不同全血过滤方法用于去白细胞血液制备的临床对照研究

目的:观察不同全血过滤方法用于去白细胞血液制备的效果。方法:采用两种全血过滤方法进行对比研究,对照组采用常规法,将采集后全血混匀后直接与白细胞滤器连接直接过滤;实验组采用湿润滤盘法,血液采集完成混匀后静置,先用上层血清10～20 m L湿润滤盘,再混匀与白细胞滤器连接后进行过滤。比较两组制备方法所用的过滤时间、血液回收率、过滤前后血液指标情况及24小时内溶血的发生情况。结果:两组全血过滤方法过滤前后白细胞、红细胞、血红蛋白、血小板及血浆游离血红蛋白水平比较差异均无明显统计学意义(P0.05)。而实验组过滤时间短于对照组,血液回收率高于对照组,且24小时内溶血比例明显低于对照组(P0.05)。结论:常规法与湿润滤盘法均能达到去白细胞血液标准,但湿润滤盘法较常规法能有效的降低过滤时间、增加血液回收率,减少去白细胞悬浮红细胞因溶血造成的血液不合格率,值得临床推广应用。     

巨细胞病毒对人胚成纤维细胞骨架的影响及其凋亡诱导作用

人胚成纤维细胞是人巨细胞病毒(human cytomegalovirus,CMV)的容许细胞,CMV可在该细胞内活化复制[1].CMV感染后对细胞骨架及细胞凋亡有何影响尚不明确.国外有学者在透射电镜下观察感染CMV的人胚成纤维细胞,发现其微丝解聚,细胞骨架破坏[2],但结果尚不完善.我们的研究分别从细胞形态学、核酸水平、细胞超微水平等方面观察了感染CMV后的人胚成纤维细胞形态、肌动蛋白基因(β-actin)mRNA以及微丝的变化,并进一步观察了其对细胞凋亡的影响.     

甲肝病毒在人血白细胞中体外连续传代和在分类白细胞中增殖的研究

1985年我们采用间接免疫荧光法(IF法)检测出甲肝患者外周血白细胞中有甲肝抗原(HAAg)存在,继而又将HAAg阳性白细胞直接种入PLC/PRF/5细胞,分离到两株甲肝病毒(HAV)NJ—3株和H—1株。为了弄清白细胞所携带的病毒究竟仅为吸附吞饮,抑或能在其中复制增殖,我们将分离到的HAV用正常人血白细胞进行体外增殖试验,现将结果报告如下。     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

血液检测中白细胞下降的病因及对飞行学员的影响

白细胞是血液中体具有保护和防御作用的有核血细胞。根据其免疫功能不同分为吞噬细胞和免疫细胞。吞噬细胞具有非特异性的免疫功能,能够吞噬各种异物,包括粒细胞和单核细胞两种。免疫细胞具有特异性的免疫功能,参与体液性和细胞性免疫,即淋巴细胞[1]。虽然白细胞的形态结构和生理功能多样化,但是彼此在机体的防护、免疫和创伤愈治过程中起协同作用,而不是相互孤立。     

S/D法灭活血液制剂中脂包膜病毒效果验证的研究

选用不同核酸的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证S/D法处理对纤维蛋白原、凝血酶原复合物、凝血因子Ⅷ、静注丙种球蛋白、免疫血浆等血液制剂的病毒灭活效果。结果该法对所有被处理的血液制剂中的PRV及VSV灭活能力分别为≥3.38~5.88和≥3.50~4.75logTCID50/0.1ml,表明S/D法对两种病毒核酸类型的脂包膜病毒有良好的灭活效果。     

Real-time quantitative polymerase chain reaction

In Charcot-Marie-Tooth type 1A disease (CMTIA), heterozygosity for the peripheral myelin protein 22 (PMP22) duplication increases the gene dose from two to three, whereas, in hereditary neuropathy with liability to pressure palsies (HNPP), heterozygosity for the PMP22 deletion reduces the gene dose from two to one. Thirty-eight Norwegian patients with CMT1, 4 patients with HNPP, 15 asymptomatic family members, and 45 normal controls were studied using the ABI 7700 sequence detection system and the TaqMan method of real-time quantitative polymerase chain reaction (PCR). Using a comparative threshold cycle (Ct) method and albumin as reference gene, the gene copy number by PMP22 gene duplication or deletion on chromosome 17p11.2-12 was quantified. The PMP22 duplication ratio ranged from 1.50 to 2.21, the PMP22 deletion ratio ranged from 0.44 to 0.55, and the PMP22 ratio in normals ranged from 0.82 to 1.27. All samples were run in triplicate, with a mean standard deviation of 0.07 (range 0.01-0.17). Thirty-four of thirty-eight CMT1 patients (89.6%) had the PMP22 duplication and the four HNPP patients had the PMP22 deletion. This was not found in any of the asymptomatic family members or the controls. Real-time quantitative PCR is a sensitive, specific, and reproducible method for diagnosing PMP22 duplication and deletion. The method is fast, allowing 13 patients to be diagnosed in 2 h. It involves no radioisotopes and requires no post-PCR handling. In our opinion, real-time quantitative PCR is the first method of choice in diagnosing PMP22 duplication and deletion.     

Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks post-partum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.     

Theoretical uncertainty of measurements using quantitative polymerase chain reaction.

Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.     

Analysis of specific bacteria from environmental samples using a quantitative polymerase chain reaction

This article describes the use of quantitative PCR for measuring bacterial abundance in environmental samples. The two approaches discussed are: 1) The use of an internal PCR standard constructed to be the same size and have the same sequence as the primary amplification target, but differing from the primary target by 2-3 bases, corresponding to a unique restriction site. This allows the amount of target amplicon to be compared with the internal standard and circumvents the problem of differential amplification efficiencies when using dissimilar targets and standard amplicons. 2) The use of Taqman technology (Applied Biosystems, Foster City, California) with a dual labeled oligonucleotide probe which binds internal to the PCR primers. The detection of Bacteroides is used as an example for both approaches.     

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection.     

Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction

Soon after discovery of the polymerase chain reaction (PCR), various laboratories have attempted to use quantitative PCR (QPCR) to detect DNA damage in specific gene segments. The development of techniques that facilitate long PCR increased the sensitivity of the assay so that biologically relevant doses of DNA-damaging agents could be assessed. QPCR has been used to survey DNA damage induced by different genotoxicants and to establish the repair kinetics of numerous genes. Current work seeks to analyze damage and repair in specific genes from animals exposed to specific DNA-damaging agents such as oxidative stress.     

Sex determination using the polymerase chain reaction

A practical class experiment on the PCR is described which has been used over several years as part of an undergraduate biochemistry and molecular biology course for science students. A major aim is to provide experience in the use of the polymerase chain reaction (PCR) and its interpretation. Students are given small coded DNA samples and use the PCR reaction to determine whether the sample is from a male or a female.