Localized versus Generalized Hemorrhage

Hemorrhage from a single location commonly indicates injury, infection, tumor, or an isolated blood vessel defect and is called localized bleeding or localized hemorrhage. A surgical site that bleeds excessively because of inadequate cauterization or an ineffective suture is an example of localized bleeding. Localized bleeding seldom implies a defect of vessels, a qualitative platelet defect, a reduced platelet count, or the coagulation cascade mechanism.

Bleeding from multiple sites, spontaneous and recurring bleeds, or a hemorrhage that requires physical intervention and transfusions is called generalized bleeding. Generalized bleeding is potential evidence for a disorder of primary hemostasis involving defects in blood vessels, defects in platelets, or reduced platelet count, or secondary hemostasis involving defects in the coagulation cascade mechanism. Disorders of primary or secondary hemostasis that exhibit generalized bleeding or hemorrhage are called coagulopathies.

Mucocutaneous versus Anatomic Hemorrhage

Generalized bleeding may exhibit a mucocutaneous or a soft tissue (anatomic) pattern.

Mucocutaneous hemorrhage may be manifested as bruises or purpura, purple lesions of the skin caused by extravasated (seeping) red blood cells (RBCs).² Petechiae and ecchymoses are purpura of less than or greater than 3 mm, respectively, and the presence of more than one such lesion may indicate a disorder of primary hemostasis. Other symptoms of defects of primary hemostasis are menorrhagia (profuse menstrual flow), bleeding from the gums, hematemesis (vomiting of blood), and epistaxis (uncontrolled nosebleed). Although nosebleeds are common and mostly innocent, especially in children, they suggest a primary hemostatic defect when they occur repeatedly, last longer than 10 minutes, involve both nostrils, or require physical intervention or transfusions.

Mucocutaneous hemorrhage tends to be associated with thrombocytopenia (platelet count lower than 150,000/mcL), qualitative platelet disorders, von Willebrand disease (VWD), or vascular disorders such as scurvy or telangiectasia (see Chapters 43 and 44). A careful history and physical examination distinguishes between mucocutaneous and anatomic bleeding, and the distinction helps direct investigative laboratory testing and treatment.

Anatomic (soft tissue) hemorrhage is seen in acquired or congenital defects in secondary hemostasis, or plasma coagulation factor deficiencies. Examples of anatomic bleeding include recurrent or excessive bleeding after minor trauma, dental extraction, or a surgical procedure. In such cases, hemorrhage may immediately follow a traumatic event, but is often delayed or recurs minutes or hours after the initial blood flow is stopped. In other patients, anatomic bleeding episodes are spontaneous. Most anatomic bleeds are internal, such as bleeds into joints, body cavities, muscles, or the central nervous system, and may initially have few visible signs. Because joint bleeds cause swelling and acute pain, they may not be immediately recognized as hemorrhages, although hemophilia patients usually know what is happening from experience. Repeated joint bleeds (hemarthroses) cause painful inflammation, with swelling and permanent cartilage damage that immobilize the joint. Repeated joint bleeds cause painful inflammation and permanent cartilage damage that swells and immobilizes the joint. Bleeds into soft tissues such as muscle or fat may cause nerve compression and subsequent temporary or permanent loss of function.³ When the bleeding involves body cavities, it causes symptoms related to the organ that is affected. Bleeding into the central nervous system, for instance, may cause headaches, confusion, seizures, and coma and must be managed as a medical emergency. Bleeds into the kidney may present as hematuria and may potentially be associated with acute kidney failure.

Whenever a soft tissue or mucocutaneous generalized hemostatic disorder is suspected, hemostasis laboratory testing is essential. Emergency department physicians often must begin to intervene in cases of acute hemorrhage, even before taking a history or waiting for the results of laboratory assays. In these instances, group-matched or O-negative RBCs, frozen plasma (FP), platelet concentrate, cryoprecipitate, recombinant activated coagulation factor VII (rFVIIa; NovoSeven, Novo Nordisk, Inc., Princeton, N.J.) or the antifibrinolytic tranexamic acid (Cyklokapron, Pfizer, New York, N.Y.) may be used to correct coagulation factor and platelet deficiencies to stop hemorrhage.4–6 Box 41-1 lists symptoms that suggest generalized hemorrhagic disorders.

Acquired versus Congenital Bleeding Disorders

Liver disease, kidney failure, chronic infections, autoimmune disorders, obstetric complications, dietary deficiencies such as vitamin C or vitamin K deficiency, blunt or penetrating trauma, and inflammatory disorders may all be associated with coagulopathy. If a patient’s bleeding episodes began after infancy, are associated with some disease or physical trauma, and are not duplicated in relatives, they probably are caused by an acquired, not a congenital, condition. When an adult patient comes for treatment of generalized hemorrhage, the physician first looks for an underlying disease or event and takes a personal and family history. The important elements of patient history are age, sex, current or past pregnancy, any systemic disorders such as diabetes or cancer, trauma, and exposure to drugs, including over the counter drugs or drugs of abuse. The physician determines the trigger, location, and volume of bleeding, then orders laboratory assays (Table 41-1). The initial coagulopathy test profile should consist of a complete blood count (CBC) that includes a platelet count, prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen assay.⁷ These tests take on clinical significance when the history and physical examination already have established the existence of abnormal bleeding. They are not effective as screens for healthy individuals.

