Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals.

Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.

Incidence of invasive ureaplasma in VLBW infants: relationship to severe intraventricular hemorrhage.

OBJECTIVE: As Ureaplasmas may be pathogens in preterm infants, this study was conducted to determine the incidence of invasive disease with Ureaplasma parvum and Ureaplasma urealyticum and the relationship with adverse outcomes in a prospective cohort of very low birth weight (VLBW) infants. STUDY DESIGN: DNA was extracted from the cord or venous blood and cerebrospinal fluid (CSF) samples obtained from 313 VLBW infants. PCR was performed using primers for the mba gene to detect all 14 serovars and then repeated for all positive samples using species-specific primers. RESULT: Ureaplasma species were detected in serum and/or CSF samples from 74 of 313 (23.6%) infants. U. parvum was the predominant species (70%). Presence of Ureaplasma was significantly associated with elevated interleukin-1beta in cord blood (odds ratio (OR) 2.6, 1.05 to 6.45, P=0.039). Ureaplasma serum-positive infants had a 2.3-fold increased risk of intraventicular hemorrhage > or =grade 3 (OR 2.50; 1.06 to 5.89, P=0.036). CONCLUSION: Invasive Ureaplasma occurs commonly in VLBW infants and may increase the risk for severe intraventricular hemorrhage.

Complete inhibition of spontaneous activity in neuronal networks in vitro by deltamethrin and permethrin.

Types I and II pyrethroid insecticides cause temporally distinct decreases in voltage-gated sodium channel (VGSC) inactivation rates that are proposed to underlie their characteristic differences in toxicity signs. How alterations in VGSC channel function give rise to the characteristic differences in signs of pyrethroid intoxication is not completely understood, particularly those changes that occur in functional networks of interconnected neurons. To characterize better pyrethroid actions at the network level, effects of the Type I pyrethroid permethrin (PM) and the Type II pyrethroid deltamethrin (DM) on spontaneous glutamate network-dependent spikes and bursts were investigated in primary cultures of frontal cortex or spinal cord neurons grown on microelectrode arrays (MEAs). Fast GABAergic transmission was blocked by BIC, and concentration-dependent effects of DM (1nM to 5microM) and PM (10nM to 50microM) were examined. Both compounds caused concentration-dependent reductions in the network spike and burst rates. DM was more potent than PM, with IC(50) values of approximately 0.13 and approximately 4microM for inhibition of spike rate in cortical and spinal cord neurons, respectively. Both compounds decreased the percentage of spikes that occurred within a burst and increased the interspike interval within bursts. Onset of effects was rapid, but recovery from total activity loss was not readily achievable. Individual neurons responded heterogeneously; activity of most declined monophasically, but activity in others exhibited biphasic responses with increases followed by decreases in activity. In spinal cord, DM caused a greater number of biphasic responses (29%) than PM (10%). These results demonstrate that both DM and PM inhibit activity of glutamatergic networks, but with different potencies.

Acute and sub-chronic functional neurotoxicity of methylphenidate on neural networks in vitro.

Methylphenidate (MPH) is the drug of choice in the treatment of attention deficit and hyperactivity disorders. Although a popular drug, concentration-dependent electrophysiological alteration or impairment (functional toxicity) and reversibility, have not been quantified. This study used spontaneously active neuronal networks growing on microelectrode arrays (MEA) to investigate functional neurotoxicity of MPH by assessing its acute and sub-chronic electrophysiologic effects on auditory cortex networks (ACN) and frontal cortex networks (FCN) at concentrations that reflect clinical doses and overdoses. Acute exposure to 1-300 microM MPH induced concentration-dependent inhibition of spontaneous activity with spike rate IC(50) values (concentration inducing 50% inhibition) of 112.9 +/- 18.6 and 108.0 +/- 18.9 microM for ACNs and FCNs respectively. Sub-chronic exposure to 1 mM MPH for 24 h blocked all activity followed by partial spontaneous recovery after 15 h. Tyrosine hydroxylase immunocytochemistry analysis indicated positive staining of neurons, confirming the presence of catecholaminergic neurons in cultured cortex networks.

Quantification of zinc toxicity using neuronal networks on microelectrode arrays.

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 microM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min and consisting of increased spiking and enhanced, coordinated bursting, followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Above 100 microM total zinc acetate, the activity loss was associated with massive cell swelling, blebbing, and even vigorous neuronal cell lysing. Glia showed stress, but did not participate in the extensive cell swelling. Network activity loss generally preceded morphological changes. Cultures pretreated with the GABA(A) receptor antagonists bicuculline (40 microM) and picrotoxin (1mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 microM). However, recovery was not complete and slow deterioration of network activity continued over 6-h periods. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss.

