Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

[Clinical performance measurement in surgery and orthopedics -- new aspects in 2004]. / Externe Qualitätssicherung Chirurgie und Orthopädie -- Was ändert sich im Jahr 2004?

In 2004, principles and practice of clinical performance measurement (CPM) in German hospitals were changed according to new legislative and administrative regulations. In many respects, focus and methods of clinical performance measurement were improved in favour of hospitals. Starting from January 1, 2004, the new Gemeinsamer Bundesausschuss (Joint Federal Board) has competence for decisions on future focus and scope of CPM. Former agreements on implementation of CPM in 2004 will be effective as long as Gemeinsamer Bundesausschuss passes new resolutions. Methods to identify relevant cases for CPM particularly changed in 2004. Until end of 2003, obligations to report case data were based on special types of hospital reimbursement. In 2004, obligations for reporting no longer derive from financial criteria, but from medical criteria such as diagnoses and procedures. In 2003, reporting for CPM covered more than 30 subjects in medicine and nursing. For 2004, the scope of CPM has been reduced by 13 subjects which need to be reconsidered in order to secure unified quality goals for out-patients as well as in-patients and to allow long-term follow-up of outcome data. For their CPM expenditure, hospitals receive an additional fee of euro 0.58 per case reimbursed by DRG. Financial sanctions will be effective for hospitals with overall CPM reporting rates below 80 %. Starting from 2005, hospitals are obliged to publish CPM reporting rates for each CPM subject in annual hospital quality reports.

Histopathology and molecular genetics of multiple cysts and microcystic (serous) adenomas of the pancreas in von Hippel-Lindau patients.

Microcystic adenoma and cysts of the pancreas occur sporadically or as a part of von Hippel-Lindau (VHL) disease. The pathology of pancreatic cystic disease in VHL patients has not been well characterized. Furthermore, it is presently unknown whether the alteration of the VHL gene is responsible for the development of the entire spectrum of pancreatic serous cystic lesions. We performed a histopathological analysis of 21 cysts and 98 microcystic adenomas in nine VHL patients with a known germline mutation. In addition, PCR-amplified DNA from 27 pancreatic cystic lesions in three informative patients was studied for allelic deletions with polymorphic markers spanning the VHL gene locus. In all patients, pancreatic lesions were multiple: 21 benign serous cysts, 63 microscopic microcystic adenomas (size <0.4 cm), and 35 macroscopic microcystic adenomas (size >0.5 cm). The average number of lesions per patient was 2.1 benign cysts (range, 0-8), 7.7 (1-37) microscopic microcystic adenomas, and 3 (0-21) macroscopic microcystic adenomas. All lesions showed similar histology and contained prominent fibrous stroma, clear and/or amphophilic, glycogen-rich epithelial cells, endothelial and smooth muscle cells. VHL deletions were detected in all types of pancreatic cystic lesions. The presence of VHL gene allelic deletions in the spectrum of multifocal pancreatic cystic lesions provides direct molecular evidence of their neoplastic nature and integral association with VHL disease. The histopathological and molecular data establish a serous cyst-microcystic adenoma continuum in the development of pancreatic cystic neoplasia in VHL disease.

Plasmapheresis combined with continuous venovenous hemofiltration in surgical patients with sepsis.

OBJECTIVE: To examine the effect of continuous venovenous hemofiltration (CVVHF) combined with plasmapheresis (TPE) in critically ill surgical patients after treatment of the septic focus. DESIGN: Observational pilot study. SETTING: University teaching hospital intensive care unit. INTERVENTIONS: TPE and CVVHF were administered 24 h after surgical and/or interventional treatment of septic focus. Arterial blood pressure, cardiac output, and systemic vascular resistance values were monitored. We examined the effect of the combined extracorporeal detoxification on outcome related to age, morbidity, organic failure rate, and initial APACHE II score. MEASUREMENTS AND RESULTS: Forty-three patients with sepsis were treated; 19 received TPE in combination with CVVHF, and 24 did not receive extracorporeal therapy. Overall mortality was 44.2%. In the therapy group mortality was lower (42.1 vs. 45.8%), but the primary organic failure rate was higher. The relationship between mortality and age was similar in the two groups. There was also no difference between the groups in the course of scores on APACHE II, multiple-organ failure, and sepsis severity. Only patients with an initial APACHE II score of 21-25 had a significant reduction in mortality after combined extracorporeal detoxification. Mortality of 17% in TPE/CVVHF patients with single- (pulmonary) and double-organ failure (renal/pulmonary) was significantly lower (P < 0.0001) than in untreated patients. CONCLUSIONS: Reduction in mortality in single- and double-organ failure was as high as 28% in septic patients with combined extracorporeal detoxification. A prospective randomized trial in sepsis and double-organ failure should be projected.

