Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay.

INTRODUCTION: Antithrombin resistance (ATR) is a novel thrombotic risk in abnormal prothrombins. A manual ATR assay using Oxyuranus scutellatus (Ox) venom as a prothrombin activator was established for detecting antithrombin-resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the ATR assay using bovine factors Xa and Va (FXa/Va) as prothrombin activators and have optimised assay conditions for an automated instrument (ACL TOP 500). METHODS: Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl2 , and bovine FXa/Va), followed by inactivation with antithrombin for 10, 20 and 30 minutes. We added a chromogenic substrate S-2238, and assessed changes in absorbance/min at 405 nm. We also adapted assay conditions for ACL TOP 500. RESULTS: Optimum conditions for FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 µg/mL FXa and 0.1 µg/mL FVa. ATR assay kinetics with the FXa/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild-type or antithrombin-resistant prothrombin. Using ACL TOP 500, optimum conditions for the FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 µg/mL FXa and 0.2 µg/mL FVa. The automated ATR assay with the FXa/Va activator demonstrated good detectability for antithrombin-resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade. CONCLUSION: We optimised the ATR assay with the FXa/Va activator and adapted the assay for ACL TOP 500; the assay showed the ability to clearly detect antithrombin-resistant prothrombin in manual and automated procedures.

Double muscle transfer for upper extremity reconstruction following complete avulsion of the brachial plexus.

Recent interest in reconstruction of the upper limb following brachial plexus injuries has focused on the restoration of prehension following complete avulsion of the brachial plexus. The authors use free muscle transfers for reconstruction of the upper limb to resolve the difficult problems in complete avulsion of the brachial plexus. This article describes the authors' updated technique--the double free muscle procedure. Reconstruction of prehension to achieve independent voluntary finger and elbow flexion and extension by the use of double free muscle and multiple nerve transfers following complete avulsion of the brachial plexus (nerve roots C5 to T1) is presented. The procedure involves transferring the first free muscle, neurotized by the spinal accessory nerve for elbow flexion and finger extension, a second free muscle transfer reinnervated by the fifth and sixth intercostal nerves for finger flexion, and neurotization of the triceps brachii via its motor nerve by the third and fourth intercostal motor nerves to extend and stabilize the elbow. Restoration of hand sensibility is obtained via the suturing of sensory rami from the intercostal nerves to the median nerve. Secondary reconstruction, including arthrodesis of the carpometacarpal joint of the thumb and glenohumeral joint, and tenolysis of the transferred muscle and distal tendons, improve the functional outcome. Based on the long-term result, selection of the patient, donor muscle, and donor motor nerve were indicated. Most patients were able to achieve prehensile functions such as holding a can and lifting a heavy box. This double free muscle transfer has provided prehension for patients with complete avulsion of the brachial plexus and has given them new hope to be able to use their otherwise useless limbs.