Before 1992, medical laboratory scientists performed assays to detect only three proven venous thrombosis risk factors: deficiencies of the coagulation control factors antithrombin, protein C, and protein S.¹ Taken together, these three deficiencies accounted for no more than 7% of cases of recurrent venous thromboembolic disease and bore no relationship to arterial thrombosis. Since the report by Dahlback et al² of activated protein C (APC) resistance in 1993 and the characterization by Bertina et al³ of the factor V Leiden (FVL) mutation as its cause in 1994, efforts devoted to thrombosis prediction and evaluation have redefined the hemostasis laboratory and increase its workload exponentially. The list of current assays includes APC resistance, FVL mutation, prothrombin G20210A mutation, factor VIII activity, lupus anticoagulant (LA), anticardiolipin (ACL) antibodies, and anti–β₂-glycoprotein I (anti–β₂-GPI) for venous thrombosis and tissue plasminogen activator (TPA), plasminogen activator inhibitor 1 (PAI-1), high-sensitivity C-reactive protein (CRP), lipoprotein (a), plasma homocysteine, and tests for platelet activity such as urinary 11-dehydrothromboxane B₂ (see Chapters 44 and 46) for arterial thrombosis. Technical improvements have also been made in the diagnostic accuracy of integrated LA identification kits, the quantitative D-dimer assay, and tests for coagulation activation markers such as prothrombin fragment 1+2 (PF 1+2) and thrombin-antithrombin (TAT) complex.⁴ In addition, old tests, such as the fibrinogen assay and the factor VIII assay, have been applied to the prediction of venous and arterial thrombotic risk.

Introduction to Thrombosis

Etiology of Thrombosis

Thrombosis is a multifaceted disorder resulting from abnormalities in blood flow, such as stasis, and abnormalities in the coagulation system, platelet function, leukocyte activation molecules, and the blood vessel wall. Thrombosis is the inappropriate formation of platelet or fibrin clots that obstruct blood vessels. These obstructions cause ischemia (loss of blood supply) and necrosis (tissue death).⁵ Thrombophilia is defined as the predisposition to thrombosis secondary to a congenital or acquired disorder. The theoretical causes of thrombophilia are the following:

• Physical, chemical, or biologic events such as chronic or acute inflammation that release prothrombotic mediators from damaged blood vessels or suppress blood vessel production of normal antithrombotic substances

• Inappropriate and uncontrolled platelet activation

• Uncontrolled triggering of the plasma coagulation system

• Inadequate control of coagulation-impaired fibrinolysis

Prevalence of Thrombosis

At least 30% of the world’s people are expected to die of a thrombotic condition, and 25% of initial thrombotic events are fatal.⁶ Many fatal thromboses go undiagnosed before autopsy.

Prevalence of Venous Thrombosis

The annual incidence of venous thrombosis in the unselected U.S. population is 1 in 1000.7,8 This includes the comparatively innocent thrombosis of superficial leg veins as well as the more dangerous deep vein thromboses that form in the iliac, popliteal, and femoral veins of the upper legs and calves.⁹ Large occlusive thrombi also may form, although less often, in the veins of the upper extremities, liver, spleen, intestines, brain, and kidneys. The symptoms of thrombosis include the sensation of heat, localized pain, redness, and swelling. In deep vein thrombosis, the entire leg swells.

Fragments of thrombi, called emboli, may separate from the proximal end of a venous thrombus, move swiftly through the right chambers of the heart, and lodge in the arterial pulmonary vasculature, causing ischemia and death of lung tissue.¹⁰ Nearly 95% of these pulmonary emboli arise from thrombi in the deep leg and calf veins. Of the 250,000 individuals who experience pulmonary embolism in the United States annually, 10% to 15% die within 3 months. Many pulmonary emboli go undiagnosed because of the ambiguity of the symptoms. Coagulation system imbalances, such as inappropriate activation, gain of coagulation factor function, inadequate control, or fibrinolysis, are the mechanisms most often implicated in venous thrombosis.¹¹

