HEMOSTASIS

I .Definitions:

a.Hemostasis is the process by which blood is changed to a solid state. It is

what stops the bleeding after an injury to the blood vessels occurs. The
blood vessels are protected by cells that prevent the formation of thrombin, a
coagulation protein that catalyzes reactions in the bloodstream. When an
injury permeates the cells and gets to the vessels, hemostasis occurs.

b.Haemostasis is the process of maintaining blood in its fluid state

while stopping bleeding in cases of trauma or disease. It is the
balance between the formation of blood clots to stop bleeding (or
haemorrhage) from injured blood vessels and the prevention of clot
formation (or thrombosis) beyond the site of vessel injury.

c.Hemostasis is a complex process which causes the bleeding process to

stop. It refers to the process of keeping blood within a damaged blood vessel
(the opposite of hemostasis is hemorrhage).

II .Four major component of hemostasis, their roles and tests used to

evaluate them.

The ability of the body to control the flow of blood following vascular injury is
paramount to continued survival. The process of blood clotting and then the
subsequent dissolution of the clot, following repair of the injured tissue, is termed
hemostasis. Hemostasis, composed of 4 major events that occur in a set order
following the loss of vascular integrity:

1. The initial phase of the process is vascular constriction. This limits the
flow of blood to the area of injury.
2. Next, platelets become activated by thrombin and aggregate at the site
of injury, forming a temporary, loose platelet plug. The protein fibrinogen
is primarily responsible for stimulating platelet clumping. Platelets clump
by binding to collagen that becomes exposed following rupture of the
endothelial lining of vessels. Upon activation, platelets release the
nucleotide, ADP and the eicosanoid, TXA2 (both of which activate
additional platelets), serotonin, phospholipids, lipoproteins, and other
 proteins important for the coagulation cascade. In addition to induced
secretion, activated platelets change their shape to accommodate the
formation of the plug.
3. To insure stability of the initially loose platelet plug, a fibrin mesh (also
called the clot) forms and entraps the plug. If the plug contains only
platelets it is termed a white thrombus; if red blood cells are present it is
called a red thrombus.

4. Finally, the clot must be dissolved in order for normal blood

flow to resume following tissue repair. The dissolution of the clot
occurs through the action of plasmin.

Blood Coagulation Tests and Interpretations

a.Bleeding time assays are used to evaluate the vascular and platelet
responses that are associated with hemostasis. The bleeding time is a frequent
assay performed on preoperative patients to ensure there is an adequate
response to vessel injury prior to surgery. As indicated above, the rapid
responses to vascular injury (occurring within seconds) are vessel constriction
and platelet adhesion to the vessel wall. The Ivy method for determining the
bleeding time involves the use of a blood pressure cuff (sphygmomanometer)
which is placed on the forearm and inflated to 40mmHg. A superficial incision is
then made on the forearm and the time it takes for bleeding to stop is recorded.
With the Ivy method bleeding should stop within 1–9 minutes. Any bleeding time
greater than 15 minutes would be indicative of a defect in the initial responses of
vessels and platelets to vascular injury. A less invasive bleeding time assay
involves the use of a lancet or special needle and a 3–4mm deep prick is made
on the fingertip or earlobe. This bleeding time assay is referred to as the Duke
method and in this assay bleeding should cease within 1–3 minutes. The
bleeding time is affected (prolonged) by any defect in platelet function, by
vascular disorders, and in von Willebrand disease but is not affected by other
coagulation factors. Disorders that are commonly associated with an increased
bleeding time include thrombocytopenia, disseminated intravascular coagulation
(DIC), Bernard-Soulier syndrome and Glanzmann thrombasthenia. Abnormal
bleeding times are also found in patients with Cushing syndrome, severe liver
disease, leukemia, and bone marrow failure.

Defects associated with factors of the pathways of blood coagulation can

also be assessed with specific assays.

b.The prothrombin time (PT) is an assay designed to screen for defects in

fibrinogen, prothrombin, and factors V, VII, and X and thus measures activities of
the extrinsic pathway of coagulation. When any of these factors is deficient then
the PT is prolonged. A normal PT is 11.0–12.5 seconds. A PT greater than 20
seconds is indicative of coagulation deficit. The PT is measured using plasma
after the blood cells are removed. A blood sample is collected in a tube
containing citrate or EDTA to chelate any calcium and thus inhibit coagulation
and then the cells are removed by centrifugation. After the cells are removed
excess calcium is added to the plasma to initiate coagulation. The most common
measure of PT is to divide the time of coagulation of a patients blood by that of a
known standard and this value is referred to as the international normalized ratio
(INR). Normal INR values range from 0.8–1.2. PT is used to determine the
correct dosage of the warfarin class of anti-coagulation drugs (e.g. Coumadin),
for the presence of liver disease or damage, and to evaluate vitamin K status.

c.The partial thromboplastin time (PTT) is used to assay for defects in the
intrinsic pathway of coagulation. The PTT assay has been modified by the
addition of activators that shorten the normal clotting time and this form of the
assay is referred to as the activated partial thromboplastin time (aPTT). The
PTT is normally prescribed in patients with unexplained bleeding or clotting. The
assay will evaluate the function of fibrinogen, prothrombin, and factors V, VIII, IX,
X, XI, and XII. A defect in any of these factors will result in a prolonged PTT (or
aPTT). A normal PTT is 60–70 seconds, whereas for the aPTT the normal range
is 30–40 seconds. The PTT is a standard assay used to assess the efficacy of
heparin anticoagulant therapy. Prolonged PTTs are associated with acquired or
congenital bleeding disorders associated with coagulation factor deficiency,
vitamin K deficiency, liver disease, DIC, von Willebrand disease, leukemia,
hemophilia, and during heparin administration.

