BTK inhibition limits microglia-perpetuated CNS inflammation and promotes myelin repair.

In multiple sclerosis (MS), persisting disability can occur independent of relapse activity or development of new central nervous system (CNS) inflammatory lesions, termed chronic progression. This process occurs early and it is mostly driven by cells within the CNS. One promising strategy to control progression of MS is the inhibition of the enzyme Bruton's tyrosine kinase (BTK), which is centrally involved in the activation of both B cells and myeloid cells, such as macrophages and microglia. The benefit of BTK inhibition by evobrutinib was shown as we observed reduced pro-inflammatory activation of microglia when treating chronic experimental autoimmune encephalomyelitis (EAE) or following the adoptive transfer of activated T cells. Additionally, in a model of toxic demyelination, evobrutinib-mediated BTK inhibition promoted the clearance of myelin debris by microglia, leading to an accelerated remyelination. These findings highlight that BTK inhibition has the potential to counteract underlying chronic progression of MS.

24/7 Therapeutic Drug Monitoring of Beta-Lactam Antibiotics with CLAM-2000.

BACKGROUND: The aim of this study was to evaluate the CLAM-2000 automated preanalytical sample preparation module with integrated liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) as a method for 24/7 therapeutic drug monitoring (TDM) of beta-lactam antibiotics in routine clinical diagnostics. METHODS: Method validation was performed using quality control samples. Method comparison was performed with routine samples from patients treated with beta-lactam antibiotics. RESULTS: The determination of piperacillin, meropenem, ceftazidime, flucloxacillin, and cefotaxime was performed using D5-piperacillin and D6-meropenem as internal standards. The linearity of the method was within the therapeutic range of beta-lactam antibiotics. The imprecision and accuracy data obtained from quality control samples were within 15%, and the imprecision of patient samples on the instrument was less than the 5% coefficient of variation (CV). Internal standards stored in the instrument at 9 °C for at least one week were stable, which facilitated reagent use and storage. CONCLUSION: The CLAM-2000 (Shimadzu, Kyoto, Japan) provides reproducible results as an established routine instrument and is a useful tool for 24/7 TDM of beta-lactam antibiotics in routine clinical diagnostics.

Feasibility of Continuous Infusion of Cefiderocol in Conjunction with the Establishment of Therapeutic Drug Monitoring in Patients with Extensively Drug-Resistant Gram-Negative Bacteria.

BACKGROUND AND OBJECTIVE: Resistance to antibacterial substances is a huge and still emerging issue, especially with regard to Gram-negative bacteria and in critically ill patients. We report a study in six patients infected with extensively drug-resistant Gram-negative bacteria in a limited outbreak who were successfully managed with a quasi-continuous infusion of cefiderocol. METHODS: Patients were initially treated with prolonged infusions of cefiderocol over 3 h every 8 h, and the application mode was then switched to a quasi-continuous infusion of 2 g over 8 h, i.e. 6 g in 24 h. Therapeutic drug monitoring (TDM) was established using an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: Determined trough plasma concentrations were a median of 50.00 mg/L [95% confidence interval (CI) 27.20, 74.60] and steady-state plasma concentrations were a median of 90.96 mg/L [95% CI 37.80, 124]. No significant differences were detected with respect to acute kidney injury/continuous renal replacement therapy. Plasma concentrations determined from different modes of storage were almost equal when frozen or cooled, but markedly reduced when stored at room temperature. CONCLUSIONS: (Quasi) continuous application of cefiderocol 6 g/24 h in conjunction with TDM is a feasible mode of application; the sample for TDM should either be immediately analyzed, cooled, or frozen prior to analysis.

Ultrasound-Induced Release of Nimodipine from Drug-Loaded Block Copolymer Micelles: In Vivo Analysis.

