PLASMA AND ITS CONSTITUENTS 123.pptx

Contents:
o Introduction
o Serum
o Composition of plasma
o Plasma Proteins
o Albumin-Globulin Ratio
o Properties of Plasma Proteins
o Functions of Plasma Proteins
o Origin of plasma proteins
o Separation of plasma proteins
o Albumin
o Globulin
o Variations in plasma proteins
o Plasmapheresis
o Plasma Expanders

PLASMA
o A straw-colored clear liquid part of blood.
o The plasma is the liquid medium of blood (55-60%), in
which the cell components namely erythrocytes,
leukocytes, platelets are suspended
o It contains 91% to 92% of water and 8% to 9% of solids.
o The solids are the organic and the inorganic
substances.
Guyton and Hall Textbook of medical physiology, Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Plasma Proteins, 3rd edition

Serum :
o A clear straw-colored fluid that oozes from blood clot.
o For clinical investigations, serum is separated from blood cells and clotting
elements by centrifuging.
o It is different from plasma only by the absence of fibrinogen, i.e. serum
contains all the other constituents of plasma except fibrinogen.
SERUM = PLASMA - FIBRINOGEN

Composition of Plasma
K Sembulingam, Essentials of Medical Physiology, Blood, 6th
Edition

Plasma Proteins:
Plasma proteins:
1. Serum albumin
2. Serum globulin
3. Fibrinogen
Normal values of the plasma proteins are:
Total proteins : 7.3 g/dL (6.4 to 8.3 g/dL)
Serum albumin : 4.7 g/dL
Serum globulin : 2.3 g/dL
Fibrinogen : 0.3 g/dL
Guyton and Hall Textbook of medical physiology,Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Plasma Proteins, 3rd edition

Albumin-Globulin Ratio:
Normal A/G ratio is 1.2 to 1.5 : 1.
The A/G ratio is lowered either due to decrease in albumin or increase in globulins,
as found in the following conditions:
1.Decreased synthesis of albumin by liver
2.Excretion of albumin in urine in kidney damage
3.Increased production of globulins associated with chronic infections, multiple
myelomas etc.

Properties of Plasma Proteins:
oMolecular Weight:
Albumin : 69,000
Globulin : 1,56,000
Fibrinogen : 4,00,000
oSpecific Gravity:
Specific gravity of the plasma proteins is 1.026.

o Oncotic Pressure:
Osmotic pressure exerted by proteins in the plasma is called colloidal osmotic
(oncotic) pressure.
Normally, it is about 25 mm Hg. Albumin plays a major role in exerting oncotic pressure.
o Buffer Action:
Acceptance of hydrogen ions is called buffer action.
The plasma proteins have 1/6 of total buffering action of the blood.

Functions of Plasma Protein
Helps in
coagulation of
blood
Helps to maintain
colloidal osmotic
pressure
Maintaining
viscosity of blood
Maintaining
systemic arterial
blood pressure
constant
Provides
suspension stability
to blood
Acid-base balance
in the body
Immune Function Transport Function
Reservoir Function
Helps in nutrition of
tissues
Role as “Reserve
Protein"

Acid-base Balance:
• Plasma proteins act as buffers. Their buffering capacity is 1/6th (about 15%-16%) of total
buffering capacity of blood.
• They are amphoteric in nature and thereby maintain the blood pH at 7.4, by accepting or
donating H+.
• At physiological blood pH of 7.4, plasma proteins exist in an 'ionised' form i.e.
(i) 'C' terminal end is in the form of COO-
(ii) 'N' terminal end is in the form of -NH3+
Guyton and Hall Textbook of medical physiology, Regulation of Acid-Base Balance,11th Edition; K Sembulingam, Essentials of Medical Physiology, Acid-Base Balance, 6th
Edition

Colloidal Osmotic Pressure (COP):
i. COP is inversely proportional to the
molecular size and shape, and is
directly related to the concentration of
molecules.
ii. COP across the capillary wall helps to
maintain the exchange of fluid at
tissue level.

Origin of Plasma Proteins:
o IN EMBRYO: In embryonic stage, the plasma proteins are synthesized by the
mesenchyme cells.
The albumin is synthesized first and other proteins are synthesized later.
o IN ADULTS: In adults, the plasma proteins are synthesized mainly from
reticuloendothelial cells of liver.
The plasma proteins are synthesized also from spleen, bone marrow, disintegrating
blood cells and general tissue cells.
Gamma globulin is synthesized from B lymphocytes.

Separation of Plasma Proteins:
1)Precipitation Method
2)Salting-out method
3)Electrophoresis
4)Crohn’s Fractional Precipitation Method
5)Ultracentrifugation Method
6)Gel Filtration Chromatography
7)Immunoelectrophorectic Method

1) Precipitation Method:
o Proteins in the serum are separated into albumin and globulin.
o This is done by precipitating globulin with 22% sodium sulfate
solution.
o Albumin remains in solution.

2) Salting out method:
Serum globulin is separated into two fractions called euglobulin and pseudoglobulin by
salting out with different solutions.
o Euglobulin is salted out by,
1) Full saturation with sodium chloride solution.
2) Half saturation with magnesium sulfate solution.
3) One
-third saturation with ammonium sulfate solution.
o Pseudoglobulin is salted out by,
1) Full saturation with magnesium sulfate.
2) Half saturation with ammonium sulfate.

3) Electrophoresis:
It is done in a Tiselius apparatus by using paper or
cellulose or starch block.
By this method, the proteins are separated into-
a) albumin (55%)
b) alpha globulin (13%)
c) beta globulin (14%)
d) gamma globulin (11%)
e) fibrinogen (7%).

