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

CHAPTER 38. Hemorrhagic disorders and laboratory assessment

George A. Fritsma*

OUTLINE

Bleeding Symptoms

Localized Versus Generalized Hemorrhage

Mucocutaneous Versus Anatomic Hemorrhage

Acquired Versus Congenital Bleeding Disorders

Acquired Coagulopathies

Acute Coagulopathy of Trauma-Shock (ACOTS)

Liver Disease Coagulopathy

Chronic Renal Failure and Hemorrhage

Vitamin K Deficiency and Hemorrhage

Autoanti-VIII Inhibitor and Acquired Hemophilia

Acquired von Willebrand Disease

Disseminated Intravascular Coagulation

Congenital Coagulopathies

Von Willebrand Disease

Hemophilia A (Factor VIII Deficiency)

Hemophilia B (Factor IX Deficiency)

Hemophilia C (Rosenthal Syndrome, Factor XI Deficiency)

Other Congenital Single-Factor Deficiencies

Objectives

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

1. Distinguish among the causes of localized versus generalized, soft tissue versus mucocutaneous, and acquired versus congenital bleeding.

2. List and interpret laboratory tests that differentiate among acquired hemorrhagic disorders of trauma, liver disease, vitamin K deficiency, and kidney failure.

3. Discuss the laboratory monitoring of therapy for acquired hemorrhagic disorders.

4. Interpret laboratory assay results that diagnose, subtype, and monitor the treatment of von Willebrand disease.

5. Use the results of laboratory tests to identify and monitor the treatment of congenital single coagulation factor deficiencies such as deficiencies of factors VIII, IX, and XI.

6. Explain the principle and rationale for the use of each laboratory test for the detection and monitoring of hemorrhagic disorders.

CASE STUDY

After studying this chapter, the reader will be able to respond to the following case study:

A 55-year-old man comes to the emergency department with epistaxis (uncontrolled nosebleed). He reports that he has “bleeder’s disease” and has had multiple episodes of inflammatory hemarthroses (joint bleeding). Physical examination reveals swollen, immobilized knees; mild jaundice; and an enlarged liver and spleen. CBC results indicate that the patient is anemic and has thrombocytopenia with a platelet count of 74,400/μL (reference interval, 150,000 to 450,000/μL). The PT is 18 seconds (reference interval, 12 to 14 seconds), and the PTT is 43 seconds (reference interval, 25 to 35 seconds).

1. What is the most likely diagnosis?

2. What treatment does the patient need?

Bleeding symptoms

Hemorrhage is severe bleeding that requires physical intervention. Hemorrhage may be localized or general, acquired or congenital. To establish the cause of a patient’s tendency to bleed or a bleeding event, the physician obtains a complete patient and family history and performs a physical examination before ordering diagnostic laboratory tests.1

Localized versus generalized hemorrhage

Bleeding from a single location commonly indicates injury, infection, tumor, or an isolated blood vessel defect and is called localized bleeding or localized hemorrhage. An example of localized bleeding is an inadequately cauterized or ineffectively sutured surgical site. Localized bleeding seldom implies a blood vessel defect, a qualitative platelet defect, a reduced platelet count (thrombocytopenia), or a coagulation factor deficiency.

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 such as blood vessel defects, platelet defects, or thrombocytopenia, or secondary hemostasis characterized by single or multiple coagulation factor deficiencies.

Mucocutaneous versus anatomic hemorrhage

Generalized bleeding may exhibit a mucocutaneous (systemic) or a soft tissue (anatomic) pattern. Mucocutaneous hemorrhage may appear as petechiae, pinpoint hemorrhages into the skin (Figure 40-1A); purpura,purple lesions of the skin greater than 3 mm caused by extravasated (seeping) red blood cells (RBCs) (Figure 40-1B); or ecchymoses (bruises) greater than 1 cm typically seen after trauma (Fig. 40-1C).2 The unprovoked presence of more than one such lesion may indicate a disorder of primary hemostasis. Other symptoms of a primary hemostasis defect include bleeding from the gums, epistaxis (uncontrolled nosebleed), hematemesis (vomiting of blood), and menorrhagia (profuse menstrual flow). 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/μL; Chapter 40), qualitative platelet disorders (Chapter 41), von Willebrand disease (VWD), or vascular disorders such as scurvy or telangiectasia (Chapters 13 and 37). A careful history and physical examination distinguish between mucocutaneous and anatomic bleeding; this distinction helps direct investigative laboratory testing and subsequent treatment.

Anatomic (soft tissue) hemorrhage is seen in acquired or congenital defects in secondary hemostasis, or plasma coagulation factor deficiencies (coagulopathies).3 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 primary event, but it is often delayed or recurs minutes or hours after the event. Anatomic bleeding episodes may even be 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 (hemarthroses) cause swelling and acute pain, they may not be immediately recognized as hemorrhages, although experienced hemophilia patients usually recognize the symptoms at their outset. Repeated hemarthroses cause painful swelling and inflammation, and they may culminate in permanent cartilage damage that immobilizes the joint. Bleeds into soft tissues such as muscle or fat may cause nerve compression and subsequent temporary or permanent loss of function.4 When 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 is managed as a medical emergency. Bleeds into the kidney may present as hematuria and may be associated with acute kidney failure.

Hemostasis laboratory testing is essential whenever a mucocutaneous or soft tissue disorder is suspected. lists symptoms that suggest generalized hemorrhagic disorders. Besides a complete blood count that includes a platelet count, most laboratory directors offer the prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen assay (Box 38-1Table 38-1).5 In 2000, laboratory practitioners began to addthromboelastography performed using the Thromboelastograph® (TEG, Haemoscope Corporation, Niles, IL) or subsequently thromboelastometry (TEM) using the rotational thromboelastometer (ROTEM®, Pentapharm GmbH, Munich, Germany; FDA-cleared 2011) (Chapter 44). TEG and TEM are coagulometers that measure whole blood clotting.6 Both report clot onset, clot strength, and fibrinolysis within 15 minutes of blood collection. Experienced laboratory practitioners must interpret TEG or TEM results.

TABLE 38-1

Screening Tests for a Generalized Hemostatic Disorder

Test

Assesses

Hemoglobin, hematocrit; reticulocyte count

Anemia associated with chronic bleeding; bone marrow response

Platelet count

Thrombocytopenia

Prothrombin time (PT)

Deficiencies of factors II (prothrombin), V, VII, or X (clotting time prolonged)

Partial thromboplastin time (PTT)

Deficiencies of all factors except VII and XIII (clotting time prolonged)

Thrombin time or fibrinogen assay

Hypofibrinogenemia and dysfibrinogenemia

BOX 38-1

Generalized Bleeding Signs Heralding a Possible Hemostatic Defect

• Purpura—recurrent, chronic bruising in multiple locations; called petechiae when less than 3 mm in diameter, ecchymoses when greater than 1 cm in diameter

• Epistaxis—nosebleeds that are recurrent, last longer than 10 minutes, or require physical intervention

• Recurrent or excessive bleeding from trauma, surgery, or dental extraction

• Bleeding into multiple body cavities, joints, or soft tissue

• Simultaneous hemorrhage from several sites

• Menorrhagia (menstrual hemorrhage)

• Bleeding that is delayed or recurrent

• Bleeding that is inappropriately brisk

• Bleeding for no apparent reason

• Hematemesis (vomiting of blood)

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 associate with bleeding. If a patient’s bleeding episodes begin after childhood, are associated with some disease or physical trauma, and are not duplicated in relatives, they are probably acquired, not congenital. When an adult patient comes for treatment of generalized hemorrhage, the physician first looks for an underlying disease or event and records 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 prescription drugs, over-the-counter drugs, alcohol abuse, or drug abuse. The physician determines the trigger, location, and volume of bleeding, and then orders laboratory assays (Box 38-2Table 38-1). These tests take on clinical significance when the history and physical examination already have established the existence of abnormal bleeding. Owing to their propensity for false positives, they are not effective when employed indiscriminately as screens for healthy individuals (Chapter 5).7

BOX 38-2

Indications of Congenital Hemorrhagic Disorders

• Relatives with similar bleeding symptoms

• Onset of bleeding in infancy or childhood

• Bleeding from umbilical cord or circumcision wound

• Repeated hemorrhages in childhood, adulthood

• Hemorrhage into joints, central nervous system, soft tissues, peritoneum

Congenital hemorrhagic disorders are uncommon, occurring in fewer than 1 per 100 people, and are usually diagnosed in infancy or during the first years of life.8 There may be relatives with similar symptoms. Congenital bleeding disorders lead to repeat hemorrhages that may be spontaneous or may occur following minor injury or 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 are rare.

