HAEMATOLOGY: COAGULATION

Four major steps in the process of coagulation: 1. Reaction of the blood vessels 2. Formation of platelet plug 3. Coagulation Cascade 4. Fibrinolysis Once injury to the vessel wall takes place, the wall constricts. This slows down the blood flow and allows platelets to adhere to the vessel wall. The process of adhesion requires the presence of Von Willebrand Factor. ADP, thromboxane A2 and other substances are released by the platelets which stimulate aggregation. The aggregated platelets provide a surface on which coagulation takes place. Fibrin, the final product of the process consolidates the plug. Fibrin is digested again by enzymes such as plasminogen, of the fibrinolytic pathway. The Coagulation Cascade: http://en.wikipedia.org/wiki/Coagulation#The_coagulation_cascade SCREENING TESTS FOR HAEMOSTATIC DISORDERS 1. Patient history: Both clinical and family; 2. Physical examination: Clinical evidence of bleeding, eg bruising and petechiae; 3. Laboratory Tests. LABORATORY TESTS:

Platelet Count Peripheral smear morphology of platelets Bleeding Time: This test is an index of the early stages of clot formation which involves blood vessel contraction and platelet aggregation. It is usually normal in patients with coagulation disorders, ie disorders of the Coagulation Cascade. Normal: up to 9 minutes. Prothrombin time / INR: PT is usually prolonged in patients with deficiencies of the extrinsic coagulation pathway (tissue factor pathway) : Factors VII, X, V, prothrombin (II ) and fibrinogen. Normal: 10 12 seconds. INR is a ratio of standardization of PT. (Eg. Warfarin therapeutic monitoring) Activated Partial Thromboplastin Time (APTT): The APTT tests for abnormalities of the intrinsic coagulation pathway : Factors XII, XI, IX, and VIII. Heparin typically raises APTT, but rate varies with FVIII and fibrinogen levels and other factors. Normal: 26 40 seconds.

Mixture When the APTT is prolonged and a mixture of the patient's and normal plasma is tested, the time will return to normal. This is not the case if an inhibitor to any of the clotting factors is present in the patient's plasma. Fibrin Degradation Products (D-Dimers): The fibrin clot is dissolved by plasmin which cleaves a series of peptide bonds in fibrin, degrading the molecule into degredation products. An increase in FDP's or D-Dimers is an indication of fibrinolysis and is usually positive in disorders of rampant clotting like DIC. Specialised Tests: 1. Specific coagulation factor assays 2. Correction studies 3. Platelet function tests: Plt rich plasma of patient is exposed to a variety of agents which stimulate normal plts to aggregate. eg. ADP, collagen, adrenaline, ristocetin. The rate of aggregation is measured by an aggregometer. Measurement of Von Willebrand Factor: Immunoassay. Inhibitor Assay

DEFECTS IN HAEMOSTASIS 1. Bleeding Disorders: Vascular Defects Platelet Disorders Coagulation Defects 2. Thrombosis

PLATELET DISORDERS

Can be divided into 2 groups: Quantitative and Qualitative QUANTITATIVE DISORDERS 1. Failure of Production Aplastic Anaemia; Leukemia and other Bone Marrow Infiltrates; Lack of Thrombopoeitin. 2. Ineffective Production Megaloblastic Anaemia; Alcohol Induced Thrombocytopaenia; Myelodisplasia.

