Williams Hematology



Hemophilia A (Classic Hemophilia, Factor Viii Deficiency)

Definition and History

Etiology and Pathogenesis


Prenatal Diagnosis and Carrier Detection

Clinical Features

Laboratory Features

Differential Diagnosis


Course and Prognosis
Hemophilia B (Factor IX Deficiency, Christmas Factor Deficiency)

Definition and History

Etiology and Pathogenesis

Clinical Features

Laboratory Features

Differential Diagnosis


Course and Prognosis

Treatment of Factor IX Inhibitors

Gene Therapy for Hemophilia B

Chapter References

The clinical manifestations of hemophilia A and B due to deficiency of factors VIII and IX, respectively, are clinically indistinguishable and occur in mild, moderate, and severe forms. They are the only blood clotting disorders inherited in a sex-linked recessive pattern. The severe forms of both hemophilia A and B are characterized by frequent hemarthroses, leading to chronic crippling hemarthropathy when not treated very early or prophylactically. Highly purified concentrates, prepared from human plasma or manufactured by recombinant technology, are available for treatment and are considered to be both safe and effective. In addition, prophylactic treatment is recommended, when feasible, for all severely affected patients. The main complication of treatment is the development of antibody inhibitors against either factor VIII or factor IX, which are more common in patients with hemophilia A than in patients with hemophilia B.

Acronyms and abbreviations that appear in this chapter include: AAV, adeno-associated vector; aPTT, activated partial thromboplastin time; CJD, Creutzfeldt-Jakob disease; DDAVP, 1,8-desamino-D-arginine vasopressin, desmopressin; PTC, plasma thromboplastin component; PT, prothrombin time; PTT, partial thromboplastin time; RFLP, restriction fragment length polymorphism; vWD, von Willebrand disease; vWF, von Willebrand factor.

Hemophilia A is an X-linked hereditary disorder that is due to defective and/or deficient factor VIII molecules. It is less common than von Willebrand disease (vWD), but it is more common than other inherited clotting factor abnormalities. However, hemophilia A is still rare, with an estimated incidence of only 1 in every 10,000 live male births. It is found in all ethnic groups in all parts of the world.1
Sex-linked hemophilia was recognized in the second century, when a rabbi correctly deduced that sons of hemophilic carriers were at risk for bleeding following circumcision.2 In the nineteenth century, several authors noted the sex-linked inheritance pattern of the disease and ascribed the hemorrhagic episodes to delayed blood coagulation. Morawitz developed the classical theory of blood coagulation, which recognized two major reactions: conversion of prothrombin to thrombin by a tissue substance that Morawitz termed thrombokinase and conversion of fibrinogen to fibrin by thrombin.3 In 1911, Addis demonstrated that thrombin formed more slowly in hemophilic blood than normal blood and that the defect could be corrected by small amounts of normal plasma.4 However, he incorrectly theorized that hemophilia was due to prothrombin deficiency. As protein purification techniques improved throughout the 1930s and 1940s, thrombokinase was resolved into several distinct components. Brinkhous demonstrated that the prothrombin content of hemophilic plasma was normal and that the basic defect in hemophilia was the delayed conversion of prothrombin to thrombin.5 The defect could be corrected by a fraction of normal plasma that contained the antihemophilic factor, later named factor VIII. In 1947 Pavlovsky observed that when blood from one patient with hemophilia he was studying was transfused into another, the prolonged clotting time in the recipient was corrected.6 At the time, Pavlovsky did not recognize that he was dealing with two different types of hemophilia. This was recognized by Aggeler and coworkers in 1952, when they described a patient deficient in “plasma thromboplastin component,” a blood-clotting factor different from factor VIII.7 A deficiency of “plasma thromboplastin component,” later termed factor IX, is expressed clinically as hemophilia B.8
In 1964 a proposal was put forth to organize the growing number of coagulation factors into a cascade, or waterfall, mechanism.9,10 In this scheme each zymogen clotting factor was activated to a protease that subsequently acted on the next zymogen until thrombin was ultimately produced. In this scheme both factor VIII and factor IX were considered to be proenzymes. Later, however, it was shown that factor VIII was not a proenzyme but rather a cofactor, which, when activated by thrombin, acted as an essential cofactor for factor IXa. Recently, the cascade hypothesis has been modified so that the primary role of the tissue factor/factor VII complex in the initiation of coagulation is emphasized (see Chap. 112).11
Hemophilia A is a heterogeneous disorder resulting from defects in the factor VIII gene that leads to a reduction in the circulating levels of functional factor VIII. The reduction in activity can be due to a decreased amount of factor VIII protein, the presence of a functionally abnormal protein, or a combination of both. For factor VIII to be an effective cofactor for factor IXa, it must first be activated by thrombin, a reaction that results in the formation of a heterotrimer composed of the A1, A2, and A3, -C1, -C2 domains of factor VIII in a complex with calcium (see Chap. 112).12 Activated factor VIII (VIIIa) and activated factor IX (IXa) associate on the surface of activated platelets to form a functional factor X-activating complex (“tenase” or “Xase”).13 In the presence of factor VIIIa, the rate of factor X activation by factor IXa is dramatically enhanced. It is not surprising that hemophilia A and B have similar clinical manifestations, since both factor VIIIa and factor IXa are required to form the Xase complex. The lack of either leads to a similar lack of platelet Xase activity. In patients with hemophilia, clot formation is delayed because thrombin generation is markedly decreased. The clot that is formed is friable and easily dislodged, leading to excessive bleeding.
Hemophilia A is an X-linked recessive disorder that occurs almost exclusively in males. About 30 percent of the mutations arise de novo. The inheritance pattern of both hemophilia A and B is shown in Fig. 123-1. Note that all the sons of affected hemophilic males are normal, while all the daughters are obligatory carriers of the factor VIII defect. Sons of carriers have a 50 percent chance of being affected, while daughters of carriers have a 50 percent chance of being carriers themselves.

FIGURE 123-1 Inheritance pattern of hemophilia A. X is normal; Xh is an abnormal X chromosome with the hemophilic gene; Y is normal; XX is a normal female; XY is a normal male; XXh is a carrier female; XhY is a hemophilic male.

The factor VIII gene is very large, about 186 kb, with about 9 kb of exons. It contains 26 exons and 25 intervening sequences or introns. The size and complexity of the gene have made it difficult to pinpoint, on a routine basis, specific mutations that result in hemophilia. Nevertheless, the factor VIII gene has now been cloned and sequenced, and numerous specific mutations have been described.14
Hemophilia A can be due to multiple alterations in the factor VIII gene, including gene rearrangements; missense mutations, in which there is a single base substitution leading to an amino acid change in the molecule; nonsense mutations, which result in a stop codon; abnormal splicing of the gene; deletions of all or portions of the gene; or insertions of genetic elements.14 A review of the genetic defects leading to hemophilia A can be found on the Internet.15 A summary of the different mutations as of 1996 are shown in Table 123-1.16


One of the most common mutations, accounting for 40 to 50 percent of patients, is a unique combined gene inversion and crossing-over that disrupts the factor VIII gene.17,18 The factor VIII gene is schematically represented in Fig. 123-2 and Fig. 123-3. Within intron 22 are two other genes, one called F8A, which is transcribed in the 5′ direction, and F8B, which is transcribed in the 3′ direction of the factor VIII gene. In addition, the hatched boxes in Fig. 123-3 show two other homologous sequences (a2,a3) 5′ to the F8A gene that lies within intron 22 (a1). The presence of an extragenic F8A sequence 5′ to the F8A gene within intron 22 is central to the inversion and translocation of the factor VIII gene from exon 1 to exon 22. The mechanism is homologous recombination between the F8A gene sequence that lies within intron 22 and one of the homologous sequences of the F8A gene 5′ to the factor VIII gene. During meiosis, there is crossing-over of homologous sequences between the F8A gene nested in intron 22 and the extragenic F8A sequence 5′ to intron 22 so that transcription of the complete factor VIII sequence is interrupted as shown in Fig. 123-3. Many of the patients with an inversion are susceptible to development of antifactor VIII inhibitor antibodies.

FIGURE 123-2 A schematic representation of the factor VIII gene. The factor VIII gene is located at q28 on the X chromosome. The figure shows the region of factor VIII gene to be enlarged on the second line. Notice that there are two genes designated a2 and a3 5′ to the factor VIII gene. The hatched area in factor VIII gene corresponds to intron 22 shown in the third line. Notice within intron 22 (fourth line) two nested genes, one of which is designated F8A, which is transcribed in a direction opposite that of the whole factor VIII gene and is homologous to the a2 a3 genes shown in line 2. (Reprinted with permission of the publisher from “Hemophilia A and Parahemophilia,” in the Metabolic and Molecular Basis of Inherited Diseases, 7th ed, vol 3, edited by CR Scriver, AL Beaudet, WS Sly, D Valle, p 3247. McGraw-Hill, New York, 1995.)

FIGURE 123-3 Schematic representation of inversion and crossing-over at intron 22. The figure depicts the inversion and crossing-over of the a3 gene with its homologous sequence a1 nested within intron 22. In the middle of the figure it can be seen that when there is a crossing-over of the a1 gene nested within intron 22 and the a3 gene extragenic to factor VIII, a portion of the factor VIII gene is transcribed in a reverse manner from exon 1 through exon 22. (Reprinted with permission of the publisher from: Antonarakis SE, Kazazian HH, Tuddenham EG: Molecular etiology of factor VIII deficiency in hemophilia A. Hum Mutat 5:1, 1995.)

