Platelets

Developmental pathway of platelets

 Origin and structure
• Megakaryocytes diameter 35‐160 μm
• Platelet diameter 2‐3 μm
• Contain an irregular ring of lobed nuclei
• Platelets are formed within the cytoplasm of
megakaryocytes and released into the circulation
• Platelet survival time 8‐12 days
• Destroyed mainly in spleen
• Blood count: 1.5‐4lakh/ µL of blood
 Morphology
• Wet preparations: colourless, moderately refractile,
discoid or elliptical
• Darkfield illumination: translucent, sharp contour, few
immobile granules
• Leishman’s stain shows a faint blue cytoplasm with
distinct reddish purple granules
• Electron microscopy reveals a cell membrane 6 nm
thick which surrounds a cytoplasmic matrix containing
Golgi apparatus, endoplasmic reticulum, 50‐100 very
dense granules, mitochondria, microvesicles,
microtubules, filaments & granules
Structure of platelet
 Structure of platelet…
• Membrane structures:surface glycoproteins serve as receptors,
facilitate platelet adhesion & contraction, and determine expression of
specific platelet antigens and antigens shared with other formed elements
• Canalicular system:numerous invaginations of the platelet surface
and, interspersed among these structures, a set of narrower channels
termed the dense tubular system
• Dense tubular system : is the major site for storage of Ca2+ and the
location of cyclooxygenase
• Cytoskeleton
• Microtubules
• System of contractile proteins
• Granules
 Platelet granules
• Dense body: ADP, ATP, GTP, GDP, serotonin, secretable
Ca++
• Alpha granules:
 Platelet specific proteins: PF‐4, thromboglobulin, PDGF,
thrombospondin
 Homologs of plasma proteins: fibrinogen, fibronectin,
albumin
• Dense tubular system: Prostaglandin converting
enzymes, contractile calcium
• Peroxisomes: Catalase
• Lysosomes: Acid hydrolase
 Function
• Hemostasis
• Blood coagulation
• Phagocytosis
• Storage & transport of substances
Events in hemostasis
Events in hemostasis….
 Hemostasis (Arrest of bleeding)
Formation of hemostatic plug due to
 Adhesviness to damaged lining of blood vessels
Promoted by Ca++ ions & ADP
 Aggregation of platelets leads to formation of
white bodies or micro thrombi which may grow
until they almost fill the lumen of a small vessel
At first the aggregation is reversible
Then it becomes irreversible, platelet granules are
discharged, leukocytes begin to adhere to
platelets, fibrin deposition occurs
 Suggested sequence of events in
hemostasis
• Platelet adherence to collagen in damaged
vessels wall
• ATP is converted to ADP by ATPase
• ADP is released and promotes aggregation of
passing platelets : formation of platelet plug
• Release of tissue factor from damaged vessels
& phospholipids from platelets will promote
thrombin formation
• Firm clot seals the vessel permanently
 Other reactions in hemostasis
• After the initial dilatation the damaged vessels constrict for
about 20 minutes, 5‐HT being important in this process
• Prostaglandin forming system is also involved in platelet
aggregation
 Arachadonic acid is released from the platelet cell
membrane by phospholipase and then rapidly oxidized by
the enzyme cyclo oxygenase to PGG2 & PGH2
 In the platelets:PGG2 is converted to TX A2 a highly potent
platelet aggregator and constrictor of arterial muscle
 In the arterial endothelium: PGG2 & PGH2 are converted to
prostacyclin a potent inhibitor of platelet aggregation and a
vasodilator
 Blood clotting
• In blood deprived of platelets
Clotting time in glass tube is prolonged
Activation of prothrombin is incomplete
Formed clots do not retract
Platelets are necessary for intrinsic clotting
process by releasing Platelet factor 3 which
causes conversion of prothrombin to thrombin
by factor X and V
 Clot retraction
• Clot retraction occurs by shortening of fibrin
fibers produced by contraction of attached
platelet pseudopodia, which contain
actomyosin like protein.
• Agents which inhibit cell metabolism or
enzyme activity also inhibit clot retraction
• Blood allowed to clot in a glass tube at 370 C
begins to show clot retraction after 30
minutes
 Phagocytosis
• Carbon particles, immune complexes and
viruses are phagocytosed by platelets
• A vestigial mechanism of clearing particulate
material from the blood
 Storage & transport
• Stores of 5‐HT and histamine which are
released by platelet disintegration
• They also take up 5‐HT by active transport
• Epinephrine & potassium
 Non hematological functions of
platelets
Inflammation
Platelets have 4 features common to all
inflammatory cells
• Posssess wide range of infammatory mediators
• Presence of receptors for other inflammatory
cells
• Ability to respond to noxious stimuli
• Cooperative with other inflammatory cells
Platelet & growth factors
PDGF, EGF, FGF are released from alpha
granules of platelets
Platelets & neurotransmitters
Despite being of different embryological origin
it has been shown that platelets behave like
serotonergic neurons in CNS. Thus they
provide a neuronal model for studying
neurons
Platelets & metastasis
Promote tumour survival in blood stream by
protecting tumour cells from attack by NK
cells
Platelets in asthma
Release of PAF & PF4 cause bronchial hyper
reactivity
 Hemostasis
• Physiology of coagulation
• Tests for clotting
• Clot retraction
• Fibrinolysis
• Anticoagulants
 Hemostasis (Prevention of blood loss)
Mechanisms by which hemostasis is achieved
1. Vascular constriction
Primary hemostasis
2. Platelet plug formation
3. Blood clot formation Secondary hemostasis
4. Clot retraction and fibrinolysis Tertiary hemostasis
 Primary hemostasis
Vascular phase: Triphasic response
1. Immediate reflex vasoconstriction :An intrinsic
response of smooth muscles in small arterioles & pre
capillary sphincters
2. Transient vasodilation: Due to PG E2, Histamine &
prostacyclin
3. Sustained vascular contraction:Due to release of local
autocoid factors from the traumatized tissue and
platelets. Ex. TX A2, Fibrinopeptide B, epinephrine ,
nor epinephrine
 Primary hemostasis…
Platelet phase of hemostasis:
Important in controlling bleeding from capillaries & small venules in
erosion of mucosal surfaces
• Adherence to exposed subendothelial structures & activation
• Burst of metabolic activity
• Shape change
• Platelet aggregation/ plugs
• ADP extrusion & other activated subtances
• Activation & avalibility of PF 3 & other procoagulants
• Initiation of blood clotting
• Consolidation of platelet plug by fibrin
• Clot retraction
 Adhesion
Defined as the attachment of platelets to non
platelet surfaces
The first detectable event after vascular injury

