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 x1012 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 O2 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 10-7m 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)