Of the different insertions in the factor VIII gene that have been reported, a few are LINE (L1) elements which are transposon sequences, i.e., sequences that have been inserted frequently throughout the genome.19 Most insertions result in severe hemophilia.
Mutations in hemophilia A occur frequently at CpG dinucleotides (see Chap. 9).19 Since TaqI recognizes the sequence TCGA, CpG mutations at this site can be directly detected by the loss of a TaqI cleavage site. Codons for the amino acid arginine (CGA) are frequently affected by mutations at CG doublets. A C®T transition often results in a stop codon as shown in Fig. 123-4. A stop codon results in synthesis of a truncated factor VIII molecule and is usually associated with severe hemophilia.

FIGURE 123-4 Examples of mutations and CG doublets. In this figure the black box denotes exon 26. Note that a C to T transition results in a stop codon (TGA) while a G to A transition results in a substitution of a glutamine for an arginine residue.

A G®A transition results in a missense mutation, which often leads to a dysfunctional factor VIII molecule and may be associated with mild, moderate, or severe hemophilia. Some missense mutations result in the production of normal or near-normal amounts of factor VIII antigen, while the coagulant activity may be dramatically or only slightly reduced. Many other single base substitutions have been described, resulting in hemophilia of varying degrees of severity.
Large deletions in the factor VIII gene are almost always associated with severe hemophilia. However, cases have been described in which a small deletion that does not change the reading frame of the gene results in milder disease. Patients with large deletions who have no detectable factor VIII antigen are also thought to be more susceptible to the development of anti-factor VIII antibodies, although it is clear that antibodies also occur in patients without deletions.14,19
Hemophilia A in females is extremely rare, although affected female offspring from an affected father and carrier mother have been reported. Hemophilia A may also occur in females with X-chromosomal abnormalities such as Turner syndrome, X mosaicism, and other X-chromosomal defects.20,21 If a carrier female has the normal X chromosome inactivated disproportionately (“imbalanced X-inactivation”), she may have factor VIII levels sufficiently low to cause bleeding manifestations. Usually, these are mild but may be serious during surgical procedures or in instances of significant trauma.
A careful and complete family history is important for carrier detection.22 All the daughters of a hemophilic father will be obligatory carriers of the hemophilic defect. If a known carrier has a daughter, there is a 50 percent chance that the daughter will be a carrier.
Carrier detection becomes important when a daughter of a known carrier or a female offspring of a patient with hemophilia wishes to become pregnant. If resources for advanced carrier detection are not available, one can take a careful family history and measure both factor VIII coagulant activity and the vWF antigen. The ratio of vWF to factor VIII is higher in carrier females than noncarriers, and, thus, determining the ratio adds to the sensitivity of the test. Carriers generally have 50 percent or less of the normal factor VIII level. When these data are added to the family history, one can calculate the probability of whether a woman is a carrier.23,24
Carriers who carry the intron 22 inversion can be identified using the Southern blot technique. Where the capability exists, analysis of the complete coding region can be performed using gradient gel electrophoresis and single strand conformation polymorphism (SSCP) technology.22
The use of markers for restriction fragment length polymorphism (RFLP) is simpler than direct sequencing of the coding region of the factor VIII gene, but the use of this technique requires that the pedigree analysis include at least one hemophilic male whose mother is heterozygous for one or more RFLP markers. An example of RFLP analysis is shown in Fig. 123-5.25,26 The polymorphic markers include the variable number of tandem repeats (VNTR) in introns 13 and 22, as well as Bcl I and Xba I restriction sites. One can readily see that the female III-2 has the same polymorphic marker in intron 13 and the same Xba I restriction site as her hemophilic brother, carrier mother, and hemophilic grandfather. In contrast, the female III-1 inherits markers that are not linked with hemophilia.

FIGURE 123-5 Use of RFLP and VNTR for carrier diagnosis of hemophilia. The carrier female (II-1) is informative for polymorphisms at intron 13 and the Xba I site but not informative for markers on Bcl I or on intron 22. As can be seen III-2 is a carrier of the hemophilic trait with markers similar to the hemophilic grandfather (I-1) and hemophilic brother (III-4). VNTR stands for variable number of tandem repeats, while the Bcl I and Xba I are sites cleaved by these restriction endonucleons. (Reprinted with permission of the publisher from Diagnosis of hemophilia A and B carriers, in Hemophilia, edited by CD Forbes, LM Aledort, R Madhok, p. 68. 1997.)

Prenatal diagnosis of hemophilia can now be carried out almost routinely. If a carrier female has a fetus that can be identified as a female by chromosomal analysis of cells obtained by amniocentesis (about the sixteenth week), or by chorionic villus sampling at the tenth week of gestation, it is usually of little worry whether the female fetus is a carrier, since carriers usually have no bleeding tendency. If the fetus is male, sufficient cells can be obtained to carry out DNA analysis using the methods described above. A decision as to whether an affected fetus is carried to term is one that should be decided by the parents after they are provided with all the necessary information.
Hemophilia A is characterized by excessive bleeding into various parts of the body. Soft-tissue hematomas and hemarthroses leading to severe, crippling hemarthropathy are highly characteristic of the disease. The disease has been broadly classified as mild, moderate, and severe, although there is overlap between categories. Table 123-2 shows a classification based on the severity of clinical manifestations. A range of plasma factor VIII concentrations in percentages of normal and in units per milliliter is given for each category of severity. Occasionally, some patients with very low factor VIII clotting activity will be mildly affected clinically. It has been suggested that some patients with factor VIII levels compatible with severe hemophilia may exhibit mild symptoms because of the coinheritance of the factor V Leiden mutation (R506Q) with the hemophilic gene.27 However, in some patients with “severe” hemophilia with a “milder” bleeding diathesis, neither the factor V Leiden mutation nor other known “prothombotic” markers have been found.28 Severely affected patients (less than 1 percent factor VIII) frequently experience bleeding without known trauma other than that associated with the usual day-to-day activities. Without effective treatment, recurrent hemarthroses, resulting in chronic hemophilic arthropathy, occur by young adulthood and are highly characteristic of the severe form of the disorder. Severely affected patients are also subject to serious hemorrhages that may dissect through tissue planes, ultimately leading to compromise of vital organs. However, bleeding episodes are intermittent, and some patients go for weeks or months without hemorrhage. Except for intracranial bleeding, sudden death due to hemorrhage is rare.


Moderately affected patients with hemophilia have occasional hematomas and hemarthroses usually, but not always, associated with known trauma. These patients have greater than 1 to 5 percent factor VIII activity. Although hemarthroses occur in moderately affected patients, hemarthropathy is less disabling than that occurring in severely affected patients.
Mildly affected patients with hemophilia (6 to 30 percent factor VIII) have infrequent bleeding episodes, and the disease may go undiagnosed, only to be discovered because of excessive hemorrhage postoperatively, following trauma, or after the toss and tumble of contact sports.
Most carriers have roughly 50 percent factor VIII activity and experience no bleeding difficulty, even with surgical procedures. Carriers with factor VIII levels below 50 percent, usually due to extremely imbalanced X-inactivation, may experience excessive bleeding after trauma (e.g., childbirth or surgery), and therefore a factor VIII level should be obtained in all carriers.
Bleeding into joints accounts for about 75 percent of bleeding episodes in severely affected patients with hemophilia A.29 The normal synovium has few cells, but below the synovial layer are numerous capillaries that can be damaged by the mechanical trauma associated with day-to-day use of joints. The joints most frequently involved, in decreasing order of frequency, are knees, elbows, ankles, shoulders, wrists, and hips. Hinge joints are much more likely to be involved than ball and socket joints. Hemarthroses usually occur when an affected child begins to walk.
Hemarthroses are sometimes heralded by an aura of mild discomfort, which, over a period of minutes to hours, becomes progressively painful. The joint usually swells, becomes warm, and exhibits limited motion. Occasionally the patient experiences a mild fever. Significant and sustained fever, however, suggests an infected joint. Bleeding in the knee joint is more easily detected by physical findings than bleeding into either the elbow or shoulder. When bleeding stops, the blood resorbs, and the symptoms subside over a period of several days. If hemarthroses are treated early and the joint is not chronically involved, pain usually subsides in 6 to 8 h and disappears in 12 to 24 h. However, repeated hemorrhage into the joints eventually results in extensive destruction of articular cartilage, synovial hyperplasia, and other reactive changes in the adjacent bone and tissues. Acute bleeding into a chronically affected joint may be difficult to distinguish from the pain of degenerative arthritis.
One of the major complications of repeated hemarthroses is joint deformity complicated by muscle atrophy and soft tissue contractures (Fig. 123-6). The various radiologic stages of progressive destruction of joint cartilage and adjacent bone are shown in Figure 123-7. Osteoporosis and cystic areas in the subchondral bone may develop, and there is progressive loss of joint space.

FIGURE 123-6 Hemophilic arthropathy. The chronic effects of a repeated hemorrhage into the knee of a severely affected hemophilic.

