PRIMARY AND SECONDARY

HEMOSTASIS
 Hemostasis
• Process in which several factors work together
or in sequence to stop the flow of blood from
an injured blood vessel

• Combination of clotting and lysing

mechanisms that maintain the integrity of the
vascular system
 3 aspects of hemostasis
1. Extravascular:
– Physical constriction of injured skin and tissues resulting in
the release of tissue juice which contains tissue
thromboplastin
2. Vascular:
– Constriction of injured blood vessel brought about by reflex
constriction and by the serotonin released by disintegrated
platelets
3. Intravascular:
– Physical and biochemical changes undergone by platelets and
the interaction of the different coagulation factors

ALL THESE ASPECTS REACT SIMULTANEOUSLY TO STOP

BLEEDING.
 3 steps of normal hemostasis

• Vasoconstriction
• Platelet aggregation
• Fibrin formation
 Coagulation Factors:
I. Fibrinogen XI. Plasma thromboplastin
II. Prothrombin antecedent (PTA)
III. Tissue thromboplastin XII. Hageman factor/ glass
IV. Calcium factor/ contact factor
V. Labile factor/ preaccelerin/ XIII. Fibrin stabilizing factor
(FSF)/ Fibrinase
AC-Globulin
VI. Obsolete • Prekallikrein/ Fletcher factor
VII. Proconvertin/ stable factor • High molecular weight
kininogen/ Fitzgerald factor/
VIII. Antihemophilic factor (AHF) Williams Factor
IX. Plasma thromboplastin
component (PTC)/ Christmas
Factor
X. Stuart-Prower
factor/autoprothrombin III
 Stages of hemostasis: Primary
Hemostasis
• Constriction of damaged blood vessels
(vasoconstriction)
• Platelet plug formation
• Blood vessels
– Intact blood vessels secrete:
• Prostacyclin: AKA prostaglandin I2 or PGI2; inhibits platelet
activation
• Adenosine: stimulates vasodilation
• Thrombomodulin: binds thrombin
• Heparan sulfate: weakly enhances the activity of AT-III
• Tissue plasminogen activator (TPA): MAJOR plasminogen
activator
• Von Willebrand Factor: carrier protein of Factor VIII; important
for platelet adhesion
• Platelets
 Laboratory tests for Primary
hemostasis:
• Bleeding time
• Capillary Fragility Test
• Platelet aggregation test
• Platelet adhesiveness Test
• Platelet count
BLEEDING TIME
• Measures the ability of small
blood vessels to control free flow
of blood after injury

• It is a measure of the function of

platelets as well as the integrity
of the blood vessel wall

• In vivo measurement of platelet

participation in small vessel
hemostasis
• Screening test for detecting
disorders of platelet function
and von Willebrand Disease

• Directly affected by platelet

count and the ability of the
platelets to form a plug
• Purpose: provide a
measure of the platelet
function in small vessel
hemostasis
• A prolonged bleeding time is
not in itself diagnostic of
underlying platelet disorders.
It indicates the need for more
quantifiable test.
• The results are affected by
the platelet count, thickness
or vascularity of the skin
(less thick or vascular =
increased BT), quality of the
blood vessels.

• It is not affected by the

• Prolonged bleeding time:
–When the platelet count is lower than
30,000-50,000/uL
–Dysfunctional platelet
–Von Willebrand Disease
–Ingestion of aspirin-containing
compounds, anti-inflammatory drugs,
anticoagulants and some antibiotics
 Methods:
• Duke’s method
• Ivy’s method:
• Copley-Lalitch Method
• Aspirin Tolerance test (Quick’s method)
 Duke’s method
• Materials:
– 70% isopropyl alcohol
– Cotton
– Blood lancet
– Filter paper
– Timer
 Duke’s method
• Procedure:
1. Cleanse the earlobe or the 3rd or 4th finger with
70% isopropyl alcohol and allow it to dry
2. Make a relatively deep puncture with a sterile
blood lancet and start timing (3mm)
3. Blot the blood using filter paper every 30 seconds.
NOTE: do not allow the filter paper to touch the
wound as this will hasten the bleeding time. (START
TIMING IMMEDIATELY AFTER THE SKIN PUNCTURE)
4. Stop timing as bleeding ceases and record the
bleeding time