Diagnosis of the above-mentioned disorders: Platelet count ; Bone marrow biopsy. 3. Increased Destruction of Platelets In these cases the megakaryocyte and platelet production is increased to compensate for the platelet destruction taking place in the circulation. Causes include: Immune; DIC; Thrombotic Thrombocytopaenic Purpura. Immune Thrombocytopaenia Pathogenesis The platelets are coated by an auto antibody; usually IgG. This occurs by one of three mechanisms: 1. IgG binds to antigen on the platelet surface 2. IgG binds to a drug which is adsorbed on the platelet surface. 3. IgG in immune complexes binds to Fc receptors on platelet surface. The antibody coated platelets are then removed by the reticuloendothelial system causing thrombocytopaenia. This disorder can be secondary to conditions such as: CLL, HIV, Lymphoma, SLE. Drugs such as quinidine also can cause this condition. There may be no known cause: Idiopathic Thrombocytic Purpura. Idiopathic Thrombocytopaenic Purpura This disorder is often seen in children and young adults. It is thought that the autoantibody with the platelet glycoproteins GP11a / GP11b. Clinical Bruising, petechial rash, increased menstrual bleeding. Usually no history of precipitating event. Laboratory FBC: Low platelets Bone Marrow: Increased platelet production Increased megakaryocytes Mild erythroid hyperplasia if bleeding has taken place. Platelet IgG antibodies: Increased detection. APTT: Needs to be done to rule out Lupus Anticoagulant. Treatment Steroids: Suppress phagocyte activity and decrease binding of IgG. Splenectomy: Stops removal of platelets. Disseminated Intravascular Coagulation Pathogenesis This disorder is caused by the exposure of the circulating blood to procoagulant activity which initiates the coagulation process. This results in the formation of fibrin and the consumption of both clotting factors and platelets.

The consumption will eventually lead to severe bleeding. Causes 1. Severe infection especially gram negative bacteria. The endotoxin damages the vascular endothelium and activates Factor VII which could initiate coagulation. 2. Complications of pregnancy: Abruptio placenta release of tissue factor into circulation. Retained dead foetus intrauterine material and tissue factor is absorbed into circulation. Septic abortion. 3. Malignancy procoagulant tumor products gain access to circulation, eg. APL-M3 4. Other causes severe trauma, snake bite. Clinical Severe bleeding Hemorrhagic Tissue Necrosis: uncommon, but can occur due to occlusion of small vessels by fibrin. Microangiopathic Hemolytic Anemia. Laboratory Low platelet count. Blood Smear: Decreased platelets Increased red cell fragmentation, due to fibrin strands crossing the vessels causing a Microangiopathic anemia. Prothrombin Time / INR: Prolonged (consumption of clotting factors) APTT: Prolonged (consumption of clotting factors) Reduced plasma fibrinogen. A positive test for Fibrin Degradation Products or D-Dimers. ( Due to increased fibrinolysis ) Other tests: Decreased Antithrombin III Decreased clotting factors. Treatment Replace clotting factors with fresh frozen plasma. Prevent clotting: Heparin Treat underlying disorder. Thrombotic Thrombocytopaenic Purpura (TTP) TTP is an acute potentially fatal disorder of unknown cause. It more often affects women and is characterized by purpura and organ damage. Pathogenesis It is thought the plasma of TTP patients contain a factor which induces platelet aggregation. The platelet aggregates lodge themselves in the microcirculation and cause vessel wall damage and often organ damage. Clinical Fever Severe thrombocytopaenia CNS involvement Liver damage; jaundice Renal damage Cardiac damage Pancreatitis

Laboratory Decreased platelets Red cell fragmentation (microangiopathic anemia) PT / INR: Normal APTT: Normal Fibrinogen: initially normal but may fall later. Treatment Transfusion of large volumes of plasma. Increased Splenic Pooling When there is splenic enlargement of any cause there is an increased proportion of platelets pooled in the spleen resulting in thrombocytopaenia.

QUALITATIVE PLATELET (FUNCTION) DISORDERS Can be divided into: Primary: Hereditary and acquired. Secondary Platelet function disorders are characterized by: Normal platelet count Long bleeding time. The abnormality may be: Intrinsic eg. A missing glycoprotein Extrinsic ie. platelets are normal but cannot function due to outside factor. Primary Hereditary Platelet Disorders: Bernard Soulier Syndrome Pathogenesis Platelets fail to adhere to the sub endothelium due to a missing cell surface glycoprotein, GP1b. GP1b binds to Von Willebrand Factor, which in turn binds to the endothelium. Laboratory Mild to moderate thrombocytopaenia. Peripheral Smear: Giant Platelets Bleeding Time: Prolonged. Disorders of Platelet Secretion Pathogenesis These disorders are caused by one of the following: 1. Storage Pool disease insufficient ADP in the dense granules of the platelet. 2. Defective responsiveness to thromboxane A2 caused by impaired release of ADP. Clinical Usually mild bleeding Easy bruising, mild post op bleeding.

