Stability studies in biological fluids during post-analysis custody. Opiate compounds derived from heroin consumption.

In Forensic Toxicology, the evidences have to be maintained under custody for, at least, one year. Depending on the conditions and duration of storage, drug concentrations might have changed considerably since the first analysis. The aim of this study is to evaluate in vitro stability of opiate compounds, derived from heroin consumption, 6-acetylmorphine (6-MAM), morphine (MOR) and codeine (COD), in blood and urine, during post-analysis custody. Parameters evaluated were: time of custody, temperature, addition of preservative (blood) and pH (urine). Blood and urine samples were spiked with the three analytes to give a final concentration of 1000 ng/mL. The prepared samples were divided into 2 groups and stored at two temperatures (4 °C and -20 °C). Each one of these groups was subsequently divided in other two groups: with and without preservative (1%NaF) for blood, and pH 4 and 8 in the case of urine. 6-MAM, MOR and COD were analyzed by GCMS after SPE and derivatization with BSTFA. Analyses were performed in triplicate every two weeks for a year. In blood samples 6-MAM is the only compound that degrades. The best storage conditions were at -20 °C with NaF, with 6-MAM recoveries, after one year of custody, of 47.1 ± 1.5%; while in the other conditions 6-MAM disappeared after 215 days (at 4 °C with NaF), 45 days (at -20 °C without NaF) and 15 days (at 4 °C without preservative). COD does not degrade, with recoveries higher than 90%, in all of the conditions. They ranged from 89.7 ± 3.6% in samples maintained at -20 °C without NaF to 95.9 ± 2.0% in those maintained at 4 °C with NaF. MOR recoveries were lower than those of COD. They ranged from 66.9 ± 3.6%, in frozen samples added with NaF, to 78.6 ± 0.5% in refrigerated samples without preservative. In urine samples the three compounds were stable in all the studied conditions, with the exception of 6-MAM in samples at pH 8 and stored at 4 °C. In these conditions, 6-MAM disappeared after 135 days of custody; while recoveries in the other conditions ranged from 93.7 ± 6.4%, at 4 °C and pH 4, to 85.1 ± 2.0% at -20 °C and pH 8. MOR and COD recoveries were similar in the four conditions. In the case of MOR, they ranged from 82.1 ± 1.2% at 4 °C and pH 4 to 89.5 ± 6.0% at -20 °C and pH 8. As far as COD is concerned, recoveries ranged from 111.6 ± 5.8% at 4 °C and pH 8 to 102.6 ± 1.2% at 4 °C and pH 4. In conclusion, the study showed that the most labile opiate compound is 6-MAM. Its stability mainly depends on urine pH or the addition of preservative, in blood samples. The best storage conditions for samples from heroin consumers are in the freezer, at -20 °C. In addition, blood samples must be added with 1%NaF and urine samples must be buffered at pH 4.

Oral health status and needs of homebound elderly in an urban home-based primary care service.

OBJECTIVES: This study assessed the oral health status, dental utilization and dental needs of the homebound elderly (HBE) care patients within the Mount Sinai Visiting Doctor program. METHODS: Of the 334 eligible patients, 57% agreed to participate and 95.4% completed the clinical examinations, the Dental Utilization and Needs survey and Geriatric Oral Health Assessment Index conducted in each subject's home by a trained research team. RESULTS: Among 75% who were dentate subjects, 40% needed restorative dental care, 45.6% needed dental extractions, and 33% complained of current oral pain. Overall, 92.0% needed some type of dental care and 96% stated that they had not seen a dentist since they became homebound (mean number of years in program = 3.2 ± 2.58). CONCLUSION: Findings show the oral health status of these homebound elderly was poor and their quality of life was significantly affected by the lack of basic dental care.

Five-year stability study of free and total prostate-specific antigen concentrations in serum specimens collected and stored at -70 degrees C or less.

