Initial testing for a coagulation disorder involves a CBC with platelet count, PT and PTT, and fibrinogen tests. Show Interpretation of Initial TestsHemophilia A or Ba,bVWDPlatelet DefectPlatelet countNormalNormal or reducedNormal or reducedPTNormalNormalNormalPTTProlongedcNormal or prolongedcNormalaThe 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 StudiesA 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 AssaysFactor 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 AssaysConsider 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 AssaySince 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 AssaysThe 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 TestingIn 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 TestingIn 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. MonitoringTrough-Level TestingTrough 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 TestingIndividuals 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 :
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 ScreeningScreening 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. ARUP Laboratory Tests0030215 Prothrombin Time 0030215 Method Electromagnetic Mechanical Clot Detection 0030235 Partial Thromboplastin Time 0030235 Method Electromagnetic Mechanical Clot Detection 0030130 Fibrinogen 0030130 Method Electromagnetic Mechanical Clot Detection 2014318 Prolonged Clot Time Reflex Panel 2014318 Method Electromagnetic Mechanical Clot Detection/Qualitative Hemagglutination/Platelet Agglutination/Microlatex Particle-Mediated Immunoassay  Factor Assays 0030095 Factor VIII, Activity 0030095 Method Electromagnetic Mechanical Clot Detection 3002343 Chromogenic Factor VIII, Activity 3002343 Method Chromogenic Assay 0030026 Factor VIII Activity with Reflex to Bethesda Quantitative, Factor VIII 0030026 Method Electromagnetic Mechanical Clot Detection 0030125 von Willebrand Panel 0030125 Method Electromagnetic Mechanical Clot Detection/Platelet Agglutination/Microlatex Particle-Mediated Immunoassay 0030100 Factor IX, Activity 0030100 Method Electromagnetic Mechanical Clot Detection 0030032 Factor IX Activity with Reflex to Bethesda Quantitative, Factor IX 0030032 Method Electromagnetic Mechanical Clot Detection Preferred Tests to Detect Causal Gene Variant For mild to moderate disease 3004241 Hemophilia A (F8) Sequencing 3004241 Method Massively Parallel Sequencing For severe disease 3004232 Hemophilia A (F8) 2 Inversions with Reflex to Sequencing and Reflex to Deletion/Duplication 3004232 Method Inverse Polymerase Chain Reaction/Massively Parallel Sequencing/Multiplex Ligation-dependent Probe Amplification
Acceptable Test to Detect Causal Gene Variant For severe disease 2001759 Hemophilia A (F8) 2 Inversions 2001759 Method Inverse Polymerase Chain Reaction/Electrophoresis Related Tests0030260 Thrombin Time with Reflex to Thrombin Time 1:1 Mix 0030260 Method Electromagnetic Mechanical Clot Detection 0030110 Factor XI, Activity 0030110 Method Electromagnetic Mechanical Clot Detection References
Additional Resources Boylan B, Rice AS, Neff AT, et al. Survey of the anti-factor IX immunoglobulin profiles in patients with hemophilia B using a fluorescence-based immunoassay. J Thromb Haemost. 2016;14(10):1931-1940. de Brasi C, El-Maarri O, Perry DJ , et al. Genetic testing in bleeding disorders. Haemophilia. 2014;20 Suppl 4(0 4):54-58. Graf L. Extended Half-Life Factor VIII and Factor IX Preparations. Transfus Med Hemother. 2018;45(2):86-91. Kitchen S, Tiefenbacher S, Gosselin R. Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies. Semin Thromb Hemost. 2017;43(3):331-337. MASAC Statement Regarding Use of Various Clotting Factor Assays to Monitor Factor Replacement Therapy. National Hemophilia Foundation. [Issued: Jun 2014; Accessed: Feb 2020] Müller J, Pekrul I, Pötzsch B, et al. Laboratory monitoring in emicizumab-treated persons with hemophilia A. Thromb Haemost. 2019;119(9):1384-1393. St Ledger K, Feussner A, Kalina U, et al. International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories. J Thromb Haemost. 2018;16(3):555-564. Related Information From ARUP LaboratoriesTopics From ARUP ConsultFunctional Platelet Disorders Prolonged Clotting Time Evaluation Uncommon Factor Deficiencies Von Willebrand Disease - VWD Test Fact Sheets From ARUP ConsultChromogenic Factor VIII, Activity Prolonged Clot Time Reflex Panel Selected Scholarly Publications From ARUP LaboratoriesHeikal NM, Murphy KK, Crist RA , et al. Elevated factor IX activity is associated with an increased odds ratio for both arterial and venous thrombotic events. Am J Clin Pathol. 2013;140(5):680-685. Nance D, Rodgers GM. Switching haemophilia products and inhibitor risk: a United States' perspective. Eur J Haematol. 2015;94(4):283. Rodgers GM. Prothrombin complex concentrates in emergency bleeding disorders. Am J Hematol. 2012;87(9):898-902. Sborov DW, Rodgers GM. How I manage patients with acquired haemophilia A. Br J Haematol. 2013;161(2):157-165. |
