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HEMOSTATIC FUNCTION

Legend
PPT
Recording
2012 trans
Hemostasis

o There should be a balance as to the


formation and dissolution of hemostatic plug.
If there is tilting of the balance it would result
to bleeding or excessive clot
formation/coagulation.
It involves 3 elements to form the Hemostatic
Plug:
o Intact Vascular System
o Cellular Elements (mainly:
Platelets but granulocytes,
monocytes play some minor role)
o Coagulation Factors

Hemostasis is the process of arresting


bleeding in which 3 important events
take place
o Reaction of the bloopd vessels to
the site of injury which is
VASOCONSTRICTION
o Formation of platelet plug in the
site of injury and release of
active substances from the
aggregated platelets
o Coagulation of Blood

Under normal conditions, the formation and


dissolution of thrombi is maintained in a
delicate balance. (fig ).
o Without this balance, the individual
may experience either excessive
bleeding (poor clot formation or
excessive Fibrinolysis)
o Vaso-occlusion (uncontrolled
formation of thrombin in vascular
system, occluding vessels and
depriving organs of blood).
Process of Hemostasis:

It is also a process of (1) retains the blood within the vascular


system during periods of injury (2) localizes the reactions
involved in the site of injury (3) repairs/ re-establishes the blood
flow through injured vessel
Importance of Balance in Hemostasis

Hemostasis
o Intricate system maintaining blood in
fluid state
reacts to vascular injury to stop
blood loss and seal vessel wall
o Tightly Regulated System

There are substances released by


the endothelial and subendothelial
cells that either prevent clot
formation or initiate platelet
formation depending on the
situation
When there is blood vessel (BV)
injury, there is a neural mechanism
that causes transient
vasoconstriction and at the same
time there is endothelin (a potent
endothelium-derived
vasoconstrictor) release.

The effect is transient and bleeding would


resume if not for activation of the platelet and
coagulation systems

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o
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o
o
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o
o

Events that take place are the


interaction of the platelets and
the vascular response to injury
Initial response in primary
hemostasis is Platelet adhesion,
whereby in requires vWF for the
platelet to adhere in the injured
area of the endothelium
There is a change in the shape
of the platelet membrane
There is also release of granules
and promote platelet
aggregation
Release of platelet factor V, in
the activated plelets, will lead
toactivation of fibrin function,
hence having a stable fibrin clot
In a normal individual,
coagulation is initiated within 20
seconds after an injury occurs to
the blood vessel damaging the
endothelial cells.
PLATELETS immediately form a
hemostatic plug at the site of
injury. This is called primary
hemostasis.
BV injury exposes collagen which
will attract platelets.
Platelets will adhere to that site of
injury thru membrane receptors
(glycoprotein Ib (GpIb) receptors)
which will link with von
Willebrand factor (vWF) on
exposed extracellular matrix
(ECM).

The platelets will then change


shape from round or discoid to
very irregular and release
granules that will recruit
additional platelets to form the
platelet plug.
The end result is the formation of
platelet plug

Plasma components called


coagulation factors respond (in a
complex cascade) to form fibrin
strands which strengthen the
platelet plug.
Plasma proteins, tissue factor and
calcium interacts on the surface
of the platelets to form a fibrin clot.
During primary hemostasis there is constriction of
damged blood vessels, which decreases the blood flow
through the injured vessels. Platelets clump together and
adhere to injured vessels to form a platelet plug and further
inhibit bleeding.
During secondary hemostais the coagulation factor
present in the blood (and tissue factor) interact, forming a
fibrin meshwork (clot) to more efficiently stop the bleeding.