[Wound dressings in chronic wounds]. / Wundauflagen bei chronischen Wunden.

Hydroactive wound dressings retain exsudate in the wound region or incorporate wound exsudate by gel formation. They create the local environment for moist wound healing which is experimentally and clinically characterized by accelerated reepithelialization, inflammatory reaction and angiogenesis as well as reduced wound pain and wound infection rates. Clinically relevant product groups of hydroactive wound dressings (hydrocolloids and hydropolymers, semipermeable films, calcium alginates) are distinct as to chemical structure, physical properties and functional characteristics in local wound treatment. Between the product groups, there are considerable differences with respect to inflammatory reactions at the wound bottom, absorption of exsudate, occlusion properties, wound edge adherence, adaptability to the wound shape and material integrity of wound dressings. Experimental and clinical results of moist wound treatment by hydroactive wound dressings such as hydrocolloids and hydropolymers, semipermeable films or calcium alginates reveal a wide range of local response on the different types of dressings. They offer the opportunity of therapeutic differentiation. To elucidate the differential indication for different product groups of hydroactive wound dressings in local treatment of chronic wounds, additional experimental and clinical research is required.

[Therapy-resistant pyoderma gangrenosum--treatment with mycophenolate mofetil and cyclosporine A]. / Therapieresistentes Pyoderma gangraenosum. Eine Behandlung mit Mycophenolatmofetil und Cyclosporin A.

A 68-year old woman presented with ulcerations on the calves that had occurred spontaneously. The very painful lesions both clinically and histologically showed the characteristics of pyoderma gangrenosum. During hospitalization she was treated with corticosteroids (oral, i.v., topically), clofazimine, cyclophosphamide, intravenous immune globulin, cyclosporine (oral, local), dapsone, thalidomide and sodium cromoglycate (topically) without any benefit. Finally, when treated with mycophenolate mofetil (CellCept) (oral) and cyclosporine (oral), her skin lesions showed continuous improvement. The topical application of thrombocytic growth factors (cytokines) probably accelerated the granulation. Eight weeks after initiating this treatment the lesions could be covered with split thickness skin grafts. Our observation suggests that mycophenolate mofetil, a novel immunosuppressive agent which has thus far been used almost exclusively in transplantation medicine, may be an effective therapeutic modality in combination with cyclosporine A for the treatment of pyoderma gangrenosum.

Microdissection-based analysis of mature ovarian teratoma.

The genotypic features of mature ovarian teratomas (MOTs) are controversial. Early studies detected a homozygous genotype in MOTs suggesting that these tumors are composed of germ cells that have undergone meiosis I. Other studies, however, revealed a heterozygous genotype in a substantial proportion of MOTs suggesting an origin either from premeiotic germ cells or from a somatic cell line. In view of the complex morphology of MOTs and to increase the sensitivity of teratoma genotyping, we applied tissue microdissection before genetic analysis of teratomatous tissue. This approach allowed selective analysis of different heterotopic tissue elements as well as the lymphoid tissues within MOTs the origin of which is unknown. After DNA extraction, the tissue samples were polymerase chain reaction amplified using a random panel of highly informative genetic markers for different chromosomes to evaluate heterozygosity versus homozygosity. In all seven cases that were analyzed, heterotopic tissues consistently revealed a homozygous genotype with several markers; in two cases, heterozygosity was detected with a single marker, indicating a meiotic recombination event. Lymphoid aggregates within MOTs were heterozygous and derived from host tissue rather than from teratomatous growth. However, well differentiated thymic tissue was consistently homozygous, suggesting lymphoid differentiation capability of MOTs. We conclude that potential pitfalls in genotyping of teratomas including meiotic recombination and host cell participation can be avoided by a microdissection-based approach in combination with a panel of genetic markers.

Posttraumatic hypertension secondary to adrenal hemorrhage mimicking pheochromocytoma: case report.