V V V
GP + W GP W GP W
F F F

VWF binding Adhesion

High molecular weight subunit of vWF acts as an initial ligand
between platelets & subendothelial structures i.e. collagen
 Von Willebrand factor
• A protein synthesised by vascular endothelial cells & megakaryocytes
• Belongs to a class of adhesive proteins .
• Adhesive proteins contain a sequence of amino acids Arg‐Gly‐Asp(RGD)
sequence
• This sequence allows adhesive proteins to bind to integrins (cell surface
proteins)
• GP IIb & GP IIIa are two integrins present on platelet surface
• Secreted into plasma and also abluminally into the superficial layers of sub
endothelium
• Sub endothelial vWF contributes to platelet adhesion but is insufficient
• After endothelial cell disruption the plasma vWF binds to the
subendothelium after which it can bind to surface of unstimulated
platelets and link them to the subendothelium
 Adhesion…
• Fibronectin
Synthesised by endothelial cells
Stored & secreted by platelets
Can bind collagen
 Platelet shape change
• Occurs within seconds of exposure to
activating agents
• From flattened discs to spheres with multiple
projecting pseudopods
• Polymerization of platelet actin
• Microtubules that are normally at the
periphery go to the center surrounding the
granules that have gone to the center prior to
release
 Platelet aggregation
Defined as the attachment of activated platelets to each other
• Primary aggregation: ADP & thrombin cause direct
aggregation
GP IIb‐IIIa is an integrin that recoginzes two RGD sequences
present on fibrinogen
Fibrinogen thus forms a bridge between two opposing
activated platelets
• Secondary aggregation: substances causing aggregation by
release of ADP or PG
Thrombospondin from alpha granules leads to ireversible
aggregation by binding to fibrinogen & GP IV on platelet
surface
 Platelet activation…
Series of progressive overlapping events
• Shape change
• Aggregation
• Liberation & oxidation of arachadonic acid
• Secretion of alpha & dense granules
• Reorganization of surface membrane
phospholipids
• Oriented centripital contraction of actomyosin
 Platelet activation…
Primary agonists triggering platelet activation
• Thrombin formed at injury site
• Sequence of collagen on subendothelium
Maintenance & amplification of platelet
activation
• ADP
• Arachadonic acid oxidation products
• Raised cytosolic Ca++ triggers
Phosphorylation of myosin light chain kinase.
Required for reorientation of cytoskeletal
proteins needed for platelet shape change,
secretion and contraction
Activation of a calcium dependent protease
called calpain which through proteolysis
activates other platelet enzymes
Activation of phospholipase A2
Prostaglandin thromboxane system
PLA2
Membrane PL AA Lipoxygenase HPETE GPX HETE
Cycloxygenase
Aspirin Prostacyclin Inhibition of release
Aggregation
PGG2+ PGH2
PGF2α, PGD2, PGE2
TX synthase
Initiation of release
Aggregation
Malonaldihyde TXA2
 Role of aspirin
• Aspirin irreversibly inactivates platelet
cycloxygenase
• Thus synthesis of PGH2 & TX A2 is prevented
• Prolongation of bleeding time may occur
• Therapeutic role in patients of coronary artery
disease
• PGH2: Acts as a cofactor enhancing collagen’s ability to
function as a platelet agonist
• TX A2: Binds to a specific platelet membrane receptor
with resultant activation of phospholipase C &
amplification of platelet activation through further
generation of DAG & IP3
• Prostacyclin: Secreted by endothelium helps to keep
platelets in an unstimulated state through activation of
platelet adenyl cyclase & a resultant rise in platelet
cAMP levels
• PGD2: Can also activate platelet adenyl cyclase
 Phosphoinositol metabolism
Platelet activation

