The preparation of leukocyte-poor red blood cells: a comparative study

Thirteen methods of prepararing leukocyte-poor red blood cells were compared for their effectiveness in removing leukocytes. When the starting material was six to ten days old, the residual leukocyte count was three to four times lower than when fresh material was used and no granulocytes could be found. The most effective manual procedure was a single upright sedimentation of six- to ten-day-old saline-diluted red cells. Inverted sedimentation was no more effective in leukocyte depletion than upright sedimentation. Automated cell processors were no more effective than several of the better manual methods. Freezing and deglycerolizing resulted in a ten-fold improvement in leukocyte removal with less red blood cell loss but at higher cost than the nonfreezing methods. The study indicates that unless the minimal leukocyte count of frozen red blood cells is essential, a single upright sedimentation of saline-diluted, six- to ten-day-old red blood cells is currently the most effective procedure for preparing leukocyte-poor red blood cells.     

A gene transfer strategy for making bone marrow cells resistant to trimetrexate

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y- containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.     

Value of a new stick-type rapid urine test for the diagnosis of Helicobacter pylori infection in the Vietnamese population

AIM:To assess the value of a new test for the diagnosis of Helicobacter pylori(H.pylori) infection,Rapirun H.pylori Antibody Stick(Rapirun Stick),in a Vietnamese population.METHODS:Eligible patients without previous history of H.pylori eradication were recruited.Rapid urease test(RUT) and histologic examination were used to diagnose the H.pylori infection.Patients were considered H.pylori positive when the RUT results were positive and/or the bacteria were detected histologically.Rapirun Stick tests were performed using urine samples,and the results were compared with the other 2 methods.RESULTS:We enrolled 200 patients with a mean age of 36(range,18-76) years.There were 116 females and 84 males.Of the 200 patients,111(55.5%) were diagnosed as being H.pylori positive.The sensitivity,specificity,and accuracy of the Stick test were 84.7%,89.9%,and 87.0%,respectively.There were 17(8.5%) falsenegative patients and 9(4.5%) false-positive patients.CONCLUSION:The Rapirun Stick test has high sensitivity,specificity,and accuracy for the diagnosis of H.pylori infection in the Vietnamese population.The test can be clinically applied in Vietnamese populations.     

Effectiveness of a prospective physician self-audit transfusion- monitoring system

BACKGROUND: The purpose of this study was to search for a more effective transfusion-monitoring system than the existing system of retrospective peer review. STUDY DESIGN AND METHODS: This research used a study-control, preintervention and postintervention design, to evaluate the effectiveness of a prospective physician self-audit transfusion-monitoring system that functioned without the direct involvement of transfusion service physicians. This research also evaluated the effectiveness of issuing to physicians a memo with transfusion guidelines. Three process indicators were used to assess physician behavior at various stages of the blood-ordering process: 1) the number of crossmatches ordered per admission, 2) the transfusion-to- crossmatch ratio, and 3) the number of blood units returned to the laboratory after physician self-auditing. The study used two outcome indicators to reflect overall blood utilization: 1) the percentage of patients who received red cell transfusions and 2) the number of blood units transfused per recipient each month. RESULTS: The prospective physician self-audit system implemented at the study hospital did not reverse physician transfusion decisions, and the process of issuing to physicians a memo with transfusion guidelines at the control hospital failed to reduce blood usage. However, a transient reduction in blood utilization was observed at the study hospital. CONCLUSION: The reduction was hypothesized to be due to a Hawthorne effect, in which observed behavior is affected by the subject's awareness of the research study.     

Identification of dihydropteridine reductase in human platelets

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.     

Cortical cerebral microinfarcts on 7T MRI: Risk factors,neuroimaging correlates and cognitive functioning – The Medea-7T study

