BLOOD

Blood is a fluid connective tissue, which circulates in the vascular channels to all
tissues of the body. It constitutes about 6 to 7% of the body weight. It is also a
complex fluid, where, in the aqueous medium, namely plasma, the three types of calls
are suspended. The three types of cells are, erythrocytes, leucocytes and
thrombocytes, leucocytes and thrombocytes.
Blood performs various functions, which include:
 Transport of respiratory gases.
 Transport of nutrients and other substances like hormones, metals, drugs etc.
 Transport of waste products to the excretory organs.
 Protection against infections.
 Maintenance of body temperature by distributing heat.
 Maintenance of acid base balance with the help of buffers.
The blood is slightly alkaline and its pH is 7.40 + 0.02. The specific gravity of blood
ranges from 1055 to 1060 and it is due to the solute concentration, especially the
proteins. The viscosity of blood is primarily related to the red cell volume and to a
lesser extent to the plasma proteins.

PLASMA
Plasma is the fluid part in the blood and it constitutes 55% by volume. It contains
93% water and 8% solids. The solids contain inorganic and organic substances.
Organic constituents form the bulk of solids present in plasma. It includes proteins,
glucose, cholesterol, lipids, nonprotien nitrogenous substances (NPN), hormones,
enzymes, etc.
Plasma proteins
Plasma proteins are albumin, globulin and fibrinogen.

Serum
It is obtained by allowing the blood to clot. The fluid that separates out from the clot
is known as serum. The plasma without fibrinogen is serum.
Separation of plasma proteins : Plasma proteins are separated by salting out process
by mixing with full or half saturation of ammonium sulphate. The method of choice
now is electrophoresis, in which, the proteins, depending on their size separate on a
strip of paper, when electric current is passed through the electrolyte solution.

The total protein level in plasma is 6 to 7 gm%

Albumin 4 to 5 gm%
Globumin 2 to 3 gm%
Fibrinogen 0.25 to 0.3 gm%

Globulin is further subdivided into alpha, beta, gama with 1 and 2 subfractions in each
category.

Albumin is the major plasma protein and its molecular weight is 68,000. Globulin has
many sub fractions and includes prothrombin, which is an important clotting factor.
Globulin has a molecular weight ranging form 90,000 to 1,55,000. Fibrinogen is the
least in concentration, but its molecular weight is the highest, which is 3,40,000.

Functions of plasma proteins

 Maintains colloidal osmotic pressure : Albumin is responsible for creating this
pressure in plasma and it helps to oppose the filtration pressure. Normal
colloidal osmotic pressure is 25 mmHg. Any decrease in this value, as seen in
conditions like, malnutrition, excessive protein loss as in renal disease and
reduced formation in liver disease, will lead to fluid loss from the capillaries
and result in edema.

 Transport of substances ; Albumin transports hormones such as thyroxine,

dyes, enzymes, bile pigment, fatty acids Globulin transports metals like iron
(transfeein), copper (ceruloplasmin) and hormones such as, thyroxine (TBG)
and cortisl (transcortin)

 Coagulation of blood : Fibrinogen, prothrombin and various other clotting

factors help in the coagulation of blood.
  Antibody formation :Gamma globulins are immunoglobulins, which are
produced against antigens. Immunoglobulins are of five types namely IgA,
IgD, IgG, IgE and IgM.

 Erythrocyte sedimentation rate: Fibrinogen is the main plasma protein, which

influences ESR by making red blood cells to form rouleaux. The next protein,
which influences ESR is globulin.

 Buffering action: Plasma proteins are anions and exist in ionic form. This
gives buffering capability for the plasma protein, which helps to maintain the
pH of blood.

 Protein reserve: Plasma proteins act as protein reserve, which can be used by
the tissues during starvation.

Formation of plasma proteins

Albumin, alpha, beta globulins and fibrinogen are produced in the liver, while gamma
globulins are produced from the lymphoid tissue.

ERYTHROCYTES

Erythrocytes are biconcave, non-nucleated and disc shaped, with cytoplasm

containing a red colour pigment haemoglobin. The cells have a mean diameter 7.5
and thickness 2 . The thickness is less at the center, which is 1.0. In venous blood,
they are slightly larger by about 0.5 , due to the osmotic changes. The cell
membrane has a bimolecular lipid structure and the lipoprotein complex on the
membrane contains, A, B and Rh antigens.

Metabolism in RBC
The cytoplasm has no mitochondria and hence no oxidative phosphorylation occurs in
the red cell. There is anaerobic glycolysis leading to Embden Meyerhof pathway of
glucose metabolism, which generates ATP and NADH. There is also formation of 2,3
DPG. To a little extent, there is also pentose phosphate pathway for glucose
oxidation. Red cells, even through non nucleated, are considered as living cells, since
they carry out the above described metabolic reactions. Red cell cytoplasm contains
more potassium and also rich in carbonic anhydrase enzyme. The membrane has an
active sodium pump mechanism to maintain a low level of its concentration inside the
cell. This prevents any change in its size, due to osmotic changes.
The average surface area of a red cell is 120 2 and the mean volume is 90 3. The
biconcave shape of the cell gives a greater surface area / volume ratio and this is
advantageous to the cell to increase its volume without rupture, if there is an osmotic
change. Another advantage is, that, there is a maximum surface area available per
unit mass of haemoglobin for gas transport. The quantity of haemoglobin, in a single
cell is about 33% of wet weight of the cell.
Red cells perform important functions such as, transport of respiratory gases, and
maintenance pH of blood. Normal count of red cell in adult male ranges from 5 to 5.5
million per cubic millimeter of blood. In females, it is slightly lower due to the low
basal metabolism and the range is 4 to 4.5 million cells per cubic millimeter of blood.
In the new born, the count is more than adults. It ranges from 6.5 to 7 million per
cubic millimeter of blood. The volume of red cells in 100ml of blood, forms the
haematocrit (Hct). Its value in males is 47% and in females it is 43%.

Life span of RBC

Average life period of a red cell is 120 days and it can be determined, by tagging red
cells to radio active isotopes P32 or Fe55 and reintroducing them into the circulation.
The radio activity count is measured until 0 value. The number of days that takes to
get 0 count gives the life period of red cell. The senile red cells are destroyed in the
reticuloendothelial system (tissue macrophages) in spleen, liver and bone marrow.
Physiologically red cell count increases in the following situations high attitude,
muscular exercise, adrenaline secretion.
Polycythemia :The rise in RBC count is called polycythemia. Accompanying the rise
in red cell count, thee is also increase in Hb%. If the abnormal increase in the count is
due to the tumor of the red bone marrow, it will be known as polycythemia vera and
described under primary polycythemia. In this type, the erythropoietin level will be
normal.
RBC number also increases in high altitude, pulmonary disorders, renal diseases and
congenital cardiac defects. In all these conditions, there is presence of hypoxia, which
stimulates the secretion of erythropoietin. The rise in RBC in such conditions is
called secondary polycythemia. Polycythemia can also result, due to the contraction
of plasma volume and it is described as relative polycythemia.

Anaemia
Reduction in red cell count occurs, in bone marrow suppression, increased destruction
of red cells and nutritional deficiencies. The condition is known as anaemia, which
causes reduced supply of oxygen to the tissues. Anaemia could result from a decrease
in haemoglobin concentration, or reduced red cell count, or both.

Erythropoiesis
During embryonic life, erythrocytes are formed from yolk sac. In early foetal life,
liver and spleen take over the function of producing blood cells and the bone marrow
joins the list of forming blood cells in the later months of foetal life. From birth until
adulthood, the entire bone marrow is involved in blood cell production. From
adulthood onwards, only the red bone marrow is capable of producing blood cells. In
the adult, the shaft of long bones is occupied by the yellow fatty tissues. Red bone
marrow at the ends of long bones, flat bones like skull, membranous bones like
sternum, vertebrae, pelvis, and ribs are active sites of blood cell production in adult.
Although, liver and spleen have lost the function of producing blood cells in adult,
they are capable of forming cells when there is prolonged stimuli (extra medullary
haemopoiesis).

Stem cell
The stem cell in the red bone marrow is uncommitted and pleuripotent. That is, it can
give rise to erythroid or myeloid series. About 75% of bone marrow cells belong to
myeloid series and the remaining 25% only is the erythroid series. These stem cells
when they divide to give rise to committed stem cells, part of the new cells that are
formed, become uncommitted multipotent, so that their number remains constant.
More white cells producing stem cells are present, since they have a short life period.
The stem cell, which enters the erythroid series, becomes unipotent, committed stem
cell, being facilitated by interleukins 1, 3 and 5 and granulocyte macrophage-colony
stimulating factor (GM-CSF). The proliferation of further stages of red cell
formation, depends on growth factors, colony stimulating factors (CSF), and the
hormone erythropoietin. The stages of erythropoiesis are described below.

Proerythroblast
The precursor cell is large up to 20 microns size, nucleus is single and large,
nucleolous is present. The cell shows active proliferation.