• Normal value: 2 – 4 minutes

 Ivy’s method:

• The puncture site is the patient’s

forearm
• It uses sphygmomanometer to
establish pressure (40 mmHg) and the
result is recorded using the wiped
blood in the filter paper for every 30
seconds (3 successive punctures in the
form of triangle; 2 – 3 mm)
• Normal value: 1 – 7 minutes
 Ivy’s method
• Procedure:
1. Apply a sphygmomanometer cuff on the patient’s upper
arm. Inflate it at 40mmHg. Maintain this pressure during
the entire process
2. Cleanse the area on the volar surface of the forearm with
70% alcohol and allow it to dry. The selected area should
be free from visible veins.
3. Make three successive punctures in the form of triangle to
a depth of 2-3 mm using a disposable lancet. Start timing
4. Blot the blood with filter paper at 30-second interval until
the bleeding stops
5. Record the time when the bleeding stops
 Extra notes
• Locate area for bleeding time:
– From the middle finger, move up
the arm to a straight line to 5 cm
below the fold in the elbow
– Select site avoiding surface veins,
bruises, and edematous areas

• The time between the inflation

and incision should be 30 – 60
 Extra notes

• Do not touch the paper directly to

the incision, so as not to disturb
the formation of the platelet plug.
• The bleeding time is determined
to the nearest 30 seconds
• BT in newborns (Ivy’s):
– 20mmHg: < 2 lbs
– 25mmHg: 2 – 4 lbs
 Copley-Lalitch Method

• The blood flowing in the

punctured finger (6mm) is
observed and timed when
immersed in a beaker of sterile
physiologic NSS prewarmed at 37
degrees Celsius.

• Normal Value: <3 minutes

• Procedure:
1. Cleanse the fingertip with alcohol
and allow it to dry
2. Make a puncture to a depth of
6mm. Start timing
3. Immerse the punctured finger in
sterile physiologic saline solution
warmed at 37 degrees Celsius
4. Record
• Observe the flowing out of
red blood against the
colorless NSS

• Stop timer as soon as blood

stops flowing out
 Aspirin Tolerance test (Quick’s
method)
• This method involves determination of the
patient’s first bleeding time initially and then if
the bleeding time is more than 6 minutes, do not
proceed.

• Otherwise, give the patient 2 tablets of aspirin

and a glass of water then determine the patient’s
second bleeding time using Duke’s method as
well after 2 hours.

• Normal value: 1st BT – 2nd BT = < 2 minutes

(Secondary Hemostasis Test)

CLOTTING TIME
 Clotting time

• It measures the
period required for
blood to clot or
solidify after it has
been extracted from
the body
• Screening test for all
stages of the intrinsic
coagulation pathway

• Monitor heparin
therapy
 Methods

• Whole blood or Lee and

White method
• Capillary blood
methods:
–Drop or slide
–Capillary tube or Dale
and Laidlaws method
 Lee and White method (whole blood)
• Procedure:
1. Label 3 tubes (1, 2, 3). Place the clean, dry tubes in a 37
degree Celsius water bath
2. Obtain 3 ml venous blood in a plastic syringe, preferably
using the 2-syringe method
3. Place 1 ml of blood into each tube starting with tube 1
4. Start timing as blood is delivered into tube 1. Put the
tubes back into the water bath
5. Gently tilt tube 3 every 30 seconds until blood solidifies.
Handle tube 2 in the same way. Finally tilt tube 2 until
blood forms a solid clot
6. Stop timing and record the coagulation time
 Lee and White method (whole blood)
• Normal value: 7 – 15 minutes
• This method is a rough measure of all intrinsic
clotting factors in the absence of tissue factors
• Variations are wide and the test sensitivity is
limited
• Whole blood, when removed from the
vascular system and exposed to a foreign
surface, will form a solid clot
 Lee and White method (whole blood)