Phosphatidyl inositol bis phosphate(PIP‐2)

Diacyl glycerol(DG)+Inositol
Co factor for
triphosphate(IP3)
Protein kinase C
Mobilises Ca ++ from
Phosphatidic acid(PA) intracellular stores
Mobilises Ca ++ from
intracellular stores
 Platelet release reaction
α granules
 Platelet proteins
Thrombospondin: Paltelet aggregation
PF4: Neutrlises anticoagulant activity of heparin, competes with
antithrombin III for bonding sites on heparan sulphate present on
endothelial cells, chemo attractant for WBC, smooth muscle cells &
fibroblasts
PDGF:chemo attractant for WBC, smooth muscle cells & fibroblasts
TGF‐β:chemo attractant for WBC, smooth muscle cells & fibroblasts
 Analogus of plasma proteins:
Albumin, IgG‐ no known hemostatic function
Fibrinogen, vWF, Factor V‐ known hemostatic function
Dense granules: ADP, ATP, Calcium, serotonin
Lysosomal granules:
Compaction & stabilization of platelet
plug
• The GP IIb‐IIIa hetrodimer traverses the
platelet membrane
• When fibrinogen binds to the external domain
of GP IIb‐IIIa the cytoplasmic domain is altered
and binds to actin filaments
• This orients centripetal contraction
 Blood coagulation
Secondary hemostasis
 Blood clotting factors
Factor I (Fibrinogen): a soluble plasma protein. In
afibrinoginemia clotting does not occur
Factor II(Prothrombin): Inactive precursor of thrombin
Factor III(Tissue factor/ Tissue thromboplastin): converts
prothrombin to thrombin in presence of factors V, VII,
X, Ca++ and phospholopids
Factor IV (Calcium): Essential for clotting.
FactorV(Labile factor/ proaccelerin):
FactorVII(Stable factor/ proconvertin): Deficiency induced
by oral anticoagulants
 Blood clotting factors…
• FactorVIII(Antihemophilic globulin/factor A):
Congenital defficiency causes Classical Hemophilia
• FactorIX (Christmas factor/ antihemophilic factor B):
Congenital deficiency leads to a hemorrhagic state
resembling Hemophila(Christmas disease)
• Factor X(Stuart‐Prower factor)
• Factor XI(Plasma thromboplastin antecedent/
Antihemophilic factor C)
• FactorXII(Hageman factor, contact factor)
• Factor XIII(fibrin stabilizing factor)
 Blood clotting factors…
HMW‐K: High molecular weight kininogens
Pre‐Ka: prekallikrein/ Fletcher factor
Ka: Kallikrein
PL: Platelet phospholipid
• Factors II, VII, IX, X, protein C & protein S
are formed in the liver
Vitamin K is necessary for some post
translational modifications in these factors
In vitamin K defficiency or
inhibition by oral anticoagulant ,Warfarin
the plasma levels of these factors are low
Basic reactions involved in coagulation

