Molina E S, Fujita A, Sogayar M C, Demasi M A
Department of Biochemistry Chemistry Institute, NUCEL-NETCEM, School of Medicine, University of São Paulo, São Paulo, SP, Brazil.
Haemophilia. 2014 Nov;20(6):e392-8. doi: 10.1111/hae.12484. Epub 2014 Jun 26.
The tail bleeding model using haemophilic mice has been used as one of the standard assays for efficacy evaluation of novel antihaemophilic therapies at the preclinical level. A number of different configurations and endpoints have been proposed in the literature for this model, hindering interlaboratory comparisons. A particular configuration, known as the tail bleeding survival assay (TBS), adopted by several groups, involves measuring the ability of conscious haemophilic mice to survive exsanguination following tail transection. Major limitations to this configuration include ethical constraints and impaired quantitative determinations. The aim of this study was to standardize and validate a quantitative haemostatic assay for evaluation of antihaemophilic therapies employing an alternative to TBS, which involves a more humane endpoint associated with stable clot formation. Haemophilic mice were treated with vehicle or different doses of two antihaemophilic reference products licensed in Brazil. The haemostatic response was evaluated by our quantitative tail bleeding haemostatic assay (qTBA) over a period of 120 min and then quantified by dose-response modelling. We demonstrate that our qTBA method allows a direct relationship between the number of animals which achieved full haemostatic response and the dosage of both antihaemophilic factors evaluated over 120 min. In addition, the method sensitivity is suitable to demonstrate the conversion from a severe to a moderate haemophilia phenotype. Our proposed qTBA is easy to implement and constitutes an alternative and more ethical endpoint, which could be effectively used as a surrogate to the commonly employed survival endpoint, allowing quantitative haemostatic response evaluation associated with stable clot formation.
使用血友病小鼠的尾部出血模型已被用作临床前水平评估新型抗血友病疗法疗效的标准试验之一。文献中针对该模型提出了许多不同的配置和终点，这妨碍了实验室间的比较。几个研究小组采用的一种特定配置，即尾部出血存活试验（TBS），涉及测量清醒的血友病小鼠在尾部横断后失血性休克存活的能力。这种配置的主要局限性包括伦理限制和定量测定受损。本研究的目的是标准化并验证一种定量止血试验，以评估抗血友病疗法，该试验采用TBS的替代方法，其涉及与稳定血栓形成相关的更人道的终点。用载体或巴西许可的两种抗血友病参考产品的不同剂量治疗血友病小鼠。通过我们的定量尾部出血止血试验（qTBA）在120分钟内评估止血反应，然后通过剂量反应模型进行量化。我们证明，我们的qTBA方法允许在120分钟内实现完全止血反应的动物数量与所评估的两种抗血友病因子的剂量之间建立直接关系。此外，该方法的灵敏度适用于证明从严重血友病表型向中度血友病表型的转变。我们提出的qTBA易于实施，构成了一种替代的、更符合伦理的终点，可有效地用作常用存活终点的替代指标，允许评估与稳定血栓形成相关的定量止血反应。
