Topic 3: Mature Erythrocyte

Learning Objectives

At the end of this topic, students will be able to:

1. Discuss the structure and physiology of erythrocytes

2. Describe the chemical composition of RBC membrane.
3. Discuss the Embden-Meyerhof anaerobic glycolytic pathway (EMP) in the erythrocyte,
with attention to adenosine triphosphate generation and consumption.
4. Name three diversion pathways from the EMP within RBCs and state the function or
purpose of each.
5. Describe the role of 2,3-bisphosphoglycerate in erythrocyte metabolism and the
sacrifice made to produce it.
6. Name the components related to the hexose monophosphate pathway that act to
detoxify peroxide and the type of chemical process that accomplishes the
detoxification.
7. Describe the methemoglobin reductase pathway that diverts from glycolysis.
8. Describe the functions of transmembranous proteins as they affect RBC function.
9. Cite the relative concentrations of sodium and potassium inside RBCs and name the
structure that maintains those concentrations.
10. Discuss the normal destruction of RBC
11. State the dimensions of the counting area of a Neubauer and other ruled
hemacytometer.
12. Describe the performance of manual cell counts for leukocytes, erythrocytes, and
platelets, including types of diluting fluids, typical dilutions, and typical areas
counted in the hemacytometer.
13. Calculate dilutions for cell counts when given appropriate data.
14. Calculate hemacytometer cell counts when given numbers of cells, area counted, and
dilution.

Activating Prior Learning

Answer/do the following:

1. __________ is the main component of RBC.
2. __________, ___________, and ____________ are the main composition of RBC
membrane.
3. __________ most commonly used dilution fluid in RBC
4. __________ is the major pathway of RBC destruction.
5. __________ most commonly used hemocytometer ruled area for counting blood
cells.

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Presentation of Contents

Structure and Physiology

} Non-nucleated, biconcave disc-like cell
} 6-8 microns in diameter
} Central area of pallor
} Nutritional Requirements
o CHON and amino acids
o Vit B12, folic acid, Vit. B6
o Fe++
o Riboflavin, panthotenic acid, nicotinic acid
} HEMOGLOBIN = main cell component
} lacks nucleus & organelles (no hemoglobin is synthesized because it lacks
mitochondria)
} 120 days = survival period
} Stimulated by EPO (Hypoxia)
} In every 100 RBCs are 3 – 8 platelets maybe seen
} In every 1000 RBCs , there is 1 WBC
} Conditions necessary for Red Cell survival:
o Red cell membrane must be deformable or flexibility; allows cell to adjust
to small vessels in the microvasculature
o Hemoglobin structure and function must be adequate
o Red cell must maintain osmotic balance and permeability
} Requirements for normal erythrocyte to form:
o adequate supply/amount of EPO, Iron, Protoporphyrin III, and Globin

Red Cell Membrane

} 3 Basic Functions of the Membrane Composition & Structure:
1. Separate the ICF environment of the cytoplasm from the ECF environment of
the plasma
2. Allow nutrient & ion passage selectively into and out of the cell
3. Let the cell to deform
} The red cell membrane consists of a bipolar lipid layer supported by structural
proteins.
1. 50% of the membrane is protein
2. 40% is lipid
3. 10% is carbohydrate.

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 Structure of the red cell membrane.
Actin Spectrin HEMOGLOBIN Ankyrin INSIDE CELL

Band 4.1

Cholesterol Lipid
Fatty bilayer
acid
chain

Phospholipid Band 3 OUTSIDE CELL

Glycophorin PLASMA

} Composition
1. Protein (50%)
 Peripheral protein: skeletal proteins
a. Spectrin – maintains BICONCAVITY of RBC’s
 gives the cell membrane its flexibility and strength.
 The red cell distorts as it passes through tiny capillaries, but once
through the capillary, it immediately returns to its biconcave
shape.
 If spectrin is denatured, e.g. by heat, the red cell assumes a
spherical shape and loses its flexibility (spherocytosis).
b. Actin
c.

