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Results for “Repeatability and Reproducibility”

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PubMed1977

Pulse wave from transit ratios in the assessment of peripheral vascular disease.

This paper presents a ratio of the pulse wave form transit times in the proximal and distal arterial tree, produced by a simple, non-invasive, repeatable and reproducible technique. It has been shown to be highly significant in separating normal from disease states and the proximal from the distal lesion. It has been shown to be independent of the effects of age. In the clinical situation the ratio is of value in the initial assessment of the patient (by separating ischaemic from now-ischaemic limb pain); in elucidation of significant occult proximal lesions in the presence of distal disease; in indicating the type of angiography required (femoral arteriography rather than translumbar aortography in suitable subjects); in following the progress of patients in operative and conservative management régimes; and in the long term follow-up of the natural history of obliterative vascular disease.

Adult↗

PubMed2004

Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection.

Tryptophan metabolism is disturbed in mental depression, and the induction of indoleamine 2,3-dioxygenase increases kynurenine production. In order to determine this disturbance in patients with chronic hepatitis C and receiving interferon-based immunotherapy, a new and specific HPLC protocol was elaborated. For tryptophan, the assay was linear from 6.25 to 100 micromol L(-1), and the limits of detection (LOD) and quantitation (LOQ) for the method were 0.7 and 8.0 micromol L(-1). For kynurenine, the linearity of calibration was from 0.0625 to 6.25 micromol L(-1), with LOD and LOQ of 2 and 3 nmol L(-1). Reproducibility and repeatability were satisfactory. The method allowed study of human blood serum.

Chromatography, High Pressure Liquid↗

PubMed1987

Multiple HLA class I-dependent cytotoxicities constitute the "non-HLA-restricted" response in infectious mononucleosis.

Primary Epstein-Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad-ranging and apparently non-HLA-restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo-preserve IM mononuclear cell preparations, after depletion of CD16+ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen-dependent reactivities against the autologous EBV-transformed lymphoblastoid cell line (LCL) and particular HLA class I-mismatched LCLs. The autologous LCL-directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV+ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus-specific HLA-restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA-mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA-mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV+ and EBV- B cell blasts from the relevant HLA-mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus-specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA-mismatched LCLs arises from fortuitous cross-recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV-specific response.

Antibodies, Monoclonal↗

PubMed1995

Rapid detection of toxoplasmic nucleic acid by enzyme-linked immunofiltration-assay after membrane transfer.

We report the use of enzyme-linked immunofiltration-assay (ELIFA) for rapid detection of Toxoplasma gondii DNA after transfer to nitrocellulose or nylon membranes. Saturation, specific labeling with a nonradioactive oligonucleotide probe and washing steps are included in an active filtration process, which is controled by microcomputer regulating reagent flow. This method provides excellent specificity, reproducibility and repeatability. Recycling in a closed circuit or by reversing the direction of flow provides the advantage of reducing the required volume of costly reagents (e.g. probes). ELIFA does not require lengthy membrane manipulation, and hybridization as well as stringency and revelation steps can be automated. The procedure can be carried out in less than 2 h, which is a major time saving compared to conventional solid-phase molecular hybridization techniques.

Animals↗

PubMed2003

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.

Cyclodextrins↗

PubMed2004

Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry.

Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.

Amino Acid Sequence↗

PubMed2002

Interlaboratory validation of oxidation-index measurement methods for UHMWPE after long-term shelf aging.

An international oxidation index standard would greatly benefit the orthopedic community by providing a universal scale for reporting oxidation data of ultra-high molecular weight polyethylene (UHMWPE). We investigated whether severe oxidation associated with long-term shelf aging affects the repeatability and reproducibility of area-based oxidation index measurement techniques based on normalization with the use of 1370- or 2022-cm(-1) infrared (IR) absorption reference peaks. Because an oxidation index is expected to be independent of sample thickness, subsurface oxidation was examined with the use of both 100- and 200-microm-thick sections from tibial components (compression-molded GUR 1120, gamma irradiated in air) that were shelf aged for up to 11.5 years. Eight institutions in the United States and Europe participated in the present study, which was administered in accordance with ASTM E691. On average, the 100-microm-thick samples were associated with significantly greater interlaboratory relative standard uncertainty (40.3%) when compared with the 200-microm samples (21.8%, p = 0.002). In contrast, the intralaboratory relative standard uncertainty was not significantly affected by the sample thickness (p = 0.21). The oxidation index method did not significantly influence either the interlaboratory or intralaboratory relative standard uncertainty (p = 0.32 or 0.75, respectively). Our interlaboratory data suggest that with the suitable choice of specimen thickness (e.g., 200 microm) and either of the two optimal oxidation index methods, interlaboratory reproducibility of the most heavily oxidized regions in long-term shelf-aged components can be quantified with a relative standard uncertainty of 21% or less. Therefore, both the 1370-cm(-1) and the 2022-cm(-1) reference peaks appear equally suitable for use in defining a standard method for calculating an oxidation index for UHMWPE.