• 2 syringe method:
– Collect at least 1-2 ml of
blood in a plastic syringe and
discard (this prevents tissue
thromboplastin from entering
the blood sample)

– Collect at least 5 ml of blood

in the second plastic syringe
 Drop or slide
• Procedure:
1. Perform a skin puncture. Discard
the first drop of blood
2. Place a drop of blood on a clean
glass slide. Start timing
3. Draw the tip of the lancet across
the drop of blood at 30-second
interval until fibrin threads cling
to the tip
4. Stop timing and record the
• Start the timer as soon as the
2nd drop of blood appears
• Fibrin threads appears as a
“thread-like” substance that
clings to the tip of the lancet
• Observe time accurately
• Do not perform both tests on
one finger
 Dale and Laidlaws method (Capillary
tube)
• Procedure:
1. Make a skin puncture. Wipe off the first drop of blood
2. Fill a non-heparinized capillary tube ¾ with blood
3. Start timing as soon as blood enters the tube
4. Set the capillary tube aside in a horizontal position for 2
minutes
5. Break off about 1 cm of the capillary tube at 30 – second
interval until fibrin thread bridges the broken ends of the
capillary tube
6. Record the time

Normal value: 2 – 4 minutes

 Extra notes
• Poor venipuncture technique:
– hemolysis or tissue thromboplastin to
mix with the blood, shortens the
clotting time
– Bubbles entering the syringe, increase
the rate of coagulation
• Unnecessary agitation shortens blood
coagulation
• Always tilt the tube in the same
direction and at the same angle so
that the blood is moving in the same
pathway up the side of the tube each
time
 Laboratory tests for Primary
hemostasis:
• Bleeding time
• Capillary Fragility Test
• Platelet aggregation test
• Platelet adhesiveness Test
• Platelet count
 Capillary Fragility Test

• Tourniquet test / Capillary Resistance Test

• Ability of the capillaries to resist pressure
• Formation of tiny spot or petechiae is
observed
• Normal:
– Capillaries in the arms will resist pressure up
to 100 mmHg
• Abnormal:
– Petechiae grade of 2+ and up
– Seen in thrombocytopenic purpura,
decreased fibrinogen and vascular purpura
• Materials:
–Sphygmomanometer
–Timer
• Tourniquet/ Rumple – Leede / Hess Test procedure
1. Use a pen to mark any tiny hemorrhages or
petechiae that are already present on the patient’s
arm
2. Wrap the sphygmomanometer around the arm
just above the elbow
3. Inflate the apparatus at (either of the following):
– 100 mmHg for 5 minutes
– 50 mmHg for 10 minutes
– Midway between the systolic and the diastolic pressure
for 5 minutes (Quick’s)
– 35 mmHg for 15 minutes (Gothlin’s)
4. Release the pressure and allow 15 – 30 minutes to
elapse
5. Count the number of fresh petechiae
• Manner of reporting:
Grade Petechiae
1+ 1 – 10
2+ 11 – 20
3+ 21 – 50
4+ > 50

Normal: 1+ = 1 – 10
petechiae
Other methods for Capillary resistance
test

• Suction or
petechiometer
–Application of negative
pressure to the skin and
to the vascular tissue
causes invasion of blood
–Normal: > 4 petechiae at
200 mmHg
 Laboratory tests for Primary
hemostasis:
• Bleeding time
• Capillary Fragility Test
• Platelet aggregation test
• Platelet adhesiveness Test
• Platelet count
 Platelet Aggregation
• Gold standard test to
determine platelet function
• During primary hemostasis
platelets clump (aggregate)
at the site of injury
• Platelet-rich plasma is treated with a known
aggregating agent.
– If aggregated, cloudiness or turbidity patterns are
determined
– Light transmitted through a suspension of aggregated
platelets with that of a suspension of nonaggregated
platelets using an aggregometer.
– The curve that is obtained can be used to assess platelet
function.
 Laboratory tests for Primary
hemostasis:
• Bleeding time
• Capillary Fragility Test
• Platelet aggregation test
• Platelet adhesiveness Test
• Platelet count
 Platelet adhesiveness
test

