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PubMed1996

Evaluation of a fluorescence polarographic immunoassay with increased sensitivity for measurement of low concentrations of tobramycin in serum.

The limits of quantitation of the assay of tobramycin in serum by the fluorescence polarization immunoassay system marketed by Abbott Laboratories (TDxFLx system) are 0.1 and 10.0 mg/L. For some pharmacokinetic studies, however, a more sensitive analysis is needed. The sensitivity of the TDxFLx system can theoretically be increased 10-fold by pipetting buffer solution into the sample well and 450 microliters serum into the predilution well. The assay modified in this way can be run with the usual calibration of the apparatus for normal analysis. The analytical performance of this modification of the TDxFLx assay was assessed. Tobramycin concentrations ranged from 0.01 to 1.0 mg/L. Analytical recovery ranged from 85 to 95%. The coefficients of variation for within-run and between-run precision ranged from 0.5% to 5% and from 2% to 6%, respectively, for control concentrations ranging from 0.05 to 0.75 mg/L. Based on recovery and precision results, the lower limit of quantitation was established as 0.025 mg/L. There was no significant detectable cross-reactivity from ceftazidime, aztreonam, flucloxacillin, cilastatin, or imipenem. There was a small, but significant cross-reactivity from gentamicin and netilmicin. Hyperbilirubinemia did not affect the assay, but hyperlipidemia gave falsely elevated results of the tobramycin assay. It was concluded that modification of the assay resulted in an acceptable method to quantify low concentrations of tobramycin in serum.

Anti-Bacterial Agents↗
PubMed2001

Therapeutic drug monitoring of haloperidol, perphenazine, and zuclopenthixol in serum by a fully automated sequential solid phase extraction followed by high-performance liquid chromatography.

In Denmark, haloperidol, perphenazine, and zuclopenthixol are among the most frequently requested antipsychotics for therapeutic drug monitoring. With the number of requests made at the authors' laboratory, the only rational analysis is one that can measure all three drugs simultaneously. The authors therefore decided to develop an automated high-performance liquid chromatography (HPLC) method. Two milliliters serum, 2.0 mL 10 mmol/L sodium phosphate buffer (pH 5.5), and 150 microL internal standard (trifluoperazine) solution were pipetted into HPLC vials and extracted on an ASPEC XL equipped with 1 mL (50 mg) Isolute C2 (EC) extraction columns and acetonitrile-methanol-ammonium acetate buffer (60:34:6) as extracting solution. Three hundred fifty microliters was analyzed by HPLC; a 150 x 4.6-mm S5CN Spherisorb column with a mobile phase of 10 mmol/L ammonium acetate buffer-methanol (1:9), a flow rate of 0.6-1.7 mL/min, and ultraviolet detection at 256 and 245 nm were used. Reproducibility was 5-12% and the lower limit of quantitation was 10, 1, and 5 nmol/L (4, 0.4, and 2 ng/mL) for haloperidol, perphenazine, and zuclopenthixol, respectively. The method was found to be sufficiently selective and robust for routine analysis.

Antipsychotic Agents↗
PubMed2004

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

STUDY DESIGN: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. OBJECTIVES: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. SUMMARY OF BACKGROUND DATA: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. METHODS: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. RESULTS: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. CONCLUSION: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Animals↗
PubMed2003

Prolongation of skin xenograft survival with modified cultured fibroblasts.

Cyclosporine A, one of the most potent immunosuppressive drugs, mediates some of its immunosuppressive and nephrotoxic effects by enhancing transforming growth factor-beta secretion and receptor expression. In this experimental study, the effect of cyclosporine pretreatment of cultured dermal fibroblasts on xenogeneic tissue rejection after microimplantation beneath skin grafts was investigated. The effects of the site-specific immunosuppressive strategy on skin xenograft survival were tested. Because the skin is an immunological indicator of the rejection of composite tissue allografts, it was considered that this strategy could be used as a supportive therapy for composite tissue allotransplantation in the future. In the first stage of the study, fibroblast cultures obtained from skin biopsy samples from five rats were treated with different single doses of cyclosporine (100 to 3000 ng/ml), and transforming growth factor-beta levels were measured in culture supernatants after 72 hours. In the second stage, 60 Sprague-Dawley rats were divided into six groups, as follows. For group I (sham), only the standard grafting procedure was performed. For group II, after the standard grafting procedure, rats were treated with intraperitoneal injections of 30 mg/kg (n = 5) or 10 mg/kg (n = 5) cyclosporine for 10 days. For group III, cultured fibroblasts obtained from skin biopsy samples from rats were treated with 100 or 500 ng/ml cyclosporine, and the cells were collected by light trypsinization and centrifugation after 72 hours. After the standard skin grafting procedure, modified fibroblasts were implanted between the graft and the recipient bed with a Pasteur pipet. For group IV, the same procedures as for group III were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. For group V, in addition to standard grafting, unmodified fibroblasts (not treated with cyclosporine) were implanted between the graft and the recipient bed. For group VI, the same procedures as for group V were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. The rejection process was observed macroscopically, and statistical significance was determined with the Mann-Whitney test (p < 0.01). Graft survival times were significantly prolonged in groups III and IV, compared with groups I, II, V, and VI (p < 0.001). No difference between groups III and IV was observed. The novel finding of this investigation is that xenogeneic skin graft survival times could be prolonged with microimplantation of cyclosporine-treated cultured fibro-blasts.