Congenital hemorrhagic disorders are uncommon, occurring in 1 per 100 people, and are usually diagnosed in infants or young children, who often have relatives with similar symptoms. Congenital bleeding disorders lead to repeated hemorrhages that may be spontaneous or occur following minor injury or may occur in unexpected locations, such as joints, body cavities, retinal veins and arteries, or the central nervous system. Patients with mild congenital hemorrhagic disorders may have no symptoms until they reach adulthood or experience some physical challenge, such as trauma, dental extraction, or a surgical procedure. The most common congenital deficiencies are VWD, factor VIII and IX deficiencies (hemophilia A and B), and platelet function disorders. Inherited deficiencies of fibrinogen, prothrombin, and factors V, VII, X, XI, and XIII also exist, but are rare (Box 41-2).

Acute Coagulopathy of Trauma-Shock

In North America, unintentional injury is the leading cause of death among those aged 1 to 45 years. The total rises when felonious, self-inflicted, and combat injuries are included: in the United States alone, trauma causes 93,000 deaths per year.⁹ Severe neurologic displacement accounts for 50% of deaths, with most occurring before the patient arrives at the hospital; however, of initial survivors, 20,000 die of hemorrhage within 48 hours.¹⁰ Acute coagulopathy of trauma-shock (ACOTS) accounts for fatal hemorrhage; 3000 to 4000 of these deaths are preventable. ACOTS is triggered by the combination of injury-related acute inflammation, platelet activation, tissue factor release, hypothermia, acidosis, and hypoperfusion (poor distribution of blood to tissues caused by low blood pressure), all of which are elements of systemic shock. For in-transit fluid resuscitation, colloid plasma expanders such as 5% dextrose are used to counter hypoperfusion.¹¹ The administration of plasma expanders has a dilutional effect that is compounded by emergency department transfusion of RBCs, and although these measures are life saving, they intensify the coagulopathy, as does subsequent surgical intervention.

Massive transfusion, defined as administration of 4 to 5 RBC units within 1 hour or 8 to 10 units within 24 hours, is essential in otherwise healthy trauma victims when the systolic blood pressure is less than 110 mm Hg, pulse is greater than 105 beats/min, pH is less than 7.25, hematocrit is less than 32%, hemoglobin is less than 10 g/dL, and PT is prolonged to greater than 1.5 times the mean of the reference interval or generates an international normalized ratio (INR) of 1.5.¹²

Liver Disease

The bleeding associated with liver disease may be localized or generalized, mucocutaneous or anatomic. Enlarged and collateral esophageal vessels called esophageal varices are a complication of chronic alcoholic cirrhosis; hemorrhaging from varices is localized bleeding, not a coagulopathy, though often fatal. Mucocutaneous bleeding occurs in liver disease–associated thrombocytopenia, often accompanied by decreased platelet function. Soft tissue bleeding is the consequence of procoagulant dysfunction and deficiency.

Renal Failure and Hemorrhage

Acute renal failure may be associated with acute gastrointestinal bleeding caused by specific anatomic lesions, but this does not necessarily indicate that a coagulopathy or platelet dysfunction is present.³¹ Chronic renal failure of any cause is commonly associated with platelet dysfunction and mild to moderate mucocutaneous bleeding.³² Platelet adhesion to blood vessels and platelet aggregation are suppressed, perhaps because the platelets are coated by guanidinosuccinic acid or dialyzable phenolic compounds.³³ Decreased RBC mass (anemia) and thrombocytopenia contribute to the bleeding and may be corrected with dialysis, erythropoietin or RBC transfusions, and interleukin-11 therapy.^34,35

Hemostasis activation syndromes that deposit fibrin in the renal microvasculature reduce the function of the glomeruli. Examples of such disorders are DIC, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Although these are by definition thrombotic disorders, they invariably cause thrombocytopenia, which may lead to mucocutaneous bleeding. Fibrin also may be deposited in renal transplant rejection and in the glomerulonephritis syndrome of systemic lupus erythematosus. This may be associated with a rise in quantitative plasma D-dimer, thrombin-antithrombin complexes, or prothrombin fragment 1 + 2, which are markers of coagulation activation (see Chapter 45).³⁶

Laboratory tests for bleeding in renal disease provide only modest information with little predictive or management value. The bleeding time may be prolonged, but is too unreliable to provide an accurate diagnosis or to assist in monitoring treatment.³⁷ Platelet aggregometry test results may predict bleeding. The PT and PTT are expected to be normal.

Management of renal failure–related bleeding typically focuses on the severity of the hemorrhage without reliance on laboratory test results. Renal dialysis improves platelet function, particularly when anemia is well controlled. Desmopressin acetate may be administered intravenously (DDAVP) or intranasally (Stimate) to increase the plasma concentration of high-molecular-weight multimers of VWF, which also aids platelet adhesion and aggregation.³⁸ Patients with renal failure should not take aspirin, clopidogrel, or other platelet inhibitors, because these drugs increase the risk of hemorrhage.

