【作者】 白晓；

【导师】 蒲晓允；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 目的:为适应高科技局部战争条件下对战地伤员(休克、出血、感染、多器官衰竭等重伤比例相应的增加)的救治保障需要,满足野战环境下急救设备便携、机动的需求,拟研制一种基于低电压启动的微型光谱仪进行检测,并可在野战条件下使用的生化检测仪器及配套试剂。其检测的指标包括血红蛋白、葡萄糖及血钾、钠、氯、二氧化碳,并将这几项指标组合在一起,形成急救生化的功能检测单元,通过一次检测获得6项检测结果。本研究重点在于对上述6种检测试剂进行方法改良与改进,使之能适用于微型光谱仪的检测要求,同时对其同步检测的可行性进行探讨,为简化操作、缩短报告时间、适应野外检测创造条件,也为提高战地急救能力提供技术支持。方法:1、为每项检测指标在微型光谱仪的检测波长范围内(360nm～780nm)选择一种合适的方法及试剂;2、分别将选定的各项指标液体检测试剂制备为干试剂:对试剂干燥方式进行选择,同时对改善试剂外观及性能的添加剂种类和浓度进行筛选;3.将制备好的干试剂通过调整试剂样品稀释比、检测时间以及对显色剂进行调整替换,进行干试剂成分及测定方法的改进;4、初步观察各项指标检测试剂的性能:对干试剂的准确性、重复性、稳定性进行初步评价。5、将六项指标检测试剂同步冻干于自制检测杯中,在微型光谱仪上进行整体检测的初步应用,同时对检测试剂性能进行再评价,对测定结果采用SPSS 11.0软件进行统计学分析处理。6、对仪器的温控系统及磁力混匀系统进行使用评价。主要结果:1、血红蛋白检测试剂由高铁氰化钾,氰化钾,离子表面活化剂等成分配置而成,并直接进行冻干。制备后的干试剂具有良好的溶解性能,降低试剂样品稀释度为125:1,使冻干后试剂具有良好溶解性能,同时获得较好的线性范围。试剂准确性实验偏倚系数为5.76%、试剂批内变异系数为2.22%,批间变异系数均值为3.37%,试剂在3个月内稳定性能良好。2、葡萄糖检测试剂由酶试剂、0.5%的自制色原TBHBA、以及10g/L的添加剂白蛋白制备而成,增加试剂样品稀释倍数为200:1,使试剂缩短检测时间为10min,且试剂准确性实验偏倚系数为3.28%,试剂批内变异系数为2.15%,批间变异系数均值为3.76%,试剂在3个月内稳定性能良好。3、钾离子检测试剂由四苯硼锂、蛋白水解酶、氢氧化钠以及20g/L的添加剂木糖醇组成,制备好的干试剂保持了较好的溶解性能及反应性能。试剂准确性实验偏倚系数为4.13%,试剂批内变异系数为2.98%,批间变异系数均值为4.06%,试剂在3个月内稳定性能良好。4、钠离子检测试剂由β-半乳糖苷酶、O-硝基酚半乳糖苷、缓冲液等组成,分两步进行冻干。制备好的干试剂溶解性能良好,且试剂准确性实验偏倚系数为2.79%,试剂批内变异系数为2.10%,批间变异系数均值为4.34%,试剂在3个月内稳定性能良好。5、氯离子检测试剂由硫氰酸汞、硝酸铁、硝酸汞等以及40g/L的添加剂右旋糖酐组成,将制备好的干试剂稀释倍数调整为100:1,获得了较好的检测性能。试剂准确性实验偏倚系数为3.05%,试剂批内变异系数为2.10%,批间变异系数均值为3.15%,试剂在3个月内稳定性能良好。6、二氧化碳检测试剂由碳酸酐酶、指示剂及20g/L的添加剂聚乙二醇组成,制备好的干试剂准确性实验偏倚系数为3.27%,试剂批内变异系数为3.57%,批间变异系数均值为5.41%,试剂在3个月内稳定性能良好。7、按照六项指标检测试剂的冻干方式,直接冻干于自制检测杯中,使用统一的稀释液,加样后自动进行磁力混匀及加热,孵育后由系统在指定波长下进行检测,通过电脑微处理快速输出报告。由检测结果显示,各指标检测试剂偏倚系数均在10%以内,除二氧化碳检测试剂批间重复性(CV%)超出5%外,其余各项指标检测试剂批内及批间重复性(CV%)均小于5%,且测定结果与干化学分析仪VITRO S-250相关性r≥0.97。8、微型光谱仪空气孵育系统在3min内能够达到试剂孵育所需的37℃,同时混匀系统92%的磁力搅拌子混匀状态良好。结论:1、通过对血红蛋白、葡萄糖及钾、钠、氯、二氧化碳,六种急救生化检测试剂方法、成分的改进与改良,研制出了适用于微型光谱仪的检测干试剂。该试剂在三个月内稳定性能良好,测定结果准确、可靠,能够基本满足临床测定的要求,试剂便于保存和携带。2、多项目急救生化指标组合式检测所形成的功能检测单元,能够通过一次检测即可获得6项测定结果,可为伤员的现场救治提供必要的检验数据,同时缩短了报告时间,为及时救治提供了保证。3、低电压启动的微型光谱仪,采用干电池作动力,无需交流电能完成自动孵育及混匀,整套装置具有良好的便携性和机动性,能够满足战场急救、现场快速检验的需要。4、微型离心机的配套研制,可为检测标本提供快速、有效的分离,同时仪器运行使用干电池,实现了不受环境限制、进行快速现场检测的目的。综上所述,由实验研究证实战地急救生化试剂的性能是可靠的,其基于微型光谱仪进行的同步检测方法是可行的。整套检测系统适于野战环境,能够满足便携、机动的战地检测需求,实现为战地急救提供快速、可靠检测数据的目的。更多还原

【Abstract】 Objective:To meet the first aid needs of battlefield wounded persons (corresponding rate increment of serious injuries like shock, bleeding, infection, multiple organ failure and so on) in the high-technology partial war, and meet the requirements of convenience and automation of first-aid equipments in field war, we plan to develop biochemical detection equipments and matched reagents, which is based on low-voltage-start miniature optic spectrometers and can be used in field war. Detection indexes include hemoglobin, glucose and kalium, sodium, chloridion, carbon dioxide combining power. Combination of these six indexes will form an examining unit for first-aid biochemical functions, and one test gives six results. This research focuses on how to improve the six detection reagents and how to make them meet the requirements of miniature optic spectrometers. And it discusses the feasibility of simultaneous detection, and creates