Early normoblast
The cell size is reduced. The nucleus condenses and chromatin threads appear.
Nucleoli disappear and the cell continues to show active proliferation.

Intermediate normoblast
Nucleus further condenses. The cell size is also reduced. The cytoplasm shows the
synthesis of haemoglobin and hence it takes both acidic and basic stains
(polychromatophilic). The cell mitosis stops in this stage.

Late normoblast
There is reduction in the cell size. Nucleus is pushed to the periphery and at the end
of this stage, it is extruded out, by a process called pyknosis. The cytoplasm shows
greater synthesis of haemoglobin.

Reticulocyte
After the extrusion of nucleus, the reticulocyte is formed and released into the
circulation. The cell size becomes 7.2. The cytoplasm has a fine reticulum, which
are the nuclear remnants of RNA. Hemoglobin synthesis is completed and within 24
to 48 hours, they mature into red cells in the circulation. Normal count of reticulocyte
is 1% of red cell count. Reticulocytosis is the increase in reticulocyte count, which is
seen during accelerated erythropoiesis from bone marrow stimulation. Treatment of
pernicious anaemia, by vit B12 extract, shows the feature of reticulocytosis.
Erythrocyte
Both reticulocytes and erythrocytes are present in the peripheral circulation. The red
cell, which is formed from reticulocyte, exhibits its normal morphology.

Regulation of erythropoiesis
Erythropoiesis is regulated by several factors and the important regulator is the red
cell number. There are a number of erythrocytes destroyed every day and equal
number of cells is produced by the red bone marrow, so that, the total red cell number
remains constant. To keep the red bone marrow in a ready state of erythropoietic
activity, there should be availability of various factors, which are grouped as
erythropoietic factors.

 Hypoxia: Oxygen lack at the tissue level is the potent stimulus for
erythopoiesis and it is brought about by the hormone erythropoietin, secreted
from kidney. In high altitude, erythropoiesis is stimulated by hypoxia.
 Erythropoietin: It is a glycoprotein hormone synthesized from kidney. The
secretion of the hormone also occurs from liver. The stimulus for secretion is
hypoxia and reduced red cell count. The reduced cell count itself gives
hypoxia, which stimulates the hormone secretion. Erythropoietin acts on the
bone marrow and causes proliferation of erythroid marrow and causes
proliferation of erythroid precursor cells. It facilitates the formation of
unipotent, committed stem cell from the totipotent uncommitted stem cell
from the totipotent uncommitted stem cell. The proliferation of the precursor
cells will accelerate erythropoiesis. Chronic renal disease usually is associated
with anaemia, as there is lack of erythropoietin.
 Thyroxine, cortisol and growth hormone: These hormones stimulate red cell
formation, due to their involvement in metabolism. They increase oxygen
consumption by the cells and the resultant hypoxia stimulates erythropoiesis.
 Androgens: They directly stimulate red bone marrow to produce red blood
cells. Lack of androgens leads to anaemia.
 Vitamin B12 (cyanocobalamine): It is extrinsic factor and for its absorption,
intrinsic factor in the gastric juice should be present. The absorption of B12
occurs from the terminal ileum. Vit B12 source comes from animal origin and
 bacterial flora. B12 is necessary for DNA and RNA synthesis and due to this
involvement, B12 brings about proliferation and maturation of erythroid
precursor cells. Lack of Vit B12, causes pernicious anaemia, which is
characterized by the presence of large immature cells called megaloblasts in
the circulation.
 Folic Acid: It is necessary for the synthesis of RNA and through this
involvement, it influences the proliferation and maturation of erythroid
precursor cells. Deficiency of folic acid causes megaloblastic anaemia, similar
to B12 deficiency but without neurological symptoms.
 Pyridoxine (vit. B6): Pyridoxine is involved in haemoglobin synthesis.
 Metals: Iron is necessary for haemoglobin synthesis. Deficiency of iron
causes microcytic hypochromic anaemia. Iron is absorbed from the upper part
of the small intestine. The absorption is regulated by apoferritin present in the
mucosal cell. When iron is bound to this compound, it is converted to ferritin.
The saturation of apoferritin in the intestinal mucosal cell regulates iron
absorption. The excess iron present in the diet is excreted in the feces. Iron is
not only needed for haemoglobin synthesis, but also, required for myoglobin
and enzymes like catalase synthesis. Iron is transported in the blood bound to
transferrin. There is also reutilization or recycling of iron, released from the
break down of red cell and haemoglobin. Excess iron, when released, is
deposited in the tissues like spleen and the condition is called haemosiderosis.
The iron from haemosiderin is not available for exchange, whereas, iron from
ferritin is readily available for exchange. Normal requirement of iron in adult
is 12 to 15 mg per day and the requirement is more in pregnant and
reproductive women. Other metals like zinc, copper, cobalt, manganese are
also necessary for erythropoiesis.

LEUCOCYTES
Leucocytes are nucleated and larger in size than red blood cells. There are two types
of leucocytes, namely, granulocytes and agranulocytes. Total count of leucoytes
varies from 4,000 to 11,000 cells per cubic millimeter of blood. These white blood
corpuscles are involved in protecting the body against infections and foreign bodies.
A differential count of leucocytes reveals five types, which are described as follows.
Neutrophil 50 – 70%
Eosinophil 2 – 5%
Basophil 0 – 1%
Lymphocyte 25 – 35%
Monocyte 3 – 8%

Granulocytes: These cells have a nucleus, which is lobulated and the cytoplasm
contains granules. It includes three types, namely neutrophil, eosinophil and basophil.

Neutrophil: It constitutes 60 to 70% of the total leucocyte count. The cells are
multilobed and usually ranges from 3 to 5. They are also known as
polymorphonuclear leucocyte. Cell size is 10 microns is diameter and the cytoplasm
contains fine granules, which appear purplish in a peripheral blood smear. The
granules contain many enzymes, which include lysozymes and peroxidase.
Lysozymal enzymes digest the bacteria and peroxidase reduces hydrogen peroxide.
The primary function of neutrophil is to provide first line of defense against the
invading microorganisms. This is achieved, by a process called phagocytosis.
Neutrophils leave the circulation and enter the tissues where the micro organisms are
present. They leave the capillary endothelial wall by binding to neutrophil adhesion
molecule called intergrin protein and through a process of diapedesis the neutrophils
leave the capillary.

Phagocytic defence
The entry of bacteria or foreign particulate matter into the body, triggers the bone
marrow to release more neutrophils into the circulation, which forms the part of
inflammatory response to the bacterial entry. The macrophages, lymphocytes,
basophils, and mast cells release leucotreins and complement proteins (C5a), which
cause chemotaxis. That is, the neutrophils are attracted to migrate to the site of
bacterial entry. The bacteria are now coated with the complement protein to be
presented to the phagocytes. This is called opsonisation. The opsonised bacteria bind
to the receptors on the neutrophil membrane and by a endocytosis process, the
bacteria are ingested into the cell. The ingested bacteria are now enclosed in a
vacuole called phagosome. Enzymes of the neutrophilic granules fuse with the
phagosome and digest the bacteria.
Release of bactericidal agents
There is increased respiratory burst in the neutrophil, where the cell shows accelerated
oxygen uptake and metabolism leading to generation of super oxide ion, hydrogen
peroxide and free radical. The free radial oxygen and hydrogen peroxide are
bactericidal agents. The enzyme myelo peroxidase converts hydrogen peroxide and
chloride ions to hypothalous acid, which is also a bactericidal agent. These lysozymal
enzymes digest the wall of the bacteria.
The process of phgocytosis, causes spillage of digestive enzymes into the surrounding
tissues as part of the inflammatory reaction, causing destruction of the tissue.
Physiological rise in neutrophil count is observed in the following
Menstruation, pregnancy, muscular exercise, diurnal rise in the afternoon, and stress
stimuli.
Pathological rise in neutrophil count can be seen in the following
Acute infections by pus forming organisms and myocardial infarction.
Neutrophils are removed through the excretion in gastrointestinal secretions. The
average life of the cell in the circulation is 12 hours and when, it enters the extra
vascular tissues, stays for 4 to 5 days. Neutrophils that leave the circulation will not
come back. They are lost after their defense against the bacteria.