We report the case of a 68-year-old man who presented with a mass 3 x 4 cm in size located in the right adrenal gland together with extreme hypertension, tripled urine levels for normetanephrine, and normal plasmatic levels of metanephrines. The patient had suffered a fall from a height of 2.5 meters before hospitalization. [123I]MIBG-scan was repeatedly positive in the area of the right adrenal gland. At laparotomy under alpha-adrenergic blocking agents, the suspected pheochromocytoma was histologically confirmed as hematoma. After resection of the adrenal gland, blood pressure returned to normal without drug therapy as did metanephrine levels in urine. Although adrenal insufficiency after distension of the gland caused by hemorrhage has been reported, there are no data available regarding the mimicking of a hormonally active pheochromocytoma. We conclude that intra-adrenal pressure rise caused by hematoma may cause partial ischemic necrosis to the gland but may also induce reactive hyperplasia with periodic excessive secretion of catecholamines. This interpretation is consistent with the finding that plasma levels of catecholamines were normal in contrast to the urinary normetanephrines in the presented case. It might be worthwhile to investigate patients with intra-adrenal hemorrhage immediately after sustaining multiple injuries and in the posttraumatic course of several months up to 1 or more years together with verification of abnormal urinary excretion of metanephrines as a sign of impaired adrenal function.

Potent vasodilatory with minor cardiodepressant actions of mibefradil in human cardiac tissue.

1. The present study compared the cardiovascular effects of mibefradil (MIB), a novel Ca2+-channel antagonist with high selectivity for T-type Ca2+-channels to the effect of the L-type Ca2+-channel-antagonists nifedipine (NIF) and diltiazem (DIL) in left ventricular myocardium and coronary arteries of hearts obtained from patients suffering from dilated cardiomyopathy (NYHA IV). Right atrial myocardium from patients undergoing aortocoronary bypass surgery without signs of cardiac failure was studied as well. 2. NIF and DIL (100 micromol l(-1)) completely depressed force of contraction (FOC) in electrically driven left ventricular myocardium (NIF 6.5+/-1.4% and DIL 7.1+/-1.2% of control), whereas a similar concentration of MIB only reduced force of contraction to 55.1+/-4.0% of the basal FOC. The negative inotropic potency as measured by the concentration needed to reduce basal FOC for 25% was NIF (0.0095 micromol l(-1))>DIL (0.041 micromol l(-1))>MIB (9.47 micromol l(-1)). 3. All three Ca2+-channel antagonists were more potent in human atrial compared to human left ventricular myocardium to reduce FOC. 4. The rank order of Ca+-antagonistic moiety as measured by the decrease of the intracellular Ca2+-transient (fura-2 ratio method) was NIF>DIL>MIB. 5. All Ca2+-channel antagonists completely relaxed human coronary arteries (% of papaverine effect: MIB 81.7+/-5.5%, DIL 91.3+/-0.9%, NIF 96.4+/-3.7%) precontracted with PGF2alpha (0.3 micromol l(-1)). The rank order of vasodilatory potency was NIF (EC50; 0.02 micromol l(-1))>DIL (0.13 micromol l(-1))>MIB (2.05 micromol l(-1)). 6. The vasoselectivity measured by the ratio of the concentration needed to achieve a 25% decrease in force and the concentration needed for 25% vasodilatation was 316 for MIB, 1.5 for NIF and 1.0 for DIL. 7. The present study provides evidence that blockade of T-type Ca2+-channels (e.g. mibefradil) results in potent vasodilatory properties with only minor cardiodepressant effects.

[Plasma separation combined with CVVHF in septic and SIRS patients]. / Plasmaseparation kombiniert mit CVVHF in Sepsis- und SIRS-Patienten.

In a prospective non-randomized trial, 59 patients with sepsis (n = 43) and SIRS (n = 16) were treated on a surgical intensive care unit. In 22 patients plasmapheresis in combination with continuous venovenous hemofiltration (CVVHF) was administered. Lethality was 56% in the sepsis group; in the therapy group lethality was significantly lower in patients with plasmapheresis, even though in this population the organic failure rate was higher. Finally the dependency of lethality and age was similar in both groups. Lethality at 22% in the plasmapheresis group with double organ failure was significantly lower (P > 0.01) than in controls. Reduction of lethality seemed to be as high as 18% in patients with sepsis, while patients with SIRS did not profit from the additional therapy. A prospective randomized trial in sepsis and double organic failure should be projected.

