A novel method using formamide for the elution of antibodies from erythrocytes

BACKGROUND AND OBJECTIVES: Accurate identification of antibodies that sensitize red blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution method that does not alter their antigen-binding properties, and analysis of the eluates against a panel of RBCs. MATERIALS AND METHODS: A method was developed that allowed efficient RBC antibody elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and known antigen-antibody disruptive reagents were tested using these RBCs. The best reagent conditions were optimized. Eluates made were tested and compared to results obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient samples that were positive with direct antiglobulin tests (DATs), and in vitro commercial antisera-sensitized RBCs representing clinically significant antibodies, were used for evaluating the new method. RESULTS: The formamide method was efficient at removing antibodies from RBCs. The patient samples with a positive DAT had antibodies recovered with the same specificity when compared to the acid-based technique. The length of preparation time was similar for both formamide and acid-based methods. Results of testing the eluates made from reagent RBCs sensitized with commercial antisera were distinct with antigen-positive and -negative erythrocytes. CONCLUSIONS: The formamide method compares well with acid techniques and may be an alternative choice of elution method.     

Transmural atrial pacing in patients with postoperative congenital heart disease

INTRODUCTION: Some patients with postoperative congenital heart disease require permanent cardiac pacing, but the use of transvenous or epicardial pacing leads may be limited by type of cardiac malformation, venous connections, body size, or fibrosis. Transmural atrial pacing may provide an alternative in difficult patients, but to date has been described in only a few articles with small patient numbers, and data from lead performance are lacking. METHODS AND RESULTS: Records were reviewed in 18 consecutive patients (4 months to 21 years old) with postoperative congenital heart disease receiving transmural atrial pacing leads from July 1994 to December 1996. Implantation materials and techniques were described. Lead sensing and capture thresholds obtained acutely and during short-term follow-up (mean: 11.0 months) were evaluated, and comparisons were made between patients with postoperative Fontan anatomy and non-Fontan patients, and between patients receiving steroid-eluting and nonsteroid leads. Overall, the median acute sensing and capture thresholds of transmural leads were 4.1 m V and 0.7 V at 0.5 msec, respectively. Median follow-up thresholds were 2.8 m V and 0.8 V, respectively. Performance of leads in Fontan patients was similar to those in non-Fontan patients. Steroid-eluting leads had a chronic capture threshold of 0.6 V versus 0.9 V for nonsteroid leads (P = 0.038). CONCLUSION: Transmural atrial pacing leads were successfully implanted in patients with diverse ages and types of postoperative congenital heart disease. Lead performance was acceptable both acutely and during the first year of follow-up.     

Intervention Versus Observation in Symptomatic Patients With Normal Flow Low Gradient Severe Aortic Stenosis

Objectives

The purpose of this study was to describe patients with severe symptomatic aortic stenosis with normal flow and low gradients and determine whether they benefit from intervention.

Xeroderma pigmentosum patients from the federal Republic of Germany: Decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity

Summary A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the UV-like carcinogen (Ac)₂ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D₀) was calculated for each cell strain.For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E₀.Comparing D₀ with E₀ values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previouly shown (Fischer et al. 1982) that D₀ values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA.A considerable similarity in sensitivity towards both UV light and (Ac)₂ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV-and (Ac)₂ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - UV light ultraviolet light - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

The change of the free energy of ATP hydrolysis during global ischemia and anoxia in the rat heart. Its possible role in the regulation of transsarcolemmal sodium and potassium gradients

The timecourse of change of the cytoplasmic free energy of ATP hydrolysis during acute global ischemia and during anoxic perfusion was determined in the isolated rat heart. The timecourse of change of transsarcolemmal Na+ and K+ gradients during anoxia, and of extracellular K+ during ischemia were measured. The free energy of ATP hydrolysis was calculated from the equilibrium of the creatinekinase reaction, taking into account the pH-dependence of the equilibrium constant, and intracellular inorganic phosphate. In control aerobic hearts the mean free energy of ATP hydrolysis was 55.2 kJ/mol. Both during ischemia and anoxia it declines biphasically. The first rapid phase terminates within 4 min into a plateau of about 46 kJ/mol. The duration of this plateau is shorter during anoxia than during ischemia. The second phase of decrease starts after 6 to 8 min during anoxia and after 15 to 20 min during ischemia. After 30 min of anoxia the free energy of ATP hydrolysis has decreased to 31 kJ/mol and after 30 min of ischemia a value of 35.5 kJ/mol is reached. The timecourses of change of measured intracellular Na+ and K during anoxia and of extracellular K+ during ischemia were also biphasic. During anoxia the loss of intracellular K+ was almost equal to the gain of intracellular Na+ at any point. Based on the assumption that the sodium pump is in thermodynamic equilibrium or near-equilibrium during anoxia and ischemia, the time-course of change of Na+ and K+ gradients during anoxia and of extracellular K+ during ischemia were calculated from the respective timecourses of change of the free energy of ATP hydrolysis. Good agreement was observed between calculated and measured changes of Na+ and K+ gradients. It is concluded that the magnitude and direction of change of transsarcolemmal ion-gradients during anoxia and ischemia may be under direct thermodynamic control of myocardial energy metabolism.     