Eosinophil
These cells have large granules, which take up eosin stain and appear orange red.
The cell is 10 microns in size and constitutes 2 to 4% of total white cell count. The
nucleus is bi lobed.
Eosinophils are concerned with the inhibition of allergic reaction. It also attacks
parasites and larvae and kills them by their toxic chemicals released from them.
Eosinophil number increases, in allergic reactions and parasitic infestations. Allergic
reaction is prevented by inhibiting histamine release.
Eosinophil also show phagocytic action similar neutrophil.
Eosinophilia indicates rise in eosinophil count and it is usually seen in the following :
* Allergic conditions like urticaria, hay fever and bronchial asthma.
* Parasitic infestations like hook worm, round worm, thread worm infestations.
Eosinophil count decreases after administration of glucocorticoid and adrenaline. The
decrease is due to sequestration of eosinophils from the circulation to the organs like
spleen and liver.
Basophil
Basophils are 10 microns in size and they have usually a bi lobed or sometimes three
lobed nucleus. The cytoplasm contains coarse meta chromatic granules and appears
as deep blue after staining the peripheral blood smear. Normal count of basophils is 0
to 1% of leucocyte count. Basophil cytoplasm granules contain heparin, hitamine and
serotonin.
Basophils mediate immediate hypersensitivity reactions including anaphylaxis.
Basophils have receptors for IgE immunoglobulin, which mediates hyper sensitivity
reactions. The release of histamine from basophils is responsible for such reactions.
Heparin release from basophils, activates lipoprotein lipase, which help triglyceride
metabolism. Besides this function, heparin also acts as an anti coagulant.
Increase in basophils is seen in anaphylactic shock and other hypersensitivity
reactions. Abnormal rise in basophil number is seen in leukemia.

Agranulocytes
These white cells have a single nucleus and the cytoplasm contains no granules. It
consists of two types namely lymphocytes and monocytes.

Lymphocytes
The cells have a large nucleus and cytoplasm does not contain granules. Size varies
from 8 to 15 microns. Its count is 25 to 30% of total white cell count.
The function of lymphocytes is in providing immunity to the body. Functionally,
there are two types of lymphocytes, namely, T lymphocytes and B lymphocytes. T
lymphocytes give cellular mediated immunity and B lymphocytes are involved in
humoral mediated immunity. The detailed description of immunity has been dealt
with separately.
Lymphocytes count increase occurs in chronic infections such as tuberculosis and
decrease in count results, when cortisol is administered.

Monocytes
Monocytes are the largest size in white cell types. Its size ranges from 15 to 18
microns. The nucleus is kidney shaped and there is relatively more cytoplasm
between nuclear membrane and cell membrane. The cell count is 3 to 8% of total
leucocytes.
Monocytes are considered as second line of defence in the body. They have the
circulation and become tissue macrophage. Tissue macrophage lives for many
months. Macrophages have many functions, which include their role in the
processing of antigens and presenting them to T and B lymphocytes.
Monocytes count rises in kala azar, malaria, typhoid and infectious mononucleosis,
bacterial endocarditis protozoan disease, and Hodgkin’s disease.

Leucopoiesis
The stem cell, which is unipotent and committed is present in the red bone marrow for
the leucocytes. The stem cell for neutrophil and monocyte arise from a common
precursor, whereas, eosinophil and basophil have different stem cells, as precursors
for each type.

Regulation of leucopoiesis
The unipotent, committed stem cells of granulocytes proliferate by the presence of
growth factors like :
 GMCSF (granulocyte macrophage colony stimulating factor) It is produced
from macrophages, fibroblasts, T-lymphocytes, and vascular endothelial cells.
This growth factor is formed by the stimulation of interleukin I and tumor
necrosis factor. GM-CSF is also required for the proliferation of erythroid
precursors and development of macrophages.
 Interleukin I This is also a growth factor for the proliferation of granulocytes
precursors in the bone marrow. It is produced from macrophages.
 Interleukin 3 It is also a multi colony stimulating factor (multi-CS). IL-3 is
produced from T-lymphocytes by the stimulatory effect of IL-I, IL-3, causes
proliferation of all types of leucocyte precursors and also erythroid and T-
lymphocytes.
 Interleukin 5 It is produced from T-lymphocytes and it is specifically required
for the proliferation of eosinophil precursor.
 Tumor necrosis factor Though its main action is to inhibit tumor growth, it
also shows haemopoietic effect. It is produced from macrophages.
The stages of granulopoiesis in the red bone marrow, shows, the committed stem cell
proliferating into myeloblast (Fig. 6.3) It is a large cell with 20 to 22  size and the
nucleus is large, occupying the whole cell. There are also nucleoli present. The cell
shows active mitosis. This stage gives rise to myelocyte, which is characterized by
reduction of cell size, disappearance of nucleoli. The cell shows active mitosis and
granules appear in the cytoplasm. The next stage is metamyelocyte, where the
nucleus shows lobulation and the granules become more in cytoplasm. The cell size
is further reduced and mitosis stops here. With the nucleus lobulation and specific
granules filling the cytoplasm, the granulocytes are released into the circulation.
Neutrophils in the blood exist in two forms namely :
a) circulating granulocyte pool (CGP)
b) marginal granulocyte (MGP)
MGP refers to the neutrophils in the lining of small blood vessels like venules. This
pool can readily enter the circulation, when there is a demand. The rise in neutrophil,
due to adrenaline and exercise, comes from the MGP.
Granulocytes live for 2 to 4 days. Some live for a few hours only. Those which leave
the circulation and enter the tissues stay for longer days. Most of the white blood
cells are lost in the gastrointestinal secretions.
Lymphocytes are produced from bone marrow. The precursor cells migrate to the
lymphoid tissues like thymus and lymph node.

The committed, unipotent stem cell proliferates into lymphoblast. This precursor cell
differentiates into lymphocytes. If processing takes place in the thymus, T-
lymphocyte is formed. On the other hand, if the cells are processed in the spleen and
liver, it develops into B lymphocyte.

Monocyte develops from a precursor stem cell in the red bone marrow, which is
common to neutrophil (Fig. 6.3). The committed, unipotent stem cell proliferates and
gives monocytoblast. This precursor cell differentiates into monocyte and released
into the circulation.

THROMBOCYTES (PLATELETS)
They are small (2 to 3 ), ovalshaped and non nucleated cells. There is mitochondria
in the cell and the characteristic feature is the presence of microtubular system,
consisting of contractile proteins (actin and myosin) and a canalicular system, which
communicates with the exterior. The cytoplasm contains two types of granules,
namely, dense granules containing serotonin, epinephrine, ADP, other adenine
nucleotides and alpha granules, consisting of several proteins like platelet derived
growth factor (PDGF) and clotting factors.
The normal count of platelets is 250,000 to 300,000 cells / mm3 of blood. Their life
period is 4 to 7 days and are destroyed in the spleen. Platelets also stay in the spleen
as reservoir pool. During bleeding and epinephrine secretion, this pool is released
into the circulation.

Formation of platelets
The production of platelets, depends on the number of cells that are destroyed in the
spleen. The substances released from lysed platelets themselves stimulate
thrombopoiesis. The uncommitted stem cell in the red bone marrow becomes a
committed stem cell by the stimulation of colony stimulating growth factors. Further
differentiation of megakaryocytes (precursor cell) requires Interleukin (IL3). The
megakaryoctyes breakes up into protoplasmic fragments and form thrombocytes.

Functions of platelets
 Formation of platelet plug
When a blood vessel is injured, the platelets undergo adhesion to the exposed
collagen. The platelet adhesion is facilitated by von Willebrand factor, which is
present in the wall of the platelets and also in the wall of the vascular endothelium.
Once the platelets adhere to the collagen, further adhesion of platelets to the site of
injury is promoted by the release of contents of platelets, like ADP and platelet
activating factor. This leads to more aggregation of platelets on the damaged vascular
endothelium. Thromboxane A2 is a potent platelet aggregator. Its formation is
facilitated by the entry of Ca++ into the cell. The aggregation of platelets causes a
plug at the site of injury and seals the wound. This arrests the bleeding. The
aggregation of platelets is limited by the formation of prostacyclin from
endoperoxides in the endothelium. The next step after aggregation is the release of
platelets contents at the site of damage in the blood vessel. Drugs like aspirin, when
administered orally in low doses (75 to 100 mg / day) inhibits thrombaxane A2
without significant impairment of prostacyclin production. Thus, by altering the
balance between platelet thrombaxane A2 and endothelium prostacylin, aspirin in low
doses helps to prevent myocardial infarction, unstable angina, stroke and transient
ischemic attacks.

 Release of vasoconstrictor substances

Platelets release vasoconstrictor substances like serotonin, epinephrine, which cause
vasoconstriction and reduce blood loss. Thromboxane A2 which is formed from
platelets also causes vasoconstriction of small blood vessels and minimize bleeding.

 Platelets help in blood coagulation

Platelets release platelet factor 3, which is a membrane phospholipids and facilitates
the intrinsic formation of thrombin. Thrombin itself causes platelet aggregation and
causes release of more platelet factor 3, thus enhancing the clotting mechanism.

 Clot retraction
After the coagulation of blood, the clot shrinks, expelling the serum. This is called
clot retraction which is helped by platelets. The process is initiated by the thrombin
acting on the platelets. It is likely that thrombin causes calcium entry into the
platelets to activate contractile proteins actin and myosin, present in platelets. The
contractile proteins contract like in muscle and gives retraction of clot. The retraction
of fibrin threads can bring together the edges of the damaged endothelium and
facilitates faster wound healing.

 Repair of damaged endothelium

The aggregation of platelets will also release platelet derived growth factor (PDGF).
It is a growth factor. Which helps in repairing of the damaged blood vessel.

