Hemophilia - Factor VIII or IX Deficiency

Educational Podcast From ARUP Laboratories

LabMind: An Interview With Dr. Karen Moser: Solving Coagulation Mysteries One Patient at a Time

Medical Experts

Contributor

Moser

Karen A. Moser, MD

Associate Professor of Pathology (Clinical), University of Utah

Medical Director, Hematopathology and Hemostasis/Thrombosis Laboratory, ARUP Laboratories

Contributor

Smock

Kristi J. Smock, MD

Professor of Pathology (Clinical), University of Utah

Vice President, ARUP Institute for Clinical and Experimental Pathology^®

Medical Director, Hemostasis/Thrombosis Laboratory, ARUP Laboratories

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 in hemophilia A or factor IX 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 factor VIII activity is reduced, but not to the extent as 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), activated partial thromboplastin time (aPTT), mixing studies, and factor assays to identify the factor deficiency.

Quick Answers for Clinicians

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 and von Willebrand factor (VWF) temporarily increase with stress. In addition, since VWF is a carrier for factor VIII, von Willebrand disease (VWD) should be ruled out in patients with decreased factor VIII levels.

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 aPTT, 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
aPTT	Prolonged^c	Normal or prolonged^c	Normal
^aThe same pattern can occur in deficiencies of factor XI, factor XII, prekallikrein, or high-molecular-weight kininogen. ^bThrombin clotting time and fibrinogen activity will also be normal in hemophilia A and B. ^caPTT results are affected by reagents/instruments used by the lab. In addition, the aPTT may yield a normal result in mild cases of hemophilia and in female hemophilia carriers. VWD, won Willebrand disease Source: Srivastava, 2020

Mixing Studies

An aPTT that corrects with a mixing study suggests a factor deficiency, whereas an aPTT that does not correct with a mixing study suggests that an inhibitor is present. An inhibitor pattern may occur as a result of 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. (Refer to the Monitoring section.)

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 factor VIII or factor IX deficiency. Normal factor levels do not rule out carrier status; definitive determination of female carrier status relies on genetic testing.

Hemophilia severity is classified based on factor activity.

Disease Classification	Expected Factor Activity
Mild	6-40%
Moderate	1-5%
Severe	<1%

Nonsevere hemophilia A may require both one-stage clot-based factor VIII activity and chromogenic factor VIII activity for diagnosis and severity classification, due to differences in how the underlying mutations affect factor VIII activity in the tests.

In infants with a factor VIII level at the lower end of normal, testing should be repeated at approximately 6 months of age. In neonates who may be mildly affected with hemophilia B, the factor IX 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.

von Willebrand Factor Assay

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

Bethesda Assay

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 receiving treatment for hemophilia for the development of an inhibitor. Refer to the Monitoring section.

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.

Prenatal and Perinatal 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 factor VIII and factor IX activity in addition to aPTT testing. However, interpretation of both aPTT and factor IX 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).

Factor VIII Assays—Extended Half-Life Replacement Products

Modified extended half-life factor VIII replacement products may lead to under- or overestimation of factor VIII activity in clot-based factor VIII assays (one-stage assays) using certain aPTT reagents.^,^,