Vitamin K Deficiency

Vitamin K is ubiquitous in foods, especially green leafy vegetables, and the daily requirement is small, so pure dietary deficiency is rare. Body stores are limited, however, and become exhausted when the usual diet is interrupted, as when patients are fed only with parenteral (intravenous) nutrition for an extended period or when patients embark upon fad diets. Also, because vitamin K is fat soluble and requires bile salts for absorption, biliary duct obstruction (atresia), fat malabsorption, and chronic diarrhea may cause vitamin K deficiency. Broad-spectrum antibiotics that disrupt normal flora may cause a slight reduction because they destroy bacteria that produce vitamin K. The degree of reduction is insignificant when the diet is otherwise normal, but may become an important issue when the patient is receiving only parenteral nutrition.

Vitamin K Antagonists

The γ-carboxylation cycle of coagulation factors is interrupted by coumarin-type oral anticoagulants such as warfarin that disrupt the vitamin K epoxide reductase and vitamin K quinone reductase reactions (Figure 41-1). In deficient γ-carboxylation, the liver releases dysfunctional des-γ-carboxyl factors II (prothrombin), VII, IX, and X, and proteins C, S, and Z; these inactive forms are called proteins in vitamin K antagonism (PIVKA). Therapeutic overdose or the accidental or felonious administration of warfarin-containing rat poisons may result in moderate to severe hemorrhage because of the lack of functional factors. The effect of brodifacoum or “superwarfarin,“ often used as a rodenticide, lasts for weeks to months, and treatment of poisoning with this substance requires repeated administration of vitamin K with follow-up PT monitoring.⁴³ Warfarin overdose is the single most common reason for hemorrhage-associated emergency department visits.

Autoanti-VIII Inhibitor and Acquired Hemophilia

Acquired autoantibodies that specifically inhibit factors II (prothrombin), V, VIII, IX, and XIII and VWF have been described in nonhemophilic patients.⁴⁴ Anti-VIII is the most common. Patients who develop an autoantibody to factor VIII, which is diagnostic of acquired hemophilia, are frequently older than age 60 and have no apparent underlying disease. Acquired hemophilia is occasionally associated with rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, or lymphoproliferative disease. It also may occur in women of childbearing age 2 to 5 months after delivery. Patients with inhibitor autoantibodies are given immunosuppressive therapy, although autoantibodies that develop after pregnancy typically disappear spontaneously. Acquired hemophilia has an overall incidence of 1 per 1 million people per year. Patients experience sudden and severe bleeding in soft tissues or bleeding in the gastrointestinal or genitourinary tract. Acquired hemophilia, even when treated, remains fatal in at least 20% of cases. Autoantibodies to other procoagulants are less frequent, but create similar symptoms.⁴⁵

Clot-Based Studies in Acquired Hemophilia

PT, PTT, and thrombin time are recommended tests for any patient with sudden onset of anatomic hemorrhage resembling acquired hemophilia. In the presence of a factor VIII inhibitor, which is the most common type, the PTT should be prolonged, whereas the PT and thrombin time are expected to be normal. A factor assay should reveal factor VIII activity to be less than 30% and often undetectable.

The presence of the inhibitor is confirmed with clot-based mixing studies. The PTT prolongation may be corrected initially by the addition of NP to the test specimen in a 1:1 ratio, but PTT again becomes prolonged after incubation of the 1:1 NP–patient plasma mixture at 37° C for 1 to 2 hours. The return of the lack of correction after incubation occurs because factor VIII autoantibodies are frequently of the immunoglobulin G4 isotype, which is time and temperature dependent. Consequently, the inhibitor effect may be evident only after the patient’s inhibitor is allowed to interact with the factor VIII in the NP for 1 to 2 hours at 37° C before testing. A few high-avidity inhibitors may cause immediate prolongation of the PTT; in this case an incubated mixing study is unnecessary.

The in vitro kinetics of factor VIII neutralization by inhibitor are nonlinear. Although there is early rapid loss of factor VIII activity, residual activity remains, which indicates that the reaction has reached equilibrium. This is called type II kinetics (Figure 41-2). In contrast, alloantibodies to factor VIII, which develop in 20% to 25% of patients with severe hemophilia in response to factor VIII therapy, exhibit type I kinetics. In the latter, there is linear in vitro neutralization of factor VIII activity over 1 to 2 hours, which results in complete inactivation. In type I kinetics in vitro measurement is relatively accurate, whereas in type II kinetics the titration of inhibitor activity is semiquantitative.⁴⁶

Quantitation of autoanti-VIII inhibitor is accomplished using the Bethesda assay, which is ordinarily employed to measure inhibitor in hemophilic patients with alloantibodies to factor VIII (see Hemophilia A and Factor VIII Inhibitors). Titer results help the clinician choose the proper therapy to control bleeding. Repeat titers are used to follow the response to immunosuppressive drugs, but are not needed for management of the bleeding symptoms.