Disorders of platelets function

Purpura: It is characterized by the spontaneous bleeding beneath the skin and mucous
membrane. This gives purplish spots on the skin. It is a bleeding disorder, caused by
the deficiency of platelets (thrombocytopenic purpura) or functional defect in
platelets. In this disorder, the bleeding time is prolonged, but the clotting time is
normal.
HAEMOSTASIS(BLOOD COAGULATION)
Haemostasis is a process in which the bleeding is arrested by the formation of clot.
Clotting of blood is an enzymatic process, which involves various plasma clotting
factors, leading to the conversion of fibrinogen to fibrin.
The clotting factors are plasma proteins, which exist in inactive proenzyme form.
During the process of coagulation, each inactive proenzyme is converted into active
enzyme form. This activated factor inturn, activates subsequent proenzyme clotting
factors to finally result in fibrin (clot). The activation of clotting begins with the
blood coming in contact with the rough surface and collagen of the damaged blood
vessel. At each step in the activation of clotting factor, the reaction is amplified and is
known as enzyme cascade mechanism.
Broadly, the process of clotting can be described in three steps.
In the first step, the prothrombin activator is formed. It is generated in the intrinsic
pathway within the blood and also in the extrinsic pathway from the damaged tissues.
This, in the next step activates prothrombin to thrombin. In the last step, the thrombin
converts fibrinogen to fibrin. Though it appears as a simple process, the involvement
of several clotting factors makes it a complex enzymatic reaction.
Clotting factors are represented by Roman nomenclature from I to XIII.

 Factor I fibrinogen: The clot is fibrin thread, formed from fibrinogen by the
enzymatic action of thrombin. Fibrinogen is one of the constituents of plasma
proteins and its concentration is 0.25 to 0.3 g%. The level of fibrinogen is
increased in pregnancy, inflammation, tissue damage, etc. An increased
concentration of fibrinogen, raises the rate of erythrocyte sedimentation, by
influencing its rouleaux formation.
 Factor II prothrombin: It belongs to globulin fraction of plasma proteins and it
is activated by prothrombin activator (activated factor X) to thrombin.
Prothrombin is synthesized from the liver, in the presence of vitamin K.
Drugs like dicoumarol competitively inhibits the action of vitamin K and
prevents clotting. Diseases affecting the liver, would cause deficiency of
prothrombin, leading to clotting disorder.
 Factor III tissue thromboplastin: It is present in the cells. It consists of
lipoprotein phospholipids complex, which is released, when the tissue is
 damaged. The released tissue phospholipids activates factor VII, which inturn
activates factor X. Tissue thromboplastin role in the coagulation is seen in the
extrinsic pathway. However, tissue thromboplastin-factor VII complex is also
observed in the activation of factor IX (Christmas factor), thereby, showing
that it has a role in the intrinsic pathway of coagulation also.
 Factor IV calcium: This divalent cation is required in almost all steps in the
coagulation, except in the initial activation of XII Factor (Hageman Factor) in
the intrinsic pathway. Chelating agents suchas, EDTA, citrate, oxalate,
fluoride act as anti coagulants.
 Factor V proaccelerin: It is converted to active from by Xth activated factor
(Stuart factor) in both intrinsic and extrinsic pathways of coagulation.
Activated factor V is crucial in the reaction, which leads to the conversion of
prothrombin to thrombin.
 Factor VII proconvertin (serum prothrombin conversion accelerator): It is a 
globulin fraction, which is synthesized from the liver in the presence of
vitamin K. Factor VII is involved in the extrinsic pathway of clotting. Tissue
thromboplastin, released from the damaged tissues, activates factor VII.
 Factor VIII antihaemophilic globulin factor (AHG): AHG has a component
called vWF (von Wille-brand factor) and both are involved in the intrinsic
pathway of clotting. The vWF is also present in platelets and helps in its
adhesion to the damaged endothelium. Deficiency of VIII factor causes
haemorrhagic disorder called haemophilia.
 Factor IX Christmas factor: This factor is also synthesized in the liver in the
presence of vitamin K. Factor IX is activated by the activated XI factor in the
intrinsic pathway. Deficiency of this factor will also cause haemophilia,
which is called Christmas disease.
 Factor X Stuart factor: This globulin protein is involved in both the intrinsic
and extrinsic pathways of coagulation. The activated factor X is also called
prothrombin activator, which activates prothrombin.
 Factor XI plasma thromboplastin antecedent (PTA): It is activated by XII
activated factor. The activation requires high molecular weight kininogen and
kallikrein. PTA is involved in the intrinsic pathway.
  Factor XII Hageman’s factor: This factor is activated by surface contact with
collagen and negatively charged surface from the damaged endothelium.
Activation of XII factor is the first step in the clotting process in intrinsic
pathway.
 Factor XIII fibrin stabilizing factor: Activation of XIII factor is caused by
thrombin. The activated XIII factor causes polymerization of fibrin, converted
it into an insoluble clot.

Process of clotting
Role of platelets: Injury to the blood vessel exposes collagen and the negatively
charged surface of the endothelium. This causes platelets adhesion and aggregation
on the damaged endothelial surface. This is followed by the release of platelets
contents, such as ADP, vasoconstrictors, platelet factor III, etc. The adhesion and
aggregation give temporary haemostatic plug. The release of platelet factor III
activates the clotting process.

Generation of prothrombin activator: The first step in the clotting process is the
formation of activated factor X, which is also called prothrombin activator. It is
formed in two ways. Intrinsic pathway (within the blood) Extrinsic pathway (from
tissues).

Intrinsic pathway: As said earlier, the clotting factors are proenzymes, which are
activated during clotting and in each step the reaction is amplified. This is known as
cascade mechanism.
The exposure of collagen and the negative charge on the injured surface, causes the
activation of Hageman’s factor (XII). This is the beginning of sequential events in the
coagulation of blood. The activated XII acts as an enzyme and converts XI to XI
activated factor. This activation also requires high molecular weight kininogen and
kallikrein.
The activated Hageman’s factor is also involved in augmenting the activity of factor
VII in the extrinsic pathway. It also activates plasminogen, which is involved in
fibrinolysis. Further, it causes the formation of complement proteins which
participate in the immune reactions.
The activated XI factor, in the presence of Ca++ activates IX into IX activated factor
(activated Christmas factor). It is in this step, calcium is first required for the
activation. Activated Christmas factor converts Stuart factor into its active from,
through the activation of VIII factor (AHG) in the presence of Ca++ and
phospholipids of plate let factor III.
Activated Stuart factor is also known as prothrombin activator. It causes the
conversion of prothrombin to thrombin, in the presence of activated proaccelerin (V
activated factor), Ca++ and phospholipids.
Thrombin acts as an enzyme protease and converts fibrinogen to fibrin.

Extrinsic pathway
Formation of prothrombin activator in extrinsic pathway occurs, when blood comes in
contact with the injured tissues. The damaged cells release phospholipids from the
cell membrance. The tissue phospholipids shows, clot promoting activity through the
activation of VII Factor, in the presence of Ca++. The activated VII factor, converts
Xinto X activated factor, in the presence of altered proaccelerin (V activated factor),
Ca++ and phospholipids. From now onwards, the steps leading to the formation of
fibrin are identical to those observed in the intrinsic pathway.

Formation of Fibrin (Role of fibrin stabilizing factor): Blood coagulation basically

involves the conversion of soluble plasma protein fibrinogen to nsoluble fibrin clot by
the enzyme thrombin. Initially, fibrin is formed as soluble monomer. This is
followed by the cross linking of fibrin threads into fibrin polymer by the action of
XIII (fibrin stabilizing factor) and thrombin. Fibrin polymer is an insoluble clot.
Calcium ions are needed for the polymerization of fibrin. The fibrin polymer appears
as strands of meshwork, in which the red cells and serum are trapped. The red colour
of clot is due to the trapped red cells.

FIBRINOLYSIS
As soon as the clot is formed, the mechanism to liquefy the clot is activated. The
dissolution of clot is called fibrinolysis. The enzyme responsible for this is plasmin,
which comes from the proenzyme plasminogen. The plasminogen is activated to
plasmin in the natural way by substances like tissue plasminogen activators such as,
urokinase, activated Hageman’s factor, activated PTA, vascular endothelium and
kallikrein. There are also enzymes that are not present in the body, but shows
plasminogen activation, when administrered. They are streptokinase and
staphylokinase enzymes. Tissue plasminogen activators or streptokinase, are used
clinically to dissolve clots, especially in coronary arteries.
Plasmin binds to the clot surface and digests fibrin, without attacking fibrinogen. As
a matter of fact, the plasminogen activation occurs on the clot surface and not in the
plasma. Plasmin, when comes in contact with the fibrinogen effectively digests it.
Inhibitors of clotting (Mechanisms which keep the blood in a fluid state).
There are mechanisms present in the blood itself to prevent clotting. The fluid state of
the blood is due to the balance between factors, which cause clotting and those, which
oppose it. The following description gives an account of inhibitors of clotting.

