Tests
1st tests to order
activated partial thromboplastin time (aPTT)
Test
Factor VIII and/or factor IX assay should be requested to confirm the diagnosis if the aPTT is prolonged and corrects with mixing.[38][61]
If the diagnosis is clinically suspected, even if the aPTT is not prolonged, factor VIII and IX assays should be ordered.
Result
usually prolonged; may not be prolonged in mild cases (factor levels >30%); aPTT will be spuriously shortened in patients with hemophilia A receiving emicizumab prophylaxis, and should not be used for monitoring (except in the rare case when the development of anti-emicizumab is suspected [aPTT will be inappropriately prolonged when the effect of emicizumab is absent]); the effect of emicizumab on shortening the aPTT will persist for up to 6 months after discontinuation
plasma factor VIII and IX assay
Test
Levels of factor VIII or IX are used to establish diagnosis and severity.
A one-stage or two-stage clotting assay is used.
A one-stage method is more commonly used as it is simple and readily automated.
A two-stage method is more difficult to perform.
Another technique is the chromogenic substrate assay, which is based upon factor VIII-dependent factor X activation (for factor VIII assay) and factor IX-dependent factor X activation (for factor IX assay). The chromogenic assay is similar to the two-stage clotting assay.
The bleeding phenotype usually correlates directly with factor VIII or IX level. However, some cases of mild hemophilia A may have discrepant factor VIII levels when assessed by the one-stage and chromogenic assays.[38][61] Correlation of the factor VIII level by these assays with the bleeding phenotype is important.
In acquired hemophilia, factor VIII levels are low.
Result
decreased or absent factor VIII or IX levels; severity is based on level of factor present; severe: <0.01 international units/mL, moderate: 0.01 to 0.05 international units/mL, mild: >0.05 but <0.40 international units/mL; in patients with hemophilia A receiving emicizumab prophylaxis, assessment of factor VIII activity should be performed by chromogenic assay using bovine reagents; the effect of emicizumab on aPTT-based assay will persist for up to 6 months after discontinuation
mixing study
Test
A mixing study (incubating patient plasma with normal plasma for 2 hours at 98.6°F [37°C] and repeating aPTT) is requested if the aPTT is prolonged.[38][61]
Correction of aPTT with mixing study suggests coagulation factor deficiency, while the lack of correction suggests the presence of a coagulation inhibitor.
In patients with factor VIII inhibitor antibodies (in congenital or acquired hemophilia), prolonged aPTT cannot be corrected by incubating patient plasma with normal plasma for 2 hours at 98.6°F (37°C) due to the presence of time- and temperature-dependent factor VIII inhibitor.
Result
aPTT corrected; in patients with hemophilia A receiving emicizumab prophylaxis, aPTT will be spuriously shortened so that the mixing study will be uninterpretable; the effect of emicizumab on aPTT will persist for up to 6 months after discontinuation
CBC
Test
Performed to rule out thrombocytopenia as a cause of bleeding.
Anemia may be present if there has been significant bleeding.
Result
usually normal; low Hb if bleeding has been severe or prolonged
prothrombin time (PT)
Test
Performed to evaluate the extrinsic and common pathways of coagulation.
Result
normal
plasma von Willebrand factor assay
Test
Performed to rule out von Willebrand disease.
Result
normal
plasma factor V, VII assay
Test
If the PT is prolonged, factor VII assay should be checked; factor V assay should also be checked to rule out combined inherited factors V and VIII deficiency (autosomal recessive).
Result
normal
plasma factor XI, XII assay
Test
If aPTT is prolonged, but factors VIII and IX are normal, then factors XII and XI assay should be checked.
Result
normal
closure time/bleeding time and platelet aggregation studies
Test
Performed to evaluate platelet function.
Result
normal
serum liver aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT])
Test
Performed as part of the initial workup because liver dysfunction can contribute to prolongation of PT and aPTT.
Result
normal
plain x-rays of specific bony sites
Test
X-rays have been traditionally used to describe the clinical progression of arthropathy.[33]
MRI and ultrasound may detect soft-tissue bleeding and its consequences at an early stage.
MRI may be helpful to evaluate whether a patient is a candidate for surgical procedures including synovectomy, joint fusion, or joint replacement.
Result
may demonstrate findings of acute joint bleeding (hemarthrosis) or bone changes more consistent with chronic arthropathy
prenatal factor VIII or IX mutation analysis by amniocentesis or chorionic villus sampling (CVS)
Test
Women who are carriers and are at risk of having a child with hemophilia may seek prenatal diagnosis.
