Effect of collection and preprocessing methods on neutrophil elastase plasma concentrations.

OBJECTIVES: Elevated plasma levels of the elastase alpha 1-proteinase inhibitor complex (E-alpha 1 PI) have been proposed as a marker of bacterial infection and neutrophil activation. Liberation of elastase from neutrophils after collection of blood may cause falsely elevated results. Collection methods have not been validated for critically ill neonates and children. We evaluated the influence of preanalytical methods on E-alpha 1 PI results including the recommended collection into EDTA tubes. DESIGN AND METHODS: First, we compared varying acceleration speeds and centrifugation times. Centrifugation at 1550 g for 3 min resulted in reliable preparation of leukocyte free plasma. Second, we evaluated all collection tubes under consideration for absorption of E-alpha 1 PI. Finally, 12 sets of samples from healthy adults and 42 sets obtained from critically ill neonates and children were distributed into the various sampling tubes. Samples were centrifuged within 15 min of collection and analyzed with a new turbidimetric assay adapted to routine laboratory analyzers. RESULTS: One of the two tubes containing a plasma-cell separation gel absorbed 22.1% of the E-alpha 1 PI content. In the remaining tubes without absorption of E-alpha 1 PI no differences were observed for samples from healthy adult patients. However, in samples from critically ill neonates or children, significantly higher results were obtained for plain Li-heparin tubes (mean = 183 micrograms/L), EDTA tubes (mean = 93 micrograms/L), and citrate tubes (mean = 88.5 micrograms/L) than for the Li-hep tube with cell-plasma separation gel and no absorption of E-alpha 1 PI (mean = 62.4 micrograms/L, p < 0.01). CONCLUSION: Contrary to healthy adults, E-alpha 1 PI results in plasma samples from critically ill neonates and children depend on the type of collection tube.

Lack of Enantioselectivity in the SULT1A3-catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study.

(1R)-Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)-catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)-normetanephrine and (1S)-normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3'-phosphoadenosine-5'-phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K(M) , V(max) , and k(cat) values for the sulfonation of (1R)-normetanephrine, (1S)-normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate-enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an "induced-fit model" in the catalytic pocket. Chirality, 00:000-000, 2012.© 2012 Wiley Periodicals, Inc.

Analytical interference of 4-hydroxy-3-methoxymethamphetamine with the measurement of plasma free normetanephrine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The diagnosis of pheochromocytoma relies on the measurement of plasma free metanephrines assay whose reliability has been considerably improved by ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Here we report an analytical interference occurring between 4-hydroxy-3-methoxymethamphetamine (HMMA), a metabolite of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), and normetanephrine (NMN) since they share a common pharmacophore resulting in the same product ion after fragmentation. DESIGN AND METHODS: Synthetic HMMA was spiked into plasma samples containing various concentrations of NMN and the intensity of the interference was determined by UPLC-MS/MS before and after improvement of the analytical method. RESULTS: Using a careful adjustment of chromatographic conditions including the change of the UPLC analytical column, we were able to distinguish both compounds. HMMA interference for NMN determination should be seriously considered since MDMA activates the sympathetic nervous system and if confounded with NMN may lead to false-positive tests when performing a differential diagnostic of pheochromocytoma.

LC-MS/MS based assay and reference intervals in children and adolescents for oxysterols elevated in Niemann-Pick diseases.

BACKGROUND: Niemann-Pick type C (NP-C) is a rare progressive neurodegenerative lipid storage disorder with heterogeneous clinical presentation and challenging diagnostic procedures. Recently oxysterols have been reported to be specific biomarkers for NP-C but knowledge on the intra-individual variation and on reference intervals in children and adolescents are lacking. METHODS: We established a LC-MS/MS assay to measure Cholestane-3β, 5α, 6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following Steglich esterification. To assess reference intervals and intra-individual variation we determined oxysterols in 148 children and adolescents from 0 to 18 years and repeat measurements in 19 of them. RESULTS: The reported method is linear (r>0.99), sensitive (detection limit of 0.03 ng/mL [0.07 nM] for C-triol, and 0.54 ng/mL [1.35 nM] for 7-KC) and precise, with an intra-day imprecision of 4.8% and 4.1%, and an inter-day imprecision of 7.0% and 11.0% for C-triol (28 ng/ml, 67 nM) and 7-KC (32 ng/ml, 80 nM), respectively. Recoveries for 7-KC and C-triol range between 93% and 107%. The upper reference limit obtained for C-triol is 40.4 ng/mL (95% CI: 26.4-61.7 ng/mL, 96.0 nM, 95% CI: 62.8-146.7 nM) and 75.0 ng/mL for 7-KC (95% CI: 55.5-102.5 ng/mL, 187.2 nM, 95% CI: 138.53-255.8 nM), with no age or gender dependency. Both oxysterols have a broad intra-individual variation of 46%±23% for C-triol and 52%±29% for 7-KC. Nevertheless, all Niemann-Pick patients showed increased C-triol levels including Niemann-Pick type A and B patients. CONCLUSIONS: The LC-MS/MS assay is a robust assay to quantify C-triol and 7-KC in plasma with well documented reference intervals in children and adolescents to screen for NP-C in the pediatric population. In addition our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases.