Coagulation Cascade

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and tissue factor), whilst the


intrinsic pathway acts
to amplify (but not initiate) the
coagulation cascade.
Two parts:
intrinsic system - activated
by coagulation factors that
are already present in the
blood,
acts to amplify (but
not initiate) the
coagulation cascade.
Two pathways
Intrinsic already found
within the blood
Amplifies coagulation
cascade
Extrinsic found outside the
blood vessel, more important
in response to injured blood
vessels
Initiates coagulation
cascade
Tissue factor adjacent to the site of
injury activates the inactive factor
VII. The activated factor VII,
together with tissue factor,
activates the inactive factor X.
the activated factor X, together
with factor V will cleave
prothrombin to thrombin. The
thrombin will then cleave fibrinogen
to fibrin. At the same time, the
thrombin will cause platelet
activation.
the coagulation cascade of
secondary hemostasis has two
pathways, the Contact Activation
pathway (formerly known as the
Intrinsic Pathway) and the Tissue
Factor pathway (formerly known as
the Extrinsic pathway) that lead to
fibrin formation.
it is now known that the main
pathway for initiation of coagulation
is the extrinsic pathway (factor VII

extrinsic system - initiated


outside of blood vessels in
the presence of injury to a
vessel.
factor VII, which is
present in the blood, is
converted to its
activated form, factor
VIIa, by binding to
tissue factor (TF).
TF/factor VIIa complex
formed in this way
converts factor X into
its activated form
factor Xa. This in turn
forms a complex with
factor Va and thus
brings about cleavage
of prothrombin to form
thrombin.
In turn, thrombin
cleaves fibrinogen to
form fibrin, which
polymerises to form
fibrin threadsIn the
past, most people
believed that either the
intrinsic or extrinsic
pathway could initiate
coagulation.
it is now known that
the main pathway for
initiation of
coagulation is the
extrinsic pathway
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(factor VII and tissue


factor), whilst the
intrinsic pathway acts
to amplify (but not
initiate) the
coagulation cascade.
The four phases of coagulation
(1) Contact phase; small amounts of factor XII
automatically activate when coming in contact with (-)
charged surface like collagen
(2) Activation of factor X via intrinsic and extrinsic
pathway; to summarize these two systems, the intrinsic
coagulation mechanism requires a contact phase, followed
by activation of factor IX and X. The extrinsic mechanisms
primary action is the activation of factor X. From this point
on, two systems will combine into common pathway. It is
most likely the systems operate simultaneously in vivo.
(3) Conversion of prothrombin to thrombin; marks the
beginning of common pathway, which adheres to platelet
surfaces along with factor Xa and factor V (cofactor). This
complex converts prothrombin to thrombin
(4) Formation of fibrin clot; fibrin formation occurs by the
action of thrombin on fibrinogen.

2
3
4
5

Tertiary phase of hemostasis

o Defined as the formation of


plasmin, which is the main
enzyme responsible for
fibrinolysis (breakdown of the
clot).
o Controls/regulates formation of
clot by way of tissue
plasminogen activator (t-PA).
o It occurs so that the clot does
not grow enough to obstruct the
pathway of blood.
o t-PA will activate Plasminogen
to form plasmin.
- While the coagulation cascade
is happening, the endothelial
cells at the same time releases
tPA, which will activate
plasminogen giving rise to
plasmin, giving rise to
breakdown of platelets in the
clot which will produce fibrin
degradation product (FDP)

PRE-ANALYTICAL VARIABLES ON
HEMOSTATIC TESTS
A coagulation test result is only good as
the sample received.
- you would want to a have a result
that is accurate, precise and
reproducible
- type of sample must be strictly
monitored in order to get the real
actual result
Pre-analytical variables
1 1. Sample specimen
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volume 9:1 ratio of blood sample
to anticoagulant-assuming with normal Hct

reject clotted, hemolyse, lipemic


samples - If with clotted specimen and you
removed the clot

result becomes prolonged


1
2 2. Specimens will be rejected in
the following circumstances:
presence of clots
visible hemolysis, lipemic
samples
incorrect anticoagulant
tubes filled to less than or
more than the recommended
volume
3
2 3. Anticoagulant- blue top ; 3.2 %
buffered sodium citrate which binds
with calcium (in the blood) to arrest
the clotting stage
3
4 4. Manner of phlebotomy
- Tourniquet should be released
once there is backflow of blood
in the syringe in order not to
activate extrinsic pathway
- Tourniquet should not stay > 2
min
- If needle punctures the blood
vessel, mere puncturing already
activates the clotting mechanism
- When the blood sample is to be
used in coagulation studies, as
much as possible trauma is
minimize thus probing is avoided
- Once blood is seen just beyond
the needle, tourniquet may be
released
5
6 5. Storage- samples frozen at -20C,
stable 2 weeks; samples frozen at
-70C, stable 6 months
- Should be done right away
- Once settled, should be
centrifuged
- Separate RBC and plasma

