一次性塑料血袋中还原物质的分析及其安全性研究
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摘要
一次性塑料血袋在贮存和使用过程中，血袋袋体中的小分子有机物(主要是还原物质)会析出到保存液或血液制品中，影响血液制品的质量和输血安全，因此血袋中的还原物质必须严格控制。国标GB14232.1—2004／ISO3826—1：2004中已经对还原物质的总量作了限制，但仍不足以说明还原物质的安全性，血袋中还原物质的组成状况尚未见有文献报道。血袋中析出的一些痕量的有毒的小分子有机物对还原物质的总量指标贡献很小，但危害却很大，对血液质量和被输血者存在潜在威胁，因此只控制还原物质的总量是不够的。本文分析了血袋中还原物质的组成，并对其安全性做出了初步评价。
在还原物质的分析方面，本文采用间接碘量滴定法测定了不同血袋样品的还原物质总量，从血袋的生产厂家(配方、工艺)、类型及储存稳定性(保存温度、时间)方面考察了还原物质的变化规律。结果表明，血袋袋体材料的配方及工艺是影响还原物质总量的主要因素；提高保存温度，还原物质的总量增加；全血袋的还原物质总量最高，其次是转移袋和未灭菌袋；血袋的还原物质总量随保存时间增加而缓慢增加。同时本文还探索了合适的SPE／GC／MS条件，用该法可以很好地鉴定出一次性塑料血袋水浸提液、保存液、袋装全血中的还原物质。结果发现，水浸提液中的还原物质主要是一些酯类和烷烃，保存液中极性较强的醇类含量较高，而袋装全血中仅检测到烷烃和较高含量的邻苯二甲酸二(2-乙基)己酯(DEHP)。
在还原物质的安全性评价方面，从血液质量的变化，溶血性能，凝血及补体性能的变化来考察还原物质含量不同的血袋的血液相容性。结果表明，还原物质对白细胞、血小板及总补体CH50有一定的破坏作用；血液中钾离子浓度和游离血红蛋白随还原物质的增加而增加；而还原物质对溶血率、红细胞、Na~+浓度、pH、补体C3、C4、凝血指标PT和Fbg的影响不明显。同时本文采用MTT法考察了还原物质含量不同的血袋的细胞毒性，结果表明，血袋材料的细胞毒性与其还原物质的总量呈明显的正相关性。还原物质的总量、种类及每种还原物质的含量都会影响其安全性。
Small molecular compounds (reducing substances) will release from blood bag materials into anticoagulants or blood products during the time of plastic blood bags reserved or used, which will have a negative effect on blood products and donee. So reducing substances of plastic blood bags must be strictly controlled. GB14232.1 -2004/ISO3826-1:2004 has limits the total amounts of reducing substances, but it is not adequate to illuminate the ponderance of reducing substances. The composing determination of reducing substances released from plastic blood bags hasn't been reported by literatures. Some trace poisonous small molecular compounds released from blood bag materials contribute little to the total amounts of reducing substances, but they do potential harm to blood products and donee. This paper analyzed the composing of reducing substances and had a primary safety assessment on them.
At the aspect of reducing substances analysis, the total amounts of reducing substances released from different type blood bags were determined by indirect iodimetry. This paper researched the reducing substances' changing rules. The result indicated that factors such as prescription, craft, type, temperature, time, et al, had different affection on total amounts of reducing substances released from plastic blood bags. Prescription and craft of blood bag materials were the basical factors of affecting the total amounts of reducing substances. Reducing substances would increase as reserving temperature heightened. The reducing substances released from whole blood bags were much more than those released from translation bags and non-sterilized bags. Reducing substances would increase as reserving time prolonged. The composing of reducing substances was determined by SPE/GC/MS released into plastic blood bags aqueous extract, anticoagulant and whole blood. The result indicated that esters and alkanes were the main reducing substances in aqueous extract, polar alcohols had high amounts in anticoagulant, while only alkanes and a great deal of DEHP were determined in whole blood.
At the aspect of safety assessment of reducing substances, haemocompatibility was investigated by testing blood quality, haemolysis, blood coagulation, complement. The result indicated that reducing substances could destroyed white blood cells, blood platelets and CH50. The concentration of K~+ and free hemoglobin in whole blood would increase quickly as reducing substances increased. But reducing substances had little effect on haemolysis, red blood cells, the concentration of Na~+, pH, complement of C3, C4, PT, Fbg. Cytotoxicity was determined by the method of MTT. The result indicated that cytotoxicity of blood bags materials had positive correlation with their total amounts of reducing substances.
