Hemophilia is due to a deficiency of coagulation factor VIII (hemophilia A) or coagulation factor IX (hemophilia B).1 One other type, hemophilia C, which is very rare, is caused by a deficiency of clotting factor XI.2 A meticulous diagnostic workup is necessary for the proper management of hemophilia, and comprehensive laboratory testing is required. According to the World Federation of Hemophilia Guidelines for the Management of Hemophilia, an accurate diagnosis is based on protocols and procedures conducted by laboratories with expertise in coagulation tests, appropriate equipment and reagents, and quality assurance protocols.1 The diagnosis of hemophilia involves screening tests and clotting factor tests. Screening tests evaluate blood clotting. Clotting factor tests, also known as factor assays, are essential for typing hemophilia and determining its severity.3

Basic Screening Tests for Hemophilia 

Complete Blood Cell (CBC) Count

Although the CBC count can be normal in hemophilia, in cases of abnormally heavy and prolonged bleeding, the hemoglobin level and red blood cell count may fall.3

Activated Partial Thromboplastin Time (aPTT)

The aPTT measures the clotting ability of factors VIII, IX, XI, and XII. When the values for any of these factors are low, clotting is prolonged. The aPTT is prolonged in individuals with hemophilia A or B.3

Prothrombin Time (PT)

The PT measures the clotting ability of factors I, II, V, VII, and X. Clotting is prolonged when the values for any of these factors are low. The PT is normal in individuals with hemophilia A or B.3

Fibrinogen Test

This test also measures the clotting ability of blood. It is recommended that a fibrinogen test be performed along with other blood clotting tests and when the PT or aPTT is abnormal.3

Confirmatory Test: Functional Coagulation Assay

To confirm a diagnosis of hemophilia and monitor treatment, factor VIII or IX deficiency is determined with a functional coagulation assay. This test also helps to rule out other entities in the differential diagnosis, such as von Willebrand disease. Additionally, coagulation assays are performed to assess disease severity, to determine a patient’s coagulation status preoperatively, and to optimize doses prophylactically whenever a reduction in factor activity during adequate treatment suggests inhibitor development.1

In factor VIII4 and factor IX5 assays, activity is compared with the normal pooled plasma standard, which has 100% activity or the equivalent of factor VIII or factor IX IU/mL respectively. Normal values are between 50% and 150%. Values are between 5% and 50%, between 1% and 5%, and below 1% in mild, moderate, and severe cases, respectively, of hemophilia A or B.4,5

The 3 main factor VIII assays in use are 1-stage and 2-stage clotting assays and 2-stage chromogenic assays. Approximately one-third of mild cases of hemophilia A show significantly higher values on the automated 1-stage factor VIII assay than on the 2-stage 

chromogenic assay.6,7

A specific assay to evaluate factor XI activity is required to confirm a diagnosis of hemophilia C.8

Imaging Studies for Acute Bleeds

After the start of coagulation therapy, imaging is required immediately even when hemorrhage is not strongly suspected. The choice of imaging technique is based on the degree of clinical suspicion and the anatomical site of involvement.4,5

Computed tomography of the head without contrast is useful to assess traumatic or spontaneous intracranial hemorrhage. Magnetic resonance imaging of the head and spinal column is also advised for the further assessment of traumatic or spontaneous hemorrhage. Magnetic resonance imaging is also important for the evaluation of cartilage, synovium, and joint spaces. Ultrasonography is an important imaging modality for the assessment of joints with acute or chronic effusions. However, it is not useful for the evaluation of bone or cartilage. Special studies like angiography and nucleotide bleeding scans are sometimes clinically indicated.4,5 

Imaging modalities are not required for a diagnosis of factor XI deficiency. However, imaging techniques can be used to evaluate the extent of bleeding for the management of bleeding in any location.8

Tests for Inhibitors

Laboratory tests to confirm the presence of a factor VIII4 or IX5 inhibitor become clinically significant when episodes of  uncontrolled bleeding occur despite the infusion of sufficient amounts of factor concentrate. For this assay, measurement of the aPTT is repeated after the patient’s plasma has been incubated with normal plasma at 37°C for 1 to 2 hours. If a prolonged aPTT remains uncorrected, the inhibitor titer is measured with the Bethesda method.4 In ideal circumstances, the Nijmegen modification of the Bethesda inhibitor assay is used to detect an inhibitor in case of a positive mixing test result.9 Conventionally, a value higher than 0.6 Bethesda unit (BU) is considered positive for the presence of an inhibitor. A value below 5 BU is a low inhibitor titer, and any value higher than 5 BU is considered a high inhibitor titer. The distinction is of clinical significance because patients with low inhibitor titers may respond to increased doses of factor VIII concentrate,4,5 whereas those with high inhibitor titers require treatment with agents that bypass factor VIII, and consideration of the induction of immune tolerance.4

