M. tuberculosis, using whole blood culture (WBA). The addition of 1,25-dihydroxy vitamin D facilitated detection of the activity of TMC207 in the 3-day cultures. Pyrazinamide failed to show significant activity against a PZA-resistant strain (M. bovis BCG), and was not further considered. Low, mid, and high therapeutic concentrations of each remaining drug were tested individually and in a paired checkerboard fashion. Observed bactericidal activity was compared to that predicted by the sum of the effects of individual drugs. Combinations of PNU-100480, TMC207, and SQ109 were fully additive, whereas those including PA-824 were less than additive or antagonistic. The cumulative activities of 2, 3, and 4 drug combinations were predicted based on the observed concentration-activity relationship, published pharmacokinetic data, and, for PNU-100480, published WBA data after oral dosing. The most active regimens, including PNU-100480, TMC207, and SQ109, were predicted to have cumulative activity comparable to standard TB therapy. Further testing of regimens including these compounds is warranted. Measurement of whole blood bactericidal activity can accelerate the development of novel TB regimens.There presently is no rapid method to assess the bactericidal activity of new regimens for tuberculosis. This study examined PNU-100480, TMC207, PA-824, SQ109, and pyrazinamide, singly and in various combinations, against intracellular  This need is greatest for extensively-drug resistant disease (XDR-TB), which presently requires at least 24 months of treatment for cure The problem of microbial persistence due to phenotypic tolerance is not unique to mycobacteria M. bovis Bacillus Calmette-Guérin (BCG), and reaching bacteriostasis in TB patients tested with their own isolates Current strategies to test new TB drug combinations have important shortcomings. MIC testing, a common first step, does not adequately predict bactericidal activity for TB drugs, even when comparisons are limited to drugs within a single class Several studies have examined the whole blood bactericidal activity (WBA) of TB drugs at multiple time points after oral drug administration. First line drugs show a rank order of cumulative WBA (its area under the curve [AUC] over 24 hrs) of rifampin>isoniazid>pyrazinamide>ethambutol when tested in healthy volunteers M. tuberculosis H37Rv was grown in mycobacterial growth indicator tubes  until 1–2 days after growth was detected, and frozen in aliquots. A titration experiment determined the relationship between inoculum size and time-to-positivity (TTP) over a 6-log range  – log, where final and initial are the volumes corresponding to TTP of the completed cultures and its inoculum, respectively, based on the titration curve. The laboratory protocol and software (version 12) developed by the author (RSW) are available on request. WBA was reported as log change/day.A single healthy volunteer served as a blood donor for this study, after providing written informed consent. The study protocol was approved by the Institutional Review Board of the Veterans Administration Connecticut Healthcare System. Drug powders were either dissolved first in dimethylsulfoxide , or RPMI-1640 tissue culture medium . Stock solutions were prepared at concentrations ranging from 10 mg/L (isoniazid) to 200 mg/L (pyrazinamide) and kept at −30°C until needed. WBA was measured as previously described Each drug was tested individually to establish its concentration – activity relationship. Drug pairs were then tested in a checkerboard fashion . The obsin vivo are twice those in ex vivo whole blood culture, due to dilution with tissue culture medium. Expected concentrations in plasma of the novel compounds were therefore reduced by half so that results could be compared to orally-administered therapies. Values for hourly time points were estimated by interpolation. The combined activities of specific drug combinations were calculated for each hourly time point by adding the activities of the individual drugs and adjusting the result according to the mean difference between observed and predicted values. Cumulative WBA was then calculated as the AUC0–24 by the trapezoidal method, and reported as log change.Drug activity after oral administration was predicted based on published PK data and the observed concentration-activity relationship . For PA-Paired values were compared using the paired t test. Comparisons among multiple values were performed by the repeated measures one-way analysis of variance (ANOVA). Post-hoc testing to examine differences between specific pairs was then performed by the Holm-Sidak method. All statistical tests were performed using SigmaPlot version 11.−bx for curve fitting, where y = time-to-positivity in days, x = log inoculum volume in µl, and e = Euler's number. Values for y0, a, and b that most closely fit experimental results were identified using the iterative non-linear regression module (Solver) provided with Microsoft Excel, using its default parameters. This exponential decay equation was selected because it provided the best fit for all of 5 different batches of M. tuberculosis stock (not shown). The adequacy of curve fitting for the batch used in this study is illustrated by the superimposition of curves for predicted and observed data in the left panel of vs. interpolation). The two sets of results are equal except for those samples showing maximal bactericidal effect, the activities of which were underestimated by the interpolation method. The exponential regression method was therefore selected for use in this study.Preliminary experiments addressed 3 concerns of this project: the method of determining the titration curve, the suitability of whole blood culture conditions to detect effects of TMC207 (bedaquiline), and the activity of pyrazinamide against pyrazinamide-resistant mycobacteria. Initial experiments had indicated that the time-to-positivity of MGIT cultures from highly bactericidal drug combinations could exceed those tested in the titration experiment. Such results cannot be analyzed by point-to-point interpolation, the method used in previous studies of this method. The WBA spreadsheet was therefore modified to perform exponential regression analysis rather than interpolation, using the equation y = y0+aein vivo and in vitro that presumably reflects the size of the ATP pool at the start of drug exposure and the net balance between ATP consumption and production. Studies in patients treated with TMC207 indicate a 4-day delay before an effect on mycobacterial viability in sputum becomes evident M. tuberculosis viability. All subsequent experiments were therefore conducted in the presence of added vitamin D.A second preliminary experiment sought to identify whole blood culture conditions that adequately detected the effect of TMC207 (bedaquiline). This compound, which acts by selective inhibition of mycobacterial ATP synthetase M. bovis BCG (ATTC TMC 1101). Although pyrazinamide has historically been a key sterilizing TB drug, recent surveys indicate resistance rates as high as 85% in MDR-TB strains M. bovis strains (including BCG) are characteristically resistant to pyrazinamide M. tuberculosis, which grew in the absence of added drug, viability of the attenuated BCG strain declined by log −0.135. Progressive addition of vitamin D, PNU-100480, and PZA (20 mg/L) resulted in progressive loss of viability, to log −0.151, −0.372, and −0.410, indicating a net incremental effect of pyrazinamide of −0.038. The effect of pyrazinamide appeared substantially reduced compared that when tested against M. tuberculosis following oral administration  A final preliminary experiment examined the activity of pyrazinamide-containing regimens against PZA-resistant The bactericidal activities of drugs tested individually and in 2 and 3-way combinations are shown in Cells other than those in the first column and row in For each combination, the observed activity was compared to that expected as the sum of each drug tested individually. The mean and standard deviation of the difference between observed and expected values appears in each panel. Positive values indicate drug combinations that overall were less than additive, and negative values, more than additive. No combination was significantly more than additive. The combinations of PNU-100480 plus TMC207 (panel A) and PNU-100480 plus SQ109 (panel E) overall were additive, each having observed and expected values that did not differ significantly. In contrast, the combination of PA-824 plus TMC207 (panel B) was significantly less active than expected . For PA-824 plus PNU-100480 (panel C), a trend toward reduced activity overall was present . Panel D shows the predicted and actual activity of the PNU-TMC-Pa combination. In this case, limited testing was performed, using only mid concentrations of PNU-100480 and TMC207. Despite the reduced number of test conditions, the combination was significantly less active than predicted . Inspection of individual cells revealed antagonism when low concentrations of PA-824 were combined with low or mid concentrations of PNU-100480. Under these conditions, the activity of the two drugs combined appeared less than that of PNU-100480 alone. Antagonism was also apparent when low concentrations of PA-824 were combined with mid concentrations of TMC207 plus PNU-100480.in vitro data, drug concentrations in whole blood culture was assumed to be half those in blood, due to dilution with tissue culture medium. Predicted cumulative WBA in these subjects  did not differ significantly from that measured directly (−0.316±0.044) according to paired t test (P = .06). However, the trend toward statistical significance indicated that cumulative WBA ex vivo might be lower than that predicted in vitro. We therefore adopted a conservative approach and used the ex vivo values for PNU-100480 for all subsequent comparisons. In each case, the activity of a regimen was predicted as the sum of the individual drugs adjusted by the mean difference between observed and predicted values for that combination.For 3 of the compounds tested , knowledge of a compound's concentration-activity relationship and pharmacokinetics in plasma should enable the prediction of its cumulative WBA after oral dosing according to the method outlined in vs. daily dosing, whether given individually, or in combination with PNU-100480.Results for combinations of PNU-100480 and TMC207 are illustrated in in vitro models Results for other 2, 3, and 4-drug combinations are summarized in i.e., reduced activity with PA-824 added). At the 600 mg dose, two of four showed antagonism. The remainder all showed less than additive effects.Antagonism was apparent when 200 mg PA-824 was added to some PNU or TMC-containing regimens. Of the five instances in M. tuberculosis strain such as H37Rv to inform activity against highly resistant strains This study examined the bactericidal activities of PNU-100480, TMC207, PA-824, and SQ109, singly and in various combinations, using whole blood culture. These compounds are each members of new classes of antimycobacterial drugs: oxazolidinone, diarylquinoline, nitroimidazole, and ethylenediamine, respectively. None exhibits cross-resistance with existing TB drugs. For each, MICs against M/XDR-TB strains equal those for fully drug-sensitive strains. As a result, new regimens comprised of these drugs will be as active against highly resistant mycobacterial strains as for drug sensitive strains. This permits experimental studies with a fully sensitive in vitro models can replicate the complexity and variability of human disease.The main findings of the present study were that combinations of PNU-100480 and TMC207 were highly active, approaching the combined activity of isoniazid plus rifampin, whereas 2 and 3-drug combinations including PA-824 showed less than fully additive activity. These observations can provide important guidance in the selection for further testing of novel regimens against tuberculosis in animal models and in patients. At the same time, the findings raise questions as to the mechanisms underlying these observations, and the extent to which The range of drug concentrations tested in this study reflected those in plasma in phase 1 and 2 clinical trials, recognizing that our understanding of optimal dosing of these drugs may yet be incomplete. PNU-100480 and PA-824 both have been reported to show time-dependent killing in vitro (in vitro based solely on concentrations of the parent. Levels of PNU-101603 reach concentrations in humans several times that of PNU-100480 PNU-101603, the sulfoxide metabolite of PNU-100480, appeared not to contribute to its parent when the two were combined in vitro ; similaret al found antagonism when PA-824 was added to the combination of TMC207 plus pyrazinamide in the mouse model, resulting in >1 log increases in CFU counts that could not be explained by drug-drug PK interactions M. tuberculosis through activation of DosR The finding that combinations of PA-824 with PNU-100480 or TMC207 were less than additive, and in some circumstances, antagonistic, was unexpected. Studies in the mouse model of other drug combinations that include PNU-100480 have not shown evidence of antagonism in vivo. However, sub-MIC concentrations of PNU-100840 and TMC207 are not anticipated for the regimens of greatest interest, minimizing the significance of this shortcoming.These experiments were intended to provide a rapid survey of potential drug combinations rather than to serve as definitive experiments. As such, their limitations should be acknowledged. Studies of antimicrobial activity conducted by adding drugs directly to cultures eliminate the greatest source of variability, that of inter-subject differences in pharmacokinetics and metabolism. Additional population-based studies will be required to better understand how these PK differences affect the efficacy of new TB regimens. Three dimensional modeling of drug interactions In summary, this study used a whole blood culture model to examine potential novel drug combinations that might ultimately be used to treat XDR-TB. The main findings were that combinations including PNU-100480, TMC207, and SQ109 were highly active and fully additive, whereas those including PA-824 were less than additive and in some instances antagonistic. Further studies of the PNU-TMC combination are warranted. The whole blood method can accelerate TB drug development.


