Percutaneous cholecystostomy in critically ill patients with acute cholecystitis: complications and late outcome.

AIM: To evaluate the outcome of percutaneous cholecystostomy in critically ill patients with acute cholecystitis. MATERIALS AND METHODS: The study group included critically ill patients who underwent percutaneous cholecystostomy for acute cholecystitis at a tertiary medical centre in 2007-2011. Data on complications, morbidities, surgical outcome, and imaging findings were collected from the medical files and radiology information system. RESULTS: There were 48 women (59.3%) and 33 men (40.7%), with a median age of 82 years (range 47-99 years). Seventy-one (88%) had calculous cholecystitis and 10 (12%), acalculous cholecystitis. The drain was successfully inserted in all cases with no immediate major procedural complications. Fifteen patients (18.5%) died in-hospital within 30 days, mainly (93%) due to septic shock (14/15), another 20 patients (24.7%) died during the study period of unrelated co-morbidities. Of the remaining 46 patients, 36 (78.2%) had surgical cholecystectomies. In patients with acalculous cholecystitis, the drain was removed after cessation of symptoms. Transcystic cholangiography identified five patients with additional stones in the common bile duct. They were managed by pushing the stones into the duodenum via the cystostomy access, sparing them the need for surgical exploration. CONCLUSIONS: Early percutaneous gallbladder drainage is safe and effective in critically ill patients in the acute phase of cholecystitis, with a high technical success rate. Surgical results in survivors are better than reported in patients treated surgically without drainage. Bile duct stones can be eliminated without creating an additional access.

Microchip electrophoresis: a method for high-speed SNP detection.

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.

High-throughput biopolymer desalting by solid-phase extraction prior to mass spectrometric analysis.

In the last 10 years mass spectrometry (MS) has become an important method for analysis of peptides, proteins and DNA. It was recently utilized for accurate high-throughput protein identification, sequencing and DNA genotyping. The presence of non-volatile buffers compromises sensitivity and accuracy of MS biopolymer analysis; it is essential to remove sample contaminants prior to analysis. We have developed a fast and efficient method for desalting of DNA oligonucleotides and peptides using 96-well solid-phase extraction plates packed with 5 mg of Waters Oasis HLB sorbent (Waters, Milford, MA, USA). This reversed-phase sorbent retains the biopolymer analytes, while non-retained inorganic ions are washed out with pure deionized water. DNA oligonucleotides or peptides are eluted using a small amount (20-100 microl) of acetonitrile-water (70:30, v/v) solution. The SPE desalting performance meets the requirements for MS applications such as protein digest analysis and DNA genotyping.

Sequencing of antisense DNA analogues by capillary gel electrophoresis with laser-induced fluorescence detection.

A method using capillary gel electrophoresis with laser-induced fluorescence detection is described which permits complete sequence determination of antisense DNA analogues of unknown sequence. This method, originally created as a tool to confirm the sequence of antisense oligonucleotides being developed as therapeutic drugs, utilizes data collected under a range of experimental conditions described by the Ogston model as applied to gel electrophoresis. A linear relationship independent of experimental conditions between the relative electrophoretic migration time and the oligonucleotide base number was observed and is shown to be consistent with a simplified version of this model and can be used to facilitate the sequence determination.

Uterine arterial embolization for the management of leiomyomas.

BACKGROUND: Leiomyoma is the common benign tumor of the female genital tract. The traditional treatment is hysterectomy, myomectomy or medical therapy by hormonal manipulation. Uterine arterial embolization, a recognized treatment for acute pelvic hemorrhage, has recently been applied to the management of non-acute uterine hemorrhage due to leiomyoma. OBJECTIVES: To describe our experience with uterine arterial embolization for the management of uterine fibroid. METHODS: Uterine arterial embolization was performed in nine patients with leiomyomas in whom medical therapy failed and who sought to avoid surgery. RESULTS: Follow-up ultrasound examination after 2 months revealed an average reduction in fibroid volume of 38%. There were no early or long-term complications. CONCLUSIONS: Uterine arterial embolization appears to be effective and safe in the management of symptomatic leiomyomas. It is a promising alternative to myomectomy or hysterectomy and warrants further investigation in this setting.

[Detection of anomalous origin of left coronary artery by spiral CT as a cause of angina pectoris].

