A newly designed flexible fiberoptic bronchoscope for use in intensive care units

A newly designed flexible fibroptic bronchoscope has been manufactured for use in intensive care units (ICU). It has an inside channel of large caliber, diameter 2.5 mm, through which pulmonary secretions can be aspirated directly via the suction tube. Furthermore, prompt bedside use is possible since handle type batteries can easily be attached to the bronchofibroscope. This new instrument is now employed in our ICU for: 1. The diagnosis and treatment of atelectasis. 2. Suction of retained secretions. 3. Detection of tracheal obstruction. 4. Evaluation of endotracheal and tracheostomy tubes whilst in position. 5. Observation of tracheal and bronchial changes. 6. Help in endotracheal intubation. It was used most frequently for the diagnosis and treatment of atelectasis and suction of retained secretions.     

显微数字成像组合组织芯片技术平台的建立及其在肿瘤标志物研究中的应用

目的:利用我室新建立的显微数字成像组合组织芯片技术平台探讨新型肿瘤标志物MDA-7/IL-24在胃癌中的表达及意义。方法:取胃癌标本50例及对应正常胃黏膜10例,从预先作好标记的供体石蜡块中取出目标组织,应用组织芯片点样仪制成一定阵列的受体石蜡块,将石蜡切片贴附于硅胶包被的玻片上,采用高敏感EnVision两步法进行MDA-7/IL-24单克隆抗体免疫组织化学染色。将染好的切片置于显微数字成像分析仪中读取图像信息并进行分析。结果:计算机软件将读取的组织芯片全貌显示在屏幕上,研究者在计算机定位下对每一标本点逐一放大分析。50例胃癌组织标本中,剔除组织脱落或取材部位欠佳的无效点,42例纳入统计分析。42例胃癌中27例MDA-7/IL-24表达消失(64.29%)。10例正常胃黏膜腺体细胞质MDA-7/IL-24染色全部阳性。MDA-7/IL-24表达消失组癌周淋巴结转移率明显增高(P=0.01),且在TNMⅢ、Ⅳ期患者明显多于TNMⅠ、Ⅱ期(P=0.003)。MDA-7/IL-24表达与否与年龄、性别、肿瘤发生部位及组织分型无明显关系。结论:显微数字成像组合组织芯片技术在筛查肿瘤标志物表达时显示出高通量、可比性强、计算机定位精确及图像清晰直观等优点,是后基因组时代大规模肿瘤标志物筛选定位分析中可以选择的一种新型技术平台。MDA-7/IL-24表达消失与胃癌的浸润生长及淋巴结转移增加有明显关系,故可将MDA-7/IL-24表达消失作为判断胃癌浸润、转移能力的一种新型肿瘤标志物。     

A simple direct assay for cyclic amp in plasma and other biological samples using an improved competitive protein binding technique

An improved cyclic AMP assay using a purified and activated binding protein from bovine skeletal muscle is described.Activation of the purified binding protein by bovine serum albumin was investigated and shown to increase the assocation constant from 0.4 · 10⁹ M^?1 to 1.0 · 10⁹ M^?1. The fully activated binding protein was not affected by the presence of large amounts of protein in biological samples such as plasma.Using the fully activated binding protein and with optimised assay conditions it has been demonstrated that the assay has a remarkable freedom from non-specific interference by materials normally found in crude biological extracts.This freedom from interference, coupled with a simple method of preparing plasma, permits the hitherto unreported rapid direct assay of plasma cyclic AMP without the need for extraction procedures or the use of cyclic AMP-free plasma controls, thus making the method more reliable and suitable for use in the clinical laboratory.Development of an assay markedly insensitive to non-specific interference increases the number of potential applications of the assay.     

A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants

The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.     

Modified TDx assay for cyclosporine and metabolites, for use with whole-blood samples

A recently introduced fluorescence polarization immunoassay (FPIA) for determination of cyclosporine A in serum and plasma is discussed with regard to its use for whole-blood samples, with and without hemolysis before the assay. The performance characteristics of the modified method are highly satisfactory (within-run CVs 2.06 to 5.50% and 1.99 to 3.39%, respectively; long-term between-run CVs under routine conditions 5.73 to 8.95%). The limit of detection is 30 micrograms/L. Results agree well with those obtained with the RIAs compared, but the modified FPIA is more convenient and faster.     