Laboratory Abnormal Platelet morphology Bleeding Time: Prolonged Impaired platelet aggregation when exposed to: collagen, adrenaline, low concentrations of ADP. Electron Microscopy: Reveals a number of dense granules. Therapy Bleeding is not severe and therefore therapy is not needed. Drugs that impair platelet function such as Asprin must be avoided. Thrombasthaenia (Glanzmans Disease) Autosomal recessive disorder. Pathogenesis Platelets are deficient in platelet associated fibrinogen and lack two glycoproteins: GPIIb and GPIIIa. Normal platelets form a calcium dependant GPIIb/IIIa complex to which fibrinogen, fibronectin and Von Willebrand Factor can bind. Failure of this binding results in defective hemostatic plug formation and a serious bleeding disorder. Laboratory Peripheral Smear: Platelets appear normal Bleeding Time: Prolonged Platelet Aggregation: Abnormal with all stimuli ADP, collagen, adrenaline, thrombin. Von Willebrands Disease (VWD) VWD is a common hereditary bleeding disorder. It is caused by an abnormality of the Von Willebrand Factor. It is autosomal dominant. The Von Willebrand Factor VWF is a glycoprotein It is synthesised by the endothelial cells and megakariocytes stored in alpha granules. Secreted from the endothelial cells and platelet alpha granules. VEF is secreted into the plasma as large multimers which become degraded as it circulates. Function of VWF is to: 1. Facilitate the adhesion of platelets to the subendothelium of the vessel wall. This function is performed by the large multimers. 2. Maintain normal levels of Factor VIII. Factor VIII circulates as a complex with VWF. Multimers of all sizes bind Factor VIII. It is thought that VWF serves as a 'carrier' for Factor VIII; stabilizing it; increasing its half life in the plasma. Tests for VWF 1. Concentration of the protein measured by electroimmunoassay. Plasma is subjected to electrophoresis through gel containing antibody to VWF. 2. The size of the different multimers can be determined by SDS agarose gel electrophoresis or crossed electrophoresis. 3. The function of VWF can be determined by: Bleeding Time Platelet aggregation Patients with VWD have abnormal aggregation with the stimulant

ristocetin. Ristocetin is an antibiotic which causes platelet aggregation in the presence of the intermediate multimers of VWF. 4. APTT: Prolonged 5. Factor VIII assay: Reduced. Types of Von Willebrands Disease Type I and Type II: Mild bleeding disorder. Patients can suffer from nose bleeds, increased menstrual bleeding and increased bleeding after minor surgery. Massive joint and tissue hemorrhage does not occur. Type I Characterized by a decreased plasma concentration of VWF. Laboratory Increased Bleeding Time Slightly prolonged APTT Decreased levels of Factor VIII Reduced VWF antigen Failure of platelets to aggregate with ristocetin. Type IIa Characterized by normal levels of VWF, yet platelets fail to aggregate with ristocetin. Reduced amounts of the large and intermediate multimers of VWF. Laboratory Increased Bleeding Time Abnormal aggregation with ristocetin Normal or slightly reduced VWF Absence of large and intermediate multimers Normal FVIII activity. Type IIb Characterized by increased sensitivity to ristocetin induced aggregation. The large multimers are decreased whereas the intermediate multimers are increased. The endothelial cells do secrete the large multimers, but these are abnormal and disappear rapidly. Laboratory Increased Bleeding Time Increased aggregation sensitivity to ristocetin Normal or slightly reduced VWF ag. Normal FVIII activity Absence of the large multimers. Type III This is a rare disorder, characterized by no measurable VWF. Clinical Severe bleeding disorder.

Skeletal and joint hemorrhage very similar to Hemophilia. Laboratory Increased Bleeding Time Reduced aggregation with ristocetin Reduced FVIII activity No measurable VWF. Pseudo Von Willebrands Disease Clinically this disorder resembles Type IIb disease. The platelets are abnormal and bind the large multimers of VWF, causing aggregation. These platelet/VWF complexes are removed from the circulation. So, Pseudo VWD feature abnormal platelets and normal VWF; whereas VWD is characterized by normal platelets and abnormal or low VWF. Treatment for VWD DDAVP: Increases VWF levels in the plasma for several hours. This treatment is useless in Type II disorders as the VWF is abnormal. Replacement therapy: Donor Plasma. DISTINGUISHING VWD FROM HEMOPHILIA VWD HEMOPHILIA BLEEDING TIME : FACTOR VIII: VON WILLEBRAND FACTOR Prolonged Moderately reduced Reduced Normal Severely reduced Normal