探讨肝硬化测定肝静脉压梯度和门静脉血管阻力与食管静脉曲张再出血之关系

对有食管静脉曲张 (EV)出血后 ,肝硬化并门静脉高压之患者 2 1例 ,测定肝静脉压梯度 (HVPG)为 (2 2 6± 0 34)kPa;继之对其中之 18例又测门静脉血管阻力 (PVR)为 (0 2 3± 0 0 6 )kPa·ml 1·min·kg。治疗分两组 :手术组(n =10 )和介入组 (n =11) ,前者其HVPG和PVR之两值显著低于后者 ,随访 18个月内再出血率 ,手术组 (30 % )显然低于介入组 (5 4 6 % ) ,但两组之再出血例如以两值中位数相对比则无差异 ,均发生于高值者。再出血例与未再出血例就两值相对比 ,前者显著高于后者 ,差异非常显著 ,PVR尤著 (P <0 0 0 1)。本研究提示HVPG >2 33kPa ,PVR >0 2 2kPa·ml 1·min·kg是EV再出血危险的域值。HVPG与PVR呈正相关。     

血清-腹水白蛋白梯度与食管-胃底静脉曲张破裂出血的关系

目的 探讨血清-腹水白蛋白梯度（Serum-ascites albumin gradient,SAAG）与门脉高压的关系,评价SAAG对预测门脉高压性食管-胃底静脉曲张破裂出血的价值.方法 测定29例肝硬化腹水患者SAAG值并行胃镜检查以了解有无食管-胃底静脉曲张,以11 g/L为界将29例患者分为高SAAG组及低SAAG组,采用Pearson相关分析法分析SAAG与食管-胃底静脉曲张之间的关系.29例患者中并发食管-胃底静脉曲张破裂出血者8例,非出血者21例,测定两组患者的SAAG值并进行比较.结果 26例高SAAG患者中17例有食管-胃底静脉曲张,而3例低SAAG患者均无食管-胃底静脉曲张.SAAG与食管-胃底静脉曲张存在正相关关系（r=0.40,P＜0.05;r=0.84,P＜0.01）;8例出血组患者其SAAG值为（24.24±7.18）g/L,21例非出血组患者其SAAG值为（18.27±6.06）g/L,两组比较差异有显著性（P＜0.05）.结论 SAAG与门脉压力密切相关且对预测门脉相关性食管-胃底静脉出血有一定临床价值.     

Automated Algorithm Using Pre-Intervention Fractional Flow Reserve Pullback Curve to Predict Post-Intervention Physiological Results

