The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.     

Variation of sex ratio and ABO blood group composition by month of birth of blood donors in Tokyo

Sex ratio of 17,273 blood donors born during the period between 1925 and 1935 was examined according to their month of birth and ABO blood groups in comparison with 5,810 healthy non-blood donors born in the 1900s to 1930s. The sex ratio of the blood donors and the non-blood donors varied similarly according to their month of birth with a prominent peak in summer births and a trough in winter births. This birth season with a high sex ratio was different from that of the general births during the period between 1921 and 1935, in which a maximum sex ratio was found in November. A possible explanation for the difference is the different rate of male and female infant deaths according to birth month. Variation of the sex ratio according to season of birth was not similar among the four ABO blood groups. Sex ratio of the donors with blood group B showed no elevation among the summer births. Non-blood donors with blood group B, on the contrary, showed a higher sex ratio than the others in the summer births. This difference can not be explained by infant or juvenile deaths. A possibility is that a tendency to become a blood donor is modified by the season of one's birth differently according to gender and ABO blood groups.     

The incidence and features of serological ABO subgroups among one million blood donors in Beijing

This study aims to determine the incidence of serological ABO subgroups from a large-scale database, along with the features of blood samples with serological ABO discrepancies. The serological ABO results of one million individuals were randomly sampled from a blood donor database in Beijing between 2009 and 2010. All samples were diagnosed by serological reverse and forward ABO typing using an automatic analyzer. The proportions of the normal ABO types were 27.28%, 31.57%, 30.56%, and 10.16% for blood types A, B, O, and AB, respectively. In samples in which ABO discrepancies or obvious weak agglutinin were identified in the forward or reverse typing, further tests to analyze the ABO subgroup were conducted. The overall incidence of ABO subgroups was 0.047%, with 14 ABO subgroups observed: A2, A3, Ax, Am, Aint, Aend, B2, B3, Bx, Bm, Bel, B(A), cisAB, and AB^h. In conclusion, this study revealed the exact normal ABO and subgroup distributions in the general, healthy population of Beijing using samples from a blood donor database.     

兰州地区无偿献血人群血清TTV-DNA的检测

为了解兰州地区人群TTV的感染状况,作者采用套式PCR法,以TTV-DNA的ORFl中的一段309nt的序列为扩增序列,对36份兰州地区无偿献血人群血清DNA进行扩增,将扩增阳性片段测序并进行序列同源性比较,结果发现兰州地区无偿献血人群TTV感染率为52．7％。10份兰州序列之间的同源性为88．6％～95．4％,兰州序列与日本TA287株的同源性为88．1％～93．3％,表明兰州地区无偿献血人群TIN感染非常普遍,兰州地区TTV流行株存在变异,至少有基因亚型存在。     

Purification of Genomic DNA with Minimal Contamination of Proteins

The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for an easy extraction and purification of genomic DNA from a wide range of samples, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. RBCs are lysed by dilution into a hypotonic solution. Tissues are broken down and digested by proteinase K in the presence of an anion detergent to release genomic DNA. After precipitation of the detergent and proteins, unique beads that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low concentration samples. No DNA-binding beads or columns are used in the method, eliminating the problem of low yield and the risk of shearing of genomic DNA. The purified samples are free of proteins, lipids, salts, and RNA contamination. Purified samples are also stable for storage and suitable for all downstream applications.     

