Artifactual Decrease Research Articles

Preventing pseudohyponatremia: Intralipid®-based lipemia cutoffs for sodium are inappropriate

BackgroundPseudohyponatremia describes an artifactual decrease in plasma sodium result in samples with high proteins and/or lipids when measured by an indirect ion-selective electrode (ISE) method. We suspected that Intralipid®-based lipemia cutoffs are inappropriate for detecting interfering lipids in human samples and a major contributing factor to the existence of pseudohyponatremia. MethodsWe evaluated 2 approaches to derive a lipemia cutoff for sodium, one in which patient plasma samples were pooled and spiked to simulate hyperlipidemia using Intralipid® (commonly used approach by in-vitro diagnostics manufacturers), and another in which endogenous hyperlipidemic samples (n = 31) were measured by methods not affected by hyperlipidemia (i.e., direct ISE and post-ultracentrifugation indirect ISE). Triglycerides, lipemic index (L-index) and indirect ISE sodium concentrations of samples were measured on Roche Cobas® 8000 and direct ISE on Radiometer® ABL835 Flex analyzers. Endogenous hyperlipidemic samples were also ultracentrifuged on Beckman Coulter® Airfuge to clear excess lipids and re-analyzed for sodium by indirect ISE. ResultsWe discovered that Intralipid® is not an accurate emulation of the lipemic interference seen in pseudohyponatremia because it showed no effect up to the maximum level of lipemia tested (L-index = 2000). By contrast, endogenous hyperlipidemic samples demonstrated significant deviations in sodium concentration (≥4 mmol/l) when L-index approached or exceeded 700, and a strong positive correlation between L-index and the difference between the indirect and direct methods (i.e., extent of pseudohyponatremia). ConclusionsClinical laboratories should lower their tolerance for lipemia from the currently recommended L-index cutoff of 2000 on Roche Cobas 8000®. We recommend reflexing to direct ISE when L-index exceeds 700. Manufacturers and laboratories with other indirect ISE methods should evaluate the effect of lipid interference on their method using hyperlipidemic human samples not Intralipid®.

Improved Diagnostic Performance of High-Sensitivity Cardiac Troponin Assays Is an Artifact of Censored Data.

High-sensitivity cardiac troponin assays with improved analytical performance at low concentrations are credited with increased diagnostic sensitivity in acute coronary syndrome patients. We investigated the relationship between analytical sensitivity (detection capability) and diagnostic accuracy and tested the effect of censoring data with a software model. We generated 4 sets of results with decreasing detection capability and compared the ROC curves with and without censored data. There was no relationship between diagnostic performance and detection capability. When data were censored the diagnostic accuracy decreased progressively with an increase in the threshold concentration for censoring. The ROC curves constructed with censored data have a characteristic appearance with a straight line between the censoring point and the top right hand corner. There is not a direct relationship between the diagnostic accuracy and the detection capability of cardiac troponin assays. The artifactual decrease in diagnostic accuracy can be added to the list of reasons why data should not be censored and this practice should be disclosed in studies on diagnostic accuracy.

Failure of Homeostatic Model Assessment of Insulin Resistance to Detect Marked Diet-Induced Insulin Resistance in Dogs

Accurate quantification of insulin resistance is essential for determining efficacy of treatments to reduce diabetes risk. Gold-standard methods to assess resistance are available (e.g., hyperinsulinemic clamp or minimal model), but surrogate indices based solely on fasting values have attractive simplicity. One such surrogate, the homeostatic model assessment of insulin resistance (HOMA-IR), is widely applied despite known inaccuracies in characterizing resistance across groups. Of greater significance is whether HOMA-IR can detect changes in insulin sensitivity induced by an intervention. We tested the ability of HOMA-IR to detect high-fat diet–induced insulin resistance in 36 healthy canines using clamp and minimal model analysis of the intravenous glucose tolerance test (IVGTT) to document progression of resistance. The influence of pancreatic function on HOMA-IR accuracy was assessed using the acute insulin response during the IVGTT (AIRG). Diet-induced resistance was confirmed by both clamp and minimal model (P < 0.0001), and measures were correlated with each other (P = 0.001). In striking contrast, HOMA-IR ([fasting insulin (μU/mL) × fasting glucose (mmol)]/22.5) did not detect reduced sensitivity induced by fat feeding (P = 0.22). In fact, 13 of 36 animals showed an artifactual decrease in HOMA-IR (i.e., increased sensitivity). The ability of HOMA-IR to detect diet-induced resistance was particularly limited under conditions when insulin secretory function (AIRG) is less than robust. In conclusion, HOMA-IR is of limited utility for detecting diet-induced deterioration of insulin sensitivity quantified by glucose clamp or minimal model. Caution should be exercised when using HOMA-IR to detect insulin resistance when pancreatic function is compromised. It is necessary to use other accurate indices to detect longitudinal changes in insulin resistance with any confidence.

Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.

Generation and Evaluation of a Simultaneous Cardiac and Respiratory Gated Rb-82 PET Simulation.

The goal is to generate and evaluate a simulated 4D Rb-82 PET dataset that realistically models simultaneous respiratory and cardiac motions for use to study the effects of the motions and their compensation using various gating schemes. Normal cardiac and respiratory (C&R) motions were simulated separately using the realistic 4D XCAT phantoms. The C&R motion cycles were divided into 24 and 48 equally-spaced time points, respectively. The simultaneous dual motions were modeled by 24 × 48 phantoms with different combinations of C&R motion phases. Almost noise-free projections of the heart, blood pool, lungs, liver, stomach, spleen, and the remaining body were simulated separately using the combined SimSET and GATE Monte Carlo simulation program which is 12 times faster than GATE alone. The projections were scaled and combined to simulate a typical Rb-82 myocardial perfusion (MP) PET patient study. The no gating, 6-frame respiratory gating only, 8-frame cardiac gating only, and simultaneous 6-frame respiratory and 8-frame cardiac gating schemes were applied. Each gated projection dataset was reconstructed using a 2D OS-EM without and with attenuation correction (AC) using an averaged and gated attenuation maps. The reconstructed images were evaluated in terms of artifactual non-uniformity in the MP polar map. Significant artifactual non-uniformity was found in the MP polar map over all gating scheme without AC. With AC, the artifactual decreases in both the anterior and inferior regions were reduced with respiratory gating. Cardiac motion alone did not cause significant artifactual non-uniformity. In addition, the combination of dual gating and AC using the gated attenuation map provided the most uniform MP polar map. We demonstrated the flexibility and utility of the 4D XCAT phantom set with simultaneous C&R motions. It is a powerful tool to study motion effects on MP PET studies and to evaluate C&R gating schemes, AC and quantitative 4D PET image reconstruction methods.

Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues

We thoroughly analyze the linear propagation effects that affect polarization-resolved Second Harmonic Generation imaging of thick anisotropic tissues such as collagenous tissues. We develop a theoretical model that fully accounts for birefringence and diattenuation along the excitation propagation, and polarization scrambling upon scattering of the harmonic signal. We obtain an excellent agreement with polarizationresolved SHG images at increasing depth within a rat-tail tendon for both polarizations of the forward SHG signal. Most notably, we observe interference fringes due to birefringence in the SHG depth profile when excited at π/4 angle from the tendon axis. We also measure artifactual decrease of ρ = Χxxx/Χxyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both ρ and the tendon birefringence and diattenuation.

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Interference of Xolair (anti-human IgE) in serological assays for total and allergen specific IgE

