HEMOSTASIS AND THROMBOSIS

HEMOSTASIS AND THROMBOSIS

Introduction

This document provides a basic overview of hemostasis and thrombosis with particular focus on the role of the clinical laboratory. Hemostasis can be defined as the physiological process that keeps blood flowing while allowing solid clot formation, or thrombosis, to prevent blood loss from sites of vascular damage.1,2 This requires a delicate balance between a potent coagulation mechanism that rapidly forms a stable clot and a regulatory system that limits clot formation to the site of injury. As the site of damage is repaired, fibrinolysis degrades the clot and restores unrestricted blood flow.

In general, there are four clinical situations that call for the use of coagulation tests in the assessment of patients. These are:

  1. Excessive bleeding: Defects in primary and secondary hemostasis are potential causes of excessive bleeding (seeHemostasis).
  2. Increased risk of thrombosis: A number of congenital and acquired conditions can lead to pathologic clot formation (seeThrombophilia).
  3. Therapeutic monitoring: More details about tests used to monitor anticoagulant therapy are provided under the following:
  4. Pathologic thrombosis: More details about tests used to evaluate thrombotic conditions are provided under the following:

Hemostasis

Primary Hemostasis. This term refers to the response to vascular injury that produces a platelet clot at the site of damage.1,3 Primary hemostasis serves to immediately limit bleeding through the formation of a loose platelet plug. Platelets play a key role in the rapid response to blood vessel injury by:

Defects in primary hemostasis are generally associated with mucocutaneous bleeding, characterized by epistaxis, ecchymosis, genitourinary bleeding, or gingival bleeding. A typical patient with defective primary hemostasis might experience profuse bleeding from small cuts and require the application of pressure for a prolonged period to stop the bleeding.2,4 This type of bleeding pattern is different from that typically seen in patients with defects in secondary hemostasis, where deep tissue bleeding and hemarthroses are more the norm.3

Platelet counts and platelet function tests are a useful aid in the assessment of primary hemostasis. von Willebrand factor (vWF) serves to attach platelets to the blood vessel walls and to each other during primary hemostasis. Defects in vWF concentration or activity are very common, affecting approximately 1% of the population.1 The role of the clinical laboratory in the assessment of vWF function is described in detail under the test descriptions below.

Secondary Hemostasis. Secondary hemostasis refers to the cascade of enzymatic reactions that ultimately results in the conversion of fibrinogen to fibrin monomers. Fibrin monomers are then cross-linked into insoluble strands that serve to stabilize the loose platelet clot formed in primary hemostasis. Secondary hemostasis is triggered by the release of tissue factor from epithelial cells that are exposed to the circulation at the site of vascular injury. Defects in secondary hemostasis decrease fibrin production and reduce the stability of the formed clot.1,2,3,5 In these conditions, bleeding is generally delayed compared to that observed in defective primary hemostasis. The loose platelet plug is not stabilized by fibrin strands and starts to leak. Typical symptoms of patients with defective secondary hemostasis include soft-tissue bleeding, hematomas, retroperitoneal bleeding, or hemarthrosis. The hemophilias are examples of defects in secondary hemostasis.2

Two simple screening tests are most often used in the initial assessment of secondary hemostasis. Both the prothrombin time (PT) and partial thromboplastin time (PTT) are performed on plasma separated from whole blood collected with sodium citrate anticoagulant. Citrate binds calcium in the blood and inhibits clot formation. Like all other clot-based coagulation tests, both the PT and PTT assays are initiated by adding excess calcium to the testing tube to overcome the effect of the citrate. The testing sequence for PT and PTT are explained below.

fig 1 - prothrombin time

In normal hemostasis, tissue factor that is released into the circulation as the result of vascular damage unleashes a coagulation cascade that ultimately results in the production of fibrin.1,2,6 Phospholipid membranes play an integral role in providing a surface for catalytic enzyme complex formation for these reactions. Platelets that form the primary hemostatic plug provide the phospholipid required to promote fibrin generation.3 The PT test brings these components (tissue factor and phospholipid) together in the test tube. Since tissue factor is essentially absent in the normal plasma and must be supplied from an external source, the cascade of enzymatic reactions triggered in the PT test is referred to as the “extrinsic pathway.”

fig 2 - partial tt

The term “partial thromboplastin” is derived from the fact that this assay differs from the PT (which uses “complete thromboplastin”) in that no tissue factor is used to initiate clotting.1,2,5 Instead, the reagent for the PTT simply includes a source of phospholipid. The clinical chemists who developed the PTT gave the name “intrinsic pathway” to the cascade of reactions triggered in the PTT test based on the mistaken perception that coagulation that occurs in the PTT test is initiated without the addition of any external factors. It is now understood that an external factor is, in fact, involved in the initiation of the PTT clotting cascade.7 The intrinsic pathway is actually activated by plasma contact with the negatively charged glass surface of the test tube. This “contact activation” can be enhanced by adding particulate matter like silica, kaolin, or ellagic acid into the test mixture in what we now refer to as an “activated” partial thromboplastin time, or aPTT.