III.Hemostasis

Sudden and severe loss of blood can lead to shock and death. When blood
vessels are damaged, Hemostasis (clot formation) will arrest bleeding. This
process is divided into three phases.

I. Vascular phase - Cutting or damaging blood vessels leads to vascular

spasm of the smooth muscle in the vessel wall. This produces a
vasoconstriction which will slow or even stop blood flow. This response
will last up to 30 minutes and is localized to the damaged area.

II. Platelet phase - Damaged endothelial cells lining the blood vessel
release von Willebrand's Factor. This substance makes the surfaces of the
endothelial cells "sticky". This condition may, by itself, be enough to close
small blood vessels. In larger blood vessels, platelets begin to stick to the
surfaces of endothelial cells. This effect is called Platelet Adhesion.

The platelets that adhere to the vessel walls now begin to secrete
Adenosine diphosphate (ADP) which is released from "stuck" platelets.
This material causes the aggregation of nearby free platelets which attach
to the fixed platelets and each other. This
aggregation of platelets leads to the formation of a platelet plug.

This clumping of platelets serves a number of functions:

1. It can plug the break in a small blood vessel.

2. Aggregated platelets release Platelet Thromboplastin (Factor III)

which activates the clotting
process.

3. Clumped platelets provide a surface essential for the clotting

process.Along with ADP, the
clumped platelets secrete thromboxane, a powerful vasoconstrictor.

III. Coagulation Phase - Begins 30 seconds to several minutes after

phases I and II have commenced.

A. The overall process involves the

formation of the insoluble protein Fibrin from
the plasma
protein Fibrinogen through the action of
the enzyme Thrombin. Fibrin forms a
network of fibers
which traps blood cells and platelets
forming a thrombus or clot.

B. This process depends on the presence in the blood of 11 different

clotting factors (proteins) and calcium (Factor IV). Ultimately, these
factors will generate the production of Prothrombin
Activator (Factor X). Depending on the initial trigger for the clotting
reactions, there are two
pathways leading to the formation of the thrombus; the Extrinsic
Pathway and the Intrinsic
Pathway.

IV.Effects on testing of sampling problems and running of coagulation

tests.

Most patients require laboratory evaluation (see Table 3: Hemostasis:

Laboratory Tests of Hemostasis by Phase ). The initial tests are

• CBC with platelet count

• Peripheral blood smear
• PT and PTT
Screening tests evaluate the components of hemostasis, including the number of
circulating platelets and the plasma coagulation pathways. The most common
screening tests for bleeding disorders are the platelet count, PT, and PTT. If
results are abnormal, a specific test can usually pinpoint the defect.
Determination of the level of fibrin degradation products measures in vivo
activation of fibrinolysis.

Prothrombin time (PT) screens for abnormalities in the extrinsic and common
pathways of coagulation (plasma factors VII, X, V, prothrombin, and fibrinogen).
The PT is reported as the international normalized ratio (INR), which reflects the
ratio of the patient's PT to the laboratory's control value; the INR controls for
differences in reagents among different laboratories. Because commercial
reagents and instrumentation vary widely, each laboratory determines its own
normal range for PT and PTT; a typical normal range for the PT is between 10
and 13 sec. An INR > 1.5 or a PT ≥ 3 sec longer than a laboratory's normal
control value is usually abnormal and requires further evaluation. The INR is
valuable in screening for abnormal coagulation in various acquired conditions
(eg, vitamin K deficiency, liver disease, DIC). It is also used to monitor therapy
with the oral vitamin K antagonist, warfarin.

Partial thromboplastin time (PTT) screens plasma for abnormalities in factors

of the intrinsic and common pathways (prekallikrein; high mol wt kininogen;
factors XII, XI, IX, VIII, X, and V; prothrombin; fibrinogen). The PTT tests for
deficiencies of all clotting factors except factor VII (measured by the PT) and
factor XIII. A typical normal range is 28 to 34 sec. A normal result indicates that
at least 30% of all coagulation factors in the pathway are present in the plasma.
Heparin prolongs the PTT, and the PTT is often used to monitor heparin therapy.
Inhibitors that prolong the PTT include an autoantibody against factor VIII (see
Coagulation Disorders: Hemophilia; see also Coagulation Disorders: Coagulation
Disorders Caused by Circulating Anticoagulants) and antibodies against protein-
phospholipid complexes (lupus anticoagulant—see Coagulation Disorders:
Coagulation Disorders Caused by Circulating Anticoagulants; see also
Thrombotic Disorders).

Prolongation of PT or PTT may reflect

• Factor deficiency
• Presence of an inhibitor of a component of the coagulation pathway
The PT and PTT do not become prolonged until one or more of the clotting
factors tested are about 70% deficient. For determining if prolongation reflects a
deficiency of one or more clotting factor or the presence of an inhibitor, the test is
repeated after mixing the patient's plasma with normal plasma in a 1:1 ratio.
Because this mixture provides about 50% of normal levels of all coagulation
factors, failure of the mixture to correct almost completely the prolongation
suggests the presence of an inhibitor in patient plasma.

