The erythrocyte lecture 2

3. ERYTHROCYTES Lecture by GK Mbassa

Introduction
Red blood cells (RBC) constitute 99 % of blood cells
They are the smallest cells in the mammalian body
They function in the transportation of oxygen to cells and tissues and carbon dioxide from cells and tissues to the lungs for exchange with O2

Erythrocyte properties
Red blood cells or erythrocytes contain haemoglobin (Hb)
Hb gives the red colour of blood
Individual erythrocytes are orange yellow
On centrifugation erythrocytes are heavier and settle to the bottom to form a packed cell pellet.
White blood cells form a layer (buffy coat) on top of the red blood cell pack.
Platelets are settled on top of the white blood cells.

Erythrocytes are non motile cells, but withstand deformation, pass though smallest capillaries and venules and are remarkably elastic
Erythrocytes are circular biconcave discs in most mammals, but oval in llamas and camels
Mammalian erythrocytes have no nuclei (anucleate).

All other vertebrates have nucleated RBC
Sizes (volumes) of erythrocytes of some mammals
Man, 55-65 fl, dog (7 μm diameter), 37–55 fl, cat, 39–55 fl, horse, 37–59 fl, ox, 40–60 fl, pig, 50–60 fl, sheep, 28–40 fl, goat, 17–38 fl (4 μm diameter).
Goat has highest erythrocyte counts
RBC counts vary with age, species, breed, physiological state, feeds and other factors in health and in disease.

Reptilian, amphibian, bird and fish erythrocytes
Large, nucleated, discoid to ovoid; bird 80–200 fl, reptiles 100–290 fl
Bird RBC 0.5 – 1.5 x10 12 cells/l.
Sizes vary inversely to counts
The smaller the size, the larger the number

RBC life span 120 days only, short because of the lack of nuclei
RBCs have semi-permeable trilaminar membrane
RBCs are elastic, alter shape to pass through capillaries
Biconcave discoid shape maintained by membrane protein spectrin
Shape affected by osmotic forces
RBC is soft colloid to change shape in various sized vessels

RBCs are affected by extraneous solutions
In isotonic solution, maintain normal form
In hypertonic media, lose water, shrivel, shrink to burr like, thorny pineapple like structure (crenate), surface membrane folds extensively.
In hypotonic solution imbibe water, swell and rupture (osmotic hemolysis).

Hypoptonicity causes RBC to be spherical, stretch their membranes
Hb oozes out leaves colourless structure called erythrocyte ghosts. RBC rupture is called haemolysis

Rouleaux formation
RBC aggregate in form of stacks, called rouleaux due to adhering to each other at broad surfaces
Rouleaux formation depends on changes in the plasma, net positive charge in the plasma changes surface charge on erythrocytes, increasing their adherence to each other.

Rouleaux formation is clinically an increased sedimentation rate of blood
Rouleaux formation more frequent in equine, felines, rare in carnivore, ruminant, porcine, man

RBC cytoplasmic contents
RBC has certain proteins and lipids
Delicate network granule (substantia reticulofilamentosa) is demonstratable in reticulocytes and proerythrocytes
RBC has Hb; (1) globin, a protein and
(2) iron containing heme pigment

Old RBCs are sequestered in the spleen and bone marrow by phagocytes
Heme iron is captured for re-use
Hb porphyrins are degraded to bilivedrin (toxic, green)
Then to bilirubin (yellow, toxic)
Bilirubin is conjugated with glucuronic acid in liver to less toxic conjugated bilirubin Bilirubin is stored as bile in gall bladder

Blood volume is 6–8 % of body weight
% of blood volume occupied by RBC is called hematocrit or packed cell volume (PCV)
PCV depends on number & size of cells and plasma volume
RBC staining (affinity for Hb) may be
normal (normochromasia)
lower (hypochromasia)
higher Hb (hyperchromasia) than normal.

