Lab Diagnosis Of Thrombosis And Thrombophilia PDF

Lab Diagnosis Of Thrombosis And Thrombophilia PhD. MD. Marsela Haruni Medicine Learning Objectives Understanding the: Mechanisms Lab Diagnosis Management of thrombosis and thrombophilia Medicine  Definitions Thrombosis: Formation of a blood clo...

Lab Diagnosis Of Thrombosis And Thrombophilia PhD. MD. Marsela Haruni Medicine Learning Objectives Understanding the: Mechanisms Lab Diagnosis Management of thrombosis and thrombophilia Medicine  Definitions Thrombosis: Formation of a blood clot inside a blood vessel, obstructing the flow of blood. Medicine  Thrombophilia: A condition that increases the risk of blood clots forming in blood vessels. Medicine  Mechanisms of Blood Clotting 1. The Coagulation Cascade o Explanation of the coagulation cascade and the role of zymogen activations. o Intrinsic and extrinsic pathways leading to the common pathway. o Formation of fibrin clot. 2. Role of Platelets o How platelets contribute to clot formation. o Interaction between platelets and coagulation factors. 3. Regulation of Coagulation o Natural anticoagulants (Protein C, Protein S, Antithrombin). o Fibrinolysis: the process of breaking down clots. Medicine  Because of its fluid nature, blood flows freely throughout the circulatory system. However, if there is a break in the “piping” of the system, blood will be lost unless steps are taken. One of the challenges for the body is to plug holes in damaged blood vessels while still maintaining blood flow through the vessel. This challenge is complicated by the fact that blood in the system is under pressure. If the repair “patch” is too weak, it is blown out by the blood pressure. For this reason, stopping blood loss involves several steps. First, the pressure in the vessel must be decreased long enough to create a secure mechanical seal in the form of a blood clot. Once the clot is in place and blood loss has been stopped, the body’s repair mechanisms can take over. Then, as the wound heals, enzymes gradually dissolve the clot while scavenger leukocytes ingest and destroy the debris. Medicine  Medicine Hemostasis has three major steps: 1. Vasoconstriction The first step in hemostasis is immediate constriction of damaged vessels to decrease blood flow and pressure within the vessel temporarily, decreasing the flow within the damaged vessel. 2. Temporary blockage of a break by a platelet plug Plug formation begins with platelet adhesion, when platelets adhere or stick to exposed collagen in the damaged area. The adhered platelets become activated, releasing cytokines into the area around the injury. These platelet factors reinforce local vasoconstriction and activate more platelets, which aggregate or stick to one another to form a loose platelet plug. Medicine 3. Coagulation, the formation of a clot that seals the hole until tissues are repaired. Simultaneously, exposed collagen and tissue factor (a protein-phospholipid mixture) initiate the third step, the formation of a fibrin protein mesh that stabilizes the platelet plug to form a clot. Fibrin is the end product of a series of enzymatic reactions known as the coagulation cascade. Eventually, as the damaged vessel repairs itself, the clot retracts when fibrin is slowly dissolved by the enzyme plasmin. The body must maintain the proper balance during hemostasis. Too little hemostasis allows excessive bleeding; too much creates a thrombus, a blood clot that adheres to the undamaged wall of a blood vessel {thrombose, a clot or lump}. Medicine  A large thrombus can block the lumen of the vessel and stop blood flow. Inappropriate blood clotting plays an important role in strokes and heart attacks. Inherited mutations affecting platelet function can lead to inappropriate clotting or excessive bleeding due to failure of hemostasis. Medicine The coagulation cascade is a complex series of events that leads to blood clot formation, crucial for stopping bleeding and initiating healing. It consists of two main pathways: THE INTRINSIC PATHWAY and the EXTRINSIC PATHWAY, both of which converge into the COMMON PATHWAY. Zymogens: Most coagulation factors are produced in an inactive form known as zymogens. These factors require activation to become functional enzymes that catalyze the next steps in the cascade. Intrinsic Pathway: Triggered by damage to blood vessels, this pathway involves several zymogens (factors XII, XI, IX, and VIII) that activate each other through a series of enzymatic reactions. The cascade amplifies the signal, leading to the activation of factor X. Extrinsic Pathway: Initiated by external trauma that exposes tissue factor (TF) at the site of injury. TF binds to factor VII, activating it. The TF-VIIa complex then activates factor X directly. Common Pathway: Once factor X is activated (Xa), it combines with