Abnormal Electrophoretic Pattern:
Electrophoresis of serum proteins is conveniently used for the diagnosis of certain
diseases
1.Multiple myeloma : A sharp and distinct M band appears in the γ-globulin
fraction.
2.Acute infections : α1- and α2- globulins are increased.
3.Nephrotic syndrome : Decreased albumin with sharp and prominent α2-globulin.
4.Primary immune deficiency : Diminished γ-globulin band.
5.α1-Antitrypsin deficiency: Diminished α1-globulin band

4) Crohn’s Fractional Precipitation Method:
By this method, plasma proteins are separated into albumin and different
fractions of globulin, depending upon their solubility
5) Ultracentrifugation Method:
In this method, albumin, globulin and fibrinogen are separated depending
upon their density.
This method is also useful in determining the molecular weight of these
proteins

6) Gel Filtration Chromatography:
o Gel filtration chromatography is a column chromatographic method by
which the proteins are separated on the basis of size.
o Protein molecules are separated by passing through a bed of porous
beads.
o The diffusion of different proteins into the beads depends upon their size

7) Immunoelectrophoretic Method:
o By this method, the proteins are separated on the basis of electrophoretic
patterns formed by precipitation at the site of antigen-
antibody reactions.
o This technique provides valuable quantitative measurement of different
proteins.

ALBUMIN:
The major constituent (60%) of plasma proteins with a concentration of
3.5-5.0 g/dl.
Human albumin has a molecular weight of 69,000.
Consists of a single polypeptide chain of 585 amino acids with 17
disulfide bonds.

 Synthesis of Albumin:
Albumin is exclusively synthesized by liver.
Liver produces about 12 g albumin per day which represents 25% of the
total hepatic protein synthesis.
Albumin has an half-life of 20 days.

 Functions of Albumin:
Osmotic function:
Due to its high concentration and low molecular weight, albumin contributes to 75-80% of the
total plasma osmotic pressure (25 mm Hg).
Transport function:
Plasma albumin binds to several biochemically important compounds and transports them in
the circulation.
Nutritive function:
Albumin serves as a source of amino acids for tissue protein synthesis to a limited extent,
particularly in nutritional deprivation of amino acids.
Buffering function:
Among the plasma proteins, albumin has the maximum buffering capacity.

 Clinical Significance of Albumins:
1. Albumin, binding to certain compounds in the plasma, prevents them from crossing
the blood-brain barrier.
2. Hypoalbuminemia (lowered plasma albumin) is observed in malnutrition, nephrotic
syndrome and cirrhosis of liver.
3. Albumin is excreted into urine (albuminuria) in nephrotic syndrome and in certain
inflammatory conditions of urinary tract.
4. Microalbuminuria (30-300 mg/day) is clinically important for predicting the future risk
of renal diseases.
5. Therapeutically useful for the treatment of burns and haemorrhage.

Globulin:
o Globulins constitute several distinct proteins that are separated into four distinct bands-
α1-globulin
α2-globulin
β-globulin
γ-globulin
o Globulins are, in general, bigger in size than albumin.

Forms of globulin:
1. Glycoprotein
2. Lipoprotein
3. Transferrin
4. Haptoglobins
5. Ceruloplasmin
6. Fetuin
7. Coagulation factors
8. Angiotensinogen
9. Haemagglutinins
10. Immunoglobulin

α1-Antitrypsin(α-anti-proteinase) :
o A glycoprotein with 394 amino acids and a molecular weight of 54,000.
o A major constituent of α1-globulin fraction of plasma proteins with a normal
concentration of about 200 mg/dl.
o α1-Antitrypsin is a serine protease inhibitor.
o It combines with trypsin, elastase and other protease enzymes and inhibits their
activity.

 Clinical significance of α1-AT:
1.Emphysema: At least 5% of emphysema cases are due to the deficiency of α1 -AT.
2.Effect of smoking on α1- AT:
3.α1-Antitrypsin deficiency and liver disease
Smoking causes oxidation of
methionine to methionine
sulfoxide
Cannot bind and inactivate
proteases
Accumulation of
mutant α1-AT
aggregates to form
polymer
Polymer causes liver
damage(hepatitis)
Accumulation of
collagen resulting in
fibrosis(cirrhosis)

α-2 Macroglobulin:
o A high molecular weight (8,00,000) protein.
o A major constituent of α2-fraction.
o α-2 Macroglobulin inhibits protease activity and serves as an anticoagulant.
o It’s concentration in plasma is elevated in nephrotic syndrome.

Haptoglobin (Hp) :
o A plasma glycoprotein with an approximate MW of 90,000.
o Hp is an acute phase protein since its plasma concentration is increased in several
inflammatory conditions.
o Haptoglobin binds with the free hemoglobin (known as extra-corpuscular hemoglobin) that
spills into the plasma due to hemolysis.
o Haptoglobin- haemoglobin (Hp-Hb) complex (mol. wt. 155,000) cannot pass through
glomeruli of kidney while free Hb (mol. wt. 65,000) can.
o Hemolytic anemia is associated with decreased plasma concentration of haptoglobin.

Ceruloplasmin:
o A blue colored, copper-containing α2-globulin with MW of 150,000.
o Plasma concentration -30 mg/dl.
o Ceruloplasmin binds with almost 9O% of plasma copper (6 atoms of Cu bind to a
molecule).
o Albumin carrying only 10% of plasma copper is the major supplier of copper to the
tissues.
o Ceruloplasmin possesses oxidase activity, and it is associated with Wilson's disease.

Transferrin:
o Glycoprotein
o Molecular weight of 76,000.
o It is associated with β-globulin fraction.
o A transporter of iron in the circulation.