Antithrombin III: It is the major inhibitor of coagulation, which inhibits thrombin and
activated Stuart factor (Xa). It forms a complex with heparin anticoagulant and shows
enhanced activation.

Protein C: It is a proenzyme and activated by thrombin – thrombomodulin complex

on the endothelial surface. Thromobomodulin is present on the endothelial surface
and also released from platelets. It combines with thrombin and activates protein C.
The activated protein C inhibits VIII and V factors and prevents clotting. It also
promotes fibrinolysis.

Heparin: Heparin is a naturally occurring anticoagulant, which is secreted from mast

cells and basophils. Heparin has to bind to antithrombin III to become an
anticoagulant. There is also another factor for heparin activation. It is heparin
cofactor II. This complex also inhibits thrombin activity.

Vascular endothelium: The initation of clotting occurs, when blood vessel injured,
exposing the collagen. This provides the rough surface for the activation of XII
Factor. In normal conditions, where there is no damage to the vascular endothelium,
the collagen is not exposed on the surface and hence no activation of XII factor.

Fibrinolysis: Fibrinolytic mechanism described earlier, also prevents clotting and

helps to keep the blood in fluid state.
Intravascular thrombosis: It is the formation of clot within the blood vessel. There are
conditions, which favour the formation of clot within the blood. They are;
 Injury to the vascular well
 Slow rate of blood flow
 Deficiency of antithrombin III, protein C, heparin confactor II.
 Presence of atherosclerotic plaques in the lumen of the vessel.
If the thrombus breaks up and carried away in the circulation, it gets lodged in a
vessel and form an embolus. Emboli in pulmonary arteries, coronary or cerebral
arteries can become severe enough to cause death.

Anticoagulants
Salts: Citrates, oxalates, fluorides and EDTA (ethylene di amine tetra acetic acid)
are called chelating agents, as they bind calcium. Since calcium is required in
many steps in the process of coagulation, the removal of calcium in the blood by
these salts, results in prevention of clotting.
Drugs
Heparin: It is a naturally occurring anticoagulant, but, not a constitutent of plasma. It
is secreted from mast cells and basophils and also present in many tissues like lungs
and liver. It is a negatively charged sulfated polysaccharide. Heparin requires
antithrombin III for its anticoagulant activity. It also combines with heparin cofactor
and shows anticoagulant action. Heparin inhibits thrombin action. It also inhibits the
formation of prothrombin activator, as it blocks the action of IX factor. Heparin is
widely used in clinical practice and laboratories as an anticoagulant. Heparin action is
inhibited by protamine sulphate, which is a positively charged polypeptide obtained
from fish sperm. Heparin can be used as an anticoagulant both in vivo and in vitro.

Dicoumarol :It is a vitamin K antagonist, as it competitively inhibits the action of

vitamin K in the liver. Vitamin K is required for the synthesis of prothrombin, factors
VII, IX and X and administration of dicoumarol, blocks the synthesis of these clotting
factors. Dicoumarol can be effective as anticoagulant only in vivo.
Tests for clotting
Clotting time: It is sually determined by the capillary tube method, where the time
taken for the appearance of fibrin threads is noted as the clotting time. The normal
duration is 3 to 8 minutes. Deficiency of clotting factors leads to increased clotting
time. In haemophilia, the clotting time is prolonged, but the bleeding time would be
normal.

Bleeding time: This can be determined by pricking the ear lobe or the finger tip and
the time taken for the arrest of bleeding is noted as bleeding time. The arrest of
bleeding is due to the formation of platelet plug. Bleeding time normally ranges from
2 to 5 minutes. Bleeding time is prolonged in purpura due to platelet deficiency.
However, the clotting time is normal as there is no deficiency of clotting factors.

Prothrombin time : Quick method – this test indicates the quantity of prothrombin
present in the plasma. A citrated blood is taken (so that the prothrombin is not
converted to thrombin). To this, tissue thrombo-plastin (brain extract) and calcium
are added. This mixture is incubated at 370 C and the time taken for the appearance of
clot denotes pro-thrombin time. Normal prothrombin time ranges from 10 to 14 sec.
Prothrombin time increases in liver diseases and vitamin K deficiency.

Thromboplastin generation test : This test specifically measures the defects in clotting
factors involved in the intrinsic pathway of formation of prothrombin activator.
Samples of adsorbed plasma containing factors VIII, V, Serum containing factors XII,
XI, IX, X, calcium and platelets are added and incubated at 37 C. These three
components will generate prothrombin activator. This mixture is made to act with
normal plasma, which contains fibrinogen and prothrombin. The time for the clot to
appear, indicates thromboplastin generation test. Normal value is 6 to 15 seconds.

Clotting disorders :
Haemophilia : It is a clotting disorder in which there is a haemorrhagic tendency, due
to the deficiency of clotting factors VIII (AHG) and IX factor (Christmas Factor).
Haemophilia is a genetic disorder, transmitted as X linked recessive character. The
males suffer from the disease and females carry the disorder. There are two types of
haemophilia and they are described as follows:
Haemophilia A or classical haemophilia : It results due to the deficiency of VIII
factor, antihaemophilic globulin (AHG). The clotting time is abnormally prolonged.
Von willebrand’s disease is also a hereditary disease, inherited as dominant trait
inboth sexes. Since vWF is a subconstituent of VIII factor, von Willerbrand’s disease
will also cause deficiency of VIII factor and symptoms of haemophilia. It also affects
platelet function in forming adhesion to the injured surface of the vessel wall.

Haemophilia B or Christmas disease : Clotting factor IX (plasma thromboplastin

antecedent) is a vitamin K dependent factor, which is synthesized from the liver.
Christmas disease is also an X linked disorder, resulting from the deficiency of IX
factor.

BLOOD GROUPS AND TRANSFUSION

Blood groups existence is based on the two types of agglutinogens on the surface of
red blood cells. These agglutinogens are A and B and they are responsible for the
four types of blood groups i.e. A, B, AB and O. The agglutinogens are
mucopolysaccharides, which are not only present on the red cells, but also, in body
secretions, such as, saliva, gastric juice and in tissues of liver, kidney and lungs. The
red cell surface has more than 30 antigens on its surface, but they do not induce any
transfusion reactions. Hence, for the purpos1e of blood transfusion, A, B, O and AB
groups cross matching is done with donor’s red cells and recipient’s plasma. It should
be kept in mind that there are agglutinogens other than A and B present in some
individuals and the blood group is named after their family name. The red cell
surface in 85% of the individuals, shows another type of agglutinogen called Rh
agglutinogen. This is present in addition to the A, B agglutinogens and such
individuals are called Rh+. The blood group will be called Rh-, if the Rh
agglutionogen is absent on the red cell membrane. In plasma, there are antibodies
called agglutinins, which act against the blood group of opposite agglutinogens.

LAW OF BLOOD GROUPING: Landsteiner, the discoverer of blood groups,

propounded the law of blood grouping based on the type of agglutinogens and
agglutinins present in the red cell and plasma respectively.
The law states, that, when an agglutinogen is present in the RBC, the plasma will not
contain the corresponding agglutinin. Conversely, when the red cell does not contain
the agglutinogen, the plasma will contain the corresponding agglutinin. The second
part of the law cannot be applicable to the Rh system. In Rh- individuals, Rh
agglutinins will not be present. It is produced only when Rh- plasma is sensitized
with Rh+ cells. Blood groups are determined by taking red cells suspension and
mixing with the three types of sera namely, anti A, anti B and anti D on a glass slide
and observed for agglutination reactions. The anti D sera is used for determining the
Rh group. The agglutinins are immuno globulins. The anti A and anti B agglutinins
belong to IgM type, The Rh agglutinin belongs to IgG type. The presence of opposite
agglutinin in plasma, can be explained, from the fact, that during infancy, blood group
antigens of non self ingested along with the food. The antibodies are developed
passively in the plasma and continue to stay in the rest of life.

INHERITANCE OF BLOOD GROUPS

Blood groups are inherited as mendelian allelomorphs. The dominance is seen in type
A and type B genes. The O type gene does not have function and there is no
agglutinogen present for it in the red cell. Hence, the inheritance of blood groups
depends on any of the three genes namely A, B and O. The phenotype of A or B
group person can have the homozygous or heterozygous genotypes. This is the
reason, why the blood group of a child can be different from that of both the parents.
For example, if one of the parents is A Group and the other is B group, then, the
genotypes in the children would be AB, AO, BO, OO, and the possible blood groups
in the offspring can be any one of the groups namely, AB, A, B, O. The blood
groups, besides, its role in the transfusion of blood, also helps in medico-legal cases to
solve the disputed parentage.
Rh+ persons will have D type gene, whereas the Rh- person gene type is d. The Rh+
phenotype will have, the genotypes DD, Dd and both will be Rh+. The Rh-
phenotype will have only dd genotype.

