Transcript The Erythrocyte Henry O. Ogedegbe, PhD., BB(ASCP), C(ASCP)SC, CC(NRCC) Department of EHMCS

The Erythrocyte
Henry O. Ogedegbe, PhD., BB(ASCP),
C(ASCP)SC, CC(NRCC)
Department of EHMCS
Learning Objectives
• Upon completion of the materials in this chapter, the
student will be able to:
• List sites of erythrocyte production and destruction
• Draw a flow diagram of the major erythrocyte
morphologic maturation process
• Describe classic morphologic features of each erythrocyte
maturation stage
• List each major erythrocyte component and its primary
functions
• Compare and contrast the destruction of normal and
severely damaged cells
Normal Erythrocyte Production,
Physiology and Destruction
• The primary function of the erythron is to deliver oxygen
to the tissues and carbon dioxide from the tissues
• To function effectively, the body needs approximately 309
X 109 circulating erythrocytes per kilogram of body weight
• The production and destruction of erythrocytes is kept in
balance
• The erythrocytes live for 120 days after which they are
removed from the circulation by the cells of the RE system
• They are promptly replaced to maintain the balance
Normal Erythrocyte Production,
Physiology and Destruction
• Origin:
• The erythrocytes originate from a pluripoten stem cell
called the colony forming unit-S (CFU-S)
• During development, the cells become specialized and
synthesize proteins needed for function and survival
• As the cell matures the morphology changes.
• There are stages in the maturation process
• These stages take place in the bone marrow and may be
differentiated by light microscopy
• The burst-forming unit erythroid is the earliest erythroid
committed cell
Normal Erythrocyte Production,
Physiology and Destruction
• The BFU-E is closely related to the CFU-S and matures
into the CFU-E
• The terms BFU-E and CFU-E are laboratory descriptions
of growth patterns in culture media
• Only a few BFU-E are present in the bone marrow and
therefore are difficult to identify morphologically
• They are different from but similar to small to medium
sized lymphocytes
• Increased numbers of BFU-E have been demonstrated in
some anemias
Normal Erythrocyte Production,
Physiology and Destruction
• Production Sites:
• The main sites of adult blood cell production include the
vertebrae, pelvis, ribs, sternum, skull and the long bones
• Radioactive imaging have been used to study the site of
blood cell production
• Iron is the radioisotope of choice because it mimics
ingested iron which is bound to transferrin in the blood
• Studies can be carried out in cases of anemia or after
radiation treatment to evaluate production sites
Erythrocyte Maturation
• Erythropoietin and other growth factors stimulate BFU-E
and CFU-E to differentiate to the rubriblast stage
• The rubriblast is the first precursor that can be recognized
by light microscopy
• The rubriblast gives rise to sixteen mature erythrocytes
through four cell divisions which take about 72 hours
• Changes take place in the rubriblast as it matures and
differentiates from a primitive nucleated cell to a mature
non-nucleated cell
Erythrocyte Maturation
• Ultrastructure:
• Organelles are present in the early erythrocyte which are
necessary for the synthesis of hemoglobin, and proteins
• As the proteins accumulate, the number of organelles
gradually diminish
• Differentiation of the maturing erythrocyte results in
alterations in morphology and membrane properties
• This results from reorganization of membrane skeletal
protein network
• An important component of the network is protein 4.1
• Protein 4.1 serves as a critical link between the
cytoskeleton and the lipid bilayer
Erythrocyte Maturation
Erythrocyte Maturation
• Nucleus:
• The nucleus is very important in the earliest stages of red
cell development
• It is the site of DNA and RNA synthesis and thus critically
involved in the red cell development and maturation
• Chromatin contains genetic material and is composed of
DNA, histones, and other proteins