Acquired Platelet Function Disorders: Myeloproliferatives and Myelo Displastic Syndromes Platelet abnormalities include: Lack of membrane adrenergic receptors which causes abnormal adrenaline aggregation. Abnormalities of the dense granules causing depletion of ADP. Antibody Mediated Damage Antibodies coating the platelets in ITP and SLE cause damage leading to increased bleeding times and abnormal aggregation. Macroglobinaemia and Myeloma Increased concentration of plasma immunoglobulins coating the platelets and interferes with aggregation. Drugs Aspirin: Aspirin acts on the prostaglandin synthetic pathway, which can result in defective ADP release. Patients on aspirin have an increased bleeding time and defective platelet aggregation.

Uraemia Accumulation of waste products interferes with platelet function. This is important in Renal disease.

COAGULATION DISORDERS COAGULATION DISORDERS COAGULATION FACTORS All coagulation factors apart from factor VIII are synthesized in the liver. Factors II ( prothrombin ), VII, IX, X are dependent on vitamin K for synthesis. Factors XIII, V, XI, XII are synthesized independently of vitamin K. http://en.wikipedia.org/wiki/Coagulation HEREDITARY COAGULATION DISORDERS HAEMOPHILIA A AND B Hemophilia A: Factor VIII deficiency Hemophilia B: Factor IX deficiency Both disorders have similar clinical symptoms and genetic inheritance. In order to differentiate between them, each specific coagulation factor needs to be measured. 85 % of Hemophilia patients are factor VIII deficient while 15 % have a factor IX deficiency. Genetic Inheritance Genetic transmission is via the X chromosome (female sex chromosome). It is a defective recessive X-linked single gene; it's X-allele usually being dominant normal. Thus, females ( XX ) are carriers but usually not affected by the disease. Men ( XY ) receiving the defective gene, have no counter allele; thus are affected by the disease, while not being able to pass it on. http://en.wikipedia.org/wiki/Hemophilia Three types: Severe: < 1 % Factor VIII or IX Moderate: 1 5 % Factor activity Mild: 5 30 % Factor activity

Clinical symptoms Patients bleed throughout life. They often bleed into joints and muscles causing haematomas and haemarthroses. The first major bleed usually occurs before 18 months of age. Mild type: easy bruising; moderate bleeding post trauma or surgery. Laboratory diagnosis APTT: Prolonged. Both factors are part of the intrinsic pathway. PT / INR: Normal Bleeding Time: Normal Factor VIII and IX assays need to be done to establish the diagnosis. If the Factor VIII assay shows deficient, it may be necessary to exclude a diagnosis of Von Willebrands Disease. Inhibitors:Approximately 15 % of Factor VIII deficient patients develop inhibitors to factor VIII. In these cases the mix (patient plasma + control plasma) fails to correct the prolonged APTT. An inhibitor screen can be performed to quantitate the inhibitor. Treatment Plasma products, cryoprecipitate, lyophilised factor concentrates. The danger in using plasma concentrates is the transmission of viruses, eg. HIV and Hepatitis. The risk has been reduced by: testing all donors; heat treatment of concentrates. Detection of carriers Carriers of Hemophilia can be detected by the calculated ratio between their Factor VIII coagulation activity and Von Willebrand Factor. Normal: 1.0 Carriers: < 1.5 Molecular techniques are now being used. Detection of the disease and carriers can be made by analyzing amniotic fluid. FACTOR XI DEFICIENCY This disorder is less common than Hemophilia and is predominant in patients of Ashkenazic Jewish descent. It is autosomal recessive. Clinical findings Mild bleeding. Post operative bleeding can be a problem. Laboratory diagnosis APTT: Prolonged Factor XI assay: Decreased Treatment Prophylactic treatment with fresh frozen plasma.