Differences may be in sizes (anisocytosis), shapes (poikilocytosis) and number (hypererythrocytosis)
Lower than normal counts is oligoerythrocytosis or anemia

Erythrocyte morphology
Mammalian erythrocytes are non nucleated
In all others erythrocytes are nucleated
On blood smears RBCs stained with Giemsa stain are pink because they bind eosin
RBC constitute 99% of blood cells
RBC are fairly rounded, 7.5 μm diameter
Thin central stains less than outer thicker annular area

Ultrastructurally mature erythrocytes have smooth surfaces
Immature RBC have rough surfaces
RBCs show small pits at sites of iron intake
Erythrocytes are entirely filled with haemoglobin
Hb, uniform granular appearance in electron micrographs
Organelles are entirely lacking in mature RBC apart from plasmalemma
In mammalian erythrocytes organelles disappear during maturation
Scanning microscopy reveals characteristic biconcave shapes

Species variations in erythrocyte morphology
Differences in RBC occur on morphology, size, shapes
Are biconcave discs with central pale area in human, canine
In cattle, sheep, goat central pale area is shallow, cells are flat discs
Feline and equine have shallow central pallor

Elliptical RBC in camellidae; camel, alpaca, llama
Sickle shaped, pliable, non fragile RBC in deer
Poikilocytes in goats; rounded, spindle shaped, rods, pears, triangular and fusiform
Hemoglobin alters shapes after polymerization
Goat sheep RBC show wart-like protuberances (senescence)

In some sheep RBC are sickle-shaped shaped.
In general healthy RBC show biconcavity in all species
Polikilocytosis in goats (shapes); spindle, rod, pear, triangular, discoid, fusi-form, match stick, spheroid.

Cells vary due to (1) polymerization of Hb to longitudinal tubular fibres (2) presence of intracytoplasmic microtubules, longitudinally oriented

Erythrocyte membrane
RBC plasmalemma is trilaminar
Cell membrane selectively permeable Na and K
Potassium prevents escape of Hb and other cell materials

RBC contains 60% H20, 40% conjugated proteins globin and heme pigment (4%) to form (hemoglobin)
RBC membrane is permeable to water, electrolytes, polysaccharides impermeable to Hb.

RBC osmolarity is determined by Hb.
Osmolarity of Hb in RBC is equal to that of plasma (isotonic), water absorption for plasma or RBC
RBC fragility test measures degree of RBC resistance to haemolysis
RBC is subjected to decreasing salt concentration until it haemolyses
Concentration of salt at which the RBC haemolyzes is fragility value
Species differences in RBC fragility are related to RBC size only
Small erythrocytes eg goat most susceptible to fragility, least for dogs
RBC fragility is a diagnostic test for certain anaemias

Functions of the erythrocyte
Primary function of erythrocytes is to carry Hb
Hb carries oxygen by being oxidized to O2―Hb (oxy-haemoglobin)
Transports O2 to cells
Then reduced to H-Hb
Hb has high affinity for O2

Advantages of Hb being within RBC
Blood viscosity not increased as when Hb is free
Free Hb increases viscosity
Hb exerts osmotic pressure 3x that caused by plasma proteins alone, enclosed Hb in RBC prevents Hb from exerting osmotic pressure in plasma which would have profound effects on movement of fluids through capillary walls and renal glomerulus
Intra RBC environment is slightly more acidic than plasma for efficient respiration
Respiratory pigment Hb is in environment of acidic pH in RBC

Hb is removed from metabolic pool, preventing its rapid turn over. T½ for Hb in RBC is months, T½ for Hb in plasma is only 3 hrs
To keep Hb in proximity of enzymes to maintain its chemical state
Hb chemical state is required for O 2 transport
Saturated Hb carries 1.3 ml oxygen per gram

Red blood cell membrane
Erythrocyte membrane is deformable and resilient, its functions include
Enclose cellular components, vital for survival and functions of red blood cell
Selectively permeable to electrolytes, particularly cations
Regulating RBC contents and ionic gradient between intracellular and extracellular environments

Membrane composition
RBC membrane is trilaminar composed of a biomolecular structure of two electron dense layers each 25 x 10 -7 m and a 20 – 30 x 10 –7 electron – lucent zone
Its chemical composition include 48% proteins, 44% lipid, 8% carbohydrates
Carbohydrate moiety associated with proteins (glycoproteins) or lipids (glycolipids)

Distribution of proteins and lipids is asymmetrical in outer and inner biomolecular layers
Lipid bilayer comprised of phospholipid molecules
Hydrophobic non polar groups directed inwards toward each other

Hydrophilic polar groups directed outwardly
Cholesterol is interspersed between phospholipid molecules, functioning to increase membrane surface, decreasing fragility

Protein molecules are asymmetrically distributed
Within the membrane as integral membrane proteins
At sub-membranous locations as peripheral membrane proteins
Some membranous proteins remain intra-membranous, others traverse membrane to outer, become glycosylated while attached to inner membranous structural proteins

