Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 39. Thrombotic disorders and laboratory assessment

George A. Fritsma*


Developments in Thrombosis Risk Testing

Etiology and Prevalence of Thrombosis

Thrombosis Etiology

Prevalence of Thrombosis

Thrombosis Risk Factors

Acquired Thrombosis Risk Factors

Thrombosis Risk Factors Associated with Systemic Diseases

Congenital Thrombosis Risk Factors

Double Hit

Laboratory Evaluation of Thrombophilia

Antiphospholipid Antibodies

Activated Protein C Resistance and Factor V Leiden Mutation

Prothrombin G20210A


Protein C Control Pathway

Arterial Thrombosis Predictors

C-Reactive Protein

Plasma Homocysteine

Fibrinogen Activity

Lipoprotein (a)

Disseminated Intravascular Coagulation




Laboratory Diagnosis

Specialized Laboratory Tests That May Aid in Diagnosis


Localized Thrombosis Monitors

Heparin-Induced Thrombocytopenia

Cause and Clinical Significance

Platelet Count

Laboratory Tests for HIT




After completion of this chapter, the reader will be able to:

1. Describe the prevalence of thrombotic disease in developed countries.

2. Define thrombophilia.

3. Distinguish between venous and arterial thrombosis.

4. Differentiate among acquired thrombosis risk factors related to lifestyle and disease, and congenital risk factors, listing which factors can be assessed in the hemostasis laboratory.

5. Discuss the frequency with which the heritable risk factors occur in various ethnic groups.

6. Offer a sequence of lupus anticoagulant antibody tests that provides the greatest diagnostic validity and interpret the results of the tests.

7. Describe the relevance of antithrombin assays, proteins C and S assays, activated protein C resistance, the factor V Leiden assay, and the prothrombin G20210A assay to assess venous thrombotic risk.

8. Describe the relevance of the tests for high-sensitivity C-reactive protein, homocysteine, fibrinogen, and lipoprotein (a) to assess arterial thrombotic risk.

9. Describe the causes and pathophysiology of disseminated intravascular coagulation (DIC).

10. Describe the assays comprising a primary test profile for diagnosis and management of DIC in an acute care facility.

11. Discuss the value of quantitative D-dimer assays.

12. Describe the cause and clinical significance of heparin-induced thrombocytopenia.

13. Describe the clinical diagnosis, laboratory diagnosis, and management of heparin-induced thrombocytopenia.


After studying the material in this chapter, the reader should be able to respond to the following case study:

A 42-year-old woman with no significant medical history developed sudden onset of shortness of breath and chest pain. She was taken to an emergency department, where a pulmonary embolism was diagnosed. After admission, she was treated with intravenous heparin and given a hypercoagulability workup.

1. For what conditions can the woman be tested while she is in the hospital?

2. List possible acquired risk factors for thrombosis that need to be excluded in this patient.

3. What would be the implications of diagnosing a congenital risk factor for thrombosis in this patient?

Developments in thrombosis risk testing

Before 1992, medical laboratory professionals performed assays to detect only three inherited venous thrombosis risk factors: deficiencies of the coagulation control factors antithrombin, protein C, and protein S.1Taken 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 and colleagues2 of activated protein C (APC) resistance in 1993 and the characterization by Bertina and colleagues3 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 increased its workload exponentially. The list of current assays includes antithrombin, protein C, protein S, APC resistance, FVL mutation, prothrombin G20210A mutation, lupus anticoagulant (LA), and several additional markers of both venous and arterial thrombotic disease described in this chapter. Technical improvements have enhanced the diagnostic efficacy of integrated LA detection kits, the quantitative D-dimer assay, and tests for localized coagulation activation markers such as prothrombin fragment 1+2 (PF 1+2, PF 1.2) and thrombin-antithrombin (TAT) complex.4

Etiology and prevalence of thrombosis

Thrombosis etiology

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 (once called hypercoagulability) 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 blood coagulation system activation

• Uncontrolled fibrinolysis suppression

Prevalence of thrombosis

From 2000 to 2010, the U.S. death rate attributable to venous and arterial thrombotic disease declined 31%, and the number of thrombosis-related deaths declined by 17% per year. Yet, in 2010 thrombosis accounted for one of every three deaths in the United States. Of these, 25% of initial thrombotic events were fatal, and many fatal thromboses went undiagnosed before autopsy.6

Prevalence of venous thromboembolic disease

The annual incidence of venous thromboembolic disease (or venous thromboembolic events, VTE) in the unselected U.S. population has remained constant for at least 25 years at 1 in 1000 and is more prevalent in African Americans and in women of childbearing age.78 The most prevalent VTE is deep vein thrombosis, caused by clots that form in the iliac, popliteal, and femoral veins of the calves and upper legs.9 Large occlusive thrombi also may form, although less often, in the veins of the upper extremities, liver, spleen, intestines, brain, and kidneys. Thrombosis symptoms include localized pain, the sensation of heat, 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 necrosis of lung tissue.10 Nearly 95% of these pulmonary emboli arise from thrombi in the deep leg and calf veins. Of the 250,000 U.S. residents per year who suffer pulmonary emboli, 10% to 15% die within 3 months. Many pulmonary emboli go undiagnosed because of the ambiguity of the symptoms, which may resemble those of heart disease or pneumonia. Predilection for deep venous thrombosis versus pulmonary embolism shows a familial distribution. Coagulation system imbalances, such as inappropriate activation, gain of coagulation factor function, inadequate control of thrombin generation, or suppressed fibrinolysis, are the mechanisms that cause VTE; components of cancer, or chronic heart, lung, or renal disease are often implicated in VTE.11

Prevalence of arterial thrombosis

Cardiovascular disease caused 380,000 premature U.S. deaths in 2010, and 790,000 strokes accounted for 1 in 19 premature deaths (deaths before 78 years of age). Approximately 80% of acute 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 vessel wall unstable atherosclerotic plaque. 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 (Chapter 37). Small plaques rupture, occluding arteries and releasing mediators that trigger thrombotic events. The mediators activate platelets, which combine with von Willebrand factor to form arterial platelet plugs—the “white thrombi” that cause ischemia and necrosis of surrounding tissue (Chapter 13).

The hemostasis-related lesions we associate with arterial thrombosis are blood vessel wall destruction and platelet activation. Often these are inseparable. Researchers continue to examine new thrombosis markers that capture pathological 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 circumstances that either help maintain or damage our hemostasis systems. Their variety and interplay make it difficult to pinpoint 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. lists the nondisease risk factors implicated in thrombosis.Table 39-113

TABLE 39-1

Nondisease Risk Factors That Contribute to Thrombotic Disease

Risk Factor


Contribution to Thrombosis

Laboratory Diagnosis


Thrombosis after age 50

Risk doubles each decade after 50



Distance driving, air travel, restriction to wheelchair or bed, obesity

Slowed blood flow raises thrombosis risk



Fatty foods; inadequate folate, vitamin B6, and vitamin B12

Homocysteinemia associated with 2× to 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 arterial 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 μg, formulation with progesterone

4× to 6× increased risk




3× to 5× increased risk


Hormone replacement therapy


2× to 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




Depends on degree




Arterial thrombosis


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 , with an indication of the laboratory’s diagnostic contribution.Table 39-214

TABLE 39-2

Diseases with Thrombotic Risk Components


Examples or Effects

Contribution to Thrombosis

Laboratory Diagnosis

Antiphospholipid syndrome

Chronic antiphospholipid antibody often secondary to autoimmune disorders

When chronic, 1.6× to 3.2× increased risk of stroke, myocardial infarction, recurrent spontaneous abortion, venous thrombosis

PTT mixing studies, lupus anticoagulant profile, anticardiolipin antibody and 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 aggregometry

Hepatic disease

Diminished production of most 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: adenocarcinoma

Trousseau syndrome, low-grade chronic DIC

20× increased risk of thrombosis; 10% to 20% of people with idiopathic venous thrombosis have cancer

DIC profile: platelet count, D-dimer, PTT, PT, fibrinogen, blood film examination


Acute promyelocytic leukemia (M3), acute monocytic leukemia, (M4-M5)

Increased risk for chronic DIC

DIC profile: platelet count, D-dimer, PTT, PT, fibrinogen, blood film examination

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, PTT, PT, fibrinogen, blood film examination

Chronic inflammation

Diabetes, cancer, infection, autoimmune disorder, obesity, smoking


Fibrinogen, HSCRP

DIC, Disseminated intravascular coagulation; PT, prothrombin time; PTT, partial thromboplastin time, HSCRP, high-sensitivity C-reactive protein.

Together, transient and chronic antiphospholipid (APL) antibodies such as the lupus anticoagulant (LA), anticardiolipin (ACL) antibodies, and anti–β 2-glycoprotein I (anti–β2-GPI) antibodies may be detected in 1% to 2% of the unselected population.15 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, venous and arterial 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.16

Myeloproliferative neoplasms such as essential thrombocythemia and polycythemia vera (Chapter 33) may trigger thrombosis, probably through platelet hyperactivity. A cardinal sign of acute promyelocytic leukemia(Chapter 35) is DIC secondary to the release of procoagulant granules from malignant promyelocytes. DIC can intensify during therapy at the time of vigorous cell lysis.17 Paroxysmal nocturnal hemoglobinuria(PNH) (Chapter 24) is caused by a stem cell mutation that modifies membrane-anchored platelet activation suppressors. Venous or arterial thromboses occur in at least 40% of PNH cases.18

Chronic inflammatory diseases cause thrombosis through a variety of mechanisms, such as elevation of fibrinogen and factor VIII, suppressed fibrinolysis, promotion of atherosclerotic plaque formation, and reduced free protein S activity secondary to raised C4b-binding protein (C4bBP) levels. Diabetes mellitus is a particularly dangerous chronic inflammatory condition, raising the risk of cardiovascular disease sixfold.19 Conditions associated with venous stasis, such as congestive heart failure, also are risk factors for venous thrombosis. Untreated atrial fibrillation increases the risk of ischemic strokes caused by clot formation in the right atrium and embolization to the brain.20 Nephrotic syndrome creates protein imbalances that lead to thrombosis through loss of plasma proteins such as antithrombin. Nephrotic syndrome may also cause hemorrhage (Chapter 38).21

Congenital thrombosis risk factors

Clinicians suspect congenital thrombophilia 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 thrombosis ().Table 39-322 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.23

TABLE 39-3

Predisposing Congenital Factors and Thrombosis Risk

Risk Factor


Risk of Thrombosis

Laboratory Tests

AT (previously AT III) deficiency

AT, enhanced by heparin, inhibits the serine proteases IIa (thrombin), IXa, Xa, and XIa

Heterozygous: increased 10× to 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 stabilizing cofactor

Heterozygous: increased 2× to 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 stabilizing cofactor for activated protein C, 40% is free, 60% circulates bound to C4bBP

Heterozygous: increased 1.6× to 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 (R506Q) 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 prothrombin gene untranslated 3′ promoter region creates moderate elevation in prothrombin activity

Heterozygous: increased 1.6× to 11.5×

Molecular assay only; phenotypic assay provides no specificity


Associated with arterial thrombosis

Under investigation: acute phase reactant

Clauss fibrinogen clotting assay, immunoassay, nephelometric assay

APC, Activated protein C; AT, antithrombin; C4bBP, complement component C4b binding protein; PC, protein C; PS, protein S; PTT, partial thromboplastin time.

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

APC resistance is found in 3% to 8% of Caucasians worldwide. APC resistance extends to Arabs and Hispanics, but the mutation is nearly absent from African and East Asian populations ().Table 39-424 APC resistance may exist in the absence of the FVL mutation and is occasionally acquired in pregnancy or in association with oral contraceptive therapy.25

TABLE 39-4

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


Unselected Population

People with at Least One Thrombotic Event

Activated protein C resistance, factor V Leiden mutation

3% to 8% of Caucasians, rare in Asians or Africans


Prothrombin G20210A

2% to 3% of Caucasians, rare in Asians or Africans


Antithrombin deficiency

1 in 2000 to 1 in 5000


Protein C deficiency

1 in 300


Protein S deficiency



Hyperhomocysteinemia associated with methylenetetrahydrofolate reductase gene mutations



The FVL gene mutation is the most common inherited thrombophilia, and the prothrombin G20210A gene mutation is the second most common inherited thrombophilia tendency in patients with a personal and family history of deep vein thrombosis.26 Altogether, protein C, protein S, and antithrombin deficiencies are found in only 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. Just because someone 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 who is heterozygous for the FVL mutation has a thirty-fivefold 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 tenfold.27

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 determines the patient’s cumulative risk of thrombosis.28 Table 39-5summarizes the commonly used thrombosis risk assays and indicates those that can be relied on while the patient is on antithrombotic therapy or while the patient is recovering from an acute thrombotic event.