Nimodipine prevents cerebral vasospasm and improves functional outcome after aneurysmal subarachnoid hemorrhage (aSAH). The beneficial effect is limited by low oral bioavailability of nimodipine, which resulted in an increasing use of nanocarriers with sustained intrathecal drug release in order to overcome this limitation. However, this approach facilitates only a continuous and not an on-demand nimodipine release during the peak time of vasospasm development. In this study, we aimed to assess the concept of controlled drug release from nimodipine-loaded copolymers by ultrasound application in the chicken chorioallantoic membrane (CAM) model. Nimodipine-loaded copolymers were produced with the direct dissolution method. Vasospasm of the CAM vessels was induced by means of ultrasound (Physiomed, continuous wave, 3 MHz, 1.0 W/cm2). The ultrasound-mediated nimodipine release (Physiomed, continuous wave, 1 MHz, 1.7 W/cm2) and its effect on the CAM vessels were evaluated. Measurements of vessel diameter before and after ultrasound-induced nimodipine release were performed using ImageJ. The CAM model could be successfully carried out in all 25 eggs. After vasospasm induction and before drug release, the mean vessel diameter was at 57% (range 44-61%) compared to the baseline diameter (set at 100%). After ultrasound-induced drug release, the mean vessel diameter of spastic vessels increased again to 89% (range 83-91%) of their baseline diameter, which was significant (p = 0.0002). We were able to provide a proof of concept for in vivo vasospasm induction by ultrasound application in the CAM model and subsequent resolution by ultrasound-mediated nimodipine release from nanocarriers. This concept merits further evaluation in a rat SAH model.

Metabolomic Profiling of Blood-Derived Microvesicles in Breast Cancer Patients.

Malignant cells differ from benign ones in their metabolome and it is largely unknown whether this difference is reflected in the metabolic profile of their microvesicles (MV), which are secreted into the blood of cancer patients. Here, they are present together with MV from the various blood and endothelial cells. Harvesting MV from 78 breast cancer patients (BC) and 30 controls, we characterized the whole blood MV metabolome using targeted and untargeted mass spectrometry. Especially (lyso)-phosphatidylcholines and sphingomyelins were detected in a relevant abundance. Eight metabolites showed a significant discriminatory power between BC and controls. High concentrations of lysoPCaC26:0 and PCaaC38:5 were associated with shorter overall survival. Comparing BC subtype-specific metabolome profiles, 24 metabolites were differentially expressed between luminal A and luminal B. Pathway analysis revealed alterations in the glycerophospholipid metabolism for the whole cancer cohort and in the ether lipid metabolism for the molecular subtype luminal B. Although this mixture of blood-derived MV contains only a minor number of tumor MV, a combination of metabolites was identified that distinguished between BC and controls as well as between molecular subtypes, and was predictive for overall survival. This suggests that these metabolites represent promising biomarkers and, moreover, that they may be functionally relevant for tumor progression.

LC-MS Based Platform Simplifies Access to Metabolomics for Peroxisomal Disorders.

Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit.

Targeted metabolomics revealed changes in phospholipids during the development of neuroinflammation in Abcd1tm1Kds mice and X-linked adrenoleukodystrophy patients.

X-linked adrenoleukodystrophy (X-ALD) is the most common leukodystrophy. Despite intensive research in recent years, it remains unclear, what drives the different clinical disease courses. Due to this missing pathophysiological link, therapy for the childhood cerebral disease course of X-ALD (CCALD) remains symptomatic; the allogenic hematopoietic stem cell transplantation or hematopoietic stem-cell gene therapy is an option for early disease stages. The inclusion of dried blood spot (DBS) C26:0-lysophosphatidylcholine to newborn screening in an increasing number of countries is leading to an increasing number of X-ALD patients diagnosed at risk for CCALD. Current follow-up in asymptomatic boys with X-ALD requires repetitive cerebral MRIs under sedation. A reliable and easily accessible biomarker that predicts CCALD would therefore be of great value. Here we report the application of targeted metabolomics by AbsoluteIDQ p180-Kit from Biocrates to search for suitable biomarkers in X-ALD. LysoPC a C20:3 and lysoPC a C20:4 were identified as metabolites that indicate neuroinflammation after induction of experimental autoimmune encephalitis in the serum of Abcd1tm1Kds mice. Analysis of serum from X-ALD patients also revealed different concentrations of these lipids at different disease stages. Further studies in a larger cohort of X-ALD patient sera are needed to prove the diagnostic value of these lipids for use as early biomarkers for neuroinflammation in CCALD patients.