The anomalous origin of the left coronary artery can lead to angina pectoris, acute myocardial infarction or even sudden death, especially during exercise. We present a patient in whom the anomalous origin of the left coronary artery is from the right sinus of Valsalva, crossing between the aorta and the pulmonary trunk and causing ischemic chest pain. The anomaly was verified by a Spiral CT, as the coronary angiographic findings were not conclusive, particularly regarding the left course in relation to the major arteries. We suggest that Spiral CT is useful for detecting this kind of anomaly, particularly in clarifying the relationship between the left main coronary artery and the major arteries.

The evaluation of a novel technique to treat saphenous vein incompetence: preclinical animal study to examine safety and efficacy of a new vein occlusion device.

OBJECTIVES: We tested a novel technique to treat great saphenous vein (GSV) incompetence in an animal model. METHODS: V-block (VVT Medical Ltd, Kfar Saba, Israel), an occlusion device composed of a nitinol frame and anchoring hooks, was percutaneously deployed at the saphenofemoral junction in 12 sheep. Four of the 12 sheep were treated with adjunctive liquid sclerotherapy. Animals underwent duplex ultrasound, venography and histopathological evaluation immediately postimplantation at 30, 60 and 90 days. RESULTS: V-block was successfully deployed in all animals without adverse events. There was no device migration at follow-up. Histopathological analysis demonstrated V-block to be lodged within the GSV and surrounded by fibrous tissue in all samples. Obliteration of the GSV lumen, widespread intimal loss and multifocal medial smooth muscle loss was noted. CONCLUSIONS: In this animal study V-block was deployed without complications, remained in stable position and led to GSV occlusion. This device has promise for future use in humans.

Use of a collagen-based device for closure of low brachial artery punctures.

PURPOSE: To report our experience with the Angioseal vascular closure device for hemostasis of distal brachial artery puncture. METHODS: Between September 2003 and August 2005, 64 Angioseal vascular closure devices were inserted in 64 patients (40 men, 24 women; mean age 65 years) immediately after diagnostic or therapeutic arterial angiographies performed through a 5 Fr to 7 Fr sheath via the distal brachial artery. Ultrasound examination of the brachial artery preceded the angiography in all cases and only arteries wider than 4 mm were closed by the Angioseal. In cases of a sonographically evident thin subcutaneous space of the cubital fossa, tissue tumescence, using 1% Lidocaine, was performed prior to the arterial closure. RESULTS: The deployment success rate was 100%. No major complications were encountered; only 2 patients developed puncture site hematoma, and these were followed conservatively. CONCLUSIONS: Closure of low brachial artery punctures with the Angioseal is simple and safe. No additional manual compression is required. We recommend its use after brachial artery access interventions, through appropriately wide arteries, to improve early patient ambulation and potentially reduce possible puncture site complications.

Polymer solutions as a pseudostationary phase for capillary electrochromatographic separation of DNA diastereomers.

The solutions of linear polymers traditionally used for DNA separation have been employed for the capillary electrophoresis (CE) of diastereomers of chemically modified DNA. The selectivity of diastereomeric separation of the phosphorothioate (PS) and 2'-O-methylated (2-OMe) PS oligonucleotides depends on the nature of the polymer additive in the CE background electrolyte. The selectivity of separation for different polymers increases in the line: linear polyacrylamide < polyethylene glycol < polyvinyl pyrrolidone. The separation of oligomer diastereomers was shown to be primarily based on the hydrophobic interaction with the polymer network that acts as a pseudostationary phase. While lowering the temperature resulted in improved separation, the addition of organic modifiers such as formamide, methanol or acetonitrile counteracts the solute adsorption on the polymer network, and decreases the selectivity of DNA diastereoseparation. The effect of molecular mass and concentration of the polymer on the separation selectivity was investigated.

Percutaneous balloon dilatation for the treatment of early and late ureteral strictures after renal transplantation: long-term follow-up.

