BLOOD CELLS FORMATION

BLOOD CONSTITUENTS
Suspended in the watery plasma are seven types of cells
and cell fragments.
• 1. red blood cells (RBCs) or erythrocytes
• 2. platelets or thrombocytes
• 3. five kinds of white blood cells (WBCs) or leukocytes
a) Three kinds of granulocytes
- neutrophils
- eosinophils
- basophils
b) Two kinds of leukocytes without granules in
their cytoplasm
- lymphocytes
- monocytes
 MAJOR COMPONENTS OF BLOOD
If one takes a sample of blood, treats it with an agent
to prevent clotting, and spins it in a centrifuge,
• the red cells settle to the bottom
• the white cells settle on top of them forming the
"buffy coat".
• The plama at the top (fraction 55%)
• The fraction occupied by the red cells is called the
hematocrit. Normally it is approximately 45%.
- Values much lower than this are a sign of
anemia.
 GENERAL PROPERTIES OF WHOLE
BLOOD
• Fraction of body weight 8%
• Volume of the adult body
Female: 4 - 5 L;
Male: 5 - 6 L
• Mean temperature 38° C (100.4° F)
• pH 7.35 - 7.45
• Viscosity (relative to water)
Whole blood: 4.5 - 5.5;
plasma: 2.0
• Osmolarity 280 - 300 mOsm/L
 BLOOD VOLUME
A 70 kg man
• Total blood volume 5-6 litres
• If 5 litres
- 1 litre in lungs
- 3 litres in veins of systematic
circulation
- 1 litre in arteries, systemic
circulation and capillaries
 ESTIMATION OF BLOOD
VOLUME
• Formula
V1 x C1 = V2 x C2
V1 volume of injected substance
C1 concentration of injected substance
V2 unknown volume of blood
C2 concentration of injected substance in blood

V2= V1 x C1 = Volume of blood

C2
 PLASMA CONSTITUENCE
Plasma consists of :
• Water

• Dissolved solutes
- Plasma Proteins

• Plasma proteins
- 7% to 9% of the plasma

• Three types of proteins:

- Albumin,
- Globulins
- Fibrinogens
Albumin
- 60% to 80% of plasma proteins
- Smallest in size
- Produced by the liver
- Maintain blood volume and pressure due to osmotic effect;
act as carriers for other substances.
Globulins
- Grouped into three subgroups: alpha, beta and gamma
- Alpha and Beta produced by liver; function in transporting
lipids and fat-soluble vitamins
- Gamma are antibodies produced by lymphocytes and
function in immunity
Fibrinogen
- Accounts for about 4% of Plasma proteins
- Produced by liver
- Important as a clotting factor
- On clotting, fibrinogen is converted into insoluble fibrin.
- Fluid from clotted blood (Serum) is plasma lacking
fibrinogen.
 BLOOD FUNCTIONS
Overall, blood performs the following functions:
1.Transports
• oxygen from the lungs to body tissues
• transports the waste products of cellular metabolism
• nutrients, hormones and enzymes.
2.Regulates
• blood clotting
• body temperature
• acid-base balance
• water and electrolytes
3.Protects against harmful organisms through white cells and
antibodies
 BLOOD DEVELOPMENT
• Formed by the process known as
Haemopoiesis
 HAEMOPOIESIS
• the process of blood cell formation
- the yolk sac;
- mesenchyme and blood vessels
 BLOOD CELLS HIERARCHY
• Stem cells
• Self renewal
- (50% of daughter cells differentiate the remaining do
not differentiate)

• Plasticity
- (reports indicate that transplanted bone marrow cells
can contribute repair & regeneration of tissue types such
as brain, muscle, lung, liver, gut epithelium and skin)

• Progenitor cells (progeny of stem cells)

• Developmentally-restricted cells

• Mature cells
• Mature cell production takes place from the more developmentally-restricted
progenitors
 PROGENITOR CELLS
• Encompasses from immediate progeny of
stem cells to cells committed to one
differentiation lineage

• Progenitor cells become progressively more

restricted in their differentiation and
proliferation capacity
• Late progenitor cells eventually restricted to one
lineage
REGULATION OF HAEMOPOIESIS

Controlled cell
production
Controlled cell
death
 HAEMOPOIETIC GROWTH FACTORS
• Cytokines
• IL 1 (Interleukin 1)
• IL 3
• IL 4
• IL 5
• IL 6
• IL 9
• IL 11
• TGF-β
• SCF (Stem cell factor, also known as kit-ligand)

Cytokines have no (e.g IL-1) or little (SCF) capacity to stimulate

cell proliferation on their own, but are able to synergise with
other cytokines to recruit nine cells into proliferation
 HAEMOPOIETIC GROWTH FACTORS
(TARGETING SPECIFIC PROGINATOR CELLS)
• GM-CSF
• Granulocyte-Macrophage colony stimulating factor
• M-CSF
• Macrophage colony stimulating factor
• Erythropoietin
• Erythropoiesis stimulating hormone
(These factors have the capacity to stimulate the proliferation of their
target progenitor cells when used as a sole source of stimulation)
• Thrombopoietin
• Stimulates megakaryopoiesis
 SITES OF HAEMOPOIESIS
• Fetus
- 0–2 months (yolk sac)
- 2–7 months (liver, spleen)
- 5–9 months (bone marrow)
• Infants
- Bone marrow (practically all bones)
• At puberty:
- Beginning of replacement with areas of fat
(becomes yellow bone marrow)
 SITES OF HAEMOPOIESIS
• Yolk sac