We determined the occurrence and association of cortical cerebral microinfarcts (CMIs) at 7 T MRI with risk factors, neuroimaging markers of small and large vessel disease, and cognitive functioning. Within the Medea-7T study, a diverse cohort of older persons with normal cognition, patients with vascular disease, and memory clinic patients, we included 386 participants (68 ± 9 years) with available 7 T and 1.5 T/3T brain MRI, and risk factor and neuropsychological data. CMIs were found in 10% of participants and were associated with older age (RR = 1.79 per +10 years, 95%CI 1.28–2.50), history of stroke or TIA (RR = 4.03, 95%CI 2.18–7.43), cortical infarcts (RR = 5.28, 95%CI 2.91–9.55), lacunes (RR = 5.66, 95%CI 2.85–11.27), cerebellar infarcts (RR = 2.73, 95%CI 1.27–5.84) and decreased cerebral blood flow (RR = 1.35 per −100 ml/min, 95%CI 1.00–1.83), after adjustment for age and sex. Furthermore, participants with >2 CMIs had 0.5 SD (95%CI 0.05–0.91) lower global cognitive performance, compared to participants without CMIs. Our results indicate that CMIs on 7 T MRI are observed in vascular and memory clinic patients with similar frequency, and are associated with older age, history of stroke or TIA, other brain infarcts, and poorer global cognitive functioning.     

胚胎嗅鞘细胞移植治疗脑性瘫痪：4例术后4周结果报告

目的:观察胚胎嗅鞘细胞移植治疗脑性瘫痪的有效性和安全性。方法:①病例资料:4例因出生时缺血缺氧确诊为脑性瘫痪的患者,男2例,女2例,年龄分别为14岁、岁、个月、岁。嗅92817鞘细胞由北京市虹天济神经科学研究院细胞中心提供,实验经医学伦理委员会批准,4例脑性瘫痪患者均签署知情同意书。②实验方法:根据术前MRI或CT片,患者均在局麻下行微创立体定向嗅鞘细胞移植术,选取双额放射冠为注射靶点,每侧注射1.0×106个细胞。术后给予止血、抗感染、康复等常规处理。③实验评估:分别于嗅鞘细胞移植前、移植后4周采用脑瘫综合功能评定量表、脑瘫日常生活能力量表评价患者神经功能及生活质量的改善。结果:①嗅鞘细胞移植术后4周,4例患者较术前均有不同程度的神经功能改善,未出现手术并发症。②脑瘫综合功能评定总分:病例1由92.5分增至94分,病例2由55分增至56分,病例3由10.5分增至11.5分,病例4由9.5分增至13分。③脑性瘫痪日常生活能力量表总分:病例1由82.0分增至83.5分,病例2无变化,为16.5分,病例3由5.0分增至7.5分,病例4由5.0分增至8分。结论:嗅鞘细胞移植治疗脑性瘫痪患者近期评价安全可行,可部分改善神经功能与生活质量,长期效果有待进一步随访。     

脂肪干细胞在体外特定培养液中向软骨细胞表型的分化

目的:应用转化生长因子β1,体外诱导脂肪干细胞向成软骨细胞表型分化,探讨其作为组织工程化软骨种子细胞的可行性。方法:实验于2005-04/2006-06在华中科技大学同济医学院公卫实验室完成。①取大鼠腹股沟处脂肪,酶消化法分离、培养脂肪干细胞,体外传代培养。②取第3代细胞通过转化生长因子β1、地塞米松和维生素C诱导脂肪干细胞向软骨细胞分化。③诱导后14d观察细胞形态变化,进行阿辛蓝染色检测软骨基质的分泌、免疫组织化学检测细胞Ⅱ型胶原的表达,采用Western-blot和反转录-聚合酶链反应检测诱导前后成软骨相关的Sox9,蛋白聚糖与Ⅱ型胶原的表达。结果:①细胞接种的最初几日,细胞呈圆形,1周后贴壁细胞呈长梭形,体积增大;14d后贴壁生长细胞基本长满单层,中心细胞排列紧密,形态与骨髓间充质干细胞相似。诱导培养后,细胞形态逐渐由梭型向多角形、多边形转变。诱导14d后多数细胞呈平坦的多边角形状细胞;其夹杂多角突起状或多角纺锤状细胞。②诱导后阿辛蓝染色示糖胺聚糖均匀分布于基质中。③免疫组织化学染色示基质中Ⅱ型胶原表达阳性。④反转录-聚合酶链反应检测成软骨相关的Sox9、蛋白聚糖、Ⅱ型胶原mRNA表达阳性。⑤Western-blot印迹检测细胞诱导后Ⅱ型胶原蛋白表达阳性。结论:脂肪干细胞在特定培养液的诱导下可向成软骨细胞表型分化,并能分泌软骨细胞特异性基质,有望成为软骨组织工程新的细胞来源。