‫كلية التقنيات الصحية والطبية‬

‫قسم تقنيات التحليالت المرضية‬

3rd Stage
Blood Banking

Lecture 11

Dr. Hussam S. Aziz

MD, MSc Medical Genetics
 An efficient and rapid mechanism for stopping
bleeding from sites of blood vessel injury is
essential for survival
 The process of stopping bleedings is called
haemostasis
 The normal haemostatic response to vascular
damage depends on a closely linked interaction
between three components:
◦ Blood vessel wall
◦ Platelets
◦ Coagulation factors
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 Defect in any of these three elements can lead to
bleeding tendency (coagulation defect)
 On the other hand, such a response needs to be
tightly controlled to:
◦ Prevent extensive clots developing (to prevent
intravascular coagulation); and
◦ Break down (desolve) such clots once damage is
repaired
 The haemostatic system thus represents a delicate
balance between pro-coagulant and anti-
coagulant mechanisms
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 The five components required to regulate the
coagulation process:
◦ Blood vessels
◦ Platelets
◦ Coagulation factors
◦ Coagulation inhibitors
◦ Fibrinolysis

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 Immediate vasoconstriction of the injured vessel
and reflex constriction of adjacent small arteries
and arterioles is responsible for an initial slowing of
blood flow to the area of injury
 When there is widespread damage this vascular
reaction prevents bleeding to death

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 Normal platelet count is approximately 250 ×
10^9/L (range 150 – 400 × 10^9/L)
 Normal platelet lifespan is 7–10 days
 Platelets are produced in the bone marrow by
fragmentation of the cytoplasm of
megakaryocytes, one of the largest cells in the
body
 The precursor of the megakaryocyte – the
megakaryoblast – arises by a process of
differentiation from the haemopoietic stem cell

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 Platelets form by fragmentation from the tips of
cytoplasmic extensions of megakaryocyte
cytoplasm
 Each megakaryocyte giving rise approximately to
1000 – 5000 platelets
 The time interval from differentiation of the stem
cell to the production of platelets averages 10 days
 Thrombopoietin is the major regulator of platelet
production and is constitutively produced by the
liver and kidneys

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 Immature Mature
Megakaryocyte Megakaryocyte

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 Platelets are extremely small and discoid, 3.0 × 0.5
μm in diameter
 The glycoproteins of the surface coat are
particularly important in the platelet reactions of
adhesion and aggregation which are the initial
events leading to platelet plug formation during
haemostasis
 Adhesion to collagen is facilitated by glycoprotein
Ia (GPIa)

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 Glycoproteins Ib and IIb/IIIa are important in the
attachment of platelets to von Willebrand factor
(VWF) and hence to vascular subendothelium
 The binding site for IIb/IIIa is also the receptor for
fibrinogen which is important in platelet – platelet
aggregation
 The membrane phospholipids are of particular
importance in the conversion of coagulation
factor X to Xa and prothrombin (factor II) to
thrombin (factor IIa)

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 The platelet contains three types of storage
granules: dense, α, and lysosomes
 α granules contain clotting factors, VWF, and
platelet - derived growth factor (PDGF)
 Dense granules are less common and contain
adenosine diphosphate (ADP), adenosine
triphosphate (ATP), serotonin and calcium
 Lysosomes contain hydrolytic enzymes
 During the release reaction, the contents of the
granules are discharged into the open canalicular
system
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 Several platelet surface proteins have been found
to be important antigens in platelet - specific
autoimmunity
 The antigens have been termed human platelet
antigens (HPA)
 Platelets also express ABO and human leucocyte
antigen (HLA) class I but not class II antigens

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 The main function of platelets is the formation of
mechanical plugs during the normal haemostatic
response to vascular injury
 In the absence of platelets, spontaneous leakage
of blood through small vessels may occur
 Platelet function falls into three:
◦ Adhesion immobilization of platelets at the sites
of vascular injury
◦ Aggregation of platelets
◦ Release reactions of VWF and other clotting
factors
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 VWF is involved in platelet adhesion to the vessel
wall and to other platelets (aggregation)
 It also carries factor VIII
 VWF is synthesized both in endothelial cells and
megakaryocytes, and stored in Weibel–Palade
bodies and platelet α granules, respectively
 Plasma VWF is almost entirely derived from
endothelial cells

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 VWF is released to the plasma by two distinct
pathways:
◦ Majority is continuously secreted
◦ Minority is stored in Weibel – Palade bodies
 The stored VWF can raise the plasma levels when
released under the influence of several factors,
such as stress, exercise, adrenaline and infusion of
desmopressin (antidiuretic hormone analogue)

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 This is characterized by cross-linking of platelets
through active GPIIb/IIIa receptors with fibrinogen
bridges
 A resting platelet has about 50,000–80,000
GPIIb/IIIa receptors, which do not bind fibrinogen,
VWF or other ligands
 Stimulation of a platelet leads to an increase in
GPIIb/IIIa molecules, enabling platelet cross-linking
with fibrinogen bridges

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 Activation by various factors, inducing intracellular
signaling, leads to the release of α granule contents
 These have an important role in platelet aggregate
formation and stabilization
 In addition, ADP, released from dense granules,
and Thromboxane A 2 (TXA2) are the two major
platelet positive feedback loops important in
secondary amplification of platelet activation to
form a stable platelet aggregate
 TXA2 also has powerful vasoconstrictive activity

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 After platelet aggregation and release, the exposed
membrane phospholipid (platelet factor 3) is
available for two reactions in the coagulation
cascade:
 Both phospholipid-mediated reactions are calcium
ion-dependent
1. Tenase: involves factors IXa, VIIIa and X in the
formation of factor Xa
2. Prothrombinase: results in the formation of
thrombin from the interaction of factors Xa, Va and
prothrombin (II)