The enzymatic reactions of the extrinsic and intrinsic pathways of coagulation are depicted in Figure 1. For many years clinical scientists felt that these two pathways, corresponding to the PT and aPTT laboratory tests respectively, represented unique physiologic mechanisms for the initiation of normal coagulation.1,2,5 It turns out, however, that while several steps of the intrinsic pathway are critical to normal hemostasis, contact activation is not a physiologically relevant mechanism for the initiation of normal coagulation. This became evident when it was discovered that even severe deficiencies of the intrinsic pathway factors (HMWK, prekallikrein, or factor XII) that will produce a markedly extended aPTT in vitro will not cause bleeding in vivo. It is now clear that extrinsic activation by tissue factor is the primary initiator of normal coagulation. Tissue factor forms a complex with activated VIIa that initiates the extrinsic pathway. This leads to the formation of thrombin, which in turn activates factor VIII. The tissue factor/factor VIIa complex also directly activates factor IX to IXa. Together, activated factors VIIIa and IXa form the potent “tenase” complex, effectively bypassing the contact activation pathway. Contact activation is just a handy trick that allows the clinical chemist to assess the functionality of several important intrinsic pathway factors in a test tube. Contact activation can occur in vivo when negatively charged surfaces, such as heart valve prostheses or vascular stents, are installed in the blood stream.7

Gamma-carboxyl glutamate (gla) residues on factors II, VII, IX, and X play a critical role in effective secondary hemostasis.1,4 These gla residues are formed by vitamin K-dependent enzymes and facilitate calcium-dependent complex-binding to phospholipid membranes. This serves to concentrate the catalytic activity at the point of injury and to prevent thrombosis from spreading throughout the vasculature. These complexes consist of a proteolytic enzyme associated with a catalytic cofactor that is anchored to a negatively charged phospholipid surface by gla residues. Vitamin K deficiency or inhibition of vitamin K-dependent enzymes by oral anticoagulants serves to block the formation of gla residues and disrupt hemostasis. Since factor VII, an extrinsic pathway factor, has the shortest half-life of the vitamin K-dependent cofactors, the PT test is more sensitive to oral anticoagulant therapy or vitamin K deficiency than the aPTT.5

Two Phases of Coagulation

It can be helpful to consider secondary hemostasis as a process that occurs in two distinct phases.6 The initiation phase, triggered by the release of tissue factor into the bloodstream, results in the production of a relatively small amount of thrombin through the extrinsic pathway. Once this first thrombin is produced, the propagation phase of coagulation begins. Thrombin drives the conversion of factors V and VIII to their activated forms. Activated factor VIII combines with activated factor IX to produce the very powerful tenase complex. This complex drives the accelerated production of thrombin in a cycle that feeds on itself in an explosive manner. The second phase of coagulation rapidly becomes the predominant mechanism of fibrin generation. In many ways, the two-phase process of coagulation can be compared to the ignition and subsequent explosion of a stick of dynamite. The first phase can be thought of as the slow-burning fuse that ignites the explosion of the second phase. This mental construct can help one to understand a fundamental limitation of one of the most common tests of coagulation: the prothrombin time (PT). The minimal amount of thrombin generated through tissue factor activation of the extrinsic pathway is adequate to produce quickly a detectable clot in the PT assay. The amplified thrombin generation associated with the second phase of coagulation is not required to produce a normal PT. This explains the fact that deficiencies of intrinsic pathway factors VIII, IX, and XI that produce the severe bleeding of hemophilia A, B, and C, respectively, do not typically produce an extended PT. Effective physiologic coagulation requires the second (explosive) phase of thrombin generation, but clot formation in PT does not. PT is only sensitive to variations of components of the extrinsic pathway: factors VII, X, V, II, and fibrinogen. A more complete review of the clinical relevance and analytic testing for individual coagulation pathway factors can be found under their individual test descriptions.


   086231 Factor II Activity
   086249 Factor V Activity
   800599 Factor VII Activity
   086264 Factor VIII Activity
   086298 Factor IX Activity
   086306 Factor X Activity - Clot Based
   086314 Factor XI Activity
   086322 Factor XII Activity
   001610 Fibrinogen Activity
   500436 Contact Factor Evaluation Profile
   500041 Extrinsic Pathway Coagulation Factor Profile
   500460 High Molecular Weight Kininogen (HMWK)
   500033 Intrinsic Pathway Coagulation Factor Profile
   500194 Prekallikrein (Fletcher Factor) Assay

coagulation cascade

Causes of Abnormal Screening Results

In many cases, a clinician must deal with an extended PT or aPTT in a patient who is not receiving anticoagulant therapy. Often, the key to identifying the cause of this laboratory finding is knowledge of the patient's clinical history. These tests are commonly ordered as part of the diagnostic work-up of a patient with a history of bleeding. This clinical situation is significantly different from the patient with no history of bleeding who is tested prior to surgery to rule out possible coagulation defects. The aPTT, or the more sensitive aPTT-LA, can be ordered as part of an antiphospholipid syndrome work-up of a patient with a history of thrombosis or recurrent miscarriage. Three distinct test algorithms should be employed for these three very different clinical situations.