Coupling of digital image processing and three-way calibration to assist a paper-based sensor for determination of nitrite in food samples

In this work, a novel and very interesting analytical methodology based on coupling of digital image processing and three-way calibration has been developed for determination of nitrite in food samples. Nitrite in contact with Griess reagent is able to produce a red-colored azo dye whose color intensity is correlated with nitrite concentration and here, a piece of Whatman filter paper impregnated with Griess reagent was used as the platform of the sensor and a SONY Xperia Z5 cell phone was used for image capturing from the sensor surface. To generate second-order data, the F-number of the camera''s sensor was changed as an instrumental parameter. Two calibration models were constructed by unfolded partial least squares-residual bilinearization (U-PLS/RBL) and multiway-PLS/RBL (N-PLS/RBL) and then, their performance for prediction of nitrite concentration in test samples was evaluated and the results confirmed a good performance for U-PLS/RBL (REP = 3.25 ppm, RMSEP = 8.82 ppm, RMSEC = 4.62 ppm, Q² = 0.99, γ⁻¹ = 0.05 and LOD = 0.1 ppm) which was better than that for N-PLS/RBL (REP = 13.98 ppm, RMSEP = 37.86 ppm, RMSEC = 6.46 ppm, Q² = 0.98, γ⁻¹ = 0.07 and LOD = 0.15 ppm) in predicting concentration of nitrite in test samples which motivated us to choose it for the analysis of cabbage, carrot, lettuce, watermelon, onion, potato, kielbasa and sausage as real samples.

In this work, a novel and very interesting analytical methodology based on coupling of digital image processing and three-way calibration has been developed for determination of nitrite in food samples.     

A two-site immunoradiometric assay for C-reactive protein in serum

An immunoradiometric assay was developed for C-reactive protein in serum. The assay had a sensitivity of 5 μg/l and good precision. Correlation with radial immunodiffusion (r = 0.916) and EMIT (r = 0.935) was close. A reference range for healthy adults of 0.05–4.0 mg/l was derived.     

A competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum and urine samples

In this solid-phase competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum or urine, antiserum to human alpha 1-acid glycoprotein is incubated with solid-phase-bound alpha 1-acid glycoprotein in the presence of standard or sample. Incubation with second antibody labeled with alkaline phosphatase then follows, before development with substrate. Results obtained correlate well with a fluorescent assay involving the dye Auramine O (r = 0.953) and with radial immunodiffusion (r = 0.921). The present assay covers the range 0.2 to 5 mg/L and 16 samples take 2.5 h to complete. This assay is useful for measuring concentrations of alpha 1-acid glycoprotein in serum and also in urine, for which other assay methods are not sufficiently sensitive.     

A LightCycler TaqMan assay for detection of Borrelia burgdorferi sensu lato in clinical samples

Lyme disease (LD) is an infection caused by an ixodid tick-borne spirochete, Borrelia burgdorferi sensu lato. LD manifests itself as a multisystem inflammatory disease that affects the skin in its early localized stage and spreads to the joints, nervous system, heart, and, to a lesser extent, other organ systems in its later disseminated stages. If diagnosed and treated early with appropriate antibiotics, LD is almost always readily cured. Developing a highly sensitive and specific real-time polymerase chain reaction assay could be very useful in improving the diagnostic accuracy and decreasing turnaround time for results. We report the development of a LightCycler TaqMan assay targeting the OspA gene for clinical detection of B. burgdorferi sensu lato in various types of biologic samples. This assay was validated by testing a variety of clinical samples including cerebrospinal fluid, synovial fluid, skin biopsies, and blood and culture isolates from skin biopsies. The TaqMan testing results were 100% concordant with previously reported results. Reference strains representing isolates from other geographic regions were also successfully amplified. The developed assay is robust, is highly sensitive and specific for B. burgdorferi sensu lato, and is suitable for clinical detection of the bacterium in biologic samples.     