The stability of total (t) and free (f) prostate-specific antigen (PSA) in male serum specimens stored at -70 degrees C or lower temperature for 4.7 to 4.9 years was studied. Until now, the stability of these analytes in serum has not been evaluated systematically beyond 2 years of storage at -70 degrees C. Aliquots of frozen serum were thawed in 2001 and 2006 and assayed for tPSA and fPSA using a Dade Behring Dimension(R) RxL analyzer and reagents. tPSA values ranged from 0.07 to 69.94 and 0.00 to 69.83 ng/mL in 2001 and 2006, respectively, whereas fPSA values for the tested specimens ranged from 0.02 to 5.72 and 0.00 to 5.92, respectively. Deming regression analyses showed agreement in assay values over time as tPSA values yielded a slope of 1.0112 and a y-intercept of 0.0195; fPSA values produced a slope 1.0538 and a y-intercept of -0.0442; f/tPSA values yielded a slope of 0.9631 and a y-intercept of 0.1195. A Bland-Altman analysis of the data demonstrated analyte and ratio stability over this time period. We conclude that serum, when collected properly and stored at -70 degrees C or lower temperature, may be used for tPSA and fPSA clinical studies for at least 5 years after collection.

Differentiating between malignant and idiopathic pleural effusions: the value of diagnostic procedures.

BACKGROUND: Which diagnostic procedures should be used to differentiate between idiopathic and malignant pleural effusions, is not well established. AIM: To identify which parameters allow differentiation between idiopathic and malignant pleural effusions. DESIGN: Case-note review. METHODS: Over a 12-year period, we treated 1014 consecutive pleural effusion patients, of whom 346 were diagnosed as having an idiopathic or malignant aetiology. We analysed medical history, chest X-ray, pleural fluid analysis (biochemical, microbiological and cytological), chest CT scan and additional examinations that were used according to clinical findings, and compared them with the eventual diagnosis and outcome. RESULTS: Eighty-three patients with idiopathic effusions and 263 with malignant effusions were included. Idiopathic pleural effusion resolved in 47 patients, improved in 20 and persisted in 16. Biochemical pleural fluid analysis did not predict these outcomes. A history of neoplasm, chest X-ray and CT features, as well as additional examinations according to clinical findings, established a diagnosis or suspicion of malignancy in 256 (97.7%) of the 263 patients who received a diagnosis of malignant effusion. Diagnostic thoracoscopy was helpful in seven patients in whom malignant disease was strongly suspected, despite the absence of other pathological findings. DISCUSSION: Non-invasive complementary examinations generally allowed accurate differentiation between malignant and idiopathic effusions. Patients with idiopathic pleural effusions generally had favourable outcomes.

Factors influencing length of hospital stay in patients with bacterial pleural effusion.

BACKGROUND: Factors influencing length of hospital stay have been poorly analyzed in parapneumonic pleural effusions (PPE). OBJECTIVES: The aim of this work is to identify the variables that determine increased hospital stay in patients with infectious pleural effusion (PE). PATIENTS AND METHODS: We analyzed 112 patients with PE: empyema, complicated parapneumonic and non-complicated parapneumonic. Epidemiologic, biochemical, therapeutic and radiological variables were analyzed. Correlations with hospital stay were studied using the Student's t test, analysis of variance, Mann-Whitney U-test and linear regression model. RESULTS: Among the 112 patients studied, there were 32 empyema, 50 complicated and 30 non-complicated parapneumonic cases. The median of length stay for all patients was 17 days. Longer hospitalization was required in patients with empyemic PE (p = 0.015), patients with underlying diseases (p = 0.003), those needing pleural drainage (p = 0.005) or decortication (p = 0.043) and those presenting unfavorable radiological outcome after treatment (p = 0.02). Biochemical parameters associated with longer hospital stay were elevated pleural fluid polymorphonuclear elastase (p = 0.001, r = 0.307) and lactate dehydrogenase (p = 0.001, r = 0.312). After linear regression analysis, only underlying disease, pleural drainage and pleural fluid polymorphonuclear elastase values remained in the model, explaining 23.1% of the variability of days of hospitalization. CONCLUSIONS: The patients with PPE and empyema who required longer hospitalization were those with purulent fluid, underlying disease, surgical drainage and/or decortication, with unfavorable radiological outcome and higher pleural fluid levels of lactate dehydrogenase and polymorphonuclear elastase.

Metalloproteinases and tissue inhibitors of metalloproteinases in exudative pleural effusions.

