An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood.

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and 13C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).

An LC-MS/MS based candidate reference method for the quantification of carbamazepine in human serum.

A validated LC-MS/MS-based candidate reference measurement procedure for the quantification of carbamazepine is presented in order to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring. Sample preparation was based on protein precipitation using acetonitrile followed by sample dilution. Since the previously listed certified reference material (CRM) SRM 1599 (anticonvulsant drug level assay standard) is no longer available, an ISO certified calibration material was used in this assay. As internal standards deuterated analyte congeners were applied. The method allows the measurement of carbamazepine, carbamazepine-10,11-epoxide and 10-hydroxy-10,11-dihydrocarbamazepine in the concentration range of 0.1 to 22.0µg/ml with LODs and LOQs of <0.1µg/ml and 0.1µg/ml, respectively. Comparative measurement of 105 native patient samples using the here presented method showed a good agreement between two independent laboratories with a mean bias of 0.6%.

Rapid detection and quantification of 35 benzodiazepines in urine by GC-TOF-MS.

A rapid and sensitive method for the screening and quantification of 35 benzodiazepines in human urine by gas chromatography/time-of-flight mass spectrometry was developed and validated. Target analytes were isolated from 1 ml urine by solid-phase extraction using Oasis MCX extraction columns (extraction recovery between 35 and 99%). With a supported liquid-liquid extraction method, a new modification of conventional liquid-liquid-extraction, a less time intensive alternative for benzodiazepine extraction is presented. The sample pretreatment entails the derivatization of the benzodiazepines with N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. Separation of all benzodiazepines was done within 9.5 min, and detection was based on full mass spectra for each analyte. A deconvolution algorithm was used for unresolved chromatographic peaks to identify coeluted substances. The subsequent quantification was done using significant masses. The limit of quantification is 10 ng/ml for most of the compounds. Linearity is in the range between 10 and 350 ng/ml. Reproducibility was observed with coefficients of variation below 2% at concentrations of 50 and 200 ng/ml. The accuracy is between 88 and 108% depending on the respective analyte and the concentration.

Screening for bone metastases: whole-body MRI using a 32-channel system versus dual-modality PET-CT.

The diagnostic accuracy of screening for bone metastases was evaluated using whole-body magnetic resonance imaging (WB-MRI) compared with combined fluorodeoxyglucose (FDG) positron emission tomography (PET) and computed tomography (CT) (FDG-PET-CT). In a prospective, blinded study, 30 consecutive patients (18 female, 12 male; 24-76 years) with different oncological diseases and suspected skeletal metastases underwent FDG-PET-CT as well as WB-MRI with the use of parallel imaging (PAT). With a 32-channel scanner, coronal imaging of the entire body and sagittal imaging of the complete spine was performed using T1-weighted and short tau inversion recovery (STIR) sequences in combination. PET-CT was conducted using a low-dose CT for attenuation correction, a PET-emission scan and diagnostic contrast-enhanced CT scan covering the thorax, abdomen and pelvis. Two radiologists read the MRI scans, another radiologist in combination with a nuclear medicine physician read the PET-CT scans, each in consensus. The standard of reference was constituted by radiological follow-up within at least 6 months. In 28 patients, 102 malignant and 25 benign bone lesions were detected and confirmed. WB-MRI showed a sensitivity of 94% (96/102), PET-CT exams achieved 78% (79/102; P<0.001). Specificities were 76% (19/25) for WB-MRI and 80% (20/25) for PET-CT (P>0.05). Diagnostic accuracy was 91% (115/127) and 78% (99/127; P<0.001), respectively. Cut-off size for the detection of malignant bone lesions was 2 mm for WB-MRI and 5 mm for PET-CT. WB-MRI revealed ten additional bone metastases due to the larger field of view. In conclusion, WB-MRI and FDG-PET-CT are robust imaging modalities for a systemic screening for metastatic bone disease. PAT allows WB-MRI bone marrow screening at high spatial resolution and with a diagnostic accuracy superior to PET-CT.

Fistula between cystic artery pseudoaneurysm and cystic bile duct cause of acute anemia one year after laparoscopic cholecystectomy.