Acquired von Willebrand Disease

Acquired VWF deficiency, with symptoms similar to those of congenital VWD, has been described in association with hypothyroidism; autoimmune, lymphoproliferative, and myeloproliferative disorders; benign monoclonal gammopathies; Wilms tumor; intestinal angiodysplasia; congenital heart disease; pesticide exposure; and hemolytic uremic syndrome.⁵³ The pathogenesis of acquired VWD varies and may involve decreased production of VWF, presence of an autoantibody, or adsorption of VWF to abnormal cell surfaces, as seen in association with lymphoproliferative disorders and Wilms tumor.⁵⁴

Acquired VWD manifests with moderate to severe mucocutaneous bleeding and may be suspected in any patient with recent onset of bleeding who has no significant medical history. Although the PT is not affected, the PTT may be moderately prolonged if the VWF reduction is severe enough to cause a deficiency of coagulation factor VIII, for which VWF serves as a carrier molecule. As in congenital VWD, the diagnosis is based on a finding of diminished VWF activity (ristocetin cofactor [VWF : RCo] assay) and the presence of VWF antigen (VWF : Ag) by immunoassay. It may be difficult to differentiate between mild, previously asymptomatic congenital VWD and acquired VWD.

If the patient requires treatment for bleeding, DDAVP or a plasma-derived factor VIII/VWF concentrate such as Humate-P (CSL Behring, King of Prussia, Pa.), Wilate (Octapharma, Hoboken, N.J.), or Alphanate (Grifols, Los Angeles, Calif.) is effective at controlling the symptoms. Cryoprecipitate is no longer recommended for treatment of VWD because it does not undergo viral inactivation.

Disseminated Intravascular Coagulation

DIC, although characteristically identified through hemorrhagic symptoms, is classified as a thrombotic disorder and is described in Chapter 42.

Molecular Biology and Functions of von Willebrand Factor

VWF is a glycoprotein whose molecular mass ranges from 800,000 to 20,000,000 D, the largest molecule in human plasma. Its plasma concentration is 0.5 to 1.0 mg/dL, but a great deal more is readily available on demand from storage organelles. VWF is synthesized in the endoplasmic reticulum of endothelial cells and stored in cytoplasmic Weibel-Palade bodies of endothelial cells. It is also synthesized in megakaryocytes and stored in the α-granules of platelets (see Chapter 13). Weibel-Palade bodies and α-granules release VWF in response to a variety of hemostatic stimuli.⁵⁶

The VWF gene consists of 52 exons spanning 178 kilobase pairs (kb) on chromosome 12.⁵⁷ The translated protein is a monomer of 2813 amino acids that, after glycosylation, forms dimers that are transferred to the aforementioned storage organelles, where the dimers polymerize to form enormous multimers. At the time of storage, a signal sequence and a propeptide, known as VWF antigen II, are cleaved so that the mature monomers, already polymerized, consist of 2050 amino acids.⁵⁸

Each VWF monomer has four functional domains that bind factor VIII, platelet glycoprotein Ib/V/IX, platelet glycoprotein IIb/IIIa, and collagen, respectively (see Chapter 13).⁵⁹ Upon release from intracellular stores, VWF forms a complex with coagulation factor VIII named VIII/VWF (Figure 41-3). VWF protects factor VIII from proteolysis, prolonging its plasma half-life from a few minutes without VWF to 8 to 12 hours with VWF. Table 41-3 lists the nomenclature for the structural and functional components of the factor VIII/VWF molecule.

Although VWF is the factor VIII carrier molecule, its primary function is to mediate platelet adhesion to subendothelial collagen in areas of high flow rate and high shear force, as in capillaries and arterioles. VWF first binds fibrillar intimal collagen exposed during the desquamation of endothelial cells. Subsequently, platelets adhere through their glycoprotein Ib/V/IX receptor to the VWF “carpet.“ The largest VWF multimers are best equipped to serve the adhesion function. When VWF binds glycoprotein Ib/V/IX, platelets become activated and express a second VWF binding site, glycoprotein IIb/IIIa. This receptor binds VWF and fibrinogen to mediate irreversible platelet-to-platelet aggregation. The adhesion and aggregation sequences are essential to normal hemostasis.

Pathophysiology of von Willebrand Disease

Structural (qualitative) or quantitative VWF abnormalities reduce platelet adhesion, which leads to mucocutaneous hemorrhage of varying severity: epistaxis, ecchymosis, menorrhagia, gastrointestinal tract bleeding, and surgical bleeding. Symptoms vary over time and within kindreds, because VWF production and release is susceptible to a variety of physiologic influences. Severe quantitative VWF deficiency creates in addition factor VIII deficiency owing to the inability to protect nonbound factor VIII from proteolysis. Many people have VWF levels in the intermediate range of 30% to 50% of normal and maintain a factor VIII level sufficient for competent coagulation. When factor VIII levels decrease to less than 30% of normal, anatomic soft tissue bleeding accompanies the typical mucocutaneous bleeding pattern of VWD.

von Willebrand Disease Types and Subtypes

Subtype 2B von Willebrand Disease

In subtype 2B VWD, rare mutations within the A1 domain raise the affinity of VWF for platelet glycoprotein Ib/V/IX, its customary binding site; these are thus “gain of function” mutations. Large VWF multimers spontaneously bind resting platelets and are unavailable for normal platelet adhesion. Consequently, the electrophoretic multimer pattern is characterized by lack of high-molecular-weight multimers but presence of intermediate-molecular-weight multimers. There also may be moderate thrombocytopenia caused by chronic platelet activation, because multimer-coated platelets indiscriminately bind the endothelium.