Ankyrin
 Integral Protein: Glycophorins A,B,C
 contains SIALIC ACID w/c gives red cells a negative charge

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 *ZETA POTENTIAL – negativity of RBC’s causing cells to repel
to one another as they move through the circulation
 important for the active transport of solutes across the membrane

2. Lipids (40%)
 External surface: phosphatidylcholine, glycolipid and sphingomyelin
 Internal surface: phosphatidylethanolamine, phosphatidylinosotil, and
phosphatidylserine
 The phospho- and glycolipids are structural with polar groups
(hydrophilic) on the external and internal surfaces of the cell.
 Non-polar groups (hydrophobic) form a barrier at the centre of the
membrane.
 The lipid bilayer forms the wall of the red cell and separates the contents
from the external environment.
 Oxygen can pass through the lipid barrier and bind to the Hb inside.
 Alteration in lipid composition can produce
target cells or Acanthocytes (increased cholesterol & phospholipids)

Target cells Acanthocytes

3. Carbohydrates (10%)
 mostly found on the external surface of the red cell membrane
 Monosaccharides are associated with specific blood group antigens, e.g.
ABH, Lewis, Duffy, etc.

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 } Transmembrane Protein Transporters or Pumps
1. Water transporter: Aquaporin 1
2. Glucose transporters: GLOTI 1 & GLOT 4; Sodium / Hydrogen exchanger 1
3. Membrane enzymes; Na – K – ATPase; Ca – Mg – ATPase
 Increased Na – lysis
 Increased K – shrinkage
 Increased Ca - Decreased deformability

} Hemoglobin Viscosity
 Normal erythrocyte Hb has a low viscosity & is fluid
 Increased RBC count = produces a high viscosity of the blood
(THROMBOSIS)

} Energy Metabolism
 Importance of Energy
o maintain various components of the erythrocyte
o preserve membrane shape, enzymatic reactions & movement of
calcium, sodium & potassium
o reduce oxidized proteins
 Sources of Energy
o GLUCOSE – principal source of energy
o Other sugars – Galactose, Fructose, Mannose

Metabolic Pathway
1. Embden – Meyerhof Pathway
 Major source of red cell energy
 90% glycolysis occurs in this pathway
 maintains cellular energy by generating ATP
 ANAEROBIC method for energy generation through w/c every glucose
broken down to lactate, yields 2 ATPs
 ATP controls Na+ and K+ and prevent oxidation of membrane lipid
 4 Intermediates in EMP:
i. Formation of 2,3 DIPHOSPHOGLYCERATE (2,3 DPG)
 regulates Hb affinity for oxygen (helps reduce hypoxia by causing
more oxygen to be released to the tissues until erythrocyte numbers
are back in balance)
 generated by the Rapoport – Luebering shuttle
ii. ATP
 high energy phosphate
 2 ATP’s = net gain (in every glucose molecule)
 Functions:
a. maintains membrane shape deformability by: phosphorylation
of spectrin and chelation of calcium

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 b. provides energy for active transport of cation pump needed for
the NA-K conc. Pump & calcium flux
c. assists in modulating the amt of 2,3 DPG generate
d. maintains membrane lipids
iii. Nicotinamide adenine dinucleotide (NAD)
 co – enzyme w/ glyceraldehyde-3-phosphate dehydrogenase in the
formation of of 1,3DPG
 co – enzyme w/lactate dehydrogenase (LDH) to reduce pyruvate to
lactate
iv. Nicotinamide adenine dinucleotide hydrogenase (NADH)
 co – enzyme w/methemoglobin reductase & reduce methemoglobin
to Hb
 PK (pyruvate – kinase) deficiency – most common hereditary enzyme
deficiency of EMP

2. Hexose Monophosphate Shunt/ Pentose Phosphate Pathway

 10% glycolysis occurs in this pathway
 AEROBIC PATHWAY; OXIDATIVE PATHWAY
 Glucose 6-phosphate is converted to 6-phospho-gluconate and so to ribulose
5-phosphate.
 NADPH is generated and linked with glutathione to protect against oxidative
stress e.g. hydrogen peroxide produced by oxidative drugs or phagocytes. It is
also used to maintain Hb in the active ferrous (Fe++) state.
 generate ribose-5-phosphate (R-5-P) = used during nucleic acid metabolism
 Provides REDUCED GLUTATHIONE (GSH) to prevent denaturation of
globin of the Hb molecule by oxidation—the principal reducing agent in the
cell.
 G6PD (glucose-6-phosphate dehydrogenase) deficiency
i. one of the commonest inherited
abnormalities in HMS in which the red
cells are susceptible to oxidant stress
ii. most common enzymopathy in
HEREDITARY HEMOLYTIC
ANEMIA
iii. yields HEINZ BODIES