FACTOR XII DEFICIENCY (PREKALLIKREIN DEFICIENCY ) Usually discovered incidentally and does not impair in vivo coagulation. The APTT is prolonged Factor XII assay is decreased. FACTOR DEFICIENCIES PROLONGING THE PT / INR Factors X, V, and II (prothrombin) prolong both the APTT and PT / INR. Factor VII prolongs only the PT / INR. Diagnosis is made by doing specific assays. FACTOR VII DEFICIENCY Disorder with minimal or mild bleeding. If factor levels drop below 3 % serious bleeding can occur. FACTOR V DEFICIENCY Patients have bleeding of varying severity. If platelet Factor V levels are also low, serious bleeding can occur. FACTOR X AND PROTHROMBIN DEFICIENCY This disorder can have serious bleeding episodes. DISORDERS OF FIBRINOGEN AFIBRINOGENAEMIA Disorder extremely rare and is autosomal recessive. Blood fails to clot with all screening tests and only traces of fibrinogen are detected. Patients have little spontaneous bleed can bleed after surgery. DISFIBRINOGENAEMIA This disorder is autosomal dominant and is extremely rare. The plasma from patients with this disorder consists of both normal and abnormal fibrinogen. Patients may have symptoms of bleeding but can also suffer from thrombosis. FACTOR XIII DEFICIENCY Clinical Bleeding from the umbilical stump. Poor wound healing. Spontaneous abortions. Laboratory All coagulation tests are normal. If the plasma clot is incubated overnight in 5 M urea solution, the clot will dissolve if Factor XIII deficiency exists.

ACQUIRED COAGULATION DISORDERS VITAMIN K DEFICIENCY Vitamin K is needed for the synthesis of the following clotting factors: Factor II ( prothrombin ) Factor VII Factor IX Factor X 2 mcg. of vit. K is needed per kilogram of body weight daily. It come sfrom two sources: diet and bacterial synthesis of vit. K in the intestine. Patients with vit. K deficiency develop a bleeding tendency within 10 days. Warfarin is a vitamin K antagonist. Laboratory features PT / INR: Always prolonged. Factor VII has the shortest half life and is therefore the first factor to be affected. APTT: Normal to prolonged. Treatment Vitamin K. To monitor response repeat the INR and APTT 24 hours later. LIVER DISEASE Liver disease may cause bleeding problems because of: 1. Impaired synthesis of clotting factors. All clotting factors except Factor VIII are synthesized in hepatic parenchymal cells. 2. Increased fibrinolysis: alpha 2 antiplasmin, the primary inhibitor of plasmin is also synthesized in the liver. (plasmin is a clotting regulator which breaks down it's products.) 3. Congestive splenomegaly can occur and therefore thrombocytopaenia can develop. 4. Disseminated intravascular coagulation (DIC) may develop. Laboratory diagnosis INR / PT: Prolonged APTT: Prolonged Fibrinogen: Reduced Fibrin Degradation Products (D dimers): Positive in DIC Therapy Vitamin K Fresh frozen plasma Cryoprecipitate

ANTIBODIES TO FACTORS These can develop in patients with hereditary clotting factor deficiencies. The patient's immune system reacts to administered clotting factors as though they were foreign. Antibodies can also develop in autoimmune disorder such as SLE. Laboratory diagnosis A mixture of normal and patient's plasma fails to correct the APTT. Factor VIII antibodies Can be seen in: Hemophilia Post partum women Patients with immune disorders such as rheumatoid arthritis. Laboratory The lab. findings resemble those found in Hemophilia A. There is a prolonged mixture and a high titre of Factor VIII antibody. Treatment Immunosuppressive agents in patients other than those with hemophilia. The Lupus Anticoagulant 5 -10 % of SLE (systemic lupus erythrematous) patients develop plasma antibodies which inhibit the phospholipid used in the APTT test. Patients with this disorder often have reduced platelets. This antibody is not only found in patients with SLE but also other diseases such as: Immune disorders Patients on certain drugs such as quinine. Clinical features Patients do not bleed abnormally but often have increased thrombosis. It has been associated with spontaneous abortions. Laboratory features APTT: Prolonged and does not correct with the mixture. PT / INR: Normal or slightly prolonged. Special tests: By adding a synthetic phospholipid mixture to the APTT system, the time can be corrected. This is the Kaolin Clotting Time. THROMBOSIS Thrombosis occurs when there is a breakdown in the balance between thrombogenic factors and protective mechanisms. This results in an abnormal mass which can lead to vascular occlusion. Thrombogenic factors include: Damage to the vessel wall Stimulation of platelet aggregation Activation of blood coagulation.