• Liver and spleen

• Bone marrow
– Gradual replacement of active (red) marrow by
inactive (fatty) tissue
– Expansion can occur during increased need for
cell production
BLOOD CELLS PRODUCTION
 HAEMOPOIETIC SITES
• Adult:
• RBM(red bone marrow) is in spongy bone of:
vertebrae; ribs; sternum; cranium;
clavicles;scapulae; pelvis.
• Little or none in long bone
• Total marrow ~ 5% of total body weight
 YELLOW BONE MARROW

• Primarily fat but contains some

concentrated areas of hemopoietic marrow
tissue
• Intergrades between red and yellow
marrow exist –
all degrees
• Normal conditions:
no hemopoietic activity in YBM
 HEMOPOIETIC TISSUES

• Red bone marrow and lymphoid organs

• Sources of Blood elements (two)

• lymphoid (for nongranular leucocytes)

• myeloid (for granulocytes and red

corpuscles)
 ERYTHROCYTES

• The production of red blood cells is referred

to as erythropoiesis.
• Erythroid cell
- Is the first haematopoietic lineage to
arise during vertebrate embryogenesis.
The various cell types in erythrocyte
development are characterised by
1. The gradual appearance of haemoglobin and
disappearance of ribonucleic acid (RNA) in the cell

2. The progressive degeneration of the cell's nucleus

which is eventually extruded from the cell

3. The cell gradual loss of cytoplasmic organelles, for

example mitochondria, gradual reduction in cell size
 STAGES IN ERYTHROPOIESIS:
• Prorubricyte (Basophilic Erythroblast)
- Smaller than hemocytoblast
- Nuclear network slightly coarser
- Cytoplasm deeply basophilic
• Rubricyte (Polychromatophilic Erythroblast)
- Half size of Prorubricyte
- Nucleus checkered (coarse chromatin)
- Cytoplasm losing RNA, gaining Hb
- Color variable: purplish; lilac; gray)
• Metarubricyte (Normoblast)
- Half size of rubricyte
- Nucleus increasingly shrunken/pycnotic
- Mitosis ceases
- Cytoplasmic Hb –strongly acidophilic
• Erythrocyte
- Nucleus lost by extrusion
- Immature forms: delicate RNA network
= Reticulocyte
- Enter sinusoids via growth pressure
 RBC SHAPE
• Is a bag-like
biconcave containing
Hb
• The bag-like shape
allows RBC to take
any shape.
• Biconcave shape
useful for oxygen
diffusion.
• Increased surface
area to trap oxygen
 Erythropoiesis
• Is regulated so that the number of
circulating erythrocytes is maintained
within a narrow range.

• Normally, a little less than l% of the body's

total red blood cells are produced per day
and these replace an equivalent number
that have reached the end of their life span.

• However that still represents a huge

200,000,000,000 cells
 Erythropoiesis
• Is stimulated by hypoxia (lack of oxygen).

• oxygen lack does not act directly on the

haemopoietic tissues but instead stimulates
the production of a hormone, erythropoietin.

• This hormone then stimulates haemopoietic

tissues to produce red cells.
 Erythropoietin
• Is a glycoprotein.

• It is inactivated by the liver and excreted in

the urine.

• It is now established that erythropoietin is

formed within the kidney by the action of a
renal erythropoietic factor (erythrogenin) on
plasma protein (erythropoietinogen).
 Erythrogenin

• Is present in the juxtaglomerular cells of the

kidneys

• Is released into the blood in response to

hypoxia in the renal arterial blood supply.
 The rate of erythropoiesis
• Factors that can affect and influence
erythropoietin production:
- Thyroid hormones,
- Thyroid-stimulating hormone,
- Adrenal cortical steroids,
- Adrenocorticotrophic hormone,
- Human growth hormone (HGH)
• All promote erythropoietin formation and so
enhance red blood cell formation (erythropoiesis).
• In thyroid deficiency and anterior pituitary
deficiency, anaemia may occur due to reduced
erythropoiesis.
 Excess Red Blood Cell Production

• Polycythaemia is often a feature of

increased RBCs production (e.g in
Cushing's syndrome).

• However, very high doses of steroid

hormones seem to inhibit erythropoiesis.
 Male/Female Hormones

• Androgens (male hormones) stimulate and

oestrogens (female hormones) depress the
erythropoietic response.

• In addition to the effects of menstrual blood

loss, this effect may explain why women
tend to have a lower haemoglobin
concentration and red cell count than men.
 Plasma levels of erythropoietin
• Plasma levels of erythropoietin are raised in
hypoxic conditions (low oxygen levels).

• This produces erythrocytosis (increase in the

number of circulating erythrocytes) and the
condition is known as secondary polycythaemia
(e.g. at high altitude and in cardiac failure)
• Physiological polycythaemia occurs in Natives who
live in at high altitude of 14,000 – 17,000 feet.
 Dietary element role in Erythropoiesis
• Protein
Required to make red blood cell proteins and also for the globin part
of haemoglobin
• Vitamin B6
Not clear what the role is but deficieny has occassionally been
associated with anaemia
• Vitamin B12 and folic acid
Needed for DNA synthesis and are essential in the process of red blood
cell formation
• Vitamin C
Required for folate metabolism and also facilitates the absorption of
iron. Extremely low levels of Vitamin C are needed before any problems
occur. Anaemia caused by lack of Vitamin C (scurvy) is now extremely rare
• Iron
Required for the haem part of haemoglobin
• Copper and Cobalt
There is some evidence that these two trace minerals are essential for
the production of red blood cells in other animals but not in humans
 Ineffective Erythropoiesis
• Not all RBCs develop and mature successfully

- Some die in marrow & removed by marrow

macrophages

- The failure to mature results in death in the

marrow

This is known as INEFFECTIVE erythropoiesis &

when RBCs develop & mature successfully is
known as EFFECTIVE erythropoiesis
 Polycythaemia Vera (Erythremia)

• Caused by gene aberrations occuring in

haemoblastic cell line.