Fatal metformin overdose: case report and postmortem biochemistry contribution.

Metformin is an oral antihyperglycemic agent used in the management of type 2 diabetes mellitus. Lactic acidosis from metformin overdose is a rare complication of metformin therapy and occurs infrequently with therapeutic use. Fatal cases, both accidental and intentional, are extremely rare in clinical practice. Metformin is eliminated by the kidneys, and impaired renal function can result in an increased plasma concentration of the drug. In this report, we describe an autopsy case involving a 70-year-old woman suffering from diabetes mellitus and impaired renal function who received metformin treatment. Metformin concentrations in the peripheral blood collected during hospitalization and femoral blood collected during autopsy were 42 and 47.3 µg/ml, respectively. Lactic acidosis (29.10 mmol/l) was objectified during hospitalization. Furthermore, postmortem biochemistry allowed ketoacidosis to be diagnosed (blood β-hydroxybutyrate, 10,500 µmol/l). Death was attributed to lactic acidosis due to metformin intoxication. Increased plasma concentrations of the drug were attributed to severely impaired renal function. The case emphasizes the usefulness of performing exhaustive toxicology and postmortem biochemistry towards the more complete understanding of the pathophysiological mechanisms that may be involved in the death process.

Diagnosing pulmonary embolism in outpatients with clinical assessment, D-dimer measurement, venous ultrasound, and helical computed tomography: a multicenter management study.

PURPOSE: To evaluate a diagnostic strategy for pulmonary embolism that combined clinical assessment, plasma D-dimer measurement, lower limb venous ultrasonography, and helical computed tomography (CT). METHODS: A cohort of 965 consecutive patients presenting to the emergency departments of three general and teaching hospitals with clinically suspected pulmonary embolism underwent sequential noninvasive testing. Clinical probability was assessed by a prediction rule combined with implicit judgment. All patients were followed for 3 months. RESULTS: A normal D-dimer level (&lt;500 microg/L by a rapid enzyme-linked immunosorbent assay) ruled out venous thromboembolism in 280 patients (29%), and finding a deep vein thrombosis by ultrasonography established the diagnosis in 92 patients (9.5%). Helical CT was required in only 593 patients (61%) and showed pulmonary embolism in 124 patients (12.8%). Pulmonary embolism was considered ruled out in the 450 patients (46.6%) with a negative ultrasound and CT scan and a low-to-intermediate clinical probability. The 8 patients with a negative ultrasound and CT scan despite a high clinical probability proceeded to pulmonary angiography (positive: 2; negative: 6). Helical CT was inconclusive in 11 patients (pulmonary embolism: 4; no pulmonary embolism: 7). The overall prevalence of pulmonary embolism was 23%. Patients classified as not having pulmonary embolism were not anticoagulated during follow-up and had a 3-month thromboembolic risk of 1.0% (95% confidence interval: 0.5% to 2.1%). CONCLUSION: A noninvasive diagnostic strategy combining clinical assessment, D-dimer measurement, ultrasonography, and helical CT yielded a diagnosis in 99% of outpatients suspected of pulmonary embolism, and appeared to be safe, provided that CT was combined with ultrasonography to rule out the disease.

Prevalence and clinical importance of mesenteric venous thrombosis in the swiss inflammatory bowel disease cohort.

