Stanley R L, Maile C, Piercy R J
Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Haffield, Hertfordshire AL9 7TA, UK.
Equine Vet J. 2009 Jan;41(1):82-6. doi: 10.2746/042516408x330374.
Muscle biopsy is increasingly used in equine veterinary practice for investigating exertional, inflammatory or immune mediated myopathies and unexplained muscle atrophy. Although formalin-fixed samples are often used, for complete evaluation, fresh-frozen tissue is required. Freezing muscle in veterinary practice is impractical: samples sent to specialist laboratories for processing are therefore susceptible to delays, potentially leading to artefact and compromising histological interpretation.
Altered temperature, duration and hydration status influence the severity of storage-induced artefact in equine muscle.
Skeletal muscle obtained immediately post euthanasia was divided into 6 independent samples from each of 8 horses. One sample per horse was frozen immediately in isopentane precooled in liquid nitrogen. Additional samples were stored in conditions designed to mimic possible situations encountered in practice, including increased storage times, temperature and hydration status. Following storage, stored samples were frozen as before. Cryosections were stained using haematoxylin and eosin and ranked for artefact on 2 occasions by 2 blinded observers. The best samples were processed subsequently with a panel of routine stains and immunolabelled for collagen V to enable the measurement of minimum fibre diameters.
Both prolonged storage and increased hydration resulted in more storage-associated artefact. Samples stored for 24 h chilled on dry gauze were ranked higher than those stored on damp gauze; however, a panel of routinely-used histochemical staining techniques was unaffected by chilled 24 h storage. There was no significant effect of storage on mean fibre diameter; however, both chilled dry and damp storage for 24 h caused a significant increase in fibre-size variability.
Caution should be exercised when interpreting fibre size profiles in shipped samples. Equine muscle biopsy samples are optimally shipped in dry gauze, sealed in plastic containers and shipped on ice packs to be processed within 24 h and can thus be interpreted by the receiving laboratory with minimal artefact.
肌肉活检在马兽医实践中越来越多地用于调查运动性、炎症性或免疫介导性肌病以及不明原因的肌肉萎缩。尽管经常使用福尔马林固定的样本，但为了进行全面评估，需要新鲜冷冻组织。在兽医实践中冷冻肌肉不切实际：因此，送往专业实验室处理的样本容易出现延迟，这可能导致人为假象并影响组织学解释。
温度、时长和水合状态的改变会影响马肌肉中储存诱导的人为假象的严重程度。
对8匹马在安乐死后立即获取的骨骼肌进行分割，每匹马分为6个独立样本。每匹马的一个样本立即在液氮预冷的异戊烷中冷冻。其他样本则在模拟实践中可能遇到的情况的条件下储存，包括延长储存时间、改变温度和水合状态。储存后，将储存的样本像之前一样冷冻。冰冻切片用苏木精和伊红染色，并由两名不知情的观察者分两次对人为假象进行评分。随后对最佳样本进行一组常规染色，并对V型胶原进行免疫标记，以测量最小纤维直径。
储存时间延长和水合增加均导致更多与储存相关的人为假象。在干纱布上冷藏24小时的样本评分高于在湿纱布上储存的样本；然而，一组常用的组织化学染色技术不受冷藏24小时储存的影响。储存对平均纤维直径没有显著影响；然而，冷藏干燥和潮湿储存24小时均导致纤维大小变异性显著增加。
在解释运输样本中的纤维大小分布时应谨慎。马肌肉活检样本最好用干纱布包装，密封在塑料容器中，并与冰袋一起运输，以便在24小时内进行处理，这样接收实验室就可以在人为假象最少的情况下进行解读。