Rationale The effect of Xolair (Omalizumab: monoclonal anti-human IgE) administration on the accuracy of serological immunoassays for IgE has not been reported. We studied the potential interference of Xolair on a widely-used, FDA-cleared immunoassay for total and allergen-specific IgE. Methods Sera from atopic adults [total serum IgE levels: 78-1186 kIU/L] were incubated (1hr,37oC; 16-18hrs,23oC) with constant volumes of buffer or 7-ten-fold concentrations of Xolair to obtain 8 anti-human IgE:human IgE molar ratios: 0-(sham control), 0.01, 0.1, 1, 10, 100, 1000, 10,000. Specimens were analyzed in duplicate in the Pharmacia FEIA CAP System for total and allergen-specific IgE antibody to 8 allergen specificities: yellow jacket venom, common ragweed, cat dander, dog dander, D. farinae, D. pteronyssinus, Alternaria alternata and Peanut. Results Minimal but reproducible artifactual decreases in measured total serum IgE up to 14% were observed when sera were incubated with 1 to 10 fold molar excess of anti-IgE to IgE. Greater interference was observed with all sera analyzed in the allergen-specific IgE assay. Allergen-specific IgE antibody levels in sham-control sera receiving no Xolair ranged from 0.82 to 65.70 kIU/L. Xolair interference was independent of allergen specificity but dependent on the initial level of allergen-specific IgE. Measured decreases in allergen-specific IgE from 10% to 54% were seen at therapeutically achieved anti-IgE:IgE molar ratios of 1 to 10. Conclusions Total serum IgE CAP analyses appear minimally altered by Xolair in serum, but allergen-specific IgE measurements on sera from allergic patients receiving Xolair should be interpreted with caution, since their accuracy may be compromised by anti-IgE interference.

Tono-Pen versus Goldmann tonometry after excimer laser photorefractive keratectomy

Purpose: To compare intraocular pressure (IOP) measurements by the Tono-Pen 2tonometer and Goldmann applanation tonometer (GAT) in post-photorefractive keratectomy (PRK) patients. Setting: Refractive Surgery Center, Assaf Harofeh Medical Center, Zerifin, Israel. Methods: In 18 patients, IOP was measured by GAT and then by Tono-Pen 2tonometer 2 to 18 months following PRK (mean 6.6 months ± 5.1 [SD]). Photorefractive keratectomy had been performed in 1 eye of each patient; the fellow eyes served as controls. Corneal curvature and thickness were evaluated. Ten of the 18 patients were treated with topical steroids. Results: In the post-PRK eyes, mean GAT IOP was 1.8 ± 3.1 mm Hg lower than meanTono-Pen IOP ( P = .012); there was no significant IOP difference in the control (fellow) eyes. In steroid-treated post-PRK eyes, mean GAT IOP (12.2 mm Hg) was 2.2 ± 1.3 mm Hg lower than mean Tono-Pen IOP (14.4 mm Hg) ( P = .0007). Mean Tono-Pen IOP in steroid-treated post-PRK eyes was 4.3 ± 3 mm Hg higher than in the fellow eyes ( P = .0014); mean GAT IOP was only 2.3 ± 3.5 mm Hg higher ( P = .04). In post-PRK eyes without topical steroid treatment, mean GAT IOP was 2.0 ± 1.18 mm Hg lower than in the fellow eyes ( P = .001); there was no significant difference in Tono-Pen OP. There was a negative correlation between the difference in IOP values (Tono-Pen minus GAT) and corneal curvature in post-PRK eyes ( r = 0.76, P = .0108, n = 15). Conclusions: The Tono-Pen tonometer appeared to be less affected than the GAT by the relative flattening, thinning, and anterior stromal decreased rigidity of the central cornea that occur following PRK. Post-PRK steroid-induced IOP elevation may be masked by the artifactual decrease in GAT IOP.

A study of possible causes of artifactual decreases in the left ventricular apex with SPECT cardiac perfusion imaging

The objectives of this investigation were to determine the impact on apparent localization of perfusion agents in the apex of the left ventricle (LV) of cardiac and respiratory motion, extra-cardiac uptake, the anisotropic spatial resolution associated with imaging, and thinning of the LV wall at the apex. The ability of attenuation compensation (AC), and of combined attenuation and spatial resolution correction (ARC) to alter the impact of these factors on apparent apex counts was also investigated. The MCAT phantom was used to simulate cardiac gated radionuclide perfusion imaging both with and without apical thinning of the LV wall. Simple respiratory motion of the heart was included in the simulation. After reconstruction using filtered backprojection (FBP) with no correction for physical degradation (NC) and using the maximum likelihood ordered subset expectation maximization (OSEM) algorithm with AC, and with ARC, polar maps were generated. Results show that cardiac motion had no impact on apex counts other than an increase in counts with myocardial thickening. Respiratory motion of a magnitude similar to that observed clinically does not influence the apex, but does have some effect on adjacent regions. A small decrease in apical counts was observed with body-contouring as opposed to imaging with a circular camera orbit. Counts in the apex varied greatly with extent of the thinning. AC increased the visibility of the apparent decrease in apical counts over NC with apical thinning, as did ARC over AC.