Acute Phase Protein:
o Acute phase response refers to a non-specific response to the stimulus of infection,
injury, various inflammatory conditions (affecting tissue/ organs), cancer etc.
o This phase is associated with a characteristic pattern of changes in certain plasma
proteins, collectively referred to as acute phase protein.
o Negative acute phase reactants-
1. Albumin
2.Transferrin.

C-reactive Protein:
o CRP is a major component of acute phase proteins.
o It is produced in the liver and is present in the
circulation in minute concentration (< 1 mg/dl).
o C-reactive protein is involved in the promotion of
immune system through the activation of complement
cascade.
o Estimation of CRP in serum is important for the
evaluation of acute phase response.

Immunoglobulins:
o Chemical nature of antibodies is globulin and they are named as
immunoglobulins.
o Serum globulins could be separated into pseudoglobulins (water soluble) and
euglobulin (water insoluble).
o Immunoglobulins constitute about 20 to 25 % of the total serum proteins.
o Based on sedimentation studies, most antibodies are sedimented at 7S (M.W
1,50,000- 1,80,000).
Guyton and Hall Textbook of medical physiology, Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Immunoglobulins, 3rd edition

o Tiselius and Kabat (1938) showed that most serum antibodies on electrophoretic
mobility, belong to gamma-globulins which is used now synonymously with antibody.
o The term 'Immunoglobulin' was proposed by expert committee of WHO in 1964.
o Immunoglobulins are mainly synthesised by plasma cells.
o Immunoglobulin includes, besides antibody globulins, the abnormal myeloma proteins,
the cryoglobulin and the macroglobulin.
o Thus, all immunoglobulins may not be antibodies but all antibodies are
immunoglobulins.
Guyton and Hall Textbook of medical physiology,Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Immunoglobulins, 3rd edition

Structure of Immunoglobulin:
o Porter, Edelman and Nisonoff (1959-64) developed a
technique for cleavage of immunoglobulin molecule.
o Structure of antibody consists of-
Two identical heavy (H) chains
Two identical Light (L) chains.
o Both type of chains are polypeptide in nature.
o The two heavy chains are held together by disulphide
(S-S) bonds.

o The H chains are structurally and antigenically distinct in different classes of
immunoglobulins.
o The L chains present in two forms kappa (K) and lambda (L).
Each immunoglobulin has either two kappa or two lambda light chains but both (K &
L) are never found together in a molecule.
Kappa and Lambda chains are present in a ratio of 2: 1 in human sera.

Effect of enzymes:
A)Papain digestion:
o Porter and colleagues split rabbit lgG antibody to egg albumin, by a proteolytic enzyme
papain in the presence of cysteine.
o Papain can digest immunoglobulin molecule into three fragments-
a) One Fc fragment (Fc crystallisable)
b) Two identical Fab (fragment antigen binding) fragments.
o Each Fab fragment consists of a light chain and a part of H chain. The portion of the
heavy chain in Fab fragment is named Fd region.
Guyton and Hall Textbook of medical physiology,Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Plasma, Immunoglobulins, 3rd edition.

B)Pepsin Digestion:
o Pepsin cleaves at a different point of
immunoglobulin molecule and gives rise
to- a)Fc portion
b)Two Fab fragments
o This Fab fragment is bivalent and can still
precipitate with antigen. It is called
F(ab’)2.
o Pepsin also degrades the Fc portion into
smaller fragments.
Guyton and Hall Textbook of medical physiology, Overview of Circulation,11th Edition; U Satyanarayan Biochemistry, Plasma Immunoglobulins, 3rd edition

Amino acid Sequences:
H-chain L-chain
M.W. 50,000 20,000
Aminoacids 420-460 210-230
o Both L and H chains consist of two portions each, a variable (V) region and a constant (C)
region.
o In the L chain the two regions are of equal length while in the H chains the variable region
constitutes approximately only a fifth of the chain.
o Variable (V) regions at aminoterminus (NH2)
Constant (C) region at carboxyterminus (COOH).

o The infinite range of the antibody specificity of immunoglobulins is due to the
'variable regions' of the H and L chains.
o There are many hypervariable regions present in variable portions of both H and L
chains and are involved with the formation of the antigen binding sites.
o Complementarity determining regions or CDRs: The sites on the hypervariable
regions which make actual contact with the epitope.
o There are three hypervariable regions in the L and four in the H chain.

Immunoglobulin Domains:
o Immunoglobulins are folded to form globular variable and constant domains.
o In each heavy chain,
1) One in variable region (VH)
2) Three in constant region (CHI, CH2, CH3).
There is one additional fourth domain on heavy chain (CH4) in lgM and lgE molecule.
o Light chain has one domain in variable region (VL) and one in constant region (CL).
o The variable region domains (VL and VH) are responsible for the formation of a specific
antigen binding site.
o The constant region domains mediate the secondary biological functions.

Immunoglobulin Antigen Determinants
1.Isotypes:
o These determinants are shared by all members of the same species.
o On the basis of isotypic markers on H chains, different classes of immunoglobulins
are differentiated .
o Various H chain markers are gamma, mu, alpha, delta and epsilon in
immunoglobulins IgG, IgM, IgA, IgD and lgE respectively.
o The light chains are also distinguished through isotypic markers into kappa and
lambda.

2. Allotypes:
o These are individual specific determinants within a species.
o The allotype markers in humans are
1) Gm on gamma heavy chains,
2) Am on alpha heavy chains
3) Km (originally designated as Inv) on kappa light chains.
o No allotypic markers have been found for lambda light chains or µ, δ or ε heavy chains.
o Allotype markers are useful in testing paternity.

3. Idiotypes:
o Idiotype markers are located in hypervariable regions of the immunoglobulin
molecule.
o Idiotypes are specific for each antibody molecule.
o By immunization with Fab fragments, antiidiotypic antibodies can be produced.