Failure of protective mechanisms: Deficiency of natural coagulation inhibitors: Antithrombin III, Protein S and C Reduced hepatic clearance of activated clotting factors. Defective fibrinolysis. Thrombosis usually occurs in vessels where the normal blood flow has been disturbed. These can be veins (Deep vein thrombosis DVT) or artery (Arterial thrombosis). There are variety of causes: Protein C deficiency Protein C is vitamin K dependant and is synthesized in the liver. It inhibits the active forms of Factor V and Factor VIII. Activation of Protein C occurs via thrombin bound to a protein called thrombomodulin. Clinical features Patients have recurrent venous thromboses from an early age. Diagnosis Protein C assay: Reduced Protein S deficiency The anticoagulant activity of protein C requires a co-factor which is Protein S. Prot. S is also vitamin K dependant. Clinically patients suffer from thromboses from an early age. Diagnosis is made by measuring the amount of Protein S. Antithrombin III deficiency Antithrombin III is a potent inhibitor of factors XII, XI, X, IX, VIII. Deficiencies can lead to recurrent thrombosis. Activated Protein C Resistance Protein C acts on Factor V and inhibits its function. If Factor V is resistant to this action, patients have an increased risk of thrombosis. This disorder is known as APC resistance or Factor V Leiden. Diagnosis is made by detecting the abnormality at molecular level using PCR. This disorder is thought to be a major cause of thrombosis in young adults. TREATMENT OF THROMBOSIS 1. Anti thrombotic therapy: Aim is to induce fibrinolysis and dissolve the clot. Examples: Streptokinase, Urokinase 2. Anti coagulants: This is the first line of therapy. Anticoagulants can be used to: Treat a current thrombosis, Prevent further thrombosis Anticoagulant therapy is monitored by the INR and APTT. The INR is used to monitor warfarin therapy: Therapeutic range is 2.0 4.0 The APTT is used to monitor Heparin therapy.

BLEEDING DISORDERS

VASCULAR DEFECTS Vascular disorders are a heterogenous group of conditions which are characterized by bruising and spontaneous vessel bleeding. The abnormality is caused either by damage to: A) The vessels themselves B) The perivascular connective tissue. Clinical Symptoms are not severe. Bleeding is usually into the skin; can also be from mucous membranes. Laboratory tests Main screening test is Bleeding Time, which can often be normal. INHERITED VASCULAR DISORDERS Inherited Telangiectasia Pathogenesis The abnormality lies in the sub-endothelial connective tissue which results in abnormally dilated blood vessels. Clinical Telangiectasia appears in both the skin and mucous membranes. Bleeding may be from the gastrointestinal tract. Anemia may develop due to blood loss. Laboratory Bleeding Time: Normal Other Inherited Vascular Disorders are extremely rare and consist of: Ehlers Danlos Syndrome Osteogenesis imperfecta Pseudoxanthoma elasticum These are all characterized by vascular fragility. ACQUIRED VASCULAR DISORDERS Simple Easy Bruising ( Purpura Simplex ) This disorder occurs mainly in women. The cause is unknown but an increase in vessel fragility is thought to be responsible.

Clinical Recurrence of unexplained bruises. Diagnosis Made on clinical features. Other bleeding disorders need to be ruled out. Senile Purpura Occurs commonly in elderly people. The histology of the affected skin shows marked atrophy of collagen. Purpura Post Infection and Drugs The Purpura is thought to be caused by toxic damage to the vascular endothelium. A number of drugs have been implicated. The Purpura clears after removal of the drug. Other rare acquired vascular defects: Hanloch Schonlein Syndrome: ? Hypersensitivity. Scurvy: Vitamin C is necessary for collagen formation. Notes are from Cape Pen. University of Technology (Fmr. Cape Technikon), 2002, ND., B.Tech.: Biomedical Technology. More Course notes at : http://www.scribd.com/document_collections/2374607

Formula for INR: Patient PT MNPT (Mean PT for 10 males and 10 females with normal coagulation)
ISI

= PT Ratio (PR)

INR = PR

Coagulation Cascade: