11/13/2016

Erythropoiesis
Professor Amany Ahmed Osman
Professor of Hematology
Ain Shams University

CP6002 Prof. Amany Ahmed Osman

ILOs of erythropoiesis:
• -to define the process of erythropoiesis, its stages & criteria of
maturation
• -to recognize reticulocytic counts & diagnostic significance
• -to list requirements of effective erythropoiesis
• -to discuss Hb synthesis
• -to know basic biochemical & physiological information regarding
RBCmetabolism, Hb structure & function
• -to list RBC abnormalities
• -to discuss ineffective erythropoiesis: causes & lab findings

CP6002 Prof. Amany Ahmed Osman

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Erythropoiesis
• Def: the process of producing red blood cells by the stem cells in the
bone marrow

• Normally: 1012 RBCs are produced/day to replace senescent cells.

• Stem cell→CFUGEMM→BFUE→CFUE→Pronormoblast.

CP6002 Prof. Amany Ahmed Osman

CP6002 Prof. Amany Ahmed Osman

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CFU-E vs BFU-E
• CFU-E colony of erythroblasts containing 16 cells. • BFU-E (group of colonies) of erythroblasts containing
over 1,000 cells.

CP6002 Prof. Amany Ahmed Osman

Pronormoblast:
• 1st recognizable eryrthroid precursor in BM.

• large cell (12-20μ)

• non-granular dark blue cytoplasm dt RNA for active protein synthesis

• central nucleus with one or more nucleoli

• open stippled chromatin

CP6002 Prof. Amany Ahmed Osman

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Maturation of erythroid series

• Smaller
• Acquisition of Hb → pink cytoplasm
• Loss of RNA → paler cytoplasm
• Eccentric nucleus → extruded
• Condensation of chromatin
• Loss of ability to divide (16 → 16)

CP6002 Prof. Amany Ahmed Osman

12-20 u

10-16 u

10-14 u

10-12 u

8.5 u

1 pronormoblast (after 4 divisions)→16 RBCs or less due to ineffective erythropoiesis

CP6002 Prof. Amany Ahmed Osman

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Schematic diagram of the ultrastructure of the normoblast

(visualized by electron microscopy)

CP6002 Prof. Amany Ahmed Osman

CP6002 Prof. Amany Ahmed Osman

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CP6002 Prof. Amany Ahmed Osman

Erythroid series

CP6002 Prof. Amany Ahmed Osman

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Erythroid series

CP6002 Prof. Amany Ahmed Osman

CP6002 Prof. Amany Ahmed Osman

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Erythroid series

CP6002 Prof. Amany Ahmed Osman

CP6002 Prof. Amany Ahmed Osman

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Reticulocytes
•Reticulocytes are immature non-nucleated RBCs that contain remnants of
ribosomal RNA which continue to synthesize Hb after loss of their nucleus.
•Spends 1-2 days in BM & circulates in PB where complete loss of RNA occurs
mainly in the spleen. This maturation process takes approximately 2 – 3 days, of
which about 24 hours are spent in the circulation to give a completely pink-staining
mature non-nucleated biconcave disc.
•Reticulocytes appear in Leishman stained films larger than mature RBCs and show
polychromasia.

CP6002 Prof. Amany Ahmed Osman

Normoblasts
• Normally, no normoblasts appear in adult PB.

• Normoblastemia occurs in:

-physiological: cord blood, neonates
-pathological: extramedullary hemopoiesis, severe anemias, some
marrow diseases.

• Normoblasts are expressed as no/100 WBCs.

CP6002 Prof. Amany Ahmed Osman

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Counting and calculations:

a) Reticulocytic %: the cells with reticulofilamentous material are counted in
at least 500 red cells and their % is obtained.
Normal range: Adults and children :0.5-2.5%,
Infants : 2-5%.
b) Corrected reticulocytic %: in order to correct the previous value as it is
related to RBC count:
reticulocytic count x Hct of patient/normal Hct(45%)

CP6002 Prof. Amany Ahmed Osman

c)Reticulocytic production index: Still this correction is not enough as in cases of increased
demands, there is an increase in erythropoietic stimulus which leads to premature release in the
circulation and these stress reticulocytes have a maturation time as long as 3 days. So further
correction is needed:
reticulocytic count x Hct of patient/normal Hct(45%) x 1/maturation time in days(acc. to Hct
value)
45% →1 day, 35%→ 1.5 days, 25% →2 days, 15%→2.5 days. (Normal RPI: 2-3)
d) Absolute reticulocytic count: is calculated by multiplying the reticulocytic count x the RBC
count.
Normal range: Birth: 120-400 x 109/L
6-12 years: 30-100 x 109/L
Adults: 50-100 x 109/L

CP6002 Prof. Amany Ahmed Osman

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Diagnostic significance:
1-The number of reticulocytes & percentage is a good indicator of bone marrow activity:
-Reticulocytosis (abnormally high numbers of reticulocytes):
When there is an increased production of red blood cells to overcome chronic or severe loss
of mature red blood cells, such as in a hemolytic anemia or hemorrhage.
-Reticulocytopenia(Abnormally low numbers of reticulocytes) can be attributed to
chemotherapy, aplastic anemia, or factors impairing normal reticulocytic response to
anemia as in: marrow disease (hypoplasia, infiltration by carcinoma, lymphoma, myeloma, AL,
TB), deficiency anemia (iron, folate, B12), lack of epo (renal disease), chronic disease,
ineffective erythropoiesis( thalassemia major, megaloblastic anemia, MDS, MF, CDA).

