High-resolution pediatric reference intervals for 15 biochemical analytes described using fractional polynomials.

OBJECTIVES: Assessment of children's laboratory test results requires consideration of the extensive changes that occur during physiological development and result in pronounced sex- and age-specific dynamics in many biochemical analytes. Pediatric reference intervals have to account for these dynamics, but ethical and practical challenges limit the availability of appropriate pediatric reference intervals that cover children from birth to adulthood. We have therefore initiated the multi-center data-driven PEDREF project (Next-Generation Pediatric Reference Intervals) to create pediatric reference intervals using data from laboratory information systems. METHODS: We analyzed laboratory test results from 638,683 patients (217,883-982,548 samples per analyte, a median of 603,745 test results per analyte, and 10,298,067 test results in total) performed during patient care in 13 German centers. Test results from children with repeat measurements were discarded, and we estimated the distribution of physiological test results using a validated statistical approach (kosmic). RESULTS: We report continuous pediatric reference intervals and percentile charts for alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl-transferase, total protein, albumin, creatinine, urea, sodium, potassium, calcium, chloride, anorganic phosphate, and magnesium. Reference intervals are provided as tables and fractional polynomial functions (i.e., mathematical equations) that can be integrated into laboratory information systems. Additionally, Z-scores and percentiles enable the normalization of test results by age and sex to facilitate their interpretation across age groups. CONCLUSIONS: The provided reference intervals and percentile charts enable precise assessment of laboratory test results in children from birth to adulthood. Our findings highlight the pronounced dynamics in many biochemical analytes in neonates, which require particular consideration in reference intervals to support clinical decision making most effectively.

Development of an in-vitro model for extracorporeal blood pumps to study the effects of artificial pulsatility on human blood.

Introduction: The application of extracorporeal circulation (ECC) systems is known to be associated with several implications regarding hemolysis, inflammation, and coagulation. In the last years, systems with pulsatile blood flow are increasingly used with the intention to improve hemodynamics in reperfusion. However, their implications on the aforementioned aspects remain largely unknown. To investigate the effects of pulsatility, this ex-vivo study was initiated. Methods: Test circuits (primed with human whole blood) were set up in accordance with the recommendations of international standards for in-vitro evaluation of new components and systems of ECC. Diagonal pumps were either set up with non-pulsatile (n = 5, NPG) or pulsatile (n = 5, PG) pump settings and evaluated for 6 h. All analyses were conducted with human whole blood. Blood samples were repeatedly drawn from the test circuits and analyzed regarding free hemoglobin, interleukin 8 (IL-8), platelet aggregation and acquired von Willebrand syndrome (AVWS). Results: After 1 h of circulation, a significant coagulation impairment (impaired platelet function and AVWS) was observed in both groups. After 6 h of circulation, increased IL-8 concentrations were measured in both groups (NPG: 0.05 ± 0.03 pg./mL, PG: 0.03 ± 0.01 pg./mL, p = 0.48). Pulsatile pump flow resulted in significantly increased hemolysis after 6 h of circulation (NPG: 37.3 ± 12.4 mg/100 L; PG: 59.6 ± 14.5 mg/100 L; p < 0.05). Conclusion: Our results indicate that the coagulative impairment takes place in the early phase of ECC. Pulsatility did not affect the occurrence of AVWS ex-vivo. Prolonged durations of pulsatile pump flow led to increased hemolysis and therefore, its prolonged use should be employed cautiously in clinical practice with appropriate monitoring.

Acute psychotropic, autonomic, and endocrine effects of 5,6-methylenedioxy-2-aminoindane (MDAI) compared with 3,4-methylenedioxymethamphetamine (MDMA) in human volunteers: A self-administration study.