factor V to form prothrombinase, which converts prothrombin (factor II) into thrombin (factor IIa). Thrombin is a key player that not only converts fibrinogen to fibrin (the main component of a clot) but also activates other factors (like V, VIII, XI) to amplify the coagulation process. Medicine Role of Zymogen Activation Sequential Activation: Each zymogen activation is a crucial step that amplifies the response. For example, one activated factor can activate multiple molecules of the next factor in the cascade, leading to a rapid response to injury. Regulation: The activation of zymogens is tightly regulated to prevent excessive clotting. Natural inhibitors (like antithrombin and protein C) help control the cascade. Clinical Relevance: Understanding zymogen activation helps in diagnosing and treating coagulation disorders, such as hemophilia, where specific zymogens (like factor VIII) are deficient or dysfunctional. The coagulation cascade is a finely-tuned system that relies on zymogen activation to ensure a rapid and effective response to vascular injury, leading to the formation of a stable blood clot while being tightly regulated to prevent pathological clotting. Medicine Medicine Medicine Medicine  Platelet production Platelets are produced in the bone marrow by fragmentation of the cytoplasm of megakaryocytes, one of the largest cells in the body. The precursor of the megakaryocyte – the megakaryoblast – arises by a process of differentiation from the haemopoietic stem cell. The megakaryocyte matures by endomitotic synchronous replication, enlarging the cytoplasmic volume as the number of nuclear lobes increases in multiples of two. Thrombopoietin (TPO) is the major regulator of platelet formation and 95% is produced by the liver. Medicine  Early on invaginations of plasma membrane are seen. At a variable stage in development the cytoplasm becomes granular. Mature megakaryocytes are extremely large, with an eccentrically placed single lobulated nucleus and a low nuclear : cytoplasmic ratio. Platelets form by fragmentation from the tips of cytoplasmic extensions of megakaryocyte cytoplasm, each megakaryocyte giving rise approximately to 1000–5000 platelets. The time interval from differentiation of Megakaryocytes: (a) immature form with basophilic the human stem cell to the production of cytoplasm; (b) mature form with many nuclear lobes and platelets averages 10 days. pronounced granulation of the cytoplasm. Medicine  Platelet Structure Platelets are extremely small and discoid, 3.0 × 0.5 μm in diameter. The glycoproteins of the surface coat are particularly important in the platelet reactions of adhesion and aggregation, which are the initial events leading to platelet plug formation during hemostasis. Adhesion to collagen is facilitated by glycoprotein Ia (GPIa). Glycoproteins Ib (defective in Bernard–Soulier syndrome) and IIb/IIIa (also called αIIb and β3, defective in Glanzmann’s thrombasthenia) are important in the attachment of platelets to von Willebrand factor (VWF) and hence to vascular sub endothelium. The binding site for IIb/IIIa is also the receptor for fibrinogen which, like VWF, is important in platelet–platelet aggregation. Medicine  The membrane phospholipids (previously known as platelet factor 3) are of particular importance in the conversion of coagulation factor X to Xa and prothrombin (factor II) to thrombin (factor IIa). A deficiency in membrane phospholipids disrupts the assembly of coagulation factors on the platelet surface, which is essential for effective coagulation. This can lead to a slower or inadequate clotting response. Laboratory tests measuring coagulation, such as prothrombin time (PT) and activated partial thromboplastin time (APTT), may show prolonged results, indicating a reduced ability to coagulate effectively. Medicine The main function of platelets is the formation of mechanical plugs during the hemostatic response to vascular injury. In the absence of platelets, spontaneous leakage of blood through small vessels may occur. There are three major platelet functions: Adhesion Aggregation Release reactions and amplification The immobilization of platelets at the sites of vascular injury requires: specific platelet–vessel wall (adhesion) and platelet–platelet (aggregation) interactions, both partly mediated through VWF. Medicine Platelets are produced from megakaryocytes in the bone marrow stimulated by thrombopoietin. They have surface glycoproteins which facilitate direct adherence to subendothelial tissues and also, via von Willebrand factor, to collagen, to other platelets (aggregation) and to fibrinogen. Platelets contain different types of storage granules which are released after platelet activation. Medicine  Von Willebrand factor (VWF) VWF is involved in shear-dependent platelet adhesion to the