3. Methemoglobin Reductase Pathway

 Prevents oxidation of heme iron
 Maintains hemoglobin in Fe2+ (Ferrous) state to be functional

4. Luebering – Rapoport Pathway

 Responsible for generation of 2,3-DPG which regulates hemoglobin affinity
for O2
- Increased 2,3-DPG – decreased O2 affinity (RIGHT)
- Increased 2,3-DPG – decreased O2 affinity (LEFT)

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  important in the oxygen – carrying capability of erythrocytes

Mechanism of RBC Destruction

} Normal
a. constant buffeting in the blood stream
b. by alterations in its tension w/c is dependent on the ionic changes
c. becomes more spherical as it age enabling to the rupture of the cell
membrane
} Abnormal
a. various types of hemolysis
b. hemoglobin alterations
c. parasitic infections
} As red blood cells age: decreased enzymes, ATP, size & increase in density
} Approximately 1% of the red blood cells leave the circulation each day and are
broken down by the mononuclear phagocytic system (MPS)
} CULLING = destruction of senescent (aged) red cells by the spleen.
} PATHWAYS: EXTRAVASCULAR & INTRAVASCULAR

Extravascular
90% aged red blood cell
destruction – MAJOR PATHWAY
Happens within the
reticuloendothelial system
Happens when complement is not
activated or incompletely activated
Leads to increased unconjugated
bilirubin & urine/fecal urobilinogen
Intravascular
10% aged red cell destruction
Happens within blood vessels
Happens when complement is
completely activated
Leads to hemoglobinuria,
haptoglobin & hemopexin
Associated with ABO hemolysis

Associated with Rh hemolysis

Red Blood Cell Count

} Transports Oxygen and removed waste products from the Body’s tissue
} Total number of red blood cells/L of whole blood
} Factors affecting RBC count:
o Posture – Low in recumbent position
o Exercise or Excitement – Higher than those under basal conditions
o Dehydration – Higher in severe hemoconcentration
o Age-Higher in newborn infants
o Sex- Lower in women

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 o Altitude – Higher in high altitude than those at sea level

} Hemocytometry
o estimation of the total number of blood cells in a given volume
o numerical evaluation of formed elements of blood
o consists of counting chamber or hemocytometer, pipettes & diluting fluid
o microscopic method
o Instruments used in hemocytometry
1. Diluting Fluid
 used mainly to disperse blood cells to facilitate counting of cells
 Characteristic of an ideal diluting fluid:
o ISOTONIC (RBC); HYPOTONIC (WBC)
o Has a high specific gravity
o Easy to prepare
o Good preservative
o Has a buffer action
o Does not initiate growth of molds
o Stable
o Non allergenic
o Non -corrosive
 RBC Diluting Fluids
o Dacie’s Fluid – Formol citrate fluid; BEST DILUTING
FLUID for RBCs; can be kept for a long period of time;
cell morphology not altered
o NSS (0.85%) – for emergency use; ideal to use in case of
excessive rouleax formation & autoagglutination
o 3.8% Na Citrate
o Hayem’s
o Gower’s
o Toissons’s
o Bethel’s
 WBC Diluting Fluids - lyze red blood cells except nucleated red
blood cells
o 2-3% Glacial Acetic Acid
o 1% HCL
o Tuerk’s solution: Glacial acetic acid & Methyl violet
 Platelet Diluting Fluids
o Rees & Ecker d.f. – light microscopy
o 1% Ammonium oxalate – phase contrast microscopy
2. Pipets/Pipettes
 Thoma Pipette

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 o Consist of graduated capillary tube, mixing bulb with glass
bead and aspirating tube
o Parts: stem, bulb, rubber tube