NMDA receptor-dependent periodic oscillations in cultured spinal cord networks.

Cultured spinal cord networks grown on microelectrode arrays display complex patterns of spontaneous burst and spike activity. During disinhibition with bicuculline and strychnine, synchronized burst patterns routinely emerge. However, the variability of both intra- and interculture burst periods and durations are typically large under these conditions. As a further step in simplification of synaptic interactions, we blocked excitatory AMPA synapses with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX), resulting in network activity mediated through the N-methyl-D-aspartate (NMDA) receptor (NMDA(ONLY)). This activity was APV sensitive. The oscillation under NMDA(ONLY) conditions at 37 degrees C was characterized by a period of 2.9 +/- 0.3 s (16 separate cultures). More than 98% of all neurons recorded participated in this highly rhythmic activity. The temporal coefficients of variation, reflecting the rhythmic nature of the oscillation, were 3.7, 4.7, and 4.9% for burst rate, burst duration, and interburst interval, respectively [mean coefficients of variation (CVs) for 16 cultures]. The oscillation persisted for at least 12 h without change (maximum observation time). Once established, it was not perturbed by agents that block mGlu receptors, GABA(B) receptors, cholinergic receptors, purinergic receptors, tachykinin receptors, serotonin (5-HT) receptors, dopamine receptors, electrical synapses, burst afterhyperpolarization, NMDA receptor desensitization, or the hyperpolarization-activated current. However, the oscillation was destroyed by bath application of NMDA (20-50 microM). These results suggest a presynaptic mechanism underlying this periodic rhythm that is solely dependent on the NMDA synapse. When the AMPA/kainate synapse was the sole driving force (n = 6), the resulting burst patterns showed much higher variability and did not develop the highly periodic, synchronized nature of the NMDA(ONLY) activity. Network size or age did not appear to influence the reliability of expression of the NMDA(ONLY) activity pattern. For this reason, we suggest that the NMDA(ONLY) condition unmasks a fundamental rhythmogenic mechanism of possible functional importance during periods of NMDA receptor-dominated activity, such as embryonic and early postnatal development.

Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors.

We have utilized cultured neuronal networks grown on microelectrode arrays to demonstrate rapid, reliable detection of a toxic compound, trimethylolpropane phosphate (TMPP). Initial experiments, which were performed blind, demonstrated rapid classification of the compound as a convulsant, a finding consistent with previous whole animal neurobehavioral studies. TMPP (2-200 microM) reorganized network spike activity into synchronous, quasi-periodic burst episodes. Integrated burst amplitudes invariably increased, reflecting higher spike frequencies within each burst. The variability of network burst parameters, quantified as coefficients of variation (CVs), was decreased. Mean CVs for burst duration, interburst interval, and burst rate were lowered by 42+/-13, 58+/-5.5, and 62+/-1.8%, respectively (mean+/-SEM, n=8 cultures, 197 channels). These changes in network activity paralleled the effects induced by bicuculline, a known disinhibitory and seizure-inducing drug, and confirmed classification of TMPP as a potential epileptogenic compound. Simple pharmacological tests permit exploration of mechanisms underlying observed activity shifts. The EC(50) for GABA inhibition of network activity was increased from 2.8 to 7.0 microM by 20 microM TMPP and to 20.5 microM by 200 microM TMPP. Parallel dose-response curves suggest that TMPP acts by a competitive antagonism of GABA inhibition, and are consistent with reported patch-clamp analysis of TMPP-induced reduction of inhibitory postsynaptic current amplitudes. The potency of TMPP in generating epileptiform activity in vitro was comparable to concentrations reported for in vivo studies. TMPP and bicuculline produced both increases and decreases in burst rate depending on native spontaneous bursting levels. These results demonstrate a need for multivariate analysis of network activity changes to yield accurate predictions of compound effects.

Detection of physiologically active compounds using cell-based biosensors.

Cell-based biosensors are portable devices that contain living biological cells that monitor physiological changes induced by exposure to environmental perturbations such as toxicants, pathogens or other agents. Methods of detecting physiological changes include extracellular electrical recordings, optical measurements, and, in the future, functional genomics and proteomics. Several technical developments are occurring that will increase the feasibility of cell-based biosensors for field applications; these developments include stem cell and 3D culture technologies. Possible scenarios for the use of cell-based biosensors include broad-range detectors of unknown threat agents and functional assessment of identified agents.

Neurophysiologic effects of chemical agent hydrolysis products on cortical neurons in vitro.

