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Molecular Testing in Coagulation

John Bartlett, Director of Diagnostic Development at the Ontario Institute for Cancer Research writes that “accurate and appropriate diagnosis is fundamental to the successful treatment of disease.” With the introduction and evolution of new technologies, we can evaluate coagulopathy at a molecular level like never before. Nevertheless, we must avoid ordering expensive and sometimes expansive molecular tests for rare disorders when alternative methods of diagnosis are…

Laboratory Support for Warfarin Monitoring

Warfarin use has declined with increased adoption of oral direct thrombin and direct factor (F) Xa inhibitors, but warfarin remains an important oral anticoagulant for patients with contraindications to or indications not currently covered by direct oral anticoagulant therapy, and for those with financial constraints that limit access to these medications. Warfarin is a vitamin K antagonist, which exerts its anticoagulant effect by interfering with γ-carboxylation…

Laboratory Monitoring for Heparins, Fondaparinux, Direct Thrombin Inhibitors, and Oral Anti-Xa Medications

Laboratory testing modalities and recommendations for monitoring heparin, heparin-like medications, direct thrombin inhibitors (DTIs), and direct anti-Xa inhibitors (DXIs), including indications, testing recommendations, testing modalities, and test limitations are discussed below. There are several options available for laboratory monitoring of these drugs, all with varying levels of utility. New DTI’s and DXI’s are in clinical trials and the number of patients undergoing direct oral anticoagulant therapy…

Laboratory Diagnosis of Lupus Anticoagulant and Antiphospholipid Antibodies

Introduction Antiphospholipid (aPL) antibodies are a heterogeneous family of autoantibodies that are primarily directed against plasma proteins complexed with anionic phospholipids that are found on damaged/activated cellular membranes, including endothelial cells, trophoblasts, platelets, and monocytes. The formation of autoantibodies against many antigenic targets, interfering with many of the biochemical and cellular functions of phospholipids, has led to various pathological clinical manifestations in patients with high levels…

Laboratory Evaluation of Thrombotic Thrombocytopenic Purpura

A rapid and accurate diagnosis of thrombotic thrombocytopenic purpura (TTP) is critical, as initiation of therapeutic plasma exchange (TPE) can be lifesaving. To date, the diagnosis of TTP is dependent on the demonstration of an otherwise unexplained microangiopathic hemolytic anemia and thrombocytopenia, which may or may not have concurrent end organ damage, such as neurologic symptoms or renal dysfunction. Laboratory findings consistent with a hemolytic anemia…

Laboratory Evaluation of Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a clinicopathologic syndrome characterized by a constellation of clinical findings and identification of antiplatelet factor 4 (PF4)/heparin antibodies. Classically in HIT, progressive thrombocytopenia is observed, reaching a nadir within 5–10 days following the immunizing heparin exposure. In addition, because the mechanism by which thrombocytopenia ensues involves platelet activation, thrombosis occurs in a significant proportion of cases. Thrombosis in HIT can be severe…

Acquired Prothrombotic Conditions

Classic Acquired Prothrombotic Conditions Although there is a growing number of known established thrombophilias with an underlying genetic basis, the majority of venous thromboembolism events are provoked in the context of an acquired hypercoagulability state, leading to venous thrombosis. These acquired cases can be associated with clinical events, such as surgery, malignancy, pregnancy, drugs, kidney disease, infection, and inflammatory states. The most classic acquired conditions include…

Chronic Elevated Levels of Factor VIII and Other Coagulation Factors

Chronic elevation of some coagulation factor levels is associated with an increased risk of venous thrombosis (factors VIII, IX, XI, II). Some of these show level dependence; the risk increases linearly with the plasma factor level (factors VIII, IX, II). For other coagulation factors, elevated levels were either not confirmed as a venous thrombotic risk factor or the risk association was not independent; it disappeared when…

Molecular Testing for Factor V Leiden and Prothrombin Gene Mutations in Inherited Thrombophilia

Factor V Leiden Factor V Leiden (FVL) refers to the specific G-to-A transition at nucleotide 1601 (c.1601G>A) in exon 10 of the factor V gene, which results in arginine to glutamine substitution at amino acid 534 of the protein (p.Arg534Gln), previously known as p.Arg506Gln. Activated factor V serves as a cofactor for the conversion of prothrombin to the active enzyme thrombin. Activated protein C (APC) is…

Testing for Activated Protein C Resistance

Activated protein C (APC) resistance has a reported prevalence of 10%–15% and is a common cause of thrombophilia. The majority of APC resistance is directly attributable to heritable mutations in coagulation factor V (FV), primarily the FV Leiden (FVL) R506Q mutation, which results in decreased efficiency of FVa inactivation by APC due to alteration of one of the APC cleavage sites. However, other less common mutations…