• The chromatin is finely dispersed and appears condensed
or granular
• The more condensed heterochromatin are inactive
Erythrocyte Maturation
• The heterochromatin take on a basophilic color (dark blue)
with basic dyes
• The active euchromatin does not stain with basic dyes
• As the cell matures chromatin becomes more dense and
metabolic and synthetic activities start to decline
• Finally the nucleus becomes inactive and it is extruded
from the cell
• Nucleoli are present in the rubriblast and they contain
RNA, proteins and DNA
• Nucleoli are involved with synthesis of ribosomal RNA
Erythrocyte Maturation
Erythrocyte Maturation
• Cytoplasm:
• Ribosomes and polyribosomes are present in the early
erythrocyte precursor
• They are the sites of globin and other protein synthesis
• Polyribosomes probably synthesize different proteins from
those synthesized by the ribosome
• Ribosomes give the cytoplasm of early precursors a deep,
dark blue color
• As hemoglobin is formed, the number of ribosomes
diminish and the blue color is replaced by a reddish pink
color
Erythrocyte Maturation
• Golgi apparatus is also present in the early precursor and is
located near the nucleus
• The Golgi is involved with protein modification within the
cell
• The mitochondria is also visible under electron microscope
as rod shaped organelles
• They are involved with aerobic generation of energy for
the maturing cell and insertion of ferrous iron into
protoporphyrin IX during heme synthesis
• Iron is present in the cytoplasm as ferritin and hemosiderin
Erythrocyte Maturation
• Maturation Stages:
• Six morphological stages of erythrocyte maturation may be
identified from a bone marrow sample with Wright stain
• Normal maturation is dependent on intake of proper
nutrients and vitamins such as folate, vitamin B12 and iron
• Nomenclature:
• There are three nomenclatures used to describe the six
stages
– Rubri (proposed by the ASCP)
– Erythroblast (proposed by Paul Ehrlich)
– Normoblast (normal precursor)
Erythrocyte Maturation
Erythrocyte Maturation
• General Guidelines:
• The ASCP published the following general guidelines for
identification of erythroid precursors:
– Progressive decrease in size and the degree of cytoplasmic
basophilia (blue color) as the cell matures
– Nuclei are round or oval in the blast stage become round thereafter
– Gradual increase in coarseness and condensation of the chromatin,
ranging from fine in the early stages to pyknotic in the stage just
before nuclear extrusion
Erythrocyte Maturation
• Rubriblast (Pronormoblast):
• This is the earliest erythrocyte precursor identifiable by
light microscopy in a Wright stained bone marrow prep
• Cell size ranges from 12 to 25 m
• The nuclear:cytoplasmic ratio is high
• Nucleus usually occupies more than 80% of cell
• The cytoplasm stains basophilic due to high RNA content
• The Golgi may be visible near the nucleus usually pale
Erythrocyte Maturation
•
•
•
•
Prorubricyte (Basophilic normoblast):
Slightly smaller (12 – 17 m) than a rubriblast
Nucleus usually occupies 75% of the cell
The cytoplasm is basophilic and the Golgi is usually
visible near the nucleus
• The nucleus is round and its chromatin is dark violet and
coarser and more clumped
• The nucleoli is absent and helps in the identification
• The prorubricyte usually divides two times giving rise to
four rubricytes
Erythrocyte Maturation
•
•
•
•
Rubricyte (Polychromatophilic normoblast:
It is usually smaller than the prorubricyte (12-15 m)
Has a round nucleus that may be eccentric
The nucleus is smaller and the cytoplasm becomes more
prominent
• There is a spectrum of blue color due to synthesis of
hemoglobin
• The RNA and hemoglobin give the cytoplasm a blue gray
violet color called polychromasia or polychromatophilia
• The cell may be confused with a lymphocyte
Erythrocyte Maturation
•
•
•
•
•
•
•
•
Metarubricyte (Orthochromic normoblast):
This is the last nucleated erythrocyte stage
It is slightly smaller than the rubricyte (8-12m)
The cytoplasm is polychromatophilic and more pinkish
than that of the rubricyte