CROSS MATCHING: Cross matching of blood groups is done to find out the
compatibility of blood groups for transfusion. If transfusion of blood is carried out
without cross matching of groups, the donor’s red cells will be clumped by the
agglutinin present in the recipient’s plasma. The clumping of red cells is called
agglutination. In cross matching, the donor’s red cells are matched with the recipient’s
plasma and observed for agglutination reactions. Normally the donor’s plasma is
diluted in the recipient and hence the agglutination of recipient’s red cells with the
donor’s agglutinin of plasma does not occur. However, in some cases, there could be
agglutination reactions in the latter. To rule out such a possibility, the cross matching
is also carried out, with the agglutinin of donor’s plasma and the red cells of the
recipient. Only if there is absence of agglutination in both the tests, the blood group is
selected for transfusion.
It will be seen from the table that O group can be given to all other groups and AB
can receive from all other groups. The O group is referred as universal donor, as it
does not have any agglutinogen. The AB group is called as universal recipient, as it
contains no agglutinins in the plasma. Since there is presence of Rh agglutinogen in
the red cells of 85% of the individuals, it is better to avoid such terms. For
transfusion of blood, Rh compatibility test should also be included. The transfusion
of Rh+ blood should never be given to Rh- person. In emergency, where the blood
typing cannot be done, O negative group can be transfused.

RH FACTOR : It is a type of agglutinogen called Rh antigen or D antigen present

on the RBC’s of 85% of the individuals. Those who have this antigen on the red cells
are called Rh+. The name is derived from the experiment performed with the rhesus
monkey’s cells mixed with the rabbit’s serum. The rabbit serum developed antibodies
against the rhesus monkey’s red cells. This immunized rbbitserum, when tested with
the human RBCs, agglutination was noticed in 85% of the samples tested. This
showed that the human RBC contained an antigen, similar to rhesus monkey red cell
antigen. Those who show this, on their red cells, are called Rh+. The remaining 15%
of the individuals show no such Rh antigen on the red cell and they are called Rh -.
Their plasma does not contain Rh antibodies, but produced, when Rh+ blood is given
and sensitized.

RH INCOMPATIBILITY : In blood trnasusion, Rh+ blood should not be given to a

Rh- person. In the first transfusion, the Rh- plasma becomes sensitized by the Rh+
red cells. Subsequent transfusion of Rh+ blood to the same person, will result in
agglutination reaction, due to Rh antibodies produced by the Rh- plasma against Rh+
red cells, Rh incompatibility also results, when an Rh- woman conceived Rh+ foetus,
due to the Rh antigen being inherited from the father. In the first pregtnancy, the
foetal red cells, which are Rh+, enter the maternal circulation during child birth, when
the placental blood vessels rupture. This causes sensitization of Rh- plasma of the
mother. In the second or third pregnancy, the Rh- plasma, which is already sensitized
with Rh+ foetal red cells, produces, enormous Rh antibodies. It crosses the placental
barrier and enters the foetal circulation. The agglutination of foetal red cells by the
Rh antibodies, results in haemolytic disease of the new born. The new born will show
severe jaundice and anemia. The peripheral blood shows the nucleated precursor red
cells and hence the name erythro blastosis foetalis. Sometimes, the child dies I utero,
giving still born births. If the jaundice is severe, it will lead to deposition of bile
pigments in the basal ganglia, resulting in kernicterus. This Rh incompatibility can be
treated by exchange transfusion of Rh- blood to the child. The mother can also be
effectively immunized against Rh antibodies, by giving anti Rh immunoglobulin,
immediately after the birth of the first child.

TRANSFUSION OF BLOOD : Transfusion of whole blood is required in severe

haemorrhage, surgery, and anemia (when Hb% falls to 40). There are conditions,
where only the components of blood need to be given, such as, albumin, clotting
factors, platelets concentrate, red cells, whole plasma, gamma globulins, etc.
The blood selected for transfusion should be free from diseases like, malaria, syphilis,
hepatitis and acquired immune deficiency syndrome. If an individual comes to know
earlier of the requirement of blood for transfusion, as in undergoing, surgery, the
safest way, is, to collect his or her own blood periodically at regular intervals and use
it during surgery. This method of transfusion is called autologous transfusion.

Precautions to be taken in transfusion of blood:

 Screening of the donor, who is free from communicable diseases.
 Cross matching of blood to ensure compatibility
 Storing of blood in aseptic conditions.
 Transfusion should not be rapid (The rate should be 100 to 200 ml/hour).
CHANGES DURING STORAGE OF BLOOD

The blood collected for transfusion is stored at 0 to 4 C in blood banks. During

storage, the red cells become spherical, due to the inhibition of sodium pump and
water moving in to the cell. Sodium pump is inhibited as the temperature is very low
during storage. The size of the red cell becomes normal, when the blood is
transfused, as the sodium pump is being restored. During storage of blood, acid
citrate dextrose is added, to provide energy to the red cell. The absence of sodium
pump, during storage of blood, causes potassium level to be more in plasma and
sodium remaining inside the red cell. The ions go back to their positions, when the
blood is transfused into the subject. If the transfusion is too rapid, there is a danger of
hyperkalemia in the recipient.

Effects of incompatible transfusion

Incompatible or mismatched blood transfusion can occur, if, cross matching is not
carried out Example of mismatched transfusion would be A group person receiving B
group blood. In such conditions, the red cells agglutinate causing haemolysis. There
is shock like state, due to the hypersensitivity reaction. There is shivering tightness of
chest, fall in blood pressure dyspnea etc. The haemolysis leads to release of
haemoglobin, haemoglobinuria and formation of increased bile pigments. This results
in post transfusion jaundice. The free haemoglobin precipitates the renal tubules
causing renal damage (lower nephron nephrosis). The renal failure can lead to
uraemia and death.

IMMUNITY
Our body is exposed to a variety of pathogenic organisms like bacteria, virus, fungi,
parasites, etc. To combat these pathogens, the body shows two types of defense
mechanisms. One is known as innate or non specific immunity and the other is
acquired or specific immunity. Both the systems are interlinked to give an effective
defense mechanism to the body.
Innate immunity
Barriers: It refers to the protective mechanisms, that are present to prevent the entry of
pathogenic organisms. There are barriers in different regions of the body, such as,
skin, and the lipid layer in it prevents entry of micro organisms.
Secretions like, tears, saliva, and acid in the stomach contain antibodies and they form
barriers for pathogens.
Mucosa lining the air ways, not only contains cilia to remove the pathogens, but also,
contains chemicals, which have bactericidal properties.
Lymphoid organs like tonsil, Peyer’s patches, appendix, form effective barrier by
trapping the bacteria, which may try to enter the body.

Phagocytosis: In spite of the body having these barriers, the micro-organisms enter
the body and in such conditions, the neutrophils and macrophages are activated to
phagocytise the invader. The process of phagocytosis has been described earlier.

COMPLEMENT SYSTEM

In addition to phagocytois, there are enzymes called complement system present in

the plasma. When activated by the micro organisms, these proteins show the
following reactions.
Pathogenic organisms are coated with the complement protein, so the neutrophil and
macrophages indentified them easily and phagocytize them.
Some complement proteins stimulate the release of histamine from mast cells and
basophils, which causes vasodilatation and migration of neutrophils to the site of
infection.
There are other complement proteins, which form membrane attack complex, where
the protein forms a hole in the wall of the bacterial and causes its lysis by allowing
entry of sodium and water into the bacterial cell.

C-REACTIVE PROTEIN: It is present in the plasma when there is entry of micro

organisms into the body. It coats the bacterial, which inturn, activates the
complement system. The complement system can promote phagocytosis.
INTERFERONS: When virus infects the cells, it releases alpha interferons. They in
turn are attached to other tissues and prevent the entry of virus.

NATURAL KILLER CELLS: It is a type of lymphocyte, which kills the virus without
any prior sensitization. It is an important mechanism to fight against virus infections
and also inhibits malignant growth of tissues.

ACQUIRED IMMUNITY: Acquired immunity is specific in nature, that is, defense

mechanism exists for each specific antigen. First exposure would cause sensitization
(primary response) of the lymhocytes and subsequent entry of the same pathogen
leads to effective immune response, by way of releasing specific antibody against it
(secondary response) . There is also a development of memory in the immune system
to the specific antigen. The immune system is represented by the activity of
Lymphocytes. Functionally, there are two types of lymphocytes namely.
T lymphocytes, give cell mediated immunity. It is responsible for delayed
hypersensitivity reactions, rejection of transplants of foreign tissue, defense against
virsuses, fungi and bacillus mycobacterium. It also prevents growth of tumors. T
lymphocytes are of different types namely, helper T cells, cyto toxic T cells,
suppressor T cells and memory T cells.
B lymphocytes are responsible for humoral mediated immunity. It releases antibodies
or immunoglobulins against antigens.
Lymphocytes are produced from bone marrow. The stem cells developing into
lymphocytes go to lymphoid organs and differentiate. The lymphocytes that are
processed in the thymus are known as T lymphocytes. Lymphocytes, which are
processed in the bone marrow and liver forms the bursa equivalent in humans, called
Blymphocytes. Both T and B lymphocytes can be seen in lymph nodes and bone
marrow, as migration of the processed lymphocytes occurs in these regions. Although
there are two types of immunity, both are interlinked for efficient functioning of the
immune system.