Prevalence of Arterial Thrombosis

Cardiovascular disease causes 500,000 premature deaths annually in the United States; 500,000 cerebrovascular accidents (strokes) result in 100,000 stroke-related deaths. About 80% of myocardial infarctions and 85% of strokes are caused by thrombi that block coronary arteries or carotid end arteries of the vertebrobasilar system, respectively.¹² Transient ischemic attacks and peripheral arterial occlusions are more frequent than strokes and coronary artery disease and, although not fatal, cause substantial morbidity.

One important mechanism for arterial thrombosis is the well-described atherosclerotic plaque formation in the vessel walls. Activated platelets, monocytes, and macrophages embed the fatty plaque within the endothelial lining, suppressing the normal release of antithrombotic molecules such as nitric oxide and exposing prothrombotic substances such as tissue factor (see Chapter 40). Small unstable plaques rupture, occluding arteries and releasing thrombotic mediators to trigger thrombotic events. Activated platelets also form arterial platelet plugs with von Willebrand factor and only minimal fibrin, the “white thrombi” that cause death of surrounding tissue (see Chapter 13).

The hemostasis-related lesions we associate with arterial thrombosis are blood vessel wall destruction and platelet activation. Often these are inseparable. Researchers currently are examining new thrombosis markers that capture pathologic events in platelets and endothelial cells before a thrombotic event occurs.

Thrombosis Risk Factors

Acquired Thrombosis Risk Factors

In life, we acquire a legion of habits and conditions that either maintain or damage our hemostasis systems. Their multiplicity makes it difficult to pinpoint precisely the factors that contribute to thrombosis or to determine which have the greatest influence. These factors seem to contribute to venous and arterial thrombosis in varying degrees. Table 42-1 lists the nondisease risk factors implicated in thrombosis.¹³

TABLE 42-1

Nondisease Risk Factors That Contribute to Thrombotic Disease

Risk Factor	Comment	Contribution to Thrombosis	Laboratory Diagnosis
Age	Thrombosis after age 50	Risk doubled each decade
Immobilization	Distance driving, air travel, restriction to wheelchair or bed, obesity	Decreased blood flow increases thrombosis risk
Diet	Fatty foods; inadequate folate, vitamin B₆, and vitamin B₁₂	Homocysteinemia associated with 2-7× increased risk for arterial or venous thrombosis	Plasma homocysteine, vitamin levels, and lipid profile
Lipid metabolism imbalance	Hyperlipidemia, hypercholesterolemia, dyslipidemia, elevated lipoprotein (a), decreased HDL-C, elevated LDL-C	Moderate thrombosis association with LDL-C elevation and hypercholesterolemia, may be congenital	Lipid profile: total cholesterol, HDL-C, LDL-C, triglycerides, and lipoprotein (a)
Oral contraceptive use	30 mcg, formulation with progesterone	4-6× increased risk
Pregnancy		3-5× increased risk
Hormone replacement therapy		2-4× increased risk
Femoral or tibial fracture		80% incidence of thrombosis if not treated with antithrombotic
Hip, knee, gynecologic, prostate surgery		50% incidence of thrombosis if not treated with antithrombotic
Smoking		Depends on degree	hsCRP, fibrinogen
Inflammation	Chronic or acute	Arterial thrombosis	hsCRP, fibrinogen
Central venous catheter	Endothelial injury and activation	33% of children with central venous lines develop venous thrombosis

HDL-C, High-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol.

