Hemophilia - Factor VIII or IX Deficiency

Hemophilia A and hemophilia B are bleeding disorders caused by genetic variants in the F8 or F9 gene that result in deficiencies of factor VIII (FVIII) in hemophilia A or factor IX (FIX) in hemophilia B, respectively. These disorders are clinically indistinguishable and present with bleeding symptoms that range from mild to severe, depending on the underlying defect (ie, the frequency and severity of bleeding is based on the level of factor activity). Hemophilia occurs in an estimated one in 10,000 births worldwide, and 80-85% of cases are hemophilia A. Both hemophilia A and B are rare in females due to X-linked inheritance. Acquired hemophilia, an autoimmune disorder, is rarer, occurring in one to two individuals per million. Female carriers of hemophilia and those with subhemophilia (in which FVIII activity is reduced, but not to the extent seen in mild hemophilia) are also at risk for bleeding complications during surgical procedures and can pass hemophilia to offspring. A workup for hemophilia typically involves tests such as prothrombin time (PT), partial thromboplastin time (PTT), mixing studies, and factor assays to identify the factor deficiency.

Quick Answers for Clinicians

Who should be tested for hemophilia?

Individuals with any of the following should be considered for testing:

Spontaneous bleeding (particularly into joints, muscles, and soft tissues)
Excessive bleeding suggestive of a coagulation disorder
Family history of hemophilia
Acute or recent onset bleeding with or without a partial thromboplastin time (PTT) result
Easy bruising in early childhood

Which lab tests should be used for hemophilia testing?

Basic coagulation tests aid in identifying the cause of bleeding, and factor assays confirm the diagnosis. Initial tests include a CBC with platelet count, prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen tests. Mixing studies help to determine the presence of a factor deficiency versus an inhibitor. Factor assays are used to definitively identify and quantify factor VIII (FVIII) or factor IX (FIX) deficiency.

What issues are important to keep in mind when testing for hemophilia?

Patients being tested for bleeding disorders should avoid vigorous exercise around the time of testing. Anxiety over testing can also interfere with results, as levels of factor VIII (FVIII) and von Willebrand factor (VWF) temporarily increase with stress. In addition, since VWF is a carrier for FVIII, von Willebrand disease (VWD) should be ruled out in patients with decreased FVIII levels.

What should I know about monitoring patients with hemophilia?

Patients being treated for hemophilia are at risk for the development of inhibitors. Inhibitor development is more common in hemophilia A than B. In mild or moderate disease, inhibitor testing should be performed annually, and also when a patient does not respond as expected to factor concentrate replacement, before and after changing factor products, 5-7 days before invasive procedures, and approximately 3 weeks after intensive treatment (>5 exposure days) or surgery. In severe disease, the frequency of inhibitor testing is related to treatment. See Monitoring.

Indications for Testing

Hemophilia testing is warranted in individuals with spontaneous bleeding (particularly into joints, muscles, and soft tissues) or prolonged/excessive bleeding that is suggestive of a coagulation disorder. Common bleeding manifestations include epistaxis, hemarthroses, muscle hemorrhage, hematomas, and menorrhagia in women. Individuals with a family history of hemophilia and those who have experienced acute or recent-onset bleeding should be tested.

Laboratory Testing

Diagnosis

Initial testing for a coagulation disorder involves a CBC with platelet count, PT and PTT, and fibrinogen tests.

Interpretation of Initial Tests

	Hemophilia A or B^a,b	VWD	Platelet Defect
Platelet count	Normal	Normal or reduced	Normal or reduced
PT	Normal	Normal	Normal
PTT	Prolonged^c	Normal or prolonged^c	Normal
^aThe same pattern can occur in deficiencies of FXI, FXII, prekallikrein, or high molecular weight kininogen. ^bThrombin clotting time and fibrinogen activity will also be normal in hemophilia A and B. ^cPTT results are affected by reagents/instruments used by lab. In addition, the PTT may yield a normal result in mild cases of hemophilia and in female hemophilia carriers. VWD, won Willebrand disease Source: Srivastava, 2014

Mixing Studies

A PTT that corrects with a mixing study suggests a factor deficiency, whereas a PTT that does not correct with a mixing study suggests that an inhibitor is present. An inhibitor pattern can be seen with factor inhibitors, lupus anticoagulants, and anticoagulant medications that inhibit coagulation factors. Incubated mixing studies (in which a mixture of patient plasma and normal pooled plasma is incubated for 1-2 hours at 37°C before testing) are often necessary to detect factor VIII inhibitors, which can develop in patients with inherited or acquired hemophilia A. (See Monitoring below.)