IgG:
o Major serum immunoglobulin with normal
serum concentration of 8-16mg/ml.
o 1,50,000 MW
o Half life of 24 days.
o It is the only immunoglobulin that is
transported through placenta.
o Provides natural passive immunity to
newborn.

o It is distributed equally between the intravascular and extravascular compartments.
o IgG appears late but persists for longer period.
It appears after the initial immune response which is IgM in nature.
o It participates in precipitation, complement fixation and neutralization of toxin and viruses.
o IgG binds to microorganisms and enhances the process of phagocytosis.

o Extracellular killing of target cells coated with IgG is due to recognition of Fe receptor of
IgG by killer cells bearing the appropriate receptors.
o Passively administered IgG suppresses the homologous antibody synthesis by feed back
mechanism.
o Based on this property, isoimmunization of women is done by administration of anti-RhD
IgG during delivery.
o It is protective against those microorganisms which are active in the blood and tissues.
Subclass IgG 1 IgG 2 IgG 3 IgG 4
Distribution 65% 23% 8% 4%

IgA :
o Second major immunoglobulin with normal serum concentration of 0.6 to 4.2 mg/ml.
o Half-life of 6-8 days
o The principal immunoglobulin present in secretions such as milk, saliva, tears, sweat,
nasal fluids, colostrum and in secretions of respiratory, intestinal and genital systems.
o IgA occurs in two forms-
1) Serum IgA
2) Secretory IgA
o Serum IgA is a monomeric 7S molecule (1,60,000 MW)

Secretory IgA:
o Found on mucosal surfaces and in secretions
o MW 400,000
o A dimer formed by two monomer units joined
together by a glycoprotein named the J chain.
o Secretory IgA and J chain are produced by the
plasma cells situated near the mucosal or
glandular epithelium
o Secretory IgA contains another glycine rich
polypeptide called the secretory piece or
secretory component.

o IgA protects the mucous membranes against microorganisms.
o IgA covers the microorganisms to inhibit their adherence to mucosal surfaces.
o IgA does not fix complement but can activate alternative complement pathway.
o IgA is mainly synthesized locally by plasma cells and little is derived from serum.
o Two subclasses of IgA (IgA1 and IgA2) are known.
o IgA2 is predominant (60%) in the secretions but constitutes a minor part of serum IgA.

IgM :
o A pentamer consisting of 5 immunoglobulin
subunits and one molecule of J chain, which joins
the Fe region of the basic subunits.
o Each H chain of IgM has four CH domains rather
than three as seen in H chain of IgG molecule.
o It constitutes about 5- 8 percent of total serum
immunoglobulins.
o Half life is about five days.
o ‘Millionaire Molecule'.

o IgM is mainly distributed intravascularly (80%).
o It is the earliest synthesized immunoglobulin by foetus in about 20 weeks of age.
o It appears early in response to infection before IgG. IgM antibodies are short lived, and
disappear earlier than IgG. Hence, its presence in serum indicates recent infection.
o It can not cross the placenta, presence of IgM antibody in serum of newborn indicates
congenital infection.
o Its detection is, therefore, useful for the diagnosis of congenital syphilis, HIV infection,
toxoplasmosis and rubella.

o It is very effective antibody in agglutination, complement fixation, opsonization and
immune hemolysis.
o IgM provides protection against blood invasion by microorganisms. Its deficiency is
often associated with septicemia.
o The isohaemagglutinins (Anti-A, Anti-B), antibodies to typhoid 'O' antigen (endotoxin),
rheumatoid factor and WR. antibodies in syphilis belong to IgM.

IgE :
o IgE is mainly produced in the linings of respiratory and intestinal tracts.
o It is mostly distributed extravascularly.
o Also referred to as reagins.
o Molecular weight is 1,90,000 (8S molecule).
o Half life is 2-3 days.
o It resembles IgG in structure.
o It is heat labile (inactivated at 56°C in one hour)

o It has affinity for surface of tissue cells, particularly mast cells of the same species
(homocytotropism).
o IgE mediates type I hypersensitivity (atopic) reaction.
o High level of IgE in serum is also seen in children with a high load of intestinal parasitism.
o It cannot cross the placental barrier or fix the complement.
o IgE is responsible for anaphylactic type of reaction.
o It may play some unidentified role in defence against intestinal parasitic infections.

Role of Different Immunoglobulins:
1. IgG - protects the body fluids
2. IgA - protects the body surfaces
3. IgM - protects the blood stream
4. IgE - mediates reaginic hypersensitivity
5. IgD - recognition molecule on the surface of B lymphocytes

Immunoglobulin Classes:

Common variable immunodeficiency:
• Also known as adult-onset agammaglobulinaemia, late-onset
hypogammaglobulinaemia or acquired agammaglobulinaemia, common
variable immunodeficiency (CVID) is relatively common.
• Serum IgG is low with a marked decrease in IgM or IgA. The serum IgM
concentration is normal in about half of the patients.
• Most patients have normal B-cell numbers, though some do have low or
absent B cells.
• Abnormalities in T-cell numbers or function are common.
Scully’s Medical Problems in Dentistry, Immunodeficiencies, 7th
Edition

Clinical Features:
• Symptoms are mainly frequent bacterial infections of ears, sinuses, bronchi and lungs.
• Gastrointestinal disease, including chronic diarrhoea, affects as many as 30%.
• There is a high incidence of inflammatory bowel disease, gluten-sensitive enteropathy
and nodular lymphoid hyperplasia..
• Hematological abnormalities may include autoimmune haemolytic anaemia, immune
thrombocytopenia, leukopenia and pernicious anaemia.
• Arthralgia in the knee, ankle, elbow or wrist is common.
• Rheumatoid arthritis and other collagen vascular diseases are also more common than
expected.
• Splenomegaly and lymphadenopathy may be seen.
Edition

