33-Hemostasis and Coagulation Profile

‫نقابة المهن العلمية بالمنوفية‬ [1]
emostasis and oagulation rofile

Overview of hemostasis
Hemostasis is a complex physiologic process that keeps circulating blood in a fluid state and then, when an injury occurs, produces a
clot to stop the bleeding, confines the clot to the site of injury, and finally dissolves the clot as the wound heals.
When hemostasis systems are out of balance, hemorrhage (bleeding) or thrombosis (pathological clotting) can be life-
threatening.
Primary hemostasis
Primary hemostasis is a rapid, short-lived response refers to the role of blood vessels and platelets in response to a vascular injury. The
blood is normally exposed to only the endothelial cell lining of the blood vessels. The intact vascular endothelium prevents thrombosis
by inhibiting platelet aggregation and preventing coagulation activation.
Endothelial cells form a physical barrier separating platelets in blood from collagen in the internal elastic lamina that promotes
platelet adhesion, and tissue factor in smooth muscle cells that activates coagulation.
Vascular injury will initiate a series of reactions help in primary hemostasis response as follow:
Local vasoconstriction
Immediately after a blood vessel has been injured, the smooth muscle inside
the vessel wall contracts. This reduces the flow of blood from the ruptured
vessel and therefore prevents leakage.
The initial vasoconstriction is mediated by:
▪ Reflex neurogenic mechanisms (initiated by local sympathetic pain

receptors)
▪ The release of endothelin by damaged endothelial cells
▪ The release of serotonin and Thromboxane A2 from the activated
platelets
Although veins and capillaries do not have smooth muscle cells, bleeding into surrounding tissues creates extravascular
pressure on the blood vessel, effectively minimizing the escape of blood.
Platelet plug formation
Platelets adhesion:
▪ Von Willebrand Factor (vWF) is released from

the damaged endothelial cells.
▪ The secreted vWF binds with subendothelial
collagen at the site of injury
▪ Platelet receptor glycoprotein Ib (GP Ib), located
on the cell wall, attaches to von Willebrand
Factor and activates the platelet.
Platelets degranulation:
When activated, platelets immediately undergo change.

They begin to swell; they assume irregular forms with
numerous pseudopods protruding from their surfaces and
they release the contents of their granules (mainly
adenosine diphosphate, serotonin, thromboxane A2 and
Von Willebrand Factor).
Platelets aggregation:
ADP and TxA2 promotes platelet aggregation through exposing the fibrinogen receptor glycoprotein IIb/IIIa thus fibrinogen cross-links
with its receptor aiding in aggregation of adjacent platelets
Platelets alone are responsible for stopping the bleeding of unnoticed wear and tear of our skin on a daily basis.
Secondary hemostasis
To control major bleeding in the long term, the platelet plug must be reinforced by fibrin.
Secondary hemostasis involves a series of blood protein reactions through a cascade-like process that concludes with the formation of
an insoluble fibrin clot.
Coagulation factors
Clotting factors (procoagulants) are glycoproteins in the blood that control bleeding. Nearly all are synthesized in the liver, although
monocytes, endothelial cells, and megakaryocytes produce a few.
Some factors are enzymes that circulate in an inactive form called zymogens, while others are cofactors (cofactors are TF, FV,
FVIII, and HMWK) that bind, stabilize, and enhance the activity of their respective enzymes.
Classification of Coagulation Factors
There are three groups in which coagulation factors can be classified:
The fibrinogen group: Consists of factors I, V, VIII, and XIII. They are consumed during coagulation.
The prothrombin group: Factors II, VII, IX, and X all are dependent on vitamin K during their synthesis.
The contact group: Consists of factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen (HMWK). They are so named
because they are activated by contact with negatively charged foreign surfaces.
Screening test if deficient
1 I Fibrinogen Prolonged PT and PTT
2 II Prothrombin Prolonged PT and PTT
3 III Thromboplastin (Tissue factor)
4 IV Ionized Calcium
5 V Proaccelerin (Labile Factor) Prolonged PT and PTT
6 VI Nonexistent (was assigned to a procoagulant that later was

determined to be activated factor V)
7 VII Proconvertin (Stable Factor) Prolonged PT
8 VIII Antihaemophilic factor A Prolonged PTT
9 IX Plasma Thromboplastin Component (Antihaemophilic factor B or Christmas factor) Prolonged PTT
10 X Stuart-Prower Prolonged PT and PTT
11 XI Plasma Thromboplastin Antecedent (Antihaemophilic factor C) Prolonged PTT
12 XII Hageman factor Prolonged PTT
13 XIII Fibrin Stabilizing Factor (Laki-Lorand factor) Normal PT and PTT
Prekallikrein (pre-K), also called Fletcher factor
high-molecular-weight kininogen (HMWK), also called Fitzgerald factor

Coagulation cascade
The initiation of clotting begins with the activation of two enzymatic pathways that will ultimately lead to fibrin formation: the intrinsic
and extrinsic pathways.
Intrinsic pathways (Contact activation pathway)
▪ The contact activation pathway begins with formation of the primary complex on collagen consists of prekallikrein, high-
molecular-weight kininogen (HMWK) and FXII (Hageman factor).
▪ Prekallikrein is converted to kallikrein using HMWK as a cofactor
▪ kallikrein converts FXII into FXIIa using HMWK as a cofactor
▪ FXIIa converts FXI into FXIa.
▪ Factor XIa activates FIX
▪ Factor IXa with its co-factor FVIIIa form the tenase complex, which activates FX to FXa.
This process is considerably slower (5 to 10 minutes) but results in the formation of larger amounts of thrombin. This
allows the formation of larger clots.
Extrinsic pathway (Tissue factor pathway)
▪ Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF), forming an
activated complex (TF-FVIIa).
▪ TF-FVIIa complex activates FX(to form FXa)
The extrinsic pathway is a very rapid process, i.e., within 12 to 15 seconds. However, the production of Thrombin is low and
the resulting clot is small.
Common pathway
▪ FXa and its co-factor FVa form the prothrombinase complex.

▪ Prothrombinase complex cleaves prothrombin to the active enzyme, thrombin.
• Thrombin converts fibrinogen to fibrin, the building block of a hemostatic plug.
• In addition, thrombin amplifies the coagulation mechanism by activating
cofactors V and VIII by a positive feedback mechanism
Formation of fibrin occurs in three phases:
1.Proteolysis: The primary function of Protease enzyme

thrombin is to cleave fibrinopeptides A and B from  and 
chains of the fibrinogen molecule resulting in a fibrin monomers.
2.Polymerization: This occurs spontaneously due to fibrin

monomer that line up end-to-end due to hydrogen bonding.
3.Stabilization: This occurs when factor XIIIa (activated by

thrombin), convert initial weak hydrogen bonds, cross-linking
fibrin polymers to a more stable covalent bond.
Factor XIII deficiency can lead to impaired wound healing

and may cause severe bleeding problems. Factor XIII is not
involved in the process of fibrin formation and, therefore, the
PT and APTT are both normal in factor XIII deficiency
Coagulation regulatory mechanisms
Coagulation regulation occurs through coagulation inhibitors, soluble plasma proteins that are natural anticoagulants. They function to
slow the activation of procoagulants and suppress thrombin production, thus ensure that coagulation is localized and is not a systemic
response.
The principal regulators are:
Tissue factor pathway inhibitor (TFPI)
TFPI is a single-chain polypeptide protease inhibitor that is

synthesized primarily by endothelial cells.
Function
▪ TFPI inhibits coagulation in a two-step process by first

binding and inactivating Xa.
▪ The TFPI:Xa complex then binds to TF:VIIa, forming a
quaternary complex and preventing further activation of F(X)
Protein C regulatory system
Protein C is a vitamin K-dependent glycoprotein synthesized in the liver
Activation
The activation of protein C is strongly promoted by endothelial protein C receptor (EPCR), which is found on endothelial cell surface.
Thrombomodulin (TM) is another integral membrane protein expressed on the surface of endothelial cells and serves as a cofactor f or
thrombin.
The presence of Thrombin–thrombomodulin complex accelerates

activation of protein C by several orders of magnitude (about thousand
fold)
APC dissociates from EPCR and binds its cofactor, free plasma protein S
Function
Stabilized APC-protein S complex functions as anticoagulant by irreversibly

proteolytically inactivating Factor Va and Factor VIIIa thus practically slowing
or blocking the production of thrombin.
Antithrombin / heparin complex
Antithrombin (AT) is a protease glycoprotein inhibitor produced by the liver
Function
The physiological target of antithrombin are those of the contact

activation pathway, namely the activated forms of Factor X (Xa),
Factor IX (IXa), Factor XI (XIa), Factor XII (XIIa) and, to a greater
extent, Factor II (thrombin) (IIa), and also the activated form of
Factor VII (VIIa) from the tissue factor pathway
Role of heparin
Heparin is usually stored within the secretory granules of mast cells

and released only into the vasculature at sites of tissue injury.
Heparin binds to antithrombin, causing a conformational change

those results in its activation through an increase in the flexibility of
its reactive site loop.
The rate of inactivation of antithrombin target can increase by up to 1000-fold due to the binding of heparin
Fibrinolytic system
The purpose of the clot is to stop the flow of blood until the damaged vessel can be repaired.
The final event of hemostasis is fibrinolysis, the gradual digestion and removal of the fibrin clot as healing occurs.
Proteins of fibrinolysis
▪ Plasminogen: is a plasma zymogen produced by the liver