• This causes over production of RBCs

including leucocytes.
 Effects of polycythaemia
• Increased blood volume
• Increased haematocrit
• Increased blood viscosity
• Blood flow is sluggish
• Decreased rate of venous return
• All these may have stressive effect on the heart
• Due to sluggish flow of blood, tissues receive less
oxygen.
 ANAEMIA
• Reduction in circulating haemoglobin

• Nutritional deficiency anaemias

– Iron deficiency
– B12 & folate deficiency anaemia
– Protein deficiency anaemia
– Scurvy & other element deficiency
 Anaemia
• Results if there is a reduction in blood
haemoglobin concentration due to a decrease in
the number of circulating erythrocytes and/or in
the amount of haemoglobin they contain.
• Anaemia can also occur when the erythropoietic
tissues cannot supply enough normal erythrocytes
to the circulation.
• Can also occur due to abnormal red cell
production, increased destruction and when
demand exceeds capacity, plasma erythropoietin
levels are increased.
• However, anaemia can also be caused by defective
production of erythropoietin as, for example, in
renal disease.
 ANAEMIA; GLOBIN CHAIN
DEFECTS
• Thalassaemias

– Reduced globin chain synthesis

• Alpha and Beta chain synthesis defects

• Haemoglobinopathies
Abnormal globin chain synthesis

• Blood Loss (Haemorrhage)

 FUNCTIONS OF ERYTHROCYTES

• Transport of respiratory gases

• Large surface area : volume ratio

• Flexible biconcave disc

• Haemoglobin for exchange of gases

• Capable of glycolysis for the source of energy for cell

survival
 WHITE BLOOD CELLS (OR LEUCOCYTES)

• Have nuclei & do not contain hemoglobin

• Typical concentration is 5,000 - 9,000 per
cubic millimeter
• Types of WBCs
– Granular (granulated) white blood cells
include:
• neutrophils (50 - 70% of WBCs)
• eosinophils (1 - 4%)
• basophils (less than 1%)
– agranular (or non-granular) white blood cells
include:
• lymphocytes (25 - 40%)
• monocytes (2 - 8%)
VARIOUS TYPES OF WBCs
 DIFFERENCES

• Granular white blood cells

- contains numerous granules in the
cytoplasm, & their nuclei are lobed.

• Agranular white blood cells

- have few or no granules in the cytoplasm
& have a large spherical nucleus.
 Leucopoiesis
Formation of Leucocytes (white blood cells)
 Location % of total marrow

• Total marrow space in an adult is 4 litres.

• Pelvis 40%
• Vertebra 28%
• Cranium - mandible 13%
• Ribs 8%
• Sternum 2%
• End of long bones 8%
 Leukocytes (WBC)
• Are the mobile units of the body’s immune
system.
• They defend against the invasion of
pathogens.
• They identify cancer cells.
• They remove the body’s litter by
phagocytosis.
• They can leave the circulation and go to the
sites of invasion and tissue damage.
• There are five kinds of leukocytes.
Five kinds of leukocytes fall into two main categories.
•The polymorphonuclear granulocytes are the neutrophils,
eosinophils,
basophils.
Their nucleii are segmented into lobes and have an abundance
of membrane-enclosed granules in the cytoplasm.

•The mononuclear agranulocytes are the

monocytes
lymphocytes
They have a single, large, nonsegmented nucleus and
few granules.
• Monocytes are larger. Lymphocytes are the smallest
leukocytes.
• Their rates change depending on the changing defense needs
of the body.
 Granulocyte
Three types of granulocytes
Neutrophils 54 – 62%
Basophils 1 – 3%
Eosinophils <1%

• They mature from their precursor cells,

Promyelocytes.

• During an infection, the rate of granulocyte

production increases.

• Granulocytes and monocytes are produced only in

the bone marrow.
 MONOCYTES IN THE BONE MARROW
• There is not a large reserve pool of
monocytes in the bone marrow.
• Monocytes are found both in the circulating
and the marginal peripheral blood pools.
• Stimulated by growth factors, monocytes
migrate into tissues and are generally called
macrophages; some fixed in connective
tissue fibers and other sites and some
wandering freely through the connective
tissue.
• Macrophages are most numerous in
"filter" organs
- the spleen,
- liver,
- lungs,
- lymph nodes collectively
known as the mononuclear phagocyte
system, (formerly the reticuloendothelial
system)

A system that serves as an important

body defense mechanism composed of
 SPECIAL NAMES OF MACROPHAGES
• Special names of macrophages are:
• histiocytes in loose connective tissue.
• Kupffer cells in the liver
• osteoclasts in bone (they absorb and remove bone)
• microglial cells in nervous tissue like the brain
• Langerhans’ cells in the epidermis - they recognize
antigens, ingest them and present them to
lymphocytes for eventual destruction
• glomerular mesangial cells in the kidney
• pulmonary alveolar macrophages in the lungs
• macrophages in the spleen and lymph nodes
• monocytes in the blood
 Functions of Monocytes
• Both monocytes and macrophages are phagocytic
like neutrophils.