vessel wall and to other platelets (aggregation). It also carries factor VIII. It is a large glycoprotein, with multimers made up on average of 2–50 dimeric subunits. VWF is synthesized both in endothelial cells and megakaryocytes. Medicine Plasma VWF is almost entirely derived from endothelial cells, with two distinct pathways of secretion. The majority is continuously secreted, and a minority is stored in Weibel Palade bodies. The stored VWF can raise the plasma levels when released under the influence of several secretagogues, such as stress, exercise, adrenaline and infusion of desmopressin. The VWF released from Weibel–Palade bodies is in the form of large and ultra-large multimers, the most adhesive and reactive form of VWF. They are in turn cleaved in plasma to smaller multimers and monomeric VWF by the specific plasma metalloprotease, ADAMTS13. Medicine Regulation of Coagulation o Natural anticoagulants (Protein C, Protein S, Antithrombin). o Fibrinolysis: the process of breaking down clots. o It is important that the effect of thrombin is limited to the site of injury. Medicine  Coagulation Factor Inhibitors The first inhibitor to act is TFPI (Tissue Factor Pathway Inhibitor), which is synthesized in endothelial cells and is present in plasma and platelets and accumulates at the site of injury caused by local platelet activation. TFPI inhibits Xa and VIIa and tissue factor to limit the main in vivo pathway. There is also direct inactivation of thrombin and other serine protease factors by other circulating inhibitors, of which antithrombin is the most potent. It inactivates serine proteases. Heparin potentiates its action markedly. Another protein, heparin cofactor II, also inhibits thrombin. α2-Macroglobulins, α2-antiplasmin, C1 esterase Medicine inhibitor and α1 antitrypsin also exert inhibitory effects on circulating serine proteases.  PROTEIN C AND PROTEIN S These are inhibitors of coagulation cofactors V and VIII. Thrombin binds to an endothelial cell surface receptor, thrombomodulin. The resulting complex activates the vitamin K dependent serine protease, protein C, which is able to destroy activated factors V and VIII, thus preventing further thrombin generation. The action of protein C is enhanced by another vitamin K-dependent protein, S, which binds protein C to the platelet surface. An endothelial protein C receptor localizes protein C to the endothelial surface, promoting protein C activation by the thrombin–thrombomodulin complex. In addition, activated protein C enhances fibrinolysis. As with other serine proteases, activated protein C is subject to inactivation by serum protease inactivators (serpins), e.g. antithrombin. Medicine Medicine  Fibrinolysis Fibrinolysis (like coagulation) is a normal hemostatic response to vascular injury. Plasminogen, a proenzyme in blood and tissue fluid, is converted to the serine protease plasmin by activators either from the vessel wall (intrinsic activation) or from the tissues (extrinsic activation). The most important route follows the release of tissue plasminogen activator (TPA) from endothelial cells. TPA is a serine protease that binds to fibrin. This enhances its capacity to convert thrombus-bound plasminogen into plasmin. This fibrin dependence of TPA action strongly localizes plasmin generation by TPA to the fibrin clot. Release of TPA occurs after such stimuli as trauma, exercise or emotional stress. Medicine Medicine Activated protein C stimulates fibrinolysis by destroying plasma inhibitors of TPA. Plasmin generation at the site of injury limits the extent of the evolving thrombus. The split products of fibrinolysis are also competitive inhibitors of thrombin and fibrin polymerization. Normally, α2-antiplasmin inhibits any local free plasmin. Fibrinolytic agents are widely used in clinical practice (streptokinase, and urokinase). Inactivation of plasmin Tissue plasminogen activator is inactivated by plasminogen activator inhibitor (PAI). Circulating plasmin is inactivated by the potent inhibitors α2-antiplasmin and α2-macroglobulin. Medicine  SCREENING TESTS OF BLOOD COAGULATION Screening tests provide an assessment of the ‘extrinsic’ and ‘intrinsic’ systems of blood coagulation and also the central conversion of fibrinogen to fibrin. The Prothrombin Time (PT) The prothrombin time (PT) measures factors VII, X, V, prothrombin and fibrinogen. Tissue thromboplastin (a brain extract) or [synthetic] tissue factor with lipids and calcium is added to citrated plasma. The normal time for clotting is 10–14 s. It may be expressed as the international normalized ratio (INR). The effect of oral anticoagulants is monitored by the PT. The INR is based on the ratio of the patient’s prothrombin time (PT) to a mean normal PT with correction for the ‘sensitivity’ of the thromboplastin used. This is