TABLE 39-5

Thrombophilia Laboratory Test Profile


Reference Value/Interval


APC resistance

Ratio ≥1.8

Clot-based screen that employs PTT with factor V-depleted plasma.

Factor V Leiden mutation


Molecular assay performed as follow-up to APC resistance ratio that is < 1.8.

Prothrombin G20210A


Molecular assay. There is no phenotypic assay for prothrombin G20210A.

LA profile*

Negative for LA

Minimum of two clot-based assays. Primary assays are based on PTT and DRVVT, secondary assays based on KCT, or dilute PT. All four include phospholipid neutralization follow-up test.

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*


Serine protease inhibitor suppresses IIa (thrombin), IXa, Xa, XIa. When consistently below reference limit, follow up with AT antigen assay.

PC activity*


Digests VIIIa and Va. When consistently below reference limit, follow up with PC antigen assay.

PS activity*


PC cofactor. When consistently below reference limit, follow up with total and free PS antigen assay, C4b-binding protein assay.


220–498 mg/dL

Clot-based assay. Elevation may be associated with arterial thrombosis.

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

ACL, anticardiolipin; APC, activated protein C; APL, antiphospholipid antibody; 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.

The presence or absence of laboratory-detected risk factors does not affect anticoagulant treatment when thrombosis is in progress.29 However, it is important to realize that current anticoagulant therapy and ongoing or recent thrombotic events interfere with the interpretation of antithrombin, protein C, protein S, factor VIII, and LA testing. These assays should be performed at least 14 days after anticoagulant therapy is discontinued.

Antiphospholipid antibodies

APL antibodies comprise a family of immunoglobulins that bind protein-phospholipid complexes.30 APL antibodies include LAs, detected by clot-based profiles, and ACL 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 recurrent pregnancy complications, including spontaneous abortions.3132

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 implies that they were once thought to directly bind phospholipids; however, their target antigens are actually the proteins that assemble on anionic phospholipid surfaces.33 The plasma protein most often bound by APL antibodies is β2-GPI, although annexin V and prothrombin are sometimes implicated as APL targets. APL antibodies probably develop in response to newly formed protein-phospholipid complexes, and laboratory scientists continue to investigate how they cause thrombosis.3435

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.36 Most APL antibodies arise in response to a bacterial, viral, fungal, or parasitic infection or to treatment with numerous drugs (Box 39-1) and disappear within 12 weeks. These are mostly transient alloimmune APL antibodies and have no clinical consequences.37 Nevertheless, the laboratory professional must follow up any positive results on APL antibody assays to determine their persistence.

BOX 39-1

Agents Known to Induce Antiphospholipid Antibodies

Various antibiotics



Quinine and quinidine

Calcium channel blockers




Elevated estrogens

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, scleroderma, 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.38 Specialized clinical hemostasis laboratories offer APL detection profiles 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 in mixing studies (Figure 39-1Chapter 42).3940


FIGURE 39-1 Mixing study employing a partial thromboplastin time (PTT) reagent with intermediate lupus anticoagulant sensitivity. Beginning at the top left, when the PTT result exceeds the upper limit of the PTT reference interval, perform a thrombin clotting time (TT) to detect unfractionated heparin. If the TT exceeds the TT reference interval, presume that heparin is present. Treat an aliquot of the specimen with Hepzyme and repeat the PTT. If the new PTT is normal, assay the original sample for heparin. If the PTT remains prolonged, or if the TT was normal, proceed by mixing the patient plasma with control normal plasma (NP) and perform a PTT on the mixture. If the PTT is still prolonged (uncorrected) in comparison to the NP, proceed to assay for the lupus anticoagulant. If the PTT mixture corrects, prepare a new 1:1 mix and incubate at 37° C for 1 to 2 hours and repeat the PTT, comparing the result to incubated NP. If the incubated PTT shows a correction, assay for a factor deficiency. If prolonged, assay for a lupus anticoagulant (Figs. 39-2 and 39-3) (a specific factor inhibitor such as anti-factor VIII associated with bleeding will also be detected in this manner).

Lupus anticoagulant test profile

Clot-based assays with reduced reagent phospholipid concentrations are sensitive to LA. There are two commonly used test systems, and both are required for an LA profile. The need for two parallel assay systems arises from the multiplicity of LA reaction characteristics: a confirmed positive result in one system is conclusive despite a negative result in the other. The two most commonly recommended test systems are the dilute Russell viper venom time (DRVVT) and the silica-based partial thromboplastin time (PTT), both formulated with low-phospholipid concentrations designed to be LA sensitive.41 Two older systems, still used in many institutions and available from specialty laboratories and coagulation reagent distributors, are the kaolin clotting time (KCT) and the dilute thromboplastin time (DTT, also named tissue thromboplastin inhibitor test, TTI).42 As illustrated in Figure 39-2, the KCT and PTT initiate coagulation at the level of factor XII; DRVVT at factor X; and DTT at factor VII.


FIGURE 39-2 Simplified coagulation pathway illustrating the activation points of the lupus anticoagulant (LA) assays. The dilute Russell viper venom time (DRVVT) assay, regarded as the primary LA detection method, activates factor X (ten). DRVVT is typically accompanied by a low-phospholipid LA-sensitive partial thromboplastin time assay (PTT) that activates factor XII. The historic kaolin clotting time (KCT) also activates factor XII, and the dilute thromboplastin time test (DTT), still available, activates factor VII.

The 2009 International Society on Thrombosis and Haemostasis (ISTH) update of guidelines for LA detection provides the following sequence of assays:43

1. Prolonged phospholipid-dependent clot formation using an initial screen assay such as a low phospholipid PTT or DRVVT.

2. Failure to correct the prolonged clot time when mixing with normal platelet-poor control plasma and repeating the test (see mixing study below).

3. Shortening or complete correction of the prolonged screen assay result by addition of a reagent formulated with excess phospholipids.

4. Exclusion of other coagulopathies.

Performing the clot-based lupus anticoagulant mixing study. 

Laboratory practitioners perform the LA profile upon clinician request, often based on adverse thrombotic or obstetric events, or when a prolonged PTT raises the presumption of an LA. Most laboratory protocols begin with a mixing study where the PTT is performed on patient plasma combined with control normal plasma (NP) (Figure 39-1Chapter 42). The mixing study includes a 37° C incubation step, as many LAs and specific inhibitors require an incubation to enhance their avidity. Practitioners typically perform the mixing study using a PTT reagent with intermediate LA sensitivity but may substitute the prothrombin time (PT) reagent, DRVVT, or an LA-sensitive low-phospholipid PTT reagent. The mixing study includes a means for detecting unfractionated heparin, most often the thrombin clotting time or the chromogenic anti-Xa heparin assay. The practitioner may add heparinase (Hepzyme®, Siemens Healthcare USA, Inc., Malvern, PA) to the sample to neutralize heparin, although this may be unnecessary because many LA detection reagents provide heparin-neutralizing polybrene. Mixing studies that use a PT, a DRVVT, or an LA-sensitive PTT reagent seldom include the 37° C incubation step.

The NP that is mixed 1:1 with patient plasma will shorten a prolonged PTT. Each laboratory director decides what degree of PTT “shortening” constitutes “correction.” Many use the Rosner index, which defines correction as a mixture result within 10% of the NP result.44 Others define correction as return to a value within 5 seconds of the NP result or return to a value within the PTT reference interval.

In performing mixing studies, the laboratory professional employs only platelet-poor plasma—plasma centrifuged so that it has a platelet count of less than 10,000/μL (Chapter 42). The use of platelet-poor plasma avoids neutralization of LA by the platelet membrane phospholipids. Platelet membrane fragments that form during freezing and thawing can likewise neutralize LA and lead to a false-negative LA result.

Performing clot-based lupus anticoagulant tests. 

Following a mixing study that suggests the presence of an LA, specific testing for the LA is performed. Most LA protocols begin with the DRVVT, considered the most specific of the LA assays (). If the DRVVT screening reagent-patient plasma result exceeds the NP result by a predetermined ratio, frequently near 1.2, LA is presumed. The practitioner then confirms LA by mixing an aliquot of the patient sample with the DRVVT high-phospholipid confirmatory reagent, comparing the result in seconds to the original DRVVT screening reagent patient plasma result. The reagent phospholipid neutralizes LA and shortens the DRVVT. If the original screening reagent result exceeds the DRVVT confirm reagent result by a predetermined ratio, again near 1.2, LA is confirmed. Figure 39-3


FIGURE 39-3 Lupus anticoagulant (LA) algorithm. When LA is suspected, perform a dilute Russell viper venom (DRVVT) screen, comparing the patient DRVVT screen result to the control normal plasma (NP) DRVVT screen result. If the ratio of patient to NP DRVVT in seconds is greater than 1.2, mix patient plasma 1:1 with high-phospholipid DRVVT confirm reagent and perform a new DRVVT. If the patient DRVVT screen result exceeds the DRVVT confirm/patient plasma result by greater than 1.2, LA is confirmed. If the ratio is 1.2 or less or if the original DRVVT screen ratio was 1.2 or less, proceed to a silica-based partial thromboplastin (PTT) screen and confirm. Compare the patient silica-based PTT screen to the NP screen result. If the ratio is 1.2 or less, no LA is present. If the ratio is greater than 1.2, mix the patient plasma with high-phospholipid silica-based PTT confirm reagent and perform a new PTT. If the patient PTT screen exceeds the patient PTT confirm by more than 1.2, LA is confirmed. If the ratio is 1.2 or less, LA is not present.

If the original DRVVT screen ratio was less than 1.2, the practitioner turns to the silica-based low-phospholipid LA-sensitive PTT and repeats the steps used for the DRVVT, again basing results on a predetermined ratio, often 1.2.4546

There exist numerous modifications to this algorithm. Many laboratory directors prefer to begin with the silica-based low-phospholipid PTT assay, and others include an intermediate NP mixing study step. Some incorporate the KCT or DTT. Some assay systems use an absolute difference, in seconds, instead of a ratio, often 8 seconds. The 1.2 ratio and the 8-second difference are examples; each institution establishes its own reference interval and threshold ratio or difference.

Some laboratory directors choose to normalize ratios using the mean of the reference interval (MRI) or the NP value. The formula for normalization using the MRI is:


Anticardiolipin antibody immunoassay

LA and ACL antibodies coexist in 60% of cases, and both may be found in APS. 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. The laboratory practitioner pipettes test sera or plasmas to the wells alongside calibrators and controls (Figure 39-4). ACL binds the solid-phase cardiolipin–β2-GPI target complex and cannot be washed from the wells. The practitioner adds enzyme-labeled anti–human IgG, IgM, or IgA conjugates subsequent to washing, followed by a color-producing substrate. A color change indicates the presence of ACL and color intensities of the patient, and control sample wells are compared with the calibrator curve wells. Results are expressed using GPL, MPL, or APL units, where 1 unit is equivalent to 1 μg/mL of an affinity-purified standard IgG, IgM, or IgA specimen.47Reference limits are established in each laboratory.48


FIGURE 39-4 Antiphospholipid antibody (APL) immunoassay algorithm. If an APL is suspected, perform an anticardiolipin (ACL) or anti–β2-glycoprotein I (anti–β2-GPI) immunoassay. If either is positive, confirm chronicity using a new specimen collected at least 12 weeks later. If negative, perform immunoassay to detect an antiphosphatidyl serine (APTS) antibody. If positive, repeat after 12 weeks.

Anti–β2-glycoprotein i immunoassay

The practitioner performs IgM and IgG anti–β2-GPI immunoassays as a part of the profile that includes ACL assays. An anti–β2-GPI result of greater than 20 IgG or IgM anti–β2-GPI units correlates with thrombosis more closely than the presence of ACL antibodies. Any ACL or anti–β2-GPI assay yielding positive results is repeated on a new specimen collected after 12 weeks to distinguish a transient alloantibody from a chronic autoantibody.