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 Platelets derived growth factor (PDGF)
found in the α granules of platelets
stimulates vascular smooth muscle cells to
multiply and this may hasten vascular
healing following injury

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 Nitric oxide (NO) is constitutively released from
endothelial cells, and also from macrophages and
platelets
 It has a short half-life of 3 – 5 seconds
 It inhibits platelet activation and promotes
vasodilatation
 Prostacyclin synthesized by endothelial cells also
inhibits platelet function and causes vasodilatation
by raising cyclic guanosine monophosphate (GMP)
levels

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 Blood coagulation involves a biological
amplification system in which relatively few
initiation substances sequentially activate, by
proteolysis, a cascade of circulating precursor
proteins (the coagulation factor enzymes) which
ends in the generation of thrombin; this, in turn,
converts soluble plasma fibrinogen into fibrin
 Fibrin enmeshes the platelet aggregates at the
sites of vascular injury and converts the unstable
primary platelet plugs to firm, definitive and
stable haemostatic plugs

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 The operation of this enzyme cascade requires local
concentration of circulating coagulation factors at the
site of injury
 Surface-mediated reactions occur on exposed
collagen, platelet phospholipid and tissue factor
 All the enzymes, except factor XIII, are serine
proteases, i.e. have ability to hydrolyse peptide bonds
depends upon the amino acid serine at their active
center
 The scale of amplification achieved in this system is
dramatic, e.g. 1 mole of activated factor XI through
sequential activation of factors IX, X and prothrombin
may generate up to 2×10^8 mole of fibrin

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 The generation of thrombin in vivo is a complex
network of amplification and negative feedback
loops to ensure a localized and limited
production
 Generation of active thrombin from prothrombin
precursor involves serial, cascade activation of
other coagulation factors
 Activated thrombin, in turn, activates fibrinogen to
the active form (fibrin)
 Thrombin is a major component in the formation of
platelets plug

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 Endothelial cell has an active role in the maintenance
of vascular integrity
 This cell provides the basement membrane that
normally separates collagen, elastin and fibronectin
of the subendothelial connective tissue from the
circulating blood
 Endothelial cell also has a potent inhibitory influence on
the haemostatic response, largely through the
synthesis of prostaglandin and NO which have vaso-
dilatation properties and inhibit platelet aggregation
 Loss or damage to the endothelium results in both
haemorrhage and activation of the haemostatic
mechanism
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 After vascular injury, the following mechanisms
help in haemostasis and stop bleeding:
◦ Vasoconstriction
◦ Platelet reactions and primary haemostatic
plug formation
◦ Activation of clotting factors (cascade)
◦ Stabilization of the platelet plug by fibrin

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 Fibrinolysis (like coagulation) is a normal
haemostatic response to vascular injury
 Plasminogen, a β - globulin proenzyme in blood
and tissue fluid, is converted to protease plasmin
by activators either from the vessel wall (intrinsic
activation) or from the tissues (extrinsic activation)
 Tissue plasminogen activator (TPA) is an
enzyme released from endothelial cells, activates
plasminogen to plasmin
 Plasmin binds and lyses the fibrin in the platelet
plug dissolving of the plug

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 Defective haemostasis with abnormal bleeding may
result from:
1. Vascular disorder
2. Thrombocytopenia or a disorder of platelet
function; or
3. Defective blood coagulation
 A number of simple tests are employed to assess
the platelet, vessel wall and coagulation
components of haemostasis

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 As thrombocytopenia is a common cause of
abnormal bleeding, patients with suspected
bleeding disorders should initially have a blood
count including platelet count and blood film
examination
 In addition to establishing the presence of
thrombocytopenia, the cause may be obvious in
the blood film, e.g. acute leukaemia

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 The bleeding time was a useful test for abnormal
platelet function including the diagnosis of VWF
deficiency
 The test involved the application of pressure to the
upper arm with a blood pressure cuff after which small
incisions are made in the flexor surface forearm skin.
Bleeding stops normally in 3 – 8 minutes
 The bleeding time is prolonged in thrombocytopenia
but is normal in vascular causes of abnormal bleeding
 It has largely been replaced by specific platelet
aggregation tests, platelet adhesion assays and the
platelet function analysis - 100 (PFA - 100) test

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 Screening tests provide an assessment of the
‘extrinsic’ and ‘intrinsic’ systems of blood
coagulation and also the conversion of fibrinogen
to fibrin
 These tests include:
◦ Prothrombin time (PT)
◦ Activated partial thromboplastin time (APTT)
◦ Thrombin (clotting) time (TT)

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 Measures factors VII, X, V, prothrombin and
fibrinogen
 Tissue thromboplastin (a brain extract) or
[synthetic] tissue factor with lipids and calcium is
added to citrated plasma
 The normal time for clotting is 10 – 14 seconds
 It may be expressed as the international
normalized ratio (INR)

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 Measures factors VIII, IX, XI and XII in addition
to factors X, V, prothrombin and fibrinogen
 Three substances – phospholipid, a surface
activator (e.g. kaolin) and calcium – are added to
citrated plasma
 The normal time for clotting is approximately 30–40
seconds

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 Sensitive to a deficiency of fibrinogen or inhibition
of thrombin
 Diluted bovine thrombin is added to citrated plasma
at a concentration giving a clotting time of 14 – 16
seconds with normal subjects

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 A number of immunological methods are available
to detect fibrinogen or fibrin degradation products
(including D-dimers) in serum
 Patient with intravascular thrombosis has elevated
fibrin degradation products

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 Assays of specific coagulation factors
 Tests of platelet function
 Fibrinogen quantification

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