Carbohydrate residues
Confer negative surface charge, serve as antigenic determinants of blood groups and binding sites for viruses and lectins
RBC membrane is negatively charged due to carboxyl groups of sialic acid residues in glycoprotein of external surface
Degree of negative charge decreases with cell age, exposure to antibody and proteolytic enzymes such as trypsin

Sialic acid molecules are highly correlated to electrophoretic mobility of the red cell
This is used to separated cells of different ages because of age dependence of negative charge strength

Red cell membrane phospholipids
Determine RBC shape and surface of RBC
Outer membrane is rich in phosphatidlycholine and sphingomyelin, has some phosphatidylethanolamine
Inner membrane has more phosphatidylserine and phosphatidyl ethanolamine.

Cholesterol to phospholipid ratio is 0.9: 1.0 in man RBC
Cholesterol and phospholipids are in dynamic state exchanging freely between membrane and plasma
There is high cholesterol in convex than in concave surface of RBC

Low RBC cholesterol results in
Decrease in membrane surface area causing RBC to be osmotically more fragile
Increase in cholesterol
Extends surface area and causes folded contour of the cell surface, decreases red cell filterability and imparts resistance to osmatic lysis

RBC cholesterol and phospholipid content and their ratios are altered in
Liver disease
Spurr cell anemia (abetalipoproteinemia)
Stress reticulocytosis of anemia
RBC lipid composition varies with spp, and age

Red blood cell membrane proteins
Red cell membrane proteins maintain the shape and cellular integrity of erythrocyte
Solubilization of RBC membrane in sodium dodecyl sulfate (SDS) and subjecting the solution to polyacrylamide gel electrophoresis (PAGE)  , that is (SDS-PAGE) produces three classes of sialoproteins

Glycophorin A, B C as major integral proteins of RBC membrane and constitute to cytoskeleton
Spectrin, maintains RBC shape and cellular integrity, linked inwardly protein ankyrin or syndein
Actin
When the configuration spectrin is changed (dephosphorylation) RBCs crenate
RBC is transformed from discocyte to echinocyte
Abnormal spectrin results in spherocytosis, elliptocytosis, sickle cell, including human sickle cell anaemia

Metabolism of erythrocytes
The erythrocyte obtains energy of non aerobic glycolysis to maintain reduced Hb
Erythrocytes mature through polychromatophilic rubricyte stage characterized by
Tricarboxylic acid (TCA) cycle or Krebs cycle
Embden – Meyerhof (EM) pathway or glycolysis
Pentose cycle (Hexose monophosphate shunt) or HMS

These metabolic processes are reduced at metarubricyte stage and very limited at reticulocyte stage
Anaerobic EM pathway accounts for 95% producing ATP which maintains active pumping out of Na+ ions, pumping in of K+. Inward flow of Na+ and outward flow of K+ are passive processes
Oxidative pentoses cycle accounts for 5% of energy of RBC

mature erythrocyte lacks TCA cycle and c apacity for oxidative phosphorylation because of lack of organelles mitochondria, ribosomes and endoplasmic reticulum

Reduced nicotinamide – adenine dinucleotide (NADH) or reduced diphosphopyridine nucleotide (DPNH) formed in EM pathway is utilized in enzymatic reduction of methaemoglobin (iron in ferric state) to functional hemoglobin (iron in ferrous state) capable of transporting oxygen under the enzyme NADH methaemoglobin reductase

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced triphosphopyridine nucleotide (TPNH) is formed in HMS and utilized for
Conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) which protects erythrocytes against haemolysis by oxidants

Conversion of methaemoglobin to functional hemoglobin.
Lack of this reaction precipitates hemoglobin to Heinz bodies

Mature erythrocytes synthesize
reduced glutathione
Glutathione reductase
Glutathione peroxide (superoxide dismutase)

Abnormalities in RBC metabolism
Deficiency of glucose – 6 – phosphate dehydrogenase
Deficiency in pyruvate kinase
Deficiency of NADH – methaemoglobin reductase

Mammalian RBC synthesize 2, 3 – Diphosphoglycerate (DPG) which
regulates energy metabolism through glycolysis pathway
Regulates release of oxygen from haemoglobin
Species where erythrocytes stay less than 100 days have reticulocytes in circulation

species where erythrocytes stay more than 120 days have no reticulucytes in circulation
Life span is measured by use of tagging erythrocytes with radioisotopes, commonly 5Cr, 55Fe, 59Fe, 15N, 32P, and 14C by a process called Cohort labeling