The acute psychoactive, autonomic, and endocrine effects of the new psychoactive substance (NPS) 5,6-methylenedioxy-2-aminoindane (MDAI; 3.0 mg/kg, range 180-228 mg) were investigated in six healthy volunteers (four males, two females) in a non-blinded fashion without placebo. Subjective, cardiovascular, and endocrine responses were compared with two different doses of 3,4-methylenedioxymethamphetamine (MDMA) (75 mg and 125 mg) described in previously published placebo-controlled studies, which used identical outcome measures including Visual Analogue Scales (VAS), the Adjective Mood Rating Scale (AMRS), and the 5 Dimensions of Altered States of Consciousness (5D-ASC) scale. MDAI was well tolerated and produced subjective effects comparable with those of 125 mg MDMA. MDAI increased blood pressure similar to 125 mg MDMA but did not increase heart rate or body temperature. MDAI increased cortisol and prolactin levels and could be detected in serum about 20 min post ingestion and remained detectable at least for 4 days. In urine, MDAI was detectable over a period of at least 6 days. Further clinical investigations are warranted to assess whether MDAI could serve as drug with medicinal properties.

Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards. METHODS: Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes. RESULTS: Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 µg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards. CONCLUSIONS: The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.

Vitreal levels of erythropoietin are increased in patients with retinal vein occlusion and correlate with vitreal VEGF and the extent of macular edema.

PURPOSE: This study compares vitreal levels of erythropoietin (EPO) in patients with retinal vein occlusion (RVO) with control subjects. In addition, it investigates different RVO disease parameters (time of vein occlusion, patient age, vitreal vascular endothelial growth factor (VEGF) levels, and extent of central macular edema) for possible correlations with vitreal EPO levels. METHODS: Serum and vitreal EPO were measured from 6 patients with branch retinal vein occlusion, 6 patients with central retinal vein occlusion, and 12 control subjects (10 macular puckers and 2 macular holes). RESULTS: Serum EPO levels (9.8 ± 4.9 mU/mL) did not differ between the RVO and control groups and were significantly lower than vitreal EPO levels in all groups. Vitreal EPO was elevated both in branch RVO (91 ± 59 mU/mL) and central RVO (182 ± 70 mU/mL) compared with controls (35 ± 24 mU/mL). Increased vitreal EPO correlated with higher vitreal VEGF (r = 0.64, P = 0.0008) and more pronounced central macular edema (r = 0.66, P = 0.001). CONCLUSION: The results from this study indicate that EPO is locally expressed in the retina and that it is upregulated together with VEGF in RVO eyes. Because of its role both in neuroprotection and angiogenesis, ocular EPO might represent an interesting target to investigate in patients with RVO, especially in light of the current anti-VEGF treatments.

Phenotyping sarcoidosis from a pulmonary perspective.

RATIONALE: Sarcoidosis is known as a disease with high heterogeneity in clinical severity and inflammatory activity. On the basis of radiologic criteria, John Guyette Scadding developed a classification system, which is widely used, but is insufficient for clinical decision making. Therefore, biomarkers and genetic markers that predict outcome are urgently needed. OBJECTIVES: To obtain a classification system based on clinical criteria to evaluate biomarker and genetic markers. METHODS: We developed a protocol for classification of various disease courses (sarcoid clinical activity classification [SCAC]) based on clinical criteria with three categories: (1) whether the disease was acute or nonacute in onset, (2) whether treatment was required, and (3) whether there was need for long-term treatment. MEASUREMENTS AND MAIN RESULTS: In total, we evaluated 225 patients with sarcoidosis, applying both classification protocols retrospectively. The described SCAC protocol based on clinical criteria was retrospectively able to stratify patients according to disease outcome. The classes of patients with chronic disease differed significantly regarding pulmonary function test parameters and bronchoalveolar lavage fluid cell composition. Most interestingly, concentrations of soluble IL-2 receptor and neopterin were particularly increased in patients with acute disease who required long-term treatment. A moderate but significant increase in soluble IL-2 receptor and neopterin was also observed in patients with nonacute disease who needed long-term treatment. In contrast to the clinical classification system, the system based on radiologic criteria separated the patients with the need for long-term therapy insufficiently, but identified patients with advanced fibrotic remodeling. CONCLUSIONS: The described SCAC protocol is practicable and gives additional information not yet acquired by radiologic typing and seems suitable for studies evaluating genetic influence and biomarkers.