Artifacts↗

PubMed1976

Growth of in vitro colony-forming cells from normal human peripheral blood leukocytes cultured in diffusion chambers.

The growth of granulopoietic progenitor cells (CFU-C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU-C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing. A marked increase in the number of CFU-C, up to 25-fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU-C throughout the diffusion chamber culture period was very poor. The growth of CFU-C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non-proliferative pattern. Evidence suggests that the increase in CFU-C in diffusion chambers is the result of both self-renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each.

Cell Division↗

PubMed2004

Optimization of quadrupole ion storage mass spectrometric conditions for the analysis of selected polybrominated diphenyl ethers. Comparative approach with negative chemical ionization and electron impact mass spectrometry.

Gas chromatography coupled to quadrupole ion storage mass spectrometry (QISTMS) operating in the non-resonant mode is presented as an innovative approach for the analysis of selected polybrominated diphenyl ethers (PBDEs). Although reductions in complexity and time needed for optimization are achieved in comparison with the resonant option, precise adjustment of the mass spectrometric conditions is required. Differences in isolation and fragmentation patterns of target species with degree of bromination were observed. The reliability of the method was confirmed by using standard solutions through the evaluation of certain quality parameters such as accuracy (92-108%), injection repeatability and reproducibility (coefficient of variation below 10% and 15%, respectively). Detection limits ranged from 62 to 621 fg, providing sensitivity similar to that of negative chemical ionisation (NCIMS) and greater than that of electron ionization mass spectrometry. The applicability of QISTMS method to real samples and matrix effects were evaluated through the analysis of some PBDE congeners in a sewage sludge sample from a Spanish waste-water treatment plant. Comparable results were obtained using QISTMS and NCIMS. According to these observations, QISTMS performed in the non-resonant mode may constitute a low-cost, rapid and reliable alternative to high-resolution devices for the analysis of selected PBDEs in environmental samples.

Environmental Health↗

PubMed1984

Collaborative study of the USP dissolution test for prednisone tablets with Apparatus 2.

Five lots of prednisone tablets that disintegrate within 5 min were collaboratively studied by 11 laboratories using USP Apparatus 2 under carefully controlled conditions. One lot gave complete dissolution. The reproducibility and repeatability of Apparatus 2 for the four lots still dissolving at the end of the test were 2.6 and 1.6% of label claim, respectively, for the 11 laboratories.

Chemistry, Pharmaceutical↗

PubMed2003

Liquid chromatography/mass spectrometric determination of patulin in apple juice using atmospheric pressure photoionization.

This paper describes a comparison between atmospheric pressure chemical ionization (APCI) and the recently introduced atmospheric pressure photoionization (APPI) technique for the liquid chromatography/mass spectrometric (LC/MS) determination of patulin in clear apple juice. A column switching technique for on-line extraction of clear apple juice was developed. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for both APCI and APPI. The results indicated that APPI provides lower chemical noise and signal suppression in comparison with APCI. The linear range for patulin in apple juice (correlation coefficient >0.999) was 0.2-100 ng mL(-1). Mean recoveries of patulin in three apple juices ranged from 94.5 to 103.2%, and the limit of detection (S/N = 3), repeatability and reproducibility were 1.03-1.50 ng mL(-1), 3.9-5.1% and 7.3-8.2%, respectively. The total analysis time was 10.0 min.

Beverages↗

PubMed2001

Rapid monitoring assay of congenital adrenal hyperplasia with microbore high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spots.

17-hydroxyprogesterone (17OHP) is the most important plasma parameter for diagnosing and monitoring congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency. A rapid, simple, and specific method based on microbore high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) was developed to determine the presence of 17OHP on dried filter-paper blood samples from patients with CAH caused by 21-hydroxylase deficiency. 17OHP from dried blood spots formed by the action of Girard reagent P (GirP) turned out to be a water-soluble hydrazone complex. Derivatization with GirP led to higher ESI sensitivity for 17OHP. The LC/MS/MS detection of GirP-derivatized 17OHP (GirP-17OHP) was rapid (<3 min). The method is repeatable and reproducible, with CVs <7% and 12%, respectively. This new method was used for direct determination of 17OHP in dried blood specimens obtained from abnormal children and infants of various ages with a detection limit of 10 ng/mL ( approximately 12 microL blood). The method described allows for rapid and reliable measurements of 17OHP in dried blood specimens from patients affected by CAH.

17-alpha-Hydroxyprogesterone↗

PubMed2002

Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry.