• Monocyte numbers increase whenever there is

increased cell damage (Cell mobilization &
migration
Substances released from damaged tissue causes
attraction to the bacteria)

• Monocytes are less selective than neutrophils and

will ingest more variable material like old or
abnormal erythrocytes. (recognition of foreign
particles by opsonisation)
• Monocytes store released iron from lysed
erythrocytes.

• They act as antigen presenters in that they process

ingested material and present the antigen on its
(the monocyte’s) surface to a T-helper (CD4+)
lymphocyte.

• Monocytes also produce and respond (growth

factors (IL 1,3,4,6; TNF, GM-CSF(granulocyte,
monocyte colony stimulating factor)and other
cytokines involved in the immune response.
• They have IgG or complement receptors.
 Lymphocytes
• Lymphocytes 25 - 38 %

• Lymphocytes are originally produced from precursor cells

in the bone marrow.

• B cells remain in the bone marrow to mature, while T cells

migrate to and mature in a distinct organ, the thymus.

• Following maturation, the lymphocytes enter the

circulation and peripheral lymphoid organs (e.g the spleen
and lymph nodes) where they survey for invading pathogens
and/or tumour cells.

• Most new ones are produced from lymphocytes in the

- thymus
- Peripheral lymphoid organ( spleen, lymph node,
tonsil etc.)
 Types of Lymphocyte
• The three major types of lymphocyte are
T cells
B cells
Natural killer (NK) cells.
 Functions of Lymphocytes
• The acquired (specific) immune system (vs. non-
specific, innate) is comprised of the humoral and the
cell-mediated immune systems.
• The lymphocytes found in each system are
preprogrammed to respond to a specific antigen.
• The innate or non-specific immune system involves
skin and other mucosal surfaces, inflammatory
response, commensal bacteria, and phagocytosis.
• The acquired (adaptive or specific) immune system
involves two systems:
• humoral: humors in medieval physiology were body
fluids that determined a person’s health and
temperament.
• Humors also were proteins that protected against
disease. B lymphocytes are primarily involved with this
type of immunity.
• cell-mediated. T lymphocytes are responsible for this
type of immunity.
 B-lymphocytes
• Are responsible for the humoral response.
• Upon antigenic encounter they transform
into plasma cells that produce antibodies
specific to the antigen.
• Production of antibodies is a complex
process.
• Memory B cells are also produced.
• These memory cells respond more quickly
than the initial response to a future
encounter with the same antigen.
• Each activated B cell gives rise to many
progeny which together are called a clone of
cells because they are all identical.
 T-lymphocytes
• Are responsible for the cell-mediated response.

• After antigenic stimulation they proliferate and

differentiate into memory cells and effector cells.

• There are at least two types of effector cells: helper T-cells

(CD4+) and cytotoxic T-cells (CD8+). CD = cluster
differentiation markers. They are identifying membrane
proteins (antigens).

• It is the CD4+ lymphocytes to which HIV has an affinity.

• The normal ratio of CD4+:CD8+ is 2:1. In HIV-infected

individuals the ratio decreases and reverses as the AIDS
virus invades and destroys the CD4+ cells.

• The ratio is used to monitor the progress of the disease.

 T-helper cells
• T-helper cells "help" the B-cells produce antibodies by
releasing cytokines.
• They also affect the action of other cells, such as CD8+.
• CD8+ cells (cytotoxic T-cells) mediate destruction of
cells infected with pathogens such as viruses, bacterial,
protozoa, or fungi; and are also responsible for rejection
in organ transplants.
• T-helper cells help B-cells produce antibodies and also
influence other T-helpers, T-suppressors and cytotoxic
T-cells.
(T suppressors help keep the immune system in control).
 Typical recognition markers for lymphocytes[3]
LYMPH
PROPORTI PHENOTYPIC
OCYTE FUNCTION OF LYMPHOCYTE
ON MARKER(S)
CLASS

Lysis of virally infected cells and

NK cells 7% (2-13%) CD16 CD56 but not CD3
tumour cells

Release cytokines and growth factors 46% (28-

Helper T c TCRαβ, CD3 and CD4
ells that regulate other immune cells 59%)

Cytotoxic Lysis of virally infected cells, tumour 19% (13-

TCRαβ, CD3 and CD8
T cells cells and allografts 32%)

γδ T cells Immunoregulation and cytotoxicity TCRγδ and CD3

23% (18- MHC class II, CD19 and

B cells Secretion of antibodies
47%) CD21
 Lymphocyte Functions
• T cells and B-cells are the major cellular components of the
adaptive immune response.

• T cells are involved in cell-mediated immunity whereas B cells

are primarily responsible for humoral immunity (relating to
antibodies).

• The function of T cells and B cells is to recognize specific

“non-self” antigens, during a process known as
antigen presentation.

• Once they have identified an invader, the cells generate

specific responses that are tailored to maximally eliminate
specific pathogens or pathogen infected cells.

• B cells respond to pathogens by producing large quantities of

antibodies which then neutralize foreign objects like bacteria
and viruses.
• In response to pathogens some T cells, called
helper T cells produce cytokines that direct the immune
response.

• T cells, called cytotoxic T cells, produce toxic granules

that induce the death of pathogen infected cells.

• Following activation, B cells and T cells leave a lasting

legacy of the antigens they have encountered, in the
form of memory cells.