The nuclear chromatin is dense, coarse and clumped
The nucleus is degenerated and pyknotic
The nucleus is extruded from the cell at this stage
Sometimes nucleus is not completely extruded resulting in
Howell-Jolly body
Erythrocyte Maturation
• Reticulocyte (diffusely basophilic erythrocyte):
• The reticulocyte is slightly larger than the mature
erythrocyte
• The cytoplasm still contains small amounts of RNA which
produces varying amount of polychromasia
• The reticulocytes are retained in the bone marrow for 2 to
3 days before release into the marrow sinisoids
• The mechanism of release is unknown
• The retic contain Golgi apparatus remnant and residual
mitochondria which permit continued aerobic metabolism
Erythrocyte Maturation
• The retic also contain RNA which may be stained
supravitally with methylene blue or brilliant cresyl blue
• The RNA precipitates and the retics can then be counted
and the reticulocyte production index determined
• Mature Erythrocyte:
• The mature erythrocyte is approximately 7.2 m in
diameter
• It is a biconcave disc and hence referred to as a discocyte
• In a Wright stain, a central pale area is revealed which
fades gradually into the reddish pink cytoplasm
Erythrocyte Maturation
Structure and Physiology of the Mature
Erythrocyte
• The mature erythrocyte lacks a nucleus or organelles
• Components necessary for function and survival are
present
• The cell has a specialized membrane that allows for O2 and
CO2 transport and for survival for 120 days
• Various factors contribute to erythrocyte membrane and
hemoglobin maintenance
• A source of energy is required
• Membrane shape and deformability are needed
Structure and Physiology of the Mature
Erythrocyte
• Shape and Deformability:
• The erythrocyte is a biconcave disc which facilitates O2
and CO2 transport by maximizing ratio of surface area to
volume
• Allows the cell to be flexible and deformable
• This allows the cell to adjust to small vessels in the
microvasculature and still maintain a constant surface area
• A less deformable cell would be subjected to fragmentation
Structure and Physiology of the Mature
Erythrocyte
• Membrane Composition and Structure:
• The composition of the membrane allows the cell to
– Separate the intracellular fluid environment of the cytoplasm from
the extracellular fluid environment
– Selectively pass nutrients and ions into and out of cell
– Deform when required
• The membrane is composed of lipids and proteins in
approximately equal proportions by weight
• The difference in the lipids and proteins in the cytoplasmic
side and the plasma side allow for selective movement of
molecules in and out of the cell
Structure and Physiology of the Mature
Erythrocyte
• Lipids:
• Phospholipids and unesterified cholesterol predominate in
the lipid fraction
• The phospholipids form the bilayer and the hydrophilic
polar heads of the phospholipids are oriented toward the
aqueous environments
• The hydrophobic tails of the phospholipids are oriented to
the interior of the bilayer
• The phospholipids are fluid and the fatty acid tails move
freely
• Cholesterol plays an important role in maintaining surface
area
Structure and Physiology of the Mature
Erythrocyte
• Protein:
• Proteins are bound to lipids throughout the membrane
• The proteins are either peripheral proteins or integral
proteins
• The peripheral proteins are present on the inner portion of
the membrane nearest the cytoplasm
• The integral proteins are in contact with both the inner and
the outer surface of the membrane
• The integral proteins act as receptors for ions and
molecules needed in the cell such as transferrin and EPO
Structure and Physiology of the Mature
Erythrocyte
• The peripheral proteins include the  and  spectrin also
called band 1 and 2 and actin
• The proteins form the cytoskeleton of the cell and regulate
membrane shape and deformability
• Their linkage is mediated by protein 4.1
• The principal integral proteins are glycoproteins
designated glycophorin A and band 3
• They span the lipid bilayer.