HUMORAL IMMUNITY
B cell lymphocytes are involved in recognizing the antigen and release of specific
antibodies against it. This is possible, only, if, the helper T cell type (TH2) activates
B cells. There are two ways of activating the helper T cells, which in turn stiumate B
cell proliferation.
In the first mechanism, the macrphages, after taking the antigen, combine with the
class II protein, which is the major histocompatibility complex (MHC). This
combination of antigen and class II protein on the surface of the macrophage, is
presented to the helper T cells.. Similar mechanism is also shown by macrophage
related cells namely dendritic cells in lymph node and Langehans cells in the skin.
Helper T cells, release cytokines (interleukins), which activate B cells to proliferate
and transform into plasma cells. The plasma cells elaborate immunoglobulins.
In the second mechanism, the antigen can directly bind to the B cell surface
receptors, which also has class II protein (MHC). The combination of antigen and
MHC on the surface of the B Cell, results in activation of helper T cells. This inturm
leads to B cell transforming into plasma cell and liberates immunoglobulins. The
activated B cells alo form memory B cells. These cells are specialized to liberate
specific antibodies against the specific antigen, when there is subsequent entry of the
antigen, when there is subsequent entry of the antigen into the body. The memory
cells store the information and hence the response. The specific antigen by way of
releasing antibodies, divide and form clones. This can respond later, when the
specific antigen re-enters the body (clonal slection). To regulate the acivity of helper
T cells, there is a suppressor T cell, which will dampen the B cell activation.
Antigen recognition by the lymphocytes, is due to the presence of gene materials on
the surface of all nucleated cells. There are class I protein and class II proteins. Class
I protein is identified by natural killer cells to destroy viruses and the virus infected
cells. Class II protein presence on the surface of the cells, is identified by
lymphocytes, as self or nonself and take steps to accept or reject the tissues. This
class II proteins are called major histo compatibility complex (MHC) and in human it
is referred as human leucocyte antigen (HLA).
Antibodies are produced, not only, when antigen is processed by marcrophages and
helper T cells, but also, without the involvement or macrophages and helper T Cells.
The latter condition is seen, when IgM antibodies are produced in response to foreign
blood group antigens. These antigens are called thymus independent antigens. The
antigens, which stimulate the B cell through the activation of macrophage and helper
t cell, are called thymus dependent antigens.
HOW DOES ANTIBODIES FUNCTION?

There are many ways of antibodies neutralizing the antigens.

1. It is observed that antigens can be directly neutralized or inactivated by the
combination of antigen and antibody. Examples of this would be the inactivation of
viruses and snake venom. The release of IgM antibody against the incompatible
blood group antigen, producing agglutination, is also an example of direct interaction
of antibody with antigen.
2. Antibodies form a coating on the antigens for the macrophages to easily
phagocytise the antigen. This function of antibody is known as opsonisation.
3. Antigen and antibody combination activate the complement system, involving
cascade of enzyme reactions to give various complement proteins.
In the classical pathway, antigen antibody binding results in C3 formation C1
through other components of the system. The C3 complement protein, leads to C8
and C9 proteins formation and these would cause holes in the cell membrane,
producing lysis of the antigen.
The sub type of C3 namely, C3b causes opsonisation and facilitates
phagocytosis,
There are also subtypes C3a, C5a, C5, C6 and C7, which cause inflammatory
response, leading to migration of macrophages to the site of invasion of antigen.
In the other alternative pathway, the complement system is activated without
the antibody binding to antigen. The activation is due to the polysaccharides on the
wall of the bacterial and facilitated by a circulating protein factor. The complement
proteins that are formed, cause lysis of invading organism. This is also called
properidin pathway.

STRUCTURE OF ANTIBODY AND ITS SUB TYPES

Immunoglobulins consists of four polypeptide chains. There are two identical light
chains (low molecular weight) and two identical heavy chains (high molecular
weight) present in the molecule. Both the chains have constant and variable regions.
In constant chain, the aminoacid sequence is similar in all types of immunoglobulins,
whereas, in variable chain, the amino acid sequence is different in each
immunoglobulin to each type of antigen. The effector function of immunoglobulin
would be present in this region.
 The different types of immunoglobulins exist, due to the difference in the
constant regions of heavy chains. There are five types of immunoglobulins are IgM,
IgG, IgA, IgD and IgE respectively.
IgG Its serum concentration is 7 to 16 mg/ml. It crosses placenta. Its function is
and bacterial and anti viral.
IgM Its concentration in serum is 0.5 to 2 mg/ml. It is produced in early immune
response. It is a good agglutinator. It has antibacterial activity.
IgA Its level in serum is n0.4 to 4 mg / ml. It is present in body secretions.
IgD its concentration in serum is 0 to 0.4 mg/ml. It is present on lymphocyte
surface and involved in antigen recognition.
IgE its level in serum is 0.00002 mg/ml. It binds to mast cells. It is involved in
hypersensitivity reactions and allergic reactions. It is also released in parasitic
infestations.

CELLULAR IMMUNITY
T cell lymphocytes are specifically involved in this type of immunity. T cell has
receptors to identify antigen presenting cells with MHC on their surface. The
cytotoxid T cell is also known as T 8 Cell. The cytotoxid T cell gets activated by the
interleukin 2. They give pores (perforins) on the target cell and cause lysis, which is
very similar to complement system destroying the antigen. The suppressor T cell
helps to dampen the activity of cytotoxic T cell and turns off the immune e response.
This is also similar to the dampening of B cell though the inhibition of helper T cells.
Helper T cells (T4) are also involved in the regulation of B cell function. It can
recognise the antigen presenting cell with the MHC on the surface. It responds by
releasing lymphokines, which help B and T cells (cytotoxid T cell) to differentiate
and give immune response.

REGULATION OF IMMUNITY
Immunity is regulated by genetic, neural and endocrine factors. Recent studies have
shown that regions of hypothalamus, limbic lobe, influence immune mechanism,
either though neural signals or through the hormonal factors.
ABNORMALITIES OF IMMUNE MECHANISM
 Immediate hypersensitivity reactions, which can lead to anaphylactic shock.
 Break down of immunological tolerance leading to antibodies being produced
against self antigen and results in auto immune disease. Examples are,
myasthenia gravis, multiple sclerosis, Grave’s disease, Systemic lupus
erythematosis, etc.
Acquired immune deficiency syndrome (AIDS). It is caused by a retrovirus
(human immune deficiency virus), which binds to CD4 and helper T cells, causing
their number to decrease. The loss of helper T cells results in the absence of
proliferation of B cells and T cells. This results in the loss of immunity, and
spread of infection from nonpathogenic bacteria. It eventually leads to death of
the infected individual.
 During development, there could be a uncontrolled proliferation of B
lymphocytes, which gives chronic lymphocyte leukemia. When plasma cells
change into malignant growth; it results is multiple myeloma.

HAEMOGLOBIN
It is a red colour pigment present in the red cell. It is a chromoprotein with a
molecular weight of 64,5000. Haemoglobin normal concentration in males ranges
from 14 to 18g% and in females it ranges from 13 to 17g%. The function of this
pigment is to carry respiratory gases and being a protein, it takes up H+ ion and
hence helps in acid base regulation.

STRUCTURE OF HAEMOGLOBIN.
Haemoglobin contains haeme and globin. The haeme is made of protoporphyrin
and iron. There are 4 sub units in each haemoglobin molecule. The globin
consists of 4 polypetides and each one is attached to one sub unit of haeme. Thus,
there are 4 iron atoms in each molecule of haemoglobin. The globin polypeptides
are arranged in two pairs. The alpha chain contains 141 amino acids and the beta
chain is made up of 146 amino acid residues. Changes in the position of amino
acids in the globin polypepide units, will result in abnormal haemoglobin such as
sickle cell anemia. In the foetus, the haemoblogin is different from the adult
haemoglogin. The unit is replaced by unit in the globin molecule. The change
 in polypeptide chain in foetal haemoglobin, has a functional advantage. That is,
the foetal haemoglobin can be saturated fully at 40 to 45 mmHg of pO2, showing
greater diffusion of oxygen from maternal circulation to foetal circulation. The
foetal haemoglobin is replaced by the adult type soon after birth.
Haemoglobin is synthesized in the progenitor nucleated red cells, in red bone
marrow and the synthesis continues even after the cells are released into the
circulation, but restricted to only reticulocyte. Low level of synthesis, for a day or
two can be seen in reticulocyte. In RBC, the Hb synthesis cannot occur.
The haeme is synthesized separately and then conjugated, with globin
molecule. The globin that is utilized in the sythesis, is usually the recycled
molecule. Haeme is synthesized in mitochondria and defects in haeme synthesis
causes porphyries. Haeme is also a constituent of myoglobin and cytochrome
enzyme.

