Effects Of Blood Transfusion On RBC NO Bioactivity And Hemodynamics In Preterm Infants

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.