WO2022089476A1 - 一种生物液体样本中病原体的核黄素光化学灭活方法 - Google Patents
一种生物液体样本中病原体的核黄素光化学灭活方法 Download PDFInfo
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- WO2022089476A1 WO2022089476A1 PCT/CN2021/126678 CN2021126678W WO2022089476A1 WO 2022089476 A1 WO2022089476 A1 WO 2022089476A1 CN 2021126678 W CN2021126678 W CN 2021126678W WO 2022089476 A1 WO2022089476 A1 WO 2022089476A1
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- liquid sample
- riboflavin
- biological liquid
- light
- pathogens
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- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 title claims abstract description 88
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 230000002779 inactivation Effects 0.000 title claims abstract description 43
- 229960002477 riboflavin Drugs 0.000 title claims abstract description 43
- 235000019192 riboflavin Nutrition 0.000 title claims abstract description 43
- 239000002151 riboflavin Substances 0.000 title claims abstract description 43
- 244000052769 pathogen Species 0.000 title claims abstract description 41
- 239000007788 liquid Substances 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000001717 pathogenic effect Effects 0.000 title abstract description 19
- 238000005286 illumination Methods 0.000 claims abstract description 26
- 238000001228 spectrum Methods 0.000 claims abstract description 15
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 14
- 239000010836 blood and blood product Substances 0.000 claims description 14
- 229940125691 blood product Drugs 0.000 claims description 14
- 238000002617 apheresis Methods 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 230000000610 leukopenic effect Effects 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims 1
- 230000005855 radiation Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 230000006378 damage Effects 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 9
- 239000011324 bead Substances 0.000 description 8
- 239000000306 component Substances 0.000 description 8
- 239000013060 biological fluid Substances 0.000 description 7
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- 230000000415 inactivating effect Effects 0.000 description 5
- 239000012503 blood component Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 231100000645 Reed–Muench method Toxicity 0.000 description 3
- 244000078885 bloodborne pathogen Species 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006552 photochemical reaction Methods 0.000 description 1
- 230000008832 photodamage Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/08—Radiation
- A61L2/10—Ultraviolet radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0029—Radiation
- A61L2/0047—Ultraviolet radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0029—Radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
- A61L2/0088—Liquid substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising
- A61L2101/32—Organic compounds
- A61L2101/44—Heterocyclic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
- A61L2202/22—Blood or products thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the technical field of pathogen inactivation, in particular to a riboflavin photochemical inactivation method for pathogens in biological liquid samples.
- pathogens are present in biological fluid samples that need to be inactivated.
- biological fluid samples For example, in blood products, blood-borne pathogens, including viruses, bacteria, protozoa, and spirochetes, are often introduced during collection and transportation.
- the development of pathogen inactivation technology can effectively reduce the risk of blood transfusion infection.
- the promising blood pathogen inactivation technologies mainly include psoralen and riboflavin, which can be used for plasma, platelet and red blood cell pathogen inactivation.
- Psoralen may be genotoxic and therefore needs to be removed after use.
- Riboflavin (vitamin B2) is a natural vitamin necessary for the human body. Its decomposition products are widely present in human blood and tissues, and have natural photochemical reactions. They do not need to be removed after use, and are widely used in blood component pathogen inactivation. middle.
- the Chinese patent application "CN201910975223.2 Equipment and method for inactivating blood component pathogens by riboflavin photochemical method” proposes a method for inactivating pathogens in blood by riboflavin photochemical method, and the conditions for inactivation are carried out.
- the optimal conditions disclosed in the patent application are to use 309nm-313nm narrow-spectrum ultraviolet light, the best illumination time range is 5-40min, and the illumination energy range is 0.4J/ml-3J/ml.
- the inactivation effect on pathogens is still not ideal, and other components in the blood product are still damaged to a certain extent. It is for the above reasons.
- the riboflavin pathogen inactivation system is used in Europe and other countries to inactivate pathogens in blood, it has not yet been approved by the FDA in the United States.