FIGURE 123-7 Various radiologic stages of hemophilic arthropathy. The stages 0 (normal joint) and 1 (fluid in the joint) are not shown. A: Stage 2 shows some osteoporosis and epiphyseal overgrowth in knee 2. The arrows show that the epiphysis is wider in knee 2 then in knee 1. B: In Stage 3 subchondral bone cysts are identified by arrows, and joint spaces exhibit irregularities.C: Stage 4. Prominent bone cysts with marked narrowing of joint space is denoted by the arrow. D: Stage 5. Obliteration of joint space with epiphyseal overgrowth is shown denoted by the arrow.

Repeated bleeding into a joint results in synovial hypertrophy and inflammation. The synovium is thickened and folded, leading to limitation of joint motion. This results in a tendency for repeated hemorrhages leading to a so-called target joint. The joints most often involved are the knees, ankles, and elbows, which become chronically swollen. Bleeding into such a joint with significant synovial hypertrophy is usually less painful than bleeding into a normal joint, but pain, nevertheless, may occur. Chronic synovitis may persist for months or years unless adequately treated.
Infection of hemophilic joints is not common but must be suspected in all patients with fever, leukocytosis, or other systemic manifestations. Rapid diagnosis is mandatory, since infection of such joints leads to rapid loss of joint architecture and function. A painful swollen joint may require aspiration, which should be performed by experienced personnel using meticulous aseptic techniques and appropriate factor replacement therapy.
Hematomas are characteristic of blood clotting factor deficiencies. They are usually not seen, for example, in uncomplicated thrombocytopenia. Hemorrhage into subcutaneous connective tissues or into muscles may occur with or without known trauma. Once formed, hematomas may stabilize and slowly resorb without treatment. However, in moderately and severely affected patients, hematomas have a tendency to enlarge progressively and to dissect in all directions. Retroperitoneal hematomas have been known to dissect through the diaphragm, into the chest, and into the soft tissues of the neck, resulting in compromise of the airway. They may also compromise renal function by causing ureteral obstruction. The computed tomographic (CT) scan of a retroperitoneal hemorrhage is shown in Fig. 123-8. Other hematomas may expand locally and cause compression of adjacent organs, blood vessels, and nerves. A rare, and often fatal, complication of an abdominal hematoma is perforation and drainage into the colon. Subcutaneous hematomas have also been known to dissect into muscle. Pharyngeal and retropharyngeal hematomas, sometimes complicating simple colds, may enlarge and obstruct the airway. Hemorrhage in or around the airway is a potentially life-threatening situation that requires prompt administration of factor VIII. Hemorrhages occur into muscle in the following order of frequency: calf, thigh, buttocks, and forearm. Bleeding into the iliopsoas muscle is also frequent. Hematomas in these areas may lead to muscle contractures, nerve palsies, and muscle atrophy. Bleeding into the tongue or frenulum is particularly frequent in young children and is usually due to trauma. Bleeding into the myocardium is extremely unusual.

FIGURE 123-8 CT scan of a retroperitoneal hematoma in a patient with severe hemophilia A. The arrows depict the extent of the hematoma.

Pseudotumors are blood cysts that occur in soft tissues or bone. They are rare but dangerous complications of hemophilia.30 They are classified into three types. One is a simple cyst that is confined by tendinous attachments within the fascial envelope of a muscle. The second type initially develops as a simple cyst in soft tissues such as a tendon, but it interferes with the vascular supply to the adjacent bone and periosteum, resulting in cyst formation and resorption of bone. The third type is thought to result from subperiosteal bleeding that separates the periosteum from the bony cortex (Fig. 123-9). The extent of periosteal stripping is limited by the aponeurotic or tendinous attachments. Most pseudotumors are not associated with pain unless there is rapid growth or nerve compression. As the volume of the cyst increases, it compresses and destroys the adjacent muscle, nerve, and/or bone. Pseudotumors usually contain either serosanguinous fluid or a viscous brownish material surrounded by a fibrous membrane. They have a tendency to expand over a period of several years and eventually become multiloculated. Some reach enormous size and involve so many structures as to be inoperable. Erosion through surrounding tissues and penetration into viscera or through the skin can occur, usually as a late event. Sinus tracts from the pseudotumor predispose to infection and septicemia. Pseudotumors develop primarily in the lower half of the body, usually in the thigh, buttock, or pelvis, but may occur anywhere, including the temporal bone. The small bones of the hands or feet are most frequently affected in younger patients. Computerized tomography or magnetic resonance imaging (MRI) are useful in diagnosis. Needle biopsies of pseudotumors should be avoided because of the risk of infection and hemorrhage. The only reliable treatment is operative removal of the entire mass. Unless the pseudotumor is completely removed, it is likely to reform.

FIGURE 123-9 Pseudotumor of the fibula in a severely affected hemophilic. Notice the virtual destruction with cysts and calcifications. There is also involvement of the tibia.

Virtually all severely affected patients with hemophilia experience episodes of hematuria. The urine may be brown or red, depending upon the rate of bleeding. Most bleeding arises from the renal pelvis, usually from one kidney, but occasionally from both. One should consider a structural lesion as a cause of hematuria. Initially, when necessary, intravenous pyelography, ultrasound, or other appropriate studies of the genitourinary tract should be used for diagnosis, although frequently no lesions are detected except for filling defects caused by clots. If the hematuria clears upon urination, bleeding from the lower genitourinary tract should be suspected. Severe renal colic may occur when clots obstruct the ureters. If the hematuria is minimal and painless, and the patient’s past history suggests no genitourinary pathology, the physician is justified in waiting a few days for bleeding to cease. If bleeding continues, treatment with factor VIII may be necessary.
Intracranial bleeding is the most dangerous hemorrhagic event in hemophilic patients.31 Hemorrhage into the central nervous system may be “spontaneous” but usually follows trauma, which may be trivial. Symptoms often occur soon after trauma, but sometimes bleeding is delayed. Symptoms of a subdural hematoma, for example, may be delayed for several weeks. Hemophilic patients with unusual headaches should always be suspected of having hemorrhage into the brain parenchyma, a subdural or an epidural hematoma (Fig. 123-10). When intracranial bleeding is suspected, the patient should be treated immediately with factor VIII. Diagnostic procedures, such as CT scans or MRI studies, should be delayed until after initiation of treatment. Although lumbar puncture has been performed safely in severe hemophilic patients without replacement therapy, it is safer to replace factor VIII to a level of about 50 percent of normal prior to the procedure.

FIGURE 123-10 CT scan of a intracerebral hematoma in a severely affected hemophilic. The arrows point to the lesion. Notice compression of the ventricles.