• Throughout the lifetime of an animal these memory cells

will “remember” each specific pathogen encountered,
and are able to mount a strong response if the pathogen
 NEUTROPHILS
• The primary function of neutrophils is
phagocytosis.
Phagocytosis:
• It is the process by which cells engulf and
disable particles
• Phagocytosis routinely takes place in the:
- respiratory system
- gastrointestinal system
- urinary system
 Toxic Change
• Toxic change:
• Appearance of neutrophils when they
are active against severe bacterial
infections includes
- toxic granulation,
- vacuolization of the cytoplasm,
- Dohle bodies and left shift.
• This is a typical reaction characterized by
an increase in digestive enzymes and
digestive vacuoles with an increased RNA
activity.
 STEPS OF PHAGOCYTOSIS
• Opsonization (Greek = to prepare for
dining)

• Neutrophils cannot efficiently recognize and

attach to most microbes;

• This process marks the organism for ingestion by

coating the particle with immunoglobulin
(antibody) and complement (a series of proteins
that cause disruption of the bacterial membrane).

• Neutrophil membrane pseudopods envelop the

microbe forming a vacuole called a phagosome
within the cytoplasm.
• The phagosome fuses with lysosomal granules
from the neutrophil’s cytoplasm. The granules
release lytic enzymes into the phagosome.
• The lytic enzymes lead to eventual killing and
digestion of the foreign agent.
• All these processes require energy that is derived
by anaerobic glycolysis (glucose breakdown).
• Myeloperoxidase, one of the enzymes in the
primary granules, catalyzes a reaction involving
H2O2 resulting in a more toxic product.
• Myeloperoxidase deficiency is reported to be the
most common congenital neutrophil disorders.
• However, the condition is fairly benign except for
patients with other problems, like diabetes where
fungal infections may occur.

• One of the products formed in the digestion of the

foreign particle is hydrogen peroxide, which is
capable of killing microorganisms
 BASOPHILS
Function
• Basophils play a role in acute allergic reactions.
• Their granules contain
- histamine,
- heparin
- and other substances that are released in response to
the presence of allergens.
These substances cause
increased vascular permeability,
- smooth muscle spasm,
- vasodilation,
- and the clinical symptoms of an allergic reaction:-
watery eyes
runny nose
and difficult breathing.
• Histamine is a vasodilator that makes
blood vessels more permeable.
• This effect is usually seen at
inflammatory sites and allows
increased cellular movement through
the vessel walls.
• Heparin prevents blood clotting.
• Both histamine and heparin enhance
the migration of leukocytes to the
inflamed site.
 EOSINOPHIL
• Eosinophil granules contain
- enzymes
- cytotoxic proteins
- cytokine mediators.
• Eosinophils are found in high numbers in
- intestinal mucosae
- pulmonary mucosae
- the dermis of the skin
• They increase in number when the body is invaded
by
- some parasites
- and during allergy attacks.
 Function of Platelets

• Stop bleeding from a damaged vessel

* Hemostasis

• Three Steps involved in Hemostasis

1. Vascular Spasm
2. Formation of a platelet plug
3. Blood coagulation (clotting)
 Steps in Hemostasis
*DAMAGE TO BLOOD VESSEL LEADS
TO:

1. Vascular Spasm:
• Immediate constriction of blood vessel

• Vessel walls pressed together – become

“sticky”/adherent to each other

• Minimize blood loss

 Steps in Hemostasis

2. Platelet Plug formation:

a. PLATELETS attach to exposed collagen
b. Aggregation of platelets causes release of
chemical mediators (ADP, Thromboxane A2)
c. ADP attracts more platelets
d. Thromboxane A2 (powerful vasoconstrictor)
* promotes aggregation & more ADP

Leads to formation of platelet plug !

(+) Feedback promotes formation of platelet Plug
 Final Step in Hemostasis

3. Blood Coagulation (clot formation):

“Clotting Cascade”
a. Transformation of blood from liquid to solid
b. Clot reinforces the plug
c. Multiple cascade steps in clot formation
d. Fibrinogen (plasma protein)
Fibrin

Thrombin
 Thrombin in Hemostasis

Factor X
 Clotting Cascade

• Participation of 12 different clotting

factors (plasma glycoproteins)
• Factors are designated by a roman
numeral
• Cascade of proteolytic reactions
• Intrinsic pathway / Extrinsic pathway
• Common Pathway leading to the
formation of a fibrin clot !
 Hageman factor (XII)

X
inactive

active

CLOT !
 Clotting Cascade

• Intrinsic Pathway:
– Stops bleeding within (internal) a cut vessel
– Foreign Substance (ie: in contact with test
tube)
– Factor XII (Hageman Factor)
• Extrinsic pathway:
– Clots blood that has escaped into tissues
– Requires tissue factors external to blood
– Factor III (Tissue Thromboplastin)
 Clotting Cascade

• Fibrin :
– Threadlike molecule-forms the meshwork of the
clot

– Entraps cellular elements of the blood forms CLOT

– Contraction of platelets pulls the damaged vessel

close together:
• Fluid squeezes out as the clot contracts (Serum)
 Clot dissolution

• Clot is slowly dissolved by the “fibrin splitting”

enzyme called Plasmin
• Plasminogen is the inactive pre-cursor that is
activated by Factor XII (Hageman Factor)
(simultaneous to clot formation)
• Plasmin gets trapped in clot and slowly
dissolves it by breaking down the fibrin
meshwork
 Clot formation:
Too much or too little of a good
thing…
• Too much:
– Inappropriate clot formation is a thrombus (free-
floating clots are emboli)
– An enlarging thrombus narrows and can occlude
vessels
• Too little:
– Hemophilia- too little clotting- can lead to life-
threatening hemorrhage (caused from lack of one
of the clotting factors)
– Thrombocyte deficiency (low platelets) can also
lead to diffuse hemorrhages
Haemoglobin synthesis &
catabolism
 Haemoglobin synthesis