Modification of the Abbott Cyclosporine Monoclonal Whole Blood Assay

Abbott Laboratories recommends that before every Cyclosporine Monoclonal Whole Blood Assay run on the TDx analyzer, a probe cleaning procedure be performed (1) to avoid an artifactual decrease of results in the first carousel position (2). We asked whether non-use of this procedure significantly affected results for the sample in the first carousel position. In our study, the TDx analyzer underwent daily, weekly, and monthly maintenance, as recommended by the manufacturer. Between …

Effects of storage on the binding of carboplatin to plasma proteins.

Plasma ultrafiltrates are routinely used in pharmacokinetic studies of carboplatin. Experiments were performed to detect and quantitate artifactual decreases in the platinum concentration of ultrafiltrates prepared from plasma samples stored at -20 degrees C and -70 degrees C. Carboplatin was added to anticoagulated, whole human blood to produce a 20 microgram/ml concentration. Plasma produced from the blood was stored frozen at either -20 degrees C or -70 degrees C. Aliquots from each storage condition were thawed and ultrafiltered once a week for up to 100 days. Platinum concentrations in ultrafiltrates and plasma were determined by flameless atomic absorption spectrometry. There was no loss of ultrafilterable platinum in plasma samples stored at -70 degrees C, whereas there was a steady decrease in free platinum concentration in ultrafiltrates prepared from plasma samples stored at -20 degrees C. These results imply that pharmacokinetic studies of carboplatin should use ultrafiltrates prepared immediately or that plasma for such studies should be stored at -70 degrees C. Storage of carboplatin-containing plasma at -20 degrees C and subsequent ultrafiltration is not acceptable, because measurement of platinum in such ultrafiltrates will be artifactually low.

Interference of recombinant soluble CD4 immunoglobulin G in flow cytometric measurement of CD4+ lymphocytes.

Recombinant soluble CD4 covalently linked to an immunoglobulin G heavy chain (rCD4-IgG) was evaluated clinically for the treatment of human immunodeficiency virus infection. The interference of rCD4-IgG with the measurement of peripheral blood CD4 lymphocytes by whole-blood lysis flow cytometric analysis was investigated using three commercial monoclonal antibody reagents. Addition of rCD4-IgG resulted in an artifactual decrease in measured CD4 number at rCD4-IgG levels of greater than or equal to 1 micrograms/mL; the threshold for this decrease was dependent on the concentration of monoclonal antibody in the commercial preparation used for the measurement of CD4. This artifactual decrease in CD4 cell count was observed in two patients who received rCD4-IgG intravenously. The apparent decrease in CD4 count was eliminated with the use of a single phosphate-buffered saline wash step before the addition of monoclonal antibody. rCD4-IgG can bind to anti-CD4 antibody and lower the measured CD4 cell count in vitro; this interference can be eliminated by a single or a double wash step and is necessary when using the whole-blood lysis flow cytometric technique of enumerating CD4 lymphocytes in patients receiving rCD4-immunoglobulin G.