- the present invention provides a riboflavin photochemical inactivation method for pathogens in biological liquid samples, the purpose of which is: by optimizing the wavelength range of illumination, the riboflavin Photochemical inactivation methods are more effective in inactivating pathogens and cause less damage in blood components.
- a method for photochemical inactivation of riboflavin in a biological liquid sample comprising the steps of adding riboflavin to the biological liquid sample and irradiating the biological liquid sample with light, wherein the light is in a wavelength range selected from 360-370 nm and/or 390 nm -Narrow spectrum UV light in the range of 400nm.
- the light is a narrow-spectrum ultraviolet light with a wavelength range selected from the range of 390-400 nm.
- the light is ultraviolet light with a peak at 395 nm.
- the light irradiation time of the biological liquid sample is 3-30min, and the light energy range is 0.2-5J/ml.
- the light irradiation time of the biological liquid sample is 3-4.9min, and the light energy range is 0.2-0.39J/ml.
- the amount of riboflavin added in the biological fluid sample is 40-60 ⁇ M.
- the amount of riboflavin added in the biological fluid sample is 50 ⁇ M.
- the biological fluid sample is a blood product, blood product, cell product or tumor cell sample.
- the blood product is whole blood, leukopenic whole blood, packaged red blood cells, manual platelets, apheresis platelets, plasma or cryoprecipitate.
- the present invention improves the method for photochemical inactivation of pathogens in biological fluid samples (such as blood products) by riboflavin, and the light wavelength is preferred.
- the illumination in the preferred wavelength range of the present invention has a better inactivation effect on pathogens.
- the narrow-spectrum ultraviolet light in this wavelength range can be used to inactivate organisms in a shorter light time and lower light intensity. The liquid sample undergoes pathogen inactivation, thereby reducing light damage to other components in the biological liquid sample.
- the method of the invention is suitable for inactivation of pathogens in blood products, decontamination treatment of various biological liquid samples, treatment of severe clinical infection and tumor patients, etc., and is widely used.
- Fig. 1 adopts the inactivation effect of narrow-spectrum ultraviolet light of different wavelength ranges on Escherichia coli in the method for photochemical inactivation of pathogens in blood products by riboflavin;
- Fig. 2 Inactivation effect of narrow-spectrum ultraviolet light with a wavelength of 395 ⁇ 5 nm and narrow-spectrum ultraviolet light with a wavelength of 309-313 nm on Staphylococcus aureus in platelets.
- the specific operation and device of the method for photochemically inactivating pathogens in a biological liquid sample by riboflavin in the present invention can be performed with reference to the content disclosed in the prior art.
- the specific operations and devices in the following examples are consistent with the methods and devices disclosed in Chinese patent application "CN201910975223.2 Device and method for inactivating blood component pathogens by riboflavin photochemical method", the differences are the wavelength range of illumination, illumination time , light energy and riboflavin concentration were changed.
- a series of LED lamp beads in a wavelength range are selected to irradiate the plasma of healthy blood donors containing 50 ⁇ M riboflavin, the illumination time is 15 min, and the illumination intensity is 1 W. E. coli growth in blood products was then detected by the Reed-Muench method.
- LED lamp beads with wavelengths of 365 ⁇ 5nm and wavelengths of 395 ⁇ 5nm were used in the riboflavin photochemical pathogen inactivation system. It has good pathogen inactivation effect on Escherichia coli.
- LED lamp beads with a wavelength of 395 ⁇ 5nm and fluorescent tubes with a wavelength of 309-313nm were selected to irradiate the apheresis platelets containing 50 ⁇ M riboflavin. 5nm) and 9W (309-313nm).
- the growth of Staphylococcus aureus in blood products was then detected by the Reed-Muench method.
- the light dose of 309-313 nm narrow-spectrum ultraviolet light and the light dose of 395 ⁇ 5 nm narrow-spectrum ultraviolet light are compared under the same inactivation effect.
- riboflavin physiological saline solution (CAS: 83-88-5; purchased from Sigma-Aldrich, St. Louis, Missouri, USA) into the bacterial plasma suspension, the final riboflavin concentration is 50 ⁇ mol/L .