Factor Assays

Factor assays are traditionally performed using clot-based tests and are used to confirm the diagnosis of hemophilia and to classify hemophilia severity by demonstrating FVIII or FIX deficiency. Normal factor levels do not rule out carrier status; definitive determination of female carrier status relies on genetic testing. Hemophilia severity is classified as follows: severe (<1% factor activity), moderate (1-5% factor activity), or mild (6-40% factor activity).

In infants with an FVIII level at the lower end of normal, testing should be repeated at about 6 months of age. In neonates who may be mildly affected with hemophilia B, the FIX activity may need to be remeasured 3-6 months after birth for a definitive result.

Chromogenic Assays

Consider confirmation of low factor activity using a chromogenic assay. A number of interfering substances (eg, heparin, lupus anticoagulants) can interfere with the first-line clot-based factor assays. Clinically significant discrepancies have also been observed between clot-based and chromogenic assay results for some forms of hemophilia.Refer to the Chromogenic Factor VIII, Activity Test Fact Sheet for additional information.

von Willebrand Factor Assay

Since von Willebrand factor (VWF) is a carrier protein for FVIII, VWD should be ruled out in patients with decreased FVIII levels. The VWF level will be normal in patients with hemophilia. A rare subtype of VWD (type 2N) can demonstrate low FVIII activity with normal VWF levels and can resemble hemophilia A. Specialized coagulation or genetic testing can be used to distinguish these disorders.

Bethesda Assays

The Bethesda assay is used to help distinguish between factor inhibitors and factor deficiencies that are not due to an inhibitor, to titer inhibitors, and to monitor treated patients with hemophilia for the development of an inhibitor. See Monitoring below.

Genetic Testing

In patients with a family history of hemophilia, individual patient risk should be calculated by a clinical geneticist based on laboratory results and family history. Genetic testing can confirm the presence of the causative F8 or F9 gene variant in affected individuals, allows for targeted testing of documented familial mutations, and can determine carrier status in girls or women at risk.

In Utero Testing

In utero genetic testing (third-trimester amniocentesis) can be performed to determine whether hemophilia is present in male fetuses. At birth, uncontaminated cord blood can be used to establish diagnosis if testing was not performed previously. Testing should include FVIII and FIX activity in addition to PTT. However, interpretation of both PTT and FIX activity is difficult in neonates, so repeat testing at several months of age or genetic testing may be necessary to establish the diagnosis.

Monitoring

Trough-Level Testing

Trough levels (last dose of factor infused plus time since last infusion, for correct interpretation) should be measured regularly to monitor factor concentrates. Trough-level tests should be performed in conjunction with inhibitor testing if breakthrough bleeds have occurred. Confirm which type of factor assay (clot based versus chromogenic) is appropriate for the factor replacement product being used (review of the package insert may provide helpful guidance in appropriate assay selection). Additional information regarding effect of different extended half-life FVIII replacement products on FVIII activity tests is available in the Chromogenic Factor VIII, Activity Test Fact Sheet.

Inhibitor Testing

Individuals receiving treatment for hemophilia are at risk for the development of inhibitors. Monitoring is required because clinical signs do not necessarily accompany inhibitor development.

Some groups recommend a washout period of 72 hours (ie, stopping factor treatment) before an inhibitor assay is performed, but heat treatment of specimens makes this unnecessary, so it is important to know the laboratory protocol that will be used.

Factor concentrates are monitored by measuring factor levels before and after infusion. Reduced half-life of the infused clotting factor or lower than expected recovery may suggest inhibitor presence.

In mild or moderately affected patients, inhibitor testing should be performed annually and also in the following circumstances:

When a patient does not respond as expected to factor concentrate replacement
Before and after changing factor products
5-7 days before elective invasive procedures
Approximately 3 weeks after intensive treatment (>5 exposure days) or surgery
After every concentrate exposure in patients with increased risk of inhibitor formation because of a particular mutation

In patients with severe hemophilia A and B, inhibitor screening should be performed on every third concentrate exposure day or every 3 months until 20 exposure days have been reached. After that, inhibitor testing should be performed every 3-6 months until 150 exposure days are reached. (In many patients with severe hemophilia, prophylaxis has been established by the 20th exposure day and then trough levels are checked approximately every 3-6 months; if FVIII/FIX is <1 IU/dL, inhibitor testing should be performed.)

In hemophilia B, inhibitor testing is unnecessary after 150 exposure days, unless there is clinical suspicion that an inhibitor is present.

Comorbidity Screening

Screening for hemophilia-specific comorbidities has become more important because patients with hemophilia are living longer. Screening might include blood count, blood coagulation tests, iron status, viral screening (hepatitis, HIV), and liver/kidney function tests.