The rise in expression and activity of 11ß-HSD1 in human mesenchymal progenitor cells induces adipogenesis through increased local cortisol synthesis.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) plays an important role in pre-receptor glucocorticoid metabolism. This enzyme is expressed in bone, increases with age, and catalyzes the conversion of the inactive glucocorticoid cortisone into the active glucocorticoid cortisol and vice versa. Here we hypothesized that the physiological activity of 11ß-HSD1 to produce cortisol in human mesenchymal progenitor cells (hMSC) is principally sufficient to shift the differentiation potential in the direction of adipogenic. We thus investigated differentiating hMSCs and the mesenchymal stem cell line SCP-1 cultured under osteogenic conditions and stimulated with supra-physiological cortisone levels. The release of active cortisol into the medium was monitored and the influence on cell differentiation analyzed. We revealed an increase in 11ß-HSD1 expression followed by increased reductive activity of the enzyme, thereby inducing a more adipogenic phenotype of the cell models via cortisol with negative effects on osteogenesis. Through inhibition experiments with the specific inhibitor 10 j, we proved the enzyme specificity for cortisol synthesis and adipogenic differentiation. Increased expression of 11ß-HSD1 followed by higher cortisol levels might thus explain bone marrow adiposity followed by reduced bone quality and stability in old age or in situations of supra-physiological glucocorticoid exposure.

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.

Systematic forensic toxicological analysis by liquid-chromatography-quadrupole-time-of-flight mass spectrometry in serum and comparison to gas chromatography-mass spectrometry.

Comprehensive screening procedures for psychoactive agents in body fluids are an essential task in clinical and forensic toxicology. With the continuous emergence and adaption of new psychoactive substances (NPS) keeping a screening method up to date is challenging. To meet these demands, hyphenated high-resolution mass spectrometry has gained interest as extensive and expandable screening approach. Here we present a comprehensive method for systematic toxicological analysis of serum by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) with data independent acquisition. The potential of this method was demonstrated by analysis of 247 authentic serum- and 12 post-mortem femoral blood samples. Thus 950 compounds, comprising 185 different drugs and metabolites could be identified. For the detected substances, including pharmaceutical substances, illicit drugs as well as NPS, serum concentrations were confirmed ranging from traces to toxic values indicating the capability for forensic toxicological requirements. Positive identification of drugs was achieved by accurate mass measurement (±5ppm for [M+H]+; ±10ppm for [M-H]-), retention time (±0.35min), isotopic pattern match (less than 10 m/z RMS [ppm]), isotope match intensity (less than 20% RMS) and the presence of at least two fragment ions. The LC-QTOF-MS procedure was shown to be superior to serum screening by GC-MS, since 240% (335 versus 141) more drugs were identified in serum samples compared to GC-MS.

Multiplex immunoassay measurement of amyloid-ß42 to amyloid-ß40 ratio in plasma discriminates between dementia due to Alzheimer's disease and dementia not due to Alzheimer's disease.

The cerebrospinal fluid (CSF) biomarkers amyloid-ß42 (Aß42), total Tau, and phospho-181-Tau represent important diagnostic tools to support the clinical diagnosis of Alzheimer's disease (AD). Acquiring CSF by lumbar puncture is considered a moderately invasive procedure, while blood sampling is minimally invasive with calculable risks and can be performed by trained non-medical staff. Thus, the identification of reliable and robust blood biomarkers of AD-related neuropathology would be significantly advantageous in daily practice and would allow more patients to be screened. In this study, we performed a multiplex amyloid-ß assay to simultaneously measure Aß40 and Aß42. We analyzed how well Aß40, Aß42, and the Aß42 to Aß40 ratio (Aß42/40) could differentiate between patients suffering from dementia either due or not due to AD. In addition, we studied different factors affecting Aß levels in plasma. Plasma Aß42/40 level was significantly lower in patients with dementia due to AD than in those with dementia due to other causes. Aß42/40 correlated weakly between plasma and CSF, but did not differ between amyloid-PET positive or negative patients. Furthermore, we found that kidney function influences Aß40 and Aß42 plasma levels, but not Aß42/40 level. Liver function, age, and sex do not affect Aß levels in plasma.

Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane.

Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g ("microvesicles" (MV)) and 100,000 g ("exosomes") are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that - despite the different vesicle biogenesis - the routes of vesicular export are adaptable.

Use of graft-derived cell-free DNA as an organ integrity biomarker to reexamine effective tacrolimus trough concentrations after liver transplantation.

