Capillary Needle Research Articles

O-Glycan Sialylation and the Structure of the Stalk-like Region of the T Cell Co-receptor CD8

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.

Fine Needle Capillary Sampling of Eyelid Masses

To study the cytomorphology of eyelid tumors, correlate it with histopathology and determine the diagnostic accuracy of fine needle capillary (FNC) sampling (nonaspiration) in the evaluation of lid tumors. A prospective study of 70 cases of eyelid masses carried out using FNC, with confirmation by histology in 66 cases and peripheral blood smear and bone marrow examination in 3. Histology was not done in 1 case, and 11 cases were inadequate on cytology. The diagnostic accuracy of cytology was 94.73%, and false negativity was 5.17%. The ages of the patients ranged from 3 to 75 years, with a mean of 40.4. The male/female ratio was nearly equal (37:33). The upper eyelid was affected in 45 cases and lower lid in 25 cases. The right eye was affected in 35 cases, left eye in 34 cases and both eyes in 1 case. Fifteen benign, 35 malignant and 9 infectious/inflammatory lesions were encountered, with 11 inadequate smears. FNC sampling is an effective method of sampling eyelid tumors because it causes little discomfort to the patient and allows the operator to maintain better control over the procedure. A distinction between inflammatory, benign and malignant lesions and between the types of malignant tumors can be made.

Comparative Study of Fine Needle Aspiration and Fine Needle Capillary Sampling of Thyroid Lesions

To investigate whether fine needle capillary (FNC) sampling gives quantitatively and qualitatively superior cytologic material as compared to the conventional technique of fine needle aspiration (FNA) when performed by a single aspirator. Cross-sectional diagnostic test evaluation study. FNA and FNC were performed by a single operator on 200 diffuse and nodular thyroid lesions. A statistically significant difference in favor of FNC was observed for the parameter amount of cellular material. For the rest of the parameters--background blood or clot, degree of cellular degeneration, degree of cellular trauma and retention of architecture--the average score favored FNC but was not statistically significant--i.e., smears prepared from FNC displayed cellular material that was more concentrated, less damaged and less likely to be obscured by blood. Although FNC sampling was diagnostic in a greater number of cases than FNA sampling, this study did not prove a clear superiority of FNC over FNA. Until greater experience shows clear sampling superiority of FNC alone, rather than performing only FNA in diffuse or nodular thyroid lesions, incorporating FNC into the second puncture will definitely improve the quality and quantity of material at the patient's first visit.

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine.

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.

Challenges in achieving a fundamental model for ESI

The relative intensities of the ions in an electrospray ionization (ESI) mass spectrum typically do not reflect the relative concentrations of the ions in solution. A partitioning equilibrium model was developed to explain in quantitative detail many of the behaviors observed in ESI in terms of matrix effects. The fundamental experiments required to validate this model presented several challenges in terms of instrumentation, interpretation, and application. The most basic requirement is a stable spray current that can be measured with accuracy and precision since several factors depend on the spray current. Modifications to the instrument used for these experiments resulted in a stable spray current that could be measured accurately, minimum drifting of the total ion current (TIC), and removal of risking damage to the instrument if arcing occurs. The presence of multiple analyte and electrolyte peaks due to fragment and cluster ions, in addition to solvent and impurity ion peaks, require care in distinguishing and accounting for each component in the solution. The dependence of the spray current on several parameters (analyte and electrolyte concentrations, flow rate, needle voltage, and needle position) as well as the interdependencies among these parameters presented other challenges. The limited the range of variability of each parameter due to these interdependencies was extended by extending the range of stability of the spray current by replacing the metal needle with a glass capillary needle. Overcoming these challenges has enabled us to carry out the quantitative fundamental experiments required to establish a working ESI model with broad applications.

Generating electrospray from solutions predeposited on a copper wire

This study presents a novel direct probe (DP) to perform electrospray ionization (ESI). The probe is constructed simply from a thin copper ring connected to a high voltage power supply, and a capillary and syringe pump are unnecessary. Approximately one microliter of the sample solution is applied directly onto the copper ring by a micropipette. Electrospray from the solution on the copper ring is induced by surface deformation by deflecting a droplet from the copper ring. The mass spectra of proteins obtained by DP–ESI are exactly the same as those from conventional ESI sources through a capillary needle. The time deemed necessary to complete an analysis is approximately 2 minutes, and sample switching is immediate. The signals of the analyte can last from 45 seconds to more than 10 minutes depending on probe design. Three different types of probes used to retain more sample solution on the probe during electrospray were designed and constructed. Moreover, cleaning the probe between different sample analysis is easy. Since a capillary is not used for sample transportation, presence of the particles in the sample solution does not interfere with the electrospray process by capillary blockage. Copyright © 1999 John Wiley & Sons, Ltd.