The previously used bleeding time test is of doubtful reliability.

Table 3
Laboratory Tests of Hemostasis by Phase
Test Purpose
Formation of initial platelet plugs

Platelet count Quantifies platelet number

Bleeding time Screens for overall adequacy of

platelet adhesion and aggregation
on injured vascular surfaces

Platelet aggregation Evaluates adequacy of platelet

responsiveness to physiologic
stimuli that activate platelets (eg,
collagen, adenosine SOME TRADE
NAMES
ADENOCARD
Click for Drug Monograph
diphosphate, arachidonic acid)
Patterns are abnormal in hereditary
or acquired platelet functional
disorders

VWF antigen Measures total concentration of

plasma VWF protein

VWF multimer Evaluates distribution of VWF

composition multimers in plasma (eg, large
multimers are missing in type II
variants of VWD)

Ristocetin agglutination Screens for large multimers of VWF

 in plasma (often done as part of
routine laboratory evaluation for
VWD—see Thrombocytopenia and
Platelet Dysfunction: Von
Willebrand's Disease)

Ristocetin cofactor Quantifies large multimers of VWF in

activity plasma (see Thrombocytopenia and
Platelet Dysfunction: Von
Willebrand's Disease)

Formation of fibrin

PT Screens for the factors in extrinsic

and common pathways (factors VII,
X, and V; prothrombin; and
fibrinogen)

PTT Screens for the factors in intrinsic

and common pathways
(prekallikrein; high mol wt
kininogen; factors XII, XI, IX, VIII,
X, and V; prothrombin; and
fibrinogen)

Specific functional assays Determines activity as a percentage

for coagulation factors of normal

Thrombin time Evaluates the last step of

coagulation (thrombin cleavage of
fibrinogen to fibrin)
Is prolonged by heparin activation
of antithrombin and in conditions
resulting in qualitative fibrinogen
abnormalities or hypofibrinogenemia

Reptilase time If it is normal and thrombin time is

prolonged, provides presumptive
evidence that a plasma sample
contains heparin (eg, residual
heparin after extracorporeal bypass
or in a sample drawn from an IV line
kept open with heparin flushes)
because reptilase time is not
 affected by heparin activation of
antithrombin

Fibrinogen level Quantifies plasma fibrinogen, which

is increased in acute phase reactions
and decreased in severe liver
disease and severe DIC

Fibrinolysis

Clot stability during 24-h Causes lysis of clots in saline if

incubation in saline and fibrinolytic activity is excessive or in
in 5M urea 5M urea if factor XIII is deficient
Should be done in patients with
defective wound healing or frequent
miscarriages

Plasminogen activity Quantifies plasma plasminogen,

which is decreased in patients with
congenital early-onset venous
thromboembolism (rare)

α2-Antiplasmin Quantifies plasma level of this

fibrinolysis inhibitor, which is
reduced in patients with excessive
bleeding due to increased
fibrinolysis (rare)

Serum fibrinogen and Screens for DIC

fibrin degradation Decreased levels when plasmin has
products acted on fibrinogen or fibrin in vivo
(eg, in DIC)
Superseded by plasma D-dimer
assay

Plasma D-dimer Is measured with a monoclonal

antibody latex agglutination test or
with an ELISA
If high, indicates that thrombin has
been generated in vivo with
resultant deposition of fibrin,
activation of the cross-linking
enzyme, factor XIII, and secondary
 fibrinolysis
Has the practical advantage that it
can be done on citrate–plasma and,
thus, unlike the test for serum fibrin
degradation products, does not
require clotting blood in a special
tube to prepare serum free of
residual fibrinogen
Is useful in the diagnosis of in vivo
thrombosis (eg, deep venous
thrombosis, pulmonary embolism),
especially the sensitive ELISA
version

DIC = disseminated intravascular coagulation; ELISA = enzyme-

linked immunosorbent assay; VWD = von Willebrand's disease; VWF =
von Willebrand's factor.

Normal results on initial tests exclude many bleeding disorders. The main
exceptions are VWD and hereditary hemorrhagic telangiectasia. VWD is a
common entity in which the associated deficiency of factor VIII is frequently
insufficient to prolong the PTT. Patients who have normal initial test results,
along with symptoms or signs of bleeding and a positive family history, should be
tested for VWD by measuring plasma von Willebrand's factor (VWF) antigen,
ristocetin cofactor activity (an indirect test for large VWF multimers), and factor
VIII levels.