亲缘供者外周血红细胞参数对造血干细胞采集影响的经验分析

目的探讨亲缘供者外周血红细胞参数对COBE Spectra血细胞分离机的自动外周血干细胞采集程序（AutoPBSC程序）与单个核细胞采集程序（MNC程序）的影响及经验分析。 方法选取河北燕达陆道培医院2019年6月至2021年2月小红细胞亲缘供者31例45次采集为小红细胞组,选取同期非小红细胞亲缘供者51例60次采集为非小红细胞组,分别应用AutoPBSC程序和MNC程序,比较两组采集情况及采集产品相关指标。采用独立样本t检验和Mann-Whitney U检验分析2组计量资料的差异。 结果与小红细胞AutoPBSC程序组比较,小红细胞MNC程序组血小板（PLT）降低率[（25.88±15.83）﹪比（36.64±10.22）﹪]、采集效率[32.65﹪（23.60﹪,73.82﹪）比63.74﹪ （59.83﹪,68.55﹪）]、采集物体积[（158.83±34.39）比（222.91±63.9）mL]、MNC总数[（218.04±117.57）×10⁸/L比（350.24±127.64）×10⁸/L]、CD34⁺细胞总数[113.83×10⁶/L （79.25×10⁶/L,154.10×10⁶/L）比233.26×10⁶/L （177.18×10⁶/L,392.51×10⁶/L）]、MNC计数[（4.04±2.61）×10⁸/kg比（5.54±2.22）×10⁸/ kg]、CD34⁺计数[1.84×10⁶/kg （1.16×10⁶/kg,4.41×10⁶/kg）比3.64×10⁶/kg （2.49×10⁶/kg,6.37×10⁶/kg）]均升高,差异有统计学意义（P < 0.05）;与非小红细胞AutoPBSC程序组比较,非小红细胞组MNC程序组采集物体积[（162.83±51.74）比（242.56±43.25）mL]升高,差异有统计学意义（P < 0.05）。 结论对造血干细胞移植供者红细胞体积偏小时,应用MNC程序采集外周血造血干细胞,比应用AutoPBSC程序更有优势。     

成都地区中老年居民血清尿酸相关危险因素分析

目的：分析成都地区中老年居民血清尿酸水平及其影响因素,为防治心脑血管事件提供策略支持。方法：利用2007代谢综合征研究调查资料（共1061人）,把人群依据血尿酸水平分为增高组（男性〉420umol/L,女性〉357umol/L）及正常组（男性≤420umol/L,女性≤357umol/L）,分析两组人群的多个代谢性指标的分布特征,并采用logistic回归分析寻找与尿酸相关的危险因素。结果：①两个分组间年龄、收缩压、男性比重、高血压家族史、人体质量指数、腰围、臀围、空腹血糖、肾功能等指标尿酸增高组明显高于尿酸正常组,舒张压则是尿酸增高组明显低于尿酸正常组。②血脂各成分中,总胆固醇、低密度脂蛋白水平尿酸增高组高于正常组,其余甘油三酯、高密度脂蛋白分组间无统计学差异。③尿酸增高组其代谢综合征、高血压、糖尿病、肥胖、腹型肥胖、血脂异常等患病率皆高于尿酸正常组,差异有统计学意义。④Logistic回归分析提示尿酸与你男性性别、年龄、收缩压、BMI、腰围、臀围、空腹血糖、肌酐、总胆固醇、低密度脂蛋白水平呈正相关,与女性性别及舒张压呈负相关。结论：成都地区尿酸与代谢性指标及肾功能相关指标关系密切,可能可以通过减少尿酸来减少心脑血管疾病、代谢性疾病及肾脏疾病的发生。     

成都地区中老年居民血清尿酸相关危险因素分析

目的:分析成都地区中老年居民血清尿酸水平及其影响因素,为防治心脑血管事件提供策略支持。方法:利用2007代谢综合征研究调查资料(共1061人),把人群依据血尿酸水平分为增高组(男性>420umol/L,女性>357umol/L)及正常组(男性≤420umol/L,女性≤357umol/L),分析两组人群的多个代谢性指标的分布特征,并采用logistic回归分析寻找与尿酸相关的危险因素。结果:①两个分组间年龄、收缩压、男性比重、高血压家族史、人体质量指数、腰围、臀围、空腹血糖、肾功能等指标尿酸增高组明显高于尿酸正常组,舒张压则是尿酸增高组明显低于尿酸正常组。②血脂各成分中,总胆固醇、低密度脂蛋白水平尿酸增高组高于正常组,其余甘油三酯、高密度脂蛋白分组间无统计学差异。③尿酸增高组其代谢综合征、高血压、糖尿病、肥胖、腹型肥胖、血脂异常等患病率皆高于尿酸正常组,差异有统计学意义。④Logistic回归分析提示尿酸与你男性性别、年龄、收缩压、BMI、腰围、臀围、空腹血糖、肌酐、总胆固醇、低密度脂蛋白水平呈正相关,与女性性别及舒张压呈负相关。结论:成都地区尿酸与代谢性指标及肾功能相关指标关系密切,可能可以通过减少尿酸来减少心脑血管疾病、代谢性疾病及肾脏疾病的发生。     

