Accurate diagnosis of haemophilia B (HB) depends upon coagulation assays, which are performed according to strict protocols and procedures, and which require specific knowledge and expertise, as well as quality assurance.1
Coagulation screening, including prothrombin time (PT) or activated partial thromboplastin time (aPTT) assessment, is performed for any patient with a suspected bleeding disorder.1 If the PT is normal, deficiencies of the extrinsic and common coagulation pathways are excluded. A prolonged aPTT in the presence of a normal PT suggests an intrinsic or common pathway deficiency, or the presence of a coagulation factor inhibitor.
A mixing test is required to differentiate whether the prolonged aPTT is due to a factor deficiency or anticoagulants, including a coagulation factor inhibitor.1 If the aPTT is normalised by the presence of the full complement of coagulation factors from the normal plasma in the mixing test, a factor deficiency is more likely than the presence of inhibitors. Factor activity assays are performed to provide a differential diagnosis of the clotting factor deficiency.1
Algorithm for the laboratory diagnosis of haemophilia B.
Activated partial thromboplastin time (aPTT)2,3
The aPTT measures the functionality of the intrinsic (or contact-activation) and common coagulation cascade pathways.2 The measured clotting time is compared to that of control plasma samples to determine whether there is a delay in clotting. The clot formation time is proportional to the extent of factor deficiency or inhibition.
Normal aPTT values depend on the individual laboratory and the reagents used, but are typically between 22 and 40 seconds.2 Inter-laboratory results may vary depending on the laboratory method, instrumentation and reagents used.2,4
Results from PT and aPTT screening tests can be interpreted in combination to identify and exclude certain disorders. A normal PT excludes deficiencies of the extrinsic and common coagulation pathways. A prolonged aPTT in the presence of a normal PT indicates a deficiency of the intrinsic coagulation pathway (i.e., FVIII, FIX, FXI or FXII), severe von Willebrand's disease, the presence of a lupus anticoagulant or factor inhibitors.
Mixing tests can be used to distinguish between factor deficiencies and the presence of an inhibitor. Test plasma is mixed 1:1 with normal plasma (containing 100% of the normal FIX levels) and incubated at 37oC for 1–2 hours. A normalised aPTT is indicative of a clotting factor deficiency. A prolonged aPTT indicates the presence of an inhibitor.
One-stage aPTT-based clotting assays
The one-stage aPTT-based clotting assay is derived from the aPTT and is performed to quantify FIX activity to classify disease severity or monitor treatment.5,6 Test plasma is mixed with a specific factor-deficient plasma (containing normal levels of all clotting factors, but deficient in the factor of interest), and it is assumed that the deficient factor in the test plasma is the rate-limiting determinant of clotting time.
The detection of reduced FIX activity in patients with mild HB may be limited with the one-stage clotting assay, even when the most sensitive assay reagent is used.7,8 A chromogenic factor activity assay should therefore be performed if normal factor activity is reported for a patient with bleeding symptoms based on one-stage clotting assay analysis.
Chromogenic factor activity assays
The two-stage chromogenic assay can also be used to measure FIX activity to classify disease severity or monitor treatment.6,9 In the first step, test plasma is mixed with co-factors, phospholipids, calcium, prothrombin or thrombin and FX. The amount of FXa produced is proportional to the level of FIX present. In the second step, a FXa-specific chromogenic substrate is added and FXa concentration is quantified by photometric monitoring of the cleaved substrate.
Currently, chromogenic FIX activity assays are CE-marked in Europe, but are available for Research Use Only (RUO) in the USA.
The World Federation of Hemophilia (WFH) recommend the use of one-stage clotting assays for the diagnosis of HB.10
Clotting factor monitoring during treatment comprises the assessment of pre- and post-infusion factor activity levels. If recovery is lower than expected, the patient may have developed a FIX inhibitor. The Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation recommends FIX chromogenic assays for treatment monitoring, when these assays have been approved by the US Food and Drug Administration (FDA).11
The use of clinical pharmacokinetics for optimising therapy is well established for patients with haemophilia.12
Monitoring during surgery
HB patients are treated and factor activity levels monitored closely during the perioperative period to ensure that factor activity is sufficient to avoid uncontrolled bleeding or rebleeding.
1. World Federation of Hemophilia (WFH): Guidelines for the Management of Hemophilia; 2012.
2. Bates SM, Weitz JI. Coagulation assays. Circulation 2005;112:e53-60.
3. Langdell RD, Wagner RH, Brinkhous KM. Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. J Lab Clin Med 1953;41:637-47.
4. Kitchen S, Kershaw G, Tiefenbacher S. Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues. Haemophilia 2016;22 Suppl 5:72-7.
5. Ingerslev I. Laboratory assays in hemophilia. In: Textbook of Hemophilia. Chichester, UK: Blackwell Publishing Ltd; 2010.
6. Kitchen S, Preston E. Assay of factor VIII and other clotting factors In: Kitchen S, Olson J, Preston E, eds. Quality in Laboratory Hemostasis and Thrombosis: Wiley-Blackwell; 2013.
7. Park CH, Seo JY, Kim HJ, Jang JH, Kim SH. A diagnostic challenge: mild hemophilia B with normal activated partial thromboplastin time. Blood Coagul Fibrinolysis 2010;21:368-71.
8. Pouplard C, Trossaert M, A LEQ, Delahousse B, Giraudeau B, Gruel Y. Influence of source of phospholipids for APTT-based factor IX assays and potential consequences for the diagnosis of mild haemophilia B. Haemophilia 2009;15:365-8.
9. Wilmot HV, Hogwood J, Gray E. Recombinant factor IX: discrepancies between one-stage clotting and chromogenic assays. Haemophilia 2014;20:891-7.
10. Kitchen S, McCraw A, Echenagucia M. Diagnosis of hemophilia and other bleeding disorders. A laboratory manual. Montreal, Canada: World Federation of Hemophilia; 2010.
11. MASAC statement regarding use of various clotting factor assays to monitor factor replacement therapy. National Hemophilia Foundation MASAC Document #228 2014: http://www.hemophilia.org/sites/default/files/document/files/masac-228.pdf last accessed March 16, 2015.
12. Bjorkman S, Berntorp E. Pharmacokinetics of coagulation factors: clinical relevance for patients with haemophilia. Clin Pharmacokinet 2001;40:815-32.