If thrombocytopenia is present, the peripheral blood smear often suggests the

cause (see Table 2: Thrombocytopenia and Platelet Dysfunction: Peripheral
Blood Findings in Thrombocytopenic Disorders ). If the smear is normal,
patients should be tested for HIV. If the result of the HIV test is negative and the
patient is not pregnant and has not taken a drug known to cause platelet
destruction, then idiopathic thrombocytopenic purpura is likely. If there are signs
of hemolysis (fragmented RBCs on smear, decreasing Hb level), thrombotic
thrombocytopenic purpura (TTP) or HUS is suspected, although sometimes other
hemolytic disorders can cause these findings. HUS occurs in children with
hemorrhagic colitis. The Coombs' test is negative in TTP and HUS. If the CBC
and peripheral blood smear demonstrate other cytopenias or abnormal WBCs, a
hematologic abnormality affecting multiple cell types is suspected, and a bone
marrow aspiration or biopsy is necessary for diagnosis.
Prolonged PTT with normal platelets and PT suggests hemophilia A or B.
Factor VIII and IX assays are indicated. Inhibitors that prolong the PTT include
an autoantibody against factor VIII and antibodies against protein-phospholipid
complexes (lupus anticoagulant). Such inhibitors are suspected when a
prolonged PTT does not correct upon 1:1 mixing with normal plasma.

Prolonged PT with normal platelets and PTT suggests factor VII deficiency.
Congenital factor VII deficiency is rare; however, the short half-life of factor VII in
plasma causes factor VII to decrease to low levels more rapidly than other
vitamin K-dependent coagulation factors (eg, in patients given warfarin SOME
TRADE NAMES
COUMADIN
Click for Drug Monograph
anticoagulation or in patients with incipient liver disease).

Prolonged PT and PTT with thrombocytopenia suggest DIC, especially in

association with obstetric complications, sepsis, cancer, or shock. Confirmation is
by finding elevated levels of D-dimers (or fibrin degradation products) and
decreasing plasma fibrinogen levels on serial testing. Prolonged PT or PTT with
normal platelet count occurs with liver disease or vitamin K deficiency or during
anticoagulation with warfarin SOME TRADE NAMES
COUMADIN
Click for Drug Monograph
or unfractionated heparin SOME TRADE NAMES
HEPFLUSH-10
Click for Drug Monograph
. Liver disease is suspected by history and confirmed by finding elevation of
serum aminotransferases and bilirubin; hepatitis testing is recommended.

Imaging tests are often required to detect occult bleeding in patients with
bleeding disorders. For example, head CT should be done in patients with severe
headaches, head injuries, or impairment of consciousness; and abdominal CT in
patients with abdominal pain or other findings compatible with intraperitoneal or
retroperitoneal hemorrhage.

References:
 .Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu

1. "Warfarin Therapy Management in Adults".

http://www.bcguidelines.ca/gpac/pdf/warfarin_management_summary.pdf.
2. ^ Fritsma, George A. "Evaluation of Hemostasis." Hematology: Clinical Principles and
Applications . Ed. Bernadette Rodak. W.B. Saunders Company: Philadelphia, 2002. 719-
53. Print
3. ^ Della Valle P, Crippa L, Garlando AM, et al. (December 1999). "Interference of lupus
anticoagulants in prothrombin time assays: implications for selection of adequate
methods to optimize the management of thrombosis in the antiphospholipid-antibody
syndrome" (PDF). Haematologica 84 (12): 1065–74. PMID 10586206.
http://www.haematologica.org/cgi/reprint/84/12/1065.
4. ^ Horsti J, Uppa H, Vilpo JA (March 2005). "Poor agreement among prothrombin time
international normalized ratio methods: comparison of seven commercial reagents". Clin.
Chem. 51 (3): 553–60. doi:10.1373/clinchem.2004.043836. PMID 15665046.
http://www.clinchem.org/cgi/content/full/51/3/553.
5. ^ a b Jackson CM, Esnouf MP (March 2005). "Has the time arrived to replace the quick
prothrombin time test for monitoring oral anticoagulant therapy?". Clin. Chem. 51 (3):
483–5. doi:10.1373/clinchem.2004.045393. PMID 15738512.
http://www.clinchem.org/cgi/content/full/51/3/483.
6. ^ Poller L, Keown M, Chauhan N, et al. (September 2003). "European Concerted Action
on Anticoagulation. Correction of displayed international normalized ratio on two point-
of-care test whole-blood prothrombin time monitors (CoaguChek Mini and TAS PT-NC)
by independent international sensitivity index calibration". Br. J. Haematol. 122 (6):
944–9. doi:10.1046/j.1365-2141.2003.04521.x. PMID 12956765.
7. ^ Heneghan C, Alonso-Coello P, Garcia-Alamino JM, Perera R, Meats E, Glasziou P
(February 2006). "Self-monitoring of oral anticoagulation: a systematic review and meta-
analysis". Lancet 367 (9508): 404–11. doi:10.1016/S0140-6736(06)68139-7.
PMID 16458764.
8. ^ Moll, S and Ortel, TL. (August 1997). "Metering Warfarin Therapy in Patients with
Lupus Anticoagulants.". Annals of Internal Medicine 127 (3): 177–185. PMID 9245222.
9. ^ Jack Ansell (10 March 2005). "Guidelines for implementation of patient self-testing
and patient self-management of oral anticoagulation. International consensus guidelines
prepared by International Self-Monitoring Association for Oral Anticoagulation".
International Journal of Cardiology. http://www.sciencedirect.com/science?
_ob=ArticleURL&_udi=B6T16-4CVR7GB-
2&_user=10&_coverDate=03%2F10%2F2005&_rdoc=1&_fmt=&_orig=search&_sort=d
&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=31d380
c38e3d5afdba3f7dbb04e8b5b7.
10. ^ "Medicare expands coverage for home blood testing of prothrombin time international
normalized ratio". The Centers for Medicare and Medicaid Services. 19 March 2008.
http://www.cms.hhs.gov/apps/media/press/release.asp?Counter=2987.
11. ^ Quick AJ, Stanley-Brown M, Bancroft FW (1935). "A study of the coagulation defect
in hemophilia and in jaundice". Am J Med Sci 190: 501. doi:10.1097/00000441-
193510000-00009.
12. ^ Owren PA, Aas K (1951). "The control of dicumarol therapy and the quantitative
determination of prothrombin and proconvertin". Scand. J. Clin. Lab. Invest. 3 (3): 201–
8. doi:10.3109/00365515109060600. PMID 14900966.
 13. ^ Campbell HA, Smith WK, Roberts WL, Link KP (1941). "Studies on the hemorrhagic
sweet clover disease. II. The bioassay of hemorrhagic concentrates by following the
prothrombin level in the plasma of rabbit blood". J Biol Chem 138: 1–20.
14. ^ Hirsh J, Bates SM (March 2001). "Clinical trials that have influenced the treatment of
venous thromboembolism: a historical perspective" (PDF). Ann. Intern. Med. 134 (5):
409–17. PMID 11242501. http://www.annals.org/cgi/content/full/134/5/409.
15. ^ Anonymous (1983). "33: Expert Committee on Biological Standardization.
Requirements for thromboplastins and plasma used to control oral anticoagulant therapy".
World Health Organ Tech Rep Ser. pp. 81–105.