In order to choose an optimal diagnostic algorithm, one should understand the many potential causes of an abnormal screening test. In the absence of prescribed anticoagulant therapy, prolongation of these tests generally can be attributed to five common causes: (1) specimen collection and transport issues, (2) medication, (3) pathologic conditions, (4) factor inhibitors, and (5) mixing studies: distinguishing factor deficiency from inhibitors.

Specimen Collection and Transport Issues

Medication

Pathologic Conditions

Factor Deficiencies

Factor Deficiency Cases per Million Individuals1 Screening Tests Specific Factor Level TestNumber Comments
vWF >100 von Willebrand Profile 084715 aPTT can sometimes be extended
XII   aPTT 086322 It should be noted that factor XII deficiency is quite common but is not associated with bleeding
VIII 60-100 aPTT 086264 Affects males predominantly
IX 10-20 aPTT 086298 Affects males predominantly
XI 1 aPTT 086314  
Fibrinogen 1 aPTT, PT 001610  
II <0.5 aPTT, PT 086231  
V <0.5 aPTT, PT 086249  
VII <0.5 PT 800599  
X <0.5 aPTT, PT 086306  
XIII <0.5   086330 Both PT and aPTT will be normal. Must test directly
1More information about clinical aspects and testing for individual factor deficiencies can be found under the individual test descriptions.

Factor Inhibitors



   117157 Factor VIII Inhibitor Profile, Comprehensive
   117892 Lupus Anticoagulant With Reflex
   117054 Lupus Anticoagulant Comprehensive

Mixing Studies: Distinguishing Factor Deficiency From Inhibitors

A mixing study is used to study the cause of a prolonged screening test. This study can determine if the cause is a deficiency of one or more factors or an inhibitor.4 In a mixing study, platelet-free, normal plasma that is replete with all coagulation factors (near 100% activity for each) is mixed with the patient's sample. For example, in a 1:1 mix, one part patient sample is mixed with one part normal plasma, and the mixture is tested. In this case, the lowest possible concentration for any individual factor in the mixture would be approximately 50% (in the case of a patient with a factor concentration of zero and the normal pool has an activity of 100%). The mixture is tested using the same test system that produced the extended screening result. Some inhibitors are time- and/or temperature-dependent. In these cases, the sample that is tested immediately after mixing will correct, while results after the sample has been incubated for 1 hour at 37° will not correct. All plasma samples that correct upon immediate mix are retested after an incubated mix to rule out time- and/or temperature-dependent inhibitors.

Three different types of results can be observed in mixing studies:

  1. No Correction for Immediate Mix
    • When the addition of normal plasma fails to correct the clotting time into the normal range, the cause of the abnormal test is likely an inhibitor.
    • Addition of normal plasma to the reaction mixture serves to dilute inhibitors, but typically does not completely neutralize their effects.
    • The most common cause of inhibition that is observed in the clinical laboratory is caused by inhibitor-type, anticoagulants that have been given therapeutically or have contaminated the sample at collection (ie, from a heparin line).
    • Extended PT values observed in patients on oral anticoagulants typically correct in a mixing study because these therapeutics are not coagulation factor inhibitors. They work by reducing vitamin K-dependent factor levels.
    • When therapeutic factor inhibitors have been ruled out, further studies can be performed to differentiate specific factor inhibitors from lupus anticoagulants. (See Lupus Anticoagulant section.)
  2. Correction for Immediate Mix – No Correction After Incubation
    • This result is consistent with the presence of a time- and/or temperature-dependent inhibitor.
    • Historically, it was felt that lupus anticoagulants were exclusively immediate-type inhibitors. Subsequent studies have shown that as many as 30% of lupus anticoagulants are time- and temperature-dependent.4 Specific inhibitors, particularly those to factor VIII, can also be time- and temperature-dependent.
  3. Correction for Immediate Mix – Correction After Incubation
    • This result rules out the presence of immediate inhibitors as well as time- and/or temperature-dependent inhibitors.
    • The normal plasma supplies the deficient factor at a concentration high enough to allow normal clotting. In the absence of inhibitors, a sample with at least 50% of all factors will produce a normal clotting time for both the PT and aPTT tests.
Note: Mixing studies performed on samples with minimally prolonged screening tests (ie, <8 seconds) will often produce confusing results. The addition of normal plasma can sometimes dilute weak inhibitors out. This causes the mixing study to correct, a result that is more consistent with a factor deficiency.