在还原物质的分析方面，本文采用间接碘量滴定法测定了不同血袋样品的还原物质总量，从血袋的生产厂家(配方、工艺)、类型及储存稳定性(保存温度、时间)方面考察了还原物质的变化规律。结果表明，血袋袋体材料的配方及工艺是影响还原物质总量的主要因素；提高保存温度，还原物质的总量增加；全血袋的还原物质总量最高，其次是转移袋和未灭菌袋；血袋的还原物质总量随保存时间增加而缓慢增加。同时本文还探索了合适的SPE／GC／MS条件，用该法可以很好地鉴定出一次性塑料血袋水浸提液、保存液、袋装全血中的还原物质。结果发现，水浸提液中的还原物质主要是一些酯类和烷烃，保存液中极性较强的醇类含量较高，而袋装全血中仅检测到烷烃和较高含量的邻苯二甲酸二(2-乙基)己酯(DEHP)。
在还原物质的安全性评价方面，从血液质量的变化，溶血性能，凝血及补体性能的变化来考察还原物质含量不同的血袋的血液相容性。结果表明，还原物质对白细胞、血小板及总补体CH50有一定的破坏作用；血液中钾离子浓度和游离血红蛋白随还原物质的增加而增加；而还原物质对溶血率、红细胞、Na~+浓度、pH、补体C3、C4、凝血指标PT和Fbg的影响不明显。同时本文采用MTT法考察了还原物质含量不同的血袋的细胞毒性，结果表明，血袋材料的细胞毒性与其还原物质的总量呈明显的正相关性。还原物质的总量、种类及每种还原物质的含量都会影响其安全性。
Small molecular compounds (reducing substances) will release from blood bag materials into anticoagulants or blood products during the time of plastic blood bags reserved or used, which will have a negative effect on blood products and donee. So reducing substances of plastic blood bags must be strictly controlled. GB14232.1 -2004/ISO3826-1:2004 has limits the total amounts of reducing substances, but it is not adequate to illuminate the ponderance of reducing substances. The composing determination of reducing substances released from plastic blood bags hasn't been reported by literatures. Some trace poisonous small molecular compounds released from blood bag materials contribute little to the total amounts of reducing substances, but they do potential harm to blood products and donee. This paper analyzed the composing of reducing substances and had a primary safety assessment on them.
At the aspect of reducing substances analysis, the total amounts of reducing substances released from different type blood bags were determined by indirect iodimetry. This paper researched the reducing substances' changing rules. The result indicated that factors such as prescription, craft, type, temperature, time, et al, had different affection on total amounts of reducing substances released from plastic blood bags. Prescription and craft of blood bag materials were the basical factors of affecting the total amounts of reducing substances. Reducing substances would increase as reserving temperature heightened. The reducing substances released from whole blood bags were much more than those released from translation bags and non-sterilized bags. Reducing substances would increase as reserving time prolonged. The composing of reducing substances was determined by SPE/GC/MS released into plastic blood bags aqueous extract, anticoagulant and whole blood. The result indicated that esters and alkanes were the main reducing substances in aqueous extract, polar alcohols had high amounts in anticoagulant, while only alkanes and a great deal of DEHP were determined in whole blood.
At the aspect of safety assessment of reducing substances, haemocompatibility was investigated by testing blood quality, haemolysis, blood coagulation, complement. The result indicated that reducing substances could destroyed white blood cells, blood platelets and CH50. The concentration of K~+ and free hemoglobin in whole blood would increase quickly as reducing substances increased. But reducing substances had little effect on haemolysis, red blood cells, the concentration of Na~+, pH, complement of C3, C4, PT, Fbg. Cytotoxicity was determined by the method of MTT. The result indicated that cytotoxicity of blood bags materials had positive correlation with their total amounts of reducing substances.