[Endosonographic demonstration of extrapancreatic and extraintestinal gastrinoma]. / Endosonographischer Nachweis eines extrapankreatischen und extraintestinalen Gastrinoms.

HISTORY AND CLINICAL FINDINGS: No cause had been found for chronic diarrhoea in a 57-year-old man. Up to 15 watery stools daily had been without relation to food intake and without blood admixture. But muscular cramps had developed, especially in the legs. The patient had a history of recurrent peptic ulcers for which a selective proximal vagotomy had been performed 13 years ago. Physical examination was unremarkable. INVESTIGATIONS: Alkaline phosphatase activity (182 U/l) and C-reactive protein (9.3 mg/l) were slightly raised; serum iron was 42 micrograms/dl, while all other routine laboratory tests, including protein electrophoresis, blood picture and differential count were within normal limits. Gastroscopy revealed ulcerative duodenitis, gastritis with erosions and numerous ulcers and reflux oesophagitis, grade III-IV. Endosonography showed enlarged gastric mucosal relief as sign of foveolar hyperplasia and a ca. 4 x 3 cm tumour next to the duodenal bulb. Gastrin level was 7537 pg/ml (normal < 150 pg/ml). Computed tomography and somatostatin receptor scintigraphy confirmed the site and size of the gastrinoma. TREATMENT AND COURSE: Treatment with omeprazole (40 mg three times daily) slightly improved the symptoms. The tumour was excised a week after diagnosis. The patient has been symptom-free since then. CONCLUSION: Chronic diarrhoea of unknown aetiology can be caused by an endocrine tumour; endosonography can often provide information on the diagnosis and location of such a tumour.

[Optimizing a conventional documentation system on the surgical intensive care unit--a contribution to cost reduction and preventive quality management?]. / Optimierung eines konventionellen Dokumentationssystems auf der chirurgischen Intensivstation--ein Beitrag zu Kostenreduktion und präventivem Qualitätsmanagement?

In Intensive Care Units (I.C.U.), the lack of staff and funds require the mobilisation of organisational reserves in order to ensure high-quality patient care. Traditional occupational-group organised documentation systems are burdened with lack of clarity, limited utilisation by the staff, insufficient information content and difficulties in synoptic patient monitoring. They cannot meet the demands of modern intensive-care medicine. At the inauguration of our Surgical I.C.U. in June 1992, an occupational-group oriented documentation system was introduced and put into operation. Because of negative repercussions on patient care, it was replaced by a patient-centered, conventional prescription and documentation system in April 1993. In April 1994, an evaluation of the patient-centered system was carried out. We report on our initial problem analysis, the subsequent developmental and introductory phases, and the results after having used the system in our I.C.U. for one year. Data condensation, standardised data recording, as well as structured prescription, examination, assessment and decision processes, saved 730 working hours for medical and nursing staff per year, reduced the cost for documentation materials by 58% and improved the extent of data recording. In our experience, improving a conventional documentation system is a suitable instrument to support cost reduction and preventive internal quality management in the I.C.U.

[Asymptomatic choledochal cyst]. / Asymptomatische grosse Choledochuszyste im Erwachsenenalter.

HISTORY AND FINDINGS: A 45-year-old woman sustained an ankle fracture in an accident. At examination she was found to have marked pallor of skin and mucosae. There was no hint for melaena or haematemesis. INVESTIGATIONS: Biochemical tests showed marked iron deficiency anaemia (haemoglobin 7.5 g/dl) and raised serum bilirubin and C-reactive protein levels (1.94 mg/dl and 85.2 mg/l, respectively). Abdominal sonography revealed a cystic space-occupying mass (8 x 4.5 cm) projecting onto the gallbladder, interpreted as a choledochal cyst of unknown origins without bleeding. After treatment of the ankle fracture an endoscopic retrograde cholangiopancreatography was performed. This showed a large cyst of the choledochal duct into which the cystic and choledochal ducts entered, without evidence of tumour or haematoma. There was also a 1 cm prepapillary common choledochal and Wirsung duct. TREATMENT AND COURSE: With these findings, the diagnosis of a congenital choledochal cyst (type Ia of Todani) could be made. After healing of the ankle fracture the cyst was removed and a Roux Y-anastomosis created. The cystic tissue was benign. At follow-up 6 months later the patient was symptom-free and no longer anaemic. CONCLUSIONS: Congenital choledochal cysts are very rare in Europeans. The symptom-free course with anaemia and no manifestation until adulthood is also very unusual.

