Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

Urinary creatinine adjusted reference ranges for homovanillic and vanillylmandelic acid in children and adults.

The relationship between urinary homovanillic acid (HVA), vanillylmandelic acid (VMA) and creatinine is investigated and reference ranges are constructed for HVA and VMA. A total of 769 urine samples were obtained from children and adults. The ratios HVA/creatinine and VMA/creatinine were highly correlated with creatinine concentration; hence, reference to creatinine was found to be an inappropriate adjustment for urinary dilution. An alternative method based upon linear regression is proposed. After allowing for differing levels of creatinine, HVA and VMA were found to be significantly correlated with age, height and weight for individuals aged less than 20 years. In those aged over 20 years, HVA was significantly correlated with weight and VMA with age. Differences between sexes were found in the levels of HVA and VMA, the exception being VMA in the over 20 age group. Reference ranges were constructed for HVA and VMA which are variously dependent upon creatinine, age, sex and weight.

False positive results in a neuroblastoma screening programme.

Twenty thousand, eight hundred and twenty-nine babies were screened for neuroblastoma at 6 months of age by measuring homovanillic (HVA) and vanillylmandelic (VMA) acid in urine and rationing these to creatinine. Using a "cut off" of the mean + 3 SD, 10 were found to be positive. Two were found on evaluation to have neuroblastoma and in the remaining 8 the raised levels of HVA and/or VMA returned to normal. Only one of the 8 false positive babies was absolutely normal, most having a chronic disorder or illness. Utilising new centiles which relate HVA and VMA to creatinine, only 3 of the 8 would have remained positive, a false positive rate of 0.01%. The false negative rate would have remained unchanged.

Phenytoin prophylaxis in severe pre-eclampsia and eclampsia.

OBJECTIVE: To determine plasma phenytoin levels and seizure outcome in women given phenytoin for seizure prophylaxis in severe pre-eclampsia and eclampsia. DESIGN: Prospective observational study comparing two phenytoin loading regimens. SETTING: Two UK teaching hospitals. SUBJECTS: Sixty-seven consecutive women with severe pre-eclampsia and five with eclampsia. INTERVENTIONS: The first 29 women were given a 15 mg/kg intravenous loading dose of phenytoin. The next 43 received 17.5 mg/kg. All were given 500 mg phenytoin 12 h after completion of the loading dose and then 250 mg every 12 h for four doses. MAIN OUTCOME MEASURES: Total plasma phenytoin levels at 30 min, 6 h and 12 h after loading dose, 6 h after first maintenance dose and on days 2 and 3 of maintenance therapy; eclamptic seizures after starting phenytoin. RESULTS: Mean plasma phenytoin levels were higher at 30 min and 6 h after the 17.5 mg/kg loading dose. Nine of 29 (31%) phenytoin levels 30 min after the loading dose were above the therapeutic range in the 15 mg/kg group compared with 26/38 (68%) in the 17.5 mg/kg group (P < 0.01). Six of 27 (22%) phenytoin levels 12 h after the loading dose were subtherapeutic in the 15 mg/kg group compared with 2/38 (5%) in the 17.5 mg/kg group (P < 0.05). Three women, two in the 17.5 mg/kg group, developed seizures after starting phenytoin. All three had plasma levels within the therapeutic range. CONCLUSIONS: Compared with a loading dose of 17.5 mg/kg, loading with 15 mg/kg phenytoin was associated with a lower incidence of high plasma levels at 30 min but a higher incidence of subtherapeutic levels at 12 h. Seizures occur in 2 to 3% of pre-eclamptics despite apparently therapeutic phenytoin levels.

Creatinine related reference ranges for urinary homovanillic acid and vanillylmandelic acid at 6 months of age.

The relationship between homovanillic acid (HVA), vanillylmandelic acid (VMA), and creatinine in the urine of 6 month old babies has been studied and reference ranges in the form of centiles constructed for HVA and VMA against creatinine. Over 10,000 urine samples were collected from babies in four health districts in the north of England. HVA and VMA concentration, either independently or when divided by creatinine concentration, were dependent upon the absolute concentration of creatinine in the sample. After adjustment for creatinine significant differences in the mean concentration of HVA were found between sexes. No such differences were found for VMA. HVA and VMA were also found to be age dependent. Centiles were constructed using a procedure which makes no distributional assumptions about the data. The net effect of utilising these centiles was to increase the predictive value of a positive screening test from 20% to 40% without any increase in the false negative rate.

Screening for neuroblastoma in the north of England.

OBJECTIVE: To determine the feasibility of establishing a system of screening for neuroblastoma. DESIGN: Prospective study of mass screening in four clearly defined geographical areas. SETTING: Four health districts of the Northern region of England. SUBJECTS: 20,829 babies aged 6 months, 92% of target population. INTERVENTIONS: Collection of urine on filter paper for analysis of content of homovanillic and vanillylmandelic acid in relation to urinary creatinine concentrations. MAIN OUTCOME MEASURES: Derivation of reference range. Identification of babies with homovanillic or vanillylmandelic acid > 3 SD above the mean (positive cases). Investigation of positive cases for evidence of neuroblastoma. RESULTS: The upper limit of normal (3 SD above the mean) for vanillylmandelic acid was 15 mumol/mmol creatinine and for homovanillic acid 24 mumol/mmol creatinine. Of the 20,829 babies screened, 2537 (12.2%) required a second sample to be taken because the first sample was inadequate. Of these, 527 (2.5%) provided a liquid urine specimen and 10 (0.04%) had positive results for neuroblastoma. Two of them had neuroblastoma (true positives) and eight did not (false positives). A further three children from the cohort were subsequently found to have neuroblastoma; they had raised homovanillic acid or vanillylmandelic acid values, or both, but screened negative at 6 months. CONCLUSIONS: Screening for neuroblastoma is possible in the health care system of the United Kingdom. Evaluation of the efficacy of screening in reducing the mortality from neuroblastoma requires a controlled trial.