Controls are test specimens to


check if machine is working or
will give you an accurate result
o if it falls within the control
then it is good
o if it falls beyond, then it
means that there is a
problem

USES:
1
2
3

1. Pre- operative assessment


of hemostasis
2. Investigate hemostatic /
bleeding disorders
3. Monitor anticoagulant
therapy such as cardiac
patients who underwent
bypass surgery and valvular
replacements

TEST OF HEMOSTASIS
Short Screening tests:
Bleeding.T - to test Platelet
& BV function, test for
primary hemostasis
Prothrombin.T Extrinsic,
common pathway
APTT Intrinsic , common
pathway
Thrombin.T common
pathway
D-dimer test
Specialized (Extended)
diagnostic tests:
Factor assays
Tests of thrombosis TT,
FDP, DDA,
Platelet function studies:
Adhesion,
Aggregation,
Release & PG
pathway tests
Bone Marrow study
SHORT SCREENING TESTS/PANEL

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defects ex. Von Willebrands ds.,


capillary defects

BLEEDING TIME
Measures interaction between
platelets and (injured) vascular
endothelium.
Test for adequacy of primary
hemostasis or platelet function and
capillary integrity
Need adequate number of platelets
for a normal bleeding time
3 methods:
o Duke crude way
o Meilke has a standard depth
and length of puncture
o Ivy - uses BP cuff and lancet
Recommended criteria prior to testing
1. platelet count: >75,000/mm3
2. only 1 bleeding time/24hrs unless
patient has
received therapy ex. platelet infusion
3. discontinue heparin 6 hrs prior to
testing
Limitations:

If thrombocytopenia is present
( <75,000/mm3) BT may be
prolonged.
Aspirin & aspirin containing drugs
may prolong BT
Interpretation:
Reference range: 1-5 min. (Dukes)
2-10 min. (Mielke)
Prolonged in conditions like
thrombocytopenia, platelet function

PLATELET COUNT
USES ( for evaluation, diagnosis & followup):

bleeding disorders
Purpura/ petechiae lesions
Drug-induced thrombocytopenia
Idiopathic thrombocytopenic
purpura
DIC
Leukemia
chemotherapeutic mgt of malignant
diseases
PLATELETS
bone marrow megakaryocytes
Life span 7-10 days
Normal.count 150-450x109/l
36 hours in spleen - 1/3 of platelets
are destroyed in spleen
Functions:
hemostatic plug formation
Coagulation factors - release,
synthesis
Methods of Platelet Count
Direct (Rees and Ecker
method)
Indirect (Slide method)
Automated platelet count
Reference range : 150,000
450,000/mm3
specimen used is anticoagulant
blood (EDTA)
Platelet count estimation on PBS is
performed under OIF
7-20 platelets/OIF is adequate.
PROTHROMBIN TIME (Protime, PT)
USES:
to detect abnormalities in the
extrinsic and common
pathway of coagulation.
monitor the effect of warfarin
anticoagulant therapy
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assess the clotting ability of


blood

Buffered citrate is the standard


anticoagulatn for collection of
samples for coagulation studies.
Optimal volume ratio of
anticoagulant to blood should be
maintained at 1:9

Sample of the patient's blood is


obtained by venipuncture.
The blood is decalcified (by
collecting it into a tube with
oxalate or citrate ions) to
prevent the clotting process
from starting before the test.
The blood cells are separated
from the liquid part of blood
(plasma) by centrifugation.
The PT test is performed by
adding the patient's plasma to
some source of Tissue Factor
(e.g.: a protein,
thromboplastin, from
homogenized brain tissue) that
converts prothrombin to
thrombin.
The mixture is then kept in a
warm water bath at 37C for
one to two minutes.