Hemorrhage into the spinal canal is a very uncommon neurologic complication in hemophilia but can result in paraplegia. Bleeding may occur within the spinal cord itself, but epidural bleeding compressing the cord is more common.31
Peripheral nerve compression is a frequent complication of muscle hematomas, particularly in the extremities. Compression of the femoral nerve by an iliopsoas muscle hematoma can result in sensory loss over the lateral and anterior thigh, weakness and atrophy of the quadriceps, and loss of the patellar reflex. The ulnar nerve is the next most frequently involved peripheral nerve. Bleeding may occur in any muscle and may compress local neural supply. This can be followed by permanent neuromuscular defects and multiple contractures.
Mucous membrane bleeding is common in hemophilia. Epistaxis and hemoptysis, often resulting from allergic reactions or trauma, can be associated with local structural lesions involving the upper and/or lower respiratory tract. Treatment of epistaxis by cautery or by nasal packing is sometimes followed by recurrent bleeding because of sloughing of the cauterized area or dislodging of a poorly formed clot when the packing is removed. Peptic ulcer disease occurs about five times more frequently in the adult hemophilia A population than in the general male population.32 Ingestion of anti-inflammatory drugs for relief of pain of hemophilic arthropathy is a frequent cause of upper gastrointestinal hemorrhage, and a history of ingestion of aspirin and other anti-inflammatory drugs should be specifically addressed when assessing the etiology of such bleeding.33
Severe hemophilia is usually diagnosed in childhood, and so patients are treated pre- and postoperatively to prevent bleeding. Mildly, or sometimes moderately, affected patients, however, may go unrecognized until surgery results in excessive bleeding at the surgical site. Bleeding may be delayed for several hours or, occasionally, for several days. Surgery in such patients is characterized by poor wound healing due to poor clot formation. Prolonged bleeding and subsequent infection of the wound hematoma may further complicate healing. With appropriate factor VIII replacement therapy, intra- and postoperative hemorrhages can be prevented.
Dental extraction is the most frequent surgical procedure performed on hemophilic patients. Loss of deciduous teeth is seldom the cause of excessive bleeding, but extraction of permanent teeth may result in excessive hemorrhage that can persist intermittently for several days to weeks unless appropriate treatment is administered. In the untreated patient with severe hemophilia, life-threatening, dissecting pharyngeal and/or sublingual hematomas may result from dental procedures or even from administration of regional block anesthesia.
Patients with severe hemophilia A characteristically have a prolonged activated partial thromboplastin time (aPTT). The prothrombin time (PT), thrombin-clotting time (TCT), and bleeding time (BT) are normal, although minor increases in BT have been reported by some investigators. Different combinations of aPTT reagents and instrumentation exhibit varying sensitivities to factor VIII levels. In mild hemophilia, the aPTT may be only slightly prolonged or at the upper limit of normal, especially if the factor VIII activity is at or above 20 percent of normal. The aPTT is corrected when hemophilic plasma is mixed with an equal volume of normal plasma and not corrected when mixed with plasma of a known patient with hemophilia A. If the hemophilic plasma contains an inhibitor antibody against factor VIII, the aPTT on a similar mixture will be prolonged, although incubation of the mixture for 1 or 2 h at 37°C may be required to detect a prolongation. A definitive diagnosis of hemophilia A should be based on a specific assay for factor VIII activity.
Functional factor VIII coagulant activity is measured by one-stage clotting assays based on aPTT.34 Chromogenic assays for factor VIII activity are also used widely but do not always agree with one-stage assays.34 Factor VIII antigen is measured by immunologic assays, which will detect normal and most abnormal factor VIII molecules. If the factor VIII antigen level is normal but the clotting activity is reduced, the patient has a dysfunctional factor VIII molecule. Such patients have antigen-positive hemophilia, also referred to as cross-reacting material–positive (CRM+).35 In other patients, both the factor VIII antigen and activity are nearly undetectable. These patients are CRM-negative.
Factor VIII activity is expressed as percent of normal or as units per milliliter of plasma. By definition, 1 unit of factor VIII is equal to the amount in 1 ml of pooled fresh normal human plasma. Also by definition, 1 unit of factor VIII/ml is 100 percent of normal.
von Willebrand disease can sometimes be confused with hemophilia A. The basic defect in vWD is reduced activity of von Willebrand factor (vWF), which acts as a carrier of factor VIII in vivo (see Chap. 135). Thus, in vWD, factor VIII levels are reduced, although there is considerable variability. Although factor VIII is synthesized normally in patients with vWD, the half-life of factor VIII is shortened because the vWF “carrier” molecule is decreased or absent. Other abnormalities in vWD that distinguish it from hemophilia A are prolonged bleeding time, decreased vWF antigen, and decreased ristocetin-induced platelet agglutination. One variant of vWD that is particularly difficult to distinguish from hemophilia A is vWD-Normandy, in which the vWF activities are normal but factor VIII levels are low. Several mutations causing vWD-Normandy have been described, but all of them result in decreased binding of factor VIII to vWF. This results in a shortening of the intravascular survival of factor VIII and thus reduced factor VIII activity. The Normandy variant should be suspected in patients with mild hemophilia that do not exhibit a sex-linked recessive inheritance pattern.36
Hemophilia A must also be distinguished from other hereditary blood-clotting factor deficiencies that exhibit a prolonged aPTT, including deficiencies of factors IX, XI, and XII; prekallikrein; and high-molecular-weight kininogen. Only deficiencies of factors VIII and IX cause chronic crippling hemarthroses with a family history suggestive of an X-linked bleeding disorder. Hemophilia A can be distinguished from factor IX deficiency (hemophilia B) only by specific assays. Factor XI deficiency occurs in both males and females and is a milder hemorrhagic disorder compared to severe hemophilia A or B. Factor XI deficiency can be confused with mild hemophilia A or B, but specific assays will distinguish them. Deficiencies of factor XII, prekallikrein, and high-molecular-weight kininogen can be distinguished from hemophilia because they are not associated with clinical bleeding. Mild hemophilia A, with factor VIII levels of about 15 percent of normal, must be distinguished from combined deficiency of both factor V and factor VIII.37 Both the PT and aPTT are moderately prolonged in the combined disorder.
General principles applicable to the therapy of hemophilia A patients include the avoidance of aspirin, nonsteroidal anti-inflammatory drugs, and other agents that interfere with platelet aggregation. There are, however, exceptions to this rule. In view of the pain of hemophilic arthropathy, nonsteroidal anti-inflammatory agents are required. In these instances the physician should choose the agent that causes the least amount of increased bleeding. Usually this requires trying agents by trial and error to find the one most suitable for the patient. Patients should be advised of the numerous over-the-counter analgesics that contain aspirin or other antiplatelet agents. Addictive narcotic agents should be used with great caution and only when clearly indicated, since drug dependency can be a major problem in this disease. In general, intramuscular injections should be avoided. In the absence of prophylactic therapy, it is important to treat patients with hemophilia A as early as possible to avoid complications of bleeding. Surgical procedures on hemophilic patients should be scheduled early in the week to avoid “weekend crises.” Ample supplies of factor VIII should be available in the blood bank or pharmacy to ensure rapid access to treatment when needed. All hemophilic patients should have access to home treatment and periodic examinations at a comprehensive hemophilia diagnostic and treatment center. Prophylactic therapy should be considered in all severely affected patients.
Hemorrhagic episodes in patients with hemophilia A can be managed by replacing factor VIII. Several plasma products are available for use in raising factor VIII to hemostatic levels. Fresh-frozen plasma and cryoprecipitate both contain factor VIII and were once the only products available for treatment. A disadvantage of plasma is that large volumes must be infused to achieve and maintain even minimal levels of factor VIII. The highest factor VIII level that can be achieved with plasma is about 20 percent of normal, and this is not always attainable nor sufficient for hemostasis. Cryoprecipitate can be used to attain normal levels of factor VIII, but individual bags of cryoprecipitate must be pooled, the factor VIII dose can only be estimated, and the product must be stored frozen. Several commercial lyophilized factor VIII concentrates, using cryoprecipitate of pooled normal human plasmas as starting material (2000 to 20,000 donors), are now available and do not have the disadvantages of plasma and cryoprecipitate (Table 123-3). Factor VIII concentrates have been sterilized, either by heating in solution, superheating to 80°C after lyophilization, or by exposure to organic solvent-detergents that inactivate enveloped viruses including HIV and hepatitis B and C viruses but do not inactivate parvovirus nor hepatitis A.38,39 Parvovirus infection is not frequent in hemophilia A patients, since it is transmitted by cellular elements of the blood. Nevertheless, seroconversion to B19 parvovirus has been observed in patients receiving plasma-derived concentrates undergoing solvent-detergent extraction or pasteurization. Hepatitis A has also occurred in patients receiving plasma-derived concentrates inactivated by solvent-detergent techniques.40


Some of these products contain significant amounts of vWF (Table 123-3). Plasma-derived factor VIII concentrates prepared by monoclonal antibody techniques, and subjected to one of the procedures mentioned above, are highly purified and, barring breakdown in manufacturing techniques, are generally safe in terms of transmission of viral diseases.
Factor VIII produced by recombinant DNA techniques is now available and is both safe and effective (Table 123-3).41 In addition to human factor VIII, porcine factor VIII is also commercially available for human use. Porcine factor VIII may be of great benefit in hemophilic patients with factor VIII antibodies, since the human anti-factor VIII antibody may not cross-react with porcine factor VIII. A comprehensive list of factor VIII concentrates can be found on the Internet.42,43
The dose of factor VIII can be ascertained as follows: If one unit of factor VIII per milliliter of plasma is considered to be 100 percent of normal, the dose required to raise the level to a given value is dependent upon the patient’s plasma volume (roughly 5 percent of body weight in kilograms) and the level to which factor VIII is to be raised. Thus, the plasma volume of a 70-kg adult is roughly equivalent to 3500 ml (5% × 70 kg = 3.5 kg = 3500g, roughly equivalent to 3500 ml). To achieve normal factor VIII levels of 1 U/ml (100 percent), 3500 U of factor VIII should be given. This assumes a 100 percent recovery of the administered dose. In recent studies recovery approaches 100 percent, but this depends upon the method of assay and the factor VIII standard used for comparison.44 After the initial dose of factor VIII, further doses of factor VIII are based on a half-life of 8 to 12 h. Thus, after a loading dose of 3500 U of factor VIII, a dose of 1750 U could be given in 12 h. However, for practical purposes, the dose of factor VIII is based on the knowledge that 1 unit of factor VIII per kilogram of body weight will raise the circulating factor VIII level about 0.02 U/ml. Thus, to raise the factor VIII level to 100 percent, that is, 1 U/ml, the dose of factor VIII required would be about 50 U per kilogram body weight, assuming that the patient’s baseline factor VIII level is less than 1 percent of normal. The site and severity of hemorrhage determine the frequency and dose of factor VIII to be infused. Table 123-4 summarizes the recommended doses of factor VIII for various types of hemorrhage. These doses are not based on rigorous randomized studies, and recommendations vary from one hemophilia center to another. Given the high cost of factor VIII, some physicians prefer the lower doses.