革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用

目的 评估血培养阳性标本直接细菌鉴定和药敏可行性.方法 将血培养瓶放入Bact/Alert 3D 60血培养系统进行培养筛选.选取78份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心血细胞.在收集到足量菌液后,用VITEK-32革兰阴性菌鉴定药敏卡做直接鉴定药敏试验.用标准方法及亚培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 VITEK-32直接鉴定试验,78株中的74株(94.9％)准确鉴定,直接药敏试验标准符合率95.6％.KB法血标本直接药敏试验标准符合率96.2％,但微小错误率高于VITEK-32直接药敏法.结论 Bact/Alert血培养阳性标本直接VITEK-32细菌鉴定和药敏对革兰阴性菌是切实可行的,可大幅度缩短时间,为临床及时修正用药提供依据,具有较高的应用价值.     

Stabilization of duplex DNA and RNA by dangling ends studied by free energy simulations

Single unpaired nucleotides at the end of double‐stranded nucleic acids, termed dangling ends, can contribute to duplex stability. Umbrella sampling free energy simulations of dangling cytosine and guanine nucleotides at the end of duplex and single stranded RNA and DNA molecules have been used to investigate the molecular origin of dangling end effects. In unrestraint simulations, the dangling end nucleotides stayed close to placements observed in experimental structures. Calculated free energy contributions associated with the presence of dangling nucleotides were in reasonable agreement with experiment predicting the general trend of a more stabilizing effect of purine vs. pyrimidine dangling ends. In addition, the calculations indicate a more significant stabilizing effect of dangling ends at the 5′‐end vs. 3′‐end in case of DNA and the opposite trend in case of RNA. Both electrostatic and van der Waals interactions contribute to the duplex stabilizing effect of dangling end nucleotides. The free energy simulation scheme could also be used to design dangling end nucleotides that result in enhanced duplex stabilization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 418–427, 2014.     

Prevalence of low positive anti-HCV antibodies in blood donors: Schistosoma mansoni co-infection and possible role of autoantibodies

Patients infected with schistosoma frequently show a high seroprevalence of anti-hepatitis C virus (anti-HCV) antibodies. The aim of this study was to find the underlying reason for this phenomenon, and to examine a possible involvement of autoantibodies. Out of 2,400 Egyptian blood donors, 192 (8%) were anti-HCV positive by ELISA. They were 133 males and 59 females with age ranging from 27 to 48 years. According to optical density ratio (ODR) of anti-HCV antibodies, 96 cases were low positive (LP) with ODR (1-2) designated as group I, and 96 were high positive (HP) with ODR (> or =2) (group II). Both groups were examined for quantitative HCV core antigen (HCVcAg), liver function (Albumin, ALT, AST) and anti-Schistosoma mansoni(anti-Sm) IgG. Group I cases were HCVcAg negative with normal liver function tests, and 44 of them were anti-Sm positive. Ninety cases (93.75%) of group II were HCVcAg positive with markedly affected liver function tests and 72 cases were anti-Sm positive. All group I cases were examined for autoimmune markers (ANA, AMA, SMA and LKM). In group I, 33 (75%) of anti-Sm positive cases were positive for one or more of the autoimmune markers examined, while none of anti-Sm negative was positive for any marker with significant difference between the two groups (P < 0.0001). Our results primarily on blood donors indicate that LP anti-HCV frequently represents false-positive reactivity with a possible role of Sm-induced autoantibodies in this phenomenon.     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

Epidemiology of typhoid carriers among blood donors and patients with biliary, gastrointestinal and other related diseases

