Erythropoiesis is the process of red blood cell production in the bone marrow. Mature red blood cells are derived from committed progenitor cells through mitotic division and maturation phases stimulated by erythropoietin. Key events in erythropoiesis include proliferation and differentiation of red blood cell precursors in the bone marrow, entry of mature red blood cells into circulation, and removal of aged red blood cells by the spleen. Proper red blood cell structure and function relies on normal hemoglobin synthesis, membrane integrity, and cellular energetics.

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 efficient red cell production, 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 inadequate
amounts to maintain proliferation and differentiation.

Defects may occur at any stage of development and
this leads 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. Basophilic normoblast or
prorubicyte

10. Polychromatophilic
normoblast or rubicyte

11. Orthochromic normoblast or
metarubicyte

12. Reticulocyte or
polychromatophilic erythrocyte

13. Mature erythrocyte

14. Erythropoiesis

15. 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.

16. Erythropoiesis
 Mature RBCs have a lifespan of 100-120
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

17. 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 cells
content.
 Any decrease in flexibility results in a decrease in
RBC deformability and a decrease in RBC survival in
passage through the spleen.

18. 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.

19. 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 and may
result in decreased RBC survival.

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

21. Acanthocytes

22. Target cells

23. The RBC membrane
 RBC membrane proteins- 10 major and over 200
minor proteins are asymmetrically organized in the
membrane.

Integral proteins- many carry RBC antigens and act as
receptors or are transport proteins. Glycophorins are the
major integral membrane proteins.
 Located in the membrane are 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.

24. 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 which leads to a loss in
membrane deformability and a decreased RBC survival
time.

25. RBC membrane structure

26. Hemoglobin Structure and
Function
 Hemoglobin occupies 33% of the RBC
volume and 90-95% 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.

27. Structure of hemoglobin

28. 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)

29. 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.
 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

30. Summary of hemoglobin
synthesis

31. Structure of heme

32. 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

33. 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 levels are
reached. All adult normal hgbs are formed as tetramers
containing 2 α chains + 2 non-α chains. Normal adult
RBCs contain:

34. 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).

35. Assembly of hemoglobin

36. Hemoglobin Structure and
Function
 Hemoglobin synthesis 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 to affect the translation of the mRNA
for ALAS, ferritin (discussed later), and transferrin
receptors (discussed later).

37. 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.

38. How iron levels affect heme
synthesis

39. Hemoglobin Structure and
Function
 If either heme or globin synthesis is impaired, iron
accumulates in the RBC. This RBC 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 and the
cell is called a ringed sideroblast.

40. Siderocyte

41. Ringed sideroblast

42. Hemoglobin Structure and
Function
 Hemoglobin function
 The primary function of hgb is gas transport. The
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 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.

43. Oxy versus deoxy hemoglobin

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

45. Hemoglobin Structure and
Function
 The sigmoid curve permits a significant amount of O2
delivery with a small drop in O2 tension.
 O2 affinity of hgb is expressed as the partial (P)O2 (in mm
Hg) at which hgb is 50% saturated with O2.
 Increased O2 affinity means that hgb does not readily give
up itsO2.
 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

46. Hemoglobin Structure and
Function

In hypoxia there is a compensatory shift to the
right in the dissociation graph. 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.
 A shift to the right also occurs in acidosis and when
the body temperature is increased.

47. Right shift in O2 dissociation
curve

48. 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.

49. Left shift in O2 dissociation
curve

50. Comparison of an O2 dissociation curve at
normal pH and with acidosis or alkalosis

51. 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
 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 that.

Increases above 2% can occur with the ingestion of
strong oxidant drugs or

As a result of enzyme deficiency.

52. 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 1 and 2 this is an
irreversible change of hgb.

53. 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.

54. Cellular Energetics
 Glycolysis- generates 90% of the required ATP-
the breakdown of 1 glucose generates 2 ATP and
2NADH.
 Hexose monophosphate shunt (pentose
phosphate shunt) - 5- 10% 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.

55. Cellular Energetics

Inherited defects in the pathway result in the formation of
Heinz bodies with subsequent extravascular hemolysis.
 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.
 Methgb Reeducate 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.

56. Heinz bodies (new methylene
blue stain)

57. Cellular Energetics
 Leubering-Rapoport shunt - causes the
accumulation of 2,3 DPG which is
important in decreasing the hgb affinity for
O2 during O2 unloading.

58. 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.

59. 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.

60. 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:

61. Erythrocyte kinetics
 High affinity hemoglobins
 Pulmonary disorders
 Occasionally this is due to a primary defect resulting
in an unregulated proliferation of RBCs
(polycythemia vera)

62. Erythrocyte destruction
 RBC destruction is normally the result of
senescsnce.
 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 production is extravascular and
occurs mainly 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

63. Extravascular destruction of
RBCs

64. Intravascular destruction of
RBCs