Spontaneous pneumothorax in a patient with systemic sclerosis.

A 28-year-old woman developed spontaneously a right- sided pneumothorax, the leading clinical symptom of an as yet undiagnosed systemic sclerosis. The diagnosis was confirmed by Raynaud's phenomenon, microstomia, arthralgia, distal oesophageal dysfunction and antinuclear antibodies. Initial treatment with pleural suction was followed by thoracoscopy and segmental pulmonary resection. Spontaneous pneumothorax is a rare complication in patients with systemic sclerosis, most likely caused by the rupture of subpleural cysts.

[Performance and cost analysis of surgical therapy of stomach carcinoma--results and sequelae]. / Leistungs- und Kostenanalyse der chirurgischen Therapie des Magenkarzinoms--Ergebnisse und Folgerungen.

In order to identify opportunities for cost containment in gastric cancer surgery, and analysis of pre-, peri-, and postoperative cost profiles was performed for 60 gastric cancer patients who underwent curative resections (76.6%), explorative laparotomies (18.3%), and palliative gastrojejunostomies (5%). While pre- and perioperative phases only offer limited opportunities for cost reduction, postoperative complications raised the mean length of hospital stay by 47%, the mean length of intensive care treatment by 865%, and the total treatment costs by 84-248% compared to an uncomplicated clinical course. Pre-, peri-, and postoperative cost-containment efforts must focus on the prevention of postoperative complications.

[Thoracic actinomycosis]. / Die thorakale Actinomykose.

Thoracic actinomycosis is a rare disease without characteristic clinical signs. Approximately 90% of patients suffering from thoracic actinomycosis have undergone diagnostic and therapeutic procedures based on a wrong diagnostic hypothesis (malignancies 35-44%, other pulmonary disorders 33-35%). The opportunities for a timely and adequate diagnosis by the use of clinical examination, laboratory studies, microbiology studies, radiologic imaging or invasive measures are limited. In 85%, thoracic actinomycosis has not been identified prior to thoracotomy, open biopsy and histological examination. Based on a wrong diagnostic hypothesis, resective thoracic surgery according to the principles of oncologic surgery can hardly be avoided. We report on a 43-year-old male suffering from actinomycosis of the left hemithorax. Clinical signs, differential diagnosis, treatment and clinical course are described. The role of surgery in the treatment protocol of thoracic actinomycosis is discussed. In pulmonary and pleural disorders of unknown origin, differential diagnosis should include thoracic actinomycosis as early as possible. Due to the considerably high mortality rate of untreated disease, the outcome of thoracic actinomycosis can only be improved by a timely and combined employment of surgical and antibiotic therapy.

[Masked course of Whipple disease with uveitis, infection, endocardial involvement and abdominal lymphomas--case report and review of the literature]. / Maskierter Verlauf eines Morbus Whipple mit Uveitis, Sepsis, Endokardbeteiligung und abdominellen Lymphomen--Fallbericht und Ubersicht.

Whipple's disease is a systemic disease which may virtually affect any organ system, but in many cases it involves the small intestine causing gastrointestinal symptoms. The differential diagnosis is difficult since symptoms may be nonspecific. We report the case of a 44-year old white male patient with a history of migrating arthralgia and chronic fatigue. The patient newly developed an uveitis and underwent a vitrectomy; the further clinical work-up including gastroscopy with intestinal biopsy revealed no sufficient diagnosis. Subsequently, the patient's condition deteriorated with marked weight loss, fever and progressive weakness. An anaerobic sepsis with a corynebacterium was confirmed and with i.v.-antibiotics the patients's condition improved markedly. The further examinations disclosed enlarged mesenteric lymph nodes and the involvement of other organs (endocard, liver). CT-guided biopsy only showed fatty degeneration, but operative adenectomy confirmed Whipple's disease. The patient remained without relapse on long-term antibiotic treatment with doxycycline until today. Obviously, in our case the intestinal biopsies failed to detect Whipple's disease after the successful initiation of antibiotic treatment. In the absence of gastrointestinal findings and with concomitant secondary diseases the definitive diagnosis can be difficult. In addition, the previous uveitis and the endocardial involvement are most interesting.