▪ Tissue Plasminogen Activator (TPA): secreted by endothelial cells in response to coagulation.
▪ Urokinase Plasminogen Activator (UPA): secreted by urinary tract epithelial cells, monocytes, and macrophages in response to
inflammation.
Fibrinolysis pathway
▪ Fibrinolytic proteins (Plasminogen, TPA and UPA) incorporated into the fibrin clot as they bind to fibrin lysine residues thereby
concentrating and localizing them to the fibrin clot.
▪ TPA and UPA activate fibrin-bound plasminogen into a two-chain active plasmin molecule
Fibrinolysis begins a few hours after fibrin polymerization and cross-linking.
▪ Plasmin is a serine protease that systematically digests fibrin polymer and restores blood vessel patency.
▪ As fibrin becomes digested, the exposed lysine residues bind additional plasminogen and TPA, which f urther accelerates clot
digestion.
▪ Plasmin degradation of fibrin begins by the breaking down of the fibrin polymer into a monomer form known as fragment X. This
fragment is identical to a fibrin monomer, consists of one E domain and two D domains, and retains clotting properties.
▪ Fragment X is further cleaved to produce FDP, which consists of:
• Fragment Y, which consists of one E domain and one D domain
• Fragment E, which consists of the E domain only
• Fragment D, which consists of the D domain only
• D dimer, which consists of two D fragments of linked monomers that have been cross-linked by factor XIIIa in the process of
fibrin formation
Fragments X, Y, D, and E are produced by digestion of either fibrin or fibrinogen by plasmin, but D-dimer is a specific product
of digestion of cross-linked fibrin only and is therefore a marker of thrombosis and fibrinolysis
Platelets disorders
Thrombocytosis
Thrombocytosis (or thrombocythemia) is the presence of high platelet counts in the blood, and can be either essential ( also termed
primary) or reactive (also termed secondary).
Essential thrombocythemia
Pathophysiology
Essential thrombocytosis is a form of myeloproliferative disease (such as CML, and polycythemia vera)
The pathologic basis for this disease is unknown. However, essential thrombocythemia resembles polycythemia vera in that cells of the
megakaryocytic series are more sensitive to growth factors. A mutation in the JAK2 kinase has been found to be associated with
essential thrombocythemia in around 40–50% of cases.
Signs and symptoms
▪ In primary thrombocythemia, blood clots can develop anywhere in the body, most often develop in the brain, hands, and feet.
▪ Often, patients with a thrombocytosis will have increased bleeding tendencies because of possible accompanying functional
abnormalities (epistaxis (nosebleeds) and bleeding from gums and gastrointestinal tract)
▪ One characteristic symptom is throbbing and burning of the hands and feet due to the occlusion of small arterioles by platele ts
(erythromelalgia)
▪ Approximately 50% of the patients at the time of diagnosis will present with an enlarged spleen and approximately 20% of patients
will present with an enlarged liver
Diagnostic criteria
▪ If platelet counts are over 750,000 or 1,000,000

▪ No cause for reactive thrombocytosis
▪ Normal RBC mean corpuscular volume
▪ Normal serum ferritin
▪ Absent Philadelphia chromosome
Reactive thrombocytosis
In reactive thrombocytosis, the platelet count is usually less than 1 million and is transient.
In reactive thrombocytosis (and in contrast to essential thrombocythemia), platelet production remains responsive to normal
regulatory stimuli (thrombopoietin)
Causes
▪ Acute infection or inflammations (such as rheumatoid arthritis, pancreatitis, osteomyelitis, inflammatory bowel disease and
malignancy)
May be due to the overproduction of pro-inflammatory cytokines, such as interleukin IL-1, IL-6, and IL-11, that occurs in
chronic inflammatory, infective, and malignant states
▪ Acute blood loss or post-surgery due to hemorrhage (Rebound thrombocytosis)

After acute hemorrhage, the platelet count may be low for 2 to 6 days but typically rebounds to elevated levels for several days
before returning to the pre-hemorrhage level.
▪ Iron deficiency anemia associated with chronic blood loss

Iron regulates thrombopoiesis by inhibiting thrombopoietin; deficiency causes increased thrombopoietin and stimulates
thrombopoiesis. Once iron therapy is initiated, platelet usually returns to normal within 7 - 10 days.
▪ Post- splenectomy (Splenectomy-associated thrombocytosis): the platelet count reaches a maximum 1 to 3 weeks after
splenectomy (can reach or exceed 1 million) and remains elevated for 1 to 3 months.
The spleen normally sequesters about one third of the circulating platelet mass. After splenectomy, one would expect an
initial increase in the platelet count of approximately 30% to 50%.
▪ Exercise-Induced thrombocytosis: Strenuous exercise is a well-known cause of relative thrombocytosis and is likely due to the
release of platelets from the splenic pool or hemoconcentration by transfer of plasma water to the extravascular compartment or
both.
Normally, the plate let count returns to its pre-exercise baseline level 30 minutes after completion of exercise.
Thrombocytopenia
The terms thrombocytopenia and thrombopenia refer to a relative decrease of platelets in blood less than 150,000 per microlitre.
One common definition of thrombocytopenia is a platelet count below 50,000 per microlitre
Signs and symptoms
▪ Platelet count drops <50,000
The ability of the blood to clot decreases. With invasive procedures, a count <50,000 increases risk of bleeding, but otherwise, risk
is not usually significant until count drops to <20,000
At which point common symptoms include:
• Small red spots on the skin look like a rash (petechiae)

• Small purplish spots on the skin (purpura) caused by bleeding under the skin
• Unexplained extensive bruising (ecchymoses)
• Prolonged bleeding from a small cut or wound
• Numerous nosebleeds (epistaxis) and/or gingival bleeding ( gums)
• Heavy menstrual bleeding (menorrhagia)
Severe risk of CNS hemorrhage and GIT hemorrhage occurs with a platelet count less than 5000
Etiology
Decreased production of platelets
Megakaryocyte hypoproliferation
• Bone marrow infiltration by malignant cells which occurs with acute leukemia, lymphoma and myeloma
• Bone marrow hypoplasia due to cancer chemotherapy
• Aplastic anemia
• Selective marrow suppression of platelet production due to infection as neonatal thrombocytopenia due to congenital infection
(e.g. CMV, toxoplasma, rubella)
Ineffective thrombopoiesis
• Megaloblastic anemia
• Decreased production of thrombopoietin by the liver in end stage liver disease
Increased destruction of platelets
Immune thrombocytopenic purpura
It is defined as isolated low platelet count (thrombocytopenia) with normal bone marrow and the absence of other causes of
thrombocytopenia.
The term idiopathic thrombocytopenic purpura (ITP) was used previously to describe cases of thrombocytopenia arising
without apparent cause or underlying disease state.
Acute ITP
This is primarily a disorder of children (2 to 6 years of age). There is a sudden onset of thrombocytopenia, which often follows viral
infections, such as mumps, rubella, chickenpox, cytomegalovirus (CMV), and toxoplasmosis.
o Duration: Acute ITP usually lasts for 2 to 6 weeks with a spontaneous remission in 80% of patients.
o Platelet count: is usually <20,000/mm3 in patients with acute ITP.
o Pathophysiology: Acute ITP is caused by viral attachment and antigenic alteration of platelet membrane proteins that result in
the formation of platelet autoantibodies, which are most often IgG.
IgG-coated platelets are removed by macrophages in the spleen.
o Therapy: acute ITP is usually self-limited, and no treatment is needed.
Chronic ITP
This disorder can be found in patients of any age, although most cases occur in patients between the ages of 20 and 50 years.
It is found in women three times more than in men and has a slow, asymptomatic onset of thrombocytopenia.
o Duration: Chronic ITP can last from months to years.

o Platelet count: usually ranges from 30,000 to 80,000/mm3.
o Pathophysiology: Chronic ITP is often associated with SLE
Overall, the life span of the platelet is shortened from the normal 7 to 10 days to a few hours
o Therapy: The initial treatment of chronic ITP depends on the urgency for increasing the platelet count. Unless there are
additional risk factors, ITP patients with platelet counts greater than 30,000/mL should not be treated. If additional risk factors
are present, such as old age, coagulation defects, recent surgery, trauma, or uncontrolled hypertension, the platelet count
should be kept at 50,000/mL or higher
• Corticosteroids: For most patients, the initial treatment of chronic ITP consists principally of prednisone. About 70%
to90% of patients respond to this therapy, with an increase in platelet count and a decrease in hemorrhagic episodes.
• Splenectomy: In some patients splenectomy may become necessary. Splenectomy eliminates the primary site of platelet
removal and destruction. Splenectomy is an effective treatment for adult chronic ITP, with 88% of patients showing
improvement and 66% having a complete and lasting response.
Splenic sequestration of platelets due to splenomegaly caused by:
• Portal hypertension
• Hemolytic anemia
• Viral infections (such as infectious mononucleosis)
• Bacterial infections (such as syphilis)
• Parasitic infections (such as malaria)
Increased consumption of platelets
Disseminated intravascular coagulation
It is a pathological process characterized by the widespread activation of the clotting cascade that results in the formation of blood clots
in the small blood vessels throughout the body.
Thrombotic thrombocytopenic purpura (TTP)
It is a rare blood disorder characterized by clotting in small blood vessels of the body (thromboses), resulting in a low pla telet count and
microangiopathic hemolytic anemia (MAHA).
The presence of schistocytes on the peripheral blood with low platelet counts and low haptoglobin are consistent with
microangiopathic hemolytic anemia.
Causes: TTP is believed to be caused by vascular wall dysfunction, which disrupts the inert basement membrane resulting in
spontaneous aggregation of platelets and activation of coagulation in the small blood vessels.
Pseudothrombocytopenia
Artifactual thrombocytopenia is caused by in vitro clumping of platelets due to various causes which include:
EDTA-induced Platelet agglutination
This in vitro phenomenon is due to the presence of naturally occurring autoantibody (IgG) against a crypt antigen on the GPIIb /IIIa
platelet receptor. When calcium is chelated by EDTA, the GPIIb protein undergoes a change that exposes the crypt antigen. The
autoantibody then binds to the exposed site and crosslink to other platelets causing agglutination.
The condition occurs in approx. 1% of hospitalized patients and must be confirmed by evaluating a repeat blood specimen drawn into
sodium citrate
The new WBC and platelet counts should then be adjusted for the sodium citrate
dilution by multiplying the results by the dilution factor 10/9 or 1.1
The platelet clumps have been falsely counted as WBCs; therefore, a manual
WBC count is indicated.
Platelet Satellitism
As the antibody coats the platelets, the platelets rosette around segmented neutrophils
and bands. These platelets that are huddled around WBCs will not be counted by
automated equipment and the platelet count will be falsely decreased.
Bleeding disorders
It is an unusual susceptibility to bleed (hemorrhage) mostly due to hypocoagulability. It may be acquired disorder (as in the case of
ESLD) or congenital (Hemophilia and von Willebrand disease are the major genetic disorders associated with coagulopathy)
Hemophilia
The hemophilias are congenital single-factor deficiencies marked by soft tissue bleeding.
Classification
Hemophilia A (classic hemophilia): 85% of patients are deficient in factor VIII. It is a recessive X-linked genetic disorder
Hemophilia B (Christmas disease): 14% of patients are deficient in factor IX. It is a recessive X-linked genetic disorder
Although the two types have very similar signs and symptoms, they are caused by mutations in different genes.
As hemophilia A and B are X-linked recessive disorders females are very rarely severely affected.
The son of a healthy female silently carrying the deficient gene will have a 50% chance of inheriting that gene from her and
with it the disease; and if his mother is affected with haemophilia, he will have a 100% chance of being a haemophiliac.
Rarely, hemophilia C (a deficiency of Factor XI) is encountered, but its effect on clotting is far less pronounced than A or B.
Symptoms
Characteristic symptoms vary with severity.
▪ People with mild hemophilia usually suffer more minor

symptoms except after surgery or serious trauma
▪ In cases of moderate hemophilia symptoms may include:
• Easy bruising and haematomas
• Heavy bleeding from a dental procedure
• Adult females may experience menorrhagia (heavy
periods) due to the bleeding tendency.
• Bleeding from the gastrointestinal tract can lead to
blood in the stool (Hematemesis and melena)
Children with mild to moderate haemophilia may not have

any signs or symptoms at birth especially if they do not
undergo circumcision
▪ Severe complications are much more common in cases of

severe hemophilia and include:
o Hemophilic arthropathy: hemarthrosis is a joint bleed where blood enters into the joint spaces with severe pain, and
even destruction of the joint
o Deep-muscle bleeding: leading to swelling, numbness or pain of a limb
o Intracranial hemorrhage: is a serious medical emergency caused by the buildup of pressure inside the skull resulting in
Headache, nausea, loss of consciousness, brain damage, and death.
Laboratory findings
Screening test
The laboratory workup for a suspected congenital single coagulation factor deficiency starts with the PT, PTT, and thrombin time and
continues with factor assays based on the results of the screening tests
Expected laboratory values are as follows:
▪ Hemoglobin/hematocrit: Normal or low