The aim of this study was to assess the expression of several metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in exudative pleural effusions, and their relationship with inflammatory and fibrinolytic mediators in parapneumonic effusions. The study included 51 parapneumonic effusions (30 empyema or complicated parapneumonic, 21 noncomplicated parapneumonic), 28 tuberculous, 30 malignant and 30 transudates. Inflammatory markers (tumour necrosis factor-alpha, interleukin-8, polymorphonuclear elastase), fibrinolytic system variables (tissue plasminogen activator (PA), urokinase PA (u-PA), plasminogen activation inhibitor (PAI)-1, PAI-2), and several MMPs (MMP-1, MMP-2, MMP-8, MMP-9) and TIMPs (TIMP-1, TIMP-2) were determined by ELISA in plasma and pleural fluid. Elevated MMP-2 and TIMP-1 concentrations were observed in all the pleural fluid samples studied. The group of empyema or complicated parapneumonic effusions showed higher MMP-1, MMP-8 and MMP-9 concentrations than the remaining exudates. There was no correlation between MMP and TIMP levels in plasma and pleural fluid in this group of effusions. In parapneumonic effusions, MMP-1, MMP-8 and MMP-9 showed a positive correlation with the inflammatory markers and with u-PA and PAI-1. Moreover, there was a relationship between MMP-8 concentration in pleural fluid and pleural thickening at the end of treatment. In conclusion, elevated metalloproteinase-1, -8 and -9 expression was found in parapneumonic pleural effusions. These metalloproteinases could be implicated in the local inflammatory response existing in this group of effusions.

Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide.

This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption.

Tissue concentrations of MDMA and its metabolite MDA in three fatal cases of overdose.

The recreational use of amphetamine derivatives has become increasingly popular in our country in past recent years. Their use is especially common among young people participating in dance parties known as "raves." As a direct consequence of their increased use, the number of fatal cases in which these compounds have been involved have increased dramatically since the second half of the last decade. In our laboratory, we have registered 25 cases related to amphetamine derivatives use since 1996. Three of them were deeply studied and the results obtained are presented in this paper. This information may be useful for the interpretation of the results obtained in toxicological analysis in the cases in which death may be attributed to MDMA use.

Screening de drogas de abuso en sangre total mediante inmunoensayo enzimático cedia originalmente diseñado para el análisis de orina. Aplicación a casos forenses

El objetivo de este trabajo es el desarrollo de un método para detectar la presencia de drogas de abuso en sangre mediante la aplicación de la técnica de inmunoensayo enzimático CEDIA (cloned enzyme donor immunoassay) originalmente desarrollado para el análisis de orina. Las drogas de abuso que se analizaron fueron: anfetaminas, benzodiazepinas, cannabis, cocaína y metabolitos, metadona, y opiáceos. Para calcular el número de falsos positivos y falsos negativos de esta técnica, se compararon los resultados obtenidos mediante inmunoensayo con los resultados cromatográficos. La utilidad principal de esta técnica es la realización del screening de drogas de abuso en sangre cuando no existe una muestra de orina. Se estudiaron 375 casos forenses (AU)

Physical exercise, use of Plantago ovata and aspirin, and reduced risk of colon cancer.

To evaluate certain risk and protective factors for colon cancer in our population, we conducted a paired case-control study where cases were all people diagnosed with colon cancer who were registered at the Cancer Data Exchange Systems of the Community of Madrid between January 1995 and December 1996, and controls were randomly taken from electoral lists. The study population consisted of 424 persons. Using SPSS for Windows, variables were adjusted by multiple logistic regression. The results indicate that lack of physical exercise is associated with an increased risk of colon cancer, with an odds ratio (OR) of 2.8 (95% confidence interval (CI) 1.28-6.21) as compared with moderate activity 1-2 days a week. The risk decreases linearly with increasing physical exercise, and this association remains after stratifying the analysis for the existence of constipation. The consumption of is associated with a reduced risk of colon cancer in constipated patients, with an OR of 0.094 (0.014-0.639), as is aspirin use, with an OR of 0.980 (0.898-0.999). These results were obtained after adjusting all the ORs for diet, smoking, alcohol consumption, family history and socio-demographic factors such as marital status and educational level.

[Recurrent episodes of acidosis with encephalopathy in a hemodialysis program patient with short bowel syndrome]. / Episodios recurrentes de acidosis con encefalopatía en un paciente en programa de hemodiálisis con síndrome del intestino corto.

We present a case of a patient with short bowel syndrome in a hemodialysis program, with recurrent episodes of serious acidosis. The presence of a D-lactic acidosis peak secondary to bacterial overgrowth in the intestine was discovered during an acute episode of acidosis, with neurological affection. The detection of acidosis in predialysis measurements and the acute episodes of acidosis, made it necessary to administer bicarbonate to the patient and give him additional hemodialysis sessions.