- the light dose of 309-313nm fluorescent tube is 9.76J/mL, and the illumination time is 30 minutes.
- the 395 ⁇ 5nm LED lamp bead light dose is 1.25J/mL, and the illumination time is 10 minutes. Two groups of experimental samples were carried out in parallel for 6 groups.
- the experimental sample and the control sample are serially diluted by 10 1 -10 6 ;
- the narrow-spectrum ultraviolet light in the preferred wavelength range of the present invention has better inactivation effect on pathogens and less damage to other components in biological fluid samples (eg, blood products).
- the narrow-spectrum ultraviolet light in the preferred wavelength range of the invention it is possible to select shorter illumination time and lower illumination energy, thereby further reducing the damage of illumination to other components in biological fluid samples (eg, blood products).
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
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- Medicinal Chemistry (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
本发明属于病原体灭活技术领域,具体涉及一种生物液体样本中病原体的核黄素光化学灭活方法。针对现有的核黄素病原体灭活方法中存在的问题,本发明的技术方案是一种生物液体样本中病原体的核黄素光化学灭活方法:包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,所述光为波长范围选自360-370nm和/或390-400nm范围内的窄谱紫外光。本发明还进一步优选了光照时间、光照强度和核黄素浓度等参数。采用本发明的技术方案,能够达到优良的病原体灭活效果,且对生物液体样本中其他成分的损伤小。
Description
本发明属于病原体灭活技术领域,具体涉及一种生物液体样本中病原体的核黄素光化学灭活方法。
在医学或生物学中,常常出现生物液体样本中存在病原体需要灭活的情况。例如在血液制品中,采集、转运等过程中常常引入血传播病原体,这些血传播病原体包括病毒、细菌、原虫和螺旋体等。
随着血液病原体检测技术的发展,输血感染风险大大降低,但病原体检测窗口期依然存在、目前常规检测方法不能覆盖所有已知经血传播病原体。新发、再发经血传播病原体依旧威胁着输血安全,特别是血小板(PLT)常规储存在20-24℃条件下,细菌污染风险更高,持续稳定的血源性细菌污染成为了对血液供应机构威胁最大的因素。
发展病原体灭活技术可以有效降低输血感染风险,目前具有发展前景的血液病原体灭活技术主要有补骨脂素和核黄素,均可用于血浆、血小板和红细胞病原体灭活。补骨脂素可能存在基因毒性,因此使用后需要去除。而核黄素(维生素B2)是人体必须的天然维生素,其分解产物本身广泛存在于人体血液和组织中,并且具有天然的光化学反应,使用后不需去除,被广泛应用于血液成分病原体灭活中。
例如,中国专利申请“CN201910975223.2利用核黄素光化学法灭活血液成分病原体的设备及方法”中提出了一种核黄素光化学法灭活血液中病原体的方法,且对灭活的条件进行了优选。该专利申请中公开的较优条件为采用309nm-313nm窄谱紫外光,光照时间范围5-40min为最佳,光照能量范围0.4J/ml-3J/ml为最佳。但是,在该条件下,对病原体的灭活效果仍然不够理想,且对血液产品中的其它成分仍然具有一定的损伤。正是由于上述原因。虽然核黄素病原体灭活系统灭活血液中病原体在欧洲等国家使用,然而其在美国尚未得到FDA批准。