▪ Platelet count: Normal
▪ Bleeding time and PT: Normal
▪ APTT: Significantly prolonged in severe hemophilia (because of the reduced level of factor VIII), but may be normal or
minimally prolonged in mild or even moderate hemophilia
Coagulation factor assays
Coagulation factor assays are based upon the ability of the patient’s plasma to correct any sp ecific factor-deficient plasma. To measure
for factor VIII activity in a patient’s plasma, diluted patient plasma is mixed with a factor VIII –deficient plasma. An APTT test is
performed on the mixture.
Management
Clotting factors replacement therapy is used for treatment (Factor VIII is used in hemophilia A and factor IX in hemophilia B)
▪ Clotting factors are usually not needed in mild hemophilia.

▪ In moderate hemophilia clotting factors are typically only needed when bleeding occurs or to prevent bleeding with certain
events.
▪ In severe hemophilia preventive use is often recommended two or three times a week and may continue for life
• Cryoprecipitate: is a plasma-fraction preparation prepared by thawing FFP at 4◦C. It contains a concentrated portion of
fibrinogen and VIII complex.
• Fresh frozen plasma: Cryoprecipitate is deficient in factor IX so FFP replacement therapy is the treatment of choice for
hemophilia B, because FFP contains active factor IX. However, it is deficient in factor VIII.
Von Willebrand disease (vWD)
VWD is first described by Finnish professor Erik von Willebrand in 1926.
Von Willebrand disease (vWD) is the most common hereditary blood-clotting disorder in humans. It arises f rom a deficiency in the
quality or quantity of von Willebrand factor (vWF), causing impaired primary hemostasis.
Synthesis: VWF is synthesized in the endoplasmic reticulum of endothelial cells and stored in cytoplasmic Weibel -Palade bodies of
endothelial cells. It is also synthesized in megakaryocytes and stored in the -granules of platelets
Function
▪ Its primary function is to mediate platelet adhesion to subendothelial collagen

Also it acts as a carrier for Factor VIII. Upon release from intracellular stores, VWF forms a complex with coagulation factor VIII.
This complex is named VIII/VWF. VWF protects factor VIII from proteolysis, prolonging its plasma half-life from a few minutes
without VWF to 8 to 12 hours with VWF
Pathophysiology
Qualitative or quantitative VWF abnormalities reduce platelet adhesion, which leads to mucocutaneous hemorrhage of varying se verity:
▪ Nosebleeds (Epistaxis)
▪ Easy bruising (Ecchymosis)
▪ Menorrhagia
▪ Gastrointestinal tract bleeding
▪ Hematemesis
▪ Mild to moderate bleeding, usually following a hemostatic challenge such as dental extraction or surgery
Severe quantitative VWF deficiency (When factor VIII levels decrease to less than 30% of normal) creates in addition factor
VIII deficiency (equivalent to that seen in severe hemophilia A) owing to the inability to protect non-bound factor VIII from
proteolysis.
Inheritance: It affects both sexes because of its autosomal dominant inheritance pattern.
Laboratory findings: Definitive diagnosis of VWD depends on the combination of a personal and family history of mucocutaneous
bleeding and the laboratory findings:
▪ Normal platelet count

▪ Prolonged bleeding time
▪ Normal prothrombin time
▪ Prolonged PTT in severe cases (due to reduction in factor VIII levels)
Platelet function assay
In this system blood is aspirated from a blood specimen into disposable test cartridges
through a microscopic aperture cut into a biologically active membrane at the end of a
capillary. The membrane of the cartridges are coated with collagen and epinephrine or
adenosine diphosphate (ADP) inducing a platelet plug to form which closes the aperture.
▪ A normal Col/Epi closure time (<180 seconds) excludes the presence of a

significant platelet function defect.
▪ Prolonged Col/Epi closure time (>180 seconds) in the presence of normal
platelet count may indicate von Willebrand disease
Quantitative VWF test (VWF:Ag assay)
Enzyme immunoassays (sandwich ELISA) that measure the concentration of the VWF protein in plasma
Von Willebrand Factor Ristocetin Cofactor [VWF: RCo] Assay
VWF: RCo assay is the standard and widely used laboratory test for von Willebrand disease (VWD) diagnosis.
Reagents: The VWF: RCo assay reagent consists of a latex particle coated with recombinant f ragment of platelet receptor (GPIb)
through a highly specific monoclonal antibody
Principle: The VWF:RCo assay is based on the ability of VWF to induce platelet agglutination in the presence of the antibiotic
ristocetin
▪ If VWF is present in the sample, it will bind the GPIb fragment in the presence of ristocetin
▪ The degree of agglutination is directly proportional to the activity of VWF in the sample and is determined by measuring the
decrease of transmitted light caused by the aggregates
Thrombophilia
Thrombophilia (sometimes hypercoagulability) is an abnormality of blood coagulation that increases the risk of thrombosis (blood clots
in blood vessels).
Effects of thrombophilia
Factors, commonly termed Virchow's triad, predispose patient to thrombosis: stasis, hypercoagulability, and blood vessel wall injury
Venous thrombosis: The most common conditions associated

with thrombophilia are deep vein thrombosis. DVT is the
formation of a blood clot (thrombus) within a deep vein, most
commonly the legs. Although about half of those with DVT have
no symptoms but many patients may shows:
▪ Pain or tenderness
▪ Swelling
▪ Warmth
▪ Redness or discoloration
▪ Distention of surface veins
Pulmonary embolism
When a blood clot breaks loose and travels in the blood, this is called a venous thromboembolism (VTE). Since the veins return blood
to the heart, if a piece of a blood clot formed in a vein breaks off it can be transported to the right side of the heart, and from there into
the lungs.
An embolism that lodges in the lungs is a pulmonary embolism (PE). Symptoms of pulmonary embolism are typically sudden in onset
and may include one or many of the following:
▪ Dyspnea (shortness of breath)

▪ Tachypnea (rapid breathing)
▪ Chest pain (worsened by breathing)
▪ Hemoptysis (coughing up blood)
▪ More severe cases can include signs such as cyanosis (blue discoloration, usually of the lips and f ingers) and circulatory
instability because of decreased blood flow through the lungs and into the left side of the heart.
Arterial thrombosis
Arterial thrombosis is the formation of a thrombus within an artery.
Whether thrombophilia also increases the risk of arterial thrombosis is less well established
In most cases, arterial thrombosis follows rupture of atheroma (a fat-rich deposit in the blood vessel wall), and is therefore
referred to as atherothrombosis
Thrombotic stroke: A stroke is the rapid decline of brain function due to a disturbance in the supply of blood to the brain .
There are two main types of stroke:
• Ischemic, due to lack of blood flow caused by a thrombus (blood clot) usually forms around atherosclerotic plaques.
• Hemorrhagic, due to bleeding within the brain.
Myocardial infarction: Heart attack is caused by ischemia, often due to the obstruction of a coronary artery by a thrombus.
Causes of thrombophilia
Thrombophilia can be congenital (refers to inborn conditions) or acquired (refers to conditions that arise later in life).
Congenital thrombophilia
Hereditary thrombophilias should be suspected in individuals with a history of recurrent spontaneous thrombosis at a young
age, or in patients with a positive family history after a single thrombotic event
Natural anticoagulants deficiency
Even small disturbance of proteins, such as the reduction of antithrombin to only 70 –80% of the normal level, can increase the
thrombosis risk; this is in contrast with bleeding disorders such as hemophilia, which only arises if levels of coagulation factors
are markedly decreased
Protein C deficiency
The main function of protein C is its anticoagulant property as an inhibitor of coagulation factors V and VIII. A deficiency results in a
loss of the normal cleaving of Factors Va and VIIIa. The majority of people with protein C deficiency lack only one copy of the
functioning genes, and are therefore heterozygous. Before 1999, only sixteen cases of homozygous protein C deficiency had been
described (two abnormal copies of the gene, leading to absence of functioning protein C in the bloodstream).
There are two types of protein C deficiencies:
▪ Type I (quantitative deficiency): is the most common form and is associated with both reduction of immunologic and functional
activity of protein C to 50% of normal.
▪ Type II (qualitative deficiency): it is characterized by a normal amount of an abnormal protein
Protein S deficiency
Protein S, a vitamin K-dependent physiological anticoagulant, acts as a nonenzymatic cofactor to activate protein C in the degradatio n
of factor Va and factor VIIIa. Decreased (antigen) levels or impaired function of protein S leads to decreased degradation of f actor Va
and factor VIIIa and an increased propensity to venous thrombosis.
Protein S deficiency can be in inherited via autosomal dominant fashion
Anti-thrombin deficiency
ATIII binds to thrombin and then forms the thrombin-anti thrombin complex or TAT complex. This is a major natural pathway of
anticoagulation. This binding of thrombin to AT is greatly enhanced in the presence of heparin.
Inheritance is usually autosomal dominant
In nephrotic syndrome, antithrombin is lost in the urine, leading to a higher activity of Factor II and Factor X and in increased
tendency to thrombosis.
Factor V Leiden mutation
It is named after the Dutch city Leiden, where it was first identified in 1994 by Prof R. Bertina et al.
Factor V Leiden is a variant (mutated form) of human factor V, which causes an increase in blood clotting (hypercoagulability).
Factor V Leiden is an autosomal dominant genetic condition