conditions to simplify operation, and cuts report time to fit in field detection, and provides technical support to enhance battlefield first-aid ability.Methods:1. Select a suitable method and reagent for each examining index in the wave length of miniature optic spectrometer (380nm～720nm).2. Make each selected index’s liquid detection reagent into dry reagent respectively: select the dry method, and filter the additive categories and concentration for the improvement of reagent appearance and performance.3. Adjust the dried reagent dilution ratio and examining time and adjust and substitute the chromogenic agent, and then improve the element and examining method for the dried reagent.4. Observe the all examining indexes initially and test the performance of dried reagent: make a primary comment on the accuracy, repeatability and stability of the dried reagent. 5. Freeze-dry all examining reagents of the six indexes in self-made detection cup synchronously for the primary application by micro spectrometers, and at the same time evaluate examining reagent performanceperformances, and statistically analyze and deal with the detection results by the software of SPSS 11.0.6. Utilize and evaluate the equipment temperature control system and magnetic mixing system.Results:1. Hemoglobin examining reagent is made up of ferrate potassium cyanide, potassium cyanide, ionic surfactant and so on, and can be freeze-dry directly. Dried reagent has good solvability. When the dilution decreased to 125:1, the dried reagent not only has good solvability, but also gets better linearity range. Coefficient of variation in group of the reagent is 2.22%, and the average interassay variation coefficient is within 3.37%,. The reagent is stable in 3 months.2. Glucose examining reagent is made up of enzyme agent, self-made chromogen TBHBA of 0.5%, and albumin addictive of 10g/L. Increase the dilution multiple of the reagent to 200:1, and cut the reagent examining time to 10min. Coefficient of variation in groupof the reagent accuracy experiment is 2.15%, and the average interassay variation coefficient is within 3.76%. The reagent is stable in 3 months.3. Potassium ion examining reagent is made up of lithium tetraphenylborate proteolytic enzyme, sodium hydroxide, and xylitol addictive of 20g/L. Finished dried reagent has good solubility and reactivity. Coefficient of variation in group of the reagentof the reagent is 2.98%, and the average interassay variation coefficient is within 4.06%. The reagent is stable in 3 months.4. Sodium ion examining reagent is made up ofβ-galactosidase, 0-nitrophenol-galactoside, buffer solution and so on. Finished dried reagent has good solubility. Coefficient of variation in group of the reagentof the reagent is 2.10%, and the average interassay variation coefficientis within 4.34%. The reagent is stable in 3 months.5. Chloride examining reagent is made up of mercury thiocyanate, ferric nitrate, mercury nitrate, and dextran addictive of 40g/L. Adjust the dilution multiple of the reagent to 100:1, and get better examining performanceperformance. Coefficient of variation in group of