The neurophysiologic effects of chemical agent hydrolysis products were examined on cultured cortical neurons using multielectrode array (MEA) recording and the whole-cell patch clamp technique. Measurement of neuronal network extracellular potentials showed that the primary hydrolysis product of soman, pinacolyl methylphosphonic acid (PMPA), inhibited network mean burst and spike rates with an EC50 of approximately 2 mM. In contrast, the degradation product of sarin, isopropyl methylphosphonic acid (IMPA), and the final common hydrolysis product of both soman and sarin, methylphosphonic acid (MPA), failed to affect neuronal network behavior at concentrations reaching 5 mM. Closer examination of the effects of PMPA (2 mM) on discriminated extracellular units revealed that mean spike amplitude was slightly diminished to 95 +/- 1% (mean +/- S.E.M., n = 6, P < 0.01) of control. Whole-cell patch clamp records under current clamp mode also showed a PMPA-induced depression of the firing rate of spontaneous action potentials (APs) to 36 +/- 6% (n = 5, P < 0.001) of control. In addition, a minor depression with exposure to PMPA was observed in spontaneous and evoked AP amplitude to 93 +/- 3% (n = 5, P < 0.05) of control with no change in either the baseline membrane potential or input resistance. Preliminary voltage clamp recordings indicated a reduction in the occurrence of spontaneous inward currents with application of PMPA. These findings suggest that PMPA, unlike MPA or IMPA, may more readily interfere with one or more aspects of excitatory synaptic transmission. Furthermore, the data demonstrate that the combination of extracellular microelectrode array and patch clamp recording techniques facilitates analysis of compounds with neuropharmacologic effects.

Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays.

Spontaneously active neuronal networks grown from embryonic murine frontal cortex on substrate integrated electrode arrays with 64 recording sites were used to assess acute neurobiological and toxic effects of a series of seven symmetrical, bifunctional alkylene-linked bis-thiocarbonate compounds designed to possess anticholinesterase activity. Acute functional neurotoxicity in the absence of cytotoxicity was defined as total collapse of spontaneous activity. All of the compounds were characterized as mixed inhibitors of AChE, with K(i)'s in the 10(-7)-10(-6) M range. The neuronal network assays revealed high repeatability for each compound, but surprisingly diverse effects among these closely related compounds. Six of the seven compounds produced changes in network activity at concentrations of 10-350 microM. Three of the compounds were excitatory, two were biphasic (excitatory at lower concentrations, inhibitory at higher), and one was solely inhibitory. Two of the inhibitory compounds produced irreversible inhibition of activity. Responses of cortical cultures to eserine were compared to the effects produced by the test compounds, with only one of seven providing a close match to the eserine profile. Matching of response patterns allows the classification of new drugs according to their response similarity to well-characterized agents. Spontaneously active neuronal networks reflect the interactions of multiple neurotransmitter and receptor systems, and can reveal unexpected side effects due to secondary binding. Utilizing such networks holds the promise of greater research efficiency through a more rapid recognition of physiological tissue responses.

When combined, early bedside head ultrasound and electroencephalography predict abnormal computerized tomography or magnetic resonance brain images obtained after extracorporeal membrane oxygenation treatment.

Definitive neuroimaging of the brain using computerized tomography (CT) or magnetic resonance imaging (MRI) in extracorporeal membrane oxygenation (ECMO)-treated infants must be delayed until after this therapy is completed. Bedside head ultrasound (HUS) and electroencephalography (EEG) studies during ECMO, if highly correlated with later definitive neuroimaging, might be used to affect the acute clinical care and early parental counseling of infants with severe cardiorespiratory failure. One hundred and sixty ECMO-treated patients had both bedside EEG and HUS studies performed during ECMO, as well as a later CT or MRI study prior to hospital discharge. There was a significant difference in CT or MRI findings among patients having normal studies on both the HUS and EEG, compared to those having an abnormality on either the HUS or the EEG, and compared to those having abnormalities on both studies. In ECMO-treated infants, the combination of a normal bedside HUS and an EEG without marked abnormalities is highly predictive of normal post-ECMO CT and MRI neuroimaging studies.

Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultured on microelectrode arrays.

