Erythropoiesis

Terminologies

• Erythrokinetics – Dynamics of rbc production and destruction

• Erythron: Collection of all stages of RBC throughout in the body in the BM, PB, Spleen

• RBC mass: refers only to the cells in circulation

• Hypoxia: Stimulus to RBC production

1. Primary oxygen-sending in the body: Peritubular Fibroblast of Kidney

2. Once detected, Peritubular cells produce Erythropoietin

3. Reasons: ↓ RBC count, Problem in Hgb but w/ Lung Diseases, Heart Dses: Normal RBC ct. but ↑
EPO → ↑ RBC

Erythropoietin

• Thermostable, nondialyzable, glycoprotein hormone that has CHO unit & terminal sialic acid
unit Mediated by JAK2/ Janus Associated Tyrosine Kinase 2 signal transducers

Actions:

✓ Early release of reticulocytes from the BM (SHIFT/ STRESS RETICS)

✓ Preventing apoptic cell death (Major)

✓ Reducing the time needed for cells to mature in the BM (↑ rate of cellular processes, ↓ cell
cycle times)

• Erythrocyte Destruction (Eryptosis)

❖ As RBC ages, enzymes decreases, decrease in ATP and size, increase in density → cellular
aging/ Senescence

Extravascular/ Macrophage-Mediated Hemolysis (summarized)

• RBC destruction outside BV,by Splenic macrophage

• 90% Aged Red cell Destruction

• ↑ Unconjugated bilirubin, Urine/ fecal urobilinogen

• Spherocytosis, RH

• Signals to macrophages: Autologous IgG to Band 3 CHON, Phosphatidyserine, CD47-

thrombospondin 1 binding

Fullversion: Page 107-108 Rodaks hematology 5th edition

Intravascular/ Mechanical Hemolysis(summarized)

• RBC rupture within the blood vessel

• 10% aged red cell destruction

• ↑ Haptoglobin, Hemopexin, Albumin

• Schistocytes

• ABO
fullversion page 108-109 rodaks hematology 5 th edition
RBC Characteristics

• 5 million rbc per microliter of blood

• Anucleated

• Biconcave disc

• 90 fl/rbc

• Reddish in color because of the heme groups of hemoglobin

• Size: approximately 7-8 microns

• Lifespan – 120 days

Rbc Membrane and Structure

• Lipids (40%)

• External surface: Phospholipids, phosphatidylcholine, glycolipid, sphingmyelin

• Internal surface: Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine

• Cholesterol content depends on plasma cholesterol, bile acids, activity of LCAT

• Acanthocyte: Abnormal L/S ratio | Codocyte: ↑ chole, phospholipids | Ovalocyte: ↓ chole

 Carbohydrate (10%)
 Protein (50%)

• Integral Protein: Glycophorin A (Sialic acid: negative charge/ zeta potential) & Component A

• Peripheral Protein: Spectrin & Actin, Ankyrin/ Protein 4.1, adducing, dematin, tropomyosin,
tropomodulin

▪ Maintains biconcave shape of RBC

METHABOLIC PATHWAYS IN RBC

Embeden-Meyerhoff Pathway

Major source of RBC energy, 90% Anaerobic glycolysis

Maintains cellular energy by generating 2 ATP PK deficiency: ↓ EMP

Note: this pathway is essential because during maturation of the rbc the nucleus,while present in
maturing marrow normoblasts, becomes extruded as the cell passes from the bone marrow to
peripheral blood. Cytoplasmic ribosomes and mitochondria disappear24 to 48 hours after bone marrow
release, eliminating thecells’ ability to produce proteins or support oxidative metabolism

Hexose Monophosphate Shunt/ Oxidative Pathway/ Pentose-Phosphate Pathway

• G6PD → NADP is reduced to NADPH → NADPH reduce oxidized gluthathione to Reduced Gluthatione
 • Reduced Gluthathione Reduces Peroxide (toxic to globin, Oxidize Fe2+ to Fe3+) to H20 + 02 by Glut
peroxidase

• Prevents denaturation of Hb molecule by oxidation

• G6PD deficiency (most common inherited RBC enzyme worldwide) → denatured globins → Heinz
Bodies