Factor VIII can also be given as a constant infusion. Following a loading dose to raise factor VIII to the desired level, 150 to 200 U of factor VIII per hour can be infused. Factor VIII levels can be conveniently monitored from blood obtained from veins other than the one in which factor VIII is infused. In selected patients, factor VIII can be given outside the hospital in a continuous infusion using pump devices.45
During the 1970s, it was found that DDAVP caused a transient rise in factor VIII in normal subjects as well as in those with mild to moderate hemophilia. Patients with severe hemophilia A do not respond.46 After a dose of DDAVP, 0.3 µg per kilogram body weight, factor VIII levels increase two- to threefold above baseline in most, but not all, mildly or moderately affected hemophilia A patients. A concentrated intranasal spray of DDAVP can also be used (150 mg in each nostril).47 The degree of response to the drug should always be determined in patients before a bleeding episode, since occasionally mildly or moderately affected patients will not respond. The peak response to DDAVP usually occurs 30 to 60 min postinfusion. In patients with mild or moderate hemophilia A, and in carriers whose baseline factor VIII levels are lower than 0.5 U/ml, DDAVP should be used in lieu of blood products. The mechanism by which DDAVP causes an increase in factor VIII is unknown.
Repeated administration of DDAVP results in a diminished response to the agent (tachyphylaxis). In many patients the response to the second dose of DDAVP is, on the average, 30 percent less than the response to the first dose, and after further doses the response rate may be even less.48 DDAVP is a potent antidiuretic, and as a result, hyponatremia has been reported in some patients whose water intake exceeds about 1 liter per 24 h. There has been no convincing evidence that administration of DDAVP is associated with thrombosis in hemophilic patients.
Antifibrinolytic agents, e.g., epsilon-aminocaproic acid (EACA) and tranexamic acid, have been used to enhance hemostasis in patients with hemophilia A.49,50 Fibrinolytic inhibitors may be given as adjunctive therapy for bleeding from mucous membranes and are particularly valuable as adjunctive therapy for dental procedures. The usual dose of tranexamic acid for adults is 1 g four times daily. Unfortunately, tranexamic acid is not available in the United States for oral therapy. EACA can be given as a loading dose of 4 to 5 g followed by 1 g/h in adults. Another regimen is 4 g every 4 to 6 h orally for 2 to 8 days depending upon the severity of the bleeding episode. Antifibrinolytic agents are particularly useful as adjunctive therapy. However, it should be emphasized that antifibrinolytic therapy is contraindicated in the presence of hematuria.
Fibrin glue, otherwise known as fibrin tissue adhesives, has been used as adjunctive therapy to factor VIII in hemophilic patients.51 Briefly, fibrin glue contains fibrinogen, thrombin, and factor XIII. In some commercial products fibrinolytic inhibitors are added. The fibrinogen-factor XIII mixture is placed on the site of injury and clotted with a thrombin solution containing calcium. As a result, the fibrin clot is cross-linked and anchored to tissue. It is especially useful for hemostasis in patients undergoing dental surgery, who receive a preextraction bolus of factor VIII followed by application of fibrin glue to the tooth socket. Fibrin glue has also been used as adjunctive therapy to factor VIII following orthopedic procedures and circumcision. It has been of particular value for controlling bleeding when applied to the bed of a surgical wound following removal of large pseudotumors. Some hemophilia centers prepare their own “homemade” fibrin glue using cryoprecipitate as a source of fibrinogen and factor XIII. In some cases bovine thrombin preparations are used for clotting the fibrinogen solution. Bovine thrombin can result in complications, since it is contaminated with small amounts of bovine factor V. As a result, human antibodies to bovine factor V and thrombin may develop in patients receiving such products. These antibodies may cross-react with human factor V and/or human thrombin, resulting in a transient hemorrhagic disorder.52
On occasion, superficial cuts and abrasions are managed with local measures, i.e., application of pressure sometimes suffices to control bleeding, even though oozing may continue off and on for several hours. Topical thrombin is of no value in this type of bleeding. In general, cautery should be avoided, since bleeding may restart when the cauterized area sloughs.
When replacement therapy for epistaxis is needed, the factor VIII level should be raised to about 0.5 U/ml (50 percent of normal). For treatment of hematuria, patients should be instructed to drink large quantities of fluids. If hematuria is mild, uncomplicated, and painless, factor VIII replacement is not necessary unless it persists. Gross or protracted hematuria may require replacement therapy, and, in these patients, factor VIII levels of at least 50 percent of normal are needed and should be continued until bleeding stops.
Hemophilic patients needing endoscopic procedures should first be treated with factor VIII to raise levels to at least 0.5 U/ml. Only one dose may be necessary if endoscopy is uncomplicated. In cases of severe abrasions or perforations following endoscopy, factor VIII replacement should be continued until healing of the lesion is complete. In the case of expanding soft-tissue hematomas, factor VIII therapy should be started immediately and maintained until the hematoma begins to resolve. With effective therapy the patient usually experiences rapid relief from pain. For treatment of acute hemarthroses, prompt administration of factor VIII decreases the occurrence of extensive degenerative joint changes, as well as deformity and muscle wasting. For chronic synovitis and for bleeding into “target” joints, daily administration of factor VIII to raise levels to 100 percent of normal for 6 to 8 weeks may be indicated.
Any hemorrhage in a patient with hemophilia A may become major, but the following are common and frequently life-threatening: retropharyngeal; retroperitoneal; and central nervous system bleeding, whether subdural, subarachnoid, or into the brain parenchyma.53
For treatment of retropharyngeal bleeding, in particular that associated with a sensation of tightness in the throat, pain in the neck, dysphagia, or difficulty breathing, patients should receive factor VIII immediately in doses sufficient to raise factor VIII levels to normal (1.0 U/ml). Near-normal levels should be maintained until bleeding has ceased and the hematoma begins to resolve. For retroperitoneal hemorrhage, early treatment is required, and therapy should be continued for 7 to 10 days; otherwise bleeding may recur upon resumption of activity. Immediate administration of factor VIII, sufficient to raise the level to normal, should be started with the first sign of an intracranial hemorrhage or following a history of head trauma. Even asymptomatic patients with a history of head trauma should receive at least one dose of factor VIII as a prophylactic measure, and this should be given before diagnostic procedures such as a CT scan. Treatment of a known intracranial hemorrhage should be maintained for a minimum of 7 to 10 days, and the circulating factor VIII level should be kept normal throughout this period. Evacuation of subdural hematomas and surgical removal of hematomas involving the brain parenchyma can be carried out depending upon location. Despite aggressive replacement therapy, however, mortality from central nervous system bleeding is high.
For major surgical procedures, factor VIII should be raised to normal levels before operation and maintained for 7 to 10 days, or until healing is well underway. Treatment can be begun a few hours prior to surgery and continued intraoperatively. Postoperatively, factor VIII levels should be monitored at least once or twice daily to ensure that adequate levels are maintained. Since factor VIII may be “consumed” during surgery, doses of factor VIII higher than normal are sometimes required. Thus, factor VIII levels should be measured during surgery as well as in the postoperative period. Bone and joint surgery may require longer periods of factor VIII coverage. Replacement of knee, hip, and elbow joints is now possible, and several weeks of replacement therapy may be needed.54
Home therapy using available factor VIII concentrates was introduced in the United States in 1977 and represented a major advance in the treatment of all forms of hemophilia.55 Children as young as 3 years of age can be treated at home by parents or other reliable adults. Patients 6 years and older can be taught to treat themselves with factor VIII in the correct dose for an appropriate length of time. The training of patients and their families for home therapy is best accomplished in a regional comprehensive hemophilia diagnostic and treatment center or an affiliate of one of these centers. Patients are given an adequate supply of factor concentrates along with the paraphernalia required for intravenous administration. The prompt treatment of hemarthroses and hematomas made possible by home therapy resulted in a marked improvement in morbidity and mortality associated with hemophilia. In addition, the quality of life of hemophilia A patients was dramatically improved.56,57
The advent of stable and safe factor VIII concentrates has made prophylactic therapy for severely affected hemophilia A patients feasible. The administration of 50 U factor VIII/kg body weight three times weekly markedly decreases the frequency of hemophilic arthropathy and other long-term effects of hemorrhagic episodes.58,59 In 1997, the Medical and Scientific Advisory Council of the National Hemophilia Foundation recommended prophylactic therapy for severely affected patients. For prophylactic therapy to be successful, patients should be selected for reliability in managing central venous catheter devices.60,61 An analysis of the economic impact of prophylactic therapy, weighing the benefits against the high costs of factor VIII concentrates, suggests that the clinical benefit of prophylaxis is warranted as evidenced by significant improvement in the clinical condition of patients and improvement in the quality of life.62
Normal livers have been transplanted successfully into patients with hemophilia, with resulting cure of the hemophilic condition.63,64 However, donor livers are not widely available, so this procedure is rarely performed. As advances in modulation of graft-versus-host disease continue, the use of liver transplantation for the cure of hemophilia may increase.
Gene replacement therapy may offer ideal prophylactic treatment for hemophilia A. (See also Chap 19.) Recent advances in molecular biology have made gene insertion therapy a real possibility. Gene replacement therapy for factor VIII and factor IX has been shown to be possible in animal models using retroviral, adeno-associated, and adenoviral vectors. The early problems included low levels of expression and short duration of expression. Immune-mediated inhibition of retroviral and adenoviral vectors has also been a problem.65 Improvements in vectors promise advances in gene therapy for hemophilia A. In addition, factors that have retarded efficient expression of factor VIII in transduced cells have been elucidated.66 In addition, it has been possible to improve the passage of factor VIII through the endoplasmic reticulum by mutation of factor VIII residues involved in interaction with BiP, a protein chaperone within the endoplasmic reticulum.67 Nonviral vectors are also being developed for gene therapy of factor VIII.68 Mouse and dog models of hemophilia A exist, thus as improvements in vectorology continue, small and large animal models are available for testing as a prelude to clinical trials in humans. Human trials using gene transfer therapy (with B-domainless factor VIII in a retroviral vector) for hemophilia A have begun, but definitive results from these studies are not yet available.
After the advent of factor VIII concentrates in the 1960s, there was a significant reduction in the morbidity and mortality from bleeding in hemophilia, at least until the AIDS crisis that began in the late 1970s until 1985.69 The lifespan of hemophilia A patients began to approach that of normal individuals by the late 1970s. However, the use of replacement therapy has not been without significant complications. Common and serious adverse side effects of treatment include the following: the development of antibodies (inhibitors, circulating anticoagulants) against factor VIII; liver disease resulting from hepatitis B and C; and from about 1978, infection with HIV.70 Factor VIII concentrates, prepared from many thousands of donors, were contaminated with HIV from about 1978 until about 1985. The vast majority of severely affected patients became infected with HIV during this period. With the introduction of heat-treated concentrates in 1985, contamination of these products with HIV has been eliminated for all practical purposes. However, AIDS is now a leading cause of death in older patients with hemophilia.71 Chronic liver disease in hemophilia A patients resulting from transfusion-related hepatitis B and C may be accelerated by HIV infection and by the associated hepatotoxicity of antiviral drug therapy.72 Fortunately, patients treated after 1985 can expect to have virtually normal life spans free of the complications of hepatitis, AIDS, and other currently recognized blood-borne viral diseases.
Other than the transmission of viral diseases by factor VIII infusions, the main complication of hemophilia A is the development of specific inhibitor antibodies that neutralize factor VIII.73 There is current debate about the true frequency of antifactor VIII inhibitors in severe hemophilia A patients, but in one study the frequency of inhibitors in a large group of patients after 18 years of follow-up was 20 percent.74 This analysis included only patients treated with low- or intermediate-purity factor VIII concentrates. Frequent testing for inhibitors in previously untreated patients receiving newer highly purified factor VIII products from plasma or by recombinant technology revealed the frequent occurrence of transient inhibitors to factor VIII, many of which were of low titer and did not necessitate cessation of treatment with the same product. Although still a matter of uncertainty, it does not appear that the risk of inhibitors is higher when using highly purified plasma or recombinant products than that reported in earlier studies using products of lower purity.75 This is not to say that development of factor VIII inhibitors may not be related to the nature of factor VIII product.76 At least one outbreak of inhibitors appeared to be related to treatment with a specific plasma-derived factor VIII product of intermediate purity; fortunately inhibitors disappeared from the affected patients when use of the product was stopped.77
Factors related to the development of inhibitors are depicted in Table 123-5. As can be seen, they arise most frequently in severely affected patients, many of whom have gross gene rearrangements or inversion of the factor VIII gene. Inhibitors usually appear early in life, after about 100 exposure days to factor VIII replacement.


