CN114225067B - 一种血液病原体灭活方法 - Google Patents
一种血液病原体灭活方法 Download PDFInfo
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Abstract
本发明公开了一种病原体灭活方法，它是低频超声同时光照含光敏剂的血液样品；所述低频超声的频率为15‑500KHz。本发明血液病原体灭活方法，通过超声与光化学法病原体灭活技术相结合相互增强，相互补充，增强病原体灭活效果，同时降低光敏剂使用量，减少光敏剂相关的血液质量损伤，降低光照能量需求和病原体灭活处理时间，增加有效病原体灭活的血液光照厚度，节约光照袋材料，缩小光照设备尺寸，节约成本，更有利于病原体灭活技术走向市场。
Description
技术领域
本发明具体涉及一种血液病原体灭活方法。
背景技术
输血是临床常规使用的治疗方法和急救手段，血液携带的病原体可传染给患者，给患者健康带来不可估量的危害。血液安全关系到人民健康和国家安全。目前控制输血传播病原体的方法包括献血者筛选、血液筛查等方法，大大降低了输血传播病原体风险，但目前血液病原体检测存在“窗口期”和常规筛查病原体种类有限等问题，血液传播病原体风险一直存在，因此急需发展一种新的技术降低输血传播病原体风险。
血液中可能携带的病原体有革兰阳性球菌：金黄色葡萄球菌、凝固酶阴性葡萄球菌、肺炎链球菌、草绿色链球菌、肠球菌；革兰阳性杆菌：结核分枝杆菌、产单核李斯特菌；革兰阴性球菌：脑膜炎奈瑟菌、卡他布兰汉菌；革兰阴性杆菌：大肠埃希菌、铜绿假单胞菌、克雷伯菌、沙雷菌、沙门菌、不动杆菌、嗜肺军团菌、嗜血杆菌；真菌：假丝酵母菌、曲霉菌、隐球菌、球孢子菌；厌氧菌：拟杆菌、产气荚膜梭菌；病毒(甲肝病毒，乙肝病毒，丙肝病毒，人类免疫缺陷病毒等；)；寄生虫(疟疾等)；真菌；阮病毒等。
病原体灭活技术可以有效解决血液病原体检测技术存在的“窗口期”问题，降低或消除了输血传播病原体风险，也能降低非常规筛查病原体和新发病原体经输血感染的风险。此外，病原体灭活技术还可以灭活白细胞，降低移植物抗宿主病的发生率。
目前研究中病原体灭活技术包括光化学法病原体灭活技术(亚甲蓝光化学法、补骨脂素光化学法、核黄素光化学法)、短波紫外线照射法、低温等离子技术、飞秒激光/超短脉冲激光技术、高静水压技术等。国外批准使用的技术包括亚甲蓝光化学法、补骨脂素光化学法和核黄素光化学法。我国市场上批准用于血液病原体灭活的方法主要是亚甲蓝光化学法。由于亚甲蓝存在残留的毒性问题，在欧美等国家限制或禁止使用。因此，我国有必要发展新的血液病原体灭活技术供血站或临床使用降低输血传播病原体的风险。
1980年超声灭活病原体技术在食品科学中作为“新兴技术”最先提出(FDA，2000)，然后以相似的用途在其他领域进行研究，1990年后发现超声可灭活目标微生物，超声对微生物的灭活作用取得重要进展。单独超声对病原体的灭活效果不能满足食品药品安全标准，应探索病原体灭活效果更好的方法，但目前还没有关于超声与抗菌剂、化学品、热或压力相结合的技术的研究。
发明内容
为解决上述问题，本发明提供了包括如下步骤：取血液，加入光敏剂，同时进行光照处理和低频超声处理，低频超声的频率为15-500KHz。
进一步地，所述光敏剂为核黄素。
进一步地，所述核黄素在血液样品中的浓度为10-100μmol/L。
更进一步地，所述核黄素在血液样品中的浓度为50μmol/L。
更进一步地，所述核黄素溶解在生理盐水中，再加入血液样品中。
更进一步地，所述生理盐水中核黄素的含量为50-500μmol/L。
更进一步地，所述生理盐水中核黄素的含量为500μmol/L。
进一步地，所述低频超声同时光照的时间3min-2h。
更进一步地，所述低频超声同时光照的时间10min-30min。
更进一步地，所述低频超声的频率为30KHz。
更进一步地，所述光照的波长为311±50nm。
进一步地，所述血液样品为血浆、血小板、悬浮红细胞或全血。
本发明血液病原体灭活方法，通过超声与光化学法病原体灭活技术相结合相互增强，相互补充，增强病原体灭活效果，同时降低光敏剂使用量，减少光敏剂相关的血液质量损伤，降低光照能量需求和病原体灭活处理时间，增加有效病原体灭活的血液光照厚度，节约光照袋材料，缩小光照设备尺寸，节约成本，更有利于病原体灭活技术走向市场。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1不同超声、紫外光和核黄素组合处理后金黄色葡萄球菌生长量(单位：log，n＝6)
具体实施方式
实施例1、本发明血液病原体的灭活方法
1)取全血，加入500μmol/L核黄素生理盐水溶液，使全血中核黄素浓度为50μmol/L；
2)将步骤1)含核黄素的全血，置于波长311±50nm的光照环境中，同时在15KHz的条件下超声30min，即得病原体灭活的全血。
实施例2、本发明血液病原体的灭活方法
1)取血浆，加入200μmol/L核黄素生理盐水溶液，使血浆中核黄素浓度为50μmol/L；
2)将步骤1)含核黄素的血浆，置于波长311±50nm的光照环境中，同时在30KHz的条件下超声20min，即得病原体灭活的血浆。
实施例3、本发明血液病原体的灭活方法
1)取血小板，加入100μmol/L核黄素生理盐水溶液，使血小板中核黄素浓度为25μmol/L；
2)将步骤1)含核黄素的血小板，置于波长311±50nm的光照环境中，同时在50KHz的条件下超声10min，即得病原体灭活的血小板。
实施例4、本发明血液病原体的灭活方法
1)取悬浮红细胞，加入50μmol/L核黄素生理盐水溶液，使悬浮红细胞中核黄素浓度为25μmol/L；