We used spontaneously active monolayer networks in vitro, cultured on thin film microelectrode arrays as experimental platforms for the determination of trimethyltin chloride (TMT) toxicity. Two different tissues of the mouse CNS (spinal cord and auditory cortex) exhibited characteristic and dose-dependent changes of their electrophysiological activity patterns after treatment with TMT, a standard neurotoxicant. Spinal cord networks began to respond to TMT at 1-2 microM and shut off activity at 4-7 microM. Auditory cortex cultures started to respond at 2-3 microM and shut off activity at 7-8 microM. Repeated applications of low doses of TMT always influenced the electrical activity in a reversible manner, with no overt cytotoxic effects. The inhibitory concentrations for a 50% reduction of activity (IC ) were 1.5+/-0.5 microM (spinal cord) and 4.3+/-0.9 microM (auditory cortex) indicating a relatively low interculture variability within one tissue type. The non-overlapping IC50 range for cortical and spinal cord cultures may suggest tissue specificity for network responses to TMT. Shut-off concentrations were found to be within a factor of two of the lethal concentrations reported for mice in vivo. Action potential amplitude and shape did not change even when complete cessation of activity was approached, suggesting that acute TMT applications did not affect neuronal metabolism that would lead to a lowering of membrane potentials. Our results suggest that spontaneously active monolayer networks in vitro are suitable for toxicological investigations since network activity can be influenced in a dose-dependent manner. These properties allow the development of neurotoxicity biosensors based on physiological responses of spontaneously active networks.

Pulsed plasma deposition of allylamine on polysiloxane: a stable surface for neuronal cell adhesion.

Allylamine was pulse-plasma polymerized onto a hydrophobic polysiloxane substrate to create cell adhesion surfaces for cell culture that would not require pretreatment with polylysine, could be sterilized via autoclaving, and could be re-used for several culture cycles. We investigated two different plasma deposition protocols at 200 W RF power: (1) a duty cycle of 3 ms on and 5 ms off; and (2) a cycle of 3 ms on and 45 ms off. Control surfaces were unmodified polysiloxane, activated polysiloxane via flaming, and flamed polysiloxane further modified with poly-D-lysine (PDL). The different surfaces were characterized with XPS analysis, water contact angle, and cell adhesion and growth using dissociated murine embryonic spinal tissue. We found that both the amine content of the 3/45 duty cycle surface and the wettability was higher than that of the 3/5 surface. Also, spinal cord cells were better dispersed 24 h after seeding on the 3/45 surface, suggesting a difference in early adhesion dynamics. However, the networks on the two types of modified surfaces revealed no obvious morphological differences after 2 weeks in vitro. The stability of allylamine-decorated surfaces after autoclaving was high with only minor changes in wettability and nitrogen content. Cell growth on such surfaces after autoclaving was comparable to that found on flamed polysiloxane, freshly modified with PDL. Allylamine surfaces were still usable as cell growth substrates after three autoclaving cycles, 4 weeks under warm culture medium, and simple cleaning procedures, indicating the achievement of a long-lasting modification that did not require the repeated use of PDL before each seeding.

Laparoscopic cholecystectomy and cirrhosis: a case-control study of outcomes.

The incidence of gallstone disease in patients with cirrhosis is greater than that in healthy patients. Previous surgical literature reported greater morbidity and mortality in patients with cirrhosis with both open and laparoscopic cholecystectomy (LC). We compared our recent experience with LC in patients with cirrhosis and controls. A retrospective review was performed using the search terms, "cirrhosis" and "laparoscopic cholecystectomy." Forty-eight patients with cirrhosis were identified and randomly matched with healthy controls by age and sex. Four controls were assigned per patient with cirrhosis. Outcomes assessed included mortality, duration of surgery, length of hospital stay, blood transfusion requirement, postoperative complications, and need for conversion to open cholecystectomy. Forty-eight patients with cirrhosis and 187 healthy controls underwent LC. Child-Pugh classification of severity of liver disease was as follows: Child's class A, 38 of 48 patients; Child's class B, 10 of 48 patients; and Child's class C, 0 of 48 patients. Patients with cirrhosis had statistically significantly lower albumin levels (P =.0001) and prolonged prothrombin times (P =. 05). Average duration of surgery for patients with cirrhosis was 1. 71 versus 1.57 hours (P =.57) for controls. Average length of hospital stay for patients with cirrhosis was 6.47 versus 4.77 days (P =.152) for controls. Average number of units of blood transfused in patients with cirrhosis was 0.156 versus 0.0 units (P =.025) in controls. Complications occurred in 6 of 48 patients with cirrhosis (12.5%) and 8 of 187 controls (4.2%; P <.05). No child's class C patient underwent LC. Four patients with cirrhosis (8.3%) and no controls were converted to open cholecystectomy. No postoperative infections were noted. There was no mortality in either group. LC in patients with Child's class A and B cirrhosis is reasonably safe and shows no increase in morbidity or mortality or worsening of outcome. Further studies are required to evaluate the management of acute gallbladder disease in Child's class C patients.

