【作者】 沈晓霞；

【导师】 杜立中；

【作者基本信息】 浙江大学 ， 儿科学， 2010， 博士

【摘要】 早产儿尤其是超低出生体重儿(extremely low birth weight, ELBW)因医源性失血和促红细胞生成素不足,出生后不久即发生贫血,约半数以上的贫血患儿都曾在住院期间接受过输血。输血作为一种特殊、基本的治疗手段,在临床医疗工作中发挥非常重要作用。输血在挽救生命、改善病情的同时,红细胞的保存病变所带来的临床并发症及不良反应也越来越受到人们的关注。红细胞的保存病变除了传统认识如三磷酸腺苷(adenosine triphosphate.ATP)、2,3-二磷酸甘油酸(2,3-diphosphoglycerate,2,3-DPG)的丢失,双凹圆盘形态的改变及免疫调节外,最近有研究发现红细胞保存过程中另一个重要功能的丢失----NO生物活性的丢失。NO作为一种重要的内皮源性舒张因子,与血液尤其是贮存红细胞输注的有效性和安全性有着密切关系,可以通过蛋白质巯基的硝基化来实现其功能。亚硝基硫醇(nitrosothiols, RSNO)是含有硫醇物质包括蛋白质或一些小分子硝基化的产物,是NO在体内主要贮存体和载体。在红细胞内NO可以和血红素上的铁或半胱氨酸残基(Cys93)结合形成亚硝基硫醇血红蛋白(S-nitrosohemoglobin, SNO-Hb)。有研究指出一旦红细胞离开机体,红细胞内NO的生物活性及SNO-Hb就会立即迅速下降,并且在保存期限内持续下降。亚硝基硫醇浓度的变化和一系列生理病理的改变有关。此外,红细胞保存过程中受氧化应激的影响,产生活性氧簇(reactive oxygen species, ROS),通过脂质过氧化等方式对细胞造成损伤。临床研究中发现输血有助于早产儿体重恢复,有利于病情恢复如减少呼吸暂停的发作次数或改善机械通气患儿的氧合。但越来越多的证据提示输血与重症监护室死亡率升高有关,并且随着保存期延长,这种关系更为紧密。在接受心脏外科手术且接受输血的病人中发现,接受陈旧血液的病人要比接受新鲜血液病人的死亡率高,同时临床并发症更多。比较限制性输血与非限制性输血不同策略时,限制性输血在减少输血次数的同时,在住院时间、发病率及死亡率等与非限制性输血无明显差异。临床上对早产儿输血前后亚硝基硫醇浓度的变化和一系列生理病理的改变关系尚无研究报道。本研究以贫血且接受输血的早产儿为研究对象,探讨输血前后早产儿外周血RSNO变化,同时观察环鸟甘单磷酸(cyclic guanosine monophosphate,cGMP)及ROS变化,以及对临床预后转归的影响,对输血的有效性及安全性进行探讨,为制定可以的输血策略,改良红细胞保存方式提供依据。第一部分输血对早产儿红细胞NO生物活性的影响目的：1.观察输血前后,早产儿动脉血亚硝基硫醇浓度的变化与NO代谢产物硝酸盐(nitrate,NO3-)及亚硝酸盐(nitrite,NO2-)的改变,了解输血前后体内NO储存的变化。2.观察输血对NO／cGMP通路的影响。3.通过检测丙二醛(Malondialdehyde,MDA)及超氧化物歧化酶(superoxide dismutase,SOD)的变化来观察探讨输血前后ROS的变化。方法：1.研究对象的选择以首次入住我院NICU,胎龄<35周,住院期间接受输血的早产儿30例为研究对象。患儿有免疫性溶血性疾病、先天性心脏病、其他重大出生缺陷导致需外科手术、染色体异常、有急性失血病史、因经济或其他原因中途放弃治疗、转入我院前在外院治疗有输血病史及参加其他临床研究可能对本研究结果有影响的早产儿不列入研究范围。输血量以15-20ml／kg于4-5小时内输入。2.血标本的采集对满足入选标准的贫血早产儿,在输血前4小时内,输血完成后2小时内,输血后24小时时收集动脉血标本,分别用肝素及乙二胺四乙酸(Ethylene Diamine Tetraacetic Acid,EDTA)抗凝,血标本采集30min内离心,红细胞内加入红细胞稳定液,将血浆和红细胞分别避光液氮保存。3.用纳米金电化学法检测贫血早产儿输血前4小时内,输血后2小时内,输血后24小时时动脉血血浆及红细胞中RSNO的变化。用硝酸盐还原酶法检测输血前4小时内、输血后2小时内、24小时,贫血早产儿动脉血血浆N03-与N02-浓度的变化。硫代巴比妥法检测MDA,黄嘌呤氧化酶发检测SOD活性,用酶联免疫吸附反竞争法(enzyme linked immunosorbent assay, ELISA)检测cGMP,观察输血前4小时内、输血后2小时内及输血后24小时血浆内的变化。结果：1.输血后2小时内血浆及红细胞RSNO比输血前显著下降(血浆：850.55±334.14pA vs 1220.0±323.2pA,P<0.05；红细胞：14.35±4.72 nA／mmo vs 21.4±6.35nA／mmol,P<0.05)。输血后2小时内、24小时血浆内N03-与N02-之和与输血前4小时相比均有显著下降(P<0.01)。输血后24小时红细胞内RSNO为(19.34±5.77)nA／mmol,与输血后2小时内相比,有上升趋势,但两者无显著性差异。与输血前水平略低,但差异无统计学意义。输血后24小时RSNO水平在(918.73±321.58)pA,明显低于输血前水平,两者差异有统计学意义(P<0.05)。与输血后2小时内相比有上升趋势,但两者差异无统计学意义。2.cGMP变化：输血前4小时内血浆内cGMP水平在(150.85±26.09)pmol／ml,输血后2小时内和24小时分别为(146.83±23.06)pmol／ml和(150.43±23.12)pmol／ml,输血前与输血后相比无显著性差异。3.MDA和SOD变化：输血前血浆内MDA水平在(5.66±2.87)nmol／ml,输血后2小时内升至(6.73±3.32)nmol／ml,与前者相比两者有显著性差异(P<0.05)。输血后24小时血浆MDA水平在(6.50±3.83)nmol／ml,与输血前相比平均水平虽有增加,但两者差异无统计学意义。与输血2小时相比两者无显著性差异输血前血浆内SOD水平在(100.00±10.44)U／ml,输血后2小时内SOD下降至(92.81±10.11)U／ml,与前者相比,两者有显著性差异(P<0.05)。输血后24小时血浆内SOD水平逐渐上升至(99.42±12.87)U／ml,与输血后2小时内相比有显著性差异(P<0.05)；与输血前水平相当,无显著性差异。结论：1.输血后会引起血浆及红细胞NO储存迅速下降,但可以随着时间延长逐渐恢复到原来水平。2.输血后NO/cGMP通路未明显变化。提示NO储备虽有减少,但NO/cGMP通路可能不受输血影响。3.输血会造成暂时性氧化应激损伤,输血后2小时,血浆内MDA升高,血浆总SOD活力下降,输血后24小时均有不同程度的改善。第二部分输血对贫血早产儿临床血流动力学变化目的：1.观察输血前后血压、心率、脉搏等生命体征的变化2.观察输血前后氧合、呼吸机参数以及血乳酸的变化3.观察输血前后心输出量的变化方法：1.研究对象的选择以首次入住我院NICU,胎龄<35周,住院期间接受输血的早产儿30例为研究对象。2.排除标准1)有明显的感染症象；2)病情严重,随时有生命危险；3)有免疫性溶血性疾病；4)先天性心脏病,包括动脉导管未闭者；5)其他重大出生缺陷导致需外科手术；6)染色体异常者或其他畸形；7)有急性失血；8)因经济或其他原因中途放弃治疗者；9)患儿参加其他临床研究可能对本研究结果有影响者。3.观察指标输血前及输血后24小时内血压、呼吸、脉搏、体温等生命体征的变化；观察输血前及输血后24小时内氧合、呼吸机参数以及乳酸变化等；观察10例输血前及输血后24小时心输血量的变化。结果：1.观察30例早产儿中,需机械辅助通气10例,CPAP依赖3例,面罩或鼻导管吸氧10例,可自主呼吸不依赖氧气7例。