• Adhesiveness of
platelets can be
measured in
vitro by their
ability to adhere
to glass
surfaces.
Principle:
• A platelet count is performed on both
specimens of blood.
• The number of platelets in the blood,
collected through the glass bead
collecting system, will be lower than
the number obtained by routine
venipuncture.
• This is because platelets have adhered
to the column of glass beads due to
their adhesive characteristics.
• The result of this procedure are
 Laboratory tests for Primary
hemostasis:
• Bleeding time
• Capillary Fragility Test
• Platelet aggregation test
• Platelet adhesiveness Test
• Platelet count
 Laboratory test for Secondary
Hemostasis:
• Clotting time
• Prothrombin time
• Activated partial thromboplastin time
• Stypven Time
• Thrombin time
• Reptilase time
• Duckert’s Test
• Mixing Studies
Laboratory test for Secondary
Hemostasis
 Laboratory test for Secondary
Hemostasis:
• Clotting time
• Prothrombin time
• Activated partial thromboplastin time
• Stypven Time
• Thrombin time
• Reptilase time
• Duckert’s Test
• Mixing Studies
 Secondary hemostasis

• Extrinsic
• Intrinsic
• Common
 Activated partial
thromboplastin time
–Measures the time required to
generate thrombin and fibrin
polymers via the intrinsic and
common pathways

–Sodium citrate is the preferred

anticoagulant and the test should
be performed within 2 hours after
blood collection
– Principle: the calcium in the whole blood is
bound by the sodium citrate anticoagulant to
prevent coagulation. The plasma, after
centrifugation, contains all intrinsic factors
except calcium and platelets (partial
thromboplastin) and an activator (to ensure
maximal activation), are added to the plasma.
The time required for the plasma to clot is the
activated partial thromboplastin time

• Heavy spin so that we can have platelet poor plasma.

–Abnormally prolonged:
• deficiency in one of the coagulation factors
• inhibitor or circulating anticoagulant
• Disseminated intravascular coagulation
(DIC)
–Abnormally shortened:
• Partial clotting of blood
• Traumatic venipuncture
• High Factor VIII levels
• Presence of platelets in the plasma
Procedure:
1. Pre-incubate the QuikCoag Calcium Chloride (0.02M) to
37 degrees Celsius for at least 10 minutes.
2. Pipette 100 uL of test or control plasma into a test
cuvette, incubate at 37 degrees Celsius for 1 to 2 minutes.
3. Add 100 uL of the QuikCoag APTT reagent to the cuvette
containing the plasma. Maintain the suspension of the
reagent by magnetic stirring or mixing by inversion
immediately prior to use.
4. Incubate the mixture at 37 degrees Celsius for 3 minutes
5. Rapidly add 100 uL of the pre-incubated QuikCoag
Calcium Chloride (0.02M) and simultaneously start the
timer.
6. Record the clotting time in seconds.
• Expected values
– Can vary from laboratory to laboratory
– Normal plasma range: 24 – 39 seconds
PROTHROMBIN TIME
• Evaluates the generation of thrombin
and the formation of fibrin via the
extrinsic and common pathway
• Thromboplastin reagent is used
• Can be prepared through: tissue
extraction of rabbit brain or lung, tissue
culture, and molecular methods
• Mixture of tissue factor, phospholipid,
and calcium ions and is used to initiate
clotting measure as the PT.
– Prolonged results
• Deficiency of one or more factors in the extrinsic
and common pathway: factors VII, X, V, and II or I
• Prolonged values will be seen if an oral
anticoagulant such as coumarin or a coumarin-
containing substance (e.g., rat poison) is ingested
• Vitamin K deficiency, certain liver diseases,
disseminated intravascular coagulation,
dysproteinemias and presence of circulating
anticoagulants and fibrin/ fibrinogen split products
– Principle: the calcium in the whole blood is
bound by sodium citrate, thus preventing
coagulation. Tissue thromboplastin to which
calcium has been added, is mixed with the
plasma and the clotting time is notes. Factor VII
reacts with the tissue factor (thromboplastin) in
the presence of calcium ions to activate Factor
X. the coagulation mechanism continues from
there.