Prothrombin(Factor II)
Ca++, factors derived from damaged tissues,
disintegrating platelets, plasma
Thrombin
Fibrinogen Fibrin
(Factor I)
 Physiology of clotting process
Thrombin‐fibrinogen reaction
Formation of fibrin clot is the only visible &measurable
part of clotting process
Type of reaction
• Proteolysis
Thrombin
Fibrinogen Fibrin monomers + Peptides
• Polymerization
Fibrin monomers Fibrin polymers
(Soluble fibrin clot)
• Clotting
Fibrin polymers Factor
Ca++
XII Insoluble fibrin clot
• Conversion of prothrombin to thrombin
requires
Prothrombin activator or thromboplastin
Prothrombin activator is formed in 2 main ways
As result of tissue damage (Extrinsic system)
Activation of blood constituents(Intrinsic
system)
Key reaction: Factor X Factor Xa
Extrinsic pathway for initiating blood clotting .
 Extrinsic system…
• Key event triggering blood coagulation during
hemostasis is exposure of blood to tissue
factor
• It also acts as a cofactor for activation of factor
VII
Intrinsic pathway for initiating blood clotting .
 Intrinsic system
• More complicated
• More prolonged
• Fibrinolytic and kinin forming systems are also
activated
Enzyme cascade hypothesis(Macfarlane):
Surface contact induces a sequence of changes in
which an inactive precursor is converted into an
active enzyme which then acts on the next
precursor to form the next active enzyme…
An amplifying system
 Intrinsic system…
• Factor XII, prekallikrein,Factor XI & High
molecular weight kininogen are known as the
contact activation factors
• Factor XII, prekalikrein & HMWK are essential for
triggering blood coagulation in a glass test tube
• However they play no role in normal hemostasis
since patients with isolated deficiencies of each
of these do not bleed abnormally
• Once Xa is formed clotting occurs within
seconds
• Contact with a foreign substance i.e. glass or
urate crystal produces clotting only after 4‐8
minutes
Overview of clotting
 Effects of thrombin
• Positive feedback effect on factors concerned
with formation of prothrombin activator
• Activates factor VIII
• Increases activity of factor V & XIII
• Promotes aggregation of platelets
• Increases the amount of available phosphlipids
• After activating these mechanisms soon
inactivates them
 Regulation of blood coagulation
• Adsorption of thrombin onto fibrin
• Neutralization of thrombin by plasma proteinase
inhibitors, Antithrombin III, α2 macroglobulin&
heparin cofactor II
• Antithrombin III also inhibits the activity of key
intermediate enzymes, Factor IXa & Xa
• Activated protein C inhibits the cofactors for
Factor VIIIa & Va
• Extrinsic pathway inhibitor(EPI) & factor Xa
neutralize the catalytic activity of Factor
VIIa/tissue factor complex
 Clot retraction

Platelet contractile proteins contract.

•Squeezes fluid (serum) out of the clot.

•Draws the edges of the torn blood vessel together.

•Sets the stage for repair.

–PDGF

–VEGF
 Clot retraction
• Freshly formed fibrin threads are extremely sticky
and adhere to each other, blood cells, tissues &
foreign substances
• Freshly shed blood sets in a soft jelly –like mass
• Gradually it contracts down(retracts) to 40% of its
original volume, squeezing out serum
• Final clot is more tougher and solid
• Clot retraction is impaired if platelets are
removed from blood
 Fibrinolysis (Dissolution of clot)
Exrtinsic / Intrinsic
Plasminogen Plasminogen Plasmin
activators

Fibrin Plasmin
Small peptides
(Fibrin degradation products)
Intrinsic plasminogen activators get activated by:
Body or mental stress, operation, violent exercise, adrenalin
injection
Extrinsic activators: Widely distributed throughout the cell
& body fluids
Tissue activators occur in microsomes, urokinase in urine
 Fibrinolytic system & its regulation by
protein C
Endothelial cells
(Thrombomodulin thrombin complex)

Protein C Activated protein C

+Protein S
VIII inactive VIIIa V inactive Va

Inactivates inhibitors of t‐PA

Plasminogen Plasmin
Thrombin
t‐PA, u‐PA
Lyses fibrin
Plasminogen (fibrinolytic) system