引文
[1] M. E. A. Faouzi, T. Dine, M. Luyckx. compatibility and plasticizer extraction of miconazole injection added to infusion solutions and stored in PVC containers, J. Pharm. Biomed. Anal. 13(1995):1363-1372
[2] A. Jimenez, J. Lopez, Vilaplana, et al. Thermal degradation of plastisols, effect of some additives on the evolution of gaseous products [J]. Analytical and Applied Pyrolysis, 1997, 40-41:201-215
[3] GB14232．1—2004／ISO3826—1：2003《人体血液及血液成分袋式塑料容器 第1部分：传统型血袋》
[4] 杨丽庭，高俊刚，李燕芳．改性聚氯乙烯新材料．北京：化学工业出版社，2002．458-468
[5] A. Marcilla, S. Garcia and J. C. Garcia-Quesada. Study of the migration of PVC plasticizers [J]. Analytical and Applied Pyrolysis, 2004, 71(2): 457-463
[6] Ignacio Aracil, Rafael Font and Juan A. Conesa.Thermo-oxidative decomposition of polyvinyl chloride Journal of Analytical and Applied Pyrolysis,74(1-2):215-223
[7] Mustafizur Rahman, Christopher S. Brazel. The plasticizer market: an assessment of traditional plasticizers and research trends to meet new challenges [J]. Progress in Polymer Science, 2004, 29(12): 1223-1248
[8] Minna Hakkarainen. New PVC materials for medical applications—the release profile of PVC/polycaprolactone- polycarbonate aged in aqueous environments[J]. Polymer Degradation and Stability, 2003, 80(3):451-458
[9] 杨天楹，杨成民，田兆嵩．临床输血学．第1版．北京：北医协和联合出版社，1993：442-445
[10] S. S. Hill, B. R. Shaw, A. H. B. Wu. The clinical effects of plasticizers, antioxidants and other contaminants in medical polyvinylchloride tubing during respiratory and non-respiratory exposure [J]. Clin Chim Acta, 2001(304):1-8
[11] Sjoberg P, Bondesson U, Sedin G, Gustafsson J. Disposition of di and mono-(2-ethylhexyl) phthalate in newborn infants subjected to exchange transfusions. Eur J Clin Invest.1985(15): 430-436
[12] Sjoberg PO, Bondesson UG, Sedin EG, Gustafsson JP. Exposure of newborn infants to plasticizers. Plasma levels of di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate during exchange transfusion. Transfusion. 1985(25): 424-428
[13] Plonait SL, Nau H, Maier RF, Wittfoht W, Obladen M. Exposure of newborn infants to di-(2-ethylhexyl)-phthalate and 2-ethylhexanoic acid following exchange transfusion with polyvinylchloride catheters. Transfusion. 1993(33): 598-605
[14] A. O. Earls, I. P. Axford and J. H. Braybrook. Gas chromatography-mass spectrometry determination of the migration of phthalate plasticisers from polyvinyl chloride toys and childcare articles. Journal of Chromatography A January 2003, 983(1-2): 237-246
[15] Dine, M. Luyckx, M. Cazin, C. Brunet, F. Goudaliez and J.C. Cazin, Rapid determination by high performance liquid chromatography of di(2-ethylhexyl) phthalate in plasma stored in plastic bags. Biomed.Chromatogr. 5 (1991): 94-97
[16] Doull, R. Cattley, C. Elcombe, B. G. Lake, J. Swenberg, C. Wilkinson, G. Williams and M. Van Gemert, A cancer risk assessment of di(2-ethylhexyl) phthalate: Application of the New US EPA risk assessment guidelines. Regul. Toxicol. Pharmacol. 29 (1999): 327-357
[17] Huber WW, Grasl-Kraupp B, Schulte-Hermann R. Hepatocarcino genicpotential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk. Crit Rev Toxicol. 1996(26): 365-481
[18] 奚廷斐，王春仁．邻苯二甲酸二异辛酯增塑聚氯乙烯医疗器械的安全性评价．中国医疗器械信息，2005，11(2)：27-29
[19] Francesca Nuti, Sibylle Hildenbrand, Mario Chelli. Synthesis of DEHP metabolites as biomarkers for GC-MS evaluation of phthalates as endocrine disrupters. Bioorganic & Medicinal Chemistry,16 May 2005, 13(10): 3461-3465
[20] Rie Ito, Fumie Seshimo, Yuji Haishima, et al. Reducing the migration of di-2-ethylhexyl phthalate from polyvinylchloride medical devices [J]. International Journal of Pharmaceutics 2005, 303 (1): 104-112