发明内容
针对现有的核黄素病原体灭活方法中存在的问题,本发明提供一种生物液体样本中病原体的核黄素光化学灭活方法,其目的在于:通过优化光照的 波长范围,使得核黄素光化学灭活方法对病原体的灭活效果更好,且对血液成分中造成的损伤更低。
一种生物液体样本中病原体的核黄素光化学灭活方法,包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,所述光为波长范围选自360-370nm和/或390-400nm范围内的窄谱紫外光。
优选的,光为波长范围选自390-400nm范围内的窄谱紫外光。
优选的,光为波峰为395nm的紫外光。
优选的,光辐射生物液体样本的光照时间为3-30min,光照能量范围为0.2-5J/ml。
优选的,光辐射生物液体样本的光照时间为3-4.9min,光照能量范围为0.2-0.39J/ml。
优选的,核黄素在生物液体样本中的加入量为40-60μM。
优选的,核黄素在生物液体样本中的加入量为50μM。
优选的,生物液体样本为血液产品、血液产品、细胞产品或肿瘤细胞样本。
优选的,血液产品为全血、白细胞减少的全血、包装红细胞、手工血小板、单采血小板、血浆或冷沉淀。
本发明对核黄素光化学灭活生物液体样本(例如血液产品)中的病原体的方法进行改进,优选了光照波长。在相同的光照时间和光照强度的条件下,本发明优选的波长范围的光照对病原体具有更好的灭活效果。此外,由于该波长范围内的光照对病原体的灭活效果更好,因而在实际操作中,选用该波长范围内的窄谱紫外光可在更短的光照时间和更低的光照强度下对生物液体样本进行病原体灭活,从而降低光照对生物液体样本中的其他成分的损伤。
本发明的方法适用于血液产品中的病原体灭活、各种生物液体样本的去污处理、临床重度感染和肿瘤患者的治疗等,应用广泛。
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。
图1核黄素光化学灭活血液产品中的病原体的方法中采用不同波长范围的窄谱紫外光对大肠杆菌的灭活效果;
图2波长为395±5nm的窄谱紫外光和波长范围为309-313nm的窄谱紫外光对血小板中的金黄色葡萄球菌的灭活效果。
下面通过具体的实施例对本发明的技术方案作进一步的说明。
本发明中核黄素光化学灭活生物液体样本中的病原体的方法的具体操作及装置可参考现有技术中已公开的内容进行。以下实施例中的具体操作和装置采用与中国专利申请“CN201910975223.2利用核黄素光化学法灭活血液成分病原体的设备及方法”中公开的方法和装置一致,区别在于光照波长范围、光照时间、光照能量和核黄素的浓度等进行了改变。
实施例1
本实施例选择了一系列波长范围的LED灯珠对含有50μM核黄素的健康献血者的血浆进行照射,光照时间为15min,光照强度为1W。然后通过Reed-Muench法检测血液产品中的大肠杆菌生长量。
结果如图1所示,相同光照时间和光照强度下,在一系列的窄谱紫外光中,波长为365±5nm和波长为395±5nm的LED灯珠在核黄素光化学病原体灭活系统中对大肠杆菌有较好的病原体灭活效果。
实施例2
本实施例选择了波长为395±5nm的LED灯珠和波长为309-313nm的荧光灯管对含有50μM核黄素的单采血小板进行照射,光照时间均为30min,光照强度分别为1W(395±5nm)和9W(309-313nm)。然后通过Reed-Muench法检测血液产品中的金黄色葡萄球菌的生长量。
结果如图2所示,图中还给出了未通过核黄素光化学灭活的对照实验组的数据。从图中可以看出,虽然395±5nm的LED灯珠和波长为309-313nm的荧光灯管都能够对金黄色葡萄球菌进行灭活,但是在相同光照时间和光照强度下,395±5nm的LED灯珠比波长为309-313nm的荧光灯管的灭活效果更好。
对比三组实验中血小板样品的性质及成分含量,结果如下:
表1 血小板保存质量
检测项目 | 395±5nm | 309-313nm | 对照(无光照) |
PH | 7.45±0.01 | 7.26 | 7.53 |
Na+mmol/L | 153.6±0.49 | 151 | 152 |
K+mmol/L | 2.7±0 | 3 | 2.6 |
Glummol/L | 26.3±0.09 | 23.6 | 26 |
Lacmmol/L | 9.24±0.08 | 10.6 | 8.1 |
HCO3-mmol/L | 8.54±0.22 | 8.5 | 10 |
HCO3std | 15.26±0.15 | 12.2 | 17.2 |
TCO2 | 8.94±0.22 | 9.1 | 10.4 |
PLT | 714.2±6.49 | 297 | 717 |
PDW | 10.84±0.16 | 17.3 | 10.9 |
MPV | 9.5±0 | 12.1 | 9.4 |