General Management:
• Diagnosis is confirmed by finding low serum IgG, IgA and possibly IgM.
• B cells still produce antibodies after a common vaccination such as tetanus.
• Antibody levels can be normalized by intravenous immunoglobulin every 3–
4 weeks.
• Bacterial infections are treated with antibiotics.
• Physical therapy and daily postural drainage may help clear respiratory
infections.
Edition

Selective IgA Deficiency:
Clinical Features:
o Many individuals with IgA deficiency are asymptomatic if the levels of other
classes of immunoglobulin are normal or raised.
o Some have an increased incidence of upper respiratory tract infections, allergies
and autoimmune disease but a normal IgG antibody response to vaccination.
o Where IgA deficiency is associated with deficiency of IgG2, there is a definite
predisposition to recurrent bacterial or viral respiratory infections, and small
intestine infections – usually with Giardia lamblia.
Edition

General management:
o IgA replacement is difficult since autoantibodies frequently form against IgA when blood
transfusions or immunoglobulins are given, and can sometimes cause anaphylactic
reactions.
o Bacterial infections should be treated with antibiotics.
o Giardiasis needs treatment with metronidazole or quinacrine hydrochloride.
Edition

Dental aspects:
o Despite the fact that IgA is the main salivary antibody, reported effects of IgA deficiency
on dental caries and other oral disease are conflicting.
o Part of the reason for this apparently anomalous situation is that other immunoglobulins
may be secreted in saliva.
o IgA deficiency may be associated occasionally with oral ulcers and herpes labialis.
Edition

X-linked Agammaglobulinaemia (Bruton Syndrome):
o Patients with X-linked agammaglobulinaemia (XLA) are male with low CD19+ B
cells and low serum IgG, IgM and IgA, no isohaemagglutinins and/or a poor
response to vaccines.
o They develop recurrent bacterial infections in the first 5 years of life, particularly
otitis, sinusitis and pneumonia. Approximately 20% of patients present with a
dramatic, overwhelming infection, often also with neutropenia.
o Another 10–15% have higher concentrations of serum immunoglobulin than
expected or are not recognized to have immunodeficiency until after 5 years of
age.
Edition

Variations in Plasma Protein:
Hyperproteinaemia:
1. Dehydration
2. Acute hepatitis
3. Rheumatid arthritis
4. Leukemia
5. Excess of alcohol
Hypoproteinaemia:
6. Diarrhea
7. Haemorrhage
8. Burns
9. Pregnancy
10. Malnutrition
11. Cirrhosis of liver
12. Chronic hepatitis

Albumin:
Conditions when increased:
1. Dehydration
2. Excess of glucocorticoids
Conditions when decreased:
4. Burns
5. Nephrotic Syndrome

Globulin:
2. Nephrotic Syndrome
3. Rheumatoid Arthritis
4. Chronic infections
5. Emphysema
6. Acute Haemolytic Anaemia
7. Glomerulonephritis
8. Hypogammaglobulinemia

Fibrinogen:
1. Acute infections
2. Glomerulonephritis
3. Rheumatoid Arthritis
4. Myocardial Infraction
5. Trauma
6. Liver Dysfunction

Variations in plasma proteins
U Satyanarayan Biochemistry, Plasma Proteins, 3rd edition

Plasmapheresis:
Plasmapheresis is an experimental procedure done in animals to demonstrate the
importance of plasma proteins.
Earlier, this was called Whipple’s experiment because it was established by George Hoyt
Whipple.
The experiment ‘plasmapheresis’ is used to demonstrate:
1. Importance of plasma proteins for survival
2. Synthesis of plasma proteins by the liver

Therapeutic Plasma Exchange:
Plasmapheresis is used as a blood purification procedure for an effective temporary treatment
of many autoimmune diseases. It is called Therapeutic Plasma Exchange.

Uses of Plasmapheresis:
Plasmapheresis is an effective temporary treatment for the following diseases:
1. Myasthenia gravis
2. Thrombocytopenic purpura
3. Paraproteinemic peripheral neuropathy
4. Chronic demyelinating polyneuropathy
5. Guillain-
Barré syndrome
6. Lambert-
Eaton myasthenic Syndrome

Plasma Expanders:
High MW substances that exert osmotic effect and retain fluid in blood vessels when
infused IV.
These are used to correct hypovolemia due to blood loss as in trauma.
• Albumin:
It does not interfere with blood grouping or coagulation and is free of risk of transmission of
hepatitis (as it is heat treated).
• Dextran:
These can interfere with blood grouping, coagulation and platelet function.
These can reduce blood viscosity, decrease rouleax formation and improve
microcirculation.

• Dextran-40 Dextran-70 Polygeline Hetastarch
• MW.- 40,000 • MW.- 70,000 • MW.- 30,000 • MW.- 4.5 Lac
• Expands
plasma for <
24 hours.
• Expands
plasma for 24
hours.
• Expands for 12
hours.
• Expands
plasma for >24
hours.
• Stable for 10
years
• Stable for 10
years.
• Stable for 3
years.
• Interfere with
blood
grouping and
cross matching
(BGCM)
• Interfere with
BGCM
• Do not
interfere with
BGCM.
• Interfere with
BGCM.
• Interfere with
coagulation
and platelet
function
• Most
commonly
used.