Factor VIII inhibitors are antibodies, most often of the IgG class and frequently restricted to the IgG4 subclass.76 Antibodies against the A2 and C domains of factor VIII are most common. These antibodies interfere with the interaction of factor VIII with its cofactors and activators.78
The early diagnosis of factor VIII inhibitors is essential. While the presence of an inhibitor can be suspected on clinical grounds, as, for example, when a patient does not respond to conventional doses of factor VIII, laboratory diagnosis is required for confirmation. Factor VIII inhibitors are time- and temperature-dependent. The prolonged aPTT of the plasma of a patient without an inhibitor is corrected when mixed 1:1 with normal plasma even after incubation at 37°C for 1 to 2 h. In contrast, the partial thromboplastin time (PTT) on a 1:1 mixture of a patient with an inhibitor and normal plasma is prolonged after incubation at 37°C for 1 to 2 h. Specific diagnosis rests upon the demonstration that an appropriate dilution of the patient’s plasma, when added to normal plasma, neutralizes specifically factor VIII and not other blood-clotting factors that influence the PTT (i.e., factors IX, XI, XII, PK, HK). The demonstration that the inhibitor is specific for factor VIII will distinguish it from inhibitors of other clotting factors, the lupus anticoagulant, and nonspecific inhibitors. A common assay for an inhibitor is the Bethesda assay, in which the patient’s plasma is diluted to a point that, when mixed with an equal volume of normal pooled human plasma and incubated for 2 h, will decrease the factor VIII activity in the mixture by 50 percent.79 A modification of the Bethesda assay is the Nijmegen assay in which the pH of the sample over the 2-h period of incubation is controlled.80
There are several approaches to the treatment of factor VIII inhibitors81 (Table 123-6). These require knowledge of whether the patient with an inhibitor is a “high” or “low” responder and whether the bleeding episode requiring treatment is considered minor or major.


High-Responder Patients About 60 percent of patients who have inhibitors are high responders. High responders are defined as those patients whose inhibitor titer is higher than 10 Bethesda units (BU) at baseline, or whose inhibitor titers rise to greater than 10 BU after administration of factor VIII. Thus, high responders who are not treated with factor VIII for long periods of time may have a sustained high level of inhibitor or may have a very low to undetectable level of inhibitor before they are challenged with factor VIII.
As depicted in Table 123-6, major bleeding episodes in a high-responder patient whose initial inhibitor titer is below 10 BU should be treated with either human or porcine factor VIII. The rationale is that when the initial titer is low, sufficient factor VIII can be administered to neutralize the inhibitor and to attain adequate factor VIII levels for hemostasis. Although factor VIII inhibitor bypassing agents can be used (see below), they are not as reliable in achieving hemostasis as factor VIII, and their effect cannot be adequately monitored with specific laboratory tests. If human factor VIII is used, a loading dose of 10,000 to 15,000 U may be required and followed by up to 1000 U factor VIII per hour depending upon the factor VIII level. All patients with inhibitors should be tested to determine whether their inhibitor cross-reacts with porcine factor VIII, as measured in a Bethesda assay, in which porcine factor VIII replaces human factor VIII. If the inhibitor does not cross-react with porcine factor VIII, it can be administered in doses of 50 to 100 U/kg per body weight every 8 to 12 h.
In high-responder patients whose initial inhibitor is less than 10 BU and who experience a minor bleeding episode, the agent of choice would be a factor VIII inhibitor bypassing agent. Recombinant factor VIIa in doses of 90 to 120 µg/kg per body weight every 2 to 3 h has been recently introduced and is safe and effective in most hemorrhagic episodes. If this agent is not available, activated or unactivated prothrombin complex concentrates may be used. Factor VIII can also be used but should be avoided in most instances in view of an anamnestic response of the inhibitor to factor VIII.
High-responder patients whose initial inhibitor titer is greater than 10 BU usually do not respond to even high doses of human factor VIII. If the inhibitor cross-reacts with porcine factor, this product too may be ineffective. Thus, in high-responder patients whose initial inhibitor titer is greater than 10 BU, and who experience a major or minor bleeding episode, recombinant factor VIIa is considered by many to be the treatment of choice. If this agent is not available, activated or unactivated prothrombin complex concentrates should be used as noted in Table 123-6.
Low-Responder Patients Low-responder patients are arbitrarily defined as those whose inhibitor titer is less than 10 BU even after challenge with factor VIII. For major bleeding episodes, high doses of human factor VIII or porcine factor VIII may be used as recommended above. For minor bleeds, recombinant factor VIIa, prothrombin complex concentrates (activated or unactivated), are recommended, since some “low” responders will convert to high responders when challenged repeatedly with factor VIII.
Nonactivated or activated prothrombin complex concentrates contain variable amounts of activated factors, including factors VIIa, IXa, and Xa. The activated products have higher concentrations of activated factors than unactivated products. It is not known how these agents “bypass” the inhibitors, but it is postulated that they enhance the tissue factor-factor VIIa pathway of coagulation. Both products have been used successfully for the therapy of hemophilic patients with inhibitors to factor VIII or factor IX.
One of the newer agents for treating patients with inhibitors is recombinant factor VIIa.82 This product has been reported to be more effective than other bypassing agents. Factor VIIa is recommended in doses of approximately 90 to 120 µg/kg per body weight every 2 to 3 h. The dosing frequency is based on a plasma half-life of factor VIIa that is about 2 to 3 h. The mechanisms of action of factor VIIa have been investigated using in vitro techniques. In the recommended doses it is hypothesized that factor VIIa can activate factor X on the surface of activated platelets in the absence of tissue factor.83 Factor Xa can then associate with factor Va and convert prothrombin to thrombin. Since activated platelets are localized to the site of vessel injury, thrombin generation by factor VIIa is localized to the site of bleeding. This may account for the reported safety of factor VIIa.82
Other approaches to the treatment of inhibitors include: immunosuppression; removal of the antibody by plasmapheresis; adsorption of the antibody on an affinity column during plasma exchange; and administration of intravenous gamma globulin. The Malmo protocol uses nearly all of these approaches in combination, including extracorporeal adsorption of antibody to a sepharose A column; the administration of cyclophosphamide; daily administration of factor VIII; and intravenous gamma globulin.84
The most promising approach to eradication of an inhibitor is the use of immune tolerance regimens. The basis of this approach is to administer daily doses of factor VIII until the inhibitor titer is undetectable.85 Low-dose and high-dose regimens have been described as shown in Table 123-7. Factor VIII inhibitor bypassing agents are used for acute bleeds that occur during immune tolerance induction. Various approaches to the treatment of factor VIII inhibitors can be found on the Internet.86