Thrombophilia

Clinical Aspects of Pathologic Thrombosis

Venous thromboembolism (VTE) is responsible for more than 300,000 hospital admissions per year, and resultant pulmonary embolism is a contributing factor in approximately 12% of deaths among hospitalized patients.13 Pulmonary embolus is the most common cause of death associated with childbirth and is, overall, the third most common cause of death in the United States.14 The coagulation laboratory plays a central roll in determining the cause of VTE and the risk of recurrence after an initial thrombotic event.

The risk factors associated with venous thromboembolism are different from those associated with arterial thrombosis that cause heart attack, stroke, and peripheral artery diseases.15 Venous and arterial thromboses are distinct clinical entities that differ in the microenvironment of clot formation and the structure of the clots formed.13 Because of their very distinct physiology, the tests used in the assessment of these two conditions are different.13

Arterial Thrombosis

  • Arterial thrombosis forms under conditions of accelerated blood flow.
  • It often occurs at the site of rupture of atherosclerotic plaques.
  • Arterial thrombosis is typically composed of platelet aggregates linked by thin fibrin strands.
  • Tests of platelet function and inflammatory markers associated with atherosclerosis are generally most useful in arterial thrombotic risk assessment.
  • Tests for hypercoagulability are not as useful in the assessment of arterial thrombosis.15

Venous Thrombosis

  • Venous thrombosis forms in regions of slow to moderate blood flow.
  • Generally, it is composed of a mixture of red cells, platelets, and fibrin.
  • Venous thrombosis often forms in the lower limbs.
  • Clinical manifestations are a response to obstructed blood flow at the original site of thrombosis or due to embolism of the pulmonary circulation.
  • Tests for congenital or acquired defects in the coagulation cascade or fibrinolysis are often useful in the assessment of venous thrombotic risk.15

It is generally thought that most cases of venous thrombosis result from the convergence of an acquired “precipitating” condition (ie, a cause of blood stasis or vessel injury) with an underlying genetic predisposition for hypercoagulability.15 For more than a century clinicians have understood that the risk of venous thrombosis is increased in individuals with any of three predisposing conditions – referred to as Virchow's triad:16

  1. Stasis of blood
  2. Vessel injury
  3. Hypercoagulability

Genetic Causes of Venous Thrombosis

Specific genetic defects should be suspected when a thrombotic event has any of the following characteristics14,16,17:

  • Spontaneous with no predisposing condition, such as prolonged immobilization or surgery
  • Patient suffers from more than one thrombotic event
  • Patient has a positive family history of thrombosis
  • Patient is <50 years old
  • Thrombosis occurs at an unusual site (eg, mesenteric or cerebral brain)

Three general categories of test methodology are useful in the assessment of congenital thrombotic conditions:

  1. Genetic tests for specific mutations
  2. Clotting-based measurements for specific factor activity levels
  3. Immunoassays to measure the concentration of specific antigens

Genetic conditions associated with thrombophilia are listed below in order of their relative frequency of occurrence.14,16,18

Table 1. Genetic Conditions Associated With Thrombosis

Genetic Condition Testing Method Test Number
Methylenetetrahydrofolate reductase (MTHFR) gene mutation Screen: Homocysteine level 706994
Confirmation: MTHFR genetic testing 511238
Factor V Leiden mutation Screen: Activated protein C resistance 117762
Confirmation: Genetic testing 511154
Prothrombin 20210 Genetic testing only 511162
Increased factor VIII1 Factor VIII activity 086264
Protein S deficiency Protein S (activity, total/free antigen profile) 117754
Protein C deficiency Protein C (activity and antigen profile) 080465
Antithrombin deficiency Antithrombin (activity and antigen profile) 015594
Dysfibrinogenemia Fibrinogen activity 001610
Fibrinogen antigen 117052
1While factor VIII elevation is associated with a number of acquired conditions, some individuals appear to have congenitally elevated factor VIII levels that are associated with increased risk of thrombosis.14

Genetic testing is useful for the diagnosis or confirmation of MTHFR, factor V Leiden, and the prothrombin 20210 mutations. Regardless of clinical status, genetic testing can be definitive because the patient's DNA remains constant. Antigen and activity levels must be measured to diagnose the other congenital thrombotic conditions. It is important to understand, however, that the results of activity and antigen level tests can be affected by the clinical state of the patient. In many clinical circumstances, such as those listed below, tests for activity or antigen levels may produce misleading results.16,18

Table 2. Clinical Conditions That Affect Tests for Congenital Thrombotic Risk

Clinical Condition Effect
Heparin therapy or contamination Decreases antithrombin
Warfarin therapy/vitamin K deficiency Decreases protein C and protein S
Recent thrombosis or surgery Decreases antithrombin, protein C, protein S Increases homocysteine
DIC, liver disease, sepsis, L-asparaginase therapy Decreases antithrombin, protein C, protein S
Kidney disease/nephrotic syndrome Decreases antithrombin and protein S
Acute phase reaction, inflammation, infection Decreases protein SIncreases factor VIII
Pregnancy or postpartum period Decreases protein S and APCRIncreases homocysteine
Oral contraceptives or estrogen replacement Decreases antithrombin and protein S
Lupus anticoagulants (LA) Decreases APCR, protein S, and factor VIII
Vitamin B12, folate, or B6 deficiencyTreatment with methotrexate, phenytoin, or theophylline Hypothyroidism, malignancy, menopause Increases homocysteine