Rapaport-Luebering Pathway

• Generates 2,3 DPG w/c regulates O2 delivery to tissues by competing w/ O2 for the O2 binding site of
Hb

• ↑ 2,3-DPG: ↓ Hb affinity to O2 (Shift to the Right)

• ↓ 2,3-DPG: ↑ Hb affinity to O2 (Shift to the Left)

Methemoglobin Reductase Pathway

• Maintenanace of the Iron in (FERROUS) by Methemoglobin reductase/ Cytochrome b5 reductase

Hemoglobin Metabolism

Hemoglobin

• It comprises approximately 95% of the cytoplasmic content of RBCs

• The concentrationof hemoglobin within RBCs is approximately 34 g/dL

Affinity of HGB for oxygen depends

1. pH

2. Partial pressure of carbon dioxide

3. Contencetration of 2,3 biphosphoglycerate (2,3 BPG)

4. Temperature

5. Presence of other hgb species that are non functional

Oxygen Dissociation Curve

• The curve produced when 2 variables are plotted on a graph

Bohr effect

– a shift in the curve due to an alteration in pH

- effect of hydrogen ions and CO2 on the affinity of hemoglobin for oxygen

Haldane effect

- Depicts the occurrence by which the binding oxygen to the hemoglobin promotes the release of CO2

Shift to the left Shift to the right

pH (increased) pH (decreased)
PCO2 (decreased) PCO2 (increased)

2,3 BPG (decreased) 2,3 BPG (increased)

Temperature (decreased) Temperature (increased)

Increased affinity to O2 Decreased affinity to O2

Hemoglobin Structure

• Heme consists of a ring of carbon, hydrogen, and nitrogenatoms called protoporphyrin IX, with a
central atom of divalent tferrous iron. Each of the four heme groups is positioned in a pocket of
the polypeptide chain near the surface of the hemoglobin molecule. The ferrous iron in each
heme molecule reversibly combines with one oxygen molecule. When the ferrous irons are
oxidized to the ferric state, they no longer can bind oxygen. Oxidized hemoglobin is also called
methemoglobin.

Simplified Explanation of HGB structure

• Consists of CHN atoms called Protoporphyrin IX + Ferrous Iron

• Ferrous in each Heme molecule combines with 1 O2 molecule

• Fe2+ → Oxidized to Fe 3+ → can’t bind O2→ Methemoglobin

• It begins in the mitochondria → ALA formation

• Then in Cytoplasm (Copro) → Mitochondria (Protoporphyrinogen IX)

HGB values

• 4 heme + 4 globin/polypeptide chains

• 1 heme= 4 Pyrolle Rings + 1 Iron 1 Hgb= 4 Iron

• 1 heme= 1 mole 0f O2 = 1.34 ml 1 Hgb= 4 moles of O2

Age group Conventional units S.I units

Children (8 to 13 y.o) 12 to 15 g/dl 120 to 150 g/L

Adult (male) 14 to 18 g/dl 140 to 180 g/L

Adult (female) 12 to 15 g/dl 120 to 150 g/L

Globin structure

• The four globin chains comprising each hemoglobin molecule consist of two identical pairs of
unlike polypeptide chains, 141to 146 amino acids each. Variations in amino acid sequences give
rise to different types of polypeptide chains. Each chain is designated by a Greek letter

Simplified explanation of globin structure

 • GLOBIN/ POLYPEPTIDE CHAIN

• Consists of 2 pairs of unlike polypeptide chains

• Produced in the ribosomes in the cytoplasm of RBC

• Alpha, zeta: Chromosome 16, 141 AA

• Beta, Gamma, Delta, Epsilon: Chromosome 11, 146 AA

• Zeta, Epsilon: Embryonic only (1st 3 months)

• Alpha is (+) charged & highest affinity To B chain (- CHARGED)

Heme biosynthesis begins in the mitochondria with the condensation of glycine and succinyl coenzyme A
(CoA) catalyzed by aminolevulinate synthase to form aminolevulinic acid (ALA) In the cytoplasm,
aminolevulinic acid dehydratase (also known as porphobilinogen synthase) converts ALA to
porphobilinogen (PBG). PBG undergoes several transformations in the cytoplasm from
hydroxylmethylbilane to coproporphyrinogen III. This pathway then continues in the mitochondria until,
in the final step of production of heme, Fe2+ combines with protoporphyrin IX in the presence of
ferrochelatase (hemesynthase) to make heme.
 • Complete Hgb Molecule