Platelets, Vascular and Clotting Factors

a brief review of their function

Objectives:
1. Describe the role platelets play in normal hemostasis.
2. Describe hemostasis and the various factors involved with coagulation.

Glossary:

Platelets -
small, anuclear cytoplasmic disks. In an unstimulated state, the shape is
discoid.
Hemostasis -
the process in circulation where the blood is maintained fluid in vessels
and without major loss in case of injury.
Coagulation factors -
Components that exist in the circulation and supply the necessary
constituents for clot formation.

Hemostasis:
The property of the circulation where the circulating fluid is maintained within the blood
vessels is referred to as hemostasis. The process depends on a delicate and complex
interplay of at least 4 systems: vascular, plasma coagulation factors, platelets and
fibrinolytic system.
Vascular System:
Blood normally flows smoothly through the vascular system without cellular adherence
to the vessel wall. The thin layer of endothelial cells lining the inner surface of the
various vessels helps to maintain a thrombo-resistant surface. When vascular injury
occurs following trauma or in certain vessel diseases, the endothelial cells interact with
platelets and clotting factors to form a blood clot at the site of injury.

Platelets and Hemostasis:

The platelet has at least a fourfold function: (1) In response to vascular injury, platelets
are stimulated to initiate the formation of a primary hemostatic plug, (2) the platelet
contributes phospholipid (sometimes referred to as platelet factor 3 or PF3) to the
coagulation cascade, (3) they help maintain vascular integrity through endothelial support
and (4) platelets may have a role in inflammatory response, possibly by activating the
fifth component of complement.

There is a sequence of events which occurs at the site of vascular injury. First, the platelet
is attracted to the exposed sub-endothelial layer of collagen and adheres to it. To
accomplish this, the platelet undergoes a shape change. Secondly, the platelets release
intrinsic adenosine diphosphate (ADP), among other substances. The released ADP
stimulates other platelets to stick together at the wound site, and, thirdly, aggregation
occurs. In this process, platelets adhere to each other to form a beginning plug. Finally,
coagulation occurs and fibrin forms around the platelet aggregate to initiate repair.(See
figure 1)

Steps

Aggregation of thrombocytes (platelets). Platelet rich human blood plasma

(left vial) is a turbid liquid. Upon addition of ADP, platelets are activated
and start to aggregate, forming white flakes (right vial)

• The first step is immediate constriction of damaged vessels caused by

vasoconstrictive paracrine released by the endothelium.
Vasoconstriction temporarily decreases blood flow and pressure
within the vessel. When you put pressure on a bleeding wound, you
also decrease flow within the damaged vessel.
• Vasoconstriction is rapidly followed by the second step,mechanical
blockage of the hole by a platelet plug. The plug forms as platelets
stick to the exposed collagen(platelet adhesion) and become activated,
releasing cytokines into the area around the injury. Platelet factors
 reinforce local vasoconstriction and activate more platelets which
stick to one another( platelet aggregation) to form a loose platelet
plug.
• Simultaneously, exposed collagen and tissue factor (a protein-
phospholipid mixture) initiate the third step, a series of reactions
known as the coagulation cascade. The cascade is a series of
enzymatic reactions that ends in the formation of a fibrin protein fiber
mesh that stabilizes the platelet plug. The reinforced platelet plug is
called a clot. Some chemical factors involved in the coagulation
cascade also promote platelet adhesion and aggregation in the
damaged region.

Eventually, as the damaged vessel repairs itself, the clot retracts and is
slowly dissolved by the enzyme plasmin.