Acquired Causes of Venous Thrombosis

The incidence of venous thrombosis increases dramatically with age. The rate of occurrences changes from approximately one per 100,000 individuals younger than 40 years of age to one per 1000 individuals older than 75.14 A number of acquired conditions that are associated with an increased risk of thrombosis have been identified. They are listed in Table 3.17

Table 3. Acquired Conditions Associated With Thrombosis17
Antiphospholipid antibodies (see below) Malignancy
Atrial fibrillation Myeloproliferative disorders
Congestive heart failure Nephrotic syndrome
Diabetes mellitus Oral contraceptive therapy
Estrogen therapy Paroxysmal nocturnal hemoglobinuria
Heparin-induced thrombocytopenia Postoperative state
Hypertension Pregnancy
Hyperviscosity Thrombotic thrombocytopenic purpura
Immobilization Trauma

Antiphospholipid Antibody Syndrome

Antiphospholipid (APL) antibodies can be detected in as many as 2% of unselected individuals,11 and they justify special consideration. These antibodies are the most common acquired cause of increased thrombotic risk.4,10,12,13,19,20,21 The term “antiphospholipid antibody syndrome” (APS) refers to a spectrum of clinical conditions that is associated with the presence of antiphospholipid antibodies. Both clinical and laboratory features must be present for the diagnosis of APS to be made.20,21,22 Any one or more of the conditions discussed below can occur in patients with the antiphospholipid antibody syndrome.

  • Venous Thrombosis. Antiphospholipid antibody syndrome can be detected in approximately 10% of patients presenting with their first venous thrombosis.19 Studies have shown that at least one thrombotic event occurs in approximately 30% of patients with persistent antiphospholipid antibodies.19 Deep venous thrombosis (DVT) occurs in approximately 40% of individuals with primary APS. The risk is even greater in individuals with other thrombotic risk factors (ie, the second hit) such as pregnancy, prolonged immobilization, or oral contraceptive therapy.19,20
  • Arterial Thrombosis. Arterial thrombosis in APS is less common than venous thrombosis but results in greater incidence of morbidity.11 More than 25% of patients with APL have evidence of arterial occlusions.11 Antiphospholipid antibodies can be detected in as many as 33% of patients with strokes prior to the age of 50, and it is observed in 7% to 10% of unselected stroke patients.11 The incidence of stroke in patients with antiphospholipid antibodies increases when other risk factors (such as hypertension, hyperlipidemia, or smoking) are present. Patients with antiphospholipid antibodies also suffer from an increased incidence of cerebral infarcts, severe vascular headaches, transient ischemic attacks, and visual disturbances.13 Arterial thrombotic events are more common in patients with antiphospholipid antibodies secondary to SLE than those with primary antiphospholipid antibodies.12
  • Infertility and Complications of Pregnancy. Antiphospholipid antibodies can cause female infertility due to impaired uterine implantation of the embryo.19 In pregnancy, antiphospholipid antibodies are associated with placental-vascular thrombosis that can lead to fetal demise, fetal growth retardation, premature delivery, and neonatal thrombosis. Fetal loss associated with APS typically occurs in the second trimester. Between 5% and 15% of cases of recurrent spontaneous abortion are linked to APS.11
  • Thrombocytopenia. Thrombocytopenia is reported in 30% to 50% of patients with primary APS, although this rarely causes bleeding; however, thrombocytopenia is not considered one of the primary diagnostic criteria for APS because it can be found in so many other conditions.19,20 Positive test results for antiphospholipid antibodies are increased in patients with idiopathic thrombocytopenia (ITP).19
  • Other Conditions. APS is sometimes associated with symptoms that can include vasculitic rashes, dermal necrosis of digits, livedo reticularis, nephropathy, arthralgias, pulmonary hypertension, chorea, and migraine headaches.20,22,23
  • Catastrophic APS. Rarely, patients with antiphospholipid antibodies will suffer from multiple thrombotic occlusions simultaneously in a sometimes fatal condition that is referred to as catastrophic APS.20

Antiphospholipid antibodies can be subclassified into several clinical categories:

  • Primary antiphospholipid antibodies
    Represent the majority of cases
    Twice as common in women as men11,19
    Occur in individuals who are otherwise healthy with no predisposing conditions
    Often characterized by antibodies to just one phospholipid or protein12
  • Secondary antiphospholipid antibodies
    Associated with systemic lupus erythematosus (SLE), other autoimmune disorders, malignanciesObserved in approximately 50% of individuals with SLE with same female-to-male incidence ratio of 9:119Secondary antiphospholipid antibodies often characterized by antibodies to multiple phospholipids or proteins12
  • Drug-induced antiphospholipid antibodies
    A variety of therapeutic drugs can induce the production of antiphospholipid antibodies

calcium channel blockers
chlorpromazine
hydralazine
hydantoin
isoniazid
methyldopa
phenytoin
phenothiazine
procainamide
quinine
quinidine
thorazine
various antibiotics
Individuals with drug-induced antiphospholipid antibodies that persist after the drug treatment is ended experience an increased risk of thrombosis12
  • Infection-induced antiphospholipid antibodies19
    Often observed during the convalescent phase of acute bacterial and viral infections
    Often observed in individuals with syphilis
    Generally not associated with an increased risk of clinical complications
    Usually transient

Note: Because it is not possible to distinguish infection-induced antiphospholipid antibodies from clinically significant antiphospholipid antibodies, all patients that test positive for antiphospholipid antibodies should be retested after 6-8 weeks to rule out transient antibodies.19

Antiphospholipid antibodies can be detected indirectly with tests that are based on their effect on clot-based, in vitro coagulation assays (ie, lupus anticoagulants) or directly by solid-phase immunoassay.24 Due to the heterogeneity of antibodies associated with APS, both clotting and solid-phase immunoassay testing is recommend when APS is suspected.10,21

Lupus Anticoagulants

Lupus anticoagulants (LA) are antiphospholipid antibodies that prolong phospholipid-dependent coagulation assays.4,10,19 Lupus anticoagulants derive their name from the fact that they were first observed in patients with systemic lupus erythematosus (SLE); however, the vast majority of individuals with lupus anticoagulants do not have SLE. These antibodies were referred to as anticoagulants because they delay clotting in in vitro clotting assays. It has subsequently been shown that lupus anticoagulants are actually associated with an increased clinical tendency toward thrombosis, not anticoagulation.4 While lupus anticoagulants are occasionally found in patients with arterial thrombosis, they are much more frequently observed in patients with venous thrombosis and/or resultant pulmonary embolism.12

In clot-based assays, lupus anticoagulants neutralize anionic phospholipids that are involved in the coagulation cascade. Coagulation screening assays such as the activated partial thromboplastin time (aPTT), Russell viper venom time (RVVT), and prothrombin time (PT) can be affected by lupus anticoagulants. The extent of clotting time prolongation is highly dependent on the sensitivity of the reagent employed. Reagents with reduced amounts of phospholipid, such as the aPTT-LA and dilute Russell viper venom time (dRVVT), have enhanced sensitivity for lupus anticoagulants.10 The standard prothrombin time (PT) is rarely affected by lupus anticoagulants because of the high concentration of phospholipid in the reagent thromboplastin.19 However, a dilute prothrombin time test can be used to detect some lupus anticoagulants.25 Many lupus anticoagulants are discovered as the result of routine screening with the standard aPTT test – despite the fact that this reagent is not as sensitive for lupus anticoagulants as the aPTT-LA reagent.4,13

Due to the heterogeneity of lupus anticoagulant antibodies, no single assay identifies all cases.10 The International Society on Thrombosis and Haemostasis (ISTH) has established criteria for the diagnosis of lupus anticoagulants (see Figure 4). The ISTH has defined the minimum diagnostic criteria for lupus anticoagulants to include:10,19,20

  • a prolonged clot time in a screening assay such as aPTT-LA, dRVVT, or dilute PT
  • mixing studies indicating the presence of an inhibitor
  • positive confirmatory studies defining phospholipid dependence of the inhibitor
  • no evidence of other coagulopathies through the use of specific factor assays if the confirmatory step is negative or there is evidence of a specific factor inhibitor

Often the evaluation for lupus anticoagulants is confounded by the acute phase response or anticoagulant therapy. This is particularly the case when a patient is hospitalized and started on anticoagulant therapy before samples for coagulation testing can be drawn. If the patient has a deep vein thrombosis, many acute phase proteins such as fibrinogen, factor VIII, and C4B-binding protein will be elevated and can affect coagulation assays.

The serendipitous detection of an extended aPTT during a preoperative screen should trigger a thorough review of the patient's history. Testing for antiphospholipid antibodies may not be indicated if the patient's clinical history is not suggestive of APS.4,13,19 Alternatively, an extended aPTT may be detected in an individual who presents with the clinical features consistent with APS. In this case solid-phase ELISA tests for antiphospholipid antibodies should also be performed in order to establish or exclude the diagnosis. If the lupus anticoagulants or antiphospholipid antibody testing is positive, the testing should be repeated in 6-8 weeks to confirm the persistence of the antibody.