OBJECTIVE. The purpose of this study was to evaluate the prevalence of mesenteric venous thrombosis (MVT) in the Swiss Inflammatory Bowel Disease Cohort Study and to correlate MVT with clinical outcome. MATERIALS AND METHODS. Abdominal portal phase CT was used to examine patients with inflammatory bowel disease (IBD). Two experienced abdominal radiologists retrospectively analyzed the images, focusing on the superior and inferior mesenteric vein branches and looking for signs of acute or chronic thrombosis. The location of abnormalities was registered. The presence of MVT was correlated with IBD-related radiologic signs and complications. RESULTS. The cases of 160 patients with IBD (89 women, 71 men; Crohn disease [CD], 121 patients; ulcerative colitis [UC], 39 patients; median age at diagnosis, 27 years for patients with CD, 32 years for patients with UC) were analyzed. MVT was detected in 43 patients with IBD (26.8%). One of these patients had acute MVT; 38, chronic MVT; and four, both. The prevalence of MVT did not differ between CD (35/121 [28.9%]) and UC (8/39 [20.5%]) (p = 0.303). The location of thrombosis was different between CD and UC (CD, jejunal or ileal veins only [p = 0.005]; UC, rectocolic veins only [p = 0.001]). Almost all (41/43) cases of thrombosis were peripheral. MVT in CD patients was more frequently associated with bowel wall thickening (p = 0.013), mesenteric fat hypertrophy (p = 0.005), ascites (p = 0.002), and mesenteric lymph node enlargement (p = 0.036) and was associated with higher rate of bowel stenosis (p < 0.001) and more intestinal IBD-related surgery (p = 0.016) in the outcome. Statistical analyses for patients with UC were not relevant because of the limited population (n = 8). CONCLUSION. MVT is frequently found in patients with IBD. Among patients with CD, MVT is associated with bowel stenosis and CD-related intestinal surgery.

Swiss clinical practice guidelines for skin cancer in organ transplant recipients.

Patients with a solid organ transplant have increased in numbers and in individual survival in Switzerland over the last decades. As a consequence of long-term immunosuppression, skin cancer in solid organ recipients (SOTRs) has been recognized as an important problem. Screening and education of potential SOTRs about prevention of sun damage and early recognition of skin cancer are important before transplantation. Once transplanted, SOTRs should be seen by a dermatologist yearly for repeat education as well as early diagnosis, prevention and treatment of skin cancer. Squamous cell carcinoma of the skin (SCC) is the most frequent cancer in the setting of long-term immunosuppression. Sun protection by behaviour, clothing and daily sun screen application is the most effective prevention. Cumulative sun damage results in field cancerisation with numerous in-situ SCC such as actinic keratosis and Bowen's disease which should be treated proactively. Invasive SCC is cured by complete surgical excision. Early removal is the best precaution against potential metastases of SCC. Reduction of immunosuppression and switch to mTOR inhibitors and potentially, mycophenolate, may reduce the incidence of further SCC. Chemoprevention with the retinoid acitretin reduces the recurrence rate of SCC. The dermatological follow-up of SOTRs should be integrated into the comprehensive post-transplant care.

Teaching clinical anatomy of the female pelvic floor to undergraduate students: A critical review of nevralgic points

Pelvic floor anatomy is complex and its three-dimensional organization is often difficult to understand for both undergrad- uate and postgraduate students. Here, we focused on several critical points that need to be considered when teaching the perineum. We have to deal with a mixed population of students and with a variety of interest. Yet, a perfect knowledge of the pelvic floor is the basis for any gynecologist and for any surgical intervention. Our objectives are several-fold; i) to estab- lish the objectives and the best way of teaching, ii) to identify and localize areas in the female pelvic floor that are suscepti- ble to generate problems in understanding the three-dimensional organization, iii) to create novel approaches by respecting the anatomical surroundings, and iv) prospectively, to identify elements that may create problems during surgery i.e. to have a closer look at nerve trajectories and on compression sites that may cause neuralgia or postoperative pain. A feedback from students concludes that they have difficulties to assimilate this much information, especially the different imaging tech- niques. Eventually, this will lead to a severe selection of what has to be taught and included in lectures or practicals. Another consequence is that more time to study prosected pelves needs to be given.

Long-term citalopram administration reduces responsiveness of HPA axis in patients with major depression: relationship with S-citalopram concentrations in plasma and cerebrospinal fluid (CSF) and clinical response.

RATIONALE: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a well-documented neurobiological finding in major depression. Moreover, clinically effective therapy with antidepressant drugs may normalize the HPA axis activity. OBJECTIVE: The aim of this study was to test whether citalopram (R/S-CIT) affects the function of the HPA axis in patients with major depression (DSM IV). METHODS: Twenty depressed patients (11 women and 9 men) were challenged with a combined dexamethasone (DEX) suppression and corticotropin-releasing hormone (CRH) stimulation test (DEX/CRH test) following a placebo week and after 2, 4, and 16 weeks of 40 mg/day R/S-CIT treatment. RESULTS: The results show a time-dependent reduction of adrenocorticotrophic hormone (ACTH) and cortisol response during the DEX/CRH test both in treatment responders and nonresponders within 16 weeks. There was a significant relationship between post-DEX baseline cortisol levels (measured before administration of CRH) and severity of depression at pretreatment baseline. Multiple linear regression analyses were performed to identify the impact of psychopathology and hormonal stress responsiveness and R/S-CIT concentrations in plasma and cerebrospinal fluid (CSF). The magnitude of decrease in cortisol responsivity from pretreatment baseline to week 4 on drug [delta-area under the curve (AUC) cortisol] was a significant predictor (p<0.0001) of the degree of symptom improvement following 16 weeks on drug (i.e., decrease in HAM-D21 total score). The model demonstrated that the interaction of CSF S-CIT concentrations and clinical improvement was the most powerful predictor of AUC cortisol responsiveness. CONCLUSION: The present study shows that decreased AUC cortisol was highly associated with S-CIT concentrations in plasma and CSF. Therefore, our data suggest that the CSF or plasma S-CIT concentrations rather than the R/S-CIT dose should be considered as an indicator of the selective serotonergic reuptake inhibitors (SSRIs) effect on HPA axis responsiveness as measured by AUC cortisol response.