The haemoglobins are red globular

proteins, which have a molecular weight of
about 68,000 and comprise almost one
third of the weight of a red cell.
The haemoglobin is composed of haem
and globin.
 Haemoglobin synthesis
• The main function of red cells is to carry
O2 to the tissues and to return carbon
dioxide (CO2) from tissues to the lungs.
• In order to achieve this gaseous exchange
the red cells contain the specialized
protein haemoglobin.
• Each red cell contains approximately 640
million Hb molecules.
 Haemoglobin synthesis
• 65% of the Hb is synthesized in the
erythroblasts, and 35% at the reticulocyte stage.
• Haem synthesis occurs largely in the
mitochondria.
• Globin synthesis occurs in the polyribosomes.
• Although haem and globin synthesis occur
separately within developing red cell precursors,
their rates of synthesis are carefully coordinated
to ensure optimal efficiency of Hb assembly.
 Globin synthesis
• The various globins that combine with haem to
form Hb are all single chain polypeptides.
• The synthesis of these globins is under genetic
control.
• Humans normally carry eight functional globin
chains, arranged in two, duplicated gene
clusters: the -like cluster (, ,  and  globin
genes) on the short arm of chromosome 11 and
the -like cluster ( and  globin genes) on the
short arm of chromosome 16.
 Ontogeny of globin synthesis
Globin synthesis is first detected in the primitive
erythroid precursors of the yolk sac at about 3
weeks’ gestation.
• Embyonic :
Haemoglobin Gower I ( 22)
Haemoglobin Portland ( 22)
Haemoglobin Gower II (2)
• Fetal : HbF (22), HbA (22)
• Adult : HbA, HbA2 ( 22), HbF.
Haemoglobin
• Each molecule of
normal adult
haemoglobin (Hb-A)
consists of four
polypeptide chains
, each with its
own haem group.
 Haemoglobin
• Normal adult blood also contains small
quantities of two other haemoglobins, Hb-
F and Hb-A2. These also contain chains
but with  and  chains respectively
instead of .
• The major switch from fetal to adult
haemoglobin occurs 3-6 months after
birth.
 Normal Hb in adult blood
Hb A Hb A2 Hb F

structure 22 22 22

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

 Iron Metabolism
Transport of iron
• Iron obtained from diet is absorbed from the
small intestine
• It combines with a -globulin (apotransferrin)
to form transferrin.
• Iron loosely combine with the globulin
molecule and can be released at any point in
the body
• Excess iron is stored as ferritin (storage iron)
• Small quantities of iron can also be stores as
insoluble iron (hemosiderin)
 Importance of Iron
• Iron is critical for a number of synthetic &
enzymatic processes

• Most of body iron is part of the Hb

• Iron is recycled and thus conserved by the

body

• Daily intake is about 1mg.

 Iron in RBCs
• Average blood volume of a physiological person
(70kg) is about 5 litres.

• Thus 150g of Hb / L of blood (normal 15g/Decl.)

• Therefore there 750g of Hb in the body

• Each gm of Hb contains appr. 3.3 mg of iron

• In the blood there is 3.3 x750 = 2475 mg (2.5g) of

iron.
 Transferrin Receptors
• Most cells have transferrin receptors (CD71)
to which Iron is ladden transferrin binds.

• The receptor-transferrin-iron complex is

imported into the cell cytosol by endocytosis

• Endocytotic vacuole fuses with a lysosome at

acid PH and Iron is released from transferrin
and taken up to mitochondria where it is
incorporated into haem( Fe++ +
protoporphyrin ix)
 Body Iron
• Haemoglobin = 2200 mg

• Ferritin & Hemosiderin = 1000 mg

• Myoglobin = 300 mg

• Other Fe (cytochromes; Enzymes = 100 mg

• Total = 3600 mg
Haemoglobin synthesis
• Haem synthesis starts
with the condensation of
glycine and succinyl
coenzyme A under the
action of a rate limiting
enzyme -aminolaevulinic
acid synthase.
 -ALA will be formed.
• Pyridoxal phosphate (vit.
B6) is a coenzyme for this
reaction.
Haemoglobin synthesis
• A series of biochemical
reactions will follow.
• Two molecules of -ALA
condense to form a
pyrrole called
porphobilinogen (PBG)
• Four PBG condense to
form a tetrapyrrole
uroporphyrinogen III.
• UPG III is then converted
to coproporphyrinogen.
Haemoglobin synthesis
• CPG then changes to
protoporphyrin which
ultimately combines with
iron in the ferrous state
(Fe2+) to form haem.
• Iron is brought to the
developing red cells by a
carrier protein
( transferrin) which
attaches to special
binding sites on the
surface of these cells.
• Transferrin releases iron
and returns back to
circulation.
Haemoglobin synthesis
• Each molecule of
haem combines with
a globin chain.
• A tetramer of four
globin chains each
with its own haem
group in a pocket is
formed to make up a
haemoglobin
molecule.
Haemoglobin structure
• Haem consists of a
protoporphyrin ring with
an iron atom at its centre.
• The protoporphyrin ring
consists of four pyrrole
groups which are united
by methane bridges
(=C-).
• The hydrogen atoms in
the pyrrole groups are
replaced by four
methylene (CH3-), two
vinyl (-C=CH2) and two
propionic acid (-CH2-
CH2-COOH) groups.
Hb Structure & Function
 Function
• Hb is a large protein (66.7 kD) coupled to four porphyrins or heme
moities.