对其中10例早产儿观察其输血前后心输血量的变化。2.输血后24小时心率明显下降,从原来的(150.63±10.87)次／分,下降至146.75±13.0次／分(P<0.05)。输血前后呼吸、血压均无显著性变化。3.输血后24小时内患儿氧合情况改善不明显,呼吸机使用参数,乳酸均无显著性变化。4.输血前及输血后24小时,心输血量虽有下降趋势,从输血前的250.4±71.3ml／kg／min下降为输血后的239.1±64.4ml/kg／min,但两者相比无统计学意义。结论：1.输血后心率明显下降,但对其他血液动力学指标无明显影响。2.输血对早产儿氧合情况改善不明显。第三部分不同输血策略对贫血早产儿临床预后的影响目的：1.通过回顾性研究比较限制性输血组与非限制性输血组的患儿院内感染、住院天数、死亡率等一些临床预后情况。方法：1.研究对象的选择对2004年至2008年首次入住我院NICU,体重〈1500g,住院时间超过2周且住院期间接受输血的早产儿为研究对象。排除标准同实验第一部分。最后入选130名例,其中限制性输血组57例,非限制性输血组73例。2.分组标准限制性输血组的各输血阈值根据美国2008年早产儿输血指南,非限制性输血组则在限制性输血阈值基础上结合患儿的实际情况及目前国内输血策略适当提高Hct阈值。对于一般病情稳定,不需要辅助通气,限制性非限制性分别为Hct<21%和<30%；对于需要吸氧,频发呼吸暂停需要气囊复苏,或心率>180次／min或呼吸>80次／min持续24 h以上,或体重增加不理想(<10g／d)的患儿,限制性输血组要确保Hct>31%,非限制性组>38%；对于需要CPAP或机械辅助通气的患儿,限制性输血组和非限制性输血组的输血阈值Hct分别为36%和46%。3.观察指标①患儿的基本情况：包括患儿的胎龄,性别,出生体重,apgar评分,入院Hct,是否辅助通气、多胎等。②患儿的输血情况：入院基础Hct,输血前Hct,输血量,输血次数,首次输血时日龄。③观察指标：输血前后血压、呼吸、脉搏、体温等生命体征以及呼吸机的使用参数的变化等；住院天数,死亡率,院内感染率,呼吸暂停次数及程度的变化,氨茶碱的使用,体重恢复,PDA是否重新开放,输血反应等。结果：1.两组患儿的基本情况相似,出生体重、孕周、Apgar评分,入院Hct均差异无统计学意义(P>0.05)。2.限制性输血组患儿接受输血前的Hct为(29.7±5.56)%,要明显低于非限制性输血组患儿(36.5±6.43)%(P<0.01)；限制性输血患儿和非限制性输血患儿每次接受输血量相当(23.7±6.1 vs 23.3±7.7,P>0.05),接受输血总量虽有增加(0.63±0.41 VS 0.56±0.54),但差异无统计学意义(P>0.05)。3.非限制性输血患者机械辅助通气天数比限制性输血组明显减少(5.9±4.7 vs7.5±6.7,P<0.05)。在院内感染、住院天数、死亡率、体重恢复、呼吸暂停次数及程度等方面两种输血策略无显著性差异。4.在限制性输血患几中发现有Ⅲ、Ⅳ度出血4例,脑白质软化3例,而在非限制性输血患儿则均只有1例。结论：1.非限制性输血可显著减少极低体重儿机械通气时间,可能更利于临床恢复,临床治疗上不要一味采取保守输血策略。2.限制性输血对神经系统是否有影响尚不能确定。更多还原

【Abstract】 Preterm infants, especially those with extremely low birth weight (ELBW) rapidly become anemic as a result of phlebotomy loss and inadequate erythropoiesis (EPO) Nearly 50% will receive at least one blood transfusion by the end of their hospitalization. Red blood cell(RBC) transfusion as a special, basic treatment, play a key role in therapeutic intervention. Used correctly, transfusion may be lifesaving. However, there is a growing appreciation that storage has associate with a negative effect on the recipient.The traditional view of storage lesions include the decrease of adenosine triphosphate(ATP)and 2,3-diphosphoglycerate(2,3-DPG),loss of RBC shape and immunomodulation. Now people find another "storage lesion"—depletion of vasodilator nitric oxide (NO).NO is an important endothelial relaxing factor. Recent studies have paid more attention to the nitrosylation of the proteins in the regulation of nitric oxide homeostasis, which is a cyclic guanosine monophosphate(cGMP) undependent signaling pathway. Nitrosothiols (RSNO) are the products of nitrosylation of protein and low molecular thiols and represent a means either for the storage or transport of NO. Hemoglobin binds NO in at least two distinct sites, the direct attachment of NO to the heme iron or at the cysteine residue (Cysp 93) as s-nitrosylated haemoglobin or S-nitrosohemoglobin(SNO-Hb). It has reported that when RBCs leave the body during blood-banking donations, nitric-oxide bioactivity and S-nitrosohemoglobin in RBCs begin breaking down almost immediately.The change in nitrosothiols concentration is associated with a series of pathophysiology processes. Oxidative damage to RBCs during storage including increase of reactive oxygen species (ROS) is a well-described mechanism contributing to the storage lesion through lipid peroxidation.Several studies have shown decreased frequency of apnea, improved oxygenation and faster