The purpose of this study was to determine the detection threshold of two clotting tests to minute quantities of factor (F) IX in the canine hemophilia B system: the whole blood clotting time (WBCT) and the activated partial thromboplastin time (aPTT). To our knowledge, the relative sensitivity of these tests for minute amounts of canine FIX has not been previously published. As these tests are relatively easy and quick to perform, they are useful and important for determining when infused FIX has been cleared, correctly identifying onset and estimating level of transgene expression in gene transfer experiments, and detecting inhibitory antibodies to FIX.

The WBCT was sensitive to as little as 0.01% FIX (Fig. 1A). The estimated level of canine FIX in normal dogs is 5 μg mL−1, thus 0.01% is estimated to be 0.5 ng FIX per mL plasma [1]. The sensitivity of the aPTT to canine FIX was 0.1%, estimated to be 5 ng mL−1 (Fig. 1B). As no activator is used with the WBCT, these data suggest coagulation assays are more sensitive to canine FIX with less activation of the coagulation system. Transfused canine FIX shortened the aPTT for an average of 12 days (range 8–17 days) and the WBCT for an average of 22 days (range 14–27 days). This difference is most likely due to the differences in sensitivity of these two assays. These data provide a reference time-point after which transfused FIX that has not yet been cleared can be distinguished from expressed FIX resulting from successful gene transfer experiments.


Figure 1.  (A). Effect of canine factor (F) IX on the WBCT in hemophilia B dog blood. The addition of 0.01% canine FIX significantly shortened the WBCT (heavy line) from baseline (*P = 0.039). (B) Effect of canine FIX on the activated partial thromboplastin time in hemophilia B dog plasma. The addition of 0.1% canine FIX significantly shortened the aPTT (heavy line) from baseline (*P = 0.0116). (C) Effect of transfused canine FIX on WBCT in a hemophilia B dog. This dog received 160 U FIX per kg over 3 days to provide hemostasis for a partial hepatectomy performed as a control for a gene therapy protocol (i.e. no gene therapy was given) [21]. The WBCT takes approximately 27 days to return to baseline > 60 min. (Range of values for hemophilia B and normal dogs is indicated by the dotted lines. Linear regression lines [light lines] are included on Panels A and B.)

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A test for the delayed clotting of whole blood in severe hemophilia was first described by Wright [2] over a century ago. The original directions of Lee and White [3] in 1913 for the WBCT are now obsolete, and many modifications have been made, including the use of siliconized test tubes to prevent activation of the clotting system [4]. Careful collection of blood samples is essential if consistency in the WBCT values is to be obtained with replicate testing [5]. In contrast to other tests, the WBCT is relatively insensitive to plasma FIX levels above about 3–5% of normal (Fig. 1A). Hence patients with moderate or mild hemophilia may have a normal WBCT even during bleeding episodes [6]. While quite useful in preclinical animal studies, the WBCT tests are infrequently used in diagnosis and monitoring of therapy in humans. The advent of gene therapy in canine hemophilia B has indicated the need for readily available clotting tests that are sensitive to low levels of plasma FIX activity and can be easily repeated over years of follow-up [7]. Additionally, sudden prolongation of the WBCT has been a harbinger of inhibitory antibodies to canine FIX [8].

The hemophilia B dogs require frequent plasma FIX therapy to treat spontaneous bleeding [9,10] and, following invasive procedures, to prevent hemorrhage and facilitate wound healing [11]. Residual plasma FIX post-transfusion was detectable by aPTT for 8–17 days and the WBCT for 14–27 days (Fig. 1C). This late phase of clearance may indicate a change in kinetics of elimination of FIX, influenced perhaps by a slow return to the blood stream of procoagulants. This is especially relevant to FIX that is known to bind to type IV collagen via lysine at position 5 or valine at position 10 [12–15]. Notably, FIX variants mutated at these sites have increased circulating levels of FIX [16]. The animal that required the longest period of time (27 days, Fig. 1C) for the WBCT to return to basal values had a partial hepatectomy and received the largest dose of FIX replacement therapy, suggesting but not proving that dose of FIX may be a factor in the late phase of elimination [17].

Other canine FIX activity assays include FIX bioassay by two-stage [18] and chromogenic methods [19]. In general, these assays are used to detect FIX levels of 1% or greater and are difficult to reliably standardize for levels lower than 1%. A canine FIX ELISA has also been established for determining protein levels [1,20,21]. All three of these assays are quantitative and require considerable laboratory time and expertise. In contrast, the WBCT and the aPTT, while qualitative, can be performed rapidly and are quite sensitive to small amounts of canine FIX that can give an early readout on how to interpret gene transfer and protein replacement experiments for inhibitors.

Disclosure of Conflict of Interests

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information

The authors state that they have no conflict of interest.


  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information
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Supporting Information

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information

Data S1. Supporting information includes details of methods, analyses, and additional results not included in print.

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