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.49 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

The activated protein C (APC)–protein S complex normally hydrolyzes activated factors V and VIII (factors Va and VIIIa). A mutation in the factor V gene substitutes glutamine for arginine at position 506 of the factor V molecule (FV R506Q). The arginine molecule is a normal cleavage site for APC, so the substitution slows or resists APC hydrolysis (Figure 39-5). The resistant factor Va remains active and raises the production of thrombin, leading to thrombosis. The factor V R506Q mutation is named for the city in The Netherlands in which it was first described: Leiden [factor V Leiden (FVL) mutation, also referred to asAPC resistance]. Between 3% and 8% of Northern European Caucasians possess the FVL mutation (Table 39-4).50 Owing to its prevalence and the associated threefold higher thrombosis risk (eighteenfold higher for homozygotes), most acute care hemostasis laboratory directors provide APC resistance detection to screen for FVL.5152


FIGURE 39-5 Factor V Leiden mutation. A point mutation within the factor V gene results in the substitution of amino acid 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 glutamine 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.53 PTT reagent is added to two aliquots of the mixture and incubated for 3 minutes (Figure 39-6). 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 time to clot formation of the second aliquot is at least 1.8 times the time to clot formation of the first (prolonged time to clot due to the increased amount of APC), so the normal ratio of PTT results between the two assays is 1.8 or greater. In APC resistance, the ratio is less than 1.8.54


FIGURE 39-6 Activated protein C resistance ratio (APCR) measurement. Patient plasma is mixed 1:4 with factor V–depleted plasma and partial thromboplastin time (PTT) reagent. Two aliquots of this mixture are then tested: one aliquot is mixed with calcium chloride (CaCl2) alone, and the other aliquot is mixed with CaCl2 plus activated protein C (APC). The reaction in the mixture with the APC should be prolonged to a clotting time at least 1.8 times longer than the mixture without APC. A ratio of 1.8 or less implies APC resistance.

Performance characteristics of the activated protein c resistance test. 

Factor V–depleted plasma compensates for potential factor deficiencies and for oral anticoagulant therapy by providing normal coagulation factors. The laboratory professional uses only platelet-poor plasma in the APC resistance test to prevent loss of sensitivity caused by the abundant release of platelet factor 4. APC resistance reagent test kits contain polybrene or heparinase to neutralize UFH. LA, however, affects the test system adversely.55 If LA is present, the molecular test for FVL is indicated.56

Factor V leiden mutation assay

Most laboratories confirm the APC resistance diagnosis using the molecular FVL mutation test. The determination of zygosity is important to predict the risk for thrombosis and establish a treatment regimen.

Prothrombin G20210A

A guanine-to-adenine mutation at base 20210 of the 3′ untranslated region of the prothrombin (factor II) gene has been associated with mildly elevated plasma prothrombin levels, averaging 130%.52 The increased risk of thrombosis in those with the mutation seems to be related to the elevated prothrombin activity.53 The prevalence of this mutation among individuals with familial thrombosis is 5% to 18%, whereas prevalence worldwide is 0.3% to 2.4%, depending on race.57 The risk of venous thrombosis in heterozygotes is only two to three times the baseline risk. Although the mutation may cause a slight prothrombin elevation, a phenotypic prothrombin activity assay is of little diagnostic value because there is considerable overlap between normal prothrombin levels and prothrombin levels in people with the mutation.5859


Antithrombin (previously named antithrombin III or ATIII) is a serine protease inhibitor (SERPIN) that neutralizes factors IIa (thrombin), IXa, Xa, XIa, and XIIa, all the serine proteases except factor VIIa. Antithrombin activity is enhanced by unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and synthetic pentasaccharide (fondaparinux) (Figures 39-7 and 39-8). Antithrombin was the first of the plasma coagulation control proteins to be identified and the first to be assayed routinely in the clinical hemostasis laboratory.55 Other members of the serpin family are heparin cofactor II, α2-macroglobulin, α2-antiplasmin, and α1-antitrypsin. Typically, hemostasis specialty laboratories or reference laboratories are the only places that assay serpins other than the more commonly ordered antithrombin activity, which has found its way into laboratories at most acute care facilities.


FIGURE 39-7 The reaction between antithrombin (AT) and activated coagulation factor II (IIa; thrombin) is supported by heparin. Standard unfractionated heparin, with a molecular weight of 5000 to 40,000 Daltons, provides polysaccharide chains of at least 17 sugar subunits. Heparin molecules of this length support the reaction between AT and IIa, as AT and IIa assemble on the heparin molecule. The AT molecule attaches to a specific pentasaccharide sequence. The IIa possesses a heparin-binding site that enables it to assemble on the heparin surface adjacent to the AT, a property called approximation. The AT becomes sterically modified (allostery), supporting a covalent reaction between the AT protease binding site and the IIa active protease site. Thrombin (IIa) and antithrombin, covalently bound, release from heparin and form measurable plasma thrombin-antithrombin (TAT) complexes, useful as a marker of coagulation activation.


FIGURE 39-8 The reaction between antithrombin (AT) and activated coagulation factor X (Xa) is supported by low-molecular-weight heparin. Low-molecular-weight heparin, with a molecular weight of less than 8000 Daltons, provides polysaccharide chains of six or seven sugar subunits. Heparin molecules of this length support the reaction between AT and Xa. The AT molecule attaches to a specific pentasaccharide sequence. The Xa does not possess a heparin-binding site and does not need to assemble on the heparin surface adjacent to the AT. The AT becomes sterically modified (allostery), supporting a covalent reaction between the AT protease binding site and the Xa active protease site.

Antithrombin deficiency

Acquired antithrombin deficiency occurs in liver disease, in nephrotic syndrome, with prolonged heparin therapy, with asparaginase therapy, with the use of oral contraceptives, and in DIC, where antithrombin is rapidly consumed. Congenital deficiency is present in 1 in 2000 to 1 in 5000 of the general population and accounts for 1.0% to 1.8% of recurrent venous thromboembolic disease cases.60 About 90% of cases of antithrombin deficiency are quantitative (reduced production), or type I; the remainder are caused by mutations creating structural abnormalities in the antithrombin protease binding site or the heparin bindingsite. Type II mutations do not reduce antithrombin production, but the molecules are nonfunctional.61

Antithrombin reference intervals

Adult plasma antithrombin activity ranges from 78% to 126%, whether measured by clot-based or chromogenic assay. Antithrombin antigen levels range from 22 to 39 mg/dL (68% to 128%) by latex microparticle immunoassay, and the plasma biologic half-life is 72 hours. Adult levels are reached by 3 months of age, and levels remain steady throughout adult life, except during periods of physiologic challenge, such as pregnancy. Antithrombin activity decreases with age.62

Antithrombin activity assay

Laboratory practitioners screen for antithrombin deficiency using a clot-based or chromogenic functional assay. The clot-based assay has been available since 1972, but most laboratory directors choose the chromogenic assay because of its stability and reproducibility. The operator mixes test plasma with a solution of heparin and factor Xa () and incubates the mixture at 37° C for several minutes. During incubation, the heparin-activated plasma antithrombin irreversibly binds a proportion of the reagent factor Xa. Residual factor Xa hydrolyzes a chromogenic substrate, added as a second reagent. The degree of hydrolysis, measurable by colored end product intensity, is inversely proportional to the activity of antithrombin in the test plasma.Figure 39-963 The chromogenic substrate test for plasma antithrombin activity detects quantitative and qualitative antithrombin deficiencies and detects mutations affecting the proteolytic site but not the heparin binding site. Clot-based antithrombin assays are affected by, and therefore detect, mutations in both the proteolytic and heparin binding sites.


FIGURE 39-9 Chromogenic antithrombin (AT) functional assay. Patient plasma is pipetted into a reagent consisting of heparin, a measured concentration of activated coagulation factor X (Xa), and a chromogenic substrate. AT is activated by heparin and binds Xa. Excess Xa hydrolyzes the substrate and produces a yellow product, para-nitroaniline (pNA). The intensity of pNA color is inversely proportional to AT activity.

Antithrombin antigen assay

Antithrombin concentration is measured in a turbidometric microparticle immunoassay using a suspension of latex microbeads coated with antibody to antithrombin. In the absence of antithrombin, the wavelength of incident monochromatic light exceeds the latex microparticle diameter, so the light passes through unabsorbed.64 In the presence of antithrombin, the particles form larger aggregates. The antithrombin concentration is directly proportional to the rate of light absorption change. Antithrombin antigen levels are diminished in quantitative (type I) but not qualitative (type II) antithrombin deficiency. Oral anticoagulant Coumadin therapy may raise the antithrombin level and mask a mild deficiency. Antithrombin activity remains decreased for 10 to 14 days after surgery or a thrombotic event, so the assay should not be used to establish a congenital deficiency during this period.65

Heparin resistance and the antithrombin assay

Antithrombin may become decreased during prolonged or intense heparin therapy and may be largely consumed if the patient has a congenital antithrombin deficiency. In this instance, heparin may be administered in therapeutic or higher dosages, but it neither exerts an anticoagulant effect nor is detected by the PTT. This is known as heparin resistance. In such cases, an antithrombin assay is necessary to confirm antithrombin deficiency. Antithrombin deficiency may be treated with antithrombin concentrate (Thrombate III; Grifols, Inc., Los Angeles, CA).66

Protein C control pathway

Thrombin is an important coagulation factor because it cleaves fibrinogen, activates platelets, and activates factors V, VIII, XI, and XIII. In the intact vessel where clotting would be pathological, thrombin binds endothelial cell membrane thrombomodulin and becomes an anticoagulant.67 How does this paradox happen? The thrombin–thrombomodulin complex activates plasma protein C, and the APC binds free plasma protein S (Figure 39-10).68 The stabilized APC–protein S complex, simultaneously bound to the endothelial protein C receptor, hydrolyzes factors Va and VIIIa to slow coagulation. Recurrent venous thrombosis is the potential consequence of protein C or protein S deficiency.69


FIGURE 39-10 Protein C pathway. Thrombin (activated coagulation factor II or IIa) binds constitutive thrombomodulin (TM) on the endothelial cell membrane. The TM-thrombin complex activates protein C (PC). Activated protein C (APC) binds protein S (PS) on endothelial cell or platelet membrane phospholipids. The bound active complex hydrolyzes (inactivates) activated coagulation factors V and VIII (Va and VIIIa). PS can be in either a free (able to interact as described above) or bound state. C4bBP,C4b-binding protein; Vi, inactivated coagulation factor V; VIIIi, inactivated coagulation factor VIII.

Protein S, the cofactor that binds and stabilizes APC, circulates in the plasma either free or covalently bound to the complement binding protein C4bBP. Bound protein S cannot participate in the protein C anticoagulant pathway; only free plasma protein S is available to serve as the APC cofactor. Protein S–C4bBP binding is of particular interest in inflammatory conditions, where acute phase reactant C4bBP level rises, binding additional protein S. Free protein S levels are proportionally decreased.

Protein C and protein S reference ranges

Heterozygous protein C or protein S deficiency leads to a 1.6-fold to 11.5-fold increased risk of recurrent deep vein thrombosis and pulmonary embolism. Protein S deficiency also has been implicated in transient ischemic attacks and strokes, particularly in the young. The reference interval for both protein C and protein S activity and antigen levels is 65% to 140%, and levels ordinarily remain between 30% and 65% for heterozygotes.

Control proteins C and S (and Z) and factors II (prothrombin), VII, IX, and X are vitamin K-dependent. Because the half-life of protein C is 6 hours, its level decreases as rapidly as that of factor VII at the onset of Coumadin (warfarin, vitamin K antagonist) therapy. In heterozygous protein C deficiency, protein C activity may drop to less than 65% (a thrombotic level) more rapidly than the coagulation factor activities reach low therapeutic levels (less than 30%). Consequently, in early Coumadin therapy a patient may experience Coumadin-induced skin necrosis, a paradoxical situation in which the anticoagulant therapy brings on thrombosis of the dermal vessels. This complication is suspected when the patient develops painful necrotic lesions that are preceded by severe itching, called pruritus. The necrosis may require surgical débridement. To avoid this risk, hematologists recommend coadministration of LMWH or synthetic pentasaccharide (fondaparinux) with Coumadin for any patient suspected of protein C deficiency or known to have previously suffered skin necrosis until a satisfactory and stable international normalized ratio (INR) is reached (Chapter 43).

Activity levels of protein C and protein S remain below normal for 10 to 14 days after cessation of Coumadin therapy. Similarly, for several days after surgery or a thrombotic event, these proteins are diminished even if Coumadin has not been used.70 Their activities are depressed in pregnancy, liver or renal disease, vitamin K deficiency, DIC, and with oral contraceptive use. Protein C and protein S assays therefore cannot be used to identify a congenital deficiency when they are employed within 14 days after thrombosis or after the cessation of Coumadin therapy, during pregnancy, or in the presence of DIC, liver disease, renal disease, vitamin K deficiency, or oral contraceptive use.71

Homozygous protein C or protein S deficiency results in neonatal purpura fulminans, a condition that is rapidly fatal when untreated. Treatment includes administration of factor concentrates and lifelong Coumadin therapy.