Protein Identification at the Low Femtomole Level from Silver-Stained Gels Using a New Fritless Electrospray Interface for Liquid Chromatography–Microspray and Nanospray Mass Spectrometry

Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low μl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (≤ 10 μm) are packed directly into laser-pulled fused silica capillary needles from which a spray originates. For analysis of proteins/peptides in solution, low femtomole sensitivity has been achieved (attomoles for selected-ion monitoring), while low nanogram sensitivity was attained for proteins derived from in-gel-digested silver-stained bands from 1-D and 2-D gels. Several applications for tandem MS protein/peptide identification using LC-microspray, LC-nanospray, or infusion nanospray are presented.

Evaluation of Fine Needle Capillary Sampling in Superficial and Deep-Seated Lesions

To study the efficacy and merits of a simplified cytologic technique of fine needle capillary (FNC) sampling in 670 cases. FNC sampling was tested on a consecutive series of 600 superficial palpable masses and 70 deep-seated lesions during the period January 1993-September 1993. Among superficial masses, sampling was done on 380 lymph nodes, 88 breast lesions, 56 thyroids, 12 salivary glands and 64 miscellaneous sites. Fifty-five lung lesions were sampled using 8 computed tomography, while 15 abdominal masses were sampled using ultrasonography. Cell samples were assessed as diagnostically adequate or inadequate. The diagnostic adequacy in lymph node, breast and thyroid lesions was 95%, 91.07% and 90.09%, respectively. The diagnostic adequacy for deep-seated lesions was 91.42%. The procedure gives a better perception of tumor consistency and control of the hand. FNC resulted in a high yield of good-quality material with retention of cellular architecture. It is recommended that the operator become familiar with the macroscopic slide appearance of a cellular smear. Doubts persist with respect to fibrotic and cystic lesions.

Overcoming Gas Sampling Problems: Analysis of Volatiles Using Capillary Column Needles

Sampling factors that could affect gas chromatograph (GC) response for volatile analysis such as syringe pumping time, injection volume, needle length, temperature, and the type of volatile were investigated. Capillary GC column segments (steel and glass) were installed in gas-tight syringes and used as needles for volatile analysis. Standard stainless-steel needles were also used. Hexylacetate, ethyl-2-methylbutyrate, 6-methyl-5-hepten-2-one, and butanol standard were measured. The number of pumps required to maximize GC response for each needle–volatile combination was determined. Maximal GC response for hexylacetate using standard stainless steel, capillary glass, and capillary steel needles required 10, 20 and 30 pumps, respectively. However, for butanol measurement, the optimal syringe pump number was 5 to 10 for all needle types. The use of a capillary needle resulted in an increase in GC response in the range of 3- to 15-fold relative to a standard stainless steel needle. Injection volume affected GC response in a needle-and volatile-dependent manner. In no case did injection volume vs. GC response extrapolate through origin. The GC response for capillary column needles increased as temperature decreased. Capillary column needles may be useful tools for analysis of volatiles that readily partition into the column coating.

Micro-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins

A “micro-electrospray” ionization source has been developed that markedly increases the sensitivity of the conventional electrospray source. This was achieved by optimization of the source to accommodate nanoliter flow rates from 300 to 800-nL/min spraying directly from a capillary needle that, for the analysis of peptides, contained C18 liquid chromatography packing as an integrated concentration—desalting device. Thus, a total of 1 fmol of methionine enkephalin was desorbed from the capillary column spray needle, loaded as a 10-μL injection of 100-amol/μL solution. The mass spectrum showed the [M + H] + ion at m/z 574.2 with a signal-to-noise ratio of better than 5:1 from a chromatographic peak with a width of about 12 s. A narrow range (15-u) tandem mass spectrum was obtained for methionine enekphalin from the injection of 500 amol, and a full-scan tandem-mass spectrum was obtained from 50 fmol. For proteins, the average mass measurement accuracy was approximately 100–200 ppm for the injection of 2.5 fmol of apomyoglobin and 20–40 ppm for 200 fmol. Carbonic anhydrase B and bovine serum albumin showed similar mass measurement accuracies.

Blood viscometer with vacuum glass suction tube and needle.

A new type of viscometer for measuring human blood viscosity is made by using a vacuum glass tube and a capillary needle. This new viscometer needs a volume of only about 5 × 10–6–8 × 10–6 m3 of blood. Simple handling, quick measurement and accurate results in a wide shear rate range are its superior features. Additionally, the whole measuring set with the viscometer is easy to use and to carry, and it is not expensive.By using various standard-viscosity Newtonian liquids, it is confirmed experimentally that this viscometer is sufficiently accurate in the range of less than about 10–2 Pa·s. The viscosity of fresh human blood collected in a hospital is measured by the new viscometer and it is confirmed that the viscosity value obtained is reasonable by comparing it with that by a traditional measuring method. Further, it is shown that the new viscometer can measure the viscosities of other liquids which have complex properties.

8- 14C]Adenine Feeding and Cytokinin Production in Pea Fruits

Summary No evidence could be found for the incorporation of [8- 14 C]adenine into the endogenous cytokinins of pea seeds when fed for three days using a capillary needle. The wounding did not cause any abnormal growth of the seeds within the pods. It would appear that more serious attention should be given to the hypothesis that tRNA breakdown results in cytokinin release.