weight gain after transfusion of anemic infants. There is growing evidence correlating RBC transfusion with increased mortality rates in the intensive care unit(ICU). Importantly, multiple studies have suggested this association becomes stronger with increasing duration of RBC storage. Patients following cardiac-surgery who were transfused with "aged blood" had an increased risk of postoperative complications and mortality and reduced chance for survival comparing with"fresh blood" transfusion. Using a restrictive transfusion strategy is as safe as using a liberal transfusion without causing hospitalization, mortality and morbidity increased, and can decrease the number of patients exposed to RBC transfusions.However, the role of blood transfusion in regulating NO pathway and clinical outcomes has not yet been clearly addressed in preterm infants with anemia. In this study, we select preterm with anemia as study subjects to investigate the change of RSNO before and after transfusion. We also observed the change of NO/cGMP pathway and ROS. The aim of this study was to weigh the safety and efficacy of blood transfusion to make the optimal decision. Part I The Influences of Blood transfusion on RBC NO bioactivity in preterm infantsAim:1. To investigate the changes of RSNO in plasma and red blood cell, and the changes of NO metabolites nitrate (NO3-) and nitrite(NO2-) in plasma before and after blood transfusion in preterm infants with anemia.2. To analyze NO/cGMP pathway before and after blood transfusion.3. To investigate the role of blood transfusion in redox equilibration by evaluating malondialdehyde (MDA) and superoxide dismutase (SOD) concentration.Methods:1. Study subjectsIn this study we enrolled 30 hospitalized preterm infants(gestational age< 35 weeks)admitted for the first time to our hospital, who were transfused during hospital stay. Infants were excluded if they had alloimmune hemolytic disease, congenital heart disease (including patent ductus arteriosus), other major birth defect requiring surgery, or a chromosomal abnormality. They also were excluded if they stopped therapy for economic or other reasons, or they had received transfusions before they could be enrolled. Infants participating in other clinical studies were excluded if their participation had the potential to interferewith this study by affecting its conduct or outcome.2. Blood collectionBlood(2mL) was collected in foil-wrapped vacutainers containing heparin and lmL was collected with Ethylene Diamine Tetraacetic Acid(EDTA) before and after transfusion (2h,24h).After centrifugation at 750g for 10minutes at 4℃within 30min of collection,100ul of RBC pellet were put into 900ul of stabilization solution. Serum and RBC were stored in liquid nitrogen without light exposure.3. The levels of RSNO in plasma and RBC were detected by electrochemical method using gold nanoparticles. The concentration of blood NO2-and NO3-was analyzed before and after transfusion by nitrate reductiase method. The change of cGMP was assessed by enzyme linked immunosorbent assay(ELISA). Thiobarbituric acid method and hydroxylamine method were used for the evaluation of oxidative stress parameters (MDA, SOD).Result1. The levels of RSNO in plasma and RBC decreased significantly 2h post-transfusion (plasma:850.55±334.14pA vs 