Contents :
o Introduction
o Morphology
o Structure and composition
o Development of platelets
o Lifespan and fate of platelets
o Investigations
o Variations in platelet count
o Properties of platelets
o Functions of platelets
o Activators and inhibitors of platelet
o Platelet Disorders
o Haematopoietic Growth Factors
o Anti-platelet Drugs

o Platelets are small colorless, non-nucleated and moderately refractive bodies.
o These formed elements of blood are considered to be the fragments of cytoplasm.
o Normal Platelet Count is about 1.5 to 4 hundred thousands/μl, in the peripheral
blood.
o A critical count of platelets –
Platelet count less than 40,000 /μl.
o When platelet count becomes < 40,000 /μl , symptoms (purpura) develop.
Guyton and Hall Textbook of medical physiology, Hemostasis and Blood Coagulation,11th Edition; K Sembulingam, Essential of Medical Physiology, Platelets, 6th
Edition
Introduction:

Morphology:
o Small( diameter between 2-4 μm)
o Volume : 7.5 cu µ (7 to 8 cu µ).
o Non-nucleated mass of protoplasm.
o When inactive, platelet has a disc like structure
o When active, it becomes spherical.
o Inactivated platelets are without processes or filopodia
and the activated platelets develop processes or
filopodia.
Edition
Inactivated Platelets
Activated Platelets

Structure and Composition:
Platelet is constituted by:
1. Cell membrane or surface membrane
2. Microtubules
3. Cytoplasm
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Cell Membrane:
o Cell membrane of platelet is 6 nm thick.
o Extensive invagination of cell membrane forms an open canalicular system.
o Cell membrane of platelet contains lipids in the form of-
1) phospholipids
2) cholesterol and glycolipids
3) carbohydrates as glycocalyx and glycoproteins and proteins.
o The membrane of platelet has 2 layers:
1) Outer glycocalyx layer
2) Inner lipoprotein layer
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Glycoproteins:
o Glycoproteins prevent the adherence of platelets to normal endothelium
o Accelerate the adherence of platelets to collagen and damaged endothelium in ruptured
blood vessels.
o Glycoproteins also form the receptors for ADP and thrombin.
Phospholipids:
o Phospholipids accelerate the clotting reactions.
o The phospholipids form the precursors of thromboxane A2 and other prostaglandin-related
substances
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Tubules within the platelet are of two varieties:
Open tubules-
Open tubules communicate with the ECF.
During activation, from ECF enter inside of
the platelets via these open tubules.
Dense tubules-
Do not communicate with the exterior.
These tubules store calcium.
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Cytoplasm:
Cytoplasm of platelets contains the cellular organelles.
Cytoplasm also contains some chemical substances such as proteins, enzymes,
hormonal substances, etc.
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Enzymes:
1. Adenosine triphosphatase (ATPase)
2. Enzymes necessary for synthesis of prostaglandins.

Proteins:
1. Contractile proteins-
i. Actin and myosin.
ii. Thrombasthenia
2. von Willebrand factor
3. Fibrin-stabilizing factor
4. Platelet-derived growth factor(PDGF)
5. Platelet-activating factor (PAF)
6. Vitronectin (serum spreading factor)
7. Thrombospondin.
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Hormonal Substances:
1. Adrenaline
2. 5-hydroxytryptamine (5-HT; serotonin)
3. Histamine
Other Chemical Substances:
1. Glycogen
2. Substances like blood group antigens
3. Inorganic substances such as calcium, copper, magnesium and iron.
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o Platelet Granules:
Granules present in cytoplasm of platelets are of two types:
1.Alpha granules
2. Dense granules.
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Pluripotent Cell CFU-M Megakaryocyte Platelet
Development of Platelet

Lifespan and Fate of platelet:
o Average lifespan of platelets is 10 days.
o It varies between 8 and 11 days.
o Platelets are destroyed by tissue macrophage system in spleen.
o Splenomegaly decreases platelet count and
o Splenectomy increases platelet count.
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Investigations:
1. Platelet Count:
Platelet count is a test that gives the average number of platelets per microliter
of blood.
2. Mean Platelet Volume:
A Mean Platelet Volume (MPV) blood test measures the average size of your
platelets.
The ideal range of number of platelets is from 200,000 to 400,000
platelets/mcL, while the average volume of a platelet is about 9.4 to 12.3 fL
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Variations in platelet count:
PHYSIOLOGICAL VARIATIONS:
1. Age: Platelets are less in infants (1,50,000 to 2,00,000/cu mm) and reaches normal
level at 3rd month after birth.
2. Sex: In females, it is reduced during menstruation.
3. High altitude: Platelet count increases.
4. After meals: After taking food, the platelet count increases.
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Properties of Platelets:
1) Adhesiveness :
Adhesiveness is the property of sticking to a rough surface.
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Injury to blood vessel
Endothelium is damaged
and subendothelial
collagen is exposed.
Platelet gets activated
Platelets get adhere to
the collagen

2) Aggregation:
o Aggregation is the grouping of platelets.
o Adhesion is followed by activation of more number of
platelets by substances released from dense granules of
platelets.
o During activation, the platelets change their shape with
elongation of long filamentous pseudopodia which are called
processes or filopodia.
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Activated Platelet

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3) Agglutination:
Agglutination is the clumping together of platelets.
Aggregated platelets are agglutinated by the actions of some platelet
agglutinins and platelet-activating factor.
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Functions of Platelets:
Activated platelets immediately release many substances. This process is known as
Platelet release reaction.
1) Role in clot retraction:
In the blood clot, blood cells including platelets are entrapped in between the fibrin
threads.
Cytoplasm of platelets contains the contractile proteins, namely actin, myosin and
thrombasthenia, which are responsible for clot retraction.
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2) Role in prevention of blood loss:
Platelets accelerate the hemostasis by three ways:
I. Platelets secrete 5-HT, which causes the constriction of blood vessels.
II. Due to the adhesive property, the platelets seal the damage in blood vessels like
capillaries.
III. By formation of temporary plug, the platelets seal the damage in blood vessels
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K Sembulingam, Essential of Medical Physiology, Hemostasis, 6th
Edition