BACKGROUND: Immunosuppressant therapeutic ranges for transplant patients have traditionally been established by indirect clinical means. However, "liquid biopsy" methods measuring graft-derived cell-free DNA (GcfDNA) in blood directly interrogate donor organ integrity. This study was performed to determine whether GcfDNA quantification could be used to reexamine minimally effective trough tacrolimus (Tacro) concentrations in liver transplantation (LTx) patients. METHODS: As part of a large prospective study to demonstrate the ability of GcfDNA to identify early graft rejection, 10 adult white LTx patients [8 men, 2 women, 3 hepatitis C virus (HCV) positive; mean ± SD age (years) = 56 ± 9.4] had simultaneous GcfDNA and whole-blood trough Tacro concentrations measured between days 5 and 30 after LTx. Samples were analyzed using droplet digital polymerase chain reaction for GcfDNA and liquid chromatography tandem mass spectrometry for Tacro. GcfDNA and trough Tacro concentrations were then compared to identify Tacro concentrations associated with intact graft integrity. RESULTS: Although there were large individual differences, there was a highly significant (Fisher P = 0.00002) segregation between whole-blood Tacro concentrations of ≥8 µg/L and normal (≤10%) GcfDNA percentages. The best discrimination in this population between effective and ineffective trough Tacro concentrations was estimated to be at 6.8 µg/L (P < 10(-7)). Compared with HCV- patients (n = 7), the 3 HCV+ patients had more variable associations between GcfDNA percentages and Tacro concentrations. CONCLUSIONS: Direct measurement of graft integrity using GcfDNA was useful to confirm the lower limit of the therapeutic ranges for trough Tacro concentrations after LTx. It would probably be useful to do so also for other immunosuppressant drugs and after other solid organ transplants. The method might be especially useful to detect graft injury during immunosuppressant dose minimization strategies.

Pharmacokinetics of meropenem in critically ill patients with severe infections.

BACKGROUND: Meropenem is an effective ß-lactam antibiotic that is frequently used to treat serious infections in both intensive care unit (ICU) and febrile neutropenic hematology/oncology (Hem/Onc) patients. Studies suggest that to be effective, meropenem concentrations must be maintained above the inhibitory concentrations for the majority of a dosing interval. However, the pharmacokinetics (PK) of meropenem seem to differ in critically ill patients compared with healthy or less ill subjects used to select labeled dosing regimens. OBJECTIVES: This study was designed to investigate meropenem PK in critically ill patients and to see how often standard dosing regimens produced adequate plasma concentrations. A secondary objective was to investigate how achieved concentrations were related to outcomes (morbidity and mortality) in these patients. METHODS: Meropenem plasma concentrations over time were measured using a high pressure liquid chromatography assay in febrile Hem/Onc and ICU patients who were treated with standard meropenem dosing schedules. Outcomes such as fever control and survival were assessed in these patients and compared with individual meropenem PK data and with recommended target concentrations. RESULTS: A total of 25 subjects including 10 febrile Hem/Onc and 15 ICU patients with a variety of serious illnesses and baseline renal function were studied. Mean peak concentrations were less variable than were pre-dose concentrations. Post peak and trough concentrations were often below recommended minimum inhibitory concentrations. Both clearance and volumes of distribution were greater than reported in less ill subjects, only in part explained by increased renal clearance. Therefore, serum concentrations often did not exceed recommended concentration targets even for moderately sensitive organisms. Inadequate concentrations were especially common in the mostly ill, febrile neutropenic Hem/Onc subjects and seemed to explain at least some therapeutic failures. Conversely, drug accumulation occurred in ICU subjects with decreased renal function. CONCLUSIONS: Standard meropenem dosing regimens were inadequate in many critically ill febrile, neutropenic Hem/Onc, and septic ICU patients. These data suggest a role for meropenem concentration monitoring in such patients.

Serotonergic overstimulation in a preterm infant after sertraline intake via breastmilk.