Detoxifying proteins such as O6-methylguanine-DNA-methyltransferase (MGMT) confer a strong intrinsic resistance to CSC in all studies. Extrinsic factors may also contribute to the resistance of CSC to TMZ. These may include TMZ concentrations in the brain parenchyma, TMZ dosing schemes, hypoxic microenvironments, niche factors, and the re-acquisition of stem cell properties by non-stem cells. Thus, the interaction of CSC and chemotherapy is more complex than may be expected and it is necessary to consider these factors in order to overcome chemoresistance in the patient.Glioblastomas (GBM) are a paradigm for the investigation of cancer stem cells (CSC) in solid malignancies. The susceptibility of GBM CSC to standard chemotherapeutic drugs is controversial as the existing literature presents conflicting experimental data. Here, we summarize the experimental evidence on the resistance of GBM CSC to alkylating chemotherapeutic agents, with a special focus on temozolomide (TMZ). The data suggests that CSC are neither resistant nor susceptible to chemotherapy  The role of chemotherapy in the treatment of glioblastoma (GBM) has undergone considerable changes in the last two decades. While alkylating substances such as nimustine (ACNU), carmustine (BCNU), and lomustine (CCNU) have been used since the late 1970s , the intCancer stem cells (CSC) are postulated mediators of chemoresistance. The CSC hypothesis proposes that tumors are driven by subpopulations of tumor cells with stem cell-like properties, referred to as CSC . It furtGBM are a paradigm for the investigation of CSC in solid malignancies. The resistance of GBM CSC towards radiotherapy and chemotherapy has been extensively studied in the last 5 years. Here, we summarize the current knowledge on the resistance of GBM CSC to chemotherapy, with a special focus on TMZ as the current standard of care.+, but not CD133-, tumor cells were able to reconstitute the initial tumor in vivo when injected into immune-deficient nude mice [+ and CD133- cells within CSC lines may have similar tumorigenic potential [- CSC have recently been identified [For several types of brain tumors, including a subgroup of primary GBM, CSC were found to express CD133. CD133ude mice ,10. Howeude mice ) has beeude mice . GBM celotential ,15. In aentified -18. Thusentified . The CSCentified ,23. The in vivo but in a persisting period of stable disease that may be followed by a late reduction of tumor volume [However, stem cell-specific therapies, although preventing further growth of the tumor, will not result in an impressive shrinkage of the lesion r volume . Becauser volume .In vitro, these implications raise questions about viability assays used to determine the survival of all tumor cells -2,5-diphenyltetrazoliumbromide (MTT) or water soluble tetrazolium (WST), apoptosis/necrosis assays like terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or propidium iodide). Such assays may fail to detect the stem cell-specific effects of a drug. Sequential in vivo transplantation experiments are the gold-standard to detect the small population of CSC within the whole of tumor cells [in vitro . Recentrized in ,27.d)-1, 2, 3, 5-tetrazin-4(3H)-one) and nitrosoureas . In contrast to CNUs, TMZ undergoes rapid non-enzymatic conversion at physiologic pH to its reactive compound 5-3-(methyl)-1-(triazen-1-yl) imidazole-4-carboxamide (MTIC). MTIC is thought to cause DNA damage mainly by methylating the O6-position of guanine (primary lesion) which then mismatches with thymine in double-stranded DNA (O6G-T) in the first cell cycle after treatment. This mismatch induces futile cycles of mismatch repair triggered by recurrent GT-mismatches resulting in either double strand breaks or a critical recombinogenic secondary lesion. This secondary lesion is assumed to be an apurinic/athymidinic site formed during faulty mismatch repair that blocks replication resulting in either DNA double strand breaks (tertiary lesion), sister chromatid exchange, or other aberrations [WAF1/Cip1 - mediated G2-M arrest beginning 2 days after treatment with a small number of cells undergoing apoptosis but most undergoing senescence over a 10-day period. In contrast, TMZ induces temporary G2-M arrest accompanied by only minor changes in p53 or p21WAF1/Cip1 expression in p53 deficient cells [To date, two groups of alkylating agents are commonly used in the clinic: TMZ  towards TMZ or other alkylating agents.In summary, there is a consensus in the literature that the clonogenic growth monitored by colony-forming assays is much more sensitive to treatment with either TMZ or CNUs However, none of the papers investigated CSC in detail or used assays specific for CSC. As non-stem cells may also show clonogenic growth without being able to proliferate infinitely, the results have to be interpreted with caution . In addiSeveral recent studies have focused on the effects of chemotherapy on CSC. The CSC hypothesis led to the development of a new cell culture model for GBM. GBM cell lines derived from freshly resected tumor specimens and cultured in serum-free medium supplemented with EGF and bFGF - conditions optimized for the growth of neural stem cells - mirror the phenotype and genotype of primary tumors more closely than serum-cultured cell lines do ,41. Thus+ CSC lines were treated with up to 100 μM TMZ, whereas only a minor increase of cell death was observed at similar concentrations [+ CSC lines derived from primary astrocytic GBM were treated with up to 500 μM TMZ over different periods of time (2 d and 42 d), there was a dose-dependent reduction of CD133+ cells (by 80%), cell proliferation (by up to 100%), as well as clonogenicity and tumorigenicity (by up to 100%), whereas cell death did not exceed 6%. Cells with stem cell-like properties were selectively depleted irrespective of the CD133 or MGMT status. However, MGMT expressing CSC lines required up to 100 fold higher TMZ concentrations. In summary, the authors interpreted their results as selective depletion of CSC from CSC lines by TMZ [Eramo et al. were the first to investigate the chemoresistance of GBM CSC. A marked resistance was observed of GBM CSC lines to different chemotherapeutic agents, amongst them TMZ. GBM CSC lines were treated with 250 μM TMZ for 48 h and minor cell death was determined as a measure of treatment efficacy . Using ttrations . The auts by TMZ . Additio+ CSC showed significant resistance to chemotherapeutic agents . The viability of CD133+ cells was significantly less decreased than the viability of CD133- tumor cells when treated with up to 2000 μM TMZ for 48 h. Notably, even this concentration did not induce cell death in more than 50% of the cells [in vitro. A correlation of CD133 expression and resistance towards TMZ was not reported [+ stem cell-like cells in a study by Lamoral-Theys et al. using oligodendroglioma HS683 cells. HS683 cells were cultured using serum, but not using medium conditions optimized for CSC. The authors found that long-term treatment of HS683 cells with increasing concentrations of TMZ (from 0.1 μM to 1000 μM) resulted in a substantially decreased tumorigenicity of long-term treated cells, despite a wash-out phase of 8 weeks after TMZ treatment. Notably, the long-term treatment reduced the tumorigenicity and CD133 expression much more effectively than short-term treatment, indicating that the dosing schema influences the depletion of CSC in vitro. The MGMT expression remained unchanged in both groups [In contrast to these reports, Liu et al. used CSC lines that have been established using serum and switched to serum-free culture conditions before the experiments. The authors found that sorted CD133he cells . Ghods ehe cells . Bleau ehe cells , the numhe cells . This trhe cells . They foreported . TMZ subh groups . Villalvh groups . Similarh groups .in vitro [Based on the high toxicity of TMZ to neurosphere-forming cells within CSC lines, Mihaliak et al.  developein vitro .in vivo: Pistollato et al. [Two recent studies investigated the susceptibility of CSC derived from the core and the periphery of GBM o et al.  focused o et al.  confirmeTo present, only one paper has thoroughly investigated the effects of CNUs on CSC lines . Based oDue to differences in experimental approaches, these studies are difficult to compare. Given that serum-cultured cell lines rapidly acquire multiple mutations and substantially differ from the original tumor, they may provide conflicting results as compared to studies using medium conditions favoring the growth of CSC. However, most recent studies used CSC lines continuously cultured under serum-free conditions. In summary, Eramo et al., Bleau et al., and Pistollato et al. reported an increased resistance of CSC while Beier et al., Bleau et al., Mihaliak et al., Gilbert et al., and Clement et al. described experimental data relating to CSC and TMZ which is consistent with an increased susceptibility of CSC towards TMZ. The data presented by Glas et al., do not allow unambiguous conclusions on the susceptibility of CSC towards chemotherapy  All papers uniformly reported that MGMT protein expression is associated with a high resistance of CSC to TMZ. However the role of other factors known to modulate chemoresistance has not been investigated in detail. (II) Whenever investigated in detail, neurosphere-forming cells without MGMT expression were susceptible to TMZ. (III) Different TMZ schedules and concentrations may result in conflicting experimental results. (IV) Environmental factors, like hypoxia in the core of GBM, may contribute to the resistance of CSC towards TMZ. (V) Several signaling pathways, e.g. Shh, IGF-1/PI-3 kinase, NOTCH, or STAT3 interfere with TMZ resistance. (VI) There is no consensus on adequate methodologies to investigate CSC In vitro, approximately half of all GBM cell lines resisted to TMZ concentrations of 50 μM [A set of papers  , TMZ wasTMZ requires an intact mismatch repair system to cause toxic double stranded breaks. Thus, mutations in the major crucial component of the mismatch repair system mutS homolog 6 (MSH6) mediated TMZ resistance especially in recurrent GBM after TMZ-based radiochemotherapy ,75. A wein vitro and in vivo remains unknown, although a loss of stem cell properties in GBM CSC lines and a pronounced sensitivity of colony-formation to treatment with TMZ has been observed in serum cultured cell lines [In addition, the fate of CSC after TMZ treatment ll lines ,45-48. Ill lines ,77, senell lines ,79, or all lines ,81. Howell lines ,44,48,82in vitro is feasible [in vivo . In theThe CSC hypothesis predicted that the selective elimination of CSC would halt the perpetual growth and tumorigenicity of tumor cells. However, this hierarchy was recently called into question ,50. It win vivo or in vitro. Still, the available experimental data clearly indicate that resistance of GBM and derived CSC against TMZ is much more complex than expected. The initial predictions of the CSC hypothesis remain true under specific experimental conditions but multiple additional aspects have to be considered to understand why GBM almost invariably relapse. This review summarizes several known aspects of chemoresistance; additional contributors are likely to be discovered. A more comprehensive view will help to better understand the multifaceted factors that contribute to the survival or reoccurrence of CSC causing fatal relapses in the patient.There is no consensus in the literature on the effects of chemotherapeutic drugs on GBM CSC CPB has received an unrestricted travel grant from Schering Plough in 2008. All other authors declare that they have no competing interests.DB, JBS, and CPB wrote and finally approved the manuscript.