CP6002 Prof. Amany Ahmed Osman

Diagnostic significance:
2-In cases of anemia, reticulocyte percentage should be higher than "normal" if the bone
marrow's ability to produce new blood cells remains intact. Calculating the reticulocytic
production index is an important step in understanding whether the reticulocyte count is
appropriate or inappropriate to the situation.
RPI < 2 indicates ineffective erythropoiesis.
3-Therapeutic trial test: reticulocytic counts can also be used to monitor the progress of
treatment for anemia where the earliest rise as a response can be recorded as early as 3
days post treatment and peaking after 7 days of therapy after recording base line
reticulocytes.

CP6002 Prof. Amany Ahmed Osman

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CP6002 Prof. Amany Ahmed Osman

Requirements of Effective Erythropoiesis

• 1- Adequate stem cells
• 2- Suitable microenvironment
• 3- Precursors: Erythropoietin
Metals: iron, cobalt
Vitamins: B12, folate, C, E, B6,
thiamine & riboflavin
Hormones: androgens
thyroxine

CP6002 Prof. Amany Ahmed Osman

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Erythropoietin(Epo)
• Epo gene.
• A heavily glycosylated polypeptide (165 aa) of MW 34 kDa.
• Production sites:
-90% in peritubular interstitial kidney cells
-10% in liver & elsewhere.
There are no preformed stores.
• Stimulus for secretion: O2 tension in kidney tissues.
• Effect: ↑ no of progenitor cells committed to erythropoiesis →
erythroid hyperplasia in BM.

CP6002 Prof. Amany Ahmed Osman

Epo Drive
• ↑ Epo: • ↓ Epo:
- Anemias -High red cell mass
- Abnormal Hb(struct or metab), -Some abnormal Hb which
unable to give up O2 easily release O2 more readily than
-Low atmospheric O2 normal
-Defective cardiac or pulmonary
function
-Damage to renal circulation

CP6002 Prof. Amany Ahmed Osman

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Erythroid hyperplasia
• Transcription factors GATA-1 & FOG-1 are activated by Epo receptor
stimulation→ enhance expression of:
-erythroid-specific genes (heme biosynthesis & RBC membrane
proteins)
-anti-apoptotic genes
-transferrin receptor (CD71)
Late BFUs & CFUs (have Epo recpetors) are stimulated to proliferate,
differentiate &produce Hb.

CP6002 Prof. Amany Ahmed Osman

Recombinant Epo therapy(IV or SC)

Indications
• Treatment of anemia due to end-stage renal disease (with or without dialysis)
• Pre-autologous blood transfusions (preoperative)
• Anemias of chronic disorders( rheumatoid arthritis or cancer)
• Anemias associated with chemotherapy
• AIDS
• Anemia of prematurity
• Some cases of MDS or MM
N.B.:- a low Epo level prior to ttt can predict a good response
- Concomitant iron therapy is needed to maximize Epo response
-Side effects: rise of blood pressure, thrombocytosis & local injection site
reactions

CP6002 Prof. Amany Ahmed Osman

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Hb synthesis in developing red cell

•Heme synthesis occurs largely in the

mitochondria by a series of biochemical reactions
starting by condensation of glycine & succinyl CoA
under rate limiting enzyme δ-aminolevulinic acid
(ALA)synthase, PP (Vit B6) is a coenzyme & Epo is
a stimulator.

CP6002 Prof. Amany Ahmed Osman

Hemoglobin
• Function:- it is a specialized protein in RBCs.
-in arterial blood, carries O2 from lung to tissues & in venous blood, carries
CO2 from tissues to lungs.
• Each RBC contains ~ 640 million Hb molecules
• MW of Hb is 68,000 dalton
• Each Hb molecule consists of 4 polypeptide chains, each binds to 4 O2 molecules
• Normal Hbs in adult blood

Hb A Hb F Hb A2
Structure α2β2 α2γ2 α2δ2
Normal(%) 96-98
CP6002
<Prof.1Amany Ahmed Osman 1.5-3.2

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Basic Biochemical & Physiological Information