calibrated against a primary World Health Organization (WHO) standard thromboplastin. Medicine  The activated partial thromboplastin time (APTT) The activated partial thromboplastin time (APTT) measures factors VIII, IX, XI and XII in addition to factors X, V, prothrombin and fibrinogen. Three substances – phospholipid, a surface activator (e.g. kaolin) and calcium – are added to citrated plasma. The normal time for clotting is approximately 30–40 s. Prolonged clotting times in the PT and APTT because of factor deficiency are corrected by the addition of normal plasma to the test plasma (50 : 50 mix). If there is no correction or incomplete correction with normal plasma, the presence of an inhibitor of coagulation is suspected. Medicine  Specific Assays Of Coagulation Factors Most factor assays are based on an APTT or PT in which all factors except the one to be measured are present in the substrate plasma. This usually requires a supply of plasma from patients with hereditary deficiency of the factor in question or artificially produced factor-deficient plasma. The corrective effect of the unknown plasma on the prolonged clotting time of the deficient substrate plasma is then compared with the corrective effect of normal plasma. Results are expressed as a percentage of normal activity. A number of chemical, chromogenic and immunological methods are available for quantification of other proteins such as fibrinogen, VWF, factor Xa and factor VIII. Medicine  BLEEDING TIME The bleeding time is not a reliable assessment of platelet function as it is insensitive and has poor reproducibility. It was used to identify abnormal platelet function, including the diagnosis of VWF deficiency, but is no longer used in routine clinical practice. It has been replaced by specific platelet aggregation tests, platelet adhesion assays and the platelet function analysis-100 (PFA-100) test(Conventional platelet aggregometry). The bleeding time is prolonged in thrombocytopenia but is normal in vascular causes of abnormal bleeding. Medicine  HEREDITARY COAGULATION DISORDERS Hereditary deficiencies of each of the coagulation factors have been described. Hemophilia A (factor VIII deficiency) Hemophilia B (Christmas disease, factor IX deficiency) Von Willebrand disease (VWD) These are the most frequent; the others are rarer. Medicine  HEMOPHILIA A Hemophilia A is the most common of the hereditary clotting factor deficiencies. The defect is an absence or low level of plasma factor VIII. Approximately half of the patients have missense or frameshift mutations or deletions in the factor VIII gene. The prevalence is of the order of 30–100 per million population. The inheritance is sex-linked, but up to one-third of patients have no family history and these cases result from recent mutation. The following tests are abnormal: 1 Activated partial thromboplastin time (APTT). 2 Factor VIII clotting assay. The platelet function analysis-100 (PFA-100) and pro thrombin time (PT) are normal. Medicine  CLINICAL FEATURES Infants may develop joint and soft tissue bleeds and excessive bruising when they start to be active. Recurrent painful hemarthroses and muscle hematomas dominate the clinical course of severely affected patients and, if inadequately treated, lead to progressive joint deformity and disability. Medicine INHIBITORS One of the most serious complications of hemophilia is the development of antibodies (inhibitors) to infused factor VIII which occurs in 30–40% of severely effected patients, usually within the first 50 days of exposure. This renders the patient refractory to further replacement therapy. Immunosuppression and immune tolerance regimens have been used in an attempt to eradicate the antibody with success (at great cost) in about two-thirds of cases. Medicine  FACTOR IX DEFICIENCY (HEMOPHILIA B, CHRISTMAS DISEASE) The inheritance and clinical features of factor IX deficiency (Christmas disease, hemophilia B) are identical to those of hemophilia A. Indeed, the two disorders can only be distinguished by specific coagulation factor assays. The incidence is one-fifth that of hemophilia A. Factor IX is coded by a gene close to the gene for factor VIII near the tip of the long arm of the X chromosome. Its synthesis is vitamin K-dependent. Carrier detection and antenatal diagnosis are performed as for hemophilia A. The principles of replacement therapy are similar to those of hemophilia A. Bleeding episodes are treated with high-purity factor IX concentrates. Because of its longer biological half-life, infusions do not have to be given as frequently as do factor VIII concentrates in hemophilia A. Medicine  Laboratory findings The following tests are abnormal: 1. APTT 2. Factor IX clotting assay. As in hemophilia A, the PFA-100 and PT tests are normal. Medicine  VON WILLEBRAND