X tighter than O2.
This may be seen with heavy smokers
This may be reversed with high concentrations of O2
 Methgb - occurs when iron is oxidized to the +3 (ferric) state. In order for hgb to carry O2 the iron must be in the +2 (ferrous) state. In the body, normally~ 2% is formed and reducing systems prevent an increase beyond 2%.
Increases above 2% can occur with the ingestion of strong oxidant drugs or
As a result of enzyme deficiency.

60. Hemoglobin Structure and Function
Methgb can be reduced by treatment with methylene blue or ascorbic acid.
Sulfhgb - occurs when the sulfur content of the blood increases due to ingestion of sulfur containing drugs or to chronic constipation.
Unlike the formation of carboxyhgb and methgb, the formation of sulfhgb is an irreversible change of hgb.

61. Cellular Energetics
Maintenance of hgb function requires active RBC metabolic pathways for ATP production. ATP is required for:
Maintaining hgb in the reduced form
Membrane integrity and deformability
Maintaining the RBC intracellular volume
Producing adequate amounts of NADH, NADPH, and GSH
RBCs generate energy almost exclusively from the anaerobic breakdown of glucose - 4 metabolic pathways are important for maintaining cellular energetics.

62. Cellular Energetics
Glycolysis- generates 90% of the required ATP- the breakdown of 1 glucose generates 2 ATP and 2 NADH.
Hexose monophosphate shunt (pentose phosphate shunt) % of the glucose is metabolized this way. It produces NADPH and GSH which protect the RBC from oxidative injury.
If the concentrations of these are too low, the globin will denature and precipitate in the cell. This is seen as Heinz bodies which attach to the membrane causing membrane damage and RBC destruction.

63. Cellular Energetics Heinz bodies
Inherited defects in the pathway result in the formation of Heinz bodies with subsequent extravascular hemolysis (more on this later).
Heinz bodies can only be seen with a supravital stain such as new ethylene blue.
The most common deficiency is Glucose-6-Phosphate Dehydrogenase deficiency.
Heinz bodies

64. CELLULAR ENERGETICS
Methgb Reductase Pathway- maintains iron in the reduced functional state.
There are 2 pathways, the NADH and the NADPH reductase pathways. They are dependent upon NADH and NADPH respectively.
In the absence of the enzymes or NADH and NADPH, methgb, which can't transport O2, is formed.
Leubering-Rapoport shunt - causes the accumulation of 2,3 DPG which is important in decreasing the hgb affinity for O2 during O2 unloading.

66. Erythrocyte kinetics
The normal erythrocyte concentration varies with age, sex, and geographic location.
There is a high RBC count at birth which decreases until the age of 2-3 months where physiologic anemia is seen due to low levels of erythropoietin production.
The RBC count will then gradually increase until adult levels are reached at about 14 years of age.
Males have higher RBC counts because testosterone stimulates erythropoietin production.

67. Erythrocyte kinetics
Individuals living at high altitudes have increased RBC levels because of the decreased partial pressure of O2 at high altitudes which leads to decreased O2 saturation.
A decrease in RBC mass and therefore, a decrease in hemoglobin concentration results in tissue hypoxia and can lead to anemia. Anemia is not necessarily a diagnosis in itself, but is a clinical sign of many different pathologies.

68. Erythrocyte kinetics
An increase in RBC mass is called polycythemia and it may lead to an increase in blood viscosity.
Polycythemia may be relative or absolute
Relative polycythemia occurs with a decreased plasma volume. This occurs with dehydration.
Absolute polycythemia results from an actual increase in RBC mass. This may occur in disorders that prevent adequate tissue oxygenation such as:
High affinity hemoglobins
Pulmonary disorders
Occasionally this is due to a primary defect resulting in an unregulated proliferation of RBCs (polycythemia vera)

69. Erythrocyte destruction
RBC destruction is normally the result of senescsence.
Each day ~ 1% of the RBCs are removed and replaced.
RBC aging is characterized by decreased glycolytic enzyme activity which leads to decreased energy production and subsequent loss of deformability and membrane integrity.
90% of aged RBC destruction is extravascular and occurs mainly in the phagocytic cells in the spleen, with a small amount occurring in the liver and bone marrow.
5-10% of RBC destruction is intravascular, occurring in the lumen of the blood vessels

70. Extravascular destruction of RBCs

71. Extravascular destruction of RBCs

72. Intravascular destruction of RBCs

73. Intravascular destruction of RBCs