We report our experience with percutaneous balloon dilatation (PBD) for the treatment of ureteral strictures in patients with renal allografts. Of the 422 consecutive patients after renal transplantation in our center 10 patients had ureteral strictures. An additional 11 patients were referred from other centers. The 21 patients included 15 men and 6 women aged 16 to 67 years. Strictures were confirmed by sonography and scintigraphy in all cases. Patients underwent 2 to 4 PBDs at 7-10-day intervals. Clinical success was defined as resolution of the stenosis and hydronephrosis on sequential ultrasound and normalization of creatinine levels. Patients were divided into two groups: those who underwent transplantation more than 3 months previously and those who underwent transplantation less than 3 months previously. PBD was successful in 13 of the 21 patients (62%). There was no statistically significant difference in success rate between the patients with early (n = 12) and those with late (n = 9) obstruction: 58.4% and 66%, respectively. No major complications were documented. PBD is a safe and simple tool for treating ureteral strictures and procedure-related morbidity is low. It can serve as an initial treatment in patients with early or late ureteral strictures after renal transplantation.

A nonradioisotope approach to study the in vivo metabolism of phosphorothioate oligonucleotides.

A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.

Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids.

The in vitro stability and metabolism of GEM[91, a 25mer phosphorothioate antisense oligonucleotide complementary to the gag mRNA region of HIV-1, was investigated using capillary electrophoresis (CE). The in vitro degradation of the parent compound at 37 degrees C was followed over the course of 120 h in human plasma. A CE method using laser-induced fluorescence detection was able to detect 5'-end intact metabolites including the parent compound extracted from biological fluids. Because the primary metabolic pathway is believed to be via 3'-exonuclease activity, the results of this study were compared with the stability of the compound in a solution containing 3'-exonuclease. The numerical solution of sequential first-order reactions was used to obtain kinetic parameters. Exonuclease digestion of the parent compound, as measured using an automated CE-UV instrument, yielded striking similarities between the two in vitro systems as well as between in vitro and in vivo systems.

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.

Detrusor resistive index evaluated by Doppler ultrasonography as a potential indicator of bladder outlet obstruction.

OBJECTIVES: To appraise detrusor blood flow by Doppler ultrasonography in men with suspected bladder outlet obstruction (BOO) to determine whether this imaging technique provides useful information for the assessment of BOO. Experimental studies have shown that BOO is associated with reduced blood flow to the detrusor. METHODS: Twenty-nine consecutive men with lower urinary tract symptoms were prospectively enrolled. A urodynamic pressure-flow study was performed by the urologist to determine BOO, and Doppler ultrasonography was subsequently performed by the radiologist. The physicians were unaware of the other's results. Scanning was performed on a filled and empty bladder. Arterial blood flow was measured at three distinct sites, the two lateral walls and the trigone, and the resistive index (RI) of each site was calculated (RI = (V(MAX) - V(MIN))/V(MAX)). For each patient, the arithmetic average of the three RIs was defined as the detrusor RI. The findings were compared between patients with and without evidence of BOO. A logistic regression model tested the predictive value of the RI. RESULTS: According to the pressure-flow study results, 22 (75%) and 7 (25%) of the 29 patients were diagnosed as having or not having BOO, respectively. A statistically significant difference was found between the detrusor RI in the obstructed versus nonobstructed patients in both full (P <0.001) and empty (P <0.03) bladder states (0.79 versus 0.68 and 0.74 versus 0.66, respectively). Our logistic regression model predicted BOO with an overall accuracy of 86%, positive predictive value of 95%, and negative predictive value of 57%. CONCLUSIONS: The RI of arterial blood flow in the detrusor measured by Doppler ultrasonography provides important predictive information for the presence of BOO. Additional studies are warranted to validate our results and explore the role of Doppler ultrasonography in the management paradigms of patients with suspected BOO.

Equivalent recognition of free and ACT-complexed PSA in a monoclonal-polyclonal sandwich assay is conferred by binding specificity of the monoclonal antibody.

The Bayer Immuno 1 PSA Assay measures free and ACT-complexed PSA on an equimolar basis, although it uses a monoclonal antibody (MM1) for capture and polyclonal antibodies for detection. Competitive inhibition studies using antibodies directed at various epitopes on PSA and PSA-ACT demonstrated that the capture antibody, MM1, does not bind to free PSA simultaneously with antibodies against Epitope E which is exposed only in free PSA. Affinity studies showed that the affinity constants of MM1 for both free PSA and PSA-ACT are similar. One explanation for the properties of MM1 is that it precludes the binding of antibodies to Epitope E due to steric hindrance. Alternatively, the binding of MM1 causes a conformation change within the free PSA molecule, so that Epitope E is altered in a way that causes a loss of binding affinity. The unusual properties of MM1 are responsible for the equimolar response of this monoclonal-polyclonal sandwich assay for free and ACT-complexed PSA.