We present a case of hemorrhage from a cystic artery pseudoaneurysm one year after laparoscopic cholecystectomy. A 78-year-old male with a history of recurrent melena, hematemesis, and right upper abdominal pain was admitted to our emergency department. His blood pressure was 60/30 mm Hg with a pulse rate of 100 beats per minute. Hemoglobin was 7.6 g/dL and white blood cell count 19500/mm(3). Computed tomography scan of the abdomen and selective digital subtraction arteriography showed a pseudoaneurysm in the region of the former bed of the gallbladder. During gastroscopy, a pulsatile bleeding out of the papilla of Vater was found. Surgery by the open approach confirmed the presence of a cystic artery pseudoaneurysm and showed an additional fistula between the pseudoaneurysm and the cystic bile duct. Resection of the pseudoaneurysm and revision of the common bile duct with implantation of a T-tube was performed. The patient recovered well and was discharged from our hospital three weeks after surgery.

Comparison of Tc-99m sestamibi SPECT with fractional flow reserve in patients with intermediate coronary artery stenoses.

BACKGROUND: Myocardial single photon emission computed tomography (SPECT) is an established noninvasive method for the assessment of the functional significance of coronary artery stenoses. Intracoronary pressure measurements to determine fractional flow reserve (FFR) are increasingly performed during coronary angiography whenever an immediate decision regarding possible intervention is required. We hypothesized that the regional summed difference score (SDSr), reflecting reversible perfusion defects in the myocardial supply area of the FFR target vessel, would be the best predictor of an abnormal FFR in patients without prior myocardial infarction. Otherwise, a regional summed stress score (SSSr) should be the best predictor of an abnormal FFR in patients with prior myocardial infarction for different patient subgroups with coronary artery disease. METHODS AND RESULTS: In this study 50 patients (mean age, 65 +/- 9.1 years; 18 women) with coronary artery disease and a 50% to 75% coronary stenosis (target vessel) were prospectively investigated. Dobutamine myocardial SPECT was performed as a single-day stress/rest protocol by use of technetium 99m sestamibi. For image interpretation, semiquantitative analysis was conducted by calculating SSSr and SDSr. Within 8 (+/-14.9) days, coronary angiography was performed and FFR was calculated by use of a pressure wire (normal FFR, > or = 0.75). The mean FFR of all patients was 0.78 +/- 0.14. Of 50 patients, 17 had an FFR lower than 0.75 in the target vessel. Receiver operating characteristic analysis identified an SDSr of 1 or greater and an SSSr of 3 or greater as the best threshold values for predicting ischemic FFR. Sensitivity, specificity, and negative and positive predictive values of SDSr and SSSr for the detection of FFR values lower than 0.75 in the target vessel were 80%, 76%, 53%, and 92%, respectively, and 70%, 93%, 78%, and 90%, respectively, in patients without prior myocardial infarction and 57%, 50%, 67%, and 40%, respectively, and 100%, 50%, 78%, and 100%, respectively, in patients with prior myocardial infarction. Weak correlation was found between the single values of FFR with both SDSr and SSSr for the different patient subgroups. CONCLUSION: Among the dobutamine myocardial scintigraphy variables studied, SDSr was the best predictor of an abnormal FFR (cutoff value of 0.75) in patients without prior myocardial infarction. As assumed, SSSr was the best predictor of an abnormal FFR in patients with prior myocardial infarction in the target region.

High-resolution whole-body magnetic resonance image tumor staging with the use of parallel imaging versus dual-modality positron emission tomography-computed tomography: experience on a 32-channel system.

OBJECTIVE: The objective of this study was to compare the accuracy in staging of various malignant tumors with whole-body magnetic resonance imaging (WB-MRI) using parallel imaging (PAT) and positron emission tomography-computed tomography (PET-CT). MATERIALS AND METHODS: In a prospective study, 41 patients withoncologic diseases underwent [F]-fluoro-2-deoxy-D-glucose PET-CT for tumor staging and WB-MRI on a 32-channel-scanner with the use of PAT. Coronal T1w and STIR sequences at 5 body levels, axial HASTE imaging of the lung, and contrast-enhanced T1w sequences of the liver, brain, and abdomen were performed. TNM stage was assessed for both modalities in a separate consensus reading using histologic results and radiologic follow up within 6 months as the standard of reference. RESULTS: Three primary and 4 recurrent tumors were detected; one recurrent tumor was missed with WB-MRI. Sixty benign and 60 malignant lymph nodes were detected with a sensitivity of 98% and specificity of 83% for PET-CT and 80%/75% for WB-MRI, respectively. One hundred ninety-one malignant and 77 benign distant lesions were detected with a sensitivity/specificity of 82% for PET-CT and 96%/82% for WB-MRI. Accuracy for correct TNM staging was 96% for PET-CT and 91% for WB-MRI. CONCLUSION: WB-MRI and PET-CT are reliable imaging modalities for tumor staging. WB-MRI is highly sensitive in detecting distant metastases; PET-CT is superior in lymph node staging. PAT makes high-resolution WB-MRI feasible within less than 1 hour.