Portal vein embolization before right hepatectomy: prospective clinical trial.

OBJECTIVE: To assess the impact of liver hypertrophy of the future liver remnant volume (FLR) induced by preoperative portal vein embolization (PVE) on the immediate postoperative complications after a standardized major liver resection. SUMMARY BACKGROUND DATA: PVE is usually indicated when FLR is estimated to be too small for major liver resection. However, few data exist regarding the exact quantification of sufficient minimal functional hepatic volume required to avoid postoperative complications in both patients with or without chronic liver disease. METHODS: All consecutive patients in whom an elective right hepatectomy was feasible and who fulfilled the inclusion and exclusion criteria between 1998 and 2000 were assigned to have alternatively either immediate surgery or surgery after PVE. Among 55 patients (25 liver metastases, 2 cholangiocarcinoma, and 28 hepatocellular carcinoma), 28 underwent right hepatectomy after PVE and 27 underwent immediate surgery. Twenty-eight patients had chronic liver disease. FLR and estimated rate of functional future liver remnant (%FFLR) volumes were assessed by computed tomography. RESULTS: The mean increase of FLR and %FFLR 4 to 8 weeks after PVE were respectively 44 +/- 19% and 16 +/- 7% for patients with normal liver and 35 +/- 28% and 9 +/- 3% for those with chronic liver disease. All patients with normal liver and 86% with chronic liver disease experienced hypertrophy after PVE. The postoperative course of patients with normal liver who underwent PVE before right hepatectomy was similar to those with immediate surgery. In contrast, PVE in patients with chronic liver disease significantly decreased the incidence of postoperative complications as well as the intensive care unit stay and total hospital stay after right hepatectomy. CONCLUSIONS: Before elective right hepatectomy, the hypertrophy of FLR induced by PVE had no beneficial effect on the postoperative course in patients with normal liver. In contrast, in patients with chronic liver disease, the hypertrophy of the FLR induced by PVE decreased significantly the rate of postoperative complications.

A Multicenter Randomized Clinical Trial of Primary Anastomosis or Hartmann's Procedure for Perforated Left Colonic Diverticulitis With Purulent or Fecal Peritonitis.

OBJECTIVES: : To evaluate the outcome after Hartmann's procedure (HP) versus primary anastomosis (PA) with diverting ileostomy for perforated left-sided diverticulitis. BACKGROUND: : The surgical management of left-sided colonic perforation with purulent or fecal peritonitis remains controversial. PA with ileostomy seems to be superior to HP; however, results in the literature are affected by a significant selection bias. No randomized clinical trial has yet compared the 2 procedures. METHODS: : Sixty-two patients with acute left-sided colonic perforation (Hinchey III and IV) from 4 centers were randomized to HP (n = 30) and to PA (with diverting ileostomy, n = 32), with a planned stoma reversal operation after 3 months in both groups. Data were analyzed on an intention-to-treat basis. The primary end point was the overall complication rate. The study was discontinued following an interim analysis that found significant differences of relevant secondary end points as well as a decreasing accrual rate (NCT01233713). RESULTS: : Patient demographics were equally distributed in both groups (Hinchey III: 76% vs 75% and Hinchey IV: 24% vs 25%, for HP vs PA, respectively). The overall complication rate for both resection and stoma reversal operations was comparable (80% vs 84%, P = 0.813). Although the outcome after the initial colon resection did not show any significant differences (mortality 13% vs 9% and morbidity 67% vs 75% in HP vs PA), the stoma reversal rate after PA with diverting ileostomy was higher (90% vs 57%, P = 0.005) and serious complications (Grades IIIb-IV: 0% vs 20%, P = 0.046), operating time (73 minutes vs 183 minutes, P < 0.001), hospital stay (6 days vs 9 days, P = 0.016), and lower in-hospital costs (US $16,717 vs US $24,014) were significantly reduced in the PA group. CONCLUSIONS: : This is the first randomized clinical trial favoring PA with diverting ileostomy over HP in patients with perforated diverticulitis.