Semiautomatic pipetting device (the governor pipet) for clinical chemistry procedures.
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Chemistry, Clinical↗
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A semi-automated radioimmunoassay technique employing a nine-channel pipetting and sample handling.
A semi-automated RIA-technique has been developed which employs simultaneous pipetting of nine aliquots of reagents with nine-channel pipettes and the processing of RIA samples in nine-tube units. This processing includes evaporation, centrifugation, and decantation of tubes at the end of separation step of the RIA. The nine-channel pipetting reduced the time necessary for pipetting to about one third as compared to the corresponding one channel pipetting, and made the pipetting less tiresome. The precision of pipetting was also improved. This nine-channel RIA-technique was applied to radioimmunoassays of plasma 17-beta-oestradiol and progesterone, in which [125I]iodohistamine-conjugated steroid-tracers were used.
Autoanalysis↗
Role of the membrane cortex in neutrophil deformation in small pipets.
The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.
Adult↗
Laboratory work with automatic pipettes: a study on how pipetting affects the thumb.
The aim of this study was to assess the strain that is exerted on the thumb when working with automatic pipettes. The study consisted of three parts: a survey concerning stress-related symptoms in general and in the thumb in particular, a study of the working conditions in the laboratory with the help of a video-film, and a power test. In the survey the Nordic Council of Ministers' questionnaire and a newly developed questionnaire about the thumb were used. A usual and frequently carried out analysis that included several different methods of pipetting was filmed on video. The power test measured two things, the power that is necessary to press down the button of the pipette into different positions and the maximal strength when performing a movement in the same direction. It was shown that the strain on the thumb for a woman with weak muscular structures is unacceptably high. The symptoms increase with the amount of time spent with pipetting and with age. In addition, pipetting is done in a position where the thumb is not stable but nevertheless has to work to stabilize the grip around the pipette and to press down the button of the pipette. Therefore the muscles have to work both as mobilizing and stabilizing structures. It is concluded, therefore, that some form of automation ought to be taken into consideration if the amount of pipetting work tends to increase. The pipettes should be constructed with as little button resistance as possible and the handle should be designed to fit different hand sizes.
Clinical Laboratory Techniques↗
[Evaluation of pipetting systems. III. Micropipette precision in a routine task].
OBJECTIVE: To establish a norm of the precision achievable with a micropipette in an IRMA assay under routine conditions. MATERIAL AND METHODS: A micropipette (Gilson) adjusted to dispense 100 microL was used by a single analyst with experience in its use. In each assay, ten aliquots of radioactive antiprolactin were pipetted in clean tubes (PRE-batch tubes), followed by pipetting of the tubes being processed in the assay, and at the end, a second pipetting of 10 aliquots in clean tubes (POST-batch tubes). The study includes the data of 15 consecutive batches during a seven month period with an overall mean of 283 tubes per batch. The PRE- and POST-tubes were read in a gamma counter (Crystal plus). The mean, SD and CV for PRE, POST and global (PRE+POST) tubes were calculated for each batch. RESULTS: The global CV of the 15 batches ranged from 1.6 to 6.9%, mean of 3.1%. We found no evidence of increased imprecision due to fatigue of the analyst, but surprisingly, we observed that in nine of the 15 batches there was a significant difference in the means of the PRE-tubes vs the POST-tubes (t test) without differences in precision. Thus, part of the global variability is due to what we have called pseudoimprecision (i.e. an increase in CV due to differences in means). In addition, the POST-tubes had higher values in the first 7 batches but the opposite occurred in the last 8 batches (table 2). This shift in the sign of the PRE-POST differences suggests the presence of opposite factors operating in time, i.e. one or more factors increased the volume of pipetting after using the pipette more than 150 times (batches 1-7) whereas other/others decreased it (batches 8-15). CONCLUSIONS: 1. Our first approximation to a norm of micropipetting precision in batches of 200-300 tubes was a CV of 3.1%. 2. This norm was influenced by a problem of pseudoimprecision detected ex-post-facto. 3. Our findings justify continuation studies to detect the pseudoimprecision and evaluate its causes prospectively.
Antibodies↗
An improved method for illuminating pipet tips for fire-polishing.
Shining light down the long axis of a pipet causes the pipet tip and walls to glow, yet leaves the background dark. The resulting contrast, and the appearance of colored bands near the pipet tip, improves visualization of the tip, affording more precise control of fire-polishing. Existing fire-polishing apparatus can be easily modified to incorporate this type of illumination.
Fiber Optic Technology↗
Dual-pipet techniques for probing ionic reactions
Novel dual-pipet electrodes prepared by pulling borosilicate theta-tubing are described. Three types of electrochemical experiments employing such devices include the following: (1) generation/collection experiments in which ions are ejected from one of two micropipets ("generator") into the external solution and collected at the second pipet ("collector"), (2) measurements of ohmic current-voltage curves, and (3) ion-transfer voltammetry "in the air". The first setup is used for probing ion transfers at the interface between two immiscible liquids and homogeneous reactions in solution involving ionic species. Such experiments are reported for two model processes, i.e., simple and facilitated transfers of potassium between aqueous and organic phases and complexation of potassium with dibenzo-18-crown-6 in organic solution. The second arrangement is used for characterization of theta-pipets. The last arrangement can be useful for preparation of gas sensors. The possibility of measuring the concentration of volatile substances (e.g., ammonia and nitric acid) in the gaseous phase has been demonstrated.