Two pathways lead to the formation of a fibrin clot: the intrinsic and extrinsic
pathway. Although they are initiated by distinct mechanisms, the two converge
on a common pathway that leads to clot formation. Both pathways are complex
and involve numerous different proteins termed clotting factors. Fibrin clot
formation in response to tissue injury is the most clinically relevant event of
hemostasis under normal physiological conditions. This process is the result of
the activation of the extrinsic pathway. The formation of a red thrombus or a clot
in response to an abnormal vessel wall in the absence of tissue injury is the
result of the intrinsic pathway. The intrinsic pathway has low significance under
normal physiological conditions. Most significant clinically is the activation of the
intrinsic pathway by contact of the vessel wall with lipoprotein particles, VLDLs
and chylomicrons. This process clearly demonstrates the role of hyperlipidemia
in the generation of atherosclerosis. The intrinsic pathway can also be activated
by vessel wall contact with bacteria.

Treatment
Treatment is directed at the underlying disorder and at any hypovolemia. For
immediate treatment of bleeding due to a coagulopathy that has not yet been
diagnosed, fresh frozen plasma, which contains all coagulation factors, should be
infused pending definitive evaluation.
Look at the Immediate Clinical History

1. Massive Transfusion
1. Physiological Status
 Temperature
 pH
 Duration of hypotension
 Extent of resuscitation
2. Clinical Diagnosis of Coagulopathy
 Diffuse oozing from surgical sites
 Bleeding around vascular access sites
3. Mechanism of Coagulopathy
 Dilution - Platelets, VIII and V
 Consumption - Fibrinogen and platelets
2. Cardiopulmonary Bypass
1. Platelet effects
 Activation and loss of GPIb - Plasmin, elastase and calpain
2. Aprotinin
 Repeat valvular surgery and septic endocarditis
 2 mKIU pre incision and 0.5 mKIU/hour during bypass

Take a Personal History

1. Family History
o The haemophilias follow a pattern of X linked recessive inheritance.
However up to 30% of case of haemophilia A are spontaneous mutations
with no family history.
o Von Willebrand's disease is difficult to diagnose because of the variability
in inheritance, autosomal dominant and recessive, and the variability
among patients in the level of von Willebrand factor present. The level
varies according to the ABO blood type, with the lowest levels present in
type O and the highest in type AB
2. History of surgical, traumatic events or other triggering events
Any patient who has had major surgery, a tonsillectomy or dental extractions
without unusual bleeding, has had the best evaluation of their coagulation system
possible.
3. Frequency of abnormal bleeding
4. Duration of abnormal bleeding
A bleeding abnormality manifests as moderate bleeding over a prolonged period,
not as bleeding at an excessive rate.
5. Location of abnormal bleeding
o Bleeding from skin and mucous membranes tends to occur with platelet
disorders.
 o Bleeding in joints and muscles tends to occur with the haemophilias
6. Medical Disease

1.Liver Disease
2.Renal Disease
3.Haematological malignancy - leukaemia, myeloproliferative disease
4.Vitamin K deficiency
5.Vitamin C deficiency
6.Solid organ malignancy - Prostate, lung, colon
2. Medication History - aspirin, coumarin, heparin

Exclude surgical causes of bleeding

Obtain help from a haematologist

Whole blood clotting time

1. 5ml of blood is placed in a glass container, kept at body temperature and

observed
1. A clot should occur in 5 to 15 minutes
Prolonged = Severe deficiency of any of the coagulation proteins
2. The clot should retract in 30 to 60 minutes
Weak friable clot = hypofibrinogenaemia
Early dissolution = enhanced fibrinolysis

Full blood count and examination

1. Red blood cell number

2. Red blood cell morphology abnormalities following intravascular
thrombosis or microangiopathy
3. Platelet count and function tests

Platelet Count

Two major techniques are used for automated platelet

measurements. They use size thresholds to distinguish
between platelets, leucoytes and erythrocytes.

 Light scattering
This measures the amount of light transmitted as blood elements
pass through an aperture
  Electronic aperture-impedance counting:
This measures the change in electrical resistance/capacitance as
blood elements stream through an aperture connecting a circuit.

Normal number 150 to 400x109/L.

The machines are accurate and reliable down to 10-
30x109/L but are subject to specimen error:

 Poor platelet preservation techniques allow adhesion and

clumping, resulting in abnormally low results
 Specimens stored for more than 24 hours allow aggregation of
platelets
 Haemolysed red cell fragments and bacterial contamination may be
identified as platelets

Genuinely low levels are due to

 Increased peripheral destruction

1. Dilutional - Massive blood transfusion
2. Sequestration - Look for liver and splenic disease
3. Destruction
1. Sepsis
2. Disseminated intravascular coagulation
3. Thrombotic thrombocytopaenic purpura /
Haemolytic uraemic syndrome
4. Post cardiopulmonary bypass
5. Mechanical prosthetic valves
6. Platelet antibodies
1. Drug induced - Penicillin
2. Post transfusion - HLA directed
3. Collagen vascular disease
4. Idiopathic thrombocytopaenic purpura =
Immune thrombocytopaenic purpura

 Decreased production
1. Hypocellular Bone marrow - Aplastic Anaemia
2. Hypercellular Bone marrow
1. Megaloblastic - B12 or folate deficiency
2. Myelodysplastic
3. Myelophthisic
1. Leukaemia
2. Metastatic cancer
3. Myelofibrosis
 Platelet Function

Platelet Aggregation
The addition of an agonist (thrombin, ADP, adrenaline, collagen,
ristocetin or arachnidonic acid) to platelet rich plasma normally
exhibits a biphasic response of reversible aggregation due to the
agonist followed by irreversible aggregation due to the
disintegration of the platelets.