An ultra-micro method for the determination of total nitrogen in biological fluids based on Kjeldahl digestion and enzymatic estimation of ammonia.

An ultra-micro method for the determination of the total nitrogen-content of biological fluids and suspensions is described, based on a digestion in sulphuric acid and a enzymatic determination of the ammonia formed with glutamate dehydrogenase (EC 1.4.1.3). The proposed method yields the same results as the classical Kjeldahl procedure, but is less time-consuming. The detection-limit of the nitrogen, without loss of precision and accuracy, is much lower than in the original Kjeldahl procedure, and is in the order of 35 ng N per sample.     

Improved assay for bismuth in biological samples by atomic absorption spectrophotometry with hydride generation

This simple, rapid, sensitive, reliable, and economical assay for bismuth in plasma, erythrocytes, and urine is based on atomic absorption spectrophotometry with hydride generation. Acid digestion eliminates the problem of foaming, which hitherto has complicated such assay of bismuth in plasma and erythrocytes. The detection limit of the assay has been improved to 0.1 micrograms/L, as compared with a previously documented limit of 2.5 micrograms/L. Average recovery exceeded 95% in all biological fluids. Economy of use derives from elimination of need for electrodeless discharge lamps and atomic absorption grade borohydride. Determination of basal concentrations of bismuth in clinical samples of body fluids gave reference intervals of 0.1-3.5 micrograms/L for plasma, 0.3-4.6 micrograms/L for urine.     

Developing a novel paper-based enzymatic biosensor assisted by digital image processing and first-order multivariate calibration for rapid determination of nitrate in food samples

For the first time, a novel analytical method based on a paper based enzymatic biosensor assisted by digital image processing and first-order multivariate calibration has been reported for rapid determination of nitrate in food samples. The platform of the biosensor includes a piece of Whatman filter paper impregnated with Griess reagent (3-nitroaniline, 1-naphthylamine and hydrochloric acid) and nitrate reductase. After dropping a distinct volume of nitrate solution onto the biosensor surface, nitrate reductase selectively reduces nitrate to nitrite and then the Griess reagent selectively reacts with nitrite to produce a red colored azo dye. Therefore, the color intensity of the produced azo dye is correlated with nitrate concentration. After image capture, the images were processed and digitized in the MATLAB environment by the use of an image processing toolbox and the vectors produced by the digital image processing step were used as inputs of the first-order multivariate calibration algorithms. Several multivariate calibration algorithms and pre-processing techniques have been used to build multivariate calibration models for verifying which technique offers the best predictions towards nitrate concentrations in synthetic samples and the best algorithm has been chosen for nitrate determination in potato, onion, carrot, cabbage and lettuce samples as real cases.

For the first time, a novel analytical method based on a paper based enzymatic biosensor assisted by digital image processing and first-order multivariate calibration has been reported for rapid determination of nitrate in food samples.     

Feasibility study of a screening assay that identifies the abnormal prion protein PrPTSE in plasma: initial results with 20,000 samples

BACKGROUND: It is likely that transmission of variant Creutzfeldt‐Jakob disease (vCJD) occurs by transfusion and that the candidate infectious agent (PrP^TSE) is present in small concentrations in the blood of infected donors in the asymptomatic phase of the disease. A new blood screening assay has been developed to detect PrP^TSE in citrated plasma samples. STUDY DESIGN AND METHODS: Three regional Blood Transfusion Establishments (ETS) in France (ETS Alsace, ETS Bourgogne Franche‐Comté, and ETS Pyrénées‐Méditerranée) will screen 60,000 plasma samples (20,000 in each ETS) over a time period of approximately 9 to 12 months. RESULTS: Results provided in this report are those of the first testing site in Strasbourg, Alsace. The preliminary results have demonstrated an initial specificity of 97.60%. Upon repeat testing the specificity rate achieved 99.90% (20 repeat‐positive samples). Based on the known epidemiology of vCJD in France, it is likely that the repeat‐reactive samples are not true‐positives. CONCLUSION: The screening assay was studied in terms of specificity and practicality and was found to be suitable for use in routine testing of blood donations. However, throughput must be enhanced by automation of the assay, and traceability would be improved if automated systems were used to distribute and identify samples.     