Journal Article↗
Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets.
The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.
Acetylcholine↗
Adaptations to keep a thyrotropin immunoradiometric assay "supersensitive" with automated pipetting.
The sensitivity of the immunoradiometric kit "RIA-gnost hTSH" (Behring) is poorer with a common automated pipetting station than with careful manual pipetting, principally because of tracer contamination of the inner wall of the tube, above the level reached by the wash solution. This can be eliminated by adapting the wash buffer volume so as to fill the entire tube. In this way, one can detect as little thyrotropin as 0.02 milli-int. unit/L, which is better than the 0.03 milli-int. unit/L claimed by the manufacturer. Cross-contamination from pipetting can induce overestimation of "suppressed" thyrotropin values. Prolonging the counting time to obtain an optimal counting error is of no practical value in the thyrotropin range where activity is low.
Autoanalysis↗
Spuriously high serum concentrations of TSH due to the use of an automatic pipetting device.
In a clinically hyperthyroid patient a TSH-value of 0.75 mU/L was measured. Upon retesting TSH was <0.1 mU/L. The patient's sample initially had been preceded, in the pipetting device, by a grossly hypothyroid patient's sample. Analogous problems were subsequently documented in 10 cases when samples of other hyperthyroid patients were preceded by samples with modestly to markedly elevated TSH. The spurious elevation of TSH (0.12 to 1.18 vs. <0.1 mU/L) appears to be due to a carryover effect from the respective previous sample within the used automatic pipetting system. This carryover was much larger than previously suggested, and occurred unpredictably.
Artifacts↗
Rapid flow of passive neutrophils into a 4 microns pipet and measurement of cytoplasmic viscosity.
Neutrophils from five different individuals are isolated with a density separation technique. A total of 151 unactivated (passive) cells are rapidly aspirated at constant suction pressure and at room temperature into a pipet with a diameter of 4 microns. The suction pressures in excess of an initial yield threshold are 0.5, 1 and 2 kPa and are comparable to those encountered in the microcirculation. These pressures are well in excess of the small suction pressure of approximately 20 Pa that is required to form a static hemispherical bump on the cell. At a given aspiration pressure, the leading edge of an individual cell is "tracked" as it flows into the pipet. A theory based on the flow of a Newtonian liquid from either a hemisphere or a spherical segment into a cylinder is used to model the entry process. Both theory and experiment show that during most of the entry process the leading edge of the cell moves at a nearly constant velocity with a rapid acceleration at the end. For cells from five different individuals at the three different excess aspiration pressures, Newtonian theory gives a cytoplasmic viscosity of 135 +/- 54 Pa.s and overall entry times of 3.3s (0.5 kPa), 1.6s (1 kPa) and 0.82s (2 kPa). These results and those of Evans and Yeung at lower aspiration pressures indicate that the complex cytoplasm inside unactivated neutrophils behaves as a nearly Newtonian fluid with a viscosity on the order of 10(2) Pa.s over almost a two order of magnitude range in aspiration pressure and, thus, rate of deformation.
Cytoplasm↗
An aqueous paracentesis pipet.
We describe a glass pipet tipped with a short, sharp, 30-gauge needle for performing aqueous paracentesis. The design eliminates the possibility of needle bending and minimizes fractional resistance during limbus penetration; it also allows quicker recovery of aqueous samples. A small plastic bulb at the upper end facilitates removal of aqueous samples from the pipet.
Aqueous Humor↗
Improved sample and reagent pipetting with the MINILAB.
MINILAB is a small photometer designed for use in small labs and physicians' offices. The instrument is preprogrammed for the determination of several analytes either in whole blood, plasma or serum. Each method is provided as a kit containing disposable cuvettes and capillaries for sample or reagent addition. We examined the precision of the MINILAB using the cholesterol and bilirubin methods for serum, following the recommendations provided for each method, and found that the precisions were not acceptable (CV up to 16% for cholesterol and 43% for bilirubin). Suspecting that these variations were due to sample and reagent pipetting, we tried a simple modification using a Microcap for pipetting and were able to improve the precisions, resulting in CVs of 1.4% for cholesterol and 3.5% for bilirubin.
Bilirubin↗
Precise, easy measurement of glass pipet tips for microinjection or electrophysiology.