Thrombosis Risk Factors Associated with Systemic Diseases

In addition to life events, several conditions and diseases threaten us with thrombosis. Some are listed in Table 42-2, with an indication of the laboratory’s diagnostic contribution.¹⁴

TABLE 42-2

Diseases with Thrombotic Risk Components

Disease	Examples or Effects	Contribution to Thrombosis	Laboratory Diagnosis
Antiphospholipid syndrome	Chronic antiphospholipid antibody often secondary to autoimmune disorders	When chronic, 1.6-3.2× increased risk of stroke, myocardial infarction, recurrent spontaneous abortion, venous thrombosis	Mixing studies, lupus anticoagulant profile, anticardiolipin antibody, anti–β₂-glycoprotein I immunoassays
Myeloproliferative neoplasms	Essential thrombocythemia, polycythemia vera, chronic myelogenous leukemia	Increased risk due to plasma viscosity, platelet activation	Platelet counts and platelet lumiaggregometry
Hepatic and renal disorders	Diminished production or loss of coagulation control proteins	Increased risk due to deranged coagulation pathways, excess thrombin production	Prothrombin time, proteins C and S, and antithrombin assays, factor assays
Cancer	Trousseau syndrome, low-grade chronic DIC	20× increased risk of thrombosis; 10-20% of people with idiopathic venous thrombosis have cancer	DIC profile: platelet count, D-dimer, Fg; markers of coagulation activation: prothrombin fragment 1+2, thrombin-antithrombin complex
Leukemia	Acute promyelocytic leukemia (M3), acute monocytic leukemia, (M4-M5)	Increased risk for chronic DIC	DIC profile: platelet count, D-dimer, Fg
Paroxysmal nocturnal hemoglobinuria	Platelet-related thrombosis	Increased risk for deep vein thrombosis, pulmonary embolism, DIC	Flow cytometry phenotyping for CD55 and CD59; DIC profile: platelet count, D-dimer, Fg
Chronic inflammation	Diabetes, infections, autoimmune disorders, smoking		Factor VIII, Fg, high-sensitivity C-reactive protein

DIC, Disseminated intravascular coagulation; Fg, fibrinogen.

Together, transient and chronic antiphospholipid (APL) antibodies such as LA, ACL, and anti–β₂-GPI may be detected in 1% to 2% of the unselected population. Chronic APL antibodies confer a risk of venous or arterial thrombosis—a condition called the antiphospholipid syndrome (APS). Chronic APL antibodies often accompany autoimmune connective tissue disorders, such as lupus erythematosus. Some appear in patients without any apparent underlying disease.

Malignancies often are implicated in venous thrombosis. One mechanism is tumor production of tissue factor analogues that trigger chronic low-grade disseminated intravascular coagulation (DIC). In addition, stasis and inflammatory effects increase the risk of thrombosis. Migratory thrombophlebitis, or Trousseau syndrome, is a sign of occult adenocarcinoma such as cancer of the pancreas or colon.¹⁵

Myeloproliferative neoplasms such as essential thrombocythemia and polycythemia vera (see Chapter 34) may trigger thrombosis, probably through platelet hyperactivity. A cardinal sign of acute promyelocytic leukemia (see Chapter 36) is DIC secondary to the release of procoagulant granules from the malignant promyelocytes. DIC can intensify during therapy at the time of vigorous cell lysis.¹⁶ Paroxysmal nocturnal hemoglobinuria (see Chapter 23) is caused by a stem cell mutation that modifies membrane-anchored platelet activation suppressors. Venous or arterial thromboses occur in at least 40% of cases.¹⁷

Chronic inflammatory diseases cause thrombosis through a variety of mechanisms, such as elevated fibrinogen and factor VIII, decreased fibrinolysis, promotion of atherosclerotic plaque formation, and reduced free protein S activity secondary to increased C4b-binding protein (C4bBP). Diabetes mellitus is a particularly dangerous chronic inflammatory condition, raising the risk of cardiovascular disease sixfold. Conditions associated with venous stasis, such as congestive heart failure, also are common risk factors for venous thrombosis. Untreated atrial fibrillation increases the risk of ischemic strokes due to clot formation in the right atrium and embolization to the brain.¹⁸ Nephrotic syndrome creates protein imbalances that lead to thrombosis through loss of plasma procoagulants. Nephrotic syndrome may also cause hemorrhage (see Chapter 41).¹⁹