Very rarely, individuals with lupus anticoagulants will present with an extended prothrombin time and significant clinical bleeding due to a decreased concentration of prothrombin.4,10,13,19 It is thought that the lupus anticoagulants in these individuals bind to prothrombin and cause it to be removed from the circulation. In these cases, lupus anticoagulants are not acting as inhibitors of prothrombin but are rather “non-neutralizing” antibodies. This can cause confusion in the assessment of lupus anticoagulants since mixing study results are consistent with a factor deficiency and can be corrected with the addition of normal plasma.4 Unlike most patients with lupus anticoagulants, these patients will have an extended protime due to the prothrombin deficiency. When the protime is extended in a patient with evidence of lupus anticoagulants, antibodies to prothrombin should be considered in the differential diagnosis.


lupus cascade

Anticardiolipin Antibodies

The first anticardiolipin antibody (ACA) tests were developed in the early 1900s as a screening test for syphilis.20 The Venereal Disease Research Laboratory (VDRL) test is a manual agglutination assay that detects antibodies to cardiolipin extracted from bovine heart tissue. Mass screening studies with the VDRL revealed that this test was not specific for syphilis. False-positive VDRL screening results were found to be significantly associated with lupus anticoagulants and risk of thrombosis. The first solid-phase immunoassays for antibodies to cardiolipin were developed in the early 1980s. These assays, designed to detect anticardiolipin IgA, IgG, and IgM isotypes, are approximately 100-fold more sensitive than the classical VDRL assay.20 The presence of anticardiolipin antibodies (especially those of moderate to high titer for IgG) is strongly associated with both arterial and venous thrombosis and recurrent pregnancy loss.12,21,26 The IgM and IgA isotypes of anticardiolipin antibody have also been shown to be associated with venous thrombosis.12,19,21 The distribution of isotypes of anticardiolipin antibody-positive patients with thrombosis has been found to be as follows:12

IgG 36%
IgM 17%
IgA 14%
Multiple 33%

In general, anticardiolipin antibodies are considered to be more sensitive than lupus anticoagulants for APS and are implicated in approximately five times more cases.12,21

  • At least one anticardiolipin antibody isotype can be detected in 80% to 90% of patients with APS.23
  • Approximately 90% of individuals with lupus anticoagulants will also be positive for at least one isotype of anticardiolipin antibody.19

Lupus anticoagulants are more specific for APS than anticardiolipin antibodies.12,23

  • The specificity of anticardiolipin antibodies for APS is increased with higher titer, especially for the IgG isotype.20,21

While there is frequent concordance between lupus anticoagulant and anticardiolipin antibody results, clinical situations occur in which one is present in the absence of the other.20,23

β2 Glycoprotein 1 Antibodies

Recently, an international consensus group of experts in the diagnosis and management of antiphospholipid syndrome (APS) concluded that β2 glycoprotein 1 (β2 GP-1) IgG and IgM antibodies should be included as diagnostic criteria for APS.22 This group determined that the presence of one or both of these antibodies is an independent risk factor for thrombosis and pregnancy complications.22

The term “antiphospholipid” does not fully describe the target of anticardiolipin antibody assays. A common aspect to all assays for anticardiolipin antibodies is the requirement that the assay system include a source of plasma proteins.21 In many cases, antiphospholipid antibodies require the presence of specific proteins to facilitate phospholipid binding.4,13,20 It has been determined that, in many patients, the required plasma factor is β2-glycoprotein 1 (β2GP-1).20,21,26 Solid phase enzyme immunoassays that detect antibodies that bind to β2GP-1 in the absence of phospholipid are now part of the diagnostic arsenal for APS.

  • β2GP-1-dependent antibody binding is frequently detected in patients with clinical symptoms of APS.21
  • Anticardiolipin antibodies associated with infections do not tend to be β2GP-1-dependent.21 This supports the conclusion that anti-β2GP-I assays may be more specific for APS than anticardiolipin antibodies.20,21,23, 26
  • While the majority of patients with lupus anticoagulants will also test positive for anticardiolipin antibodies and/or β2GP-1 antibodies, approximately 30% of patients tested will have discordant results.4,13
  • Approximately 20% of patients that test negative for anticardiolipin antibodies will test positive for β2GP-1.4
  • Positive anti-β2GP-1 results tend to support the diagnosis of APS in patients with a strong clinical picture for APS with negative lupus anticoagulants and anticardiolipin antibody results.23
  • Anti-β2GP-1 testing can be useful in the evaluation of patients with positive anticardiolipin antibody results and a clinical picture that is not consistent with APS.21,23,26 A negative anti-β2GP-1 result in this context would not support a diagnosis of APS.