Hepatitis Almost all multitransfused patients with hemophilia treated before 1985 were infected with one or more agents of viral hepatitis. While many infected patients did not suffer acute symptoms, at least 50 percent can be expected to develop chronic persistent or chronic active hepatitis that may lead to frank cirrhosis.87 Hepatitis C and B viruses are commonly associated with chronic liver disease. About 90 percent of adult hemophilic patients have antibodies to hepatitis B surface antigen (HBsAg), and at least 10 percent of severely affected adult patients have circulating HBsAg. The antigen-positive adult patients frequently have a superimposed infection with the delta agent, leading to severe active hepatitis and cirrhosis and an increased risk of hepatocellular carcinoma.88,89 and 90 Therapy with recombinant interferon a can improve biochemical markers of hepatocellular damage and liver histology in some patients with chronic hepatitis C,91 but the long-term benefits of a-interferon therapy are not established.
HIV Currently, about 80 percent of older, severely affected hemophilia A patients have antibodies to HIV, indicating infection with the virus. The incidence of HIV antibodies in mildly affected patients is much lower and correlates with treatment with factor VIII concentrates before viral inactivation procedures were used. In one study, 14 percent of patients treated only with cryoprecipitate during the period from 1979 to 1985 were infected with HIV, while 88 percent of patients treated with factor VIII concentrates became infected.92 Screening of donor populations and new techniques for preparing factor VIII concentrates since 1985 have nearly eliminated the risk of HIV transmission. Many HIV-infected patients with hemophilia developed AIDS, and treatment of AIDS has become an integral part of the management of severe hemophilia. In one report, AIDS was the primary cause of deaths in hemophilia A patients between the years 1976 and 1991.71 Bleeding was responsible for only 5 percent of deaths.
Perhaps related to HIV infection, immune suppression has been observed in many recipients of blood products, including factor VIII concentrates.93 Evidence of a depressed cellular immune system can be found in most patients with hemophilia A who have been treated with factor concentrates.94 There is often a reduced ratio of T-helper lymphocytes to T-suppressor lymphocytes, in addition to a decrease in natural killer cells. Anergy to cutaneously applied antigens is also a common finding in patients who have been multiply transfused. The degree of immune suppression may be related in part to the purity of the transfused product.95,96 Improved methods of purification have led to the development of products that appear to be less immunosuppressive.
Risk of Transmission of Viral Diseases by New Factor VIII Products Available factor VIII concentrates are considered to be safe and effective, and there is virtually no risk of transmitting currently known viral diseases with these products. There have, however, been occasional exceptions. For example, solvent-detergent extraction does not inactivate viruses without lipid envelopes, including hepatitis A virus and parvovirus. As a result, outbreaks of hepatitis A have been reported in patients receiving some solvent-detergent-treated products. These outbreaks of viral diseases are usually related to breakdowns during the manufacturing process.
Prions Prions are infectious particles that consist of proteinaceous material that is devoid of a nucleic acid genome.97 They are thought to be variant forms of a normal protein with an altered conformation. The “infectious” nature of prions may be due to their ability to bind to other proteins and induce similar conformational changes in them such that new “infectious” particles can be generated. Prions are responsible for several neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and spongiform encephalopathy in cows. Prions are resistant to all currently available viral inactivation techniques. Although prion diseases are generally transmitted by ingestion of infected neural tissues, there is a new variant of CJD which appears to occur in people who have eaten beef from cows infected with a form of prion causing bovine spongiform encephalopathy.98 This form of CJD has been reported mainly in the United Kingdom and has been related to the bovine disease. For example, prions have been found in tonsillar tissue of patients with new-variant CJD, heightening the concern about whether prions of this type might be transmitted by blood products. To date there is no evidence that new variant CJD or, for that matter, the usual form of CJD is transmitted in this way. However, conclusive data are lacking, since the incubation period for the disease may be many years. For this reason certain plasma products prepared from blood of donors in the United Kingdom have been withdrawn until more data are available. There is, however, encouraging preliminary data in that autopsies of 33 hemophilics in the United Kingdom showed no evidence of prion disease.98
Hemophilia B is clinically indistinguishable from hemophilia A. It is a sex-linked, recessive hemorrhagic disease characterized by a decrease in factor IX clotting activity. In 1952 Aggeler and colleagues and Biggs and colleagues observed the existence of another X-linked bleeding disorder that was clinically similar to classic hemophilia.7,99 The deficient factor has been designated as factor IX, and the disease is called hemophilia B. Other synonyms for factor IX include plasma thromboplastin component (PTC) and Christmas factor, named after the family in which it was described.
Hemophilia B occurs in 1 out of every 25,000 to 30,000 male births. Just as with hemophilia A, hemophilia B is found in all ethnic groups and has no geographic predilection.
Factor IX is a vitamin-K-dependent, single-chain glycoprotein consisting of 415 amino acids. It is activated by the factor VIIa/tissue factor complex, or factor XIa, to form the active enzyme, factor IXa. Once activated, factor IXa activates factor X in the presence of activated factor VIII, phospholipid (activated platelets), and calcium. Factor VIIIa is a necessary cofactor for the activity of factor IXa. Therefore, deficiency of either factor IX or VIII leads to a similar lack of factor X-activating activity. Factor Xa converts prothrombin to thrombin in the presence of activated factor V phospholipid and calcium. Thus, deficiency of factor IX results in delayed conversion of prothrombin to thrombin, which is the cause of the bleeding tendency. Hemophilia B can result from either the absence or dysfunction of factor IX molecules. Clinical severity of hemophilia B is roughly correlated with factor IX functional activity.
The factor IX gene is on the long arm of the X chromosome and is approximately 33 kb in length, much smaller than the gene for factor VIII.100 Because it is less complex, the factor IX gene has been studied in greater detail than the factor VIII gene. A schematic diagram of the gene and the protein product is depicted in Fig. 123-11. The protein consists of a signal peptide that targets the protein for secretion from the hepatocyte to the circulation. The propeptide is necessary for posttranslational modification of 12 amino-terminal glutamic acid residues by an intracellular vitamin-K-dependent carboxylase. The propeptide is cleaved from the mature protein before it enters the circulation. The next domain contains the 12 g-carboxyglutamic acid (Gla) residues that are necessary for calcium-dependent lipid binding. The activation peptide is cleaved from the zymogen form of factor IX either by factor VIIa/TF or by factor XIa, resulting in a two-chain active enzyme, factor IXab. The catalytic triad (histidine 221, aspartic acid 229, and serine 365) resides on the heavy chain.

FIGURE 123-11 Schematic diagram of the factor IX gene, the messenger RNA, and the protein. The exons are depicted by the black boxes. The white 3′ portion of the RNA is untranslated. The diagram of the protein shows the domains and the exons that encode each portion of the protein. The cleavage sites by factor XIa or factor VIIa/tissue factor are depicted by asterisks.

Many genetic variants of hemophilia B have been described. They include point mutations, frameshifts, deletions, and other abnormalities that cause structural and/or functional changes in the factor IX protein.101,102 and 103 Several hundred unique mutations have been reported, and a database of mutations has been developed and is updated yearly.104 It can also be found on the Internet.105
Over 30 percent of factor IX mutations occur at CG dinucleotides. These mutations often involve critical arginine residues that result in a dysfunctional molecule.106,107,108 and 109 Many mutations have been reported in more than one kindred, and some of these derive from the same “founder.”109,110 As predicted by genetic theory of X-linked recessive disorders, approximately one-third of mutations resulting in hemophilia B arise de novo.
Mutations in regulatory regions of the factor IX gene have also been identified. Particularly interesting examples are mutations in the 5′ promoter region that lead to the hemophilia B Leiden phenotype (Table 123-8). This disorder is characterized by very low levels of factor IX antigen and activity at birth and during early childhood. The levels gradually rise to 60 percent of normal or greater following puberty, apparently in response to endogenous androgen synthesis. Several different mutations in the promoter region of the factor IX gene disrupt binding of transcription factors, resulting in reduced transcription of the factor IX gene.111,112 and 113 The hormonal changes occurring at puberty are apparently able to overcome the transcription defect and maintain hemostatically adequate levels of factor IX.