Protein C assays

Functional assays detect both quantitative and qualitative protein C deficiencies.72 Either chromogenic or clot-based protein C activity assays are available. In the former, the laboratory professional first mixes the patient’s plasma with Protac (Pentapharm, Inc., Basel, Switzerland), derived from the venom of the southern copperhead serpent Agkistrodon contortrix, which activates protein C. Subsequently, a chromogenic substrate is added, and its hydrolysis by the recently generated APC is measured by assessing the intensity of colored product, which is proportional to the activity of protein C (Figure 39-11). The assay detects abnormalities that affect the molecule’s proteolytic properties (active serine protease site) but misses those that affect protein C’s phospholipid binding site or protein S binding site. In cases in which protein C chromogenic assays and immunoassays generate normal results but the clinical condition continues to indicate possible protein C deficiency, a clot-based protein C assay may detect abnormalities at these additional sites on the molecule.


FIGURE 39-11 Chromogenic protein C (PC) assay. Patient plasma is pipetted into a reagent composed of Agkistrodon contortrix venom activator and chromogenic substrate (S-2366) specific to activated protein C (APC). The APC hydrolyzes the substrate to produce para-nitroaniline (pNA), a yellow product. The intensity of color is proportional to APC activity.

The clot-based protein C assay is based on the ability of APC to prolong the PTT. The laboratory professional mixes plasma with protein C–depleted normal plasma to ensure normal levels of all factors except protein C. PTT reagent mixed with Protac and a heparin neutralizer is added, followed by calcium chloride, and the interval to clot formation is measured. Prolongation is proportional to plasma protein C activity. The clot-based protein C activity assay may be performed using an automated coagulation analyzer. Therapeutic heparin concentrations greater than 1 IU/mL consume the heparin neutralizer, prolong the PTT, and lead to overestimation of protein C. APC resistance, LA, and the presence of therapeutic direct thrombin inhibitors such as argatroban or dabigatran may prolong the PTT and falsely raise the protein C activity level in clot-based assays.

Enzyme immunoassay is used to measure protein C antigen when the functional activity is low and acquired causes have been ruled out. Microtiter plates coated with rabbit anti–human protein C antibody are used to capture test plasma protein C, and the concentration of antigen is measured by color development after the sequential addition of peroxidase-conjugated anti–human protein C and orthophenylenediamine substrate. The protein C antigen concentration assay detects most acquired deficiencies and quantitative congenital deficiencies, but it does not detect qualitative congenital abnormalities, which is why it is used only in response to an abnormally reduced protein C functional assay result.

Protein S assays

As with testing for antithrombin and protein C deficiencies, protein S deficiency screening requires a functional assay. No chromogenic assay is available. The laboratory practitioner performs a clot-based assay by mixing the patient’s plasma with protein S–depleted normal plasma to ensure normal levels of all factors other than protein S. APC and Russell viper venom are added in a buffer that contains a heparin neutralizer, followed by calcium chloride. The practitioner records the interval to clot formation. The more prolonged the test result, the higher the protein S activity (). The clot-based protein S assay can be automated. Figure 39-12


FIGURE 39-12 Clot-based protein S (PS) assay. Patient plasma is diluted and pipetted into a reagent composed of PS-depleted plasma. A second reagent is composed of Russell viper venom (RVV), which activates clotting at the level of factor X, and activated protein C (APC). The patient PS binds the reagent APC to prolong the clotting time. Clotting time is proportional to PS activity.

Therapeutic heparin levels greater than 1 IU/mL consume the heparin neutralizer and lead to overestimation of protein S activity. APC resistance, LA, and the presence of the therapeutic direct thrombin inhibitors argatroban or dabigatran may prolong the PTT and falsely raise the activity levels in clot-based protein S assays.73 Coagulation factor VII activation may occur during prolonged refrigeration of plasma at 4° C to 10° C; this may cause underestimation of protein S activity and may affect prothrombin time-based coagulation factor assays such as factor V or factor X assays.74

When there is clinical suspicion of primary protein S deficiency based on a low activity level, enzyme immunoassays are employed to measure both total and free protein S antigen. These assays detect most quantitative congenital deficiencies and aid in the diagnosis of qualitative type II congenital deficiencies characterized by normal antigen but decreased activity of protein S. In type III deficiency, the concentration of free antigen protein S and its activity, but not the total antigen, are reduced (). The concentration of plasma C4bBP, measured with an immunoassay available for research use only, aids in the classification of the type of protein S deficiency. Table 39-6

TABLE 39-6

Anticipated Protein S (PS) Test Results in Qualitative and Quantitative Deficiencies

Type of PS Deficiency

PS Activity

PS Free Antigen

PS Total Antigen




< 65%

< 65%

< 65%




< 65%

> 65%

> 65%




< 65%

< 65%

> 65%


C4bBP, C4b-binding protein.

Arterial thrombosis predictors

Arterial thrombotic disease, including peripheral vascular occlusion, myocardial infarction (heart attack), and cerebrovascular ischemia (stroke), arises from atherosclerotic plaque. The traditional predictors of arterial thrombosis risk are elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), or an elevated ratio of total cholesterol to high-density lipoprotein cholesterol (TC:HDL-C) secondary to HDL-C deficiency. One third of primary cardiovascular and cerebrovascular events occur in patients whose lipid profiles are normal, however, and half of people with proven lipid risk factors never experience an arterial thrombotic event.75

Researchers have sought additional arterial thrombosis predictors by performing prospective randomized studies of lipoprotein subtypes, fibrinolytic pathway components, and markers of inflammation. The results of these studies have led to the identification of potential markers of arterial thrombosis risk (), of which Table 39-7homocysteine and high-sensitivity CRP have become a part of many institution’s arterial thrombosis risk profiles. The role of platelets in arterial thrombosis is also under investigation.

TABLE 39-7

Markers of Arterial Thrombosis Risk


Reference Interval


High-sensitivity C-reactive protein

0.3–1.7 mg/L

Marker of inflammation; stable, reproducible


220–498 mg/dL

Chronic level > 300 mg/dL increases thrombotic risk; inadequate reproducibility with numerous test platforms


4.6–12.1 μmol/L

Reference interval and predictive values vary with population; may be reduced with vitamin B6, B12, and folate supplementation

Total cholesterol (TC)

< 200 mg/dL

Reproducible; some relationship with diet, exercise; risk prediction is partially dependent on inflammation

Ratio of total cholesterol to high-density lipoprotein cholesterol (TC : HDL-C)

< 10

Reproducible; elevated ratio relates to diet, exercise; risk prediction is partially dependent on inflammation

Low-density lipoprotein cholesterol (LDL-C)

< 130 mg/dL

Reproducible; may be significantly lowered with statin therapy

Lipoprotein (a)

2.2–49.4 mg/dL

Varies with race and age; lowered with statin therapy; inadequate reproducibility

C-reactive protein

CRP is a calcium-dependent pentameric ligand-binding member of the pentraxin family produced in the liver that circulates in plasma at a concentration below 0.55 mg/L. First described in 1930, CRP is an acute-phase reactant whose plasma concentration rises 1000-fold 6 to 8 hours after the onset of an inflammatory event such as an infection, trauma, or surgery. This rise remains stable over several days in vivo, and the protein is resistant to in vitro degradation.76 Extremely high CRP levels are identified using one of several time-honored manual semiquantitative laboratory assays, all of which employ polyclonal anti-CRP antibodies that coats a suspension of visible latex particles. The test takes place on a slide or card. Laboratory professionals continue to use this simple and inexpensive assay to confirm inflammation and monitor the effectiveness of anti-inflammatory therapy.

A second CRP assay, high-sensitivity CRP (HSCRP), was developed in the late 1990s and is used to document modest but chronic CRP elevation. Both the manual HSCRP enzyme immunoassay and an automated latex microparticle immunoassay employ sensitive monoclonal antibodies that detect CRP at normal or slightly elevated concentrations. Chronic plasma HSCRP concentrations that remain at 1.5 mg/L or above indicate atherosclerosis secondary to low-grade inflammation that correlates to increased risk of myocardial infarction and stroke.77 Consequently, HSCRP is a clinical measure, independent from the lipid profile, employed to predict cardiovascular or cerebrovascular disease (Table 39-8).78 HSCRP may also be used in relationship with total cholesterol (Table 39-9) and the TC:HDL-C ratio (Table 39-10) to predict the risk of myocardial infarction. Laboratory professionals may also use HSCRP to monitor the anti-inflammatory effects of statins.79-82

TABLE 39-8

Relative Risk for Myocardial Infarction or Stroke at Four Levels of High-Sensitivity C-Reactive Protein (HSCRP) Independent of Lipid Levels






≤0.55 mg/L




0.56–1.14 mg/L




1.15–2.10 mg/L




≥2.11 mg/L



TABLE 39-9

Relative Risk for Myocardial Infarction at Three Levels of High-Sensitivity C-Reactive Protein (HSCRP) Related to Total Cholesterol



Low: ≤191 mg/dL

Medium: 192–223 mg/dL

High: ≥224 mg/dL

Low: ≤0.72 mg/L




Medium: 0.73–1.69 mg/L




High: ≥1.70 mg/L




TABLE 39-10

Relative Risk for Myocardial Infarction at Three Levels of High-Sensitivity C-Reactive Protein (HSCRP) Related to Ratio of Total Cholesterol to High-Density Lipoprotein Cholesterol (TC : HDL-C Ratio)



Low: ≤3.78

Medium: 3.79–5.01

High: ≥5.02

Low: ≤0.72 mg/L




Medium: 0.73–1.69 mg/L




High: ≥1.70 mg/L




Plasma homocysteine

Homocysteine is a naturally occurring sulfur-containing amino acid formed in the metabolism of dietary methionine.8384 The homocysteine concentration in plasma depends on adequate protein intake and adequate levels of vitamin B6, vitamin B12, and folate. Its concentration is regulated by three enzymes: cystathionine β-synthase, which converts homocysteine to cystathionine in the presence of vitamin B6; 5,10-methylenetetrahydrofolate reductase (MTHFR), required for the remethylation of homocysteine to methionine in the folic acid cycle; and methionine synthase, which requires vitamin B12. Folate, B6, or B12deficiencies; common functional polymorphisms of the MTHFR gene; or an inherited deficiency of either cystathionine β-synthase or methionine synthase yields increased plasma homocysteine, homocysteinemia.85

Clinical significance of homocysteinemia

Fasting homocysteinemia is an independent risk factor for arterial thrombosis, with relative risk ratios of 1.7 for coronary artery disease, 2.5 for cerebrovascular disease, and 6.8 for peripheral artery disease.86,87Homozygosity for the MTHFR C677T mutation is associated with homocysteinemia but is not an independent thrombosis risk factor. Several theories link homocysteinemia with coronary artery disease, most citing damage to the endothelial cell.88

Homocysteine reference range and therapy

The reference ranges for homocysteine differ for men and women, and they rise with age, as shown in . No clinical outcomes studies have correlated homocysteine reduction with reductions in adverse arterial thrombosis events. Table 39-11

TABLE 39-11

Homocysteine Enzyme Immunoassay Reference Intervals


Reference Interval (μmol/L)

Females ≤60 yr


Females > 60 yr


Males ≤60 yr


Males > 60 yr


Fibrinogen activity

Laboratory professionals measure fibrinogen using immunoassay, nephelometry, or the Clauss clot-based method to detect dysfibrinogenemia, hypofibrinogenemia, or afibrinogenemia (Chapter 42). The same assays may be used to detect chronic hyperfibrinogenemia. Fibrinogen concentration correlates with relative risk of myocardial infarction in asymptomatic persons or patients with angina pectoris, as shown in Table 39-12.89 The relative risk triples from the first to the fifth quintiles, and even chronic high-normal levels predict increased risk. There also exists a direct correlation between fibrinogen and total cholesterol (Figure 39-13). High fibrinogen concentrations can be used to predict hypercholesterolemia and identify patients who are at high risk for new coronary events. In contrast, low normal fibrinogen levels are associated with low risk of cardiovascular events, even in people with high total cholesterol levels.


FIGURE 39-13 Coronary risk prediction by fibrinogen and cholesterol concentrations. Fibrinogen and total cholesterol synergistically predict coronary risk. Tertiles of fibrinogen concentration are shown in relation to tertiles of total cholesterol concentration with the relative risk of coronary events indicated for each combination.