Episodios recurrentes de acidosis con encefalopatía en un paciente en programa de hemodiálisis con síndrome del intestino corto / Recurrent episodes of acidosis with encephalopathy in a hemodialysis patient with short bowel syndrome

Presentamos un caso de un paciente afecto de síndrome de intestino corto, en programa de hemodiálisis por insuficiencia renal terminal secundaria a oxalosis, con episodios recurrentes de acidosis graves. Se demostró la presencia de un pico de D-lactoacidosis secundario a sobrecrecimiento bacteriano en la flora intestinal, en el curso de un episodio agudo de acidosis, con clínica de afectación neurológica. La detección de acidosis en controles prediálisis y los episodios agudos de acidosis, obligaban a administrar bicarbonato y efectuar hemodiálisis adicionales. (AU)

Improved solid-phase extraction method for systematic toxicological analysis in biological fluids.

A method for the simultaneous qualitative and quantitative determination of drugs of abuse (opiates, cocaine, or amphetamines) and prescribed drugs (tricyclic antidepressants, phenotiazines, benzodiazepines, etc.) in biological fluids--blood, urine, bile, and gastric contents--was developed. This procedure involves solid-phase extraction with Bond-Elut Certify columns followed by analysis by gas chromatography-nitrogen-phosphorus detection (GC-NPD) and confirmation by gas chromatography-mass spectrometry (GC-MS), after derivatization, when necessary. Pretreatment was performed on all samples: sonication for 15 min plus enzymatic hydrolysis with beta-glucuronidase in urine. With respect to the internal standards, nalorphine and trihexylamine were used for basic substances, allobarbital for acidic drugs, and prazepam for benzodiazepines. Acidic and basic compounds were extracted from different aliquots of samples at different pH levels: 6-6.5 for the acidic and neutral and 8-8.5 for the basic and the benzodiazepines. Several areas of experimental design were considered in the process of method optimization. These included internal standards, pH, sonication, flow rate and washing solvents. It was found that systematic analysis could be reliably performed using optimized extraction conditions. The recovery rates for the compounds tested were always higher than 61.02%.

Rapid analysis of amphetamine, methamphetamine, MDA, and MDMA in urine using solid-phase microextraction, direct on-fiber derivatization, and analysis by GC-MS.

A rapid, sensitive, and solvent-free procedure for the simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine was developed using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. A headspace vial containing the urine sample, NaOH, NaCl, and amphetamine-d3 as the internal standard was heated at 100 degrees C for 20 min. A polydimethylsiloxane fiber was maintained in the vial headspace for 10 min in order to adsorb the amphetaminic compounds, which were subsequently derivatized by exposing the fiber to trifluoroacetic anhydride for 20 min in the headspace of another vial maintained at 60 degrees C for 20 min. The trifluoroacetyl derivatives were desorbed in the GC injection port for 5 min. Several parameters were considered during the method optimization process. These included a comparison of SPME with or without headspace, the required derivatization procedure, and the influence of temperature on the headspace extraction and derivatization methods. The optimized method was validated for the four compounds tested. Calibration curves showed linearity in the range 50-1000 ng/mL (r = 0.9946-0.9999). Recovery data were 71.89-103.24%. The quantitation limits were 10 ng/mL for amphetamine and methamphetamine and 20 ng/mL for MDA and MDMA. All of these data recommend the applicability of the method for use in the analytical routine of a forensic laboratory.

Treatment of port wine stains using the pulsed-dye laser at 585 nm with the dynamic cooling device.

Port wine stains (PWS) are common vascular malformations appearing more frequently on the face and neck. One of the most prevalent treatment modalities for PWS is the pulsed-dye laser (PDL). The first generation PDL was limited to a 450 microseconds pulse width which was inadequate for the treatment of larger caliber vessels. Second generation PDLs have pulsed widths approximately three times longer (1.5 ms). This, along with the dynamic cooling device (DCD), which allows the safe use of higher fluences, should result in more clinical improvement in the treatment of PWS that were previously resistant or minimally responsive to first generation PDL treatment. We report a case of a 29-year-old white male with extensive PWS on the left face, left neck, and back, which displayed only mild changes with the first generation PDL. However, the use of the 1.5 ms PDL at 585 nm at high fluences in conjunction with the DCD resulted in marked improvement of the patient's PWS.