The selective serotonin reuptake inhibitor (SSRI) sertraline is widely used as an antidepressant agent during pregnancy and lactation because of its low placental transfer and low level of excretion into breastmilk. Symptoms such as neonatal abstinence syndrome and serotonergic overstimulation have been reported after in utero exposure to SSRIs. These symptoms are self-limiting and usually peak within the first 48 hours after birth. In our case, a preterm infant was exposed to sertraline and its main metabolite desmethylsertraline in utero and via breastmilk. Beyond the first 48 hours after birth, the infant developed increasing clinical signs of serotonergic overstimulation associated with substance intake via breastmilk, until breastfeeding was discontinued on postnatal Day 9. In spite of a low calculated daily substance intake via breastmilk, the serum substance levels of the preterm infant were within the therapeutic range of adults. The serotonergic overstimulation may be explained by the limited metabolic capacity of the infant and the immaturity of the blood-brain barrier.

Low vitamin D and elevated immunoreactivity against Epstein-Barr virus before first clinical manifestation of multiple sclerosis.

OBJECTIVE: Vitamin D deficiency and Epstein-Barr virus (EBV) infection may be associated with the development of multiple sclerosis (MS). We investigated serum 25-hydroxyvitamin D (25-OH-D) levels and anti-EBV immunoreactivity in 25 individuals before the first clinical manifestation of MS. PATIENTS AND METHODS: 56 serum samples of 25 individuals who had donated blood prior to the first clinical MS manifestation (clinically isolated syndrome (CIS)) (four male subjects, 21 female subjects, mean age 31.5 years at time of pre-CIS blood sampling; mean age at disease onset 33.4 years) were available, covering an interval of 7.3 years-2 months (mean 31.5 months) before CIS. In 18 of 25 patients serum samples were also obtained after established diagnosis of MS. Longitudinal age- and gender-matched healthy blood donors (four male subjects, 21 female subjects, 39 samples, mean age 32.5 years) served as controls. Serum 25-OH-D was measured by isotope dilution-liquid chromatography-tandem mass spectrometry. 25-OH-D levels were deconvoluted using published seasonal coefficients from a German population. Immunoglobulin G (IgG) against Epstein-Barr virus nuclear antigen-1 (EBNA1) were assessed using commercially available ELISA. RESULTS: Low 25-OH-D levels were observed during the 24-month pre-CIS interval (47.8 (32.5-77.2) nmol/l, median (IQR); healthy controls: 81.6 (57.7-98.5), p=0.004, however, still higher than after established diagnosis (24.5 (13.7-47.7), p<0.0001 compared with controls). IgG against EBNA1 during the 36-month pre-CIS interval was increased (185.9 (91.2-460.0) IU/ml, median (IQR); healthy controls 63.7 (29.5-121.6), p=0.002). CONCLUSIONS: Low vitamin D and remote EBV infection may be associated with clinical MS breakthrough within 2-3 years.

Use of total and unbound imatinib and metabolite LC-MS/MS assay to understand individual responses in CML and GIST patients.

OBJECTIVES: Trough total imatinib (t-IM) concentrations have been reported to be associated with therapeutic and toxic responses in patients with chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST). Little is known about the relationships between effects and concentrations of either unbound imatinib (f-IM) or imatinib's major metabolite, N-desmethyl imatinib (NDI). In part, this is because of the lack of a single, validated, well-described clinically useful assay for these measurements. The authors report the development and application of such an assay. MATERIALS AND METHODS: A single liquid-chromatography tandem-mass-spectrometry assay was used to monitor t-IM, f-IM, and t-NDI concentrations in CML and GIST patients treated at a tertiary German teaching hospital. The assay was also validated for measuring other kinase inhibitors, including t-nilotinib, sunitinib, and erlotinib. Ultrafiltration assays were validated and used to measure f-IM and to compare free fractions to plasma α1-acid glycoprotein concentrations (AGP). RESULTS: The assays were linear over a working range (in micrograms per liter) of 8.4-8370, 8.3-4165, and 1.0-250 and had within- and between-run coefficient of variance of <7%, <12%, and <9% for t-IM, t-NDI, and f-IM, respectively. The f-IM assay was reproducible despite high (25.2%-31.6%) but concentration-independent binding to ultrafiltration devices. Clinically relevant results, such as nondetectable (ND) t-IM (<8.4 µg/L) in non-responders and >1500 µg/L in patients with major toxicity, were found. Of 156 total samples from 68 adult CML patients and 127 total samples from 42 adult GIST, only 48 samples from 22 CML patients and 40 samples from 20 GIST patients were trough samples with adequate dosing and collection information. More than half (27 of 48 CML and 24 of 40 GIST) had t-IM concentrations ≥10% below recommended target concentrations (1002 µg/L for CML and 1100 µg/L for GIST). Concentrations >50% over targets were also found in 6 of 48 CML and 4 of 40 GIST samples. Wide variations in concentrations of t-IM (range, ND to 2973 µg/L), t-NDI (range, ND to 659 µg/L), f-IM (range, 8.3-262 µg/L), and t-IM:f-IM ratios (range, 2.6%-14%) were found both between and within patients. A statistically significant association (Spearman correlation coefficient and P value for all samples, r = 0.290 and P = 0.023; for trough only, r = -0.585 and P = 0.028) was found between AGP and f-IM concentrations but wide interpatient and intrapatient variations made individual predictions unreliable. CONCLUSIONS: The liquid-chromatography tandem-mass-spectrometry methods developed provided information useful to understand individual responses to therapy even though necessary sampling and dosing information was often not available. Wide unpredictable variations in t-IM, t-NDI, and f-IM were found. Clinical outcome trials are needed to examine whether f-IM or NDI monitoring can improve the ability to predict individual responses.