TABLE 39-12

Relative Risk of Coronary Events According to Concentration of Fibrinogen*

Fibrinogen Concentration Quintile

Relative Risk of Coronary Event











* The relative risks are shown for each of five quintiles of subjects defined according to the concentrations of each fibrinogen from 1, the group with the lowest concentration, to 5, the group with the highest concentration. Relative risks have been adjusted for all confounding factors. The group with the lowest values serves as the reference group.

Elevated fibrinogen supports coagulation and activates platelets by binding to their glycoprotein IIb/IIIa membrane receptors. Fibrinogen becomes integrated into atherothrombotic lesions and contributes to their thrombotic potential.

Although hyperfibrinogenemia predicts arterial thrombosis, the use of the fibrinogen assay for this purpose is limited. There are no independent therapeutic regimens that specifically lower fibrinogen, and no clinical trials suggest that fibrinogen reduction reverses the odds of thrombosis. Further, the various fibrinogen assay methods are not normalized. Nevertheless, statin therapy, smoking cessation, and exercise lower fibrinogen levels alongside LDL-C and total cholesterol levels, and the assay results parallel those for the other members of the risk prediction profile.

Lipoprotein (a)

Lipoprotein (a) is low-density lipoprotein that may be used to predict arterial thrombosis. The plasma level is measured by enzyme immunoassay, and its reference ranges are shown in . Although lipoprotein (a) concentrations are Table 39-13 higher in African Americans than in European Americans, the level is a stronger predictor of thrombosis in Caucasians.90

TABLE 39-13

Normal Ranges for Lipoprotein (a) by Race and Sex




African American

4.7–71.8 mg/dL

4.4–75.0 mg/dL

European American

2.2–49.4 mg/dL

2.1–57.3 mg/dL

Lipoprotein (a) may contribute to thrombosis by its antifibrinolytic property. The molecule competes with plasminogen for binding sites on newly formed fibrin polymer, decreasing the plasmin activity available for clot degradation. It also may contribute to the overall concentration of LDL-C. Levels can be lowered with statin drugs, as can LDL-C levels.

Disseminated intravascular coagulation

DIC, also named defibrination syndrome or consumption coagulopathy, is the generalized activation of hemostasis secondary to a systemic disease. DIC involves all hemostatic systems: vascular intima, platelets, leukocytes, coagulation, coagulation control pathways, and fibrinolysis.91 In DIC, fibrin microthrombi partially occlude small vessels and consume platelets, coagulation factors, coagulation control proteins, and fibrinolytic enzymes. Fibrin/fibrinogen degradation products (FDPs, traditionally called fibrin split products, FSPs), including D-dimer, become elevated and interfere with normal fibrin formation.92 This combination of events sets loose a series of toxic and inflammatory processes.93

DIC may be acute and uncompensated, with deficiencies of multiple hemostasis components, or chronic, with normal or even elevated clotting factor levels. In chronic DIC, liver coagulation factor production and bone marrow platelet production compensate for increased consumption.

Although DIC is a thrombotic process, the thrombi that form are small and ineffective, so systemic hemorrhage is often the first or most apparent sign. Acute DIC is often fatal and requires immediate medical intervention. The diagnosis relies heavily on the hemostasis laboratory, and medical laboratory professionals often perform a DIC profile under emergent circumstances.


Any disorder that contributes hemostatic molecules or promotes their endogenous secretion may cause DIC. Classifying and listing all the causes of DIC are impossible, but the major triggering mechanisms and examples of each are listed in . Table 39-14

TABLE 39-14

Conditions Associated with Disseminated Intravascular Coagulation Grouped by Mechanism


Examples of Conditions

Tissue factor is released into circulation through endothelial cell damage or monocyte activation

Physical trauma: crush or brain injuries, surgery 

Degradation of muscle; rhabdomyolysis 

Tissue ischemia; myocardial infarction 

Thermal injuries: burns or cold 


Exposure of subendothelial tissue factor during vasodilatation

Hypovolemic and hemorrhagic shock 

Malignant hypertension 

Asphyxia and hypoxia 

Heat stroke 


Endotoxins that activate cytokines

Bacterial, protozoal, fungal, and viral infections, septicemia 

Toxic shock syndrome

Circulating immune complexes

Heparin-induced thrombocytopenia with thrombosis 

Drugs that trigger an immune response 

Acute hemolytic transfusion reactions 

Allergic reactions and anaphylaxis 

Bacterial and viral infections 

Graft rejection

Particulate matter from tissue injury

Eclampsia, preeclampsia, HELLP syndrome 

Retained dead fetus or missed abortion 

Amniotic fluid embolism 

Abruptio placentae 

Rupture of uterus 

Tubal pregnancy 

Fat embolism 


Infusion of activated clotting factors

Activated prothrombin complex concentrate therapy

Secretion of proteolytic enzymes

Acute promyelocytic or myelomonocytic leukemia 

Bacterial, protozoal, fungal, and viral infections 


Toxins that trigger coagulation

Snake or spider envenomation Pancreatitis

Thrombotic disease or thrombogenic conditions

Thrombotic thrombocytopenic purpura, hemolytic uremic syndrome 

Pregnancy, postpartum period, estrogen therapy 

Deep vein thrombosis, pulmonary embolus 

Coagulation control system deficiencies 

Purpura fulminans, skin necrosis

Severe hypoxia or acidosis

Acute coagulopathy of trauma-shock 

Chronic inflammation 

Diabetes mellitus

Platelet activation

Vascular surgery, coronary artery bypass surgery 

Thrombocytosis, thrombocythemia, polycythemia 

Vascular tumors 

Vascular prostheses 

Aortic aneurysm

HELLP, Hemolysis, elevated liver enzymes, and low platelet count.

The more acutely ill the patient, the more dangerous the symptoms. Chronic DIC may be associated with vascular tumors, tissue necrosis, liver disease, renal disease, chronic inflammation, use of prosthetic devices, and adenocarcinoma. The malignancies most associated with DIC are pancreatic, prostatic, ovarian, and lung cancers; multiple myeloma; and myeloproliferative diseases. Acute DIC is seen in association with obstetric emergencies, intravascular hemolysis, septicemia, viremia, burns, acute inflammation, crush injuries, dissecting aortic aneurysms, and cardiac disorders.


Triggering events may activate coagulation at any point in the pathway. When triggered, however, DIC proceeds in a predictable sequence of events. Circulating thrombin is the primary culprit because it activates platelets, activates coagulation proteins (positive feedback loops within the coagulation cascade), and catalyzes fibrin formation, of which the ensuing clots consume control proteins. The fibrinolytic system may become activated at the level of plasminogen or TPA subsequent to fibrin formation, and endothelial cells may become damaged, releasing coagulation-active substances. Finally, leukocytes—particularly monocytes—may be induced to secrete tissue factor by the cytokines released during inflammation.

Thrombin cleaves fibrinogen, creating fibrin monomers. In normal hemostasis, fibrin monomers spontaneously polymerize to form an insoluble gel. The polymer becomes strengthened through cross-linking, binding plasminogen as it forms. In DIC, a percentage of fibrin monomers fail to polymerize and circulate in plasma as soluble fibrin monomers. The monomers coat platelets and coagulation proteins, creating an anticoagulant effect (Figures 37-12 and 37-14).

Soluble fibrin monomers, fibrin polymer, and cross-linked fibrin all activate plasminogen. Normally, the active form of plasminogen—plasmin—acts locally to digest only the solid fibrin clot to which it is bound (Chapter 37). In DIC, plasmin circulates in the plasma and digests all forms of fibrinogen and fibrin.94 Consequently, fibrin degradation products labeled X, Y, D, E, and D-dimer are detectable in the plasma in concentrations exceeding 20,000 ng/mL. D-dimer arises from cross-linked fibrin polymer, whereas the other fibrin degradation products may be produced from fibrinogen or fibrin monomers or polymers.95

Platelets become enmeshed in the fibrin polymer or are exposed to thrombin; both events trigger platelet activation, which further drives the coagulation system and produces thrombocytopenia. At the same time, coagulation pathway control is lost as protein C, protein S, and antithrombin are consumed. The combination of thrombin activation, circulating plasmin, loss of control, and thrombocytopenia contributes to the overall hemorrhagic outcome of DIC.

Free plasmin digests factors V, VIII, IX, and XI, as well as other plasma proteins. Plasmin also may trigger complement, which leads to hemolysis, and the kinin system, which results in inflammation, hypotension, and shock. During fibrinolytic therapy, in amyloidosis, and sometimes in liver disease, plasminogen becomes activated independently of the coagulation pathway. This condition, called systemic fibrinolysis or primary fibrinolysis, produces laboratory-measurable fibrinogen and fibrin degradation products, including D-dimer, and prolonged prothrombin time (PT) and PTT with a normal platelet count.96


The symptoms signaling DIC frequently are masked by the symptoms of the underlying disorder and may be chronic, acute, or even fulminant. Thrombosis in the microvasculature of major organs may produce symptoms of organ failure, such as renal function impairment, adult respiratory distress syndrome, and central nervous system manifestations. Skin, bone, and bone marrow necrosis may be seen. Purpura fulminans is seen in meningococcemia, chickenpox, and spirochete infections.

Laboratory diagnosis

DIC is a clinical diagnosis that requires laboratory confirmation (Chapter 42). The initial laboratory profile includes a platelet count, blood film examination, PT, PTT, D-dimer, and fibrinogen assay. Table 39-15lists anticipated DIC results. Prolonged PT and PTT reflect coagulation factor consumption. Fibrinogen concentrations may decrease in DIC, but because fibrinogen is an acute phase reactant that rises in inflammation, the fibrinogen concentration alone provides little reliable information and may exceed 400 mg/dL. The peripheral blood film platelet estimate confirms thrombocytopenia in nearly all cases, and the presence of schistocytes helps establish the diagnosis of DIC in about 50% of cases.9798 An elevated D-dimer level is essential to the diagnosis. D-dimer also may be elevated in inflammatory conditions, localized thrombosis, or renal disease in the absence of DIC; the PT, PTT, platelet count, blood film examination, and other laboratory assays must be performed alongside the D-dimer to rule out these disorders.

TABLE 39-15

Anticipated Results of Disseminated Intravascular Coagulation (DIC) Primary Laboratory Profile


Reference Interval

Value in DIC

Platelet count


< 150,000/μL

Prothrombin time

11–14 sec

> 14 sec

Partial thromboplastin time

25–35 sec

> 35 sec


0–240 ng/mL

> 240 ng/mL, often 10,000 to 20,000 ng/mL


220–498 mg/dL

< 220 mg/dL, often higher, because fibrinogen is an acute phase reactant

The D-dimer reference limit is typically 240 ng/mL, although the interval varies with location and technology. In 85% of DIC cases, D-dimer levels reach 10,000 to 20,000 ng/mL. A normal D-dimer assay result rules out DIC and rules out localized venous thromboembolic disease such as deep vein thrombosis or pulmonary emboli, in which levels rise to greater than 500 ng/mL but not to DIC levels.99 D-dimer concentrations are typically elevated in inflammation, sickle cell crisis, pregnancy, and renal disease, so an abnormal result alone cannot be used to definitively diagnose venous thromboembolism.100 The judicious use of the D-dimer assay reduces the requirement for invasive diagnostic tests such as pulmonary angiography when pulmonary embolism is suspected.101

Specialized laboratory tests that may aid in diagnosis

lists specialized laboratory tests that may be used to diagnose and classify DIC in special circumstances. Results consistent with DIC and clinical comments are included. Many of these tests are available in acute care facilities, but they are not routinely applied to the diagnosis of DIC. Others are available only in tertiary care facilities and hemostasis reference laboratories.Table 39-16102-104

TABLE 39-16

Specialized Hemostasis Laboratory Assays Used to Diagnose and Classify Disseminated Intravascular Coagulation (DIC)


Value in DIC


Serum fibrin degradation products (FDP)

> 10 μg/mL

Obsolete, replaced by quantitative D-dimer.

Soluble fibrin monomer


Hemagglutination assay provides valid measure of fibrin monomer. Avoid obsolete tests such as protamine sulfate solubility or ethanol gelation.

Thrombus precursor protein

> 3.5 μg/mL

Immunoassay with no interference from fibrinogen or FDPs.

Protein C, protein S, and AT activity assays

< 50%

Use to monitor therapy: plasma, AT concentrate, recombinant thrombomodulin.

Plasminogen, tissue plasminogen activator


May be useful for analyzing systemic fibrinolysis. Specimen management protocol must be strictly observed.