DISEASE In this disorder there is either a reduced level or abnormal function of von Willebrand factor (VWF) resulting from a wide variety of mainly missense mutations in different parts of the gene. VWF is produced in endothelial cells and megakaryocytes. It has two roles. It promotes platelet adhesion to sub endothelium and each other at high shear rates and it is the carrier molecule for factor VIII, protecting it from premature destruction. The latter property explains the reduced factor VIII levels found in VWD. VWF has a half-life in plasma of about 16 hours. Chronic elevation of VWF is part of the acute phase response to injury, inflammation, neoplasia or pregnancy. Medicine VWD is the most common inherited bleeding disorder. Usually, the inheritance is autosomal dominant. The severity of the bleeding is highly variable, depending on mutation type and epistatic genetic effect, such as ABO blood group. Women are more badly affected than men at a given VWF level. Typically, there is mucous membrane bleeding (e.g. epistaxis, menorrhagia), excessive blood loss from superficial cuts and abrasions, and operative and post-traumatic hemorrhage. The severity is variable in the different types. Hemarthroses and muscle hematomas are rare, except in type 3 disease. Medicine  LABORATORY FINDINGS 1. The PFA-100 test is abnormal. This has largely replaced the bleeding time test. 2. Factor VIII levels are often low. If low, a factor VIII/VWF binding assay is performed. 3. The APTT may be prolonged. 4. VWF levels are usually low. 5. There is defective platelet aggregation by patient plasma in the presence of ristocetin (VWF: Rco). Aggregation to other agents (adenosine diphosphate (ADP), thrombin or adrenaline) is usually normal. 6. Collagen-binding function (VWF: CB) is usually reduced (but rarely measured). 7. Multimer analysis is useful for diagnosing different subtypes. 8. The platelet count is normal except for type 2B disease (where it is low) Medicine  THROMBOSIS Thrombosis is the formation of solid masses of platelets and fibrin in the circulation. It may be arterial or venous. Arterial thrombosis is mainly related to atherosclerosis of the vessel wall with risk factors such as hypertension, hyperlipidemia, smoking and diabetes. Venous thrombosis is related to genetic coagulation factor abnormalities (e.g. factor V Leiden), stasis of the circulation or to an acquired increase in coagulation factors (e.g. estrogen therapy, postoperative, pregnancy) or to unknown factors (e.g. age or obesity). Medicine  ARTERIAL THROMBOSIS Pathogenesis Atherosclerosis of the arterial wall, plaque rupture and endothelial injury expose blood to subendothelial collagen and tissue factor. This initiates the formation of a platelet nidus on which platelets adhere and aggregate. Platelet deposition and thrombus formation are important in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) stimulates the migration and proliferation of smooth muscle cells and fibroblasts in the arterial intima. Regrowth of endothelium and repair at the site of arterial damage results in thickening of the vessel wall. The intrinsic pathway of fibrin formation is involved in pathological thrombosis in vivo by contact activation on damaged blood vessels. Medicine CLINICAL RISK FACTORS As well as blocking arteries locally, emboli of platelets and fibrin may break away from the primary thrombus to occlude distal arteries. Examples are carotid artery thrombi leading to cerebral thrombosis and transient ischemic attacks, and heart valve and chamber thrombi leading to systemic emboli and infarcts. Medicine  Venous thrombosis Pathogenesis and risk factors Virchow’s triad suggests that there are three components that are important in thrombus formation: 1. Slowing down of blood flow; 2. Hypercoagulability of the blood; 3. Vessel wall damage. For venous thrombosis, increased systemic coagulability and stasis are most important, with vessel wall damage being somewhat less important than in arterial thrombosis. Medicine Medicine  HEREDITARY DISORDERS OF HEMOSTASIS The prevalence of inherited disorders associated with increased risk of thrombosis is higher than that of hereditary bleeding disorders. Approximately one third of patients who suffer deep vein thrombosis (DVT) or pulmonary embolus (PE) have an identifiable heritable risk factor: rare deficiencies of antithrombin, protein C or protein S, or common mutations affecting factor V (factor V Leiden) or prothrombin. Medicine  FACTOR V LEIDEN GENE MUTATION This is the most common inherited cause of an increased risk of venous thrombosis. It occurs in approximately 3–7% of factor V alleles. There is failure of activated protein C (APC) to prolong the activated partial thromboplastin time (APTT) test when added to plasma and the phenotype is sometimes referred