3)Role in blood clotting:
Platelets are responsible for the formation of intrinsic prothrombin activator.
This substance is responsible for the onset of blood clotting.
4)Phagocytic function:
Platelets help in 'phagocytosis' of carbon particles, viruses and immune complexes.
5)Storage and transport function:
Platelets store 5-HT and histamine, which are released when the platelets
disintegrate and act on the blood vessel.
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Activators and Inhibitors of Platelets:
Activators of Platelets
1. Collagen, which is exposed during
damage of blood vessels
2. von Willebrand factor
3. Thromboxane A2
4. Platelet-activating factor
5. Thrombin
6. ADP
7. Calcium ions
8. P-selectin
9. Convulxin
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Inhibitors of Platelets
1. Nitric oxide
2. Prostacyclin
3. Nucleotidases which breakdown
the ADP

Platelet Disorders:
Disorders of platelets produce bleeding disorders by one of the following 3
mechanisms:
A. Due to reduction in the number of platelets i.e. various forms of
thrombocytopenia.
B. Due to rise in platelet count i.e. thrombocytosis.
C. Due to defective platelet functions
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A) Thrombocytopenia:
o Thrombocytopenia is defined as a reduction in the peripheral blood platelet count below
the lower limit of normal i.e. below 150,000/µl.
o However, spontaneous hemorrhagic tendency becomes clinically evident only after
severe depletion of the platelet count to level below 20,000/µl.
o Thrombocytopenia may result from 4 main groups of causes:
1. Impaired platelet production
2. Accelerated platelet destruction
3. Splenic sequestration
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Causes of Thrombocytopenia
Davidson's Principle and Practice of Medicine, Presenting Problems In Blood Diseases, 23rd Edition

Drug induced thrombocytopenia:
Drug-induced thrombocytopenia is associated with many commonly used drugs
and includes:
1.Chemotherapeutic agents (alkylating agents, anthracyclines, antimetabolites)
2.Certain antibiotics (sulfonamides, PAS, rifampicin, penicillin)
3.Drugs used in cardiovascular diseases (digitoxin, thiazide diuretics)
4.Diclofenac
5.Acyclovir
6.Heparin
7.Excessive consumption of ethanol.
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o Clinically, the patient presents with acute purpura.
o The platelet count is markedly lowered, often below 10,000/µl
o The bone marrow shows normal or increased number of megakaryocytes.
o The immediate treatment is to stop or replace the suspected drug with instruction to the
patient to avoid taking the offending drug in future.
o Occasional patients may require temporary support with glucocorticoids,
plasmapheresis or platelet transfusions.
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Heparin-induced thrombocytopenia:
Thrombocytopenia due to administration of heparin is distinct from that caused by other
drugs in following ways:
i) Thrombocytopenia is generally not so severe to fall to level below 20,000/µl.
ii) Unlike drug-induced thrombocytopenia, heparin-induced thrombocytopenia is not
associated with bleeding but instead these patients are more prone to develop
thrombosis.
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o The underlying mechanism of heparin-induced thrombocytopenia is formation
of antibody against platelet factor 4 (PF-4)-heparin complex.
o Diagnosis is made by a combination of laboratory and clinical features with 4
T’s:
1.Thrombocytopenia,
2.Thrombosis,
3.Time of fall of platelet count,
4.Absence of other causes of thrombocytopenia.
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Immune Thrombocytopenic Purpura (ITP):
o ITP includes two clinical subtypes:
1) Chronic ITP is a relatively common disorder that tends to affect women
between the ages of 20 and 40 years.
2) Acute ITP is a self-limited form seen mostly in children after viral infections.
o Antibodies directed against platelet membrane glycoproteins IIb/IIIa or Ib/IX
complexes can be detected in roughly 80% of cases of chronic ITP.
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 Clinical Manifestations:
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 LABORATORY FINDINGS:
1.Platelet count is markedly reduced (10,000-50,000/µl).
2.Blood film shows only occasional platelets which are often large in size
3.Bone marrow shows increased number of megakaryocytes which have large non-
lobulated single nuclei and may have reduced cytoplasmic granularity and presence of
vacuoles
4.With sensitive techniques, anti-platelet IgG antibody can be demonstrated on platelet
surface or in the serum of patients.
5.Platelet survival studies reveal markedly reduced platelet lifespan, sometimes less than
one hour, as compared with normal lifespan of 7-10 days
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 Management:
First-line therapy-
o Glucocorticoids, either prednisolone (1 mg/kg daily) or dexamethasone (40 mg
daily for 4 days).
o Administration of intravenous immunoglobulin can raise the platelet count by
blocking antibody receptors on reticulo-endothelial cells.
o Persistent or potentially life-threatening bleeding should be treated with platelet
transfusion in addition to the other therapies.
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Second line therapy-
o If a patient has two relapses or primary refractory disease, second-line therapies
are considered.
o The options for second-line therapy include –
1) Thrombopoietin receptor agonists (TPO-RA) eltrombopag and romiplostim
2) Splenectomy
3) Immunosuppression.
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B) Thrombocytosis:
Thrombocytosis is defined as platelet count in excess of 4,00,000/µl.
Essential Thrombocytosis-
A clonal disorder characterized by markedly elevated platelet count in the
absence of any recognizable stimulus.
Reactive Thrombocytosis-
Secondary or reactive thrombocytosis, on the other hand, occurs in response
to known stimuli such as: chronic infection, hemorrhage, postoperative state, chronic
iron deficiency, malignancy, rheumatoid arthritis and post splenectomy.
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Essential Thrombocytosis-
ET is an uncommon disorder and represents an overproduction of platelets from
megakaryocyte colonies without any added stimulus but no clonal marker is
available to distinguish primary from secondary thrombocytosis.
Pathophysiology:
There is a probable role of heredity in ET since families with ET have been reported.
Edition
ET is the absence of control by
thrombopoietin
Uncontrolled proliferation of not
only megakaryocytes but also of
the platelets