[21] Center for Devices and Radiological, U. S. Health, Food and Drug Administration: safety assessment of di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices. FDA report (2001) September
[22] Minna Hakkarainen. Qualitative and quantitative solid-phase micro extraction gas chromatographic-mass spectrometric determination of the low-molecular-mass compounds released from poly vinyl chloride/polycaprolactone-polycarbonate during ageing [J]. Chromatography A, 2003, 1010(1): 9-16
[23] K. Inoue, H. Okumura, T. Higuchi, et al. Characterization of estrogenic compounds in medical polyvinyl chloride tubing by gas chromatographymass spectrometry and estrogen receptor binding assay[J]. Clin Chim Acta, 2002(325): 157-163
[24] Koichi Inoue. Evaluation and analysis of exposure levels of di(2-ethylhexyl) phthalate from blood bags [J]. Clinica Chimica Acta, 2005, 358(1-2): 159-166
[25] 王立，汪正范，谋世芬等．色谱分析样品处理．第1版．北京：化学工业出版社，2002：94-97
[26] 郑和辉，赵立文等．饮用水中邻苯二甲酸酯的气相色谱—质谱测定法．环境与健康杂志，2005，9(5)：377—378
[27] Rie Ito, Fumie Seshimo, Naoko Miura, et al. High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry[J]. Pharmaceutical and Biomedical Analysis, 2005, 39(5): 1036-1041
[28] K. Inoue,M. Kawaguchi, F. Okada, Y. Yoshimura and H. Nakazawa, Column switching high-performance liquid chromatography electrospray mass spectrometry coupled with on-line extraction for the determination of mono and di-(2-ethylhexyl) phthalate in blood samples[J]. Anal Bioanal Chem, 2003(375): 527-533
[29] GB／T14233.2—2005《医用输液、输血、注射器具检验方法 第二部分：生物实验方法》
[30] GB／T16886.4-2005《医疗器械生物学评价第4部分：与血液相互作用实验选择》
[31] GB18469-2001《全血及成分血质量要求》
[32] B. D. Ratnar(Eeds). The blood compatibility catastrophe, J Biomed Mater Res. 1993 Mar, 27(3):283-287
[33] J.M.Ccourtney, X.Zhao and H.Qian. Biomaterials in cardiopulmonary bypass. Perfusion, 1999,14:263
[34] 郝和平．医疗器械生物学评价标准实施指南．中国标准出版社，2000：81—90
[35] 孙皎，顾国珍，钱云芳．《生物医学工程学杂志》2003，20(1)：8-10
[36] MTT mosmann T. Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65(1-2): 55-63
[2] A. Jimenez, J. Lopez, Vilaplana, et al. Thermal degradation of plastisols, effect of some additives on the evolution of gaseous products [J]. Analytical and Applied Pyrolysis, 1997, 40-41:201-215
[3] GB14232．1—2004／ISO3826—1：2003《人体血液及血液成分袋式塑料容器 第1部分：传统型血袋》
[4] 杨丽庭，高俊刚，李燕芳．改性聚氯乙烯新材料．北京：化学工业出版社，2002．458-468
[5] A. Marcilla, S. Garcia and J. C. Garcia-Quesada. Study of the migration of PVC plasticizers [J]. Analytical and Applied Pyrolysis, 2004, 71(2): 457-463
[6] Ignacio Aracil, Rafael Font and Juan A. Conesa.Thermo-oxidative decomposition of polyvinyl chloride Journal of Analytical and Applied Pyrolysis,74(1-2):215-223
[7] Mustafizur Rahman, Christopher S. Brazel. The plasticizer market: an assessment of traditional plasticizers and research trends to meet new challenges [J]. Progress in Polymer Science, 2004, 29(12): 1223-1248
[8] Minna Hakkarainen. New PVC materials for medical applications—the release profile of PVC/polycaprolactone- polycarbonate aged in aqueous environments[J]. Polymer Degradation and Stability, 2003, 80(3):451-458
[9] 杨天楹，杨成民，田兆嵩．临床输血学．第1版．北京：北医协和联合出版社，1993：442-445
[10] S. S. Hill, B. R. Shaw, A. H. B. Wu. The clinical effects of plasticizers, antioxidants and other contaminants in medical polyvinylchloride tubing during respiratory and non-respiratory exposure [J]. Clin Chim Acta, 2001(304):1-8
[11] Sjoberg P, Bondesson U, Sedin G, Gustafsson J. Disposition of di and mono-(2-ethylhexyl) phthalate in newborn infants subjected to exchange transfusions. Eur J Clin Invest.1985(15): 430-436
[12] Sjoberg PO, Bondesson UG, Sedin EG, Gustafsson JP. Exposure of newborn infants to plasticizers. Plasma levels of di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate during exchange transfusion. Transfusion. 1985(25): 424-428