Peripheral blood film exam

Anemia with schistocytes

Schistocytes (microangiopathic hemolytic anemia) are present in 50% of DIC cases; leukocytosis is common.

Localized thrombosis markers: prothrombin fragment 1+2, thrombin-antithrombin


Most useful in diagnosis of localized thrombotic events, but may be used to monitor DIC therapy. Used in clinical trials.

Factor assays: II (prothrombin), V, VIII, X

< 30%

Factors V and VIII rise in inflammation; assays may give misleading results.

Thrombin time, reptilase time


Fibrinogen levels < 80 mg/dL, elevated FDPs, and soluble fibrin monomer all prolong thrombin time and reptilase time.

AT, Antithrombin; FDP, fibrin degradation product.

Protein C, protein S, and antithrombin typically are consumed in DIC, and their assay may contribute to the diagnosis. When thawed frozen plasma or antithrombin concentrate (Thrombate III) is used to treat DIC, these assays are useful in establishing the necessity for therapy and monitoring its effect.105 Factor assays may clarify PT and PTT results. The thrombin time and the reptilase time also are sensitive DIC screens.

Tests of the fibrinolytic pathway include serum fibrin degradation products, now obsolete; chromogenic plasminogen activity assay; and TPA and PAI-1 immunoassays.106 These tests are seldom offered in the acute care hemostasis laboratory because they require careful specimen management, but they may help in the diagnosis of primary fibrinolysis, which may occur after fibrinolytic therapy.


To arrest DIC, the physician must diagnose and treat the underlying disorder. Surgery, anti-inflammatory agents, antibiotics, or obstetric procedures as appropriate may normalize hemostasis, particularly in chronic DIC. Supportive therapy, such as maintenance of fluid and electrolyte balance, always accompanies medical and surgical management.

In acute DIC, in which multiorgan failure from microthrombosis and bleeding threatens the life of the patient, heroic measures are necessary. Treatment falls into two categories: therapies that slow the clotting process and therapies that replace missing platelets and coagulation factors.

UFH may be used for its antithrombotic properties to stop the uncontrolled activation of the coagulation cascade. Because UFH may aggravate bleeding, careful observation and support are required. Repeated chromogenic anti-factor Xa heparin assays may be necessary to control heparin dosage because in DIC the PTT is ineffective for monitoring heparin therapy.

Thawed frozen plasma provides all the necessary coagulation factors and replaces blood volume lost during acute DIC hemorrhage. Prothrombin complex concentrate (Proplex T complex, Baxter Healthcare Corporation, Deerfield, IL; or Kcentra, CSL Behring, King of Prussia, PA), fibrinogen concentrate (RiaSTAP, CSL Behring, King of Prussia, PA), and factor VIII concentrate (ADVATE, Baxter, Deerfield, IL) may be used in place of plasma, particularly if there is concern for transfusion-associated circulatory overload. Repeated measurements of fibrinogen, PT, and PTT are necessary to confirm the effectiveness of these therapeutics. Platelet transfusions are necessary if thrombocytopenia is severe. The effectiveness of platelet concentrate and platelet consumption are monitored with platelet counts (Chapters 15 and 16). Red blood cells are administered as necessary to treat the resulting anemia. Antifibrinolytic therapy is contraindicated, except in proven systemic fibrinolysis.107108

Localized thrombosis monitors

In addition to D-dimer, several peptides and coagulation factor complexes are released into the plasma during coagulation. One complex, thrombin-antithrombin complex (TAT), and one peptide, prothrombin fragment F 1+2 (PF 1+2 or PF 1.2), may be assayed as a means to detect and monitor localized venous or arterial thromboses. TAT and PF 1+2 immunoassays are sensitive and specific for thrombosis, which occurs in DIC, septicemia, eclampsia, pancreatitis, leukemia, liver disease, and trauma.108 These assays are of particular value in clinical trials of anticoagulants.

PF 1+2 is released from prothrombin at the time of its conversion to thrombin by the prothrombinase complex. It has a plasma half-life of 90 minutes and a reference range of 0.3 to 1.5 nmol/L. Elevated PF 1+2 may be seen in venous thromboembolism. Heparin or oral anticoagulant therapy reduces its plasma concentration.

The TAT covalent complex is formed when antithrombin neutralizes thrombin. This reaction is enhanced by the presence of heparin. TAT has a half-life of 3 minutes and a normal range of 0.5 to 5 ng/mL.

To avoid in vitro release or activation of PF 1+2 or TAT, plasma specimens are collected in 3.2% sodium citrate and are centrifuged and separated within minutes of collection; then the plasma is frozen until ready for assay.

Heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT), also called heparin-induced thrombocytopenia with thrombosis, is an adverse effect of heparin treatment.

Cause and clinical significance

Between 1% and 5% of patients receiving unfractionated heparin (UFH) for more than 5 days develop an IgG antibody specific for heparin–platelet factor 4 complexes. In 30% to 50% of these cases, the immune complexes that are formed bind platelet Fc receptors, which leads to platelet activation, thrombocytopenia, and formation of microvascular thrombi.109 HIT occurs with UFH administration at both prophylactic and therapeutic dosages, although it is more frequent with therapeutic doses.110 Venous thrombosis predominates 5:1, but arterial thrombosis accounts for the most disturbing symptoms. Patients may develop pulmonary emboli, limb gangrene requiring amputation, stroke, and myocardial infarction. HIT is often a medical emergency, and the mortality rate is 20%.111 LMWH also causes HIT. In most cases, HIT during LMWH therapy turns out to be a cross-reaction in a patient recently exposed to UFH; however, LMWH has been implicated as a primary cause at a rate of 1% of UFH-caused HIT. Likewise, protamine sulfate, a salmon sperm derivative that cardiac surgeons use to rapidly reduce UFH’s anticoagulant may itself generate antibodies similar to anti–heparin-PF4 antibodies and cause thrombocytopenia and thrombosis symptoms indistinguishable from UFH-caused HIT.

Platelet count

Patients receiving heparin must have platelet counts performed every other day. A platelet count decrease during heparin administration indicates HIT, but interpretation of the thrombocytopenia is confounded because 30% of patients receiving heparin develop an immediate, benign, and limited thrombocytopenia, sometimes called HIT type I.112 This benign form of thrombocytopenia usually develops in 1 to 3 days, whereas thrombotic HIT, sometimes called HIT type II, develops after 5 days. However, thrombotic HIT may develop in 1 to 3 days in patients recently exposed to heparin. In HIT (HIT type II), the decrease in platelet count may exceed 40%, whereas in benign thrombocytopenia the decrease is relatively small; however, in both cases, the platelet count may remain within the normal range. The HIT diagnosis is made using the “4Ts” approach, provided in Table 39-17.

TABLE 39-17

The “4Ts” Scoring System: Laboratory and Clinical Pretest Probability of Heparin-Induced Thrombocytopenia (HIT)







Acute thrombocytopenia

> 50% decrease in platelet count to nadir of ≥20,000/μL

30%–50% decrease in platelet count, > 50% if directly resulting from surgery, or to nadir of 10,000–19,000/μL

< 30% decrease in platelet count, or to nadir of < 10,000/μL

Timing of platelet count decrease, thrombosis, or other sequelae of HIT (first day of heparin therapy is day 0)

Onset of decrease on days 5–10, or onset of decrease on day 1 if previous heparin exposure within past 5–30 days

Apparent decrease on days 5–10, but unclear due to missing platelet counts; or decrease after day 10; or decrease on day 1 if previous heparin exposure within past 31–100 days

Decrease at ≤4 days without recent heparin exposure

Thrombosis, skin lesions, acute system reaction

Proven new thrombosis or skin necrosis; acute systemic reaction after heparin exposure

Progressive, recurrent, or suspected thrombosis; erythematous skin lesions


Other causes for thrombocytopenia

No explanation for platelet count decrease is evident

Possible other cause is evident

Probable other cause is evident

From Crowther MA, Cook DJ, Albert M, et al: Canadian Critical Care Trials Group: the 4Ts scoring system for heparin-induced thrombocytopenia in medical-surgical intensive care unit patients, J Crit Care 25:287-293, 2010.

Maximum pretest probability score is 8; a score of 6 to 8 indicates high probability of HIT; 4 to 5, intermediate probability; 0 to 3, low probability.The most immunizing heparin exposure is considered first; unfractionated heparin (UFH) received during cardiac surgery is more immunogenic than UFH or low-molecular-weight heparin received for acute coronary syndrome. The day the platelet count begins to fall is considered the day of onset of thrombocytopenia. It generally takes 1 to 3 days before an arbitrary threshold that defines thrombocytopenia, such as 150,000 platelets/μL, is passed.

Laboratory tests for hit

Because at least 10% of hospitalized patients receive heparin, the acute care laboratory must provide a procedure to confirm HIT and differentiate it from benign thrombocytopenia. Most acute care laboratories provide a heparin-induced antibody immunoassay but seldom as a stat assay (Chapter 40).113 Owing to its sensitivity, the immunoassay result may be positive before clinical signs of HIT become evident. Further, the immunoassay result may be negative in a few HIT patients, possibly because peptides such as protamine sulfate may also form complexes with PF4. Microbial contamination, lipemia, and hemolysis may also invalidate immunoassay results. Conversely, patient specimen immune complexes or immunoglobulin aggregates may cause nonspecific binding and produce false-positive results.

Many laboratories provide aggregometry or lumiaggregometry methods to confirm HIT (Chapter 40).114 Aggregometry is technically demanding and is only 50% sensitive for HIT. Also technically demanding, but perhaps more sensitive, is washed platelet suspension lumiaggregometry. The reference method is the washed platelet14C-serotonin release assay (serotonin release assay, SRA), provided by reference laboratories that possess radionuclide licenses.


When heparin-induced antibodies are detected, the administration of UFH or LMWH is immediately discontinued, and the physician chooses an alternate form of anticoagulation. Complete cessation of anticoagulant therapy is risky because additional thrombotic events are likely to occur. Although LMWH causes HIT in only 1% of cases in which it is the sole anticoagulant, its use as a substitute for UFH is contraindicated owing to its tendency to react with the existing antibody. Likewise, Coumadin is discouraged; it may precipitate a potentially severe skin necrosis if given in high bolus dosages, but more importantly its onset is too slow to be practical in this setting. Synthetic pentasaccharide (fondaparinux), which mimics heparin’s antithrombin-binding sequence, has been shown in several small case series to be useful for the management of patients with suspected HIT.115

Recombinant bivalirudin (Angiomax, The Medicines Company, Parsippany, NJ) is a direct thrombin inhibitor modeled after leech saliva. Argatroban (Mitsubishi Pharma Corporation, Tokyo, Japan) is an amino acid analogue direct thrombin inhibitor. Both bind the active thrombin protease site, and, in HIT, physicians administer either argatroban or bivalirudin as a continuous infusion. Laboratory practitioners may monitor either using the PTT. The activated clotting time (ACT); the ecarin clotting time, which uses a reagent derived from Echis carinatus snake venom (Diagnostica Stago, Inc., Parsippany, NJ); or the plasma-diluted thrombin time (HEMOCLOT Thrombin, Aniara, Hyphen Biomed, West Chester, OH) may also be employed to monitor these direct thrombin inhibitors (Chapter 43).


The importance of laboratory diagnosis has become evident in all forms of thrombotic disease: chronic and acute, arterial and venous, primary and secondary, and acquired and congenital. The future may give us markers of endothelial cell disease, measures of leukocyte adhesion, and specific markers of inflammation and platelet activity that will further enable us to predict and prevent thrombosis.


• Thrombosis is the most prevalent condition in developed countries and accounts for most illnesses and premature death.

• Thrombosis may be arterial, causing peripheral artery disease, heart disease, and stroke, or venous, causing deep vein thrombosis and pulmonary emboli.

• Most thrombosis occurs as a result of lifestyle habits and aging, but many thrombotic disorders are related to congenital risk factors.

• Thrombosis risk profiles may be offered to clinicians for screening purposes in high-risk populations.

• The main hemostasis predictors of arterial thrombotic disease are elevated levels of CRP (measured by high-sensitivity assay), homocysteine, fibrinogen, lipoprotein (a), and coagulation factors.

• The main hemostasis predictors of venous thromboembolic disease are APL antibodies, antithrombin, PC, and PS deficiency, FVL mutation, and prothrombin G20210A.

• APL antibody testing requires a series of essential hemostasis laboratory assays—clot-based tests and immunoassays.

• Antithrombin may be assayed using chromogenic substrate and enzyme immunoassay analyses.