Congenital Thrombosis Risk Factors

Congenital thrombophilia is suspected when a thrombotic event occurs in young adults; occurs in unusual sites such as the mesenteric, renal, or axillary veins; is recurrent; or occurs in a patient who has a family history of the disorder (Table 42-3).²⁰ Because thrombosis is multifactorial, however, even patients with congenital thrombophilia are most likely to experience thrombotic events because of a combination of constitutional and acquired conditions.²¹

TABLE 42-3

Predisposing Congenital Factors and Thrombosis Risk

Risk Factor	Comment	Risk of Thrombosis	Laboratory Tests
AT deficiency (previously called AT III deficiency)	AT inhibits the serine proteases IIa (thrombin), IXa, Xa, and XIa. Antithrombin function is enhanced by heparin.	Heterozygous: increased 10-20× Homozygous: 100%, rarely reported	Clot-based and chromogenic AT activity assays, immunoassays for AT concentration (antigen)
PC deficiency	Activated PC is a serine protease that hydrolyzes factors Va and VIIIa, requires protein S as a cofactor.	Heterozygous: increased 2-5× Homozygous: 100%; causes neonatal purpura fulminans	Clot-based and chromogenic PC activity assays, immunoassay for PC concentration (antigen)
Free PS deficiency	PS is a cofactor for activated protein C, 40% is free, 60% circulates bound to C4bBP.	Heterozygous: increased 1.6-11.5× Homozygous: 100% but rarely reported; causes neonatal purpura fulminans	Clot-based free PS activity assays, free and total PS immunoassays for PS concentration (antigen)
APC resistance	Factor V Leiden (T506Q) mutation gain of function renders factor V resistant to APC.	Heterozygous: increased 3× Homozygous: increased 18×	PTT-based APC resistance test and confirmatory molecular assay
Prothrombin G20210A	Mutation in gene’s untranslated 3′ promoter region creates moderate elevation in prothrombin activity.	Heterozygous: increased 1.6-11.5×	Molecular assay only; phenotypic assay provides no specificity
Dysfibrinogenemia and afibrinogenemia	Association is seen with arterial thrombosis.	Under investigation: acute phase reactant	Fibrinogen clotting assay, thrombin time, reptilase time
Plasminogen mutations	Rare cases have been described.		Chromogenic substrate
Tissue plasminogen activator deficiency		Under investigation	Chromogenic substrate assay
PAI-1 elevation	PAI-1 increase may be common.		Chromogenic substrate assay

APC, Activated protein C; AT, antithrombin; C4bBP, C4b-binding protein; PAI-1, plasminogen activator inhibitor 1; PC, protein C; PS, protein S; PTT, partial thromboplastin time.

The antithrombin test (previously called the antithrombin III or AT III test) has been available since 1972, and protein C and protein S activity assays became available in the mid-1980s. The 1990s brought the APC resistance assay, the confirmatory FVL mutation molecular assay, the prothrombin G20210A mutation molecular assay, and tests for dysfibrinogenemia, plasminogen deficiency, plasma TPA, and plasma PAI-1.

APC resistance is found in 3% to 8% of whites. Resistance extends to Arabs and Hispanics, but the mutation is absent from African and East Asian populations (Table 42-4).²² APC resistance may exist in the absence of the FVL mutation and occasionally is acquired in pregnancy or in association with oral contraceptive therapy.