Protein S Deficiency Evaluation

Protein S (PS) is a vitamin K–dependent plasma glycoprotein that serves as a cofactor of activated protein C (APC). When bound to APC, it accelerates protein C–dependent degradation of factor (F) Va and FVIIIa. PS deficiency is inherited in an autosomal dominant fashion and has highly variable penetrance. It is a moderately strong risk factor for venous thrombosis, with a reported adjusted lifetime relative risk increase…

Protein C Deficiency Evaluation

Protein C (PC) is a vitamin K–dependent, heterodimeric, plasma glycoprotein that is synthesized as a zymogen in the liver. It is activated on the endothelial surface by the thrombin–thrombomodulin (TM) complex. Once produced, the activated protein C (APC) cleaves and inactivates factors Va and VIIIa. Protein S (PS) cofactor activity is required for optimal proteolytic activity of APC in vivo . PC deficiency is rare in healthy…

Antithrombin Testing

Congenital antithrombin (AT) deficiency occurs in 0.2%–0.02% of the general population. However, in patients who had suffered a venous thromboembolic event (VTE), the prevalence is approximately 1%–3%. Preanalytical variables must be taken into account when deciding on the timing of AT testing and pretest probability, as false-positive and false-negative results may occur if testing is done in the wrong patient or wrong time. Common causes of…

Thrombophilia Testing in the Pediatric Population

Introduction Thrombophilia refers to the propensity to develop thrombosis and can be a result of acquired and/or inherited conditions. Inherited conditions are usually identified by a blood test, where acquired conditions are characterized by a patient’s clinical state or associated comorbidities. There has been a documented increase in the incidence of venous thromboembolism (VTE) in the pediatric population over the last two decades. The incidence follows…

Laboratory Evaluation of Long-Term Thrombophilic Disorders

Abnormal thrombosis is a result of a pathologic shift in the balance between antihemorrhagic (prothrombotic) and antithrombotic factors toward conditions that favor thrombus formation at the wrong time and/or in the wrong location. The Virchow triad of blood stasis, inflammation, endothelial injury, and alterations of blood components, can all contribute to the pathologic event. Frequently the cause of a venous clot is not difficult to discern…

Laboratory Techniques in Fibrinolysis Testing

The purpose of this chapter is to provide an overview of laboratory techniques involved in fibrinolysis testing. Chapter 145 provides a general introduction to fibrinolytic testing and common fibrinolytic disorders ( Fig. 146.1 ). Open full size image Figure 146.1 The fibrinolytic pathway in coagulation. α 2 AP , α 2 antiplasmin; α 2 MG , α 2 macroglobulin; ECM , extracellular matrix; FDP , fibrin…

Fibrinolytic Testing

Physiology In healthy subjects, there is a dynamic equilibrium between coagulation and platelets forming a hemostatic plug to close the wound and the fibrinolytic system removing fibrin to prevent vascular occlusion. Fibrinolysis is initiated by the formation of fibrin. Tissue plasminogen activator (tPA) and plasminogen bind to fibrin. Once on the fibrin surface, tPA converts plasminogen into the active enzyme plasmin, which in turn lyses the…

Laboratory Evaluation of Factor XIII Deficiency

FXIII is a hemostatic protein that plays a critical role in stabilizing fibrin networks at the site of injury and thus preventing premature fibrinolysis. In the plasma, FXIII circulates as a tetrameric zymogen composed of two catalytic alpha chains (FXIII-A) and two carrier/inhibitory beta chains (FXIII-B) that serve to stabilize FXIII-A in the plasma pool. In addition to the plasma pool, platelet FXIII pool composed only…

Antiplatelet Therapy Monitoring

Antiplatelet medications are widely used to treat or prevent thrombosis in a variety of cardiovascular disorders. Currently available antiplatelet drugs comprise four categories: 1. Acetylsalicylic acid (aspirin) inhibits cyclooxygenase (COX-1), 2. P2Y12 receptor antagonists (clopidogrel, ticlopidine, prasugrel, cangrelor, ticagrelor) block the platelet adenosine diphosphate (ADP) receptor, 3. Phosphodiesterase inhibitors (dipyridamole, anagrelide, cilostazol) inhibit the platelet phosphodiesterases, and 4. Glycoprotein (GP)IIb/IIIa inhibitors (abciximab, eptifibatide, tirofiban, lamifiban) block…

Confirmatory Testing for Diagnosis of Platelet Disorders

Laboratory investigation of a suspected inherited platelet disorder (IPD) requires both standard and confirmatory platelet laboratory testing. The former includes platelet count, mean platelet volume and other indices by a hematology analyzer, peripheral blood smear review by light microscopy, and platelet functional analysis. The latter encompasses platelet flow cytometry, platelet transmission electron microscopy (PTEM), and molecular genetic testing. Although the standard laboratory testing is an important…