the reagent of the reagent is 2.10%, and the average interassay variation coefficient is within 3.15%. The reagent is stable in 3 months.6. Examining reagent of carbon dioxide is made up of carbonic anhydrase, indicator, and addictive of polyethylene glycol of 20g/L. The accuracy experiment’s bias coefficient the finished dried reagent is 3.27%, and the coefficient of variation in group of the reagent is 3.57%, and average interassay variation coefficient 5.41%. has good solubility and repeatability. The reagent is stable in 3 months.7. According to the freeze-drying method for examining reagents of the six indexes, freeze-dry them in the self-made cup directly, and use uniform diluent, and mix magnetically and heat automatically after loading, and test in designated wave length by the system after incubating, and output the report quickly by computer microprocessing. Examine results show that bias coefficient of each reagent is within 10%, within-batch and inter-batch repeatability (CV%) of each index reagent is less than 5%, except the reagent of carbon dioxide combining power more than 5%. All reagents are stable in 3 months. Relevance between the examining results and dry chemistry analyzer VITRO S-250 is more than 0.97.8. Air incubation system of miniature optic spectrometers can reach 37℃in 3min for reagent incubation, at the same time 92% of mixing system has good mixing status.Conclusions:1. By the improvement of the method and composition of the six first-aid biochemical detection of hexokinase, glucose and kalium, sodium, chloridion, carbon dioxide combining power, we developed the examining dry reagent for miniature optic spectrometers. The reagent is stable within 3 months with accurate and reliable results which can meet basic requirements in clinical examination. The reagent is easy for storage and carrying.2. Performance examining unit formed by the composite examination of multi-project first-aid biochemical indexes, can gain 6 examining results from one examination. It can provide necessary data for the on-site first aid of the wounded, and cut the reporting time which ensures the timely treatment.3. Low-voltage-start miniature optic spectrometers use dry batteries for energy, and can finish automate incubation and mixing without alternating current. The overall equipments have good portability and automation, which can meet the needs of battlefield first aid and on-site quick examination. 4. The development of matched miniature centrifuges can separate examining samples quickly and effectively. The equipments run by dry batteries, which breaks the environmental limitation and realized the purpose of on-site quick examination.In conclusion, the experimental research proves that the biochemical reagent performance for the battlefield first aid is reliable, and the simultaneous examining method is feasible on the basis of miniature optic spectrometers. The overall examining system is suitable for battlefield and can meet the portable and automate needs in battlefield examination, and realizes the purpose to provide quick and reliable information for battlefield first aid.更多还原