A platelet mutation that increases glycoprotein Ib/V/IX affinity for normal VWF multimers creates a clinically similar disorder called platelet-type VWD or pseudo-VWD. In this instance, the large multimers also are lost from the plasma, and platelets become adhesive (Figure 41-4). Clinically and in the laboratory, the two entities are indistinguishable.

Laboratory Detection and Classification of von Willebrand Disease

Definitive diagnosis of VWD depends on the combination of a personal and family history of mucocutaneous bleeding and the laboratory demonstration of decreased VWF activity. A CBC is necessary to rule out thrombocytopenia as the cause of mucocutaneous bleeding, and PT and PTT, which assess the coagulation system, are part of the initial VWD workup. No longer recommended, according to the 2009 NLHBI VWD guidelines, are the bleeding time test and the “PFA-100 or other automated functional platelet assays.“ These traditional screening tests generate “conflicting” sensitivity and specificity data.

The standard VWD test panel includes three assays: a quantitative VWF test (VWF : Ag assay) employing an enzyme immunoassay or automated latex immunoassay technique, such as the Liatest (Diagnostica Stago, Asnières-sur-Seine, France); the VWF activity test, which determines the factor’s ability to bind to platelets, also known as the VWF : RCo assay; and a factor VIII activity assay. The VWF : RCo assay employs preserved reagent platelets. The agonist ristocetin supports platelet agglutination in the presence of VWF.

When the ratio of the VWF : RCo assay value to the VWF : Ag concentration is less than 0.5, 0.6, or 0.7 (ratio cutoff is generated from reference interval studies by local laboratories), qualitative or type 2 VWD is inferred. Additional tests are needed to confirm type 2 VWD and to differentiate subtype 2A from subtype 2B. Low-dose ristocetin-induced platelet aggregometry (RIPA), also called the ristocetin response curve, identifies subtype 2B. The low-dose RIPA test is performed on platelet-rich plasma. In subtype 2B the patient’s platelets, because they are coated with abnormal VWF multimers, agglutinate in response to less than 0.5 mg/mL ristocetin; sometimes they even agglutinate in response to 0.1 mg/mL ristocetin. In comparison, normal platelets or platelets from a patient with subtype 2A agglutinate only at ristocetin concentrations greater than 0.5 mg/mL.

VWF multimer analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis further differentiates between VWD subtypes 2A and 2B. Although both 2A and 2B lack high-molecular-weight multimers, intermediate multimers are present in the electrophoretic pattern of subtype 2B VWD samples but are absent from the pattern of a patient with subtype 2A VWD. Multimer analysis is technically demanding and is performed mainly in reference laboratories for differentiation of VWD subtypes. Table 41-4 lists the expected test results for all VWD types and subtypes.

Because acquired VWD may mimic any one of the VWD types and subtypes, results of the laboratory workup do not help to differentiate it from the inherited condition. Review of the clinical history with emphasis on age at onset of bleeding, comorbid conditions, and family history may signal acquired disease rather than the congenital form.

Pitfalls in the Diagnosis of von Willebrand Disease

VWF activity is influenced by varying genetic penetrance, ABO blood group, inflammation, hormones, age, and physical stress.⁶⁴ Increased estrogen levels during the second and third trimesters of pregnancy nearly normalize plasma VWF activity even in women with moderate VWF deficiency. However, VWF function and concentration decrease rapidly after delivery, which may lead to acute postpartum hemorrhage, for which the obstetrician is watchful. Oral contraceptive use and hormone replacement therapy also raise VWF activity, and activity waxes and wanes with the menstrual cycle.

VWF activity rises substantially in acute inflammation such as occurs postoperatively, subsequent to trauma, or during an infection. Physical stress such as cold, exertion, or children’s crying or struggling during venipuncture causes VWF activity to rise. VWF activity rises when the tourniquet remains tied for more than 1 minute prior to venipuncture and drops if the specimen is stored in the refrigerator prior to testing. VWD patients experience fluctuation in disease severity over time, and the clinical manifestations of the disease vary from person to person within kindreds, despite the assumption that everyone in the family possesses the same mutation. When the clinical presentation suggests VWD, the VWD laboratory assay panel should be repeated until the results are conclusive.⁶⁵

Adding to VWD diagnostic confusion is concern over the variability in the results of the VWF : RCo assay, which is based on platelet aggregometry but which is performed using a variety of instruments and methods, including at least one automated device, the BCS XP system with von Willebrand reagent (Siemens Healthcare Diagnostics, Inc., Deerfield, Ill.). Ristocetin avidity varies from lot to lot. In the United States, proficiency surveys consistently reveal VWF : RCo assays to have an unimpressive interlaboratory coefficient of variation of 30% and a least detectable activity range of 6% to 12%.⁶⁶

Concern over the poor reproducibility of results of the VWF activity assay has led to attempts at developing alternative assays, the most promising of which is the VWF collagen binding (VWF : CB) assay. VWF : CB employs type III collagen as its solid-phase target antigen. Developed in 1990, the VWF : CB assay produces results that more closely match those of the VWF : RCo assay than of VWF : Ag assays, because collagen type III binds predominantly large VWF multimers; however, the target collagen composition needs to be standardized before this assay achieves standard screening status.⁶⁷ The VWF : CB assay has better precision than the VWF : RCo assay.⁶⁸

Many reference laboratories add the VWF : CB assay to their initial VWD screening profile. They argue that the VWF : CB assay detects abnormalities of VWF collagen binding, whereas the VWF : RCo assay detects abnormalities of VWF platelet binding and cite instances in which the VWF : CB value was abnormal when the VWF : RCo value was normal, and vice-versa.