Kringles : Lysine binding sites

t‐PA: Produced by recombinant DNA technology

Used in myocardial infarction and stroke
 Tests for defects in blood clotting
Screening for adequacy of hemostatic plug
formation
• Platelet count
• Bleeding time: for conditions other than
thrombocytopenia that can impair the
formation of hemostatic plug
Tests for defects in blood clotting…
Screening for the adequacy of blood coagulation
• Prothrombin time: Measures the adequacy of
reactions that clot plasma when a very high
concentration of tissue factor is present.
Tests the adequacy of extrinsic system
• Activated partial thromboplastin time: Measures
the adequacy of clotting reactions that clot
plasma when a reagent optimizing the contact
activation reactions & providing procoagulant
phospholipid is present
Tests adequacy of intrinsic system
Activated partial thromboplastin time
 Disorders affecting formation of
platelet plug
Thrombocytopenia
• Physiological: New born, Menstruation
• Pathological:
 Decreased marrow production: Tumours, fibrosis,
aplasia, drugs(thiazide diuretics)
 Spleenic sequestration: Spleenomegaly
 Increased destruction: Auto antibody formation,
aspirin, insecticides, cardiac valves, Sepsis,
vasculitis
 Disorders affecting formation of
platelet plug…
Functional platelet defects
 Disordered adhesion
Von Willebrand’s disease: vWF, Factor VIII activity, BT
Most common hereditary hemostatic disorder
 Disordered aggregation
Thrombasthenia(Glanzmann’s syndrome)
Afibrinoginemia
 Disordered granule release
Chediak Higashi syndrome
Cardiopulmonary bypass
Myeloproliferative disorders
Aspirin & other NSAIDS
• Platelet count
• >1 lakh: Asymptomatic, Normal bleeding time
• 50000‐1 lakh: Bleeding occurs after severe
trauma, Bleeding time slightly prolonged
• <50000: Easy bruising manifested by skin
purpura after minor trauma
• <20000: Spontaneous bleeding, usually have
petechiae, intracranial/ spontaneous bleeding
 Disorders affecting blood coagulation
Hereditary disorders
• Factor VIII deficiency(Hemophilia A) X linked
• Factor IX defficiency(Hemophilia B) recessive disorders
• Factor XI deficiency: Autosomal recessive
Acquired disorders
• Vitamin K deficiency
• Liver disorders: Fall in all factors except factor VIII
• Disseminated intravascular coagulation
• Acquired antibodies against clotting factors
Transmission of Hemophilia
Normal Carrier
male female
Transmission of Hemophilia
Diseased Carrier
male female
 Anticoagulants in vivo
• Antithrombin III: Serine protease inhibitor
• Heparin
• Thrombomodulin: Thrombin binding protein
• Thrombomodulin‐thrombin complex: Activates
protein C
• Activated protein C and S
• Plasmin
• Dicumarol & Warfarin: Inhibit action of vitamin K
 Anticoagulants in vitro