Enteric fever due to Salmonella Typhi is a major public health problem. Typhoid carriers have high titres of Vi agglutinins in their sera. We worked out the baseline data for Vi agglutinins from 705 healthy blood donors (controls) by ELISA and compared it with 446 patients with biliary, gastrointestinal and other related diseases (cases). The samples were divided into five groups based on the disease condition of the patients from whom they were collected. Group A (n=196) consisted of patients with stones in the gall bladder/common bile duct and Group B (n=27) with gall bladder carcinoma. Group C (n=33) comprised patients with carcinoma of the pancreas/ampulla, obstructive jaundice and/or cholangiocarcinoma. Group D (n=112) had patients with acute/chronic pancreatitis, abdominal pain, intestinal obstruction, peritonitis, carcinoma oesophagus, chronic diarrhoea, gastrointestinal bleeding and dyspepsia. Group E (n=78) included patients with miscellaneous diseases. The mean absorbance value obtained for healthy subjects +3 standard deviations was taken as the cut-off value for a positive typhoid carrier. In Group A, 10.2% samples were positive; in Group B, 7.4%; in Group C, 12.0%; in Group D, 9.8% and in Group E, 9.0%. There was a highly significant (P <0.001) increase in the presence of Vi agglutinins in the cases compared to the controls. High prevalence of typhoid carriers occurs in patients with biliary, gastrointestinal and other related diseases. Vi serology employing highly purified Vi antigen offers a practical and cost-effective way of screening for S. Typhi carriers.     

Analysis of possible factors relating to prognosis in autologous peripheral blood mononuclear cell transplantation for critical limb ischemia

Background aimsAutologous transplantation of granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cells (M-PBMNCs) has been shown to be effective in treating critical limb ischemia (CLI); however, the studies of the possible prognosis predictors after autologous M-PBMNC transplantation are inadequate. The objective of the study was to assess the possible factors affecting the results of M-PBMNC transplantation for CLI.MethodsWe reviewed the clinical profiles of 87 patients with CLI who were treated with M-PBMNC implantation in the Blood Diseases Hospital, Chinese Academy of Medical Sciences, between December 2002 and December 2011, and we followed these patients. The patients were divided into a good prognosis group and a poor prognosis group on the basis of whether amputation was performed. The significant differences of clinical variables between two groups were analyzed by means of the Mann-Whitney test and χ² test, and logistic regression analysis was used to study the variables representing the possible prognostic factors for amputation.ResultsOf the 87 patients, three patients died and one patient was lost during the follow-up period. We analyzed 83 patients. The diseases included CLI complicated by diabetes mellitus gangrene (35 cases, 42.2%), arteriosclerosis obliterans (31 cases, 37.3%) and thromboangiitis (17 cases, 20.5%). The mean age was 62 years (range, 30–87). The median follow-up time for the surviving patients was 5 years. The 5-year amputation-free rate was 72.2%, and no adverse effects related to M-PBMNC transplantation were observed.ConclusionsThe significant prognostic factors associated with poor angiogenesis were fibrinogen >4 g/L and fasting blood glucose >6 mmol/L.     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin–streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.     

Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA

A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA using a peptide nucleic acid (PNA) as a linker molecule. The terminal cysteine thiol group on the PNA peptide backbone is reacted with a maleimide moiety on the fluorochrome to produce a fluorescent conjugate which is in turn hybridized to a plasmid expression vector containing an 11-bp target sequence. Spectroscopic evaluation and an electrophoretic mobility shift assay showed that the pDNA hybridized to one PNA-fluorochrome conjugate molecule. The fluorescence signal comigrated with pDNA on acrylamide gels, confirming the stable attachment of the fluorescent conjugate to the pDNA. The utility of one of the conjugates, PNA-Oregon green 488/pCMVbeta-DTS, to probe pDNA transport across the nuclear envelope, a significant barrier to gene transfer, was undertaken using a digitonin-permeabilized HeLa cell assay. The PNA-Oregon green 488/pCMVbeta-DTS conjugate is able to efficiently traverse the nuclear membrane of the permeabilized cells, accumulating in the nuclei within 30 min and reaching maximal levels by 1h. When transfected into HeLa cells, the PNA-Oregon green 488/pCMVbeta-DTS conjugate retained 55% of the native plasmid's biological activity, as determined by a beta-galactosidase assay. Thus, this method allows for the sequence-specific coupling of commercially available fluorochromes to DNA expression vectors while retaining biological function.