TABLE 42-4

Prevalence of Congenital Thrombosis Risk Factors in the General Population and in Individuals with Recurrent Thrombotic Disease

Factor	Unselected Population	People with at Least One Thrombotic Event
Activated protein C resistance, factor V Leiden mutation	3-8% of whites, absent in Asians or Africans	20-25%
Prothrombin G20210A	2-3% of whites, absent in Asians or Africans	4-8%
Antithrombin deficiency	1 in 2000 to 1 in 5000	1-1.8%
Protein C deficiency	1 in 300	2.5-5.0%
Protein S deficiency	Unknown	2.8-5.0%
Hyperhomocysteinemia associated with methylenetetrahydrofolate reductase gene mutations	11%	13.1-26.7%
Dysfibrinogenemia	Unknown	1%
Abnormal fibrinolysis	Unknown	2%

The prothrombin G20210A gene mutation is the second most common inherited thrombophilic tendency in patients with a personal and family history of deep vein thrombosis.²³ All together, protein C, protein S, and antithrombin deficiencies are found in 0.2% to 1.0% of the world population. The incidences of dysfibrinogenemia and the various forms of abnormal fibrinolysis (plasminogen deficiency, TPA deficiency, and PAI-1 excess) are under investigation.

Double Hit

Thrombosis often is associated with a combination of genetic defect, disease, and lifestyle influences. The fact that an individual possesses protein C, protein S, or antithrombin deficiency does not mean that thrombosis is inevitable. Many heterozygotes experience no thrombotic event during their lifetimes, whereas others experience clotting only when two or more risk factors converge. A young woman heterozygous for the FVL mutation has a 35-fold increase in thrombosis risk upon starting oral contraceptive therapy. In the Physicians’ Health Study, homocysteinemia tripled the risk of idiopathic venous thrombosis, and the FVL mutation doubled it. When both were present, the risk of venous thrombosis was increased 10-fold.²⁴

Laboratory Evaluation of Thrombophilia

When thrombophilia is suspected, it is important to assess all known risk factors, because it is the combination of positive results that determine the patient’s cumulative risk of thrombosis.²⁵ The presence or absence of laboratory-detected risk factors does not affect anticoagulant treatment, however, when thrombosis is in progress. Current anticoagulant therapy and ongoing or recent thrombotic events affect the interpretation of antithrombin, protein C, protein S, factor VIII, and LA testing. These assays should be performed 10 to 14 days after anticoagulant therapy is withdrawn (Table 42-5).

TABLE 42-5

Complete Thrombophilia Laboratory Test Profile

Assay	Reference Value/Interval	Comments
APC resistance	Ratio ≥1.8	Clot-based screen based on PTT and factor V–depleted plasma.
Factor V Leiden mutation	Wild-type	Molecular assay performed as follow-up to APC resistance ratio of <1.8
Prothrombin G20210A	Wild-type	Molecular assay. There is no phenotypic assay for prothrombin G20210A.
LA profile*	Negative for LA	Minimum of two clot-based assays such as PTT, DRVVT, KCT, or dilute PT with phospholipid neutralization test for immunoglobulins of APL family.
ACL antibody	IgG: <12 GPL IgM: <10 MPL	Immunoassay for immunoglobulins of APL family. ACL depends on β₂-GPI in reaction mix.
Anti–β₂-GPI antibody	<20 G units	Immunoassay for an immunoglobulin of APL family. β₂-GPI is key phospholipid-binding protein in family.
AT activity*	78-126%	Serine protease inhibitor suppresses IIa (thrombin), IXa, Xa, XIa. When consistently below reference limit, follow up with AT antigen assay.
PC activity*	70-140%	Digests VIIIa and Va. When consistently below reference limit, follow up with PC antigen assay.
PS activity*	65-140%	PC cofactor. When consistently below reference limit, follow up with total and free PS antigen assay, C4b-binding protein assay.
Factor VIII activity	50-186%	Confirm elevated factor VIII after 12 wk.
Fibrinogen	220-498 mg/dL	Clot-based assay. Elevation may be associated with arterial thrombosis.
Plasminogen activator inhibitor 1	<30 units/mL	Elevation may be associated with venous thrombosis.