• Band 3 is an inorganic anion transport channel
• Integral proteins contain sialic acid which gives
erythrocytes a negative charge
Structure and Physiology of the Mature
Erythrocyte
• The negativity between cells called zeta potential cause
cells to repel one another as they move through the
circulation
• Membrane proteins facilitate movement of substrates and
cofactors in and out cell
• Examples include the Na+, K+ - ATPase and Ca2+, Mg2+ ATPase
• Calcium is involved in regulation of and stabilization of
membrane phospholipid structure
• High intracellular concentration of calcium, cause cell
deformability
Structure and Physiology of the Mature
Erythrocyte
• Energy Metabolism:
• The cell requires energy for cell metabolism and to
preserve the membrane integrity
• Various enzymatic reactions in the cell require energy
• Energy is required to reduce proteins and maintain
hemoglobin in its reduced state for proper functioning
• Two site prone to oxidation are the iron atom in the heme
ring and the sulfhydryl groups on the globin molecule
• Oxidation of the normal ferrous state to the ferric state
results in methemoglobin which does not deliver oxygen
Structure and Physiology of the Mature
Erythrocyte
• Normally 1% to 3% of oxygen is oxidized to
methemoglobin
• Oxidation of sulfhydryl groups causes hemoglobin
precipitation (Heinz body formation)
• Sources of Energy:
• The Embden-Meyerhof Pathway (EMP):
• This is an anaerobic process for energy generation through
glucose catabolism to lactate
• About 90% to 95% of glucose used by the cells is
metabolized by the EMP
Structure and Physiology of the
Mature Erythrocyte
• ATP is generated during the glycolysis of glucose to lactate
• ATP is needed to maintain membrane shape and
deformability
– Through phosphorylation of spectrin and calcium chelation
– Provide energy for active transport of cations
– And to modulate the amount of 2,3 DPG generated
• There is a net yield of two ATP molecules per molecule of
glucose catabolized
• 2,3 DPG is formed from the Rapoport-Luebering shunt
• Helps modulate O2 transport in the cell
Structure and Physiology of the
Mature Erythrocyte
• Hexose Monophosphate Shunt and Glutathione Reduction
pathway:
• Also called the pentose phosphate pathway is an aerobic
method of erythrocyte glycolysis
• Processes about 10% of erythrocyte glucose
• Purpose is to provide reducing potential by generating
reduced nicotinamide adenine dinucleotide phosphate
(NADPH)
• It is an oxidative pathway
Erythrocyte Destruction
• As the red cell ages, changes occur that make it susceptible
to destruction
• Alteration in the membrane integrity takes place
• Loss of sialic acid and lipids, decreased ATP and increased
Calcium have been implicated in the aging process
• At 120 days the erythrocytes are recognized as abnormal
and are removed by phagocytic cell in the RES
• As the cell ages it is depleted of glucose and their surface
area decreases
• The spleen recognizes abnormalities in the cell and
sequester it for removal
Regulation of Erythropoiesis
• A balance between production and destruction keeps the
the erythrocyte number constant
• Production of erythron requires a normal functioning
competent bone marrow
• Adequate levels of EPO, growth factors and nutrients such
as iron, folate, vitamin B12
• Between 3 X 109 and 8.5 X 109 erythrocytes are produced
daily
• Cytokines or growth factors play a important part in the
process
Regulation of Erythropoiesis
• Erythropoietin Production and Regulation:
• EPO is a glycoprotein hormone with a molecular weight of
34,000
• It is an erythroid growth factor
• It is produced in the kidney
• It is regulated by renal O2 tension which when decreased
induces expression of the EPO gene and the release of
EPO
• Prostaglandins help regulate EPO production
Regulation of Erythropoiesis
Regulation of Erythropoiesis
• Growth Factors:
• Many other hormones and cytokines secreted by various cells have
been found to stimulate erythropoiesis
• These cytokines have been identified in cell cultures and they include
–
–
–
–
–
–
–
–
EPO
Insulin
Growth hormone
Steroid hormone
Nonandrogenic thyroid hormone
IL-1, IL-4, IL-6, IL-7, IL-11, IL-12,
G-CSF
Macrophage inflammatory protein (MIP) and steel factor (SF)