We describe a simple procedure to measure precisely the tip diameter of glass micropipets. This procedure can improve the precision of microinjection and some electrophysiological measurements due to their extreme sensitivity to the pipet's exact geometric dimensions. The technique is nondestructive and readily carried out as a brief, extra step in these applications. Digital measurements of the threshold pressures for gas bubbling from (gas filled) pipets submerged in methanol were used to calculate the corresponding inner tip diameters using the LaPlace equation. Direct measurements of the inner tip diameters (from 0.2-5.0 mu) using scanning electron microscopy confirmed the validity of this indirect, simple procedure.
Electrophysiology↗
[Evaluation of pipetting systems. II. Precision and accuracy of precision dispensers].
OBJECTIVE: To evaluate the precision and accuracy of the liquid dispensers being used in our institution. MATERIAL: A total of 15 dispensers (8 micro and 7 macro) of automatic, semiautomatic or manual types were evaluated. They had been in use from 0.5 to 7 years. METHODS: The volume dispensed was established by gravimetry using an analytical balance (Chio JP-160) and deionized distilled water as previously described. The microdispensers were evaluated using volumes of 20 and 100 microL, and the macrodispensers with 1 and 5 mL. Each dispenser was evaluated using 10 replicates: the mean and the CV (coefficient of variation) were calculated with the 10 replicates. The mean was transformed to per cent of the theoretical volume. Thus a 100% ratio of accuracy corresponds to a perfect accuracy. An important methodological aspect was that the 10 dispensing replicates were performed personally by the habitual user of the dispenser. RESULTS: The mean and CV are shown in table 2 for the microdispensers, and in table 3 for the macrodispenser. In the total 25 evaluation in tables 2 and 3 there were 8 instances in which precision was poor (CV above 3%) and also 8 cases of sizable inaccuracy (ratio outside of 100 +/- 5%). In 4 (2 micro and 2 macro) of these 8 inaccuracies, the error ranged from 10% to 22%. A minority was within the specifications of precision and accuracy claimed by the manufacturers of the dispensers with the exception of the manual microdispensers which were within specifications in most instances. We attribute the latter to the lack of a mechanical device in the manual dispensers. CONCLUSIONS: As in a previous evaluation of automatic micropipettes, we detected a high proportion of pipetting systems out of specifications in both precision and accuracy. The magnitude of error in the accuracy of 4 dispensers (10 to 22%) would be catastrophic in methods demanding good pipetting.
Calibration↗
Glycosylphosphatidylinositol-alkaline phosphatase from calf intestine as substrate for glycosylphosphatidylinositol-specific phospholipases--microassay using hydrophobic chromatography in pipet tips.
An electrophoretically homogeneous glycosylphosphatidylinositol- alkaline phosphatase fraction from calf intestine, obtained by hydrophobic chromatography, was used as "enzyme-labeled" substrate for testing phospholipase activity. The reaction products were separated by (i) hydrophobic chromatography in pipet tips and (ii) Triton X-114 phase partitioning. The chromatographic method presented permits high test frequencies, does not need temperature-controlled sample handling, and is only slightly disturbed by detergents, organic solvents, and proteins. The method was used to characterize phosphatidylinositol- specific phospholipase C from Bacillus cereus and phospholipase D from calf serum. Measurement of substrate hydrolysis by phospholipases is apparently linear to enzyme concentration and time. Relative activity of both enzymes is maximum at pH 6.5, corresponding to the optimal pH range found with other glycosylphosphatidylinositol substrates and phosphatidylinositol-specific phospholipases of other sources. Maximum activity of phospholipase C was found at 0.03% Triton X-100, 0.01% Brij 35, and 0.2% n-octylglucoside. The activity is not affected by Ca(2+), NaHCO(3), o-phenanthroline, or EDTA, increasingly inhibited by MgCl(2), MnCl(2), and ZnCl(2), and slightly activated by Na+ and K+. Calf serum phospholipase D shows maximum activity at 0.05% Triton X-100, 0.02% Brij 35, and 0.4% n-octylglucoside. The apparent Km values for phospholipase C (12.25 micron) and phospholipase D (4.94 micron) found with glycosylphosphatidylinositol-alkaline phosphatase are compared with values published for other glycosylphosphatidylinositol substrates.
Alkaline Phosphatase↗
A polisher for patch pipets.
The design and construction of a novel apparatus to fire polish patch-clamp recording pipets is described. The device positions the polishing filament in the field of view of the high-power polishing objective and uses the mechanical stage of the microscope to hold the electrode, eliminating the need for a micromanipulator to hold either the filament or electrode.
Electrodes↗
Chromosomal aberrations induced in human whole blood cultures by pipetting cell pellets in the presence of AluI.
A physical method is described to permeabilize human peripheral lymphocytes in culture for the restriction endonuclease AluI. Blood cultures are incubated for 20 h, pelleted, strongly pipetted with different types of Pasteur pipettes in the presence of AluI and recovered for up to 54 h in the presence of 5-bromodeoxyuridine. Up to 20% of aberrant first posttreatment (M1) metaphases with chromosome-type aberrations are found. The method works with whole blood cultures and is therefore easy to perform.
Bromodeoxyuridine↗