– EDTA
– Oxalate
– Heparin
– Sodium Citrate
– Sodium Fluoride/Potassium Oxalate
 Characteristics
• It must not alter the size of the red cells
• It must not cause hemolysis
• It must minimize platelet aggregation
• It must minimize disruption of the staining
and morphology of leukocytes
• It must be readily soluble in blood
 EDTA
(Ethylenediaminetetraacetic acid)
• Best meets the above requirements, and is used most frequently.
• The tripotassium salt (K3EDTA), and the disodium salt (Na2EDTA).
• Calcium EDTA is not used as an anticoagulant, but in the
treatment of lead poisoning
• Mode of action: It forms insoluble calcium salts by chelation
• Uses: Making a blood smear for cell morphology studies, test for
microfilaria, perform cell counts, BUN, plasma protein, fibrinogen,
glucose, determination of Coomb's Test
• EDTA preserves the staining and morphological characteristics of
leukocytes
• Disadvantages
Excessive concentrations of EDTA will cause shrinkage of RBC's and
erroneous PCV, MCV and MCHC results
 Oxalate
• A mixture of dry ammonium oxalate and potassium oxalate in the
ratio of 3:2 is used
• Mode of action: It combines with calcium to form insoluble Ca
oxalate.
• Potassium oxalate alone causes red cells to shrink; ammonium
oxalate alone causes red cells to swell. Used together, little cellular
distortion occurs in the first hour after collection
• Uses: The oxalate mixture may be used for hematological
sedimentation studies. Potassium oxalate alone is valid for
immediate glucose determinations. Ammonium oxalate is used as a
diluent in some methods for manually counting WBCs and platelets
• Disadvantages
– 1) It does not prevent platelet aggregation in vitro as effectively as
EDTA.
– 2) It is poisonous and should not be used for blood transfusion.
 Heparin
• Chemical structure: A polysaccharide derived
from glucosamine and glucuronic acid.
Contains many sulphate groups.
• Molecular weight averages 15000‐18000
• Anticoagulant action due to its strong
electronegative charge due its sulphuric acid
group
• Antagonists: Toludene blue & protamine by
antagonizing the negative charge of heparin
 Heparin…
• First isolated from Liver
• Subsequently demonstrated in extract if many
other organs
• Inhibits blood coagulation both in vitro & in vivo
• Mechanism of action:
 Prevents activation of prothrombin to thrombin
 Neutralizes action of thrombin on fibrinogen
 Facilitates the action of antithrombin III
 Heparin…
• Heparin is the anticoagulant of choice for blood
pH and blood gas analysis for acid‐base balance.
• Disadvantages
1) It causes clumping of leukocytes.
2) It interferes with the staining of leukocytes.
3) It is the most expensive of the anticoagulants.
4) Blood will clot in 8‐12 hours because clotting is only
delayed and not prevented.
5) It is not suitable for agglutination tests, coagulation
studies (prothrombin time tests) or plasma fibrinogen
determination
 Sodium citrate
• Acid citrate dextrose (ACD) is prepared from disodium hydrogen
citrate and is the anticoagulant of choice for blood transfusions.
used in the ratio of 1 part ACD to 4 parts of blood
• Mode of action: It combines with calcium to form an insoluble salt
of calcium citrate.
• Sodium citrate is the anticoagulant of choice for studies of platelet
function and morphology
• e. Disadvantages:
– 1) It interferes with many chemical tests.
– 2) Used alone it preserves blood for only a few hours.
– 3) It has a tendency to shrink cells.
– 4) Because of a 10% dilution of blood, Na‐citrate is generally not used
for CBC.
Blood groups
 Blood groups
• Agglutinogen : The antigen present on RBC
surface
• Major 0‐A‐B blood types
Blood group A: A antigen A present on surface
Blood group B: B antigen present on surface
Blood group AB: A & B antigen present on surf.
Blood group O: No antigen present on surface
 Genetic determination of
agglutinogens
• Genes that determine the A& B phenotypes
are found on chromosome 9p
• They are expressed in a mendelian co‐
dominant manner
• The gene products are glycosyl transferases, A
or B, which confer the enzymatic ability of
attaching the specific antigenic carbohydrate
groups
 Genetic determination of
agglutinogens…
• Glycosyl transferase A directs the formation of
antigen A on RBC surface
• Glycosyl transferase B directs the formation of
antigen B on RBC surface
• When A & B transferases are absent then no
antigen is present on RBC surface and blood
group is said to be O
 Agglutinins
• Antibodies directed against RBC antigen may
result from “natural exposure” particularly to
carbohydrate that mimic some blood group
antigens
 Blood types with their genotypes