1220.0±323.2pA, P<0.05; RBC:14.35±4.72 nA/mmol vs 21.4±6.35 nA/mmol, P<0.05). Twenty four hours after transfusion,RSNO in plasma was (918.73±321.58) pA,which was still lower than that before tansfusion(P <0.05),while RSNO in RBC increased gradually but showed no difference.The concentration of nitrite/nitrate decreased significantly in plasma after transfusion(P<0.01).2. The change of cGMP:The level of cGMP was (150.85±26.09) pmol/ml before transfusion and were (146.83±23.06) pmol/ml and (150.43±23.12) pmol/ml respectively 2h and 24h post-transfusion. There was no difference in the level of cGMP before and after transfusion.3.The change of MDA and SOD levels:The concentration of MDA in plasma increased significantly 2 h post-transfusion (6.73±3.32nmol/ml vs 5.66±2.87 nmol/ml, P <0.05),and decreased markedly 24 h post-transfusion comparing with 2h post-transfusion(P<0.05). SOD levels were decreased significantly 2 hours following the transfusion (92.81±10.11U/ml vs 100.00±10.44 U/ml, P<0.05) and returned to pre-transfusion level by 24 hours after transfusion. Conclusions:1.Transfusion of stored blood is likely to reduce the level of NO store both in plasma and RBC, which may gradually return to the normal level.2.The NO/cGMP was not affected by transfusion.3. Increased the concentration of MDA and decreased the activity of SOD suggest that the recipient may suffer from the oxidative damage after transfusion.PartⅡThe Influences of Blood Transfusion on Hemodynamics in preterm InfantsAim:1. To investigate the change of vital signs such as blood pressure, heart rate and respiratory rate before and after blood transfusion.2. To investigate the change of oxygenation, ventilation indexes and plasma lactic acid before and after transfusion.3. To investigate the influences of blood transfusion on cardiac output.Methods:1. Study subjectsIn this study we enrolled 30 hospitalized preterm infants(gestational age< 35 weeks)admitted for the first time to our hospital, who were transfused during hospital stay.2. Exclusion criteria 1) evidence of infection. 2) face imminent death. 3) alloimmune hemolytic disease. 4) congenital heart disease, including patent ductus arteriosus. 5) other major birth defect requiring surgery. 6) chromosomal abnormality. 7) acute blood loss. 8) stop therapy for economic or other reasons. 9) participate in other clinical studies may have the potential to interfere with this study by affecting its conduct or outcome.3. MonitoringEach infant had 2 sets of vital signs and echocardiographic evaluations:at 1to 2 hours before and 24 hours after transfusion. The following data were collected:heart rate (HR) in beats/min; respiratory rate (RR) in breaths/min; systolic, diastolic, and mean arterial blood pressures (SBP, DBP, and MBP) in mmHg; oxygenation, ventilation indexes and lactic acid; left ventricular output (LVO) in ml/min/kg.Results:1. In the 30 enrolled infants:10 infants received ventilator support,3 infants were on CPAP support,10 infants needed oxygen supplementation.The changes of cardiac output were observed in 10 infants.2. The heart rate decreased significantly from 150bpm before transfusion to 146 bpm after transfusion (P<0.05). Respiratory rate and blood pressures were not statistically different before and after transfusion.3. The oxygenation, ventilation indexes and plasma lactic acid were not changed after transfusion.4.Left ventricular output decreased from 250.4±71.3 ml/kg/min to 239.1±64.4ml/kg/min in comparison with that before transfusion, but no statistical differences.Conclusions:1. Blood transfusion can decrease heart rate in preterm infants. 