Vitamin D Deficiency in Adult Patients with Schizophreniform and Autism Spectrum Syndromes: A One-Year Cohort Study at a German Tertiary Care Hospital.

INTRODUCTION: Vitamin D has many immunomodulatory, anti-inflammatory, and neuroprotective functions, and previous studies have demonstrated an association between vitamin D deficiency and neuropsychiatric disease. The aim of our study was to analyze the prevalence of vitamin D deficiency in a 1-year cohort of adult inpatients with schizophreniform and autism spectrum syndromes in a naturalistic inpatient setting in Germany. PARTICIPANTS AND METHODS: Our study was comprised of 60 adult schizophreniform and 23 adult high-functioning autism spectrum patients who were hospitalized between January and December of 2015. We compared our findings with a historical German reference cohort of 3,917 adults using Pearson's two-sided chi-squared test. The laboratory measurements of 25-hydroxyvitamin D2/3 [25(OH)vitamin D] were obtained using a chemiluminescence immunoassay. RESULTS: In the schizophreniform group, we found decreased (<20 ng/ml) 25(OH)vitamin D levels in 48/60 (80.0%) of the patients. In the autism spectrum group, decreased levels were detected in 18/23 (78.3%) of the patients. 25(OH)vitamin D deficiencies were found in 57.3% of the historical control group. Particularly, severe deficiencies (<10 ng/ml) occurred much more frequently in the schizophreniform (38.3%) and autism spectrum groups (52.2%), when compared to the control group (16.3%). The recommended 25(OH)vitamin D values of >30 ng/ml were observed in only 5% of the schizophreniform patients, 8.7% of the autism spectrum patients, and 21.9% of the healthy controls. DISCUSSION: We found very high rates of 25(OH)vitamin D deficiencies in both patient groups and have discussed whether our findings might be related to alterations in the immunological mechanisms. Irrespective of the possible pathophysiological links between vitamin D deficiency and schizophrenia or autism spectrum disorders, a more frequent measurement of vitamin D levels seems to be justified in these patient groups. Further prospective, controlled, blinded, and randomized research should be conducted to analyze the effectiveness of vitamin D supplementation on the improvement of psychiatric symptoms.

Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics.

The appearance of pyrazolam in Internet shops selling 'research chemicals' in 2012 marked the beginning of designer benzodiazepines being sold as recreational drugs or 'self medication'. With recent changes in national narcotics laws in many countries, where two uncontrolled benzodiazepines (phenazepam and etizolam), which were marketed by pharmaceutical companies in some countries, were scheduled, clandestine laboratories seem to turn to poorly characterized research drug candidates as legal substitutes. Following the appearance of pyrazolam, it comes with no surprise that recently, flubromazepam (7-bromo-5-(2-fluorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one), a second designer benzodiazepine, was offered on the market. In this article, this new compound was characterized using nuclear magnetic resonance, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS/MS) and liquid chromatography quadrupole time-of-flight MS (LC-Q-ToF-MS). Additionally, a study was carried out, in which one of the authors consumed 4 mg of flubromazepam to gain preliminary data on the pharmacokinetic properties and the metabolism of this compound. For this purpose, serum as well as urine samples were collected for up to 31 days post-ingestion and analyzed applying LC-MS/MS and LC-Q-ToF-MS techniques. On the basis of this study, flubromazepam appears to have an extremely long elimination half-life of more than 100 h. One monohydroxylated compound and the debrominated compound could be identified as the predominant metabolites, the first allowing a detection of a consumption for up to 28 days post-ingestion when analyzing urine samples in our case. Additionally, various immunochemical assays were evaluated, showing that the cross-reactivity of the used assay seems not to be sufficient for safe detection of the applied dose in urine samples, bearing the risk that it could be misused in drug-withdrawal settings or in other circumstances requiring regular drug testing. Furthermore, it may be used in drug-facilitated crimes without being detected.

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes.

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.