[13] Plonait SL, Nau H, Maier RF, Wittfoht W, Obladen M. Exposure of newborn infants to di-(2-ethylhexyl)-phthalate and 2-ethylhexanoic acid following exchange transfusion with polyvinylchloride catheters. Transfusion. 1993(33): 598-605
[14] A. O. Earls, I. P. Axford and J. H. Braybrook. Gas chromatography-mass spectrometry determination of the migration of phthalate plasticisers from polyvinyl chloride toys and childcare articles. Journal of Chromatography A January 2003, 983(1-2): 237-246
[15] Dine, M. Luyckx, M. Cazin, C. Brunet, F. Goudaliez and J.C. Cazin, Rapid determination by high performance liquid chromatography of di(2-ethylhexyl) phthalate in plasma stored in plastic bags. Biomed.Chromatogr. 5 (1991): 94-97
[16] Doull, R. Cattley, C. Elcombe, B. G. Lake, J. Swenberg, C. Wilkinson, G. Williams and M. Van Gemert, A cancer risk assessment of di(2-ethylhexyl) phthalate: Application of the New US EPA risk assessment guidelines. Regul. Toxicol. Pharmacol. 29 (1999): 327-357
[17] Huber WW, Grasl-Kraupp B, Schulte-Hermann R. Hepatocarcino genicpotential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk. Crit Rev Toxicol. 1996(26): 365-481
[18] 奚廷斐，王春仁．邻苯二甲酸二异辛酯增塑聚氯乙烯医疗器械的安全性评价．中国医疗器械信息，2005，11(2)：27-29
[19] Francesca Nuti, Sibylle Hildenbrand, Mario Chelli. Synthesis of DEHP metabolites as biomarkers for GC-MS evaluation of phthalates as endocrine disrupters. Bioorganic & Medicinal Chemistry,16 May 2005, 13(10): 3461-3465
[20] Rie Ito, Fumie Seshimo, Yuji Haishima, et al. Reducing the migration of di-2-ethylhexyl phthalate from polyvinylchloride medical devices [J]. International Journal of Pharmaceutics 2005, 303 (1): 104-112
[21] Center for Devices and Radiological, U. S. Health, Food and Drug Administration: safety assessment of di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices. FDA report (2001) September
[22] Minna Hakkarainen. Qualitative and quantitative solid-phase micro extraction gas chromatographic-mass spectrometric determination of the low-molecular-mass compounds released from poly vinyl chloride/polycaprolactone-polycarbonate during ageing [J]. Chromatography A, 2003, 1010(1): 9-16
[23] K. Inoue, H. Okumura, T. Higuchi, et al. Characterization of estrogenic compounds in medical polyvinyl chloride tubing by gas chromatographymass spectrometry and estrogen receptor binding assay[J]. Clin Chim Acta, 2002(325): 157-163
[24] Koichi Inoue. Evaluation and analysis of exposure levels of di(2-ethylhexyl) phthalate from blood bags [J]. Clinica Chimica Acta, 2005, 358(1-2): 159-166
[25] 王立，汪正范，谋世芬等．色谱分析样品处理．第1版．北京：化学工业出版社，2002：94-97
[26] 郑和辉，赵立文等．饮用水中邻苯二甲酸酯的气相色谱—质谱测定法．环境与健康杂志，2005，9(5)：377—378
[27] Rie Ito, Fumie Seshimo, Naoko Miura, et al. High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry[J]. Pharmaceutical and Biomedical Analysis, 2005, 39(5): 1036-1041
[28] K. Inoue,M. Kawaguchi, F. Okada, Y. Yoshimura and H. Nakazawa, Column switching high-performance liquid chromatography electrospray mass spectrometry coupled with on-line extraction for the determination of mono and di-(2-ethylhexyl) phthalate in blood samples[J]. Anal Bioanal Chem, 2003(375): 527-533
[29] GB／T14233.2—2005《医用输液、输血、注射器具检验方法 第二部分：生物实验方法》
[30] GB／T16886.4-2005《医疗器械生物学评价第4部分：与血液相互作用实验选择》
[31] GB18469-2001《全血及成分血质量要求》
[32] B. D. Ratnar(Eeds). The blood compatibility catastrophe, J Biomed Mater Res. 1993 Mar, 27(3):283-287
[33] J.M.Ccourtney, X.Zhao and H.Qian. Biomaterials in cardiopulmonary bypass. Perfusion, 1999,14:263
[34] 郝和平．医疗器械生物学评价标准实施指南．中国标准出版社，2000：81—90
[35] 孙皎，顾国珍，钱云芳．《生物医学工程学杂志》2003，20(1)：8-10
[36] MTT mosmann T. Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65(1-2): 55-63