Screening for neuroblastoma in the northern region of England. Laboratory aspects.

Our pilot study for neuroblastoma screening started in 1986. The study has progressed through several phases, with use of several analytical methods to a procedure based primarily on the use of automated gas chromatography mass spectrometry. The northern region of England has a relatively static population of approximately 3.5 million, with an annual birth rate of 41,000. The region consists of 16 administrative health districts. In 4 years, we have screened 20,829 infants from four health districts. In this program, we screen all children at 6 months of age. A urine sample is collected on filter paper by a health visitor, either at the time of the infant's routine clinic visit or during the health visitor's follow-up visit at home. In the laboratory, the sample is dried and processed for analysis of homovanillic acid (HVA) and vanillylmandelic acid (VMA), using a benchtop Hewlett Packard gas chromatograph mass spectrometer. The results are related to the creatinine content of the urine. Using cut-off limits of 39 micrograms/mg of creatinine for HVA and 25 micrograms/mg of creatinine for VMA, 2,537 infants (12.2%) required a second paper sample and 527 infants (2.5%) were observed with a liquid urine collection. Of these, the conditions of nine infants with elevated levels of HVA or VMA were investigated clinically for the possible presence of neuroblastoma. Two infants were found to have neuroblastoma; the other seven showed no evidence of tumor. In addition, there were three children who, when screened at 6 months of age, had normal levels of HVA and VMA but in whom neuroblastoma subsequently developed.

A pilot study of screening for neuroblastoma in the north of England.

A pilot study has been carried out to assess the feasibility of screening for neuroblastoma in 6-month-old infants in the north of England. A total of 20,829 infants were screened. Two true-positive cases were found, along with eight false-positive and three false-negative cases. It was shown that the concentrations of catecholamine metabolites in the urine are dependent on the creatinine content; centiles have been established to allow this relationship to be taken into account. Five of the eight false-positive cases would have been correctly assessed as normal if the new centiles had been used. Preliminary results lend support to the need for a well-designed study of neuroblastoma screening to be carried out, with death from this disease as the only end point.

Strychnine poisoning as an unusual cause of convulsions.

A fatal case of strychnine poisoning is presented. The patient vomited then suffered a series of tonic convulsions which were triggered by tactile stimulation. In between paroxysms he was initially alert. Eventually the patient became comatosed due to anoxia and had a cardiac arrest. He presented with a marked metabolic acidosis and rapidly developed renal failure caused by acute rhabdomyolysis. This clinical picture is classical for strychnine poisoning and the complications which the intoxication produces. Attention is drawn to the fact that survival can even follow the ingestion of very large doses of strychnine providing there is no delay in diagnosis and treatment.

A rapid, qualitative ELISA test for the specific detection of morphine in serum or urine.

We describe a competitive inhibition ELISA technique, with a visual end-point, to detect free morphine in blood or urine. It has a sensitivity of 2 X 10(-7) mol/l using 5 microliter samples. No significant cross-reactivity was observed with other opiate derivatives. The assay has applications as a specific screen for morphine in drug abusers, or to study the metabolism of the drug in the body (as the metabolite, morphine-3-glucuronide, does not cross-react significantly with morphine in the assay).

Biochemical screening for neuroblastoma in infants: a feasibility study.

Neuroblastoma is the most common solid tumour of childhood with an incidence of about 1:10,000 births. Prognosis depends on age and stage at presentation and for disseminated disease presenting after the age of 1 year has scarcely altered at all in the past 20 years. In Japan a method has been pioneered of mass population screening of 6-month-old babies using a urine test for the catecholamine metabolites vanillylmandelic acid and homovanillic acid. They have been able to detect the majority of cases at 6 months and have improved the overall prognosis from 25% to 75% survival. A pilot study to assess the feasibility of such a study in the UK has been carried out in the north of England. Methodology has been established for the collection of urine by health visitors and biochemical analysis by gas chromatography with both flame ionisation and mass spectrometry detection; 4,897 samples have been collected, and reference ranges have been established.

Urinary excretion of HMMA and HVA in infants.

Gas chromatography with flame ionisation detection has been used to establish the normal urinary excretion values of homovanillic acid and 4-hydroxy-3-methoxymandelic acid in 808 infants aged 6 months. This study, the essential pre-requisite for population screening for neuroblastoma in babies, reveals a mean homovanillic acid of 10.9 mumol/mmol creatinine and mean 4-hydroxy-3-methoxymandelic of 6.8 mumol/mmol creatinine. The upper 95% confidence interval were 25.5 mumol/mmol creatinine for homovanillic acid and 15.0 mumol/mmol creatinine for 4-hydroxy-3-methoxymandelic. Gas chromatography/mass spectrometry analysis of the 39 samples, (4.8%) with apparent increased excretion of one or both metabolites, revealed that this was, in each case, due to interfering peaks on chromatography.

Plasma morphine levels produced by continuous infusion in children.

Blood samples were taken from six children aged between 10 months and 15 years, at intervals over a period of 40 hours while they were receiving continuous morphine infusions. The plasma morphine values obtained showed similar and consistent levels 15-30 minutes after starting the infusions.

The stability of preservative-free morphine in plastic syringes.

The stability of preservative-free morphine in plastic syringes over 36 hours in the range of concentrations commonly used in our hospital to provide continuous morphine infusions was investigated. The morphine concentration remained at 100% (SD 1.5 percent) of the control values over 36 hours and demonstrates that the concentration of morphine is not reduced with time in these circumstances.