Acquired coagulopathies

Far more patients experience acquired bleeding disorders secondary to trauma, drug exposure, or chronic disease than possess inherited coagulopathies. Chronic disorders commonly associated with bleeding are liver disease, vitamin K deficiency, and renal failure. In all cases, laboratory test results are necessary to confirm the diagnosis and guide the management of acquired hemorrhagic events.

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 statisticians include self-inflicted, felonious, and combat injuries. In the United States alone, trauma causes 93,000 deaths per year.10 Severe neurologic displacement accounts for 50% of trauma deaths, with most occurring before the patient arrives at the hospital; however, of initial survivors, 20,000 die of hemorrhage within 48 hours.11 Acute coagulopathy of trauma-shock (ACOTS) accounts for most fatal hemorrhage, and 3000 to 4000 of these deaths are preventable. Coagulopathy is defined as any hemostasis deficiency, and 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 by most emergency medical technicians to counter hypoperfusion, though in 2013 some trauma specialists advocate for plasma instead of colloids as a means to control coagulopathy.12 Colloids dilute plasma procoagulants, which become further diluted by emergency department transfusion of RBCs. Although colloids and RBCs are essential, they intensify the coagulopathy, as does subsequent surgical intervention.

Massive transfusion, defined as administration of more than 3 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 below 110 mm Hg, pulse is greater than 105 beats/min, pH is less than 7.25, hematocrit is below 32%, hemoglobin is below 10 g/dL, and PT is prolonged to more than 1.5 times the mean of the reference interval or generates an international normalized ratio (INR) of 1.5.13

Acute coagulopathy of trauma-shock management: Rbcs

According to the American Society of Anesthesiologists surgical practice guidelines, RBC transfusions are required when the hemoglobin is less than 6.0 g/dL and are contraindicated when the hemoglobin is greater than 10.0 g/dL.14 For hemoglobin concentrations between 6.0 and 10.0 g/dL, the decision to transfuse is based upon the acuity of the patient’s condition as determined by physical evidence of blood loss; current blood loss rate; blood pressure; arterial blood gas values, especially pH and oxygen saturation; urine output; and laboratory evidence for coagulopathy provided th ere is time for specimens to be collected and laboratory assays completed.

Acute coagulopathy of trauma-shock management: Plasma

Plasma remains a key component of ACOTS management. Before 2005, donor services centrifuged donor blood units and separated and froze the supernatant plasma within 8 hours of collection, yielding fresh-frozen plasma (FFP). Most donor services now separate plasma within 24 hours of donor collection, officially naming the product FP-24 instead of FFP; however, laboratory practitioners, nurses, and physicians still say and write “FFP” from habit.15 FP-24 or FFP may also be thawed and stored at 1° C to 6° C for up to 5 days, a product called “thawed plasma.”16 Von Willebrand factor (VWF) and coagulation factor V and VIII activities decline to approximately 60% after 5 days of refrigerator storage, so thawed plasma may be ineffective in von Willebrand disease or hemophilia. High-volume trauma centers and mobile emergency services may maintain a small inventory of thawed plasma ready for emergency administration.

Frozen plasma is thawed, warmed to 37° C, and transfused when there is microvascular bleeding and the PT is prolonged to greater than 1.5 times the mean of the PT reference interval or if the PTT is prolonged to greater than 2 times the mean of the PTT reference interval. Plasma is also transfused when the patient is known to have a preexisting single coagulation factor deficiency such as factor VIII deficiency and no factor concentrate is available, when there has been significant volume replacement with colloid plasma expanders and RBCs, and when hemorrhage is complicated by or caused by Coumadin overdose (Chapter 43).

Most transfusion service directors recommend that 1 plasma unit be administered per 4 RBC units to retain coagulation stability; however, evidence from retrospective studies on battlefield casualties in Iraq and Afghanistan have led to consideration of a ratio of 1 plasma unit to each RBC unit. The 1:1 ratio appears to provide better coagulation stability, and many transfusion services have modified their massive transfusion protocols to approximate the new ratios.1718

The standard adult plasma dosage is 10 to 15 mL/kg in continuous infusion. Theoretically, plasma should be transfused until 30% coagulation factor activity has been reached for all factors; however, the actual volume that may be administered is limited by the risk of transfusion-associated circulatory overload (TACO).19

TACO and transfusion-related acute lung injury (TRALI) are potential adverse effects of plasma administration.20 TACO and TRALI can lead to potentially fatal acute adult respiratory distress syndrome (ARDS). Plasma therapy may also cause thrombosis, anaphylaxis, and multiple organ failure.

Acute coagulopathy of trauma-shock management: Platelet concentrate

During surgery, coagulopathy is assessed based on microvascular bleeding, which is evaluated by estimating the volume of blood that fills suction canisters, surgical sponges, and surgical drains. Platelet concentrateis ordered when the platelet count is less than 50,000/μL or higher when the surgeon anticipates blood loss.21 Platelet concentrate therapy is generally ineffective when the patient is known to have immune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, or heparin-induced thrombocytopenia. In patients with these conditions, therapeutic platelets are rapidly consumed, and their administration may therefore be contraindicated, although they may provide temporary rescue. Platelet concentrate is never ordered when the platelet count is greater than 100,000/μL. Platelet administration may be necessary when the platelet count is between 50,000 and 100,000/μL and there is bleeding into a confined space such as the brain or eye; if the patient is taking antiplatelet agents, aspirin, clopidogrel, prasugrel, or ticagrelor; if there is a known platelet disorder such as a release defect, Glanzmann thrombasthenia, or Bernard-Soulier syndrome (Chapters 40 and 41); or if the surgery involves cardiopulmonary bypass, which suppresses platelet activity.14

Acute coagulopathy of trauma-shock management: Components and concentrates

In an effort to reduce the risk of TACO and TRALI, improve patient outcomes, and conserve resources, transfusion service directors employ components and concentrates to augment or even replace plasma administration. Activated prothrombin complex concentrate (PCC, FEIBA® [factor eight inhibitor bypassing activity], Baxter Healthcare Corp., Deerfield, IL, or Autoplex T®, Nabi Biopharmaceuticals, Inc., Boca Raton, FL) may be used at a dosage of 50 units/kg every 12 hours, not to exceed 200 units/kg in 24 hours.2223 The dose-response relationship of FEIBA or Autoplex T varies among recipients, and because both contain activated coagulation factors, their use raises the risk of disseminated intravascular coagulation (DIC). Nonactivated PCCs such as four-factor concentrate Kcentra® (CLS Behring, King of Prussia, PA) are safer and may also be employed. Kcentra was FDA-cleared in 2013 to treat hemorrhage in Coumadin overdose, but its use in ACOTS is off-label (not FDA cleared).2425

PCCs, either activated or nonactivated, may be used in conjunction with the antifibrinolytic lysine analogue tranexamic acid (TXA, Cyklokapron®, Pharmacia & Upjohn Co., Division of Pfizer Inc., New York, NY).26TXA may be infused at a loading dose of 1 g administered over 10 minutes followed by infusion of 1 g over 8 hours.27 First FDA-cleared in 1986 to prevent bleeding in hemophilic patients who are about to undergo invasive procedures, TXA is effective for ACOTS, but this is an off-label use.