• Primary Structures: AA in the polypeptide chains

• Secondary Structure: chain arrangements in helixes

• Tertiary Structures: arrangements of helixes into pretzel configuratio

• 8 separate helical segments (A-H) Heme bind bet. E & F

• Quaternary Structures: Tetramer: complete Hgb Molecule

• Hb F are called A/F cells

Time line of HGB in the body

• 1st 3 months of embryonic development: Zeta & Epsilon

• 2nd/ 3rd trimesters of fetal life & birth: Hb F is the predominant

• 6th month of age and through adulthood: Hb A is the predominant

Oxygen Transport of Hemoglobin

• The function of hemoglobin is to readily bind oxygen molecules in the lung, which requires high
oxygen affinity; to transport oxygen; and to efficiently unload oxygen to the tissues, which
require low oxygen affinity.

Part 2

• The affinity of Hb for O2 relates to the partial pressure of O2 (PO2), often defined in terms of the
amount of O2 needed to saturate 50% of Hb (P50 Value)

• The curve is sigmoidal. Low Hb affinity for O2 at low oxygen tension & high affinity for O2 at
high oxygen tension

• Normally PO2 of 27 mmHg = p50| Shift to the left: p50< 27 mmHg Shift to the right:>27mmHg
 • Myoglobin: hyperbolic(shape)/ binds O2 w/ greater affinity than Hb but not effective in
releasing O2 to tissues

1.Carbon dioxide transport

• In venous blood, the carbon dioxide diffuses into the red blood cells and combines with water to
form carbonic acid (H2CO3). This reaction is facilitated by the RBC enzyme carbonic anhydrase.
Carbonic acid then dissociates to release H1 and bicarbonate (HCO32)

• The H1 from the second reaction binds oxygenated hemoglobin (HbO2), and the oxygen is
released from the hemoglobin due to the Bohr effect. The oxygen then diffuses out of the cell
into the tissues. As the concentration of the negatively charged bicarbonate increases, it diffuses
across the RBC membrane intothe plasma. Chloride (Cl2), also negatively charged, diffuses from
the plasma into the cell to maintain electroneutrality acrossthe membrane; this is called the
chloride shift

• In the lungs, oxygen diffuses into the cell and binds to deoxygenated hemoglobin (HHb) due to
the high oxygen tension. H1 is released from hemoglobin and combines with bicarbonate to
form carbonic acid. Carbonic acid is converted to water and CO2; the latter diffuses out of the
cells and is expelled by the lungs. As more bicarbonate diffuses into thecell to produce carbonic
acid, chloride diffuses back out into the plasma. Approximately 85% of the CO2 produced in the
tissues is transported by hemoglobin as H1. In this capacity, hemoglob inprovides a buffering
effect by binding and releasingH1. A small percentage of CO2 remains in the cytoplasm andthe
remainder binds to the globin chains as a carbamino group.

2. Carbon Dioxide Transport

• CO2 diffuses into RBC → + H20 → Carbonic Acid (H2CO3) → Carbonic anhydrase (caused by
enzyme) → H + HCO3 H + HbO2 (Oxygenated Hgb) → O2 diffuses out of the cell (Bohr effect:
shift to the right) HCO3 diffuses out of the cell as it increases and replaced by Cl to maintain
electroneutrality (CHLORIDE SHIFT) In lungs, O2 → Deoxygenated RBC → H is released + HCO3
→ Carbonic acid → Carbonic anhydrase → H2O + CO2 → CO2 is excreted in the lungs

Nitric Oxide Transport

• A third function of hemoglobin involves the binding, inactivation, and transport of nitric oxide
(NO). Nitric oxide is secreted by vascular endothelial cells and causes relaxation of thevascular
wall smooth muscle and vasodilation. When released,free nitric oxide has a very short half-life,
but some enters the RBCs and can bind to cysteine in the b chain of hemoglobin,forming S-
nitrosohemoglobin.

• Nitric oxide + Hgb → S-nitrosohemoglobin

Dyshemoglobin

• Dysfunctional hemoglobins that are not able to carry or transport oxygen

1. Carboxyhemoglobin (HbCO) –

• results from the combination of carbon monoxide with heme iron.