Hemophilia Bm is characterized by a deficiency of factor IX clotting activity and a prolonged ox-brain prothrombin time. The original hemophilia B patient with a prolonged ox-brain prothrombin time had the surname Martin and this led to the term hemophilia Bm.102,114 A number of missense mutations affecting amino acid residues at positions 180, 181, and 182 of the protein as well as several residues close to the active site region have been identified in the patients having the characteristic findings of hemophilia Bm. These mutations result in a factor IX molecule that exhibits abnormal interaction with ox-brain tissue factor.114
Hemophilia B inheritance is similar to that of hemophilia A. All daughters of affected males are obligatory carriers, while all sons are normal. Female carriers may have factor IX levels ranging from less than 10 to 100 percent of normal, but the mean level is about 50 percent of normal. Carriers of hemophilia B are usually asymptomatic, except in the cases of extreme X-chromosome inactivation, X-mosaicism, Turner syndrome, or testicular feminization.115 When the level of factor IX activity is less than 25 percent of normal, abnormal bleeding may occur, especially after trauma.
Carrier detection and genetic screening are sometimes possible through the use of DNA probes to directly identify mutations. As with factor VIII, mutations at CpG nucleotide pairs disrupt TaqI cleavage sites and can therefore be directly detected by restriction endonuclease mapping. More commonly, RFLP analysis is used. Prenatal diagnosis has been reliably accomplished by RFLP analysis of DNA obtained by chorionic villus sampling as early as 8 to 10 weeks after conception.116 This procedure can also be performed on fetal cells obtained by amniocentesis and is more accurate than fetal blood sampling for factor IX activity and factor IX antigenic material. Direct sequencing of the factor IX gene can also be used for carrier detection, but this is not available in most laboratories.
Bleeding episodes in patients with hemophilia B are clinically identical to those in hemophilia A, as described in the previous section. When patients are inadequately treated, repeated hemarthroses leading to chronic, crippling hemarthropathy occur. Hematoma formation with dissection into surrounding tissues is also common. Hematuria, bleeding from mucous membranes, and other bleeding manifestations are as described under the section on hemophilia A. The physical, psychological, vocational, and social aspects of the disease are similar to those encountered with hemophilia A. Classification of hemophilia B is based on clinical severity and roughly correlates with the level of factor IX coagulant activity. Severe disease is usually associated with factor IX levels of less than 1 percent of normal; moderate disease is associated with factor IX levels of 1 to 5 percent; and mild disease is associated with factor IX levels ranging from 5 to 40 percent of normal.
The occurrence of factor IX inhibitor antibodies is much less common in hemophilia B patients than in hemophilia A patients. Only about 3 percent of severely affected patients develop inhibitors.
The screening tests used in the diagnosis of hemophilia A are also employed in the diagnosis of hemophilia B. In most cases of hemophilia B, the prothrombin time is normal, and the partial thromboplastin time is prolonged. However, specific assay of factor IX coagulant activity is required for the definitive diagnosis. The most commonly used test is a one-stage clotting assay based on the PTT. Determination of factor IX antigen levels is of value in further classification of the disorder. Even though prothrombin times are usually normal in hemophilia B, they are occasionally prolonged, especially when ox-brain thromboplastin is the source of tissue factor. The factor IX in patients with hemophilia Bm competes with factor X for activation by the factor VIIa/tissue factor complex, thus resulting in a long PT.117 Since most American prothrombin time reagents contain rabbit brain tissue factor, the prothrombin times recorded for American patients with hemophilia Bm are usually normal, and the hemophilia Bm subtype will not be identified. In all forms of hemophilia B, the bleeding time is normal.
Hemophilia B must be distinguished from hemophilia A. Both are inherited as X-linked recessive disorders, and both have virtually identical hemorrhagic manifestations. The only way to differentiate hemophilia B from hemophilia A is to perform specific assays for factors VIII and IX on the patient’s plasma.
Inherited and acquired deficiencies of other vitamin-K-dependent factors, liver disease, and warfarin overdose must also be distinguished from hemophilia B. In these cases, not only factor IX but all other vitamin-K-dependent clotting factors will be decreased including prothrombin, factor VII, and factor X. Acquired antibodies specific for factor IX occur in nonhemophilic patients, but these are very rare.
The basic treatment of hemophilia B is replacement of factor IX. There are several products available for use, and they are listed in Table 123-9. The older factor-IX-containing products are often referred to as prothrombin complex concentrates. These products, prepared from large pools of human plasma (several thousand donors), contain not only factor IX but also prothrombin, factors VII and X, as well as proteins C and S. In addition, the products may contain small amounts of activated factors such as factors VIIa, IXa, and Xa. Some of these products have been associated with thromboembolic events, presumably due to contamination with the activated components. Deep venous thrombosis and disseminated intravascular coagulation have been reported in some patients receiving large doses of prothrombin complex concentrates. Therefore, they are not the best choice for replacement therapy in hemophilia B, even though they are much cheaper than the highly purified factor IX concentrates. When prothrombin complex concentrates are used for replacement therapy, factor IX levels of greater than 50 percent of normal should not be exceeded in order to minimize the risk of thrombosis. The use of these products in factor-IX-deficient patients with liver dysfunction is especially hazardous, since it has been shown that activated factors contaminating these preparations are not cleared efficiently by a diseased liver and as a result thrombosis can be induced.


The highly purified factor IX products are also listed in Table 123-9. Some are prepared from human plasma while one product (BeneFix) is produced by recombinant DNA technology. Although all available factor IX concentrates are now considered to be safe and effective, the recombinant product undergoes a final viral inactivation step. In addition, it is not exposed to human albumin or bovine serum during preparation. Thus, even the theoretical risk of transmission of prion diseases is averted with this preparation. Some clinicians consider the recombinant product to be the agent of choice, although it has a major drawback in that the intravascular recovery of factor IX is generally lower than the recovery of highly purified factor IX product prepared from plasma.118
The dose calculations for all factor IX products are different from those used in hemophilia A. The reason is that intravascular recovery of factor IX is usually only about 50 percent, and, in the case of the recombinant product, the recovery is even lower. The reason for this is unclear, but it has been proposed that factor IX binds to elements on the vessel wall. In fact, factor IX binds specifically to collagen type IV, a component of the vessel wall.119 The dose of factor IX can be estimated by assuming that one unit of factor IX per kilogram body weight will increase circulating factor IX by 1 percent of normal or 0.01 U/ml. Thus, in a severely affected patient, to achieve a level of 100 percent of normal (using only highly purified factor IX products), 100 U of factor IX per kilogram body weight should be given as a bolus, followed by one-half this amount every 12 to 18 h. Dosing should be monitored by assays of factor IX before and after bolus administration. Factor IX can also be administered as a constant infusion after the bolus administration. The dose of factor IX to be infused per hour can be estimated on the basis of a factor IX half-life of 12 to 18 h. Thus, in a 60-kg adult, who receives highly purified factor IX, 6000 U of the factor should raise the factor IX level to about 100 percent of normal. Over the next 12 to 18 h, the level will decrease by about 50 percent, and thus the patient will need about 3000 U of factor IX during that period or 250 U of factor IX per hour as an infusion. These calculations are only estimates of average responses, and so factor IX dosing should be monitored by factor IX assays and the dose adjusted appropriately. Prophylactic therapy for hemophilia B can also be attempted in individuals selected in the same manner as that described for hemophilia A patients. The dose of factor IX is 25 to 40 U/kg of body weight twice weekly.
Although currently available factor IX concentrates are safe in terms of transmission of HIV and hepatitis B and C viruses, patients treated prior to 1985 may have been infected with these agents.
Unless treated properly, hemophilia B is fraught with the same complications of recurrent hemorrhages as hemophilia A. Thus, in inadequately treated patients, hemarthroses and chronic hemophilic arthropathy are common. In addition to joint deformities, chronic active hepatitis and chronic persistent hepatitis are common in patients treated before 1985. About 50 percent of older and severely affected patients are now HIV-positive. Patients treated after 1985 are unlikely to have contracted HIV and can expect to have a relatively normal life span.
Patients with severe hemophilia B may develop inhibitory antibodies against factor IX, making treatment very difficult.120,121 About 3 percent of patients with severe hemophilia B develop specific inhibitor antibodies, frequently restricted in immunoglobulin composition to the IgG4 subclass and kappa light chains.122 Most inhibitors can be detected when the aPTT on a mixture of normal and patient’s plasma is prolonged. In contrast to the inhibitors in hemophilia A patients, inhibitor antibodies against factor IX are not time- and temperature-dependent, and, thus, it is usually not necessary to incubate the mixtures for 2 h at 37°C. Inhibitors to factor IX can be quantitated by modifying the Bethesda method for detecting factor VIII inhibitors. Many patients with inhibitors have mutations that result in the absence of circulating factor IX antigen, most commonly deletions and nonsense mutations.
When the inhibitor titer is below 10 BU/ml, it is sometimes possible to neutralize the factor IX inhibitor using large doses of highly purified factor IX concentrates. However, when the inhibitor titer is greater than 10 BU/ml, acute bleeding in patients should be treated with the same agents used to bypass factor VIII inhibitors as shown in Table 123-6. Recombinant factor VIIa in doses of 90 to 120 µg/kg body weight intravenously every 2 to 3 h may be used. Alternatively, activated or nonactivated prothrombin complex concentrates can be used as noted in Table 123-6.
Induction of immune tolerance can also be tried in hemophilia B patients using daily infusions of highly purified factor IX preparations. However, significant adverse reactions have been reported in severely affected patients, including anaphylaxis and the nephrotic syndrome.123 Of the reported cases, many patients were less than 12 years of age and suffered from severe hemophilia B due to large deletions of the factor IX gene. The nephrotic syndrome may be transient and remit upon cessation of factor IX replacement. The etiology of the nephrotic syndrome is not known. Patients with hemophilia B and factor IX antibodies who experience anaphylaxis with factor IX infusions should be treated with factor VIIa concentrates.
Long-term correction of hemophilia B has been achieved in animal models.124,125 and 126 Transduction of muscle cells by an adeno-associated viral (AAV) vector containing a factor IX construct resulted in phenotypic correction of the clotting defect in hemophilia B dogs for greater than 17 months.124 Likewise, transduction of hepatocytes with an AAV vector containing factor IX DNA has also been reported to correct the hemophilia defect in hemophilia B mice and dogs for about 7 and 8 months, respectively. Sustained factor IX levels of up to 25 percent of normal were obtained in one study in mice,125 while levels exceeding 100 percent were achieved in another study.126 The results in animals are encouraging and suggest that permanent corrections of the hemophilic defect using gene transfer technology in humans may be possible (see also Chap. 19). Clinical trials of gene transfer therapy for hemophilia B using adeno-associated viral vectors containing factor IX cDNA have been started, but the studies are preliminary.127

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology



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