A gas mixer for computer calibration of an anesthetic mass spectrometer.

To calibrate an anesthetic mass spectrometer without the use of premixed gases and vapors in cylinders, we devised a gas mixer using fixed resistances of capillary needle tubings and adjustable needle valves to dilute test gases and vapors with oxygen. The dilution ratio was determined during each calibration by diluting air with oxygen and noting the reduction in the ratio of nitrogen to oxygen. Empiric correction was made by the computer for the effects of density and viscosity, relative to air, on the flow of nitrous oxide, carbon dioxide, and the saturated vapors of the three anesthetics through the capillary resistor. The computer was programmed to control solenoid valves both for calibration and for the multiplexed sampling of operating rooms. Oxygen, nitrous oxide, and carbon dioxide were used as pure gases, and halothane, enflurane, and isoflurane were vaporized at room temperature in 50-ml vaporizers. The resulting calibrations were found to be accurate to within +/- 2%.

New injection syringe for gas chromatography

A new syringe construction with a thin glass or fused-silica capillary needle is described. A gas-tight retractable needle shield allows injections through a silicone rubber membrane, without mechanically affecting the needle. Thus, sampling from closed vials can be performed as well as leak-free injections into pressurised systems. The new sampling device was compared with a conventional syringe, equipped with a stainless steel needle, with respect to inertness in a heated inlet system. The thermolabile compound dibenzothiophene-5-oxide was injected in the splitless mode at 250°C and the reaction products was quantified. Considerably less degradation was observed when the new syringe was used, which reveals the catalytic activity of the metal needle, and shows the need for inertness of the complete sampling system. With only a few modifications, the construction presented can be used for automated on-column injection, which will be described in a subsequent article.

Hemolymph volume of noninfected and Plasmodium berghei-infected Anopheles stephensi

The hemolymph volume of Anopheles stephensi adult female mosquitoes was determined by a radioisotope dilution technique. [ carboxy- 14C]Inulin was injected into the hemocoels of mosquitoes with a calibrated capillary needle. After sufficient time for thorough mixing, the labeled hemolymph was collected from groups of 50 mosquitoes by a centrifugation technique. Total hemolymph volume was calculated by a conventional formula for radioisotope dilution. The mean hemolymph volume of the newly emerged adult female mosquitoes was 336 nl/mosquito. The ratio of hemolymph volume to body weight was 0.25 μl/mg body wt. By 14 days after emergence, hemolymph volume had dropped to 190 nl/mosquito. Infection of mosquitoes with the rodent malaria parasite, Plasmodium berghei, had no significant effect on hemolymph volume of the mosquito.

Electrohydrodynamic Ion Source

An electrohydrodynamic (EHD) technique is used to generate ions from liquid metals. Liquid metal is fed to the tip of a capillary needle emitter with a voltage difference applied between the emitter and an extractor electrode to produce an intense electric field at the liquid surface. Electrostatic forces overcome surface tension forces to produce ions by field emission. When using liquid cesium, time-of-flight mass analysis showed the ion current to be primarily Cs+ with a small percentage of Cs2+ and Cs3+. Electron currents of over 1 mA have been produced by operating the emitter at 2 kV negative. Besides cesium, alkali ion beams have been generated using NaK/cesium alloy and sodium. Calculations show that liquid metals of low work functions appear more suitable for production of atomic ions while higher work functions metals may produce multiatomic ions and charged droplets in addition to atomic ions.

Production of Monodisperse Sprays

A technique for producing monodisperse sprays is described. The technique makes use of Rayleigh's criterion for the breakup of capillary jets by mechanically vibrating a number of uniform size capillary needles arranged in parallel. It was used to produce sprays in the size range of 290–950 μ, but in principle there is no reason why this range cannot be extended. The regime of spray sizes where drop coalescence is likely to take place is delineated, and methods of minimizing coalescence are described.

The shape of low-speed capillary jets of Newtonian liquids

The shape of a jet of Newtonian liquid issuing from a capillary needle into air is considered. The results of two theoretical approaches are presented. One approach is a perturbation analysis about the final state of the jet and the other is a boundary-layer analysis near the point of jet formation. Comparison of the predictions with experimental jet shapes shows them to be in semi-quantitative agreement. Especially interesting is the presence of a ‘discontinuity’ in the empirical exponential decay rate of the jet radius occurring at a Reynolds number somewhere between 14 and 20 and the correspondence of this discontinuity with the peculiar behaviour in this range of the Reynolds number of the theoretical eigenvalue.

Apparatus for continuous infusion of microvolumes of solution into organs and tissue.

SummaryAn apparatus is described for infusion of small volumes of test solutions directly into tissues and organs. In more than 3000 successful infusions of the early regenerating limb of salamanders, volumes of 0.001 cc per hour have been introduced for periods of 5 or more hours. The apparatus consists of an advancing bar which is driven forward by a screw shaft attached to a clock motor. The advancing bar presses against the plungers of small syringes forcing fluid into fine polyethylene tubing and thence through a glass capillary needle into the tissues.