A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the limit of detection was 0.125 ng/mL. The excretion profile was determined after administration of 500 microg salmeterol (Serevent) to four standard-bred mares via a metered dose inhaler (MDI) with an Equinehaler adapter. Salmeterol was detected from 1 h until 12 h post-administration. Maximum urinary concentrations varied between 2.3 and 14.9 ng/mL.

Administration, Inhalation↗

PubMed2006

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains.

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.

Cardiolipins↗

PubMed1996

Limits of titin extension in single cardiac myofibrils.

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from approximately 2-5 microns. Myofibril stretch up to sarcomere lengths of approximately 3 microns resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of approximately 2.7 microns. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond approximately 3 microns sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 microns. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

Animals↗

PubMed1992

Value of a radionuclide limb blood flow technique in the assessment of percutaneous balloon and dynamic angioplasty.

In a prospective study, a radionuclide technique was used to evaluate the limb blood flow (LBF) changes in 30 patients undergoing dynamic (n = 15) or balloon (n = 15) angioplasty for arterial occlusions or stenoses, respectively. The results were compared with Doppler Ankle Brachial Index (DABI) and treadmill exercise tests. Whilst LBF values (ml of blood flow per 100 ml of limb volume per min) were significantly lower in limbs with arterial occlusion than stenosis (4.5 +/- 0.46 and 6.4 +/- 0.74, respectively; P less than 0.05). DABI provided no discrimination. Immediately after balloon angioplasty, there was a fall in DABI, from 0.60 +/- 0.05 to 0.47 +/- 0.04 (P less than 0.05), which rose 24 h later to 0.73 +/- 0.02 (P less than 0.01). Following dynamic angioplasty, DABI improved from 0.60 +/- 0.05 to 0.66 +/- 0.02 (P less than 0.05). At 3 weeks, the LBF improved from 4.6 +/- 0.66 to 11.1 +/- 0.53 (P less than 0.001) following dynamic angioplasty and from 6.2 +/- 0.68 to 8.53 +/- 0.81 (P less than 0.001) following balloon angioplasty. "Normal" LBF (greater than 10 ml/100 ml per min) was achieved in 80% of patients who underwent successful dynamic angioplasty but in only 36% of the balloon group (P less than 0.05, chi 2-test). Reproducibility of repeated LBF measurements in control limbs was superior to that of DABI. This was indicated by a lower coefficient of variation, 13.8% compared with 25.2%, and a higher correlation coefficient, r = 0.79 compared with 0.27. Treadmill exercise tests were invalid or impossible in 30% of all occasions.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Balloon↗

PubMed1989

Quantitative distribution of radiolabeled 5-aminosalicylic acid enemas in patients with left-sided ulcerative colitis.

Rectally administered suspensions of 5-aminosalicylic acid (5-ASA) are topically effective in treating left-sided ulcerative colitis. The extent to which the contents of these enemas are distributed to inflamed mucosal linings has not previously been determined. This study was undertaken to validate a technique for labeling 5-ASA with 99mTc and to quantitate the distribution of [99mTc]5-ASA in eight patients with left-sided ulcerative colitis. Eight patients underwent three colonic scintigraphic exams within five days, receiving a 60-ml radiolabeled 5-ASA enema into the unprepared rectum for each study, with sequential anterior abdominal images obtained for 4 hr. Activity within the rectum, sigmoid, descending, transverse, and ascending colon was quantitated. Over 50% of the labeled enema had advanced beyond the rectum in five of eight patients and in six of eight patients by 30 min and 60 min, respectively. The distribution of [99mTc]5-ASA was quantitatively reproducible when repeated in the same patient on different days, despite apparent visual differences. By 2 hr, the amount of the enema present within the rectum decreased significantly (P less than 0.05) compared to the initial distribution. The amount of enema present within the descending colon was increased significantly at 0.5 hr (P less than 0.05) and at 2 hr (P less than 0.01). There were no significant changes in the distribution from initial values for the sigmoid, transverse, or ascending colon at any time. In each of these cases the spread of the enema to or beyond the extent of disease was documented. In patients with left-sided ulcerative colitis, small volume [99mTc]5-ASA enemas reliably reach the area of inflammation.

Adult↗

PubMed1984

Transient intraluminal diverticulum of the esophagus: a significant flow artifact.

Esophagography in 12 patients showed a characteristic oval- to oblong-shaped barium collection surrounded by a thin radiolucent margin. The collection closely resembled an intraluminal diverticulum. Twelve patients demonstrated these findings, 6 on single-contrast and 6 on double-contrast examinations. These findings were not reproducible on repeat barium swallows, and endoscopy failed to demonstrate an intraluminal diverticulum. Nine of the patients underwent esophagectomy and esophageal substitution. Pathologic examination of the gross specimen did not reveal evidence of intraluminal diverticula. The association of this radiologic appearance in patients with moderately severe motility disorder of the esophagus is emphasized.

Adult↗