to as ‘activated protein C resistance’. Activated protein C normally breaks down activated factor V and so it should slow the clotting reaction and prolong the APTT. APC resistance is caused by a genetic polymorphism in the factor V gene, which makes factor V less susceptible to cleavage by APC. Medicine GENOTYPE INTERPRETATION Factor V Leiden: Patients who are heterozygous for factor V Leiden are at an approximately five- to eight-fold increased risk of venous thrombosis compared to the general population, but only 10% of carriers develop thrombosis during their lifetime. Individuals who are homozygous have a 30–140-fold risk. Also, this mutation is associated with the serious condition of preeclampsia. The risk of thrombosis in heterozygous people is 1 in 500 pregnancies. The incidence of factor V Leiden in patients with venous thrombosis is approximately 20–40%. Genetic screening is relatively easy and is widely performed. However, even if a patient with DVT is a carrier of factor V Leiden their absolute risk of thrombosis is still very low in the absence of other risk factors. At present it is not recommended to start anticoagulation therapy in individuals with the Leiden mutation, even if homozygous, if they have no history of thrombosis. Medicine  ANTITHROMBIN DEFICIENCY Antithrombin III (ATIII) is an important protein in the regulation of blood coagulation. A lab test for antithrombin III measures its level in the blood and can help diagnose various conditions related to abnormal clotting. Interpretation of Results Typically range from 80% to 120% of the normal value, depending on the lab. Low Levels: May indicate: 1. Antithrombin deficiency (either inherited or acquired). 2. Liver disease, since ATIII is produced in the liver. 3. Consumption due to extensive clotting (such as in disseminated intravascular coagulation - DIC). High Levels: Less common but can occur in certain inflammatory conditions. Inheritance is autosomal dominant. There are recurrent venous thromboses usually starting in early adult life and arterial thrombi may occur. Antithrombin concentrates are available and are occasionally used to prevent thrombosis during surgery or childbirth. Medicine  PROTEIN C DEFICIENCY Protein C is a critical anticoagulant protein in the blood that helps regulate clotting by inactivating factors Va and VIIIa, thereby preventing excessive clot formation. A deficiency in protein C can lead to an increased risk of thromboembolic events. Interpretation of Results Typically range from 70% to 130% of normal, depending on the laboratory's reference range. Low Levels: Indicate: 1. Inherited Protein C Deficiency: Genetic mutations affecting protein synthesis or function. 2. Acquired Protein C Deficiency: Conditions such as liver disease, vitamin K deficiency, or during acute thrombotic events. High Levels: Generally, not clinically significant but could indicate an acute phase reaction. Types of Protein C Deficiency Type I: Quantitative deficiency where total protein C levels are low. Type II: Qualitative deficiency where levels are normal, but the protein is dysfunctional. Medicine Inheritance is autosomal dominant with variable penetrance. Protein C levels in heterozygote individuals are approximately 50% of normal. Characteristically, many patients develop skin necrosis as a result of dermal vessel occlusion when treated with warfarin, thought to be caused by a further reduction of protein C levels in the first day or two of warfarin therapy. Rarely, infants may be born with homozygous deficiency and characteristically present with severe disseminated intravascular coagulation (DIC) or purpura fulminans in infancy. Medicine  PROTEIN S DEFICIENCY Protein S is a vitamin K-dependent plasma protein that plays a critical role in the regulation of blood coagulation. It acts as a cofactor to activated Protein C (APC) in inactivating coagulation factors Va and VIIIa, helping to prevent excessive clot formation. A deficiency in Protein S can lead to an increased risk of thrombosis. Interpretation of Results Typically range from 60% to 150% of the normal value, depending on the lab. Low Levels: Indicate: 1. Inherited Protein S Deficiency: Genetic mutations affecting protein synthesis or function. 