Destruction of erythrocytes
Cell decrease deformability in microcirculation is associated with;
increase in red cell rigidity
increase in blood viscosity,
impeded blood flow and
cell fragmentation

The changes in deformability depends on
Maintenance of cell geometry or biconcave shape
Normal internal or hemoglobin fluidity
Intrinsic membrane deformability or visco-elastic properties

Modes of erythrocyte destruction
Change in membrane permeability
Phagocytosis where (Na+, K+) levels are altered leading to increased osmotic fragility and haemolysis

Macrophage phagocytosis is extra-vascular haemolysis with increased un-conjugated bilirubin
Fragmentation, within circulation is intravascular haemolysis, followed by haemoglobinemia and haemoglobunuria

Transformations and abnormalities of the erythrocyte shapes
The red cell shape is in equilibrium determined by the structural properties of
1) cell membrane
2) hemoglobin

The common shape is biconcave disc
The cell regains its shape after passage through microcirculation. Membrane phospholipids, cholesterol and proteins, ATP and Ca2+ are essential for maintaining the normal shapes

Various erythrocyte shapes
The variations are shapes of erythrocytes arise due pathologic or environmental changes
Acanthocytes (spur cells) are erythrocytes with thorn spicules, spines or surface projections
Acuminocytes (fusocytes) are thin, cup shaped erythrocytes or helmet shaped

Dacryocytes are erythrocytes shaped like tear drops
Cryohydrocytes are erythrocytes that are coldy and frozen (frost erythrocytes)
Discocytes (normocytes) are normal biconcave erythrocytes (normocytes)

Drepanocytes are sickle shaped erythrocytes produced after polymerization of hemoglobin
Eccentrocytes (pyknocytes) are erythrocytes with condensed hemoglobin in one area of the cell

Echinocytes (created cells or burr cells) are erythrocytes with several blunt or pointed evenly spaced surface projections
Elliptocytes (Ovalocytes) – are erythrocytes with ellipsoid or oval shapes (e.g. match stick, cigar shaped)

Cigantocytes are large red cells larger than macrocytes and megalocytes
Keratocytes are erythrocytes with one or more pointed projection like horns, slightly notched or somewhat flat surface between projections

Knizocytes are erythorocytes with central bar of memoglobin and somewhat clear spaces on either sides appearing as triconcave cell
Leptocytes are erythorocytes with increased diameter and decreased cell thickness, thin, folderd, surface area greater than contents, appear as target cells or knizocytes. They ma be orthrochromic or polychromic

Macrocytes and megalocytes are morphologically normal erythrocytes except for an MCV grater than normal. Their appearance indicates intensified erythropoiesis
Microcytes are erythrocytes with MVC smaller than normal. They have increase central polar due to low hemoglobin content

Poikilocytes are erythrocytes having any morphology other than normal. They are of various shapes
Schizocytes (schistocytes) are erythrocytes with irregular fragments (triangular, rod, half moon, spiculated, bizarre forms)
Selenocytes are crescent shaped poorly stained red cells

Siderocytes are nucleated erythrocytes containing prossian blue. Positive iron granules visible with the light microscope
Sideroblasts are nucleated erythrocytes containing iron granules in the cytoplasm (ferritin aggregates in the cytoplasm)

Ringed sideroblasts contain iron filled mitochondria arranged in circular pattern around the nucleus
Spherocytes (Microspherocytes) are intensely stained, small spherical erythrocytes with reduced surface to volume ratio

Spiculated erythrocytes are red cells with one or more surface spicules. They generally include echninocytes, acanthocytes, dacrocytes, drepanocytes, kerotocytes and schizocytes

Stomatocytes are erythrocytes that in Wright stained films appear to have a slit or mouth like clear opening near the cell centre. They have uniconcave morphology in wet preparations

Torocytes are ring shaped erythrocytes with sharply defined clear central area and a thicknened peripheral rim of hemoglobin (also called punched out cells)

Abnormalities of iron transport and thermo-reactivity
1. Alternations in cell water resulting from changes in cell membrane and deformability
Hydrocytosis (surface area to volume ratio).
Increase in MCHC leads to increase in internal viscosity (xerocytosis).

2. Increased thermal reactivity (pyropoikilocytosis), the results are
hydrocytosis leading to hemolysis
Xerocytosis (osmotically resistant hemolysis)

The erythrocyte lecture 2

by BRUNO THADEUS

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