Administration of cryoprecipitate or fibrinogen concentrate (RiaSTAP®, CLS Behring, King of Prussia, PA) is indicated when there is microvascular bleeding and the fibrinogen concentration is less than 100 mg/dL.28 A 15 to 20 mL unit of cryoprecipitate provides 150 to 250 mg of fibrinogen, and the risk of TACO is lower than that with colloids or plasma. The fibrinogen concentrate dose is 70 mg/kg of body weight infused at less than 5 mL/minute, and a target fibrinogen level of 100 mg/dL should be maintained. Von Willebrand factor and factor VIII concentrates may also be used when the patient is deficient. Plasma and PCCs may all provide reduced factor VIII levels.2930

Recombinant activated coagulation factor VII (rVIIa, NovoSeven, Novo Nordisk Inc., Princeton, NJ) was FDA-cleared in 1999 for treating hemophilia A or B when factor VIII or factor IX inhibitors are present, respectively; its application in the treatment of ACOTS is off-label. A NovoSeven dosage of 30 μg/kg is rapidly effective in halting microvascular hemorrhage in nonhemophilic trauma victims, and NovoSeven does not cause DIC.9,31-33 However, one study found a possible link between off-label NovoSeven use and arterial and venous thrombosis in patients with existing thrombotic risk factors.34

Acute coagulopathy of trauma-shock: Monitoring therapy

A skilled operator employing TEG or TEM technology may monitor plasma, PCCs, activated PCC, four-factor PCC, TXA, and rFVIIa. Fibrinogen concentrate or cryoprecipitate efficacy may be measured using the fibrinogen assay. Also, laboratory directors characteristically advise surgeons and emergency department physicians to monitor the effectiveness of all ACOTS therapy indirectly by checking for the correction of platelet count, PT, and PTT to within their respective reference intervals. Platelet aggregometry may be used to measure post-therapy platelet function, and coagulation factor assays are valuable as follow-ups to PT and PTT to determine if the target activity of 30% has been met. While PT, PTT, platelet count, and platelet function assays are accepted approaches, TEG and TEM provide immediate feedback and may be more sensitive to small physiologic improvements.35 Once ACOTS has been stabilized, additional hemostasis-related therapy is seldom required.

Liver disease coagulopathy

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.

Procoagulant deficiency in liver disease

The liver produces nearly all of the plasma coagulation factors and regulatory proteins. Hepatitis, cirrhosis, obstructive jaundice, cancer, poisoning, and congenital disorders of bilirubin metabolism may suppress the synthetic function of hepatocytes, reducing either the concentrations or activities of the plasma coagulation factors to below hemostatic levels (less than 30% of normal).

Liver disease predominantly alters the production of the vitamin K–dependent factors II (prothrombin), VII, IX, and X and control proteins C, S, and Z. In liver disease, these seven factors are produced in their des- γ-carboxyl forms, which cannot participate in coagulation (Chapter 37). At the onset of liver disease, factor VII, which has the shortest plasma half-life at 6 hours, is the first coagulation factor to exhibit decreased activity. Because the PT is particularly sensitive to factor VII activity, it is characteristically prolonged in mild liver disease, serving as a sensitive early marker.36 Vitamin K deficiency caused by dietary insufficiency independent of liver disease produces a similar effect on the PT (see the case study in this chapter).

Declining coagulation factor V activity is a more specific marker of liver disease than deficient factor II, VII, IX, or X because factor V is non–vitamin K dependent and is not affected by dietary vitamin K deficiency. The factor V activity assay, performed in conjunction with the factor VII assay, may be used to distinguish liver disease from vitamin K deficiency.37

Fibrinogen is an acute phase reactant that is frequently elevated in early or mild liver disease. Moderately and severely diseased liver produces fibrinogen that is coated with excessive sialic acid, a condition called dysfibrinogenemia, in which the fibrinogen functions poorly. Dysfibrinogenemia causes generalized soft tissue bleeding associated with a prolonged thrombin time and an exceptionally prolonged reptilase clotting time.38 In end-stage liver disease, the fibrinogen level may fall to less than 100 mg/dL, which is a mark of liver failure.39

VWF and factors VIII and XIII are acute phase reactants that may be unaffected or elevated in mild to moderate liver disease.4041 In contrast to the other coagulation factors, VWF is produced from endothelial cells and megakaryocytes and is stored in endothelial cells and platelets.

Platelet abnormalities in liver disease

Moderate thrombocytopenia occurs in a third of patients with liver disease. Platelet counts of less than 150,000/μL may result from sequestration and shortened platelet survival associated with portal hypertension and resultant hepatosplenomegaly.42 In alcoholism-related hepatic cirrhosis, acute alcohol toxicity also suppresses platelet production. Platelet aggregation and secretion properties are often suppressed; this is reflected in reduced platelet aggregometry and lumiaggregometry results (Chapter 42). Occasionally platelets are hyperreactive. Aggregometry may be used to predict bleeding and thrombosis risk.43

Disseminated intravascular coagulation in liver disease

Chronic or compensated DIC (Chapter 39) is a significant complication of liver disease that is caused by decreased liver production of regulatory antithrombin, protein C, or protein S and by the release of activated procoagulants from degenerating liver cells. The failing liver cannot clear activated coagulation factors. In primary or metastatic liver cancer, hepatocytes may also produce procoagulant substances that trigger chronic DIC, leading to ischemic complications.

In acute, uncompensated DIC, the PT, PTT, and thrombin time are prolonged; the fibrinogen level is reduced to less than 100 mg/dL; and fibrin degradation products, including D-dimers, are significantly increased.44 If the DIC is chronic and compensated, the only elevated test result may be the D-dimer assay value, a hallmark of unregulated coagulation and fibrinolysis. Although DIC can be resolved only by removing its underlying cause, its hemostatic deficiencies may be corrected temporarily by administering RBCs, plasma, activated or nonactivated PCC, TXA, platelet concentrates, or antithrombin concentrates.45

Hemostasis laboratory tests in liver disease

The PT, PTT, thrombin time, fibrinogen concentration, platelet count, and D-dimer concentration are used to characterize the hemostatic abnormalities in liver disease (). Factor V and VII assays may be used in combination to differentiate liver disease from vitamin K deficiency. Both factors are decreased in liver disease, but factor V is not decreased in vitamin K deficiency. Table 38-2

TABLE 38-2

Hemostasis Laboratory Tests in Liver Disease

Assay

Interpretation

Fibrinogen

> 400 mg/dL in early, mild liver disease; < 200 mg/dL in moderate to severe liver disease, causing hypofibrinogenemia or dysfibrinogenemia

Thrombin time

Prolonged in dysfibrinogenemia, hypofibrinogenemia, elevation of fibrin degradation products, and therapy with unfractionated heparin

Reptilase time

Prolonged in hypofibrinogenemia, significantly prolonged in dysfibrinogenemia; not affected by heparin; assay rarely used

Prothrombin time (PT)

Prolonged even in mild liver disease due to des-γ-carboxyl factors replacing normal factors II (prothrombin), VII, IX, and X. Report PT in seconds, not international normalized ratio (INR), when testing for liver disease.