Multicenter evaluation of a new automated electrochemiluminescence immunoassay for the quantification of testosterone compared to liquid chromatography tandem mass spectrometry.

OBJECTIVES: A new electrochemiluminescence testosterone immunoassay (Elecsys® Testo II) was evaluated. DESIGN AND METHODS: Within-run precision, total precision, and method comparisons to the former immunoassay and LC-MS/MS were investigated. Reference intervals were established for adults and children. RESULTS: Imprecision ranged from 0.9% to 8.5%. Method comparisons revealed slopes from 0.68 to 1.32 and coefficients of correlation from 0.893 to 0.991. CONCLUSIONS: Improved agreement with LC-MS/MS was demonstrated, especially for female patients. The assay may be a suitable alternative for laboratories with no access to MS techniques.

Formation of monoethylglycinexylidide (MEGX) in clinically healthy dogs.

Liver function tests help to investigate actual liver function. In dogs, only a few tests are available. We evaluated the formation of monoethylglycinexylidide (MEGX) in clinically healthy dogs to assess the usefulness of this liver function test in dogs. Twenty-five healthy dogs were used in this study. The MEGX test was done according to human protocols. The results of our study showed that dogs synthesize MEGX after the administration of lidocaine. There was no age dependence of this test in dogs and no significant difference between measurements obtained at 15 and 30 min after administration of lidocaine. Female dogs had significantly (P < 0.05) higher concentrations of MEGX 15 min after administration. The reference interval for dogs after 15 min is 34 to 79 µg/L and after 30 min 39 to 89 µg/L. In conclusion, the MEGX test may be an additional liver function test in dogs.

Performance evaluation of the new ADVIA Centaur system cyclosporine assay (single-step extraction).

BACKGROUND: Cyclosporine (CsA) monitoring is essential for transplant success. We report a performance study of the recently released, fully automated Siemens ADVIA Centaur CsA assay. METHODS: Whole blood samples from 248 transplant patients were prepared using a new 1-step extraction method. Performance evaluations vs. HPLC-tandem MS (LC-MS/MS), Abbott TDx and AxSYM assays were conducted according to CLSI EP5-A2 and EP9-A2 guidelines. RESULTS: The correlation coefficient for LC-MS/MS and ADVIA Centaur was > or = 0.97 at each site, and for each transplant type. Regression analysis yielded y=0.94x+19 for all sites: 95% CI=0.91-0.96 (slope) and 10-28 (intercept). Absolute and relative bias was minimal for C0 and C2 sampling. Centaur vs. Abbott TDx and AxSYM assays: y = 0.72x+6, r = 0.98, 95% CI = 0.70-0.73 (slope), 3-9 (intercept); and y = 0.69x+18, r = 0.97, 95% CI = 0.67-0.71 (slope), 8-27(intercept). Within run CVs were 4.5%-7.1%, total CVs were 5.3%-7.7%. CONCLUSIONS: The ADVIA Centaur assay compared favorably with LC-MS/MS and Abbott assays, displaying good correlation for all transplant types and methods.