ObjectivesThis study sought to develop an automated algorithm using pre-percutaneous coronary intervention (PCI) fractional flow reserve (FFR) pullback recordings to predict post-PCI physiological results in the pre-PCI phase.BackgroundBoth FFR and percent FFR increase measured after PCI showed incremental prognostic implications. However, there is no current method to predict post-PCI physiological results using physiological assessment in the pre-PCI phase.MethodsAn automated algorithm that analyzes instantaneous FFR gradient per unit time (dFFR(t)/dt) was developed from the derivation cohort (n = 30). Using dFFR(t)/dt, the pattern of atherosclerotic disease in each patient was classified into 3 groups (major, mixed, and minor FFR gradient groups) in both the internal validation cohort with constant pullback method (n = 234) and the external validation cohort with nonstandardized pullback methods (n = 252). All patients in the validation cohorts underwent PCI on the basis of pre-PCI FFR ≤0.80. Suboptimal post-PCI physiological results were defined as both post-PCI FFR <0.84 and percent FFR increase ≤15%. From the derivation cohort, cutoffs of dFFR(t)/dt for major and minor FFR gradient were 0.035/s and 0.015/s, respectively.ResultsIn validation cohorts, dFFR(t)/dt showed significant correlations with percent FFR increase (R = 0.801; p < 0.001) and post-PCI FFR (R = 0.099; p = 0.029). In both the internal and external validation cohorts, the major FFR gradient group showed significantly higher post-PCI FFR and percent FFR increase compared with those in the mixed or minor FFR gradient groups (all p values <0.001). The proportions of suboptimal post-PCI physiological results were significantly different among 3 groups (10.4% vs. 25.8% vs. 45.7% for the major, mixed, and minor FFR gradient groups, respectively; p < 0.001) in validation cohorts. Absence of major FFR gradient lesion (odds ratio: 2.435, 95% [CI]: 1.252 to 4.734; p = 0.009) and presence of minor FFR gradient lesion (odds ratio: 2.756, 95% confidence interval: 1.629 to 4.664; p < 0.001) were independent predictors for suboptimal post-PCI physiological results.ConclusionsThe automated algorithm analyzing pre-PCI pullback curve was able to predict post-PCI physiological results. The incidence of suboptimal post-PCI physiological results was significantly different according to algorithm-based classifications in the pre-PCI physiological assessment. (Automated Algorithm Detecting Physiologic Major Stenosis and Its Relationship with Post-PCI Clinical Outcomes [Algorithm-PCI]; NCT04304677)     

三维扫描定制压力袜结合康复训练对老年患者下肢水肿的疗效观察

目的:评价三维扫描定制压力袜、传统市售医用压力袜在老年下肢水肿患者中的康复疗效。方法:将60例老年下肢水肿患者按随机数字表分为观察组和对照组,每组30例,两组患者均予常规康复治疗(包括手法牵拉肌肉、向心性肢体按摩、无痛范围内关节活动等训练)。对照组在常规康复基础上患肢穿戴市售批量生产传统医用压力袜,观察组在常规康复基础上穿戴三维扫描定制梯级压力袜,每天均穿戴16h。于治疗前、治疗3d、7d、14d、28d时分别采用圆桶溢水测量法测患肢下肢体积、采用Biomaster评估系统测关节活动度、语义差异标尺法评测压力舒适性。结果:①观察组在治疗7d、14d、28d时患肢体积减少值与对照组相比有明显差异(P0.01),观察组随治疗时间延长患肢体积减少值依次增加(P0.05),对照组不同治疗时间点间无明显差异(P0.05);②观察组在治疗7d、14d、28d时患肢踝关节被动背屈活动度增加值与对照组相比有明显差异(P0.05—0.001),观察组在治疗14d、28d时患肢踝关节被动跖屈活动度增加差值与对照组相比有明显差异(P0.001),观察组随治疗时间延长患肢踝关节背屈和跖屈被动活动度增加值依次升高(P0.001),对照组不同治疗时间点间无明显差异(P0.05);③观察组在治疗7d、14d、28d时压迫感评分明显高于对照组(P0.05),在治疗3d、7d、14d、28d时舒适感评分高于对照组(P0.001)。对照组随治疗时间延长压迫感和舒适度均依次降低(P0.05),观察组不同时间点间无明显差异(P0.05)。结论:三维扫描定制梯级压力袜能持续作用于水肿肢体,减轻下肢水肿的同时改善关节活动度,舒适安全有效。     

一种新型肠上皮干细胞分离方法的建立

目的 探讨密度梯度离心法作为一种新型肠上皮干细胞分离方法的可行性.方法 通过腹腔注射大剂量5-氟尿嘧啶(5-FU)制备肠黏膜严重损伤小鼠模型,取出损伤的肠黏膜消化成单细胞悬液,利用Percoll密度梯度离心法分离细胞体积大小不同的细胞群.HE染色观察各群细胞形态特征,免疫细胞化学及RT-PCR检测各群细胞musashi-1(msi-1)的表达.结果 分离的大部分细胞位于50%和70%密度梯度的Percoll分离液体层,50%密度梯度分离液体层中的细胞符合干细胞的形态特征,免疫细胞化学检测其msi-1表达阳性率约为93%,RT-PCR显示该群细胞msi-1 mRNA表达较强.结论 建立了一种简便、有效的分离具有活性的肠上皮干细胞的方法.