Calcium chloride (excess


quantities of ionized calcium) is
added to the mixture in order
to counteract the sodium
citrate and allow clotting to
start.
- The test is timed from the
addition of the calcium chloride
until the plasma clots.
Interpretation:
Normal: 11-15 seconds ; this
means the patient has normal
amounts of clotting factors VII
and X
Note: a prolong PT is
considered abnormal
Abnormal: prolonged PT
indicates deficiency in
Factors VII, X, V, prothrombin
or fibrinogen.
It may also indicate vit
K deficiency, liver
disease or DIC
Coumadin (Warfarin) drug
primarily used to treat many
thromboembolic conditions.
MOA: inhibit the synthesis of
vit.K-dependent clotting
factors which include II, V, VII
and X and the anticoagulant
proteins C and S
narrow therapeutic index
drug narrow window where
coumadin is considered safe
and effective.
-

International Normalized Ratio (INR)


ISI
INR = Patient PT_____
Mean Normal PT
INR system is intended only for
patients on stable oral
anticoagulant therapy
ISI - International sensitivity
index, it pertains to the
responsiveness of the
prothrombin in relation to
the reduction of vit K
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dependent clotting factor


upon addition of
thromboplastin
a value that is
provided by the
manufacturers kit that
supplies the
thromboplastin (usually
from animals of
different sources ; one
thromboplastin or
recombinant
thromboplastin (from
humans) would be
superior over the other
in order to
standardize this factor
An INR of 1.0 means that the
patients protime is normal
An INR greater than 1.0 means
the clotting time is elevated
Patients on oral anticoagulant
therapy - target INR of 2.0 to
3.0
Limitations
Anticoagulant to blood ratio is
NOT 1:9
Intake of drugs/substances
Prolongedcorticosteroids, OCP,
asparaginase,clofibrate
,
erythromycin,
ethanol
- Shortened
antihistamines,
phenobarbital, OCP,
vit. K, caffeine
ACTIVATED PARTIAL
THROMBOPLASTIN TIME (APTT)
assesses the coagulation
proteins of the intrinsic
system and common
pathways

useful screening test for


detecting deficiencies of
factors VIII, IX, XI, and XII.
use to monitor heparin
therapy whose therapeutic
range is 60-100 seconds.
detect congenital and
acquired abnormalities of the
intrinsic coagulation pathway

The aPTT test uses blood which


is decalcified to prevent
clotting before the test begins.
The plasma is separated by
centrifugation.
(Ionized) Calcium and
activating substances are
added to the plasma to start
the intrinsic pathway of the
coagulation cascade.
The substances are: kaolin
(hydrated aluminum silicate)
and cephalin.
Kaolin serves to
activate the contactdependent Factor XII
cephalin substitutes for
platelet phospholipids.
The partial thromboplastin
time is the time it takes for a
clot to form, measured in
seconds.
Normally, the sample
will clot in 35 seconds.
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Limitations:
o Lipemic samples may
interfere with photo-electric
measurements of clot
formation
o Specimens should not be
obtained after a meal
o Patients receiving
intermittent heparin
injections
APTT test should be
obtained one hour
before the next dose of
heparin is scheduled.
The specimen should
not be drawn from an
arm with a heparinized
catheter or heparin
lock
Ref.range : 25-35 sec.

THROMBIN TIME
assay to determine the time for clot
formation.
Measures the common
pathway
direct measure of fibrinogen
function and is used to ascertain if
there is a defect in fibrinogen
function.

The thrombin time will be


prolonged in
hypofibrinogenemic states
and if an abnormal protein
fibrinogen
(dysfibrinogenemia) is
present.
Ref.range : 13-15 sec.
measurement of conversion of
fibrinogen to fibrin
measures the common pathway
Procedure:
1 - Add thrombin to patients
plasma
2 - Measure time to clot

D-Dimer Test (XDP, Fibrin degradation


fragment)
assessment of the tertiary phase of
hemostasis
specific measure of fibrinolysis
Elevated D dimers are seen in DIC,
pulmonary embolism, arterial and
venous thrombosis, septicemia,
cirrhosis, carcinoma, sickle cell
crisis, and following operative
procedures.
Test methodology: immunoassay
(Elisa or
Latex agglutination)

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