In the early recognition of portal vein ligation (PVL) induced tumor progression, positron emission tomography and magnetic resonance imaging (PET/MRI) could improve diagnostic accuracy of conventionally used methods. It is unknown how PVL affects metabolic patterns of tumor free hepatic tissues. The aim of this preliminary study is to evaluate the effect of PVL on glucose metabolism, using PET/MRI imaging in healthy rat liver.18F)fluoro-D-glucose (FDG) PET/MRI imaging (nanoScan PET/MRI) and morphological/histological examination were performed before (Day 0) and 1, 2, 3, and 7 days after PVL. Dynamic PET data were collected and the standardized uptake values (SUV) for ligated and non-ligated liver lobes were calculated in relation to cardiac left ventricle (SUVVOI/SUVCLV) and mean liver SUV (SUVVOI/SUVLiver).Male Wistar rats (n = 30) underwent PVL. 2-deoxy-2-. The homogenous tracer biodistribution observed before PVL reappeared by 7th postoperative day.PVL induced atrophy of ligated lobes, while non-ligated liver tissue showed compensatory hypertrophy. Dynamic PET scan revealed altered FDG kinetics in both ligated and non-ligated liver lobes. SUVThe observed alterations in FDG uptake dynamics should be taken into account during the assessment of PET data until the PVL induced atrophic and regenerative processes are completed. Despite advances in chemotherapy regimens and ablative techniques, extended hepatectomy is considered as a gold standard for the treatment of primary and secondary liver cancers. Although, the above detailed two-stage procedure is more widely used, there is increasing evidence that PVL/PVE-induced changes have a strong stimulatory effect on tumor growth as well. Thus, there might be an increased risk, that portal vein occlusion could stimulate not only the growth of future remnant liver, but also significantly affects tumor size and tumor spread in both occluded and non-occluded liver segments. The average time to achieve optimal FRLV  is approximately 1 month after PVL/PVE. 11C]-acetate, [11C]-choline and 2-deoxy-2-(18F)fluoro-D-glucose (FDG). In the oncologic use of PET, however, FDG is the most commonly used tracer, which is able to assess the typically enhanced glucose transport and metabolism in malignant tumors. FDG was found to be superior in detecting residual tumor growth and tumor recurrence compared to morphologic imaging procedures, furthermore, FDG-PET is the primary imaging modality for detection of distant (extrahepatic) metastasis. In clinical oncology positron emission tomography (PET) is an extremely valuable tool for tumor diagnosis, staging, restaging and for treatment response follow-up. There is no study in the literature that assesses the changes in FDG uptake pattern of normal liver after PVL/PVE. Kinetic FDG data combined with volumetric information available from MR imaging of the liver, are more reliable than PET alone or PET/CT. Thus, the aim of our present study was to selectively evaluate dynamics of FDG uptake in occluded and non-occluded liver lobes by in vivo multimodal FDG PET-MRI (nanoScan PET/MRI) in a well-established rat model of portal vein ligation.ad libitum in specific, pathogen-free conditions at 22–24°C with a 12-hour light-dark cycles. Animals were allowed to acclimatize for 1 week before starting experiments. After intra-peritoneal injection of ketamine (75 mg/kg) and xylazine (7.5 mg/kg), animals were placed in a supine position and median laparotomy was performed. The median and left lateral lobes were gently mobilized to reveal the portal pedicels. Under operating microscope  the portal branches feeding the median, left lateral and caudate lobes  were dissected and completely ligated with 6–0 silk. Great care was taken to avoid damaging the vulnerable hepatic artery and bile duct. At the end of the procedure the liver lobes were gently repositioned anatomically, the peritoneal cavity was closed in two layers by running suture (4–0) and the animals were returned to their cages until further examination. Before (0 day) and 1, 2, 3, 7 days after portal vein ligation, rats were sacrificed by exsanguination for histological analysis. The liver was removed and the ligated and non-ligated lobes were weighted separately after expulsion of blood by gentle compression with gauze. Liver specimens were taken from the excised left lateral lobe (ligated lobe) and the superior right lobe (non-ligated lobe). Samples were fixed in 4% neutral-buffered formalin and embedded in paraffin for further histological and immunohistochemical examination (see below). At each time point, six animals were used .The experimental design was regulated in strict adherence to the National Institutes of Health guidelines for animal care and approved by Committee on Animal Experimentation of Semmelweis University (permit number: 22.1/2408/3/2011 and XIV-I-001/29-7/2012). 200–250 g male Wistar rats  were kept on standard chow and water 2 gas). To prevent movement, the animals were fixed to a rat chamber  and positioned in the center of field of view (FOV). Dynamic list-mode PET data were collected in three-dimensional acquisition mode. The MRI images  were acquired for 50 minutes (25 minutes for each imaging) with T1-weighted , T2-weighted  sequences. PET volumes were reconstructed using a three-dimensional Ordered Subsets Expectation Maximization (3D-OSEM) algorithm  into dynamic frames of 10×30 s, 10×60 s, 10×270 s  for all scans with a voxel size of 0.3 mm. Static images were generated based on frame 21 (15th to 19th minutes were integrated after tracer injection). PET and MRI images were automatically coregistered by the PET/MRI instrument’s acquisition software. Reconstructed, reoriented and coregistered images were further analyzed with Fusion  and VivoQuant  dedicated image analysis softwares by placing appropriate Volume of Interests (VOI) on the organs. Two volume of interests were delineated manually slice by slice on each T1 weighted scans. One VOI consisted of the ligated liver lobes and the other of the non-ligated liver lobes. For volumetric analysis the built-in VOI statistics of the VivoQuant software was used, which determines the volume from voxel size and number of voxels in the VOIs. In order to get more precise values of the volumes, the T1 weighted slices were interpolated through the slice direction to create an isovoxel data set. For FGD uptake, the standardized uptake value (SUV) was calculated by dividing the mean radioactivity concentration (MBq/mL) by injected dose per body weight (MBq/g). The mean SUV for ligated and for non-ligated liver lobes were selectively recorded. The mean SUV of corresponding liver lobes was expressed in relation to a reference tissue SUV  and to the mean liver SUV (SUVVOI/SUVLiver).In a separate study additional 6 animals underwent imaging 12 hours before and 1, 2, 3 and 7 days after portal vein ligation. Images were acquired with a PET/MRI  sequential animal imaging system. Blood glucose levels were confirmed to be within the normal range before injection of the radioactive tracer and throughout the study. In the PET experiment 6.5±1.0 MBq of FDG in 0.2 mL volume was injected into the tail vein, under anesthesia . The relative weight of ligated and non-ligated lobes to the body weight (BW) was then calculated and expressed as follows: liver weight (g)/100 g BW. The liver volume change was measured by MRI and the relative liver volume was expressed as mmNecrosis was assessed by morphological features  on each section. The extent of necrosis was graded on a scale of 0–4 described by Suzuki apoptotic cell death was identified also by morphological criteria  and was counted in 10 consecutive high-power fields  and expressed in absolute numbers.After liver weight measurement, one slice was removed for pathological examination from the same anatomic position in each animal. The formalin fixed, paraffin embedded 3–5 micrometer thick sections underwent light microscopic observation after hematoxylin and eosin staining. mitotic activity Ki-67 immunohistochemical staining was applied on formalin-fixed, paraffin-embedded tissues. Avidin–biotin–peroxidase complex immunohistochemical method was performed for antigen retrieval. For the detection of cellular glycogen content, periodic acid and Schiff (PAS) staining was used. The intensity of PAS staining was graded on a five tier scale (0: no staining –5: highly intense). The level of glycogen content was determined by scoring at least three independent microscopic fields for each sample.To determine The above mentioned histological and immunohistochemical evaluations were performed in blinded fashion by two experienced pathologists.Mann–Whitney U test and two sample Student t-test were used to compare data between groups. Normality was tested by Kolmogorov-Smirnov test. Correlation between variables was tested by Spearman’s method. All tests were performed using the Statistical Package for Social Sciences . Values are expressed as means ± S.D. A p<0.05 confidence interval was considered as statistically significant.The weight of the portal-deprived lobes decreased rapidly after the operation and reached approximately one-fourth its original weight on postoperative day 7. On the other hand the non-ligated liver lobes underwent compensatory gain in weight. By a balance