Pathophysiology
In the normal person, factor V functions as a cofactor

to allow factor Xa to activate prothrombin, resulting
in the enzyme thrombin.
Activated protein C (aPC) is a natural anticoagulant

that acts to limit the extent of clotting by cleaving
and degrading factor V.
With this mutation, the anticoagulant protein C is not

able to bind normally to Factor V, leading to a
hypercoagulable state. Thus it is also as activated
protein C resistance
Laboratory Diagnosis of APCR
APCR may be evaluated by the two-part aPTT test. The principle of the test is the inhibition of factor Va by APC, which will cause
prolongation of aPTT. Therefore, the aPTT is performed on patient plasma with and without APC.
The results are expressed in a ratio of aPTT with APC: aPTT without APC.
aPTT is decreased when is APC is added to the normal plasma. Plasma from patients with APCR has a lower ratio than the reference
ranges established for normal patients.
Ratio less than two indicates that aPTT is not being significantly prolonged with addition of APC and therefore suggests APC
resistance.
Prothrombin G20210A mutation
Prothrombin G20210A was identified in the 1990s, is almost exclusively present in Caucasians. It is an autosomal dominant disorder
caused by a single point mutation.
The "G20210A" refers to the fact that the mutation is a guanine (G) to adenine (A) substitution at position 20210 of the DNA of
the prothrombin gene.
Pathophysiology
The variant causes elevated plasma prothrombin levels (hyperprothrombinemia), possibly due to increased pre-mRNA stability.
Prothrombin is the precursor to thrombin, which plays a key role in causing blood to clot (blood coagulation). G20210A can thus
contribute to a state of hypercoagulability
MTHFR mutation
Biochemistry
Methylene tetrahydrofolate reductase (MTHFR) is the

rate-limiting enzyme in the methyl cycle.
▪ Methylenetetrahydrofolate reductase catalyzes the

conversion of 5,10-methylenetetrahydrofolate to 5-
methyltetrahydrofolate.
▪ 5-Methyltetrahydrofolate is used to convert homocysteine
(through remethylation) to methionine by the enzyme
methionine synthase.
Mutation
MTHFR mutations that is lead to methylenetetrahydrofolate reductase deficiency leads to elevated homocysteine levels
(hyperhomocysteinemia) which has been shown to be an independent risk factor for coronary heart disease and strokes
Hyperhomocysteinemia can also cause pre-eclampsia and recurrent abortion
L-Methylfolate (5-MTHF)
Unfortunately, many who have a genetic MTHFR mutation are unable to

utilize Folate and folic acid nutrients properly. L-Methylfolate (5-MTHF)
has emerged as a popular alternative, and has been used as a complementary
medicine in several recent clinical trials. It bypasses any MTHFR defects,
and is shown to be equally (if not more) effective at increasing plasma folate
levels and reducing homocysteine concentrations
Acquired thrombophilia
Anti-phospholipids syndrome
Antiphospholipid syndrome (also known as lupus anticoagulant syndrome or Hugh syndrome) is an autoimmune, hypercoagulable
state happens when the immune system makes abnormal antibodies that attack proteins that assemble on phospholipid surfaces.
It was thought that Anti-phospholipid antibodies directly bind phospholipids but later, it was demonstrated that aPL antibodies
are directed against plasma proteins bound to anionic phospholipids
Antiphospholipid syndrome can be primary or secondary. Primary antiphospholipid syndrome occurs in the absence of any other related
disease. Secondary antiphospholipid syndrome occurs with other autoimmune diseases, such as systemic lupus erythematosus (SLE).
Antiphospholipid antibodies
These antibodies hamper the anticoagulant activity of the protein C so inhibits the degradation of the active factor V which helps the
conversion of prothrombin to thrombin
There are three classes of cardiolipin antibodies that may be present in the blood: IgG, IgM and IgA but the two most
commonly tested are IgG and IgM.
These antibodies bind to prothrombin, thus increasing its cleavage to thrombin, its active form
Lupus anticoagulants were so-named because they were first found among patients with systemic lupus and interfere with the
clotting process in a test tube (they inhibit the chemical reactions that lead to clotting in the PTT test so -called anticoagulant)
β2-glycoprotein I or Apolipoprotein H (Apo-H) normally assumes an anti-coagulation activity in serum (by inhibiting coagulation
factors). So anti-β2 glycoprotein I antibodies inhibits the anti-coagulation activity and strongly implicated in autoimmune deep vein
thrombosis
There are also three classes of β2GPI antibodies IgG, IgM and IgA
Diagnostic criteria of APS
The diagnosis of APS is confirmed in the presence of at least one clinical and one laboratory criterion
Clinical criteria
▪ Vascular thrombosis: one or more venous, arterial, or small vessel thrombotic events in any tissue or organ confirmed by imaging
▪ Pregnancy morbidity
• Three or more consecutive spontaneous abortions in the first trimester
• Or one or more unexplained deaths of a morphologically normal fetus beyond the first trimester
• Or severely one or more premature births due to eclampsia or severe preeclampsia
Laboratory criteria
▪ LA (lupus anticoagulant) present in plasma on 2

or more occasions at least 12 weeks apart
demonstrating persistent positivity and exclude
transient positive tests (measured according to
LA panel)
The Lupus anticoagulant antibodies are those

that show the closest association with
thrombosis
▪ aCL-Abs (IgG and/or IgM) present in serum or

plasma on 2 or more occasions at least 12 weeks
apart (measured by a standardized ELISA assay)
Anticardiolipin antibodies are associated with

thrombosis at moderate to high titres
▪ Anti-β2GPI antibody (IgG and/or IgM) present in serum or plasma on 2 or more occasions at least 12 weeks apart (measured by a
standardized ELISA assay)
Anti-β2GPI antibody test may sometimes be ordered when initial anti-phospholipid antibody testing (aCL and LA) is
negative but suspicion of APS is still strong.
Although anti-β2GPI is not yet accepted as a serological criterion for APS, but most investigators would consider a patient
with anti-β2GPI antibodies and clinical features of APS to have the syndrome.
Lupus anticoagulant panel
The presence of LA is usually determined by using a panel of sequential tests performed together to maximize diagnostic potential
The lupus anticoagulant does not inhibit in vivo coagulation but it interferes with in vitro phospholipid -dependent coagulation assays
In the presence of phospholipid autoantibodies such as lupus anticoagulant, the reagent phospholipid is partially neutralized
causing prolongation of the clotting time.
Partial thromboplastin time (PTT)
The lupus anticoagulant produces a prolonged PTT by binding to the phospholipid used in the test.
LA activity is identified by in vitro prolongation of PTT
Dilute Russell viper venom test (DRVVT)
DRVVT is based on the activation of factor X which turns prothrombin into thrombin in the presence of factor V by Russell's viper
venom in combination with low concentrations of phospholipid (that is neutralized in the presence of LA)
DRVVT is widely used in clinical laboratories and is believed to be specific and more sensitive for detecting LA
Once an abnormal screening test result for LA is identified, the next step is to perform mixing
studies.
▪ In mixing studies, the patient’s plasma is mixed with normal plasma (initially at a
50:50 dilution).
Many laboratories use 80:20 mixtures with platelet-free double-centrifuged normal

human plasma because 50:50 are insensitive to all but the highest antibody levels mixes
▪ A new PTT or DRVVT test is performed immediately on the mixture and once again
after 2-hour incubation at 37°C
Results
Immediate Incubated Interpretation

test test
Coagulation The normal plasma corrects this deficiency and the resultant
Corrected Corrected clotting time will be normal and specific coagulation factor testing
factor
(≥50%) (>10%) is performed to determine which factor(s) is deficient.
deficiency
factor deficiency is associated with hemorrhagic problems
Specific inhibitors require an incubation to enhance their avidity.
Factor VIII inhibitor is found in 10%–20% of hemophilia patients
Prolonged
Corrected Factor VIII receiving replacement therapy. It may also develop in patients
(≤10%
(≥50%) inhibitor with immunologic problems, women after childbirth, and patients
correction)
with lymphoproliferative disorders
factor VIII inhibitor is associated with hemorrhagic problems
The APTT would not be corrected by mixing studies if lupus
Prolonged Prolonged
lupus anticoagulant was present.
(<50% (≤10%
anticoagulant it is associated with thrombosis, not severe hemorrhagic
correction) correction)
problems
Disseminated intravascular coagulation
DIC is characterized by systemic activation of blood coagulation, which results in the formation of microvascular thrombi in small
blood vessels of various organs and contributing to multiple organ dysfunction syndrome (multiple organ failure such as acute kidney
injury, hepatic dysfunction, and respiratory dysfunction).
Etiology
DIC does not occur by itself but it is always secondary to an underlying disorder and is associated with a number of clinical conditions,
generally involving activation of systemic inflammation such as:
▪ Sepsis or severe infection of any kind (infections by nearly all microorganisms bacterial, viral or parasitic can cause DIC,
though bacterial infections are the most common)
▪ Massive tissue injury as severe trauma, burns and rhabdomyolysis (due to release of tissue factor (thromboplastin) into the
circulation)
▪ Hematologic malignancy (particularly acute myeloid leukemia) and solid tumors metastasis
▪ Obstetric complications such as Preeclampsia and eclampsia
▪ Chronic liver failure
▪ Transfusion reactions (ABO incompatibility )
▪ Severe allergic or toxic reactions (i.e. snake venom)
Classification
DIC exists in both acute and chronic forms. Acute DIC develops when sudden exposure of blood to procoagulants generates
intravascular coagulation. In contrast, chronic DIC develops when blood is continuously or intermittently exposed to small am ounts of
procoagulants.
Cancer is the most common cause of chronic DIC
Pathophysiology
In DIC, the processes of coagulation and fibrinolysis are dysregulated, and the result is widespread clotting with resultant bleeding.
Regardless of the triggering event of DIC, once initiated, the pathophysiology of DIC is similar in all conditions.
▪ One critical mediator of DIC is the release of the transmembrane tissue factor (TF). TF is present on the surface of many cell types
(including endothelial cells, macrophages, and monocytes) and is not normally in contact with the general circulation, but is
exposed to the circulation after vascular damage (thus activating the extrinsic pathway).
▪ The conditions that lead to DIC are associated with systemic inflammatory response that is characterized by activation of cytokine,
interleukins 1 and 6 and tumor necrosis factor (TNF).
▪ These cytokines stimulate macrophages to express several mainly tissue factor molecules on their outer surface
▪ Upon exposure to blood and platelets, TF binds with activated factor VIIa, forming the extrinsic tenase complex. This complex
further activates factor X, leading to the common coagulation
pathway and the subsequent formation of thrombin and fibrin
▪ Excess circulating thrombin results from the excess activation of
the coagulation cascade cleaves fibrinogen, which ultimately
leaves behind multiple fibrin clots in the circulation.
▪ These excess clots trap platelets to become larger clots, which
leads to microvascular thrombosis.
▪ This lodging of clots in the microcirculation, in the large vessels,
and in the organs is what leads to the ischemia, impaired organ
perfusion, and end-organ damage that occur with DIC.
▪ Clotting factors are consumed in the development of multiple
clots, which contributes with thrombocytopenia to the bleeding
seen with DIC
▪ The red blood cells get trapped in the

fibrin strands and the sheer force of
the blood flow causes the red blood
cell to break resulting in
microangiopathic hemolytic anemia
(MAHA) with fragmented RBCs
(schistocyte) in the blood film
▪ Simultaneously, excess circulating
thrombin assists in the conversion of
plasminogen to plasmin, resulting in
fibrinolysis.
▪ The breakdown of clots results in an
excess of FDPs that is have powerful
anticoagulant properties, contributing
to hemorrhage.
▪ The excess plasmin also activates the
kinin systems (its important mediator
bradykinin is vasodilator) leading to
the clinical symptoms that patients
with acute form of DIC exhibit, such
as increased vascular permeability,
hypotension and shock.
Laboratory findings
▪ Prolongation of the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) reflect the underlying consumption
and impaired synthesis of the coagulation cascade.
Fibrinogen level has initially thought to be useful in the diagnosis of DIC but because it is an acute phase reactant, it will be
elevated due to the underlying inflammatory condition.
▪ A rapidly declining platelet count with Prolongation of bleeding time