ACL, Anticardiolipin; APC, activated protein C; APL, antiphospholipid; AT, antithrombin; β₂-GPI, β₂-glycoprotein I; DRVVT, dilute Russell viper venom time; GPL, IgG antiphospholipid antibody unit; Ig, immunoglobulin; KCT, kaolin clotting time; LA, lupus anticoagulant; MPL, IgM antiphospholipid antibody unit; PC, protein C, PS, protein S; PT, prothrombin time; PTT, partial thromboplastin time.

^*Inaccurate during active thrombosis or anticoagulant therapy. Perform 14 days after anticoagulant therapy is discontinued.

Antiphospholipid Antibodies

APL antibodies are a family of immunoglobulins that bind protein-phospholipid complexes.²⁶ APL antibodies include LAs, detected by clot-based profiles, and ACL antibodies and anti–β₂-GPI antibodies, detected by immunoassay. Chronic autoimmune APL antibodies are associated with APS, which is characterized by transient ischemic attacks, strokes, coronary and peripheral artery disease, venous thromboembolism, and repeated pregnancy complications.^27,28

APL antibodies arise as immunoglobulin M (IgM), IgG, or IgA isotypes. Because they may bind a variety of protein-phospholipid complexes, they are called nonspecific inhibitors. Their name reflects the fact that they previously were thought to bind phospholipids directly; however, their target antigens are the proteins assembled on anionic phospholipid surfaces.²⁹ The plasma protein most often bound by APL antibodies is β₂-GPI, although annexin V and prothrombin are sometimes implicated. APL antibodies probably develop in response to newly formed protein-phospholipid complexes, and investigators still are learning exactly how they cause thrombosis.^30,31

Clinical Consequences of Antiphospholipid Antibodies

Between 1% and 2% of unselected individuals of both sexes and all races, and 5% to 15% of individuals with recurrent venous or arterial thrombotic disease have APL antibodies.³² Most APL antibodies arise in response to a bacterial, viral, fungal, or parasitic infection or to treatment with numerous drugs (Box 42-1) and disappear within 12 weeks. These are mostly transient alloimmune APL antibodies and have no clinical consequences.³³ Nevertheless, the laboratory scientist must follow up any positive results on APL antibody assays to determine their persistence.

BOX 42-1 Agents Known to Induce Antiphospholipid Antibodies

Various antibiotics

Phenothiazine

Hydralazine

Quinine and quinidine

Calcium channel blockers

Autoimmune APL antibodies are part of the family of autoantibodies that arise in collagen vascular diseases; 50% of patients with systemic lupus erythematosus have autoimmune APL antibodies. Autoimmune APL antibodies are also detected in patients with rheumatoid arthritis and Sjögren syndrome, but may arise spontaneously, a disorder called primary APS. Autoimmune APL antibodies may persist, and fully 30% are associated with arterial and venous thrombosis. Chronic presence of an autoimmune APL antibody not associated with a known underlying autoimmune disorder confers a 1.8-fold to 3.2-fold increased risk of thrombosis.

Detection and Confirmation of Antiphospholipid Antibodies

Clinicians suspect APS in unexplained venous or arterial thrombosis, thrombocytopenia, or recurrent fetal loss.³⁴ Specialized clinical hemostasis laboratories offer APL detection systems that include clot-based assays for LA and immunoassays for ACL and anti–β₂-GPI antibodies. Occasionally, an LA is suspected because of an unexplained prolonged partial thromboplastin time (PTT) that does not correct when the test is repeated in mixing studies (see Chapter 45).^35,36

Lupus Anticoagulant Test Profile

Test systems that employ low-reagent phospholipid concentrations are sensitive to LA.³⁷ There are four such systems, at least two of which are required to make up an LA profile. The need for multiple test systems arises from the multiplicity of LA reaction characteristics, and a confirmed positive result in one system is conclusive despite a negative result in another. The four available systems are PTTs formulated with low reagent phospholipid concentrations and silica-based particulate activators designed to be LA sensitive: the dilute Russell viper venom time (DRVVT), the kaolin clotting time (KCT), and the dilute thromboplastin time (DTT).³⁸ As illustrated in Figure 42-1, the KCT and PTT trigger coagulation at the level of factor XII; DRVVT, at factor X; and DTT, at factor VII (see Chapter 45). The 2009 International Society on Thrombosis and Haemostasis (ISTH) update of guidelines for LA detection recommends using the DRVVT and LA-sensitive PPT as the two primary screening tests (Figures 42-2 and 42-3). This combination of assays updates the following 1995 ISTH requirements for LA identification³⁹:

Figure 42-1 Kaolin clotting time (KCT), partial thromboplastin time (PTT), dilute prothrombin time (DPT), and dilute Russell viper venom time (DRVVT). The simplified coagulation mechanism is used to illustrate that the KCT and PTT initiate coagulation at the level of factor XII, DRVVT at the level of factor X, and DPT at the level of factor VII.

Figure 42-3 Lupus anticoagulant (LA) detection using the dilute Russell viper venom time (DRVVT) system. Use in conjunction with the LA-sensitive partial thromboplastin time detection system (see Figure 42-2). If the DRVVT is prolonged, mix the patient plasma with pooled platelet-free normal plasma (NP), incubate 1 to 2 hours, and repeat. If the DRVVT for the mix is normal, suspect a factor deficiency and confirm. If the DRVVT for the mix is prolonged, proceed to the phospholipid neutralization step to confirm.

1. Prolonged phospholipid-dependent clot formation using a screen such as the low phospholipid PTT, DRVVT, KCT, or DTT

2. Failure to correct the prolonged clot formation by mixing with normal platelet-poor plasma and repeating the test (mixing study)

3. Shortening or correction of the prolonged screen test result by addition of excess phospholipids

4. Exclusion of other coagulopathies

In performing mixing studies, it is essential to use only platelet-poor plasma (plasma centrifuged so that it has a platelet count of less than 10,000/mcL). The use of platelet-poor plasma avoids neutralization of LA by the platelet membrane phospholipids. Platelet membrane fragments formed during freezing and thawing can likewise neutralize LA and lead to a false-negative LA result.

Performing the Clot-Based Lupus Anticoagulant Mixing Study

When the PTT or DRVVT is prolonged and anticoagulant therapy, particularly treatment with unfractionated heparin, has been ruled out, a new aliquot of patient platelet-poor plasma is mixed 1 : 1 with pooled normal platelet-poor plasma, and the assay is repeated on the mixture using the same test system (immediate mix). Some LAs are time and temperature dependent, so if the result for the mixture shows correction to within 10% or to within 5 seconds of the result for the normal platelet-poor plasma, the test should be repeated again after incubating the mix at 37° C for 1 to 2 hours. Each laboratory manager or director must decide what degree of shortening constitutes correction. Many use the Rosner index, which defines correction as a mixture result within 10% of the result of the platelet-poor normal plasma.⁴⁰ Many others define correction as return to a value within the PTT reference interval.

If either the PTT or the DRVVT remains prolonged (that is, uncorrected), the presence of LA is presumed, and confirmation is necessary. Confirmatory tests employ the same assay that originally yielded prolonged results, adding a high-concentration phospholipid reagent such as hexagonal phase phospholipid. If LA was the cause of prolongation, the high-phospholipid test result shortens (corrects).⁴¹

Anticardiolipin Antibody Immunoassay

LA and ACL antibodies coexist in 60% of cases, and both may be found in APS.⁴² The ACL test is an immunoassay that may be normalized among laboratories and is not affected by heparin therapy, oral anticoagulant therapy, current thrombosis, or factor deficiencies.