of atrophy and hypertrophy, the weight of total liver was maintained near to the baseline throughout the entire experiment. The volume of liver lobes changed in parallel with the liver weights. The volume change, based on MRI volumetry, correlated well with the weight change derived from autopsy ; ..th postoperative day. In contrast, in the non-ligated liver lobes, the presence of necrosis and apoptosis was not typical  PVL. Day 1 after ligation, PAS positive glycogen granules almost completely disappeared in ligated lobes and the glycogen content of cells returned to baseline level just at day 7. In contrast, the PAS staining in non-ligated liver lobes did not show significant alterations compared to the baseline level (day 0) in any examined time point .VOI/SUVCLV showed a significantly increased FDG uptake in ligated liver lobes. That value was maximal on 2nd postoperative day and returned near to the baseline at the 7th day. In non-ligated lobes the SUVVOI/SUVCLV also increased, but in a significantly lower extent compared to the ligated lobes. The increment in FDG uptake of non-ligated lobes reached a statistically significant level on 2nd and 3rd postoperative day only, as compared to the baseline  is required for the activation of the efferent molecules of caspase family. However, in these areas around the necrotic fields, a moderate degree of hypoxia is still present. Inflammation: Besides apoptosis, a massive inflammatory reaction was also present around the necrotic regions in the slides. At postoperative day one, macrophages appeared at the rim of the necrotic areas and began to remove the debris. Activated macrophages are characterized with high glucose turnover and consequently increased FDG uptake. The atrophy of portal deprived liver lobes is mainly caused by coagulation necrosis, as a result of the cessation of portal vascular inflow and the consequent hypoxic condition around the central vein. The decreased FDG release from hepatocytes- caused by the elevated glycogenolysis – can also contribute to the increased FDG accumulation in the ligated liver lobes. FDG is a glucose analog, which is normally trapped inside the cells after phosphorylation as FDG-6-phosphate. However, in the liver, FDG-6-phosphate is dephosphorylated and rapidly released, presumably due to the high activity of glucose-6-phosphatase (G-6-Pase). In non-ligated liver lobes FDG uptake also increased, but to a significantly lower extent than in ligated lobes, despite the presence of massive regeneration characterized by high mitotic activity of cells. Cell division would supposedly be associated with high glucose/FDG-uptake. nd and 3rd postoperative days. This lower-grade lobar FDG activity might make possible the early recognition of residual tumorous lesions in non-ligated liver shortly after the operation. Considering that, in terms of treatment decision, the critical points are the tumor progression in the future remnant liver (non-ligated lobes) or the formation of distant metastases, the use of FDG-PET/MRI could be beneficial in the early assessment of tumor response to PVL.The above detailed alteration in FDG uptake pattern could have a significant impact on the evaluation of PET studies after PVL. The increased FDG accumulation, especially in the case of ligated lobes, could make difficult the assessment of tumor progression by increased background activity until the PVL induced alterations are completed. However, it also should be noted that, the alteration in FDG uptake of non-ligated lobes was moderate and reached significant level only at the 211C]-acetate should be used. Nevertheless, our study has some limitations. The main limitation is the use of normal, healthy liver. In a clinical scenario, the primary and secondary liver tumors usually appear in cirrhotic or fatty livers, in which circumstances the PVL induced alterations and consequently the imaging characteristics of liver lobes may vary. Furthermore, this study examined only tumor-free animals, however, the altered circumstances caused by PVL may result in alteration of tumor FDG uptake as well. Further research on tumor-bearing animals is needed to evaluate this potential tumor response regarding to FDG-uptake after PVL. Finally, it should be noted that FDG is appropriate for the diagnosis only of FDG positive tumors. However, some liver cancer, such as hepatocellular carcinoma is characterized by variable FDG uptake caused by the variation in the expression of G-6Pase. In such case, the results from FDG-PET/MRI could become uncertain, therefore more specific tracers, such as [The aim of the study was to evaluate the FDG uptake pattern in healthy liver after PVL in small animals. Our result showed an altered FDG uptake pattern in both ligated and non-ligated liver lobes after PVL. These alterations in FDG uptake should be taken into account during the assessment of PET data until the PVL induced atrophic and regenerative processes are completed. Because this initial study merely examined healthy animals, further preclinical and clinical studies are needed to define the impact of FDG-PET in therapeutic follow-up after a two stage-hepatectomy combined with PVL.


Mycobacterium tuberculosis CRISPR locus, is used world-wide . However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution.The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results.The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus .et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan  Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a family of DNA repeats of 21 to 37 bp that are separated by regularly sized, non repetitive unique DNA spacer sequences .Mycobacterium tuberculosis complex (MTC) strains started in 1993 [Mycobacterium tuberculosis [et al. provided a standardized reverse line blot hybridization method (spacer oligonucleotide typing = spoligotyping) to genotype MTC complex strains based on polymorphism of this region, and soon after, corresponding membranes were commercialized [http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/ that has already been updated 4 times [Studies on the CRISPR region of  in 1993 . In 1995rculosis . In 1997cialized . Since tM. tuberculosis isolates with the same level of discrimination as IS6110-RFLP (Restriction Fragment Length Polymorphism) [6110-RFLP so that even smear-positive sputum samples can be directly analyzed, ii) up to 40 DNA samples can be analyzed within one day with the classical method and up to 186 with the novel microbead-based spoligotyping that has proven very reliable [6110 insertion element are better discriminated by spoligotyping than by IS6110-RFLP typing, and iv) spoligotyping patterns can distinguish between main lineages and sublineages within the M. tuberculosis complex (MTC) making them phylogenetically informative except in the rare cases where the exact same deletions (covering the exact same spacers) occurred by convergent evolution [While spoligotyping does not differentiate orphism) : i) it rreliable ,14, iii)volution ,14-17.et al. revised by van Embden et al. is a robust method with an intra-laboratory reproducibility over 90% in well-trained laboratories [The classical spoligotyping described by Kamerbeek ratories ,18. Howeratories . All theThe transfer of the classical spoligotyping method into an advanced platform represents a technical progress because it allows to produce a numerical raw result format and a standardized signal/noise cut-off determination, ensuring both a high throughput and an high quality ,14.Hence, the aims of this study were to: (i) retrospectively assess the global quality of spoligotyping results that have been produced in various laboratories worldwide during a decade by comparing their data with those provided by the new, highly reliable system, (ii) give a feedback to the laboratories about their data production quality, (iii) solve uncertainties on samples in which centers are not confident, (iv) study if the DNA extraction procedure may influence membrane-based spoligotyping results, (v) assess the possible contribution of the microbead-based technique to an increased quality of services in molecular epidemiological studies. Altogether, this article identifies the main pitfalls in CRISPR data production, at a time when tuberculosis spoligotyping still inflates and is transferred towards other micro-organisms.The capture probes for microbead-based spoligotyping are identical to the ones from the membrane-based spoligotyping technique with minor modifications to correct some sequences from the original set ,18. All PCR  was performed for 20 cycles for CTAB DNA  and 25 cet al. [Was done in TMAC buffer at 52°C for 10 minutes as described before by Zhang et al. .pm), i.e. obtaining exactly the same spoligotype by both methods . The percentage of perfect matches in a center is calculated as the number of isolates with pm divided by the total number of isolates provided by the center. The second comparison dealing with individual datapoints (or spacers) was represented by the rate of difference (rd). This rd index indicates the number of discordant data points for the total number of data points analyzed per center: e.g. if 8 of 100 DNAs tested show discordant results in 2 spacers the rd value equals 8*2*100/(100*43) = 0.37%.For each spacer, hybridization signals were recorded as RFI (Relative Fluorescence Intensity) values and were transformed in a binary code (presence/absence) using a signal/noise cut-off value of 2 times the lower values' group. Octal codification and SpolDB4 lineage identification were assigned to each spoligotype. Spoligotypes generated by the membrane technique were compared to the Luminex generated spoligotypes, spoligotype by spoligotype and spacer by spacer. The first comparison allowed determining perfect matches  Buenos Aires - Argentina ; ii) Pisa-Italy  and the Regional Reference Center for Mycobacteria, Firenze; iii) Bilthoven - The Netherlands ; iv) Paris-France ; v) Madrid-Spain ; vi) Pointe-à-Pitre - Guadeloupe ; vii) Antananarivo-Madagascar ; viii) Riyadh-Saudi Arabia ; ix) Rio de Janeiro, Oswaldo Cruz Institute Laboratory of Molecular Biology applied to Mycobacteria, Brazil; x) Antwerp-Belgium, Mycobacteriology Unit, Prince Leopold Institute of Tropical Medicine, and xi) Brussels-Belgium, Scientific Institute of Public Health, National Reference Centre of Tuberculosis and Mycobacteria.These selected strains have a worldwide origin and represent a wide range of MTC genotypes. DNA was extracted