▪ Decreased fibrinogen and antithrombin level
Differentiation between DIC and TTP
Thrombotic thrombocytopenic purpura Disseminated intravascular coagulation

Platelets Decreased Decreased
PT Normal Prolonged
PTT Normal Prolonged
Fibrinogen Normal Decreased
FDPs Absent Present
Schistocytes Marked Mild
Fibrin degradation products (FDPs)
Overview
Coagulation, at the wound site produces a mass of fibrin

threads that remains in place until the cut is healed. As a cut
heals, the clot is broken down and dissolved by plasmin.
When the clot and fibrin threads dissolve, fragments of protein
are released into the body. These fragments are fibrin
degradation products or FDPs.
Sample: Blood should be collected in a blue top containing 3.2% sodium citrate.
Procedure: Rapid latex agglutination
Interpretation: Fibrinogen degradation product (FDP) testing is commonly used to

aid in the diagnosis of disseminated intravascular coagulation (DIC). The reference
range of FDP levels is less than 10 m/ml. An FDP level of more than 40 mg/mL is
considered critical
Chronic conditions (e.g. renal failure, liver failure) in elderly persons may
cause an increase in FDP levels.
D-dimer test
Overview
D-dimers are products of cross-linked fibrin degradation.

Factor XIIIa catalyzes the γ-chain cross-linkage of adjacent
terminal domains (D-domains) of fibrin monomers.
Indications
▪ D-dimer tests are used to help rule out the presence of

an inappropriate blood clot (thrombus). Some of the
conditions that the D-dimer test is used to help rule out
include:
• Deep vein thrombosis (DVT)

• Pulmonary embolism (PE)
• Stroke
D-dimer is especially useful when a health practitioner

thinks that something other than deep vein thrombosis
or pulmonary embolism is causing the symptoms. It is a quick, non-invasive way to help rule out abnormal or excess clotting
▪ A D-dimer level may be used to help diagnose disseminated intravascular coagulation (DIC) and to monitor the effectiveness of
DIC treatment.
Typically, the D-dimer level is elevated in DIC
D-dimer test may be ordered, along with a PT, PTT, fibrinogen, and platelet count, to help diagnose the condition. D-dimer may
also be ordered at intervals when someone is undergoing treatment for DIC to help monitor its progress.
When used to monitor DIC treatment, decreasing levels indicate that treatment is effective while increasing levels may indicate
that treatment is not working.
Sample
Blood should be collected in a blue top containing 3.2% sodium citrate.
Specimens should be tested within 4 hours from the time of specimen collection. If testing will take place after 4 hours,
specimens must be refrigerated at 2 to 8C and are good up to 24 hours
Procedure
▪ Latex-enhanced turbidimetric assay

▪ Enzyme-Linked Immunosorbent Assay
Interpretation
A negative D-dimer test (D-dimer level is below a predetermined threshold) indicates that it is highly unlikely that a thrombus is
present.
A positive D-dimer result may indicate the presence of an abnormally high level of fibrin degradation products. It indicates that there
may be significant blood clot (thrombus) formation and breakdown in the body, but it does not tell the location or cause. For example, it
may be due to a venous thromboembolism (VTE) or disseminated intravascular coagulation (DIC).
Advantage
The D-dimer epitope is exposed when fibrin is lysed by plasmin. Thus, the presence of D-dimers is evidence of the combined
actions of thrombin, factor XIIIa, and plasmin and is specific for the combined presence of coagulation and f ibrinolysis
(physiologic or pathologic). In contrast, an increase in FDPs is not specific evidence of active coagulation because FDPs
include both fibrin and fibrinogen degradation products and thus may be increased exclusively by plasmin activity on
fibrinogen.
The D-dimer is a specific marker of fibrinolysis. A normal D-dimer test can be used to rule out the formation of a clot.
FDPs D-dimer Interpretation
Positive Positive DIC, DVT
Negative Negative Exclude thrombosis

Due to activation of fibrinolytic system as seen in severe liver disease (Primary
fibrinogenolysis)
All the proteins involved in fibrinolysis, except for tPA (tissue plasminogen activator)
and PAI-1 (plasminogen activator inhibitor-1), are synthesized in the liver. Reduced
plasma levels of plasminogen, alpha 2-antiplasmin, histidine-rich glycoprotein (HRG),
Positive Negative
factor XIII, and thrombin-activable fibrinolysis inhibitor (TAFI, a carboxypeptidase)
occur in patients with liver cirrhosis. Conversely, tPA levels are increased in liver
disease due to decreased clearance, and the tPA inhibitor, PAI-1, is normal or only
slightly increased in plasma. The inhibitor concentrations are insufficient to counteract
the increase in tPA, accounting for a net increase in fibrinolysis.
Management
Treatment of DIC is centered on treating the underlying condition.
Supportive therapy with blood products is also needed:
▪ Platelet transfusion to keep the platelet count greater than 50 × 109/L if there is a high risk of bleeding.
▪ Fresh frozen plasma (FFP) is a source of clotting factors, except fibrinogen. It can be considered in cases of significant
bleeding
Treatment of thrombosis with anticoagulants such as heparin is rarely used due to the risk of bleeding.
The prognosis for those with DIC, regardless of cause, is often grim: Between 20% and 50% of patients will die
DIC with sepsis (infection) has a significantly higher rate of death than DIC associated with other causes
Anticoagulation Therapy
Heparin
Trade name
▪ Unfractionated heparin (UFH) is high molecular weight heparins, such as calcium

heparin and sodium heparin.
▪ Low molecular weight heparins (LMWHs) are derived from UFH by de -
polymerization; it is more effective in treatment such as Clexane and fraxiparine
Indications
Heparin is generally used for anticoagulation for the following conditions:
▪ Acute cases of deep-vein thrombosis and pulmonary embolism (it is a rapid anticoagulant)
▪ Acute coronary syndrome due to unstable angina
▪ Pregnant women with anti-phospholipid syndrome
Mode of administration
Heparin is given parenterally because it is not absorbed from the gut, due to its high negative charge and large size. It can be injected
intravenously or subcutaneously (under the skin)
Intramuscular injections (into muscle) are avoided because of the potential for forming hematomas.
UFH has a half-life of about one to two hours after infusion. Whereas LMWH has a half-life of four to five hours
Heparin need to be given every 8-12 hours.
Mechanism of action
Heparin is a naturally occurring anticoagulant produced by

basophils and mast cells. In therapeutic doses, it acts as an
anticoagulant, preventing the formation of clots within the blood.
Heparin binds to the enzyme inhibitor antithrombin III (AT),

causing a conformational change that result in its activation through
an increase in the flexibility of its reactive site loop. The activated
AT then inactivates factors IIa (thrombin), Xa, IXa, XIa, and XIIa.
The rate of inactivation of these proteases by AT can increase

by up to 1000-fold due to the binding of heparin
Antithrombin deficiency is associated with a poor response to

heparin therapy (heparin resistance)
Side effects
▪ Bleeding
▪ Pain at the injection site
▪ Elevation of serum aminotransferase levels, which has been reported in as many as 80% of patients receiving heparin
This abnormality is not associated with liver dysfunction, and it disappears after the drug is discontinued
▪ Hyperkalemia, occurs in 5 to 10% of patients receiving heparin, and is the result of heparin-induced aldosterone suppression.
▪ Heparin-induced thrombocytopenia
It is a serious side-effect of heparin therapy. In HIT, the immune system

forms antibodies against heparin when it is bound to a protein called
platelet factor 4 (PF4). These antibodies are usually of the IgG class and
their development usually takes about five days.
The IgG antibodies form a complex with heparin and PF4 in the
bloodstream. The tail of the antibody then binds to the Fc receptor on the
surface of the platelet. This results in platelet activation and the
formation of platelet microparticles, which initiate the formation of blood
clots (thrombosis); the platelet count falls as a result, leading to
thrombocytopenia.
This condition is usually reversed on discontinuation
The platelet count should be checked every other day in patients receiving
heparin therapy. HIT should be suspected in patients who are not responding
to heparin therapy and/or are developing thrombocytopenia (50% below the
baseline value) and thrombotic complications while on heparin therapy.
Monitoring Heparin therapy
When heparin is administered for therapeutic purposes, it must be closely monitored. If too much is given, the treated person may bleed
excessively; with too little, the treated person may continue to clot.
Heparin effectiveness
The response to heparin therapy varies among different patients because its activity change based upon nonspecific binding of heparin
to plasma proteins. Therefore, heparin therapy should be closely monitored.
Laboratory assays
Current clinical laboratory assays for heparin rely on an indirect measurement of the effect of the drug, rather than on a direct measure
of its chemical presence. These include partial thromboplastin time (PTT) and anti-factor Xa activity.
Protocol
A baseline PTT and platelet count; PTT testing every 6 hours after the initial dose until the target is reached
Peripheral blood for PTT analysis should be obtained one hour before the next dose of heparin
Therapeutic range
Therapeutic range is approximately 1.5-2 times patient's baseline PTT value prior to treatment
So if the control is 36 seconds
▪ A report greater than 90 seconds indicates more-than-adequate anticoagulation for the moment the blood is drawn.
▪ A report of less than 53 seconds indicates inadequate anticoagulation for the moment the blood is drawn.
Antidote to heparin
Protamine sulfate has been given to counteract the anticoagulant effect of heparin (1 mg per 100 units of heparin that had been given
over the past four hours).
It may be used in those who overdose on heparin or to reverse heparin's effect when it is no longer needed
Warfarin
Trade name
Marevan is the most common oral anticoagulant therapy
Indications
Warfarin is commonly used to
▪ decrease the tendency for thrombosis or as secondary prophylaxis (prevention of further episodes) in those individuals who have
already formed deep vein thrombosis or pulmonary embolism
▪ To prevent stroke in people who have artificial heart valves
Warfarin is best suited for anticoagulation in areas of slowly running blood (such as in the pooled blood behind
artificial valves)
▪ Treat non-Pregnant women with anti-phospholipid syndrome (use is not generally recommended during pregnancy)
▪ In patient with chronic heart failure to reduce the likelihood of blood clots
Mechanism of action
Warfarin antagonises vitamin K
Warfarin decreases blood clotting by blocking an enzyme called vitamin K