• CO has 240x more affinity for hemoglobin than oxygen

• Once CO binds to hgb shift to the left occurs which increases its affinity and impairs oxygen
release to tissues

• Silent killer (colorless, odorless) causing hypoxia (cherry red color of blood

2. Methemoglobin (MetHb)

• Methemoglobin reductase deficiency → Ferrous Iron is oxidized to Ferric State → Unable to

combine w/ O2

• Chocolate Brown Blood But Reversible → Cyanosis

• Toxic methemoglobeinemia: Nitrites, Primaquine, Dapsone, Benzocaine | Treatment:

Methylene Blue

• Inherited meth: Deficiency of Cytochrome b5 Reductase → Hb M

• Quantitated by spectro at 630 nm

3. Sulfhemoglobin

• Formed by Irreversible oxidation of Hb by drugs (sulfanilamides, phenacetin, nitrites,

phenylhydrazine) or exposure to Sulfur chemicals → Green Pigment/ Mauve Lavender

• C. perfringens → enterogenous cyanosis

• Combine w/ Carbon monoxide → Carboxysulfhemoglobin

• Cannot be detected by cyanmethhgb

• Transferrin - a blood-plasma glycoprotein, which plays a central role in iron metabolism and is
responsible for ferric-ion delivery

• Ferritin - a blood protein that contains/stores iron

• Hemosiderin – second storage form of iron

• Haptoglobin - a protein present in blood serum which binds to and removes free hemoglobin
from the bloodstream.

• Hemopexin - is to bind and transport free heme to the liver

• Ferroportin - is a transmembrane protein that transports iron from the inside of a cell to the

outside of the cell

• Myoglobin – iron found in tissue

Iron transport

• Iron exported from the enterocyte into the blood is ferrous and must be converted to the ferric
form for transport in the blood. Hephaestin, a protein on the basolaminal enterocyte
membrane, is able to oxidize iron as it exits the enterocyte. Once oxidized, the iron is ready for
plasma transport, carried by a specific protein, apotransferrin (ApoTf). Once iron binds, the
molecule is known as transferrin (Tf). Apotransferrin binds two molecules of ferric iron.

• Ferric iron is stored in a cage-like protein called apoferritin. Once iron binds, it is known as
ferritin

Iron recycling

• When cells die, their iron is recycled. Multiple mechanisms salvage iron from dying cells. The
largest percentage of recycled iron comes from red blood cells. Senescent (aging) red blood cells
 are ingested by macrophages in the spleen. The hemoglobin is degraded, with the iron being
held by the macrophages as ferritin. Like enterocytes, macrophages possess ferroportin in their
membranes. This allows macrophages to be iron exporters so that the salvaged iron can be used
by other cells.

• Haptoglobin and hemopexin are plasma proteins able to salvage free hemoglobin or heme,
respectively, preventing them from urinary loss at the glomerulus and returning the iron to the
liver. Like macrophages, hepatocytes are important to iron salvage. They also possess
ferroportin so that the salvaged iron can be exported to transferrin and ultimately to other body
cells

Sources of Iron

1. Red meats

2. Legumes

3. Green leafy vegetables

Echinocytes

• Have short, scalloped, or spike-like projections that are regularly distributed around the cell
membrane. The projections can vary in number and appearance. Crenation can occur as the
result of the physical loss of intracorpuscular water. No disease states are related to crenation,
but this cellular distortion results from an osmotic imbalance.

Elliptocyte

• are generally narrower and more elongated than megalocytes. These cells have a rod, cigar, or
sausage shape. They represent a membrane defect in which the membrane is radically affected
and suffers a loss of integrity. Associated clinical disorders include hereditary elliptocytosis,
anemias associated with malignancy, hemoglobin (Hb) C disease, hemolytic anemias
(occasionally), iron deficiency anemia, pernicious anemia, sickle cell trait, and thalassemia.

Helmet Cell

• are usually the larger scooped out part of the cell that remains after the rupturing of a blister
cell and are formed as a result of the physical process of fragmentation. These cell fragments
are formed in the spleen and intravascular fibrin clots
 Keratocytes/Bite Cell

• are erythrocytes that are partially deformed but not cut. The spicules, resembling two horns,
result from a ruptured vacuole. Usually the cell appears like a half-moon or spindle. These cells
are seen in conditions such as disseminated (diffuse) intravascular coagulation (DIC).