using a thermolyzate or a cetyl-trimethyl-ammonium bromide (CTAB) procedure. DNA samples fulfilled the following criterion: samples representative of hard to interpret spoligotypes and samples representative of the work flow, so we could evaluate the benefits of a new platform regarding the classical one on these samples. Most centers had performed spoligotyping using commercial membranes  . Samples were analyzed in a blind way which means that we did not know the profiles from the membrane-based spoligotyping until we had everything processed by the microbead spoligotyping. We named the centers from 1 to 11 for convenience ranked according to their spoligotyping quality, so this numbering is not correlated with the order in which they are described by name to respect confidentiality. The number of samples included in this study by Center are as follows: 1 (49 samples), 2 (95 samples), 3 (98 samples), 4 (97 samples), 5 (87 samples), 6 (82 samples), 7 (120 samples), 8 (70 samples), 9 (59 samples), 10 (85 samples) and 11 (85 samples).http://www.u707.jussieu.fr/biostatgv. df = degrees of freedom, relating the number of independent observations used to compute the statistical parameters.Homogeneity "Mann-Whitney" non-parametric U test was performed in an Excel sheet and "Spearman rank correlation" test  was performed using the online tool at perfect match (pm = exactly the same spoligotype pattern over the 43 spacers), three centers obtained between 87 and 84% concordance and one laboratory only 51% .Eight centers using commercial membranes and three using in-house made membranes were included in the study, totaling 927 isolates. We performed the microbead-based spoligotyping on Luminex on the same set of DNA samples as a new standard, even though it is clear that only sequencing would provide full reference information on spacer sequences and genomic structure of the Direct Repeat locus, especially in case of doubtful hybridization results for some spacers ,14,18. C6110-RFLP for which CTAB is mandatory), whereas thermolyzates are used in other cases due to its swiftness and ease of implementation. Surprisingly, the best performing center (Center 1) used only thermolyzate DNA, and Center 11 used only CTAB DNA .Participating centers used either Cetyl-Trimethyl-Ammonium-Bromide (CTAB) extracted or heat-shock extracted (thermolyzates) DNA, depending on the type of studies they usually use the DNA for: the CTAB-based extraction method is preferred for molecular analyses demanding high purity (e.g. ISrd) index as the number of mismatches divided by the total number of analyzed data points in each center . All centers except one exhibited a "rd" below 1% : centers having the highest numbers of erroneous spoligotype patterns also had the highest number of individual spacers errors. Still, specific centers had a relative low rd as expected from their pm: centers 6 and 9 exhibited slightly higher global quality (as measured by the pm) than centers 7 and 10 respectively .Each spoligotype is a string of 43 characters so that in total 39,861 data points were analyzed in this study. Our comparative results showed 157 (0.39 %) data point discrepancies. We defined the rate of difference , hence the discrepancies reported here are upper limits and could have been lower if only random selection had occurred.We show in this study that different centers performing membrane-based spoligotyping exhibit different inter laboratory reproducibility rates ranging from 51 to 100% and most of them reaching more than 90% of quality as represented by their The reasons why spoligotyping errors occur can be diverse. First, DNA quality associated to the extraction procedure could be critical. In this study, however, the DNA extraction procedure did not influence the performance of the membrane-based spoligotyping. Nevertheless, we have to keep in mind that we only tested CTAB and thermolyzates and other extraction procedures were not tested. Some centers repeatedly introduced genotyping errors at multiple spacers in the same samples. Some samples may have been of inadequate quality for all spacers to be sufficiently amplified. However, this occurred both for centers using CTAB and thermolyzate DNA, confirming that factors other than extraction procedure, such as the amount of extracted DNA, inclusion of culture medium when preparing bacterial suspensions or possible culture contaminations, may have influenced PCR quality and hence modified the spoligotypes of some samples.Regarding artisanal process of membrane production, an adequate quantity of specific probe concentrations on the membrane could sometimes be defective which would lead to the same spacer being recurrently problematic in the same center. Also, commercial membranes might experience some spacer problems so internal quality control must be done by each lab. Indeed, we have observed that in Center 11, spacer 39 was affecting the results of 42 strains. However, in other centers, such problems were not observed. We advise positive and negative controls to be included at randomly chosen positions to detect such possible membrane production problems; centers producing their own membranes should also check their production adequately before use.Third, operator-dependent washing problems could occur on any part of the membrane so that any spacer could be wrongly scored for some isolates. Insufficient washing can lead to false positive detection of a spacer. As an example, spacers 14 or 37 were detected, although they proved to be absent. In contrast, excessive washing will level the signals of several probes down. All centers used their membranes between 8 to 14 times which is under the limit advised by membranes' manufacturer (up to 20 times). However, this might still be too high. We advise users to always record the results with positive and negative controls included at randomly chosen positions to detect the membrane fall down in quality, and possible insufficient washes.Fourth, there can be intrinsic problem due to the CRISPR structure in MTC: some spacers constantly provide weaker signals that make the distinction between positive and negative values more difficult. For instance, DVR next to spacer 15 (DVR26) harbors a deletion of 4 nucleotides at its 5' end as shown in highly diverse strains . ConsequThe high-throughput spoligotyping method has the advantage of being more sensitive to detect these slight variations, given the 3D nature of the immobilized probes, which provides more surface to attach more PCR products. Moreover, unspecific hybridization is removed more easily with the microbead technology, making the detection of spacer 15 possible in any sample. Contrarily, in our study, spacer 40 was detected several times by membrane-based spoligotyping, but not by the microbead-based technique .Fifth, the hybridization reading from the film, and the transfer of this information to a digital format have to be handled manually in the membrane-based spoligotyping increasing the transcription error risk; the best procedure to reduce this risk is to have the results read in duplicate by two independent readers, to check reading mistakes and to solve potential discrepancies.Reading errors are suppressed in the high-throughput method due to an in-house designed automatized routine, which already provides a digital file with a suggested and objective interpretation according to predefined thresholds (cut-off).As the distribution of wrongly typed spacers is random . This issue was due to a problem in membrane production. Other centers yielded errors slightly more frequently at spacer 15, that is known to be less amplified because of its neighboring modified CRISPR sequence around this spacer. In contrast, the increased sensitivity of the high-throughput method was validated by the proper detection of this spacer ,14. OtheThe high-throughput microbead-based method was shown to have a better sensitivity due to proper detection of some spacers that can be misinterpreted by the classical method, and a lower error rate due to automated interpretation and transcription. A new 68 spacers microbead-based spoligotyping format provides an increase in discriminatory power for Principal Genetic Group I clinical isolates, a feature that could be especially useful in South-East Asia where the East-African Indian (EAI) clade predominates .The launch of a new Luminex device (MagPix), with a price drop, yet lower-plex, molecular diagnostics instrument (50Plex) that would be interesting for fields labs (e.g. without requirement of air conditioning) could facilitate the spreading of spoligotyping; however this will only happen if routine utilization (through a concomitant drop in reagents prices) is done within acceptable cost limits for public health and an improved quality of service.The authors declare that they have no competing interests.EA, JZ, GR and CS declare that they never received any direct financial support by the Luminex Corp. or Luminex BV companies as well as no staff support was provided by Luminex Corp. nor Luminex BV to perform this study.EA and JZ performed and analyzed the microbead-based spoligotypes, VR, KK, RR, LR, HMG, AR, PS, MFD, KS, MH, VR, MH, DGV, SAAN, NR, CG, ET, DvS, participated in sample recruitment and performed membrane-based spoligotyping in their respective laboratories, GR performed the Statistical analyses, CS conceived and coordinated the study, EA, GR and CS wrote the manuscript. LR, VR, DvS, DGV contributed to the manuscript revision and all authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/110/prepubCenter's distribution of problematic spacers. This file highlights the problematic spacers shared by several centers (lines).Click here for file