In an effort to more closely reflect clinical reality and reduce false-positive type 1 VWD diagnoses, the 2009 NHLBI VWD guidelines have coined the nondisease description “low VWF” for the condition in which VWF activity and antigen concentrations are between 30% and 50% of normal, the ratio of VWF : RCo to VWF : Ag is greater than 0.5, and factor VIII activity is greater than 50% of normal. This suggested category reflects the difficulty in distinguishing mucocutaneous bleeding from self-reported “easy bruising” and recognizes that low VWF activity and bleeding often associate coincidentally. To make a definite type 1 VWD diagnosis, 30% of normal VWF activity is used as the cutoff. Molecular confirmation of type 1 VWD, currently but a dream because of the plethora of contributing mutations, may soon become routine as molecular microchip technology meets the challenge.

VWF activity varies by ABO blood group, and expert panels have in the past recommended that laboratory directors maintain separate reference intervals for each group, as provided in Table 41-5. The 2009 NHLBI VWD guidelines have recommended against this practice, noting that “despite the ABO blood grouping and associated reference ranges, the major determinant of bleeding risk is low VWF.“ Therefore, VWF test result reference intervals are now population based rather than ABO stratified. The internationally generalized cutoff of 50% for low VWF and 30% for VWD is clinically reasonable, although laboratory directors may choose to validate and adjust this cutoff in internal reference interval studies.

In a well-managed tertiary care facility, laboratory scientists and physicians communicate regularly with medical staff who are challenged with VWD management—for example, nurses, pharmacists, emergency department and primary care physicians, internists, surgeons, gynecologists, and hematologists. Laboratory professionals ensure that those who manage VWD patients are aware of the effects of ABO group, estrogen levels, inflammation, and physical stress on VWF activity, and that they understand the strengths and limitations of VWF laboratory assay panels, the proper interpretation of assay results, and the availability of follow-up and confirmatory assays.

Treatment of von Willebrand Disease

Mild bleeding may resolve with the use of local measures, such as limb elevation, pressure, and application of ice packs (the athlete’s acronym is RICE for rest, ice, compression, and elevation).⁶⁹ Moderate bleeding may respond to estrogen and desmopressin acetate, which trigger the release of VWF from storage organelles. Therapeutic dosages are monitored when necessary using serial VWF : Ag concentration assays. Desmopressin acetate (1-desamino-8-D arginine vasopressin) is an antidiuretic hormone analogue used to control incontinence in diabetes mellitus and bedwetting; release of VWF from storage organelles is a side-effect. Desmopressin acetate in its oral form, DDAVP, or nasal spray form, Stimate (both from CSL Behring, King of Prussia, Pa.), is consistently effective for type 1 VWD and generally useful for subtype 2A. It is contraindicated for subtype 2B, however, because it causes the release of abnormal VWF with increased affinity for platelet receptors, which may intensify thrombocytopenia and lead to increased platelet activation and thrombosis. Because of its antidiuretic property, repeated doses may lead to hyponatremia (low serum sodium). For this reason, it is necessary to monitor and regulate electrolytes during desmopressin acetate therapy.

ε-Aminocaproic acid (EACA; Amicar) or tranexamic acid (Cyklokapron) inhibits fibrinolysis and may help control bleeding when used alone or in conjunction with desmopressin acetate. Therapy using nonbiologic preparations is preferred over human plasma–derived biologic therapy, because nonbiologicals eliminate the risk of viral disease transmission and circumvent religious objections to receipt of human blood products.

For treatment of severe VWD (type 3) and subtype 2B, three commercially prepared human plasma–derived high-purity preparations are available that provide a mixture of VWF and coagulation factor VIII. These are Humate-P, Alphanate, and Wilate (FDA-cleared December 2009).⁷⁰ The calculation of the proper dosage follows principles identical to those used for treatment of hemophilia A provided in the next section. Laboratory monitoring by the VWF : Ag assay is essential to determine if the given amount produced the target level of VWF and to follow its degradation between doses.

Recombinant and affinity-purified factor VIII preparations contain no VWF and cannot be used to treat VWD. Cryoprecipitate and FP are less desirable alternatives because of the risk of virus transmission, and the necessary FP volume per dose may cause TACO. Therapy for bleeding secondary to acquired VWD follows the same principles as delineated previously plus treatment of the primary disease, if applicable. Therapeutic recommendations for VWD are summarized in Table 41-6.