Knizocyte

• resemble a pinched bottle. This abnormality is associated with hemolytic anemias, including
hereditary spherocytosis.

Leptocyte/stomatocyte/mouthcell

• resemble target cells (codocytes) but the inner,

central portion is not completely detached from the outer

membrane. This variation of the target cell is clinically associated

with hepatic disorders, iron defi ciency anemia, and

thalassemia.
Ovalocytes

• have an oval or egg-like appearancethese cells are similar in appearance to elliptocytes,

megalocytes are macrocytic and have a fuller and rounder appearance. In contrast, elliptocytes
tend to have a normal cell-size volume. Increases in this abnormality are seen in vitamin B12
and folate defi ciencies and may be observed in erythrocytes that are in the reticulocyte stage.

Pyknocyte/blister cell

are distorted, contracted erythrocytes that are similar to burr cells. These cells are seen in acute,
severe hemolytic anemia; glucose-6-phosphate dehydrogenase (G6PD) deficiency; and hereditary
lipoprotein defi ciency and may be seen in small numbers during the fi rst 2 to 3 months of life as
infantile pyknocytes.

Schistocytes

• are fragments of erythrocytes that are small and irregularly shaped. Because these cells are
produced as the result of the breaking apart of an erythrocyte, the schistocyte is about half the
size of a normal erythrocyte and may have a deeper red appearance. Increased numbers of
schistocytes can be seen in hemolytic anemias related to burns and prosthetic implants as well
as renal transplant rejections.
Sickle Cell/Drepanocyte

• resemble a crescent At least one of the ends of the cell must be pointed. Generally, the
membrane is smooth and the cell stains uniformly throughout. Sickle cells result from the
gelation of polymerized deoxygenated Hb S. Polymerization of Hb S is influenced by both
lowered oxygen levels and decreased blood pH.

• Seen in sickle cell anemia

• Spherocyte

• are erythrocytes that have lost their normal biconcave shape Extremely compact, round shape

• smaller than 6 mm and has an intense orange-red color when stained

• Clinical disorders associated with spherocytes include acquired hemolytic anemia, blood
transfusion reactions, congenital spherocytosis, and DIC

Target Cell/Codocyte

• are erythrocytes that resemble a shooting target

• A central red bull’s-eye is surrounded by a clear ring and then an outer red ring

• cells are thinner than normal, which may be because of an excessive ratio of membrane lipid to
cell volume

• seen in the hemoglobinopathies (Hb C disease, S-C and S-S disease, sickle cell thalassemia, and
thalassemia), hemolytic anemias, hepatic disease with or without jaundice, and iron deficiency
anemia as well as after a splenectomy
Teardrop Cells/Dacryocyte

• are usually smaller than normal erythrocyte

• As the term implies, teardrop cells resemble tears. This cellular abnormality is associated with
homozygous beta-thalassemia, myeloproliferative syndromes, pernicious anemia, and severe
anemias.

Anisochromia

• General term for a variation in the normal coloration

• Normally, RBC’s gave a central area of pallor of approximately 1/3 the diameter

• May also mean the occurrence of hypocromic cell and normochromic cells in the same blood
smear

• May be found in sideroblastic anemias, also in a hypochromic anemia after transfusion with
normal cells and some weeks after iron therapy for iron deficiency anemia

Hypochromic cells

• Central palor >1/3 of diameter

• Usually microcytic

Grading of Hypochromia

1+ Area of central Pallor = ½

2+ Area of central Pallor = 2/3

3+ Area of central Pallor = ¾

4+ Thin Rim of Hgb

 Anylocyte

1. Aka ghost cell/pessary cell

2. Rbc with a thin rim of hgb and a large clear center

3. May be observed in iron deficiency anemia

 Hyperchromic cells

• Rbc that lack central pallor even though they lie in adesirablearea for evaluation

• These rbcs are usually caused by a shape change

• True hyperchromia only occurs when MCHC is high

• Hereditary Spherocytosis

 Only disease in which the MCHC is high

 Has clinical manifestations namely

1. Splenomegaly

2. Anemia

3. Jaundice

Polychromatophilic erythrocytes

• Larger than normal red cells with bluish tinge (Wright’s stain)