P-LCR | 21.2±0.2 | 36.7 | 20.7 |
PCT | 0.68±0.01 | 0.36 | 0.68 |
从表1中的数据可以看到,在利用核黄素光化学法对血小板进行病原体灭活后,各项性质和成分含量的参数与未光照的对照组均有一定的偏离,但是,采用395±5nm的LED灯珠光照对各项性质和成分含量的影响明显小于采用309-313nm的荧光灯管光照对各项性质和成分含量的影响,可见,395±5nm的光照对血小板样品的损伤显著更小。
实施例3
本实施例对相同灭活效果下,采用309-313nm窄谱紫外光的光剂量和395±5nm窄谱紫外光的光剂量进行对比。
具体实验步骤为:
1、取一袋健康献血者血浆(获得当地伦理委员会支持)150ml,加入10uL金黄色葡萄球菌培养液,获得大约4-5log的细菌血浆悬浮液;
2、取9mL细菌血浆悬浮液与1mL生理盐水混合作为对照,放置于4℃冰箱中;
3、然后将500μmol/L的核黄素生理盐水溶液(CAS:83-88-5;购自美国密苏里州圣路易斯Sigma-Aldrich公司)加入细菌血浆悬浮液中,最终核黄素浓度为50μmol/L。
4、分别取300uL加核黄素的细菌血浆悬液转移到无菌24孔板中(孔直径为1.5cm),然后分别暴露于9W 309-313nm荧光灯管(UVB窄带PL-L/PL-S,飞利浦,阿姆斯特丹,荷兰)和1W 395±5nm LED灯珠下,在温度控制的环境中(20-24℃)照射样本。
309-313nm荧光灯管光剂量为9.76J/mL,光照时间为30分钟。395±5nm LED灯珠光剂量为1.25J/mL,光照时间为10分钟。两组实验样本各平行进行6组。
5、光照结束后实验样本和对照样本连续稀释10
1-10
6;
6、每个稀释后的样品取100μL加入无菌板中心,每个稀释度样本重复接种8次,测定细菌生长量。
7、37℃生化培养箱(中国上海景洪SHP-080)培养24-48h后,观察各孔细菌生长情况并记录,采用Reed-Muench法计算细菌滴度。
实验结果表明:采用309-313nm紫外光较高光剂量进行照射后与采用395±5nm紫外光较低光剂量进行照射后,309-313nm和395±5nm细菌生长量分别为2.01±1.99log和2.22±1.80log,无统计学差异(P=0.568),即对病原体的灭活效果相当。
从上述实施例中可以看到,本发明优选的波长范围的窄谱紫外光对病原体的灭活效果更好,且对生物液体样本(例如血液产品)中的其他成分的损伤更小。此外,采用发明优选的波长范围的窄谱紫外光,能够选择更短的光照时间和更低的光照能量,从而进一步降低光照对生物液体样本(例如血液产品)中的其他成分的损伤。
Claims (9)
- 一种生物液体样本中病原体的核黄素光化学灭活方法,包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,其特征在于:所述光为波长范围选自360-370nm和/或390-400nm范围内的窄谱紫外光。
- 按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述光为波长范围选自390-400nm范围内的窄谱紫外光。
- 按照权利要求2所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述光为波峰为395nm的紫外光。
- 按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述光辐射生物液体样本的光照时间为3-30min,光照能量范围为0.2-5J/ml。
- 按照权利要求4所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述光辐射生物液体样本的光照时间为3-4.9min,光照能量范围为0.2-0.39J/ml。
- 按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述核黄素在生物液体样本中的加入量为40-60μM。
- 按照权利要求6所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述核黄素在生物液体样本中的加入量为50μM。
- 按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述生物液体样本为血液产品、细胞产品或肿瘤细胞样本。
- 按照权利要求8所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述血液产品为全血、白细胞减少的全血、包装红细胞、手工血小板、单采血小板、血浆或冷沉淀。
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