• The globin portion of Hb consists of:

- 4 polypeptide chains ( a with 141aa and ß with
146aa)arranged in pairs forming a tetramer.

• Each globin chain is covalently attached to a heme moiety.

• The bonds between a and ß chains are weaker than between similar
globin chains, forming a natural cleavage plane, the a1ß2 interface,
important for oxygen binding and release.
- When this cleavage is open (relaxed state) oxygen can bind
(high oxygen affinity).
- When the two a1ß2 interfaces are closely bound (taut state)
the Hb molecule has a low affinity for oxygen.

• The binding of oxygen rotates the globin chains, moving the ß chains
together and sliding the a1ß2 interfaces apart (the relaxed state) thus
increasing the oxygen affinity of Hb.
Dissociation Curve
Oxygen Tension
 Oxygen Tension
• Hemoglobin must bind O2 at high O2 tension
and release it at low O2 tension.
• With deoxygenation the a1ß2 interface tightens
lessening the affinity of Hb for oxygen. This
conformation is stabilized by proton binding and
2,3-DPG.
• Decreasing pH strengthens the a1ß2 interface,
stabilizing the low-affinity conformation and
releasing O2 . This is the Bohr effect.
• 2,3-DPG binds to hemoglobin, forming a link at
the a1ß2 interface. This results in a stable low
affinity conformation promoting the release of
O2 . 2,3-DPG also lowers the pH.
Carbon dioxide Tension
 Carbon dioxide Tension
• Where the O2 tension is low, as in the
venous blood:
- the Bohr effect and 2,3-DPG combine
to reduce the affinity of Hb for O2 ,
releasing O2 to tissues.

• CO2 then is loaded

 Factors that Affect the Standard
Dissociation Curve
• The effectiveness of hemoglobin-oxygen binding
can be affected by several factors
• The factors can be viewed as having the effect of
shifting or reshaping the oxyhaemoglobin curve.
• Variation of the hydrogen ion concentration.
• This changes the blood's pH. A decrease in pH
shifts the standard curve to the right, while an
increase shifts it to the left. This is known as the
Bohr effect.
• Effects of carbon dioxide.
• Carbon dioxide affects the curve in two ways: first, it influences
intracellular pH (the Bohr effect), and second, CO2 accumulation
causes carbamino compounds to be generated through chemical
interactions. Low levels of carbamino compounds have the effect
of shifting the curve to the right, while higher levels cause a
leftward shift.
• Effects of 2,3-DPG. 2,3-diphosphoglycerate, or 2,3-DPG, is an
organophosphate, which are created in erythrocytes during
glycolysis.
• The production of 2,3-DPG is likely an important adaptive
mechanism, because the production increases for several
conditions in the presence of diminished peripheral tissue O2
availability, such as hypoxemia, chronic lung disease, anemia, and
congestive heart failure, among others.
• High levels of 2,3-DPG shift the curve to the right, while low levels
of 2,3-DPG cause a leftward shift, seen in states such as septic
shock and hypophosphatemia.
• are low, and hence the leftward shift enhances the placental
uptake of oxygen.
• Temperature.
- Temperature does not have so dramatic effect as the
previous factors, but hyperthermia causes a rightward
shift, while hypothermia causes a leftward shift.
• Carbon Monoxide.
Hemoglobin binds with carbon monoxide 240 times
more readily than with oxygen, and therefore the presence of
carbon monoxide can interfere with the hemoglobin's
acquisition of oxygen.
In addition to lowering the potential for haemoglobin
to bind to oxygen, carbon monoxide also has the effect of
shifting the curve to the left.
With an increased level of carbon monoxide, a person
can suffer from severe hypoxemia while maintaining a
normal PO2.
• Effects of Methemoglobinemia
- Methemoglobinemia causes a leftward shift
in the curve.
• Fetal Hemoglobin.
- Foetal hemoglobin (HbF) is structurally
different from normal haemoglobin (Hb).

-The fetal dissociation curve is shifted to the

left relative to the curve for the normal adult.


-Typically, foetal arterial oxygen pressures
are low, and hence the leftward shift enhances
the placental uptake of oxygen.
 Haemoglobin catabolism
*normal red cell destruction*
• Red cell destruction usually occurs after a mean
life span of 120 days.
• The cells are removed extravascularly by
macrophages of the reticuloendothelial system
(RES), specially in the bone marrow but also in
the liver and spleen.
• Red cell metabolism gradually deteriorates as
enzymes are degraded and not replaced, until the
cells become non viable, but the exact reason why
the red cells die is obscure.
 Haemoglobin catabolism
*normal red cell destruction*
• The breakdown of red cells liberates

1- iron for recirculation via plasma transferrin to

marrow erythroblasts

2- protoporphyrin which is broken down to

bilirubin.

3- globins which are converted to amino acids.

 Normal red cell destruction
- The bilirubin circulates to the liver where it is
conjugated to glucuronides which are excreted
into the gut via bile and converted to
stercobilinogen and stercobilin(excreted in
faeces).

- Stercobilinogen and stercobilin are partly

reabsorbed and excreted in urine as urobilinogen
and urobilin.
 Normal red cell destruction

• A small fraction of protoporphyrin is converted

to carbon monoxide (CO) and excreted via the
lungs.