epoxide reductase that reactivates vitamin K. Without sufficient active
vitamin K, clotting factors II, VII, IX, and X have decreased clotting ability.
The anticlotting protein C and protein S are also inhibited but to a

lesser degree
A few days (4-5 days) are required for full effect to occur (until vitamin
K reserve finished) since the circulating coagulation factors are not
affected by the drug (anticoagulation becomes therapeutic only when
the activities of factors II and X decrease to less than 50% of normal)
For the first three days of "warfarinization", the levels of protein C and
protein S (anticoagulation factors) drop faster than procoagulation
proteins. Therefore, bridging anticoagulant therapies (usually heparin)
are often used to reverse this temporary hypercoagulable state.
Mode of administration
It is generally taken by mouth but may also be used by injection into a vein
Warfarin has a long half-life and need only be given once a day.
Initial dose: 2 to 5 mg orally or intravenously once a day for 1 to 2 days, then adjust dose according to results o f the International
Normalized Ratio (INR).
Duration
In case of DVT or Pulmonary embolus: At least three months if risk factors are temporary but at least six months if they are
permanent or cause unknown
▪ Long-term in case of artificial valves (possibly lifelong)
Side effects
Bleeding
The only common side effect of warfarin is bleeding which include:
• Bleeding from the gums or nosebleed

• Blood in the urine dark stool
• Vomiting blood
Drug interaction that may increase the risk of bleeding include NSAIDs and Anti-platelets as Aspirin
Osteoporosis
Warfarin use for more than one year was linked with a 60% increased risk of osteoporosis-related fracture. The mechanism was thought
to be due to inhibition by warfarin of vitamin K-mediated carboxylation of certain bone proteins, rendering them nonfunctional
Contraindications
▪ Warfarin is generally contraindicated in situations where the reduction in clotting that they cause might lead to serious and
potentially life-threatening bleeds such as active gastrointestinal ulcers, low platelets, severe liver disease and for patie nts
undergoing surgery
▪ Warfarin is contraindicated in pregnancy, as it passes through the placental barrier and may cause bleeding in the f etus causing
spontaneous abortion, stillbirth and birth. Warfarin is also teratogens, that is, they cause birth defects
Monitoring Warfarin therapy
To optimize the therapeutic effect without risking dangerous side effects, close monitoring of the degree of anticoagulation is required
Warfarin effectiveness
Dosing of warfarin is complicated because it is known to interact with many commonly used medications and certain foods so it must
be adjusted for every patient
Factors decrease warfarin effectiveness (lower INR)
▪ Common food that contains large quantities of vitamin K will reduce the warfarin effect, these include:
• Broccoli
• Cabbage
• Spinach
• Beet greens
• Legumes
• Soybean oils
• Green onions
• Turnip greens
• Beef liver
• Green tea
• Lettuce
▪ Hypothyroidism makes people less responsive to warfarin treatment
▪ The PT may be shortened during acute inflammatory conditions which are accompanied by increase in fibrinogen levels
Factors increase warfarin effectiveness (higher INR)
▪ Chronic diarrhea and malabsorption affect vitamin K absorption so potentiate warfarin effect
▪ Hyperthyroidism boosts the warfarin effect.
▪ Grapefruit or Liquorice inhibits metabolism of warfarin and may increase INR
Warfarin Interactions
Warfarin is metabolized in the mitochondrial cytochrome P-450 (CYP 2C9) pathway of hepatocytes. Theoretically, use of any drug
metabolized through the CYP 2C9 pathway may unpredictably suppress or enhance the effects of warfarin.
Medications suppress warfarin effect (lower INR)
▪ Carbamazepine
▪ Phenobarbital
▪ Phenytoin
▪ Cholestyramine
▪ Ribavirin
▪ Rifampin
Medications enhance warfarin effect (higher INR)
▪ Acetaminophen
▪ Aspirin
▪ Allopurinol
▪ Amiodarone
▪ Omeprazole
▪ Metronidazole
▪ Chloramphenicol
▪ Quinolones (Ciprofloxacin, Ofloxacin, Levofloxacin, Norfloxacin)
▪ Macrolide (Azithromycin, Clarithromycin, Erythromycin)
▪ Tetracycline
The broad-spectrum antibiotics can reduce the amount of the normal bacterial flora in the bowel, which make significant
quantities of vitamin K, thus potentiating the effect of warfarin
Laboratory assays
The PT effectively monitors warfarin therapy because it is sensitive to reductions of factors II, VII, and X
Protocol
▪ The first PT is collected and performed 24 hours after therapy is initiated; subsequent PTs are performed daily until at leas t two
consecutive results are within the target therapeutic range.
▪ Monitoring continues twice a week for 1-2 weeks, then every 4 to 12 weeks until the completion of therapy, which often lasts for 6
months following a thrombotic event.
Therapeutic range
The target INR level varies from case to case depending on the clinical indicators
▪ Target INR may be 2.0–3.0 in patients with thrombosis

▪ Target INR may be 2.5–3.5 in patients with one or more mechanical heart valves
Risk of bleeding is increased once the INR exceeds 4.5
When warfarin is being given and INR is in therapeutic range, simple discontinuation of the drug for five days is usually
enough to reverse the effect and cause INR to drop below 1.5
Antidote to warfarin
For people who need rapid reversal of warfarin such as due to serious bleeding or need emergency surgery, the effects of warf arin can
be reversed with vitamin K (such as Konakion), or fresh frozen plasma (FFP)
Coagulation profile
Bleeding time (BT)
Indications
Bleeding time is a screening test that investigate the efficiency of hemostatic mechanisms other than blood clotting (vascular spasm and
platelets plug formation)
Bleeding time indicates how well platelets interact with blood vessel walls to form blood clots
It is useful before surgery to assess the risk of bleeding during an operation
Procedure
Duke’s method
With the Duke method, the patient is pricked with a special lancet, preferably on the
earlobe or fingertip to measure how long it taken for bleeding to stop
▪ Clean the finger using 70% ethanol and allow it to dry.

▪ Using a sterile disposable lancet, make a quick skin puncture
▪ Start the stopwatch as soon as the incisions are made and bleeding starts
▪ Wipe away the drop of blood every 30 seconds with a filter paper or piece of
cotton
The test ceases when bleeding ceases, the normal bleeding time is about 2-5 minutes
Do not use Index finger (frequently used for touch) or little finger (decreased
skin surface to bone depth)
Ivy’s method
Ivy, a surgeon observed that bleeding time by the Duke’s method was normal in
many patients. He attributed this to variability in the “tone” of capillaries. He
modified the bleeding time by applying a standard pressure with a
sphygmomanometer cuff proximal to the test wound to counter the inter-patient
variability in capillary “tone”.
▪ Tie a blood pressure cuff (Sphygmomanometer) on

the upper arm and a constant pressure of 40 mm Hg is
applied (maintain this pressure throughout the period
of the test)
▪ Clean the lateral aspect of the surface of the f orearm
with 70% ethanol and allow it to dry.
▪ Make a 3 skin punctures 3 cm apart (2.5 mm deep)
using the disposable lancet (avoid vein puncture).
▪ Start the stopwatch
▪ Gently touch the drop of the blood which forms over the wound, with the edge of a filter paper every
30 seconds until the bleeding stops completely.
▪ The Bleeding time is reported when no bloodstain is seen on the filter paper after a gentle touch.
▪ Record the mean of the three readings
Quality control
▪ Do not use the tip of the finger or the center of the finger (it is more sensitive and
cause much pain).
▪ Puncture should be 2 mm deep but not exceed 2.5 mm (too deep incision give false
positive results)
▪ Do not squeeze the finger, Squeezing lead to blood dilution with interstitial f luid
and help blood clotting
▪ Do not touch the skin or the incision point at any time (any disruption of the fibrin
formed or clot will prolong the bleeding time)
Clotting time (CT)
Indications
Clotting time is a screening test that investigate the efficiency of blood clotting factors
This test can be used in addition to BT to assess the risk of bleeding during an operation
This test is non-specific and insensitive to mild clotting factors defects
Procedure
Lee-white method
▪ Label three glass test tubes as 1, 2, and 3

▪ Clean the blood collection site and allow it to dry completely
▪ Collect 3.0 mL of venous blood in a disposable syringe and start the stop watch the
moment blood enters the syringe
▪ Dispense 1 mL of the blood each into the 3 test tubes
▪ Keep the tubes in water bath (or incubator) maintained at 37oC for 3 minutes
▪ Then after every 30 seconds tilt the tubes slowly to the horizontal level one after another
and see whether blood has clotted
▪ Repeat this till clotting has occurred in test tubes, such that blood does not flow out even
when the tube is tilted to 90 degrees
▪ Record the mean of the three readings
The normal time is about 5-10 minutes.
Quality control
▪ Never use plastic test tubes

▪ Low blood volume will give short clotting time
▪ Low incubation temperature will shorten clotting time
▪ Unnecessary agitation of blood sample shorten clotting time
Slide method
▪ Obtain blood in the syringe and start stop watch since appearance of the blood in the syringe.
▪ Put a drop of the blood on the slide
▪ The slide is placed on a warm plate at 37oC (incubator)
▪ Every 30 seconds put a needle in the middle of the blood drop and try to elevate it
Blood coagulation start when fibrin threads appear attached to the needles
Capillary tube method: (Wright’s method)
▪ Finger of a subject is sterilized with spirit and pricked with sterilized needle (time
of pricking is noted).
▪ Sufficient blood is made to flow inside the glass capillary tube by capillary action.
▪ After about two minutes start snapping off small lengths of the tube at intervals of
20 seconds
▪ Each time noting whether the fibrin thread is formed between the snapped ends
▪ Note the time (stop the stop watch) when the fibrin thread is first seen; it is the
clotting time of the subject.
Why CT is normally longer than BT?
Normal clotting time and bleeding time values differ because bleeding time is the time for stopping bleeding by the formation of f ibrin
network on the surface of punctured skin; that is it is the surface phenomenon. But the clotting time is the time for clotting the whole
blood, collected in the tube; therefore it is a volume phenomenon. For this reason clotting time is more than the bleeding time, when
determining by conventional methods.
Prothrombin time (PT)
Indications
▪ Before surgery, to investigate the efficiency of blood coagulation factors (evaluate extrinsic and common pathway)
PT test evaluates the coagulation factors VII, X, V, II, and I
The procedure is most sensitive to factor VII deficiencies, moderately sensitive to factor V and X deficiencies, sensitive to severe
fibrinogen and prothrombin deficiencies
▪ It is useful in monitoring anticoagulant medication (warfarin anticoagulant therapy)