• Red cell metabolism

• Hb : -Structure of Hb molecule in relation to

function (O2 – dissociation curve)
-Hb synthesis

• Iron metabolism

CP6002 Prof. Amany Ahmed Osman

Background
• Hb is the primary vehicle for transporting oxygen in the blood.
• Oxygen is also carried dissolved in the blood's plasma, but to a lesser degree.
• Each Hb molecule has the capacity to carry four oxygen molecules. How much of that
capacity is filled by oxygen at any time is called the oxygen saturation. Expressed as a
percentage, the oxygen saturation is the ratio of the amount of oxygen bound to the Hb,
to the oxygen carrying capacity of the Hb.
• The oxygen carrying capacity is determined by the amount of Hb present in the blood.
• The amount of oxygen bound to the Hb at any time is related, in large part, to the partial
pressure of oxygen to which the Hb is exposed. In the lungs, at the alveolar-capillary
interface, the partial pressure of oxygen is typically high, and therefore the oxygen binds
readily to Hb that is present.
• As the blood circulates to other body tissue in which the partial pressure of oxygen is less,
the Hb releases the oxygen into the tissue because the Hb cannot maintain its full bound
capacity of oxygen in the presence of lower oxygen partial pressures.

CP6002 Prof. Amany Ahmed Osman

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O2-Dissociation Curve
• The affinity of Hb to O2 is nicely balanced so that: -in lungs, Hb is 95-
96% oxygenated
-in tissues, Hb can give up O2 as much as possible
• Physiologically: -if affinity to O2 was less → complete oxygenation in
the lungs could not be reached
-if affinity to O2 was more → complete release of O2 to
tissues could not be reached

CP6002 Prof. Amany Ahmed Osman

•P50: partial pressure of O2 at which Hb is ½ saturated with O2. N, P50= 26.6 mmHg
•It is a conventional measure of Hb affinity for oxygen.
•Normal position of curve depends on:
-2-3 DPG conc -H+ conc -CO2 conc -Hb molecule structure

Shift to Left=↑affinity:↓ P50

= O2 is given up less easily:
-↓ 2-3 DPG
- ↓ H+ conc
- Hb F (γ chains are unable to bind 2-3 DPG)

Shift to Right=↓ affinity: ↑ P50

= O2 is given up easily:
-↑ 2-3 DPG
-↑ H+ conc
-↑ CO2 conc (both ↓ O2 affinity → Bohr
effect)
-Hb S (easily gives up O2 than HbA)

CP6002 Prof. Amany Ahmed Osman

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Red Cell Metabolism

-RBC, 8 μ in diameter, must be able to:
-pass repeatedly through the microcirculation of min diameter 3.5 μ
(total journey throughout its 120-day lifespan is estimated to be ~ 480
km
1. -carry Hb with close contact to tissues for successful gaseous exchange
2. -Maintain Hb in a reduced (ferrous) state
3. -Maintain osmotic equilibrium despite high conc of Hb protein in the cell.
4. -To fulfill these functions, the cell is a flexible biconcave disc with the
ability to generate energy as ATP by the anerobic glycolytic (Embden-
Meyerhof) pathway.
CP6002 Prof. Amany Ahmed Osman

Red Cell Membrane Structure

A lipid bilayer: integral membrane proteins & skeleton, 50% protein, 20% PL, 20% cholesterol & 10% CHO. CHO occur on outer
surface (ags) & proteins either peripheral or integral (penetrating the bilayer)
CP6002 Prof. Amany Ahmed Osman

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RBC abnormalities

CP6002 Prof. Amany Ahmed Osman

RBC inclusions

CP6002 Prof. Amany Ahmed Osman

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Ineffective erythropoiesis
• Def: inefficient erythropoiesis as 10-15% of developing erythroblasts
die within the marrow (intramedullary hemolysis) or other sites of
production and/or production of non viable RBCs which survive only
for a short time in the circulation.

• There is active erythropoiesis, but with a decreased RBC output.

Consequently, there is anemia.

CP6002 Prof. Amany Ahmed Osman

Causes
• Extramedullary erythropoiesis : MF
• Hyperplastic but functionally abnormal BM:
-Thalassemia major & intermedia (free globin chains)
- Megaloblastic anemia (slowed DNA synthesis)
-IDA
-MDS
-Sideroblastic anemias
-Congenital dyserythropoietic anemias
-Erythroleukemias

CP6002 Prof. Amany Ahmed Osman

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When to suspect ineffective erythropoiesis?

In the presence of the triad:

Anemia

↓ Reticulocytes BM erythroid hyperplasia

CP6002 Prof. Amany Ahmed Osman

Lab findings of ineffective erythropoiesis?

• ↑ S indirect Bilirubin, uro & stercobilinogen(Hb breakdown)

• ↑ S LDH, aldolase (cellular destruction & release of enzymes)
• Low reticulocytic index: < 2 (N: 2-3)
(low retics in relation to degree of anemia & BM erythroblasts)
• ↓ M/E ratio due to erythroid hyperplasia
• Ferrokinetic studies

CP6002 Prof. Amany Ahmed Osman

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