ICC患者血浆ctDNA突变检测数字PCR平台的建立及临床应用价值

目的建立KRAS G12D、TP53 C242S、IDH1 R132C突变数字聚合酶链反应(dPCR)检测平台,并初步评估其检测性能及临床应用价值。方法选取行切除手术的肝内胆管细胞癌(ICC)患者22例,设计KRAS G12D、TP53 C242S、IDH1 R132C突变位点的引物及探针,建立dPCR突变检测平台。并采用不同浓度的自配标准品质粒验证该平台的准确性、精密度、空白限、功能灵敏度及线性范围。将外周血dPCR检测结果与外周血Oseq-ctDNA及组织Oseq-T靶向测序结果进行比较。ICC患者行切除术后,每6个月采集1次外周血并跟踪随访,评估该突变检测平台对ICC患者术后疗效监测的作用。结果 dPCR平台的准确性良好[3种突变(KRAS G12D、TP53 C242S和IDH1 R132C)3个丰度的检测结果与理论值的偏差均<±15%],批内和批间精密度[变异系数(CV)]均<20%,空白限为4拷贝,功能灵敏度为0.1%,且在0.1%~10.0%范围内线性良好。ROC曲线分析结果显示,ctDNA突变谱诊断ICC的曲线下面积(AUC)为0.659,敏感性为31.8%,特异性为100%;与糖类抗原19-9(CA19-9)联合检测诊断ICC的AUC为0.841,敏感性为68.2%,特异性为100%。dPCR平台的检测结果与Oseq-ctDNA测序结果有较高的一致性(kappa=0.792,P=0.007)。随访结果显示,有1例ICC患者术后18个月KRAS G12D突变升高,与影像学检查确认的复发时间一致。结论建立了检测KRAS G12D、TP53C242S、IDH1 R132C突变的dPCR平台,可用于ICC患者的辅助诊断、疗效评估及术后动态监测。     

A colony blot immune assay to identify enteroinvasive Escherichia coli and Shigella in stool samples

Using an IpaC protein-specific monoclonal antibody a colony blot immune assay was developed for the identification of enteroinvasive Escherichia coli (EIEC) and Shigella in fecal specimens, and was evaluated in a field study. By screening the entire culture plates the colony blot assay was significantly more sensitive than the investigation of 16 randomly selected colonies from artificially contaminated fecal specimens. Among the 165 stool samples from 121 patients with diarrhea the immune assay detected IpaC expressing colonies in 16 out of the 17 specimens positive with a Shigella-, and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the colony blots, Shigella was isolated from 12, while EIEC from four of the samples. The IpaC-specific colony blot immune assay is a simple screening method to detect EIEC in stool samples for laboratories not equipped with molecular techniques.     

A micromethod for assay of lipoprotein lipase activity in needle biopsy samples of human adipose tissue and skeletal muscle

A rapid and simple procedure for assay of lipoprotein lipase (LPL) activity in small amounts of human adipose tissue and skeletal muscle is described and validated. The enzyme is eluted from tissues with heparin and the activity is determined from the eluate by measuring the release of [¹⁴C]oleic acid from a gum arabic stabilized emulsion of glycerol-tri[¹⁴C]oleate in a Tris-buffer medium containing albumin and pooled normal human serum. Reproducible results are obtained with amounts of tissue ranging from 2 to 25 mg.The K_m values of the adipose tissue and skeletal muscle LPL for the triolein substrate were 0.74 ± 0.06 and 0.77 ± 0.05 mmol/l, respectively. The standard radioactive triolein emulsion was hydrolyzed by adipose tissue LPL at a rate closely similar to rat VLDL-triglyceride labeled in vivo with [1-¹⁴C]palmitic acid, suggesting that the experimental substrate behaved in a similar manner to the natural substrate. The LPL activity was much higher in adipose tissue than in muscle. In adipose tissue the LPL activity was 2–4 times higher in women than in men whereas no sex difference was present in the LPL activity of muscle.