Partial thromboplastin time (PTT)

Mildly prolonged in severe liver disease due to disseminated intravascular coagulation (DIC) or des-γ-carboxyl factors II (prothrombin), IX, and X

Factor V assay

Factor V level becomes reduced in liver disease, but is unaffected by vitamin K deficiency so the factor V level helps distinguish the conditions

Platelet count

Mild thrombocytopenia, platelet count < 150,000/μL

Platelet aggregometry

Mild suppression of platelet aggregation and secretion in response to most agonists

D-dimer

> 240 ng/mL by quantitative assay

Plasminogen deficiency and an elevated D-dimer confirm systemic fibrinolysis. The reptilase time test occasionally may be useful to confirm dysfibrinogenemia. This test duplicates the thrombin time test except that venom of the reptile Bothrops atrox (common lancehead viper) is substituted for thrombin reagent. The Bothrops venom triggers fibrin polymerization by cleaving fibrinopeptide A but not fibrinopeptide B from the fibrinogen molecule. The subsequent polymerization is slowed by structural defects, which prolong the time interval to clot formation. The reptilase time test is unaffected by standard unfractionated heparin therapy and can be used to assess fibrinogen function even when there is heparin in the sample.46

Hemostatic treatment to resolve liver disease–related hemorrhage

Oral or intravenous vitamin K therapy may correct the bleeding associated with nonfunctional des-γ-carboxyl factors II (prothrombin), VII, IX, and X; however, the therapeutic effect of vitamin K is short-lived compared to its effect in dietary vitamin K deficiency because of the liver’s impaired synthetic ability. In severe liver disease, plasma transfusion provides all of the coagulation factors in hemostatic concentrations, although VWF and factors V and VIII may be reduced. Owing to its small concentration and short half-life of factor VII, plasma is unlikely to return the PT to within the reference interval.

A unit of plasma has a volume of 200 to 280 mL. The typical adult plasma dose for liver disease is 2 units, but the dose varies widely, depending on the indication and the ability of the patient’s cardiac and renal system to rapidly excrete excess fluid. TACO is likely to occur when 30 mL/kg has been administered, but it may occur with even smaller volumes in patients with compromised cardiac or kidney function.

If the fibrinogen level is less than 50 mg/dL, spontaneous bleeding is imminent, and cryoprecipitate or fibrinogen concentrate may be selected for therapy. Plasma and cryoprecipitate present a theoretical risk of virus transmission, as do other untreated single-donor biologic blood products, and allergic transfusion reactions are more common with plasma-containing products. Other therapeutic options for patients with liver disease–related bleeding are platelet concentrates, PCC, antithrombin concentrate (Thrombate III, Telacris® Biotherapeutics, Inc., Research Triangle Park, NC), rFVIIa, and TXA.

Chronic renal failure and hemorrhage

Chronic renal failure of any cause is often associated with platelet dysfunction and mild to moderate mucocutaneous bleeding.47 Platelet adhesion to blood vessels and platelet aggregation are suppressed, perhaps because the platelets become coated by guanidinosuccinic acid or dialyzable phenolic compounds.48 Decreased RBC mass (anemia) and thrombocytopenia contribute to the bleeding and may be corrected with dialysis, erythropoietin or RBC transfusions, and interleukin-11 therapy.49-52

Hemostasis activation syndromes that deposit fibrin in the renal microvasculature reduce glomerular function. 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 fragments 1 + 2, which are markers of coagulation activation (Chapter 42).53

Laboratory tests for bleeding in renal disease provide only modest information with little predictive or management value. The bleeding time test may be prolonged, but it is too unreliable to provide an accurate diagnosis or to assist in monitoring treatment.54 Platelet aggregometry test results may predict bleeding.55 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 temporarily activates platelets and may ultimately improve platelet function, particularly when anemia is well controlled.5657 Desmopressin acetate may be administered intravenously (DDAVP) or intranasally (Stimate®, CSL Behring, King of Prussia, PA) to increase the plasma concentration of high-molecular-weight multimers of VWF, which also aids platelet adhesion and aggregation.58 Patients with renal failure should not take aspirin, clopidogrel, prasugrel, ticagrelor, or other platelet inhibitors, because these drugs increase the risk of hemorrhage.

Nephrotic syndrome and hemorrhage

Nephrotic syndrome is a state of increased glomerular permeability associated with a variety of conditions, such as chronic glomerulonephritis, diabetic glomerulosclerosis, systemic lupus erythematosus, amyloidosis, and renal vein thrombosis.59 In nephrotic syndrome, low-molecular-weight proteins are lost through the glomerulus into the glomerular filtrate and the urine. Coagulation factors II (prothrombin), VII, IX, X, and XII have been detected in the urine, as have the coagulation regulatory proteins antithrombin and protein C. In 25% of cases, loss of regulatory proteins takes precedence over loss of procoagulants and leads to a tendency toward venous thrombosis.

Vitamin k deficiency and hemorrhage

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 people 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 gut 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.60

Hemorrhagic disease of the newborn caused by vitamin k deficiency

Because of their sterile intestines and the minimal concentration of vitamin K in human milk, newborns are constitutionally vitamin K deficient.61 Hemorrhagic disease of the newborn was common in the United States before routine administration of vitamin K to infants was legislated in the 1960s, and it still occurs in developing countries. The activity levels of factors II (prothrombin), VII, IX, and X are lower in normal newborns than in adults, and premature infants have even lower concentrations of these factors. Breastfeeding prolongs the deficiency because passively acquired maternal antibodies delay the establishment of gut flora.

Vitamin k antagonists: Coumadin

The γ-carboxylation cycle of coagulation factors is interrupted by coumarin-type oral anticoagulants such as Coumadin (warfarin) that disrupt the vitamin K epoxide reductase and vitamin K quinone reductase reactions ().Figure 38-162 In this situation, 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.63 Coumadin overdose is the single most common reason for hemorrhage-associated emergency department visits.

Image 

FIGURE 38-1 Glutamic acid (GLU) gains a carboxyl group to become γ-carboxyglutamic acid (GLA) when catalyzed by γ-carboxylase. This posttranslational modification enables the vitamin K-dependent coagulation factors II (prothrombin), VII, IX, and X and proteins C, S, and Z to bind ionic calcium necessary for normal coagulation. Vitamin K donates the carboxyl group through the quinone reductase pathway. Warfarin inactivates quinone reductase and epoxide reductase to prevent carboxylation. NADP,Nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; R, any side chain.

Detection of vitamin K deficiency or proteins in vitamin K antagonism

Clinical suspicion of vitamin K deficiency is supported by a prolonged PT with or without a prolonged PTT. In PT and PTT mixing studies, if pooled platelet-free normal plasma (NP; Cryocheck, Precision BioLogic, Inc., Dartmouth, Nova Scotia) is combined with patient plasma, the mixture yields normal (corrected) PT and PTT results, which indicates that factor deficiencies were the cause of the prolonged screening test times. Specific single-factor assays always detect low factor VII because of its short half-life, followed in turn by decreases in factors IX, X, and II (prothrombin).

The standard therapy for vitamin K deficiency is oral—or, in an emergency, intravenous—vitamin K. Because synthesis of functional vitamin K–dependent coagulation factors requires at least 3 hours, in the case of severe bleeding, plasma, activated or nonactivated PCC, four-factor PCC, or rFVIIa may be administered. The only assays for plasma, PCC, or rFVIIa efficacy are TEG or TEM, which are not available everywhere, but the patient’s recovery may be monitored indirectly using the PT/INR.

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.64 Autoanti-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. Pregnancy appears to trigger acquired hemophilia 2 to 5 months after delivery. Patients with inhibitor autoantibodies are prescribed immunosuppressive therapy, although autoantibodies that develop after pregnancy typically disappear spontaneously. Acquired hemophilia has an overall incidence of 1 per 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.65

Clot-based assays in acquired hemophilia

Laboratory directors recommend the PT, PTT, and thrombin time for any patient with sudden onset of anatomic hemorrhage that resembles acquired hemophilia. In the presence of a factor VIII inhibitor, which is the most common inhibitor, the PTT is likely to be prolonged, whereas the lab practitioner expects the PT and thrombin time to be normal. A factor assay should reveal factor VIII activity to be less than 30% and often undetectable.

The laboratory professional confirms the presence of the inhibitor with clot-based mixing studies. The PTT prolongation may be corrected initially by the addition of normal plasma (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 prolongation after incubation occurs because factor VIII autoantibodies are frequently of the immunoglobulin G4 isotype, which are 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 an autoantibody 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 38-2). 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.66

Image 

FIGURE 38-2 In type I linear kinetics, the inhibitor fully inactivates the factor in vitro. This is typical of alloantibodies such as factor VIII inhibitors in patients with severe hemophilia A. In type II kinetics, the inhibitor and factor reach equilibrium. This is typical of autoantibodies such as the factor VIII inhibitor in acquired hemophilia A. Because of the type II kinetics, the Bethesda titer is considered a semiquantitative measure in acquired hemophilia, although the results are commonly used to distinguish low-titer from high-titer antibodies.