• The tests for evaluating the protein C pathway include protein C and protein S activity and concentration, APC resistance, FVL assay, and C4bBP assay.

• The molecular test for the prothrombin G20210A mutation predicts the risk of venous thrombosis.

• DIC is a clinical diagnosis confirmed by a series of assays in the acute care facility.

• Chronic thrombosis may be identified using the PF 1+2, TAT complex, and quantitative D-dimer assays.

• The laboratory provides confirmatory tests for HIT with thrombosis.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. What is the prevalence of venous thrombosis in the United States?

a. 0.01

b. 1 in 1000

c. 10% to 15%

d. 500,000 cases per year

2. What is thrombophilia?

a. Predisposition to thrombosis secondary to a congenital or acquired disorder

b. Inappropriate triggering of the plasma coagulation system

c. A condition in which clots form uncontrollably

d. Inadequate fibrinolysis

3. What acquired thrombosis risk factor is assessed in the hemostasis laboratory?

a. Smoking

b. Immobilization

c. Body mass index

d. Lupus anticoagulant

4. Trousseau syndrome, a low-grade chronic DIC, is often associated with what type of disorder?

a. Renal disease

b. Hepatic disease

c. Adenocarcinoma

d. Chronic inflammation

5. What is the most common heritable thrombosis risk factor in Caucasians?

a. APC resistance (factor V Leiden mutation)

b. Prothrombin G20210A mutation

c. Antithrombin deficiency

d. Protein S deficiency

6. In most LA profiles, what screening test is primary because it detects LA with the fewest interferences?

a. Low-phospholipid PTT


c. KCT

d. PT

7. A patient with venous thrombosis is tested for protein S deficiency. The protein S activity, antigen, and free antigen all are less than 65%, and the C4bBP level is normal. What type of deficiency is likely?

a. Type I

b. Type II

c. Type III

d. No deficiency is indicated, because the reference range includes 65%.

8. An elevated level of what fibrinolytic system assay is associated with arterial thrombotic risk?

a. PAI-1

b. TPA

c. Factor VIIa

d. Factor XII

9. How does lipoprotein (a) cause thrombosis?

a. It causes elevated factor VIII levels.

b. It coats the endothelial lining of arteries.

c. It substitutes for plasminogen or TPA in the forming clot.

d. It contributes additional phospholipid in vivo for formation of the Xase complex.

10. What test may be used to confirm the presence of LA?

a. PT

b. Bethesda titer

c. Antinuclear antibody

d. PTT using high-phospholipid reagent

11. What molecular test may be used to confirm APC resistance?

a. Prothrombin G20210A

b. MTHFR 1298

c. MTHFR 677

d. FVL

12. What therapeutic agent may occasionally cause DIC?

a. Factor VIII

b. Factor VIIa

c. Antithrombin concentrate

d. Activated prothrombin complex concentrate

13. Which is not a fibrinolysis control protein?

a. Thrombin-activatable fibrinolysis inhibitor

b. Plasminogen activator inhibitor-1

c. α2-antiplasmin

d. D-dimer

14. What is the most important application of the quantitative D-dimer test?

a. Diagnose primary fibrinolysis

b. Diagnose liver and renal disease

c. Rule out deep venous thrombosis

d. Diagnose acute myocardial infarction


1.  Francis J.L. Laboratory investigation of hypercoagulabilitySemin Thromb Hemost; 1998; 24:111-126.

2.  Dahlback B, Carlsson M, Svensson P.J. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A; 1993; 90:1004-1008.

3.  Bertina R.M, Koeleman B.P.C, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein CNature; 1994; 369:64-67.

4.  Nieuwdorp M, Stroes E.S, Meijers J.C, et al. Hypercoagulability in the metabolic syndromeCurr Opin Pharmacol; 2005; 5:155-159.

5.  Heit J.A. Thrombophilia clinical and laboratory assessment and management. In: Kitchens C.S, Kessler C.M, Konkle B.A. Consultative Hemostasis and Thrombosis. 3rd ed. Philadelphia : Saunders 2013; 205-239.

6.  Go A.S, Mozaffarian D, Roger V.L, et al. on behalf of the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics—2014 update a report from the American Heart Association. Circulation, Published online December 18, 2013. Available at: doi:10.1161/01.cir0000441139.02102.80 2014.

7.  Heit J.A. The epidemiology of venous thromboembolism in the communityArterioscler Thromb Vasc Biol; 2008; 28:370-372.

8.  Zöller B, Li X, Sundquist J, Sundquist K. Shared familial aggregation of susceptibility to different manifestations of venous thromboembolism a nationwide family study in Sweden. Br J Haematol; 2012; 157:146-148.

9.  Jensen R. The interface of the physician and the laboratory in the detection of venous thrombotic risk—part IClin Hemost Rev; 1997; 11:1-4.

10.  Stein P.D, Matta F, Musani M.H, et al. Silent pulmonary embolism in patients with deep venous thrombosis a systematic review. Am J Med; 2010; 123:426-431.

11.  Rosendaal F.R, Reitsma P.H. Genetics of venous thrombosisJ Thromb Haemost; 2009; 7(Suppl. 1):301-304.

12.  Vemulapalli S, Becker R.C. Hemostatic aspects of cardiovascular medicine. In: Kitchens C.S, Kessler C.M, Konkle B.A. Consultative Hemostasis and Thrombosis. 3rd ed. Philadelphia : Saunders 2013; 342-394.

13.  The Surgeon General’s Call to Action to prevent deep vein thrombosis and pulmonary embolism. US Department of Health and Human Services. Office of the Surgeon General, US. Available at: 2008 Accessed 21.11.14.

14.  Piazza G, Goldhaber S.Z. Venous thromboembolism and atherothrombosis an integrated approach. Circulation; 2010; 121:2146-2150.

15.  Rand J.H, Wolgast L.R. Antiphospholipid syndrome pathogenesis, clinical presentation, diagnosis, and patient management. In: Kitchens C.S, Kessler C.M, Konkle B.A. Consultative Hemostasis and Thrombosis. 3rd ed. Philadelphia : Saunders 2013; 321-341.

16.  Rak J, Milsom C, May L, et al. Tissue factor in cancer and angiogenesis the molecular link between genetic tumor progression, tumor neovascularization, and cancer coagulopathy. Semin Thromb Hemost; 2006; 32:54-70.

17.  Sanz M.A, Grimwade D, Tallman M.S. Management of acute promyelocytic leukemia recommendations from an expert panel on behalf of the European Leukemia Net. Blood; 2009; 13:1875-1891.

18.  Rosse W.F, Ware R.E. The molecular basis of paroxysmal nocturnal hemoglobinuriaBlood; 1995; 86:3277-3286.

19.  Gröntved A, Hu F.B. Television viewing and risk of type 2 diabetes, cardiovascular disease, and all-cause mortality a meta-analysis. JAMA; 2011; 305:2448-2455.

20.  Garg N, Kumar A, Flaker G.C. Antiplatelet therapy for stroke prevention in atrial fibrillationMo Med; 2010; 107:44-47.

21.  Bramham K, Hunt B.J, Goldsmith D. Thrombophilia of nephrotic syndrome in adultsClin Adv Hematol Oncol; 2009; 7:368-372.

22.  Lane D.A, Mannuci P.M, Bauer K.A, et al. Inherited thrombophilia part 2. Thromb Haemost; 1996; 76:824-834.

23.  Johnson C.M, Mureebe L, Silver D. Hypercoagulable states a review. Vasc Endovasc Surg; 2005; 39:123-133.

24.  Itakkura H. Racial disparities in risk factors for thrombosisCurr Opin Hematol; 2005; 12:364-369.

25.  Sedano-Balbás S, Lyons M, Cleary B, et al. Acquired activated protein C resistance, thrombophilia and adverse pregnancy outcomes a study performed in an Irish cohort of pregnant women. J Pregnancy; 2011; 2011:1-9.

26.  Berg A.O, Botkin J, Calonge N, et al. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group routine testing for Factor V Leiden (R506Q) and prothrombin (G20210A) mutations in adults with a history of idiopathic venous thromboembolism and their adult family members. Genet Med; 2011; 13:67-76.

27.  Ridker P.M, Hennekens C.H, Selhub J, et al. Interrelation of hyperhomocysteinemia, factor V Leiden, and risk of future venous thromboembolismCirculation; 1997; 95:1777-1782.

28.  Heit J.A, O’Fallon W.M, Petterson T.M, et al. Relative impact of risk factors for deep vein thrombosis and pulmonary embolism a population-based study. Arch Intern Med; 2002; 162:1245-1248.

29.  Reitsma P.H, Versteeg H.H, Middeldorp S. Mechanistic view of risk factors for venous thromboembolismArterioscler Thromb Vasc Biol; 2012; 32:563-568.

30.  Alarcon-Segovia D, Cabral A.R. The concept and classification of antiphospholipid/cofactor syndromesLupus; 2006; 5:364-367.

31.  Lim W, Crowther M.A, Eikelboom J.W. Management of antiphospholipid antibody syndrome a systematic review. JAMA; 2006; 295:1050-1057.

32.  Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and HaemostasisJ Thromb Haemost; 2009; 7:1737-1740.

33.  Kandiah D.A, Sheng Y.H, Krilis S.A. Beta-2 glycoprotein I target antigen for autoantibodies in the “antiphospholipid syndrome. Lupus; 1996; 5:381-386.

34.  Lie J.T. Vasculopathy of the antiphospholipid syndromes revisited thrombosis is the culprit and vasculitis the consort. Lupus; 1966; 5:368-371.

35.  Lopez-Pedrera C, Buendia P, Barbarroja N, et al. Antiphospholipid-mediated thrombosis interplay between anticardiolipin antibodies and vascular cells. Clin Appl Thromb Hemost; 2006; 12:41-45.

36.  Hughes G.R.V. The antiphospholipid syndromeLupus; 1996; 5:345-346.

37.  Cohen D, Berger S.P, Steup-Beekman G.M, et al. Diagnosis and management of the antiphospholipid syndromeBMJ; 2010; 340:c2541.

38.  Devreese K, Hoylaerts M.F. Challenges in the diagnosis of the antiphospholipid syndromeClin Chem; 2010; 56:930-940.

39.  Marques M.B, Fritsma G.A. Quick guide to coagulation testing. 2nd ed. Washington, DC : AACC Press 2009.

40.  Fritsma G.A, Dembitzer F.R, Randhawa A, Marques M.B, Van Cott E.M, Adcock-Funk D, Peerschke E.I. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilizationAm J Clin Pathol; 2012; 137:904-908.

41.  Sato K, Kagami K, Asai M, et al. Detection of lupus anticoagulant using a modified diluted Russell’s viper venom testRinsho Byori; 1995; 43:263-268.

42.  Brandt J.T, Triplett D.A, Alving B, et al. Criteria for the diagnosis of lupus anticoagulants an update. Thromb Haemost; 1995; 74:1185-1190.

43.  Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and HaemostasisJ Thromb Haemost; 2009; 7:1737-1740.

44.  Devreese K.M. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulantsThromb Res; 2007; 119:369-376.

45.  Rauch J, Tannenbaum M, Neville C, Fortin P.R. Inhibition of lupus anticoagulant activity by hexagonal phase phosphatidylethanolamine in the presence of prothrombinThromb Haemost; 1998; 80:936-941.

46.  Schouwers S.M.E, Devreese K.M. J. Lupus anticoagulant testing in patients with inflammatory status does C-reactive protein interfere with LAC test results. Thrombosis Research; 2010; 125:102-104.

47.  Tebo A.E, Jaskowski T.D, Phansalkar A.R, et al. Diagnostic performance of phospholipid-specific assays for the evaluation of antiphospholipid syndromeAm J Clin Pathol; 2008; 129:870-875.

48.  Forastiero R, Martinuzzo M, Pombo G, et al. A prospective study of antibodies to beta2-glycoprotein I and prothrombin, and risk of thrombosisJ Thromb Haemost; 2005; 3:1231-1238.

49.  Blank M, Shoenfeld Y. Antiphosphatidylserine antibodies and reproductive failureLupus; 2004; 13:661-665.

50.  Bouaziz-Borgi L, Nguyen P, Hazard N, et al. A case control study of deep venous thrombosis in relation to factor V G1691A (Leiden) and A4070G (HR2 Haplotype) polymorphismsExp Mol Pathol; 2007; 83:480-483.

51.  Favaloro E.J, McDonald D, Lippi G. Laboratory investigation of thrombophilia the good, the bad, and the ugly. Semin Thromb Hemost; 2009; 35:695-710.