Polymicrobial cholangitis and liver abscess in a patient with the acquired immunodeficiency syndrome.

Cholangitis/cholangiopathy associated with the human immunodeficiency virus (HIV) infection is characterized by chronic abdominal pain, low-grade fever, cholestasis, and sometimes areas of focal or diffuse dilatation of the bile ducts that may be apparent on noninvasive imaging studies. Although the etiology of this biliary disease may be multifactorial, it appears to be the result of immunosuppression and/or secondary opportunistic infections rather than a direct cytopathic effect of HIV itself. Various opportunistic pathogens, including cytomegalovirus, Cryptosporidium, Campylobacter fetus, and Candida albicans, have been implicated as causes of HIV-associated cholangitis. We report an unusual case of polymicrobial cholangitis and liver abscess in a patient with HIV infection.

Drug evaluations using neuronal networks cultured on microelectrode arrays.

We used spontaneously active neuronal networks derived from dissociated embryonic murine spinal cord and auditory cortex and grown on substrate-integrated thin-film microelectrodes to determine characteristic responses to the cannabinoid agonists anandamide (AN) and methanandamide (MA). AN and MA reversibly inhibited spike and burst production in both tissue types. Responses of 21 cultures ranging in age from 23 to 111 days in vitro (d.i.v.) showed high intra- and inter-culture reproducibility at all ages. However, responses were tissue and substance-dependent. AN and MA were equipotent in cortical cultures and terminated bursting and spiking at 2.5 +/- 0.9 microM (n = 10). Spinal cultures were shut-off by 1.3 +/- 0.7 microM (n = 15) AN, but required 5.8 +/- 1.2 microM MA for activity cessation. MA, but not AN, demonstrated a biphasic influence: excitation at 0.25-3.5 microM and suppression at 4-7.1 microM. Palmitoylethanolamide, a related lipophilic molecule with no reported binding to the CBI receptor (to which AN and MA bind in the central nervous system), did not affect network activity at concentrations up to 6.5 microM. Irreversible cessation of activity was observed after 30 min applications of AN or MA at > 7 microM.

Cost-effective imaging approach to the nonbilious vomiting infant.

OBJECTIVE: To develop a cost- and time-effective algorithm for differentiating hypertrophic pyloric stenosis (HPS) from other medical causes of emesis in infants referred from community-based pediatricians and family practitioners to the imaging department of a tertiary children's care facility. METHODS: Eighty-nine vomiting infants (22 females, 67 males) between the ages of 11 and 120 days (mean, 43.5 days) had received nothing by mouth for at least 1 hour before the study. Each child was assessed for duration of vomiting, status of body weight, time and volume of last ingestion, and time of last emesis. A #8 French (Sherwood Medical, St Louis, MO) nasogastric feeding tube was placed in the child's stomach. The contents were aspirated and measured to determine likelihood of HPS. An aspirated volume >/=5 mL implicated gastric outlet obstruction, and ultrasonography (US) was performed. If this study was positive for HPS, the patient was referred for surgery. If US was negative, an upper gastrointestinal series (UGI) was performed. An aspirated stomach contents volume <5 mL suggested a medical cause for the emesis, and UGI was performed. Pediatric surgeons with no knowledge of the volume results palpated the abdomens of 73 of 89 infants (82%). RESULTS: Twenty-three of 89 patients (25%) had HPS. The aspirate criteria for HPS had a sensitivity of 91%, a specificity of 88%, and an accuracy of 89%. Of the false-positive studies (total = 8), six were related to recent significant ingestion (within 2 hours of the study), and two were attributable to antral dysmotility. The surgeons palpated the mass in 10 of 19 patients (53%). Sensitivity and specificity were 53% and 93%, respectively. Only 6 of 89 infants (7%) required both US and UGI to determine the etiology of the nonbilious vomiting. By performing the UGI in 66 patients, it was also found that 14% had slow gastric emptying and 79% had gastroesophageal reflux. Eighty-one percent of the gastroesophageal reflux was significant. CONCLUSION: The volumetric method of determining the proper imaging study is cost- and time-effective in the evaluation of the nonbilious vomiting infant for pyloric stenosis. If US was performed initially in all patients referred for imaging, two studies would have been performed in 68 of 89 patients (76%) to define the etiology of the emesis. Because we used the volumetric method, 62 fewer imaging studies were performed, representing a savings of $4464 and 30 hours of physician time. If children are given nothing by mouth for 3 to 4 hours before gastric aspiration, the specificity of the volumetric method improves to 94%, and the accuracy improves to 96%.