Quantitation of autoanti-VIII inhibitor is accomplished using the Bethesda assay, which is ordinarily employed to measure inhibitors 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.

Factor inhibitors other than autoanti-factor VIII

Anticoagulation factor II (anti-prothrombin) antibodies, detectable by immunoassay, develop in approximately 30% of patients with lupus anticoagulant.67 Although lupus anticoagulant is associated with thrombosis, some patients with antiprothrombin antibodies experience bleeding and have a prolonged PT. A finding of reduced activity on prothrombin assay and a positive test result for lupus anticoagulant confirm the diagnosis. Antiprothrombin antibodies not associated with lupus anticoagulant are rare.

Factor V and XIII inhibitors have been documented in patients receiving isoniazid treatment for tuberculosis.6869 Antibodies to factor IIa (thrombin) and factor V may arise after exposure to topical bovine thrombin or fibrin glue.70 Fibrin glue–generated autoantibodies have become less prevalent since 2008 when fibrin glue preparations began to use recombinant human thrombin instead of bovine thrombin. Autoanti-X antibodies are rare; however, factor X deficiency in amyloidosis may be caused by what seems to be an absorptive mechanism.71 In many cases of acquired inhibitors, mixing studies show uncorrected prolongation without incubation (immediate mixing study), and inhibitor titers may be determined by the Bethesda procedure.

Acquired hemophilia management

Activated PCC or rFVIIa may bypass the coagulation factor VIII inhibitor in acquired hemophilia and thereby control acute bleeding. Patients who have low titers of inhibitor (less than 5 Bethesda units) may respond to administration of desmopressin acetate (DDAVP) or factor VIII concentrates, but close monitoring of their response to therapy with serial coagulation factor VIII activity assays is warranted. Once bleeding is controlled, immunosuppressive therapy may reduce the inhibitor titer.72 Plasma exchange may also be used in severe cases, but the response is less reliable than the response to immunosuppression.

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.73 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.74

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 diminished 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, NJ), or Alphanate® (Grifols, Los Angeles, CA) 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 its hemorrhagic symptoms, is classified as a thrombotic disorder and is described in Chapter 39.

Congenital coagulopathies

Von willebrand disease

VWD is a common mucocutaneous bleeding disorder first described by Finnish professor Erik von Willebrand in 1926. VWD is caused by any one of dozens of germline mutations that result in quantitative or structural abnormalities of VWF. Both quantitative and structural abnormalities lead to decreased adhesion by platelets to injured vessel walls, causing impaired primary hemostasis. VWD is the most prevalent of the congenital bleeding disorders and is found in approximately 1% of the population. It affects both sexes because of its autosomal dominant inheritance pattern. The parameters of and laboratory testing guidelines for VWD are established and defined in the National Heart, Lung, and Blood Institute (NHLBI) publication The Diagnosis, Evaluation, and Management of von Willebrand Disease.75

Molecular biology and functions of von willebrand factor

VWF is a glycoprotein whose molecular mass ranges from 800,000 to 20,000,000 Daltons, 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 (Chapter 13). Weibel-Palade bodies and α-granules release VWF in response to a variety of hemostatic stimuli.76

The VWF gene consists of 52 exons spanning 178 kilobase pairs (kb) on chromosome 12.77 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.78

As described in Chapter 37, VWF-cleaving protease (ADAMTS-13) cleaves the ultra-large VWF multimers of the Weibel-Palade bodies at their moment of release into monomers of various size. ADAMTS-13 deficiency allows for release of the ultra-large multimers, the basis for the devastating disorder, thrombotic thrombocytopenic purpura (Chapter 40)

Each VWF monomer has four functional domains that bind factor VIII, platelet glycoprotein Ib/IX/V, platelet glycoprotein IIb/IIIa, and collagen (Chapter 13). Upon release from intracellular stores, VWF forms a complex with coagulation factor VIII. This complex is named VIII/VWF (Figure 38-3). 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 38-3 lists the nomenclature entries for the structural and functional components of the factor VIII/VWF molecule.

Image 

FIGURE 38-3 Large von Willebrand factor (VWF) multimers bind intimal collagen upon desquamation of endothelial cells or more extensive injury and expand under shear force to form a “carpet” to which platelets adhere through platelet glycoprotein (GP) Ib/IX/V binding sites. VWF binds and stabilizes factor VIII, serving as a carrier protein. The VWF antigen (VWF:Ag) is the target of quantitative immunoassays.

TABLE 38-3

Nomenclature Relating to the Factor VIII/von Willebrand Factor Complex

Term

Meaning

VIII/VWF

Customary term for the plasma combination of factor VIII and VWF.

VIII

Procoagulant factor VIII, the protein transported on VWF. Factor VIII binds activated factor IX to form the complex of VIIIa-IXa, which digests and activates factor X. Factor VIII deficiency is called hemophilia A.

VWF:Ag

Epitope that is the antigenic target for the VWF immunoassay.

VWF:RCo

Ristocetin cofactor activity, also called VWF activity. VWF activity is measured by the ability of ristocetin to cause agglutination of reagent platelets by the patient’s VWF.

VWF:CBA

Collagen binding assay, a second method for assaying VWF activity. Large VWF multimers bind immobilized target collagen, predominantly collagen III.

VWF:Immunoactivity

Automated nephelometric activity assay that employs latex microparticles and monoclonal anti-glycoprotein I-VWF receptor, a third method for assaying VWF activity.

VIII:C

Factor VIII coagulant activity as measured in factor-specific clot-based assays.

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/IX/V receptor to the VWF “carpet.” The largest VWF multimers are best equipped to serve the adhesion function. When VWF binds glycoprotein Ib/IX/V, 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, hematemesis, and surgical bleeding. Symptoms vary over time and within kindreds because VWF production and release are 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.79 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

Type 1 von willebrand disease. 

Type 1 VWD is a quantitative VWF deficiency caused by one of several autosomal dominant frameshifts, nonsense mutations, or deletions.8081 Type 1 is seen in approximately 75% of VWD patients. The plasma concentrations of all VWF multimers and factor VIII are variably, although proportionally, reduced (Figure 38-4).82 There is mild to moderate bleeding, usually following a hemostatic challenge such as dental extraction or surgery. In women, menorrhagia is a common complaint that leads to the diagnosis of VWD.

Image 

FIGURE 38-4 Schematic of von Willebrand factor multimer analysis by polyacrylamide gel electrophoresis shows diminished concentration but normal ratios in von Willebrand disease (VWD) type 1, absence of large and intermediate multimers in subtype 2A, absence of large forms in subtype 2B, and absence of all multimers in type 3.

Type 2 von willebrand disease. 

Type 2 VWD comprises a variety of qualitative VWF abnormalities. VWF levels may be normal or moderately decreased, but VWF function is consistently reduced.

Subtype 2A von willebrand disease. 

Ten percent to 20% of VWD patients have subtype 2A, which arises from well-characterized autosomal dominant point mutations in the A2 structural domain of the VWF molecule. These mutations render VWF more susceptible to proteolysis by ADAMTS-13, which leads to a predominance of small-molecular-weight multimers in the plasma (Figure 38-4). The smaller multimers support less platelet adhesion activity than the normal high- or intermediate-molecular-weight multimers. Patients with subtype 2A VWD have normal or slightly reduced VWF:Ag levels, with markedly reduced VWF activity as a result of the loss of the high-molecular-weight and intermediate-molecular-weight multimers essential for platelet adhesion.83

Subtype 2B von willebrand disease. 

In subtype 2B VWD, rare mutations within the A1 domain raise the affinity of VWF for platelet glycoprotein Ib/IX/V, 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 (Figure 38-4). 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/IX/V 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. Both clinically and in the laboratory, the two entities are indistinguishable.