• Bluish tinged – caused by the residual presence of RNA

• Large numbers : associated with decreased rbc survival, hemorrgage or erythroid hyperplastic
marrow

Grading of Polychromasia

Slight 1%

1+ 3%

2+ 5%

3+ 10%

4+ >11%

Anisocytosis

• Incrreased number of red cells with variation in size

• Normal rbc: 7-8 um in diameter (usually seen when MCV is 80-100fl)

RELATED TERMS

Macrocytes Microcytes

- Larger than normal RBCs (>8um) - Smaller than normal rbcs (<7um)

- Usually seen when MCV is > 100fl - Usually seen when the MCV is <80fl

- Associated with impaired DNA synthesis - Associated with defective hemoglobin

formation

RBC Inclusions
 Inclusion Content Visualization Associated Conditions

Basophilic stippling Aggregated RNA Wright stain (deep Lead Poisoning a.k.a
blue to purple) plumbism
a.k.a punctate basophilia
(described as irregular, Supravital stain Arsenic poisoniong
dark blue to purple
granules evenly distributed Pyrimidine 5’
nucleotidase deficiency
within the rbc)
Anemias with impaired
hemoglobin synthesis

Alcoholism

Megalobaslic anemia

Siderotic Intra-erythrocytic Siderotic granules – Sideroblastic anemias –

granules collection of iron iron stains characteriezed by
dimorphic peripheral
Pappenheimer blood picture (presense of
bodies – new both normochromic and
methylene blue, hypochromic rbc in the
wright stain blood

Thalassemia

Hemochropmatosis or
hemosiderosis

Howell-jolly bodies – Remnants of nuclear Wright stain - Megaloblastic anemias

frequently appears singly chromatin
New - After splenectomy
in a cell usually round and
<1 um in diameter, blue to methylene blue
- thalassemia
purple in color Feulgen
reaction (+)

(histochem
staining rxn for
DNA)

Cabot rings – described as Mitotic splindle Wright stain - Megaloblastic anemias

threadlike structures that remnants
- Refractory anemia
appear as purple-blue loops
or rings - Lead poisoning
Heinz bodies – most of the Denatured and Supravital - G6PD deficiency
time appear eccentrically precipitated stains – brilliant
along the inner RBC hemoglobin cresyl blue - Favism – inherited condition
resulting from sensitivity from
membrane large, round
purple to blue materials New methylene fava beans
blue
- Drug induced hemolytic
Methyl violet anemia

Crystal violet - Unstable hemolytic disease

Hgb H inclusion bodies Precipitated hgb h Supravital Hgb h disease

stains (new
methylene
blue,
bromcresol
blue)

Note:
appearance of
RBCs with hgb h
= pitted golf
ball

Parasites Protozoa/protozoans Wright stain Parasitic infection

Ex. Malaria Giemsa stain

Rouleaux formation (psuedoagglutination) 1+ = aggregates of 3 to 4 rbcs

stack of coins appearance of cells 2+ = aggregates of 5 to 10 rbcs

Remember 3+ = many aggregates with only a few free rbcs

- Caused by serum protein abnormality (either

increased globulin or fibrinogen)

- May be observed in multiple myeloma (now called

plasma cell myeloma)

Morphology Grade as:

Polychromatophilia 1+ = 1 to 5 per field

Helmet cell 2+ = 6 to 10 per field

Dacryocyte (tear drop cell) 3+ = greater than 10 per field

Acanthocyte (thorn or spur cell)

Schistocytes (fragmented RBCs)

Spherocytes
Morphology Grade as:

Poikilocytosis 1+ = 3 to 10 per field

Ovalocytes 2+ = 11 to 20 per field

Elliptocytes 3+ = greater than 20

Burr cells

Bizarre-shaped RBC

Target Cell (codocyte/leptocyte/bull’s eye

cell/Mexican hat cell)

Stomatocyte

Sickle cells Whenever present, grade as POSITIVE ONLY!

Basophillic stippling

Pappenheimer bodies

Howell-jolly bodies

• 129 tuergeon

• 288 rodaks