Genotypes Blood types Agglutinogen Agglutinin

OO O ‐ Anti a & Anti b
OA or AA A A Anti b
OB or BB B B Anti a
AB AB A&B ‐
Relative frequencies of different blood
groups
Western countries India
O 47% 32%
A 41% 20%
B 9% 40%
AB 3% 8%
 Landsteiner’s law
States that if an agglutinogen is present in the
RBCs of an individual the corresponding
agglutinin must be absent in the plasma
Or conversely if an agglutinogen is absent in
the RBCs of an individual the corresponding
agglutinin must be present in the plasma
 Rh system of blood grouping
• RBC of rhesus monkey when injected into a rabbit, the
rabbit developed antibodies to the rhesus RBC
• Further it was discovered that rabbit serum containing
anti rhesus antibodies could agglutinate not only
rhesus RBC but also human RBC in 85% cases
• The RBCs of these humans have on their surface an
antigen identical to or similar to rhesus antigen
• Thus humans having this antigen were termed as Rh
positive
 Rh blood group
• Three Rh genes are present
• Rh genes are located on chromosome 1
• Types of Rh antigens/ factor: C, D, E
• D is most antigenic
• The term Rh positive is generally used to
mean that the individual has agglutinogen D
• 85% Caucasians are Rh positive
• 99% Asians are Rh positive
 Difference between ABO and Rh
groups
• ABO antigens are present on cells other than
RBCs whereas the Rh antigen is not present on
any other cell except RBCs
• In the ABO system the plasma antibodies
responsible for transfusion reaction develop
spontaneously whereas in the Rh system
antibodies only develop in Rh negative
persons when they have been previously
sensitized
• Knowledge of the mode of inheritance of
blood groups is useful in cases of disputed
paternity
• The negative verdict is definitive: It rules out
the possibility of a given man being the father
Child’s Child’s Mother’s Possible Possible Father Father
blood genotype blood contribution contribution might could not
group group of mother of father have been have been
A AA A or AB A A A or AB O or B
AO B O A A or AB O or B
B BB B or AB B B B or AB O or A
BO O O B B or AB O or A
AB AB A A B B O or A
AB B B A A or AB O or B
O OO A O O A or B or AB
OO B O O O AB
 Rh incompatibility
• If a Rh negative person is transfused with Rh
positive blood for the first time: No immediate
adverse reaction
• If the same individual receives a second
transfusion later anti Rh antibodies are
synthesized promptly in large amounts
 Rh incompatibility in pregnancy…
• Rh negative female
• Rh positive male
• Fetus is Rh positive
Chances of an apparent abnormality resulting
from Rh incompatibility are
Negligible in first pregnancy
3% during second pregnancy
10% during third pregnancy
 Rh incompatibility in pregnancy…
Erythroblastosis foetalis/ Hemolytic disease of newborn
Fetal RBCs undergo massive hemolysis
Compensatory increase in erythropoietic activity leading
to immature cells in circulation
Enlargement of extramedullary sites of hematopoiesis
leads to hepatomegaly & spleenomegaly
Child is anemic, Jaundice (Hemolytic) is present, edema
Hydrops fetalis: Death in utero
Kernicterus: Deposition of unconjugated bilirubin in the
basal ganglia
If bilirubin > 20 mg % then brain damage
 Erythroblastosis fetalis…
Preventive treatment: Giving anti Rh antibodies to
Rh negative pregnant mother
Mechanism by which anti Rh antibody prevent
the complication of Rh incompatibility
 They destroy fetal red cells which cross over to
the maternal circulation. Thus antigenic stimulus
is reduced
 High antibody levels inhibit production of the
same antibody molecules
Definitive treatment: Exchange transfusion with Rh
negative blood
 Other blood group systems
• Lewis system
• I System
• P system
• MNSSU sysetm
• Kell system: Immunogenicity is third behind
the ABO & Rh systems
• Duffy system
• Kidd system
 Blood transfusion: Compatibility
testing
• ABO‐Rh typing: Antibodies are directed against
those antigens that lack in the individual’s own
blood
• Cross matching: A trial transfusion within a test
tube in which donor RBC are mixed with recipient
serum to detect a potential for serious
transfusion
• Antibody screen: A trial transfusion between the
receipient’s serum & commercially supplied RBC’s
that are specifically selected to contain optimal
number of RBC antigens
 Storage of blood
• CPDA‐1
Citrate : Anticoagulant
Phosphate : Buffer
Dextrose: energy source
Adenine: For resynthesis of ATP so as to extend the
storage time from 21 to 35 days
AS‐1: (Adsol): Adenine, glucose, mannitol & NaCl
Increases shelf life to 42 days
AS‐3: (Nutricel): Contains adenine,glucose, citrate,
phosphate, NaCl
 Storage of blood
Duration of storage
Set by the US federal regualtion
At least 70% of the transfused RBC’s remain in
circulation for 24 hours after infusion
Since RBC’s that survive 24 hours after
transfusion disappear from circulation at the
normal rate
Blood transfusion
 Storage of blood
Three problems
• Anticoagulation
• Preservation of cell viability
• Preservation of cell function

Criteria for assessing the adequacy of banked

blood
70% survival at 24 hours
 RBC storage lesion
Stored RBCs undergo a series of biochemical & structural
changes
• RBC nucleotide depletion
ATP