Subtype 2M von willebrand disease. 

Subtype 2M VWD describes a qualitative variant of VWF that has decreased platelet receptor binding but a normal multimeric pattern in electrophoresis. The distinguishing feature of subtype 2M that separates it from type 1 is a discrepancy between the concentration of VWF:Ag and its activity as measured using the VWF: RCo assay.

Subtype 2N von willebrand disease (normandy variant; autosomal hemophilia). 

A rare autosomal VWF missense mutation impairs its factor VIII binding site. This condition results in factor VIII deficiency despite normal VWF:Ag concentration and activity and a normal multimeric pattern. The disorder is also known as autosomal hemophilia because its clinical symptoms are indistinguishable from the symptoms of hemophilia except that it affects both males and females. Subtype 2N is suspected when a girl or woman is diagnosed with hemophilia after soft tissue bleeding symptoms. In boys or men, subtype 2N is suspected when a patient misdiagnosed with hemophilia A fails to respond to factor VIII concentrate therapy. The poor response occurs because the factor has a plasma half-life of mere minutes when it cannot be bound by VWF. The diagnosis of VWD subtype 2N is confirmed using a molecular assay that detects the specific mutation responsible for the abnormal factor VIII binding to VWF.

Type 3 von willebrand disease. 

Autosomal recessive VWF gene translation or deletion mutations produce severe mucocutaneous and anatomic hemorrhage in compound heterozygotes or, in consanguinity, homozygotes. In this rare disorder, VWF is absent or nearly absent from plasma (Figure 38-4). Factor VIII is also proportionally diminished or absent, and primary and secondary hemostasis is impaired.

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 NHLBI VWD guidelines, are the bleeding time test and the PFA-100 or other automated functional platelet assays (Chapters 42 and 44). These traditional screening tests generate “conflicting” sensitivity and specificity data.84

The standard VWD test panel includes three assays: a quantitative VWF test (VWF:Ag assay) employing enzyme immunoassay or automated latex immunoassay methodology, 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), the laboratory professional infers qualitative or type 2 VWD. 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 38-4 lists the expected test results for all VWD types and subtypes.

TABLE 38-4

Laboratory Detection and Classification of von Willebrand Disease: Typical Results*

Laboratory Test

Type 1

Type 2A

Type 2B

Type 3

VWF activity via ristocetin cofactor, collagen binding, or immunoactivity assay

Low

Low

Low

Very low or absent

VWF antigen

Low

Normal to slightly decreased

Normal to slightly decreased

Very low or absent

VWF activity to VWF antigen ratio

> 0.5

< 0.5

< 0.5

N/A

Platelet count

Normal

Normal

Decreased

Normal

Partial thromboplastin time (PTT)

Normal to slightly prolonged

Normal

Normal

Prolonged

RIPA

Decreased

Decreased

Increased

Absent

Factor VIII activity

Mildly low

Normal

Normal

< 5%

VWF multimers

Normal pattern

Large and intermediate forms absent

Large forms absent

All forms absent

* Expected results are given, but results vary over time and within affected kindred.

N/A, Not applicable; RIPA, ristocetin-induced platelet aggregometry with 1 mg/mL ristocetin; VWF, von Willebrand factor.

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 von willebrand disease diagnosis

Varying genetic penetrance, ABO blood group, inflammation, hormones, age, and physical stress influence VWF activity.85 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 phlebotomist allows the tourniquet to remain 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 kindred, 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.86

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 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, IL). 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%.87

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 requires standardization before this assay achieves standard assay status.88 The VWF:CB assay has better precision than the VWF:RCo assay.89

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.90

A second alternative to VWF:RCo is the automated HemosIL von Willebrand Factor Activity assay (Instrumentation Laboratory Company [IL], Bedford, MA). This nephelometric immunoassay employs latex microparticles coated with monoclonal antibodies directed to the VWF glycoprotein I binding site. Results compare well with the cofactor assay when the immunoassay is performed on IL instrumentation.91

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 . 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. Table 38-5

TABLE 38-5

Von Willebrand Factor (VWF) Reference Intervals by Blood Group

Blood Group

Reference Interval

O

36%–157%

A

48%–234%

B

57%–241%

AB

64%–238%

Population based: “Low VWF”

< 50%

Population based: von Willebrand disease

< 30%

In a well-managed tertiary care facility, laboratory professionals 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 localized measures, such as limb elevation, pressure, and application of ice packs (the athlete’s acronym is RICE for rest, ice, compression, and elevation).92 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 bed-wetting; 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 consumption. 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.

The lysine analogues ε-aminocaproic acid (EACA; Amicar) and TXA (Cyklokapron) inhibit fibrinolysis and may help control bleeding when used alone or in conjunction with desmopressin acetate. Therapy using nonbiological 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: Humate-P, Alphanate, and Wilate (Wilate was FDA-cleared in December 2009).92 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 plasma are less desirable alternatives because of the risk of virus transmission, and the necessary plasma 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.93 Therapeutic recommendations for VWD are summarized in Table 38-6.

TABLE 38-6

Therapeutic Strategies in von Willebrand Disease

Type

Primary Approach

Other Options

1

Estrogen, DDAVP, EACA or TXA

Factor VIII/VWF concentrate

2A

Estrogen, DDAVP, EACA or TXA

Factor VIII/VWF concentrate

2B

Factor VIII/VWF concentrate

EACA

2N

Factor VIII/VWF concentrate

EACA

3

Factor VIII/VWF concentrate

Platelet transfusions

DDAVP, Desmopressin acetate; EACA, ε-aminocaproic acid; TXA, tranexamic acid; VWF, von Willebrand factor.

Hemophilia a (factor VIII deficiency)

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.94

Factor VIII structure and function

Factor VIII is a two-chain, 285,000 Dalton protein translated from the X chromosome.95 When the coagulation cascade is activated, thrombin cleaves plasma factor VIII and releases a large polypeptide called theB domain that dissociates from the molecule. This leaves behind a calcium-dependent heterodimer that detaches from its VWF carrier molecule to bind phospholipid and factor IXa. The VIIIa/IXa complex, sometimes called tenase complex, cleaves and activates coagulation factor X at a rate 10,000 times faster than free IXa can cleave factor X. Consequently, factor VIII deficiency significantly slows the coagulation pathway’s production of thrombin and leads to hemorrhage. In vitro, factor VIII (like factor V) is labile and deteriorates at about 5% an hour at room temperature.

Hemophilia a genetics

The gene for factor VIII spans 186 kb of the X chromosome and is the site of various deletions, stop codons, and nonsense and missense mutations. Most of these mutations result in quantitative disorders in which the factor VIII coagulant activity and antigen concentration match, but in rare cases low activity is seen despite normal antigen concentration.96 The latter cases are due to qualitative or structural factor VIII abnormalities traditionally known as cross-reacting material positive disorders.97

Male hemizygotes, whose sole X chromosome contains a factor VIII gene mutation, experience anatomic bleeding, but female heterozygotes, who are carriers, do not.98 When a female carrier has children with an unaffected man, the chances of hemophilia A inheritance are 25% chance of a normal daughter, 25% chance of a carrier daughter, 25% chance of a normal son, and 25% chance of a hemophilic son. All sons of men with hemophilia A and noncarrier women are normal, whereas all daughters are obligate carriers of the disease.99 In addition, approximately 30% of newly diagnosed cases arise as a result of spontaneous germline mutations in individuals who have no family history of hemophilia A.100 Rarely, the symptoms of hemophilia A may be seen in females. This phenomenon could be due to true homozygosity or double heterozygosity, such as in the female offspring of a hemophilic father and a carrier mother. Other possibilities include a spontaneous germline mutation in the otherwise normal allele of a heterozygous female or a disproportional inactivation of the X chromosome with the normal gene, termed extreme lyonization. Finally, VWD of the Normandy subtype may present as mild hemophilia A in males and females.