▪ It can be used as a liver function test
▪ May be ordered to evaluate patient (who is not taking anticoagulant drugs) with symptoms of excessive bleeding (easy bruising,
nosebleeds, bleeding gums) or clotting (blood clot in a vein or artery)
Procedure
Sample
▪ Whole blood anti-coagulated with 3.2% buffered sodium citrate is the specimen of choice.
Sodium citrate binds calcium ions to prevent coagulation, and the buffer stabilizes specimen pH as long as the tube stopper
remains in place
Precautions
▪ The venipuncture must be a clean one and, if there is any difficulty, take a new syringe and try another vein
▪ During blood collection, the phlebotomist must remove the tourniquet within 1 minute of its application to avoid blood s tasis.
Stasis results in the local accumulation of coagulation factor VIII and von Willebrand factor (VWF), which may result in f als e
shortening of clot-based coagulation test results.
▪ A blood sample obtained from a site above heparin infusion, or through heparin-coated catheters may affect the PT (Falsely
prolonged PT).
▪ The test results from visibly hemolyzed specimens are unreliable, and the specimen must be recollected
▪ lipemic or icteric specimen must be avoided (falsely increase results due to excess plasma turbidity)
Fasting or only light non-fatty meals prior to blood collection provide samples with a desirable lower opacity.
▪ It is important that the tube be mixed by gentle inversion soon after blood collection (Inadequate mixing causes the sample to clot,
rendering it unsuitable for testing.)
Excessive specimen agitation causes hemolysis, procoagulant activation, and platelet activation (falsely shorten PT) so the
phlebotomist must never shake the tube
▪ A short draw—that is, a specimen with a smaller volume than the minimum specified by the manufacturer—generates erroneously
prolonged clot-based coagulation test results because the excess anticoagulant relative to blood volume neutralizes test reagent
calcium.
In most cases, the volume of blood collected must be within 90% of the calibrated volume
Adjustment of Sodium Citrate Volume
The 9:1 blood-to-anticoagulant ratio is required for accurate coagulation testing, provided the patient’s hematocrit is between
20 - 55%.
In polycythemia, the volume of blood contains so little plasma that excess anticoagulant remains and is available to bind to reagent
calcium, thereby resulting in prolongation of the PT and APTT.
For more accurate results, the plasma: anticoagulant ratio can be modified by decreasing the amount of anticoagulant in the collection
tube using the following formula:
C = (1.85 x 10-3) (100 - H) V >>>> Where
▪ C = volume (mL) of anticoagulant.

▪ H = patient’s HCT
▪ V = blood volume in mL
For example, to collect 3 mL of blood and anticoagulant mixture from a patient who has a hematocrit of 65%, calculate the volume of
sodium citrate as follows:
C = (1.85 x 10 -3) x (100 - 65) x 3
C = (1.85 x 10 -3) x (35) x 3
C = 0.19 mL of 3.2% sodium citrate
The anticoagulant-to-blood ratio also should be adjusted for PT in patients with a severe anemia (hematocrit less than 20%)
using the same formula to avoid falsely shortend PT
Preparation of Specimens
▪ Each specimen must be visually inspected prior to centrifugation; the presence of even a small clot requires that the specimen be
recollected.
▪ Plasma should be separated as soon as possible
Centrifuge immediately for 15 minutes at 3000 rpm on laboratory centrifuge and transfer the plasma into a clean plastic test
tube (glass tube are never used with plasma handling as it activates the coagulation cascade). It should be ensured that the
plasma is free from platelets (PPP).
High platelets count in the sample (due to inadequate centrifugation) affects PT results. Platelets secrete fibrinogen, factors V
and VIII, and VWF which may falsely shorten PT assay
Preservation of Specimens
According to Clinical Laboratory Standards Institute (CLSI) guidelines, plasma samples for PT testing are stable for 24 hours at room
temperature (18° C to 24° C) if capped.
• Refrigerating the sample (Storage at 1° C to 6° C) causes cold activation of factor VII and, therefore, shortened PT results.
• Also, specimens should never be stored at temperatures greater than 24° C because heat causes deterioration of coagu lation
factors V and VIII which causes prolongation of the PT
If the hemostasis test cannot be completed within the pre scribed interval, the laboratory practitioner must immediately cent rifuge the
specimen. The supernatant PPP must be transferred by plastic pipette to a plastic tube, sealed, and frozen and may be stored at –20° C
for up to 2 weeks or at–70° C for up to 6 months.
Reagent
Calcium Thromboplastin reagent, which is derived from rabbit brain serves as the reagent
source of tissue factor, in the presence of excess calcium, the activator which initiates the
extrinsic pathway of coagulation.
Store the reagent at 2 – 8 oC (Do not freeze). The uncontaminated reagent is stable
for: 1 year at 2 – 8 oC, 1 week at 18 – 25 oC, and 2 days at 37 oC.
Principle
▪ Citrated plasma is added to the reagent with calcium (thereby reversing the effects of
citrate), and the time required for fibrin clot formation is measured.
▪ When mixed with citrated PPP, the PT reagent triggers fibrin polymerization by
activating plasma factor VII. Calcium and phospholipids participate in the formation of the tissue factor–factor VIIa complex, the
factor VIIIa–factor IXa complex, and the factor Va–factor Xa complex.
Manual test
Bring this reagent to room temperature
Homogenization of reagent suspension before use is important to achieve accurate and consistent results
▪ Aspirate from the reagent vial enough reagent for immediate testing requirements in a thoroughly clean and dry test tube
(Plastic test tubes are preferred).
Recap the reagent vial and replace immediately to 2 – 8 oC
▪ Pre-warming the reagent at 37oC for 5 minutes.

The PT test functions correctly only at 37 ± 0.5 oC
▪ 100 l of plasma and place the tube in a water bath at 37 oC for at least 3 and for no more than 10 minutes.
Aliquots that are incubated longer than10 minutes become prolonged as coagulation factors begin to deteriorate or are
affected by evaporation and pH change.
▪ To the tube forcibly add 200 l of reagent and simultaneously start a stop watch.
▪ Mix in a water bath (37°C) for 8 seconds, then record the time required for clot formation.
▪ Shake the tube gently to mix contents.
▪ Gently tilt the tube back and forth and stop the stopwatch as soon as the first fibrin strand is visible and the gel / clot f ormation
begins.
▪ Record the time in seconds.

Perform each test on the same sample in duplicate, the duplicate
values must be within 10% of their mean or the test is repeated
for a third time.
Results
Prothrombin time result is expressed as follow:
▪ Patient PT in seconds
▪ PT control (mean normal plasma time - MNPT)
▪ PT % activity
▪ The international normalized ratio (INR)
Prothrombin time
Defined as the time (seconds) needed to clot platelet-poor plasma upon

addition of coagulation triggers, such as tissue factor in complex with
phospholipids and calcium
Normal plasma in the PT clots approximately within 10–13 s upon

triggering coagulation
Prothrombin time control
PT control is the normal population range (reference range) that consists of

data generated from a group of individuals judged to be free of any known
abnormalities.
▪ Theoretically plasma from at least 20 normal healthy individuals (include

equal number of males and females in the age group of 18-45) should be
used to establish the MNPT. The average of such PT results in
seconds=MNPT.
▪ Practically plasma from 3 normal healthy individuals are collected
• PT is done for the three samples and calculates the mean (PT1 result)
• Equal amounts of the three samples are pooled together (pooled plasma) and PT is done (PT2 result)
• The PT control is the mean of the two PT results
In case of major changes in type of reagents, or instrumentation, MNPT should be reestablished.
PT % activity
▪ Pooled normal plasma (arbitrarily assigned 100% activity)

was progressively diluted with saline (1:2, 1:4, and 1:8 which
corresponds to concentrations 50%, 25%, and 12.5%) and
tested for the Prothrombin time.
▪ Plots of paired data (percentage activities vs. clotting times)
allow the preparation of a calibration curve that can be used to
interpolate the percentage activity from the patient PT as
follow:
The international normalized ratio (INR)
The INR was invented in the early 1980s by Tom Kirkwood working at the UK National Institute for Biological Standards and
Control
▪ The result (in seconds) for a PT performed on a normal individual will vary between laboratories due to the variations between
different tissue factors used in the reagent to perform the test.
▪ Commercially available thromboplastins were derived from animal tissue (e.g. rabbit, human, bovine etc.). Contamination by
serum resulted in the presence of some coagulation factors in the commercially available thromboplastin.
Use of thromboplastins contaminated with coagulation factors made the test less sensitive to the decrease in coagulation
factors in the test plasma.
▪ World Health Organization (WHO) developed and recommended the use of the INR, calculated based on the PT ratio, f or people
who are receiving the anticoagulant warfarin
The INR is a calculation that adjusts for changes in the PT reagent sensitivity and allows for results from different
laboratories to be compared.
The INR is the Prothrombin ratio (patient's PT to a normal (control) sample), raised to the power of the ISI
value for the analytical system used.
International Sensitivity Index
Each manufacturer assigns an ISI value (International Sensitivity Index) for any tissue factor they manufacture. The ISI value
indicates how a particular batch of tissue factor compares to a WHO internationally standardized thromboplastin.
▪ Prothrombin Times are performed in duplicate for each thromboplastin and the
mean for each pair of tests derived. Tests are historically performed on 20 normal
donors not on anticoagulants and 60 patients who have been on oral anticoagulant
treatment for at least 6 weeks.
▪ The mean of each pair of PT results are plotted with the reference sample on the
Y axis and the test plasma on the X-axis.
▪ A line of best fit is drawn and the slope of this line is the ISI
The ISI is usually between 1 and 2 and attests to the purity of the reagent
The higher the ISI, the less sensitive the thromboplastin, and the shorter the PT result
The lower the manufacture ISI value, the more desirable the reagent for use
INR in absence of anticoagulation therapy is 0.8-1.2
The INR is particularly important when monitoring warfarin

anticoagulant therapy.
▪ When the INR is correctly used for monitoring a patient’s oral

anticoagulant therapy, the physician gives a standard dose of
coumarin to achieve a target INR between 2.0 and 3.0
▪ If the physician chooses to use a high dose of coumarin as indicated in mechanical heart valves or prevention of recurrent
myocardial infarction, the target value INR is between 2.5 and 3.5
An INR value >3.5 constitutes a panic value and should be reported immediately
Quality control
▪ The first step in evaluation of a prolonged PT in a patient not on warfarin, is to repeat the test to rule out a lab error.
▪ In case of prolonged PT in a patient not on warfarin, platelet count should be done, low count may indicate clot formation and new
sample must be re-collected
▪ In case of prolonged PT in a patient not on warfarin, mixing study may be done (plasma is mixed with normal plasma initially at a
50:50 dilution). If mixing corrects the PT result, reagent and procedure are adequate
Partial thromboplastin time (PTT)
Indications
▪ Before surgery, to investigate the efficiency of blood coagulation factors (evaluate intrinsic and common pathway)
The APTT test evaluates the clotting factors in the intrinsic pathway (XII, XI, IX, and VIII) as well as the common pathway
(X, V, II, and I)
Deficiencies of prekallikrein, or high-molecular-weight kininogen prolong the PTT but do not cause bleeding.
▪ It is useful in monitoring anticoagulant medication (heparin anticoagulant therapy)