Alcohol and drug use is known to be a major factor affecting the incidence of traumatic injury. However, the ways in which immediate pre-injury substance use affects patients' clinical care and outcomes remains unclear. The goal of the present study is to determine the associations between pre-injury use of alcohol or drugs and patient injury severity, hospital course, and clinical outcome.This study used more than 200,000 records from the National Trauma Data Bank (NTDB), which is the largest trauma registry in the United States. Incidents in the NTDB were placed into one of four classes: alcohol related, drug related, alcohol-and-drug related, and substance negative. Logistic regression models were used to determine comorbid conditions or treatment complications that were significantly associated with pre-injury substance use. Hospital charges were associated with the presence or absence of drugs and alcohol, and patient outcomes were assessed using discharge disposition as delimited by the NTDB.The rates of complications arising during treatment were 8.3, 10.9, 9.9 and 8.6 per one hundred incidents in the alcohol related, drug related, alcohol-and-drug related, and substance-negative classes, respectively. Regression models suggested that pre-injury alcohol use is associated with a 15% higher risk of infection, whereas pre-injury drug use is associated with a 30% higher risk of infection. Pre-injury substance use did not appear to significantly impact clinical outcomes following treatment for traumatic injury, however.This study suggests that pre-injury drug use is associated with a significantly higher complication rate. In particular, infection during hospitalization is a significant risk for both alcohol and drug related trauma visits, and drug-related trauma incidents are associated with increased risk for additional circulatory complications. Although drug and alcohol related trauma incidents are not associated with appreciably worse clinical outcomes, patients experiencing such complications are associated with significantly greater length of stay and higher hospitalization costs. Therefore significant benefits to trauma patients could be gained with enhanced surveillance for pre-injury substance use upon admission to the ED, and closer monitoring for infection or circulatory complications during their period of hospitalization. Traumatic injury is the most frequent cause of admission to the emergency department (ED) in U.S. hospital systems . The Uniincidence of traumatic injury, [It has been estimated that as many as 50% to 70% of patients with traumatic injuries have detectable levels of alcohol in their blood at the time of admission -5. The f injury, ,11-19 hoPrevious studies have considered the effects of pre-injury drug and alcohol use on hospital treatment and clinical outcomes ,16,20-34The present study represents the largest retrospective study yet to consider the impact of immediate pre-injury substance use on hospital course and outcomes following traumatic injury. The goals of this study are to determine if pre-injury substance use is associated with (i) significantly less healthy patient populations, (ii) more severe injuries, (iii) significantly different hospital course, and (iv) significantly worse clinical outcomes.The National Trauma Data Bank (NTDB) is a database maintained by the American College of Surgeons (ACS) . The NTDInjury incidents were included if and only if we could determine the presence or absence of both drugs and alcohol upon admission to the emergency department (ED). Incidents were excluded if the drug or alcohol status was either not determined or not reported. Injury incidents were placed into one of four classes: (i) "alcohol related" incidents are those in which alcohol use was present and drug use was absent; (ii) "drug related" incidents are those in which drug use was present and alcohol use was absent; (iii) "alcohol-and-drug related" incidents involved pre-injury use of both alcohol and drugs; and, (iv) "substance negative" incidents are those in which there was no reported pre-injury use of drugs or alcohol. These classes were created to distinguish the between effects associated with alcohol and drugs used individually and in combination.Demographic variables included in the study were age and gender. Age was modeled as a continuous variable, whereas gender was modeled as a dichotomous variable. Clinical variables included injury severity score (ISS), pre-existing comorbidities, secondary complications and discharge disposition (outcome). ISS was modeled as a continuous variable, whereas comorbidities, complications, and outcomes were modeled as dichotomous variables indicating the presence or absence of the condition. All incidents were required to meet the following inclusion criteria: (i) age must be at least 16; (ii) ISS must be between 0 and 75; and (iii) the gender must be delimited either "male" or "female". These criteria primarily check the validity of the data; the age validation ensures that we exclude potential pediatric trauma cases. These checks were performed to ensure the integrity of the data, and thereby increase the accuracy of the primary study variables.The pre-existing comorbidities contained in the NTDB were used to test whether the substance-abusing trauma population is significantly less healthy than the trauma population as a whole. The comorbid conditions delimited by the NTDB cover many common health conditions, such as diabetes, hypertension, obesity, and depression. Each comorbid condition was tested independently using logistic regression models in which the comorbid condition was modeled as a dichotomous response variable and regressed on substance-use status, age, gender, and ISS.The complications reported in the NTDB were used to test for the presence of significant differences in hospital course associated with pre-injury substance use. Complications positively associated with pre-injury substance use would suggest that hospital course is worse for substance-related trauma incidents than incidents without pre-injury substance use. The NTDB delimits several complication descriptions that cover a wide range of complications such as pneumonia, deep-venous thrombosis (DVT), cardiac arrest, and renal failure. Each complication was tested for its association with pre-injury substance use with logistic regression models in which the complication was modeled as a dichotomous response variable and regressed on substance-use status, age, gender, and ISS.Pre-injury substance abuse may be associated with poorer outcomes following hospitalization. To simplify the outcomes analysis, the twelve different outcome descriptions contained in the NTDB were grouped into a more compact and less redundant classification scheme. The classification scheme is as follows: (i) NTDB outcomes "Death", "DOA", "Died during treatment" were grouped together as "death"; (ii) "Home", "Home Health", and "Jail" were grouped together as "home"; (iii) "Nursing home" and "SNF" were aggregated into "nursing home"; and (iv) "Rehab" remained a separate group. Incidents in which the description was either "Burn" or "Other" were not considered because of the difficulty in interpreting these outcomes. Each outcome was tested for its association with pre-injury substance use with Chi-Square tests on contingency tables in which both the specific outcome and substance-use status were modeled as dichotomous variables.α ≤ 0.05 was accepted as significant; when necessary, a Bonferroni-corrected value of α ≤ 1/N was used to assess significance, where N is the number of statistical tests.All statistical analyses were performed in R, version 2.8.1 . Comparisons among groups were made using Wilcoxon-Mann-Whitney or Kruskal-Wallis rank sum tests. Categorical data were analyzed using Chi-square tests on contingency tables, or by using logistic regression models to estimate odds ratios for the risk associated with particular variables of interest. A value of An ANOVA model was used to test the significance of observed differences in complication rates among the four substance-use groups, while controlling for differences in age, gender, and injury severity (ISS). Tukey's "Honest Significant Differences" were computed to estimate confidence intervals on the coefficients of each substance-use group and control for the multiple hypothesis tests performed. The overall false-positive rate was maintained at α = 0.05.http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/Design) which is freely available from CRAN http://cran.r-project.org/. We used logistic regression modeling to estimate odds ratios for the association of pre-injury drug or alcohol use with (i) pre-existing comorbidities, (ii) complications arising during treatment, and (iii) outcomes following hospitalization. We state that a condition is associated with substance use when the estimated odds ratio was statistically significantly greater than one. Logistic regression models controlled for substance-use status, age, gender, and injury severity score (ISS). The fit of the regression model to the data was assessed using Chi-Square tests (α = 0.05); the full regression model was found to fit the data significantly well for all results presented herein.Logistic regressions were carried out using the `lrm` function in the 'Design' package  than females . There were significantly more males than females in the trauma incidents with pre-injury drug or alcohol use . Drugs and alcohol were significantly more likely to co-occur in the same incident than expected from their individual frequencies . The mean age of patients was 36.3 years for alcohol related incidents, 37.1 for drug related incidents, 34.3 for alcohol-and-drug related incidents, and 42.1 years for substance negative incidents, respectively .Version 7.1 of the NTDB contains 1,926,244 patient records, of which 225,081 (11.6%) met our inclusion criteria (see Methods for details). The demographics of the patients are summarized in Table P < 0.001). Pairwise Wilcoxon-Mann-Whitney tests suggested that drug use is associated with more severe injuries than those experienced in substance negative incidents (P << 0.001), whereas alcohol is not (P = 0.08). Major trauma (defined as ISS ≥ 15) was found to occur in 28.0%, 31.4%, 28.5%, and 29.0% of the alcohol related, drug related, alcohol-and-drug related, and substance negative incidents, respectively .Pre-injury substance use has been proposed to cause more severe injuries, however prior studies have not consistently established such a relationship. Therefore, the ISS score for each incident was used to determine if drug or alcohol related trauma incidents were associated with more severe injuries. The median ISS score was 9 for all four classes of trauma incidents. The mean ISS scores were 11.2 for alcohol related, 12.5 for drug related, 11.5 for drug-and-alcohol related, and 11.7 for substance-negative trauma incidents, respectively .There are 52 comorbid conditions reported in the NTDB, 44 of which were found to occur in the incidents included in this study. Eighty percent (35/44) of the reported comorbid conditions were not significantly associated with pre-injury drug or alcohol use. Six of the comorbid conditions were significantly associated with alcohol-related