TYPES OF HAEMOGLOBIN
 Adult Hb: It contains the globin as and units.
 Foetal Hb: The globin has and units.
 Thalassemia It is inherited as autosomal recessive character, in which, there is
deficient synthesis of and chains.
 Haemoglobin S (sickle cell anaemia) It is an abnormal Hb, formed from a
mutant gene and inherited as a trait in population. It is common in west
African population. The red cells are sickle shaped and this shape provides
osmotic resistance to the red cell against malarial infection. The hemolysis of
sickle cells when occurs, gives sickle cell anaemia.

DERIVATIVES OF HAEMOGLOBIN
Oxyhaemoglobin: When Hb combines with oxygen this is formed.
Deoxygenated Hb: The dissociation of oxygen from Hb gives this derivative.
Carboxy Hb: It is produced, when Hb combines with carbon mono oxide. The
affinity of Hb for CO is very high. Its affinity is 200 times greater than the affinity it
has for oxygen.
Methemoglobin: When ferrous (Fe++) in Hb is oxidized to ferric (Fe+++), it is called
methamoglobin. Exposure of red cells to drugs like phenacetin, acetanilide and
chemicals like nitrates, aniline dye, etc. will cause methaemoglobin formation.
Sulfhaemoglobin: The reaction of Hb with hydrogen sulphide gives this compound.

FATE OF HAEMOGLOBIN
When haemoglobin is released from red cells, as result of haemolysis, it is taken up
by a carrier protein haptoglobin in plasma. In the liver and spleen, the Hb is separated
from haptoglobin and catabolised.
Haemoglobin is also released from red cells, when hemolysis occurs in tissue
macrophages in spleen, liver and bone marrow. The Figure 6.11 shows the break
down of haemoglobin and formation of bile pigments (bilirubin and biliverdin). The
quantity of Hb, destroyed normally in a day is yg, which forms around 200 mg of bile
pigments (Normal concentration in plasma is 0.5 to 2mg%). Increased bile pigments
in the plasma is observed in jaundice.

BLOOD INDICES
A variation in the size and number of red cells or reduction in the concentration of
haemoglobin assumes clinical significance. The evaluation of blood indices helps to
identify the type of anaemia and plan appropriate treatment. Colour index is the ratio
between the Hb% and RBC%. Its normal value is 0.8 to 1.10. The value is low in
iron deficiency anaemia.

MEAN CORPUSCULAR VOLUME (MCV): It is the average volume of a single red

cell and it ranges from 90 to 93 3. In iron deficiency anaemia, the MCV is
decreased, while it is increased in megaloblastic anaemia.
MEAN CORPUSCULAR HEMOGLOBIN (MCH): It indicates the average content
of Hb in a single red cell. The value ranges from 28 to 32 picograms.
In iron deficiency, MCH is decreased.
MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC): It is the
relative percentage of Hb in a single red cell. Its normal value is 33%.
ANAEMIAS
Anaemia is defined as reduction in Hb% below normal for his or her age or decrease
in the circulating red cells or combination of both the factors.
Clinically, a person is said to be suffering from anaemia, when Hb% falls
below 13g% in males and 11.5g% in females. Anaemia itself is not a disease, but a
symptom of some underlying disorder and its correction depends on the treatment of
the underlying cause.

CLASSIFICATION OF ANAEMIAS
Anaemias can be classified based on their cause. Broadly it can be classified as:
 Anaemia resulting from decreased formation of red cells.
 Anaemia resulting from increased destruction of red cells.
 Anaemia resulting from blood loss.
LABORATORY INVESTIGATIONS AND CELL PICTURE
To find out the type of anaemia, the total red cell count, packed cell volume, Hb%,
colour index, MCV, MCH, MCHC, are to be carried out in a laboratory. Based on the
blood indices, the cell picture shows the following.
Normocytic Red cell volume is normal
Macrocytic Red cell volume is increased
Microcytic Red cell volume is decreased
Normochromic MCHC is normal
Hyperchromic MCHC is more than normal
Hypochromic MCHC is below normal
The cell picture in the peripheral circulation shows characteristic features
depending the type of anaemia. They are described in the discussion of various types
of anaemias.

ANAEMIA DUE TO DECREASED FORMATION OF RED CELLS

NUTRITIONAL ANAEMIAS: This type of anaemia is encountered when there is

deficiency of dietary factors such as vitamins, metals, and trace elements.
LACK OF VIT B 12: Vitamin B12 is the extrinsic factor (cyanocobalamin) and
requires the intrinsic factor presence in the gastric juice, for its absorption from the
terminal ileum. The lack of B12 in the diet, or auto immune disease destroying the
gastric parietal cells, will lead to pernicious anaemia. It is a megaloblastic anaemia,
in which large red cells appear inh the circulation. The cells also will be of unequal
sizes (Poikilocytes), the MCHC remain normal. The cell picture shows macrocytic
anaemia. That is macrocytic normochromic anaemia. Vitamin B12 is required for the
proliferation and maturation of red cells progenitors. Lack of this vitamin leads to
decreased production of red cells in the red bone marrow and arrest of maturation. In
this type of anaemia there is also involvement of neurological deficit, affecting the
myelin sheath of dorsal and lateral columns of the spinal cord. This condition is
called subacute combined degeneration of spinal cord.

FOLIC ACID DEFICIENCY: Folic acid is required for the proliferation and
maturation of erythroid cells. Lack of this vitamin causes megaloblastic anaemia,
similar to vit B12 deficiency, but without any neurological symptoms.

IRON DEFICIENCY: Iron is required for the haemoglobin formation. Iron is a

constituent of haeme. The protoporphyrin, which is formed, combines with iron to
form the haeme. The iron in the haemoblobin is necessary, as the oxygen is taken by
it and transported in the blood. Deficiency of iron causes hypochromic microcytic
anaemia. This type of anaemia is common in reproductive women, as there is loss of
iron in the menstrual blood. During pregnancy also iron deficiency anaemia is seen.

SUPPRESSED BONE MARROW FUNCTION (APLASTIC ANAEMIA):

Supression of bone marrow by drugs. X ray radiation, toxic agents etc., can cause
decreased production of all types of blood cells (panchytopenia).

CHRONIC DISEASES: Chronic infections, chronic inflammation or neoplasia cause

depression of erythropoiesis. The blood cell picture shows normochromic
normocytuc anaemia.

RENAL DISEASE: Chronic renal disease is usually associated with anaemia due to
lack of erythropoietin.
HYPOTHRODIDISM: Hypothroidism causes anaemia with the blood cell picture
being normocytic and normochromic. The anaemia is due to depressed metabolism of
the bone marrow.

II ANAEMIA ARISING FROM INCREASED DESTRUCTION OF RBC: They are

called hemolytic anaemias. Abnormal fed cells are produced, when there is a
hereditary defect. These abnormal red cells show a greater fragility. That is, these
red cells rupture easily and undergo hemolysis, thus reducing the life span of the cells
(corpuscular defect). It is also possible, that without any hereditary abnormality,
hemolysis can take place due to external factors (extracorpscular defect).

CORPUSCULAR DEFECT

HEREDITARY SPHEROCYTOSIS: The red cells are not biconcave, but spherical.
They are easily hemolysed when they pass through the small capillaries.

SICKLE CELL ANAEMIA: The RBC contains abnormal haemoglobin, where the
amino acid sequence in one of the globin chains is changed (valine instead of
glutamic acid in the sixth position of the chain). The haemoglobin is called HbS.
This gives sickle shaped red cells, which arte haemolysed easily in the vascular
system. The abnormal shape of the red cells also makes it susceptible for
phagocytosis. Sickle cell anaemia is common in West African population and gives
resistance to malarial parasite. That is, its schizont stage in the development can not
be formed in the red cell.

THALASSEMIA: The haemoglobin present in the red cell is an abnormal variant. It

could be due to the defective synthesis of globin chain. The red cells are small and
have a greater fragility causing haemolysis.

G-6-P-D (GLUCOSE-6-PHOSPHATE DEHYDROGENASE) DEFICIENCY:

Deficiency of this enzyme in RBC, causes the damage of the red cell membrane by
peroxidation. This is due to the absence of formation of reduced glutathione in the
red cell.

EXTRA CORPUSCULAR DEFECT: Haemolysis occurs, when red cells are exposed
to certain chemicals, drugs, snake venoms .
Hemolytic anaemia also can be seen by malarial parasite, viruses, bacterial
septicemia and nonprotozoam parasites.
To detect the presence of isoagglutinins and antibodies on the surface of the
red cells Coombs test is performed.

ANAEMIA RESULTING FROM BLOOD LOSS: Acute blood loss, as in

haemorrhage gives normocytic normochromic anemia. Chronic loss of blood as in
heavy menstrual blood flow, loss of blood in peptic ulcer, piles and hook worm
infestation, leads to microcytic hypochromic anaemia, similar to iron deficiency.