52.  Dahlback B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor VProc Natl Acad Sci U S A; 1994; 91:1396-1400.

53.  De Ronde H, Bertina R.M. Laboratory diagnosis of APC-resistance a critical evaluation of the test and the development of diagnostic criteria. Thromb Haemost; 1994; 72:880-886.

54.  Johnson N.V, Khor B, Van Cott E.M. Advances in laboratory testing for thrombophiliaAm J Hematol; 2012; 87(Suppl. 1):S108-S112.

55.  Ragland B.D, Reed C.E, Eiland B.M, et al. The effect of lupus anticoagulant in the second-generation assay for activated protein C resistanceAm J Clin Pathol; 2003; 119:66-71.

56.  Emadi A, Crim M.T, Brotman D.J, et al. Analytic validity of genetic tests to identify factor V Leiden and prothrombin G20210AAm J Hematol; 2010; 85:264-270.

57.  Poort S.R, Rosendaal F.R, Reitsma P.Y, et al. A common genetic variation in the 39 untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosisBlood; 1996; 88:3698-3703.

58.  Danckwardt S, Hartmann K, Gehring N.H, et al. 39 end processing of the prothrombin mRNA in thrombophiliaActa Haematol; 2006; 115:192-197.

59.  Naeem M.A, Anwar M, Ali W, et al. Prevalence of prothrombin gene mutation (G-A 20210 A) in general population a pilot study. Clin Appl Thromb Hemost; 2006; 12:223-226.

60.  Thaler E, Lechner K. Antithrombin III deficiency and thromboembolismClin Haematol; 1981; 10:369-390.

61.  De Stefano V, Rossi E. Testing for inherited thrombophilia and consequences for antithrombotic prophylaxis in patients with venous thromboembolism and their relatives. A review of the Guidelines from Scientific Societies and Working GroupsThromb Haemost; 2013, Oct; 110:697-705.

62.  Cooper P.C, Coath F, Daly M.E, Makris M. The phenotypic and genetic assessment of antithrombin deficiencyInt J Lab Hematol; 2011; 33:227-237.

63.  Rosen S. Chromogenic methods in coagulation diagnosticsHamostaseologie; 2005; 25:259-266.

64.  Antovic J, Söderström J, Karlman B, et al. Evaluation of a new immunoturbidimetric test (Liatest antithrombin III) for determination of antithrombin antigenClin Lab Haematol; 2001; 23:313-316.

65.  Wu O, Robertson L, Twaddle S, et al. Screening for thrombophilia in high-risk situations systematic review and cost-effectiveness analysis. The Thrombosis Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Health Technol Assess; 2006; 10:1-110.

66.  Spiess B.D. Treating heparin resistance with antithrombin or fresh frozen plasmaAnn Thorac Surg; 2008; 85:2153-2160.

67.  Dahlback B. The protein C anticoagulant system inherited defects as basis for venous thrombosis. Thromb Res; 1995; 77:1-43.

68.  Heit J.A. Epidemiology and risk factors for venous thromboembolism. In: Marder V.J, Aird W.C, Bennett J.S, Schulman S, White G.C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia, PA : Lippincott, Williams, and Wilkins 2013; 973-976.

69.  Mann H.J, Short M.A, Schlichting D.E. Protein C in critical illnessAm J Health Syst Pharm; 2009; 15:1089-1096.

70.  Desancho M.T, Dorff T, Rand J.H. Thrombophilia and the risk of thromboembolic events in women on oral contraceptives and hormone replacement therapyBlood Coagul Fibrinolysis; 2010; 21:534-538.

71.  Khor B, Van Cott E.M. Laboratory tests for protein C deficiencyAm J Hematol; 2010; 85:440-442.

72.  Van Cott E.M, Ledford-Kraemer M, Meijer P, et al. NASCOLA Proficiency Testing Committee. Protein S assays an analysis of North American Specialized Coagulation Laboratory Association proficiency testing. Am J Clin Pathol; 2005; 123:778-785.

73.  Clinical Laboratory and Standards Institute. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Base Coagulation Assays, Approved Guideline. 5th ed. Document H21–A5, Wayne, Pa : Clinical Laboratory and Standards Institute 2008.

74.  Ens G.E, Newlin F. Spurious protein S deficiency as a result of elevated factor VII levelsClin Hemost Rev; 1995; 9:18.

75.  Ridker P.M. Evaluating novel cardiovascular risk factors can we better predict heart attacks. Ann Intern Med; 1999; 130:933-937.

76.  Yousuf O, Mohanty B.D, Martin S.S, et al. High-sensitivity C-reactive protein and cardiovascular disease a resolute belief or an elusive link. J Am Coll Cardiol; 2013; 62:397-408.

77.  Lelubre C, Anselin S, Buodjeltia K.Z, et al. Interpretation of C-reactive protein concentrations in critically ill patientsBioMed Research International; 2013; 2013:1-9.

78.  Pepys M.B, Baltz M.L. Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A proteinsAdv Immunol; 1983; 34:141-212.

79.  Wilkins J, Gallimore R, Moore E, et al. Rapid automated high sensitivity enzyme immunoassay of C-reactive proteinClin Chem; 1998; 44:1358-1361.

80.  Ridker P.M, Cushman M, Stampfer M.J, et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy menN Engl J Med; 1997; 336:97-99.

81.  Genest J. C-reactive protein risk factor, biomarker and/or therapeutic target. Can J Cardiol; 2010; 26(Suppl. A):41A-44A

82.  Glynn R.J, Koenig W, Nordestgaard B.G, et al. Rosuvastatin for primary prevention in older persons with elevated C-reactive protein and low to average low-density lipoprotein cholesterol levels exploratory analysis of a randomized trial. Ann Intern Med; 2010; 152:488-496.

83.  Williams R.H, Maggiore J.A. Hyperhomocysteinemia pathogenesis, clinical significance, laboratory assessment, and treatmentLab Med; 1999; 30:468-474.

84.  Cacciapuoti F. Hyper-homocysteinemia a novel risk factor or a powerful marker for cardiovascular diseases? Pathogenetic and therapeutical uncertainties. J Thromb Thrombolysis; 2011; 32:82-88.

85.  Vizzardi E, Bonadei I, Zanini G, et al. Homocysteine and heart failure an overview. Recent Pat Cardiovasc Drug Discov; 2009; 4:15-21.

86.  Guba S.C, Fonseca V, Fink L.M. Hyperhomocysteinemia and thrombosisSemin Thromb Hemost; 1999; 25:291-309.

87.  Ciaccio M, Bellia C. Hyperhomocysteinemia and cardiovascular risk effect of vitamin supplementation in risk reduction. Curr Clin Pharmacol; 2010; 5:30-36.

88.  Sainani G.S, Sainani R. Homocysteine and its role in the pathogenesis of atherosclerotic vascular diseaseJ Assoc Physicians India; 2002; 50(Suppl. 1):16-23.

89.  Franchini M, Veneri D. Inherited thrombophilia an update. Clin Lab; 2005; 51:357-365.

90.  Deb A, Caplice N.M. Lipoprotein(a) new insights into mechanisms of atherogenesis and thrombosis. Clin Cardiol; 2004; 27:258-264.

91.  Mandernach M.W, Kitchens C.S. Disseminated intravascular coagulation. In: Kitchens C.S, Kessler C.M, Konkle B.A. Consultative Hemostasis and Thrombosis. 3rd ed. Philadelphia : Elsevier Saunders 2013; 174-189.

92.  Giles A.R. Disseminated intravascular coagulation. In: Bloom A.L, Forbes C.D, Thomas D.P, et al. Haemostasis and Thrombosis. New York : Churchill Livingstone 1994; 969-986.

93.  Levi M, Feinstein D.I, Colman R.W, Marder V.J. Consumptive thrombohemorrhagic disorders. In: Marder V.J, Aird W.C, Bennett J.S, Schulman S, Gilbert C, White G.C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia : Lippincott Williams & Wilkins 2013; 1178-1196.

94.  Vaughan D.E, Declerck P.J. Regulation of fibrinolysis. In: Loscalzo J, Schafer A.I. Thrombosis and hemorrhage. Philadelphia : Lippincott Williams & Wilkins 2003; 105-120.

95.  Jensen R. The diagnostic use of fibrin breakdown productsClin Hemost Rev; 1998; 12:1-2.

96.  Wada H, Kobayashi T, Abe Y, et al. Elevated levels of soluble fibrin or D-dimer indicate high risk of thrombosisJ Thromb Haemost; 2006; 4:1253-1258.

97.  Labelle C.A, Kitchens C.S. Disseminated intravascular coagulation treat the cause, not the lab values. Cleve Clin J Med; 2005; 72:377-390.

98.  Mos I.C, Klok F.A, Kroft L.J, et al. Safety of ruling out acute pulmonary embolism by normal computed tomography pulmonary angiography in patients with an indication for computed tomography systematic review and meta-analysis. J Thromb Haemost; 2009; 7:1491-1498.

99.  Khoury J.D, Adcock D.M, Chan F, et al. Increases in quantitative D-dimer levels correlate with progressive disease better than circulating tumor cell counts in patients with refractory prostate cancerAm J Clin Pathol; 2010; 134:964-969.

100.  Wada H, Wakita Y, Nakase T, et al. Hemostatic molecular markers before the onset of disseminated intravascular coagulationAm J Hematol; 1999; 60:273-278.

101.  Huisman M.V, Klok F.A. Diagnostic management of acute deep vein thrombosis and pulmonary embolismJ Thromb Haemost; 2013; 11:412-422.

102.  Sawamura A, Hayakawa M, Gando S, et al. Disseminated intravascular coagulation with a fibrinolytic phenotype at an early phase of trauma predicts mortalityThromb Res; 2009; 124:608-613.

103.  Moresco R.N, Vargas L.C, Voegeli C.F, et al. D-dimer and its relationship to fibrinogen/fibrin degradation products (FDPs) in disorders associated with activation of coagulation or fibrinolytic systemsJ Clin Lab Anal; 2003; 17:77-79.

104.  Boisclair M.D, Lane D.A, Wilde J.T, et al. A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen. Br J Haematol; 1990; 74:471-479.

105.  Gando S, Saitoh D, Ishikura H, et al. A randomized, controlled, multicenter trial of the effects of antithrombin on disseminated intravascular coagulation in patients with sepsisCrit Care; 2013; 17:R297.

106.  Favaloro E.J. Laboratory testing in disseminated intravascular coagulationSemin Thromb Hemost; 2010; 36:458-467.

107.  Levi M, de Jonge E, van der Poll T. Plasma and plasma components in the management of disseminated intravascular coagulationBest Pract Res Clin Haematol; 2006; 19:127-142.

108.  Gorog D.A. Prognostic value of plasma fibrinolysis activation markers in cardiovascular diseaseJ Am Coll Cardiol; 2010; 15:2701-2709.

109.  Brace L.D. Testing for heparin-induced thrombocytopenia by platelet aggregometryClin Lab Sci,; 1992; 5:80-81.

110.  Vermylen J, Hoylaerts M.F, Arnout J. Antibody-mediated thrombosisThromb Haemost; 1997; 78:420-426.

111.  Crowther M.A, Cook D.J, Albert M, et al. Canadian Critical Care Trials Group. The 4Ts scoring system for heparin-induced thrombocytopenia in medical-surgical intensive care unit patientsJ Crit Care; 2010; 25:287-293.

112.  Warkentin T.E, Greinacher A. Heparin-induced thrombocytopenia. In: Marder V.J, Aird W.C, Bennett J.S, Schulman S, White G.C. Basic Principles and Clinical Practice. 6th ed. Philadelphia : Lippincott Williams & Wilkins 2013; 1293-1307.

113.  Warkentin T.E, Sheppard J.A. Testing for heparin-induced thrombocytopenia antibodiesTransfus Med Rev; 2006; 20:259-272.

114.  Tan C.W, Ward C.M, Morel-Kopp M.C. Evaluating heparin-induced thrombocytopenia the old and the new. Semin Thromb Hemost; 2012; 38:135-143.

115.  Warkentin T.E, Pai M, Sheppard J.I, Schulman S, Spyropoulos A.C, Eikelboom J.W. Fondaparinux treatment of acute heparin-induced thrombocytopenia confirmed by the serotonin-release assay a 30-month, 16-patient case series. J Thromb Haemost; 2011; 9:2389-2396.

*The author acknowledges the substantial contributions of Marisa B. Marques, MD, to Chapter 42 of the third edition of this textbook, many of which continue in this edition.