trauma visits, seven were significantly associated with drug related incidents, and 4 were significantly associated with alcohol-and-drug related visits .P << 0.001) increases in the number of complications arising during treatment, but pre-injury alcohol use does not appear to be associated with significantly higher rates of complications during treatment.The vast majority of incidents - regardless of pre-injury drug or alcohol use - experienced no complications during treatment. The mean number of complications per incident was found to be 8.6, 11.3, 9.2, and 8.9 per one hundred alcohol related, drug related, alcohol-and-drug related, and substance negative trauma incidents, respectively. This corresponds to rates of complications arising during treatment of 8.3, 10.9, 9.9 and 8.6 per one hundred incidents in the alcohol related, drug related, alcohol-and-drug related, and substance-negative classes, respectively. Pre-injury drug use, is associated with small, but significant . In addition to infection, 42% (5/12) of the complications associated with pre-injury drug use were related to circulatory conditions (e.g. deep-venous thrombosis).There are a total of 24 unique complication descriptions delimited by the NTDB. Five were associated with pre-injury alcohol use, twelve were associated with pre-injury drug use, and 7 were associated with pre-injury use of both alcohol and drugs Table . InfectiChronic drug and alcohol use may be associated with patients experiencing a different set of complications than those experienced by acute users. This hypothesis was addressed by removing 18,451 (8.2%) incidents in which the patient was reported to have either "chronic alcohol abuse" or "chronic drug abuse" as a pre-existing comorbidity. The logistic regression models were subsequently reconstructed and revised odds ratios were estimated. The results shown in Table P << 0.001). The complications linked to pre-injury drug or alcohol use were associated with significantly larger hospital charges  of the incidents in our dataset did not have a valid charge entry . Using the remaining 89,279 incidents, the mean charges were $60,407.90 for alcohol-related trauma incidents, $67,991.82 for drug-related trauma incidents, $96,739.45 for alcohol-and-drug-related incidents, and $54,981.23 for substance-negative incidents. Pre-injury drug and alcohol use is associated with significantly greater hospital charges for the treatment of trauma patients . Discharge to "Home" occurred most frequently in the alcohol and drug related incidents, occurring in 73.4%, 70.5%, 72.0, 70.0% of the alcohol related, drug related, alcohol-and-drug related, and substance negative trauma visits, respectively . The increased association of "Home" with alcohol and drug use is partly caused by discharge to "Jail" (a component of "Home"), which is 1.8 times more likely to occur in the population of drug and alcohol users . Discharge to "nursing home" occurred more frequently in the substance-negative incidents (4.8%) than the alcohol related (2.5%), drug related (4.0%), and alcohol-and-drug related (2.9%) incidents . A similar trend was observed for discharge to "rehab", which occurred in 8.1%, 9.5%, 7.3%, and 10.8% of the alcohol related, drug related, alcohol-and-drug related, and substance-negative incidents, respectively . "Death" occurred in 3.6%, 3.4%, 3.0%, 4.5% of the alcohol related, drug related, alcohol-and-drug related, and substance-negative incidents, respectively .While advances such as the development of specialized trauma centers have helped to decrease mortality by as much as 20%, the health and economic costs associated with traumatic injury remain staggering. Traumatic injury is the leading cause of death for Americans under the age of 45 and the fifth most common cause of death for the nation as a whole [Drug and alcohol use has been shown to have a strong association with traumatic injury. Between 30% and 40% of trauma patients test positive for drug use and 27% to 47% of patients test positive for alcohol use at the time of admission . In an eIn the present study, we used the National Trauma Data Bank to determine the whether pre-injury alcohol or drug use is associated with (i) patient health prior to injury, (ii) injury severity, (iii) hospital course following injury, and (iv) outcome following hospitalization. In general, patients involved in drug or alcohol related trauma incidents do not appear to be appreciably less healthy prior to injury than the trauma population at large. The majority (80%) of the comorbid conditions were found to occur at indistinguishable frequencies among the four classes of trauma incidents. Furthermore, many of the comorbid conditions associated with pre injury alcohol and drug use, such as "Chronic Alcohol Abuse", "Chronic Drug Abuse", and "Documented History of Cirrhosis", are expected to be overrepresented in this population of patients.Pre-injury alcohol or drug use was not associated with more severe injures than incidents where substance use was not a factor. The median ISS score was the same for all four classes of trauma incidents, and only slight differences were observed in the mean ISS score among groups. Drug use  may be associated with slightly higher frequencies of major trauma (ISS ≥ 15). Pre-injury drug use may therefore be associated with small, but significant, increases in injury severity, whereas pre-injury alcohol use is not.Substance use does not appear to have a dramatic adverse effect on patient outcomes following hospitalization, which is consistent with a recent study finding that intoxicated drivers may have more favorable outcomes and reduced mortality . The druThough pre-injury alcohol and drug use was associated with neither less-healthy patients nor worse patient outcomes, important differences were observed with respect to treatment course, and these differences were found to be associated with pre-injury drug and alcohol use. Patients testing positive for alcohol or drugs developed infection-related complications while hospitalized at a significantly higher rate than those testing negative Table . In addiThe deleterious effects of alcohol use on the body's ability to combat infection after trauma include respiratory depression and an impaired cardiovascular response to injury ,29,41,42The link between drug use and infectious complications after trauma can also be explained by an impaired respiratory and cardiovascular response to injury . PreviouThe findings in this study illustrate that alcohol and drug use has appreciable effects on both biological and economic aspects of patient care. The hospital charges associated with drug and alcohol related trauma incidents were significantly greater than those for substance negative incidents. We noted that a large number of cases did not contain valid charge information and were therefore excluded from the analysis. We caution that there may be some bias in the cases included in this particular analysis. The length of hospitalization was also found to be longer in the drug and alcohol related trauma incidents. This may, in part, be related to the greater association of infection complications with pre-injury drug and alcohol use because incidents in which infection complications resulted in significantly greater hospital charges.For example, knowledge of a patient's drug and alcohol use could help trauma care providers manage patients,22,36. PFor example, knowledge of a patient's drug and alcohol use could help trauma care providers manage patients,22,36. PThere are some caveats to this study that should be considered. The NTDB is a database of voluntary submissions by contributing hospitals, and thus cannot be considered a representative sample of trauma incidents. We attempted to minimize biases inherent in the database as much as possible. The results obtained herein used incidents from 490/770 (64%) of the contributing hospitals, including 81/101 (80%) of the ACS level-1 trauma centers and all four geographic regions. The quality of the NTDB data is subject to the efforts of the contributing hospitals. To overcome this limitation, we strictly checked our data to make sure that the fields of interest contained reasonable values. Another data-quality limitation is evident in the 'ALCOHOLPRE' and 'DRUGSPRE' variables, which only capture the presence or absence of drugs or alcohol, and not the quantity present. Our final dataset therefore likely contains patients with varying amounts of these substances in their bodies at the time of injury. Additionally, the 'DRUGSPRE' variable only indicates if "controlled substances other than alcohol" were detected in the patient's blood or urine; the definition specifically excludes drugs administered "during any phase of resuscitation". Unfortunately, what constitutes a controlled substance is left to the discretion of the reporting hospital, and thus, while the 'DRUGSPRE' variable is intended to capture illegal drug use (e.g. cocaine or heroine), some of the incidents studied herein may not be related to the pre injury use of such substances . Voluntary controlled-substance testing and reporting also means that some patients in the substance-negative category may actually presented to the ED with alcohol or drugs in their blood at the time of injury. It's unclear the impact of such substance negative patients on our results, however our results may be conservative if such patients experienced more complications that most truly substance-negative patients.Alcohol and drug use have significant negative health effects that are magnified following trauma. Pre-injury alcohol and drug use was found to be associated with significant risk for developing infection or circulatory complications during hospitalization. The charges associated with treating these incidents were found to be significantly greater than incidents in which drugs or alcohol was not a factor. Every effort must therefore be made to encourage screening at the time of admission so practitioners can treat patients as effectively as possible. This may mean that practitioners will have to work to overturn hindering legislation such as the UPPL.The authors declare that they have no competing interests.MC participated in the design of the studies, performed the biostatistical analysis, interpreted the data and drafted the manuscript. MB participated in the design of the studies, assisted with the data analysis and drafted the manuscript. Both authors read and approved the final manuscript.