Footnotes


   1. Brandt JT, “Overview of Hemostasis,” McClatchey KD, ed, Clinical Laboratory Medicine, 2nd ed, Baltimore, MD: Lippincott Williams and Wilkins, 2002, 987-1009.
   2. Liu MC and Kessler CM, “A Systemic Approach to the Bleeding Patient,” Kitchens CS, Alving BM, and Kessler CM, eds, Consultative Hemostasis and Thrombosis, Philadelphia, PA: WB Saunders, 2002, 181-96.
   3. Kottke-Marchant K and Corcoran G, “The Laboratory Diagnosis of Platelet Disorders,” Arch Pathol Lab Med, 2002, 126(2):133-46.
   4. Triplett DA, “Coagulation Abnormalities,” McClatchey KD, ed, Clinical Laboratory Medicine, 2nd ed, Baltimore, MD: Lippincott Williams and Wilkins, 2002, 1033-49.
   5. Van Cott EM and Laposata M, “Coagulation,” Jacobs DS, Oxley DK, and DeMott WR, eds, Laboratory Test Handbook, Hudson, OH: Lexi-Comp, 2001, 327-58.
   6. Mann KG, Brummel K, and Butenas S, “What Is All That Thrombin For?” J Thromb Haemost, 2003, 1(7):1504-14.
   7. Adcock DM, Jensen R, Johns CS, et al, Coagulation Handbook, Austin, TX: Esoterix Coagluation, 2002.
   8. Adcock DM, Kressin DC, and Marlar RA, “Effect of 3.2% vs 3.8% Sodium Citrate Concentration on Routine Coagulation Testing,” Am J Clin Pathol, 1997, 107(1):105-10.
   9. Reneke J, Etzell J, Leslie S, et al, “Prolonged Prothrombin Time and Activated Partial Thromboplastin Time Due to Underfilled Specimen Tubes With 109 mmol/L (3.2%) Citrate Anticoagulant,” Am J Clin Pathol, 1998, 109(6):754-7.
   10. Brandt JT, Triplett DA, Alving B, et al, “Criteria for the Diagnosis of Lupus Anticoagulants: An Update. On Behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardization Committee of the ISTH,” Thromb Haemost, 1995, 74(4):1185-90.
   11. Jenson R, “The Antiphospholipid Antibody Syndrome,” Clin Hemost Rev, 2001, 15(11):1-4.
   12. Bick RL, “Antiphospholipid Thrombosis Syndromes,” Clin Appl Thromb Hemost, 2001, 7(4):241-58.
   13. Hirsh J, Anand SS, Halperin JL, et al, “Guide to Anticoagulant Therapy. Heparin: A Statement for Healthcare Professionals From the American Heart Association,” Circulation, 2001, 103(24):2994-3018.
   14. Adcock DM, “Laboratory Evaluation of Venous Thrombosis Risk,” Clin Hemost Rev, 2003, 17(12):1,2,5,6,8.
   15. Schafer AI, Levine MN, Konkle BA, et al, “Thrombotic Disorders: Diagnosis and Treatment,” Hematology (Am Soc Hematol Educ Program), 2003:520-39.
   16. Marques MB, “Testing for Genetic Predisposition to Venous Thrombosis,” MLO, 2002, 34(1):8-13.
   17. Marlar RA and Adcock DM, “The Multifactorial Threshold Model of Thrombotic Risk,” Clin Hemost Rev, 2003, 17(6):1,2,4-6.
   18. Triplett DA, “Thrombophilia,” McClatchey KD, ed, Clinical Laboratory Medicine, 2nd ed, Baltimore, MD: Lippincott Williams and Wilkins, 2002, 1033-49.
   19. Alving BM, “The Antiphospholipid Syndrome: Clinical Presentation, Diagnosis, and Patient Management,” Kitchens CS, Alving BM, and Kessler CM, eds, Consultative Hemostasis and Thrombosis, Philadelphia, PA: WB Saunders, 2002, 181-96.
   20. Levine JS, Branch DW, and Rauch J, “The Antiphospholipid Syndrome,” N Engl J Med, 2002, 346(10):752-63.
   21. Carreras LO, Forastiero RR, and Martinuzzo ME, “Which Are the Best Biological Markers of the Antiphospholipid Syndrome?” J Autoimmun, 2000, 15(2):163-72.
   22. Miyakis S, Lockshin MD, Atsumi T, et al, “International Consensus Statement on an Update of the Classification Criteria for Definite Antiphospholipid Syndrome (APS),” J Thromb Haemost, 2006, 4(2):295-306..
   23. Harris EN, Pierangeli SS, and Gharavi AE, “Diagnosis of the Antiphospholipid Syndrome: A Proposal for Use of Laboratory Tests,” Lupus, 1998, 7(Suppl 2):S144-8.
   24. Schjetlein R and Wisloff F, “An Evaluation of Two Commercial Test Procedures for the Detection of Lupus Anticoagulant,” Am J Clin Pathol, 1995, 103(1):108-11.
   25. Liestol S, Jacobsen EM, and Wisloff F, “Dilute Prothrombin TIme-Based Lupus Ratio Test. Integrated LA Testing With Recombinant Tissue Thromboplastin,” Thromb Res, 2002, 105(2):177-82.
   26. Reddel SW and Krilis SA, “Testing for and Clinical Significance of Anticardiolipin Antibodies,” Clin Diagn Lab Immunol, 1999, 6(6):775-82.


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