The manufacturer coats microplate wells with bovine heart cardiolipin and blocks (fills open receptor sites) with a bovine serum solution containing β₂-GPI. Test sera or plasmas are serially diluted and pipetted to the wells along with calibrators and controls. ACL binds the solid-phase cardiolipin–β₂-GPI target complex and cannot be washed from the wells. Enzyme-labeled anti–human IgG, IgM, or IgA conjugates are added. A color-producing substrate is then added, and a color change indicates the presence of ACL. Color intensities of the patient and control samples are compared with the calibrator curve. Results are expressed using GPL, MPL, or APL units, where 1 unit is equivalent to 1 mcg/mL of an affinity-purified standard IgG, IgM, or IgA specimen.⁴³ Reference limits are established in each laboratory.

Anti–β₂-Glycoprotein I Immunoassay

IgM and IgG anti–β₂-GPI immunoassays are performed as a part of the profile that includes ACL assays.⁴⁴ An anti–β₂-GPI result of greater than 20 GPL or MPL units correlates with thrombosis more closely than the presence of ACL antibodies. Any ACL or β₂-GPI assay yielding positive results should be repeated on a new specimen collected after 12 weeks to distinguish a transient alloantibody from a chronic autoantibody (Figure 42-4).

Figure 42-4 Reflexive immunoassay sequence for antiphospholipid (APL) antibodies. If either the immunoglobulin M (IgM) or IgG anticardiolipin (ACL) immunoassay or the IgM or IgG anti–β₂-glycoprotein I (anti–β₂-GPI) immunoassay yields a positive result, confirm to establish chronicity after 12 weeks. If either is confirmed, the patient has a clinically significant APL antibody. If the initial ACL and anti–β₂-GPI results are negative but an APL antibody is suspected, perform the antiphosphatidylserine (APTS) immunoassay. If the result is positive, repeat after 12 weeks to establish chronicity.

Antiphosphatidylserine Immunoassay

For cases in which an APL antibody is suspected but the routine LA, ACL, and β₂-GPI assay results are negative, the clinician may wish to order the antiphosphatidylserine immunoassay to detect APL antibodies specific for phosphatidylserine.⁴⁵ A result greater than or equal to 16 IgG or 22 IgM antiphosphatidylserine units is considered positive. The antiphosphatidylserine assay is available from specialty reference laboratories.

Activated Protein C Resistance and Factor V Leiden Mutation

Clinical Importance of Activated Protein C Resistance

Activated factors V and VIII (factors Va and VIIIa) are inactivated by the APC–protein S complex. A mutation in the factor V gene substitutes glutamine for arginine at position 506 of the factor V molecule (FV R506Q). The arginine is a cleavage site for APC, so the substitution slows or prevents hydrolysis of the factor V molecule (Figure 42-5). Resistant factor Va remains active and increases the production of thrombin. The factor V R506Q mutation is named for the city in the Netherlands in which it was first described, Leiden (FVL mutation). Between 3% and 8% of Northern European whites possess FVL (see Table 42-4).⁴⁶ Owing to its prevalence and the associated 3-fold higher thrombosis risk (18-fold higher for homozygotes), the acute care hemostasis laboratory must provide APC resistance detection to screen for FVL.⁴⁷

Figure 42-5 Factor V Leiden mutation. A point mutation of the factor V gene results in the substitution of glutamine for arginine at position 506 (R506Q) of the factor V molecule. The normal arginine at position 506 is a cleavage site for activated protein C (APC), so the substitution slows or prevents cleavage of the factor V molecule. D, Aspartic acid; G, glycine; I, isoleucine; L, leucine; R, arginine, Q, glutamine.

Activated Protein C Resistance Clot-Based Assay

In the APC resistance clot-based assay, patient plasma is mixed 1 : 4 with factor V–depleted plasma.⁴⁸ PTT reagent is added to two aliquots of the mixture and incubated for 3 minutes (Figure 42-6). A solution of calcium chloride is pipetted into one mixture, and the clot formation is timed. A solution of calcium chloride with APC is added to the second mixture and clotting is timed. The normal ratio of PTT results between the two assays is 1.8 or greater. In APC resistance, the ratio is less than 1.8.⁴⁹

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