RBC Thoma Pipette

WBC Thoma Pipette

Thoma Pipette RBC WBC

Color of bead RED WHITE
Markings 0.5, 1.0, 101 0.5, 1.0, 11
Volume of the bulb 100 10
Dilution Range 1:100 to 1:1000 1:10 to 1:100
Size of bore Smaller Larger

o Dilution Factor Formula:

Volume∈the bulb(constant )
DF =
Volume of blood used ( variable)

100
Example: RBC= =200
0.5
10
WBC= =200
0.5

o Manual Method Dilution:

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  1:200 (RBC)
 1:20 (WBC)
o Coulter Counter Dilution:
 1:50,000 (RBC)
 1:500 (WBC)
3. Hemocytometer
 an instrument used to count the blood cells
According to TYPE: According to RULINGS
Open type (Spencer, Burker, Thoma
Levy, Levy-Hausser
Closed type (Thoma – Zeiss) Tuerk
Addis Fuchs – Rosenthal
Exton Neubauer
Petroff Improved Neubauer
Bass - Jones

 Open type – Spencer,

Improved Neubauer =
most commonly used
counting chamber
 invented by Louis-
Charles Malassez
 consists of a thick glass
microscope slide.
 Depth = 0.1 mm
 Grids/Ruled Areas:
o RBC use 5
small squares in
the center large square
o WBC use 4 corner large squares
o Unopette for WBC use all 9 large squares
o Platelet use 25 small squares in the center large square

 Neubauer Counting Chamber

o Total Area – 9 mm2
o Depth – 0.1 mm
o Total volume – 0.9 mm3
o Area of the large square – 1
mm2
o Area of the smaller central
square – 0.04 mm2
o Area of the smallest square –
0.0025 mm2
o Dimensions – 3 x 3 x 0.1mm
o also used for counting sperm cells

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 Speirs - Levy Counting Chamber:
o Total Area – 10 mm2
o Depth – 0.2 mm
o Total volume – 2.0 mm3
o Dimensions – 2 x 5 x 0.2mm
o Use: for rapid, direct counts of eosinophil cells ( &
basophils) in peripheral blood for the assay of adrenal
cortical hormone level in clinical experiments and for use
in the administration of ACTH for the treatment of arthritis

 Fuchs – Rosenthal Counting Chamber:

o Total Area – 16 mm2
o Depth – 0.2 mm
o Total volume – 3.2 mm3
o Dimensions – 4 x 4 x
0.2 mm
o Use: gold standard for
quantification of
leukocytes
(eosinophils,
basophils) in cerebrospinal fluid (CSF).

 Rule in Counting in Hemocytometer

o Count cells: Top or left
triple boundary lines
o Do not count cells
touching: Bottom or right
boundary lines

 Procedure
1. Mix blood
2. Aspirate to 0.5 mark
3. Wipe excess blood
4. Suck RBC diluting fluid up to 101 mark
5. Mix blood and diluting fluid
6. Discard first few drops
7. Charge
8. Stand for 3 minutes
9. Count!

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 General Formula
o RBC Count (RBC in millions / cu.mm)
= Number of red blood cells counted X area correction
factor X Depth factor X Dilution
= Number of red blood cell s counted X 5 X 10 X 200
or
= Number of red blood cell s counted X 10,000
 To convert values to SI unit, multiply answer by
0.000001
o SI unit RBC count = X 10 12/ L
 Reference Range
o Male: 4.6 – 6.0 M / cu.mm (5.5 – 6.5)
4.6 – 6.0 x 1012/L
o Female: 4.0 – 5.4 M / cu.mm (4.5 – 5.5)
4.0 – 5.4 x 1012/L
o At birth: 5.0 – 6.5 M/cu.mm
5.0 – 6.5 x 1012/L

 Clinical Significance
Low Value High Values
} Blood loss } High altitude
(Hemorrhage) } Congenital heart
} BM failure disease
} Iron, Folate, Vitamin } Polycythemia vera
deficiencies } Dehydration
} Hemolysis
} Certain cancers
} Anemia
} After age 50
} In recumbency
} After meals (as much
as 10% lower)

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