Selective internal radiation therapy with SIR-Spheres in patients with nonresectable liver tumors.

AIM: Transarterial embolization of branches of the hepatic artery with biocompatible 90Y-labeled microspheres (SIR-Spheres) is a local treatment modality for patients with liver tumors, which, most recently, has become available in Europe. The aim of this study was to evaluate the feasibility and efficacy of this selective internal radiation therapy (SIRT). METHODS: Twenty-three patients with nonresectable hepatic metastases or hepatocellular carcinoma nonresponding to polychemotherapy and/or other local treatment were treated with SIRT. SIR-Spheres (mean activity, 2270 MBq) were administered by gentle intra-arterial infusion in the hepatic artery. A follow-up was documented by fluorodeoxyglucose-positron emission tomography (FDG-PET), course of tumor markers, and computed tomography (CT). RESULTS: Common minor side-effects were abdominal pain, nausea, and fever. Mild pancreatitis and peptic ulceration were observed once each. Currently, all patients are still alive, with survival times ranging from 11 to 518 days from SIRT up to the present. Three-month follow-up investigations are available in 13 of 23 patients, which, so far, are showing a marked decrease of FDG uptake, a drop of tumor markers, and unchanged or slightly decreasing lesion size (CT) in 10 of 13 patients. Two patients showed stable findings, while another patient showed progressive disease. Long-term follow-up investigations are available in 2 of 23 patients, showing hepatic and extrahepatic progression 6 and 9 months after SIRT. CONCLUSIONS: Our initial experience confirms that SIRT is a promising local therapeutic approach in patients with nonresectable liver tumors which is feasible and has an acceptable toxicity profile. Prospective data on comparing this treatment alone or in combination with other modalities are needed to answer whether long-term survival in this unfavorable stage of disease can be markedly improved.

Sentinel node mapping in patients with malignant melanoma using melanoma Tc-99m colloidal rhenium sulfide.

PURPOSE: The aim of the study was to localize the sentinel lymph node using lymphoscintigraphy aided by Tc-99m colloidal rhenium sulfide. MATERIALS AND METHODS: Thirty consecutive patients with histologically proved melanoma, but no clinical evidence of metastases, were examined before operation by injecting 20 to 40 MBq (0.5 to 1.1 mCi) Tc-99m colloidal rhenium sulfide with a mean particle size of 100 nm (range, 50 to 200 nm) intradermally around the lesion. Lymphoscintigraphy was performed immediately after injection. In addition, blue dye was injected before operation. A hand-held gamma probe guided the sentinel node biopsy. RESULTS: Lymphoscintigraphy revealed hot spots in all patients. During surgery, the sentinel node was identified in all 30 patients. The number of sentinel nodes per patient ranged from 1 to 4 (mean, 1.9). Histologic examination confirmed the metastatic involvement of the sentinel lymph node in 11 of 30 patients. The sentinel lymph node-positive rate was 22.4%, which was comparable to findings using Tc-99m-labeled nanocolloids. CONCLUSION: The findings indicate that Tc-99m-bound colloidal rhenium sulfide is suitable for sentinel node mapping.

[Can supplemental imaging with SPECT technique improve the diagnostic value of preoperative Tc-99m-MIBI scintigraphy in primary hyperparathyroidism?]. / Kann durch den zusätzlichen Einsatz der SPECT-Technik die diagnostische Aussage der präoperativen Tc-99m-MIBI-Szintigraphie beim primären Hyperparathyreoidismus verbessert werden?