This review aims to establish the impact on conventional angiography and endovascular intervention of contrast-enhanced magnetic resonance angiography (CE-MRA) and contrast-enhanced computed tomography angiography (CE-CTA) on a background of evolving technology, changing clinical requirements and resulting clinical repercussions.The angiographic and interventional caseload was prospectively recorded between 1997 and 2010, along with the CE-MRA and CE-CTA caseload. Waiting times and the marginal cost analyses for 2001 and 2009 were also prospectively established.Conventional diagnostic angiographies declined from a peak of 847 to 121 per year while endovascular interventions continue in similar numbers. CE-MRA increased from effectively none initially to 620 per year while CE-CTA has currently risen to 396 per year. Total diagnostic study numbers have increased but at reduced cost. Various influences are clear, including on-site modality availability, capability and accuracy along with impact of new therapies, research studies and adverse events.Vascular imaging has undergone a metamorphosis in little over a decade because of CE-MRA and CE-CTA. With waiting times significantly reduced since the start of the study and the cost-effectiveness of both CE-MRA and CE-CTA as primary diagnostic investigations established, further development of these services is inevitable.The availability of CE-MRA and CE-CTA has reduced the need for conventional angiography.• Both waiting times and the marginal cost analyses for CE-MRA and CE-CTA have reduced.• The impact of new therapies, research studies (e.g. ASTRAL) and adverse events is illustrated.•  The imaging modalities of choice in vascular imaging have changed considerably over the last decade, with advances in magnetic resonance imaging (MRI) and computed tomography (CT) technology coupled with the increasing availability of MRI and multislice CT scanners capable of these forms of non-invasive angiography. Our aim in this work has been to establish the impact of the evolving technologies of conventional angiography, interventional angiography, contrast-enhanced MRI angiography (CE-MRA) and contrast-enhanced CT angiography (CE-CTA) on the respective workloads of conventional invasive angiography and vascular interventional procedures. We have also related this to the clinical requirements with emerging clinical repercussions in a referral centre along with the impact on clinical practice.CE-MRA with gadolinium-based contrast agents has emerged as a realistic non-invasive alternative to traditional invasive diagnostic angiography. As well as the development of contrast-enhanced techniques, the ancillary technological advances in MRI software and hardware (gradient coils and phased array coils) plus bolus detection methods, stepping table techniques and parallel imaging have shortened examination times and improved image quality significantly. The use of MRI fulfils the ALARA principle (‘as low as reasonably achievable’) as regards minimising exposure to ionising radiation but there is also a favourable side effect profile in respect of the contrast agents used with extremely few patients experiencing adverse events , 2, withInvestigations with surgical/angiographic correlation have shown CE-MRA to have a high sensitivity and specificity for detection of peripheral arterial steno-occlusive disease, and 100 % sensitivity and specificity for detection of aortic or iliac aneurysms . CE-MRA CE-CTA has also advanced rapidly in the last decade with the advent of multislice scanners with increasing numbers of detector rows, accompanied by bolus detection methods and increasingly rapid rotation of CT gantries that have allowed increased spatial resolution and extended coverage. These technological advances have improved CE-CTA accuracy, and 64-detector row CE-CTA has been shown to be superior to 4- or 16-detector row studies demonstrating high sensitivity and specificity, in comparison with digital subtraction angiography . CT has From an interpretation perspective heavily calcified arterial disease can be challenging to accurately evaluate on CTA; metal prostheses may produce obscuring artefact and although CE-CTA generally assesses stents well, small-calibre stents remain challenging to analyse. In many institutions, CE-CTA is employed as the initial study of choice for investigation for vascular disease, particularly given the now widespread availability of multislice technology.This study was conducted in a 900-bed teaching hospital providing vascular surgical services as well as a regional renal medicine unit over a 12-year period. The contrast-enhanced MRI angiography (CE-MRA) service was initially introduced in June 1998 after a CE-MRA-capable MRI system was installed—a 1.5-T Philips Gyroscan ACS-NT MRI Scanner , including a contrast pump injector . Following an upgrade in 2002, the scanner was enhanced with a software and table upgrade to allow moving table peripheral angiography plus phased array coils enabling implementation of parallel imaging techniques. A second MRI scanner was added in 2009—a 1.5-T Siemens Magnetom Avanto , again with a Medrad contrast pump injector and a dedicated peripheral vascular phased array coil. Several generations of CT scanners have been employed in this time, the single detector row scanner initially installed in 1997 that was effectively unable to perform extended CTA studies being supplemented by a four-detetctor row Siemens Somatom 2 in 2002 upon which lower limb angiographic studies could be performed, although with limited slice resolution. A 64-detector row Toshiba Aquilion CT scanner  equipped with a Medrad Stellant dual-head contrast pump injector was installed in 2007, replacing the original single-detector-row machine and allowing thin collimation peripheral run-off studies. A second identical 64-slice Toshiba scanner was installed in 2009, bringing the complement of CT scanners to three: one 4 detector row and two 64 detector row.The baseline angiography and interventional vascular caseload was established for the year June 1997 to the end of May 1998. The subsequent workload to the end of May 2010 was prospectively collated each year and comparison drawn with the CE-MRA and latterly CE-CTA numbers. Latterly CE-CTA numbers were also collected for additional comparison. The numbers of investigations, procedures performed and body area examined  were recorded. CT pulmonary angiograms and more recently CT coronary angiograms have been specifically excluded, with only CT angiographic vascular studies generated via the vascular service evaluated. Ultrasound is not employed for primary investigation of lower limb peripheral vascular disease in our institution (ultrasound is reserved for graft surveillance), and hence has not been studied.Changes in waiting times over the decade have been compared to baseline, whilst a marginal cost analysis for conventional diagnostic angiographic procedures was made accounting for consumables , staffing  and hospital bed costs. Marginal cost for CE-CTA and CE-MRA was also calculated taking similar relevant parameters into consideration.The equivalent radiation dose for CE-CTA performed on our 64-detector-row scanner was estimated by calculating the mean of the effective dose of all such studies performed in the final month of data acquisition , 10.The angiographic, CT and MRI workload was evaluated up to May 2010 using the Radiology Information System (RIS) and compared with the workload in May 1998 using the vascular theatre logs. Alterations in workload pattern have been correlated historically to the changes in the imaging environment at the local and national/international level that have been perceived to have had an impact.The study period, numbering 13 years in total (including baseline years), runs from 1 June 1997 to 31 May 2010, during which time there have been 8,769 invasive angiographic procedures, of which 5,105 were diagnostic vascular and 3,664 were interventional vascular procedures. In the same period there were 6,859 CE-MRA procedures, with the overall number of vascular studies numbering 16,872. There was a year-on-year rise in the number of CE-MRA studies performed each year from 1997/98 (13 studies) to 2002/03 (760 studies) with a reciprocal 50.4 % drop in conventional diagnostic angiography during the same period  per study to around £100 (€125) currently. Conventional diagnostic angiography, including the cost of a day or overnight bed, cost £515 €640) in 2001, and this was similar in 2009. The CE-CTA marginal cost was estimated at £68 (€85) per study in 2009. The overall cost of the diagnostic angiography service is demonstrated in Fig. 40 in 200The mean effective dose for a selection of peripheral angiograms was performed using the dose-length product , with a The advent of non-invasive vascular imaging using CE-MRA and CE-CTA has resulted in non-invasive alternatives to invasive diagnostic angiography providing excellent quality three-dimensional images whilst avoiding the potential complications associated with invasive procedures. Both CE-MRA and CE-CTA are superior to ultrasound in their ability to image the entirety of the pertinent vascular tree, avoiding compromise of image quality by depth in the body or interposed bowel gas. Another major advantage over ultrasound is the depiction of the vascular tree in a familiar angiographic format with very low inter-observer variability enhancing clinical credibility and allowing straightforward discussion in the multidisciplinary team environment. The overall impact of CE-MRA and CE-CTA in our institution has been a marked reduction in the conventional diagnostic angiography workload, with extensive use of CE-MRA and more recently CE-CTA in a wide range of clinical scenarios, with tailoring of modality to individual cases bearing in mind the ALARA principle. The volume and choice of the type of examinations performed have been influenced by modality availability, clinical trends and technical developments, as well as consequential theoretical and real adverse effects. The impact has varied over time in relation to various factors as per the following discussions.A steady increase in diagnostic vascular imaging activity was apparent up until 2002/03, with an additional 382 patients (32.4 %) examined overall in the year 2001/02 as compared to 1997/98. This is in part due to a proliferation in renovascular imaging in this period. Whilst only 22 conventional renal angiograms were performed in the year prior to the advent of CE-MRA in 1997/98 for investigation of suspected renal artery stenosis, in 2002/03 377 renovascular studies were undertaken, accounting for almost all the increase in diagnostic vascular imaging. The majority were for investigation of possible atherosclerotic renovascular disease as a cause of renal impairment with or without hypertension, mainly from the renal medicine service and cardiology. Indeed referrals also came from renal physicians and cardiologists based at other institutions in and around the city. This was due to the non-invasive nature, good sensitivity and specificity relative to digital subtraction angiography, and great superiority of CE-MRA compared to Doppler ultrasound . These fscleromyxoedema-like skin thickening’ affecting the limbs and trunk [A decrease in the total number of CE-MRA studies was observed in 2007/08, reducing from 580 to 509 studies (a 12.2 % drop). This was largely due to the large relative reduction in renal studies in this period, demonstrated in Fig. nd trunk , it was nd trunk , subseqund trunk . Prior tnd trunk . This lend trunk . Furthernd trunk , 26. A Und trunk . Currentnd trunk . Recent nd trunk . This rend trunk , 31 for The increase in the use of CE-CTA over the final 5 years of the study period merits discussion, with an increase in CE-CTA (excluding CT pulmonary angiography and CT coronary angiography) from 122 studies in 2005/06 to 396 studies in the final year of data collection. This is a technology-driven phenomenon with utilisation having been revolutionised by multidetector row scanners with 16 or more detector rows. A wide range of clinical applications are described, including traumatic injuries, embolic phenomena, aneurysms and atherosclerotic disease . Figure Interestingly, despite large variations in angiographic modality choice and availability over a decade, then bar the renal angiographic threshold being reduced, the overall number of diagnostic investigations through the vascular surgical service has remained largely constant, now with a total number of studies usually between 1,200 and 1,400 per year. The only significant departure from this, 1,014 studies performed in 2004/05, was a result of transfer of a proportion of CE-MRA services to two further city sites within the health board, which commenced a CE-MRA service. This suggests that demand for services has not significantly altered, but evolution of technology and its availability has necessitated change.The significant shift to non-invasive vascular imaging is well demonstrated by comparison of monthly diagnostic workloads at the start and end of data acquisition, with minimal numbers of invasive conventional angiography now performed, largely replaced by non-invasive CE-MRA and CE-CTA. Although the number of interventional vascular cases has remained broadly constant over the study period, there has been a significant trend to an increase in the complexity of time-consuming cases, such as endovascular aneurysm repair (EVAR). Meanwhile there has been a reduction in more straightforward angioplasty  and thrombolysis. Despite the considerable increase in the time required in the interventional suite to perform procedures such as EVAR, the uptake of non-invasive angiography techniques has helped free up the time in the vascular radiology theatre suite previously reserved for routine diagnostic angiography, allowing the more extensive access required for complex interventional procedures. The relatively constant number of interventional procedures is likely a combination of the reduction in infra-inguinal angioplasties for claudication in line with local practice and the reduction in thrombolysis.Figure The potential for marked changes in diagnostic practice is demonstrated in Fig. Vascular imaging has undergone a significant metamorphosis in little over a decade, with a major decline in conventional diagnostic angiography and a corresponding boom in CE-MRA and more recently CE-CTA. The modalities each have their strengths and weaknesses but they have become firmly established and we have demonstrated cost-effective, time-effective and safe diagnostic angiographic use.