Heparin induced platelet aggregation

Used in the diagnosis of heparin-associated thrombocytopaenia.

Hess Test
In vivo assessment of collagen matrix, vascular endothelium and
platelet adhesion and aggregation
A syphgomomanometer is inflated to between the systolic and the
diastolic pressures for 10 minutes. Normal less than 15
petechiae would occur in a 5cm diameter circle.

International Normalised Ratio

Quick labeled the test prothrombin time when he devised it in

1935 as he believed it was a simple, accurate, and sensitive
measure of prothrombin. He was wrong.

The specimen should be a 3.8% trisodium citrate anticoagulant in

a 9:1 ratio with the blood, which is centrifuged to produce platelet
poor plasma. A complete thromboplastin (typically from rabbit
brain) is then added with calcium. The time to fibrin strand
formation is then measured automatically by:

o Photo-optical device.
This depends on the increase in light scattering associated with the
conversion of soluble fibrinogen to soluble fibrin. A photocell detects the
decrease in transmitted light as the clot forms and an algorithm analyses
the data to produce an endpoint.
o Electromechanical device

Many variables affect the prothrombin time

o The thromboplastin reagent. Poller in 1987 proposed The International

Normalised Ratio.
  All commercial thromboplastins are compared to an International
Reference preparation. This determines a "calibration value",
called an International Sensitivity Index which must be supplied
with every batch of thrompoplastin reagent.
 The INR = (Patient's prothrombin time / Laboratory's control
prothrombin time)ISI
o Specimen collection
 The first specimen drawn is contaminated by tissue thromboplastin
 EDTA contamination interferes with the activation process.
 Underfilling alters the anticoagulant:blood ration of 9:1
 Polycythaemia alters the anticoagulant:blood ration of 9:1
 Haemolysis, lipaemia, hyperbilirubinaemia and hyperproteinaemia
produce errors in the optical end point analysis.

A Normal INR is between 0.9 and 1.2

Prolonged = Deficiency of factor I, II, V, VII or X. The test is

most sensitive to decreases in factor VII which is one of the
vitamin K dependant factors.

1. Coumarin anticoagulation therapy

2. Vitamin K deficiency
3. Severe Liver disease
4. Massive blood transfusions
5. Disseminated intravascular coagulation
6. High dose heparin therapy

Activated partial thromboplastin time

This test derives its name from the use of a partial
thromboplastin, or "cephalin" which is a phospholipid component
that is added to a specimen that has been "activated" by exposure
to a negatively charged substance (kaolin, celite or ellagic acid).

Normally 25 to 35 seconds

Prolonged = A decrease to less than 30% activity of all the

coagulation factors

7. Heparin therapy
8. Haemophilia
9. Massive blood transfusions
10. High dose coumarin anticoagulation
 Errors in specimen collection will also affect the result

Factor activity assay

Normal plasma and the patient's plasma are compared in their
ability to correct the INR or aPTT of plasma from an individual
severely deficient in the factor of interest.

By convention normal plasma is said to have 100% or 1 unit

per ml activity. If a 1:10 dilution of a specimen has the correcting
power of a 1:100 dilution of normal plasma, the specimen has
10% or 0.1 unit per ml activity.

11.Antithrombin III activity assay

11 Decreased by consumption
1. Sepsis
2. Disseminated intravascular coagulation
3. Deep vein thrombosis or pulmonary embolism
11 Decreased due to low levels of the molecule
1. Decreased synthesis of a normal AT III molecule -
Autosomal dominant
2. Production of a dysfunctional AT III molecule - Autosomal
dominant
2. Factors II, V, VII, VIII, IX, X, XI, XII
3. von Willebrand factor
4. Fibrinogen
The Clauss clottable protein method.
Thrombin times are performed using a series of serial dilutions.
As fibrinogen concentration is the rate-limiting step, a graph of
the results is used to extrapolate fibrinogen concentration.
Specimen collection can affect the result.
5. Fitzgerald Factor Assay - High Molecular Weight Kininogen
deficiency
A rare disease manifest by a prolonged PTT with no bleeding
manifestations and not explained by a lupus anticoagulant, haemophilia,
von Willebrand's disease or heparin administration. Some patients are
reported to have thromboembolic episodes.
6. Fletcher Factor Assay - Prekallikrein factor deficiency
Deficiency is an autosomal recessive pattern with a prolonged PTT and no
bleeding tendency. Some patients may have thromboembolic episodes.
Factor Antigen assay (VII, X)

Factor Inhibitor assay (II, V, VII, VIII, IX, X, XI,

XII)
The mixing study
The patient's serum is mixed with normal serum and the aPTT of
this mixture is measured. A 50:50 mixture will correct a factor
deficiency (only 30% activity is needed for a normal aPTT). In the
presence of an inhibitor a 50:50 mix will not correct the abnormal
coagulation test.