Rayon balls and disposable-diaper material selectively adsorb creatinine

One method for the collection of urine samples from infants involves absorption of the urine on cotton balls placed in the diaper. Such samples are not timed and excretions are often expressed per mg of urinary creatinine. An assumption in this method is that the creatinine concentration is not changed by the absorption process. The concentration of creatinine in urine samples was measured before and after absorption of the urine by cotton balls, rayon balls, or diaper material over a range of wetness. For urine from both adults and infants, absorption on rayon balls and diaper material caused an important artifactual decrease in the concentration of creatinine. The effect was particularly striking in lightly wetted samples; the mean percent decrease was only 3 +/- 2% for cotton but was 9 +/- 5% for rayon (n = 10) and 13 +/- 4% for diaper material (n = 7). These data provide evidence that rayon balls and diaper material (and to a lesser extent cotton balls) selectively adsorb creatinine from human urine.

Analysis of oxidation of the membrane potential probe, Di-S-C 3(5), during granulocyte activation

Measurement of membrane potential with the fluorescent probe, Di-S-C 3(5), suggests that the chemotactic factor, FMLP causes a rapid depolarization followed by an apparent hyperpolarization of human granulocytes. This apparent hyperpolarization seems to be an artifact due to oxidation of Di-S-C 3(5) by a reactive product of the H 2O 2 and myeloperoxidase secreted from activated granulocytes. The artifactual decrease in fluorescence can be prevented by addition of a H 2O 2 scavenger, catalase, or an antioxidant, ascorbate. The calcium ionophore, A23187, causes depolarization but no hyperpolarization of human granulocytes because of its failure to stimulate the release of H 2O 2. Ouabain prevents the delayed hyperpolarization in response to FMLP. Ouabain acts via the Na-K pump to inhibit FMLP-stimulated release of peroxidase from human granulocytes. We conclude that assays of membrane potential should be conducted in the presence of 1 × 10 −4 M ascorbate to prevent oxidation of the fluorescent probe, Di-S-C 3(5).

Population dynamics of snail infection by miracidia.

The essential biological features of snail infection by miracidia are incorporated into a simple model which describes the rate of change with respect to time of the number of miracidial infections/host. The model is based on the assumption that the net rate of infection is directly proportional to the density of both miracidia and hosts. Empirical evidence is provided to support this assumption. The basic framework of the model is expanded to take into account demographic stochasticity in infection and is used to predict the percentage of snails that become infected after exposure to a known number of miracidia for a set period of time. The influence of miracidial mortalities and age-dependent infectivity are examined and theoretical predictions are compared with a range of experimental results. Underlying heterogeneity in the distribution of the number of infections/snail is shown to generate an artifactual decrease in infection rates as exposure density rises, if rate estimation procedures are based on an assumption of randomness. Empirical evidence is presented to illustrate the generation of over-dispersion in the number of miracidial infections/snail under tightly controlled laboratory conditions, using supposedly homogeneous snail populations. Biological causes for underlying patterns of heterogeneity are discussed in relation to snail susceptibility to infection and 'attractiveness' to infective stages.

A QUANTITATIVE MICROIONTOPHORETIC ANALYSIS OF THE RESPONSES OF CENTRAL NEURONES TO NORADRENALINE: INTERACTIONS WITH COBALT, MANGANESE, VERAPAMIL AND DICHLOROISOPRENALINE

1. A new experimental procedure has been devised for the study of pharmacological antagonism in the central nervous sytem using automated microiontophoresis to deliver pulses of agonists and computer-generated histograms to quantify the neuronal response. The system allows study of potential antagonists having direct depressant effects and also of neurones with irregular or slow discharge rates. 2. The histogram analysis reveals the necessity for regular, periodic delivery of agonists during the assessment of agonist-antagonist interactions. Without regular repetitive delivery, many agonists, such as noradrenaline, exhibit an apparent but artifactual decrease in inhibitory potency after an interruption of agonist pulses. Examples of this phenomenon are shown, using cerebellar Punkinje cells and cerebral cortical neurones in rats anaesthetized with halothane. 3. Preliminary results with these computer-generated drug response histograms revealed manganese, cobalt, and verapamil to be generally ineffective as antagonists of noradrenaline, despite their direct depressant effects. 4. Conversely, dichloroisoprenaline (DCI), a beta-adrenoceptor antagonist, was effective in blocking noradrenaline-induced depressions of firing in the cerebral cortex at doses which caused over 50% decrease in spontaneous discharge.