▪ PTT testing may be performed as part of a clotting disorder panel to help investigate recurrent miscarriages or diagnose anti-
phospholipid syndrome (APS)
▪ May be ordered to evaluate patient (who is not taking anticoagulant drugs) with symptoms of excessive bleeding or clotting (to
detect congenital and acquired abnormalities of the intrinsic coagulation pathway)
Procedure
Sample: Citrated, platelet-poor plasma is used for the PTT.
Samples for APTT should be centrifuged and tested within 2 hours after collection.
According to Clinical Laboratory Standards Institute (CLSI) guidelines APTT samples are stable for 4 hours if stored at 4°C.
Factors VIII and V are labile factors so storage beyond 4 hours causes falsely elevated APTT results
The presence of greater than 10,000 platelets/mL in plasma affect results as platelets release platelet factor 4 (PF4), a pro tein
that binds and neutralizes therapeutic heparin in vitro , falsely shorte ning the PTT and interfering with heparin management
Reagent
▪ Cephaloplastin reagent: a phospholipid preparation derived from rabbit brain (used as a platelet-membrane substitute) with a
factor XII activator (such as silica, ellagic acid or Kaolin)
The activator provides a surface that mediates a conformational change in plasma factor XII that results in its activation
▪ Calcium chloride: to reverse the anticoagulant effect of the citrate
The test is termed "partial" due to the absence of tissue factor from the reaction mixture.
Principle
In order to activate the intrinsic pathway, the contact depending factor XII activator, phospholipids and calcium chloride are mixed into
the plasma sample. The time is measured until a thrombus (clot) forms.
Manual test
1. Before use, the reagent should be mixed well by gentle swirling, do not shake.
2. Pre-incubate PTT reagent calcium chloride solution and plasma to 37 °C for at least 10
minutes before use.
3. To test tube, add 0.1 ml test plasma and 0.1 ml of factor XII activator
4. Shake tube briefly to mix the reagent and plasma, place tube at 37 °C for 3 minutes.
5. Add forcibly o.1 ml pre-warmed calcium chloride and simultaneously start stop watch.
6. Shake tube briefly to mix contents, keep at 37°C for 20 seconds.
7. Following 20 seconds incubation, remove the tube, gently tilt back and forth until a gel
clot forms, stop the watch and record the time.
Results
▪ The PTT result is reported in seconds as the time required f or

clot formation after the addition of CaCl2.
▪ PTT ratio can be calculated as follow: (PTT of patient plasma)/
(PTT of control)
Normal plasma in the PTT clots approximately within 24–37 s

upon triggering coagulation
Coagulation analyzer
Automated coagulation machines or Coagulometers measure the polymerization of f ibrin

monomers.
Detection techniques
Mechanical End-Point Detection
▪ This method uses a vibrating steel ball which is suspended in a reaction cup (plasma-
reagent solution).
▪ A magnetic sensor monitors the oscillation of a steel ball within the test plasma.
▪ As fibrin strands form, the viscosity starts to increase, slowing the movement.
▪ When the oscillation decreases to a predefined rate, the timer stops, indicating the
clotting time of the plasma
• Mechanical methods are not affected by icteric or lipemic plasma.

• Mechanical methods also provide a sensitive end-point able to detect weak clots such as those formed in plasmas with low
fibrinogen or a factor XIII deficiency where clots are not stabilized.
Photo-Optical End-Point Detection
▪ Photo-optical (turbidometric) coagulometers detect a change in plasma optical density (OD, light transmittance) during clotting.
Light of a specified wavelength passes through plasma, and its intensity (OD) is recorded by a photodetector.
▪ The OD depends on the color and clarity of the sample and is established as the baseline.
▪ Formation of fibrin strands causes light to scatter, allowing less light to fall on the photodetector, thus generating an increase in
OD.
▪ When the OD rises to a predetermined variance from baseline, the timer stops indicating clot formation.
• The lipemic plasma may interfere with the detection of fibrin clot formation by instruments measuring a change in optical
density. The change in optical density may be insufficient for detection.
Nephelometric End-Point Detection
▪ Nephelometry is a modification of photo-optical end-point detection in which 90-degree or forward-angle light scatter, rather than
OD, is measured.
▪ A light-emitting diode produces incident light at approximately 600 nm, and a photodetector detects variations in light scatter at 90
degrees (side scatter) and 180 degrees (forward-angle scatter).
▪ As fibrin polymers form, side scatter and forward-angle scatter rise.
▪ The timer stops when scatter reaches a predetermined intensity, and the interval is recorded.
Interpretation of coagulation profile
Condition BT CT PT PTT Platelets
Thrombocytosis Normal Normal Normal Normal Increased
Thrombocytopenia Prolonged Normal Normal Normal Decreased
Normal or
Von Willebrand disease Prolonged insensitive Normal Normal
Prolonged
Hemophilia (factor VIII or factor IX deficiency) Normal insensitive Normal Prolonged Normal
Intrinsic pathway deficiency

Normal insensitive Normal Prolonged Normal
factor VIII, IX, XI, or XII
Extrinsic pathway deficiency
Normal insensitive Prolonged Normal Normal
factor VII deficiency
Common pathway deficiency
Normal insensitive Prolonged Prolonged Normal
factor I, II, V, or X
FXIII deficiency
(a rare autosomal recessive disorder cause of a
Normal Normal Normal Normal Normal
hemorrhagic diathesis and may also predispose to
thrombosis)
Congenital thrombophilia Normal Normal Normal Normal Normal
Anti-phospholipids syndrome Normal Normal Normal Prolonged Normal
Disseminated intravascular coagulation Prolonged insensitive Prolonged Prolonged Decreased

Vitamin K deficiency Normal or
(due to an extremely poor diet, malabsorption , or Normal Normal Prolonged mildly Normal
prolonged use of antibiotics) prolonged
Liver failure (chronic) Normal Normal Prolonged Normal Normal
Liver failure (ESLD) Prolonged insensitive Prolonged Prolonged Decreased
Renal failure (uremia) Prolonged Normal Normal Normal Normal
Aspirin Prolonged Normal Normal Normal Normal

Normal or
Heparin Normal Prolonged mildly Prolonged Normal
prolonged
Normal or
warfarin Normal Prolonged Prolonged mildly Normal
prolonged
Bernard–Soulier syndrome
Normal or
(glycoprotein Ib deficiency, the receptor for von Prolonged Normal Normal Normal
Decreased
Willebrand factor lead to platelet adhesion defect)
Glanzmann's thrombasthenia
(glycoprotein IIb/IIIa deficiency, the receptor for Prolonged Normal Normal Normal Normal
fibrinogen lead to platelet aggregation defect)

33-Hemostasis and Coagulation Profile

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‫نقابة المهن العلمية بالمنوفية‬ [1]

emostasis and oagulation rofile

The initial vasoconstriction is mediated by:

▪ Reflex neurogenic mechanisms (initiated by local sympathetic pain

Platelet plug formation

▪ Von Willebrand Factor (vWF) is released from

When activated, platelets immediately undergo change.

Classification of Coagulation Factors

There are three groups in which coagulation factors can be classified:

Screening test if deficient

1 I Fibrinogen Prolonged PT and PTT

2 II Prothrombin Prolonged PT and PTT

3 III Thromboplastin (Tissue factor)

5 V Proaccelerin (Labile Factor) Prolonged PT and PTT

6 VI Nonexistent (was assigned to a procoagulant that later was

8 VIII Antihaemophilic factor A Prolonged PTT

9 IX Plasma Thromboplastin Component (Antihaemophilic factor B or Christmas factor) Prolonged PTT

10 X Stuart-Prower Prolonged PT and PTT

11 XI Plasma Thromboplastin Antecedent (Antihaemophilic factor C) Prolonged PTT

12 XII Hageman factor Prolonged PTT

13 XIII Fibrin Stabilizing Factor (Laki-Lorand factor) Normal PT and PTT

Prekallikrein (pre-K), also called Fletcher factor

high-molecular-weight kininogen (HMWK), also called Fitzgerald factor

Intrinsic pathways (Contact activation pathway)

Extrinsic pathway (Tissue factor pathway)

▪ FXa and its co-factor FVa form the prothrombinase complex.

Formation of fibrin occurs in three phases:

1.Proteolysis: The primary function of Protease enzyme

2.Polymerization: This occurs spontaneously due to fibrin

3.Stabilization: This occurs when factor XIIIa (activated by

Factor XIII deficiency can lead to impaired wound healing

Coagulation regulatory mechanisms

The principal regulators are:

Tissue factor pathway inhibitor (TFPI)

TFPI is a single-chain polypeptide protease inhibitor that is

▪ TFPI inhibits coagulation in a two-step process by first

Protein C regulatory system

Protein C is a vitamin K-dependent glycoprotein synthesized in the liver

The presence of Thrombin–thrombomodulin complex accelerates

Stabilized APC-protein S complex functions as anticoagulant by irreversibly

Antithrombin / heparin complex

Antithrombin (AT) is a protease glycoprotein inhibitor produced by the liver

The physiological target of antithrombin are those of the contact

Heparin is usually stored within the secretory granules of mast cells

Heparin binds to antithrombin, causing a conformational change

▪ Plasminogen: is a plasma zymogen produced by the liver

Fibrinolysis begins a few hours after fibrin polymerization and cross-linking.

Signs and symptoms

▪ If platelet counts are over 750,000 or 1,000,000

▪ Acute blood loss or post-surgery due to hemorrhage (Rebound thrombocytosis)

▪ Iron deficiency anemia associated with chronic blood loss

Signs and symptoms

▪ Platelet count drops <50,000

▪ Platelet count drops <20,000

At which point common symptoms include:

• Small red spots on the skin look like a rash (petechiae)

Decreased production of platelets

Immune thrombocytopenic purpura