Evaluation of new putative tumor markers for melanoma.

BACKGROUND: The early diagnosis of recurrent melanoma can contribute to better outcome if the disease can be surgically resected or if the metastases are responsive to systemic therapies. Lipid-associated sialic acid (LASA-P) and the S-100 protein (S-100) were evaluated as tumor markers for melanoma with the goal of early detection of recurrence. METHODS: Sixty-seven patients were identified who had levels of S-100 and LASA-P drawn during their clinical course. A multivariate regression analysis was performed to determine the significance of the serum markers in relation to other prognostic factors for melanoma. RESULTS: After a median follow-up of 30 months, 58 patients had recurrences, and 49 patients died of disease. LASA-P elevation was not associated with the time to recurrence (p = 0.2176) or survival (p = 0.2507). S-100 positivity was a significant predictor of recurrence (p < 0.0001) and survival (p = 0.0059). The median time to recurrence for S-100-positive and S-100-negative patients was 7.6 and 33.8 months, respectively. The median survival time was 59.2 months for S-100-negative patients and 29.6 months for patients positive for S-100. CONCLUSIONS: Serum S-100 shows significant correlations to both time to recurrence and survival and could be useful in the clinical detection of malignant melanoma.

Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen.

Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum.

Method for the simultaneous quantification of n-hexane metabolites: application to n-hexane metabolism determination.

1. The described analytical procedure permits the simultaneous determination of the main n-hexane metabolites in urine. 2-Hexanone, 2-hexanol, 2, 5-hexanediol and 2, 5-hexanedione, were chosen to dose the rats used in this study. All urine samples were collected and analysed on a daily basis, before and after acidic hydrolysis (pH 0.1) by GC/MS. 2-Hexanone, 2, 5-dimethylfurane, gamma-valerolactone and 2, 5-hexanedione were determined before hydrolysis: 2-hexanol and 2, 5-hexanediol, after hydrolysis; and 5-hydroxy-2-hexanone and 4, 5-dihydroxy-2-hexanone were calculated by the difference between gamma-valerolactone and 2, 5-hexanedione with and without hydrolysis, respectively. 2. A metabolic scheme was proposed reflecting the biotransformations undergone by the four compounds assayed. We consider 2, 5-dimethylfurane as a "true metabolite' because the quantities detected were always greater before hydrolysis. 3. It has been reported that human and rat n-hexane metabolism follow a similar pattern. Therefore, as a practical application and without increasing either sample or time requirements, the simultaneous quantification of the different metabolites and their excretion profile could provide better information about the metabolic situation of exposed workers than the determination of 2, 5-hexanedione alone. According to our experimental results, 4, 5-dihydroxy-2-hexanone itself would be a good toxicity indicator.

Using proportions of free to total prostate-specific antigen, age, and total prostate-specific antigen to predict the probability of prostate cancer.

OBJECTIVES: This study was undertaken to define the probability of prostate cancer as a function of the proportion of free to total prostate-specific antigen (FTPSA), total PSA, and age for those patients with total PSA levels between 2.5 and 20.0 ng/mL. METHODS: Prebiopsy serums were obtained from 428 untreated patients (165 malignant, 263 benign) who had undergone sextant six-core biopsy. Each patient had no prior history of prostate cancer and a prebiopsy total PSA value between 2.5 and 20.0 ng/mL. Total PSA levels were determined using the PA immunoassay performed on the TOSOH AIA-1200 automated immunoassay instrument. Free PSA levels were determined using a monoclonal-polyclonal antibody sandwich radioimmunoassay. RESULTS: In men with total PSA values between 2.5 and 20.0 ng/mL, the FTPSA significantly differentiated between patients with benign and malignant histologic states. Log linear modeling indicated distinct differences in the risk for cancer as a function of FTPSA, total PSA, and age. The highest probability for cancer was observed in men greater than 70 years of age who had a FTPSA less than 7% and total PSA more than 10.0 ng/mL. Conversely, the lowest probability for cancer was observed in patients less than 60 years of age who had a FTPSA more than 25% and a total PSA less than 4 ng/mL. CONCLUSIONS: The probability that prostate cancer will be found on biopsy has a marked gradient that is associated with age, total PSA, and FTPSA. The extreme ends of FTPSA of less than 7% and more than 25% are diagnostic for prostate cancer and benign prostatic disease, respectively.