Study of phosphorothioate-modified oligonucleotide resistance to 3'-exonuclease using capillary electrophoresis.

The effect of phosphorothioate (PS) internucleotide linkages on the stability of phosphodiester oligodeoxyribonucleotides (ODNs) was investigated using 25-mer ODNs containing single or multiple PS backbone modifications. The in vitro stability of the oligomers was measured both in 3'-exonuclease solution and in plasma. For the separation of ODNs, capillary electrophoresis with a replaceable polymer separation matrix was used. As expected, DNA fragments with PS linkages at the 3'-end were found to be more resistant to 3'-exonuclease hydrolysis. Also increasing exonuclease resistance was the non-specific adsorption of phosphorothioate ODNs to enzyme.

Impact of 3'-exonuclease stereoselectivity on the kinetics of phosphorothioate oligonucleotide metabolism.

For the enzymatic digestion of a 25-mer phosphorothioate (PS) oligonucleotide, the reaction kinetics was previously determined to be the sum of two parallel processes: a fast and a very slow phase of digestion suggesting a two-exponential model. A characteristic metabolite profile was observed both in vitro and in vivo. This behavior is shown to be the result of the stereoselective cleavage of chiral R-configuration and S-configuration PS internucleotide linkages by 3'-exonucleases. The stereoselective nature of 3'-exonuclease action was analyzed by reverse-phase HPLC. The separation of eight diastereomers of the tetramer TTCT (5'-3') was used to follow the stereoselective course of exonuclease hydrolysis of PS internucleotide linkages. Degradation of the 25-mer parent compound having a 3' S-terminal internucleotide linkage was calculated to be more than 300 times slower than an analog with a 3'-terminal R-configuration. These results support an approach for protecting antisense oligonucleotides based on the chirality of only the 3'-end internucleotide linkage.

Focal testicular lesion after sperm extraction or aspiration: sonographic appearance simulating testicular tumor.

OBJECTIVE. We describe the sonographic features of focal intratesticular lesions seen in men who underwent sperm retrieval procedures. CONCLUSION. Although many urologists believe that solid intratesticular masses are malignant until proven otherwise, a growing number of benign focal testicular lesions have been described. Awareness of the cause and sonographic appearance of focal abnormalities in men who have undergone testicular aspiration or extraction should help radiologists suggest the correct diagnosis and advise a conservative approach on the basis of close surveillance by serial physical, laboratory, and imaging studies.

Ultrasound-guided testicular sperm aspiration in azoospermic patients: a new sperm retrieval method for intracytoplasmic sperm injection.

PURPOSE: We investigated the technique of ultrasound-guided testicular sperm aspiration (USTSA) and compared it with "blind" testicular sperm aspiration (TSA) in patients with nonobstructive azoospermia. METHODS: Thirty-nine consecutive azoospermic men underwent TSA, 16 under sonographic guidance (USTSA group) and 23 with no imaging guidance (TSA group). Clinical and hormonal evaluation and sonography of the scrotum and testes were performed 1-2 days before the procedure. The aspiration was done using short-term general anesthesia. Follow-up consisted of sonographic reexamination of the scrotum and testes immediately and 1 month after the procedure. RESULTS: Intraoperative sonography with power Doppler imaging enabled good visualization of the testicular parenchyma, easy sampling, and avoidance of prominent vessels. Sufficient material was retrieved in 15 USTSA patients (94%) and 19 TSA patients (83%). No patients needed more than 4 hours' ambulatory hospitalization after the procedure. In the remaining 5 patients, aspiration failed to yield sperm, so open biopsy was performed. In those patients, postaspiration surgical exploration revealed subtunical bleeding in 3 patients after TSA but none after USTSA. Late minor complications occurred in 2 patients (13%) in the USTSA group and 7 (30%) in the TSA group. No difference was found between the 2 groups in pregnancy rate in the patients' female partners. CONCLUSIONS: USTSA is a safe and accurate method for sperm retrieval in azoospermic patients.

Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.