Hemophilia A

The hemophilias are congenital single-factor deficiencies marked by anatomic soft tissue bleeding. Second to VWD in prevalence among congenital bleeding disorders, hemophilias occur in 1 in 10,000 individuals. Of those affected, 85% are deficient in factor VIII, 14% are deficient in factor IX, and 1% are deficient in factor XI or one of the other coagulation factors, such as factor II (prothrombin), V, VII, X, or XIII. Congenital deficiency of factor VIII is called classic hemophilia or hemophilia A.⁷¹

Hemophilia A Treatment

The goal of on-demand hemophilia A treatment is to raise the patient’s factor VIII activity to hemostatic levels whenever he experiences or suspects a bleeding episode or anticipates a hemostatic challenge such as a surgical procedure. The target activity level depends on the nature of the bleeding, but it is seldom necessary to reach activity greater than 75%. Target activity should be maintained until the threat is resolved. In the case of a bleed into soft tissue or a body cavity, the sooner the target factor level is reached, the less painful is the episode, and the less likely is the patient to experience inflammation or nerve damage. Because factor VIII has a half-life of 8 to 12 hours, twice-a-day infusions are required.

With the availability of abundant recombinant factor VIII concentrate, many hemophilic patients maintain themselves on a steady prophylactic dosage designed to constantly keep their factor VIII activity at hemostatic levels.⁷⁸ Although it is initially more expensive, the prophylactic approach saves downstream resources by ameliorating the adverse effects of repeated hemorrhages and their long-term consequences.⁷⁹

Many hemophilic patients’ factor VIII activity rises upon administration of desmopressin acetate in the form of DDAVP or Stimate (nasal formulation), alone or in combination with an antifibrinolytic such as Amicar or Cyklokapron. When desmopressin acetate treatment proves ineffective, intravenous factor VIII concentrates are the next option. High-purity synthetic factor VIII concentrates are produced from mammalian cells using recombinant deoxyribonucleic acid (DNA) technology or from human plasma using factor VIII–specific monoclonal antibodies and column separation.80,81 The human plasma–derived concentrates Alphanate, Humate-P, and Wilate are prepared by chemical fractionation of human plasma and contain VWF, fibrinogen, and noncoagulant proteins, in addition to factor VIII. All plasma-derived concentrates undergo viral inactivation steps, and since 1985, none has transmitted lipid-envelope viruses such as HIV, hepatitis B virus, or hepatitis C virus. Plasma-derived factor VIII concentrates may transmit nonlipid viruses such as parvovirus B19 and hepatitis A virus, however.⁸² Recombinant products may use human albumin in the manufacturing process, which introduces the theoretical risk of Creutzfeldt-Jakob disease transmission; however, manufacturers more recently have developed products free of all human protein, such as Bioclate (Pfizer, New York, N.Y.).⁸³

When hematologists treat a hemophilic patient, they base their factor VIII concentrate dosage calculations on the definition of a unit of factor VIII activity, which is taken as the amount present in 1 mL of normal plasma and is synonymous with 100% activity. They further calculate the desired increase after factor VIII concentrate infusion by subtracting the patient’s preinfusion factor activity from the target activity level. The desired increase is multiplied by the patient’s plasma volume to compute the dosage. The patient’s plasma volume may be estimated from his or her blood volume and hematocrit.⁸⁴ The blood volume is approximately 65 mL/kg of body weight, and the plasma volume is the plasmacrit (100% − hematocrit %) as in the following formulas:

< ?xml:namespace prefix = "mml" />Plasma volume=weight in kilograms×65mL/kg×(1−hematocrit)

Factor VIII concentrate dose=plasma volume×(target factor VIII level−initial factor VIII level)

This formula is also used in the treatment of VWD. Overdosing seems to confer no thrombotic risk, but wastes resources. Regardless of the dose administered, laboratory monitoring and close clinical observation are essential to prevent or halt bleeding and its complications. Repeat dosing is done on an 8- to 12-hour schedule reflecting the half-life of factor VIII. The second administration of factor VIII uses half the concentration of the first dose.

Hemophilia B (Factor IX Deficiency)

Hemophilia B, also called Christmas disease, represents approximately 14% of hemophilia cases in the United States, although its incidence in India nearly equals that of hemophilia A.⁸⁷ Hemophilia B is caused by deficiency of factor IX, one of the vitamin K–dependent serine proteases. Factor IX is a substrate for both factors XIa and VIIa because it is cleaved by either to form dimeric factor IXa (Figure 41-5). Subsequently, factor IXa complexes with factor VIIIa to cleave and activate its substrate, factor X. Factor IX deficiency reduces thrombin production and causes soft tissue bleeding that is indistinguishable from that in hemophilia A. It also is a sex-linked, markedly heterogeneous disorder involving more than 220 separate mutations resulting in a range of mild to severe bleeding manifestations. Determination of carrier status is less successful in hemophilia B than in hemophilia A because of the large number of factor IX mutations and the lack of a linked molecule such as VWF that can be used as a normalization index. DNA analysis occasionally may be used to establish carrier status when hemophilia B has been diagnosed and its specific mutation identified in a relative.

The laboratory is essential to the diagnosis of hemophilia B. The PTT typically is prolonged, whereas the PT is normal. If the clinical symptoms suggest hemophilia B, the factor IX assay should be performed even if PTT is within the reference range, because the PTT reagent may be insensitive to mild factor IX deficiency.