ADP

AMP

IMP

Hypoxanthine
 RBC storage lesion…
• RBC membrane changes
Normal disc shaped

Spherical with surface projections

Loss of membrane proteins

Loss of deformability
 RBC storage lesion…
• 2,3 DPG depletion
Earliest detectable change
With acid citrate dextran(ACD)
DPG drops to < 50% within 48 hours
With citrate phosphate dextran(CPD)
DPG falls significantly after 2 weeks
 RBC storage lesion…
• Decreased DPG
• Increased affinity of Hb for oxygen
• Increased Hb but no proportionate increase in
oxygen avability
 RBC storage lesion…
• Accumulation of lactate
• Accumulation of H+ ions
• Decrease in pH
• Loss of K+ and gain of Na+
• Increased osmotic fragility
• Some cells undergo lysis
 Use of anticoagulant
• Citrate
Most important anticoagulant
Mode of action
After transfusion it is readily metabolized by the
recipient
Preserves RBC’s in a viable state for 1 week only
• ACD(Acid citrate dextran)
Glucose is present
RBC storage for 3 weeks
• CPD(Citrate phosphate dextran): Modified ACD with
added NaH2PO4
Viability after first 24 hours is better
At 4oC blood may be stored for 21 days
• CPDA‐1: CPD supplemented with adenine and with
25% more glucose than CPD(Standard anticoagulant
preservative)
RBC storage up to 35 days
 2,3 DPG preservation
• In ACD, CPD, CPDA‐1, 2, 3 DPG disappears by 2
weeks
• Chemical agents capable of maintaining
normal levels during storage
dihydroxyacetone, pyruvate, ascorbic acid
 Indications of blood transfusion
• Hypovolemia: Blood loss < 20 % of total blood
volume does not require RBC transfusion
fluid replacement is sufficient
• Surgery
• Anemia: Hb levels < 6 gm % require
transfusion
 Pre transfusion testing
Pre transfusion testing of a potential recipient consists of
the following
• Forward type: Determines the ABO & Rh phenotype of
the recipient
• Reverse type: Detects isoagglutinins in the patients
serum and should correlate with the forward type
testing.
The alloantibody screen identifies antibodies directed
against other RBC antigens
• Cross matching: Donor RBC’s are mixed with recipient
plasma on a slide and checked for agglutination
 Adverse effects of transfusion
• Immune mediated reactions
Acute hemolytic transfusion reactions
Occur due to pre formed antibodies that bind & lyse
donor RBC
ABO isoagglutinins are responsible for the majority
of these reactions
Presentation: Hypotension, tachypnea, tachycardia,
fever, chills, hemoglobinemia, hemoglobinuria,
chest/flank pain, discomfort at catheter infusion
site
 Adverse effects of transfusion…
• Immune mediated reactions…
Delayed hemolytic & serological transfusion
reaction:
Occurs in patients previously sensitized to RBC
alloantigen who have a negative alloantibody
screen due to low antibody levels.
When transfused with antigen positive blood an
anamnestic response results in the early
production of alloantibody that binds to donor
RBC
 Adverse effects of transfusion…
• Immune mediated reactions…
Febrile non hemolytic transfusion reaction
Most frequent reaction
S& S; Chills & rigor & 10C or more rise in
temperature
A diagnosis of exclusion
Antibodies against donor WBC’s & HLA
antigens
 Adverse effects of transfusion…
• Immune mediated reactions…
Allergic reactions
Urticarial reactions characterized by pruritic
rash, edema, headache & dizziness
Related to plasma proteins found in
transfused components
 Adverse effects of transfusion…
• Immune mediated reactions…
Anaphylactic reactions
S& S: difficulty in breathing, coughing, nausea
& vomiting, hypotension, bronchospasm,
respiratory arrest, shock & loss of
consciousness
Treatment; Epinephrine, glucocorticoids
 Adverse effects of transfusion…
• Immune mediated reactions…
Graft versus host disease
Mediated by donor T lymphocytes that
recognize host HLA as foreign and mount an
immune response
S&S: fever, cutaneous erruptions, diarrhoea &
liver abnormalities
Clinical manifestations occur 8‐10 days post
transfusion
 Adverse effects of transfusion…
• Immune mediated reactions…
Transfusion related acute lung injury
Uncommon reaction
Due to transfusion of donor plasma that contain
high titre anti HLA antibody that bind
corresponding antigen on recipient leukocytes.
These leukocytes then aggregate in pulmonary
vasculature & release mediators causing an
increase in capillary permeability
 Adverse effects of transfusion…
• Immune mediated reactions…
Post transfusion purpura
Presents as thrombocytopenia 7‐10 days after
platelet transfusion
Due to production of antibodies that react to
both donor & recipient platelets
 Adverse effects of transfusion…
• Non immune reactions
Fluid overload
Hypothermia
Electrolyte toxicity
Iron overload
 Adverse effects of transfusion…
• Infectious complications
Viral: HCV(Most common)
HBV, HIV, CMV, HTLV(I),Parvovirus B19
Bacterial: Pseudomonas & Yersenia(Can grow a
1‐60C)
Parasitic: Malaria, Babesiosis, Chaga’s disease
 Adverse effects of transfusion…
• Low factor V & VIII