Animals↗
PubMed1991

Invasion of HEp-2 cells by strains of Salmonella typhimurium of different virulence in relation to gastroenteritis.

Experiments to measure the invasiveness of seven strains of Salmonella typhimurium for HEp-2 cells showed that high inocula (100 bacteria/HEp-2 cell), as used by most workers to synchronise events and to increase the number of bacteria which invade, resulted in recovery of significantly less than 1% of the original inoculum after treatment with gentamicin to kill extracellular bacteria. Also, the cell culture medium became acidic, and microscopic examination of Giemsa-stained monolayers immediately following gentamicin treatment revealed high concentrations of bacteria associated with the cells. Moreover, with bacterium-cell interaction beyond 2 h, many HEp-2 cells became rounded, especially with virulent strains W118 and TML. Thus, the biological significance of the quantitative data was uncertain. The fall in pH and the rounding of HEp-2 cells were prevented by the use of a low (1:1) bacterium: cell ratio; but the recovery of bacteria after treatment with gentamicin was still lower than expected by microscopic examination. After treatment of cells with Triton X-100 to release bacteria, many remained bound to residual cell nuclei. Additional treatment with a rubber policeman, and vigorous pipetting to disperse aggregates of bacteria and cell debris, increased the recovery to c. 10% of the initial inoculum after interaction for 2 h, and 30-80% after 4 h, depending on the strain and experimental conditions. The pattern of invasiveness, but not the absolute count, was highly reproducible on different days and in different hands. However, after interaction exceeding 2 h, the distribution of bacteria was uneven, many cells having no associated organisms, others showing microcolonies.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Adhesion↗
PubMed2001

High-throughput genotyping with single nucleotide polymorphisms.

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

Alleles↗
PubMed2001

Phenotype microarrays for high-throughput phenotypic testing and assay of gene function.

The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes.

Escherichia coli↗
PubMed1996

Laser microsurgery of higher plant cell walls permits patch-clamp access.

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Cell Membrane↗
PubMed1982

Growth of Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) in Still Nutrient Medium.

Cell suspension cultures were established from the callus proliferation of leaf explants of 10- to 12-day-old seedlings of the peanut (Arachis hypogaea L. var. TMV-3). The cells could be cultivated in both agitated and still media, the latter promoting more of chlorophyll (Chl) synthesis. High Chl content (210-240 micrograms Chl per gram fresh weight), yield of free and pipetable cells, presence of all the pigments in the same ratio as that of the leaf tissue, and high rates of O(2) evolution (140-170 micromoles O(2) per milligram Chl per hour) were some of the desirable features of the still-grown cell cultures. However, considerable variations with regard to the above characters were observed between the cell cultures of different varieties of the peanut.O(2) evolution by the cultured cells was dependent on exogenous supply of HCO(3) (-). A well-developed photosynthetic apparatus as evidenced from photosystem I and photosystem II activities of the isolated chloroplasts and variable fluorescence measurements with the cell cultures was further documented by electron microscopic evidence of distinct granal stackings in chloroplasts and sodium dodecyl sulfate-polyacrylamide gel separation of thylakoid membranes into P700 Chl a protein complex and light-harvesting Chl a/b complex. Evidence is presented for the relative increase in the Chl associated with P700 Chl a protein complex in contrast to the light-harvesting Chl a/b complex in the cultured cells as compared to intact leaf.

Journal Article↗
PubMed1984

Heat stress responses in cultured plant cells : effect of culture handling and age.

The pipetting of pear (Pyrus communis cv Bartlett) suspension cultures was followed by a substantial but transient decrease in heat sensitivity. During a culture cycle, pear cells were most sensitive to heat at day 3, which coincided with the period of most active cell division. To minimize serious artifacts, the influence of culture handling and age on parameters such as heat sensitivity must be standardized.

Journal Article↗
PubMed2002

Semi-automatic protein crystallization system that allows in situ observation of X-ray diffraction from crystals in the drop.

Semi-automatic protein crystallization device was developed using 96 format Hydra dispenser. Eight extra syringes were added for pipetting protein solution with its own dispensing mechanism. This device allows setting up 96 drops in about three minutes. New crystallization plate has also been developed. The plate allows collecting an oscillation photograph of the crystal directly in the drop. The diffraction quality of the crystals in the drops can be inspected without causing any other physical perturbations to the crystals. With the modified dispenser and this plate, we can perform both hanging and sitting drop vapour diffusion method.

Archaeal Proteins↗
PubMed2005

Stimulation of isolated ventricular myocytes within an open architecture microarray.

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.

Action Potentials↗
PubMed2005

Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched-flow setup.

Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor sigma32 free in solution. Our results indicate that the C-terminal sigma4 domain of sigma32, which is responsible for the recognition of the -35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous sigma-factor sigmaA in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.

Chemistry Techniques, Analytical↗
PubMed2005

In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.

Adult↗
PubMed2004

Human scalp irritation compared to that of the arm and back.

Large-scale data comparing reactions to surfactants between scalp and back and arm are lacking. The sensitivity of responses between scalp and back and arm were explored utilizing an open-application model for testing the potential irritancy of sodium lauryl sulphate (SLS). 10 bald male Caucasians (mean age 56 +/- 9 years) were enrolled. We conducted 5 successive washings: for each wash, the technician pipetted 1 ml of 20% SLS solution into a glass cylinder placed on the designated area with hand pressure that prevented the cylinder leaking. The test area was then rubbed with a Teflon Policeman scrubber for 1 min. Post scrubbing, the solution was absorbed dry with a plastic pipette and blotted by gently applying paper tissues. After a 5-min rest, the procedure was repeated for 4 more times for a total of 5 times. Skin-irritancy assessments by visual scoring and instrumental measurements were made at 30 min and 24 h thereafter and squamometry at the end of last washing. Results indicated that most param- eters revealed that the back was most sensitive to the SLS challenge. Thus, these data support the current standard skin-compatibility testing procedure, employing the back for potential irritation testing of hair care products.

Arm↗
PubMed2005

Induction of multicellular 3-D spheroids of MCF-7 breast carcinoma cells by neutrophil-derived cathepsin G and elastase.

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF-7 human breast carcinoma cells adhering to culture substrates. When MCF-7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N-formyl-Met-Leu-Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3-D spheroids formed when the cultures were prolonged. The spheroid-inducing reaction was reversible and energy-dependent. The MCF-7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E-cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti-E-cadherin antibody following pipetting. The aggregation-inducing activity in neutrophils was completely inhibited by soybean trypsin-chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF-7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites.

Adenocarcinoma↗
PubMed2004

Induction of the mar operon by miscellaneous groceries.

AIM: To investigate the potential of non-antibacterial consumer products to act as inducers of the multiple antibiotic resistance (mar) operon of Escherichia coli SPC105. METHODS AND RESULTS: Wells were cut into chemically defined agar medium (CDM) contained within Petri dishes. Molten agar slurries were prepared by mixing known quantities of 35 consumer products with molten CDM and these were pipetted into each well. Plates were overlaid with molten CDM (5 ml), containing 40 microg ml(-1) X-gal and approx. 1000 CFU ml(-1) of an overnight culture of E. coli SPC105 containing a chromosomal marOII::lacZ fusion. After incubation (37 degrees C, 24 h), plates were examined for zones of growth inhibition and the presence of a blue coloration, indicative of mar (marOII::lacZ) induction. Of the 35 products tested (nine herbs and spices, 19 food and drinks and seven household products), 24 (69%) of the items produced inhibitory zones and 22 (63%) of the items induced mar expression. Apple puree was inhibitory but did not induce marOII::lacZ. Mustard, chilli and garlic were shown to be powerful inducers of marOII::lacZ. Overall six products were shown to be powerful marOII::lacZ inducers. None of these made hygiene claims. CONCLUSIONS: In addition to induction by specific biocides and antibiotics, mar is induced by the exposure of bacteria to natural substances, many of which are common to a domiciliary setting. SIGNIFICANCE AND IMPACT OF THE STUDY: Concern that the overuse of antibacterials within consumer products might select for mar-mediated resistance is shortsighted and fails to recognize the ubiquity of inducers in our environment.

Drug Resistance, Multiple, Bacterial↗
PubMed1988

Energy-dispersive X-ray microanalysis of air-dried microdroplets containing a macromolecular solute.

In the preparation of microdroplets of biological fluids for X-ray microanalysis, we have found that incorporation of a macromolecular solute, dextran, to a final concentration of 1.5-2.5% retards crystal formation and produces sufficiently uniform deposits on thin films to allow droplets to be analysed without prior freeze-drying. Analyses have been carried out at 20 kV in a scanning electron microscope, using energy-dispersive spectrometry. Absorption of Na X-rays by the added solute can be significant but its effect is minimized by preparing droplets as thin as possible, and by using standards of similar composition. The minimum detectable concentrations are increased because of the extra background contribution, and for a single determination are about 6 mM for Na and 2 mM for Cl and K. These concentrations can be further reduced by measuring replicates. The reproducibility of analysis is significantly improved (to less than 5% for Na and K) over the use of calibration curves by calculating the element concentrations from a known element in the sample, chlorine. Under our analytical conditions loss of Cl did not occur. This method requires that the Cl is measured separately by microcoulometry, but eliminates the need for a range of standard droplets on the grid, and determination of the unknowns is then independent of droplet volume, beam current, counting time and magnification. We have compared, with biological samples, the results from using Cl as an internal standard with those obtained using an added standard element, cobalt. The reproducibility using Cl was approximately two-times better than that obtained with Co, probably because of unavoidable volumetric errors when the Co is pipetted separately.

Calcium↗