Recombinant or column-purified plasma-derived factor IX concentrates are used to treat hemophilia B. Dosing is calculated the same way as for factor VIII concentrates in hemophilia A and VWD, with the exception that the calculated initial dose is doubled to compensate for factor IX distribution into the extravascular space. Repeat doses of factor IX are given every 24 hours, which reflects the half-life of the factor. The second and subsequent doses, if needed, are half the initial dose, provided that factor assays determine that the target level of factor IX was achieved.

Inhibitors to factor IX arise in only 3% of hemophilia B patients. Anti–factor IX alloantibodies react avidly with factor IX and may be detected using the Bethesda assay. Bleeding in patients with inhibitors is treated as in patients with factor VIII inhibitors using Autoplex T, FEIBA, or NovoSeven.

Hemophilia C (Rosenthal Syndrome, Factor XI Deficiency)

Factor XI deficiency is an autosomal dominant hemophilia with mild to moderate bleeding symptoms. More than half of the cases have been described in Ashkenazi Jews, but individuals of any ethnic group may be affected. The frequency and severity of bleeding episodes do not correlate with factor XI levels, and laboratory monitoring of treatment serves little purpose after the diagnosis is established. The physician treats hemophilia C with frequent infusions of FP during times of hemostatic challenge.⁸⁸ In the laboratory, the PTT is prolonged and the PT is normal.

Other Congenital Single-Factor Deficiencies

The remaining congenital single-factor deficiencies listed in Table 41-8 are rare, are caused by autosomal recessive mutations, and are often associated with consanguinity. The PT, PTT, and thrombin time may be employed to distinguish among these disorders, as shown in Table 41-7. In addition, immunoassays may be performed to distinguish among the more prevalent quantitative and the less prevalent qualitative abnormalities. In qualitative disorders, often called dysproteinemias, the ratio of factor activity to antigen is less than 0.7. The bleeding symptoms in the dysproteinemias may be more severe than in quantitative deficiencies, but the risk of inhibitor formation is theoretically lower. The clot-based measurement of factors II (prothrombin) and X may be supplemented or replaced by more reproducible chromogenic substrate assays. Fibrinogen is usually measured using the Clauss assay, a modification of the thrombin time, but may be measured by turbidimetry or immunoassay.

Because platelets transport about 20% of circulating factor V, the platelet function in factor V deficiency may be diminished, which is reflected in a prolonged bleeding time but normal platelet aggregation.⁸⁹ The PT and PTT are prolonged. Because of the concentration of factor V in platelet α-granules, normal platelet concentrate is an effective form of therapy for factor V deficiency. A combined factor V and VIII deficiency may be caused by a genetic defect traced to chromosome 18 that affects transport of both factors by a common protein in the Golgi apparatus.⁹⁰

Factor VII deficiency causes moderate to severe anatomic hemorrhage. The bleeding does not necessarily reflect the factor VII activity level. The half-life of factor VII is approximately 6 hours, which affects the frequency of therapy. NovoSeven at 30 mcg/mL and prothrombin complex concentrate such as Autoplex T preparations are effective and may provide a target level of 10% to 30%. Many factor VII deficiencies are dysproteinemias.⁹¹ The PT, but not the PTT, is prolonged in factor VII deficiency.

Factor X deficiency causes moderate to severe anatomic hemorrhage that may be treated with FP or prothrombin complex concentrate to produce therapeutic levels of 10% to 40%.⁹² The half-life of factor X is 24 to 40 hours. Acquired factor X deficiency has been described in amyloidosis, in paraproteinemia, and in association with antifungal drug therapy. The hemorrhagic symptoms are severe and life-threatening. The PT and PTT are both prolonged in factor X deficiency. In the time-honored Russell viper venom time test, which activates the coagulation mechanism at the level of factor X, clotting time is prolonged in deficiencies of factors X and V, prothrombin, and fibrinogen. The venom used is harvested from the Russell viper, the most dangerous snake in Asia. This test may be useful in distinguishing a factor VII deficiency, which does not affect the results, from deficiencies in the common pathway, although specific factor assays are the standard approach.

Plasma factor XIII is a tetramer of paired α and β monomers. The intracellular form is a homodimer (two α chains) and is stored in platelets, monocytes, placenta, prostate, and uterus. The α chain contains the active enzyme site, and the β chain is a binding and stabilizing portion. Factor XIII deficiency occurs in three forms related to the affected chain, as shown in Table 41-9. Patients with factor XIII deficiency have a normal PT, PTT, and thrombin time despite anatomic bleeds and poor wound healing. They form weak (non–cross-linked) clots that dissolve within 2 hours when suspended in a 5-M urea solution, a traditional factor XIII screening assay.⁹³ To confirm factor XIII deficiency, factor activity may be measured accurately using a chromogenic substrate assay such as the Behring Behrichrom FXIII assay (Behring Diagnostics, King of Prussia, Pa.).

Finally, autosomally inherited deficiencies of the fibrinolytic regulatory proteins α₂-antiplasmin and plasminogen activator inhibitor-1 have been reported to cause moderate to severe bleeding. Both are rare and may be diagnosed using chromogenic substrate assays.