Presence of fetal DNA in maternal blood can be detected as early as week 4 to 5 gestation, allowing male sex determination. Sex can also be determined by ultrasound beginning week 11 to 13 gestation.
DNA-based analysis of the X chromosome to identify a specific genetic mutation may be performed on amniocentesis or chorionic villus samples (CVS). Amniocentesis is usually done after week 15 of pregnancy, whereas CVS can be done between weeks 10 and 14. Limb abnormality has been associated with CVS taken before 10 weeks' gestation, and the mother should be counseled about the small risk of spontaneous abortion with both CVS and amniocentesis. If the mutation is known, direct mutation analysis can be performed. If the mutation is not already known, gene sequencing can be done to determine whether a mutation is present. Pre-implantation genetic diagnosis on early embryos is also possible.[38][59]
Result
specific genetic mutation identified
Tests to consider
head or neck CT
Test
May be required for evaluation of acute bleeding (e.g., for evaluation of suspected intracranial hemorrhage, or of bleeding near the airway).
Result
may demonstrate acute bleeding
head or neck MRI
Test
May be required for evaluation of acute bleeding (e.g., for evaluation of suspected intracranial hemorrhage, or of bleeding near the airway).
Result
may demonstrate acute bleeding
abdominal ultrasound or abdominopelvic CT scan
Test
May be required for evaluation of acute intra-abdominal bleeding or if iliopsoas muscle bleed is suspected.
Result
may demonstrate acute intra-abdominal bleeding
esophagogastroduodenoscopy or colonoscopy
Test
May be required for evaluation of acute upper or lower gastrointestinal bleed.
Result
may demonstrate acute gastrointestinal bleeding
blood factor VIII or IX mutation analysis
Test
DNA-based analysis of the factor VIII or IX genes to identify a specific genetic mutation.
To establish diagnostic accuracy, clinical severity, and risk of inhibitor development.
Recommended for patients with a diagnosis of hemophilia A or B, affected male relatives and female relatives at risk of being carriers. Genetic counseling should be provided before and after testing.[34]
Hemophilia B patients with genetic mutations resulting from deletions need careful observation, as these patients may develop anaphylaxis and/or nephrotic syndrome when factor IX is infused.[36] The allergic reaction to factor IX generally indicates that the patient is developing or has developed a factor IX inhibitor.
Mutation analysis should be carried out at specialized centers, experienced in hemophilia genetic testing.[34] If there are existing genetic test results, do not order a duplicate test unless there is uncertainty about the existing result, e.g., the result is inconsistent with the patient’s clinical presentation or the test methodology has changed.[35]
Result
specific genetic mutation identified
plasma factor VIII or IX inhibitor screen
Test
Comparison of factor VIII or factor IX activity in the patient and control mixtures demonstrates either the absence or presence of an inhibitor.
A screening test should be performed every 3 to 12 months, or sooner if clinical picture suggests inhibitor development.
In acquired hemophilia, factor VIII levels are low, with detectable factor VIII inhibitor.
Result
may be negative or positive
Bethesda assay/modified Bethesda assay (on plasma sample)
Test
Performed if the blood factor VIII or IX inhibitor screen is positive.[38][61]
A Bethesda assay (or one of the modified assays with improved sensitivity, such as the Nijmegen-modified Bethesda assay, or the Centers for Disease Control and Prevention [CDC]-modified Nijmegen-Bethesda assay incorporating preheating of the test plasma at 132.8°F [56°C] for 30 minutes) measures the amount of antibody present in the patient's sample (reported in Bethesda units [BU]).[55][56]
The assay is performed by mixing the patient's plasma with a known amount of factor VIII or factor IX. After a 2-hour incubation period at 98.6°F (37°C), the residual factor VIII or factor IX activity is determined. The CDC-modified Nijmegen-Bethesda assay accurately quantifies inhibitors, even when the patient is receiving factor concentrates, without the need for a washout period.
Inhibitory antibodies are classified as low-titer (<5 BU) or high-titer (>5 BU), based on the amount of inhibitor present.
Inhibitors can also be quantified using the chromogenic substrate Bethesda assay method, which has the advantage of reducing false positivity.[57]
Result
low-responder inhibitor: persistently <5 Bethesda units/mL (BU/mL); high-responder inhibitor: ≥5 BU/mL at any time in the past in the absence of immune tolerance induction; in patients receiving emicizumab, the Bethesda assay should be performed by chromogenic assay using bovine reagents
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