Factor VIII inhibitors

o IgG
o Occur primarily in haemophilia A patients who have received blood
component transfusions. They develop a severe coagulopathy that is very
difficult to manage
o May occur in a variety of unrelated conditions with a coagulopathy that is
variable and usually disappears spontaneously

Lupus Anticoagulant

o Majority of patients do not have systemic lupus erythematosus nor

any tendency towards increased bleeding
o IgG directed against the Xa-V-Phospholipid complex
o Markedly prolonges the aPTT
o Clinical tendency towards excessive thrombosis
o Occurs in a variety of conditions

 Essentially normal patients

 Lupus 5-10% of patients
 Infectious diseases
 Rheumatoid arthritis
 Lymphoma
 Prostatic cancer
 Acquired immunde deficiency syndrome
 Drug exposure - chlorpromazine, procainamide and antibiotics
o Laboratory tests to isolate the lupus anticoagulant
 Cardiolipin adsorption
 Reptilase Test
 Dilute aPTT
  Kaolin clotting time
 Platelet neutralisation procedure.

Thrombin Time
Thrombin is added to plasma and the time taken to form a clot
is recorded Normal is less than 15 seconds

Prolonged due to inhibition of thrombin

4. Heparin
5. Fibrin degradation products
6. Lupus anticoagulant

Prolonged due to abnormal fibrinogen

Reptilase Time
Reptilase is added to plasma and the time taken to form
a clot is recorded. Normal is less than 14-19 seconds

11 Prolonged due to inhibition of Reptilase

1 Lupus anticoagulant
11 Prolonged due to abnormal fibrinogen

Template bleeding time

A sphygmomanometer on the upper arm is inflated to
40mmHg. A skin incision 5mm long and 1mm deep is made on
the extensor surface of the forearm, using a spring loaded
template device.. The wound avoids scar tissue and superficial
vessels, and must be done within 60 seconds of inflating the
sphygmomanometer. Filter paper is used to blot the edges of the
wound at 30 second intervals until the bleeding stops. Normal is
two to nine minutes

According to Rodgers & Levin:

[A critical reappraisal of the bleeding time. Sem Thromb Haemost 1990:
16: pp1-19]
 o The bleeding time is unable to predict aspirin usage
o There is no predictive correlation with the platelet count

 The only study that claims a correlation is by Harker &

Slickter [ The bleeding time as a screening test for evaluation of
platelet function. N Engl J Med 1972; 287: pp155-159]
o There is no correlation with surgical bleeding.

Conventional theory alleges that a prolonged bleeding time is

due to

11 von Willebrand factor abnormality or deficiency

11 Platelet deficiency or abnormality - Heparin, Aspirin
11 Anaemia

Activated Coagulation Time

Fresh whole blood is added to a tube containing negatively
charged particles and timed for the formation of a clot.

The type of negatively charged particle affects the "normal"

length of ACT

Celite = Diatomaceous Earth: normal is 100-170

seconds

11 Used with high circulating levels of heparin -

cardiopulmonary bypass
11 Aprotinin prolongs the "normal" ACT
11 Black top glass test tube

Kaolin: normal is 90-150 seconds

11 Used with high circulating levels of heparin -

cardiopulmonary bypass
11 ACT not prolonged by aprotinin
11 Gold top glass test tube

Glass particles: normal 110-190 seconds

11 Used with medium circulating levels of heparin -

haemodialysis
11 Clear top plastic test tube
 None: normal 190-300 seconds

11 Used with low to no circulating levels of heparin - Vascular

and general surgery
111 White top glass test tube

Type of machine affects normal and therapeutic values

Hemochron
A warmed test tube is rotated inside the machine. As the blood clots, it
displaces the magnet within the test tube. The clotting time is determined
when the magnet has displaced enough to activate a proximity switch

Medtronic HemoTec
A mechanical plunger is dipped in and out of a kaolin activated blood
sample. The machine optically senses the time it takes the plunger to
move through the specimen. Clotting is defined by the "drop time"
threshold for the plunger

The machines do not correlate with one another but in general

the times for the Hemochron are 30% greater than the Medtronic
HemoTec

Specimen quality affects the values

1. The specimen should never be taken from a line in which heparin

is used
2. Sampling from an indwelling line must involve a two syringe
technique, with the infusion stopped
11 Initially withdraw three times the dead space volume to
eliminate any dilution from the drip fluid
11 Obtain the sample with a second syringe
3. Sampling from venipuncture must involve a two syringe
technique
11 Discard the first 2-5ml withdrawn to avoid contamination
with tissue factor
11 Obtain the sample with a second syringe

Daily calibration checks are imperative

4. Three calibration time checks ensure linearity of response

11 100 Seconds calibration instrument
11 250 Seconds calibration instrument
11 500 Seconds calibration instrument
 5. Temperature calibration of 37oC is carried out with a magnetised
thermometer.

Clinical use of the ACT in assessing adequacy of

heparinisation

1. A linear heparin/dose response curve has been well documented

2. Many reports have shown less blood loss with no increase in
fibrin formation using ACT guided heparin dosing as opposed to
protocol dosing in cardiopulmonary bypass surgery
3. Serial ACT measurements to develop individual heparin/dose
response curves should be used.
4. "Target" ACT is very system and unit dependent

Prolonged times may be due to

5. Heparin effect
6. Hypothermia
7. Platelet dysfunction
8. Haemodilution
9. Cardioplegic solutions
10. Hypofibrinogenaemia
11. Factor deficiencies