– Prothrombin 20210: discovered 1996

• Point mutation in the prothrombin gene,
• Increased prothrombin activity
• No screening test but can be investigated using
molecular techniques
• Procedure:
1. Pre-incubate the QuikCoag PT reagent to 37 degrees
Celsius for at least 10 minutes. Maintain the
suspension of the reagent by magnetic stirring or
mixing by inversion immediately prior to use.
2. Pipette 100 uL of test or control plasma to the test
cuvette
3. Incubate at 37 degrees Celsius for 1 minute
4. Rapidly add 200 uL of the pre-incubated QuikCoag PT
reagent simultaneously starting the timer.
5. Record the clotting time in seconds.
• Expected Values:
– Varies from laboratory to laboratory
– Normal Range: 10 – 15 seconds
 International normalized ratio (INR):
• The sensitivity of an individual thromboplastin
to another can vary greatly between and
within lots and maybe on single batch of
reagents depending on the shelf time of
reagents
• The more sensitive the thromboplastin
reagent, the longer the resulting PT; the less
sensitive the reagent, the shorter the resulting
PT
International normalized ratio (INR) is a
calculation made to standardize prothrombin
time. INR is based on the ratio of the patient's
prothrombin time and the normal mean
prothrombin time. Prothrombin time is a test to
learn how fast the blood clots in patients
receiving oral anticoagulant medication.
• INR range of 2.0 to 3.0: recommended
for most indications (e.g., treatment or
prophylaxis of deep venous thrombosis
[DVT], or prevention of further clotting
in patients who have had a myocardial
infarction)
• INR of 2.5 to 3.5 is recommended for
patients with prosthetic heart valves
• This varies to different laboratories
 Stypven time (obsolete)

• AKA Russell Viper Venom time – Daboia

russelii viper venom is used
• Used to detect deficiencies in prothrombin,
fibrinogen, and factors V and X (common
pathway)
• This test may be useful in distinguishing a
factor VII deficiency
Principle:
• Stypven is a thromboplastin-like substance that
activates factor X.
• When this reagent is added to plasma, together
with platelets and calcium chloride, the
coagulation process is begun at the point of
factor X activation with subsequent conversion
of prothrombin to thrombin and fibrinogen to
fibrin.
• In this way, deficiencies in factors V, X,
prothrombin and fibrinogen may be detected
 Thrombin time
• Measures the availability of functional
fibrinogen
• Principle: a measured amount of
thrombin is added to plasma. The
length of time for a fibrin clot to form
is recorded as the thrombin time
 Reptilase time
• Reptilase: enzyme from the venom of
Bothorps atrox snake; capable of converting
fibrinogen to fibrin and unaffected by heparin
• Both thrombin and reptilase times will be
prolonged when fibrinogen level is decreased
in:
i. Dysfibrinogenemia
ii. Streptokinase therapy
iii. Presence of fibrinogen degradation
products
Principle:
• When reptilase is added to the
plasma, it acts by releasing
fibrinopeptide A from the fibrinogen
molecule. The resultant monomers
polymerize end-to-end, forming a clot
 Duckert’s test (5 M urea clot solubility
test)
Principle:
• Factor XIII screening test • Patient plasma is clotted
• Fibrin clot is insoluble in by the addition of calcium
5M Urea and 1% chloride.
monochloroacetic acid • 5M urea is added to the
when left standing for clot.
24 hours • If factor XIII is absent in the
• Alternate procedure: patient’s plasma, the clot is
use 2% acetic acid or 1% dissolved in less than 24
monochloroacetic acid hours by the urea.
in place of 5M urea