• Globin chains are broken down to amino acids

which are reutilized for general protein
synthesis in the body.
 Normal red cell breakdown
haemoglobin

haem globin
iron protoporphyrin
Amino acids

CO Bilirubin
transferrin Expired air (free)
Liver
conjugation
erythroblast
Bilirubin glucuronides

Urobilin(ogen) Stercobilin(ogen)

Urine faeces
 Haemoglobin abnormalities
There are mainly two types of abnormalities,
these are :
• Quantitative abnormalities: where there is
reduction in the production of certain types
of globins e.g. a thalassaemia
b thalassaemia
• Qualitative abnormalities: where there is
production of abnormal haemoglobin e.g.
sickle cell anaemia.
BLOOD TYPES
 BLOOD GROUPS
• Blood groups discovered in 1900 and
1901 at the University of Vienna by Karl
Landsteiner:

- In the process of trying to learn why

blood transfusions sometimes cause
death and at other times save a
patient.

• In 1930, he belatedly received the Nobel

Prize for this discovery.
 HUMAN BLOOD GROUP SYSTEMS
The International Society of Blood Transfusion (ISBT) currently
recognises 29 major blood group systems (including the ABO
and Rh systems).

• Thus, in addition to the ABO antigens and Rhesus antigens,

many other antigens are expressed on the red blood cell surface
membrane.

• For example, an individual can be AB RhD positive,

-and at the same time M and N positive (MNS system), K
positive (Kell system) and Lea or Leb positive (Lewis system).

• Many of the blood group systems were named after the

patients in whom the corresponding antibodies were initially
encountered.

• The ISBT definition of a major blood group system is where

one or more antigens are "controlled at a single gene locus

- or by two or more very closely linked homologous genes with

little or no observable recombination between them".
 THE 29 BLOOD GROUPS (OCTOBER 2006)
1ABO
2MNS
3P
4Rhesus
5Lutheran
6Kell
7Lewis
8Duffy
9Kidd
10Diego
11Yt or Cartwright
12XG
13Scianna
14Dombrock
15Colton
16Landsteiner-Wiener
17Chido/Rodgers
18Hh
19Kx
20Gerbich
21Cromer
22Knops
23Indian
24Ok
25Raph
26JMH
27Ii
28Globoside
29GIL
 COMMON BLOOD GROUPS

• ABO SYSTEM
• RHESUS SYSTEM
 ABO & Rh BLOOD GROUPS
• ABO and Rh

• Antigens (glycoproteins and glycolipids)

called agglutinogens on surface of RBC’S
determine blood types.

• Blood types are inherited

• The greatest concern with blood types

involves transfussion
• During a transfusion, the person giving
the blood is a DONOR the person
getting the blood is a RECIPIENT

• The immune system may develop

antibodies called AGGLUTININS
against certain antigens. These
antibodies are in the plasma.
UNIVERSAL DONOR & RECIPIENT
Plasma
compatibility
 INCOMPATIBILITY
• If RBC’s of a donor are incompatible with
the blood of a recipient, antibodies in the
plasma of the recipient will bind to the
antigens of the donated RBC’s.
• This reaction will clump (agglutinate) and
destroy (cause hemolysis ) the donated
RBC’s, resulting in a serious and possibly
fatal reaction.
 ABO BLOOD TYPES

ABO Antige Antibodies in Plasma

Type n on
RBC’s

A A Anti-B

B B Anti-A

AB A&B Neither

O Neither Both
ABO Blood Types
ABO Antigen Antibodies Acceptable
Type on in Transfusion
RBC’s Plasma

Donate to AB
A A Anti-B
Receive from O
Donate to AB
B B Anti-A Receive from O
Donate to AB
AB A&B Neither Receive from AB
Donate to O
O Neither Both Receive from O
 RH BLOOD TYPE AND HEMOLYTIC
DISEASE OF THE NEWBORN

• Rh blood type is determined by presence of

absence of Rh agglutinogen (antigens) on
the surface of RBC’s

• If Rh agglutinogens are present, the type is

Rh +

• If no agglutinogens are present, the type is

Rh -
•People with Rh- type lack anti-Rh
agglutinins (antibodies), but if they receive
Rh+ blood, their immune systems will be
stimulated to produce them, and they are
then sensitized

•Future exposure to Rh+ blood will cause a

dangerous blood reaction.
 RHESUS BLOOD GROUP
Rhesus blood group is simpler than ABO, having only two groups:
- positive and negative.

1. Rhesus-negative blood can be given to anyone,

2. Rhesus-positive blood can only be given to other rhesus-positive

people.

3. Unlike the ABO system, antibodies to rhesus antigens do not develop

until the first time a rhesus-negative person is exposed.

4. Any subsequent exposure to rhesus antigens leads to severe side effects.

5. The rhesus blood group is actually quite complicated,

- consists of three different genes which produce the positive and
negative groups.
6. Around 85% of the UK population are rhesus-positive.
- values vary around the world.
- People of Afro-Caribbean origin have only a 5% chance of being
rhesus-negative,
- Rhesus-negative individuals are almost unknown in Chinese
populations
 HEMOLYTIC DISEASE OF THE
NEWBORN (HDN)
• Results from Rh incompatibility between
Rh- mother and her Rh+ child

• If Rh+ RBC’s of first born child enter

mother’s circulation, mother will be
sensitised, and her plasma will carry
anti-Rh agglutinins (antibodies)
 FIRST CHILD
•The first child is not harmed

•But the mother’s agglutinins, acquired by

exposure to the first child’s blood, easily pass
across the placenta where they agglutinate and
destroy the second child’s RBC’s.

How is it prevented?
HEMOLYTIC DISEASE OF THE NEWBORN