2. Acquired Protein S Deficiency: Can occur in conditions such as liver disease, vitamin K deficiency, or during acute thrombosis. High Levels: Generally, not clinically significant, but could indicate an acute phase response. Types of Protein S Deficiency Type I: Quantitative deficiency, where total Protein S levels are low. Type II: Qualitative deficiency, where levels are normal, but the protein is dysfunctional. Protein S deficiency has been found in a number of families with a thrombotic tendency. It is a cofactor for protein C. The clinical features of protein S deficiency are similar to those of protein C deficiency, including a tendency to skin necrosis with warfarin therapy. The inheritance is autosomal dominant. Medicine  PROTHROMBIN ALLELE G20210A G20210A is an allele of the prothrombin gene that has a prevalence of 2–3% in the population. It leads to increased plasma prothrombin levels and increases thrombotic risk five-fold. It is probable that the cause of venous thrombosis with this mutation is that sustained generation of thrombin results in prolonged down-regulation of fibrinolysis through activation of thrombin-activated fibrinolysis inhibitor. This mutation is also associated with an increased risk of myocardial infarction in women by 4 times, while in men the risk is increased by 1.5 times. Medicine  HYPERHOMOCYSTEINEMIA High levels of plasma homocysteine may be genetic or acquired and are associated with increased risk for both venous and arterial thrombosis. However, trials show little evidence that lowering the levels reduces these risks. If elevated homocysteine levels are found, additional tests may include: Vitamin B12 and Folate Levels: To assess nutritional status. DEFECTS OF FIBRINOGEN Defects of fibrinogen are usually clinically silent or cause excess bleeding. Thrombosis is a rare association. Medicine  HEREDITARY OR ACQUIRED DISORDERS OF HEMOSTASIS High plasma factor VIII or fibrinogen levels are also associated with venous thrombosis. The combination of multiple risk factors is associated with increased risk of thrombosis. If these are persistent they may represent a reason for extended anticoagulation. BLOOD DISORDERS Increased viscosity, thrombocytosis and enhanced platelet functional responses are possible factors for the high incidence of thrombosis in patients with polycythemia vera and essential thrombocythemia. Testing for the JAK2 V617F mutation may indicate an otherwise unsuspected myeloproliferative disease in patients with hepatic or portal vein thrombosis. Medicine  THE ANTIPHOSPHOLIPID SYNDROME Antiphospholipid Syndrome (APS) is an autoimmune disorder characterized by the presence of antiphospholipid antibodies, which can lead to an increased risk of thrombosis (blood clots) and pregnancy complications. APS can occur as a primary condition or secondary to other autoimmune diseases, such as lupus. The antiphospholipid syndrome (APS) can be defined as the occurrence of venous and arterial thrombosis and/or recurrent miscarriage in association with laboratory evidence of persistent antiphospholipid antibody. Anticardiolipin Antibodies (aCL): Measures the presence of antibodies against cardiolipin, a phospholipid. One antiphospholipid is the lupus anticoagulant (LA) which was initially detected in patients with SLE and is identified by a prolonged plasma APTT which does not correct with a 50 : 50 mixture of normal plasma. Medicine Paradoxically, in view of its name, it is associated with venous and arterial thrombosis. A second test dependent on limiting quantities of phospholipid (such as the dilute Russell’s viper venom test) is also used in diagnosis. Whereas lupus anticoagulants are reactive in the fluid phase, other antiphospholipid antibodies, such as anticardiolipin antibodies and antibodies to β2-GPI-1, are identified by solid phase immunoassay. Both solid phase assays and coagulation tests for LA should be used in the diagnosis of APS. A diagnosis of APS requires the presence of at least one of the antiphospholipid antibodies (anticardiolipin, lupus anticoagulant, or anti-β2 glycoprotein I) on two separate occasions, along with clinical criteria (thrombosis or pregnancy complications). Medicine  Investigation Of Thrombophilia Tests that may be abnormal in patients with a tendency to venous thrombosis include: Blood count and erythrocyte sedimentation rate – to detect elevation in hematocrit, white cell count, platelet count, fibrinogen and globulins. Blood film examination – may provide evidence of myeloproliferative disorder; leucoerythroblastic features may indicate malignant disease Prothrombin time (PT) and APTT – a shortened APPT is often seen in thrombotic states and may indicate the presence of activated clotting factors. A prolonged APTT test, not corrected by the addition of normal plasma, suggests an LA or an acquired inhibitor to a coagulation factor. Anticardiolipin and anti-β2-GPI antibodies. Fibrinogen assay. Medicine  DNA analysis for factor V Leiden. Antithrombin – immunological and functional assays. Protein C and protein S – immunological and functional assays. Prothrombin gene analysis for the G20210A variant. Plasma homocysteine estimation. Test for JAK2 (V617F) mutation if portal or hepatic vein thrombosis. Protein electrophoresis for paraprotein. Medicine Medicine Recommended Literature Hoffbrand’s Essential Haematology Chapter 25 -28 Medicine