Carrier Testing and Fetal Testing

A patient’s carrier status is screened by determining the ratio of factor VIII coagulant activity to the concentration of von Willebrand factor antigen. A ratio below 0.7 indicates carrier status.4

More precise results are obtained with direct testing for known gene mutations. Linkage analysis with restriction fragment length polymorphism (RFLP) in multiple family members may be used. Many laboratories perform direct mutation analysis for unknown factor VIII mutations. Inversion of the F8 gene can be detected with Southern blot.4

For prenatal testing, carriers with identifiable mutations may undergo chorionic villous sampling at approximately 10 to 12 weeks’ gestation or amniocentesis at 16 to 20 weeks’ gestation to obtain fetal cells for DNA analysis or linkage studies. When DNA analysis is not possible, fetal blood procured by means of fetoscopy at approximately 20 weeks’ gestation may be assayed for the factor VIII level.4

All these procedures carry risks varying from a low of 0.5% for maternal and/or fetal complications to a high of 1% to 6% for fetal death from fetoscopy. Such procedures must be undertaken only after patients have received complete genetic and obstetric counseling. Genetic counseling is ideally provided before a woman becomes pregnant and helps couples to make informed decisions before conception.4

Studies in the literature have described noninvasive prenatal diagnostic testing with a quantitative digital polymerase chain reaction assay of free fetal DNA in the maternal circulation. However, this method is still considered a research tool.10

In the case of a female fetus, pregnancy can be taken to term because it is unusual for carriers to have bleeding problems. If the fetus is a severely affected male, the couple must decide whether to continue the pregnancy to term. If the pregnancy is taken to term, a prenatal diagnosis makes it possible to formulate a delivery plan that will reduce the risk for intracranial hemorrhage, including avoidance of the use of vacuum devices.10

The factor IX level is usually normal in carriers of an F9 mutation. If a specific F9 mutation is known, accurate results can be obtained with direct genetic testing by RFLP.5 For fetal testing in the case of a known mutation, RFLP may be done on chorionic villous or amniocentesis samples. In the case of an unknown mutation, gene sequencing may be done.4 Genetic analysis for F9 mutations is useful to identify which mutation is causing the deficiency.8

References

  1. Peyvandi F, Kenet G, Pekrul I, Pruthi RK, Ramge P, Spannagl M. Laboratory testing in hemophilia: impact of factor and non-factor replacement therapy on coagulation assays. J Thromb Haemost. 2020;18(6):1242-1255. doi:10.1111/jth.14784
  2. Mehta P, Reddivari AKR. Hemophilia. StatPearls. Accessed July 31, 2021.
  3. Diagnosis of hemophilia. Centers for Disease Control and Prevention. Updated 17 July 2020. Accessed July 31, 2021.
  4. Drelich DA. Hemophilia A workup: approach considerations, Testing for inhibitors, carrier testing and fetal testing, Medscape. Updated June 5, 2020. Accessed July 31, 2021.
  5. Zaiden RA. Hemophilia B workup: approach considerations, testing for inhibitors, carrier testing and fetal testing, Medscape. Updated October 2, 2020. Accessed July 31, 2021.
  6. Duncan EM, Rodgers SE, McRae SJ. Diagnostic testing for mild hemophilia A in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays. Semin Thromb Hemost. 2013;39(3):272-282. doi:10.1055/s-0033-1334863
  7. Armstrong E, Hillarp A. Assay discrepancy in mild haemophilia A. Eur J Haematol Suppl. 2014;76:48-50. doi:10.1111/ejh.12374
  8. Gupta V. Hemophilia C workup: approach considerations, Medscape. Updated December 18, 2020. Accessed July 31, 2021.
  9. Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M, Mauser-Bunschoten E. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. 1995;73(2):247-251.
  10. Abdul-Kadir R, Davies J, Halimeh S, Chi C. Advances in pregnancy management in carriers of hemophilia. J Appl Hematol. 2013 Oct 1;4(4):125.

Reviewed by Debjyoti Talukdar, MD, on 8/11/2021.