Davidson's Principle and Practice of Medicine, Presenting problems in blood diseases, 23rd Edition

C) Due to defective platelet function:
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Defective
platelet
function
Hereditary
Defective
platelet
adhesion
Defective platelet
aggregation
Disorders of
platelet release
reaction
Acquired
Aspirin
Therapy
Others

Defective platelet adhesion:
a) Bernard-Soulier Syndrome-
An autosomal recessive disorder with inherited deficiency of a platelet
membrane glycoprotein which is essential for adhesion of platelets to vessel wall..
A full blood count reveals giant platelets.
Platelets fail to aggregate in response to vWF.
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b) von Willebrand’s Disease-
• In Von Willebrand’s disease, there is defective platelet adhesion as well as deficiency of
factor VIII.
• Excessive tendency to bleed
• Dental extraction in any patient with clotting factor defects can result in a delayed
bleeding episode.
• Local hemostatic measures provide effective results in a majority of cases but are
insufficient in patients with severe hemophilia A and vWF.
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Defective platelet aggregation:
a)Glanzmann’s thrombasthenia-
It is an inherited hemorrhagic disorder, caused by structural or functional abnormality
of platelets.
It leads to thrombasthenia purpura.
However, the platelet count is normal.
Normal clotting time, normal or prolonged bleeding time but defective clot retraction.
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Clinical Manifestations:
Excessive bleeding (often manifested as Gingival Bleeding, Epistaxis, Menorrhagia)
Gingival Bleeding Epistaxis Menorrhagia

Management:
o Management includes avoiding trauma and avoiding analgesics such as aspirin.
o Minor bleeding intraorally can be managed with tranexamic acid. Major bleeding
may require platelet transfusion.
o Recombinant human-activated factor VII (rFVII) used in patients with antibodies to
GP IIb/IIIa and/or human leukocyte antigen (HLA) that render transfusions
ineffective.
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Disorders of platelet release reaction:
These disorders are characterized by normal initial aggregation of platelets with ADP
or collagen but the subsequent release of ADP, prostaglandins and 5-HT is defective
due to complex intrinsic deficiencies
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Acquired Disorders:
Acquired defects of platelet functions include the following clinically significant
examples:
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Aspirin therapy
• Prolonged use of aspirin leads to easy
bruising and abnormal bleeding time.
• The anti-platelet effect of aspirin is
clinically applied in preventing major
thromboembolic disease in recurrent
myocardial infarction.
Others
• Several other acquired disorders are associated
with various abnormalities in platelet functions at
different levels.
• These include: Uremia, Liver Disease, Multiple
Myeloma, Waldenstrom’s Macroglobulinaemia and
various myeloproliferative disorders.

Pancytopenia:
o Pancytopenia refers to the combination of anaemia, leucopenia and
thrombocytopenia.
o It may be due to reduced production of blood cells as a consequence of bone
marrow suppression or infiltration, or there may be peripheral destruction or splenic
pooling of mature cells.
o A bone marrow aspirate and trephine are usually required to establish the diagnosis.
Davidson's Principle and Practice of Medicine, Presenting problems in blood diseases, 23rd Edition

Scully’s Medical Problems in Dentistry, Platelet Disorders, 7th
Edition

Dental Aspects of Platelet Disorders:
• LA can be given if the platelet levels are above 30 × 109
/L.
• Hemostasis after dentoalveolar surgery is usually adequate if platelet levels are above
50 × 109
/L.
• Major surgery requires platelet levels above 75 × 109
/L. CS can be given but if it is
delivered by the intravenous route, care must be taken not to damage the vein.
• GA can be given in hospital but expert intubation is needed to avoid the risk of
submucous bleeding into the airway.
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• Platelets can be replaced or supplemented by platelet transfusions (1 unit of platelets
should raise the count by around 10 × 109
platelets/L).
• But sequestration of platelets is very rapid.
• Platelet transfusions are best used for controlling already established thrombocytopenic
bleeding.
• When given prophylactically, platelets should be given in the following way:
1.Half just before surgery to control capillary bleeding
2.Half at the end of the operation to facilitate placement of adequate sutures.
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• Suitable preparations include,
1. Platelet-rich plasma (PRP)
2. Platelet-rich concentrate (PRC)
PRC is the best source of platelets.
• The need for platelet transfusions can be reduced by local hemostatic measures and
use of desmopressin (DDAVP) or tranexamic acid or topical administration of platelet
concentrates.
• Absorbable hemostatic agents, such as oxidized regenerated cellulose, synthetic
collagen or microcrystalline collagen, may be put in the socket to assist clotting.
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• The risk of bleeding is greater with aspirin–clopidogrel or prasugrel–aspirin
combinations, but patients taking these drugs are generally already at a higher risk of
thromboembolic events.
• Therefore, advice from a specialist should be sought before withholding treatment, or
the patient may need to be referred to a hospital setting for the procedure.
• Patients who should not have their medications stopped or altered prior to dental
surgical procedures in primary care include those taking:
1.Low-dose aspirin
2.Clopidogrel
3.Dipyridamole.
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Platelet Concentrate:
Davidson's Principle and Practice of Medicine, Principles of management of haematological disease, 23rd Edition

Hematopoietic Growth Factors:
Growth Factor Drug Indications
1. IL- 11 Oprelvekin Thrombocytopenia
induced by
chemotherapy
2. Thrombopoietin Romiplastin
Eltrombopag
ITP

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