Effects of Extended Half-Life Factor VIII Replacement Products on Factor VIII Activity
Extended Half-Life Factor VIII Replacement Product (Manufacturer)	Modification Type	Effect on Factor VIII Activity
Adynovate/rurioctocog alfa pegol (Shire/Baxalta, Baxter)	PEGylated (random) rFactor VIII	Acceptable recovery with chromogenic or 1-stage factor VIII activity (most reagents) Relatively higher recovery with aPTT reagents with ellagic acid activators (eg, Actin FS, Actin FSL) that may be clinically significant (>30% different from expected)
Afstyla/lonoctocog alfa (CSL Behring)	Single chain, B-domain truncated rFactor VIII	Underestimated with all 1-stage factor VIII activity assays^a Acceptable recovery with chromogenic factor VIII activity assays^b
Altuviiio/efanesoctocog alfa (Sanofi)	Single chain, B-domain deleted, IgG1-Fc-VWF-XTEN fusion	2- to 3-fold overestimation with chromogenic assays and aPTT reagent Actin FS Acceptable recovery with most 1-stage aPTT reagents: Actin FSL, SynthAFax, Thrombocheck SLA, Pathromtin SL, STA-PTT-A, aPTT-HS, and STA-CK Prest) Under- or overestimation with specific 1-stage aPTT reagents (eg, SynthASil, APTT SP, Actin Cephascreen, Cephen), although numbers of laboratories using some of these reagents were low in the field study
Eloctate/efmoroctocog alfa (Biogen Idec)	Fc fusion, B-domain deleted rFactor VIII	Acceptable recovery with chromogenic or 1-stage factor VIII activity for all aPTT reagents tested to date Results 20% higher with chromogenic assays (may not be clinically significant)
Esperoct/turoctocog alfa pegol (Novo Nordisk)	B-domain truncated, glycoPEGylated (site directed) rFactor VIII	Underestimated with aPTT reagents STA-PTT A, SynthAFax, SynthASil, TriniCLOT APTTS, APTT SP^c Acceptable recovery with 1-stage factor VIII activity for all other aPTT reagents tested to date
Jivi/damoctocog alfa pegol (Bayer Healthcare)	PEGylated (site directed) B-domain deleted rFactor VIII	Underestimated with aPTT reagents with silica activators (eg, STA-PTT A, APTT SP) Overestimated with Actin FS (ellagic acid) and STA-CK Prest (kaolin) Few other aPTT reagents studied to date Recovery appears acceptable with most aPTT reagents with ellagic acid activators Acceptable recovery with chromogenic assays
^aThe package insert for Afstyla recommends use of a correction factor (multiply by 2) for 1-stage factor VIII assays, which is supported by a multicenter field study; this correction factor may lead to overestimation in the low range. ^bChromogenic factor VIII assays recommended instead of 1-stage factor VIII assays. ^cChromogenic factor VIII assays recommended instead of 1-stage factor VIII assays for these reagents. IgG1, immunoglobulin G1; rFactor, recombinant factor Sources: Srivastava, 2020; Abraham, 2023; Pipe, 2024

Factor VIII Assays—Emicizumab

Factor VIII activity cannot be accurately measured using a one-stage clot-based factor VIII activity assay in the presence of emicizumab. Emicizumab is a bispecific antibody that bridges factor IX and factor X to produce activated factor X (factor Xa). Emicizumab effectively replaces the function of factor VIII in secondary hemostasis and will substitute for factor VIII function in one-stage clot-based factor VIII activity assays; therefore, the presence of emicizumab will result in overestimation of factor VIII activity (either native factor VIII or factor VIII concentrate administered in an acute care setting). Emicizumab interference may last for up to 6 months following the conclusion of therapy.

To accurately measure patient factor VIII activity (endogenous or from factor VIII concentrates) in patients receiving emicizumab, a chromogenic factor VIII activity assay should be used. Emicizumab can bind to factor IX and X in chromogenic assays using human factor-derived reagents and will still overestimate factor VIII activity. Refer to the Inhibitor Testing section for considerations for Bethesda assays in patients receiving emicizumab.

Factor IX Assays

As with factor VIII, modified extended half-life factor IX replacement products may lead to under- or overestimation of factor IX activity in clot-based factor IX assays using certain aPTT reagents.

Effects of Extended Half-Life Factor IX Replacement Products on Factor IX Activity
Extended Half-Life Factor IX Replacement Product	Modification Type	Effect on Factor IX Activity
Alprolix/eftrenonacog alfa (Sanofi Genzyme)	Fc fusion	Chromogenic assays show acceptable recovery Certain ellagic acid and silica aPTT activators preferred in 1-stage assay (except Actin FS and SynthAFax) CK Prest causes underestimation of 20-40%; APTT SP, SynthASil, and SynthAFax cause underestimation of 30%
Rebinyn/Refixia/nonacog beta pegol (Novo Nordisk)	GlycoPEGylated	Chromogenic assays show acceptable recovery Most 1-stage assays significantly over- or underestimate activity and should not be used aPTT reagents SynthAFax and Cephascreen demonstrate acceptable recovery
Idelvion/albutrepenonacog alfa (CSL Behring)	Recombinant human albumin fusion	Certain 1-stage assays significantly over- or underestimate activity (eg, Actin FS, STA-CK Prest, SynthAFax), and WFH recommends against their use aPTT reagents Pathromtin SL, SynthASil, Actin, APTT SP, and STA-PTT-A demonstrate acceptable recovery Chromogenic assays significantly overestimate activity
WFH, World Federation of Hemophilia Sources: Srivastava, 2020; Abraham 2023

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
Five to 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 factor VIII/factor IX is <1 IU/dL, inhibitor testing should be performed.)

In patients with hemophilia A who are receiving emicizumab, inhibitor testing should be performed using a chromogenic factor VIII Bethesda assay with bovine reagents to avoid emicizumab interference.

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.