Hemophilia a clinical manifestations

Hemophilia A causes anatomic bleeds with deep muscle and joint hemorrhages; hematomas; wound oozing after trauma or surgery; and bleeding into the central nervous system, peritoneum, gastrointestinal tract, and kidneys. Acute joint bleeds (hemarthroses) are exquisitely painful and cause temporary immobilization. Chronic joint bleeds cause inflammation and eventual permanent loss of mobility, whereas bleeding into muscles may cause nerve compression injury, with first temporary and then lasting disability. Cranial bleeds lead to severe, debilitating, and durable neurologic symptoms, such as loss of memory, paralysis, seizures, and coma, and may be rapidly fatal. Bleeding may begin immediately after a triggering event or may become manifest after a delay of several hours. Some bleeding seems to be spontaneous.

The diagnosis of hemophilia A begins with laboratory testing after the birth of an infant to a mother who has a family history of hemophilia. In the absence of a family history, abnormal bleeding in the neonatal period, which may appear as easy bruising, bleeding from the umbilical stump, postcircumcision bleeding, hematuria, or intracranial bleeding, is considered suspicious for hemophilia. Severe hemophilia usually is diagnosed in the first year of life, whereas mild hemophilia may not become apparent until a triggering event such as trauma, surgery, or dental extraction occurs in late childhood, adolescence, or adulthood.

The laboratory diagnosis of coagulopathies in the newborn or older infant is complicated by the requirement for an unhemolyzed specimen of at least 2 mL in volume from tiny veins and by the typically low newborn levels of some coagulation factors. Medical laboratory practitioners expect the PT and PTT to be prolonged because of physiologically low levels of factors II (prothrombin), VII, IX, and X, even in full-term infants. Factor VIII levels are similar to the levels in adults, even in infants who are born prematurely; this allows skillful laboratory staff to provide the correct diagnosis of hemophilia A using a factor VIII activity assay.

The severity of hemophilia A symptoms is inversely proportional to factor VIII activity. Laboratory professionals classify an activity level of less than 1% as severe, associated with spontaneous or exaggerated bleeding in the neonatal period. Activity levels of 1% to 5% are seen in moderate hemophilia, which is usually diagnosed in early childhood after symptoms become apparent. In mild hemophilia, with activity levels of 5% to 30%, hemorrhage follows significant trauma and becomes a risk factor mainly in surgery or dental extractions, and patients may go for long periods without symptoms.

Hemophilia a complications

As a result of frequent bleeds, hemophilia patients often have debilitating and progressive musculoskeletal lesions and deformities and neurologic deficiencies after intracranial hemorrhage. In addition, other effects of chronic diseases, such as limited productivity, low self-esteem, poverty, drug dependency, and depression, are common problems. Before the advent of sterilized and recombinant factor concentrates, chronic hepatitis often resulted from repeated exposure to blood products. Tragically, 70% of hemophiliacs treated before 1984 are human immunodeficiency virus (HIV) positive or have died from acquired immunodeficiency syndrome.101

Hemophilia a laboratory diagnosis

The laboratory workup for a suspected congenital single coagulation factor deficiency starts with the PT, PTT, and thrombin time and continues with factor assays based on the results of the screening tests. Before the physician initiates laboratory testing, however, he or she records a history of the patient’s hemorrhagic symptoms. In hemophilia A, the PT and thrombin time are likely to be normal, and the PTT is prolonged, provided that the PTT reagent is sensitive to factor VIII deficiencies at or less than the 30% plasma level. lists the expected results for each clot-based screening assay for any single-factor deficiency associated with bleeding, including deficiencies of fibrinogen and factors II (prothrombin), V, VII, VIII, IX, X, and XI. Deficiencies of contact factors (factor XII, high-molecular-weight kininogen, and prekallikrein) have no relationship to bleeding, and these factors do not appear in Table 38-7Table 38-7.

TABLE 38-7

Results of Clot-Based Screening Assays in Congenital Single-Factor Deficiencies*

Deficient Factor

PT

PTT

TCT

Reflex Test

Fibrinogen

Prolonged

Prolonged

Prolonged

Fibrinogen assay

Prothrombin

Prolonged

Prolonged

Normal

Prothrombin, V, VII, and X assays

V

Prolonged

Prolonged

Normal

Prothrombin, V, VII, and X assays

VII

Prolonged

Normal

Normal

VII assay

VIII

Normal

Prolonged

Normal

VIII, IX, and XI assays

IX

Normal

Prolonged

Normal

VIII, IX, and XI assays

X

Prolonged

Prolonged

Normal

Prothrombin, V, VII, and X assays

XI

Normal

Prolonged

Normal

VIII, IX, and XI assays

XIII

Normal

Normal

Normal

Factor XIII quantitative assay

* Results are valid when no anticoagulants are in use. Results may vary in response to reagent sensitivities.

PT, Prothrombin time; PTT, partial thromboplastin time; TCT, thrombin clotting time.

Hemophilia a carrier detection

Approximately 90% of female carriers of hemophilia A are detected by measuring the ratio of factor VIII activity to VWF:Ag value (VIII:VWF). This ratio is effective because VWF production is unaffected by factor VIII deficiency. Using a ratio, rather than a factor VIII assay value alone, normalizes for some of the physiologic variables that affect factor VIII activity and VWF:Ag assays, such as estrogen levels, inflammation, stress, and exercise.

The laboratory professional establishes a reference interval for the VIII:VWF ratio using plasmas from at least 30 women who do not have factor VIII deficiency. If the ratio of the individual being tested is below the lower limit of the interval, she is likely to be a carrier. These results may be influenced by excessive lyonization, variation in VWF production, and analytical variables; consequently, if carrier status is suspected and the VIII:VWF ratio is greater than the lower limit of the reference range, genetic testing may be necessary to detect one of the many polymorphisms associated with factor VIII deficiency.

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 or she 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 and the procedure, 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 the episode, and the less likely the patient is 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.102 Although it is initially more expensive, the prophylactic approach conserves downstream resources by ameliorating the adverse effects of repeated hemorrhages and their long-term consequences.103

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 EACA or TXA. When desmopressin acetate treatment proves ineffective, intravenous factor VIII concentrates are the next option. High-purity factor VIII concentrates are produced from mammalian cells using recombinant DNA technology or are derived from human plasma using factor VIII–specific monoclonal antibodies and column separation.104105 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, however, may transmit nonlipid viruses such as parvovirus B19 and hepatitis A virus.106 Recombinant products may use human albumin in the manufacturing process, which introduces the theoretical risk of Creutzfeldt-Jakob disease transmission; however, manufacturers now provide products free of all human protein, such as Bioclate (Pfizer, New York, NY).107

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 the mean 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 blood volume and hematocrit.108 The blood volume is approximately 65 mL/kg of body weight, and the plasma volume is the plasmacrit (100% − hematocrit %) × blood volume, as in the following formulas:

Image

Image

This formula is also used in the treatment of VWD. Overdosing seems to confer no thrombotic risk, but it 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 a and factor VIII inhibitors

Alloantibody inhibitors of factor VIII arise in response to treatment in 30% of patients with severe hemophilia and 3% of those with moderate hemophilia. The laboratory practitioner suspects the presence of an inhibitor when bleeding persists or when the plasma factor VIII activity fails to rise to the target level after appropriate concentrate administration. Most factor VIII inhibitors are immunoglobulin G4, non-complement-fixing, warm-reacting antibodies. It is impossible to predict which patients are likely to develop inhibitors based on genetics, demographics, or the type of concentrate used.109

The first step in inhibitor detection is a factor VIII assay. If the factor VIII activity exceeds 30%, no inhibitor is present. If the level is less than 30%, the laboratory practitioner proceeds to mixing studies. When the test plasma from the bleeding patient has a prolonged PTT, it is mixed 1:1 with normal plasma (NP), it is incubated 1 to 2 hours at 37° C, and the PTT of the mixture is measured. If no inhibitor is present, the incubated mixture should produce a PTT result within 10% of the incubated NP PTT. If an inhibitor is present, however, the factor VIII from the normal plasma is partially neutralized, and the mixture’s PTT remains prolonged or “uncorrected,” presumptive evidence of the inhibitor.