BACKGROUND: The focus of this retrospective study was to evaluate if the additional use of SPECT improves the diagnostic value of the preoperative Tc-99m-sestamibi scintigraphy combined with sonography in patients presenting with primary hyperparathyroidism. PATIENTS AND METHODS: 62 patients (43 female, 19 male) were examined by visual analysis of planar Tc-99m-sestamibi scintigraphy, SPECT and ultrasound. 14/62 were suffering from superimposed thyroid disease, 22/62 patients underwent previous neck surgery. 12/62 presented with recurrent hyperthyroidism, in 8/62 cases dystopic parathyroid tissue had been localized. For statistic evaluation these imaging modalities were correlated with the histopathological examinations of the surgical specimens for single use as well as combined use for all patients (group 1) and for the subgroups "histologically confirmed adenoma" (group 2), hyperplastic disease (group 3) "recurrency and/or dystopic parathyroid tissue" (group 4) and "patients presenting with thyroid nodules and/or previous neck surgery" (group 5), respectively. RESULTS: For single use planar scintigraphy showed the highest sensitivity (72%) followed by SPECT (67%) and ultrasound (58%). The combination of planar imaging with ultrasound achieved 83% whereas the combined use of all three modalities increased the sensitivity up to a maximum of 85%. The combined use of the imaging modalities leads to the following results: The histologically proven parathyroid adenomas (group 2) could be detected by planar scintigraphy and sonography with a sensitivity of 87%, for group 3 the combined use of all three modalities reached 69% sensitivity. Group 4 achieved the highest sensitivity (69%) by means of planar scintigraphy and SPECT, whereas group 5 reached 82% by means of planar scintigraphy and ultrasound. CONCLUSION: Planar Tc-99m-sestamibi scintigraphy combined with ultrasound is superior regarding to sensitivity and to the costs for the noninvasive preoperative detection in patients presenting with primary hyperparathyroidism. Due to economic reasons the additional use of SPECT might be limited to patients presenting with recurrent hyperparathyroidism or atypically located parathyroid tissue.

Early detection of myocardial microcirculatory disturbances after primary PTCA in patients with acute myocardial infarction: coronary blood flow velocity versus sestamibi perfusion imaging.

BACKGROUND: The extent of myocardial salvage after primary percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI) is variable and can not be predicted on the basis of vessel patency. The aim of this study was to evaluate the tissue salvage and the microvascular integrity after successful intervention in AMI by coronary blood flow velocity and sestamibi perfusion imaging. METHODS: Twenty-two patients (17 m, 5f; mean age 57 +/- 14 yrs.) undergoing primary PTCA and stent implantation for AMI were studied. 99mTc Sestamibi was injected intravenously before intervention and single photon emission computed tomography (SPECT) was performed immediately after successful reperfusion to determine the area at risk before PTCA due to the minimal 99mTc Sestamibi redistribution. Sestamibi SPECT was repeated 3 days and 6 months after AMI. Area at risk (%) was determined automatically by myocardial perfusion tomography (PERFIT) with the use of a multistage, 3D iterative inter-subject registration of patient images to normal templates (2SD) and myocardial salvage was calculated. Coronary flow velocity was measured using a Doppler-tipped guidewire in the infarct-related artery after successful completion of primary PTCA and in an angiographically normal reference vessel. Absolute coronary flow reserve (CFR) and relative CFR (rCFR) were calculated using hyperemic to basal average peak velocity. RESULTS: Despite successful reperfusion of the target vessel (TIMI grade III flow) CFR and rCFR remained impaired (1.8 +/- 0.9 and 0.77 +/- 0.21). Area at risk decreased significantly from 21 +/- 9% to 9 +/- 10% (p < 0.05) corresponding to 11 +/- 8% myocardial salvage. Acute CFR and rCFR showed no correlation with the area at risk before and after primary PTCA. The increase of CFR within 6 months correlated with the myocardial salvage (p < 0.05). CONCLUSIONS: Despite successful primary PTCA in AMI, CFR and rCFR often remain impaired because of a significant loss of microvascular integrity. The long-term success of primary PTCA can be assessed by myocardial salvage and the change of CFR which might be a useful parameter for additional reperfusion strategies such as glycoprotein IIb/IIIA receptor inhibition.