2)将步骤1)含核黄素的悬浮红细胞，置于波长311±50nm的光照环境中，同时在30KHz的条件下超声3min，即得病原体灭活的悬浮红细胞。
实施例5、本发明血液病原体的灭活方法
1)取全血，加入500μmol/L核黄素生理盐水溶液，使全血中核黄素浓度为50μmol/L；
2)将步骤1)含核黄素的全血，置于波长311±50nm的光照环境中，同时在30KHz的条件下超声30min时间，即得病原体灭活的全血。
以下通过试验例来说明本发明的有益效果。
试验例1评价低频超声与光化学法结合的最佳组合方式
1、方法
1.1取2袋ABO同型新鲜冰冻血浆，共约400ml，解冻后混匀；
1.2加入不超过10％的指示病原体(金黄色葡萄球菌或VSV)，病原体最终浓度大约为5-6log；
1.3将样本平均分为两份，一份加入生理盐水(占最终样本10％)，一份加入500μmol/L核黄素生理盐水溶液(占最终样本10％)，加入血浆后浓度为50μmol/L；
1.4将两份样本分装入直径为(3.5cm)的无菌培养板，每孔3mL,每板6孔；每份样本最后有9板，共18板；
1.5将样本分为3组进行不同光照时间(能量)处理；每组6板样本(有核黄素样本3板，无核黄素样本组3板)，每组6板的处理条件分为：
a.有核黄素样本+光照+低频超声；
b.有核黄素样本+光照；
c.有核黄素样本+低频超声；
d.无核黄素样本+光照+低频超声；
e.无核黄素样本+光照；
f.无核黄素样本+低频超声；
不同光照时间(能量)处理方法为：光波长为311±50nm,3组光照时间(能量)分别为10min，20min，30min(光照能量分别为0.27J/ml,0.54J/ml,0.81J/ml)；
低频超声频率30KHz，3组超声时间与光照时间同步，分别为10min,20min，30min；
对照为有核黄素样本无光照和无核黄素样本无光照，每组数据重复次数N＝6；
1.6灭活处理结束后取样本，1:10倍比稀释后培养，按照Reed-Muench法计算病原体生长浓度。
1.7分析最佳低频超声和光化学法组合病原体灭活效果。
2、结果
具体结果见表1和图1。
表1不同超声、紫外光和核黄素组合处理后金黄色葡萄球菌生长量(单位：log，n＝6)
从结果可见：超声+紫外光+核黄素组病原体灭活效果很强，光照强度达到0.81J/mL后金黄色葡萄球菌生长量几乎接近于检测限制水平1.28±0.66log(由于检测方法的限制，当样本细菌浓度低于0.5log后不能测出，因此不能测出的样本病原体浓度标记为0.5log)，而超声+紫外光组光照能量达到0.81J/mL灭活效果差；紫外光+核黄素组,超声+核黄素组和紫外光组当光照能量达到0.81J/mL时没有任何灭活效果。说明超声可以增强紫外光的病原体灭活效果，大大增强紫外光与核黄素的光化学法病原体灭活效果。因此本发明低频超声和核黄素光化学法结合具有协同增效的作用。
综上，本发明血液病原体灭活方法，通过超声与光化学法病原体灭活技术相结合相互增强，相互补充，增强病原体灭活效果，节约病原体灭活成本，具有实际推广应用价值。
Claims (8)
1.一种血液病原体灭活方法，其特征在于：包括如下步骤：
取血液，加入核黄素，同时进行光照处理和低频超声处理3min-2h，低频超声的频率为15-500KHz；
所述核黄素在血液样品中的浓度为10-100μmol/L；
所述光照的波长为311±50nm。
2.根据权利要求1所述的血液病原体灭活方法，其特征在于：所述核黄素在血液样品中的浓度为50μmol/L。
3.根据权利要求1所述的血液病原体灭活方法，其特征在于：所述核黄素溶解在生理盐水中，再加入血液样品。
4.根据权利要求3所述的血液病原体灭活方法，其特征在于：所述生理盐水中核黄素的含量为50-500μmol/L。
5.根据权利要求4所述的血液病原体灭活方法，其特征在于：所述生理盐水中核黄素的含量为500μmol/L。
6.根据权利要求1所述的血液病原体灭活方法，其特征在于：所述低频超声同时光照的时间10min-30min。
7.根据权利要求1或6所述的血液病原体灭活方法，其特征在于：所述低频超声的频率为30KHz。
8.根据权利要求1所述的血液病原体灭活方法，其特征在于： 所述血液样品为血浆、血小板、悬浮红细胞或全血。
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| CN112316166A (zh) * | 2020-10-30 | 2021-02-05 | 中国医学科学院输血研究所 | 一种生物液体样本中病原体的核黄素光化学灭活方法 |
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| AU2002306757B2 (en) * | 2002-04-12 | 2009-07-02 | Throwleigh Technologies, L.L.C. | Methods and apparatus for decontaminating fluids |
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| CN1329510A (zh) * | 1998-06-23 | 2002-01-02 | 新药研究(澳大利亚)有限公司 | 改进的消毒方法 |
| DE20319390U1 (de) * | 2003-12-11 | 2005-06-09 | Bender, Hans-Werner, Dr. | Vorrichtung aus Ultraschallapplikator und Trägerelement |
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