2. There is no significant improvement in oxygenation after transfusion in preterm infants.3. The effect of blood transfusion on cardiac output and other hemodynamic parameters in preterm infants should be evaluated.PartⅢComparison of Clinical Outcomes by Different Transfusion Strategies in PretermAim:To systematically evaluate the risks and benefits with two transfusion strategies (liberal-transfusion or restrictive-transfusion).Methods:1. Study subjectsIn this retrospective cohort study, we enrolled 130 hospitalized preterm infants(birth weight< 1500g)admitted within the first 24 hours after birth from 2004 to 2008,who were transfused during hospital stay and whose lengths of stay were over two weeks. The infants were assigned to either the liberal group(n=57),with higher hematocrit levels,or the restrictive-transfusion group (n=73),with lower hematocrit levels. Exclusion criteria were seen in PartⅠ.2. Group CriteriaWe use the U.S. Blood transfusion guidelines as the lower transfusion threshold levels in preterm infants. If requiring neither positive pressure nor oxygen, infants in the liberal- and restrictive-transfusion groups received an RBC transfusion if their hematocrit levels fell to 30% and 21%, respectively. While receiving supplemental oxygen or HR>180bpm or RR>80bpm or with poor weight gain(<10g/d), their hematocrit levels were kept at>38% and>31%, respectively. While tracheally intubated for assisted ventilation or continuous positive airway pressure(CPAP) support, the hematocrit were kept at>46% and>36%, respectively.3. Monitoring①Demographic Characteristics:gestational age; sex; birth weight; Apgar scores; multiple birth.②RBC transfusion:initial hematocrit, hematocrit before transfusion, the number of transfusion, and the volume of transfusion.③Clinical outcomes:duration on ventilator, CPAP or supplemental oxygen, time to regain birth weight,length of hospitalization, BPD,ROP,and survived to discharge.Results:1. The demographic characteristics of the infants in the 2 treatment groups were similar.2. Infants in the restrictive-transfusion group had lower hematocrit than in liberal-transfusion group(29.7±5.56% vs 36.5±6.43%,P<0.01).3. Infants in liberal-transfusion group had shorter duration of mechanical ventilation than in restrictive-transfusion group (mean±SD:5.9±4.7 vs 7.5±6.7,P<0.05). No significant differences were found in other outcomes, including apnea or nosocomial infections between two groups.4. Infants in the restrictive-transfusion group were more likely to have periventricular leukomalacia(3 infants), which were only one infant found in liberal-transfusion group.Conclusions:1. The study suggests the possible benefits from liberal-transfusion for decreasing the duration of ventilatory support in preterm infants (<1500g). At least for decreasing the ventilation support, the liberal transfusion regime may be helpful for preterm infants.2. Whether restrictive transfusion would affect the outcome of central nervous system in preterm infants needs further study.更多还原