If mixing studies and the therapeutic results suggest the presence of a factor VIII inhibitor, a Bethesda assay is used to quantitate the inhibitor. NP with 100% factor activity is mixed at increasing dilutions in a series of tubes with the full-strength patient plasma. Factor VIII assays are performed on each mixture. The operator then compares the results of the various dilutions and expresses the titer as Bethesda units. One Bethesda unit is the reciprocal of the dilution that caused neutralization of 50% of the factor VIII from the NP. The same assay is employed to measure factor VIII inhibitors in acquired hemophilia. Although the complex kinetics of acquired autoantibodies diminishes the accuracy of the results in acquired hemophilia, this method is adequate to monitor therapy.

Hemophilia patients with inhibitors are classified as low or high responders. Low responders generate inhibitor titers of 5 Bethesda units or lower and their inhibitor titers do not increase significantly following factor VIII administration. High responders generate inhibitor titers that exceed 5 Bethesda units and their antibody titers further rise in response to therapy. Low responders may be managed with raised factor VIII doses alone, whereas high responders may require activated prothrombin complex concentrate such as FEIBA (factor eight inhibitor bypassing activity) to control bleeding plus steroid or immunomodulation therapy to reduce the inhibitor antibody titer. Each laboratory director may choose to maintain a database of hemophilia patients who have inhibitors because previous titers often predict future inhibitor behavior.

Hemophilia a treatment in patients with inhibitors

Every hemophilic patient with an inhibitor needs an individualized treatment plan to control bleeding episodes. Low responders often experience cessation of bleeding upon administration of large doses of factor VIII concentrate and may be so maintained. High responders may gain no benefit from factor VIII concentrates and instead are treated with activated PCC, Autoplex T or FEIBA, which generates thrombin in the presence of factor VIII inhibitors. The activated PCC dosage should not exceed 200 units/kg per day, distributed in two to four injections, because the activated factors may trigger DIC. NovoSeven also bypasses the physiologic factor VIII requirement, because it promotes thrombin formation through the tissue factor pathway.110 The discussions in this chapter of ACOTS and acquired hemophilia provide additional detail on activated PCC dosages.

Hemophilia B (factor IX deficiency)

Hemophilia B, also called Christmas disease, totals approximately 14% of hemophilia cases in the United States, although its incidence in India nearly equals that of hemophilia A.94 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 38-5). 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 numerous separate mutations resulting in a range of mild to severe bleeding manifestations. Determination of female 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.

Image 

FIGURE 38-5 Simplified coagulation cascade mechanism showing positions of all factors whose absence may cause hemorrhage. TF, Tissue factor.

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, reflecting 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 activated PCC or rFVIIa.

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 plasma infusions during times of hemostatic challenge.111 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 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 38-8Table 38-7. 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 clot-based assay, a modification of the thrombin time, but it also may be measured by turbidimetry or immunoassay.

TABLE 38-8

Rare Congenital Single-Factor Deficiencies

Deficiency

Factor Levels

Symptoms

Therapy

Afibrinogenemia

No measurable fibrinogen

Severe anatomic bleeding

CRYO or fibrinogen concentrate to raise to > 100 mg/dL

Hypofibrinogenemia

Fibrinogen activity assay < 100 mg/dL

Moderate systemic bleeding

Dysfibrinogenemia

Fibrinogen activity assay < 100 mg/dL

Mild systemic bleeding

Prothrombin deficiency

Factor II < 30%

Mild systemic bleeding

PCC or plasma to raise to 75%

Factor V deficiency

Factor V < 30%

Mild systemic bleeding

Plasma to raise to 75%

Factor VII deficiency

Factor VII < 30%

Moderate to severe anatomic bleeding

NovoSeven, PCC, four-factor PCC, plasma

Factor X deficiency

Factor X < 30%

Severe anatomic bleeding

PCC or plasma to raise to 75%

Factor XI deficiency

Factor XI < 50%

Anatomic bleeding

Plasma to raise to 75%

Factor XIII deficiency

Factor XIII < 1%

Moderate to severe systemic bleeding, poor wound healing

Plasma or CRYO every 3 weeks

CRYO, Cryoprecipitate; PCC, prothrombin complex concentrate.

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.112 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.113

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 μg/mL and nonactivated four-factor PCC preparations are effective and may provide a target factor VII level of 10% to 30%. Many factor VII deficiencies are dysproteinemias.114 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 plasma or nonactivated PCC to produce therapeutic levels of 10% to 40%.115 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 may be life-threatening. The PT and PTT are both prolonged in factor X deficiency. In the 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 . 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.Table 38-9116 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).

TABLE 38-9

Factor XIII Deficiency

Type of Deficiency

Incidence

Factor XIII Activity

β-Protein

α-Protein

Type I

Rare

Absent

Absent

Absent

Type II

Frequent

Absent

Normal

Low

Type III

Rare

Low

Absent

Low

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

Summary

• Hemorrhage is classified as localized versus generalized, acquired versus congenital, and anatomic soft tissue hemorrhage versus systemic mucocutaneous hemorrhage.

• Acquired hemorrhagic disorders that are diagnosed in the hemostasis laboratory include thrombocytopenia of various etiologies (Chapter 40), the acute coagulopathy of trauma-shock, liver and renal disease, vitamin K deficiency, acquired hemophilia or VWD, and DIC.

• VWD is the most common congenital bleeding disorder, and the diagnosis and classification of the type and subtypes require a series of clinical laboratory assays.

• The hemophilias are congenital single-factor deficiencies that cause moderate to severe anatomic hemorrhage. The clinical laboratory plays a key role in diagnosis, classification, and treatment monitoring in hemophilia.

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 most common acquired bleeding disorder?

a. Vitamin K deficiency

b. Liver disease

c. ACOTS

d. VWD

2. Which is a typical form of anatomic bleeding?

a. Epistaxis

b. Menorrhagia

c. Hematemesis

d. Soft tissue bleed

3. What factor deficiency has the speediest effect on the prothrombin time?

a. Prothrombin deficiency

b. Factor VII deficiency

c. Factor VIII deficiency

d. Factor IX deficiency

4. Which of the following conditions causes a prolonged thrombin time?

a. Antithrombin deficiency

b. Prothrombin deficiency

c. Hypofibrinogenemia

d. Warfarin therapy

5. In what subtype of VWD is the RIPA test result positive when ristocetin is used at a concentration of less than 0.5 mg/mL?

a. Subtype 2A

b. Subtype 2B

c. Subtype 2N

d. Type 3

6. What is the typical treatment for vitamin K deficiency when the patient is bleeding?

a. Vitamin K and PCC

b. Vitamin K and plasma

c. Vitamin K and platelet concentrate

d. Vitamin K and factor VIII concentrate

7. If a patient has anatomic soft tissue bleeding and poor wound healing, but the PT, PTT, thrombin time, platelet count, and platelet functional assay results are normal, what factor deficiency is indicated?

a. Fibrinogen

b. Prothrombin

c. Factor XII

d. Factor XIII

8. What therapy may be used for a hemophilic boy who is bleeding and who has a high titer of factor VIII inhibitor?

a. rFVIIa

b. Plasma

c. Cryoprecipitate

d. Factor VIII concentrate

9. What is the most prevalent form of VWD?

a. Type 1

b. Type 2A

c. Type 2B

d. Type 3

10. Which of the following assays is used to distinguish vitamin K deficiency from liver disease?

a. PT

b. Protein C assay

c. Factor V assay

d. Factor VII assay

11. Mucocutaneous hemorrhage is typical of:

a. Acquired hemorrhagic disorders

b. Localized hemorrhagic disorders

c. Defects in primary hemostasis

d. Defects in fibrinolysis

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*The author acknowledges the substantial contributions of Marisa B. Marques, MD, to Chapter 41 of the third edition of this textbook, many of which remain in this edition.