Single, short in-del, and copy number variations detection in monogenic dyslipidemia using a next-generation sequencing strategy.

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next-generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia-causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype-phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia.

Verification of the drug history given by potential blood donors: results of drug screening that combines hair and urine analysis.

BACKGROUND: Besides modern virus-screening methods, the avoidance of transfusion transmission of viral diseases is based on the best possible selection of healthy donors. Unfortunately, most of the relevant behavior-related risk factors are not accessible to objective verification. Drug screening can be used to validate a defined section of donor statements. It may be assumed that donors who conceal drug consumption may also conceal other relevant risk factors. STUDY DESIGN AND METHODS: Hair and urine samples from 186 young potential donors who denied having consumed drugs were investigated by gas chromatography with mass selective detection and a urine fluorescence polarization immunoassay for cannabinoids, amphetamine and amphetamine derivatives, cocaine, and opiates. RESULTS: Ten potential donors with 14 positive results on hair and urine analyses (6x cannabinoids, 4x cocaine, 1x opiates, 3x dihydrocodeine) could be identified in the population investigated. CONCLUSIONS: The donor history is not adequate for identifying potential donors with risk factors. Deliberately false statements concerning risk factors are a clear breach of trust between the blood bank and potential donors. These unreliable donors represent an incalculable risk for the transfusion recipient. Therefore, it is appropriate to validate donor statements about drug consumption by random hair and urine analyses and to exclude from the donor pool all persons revealed as drug users.

[Hepatitis after blood transfusion in inpatients. Post-transfusion hepatitis, or infection of other etiology?]. / Hepatitis nach stationärer Bluttransfusion. Posttransfusionshepatitis oder Infektion anderer Atiologie?

BACKGROUND AND OBJECTIVE: Hepatitis after a transfusion of blood products is often uncritically called post-transfusion hepatitis (PTH). It was the aim of this study to ascertain in how many reported cases of suspected PTH a causal relationship between transfusion and the infection in the recipient can be proven. PATIENTS AND METHODS: Full investigations (look-back method) were made of 23 cases of PTH reported in the 10-year period of 1987-1997 (11 cases of PTH B and 12 of PTH non-A-non-B or C). The recipients had been given a mean of 7.7 blood components (range 1-57). The clinical diagnosis had been made a mean of 5.2 (range 1-27) months after transfusion. RESULTS: One case each of hepatitis B and C had been caused by transfusion of blood products. In 5 of 12 suspected cases of PTH non-A-non-B hepatitis C, PTH could not be definitively excluded (despite absent seroconversion for anti-hepatitis C virus [HCV]), because HCV-RNA findings were not available. In 16 of 23 investigated cases serological and molecular-biological tests firmly excluded PTH B or C. CONCLUSIONS: The small number of confirmed cases of PTH indicates that hepatitis that has occurred post-transfusion is frequently due to a cause not related to the transfusion. The number of transfusion-associated cases will be further reduced with the introduction (standard since 1.4.1999) of VCH-RNA screening of donors. This will raise the importance of hepatitis cases not associated with transfusion.

Conventional and quantitative liver function tests after hepatic transplantation: a prospective long-term follow-up.

In long-term survivors of liver transplantation, hepatic function is obviously of vital importance. Therefore, we prospectively performed conventional and quantitative liver function tests in patients who had survived a first transplantation for at least 4 years. Compared to 6 months after transplantation, serum bilirubin concentration and gamma GT activity were significantly lower after 3, 4, and 5 years (bilirubin 1.2 +/- 0.2 mg/dl at 6 months vs 1.0 +/- 0.1, 1.0 +/- 0.2, and 0.8 +/- 0.1 mg/dl respectively; gamma GT 106 +/- 0 33 U/l at 6 months vs 56 +/- 17, 67 +/- 35, 39 +/- 10 U/l respectively). At these points in time, blood levels of cyclosporin A were also significantly lower. Other parameters of liver cell function and liver cell integrity (AP, AST, ALT, GLDH, total protein, thromboplastin time, partial thromboplastin time) were unchanged over time. Serial quantitative liver function tests (indocyanine green half-life, galactose elimination capacity, lidocaine half-life, and MEGX formation) also remained stable. Thus, we conclude that hepatic function remains stable in long-term survivors of liver transplantation for at least several years.

[Carcinoid syndrome. Recurrent upper abdominal pain, diarrhea and flush in a 15-year-old girl]. / Karzinoidsyndrom. Rezidivierende Oberbauchbeschwerden, Durchfall und Flush bei einer 15jährigen Patientin.

BACKGROUND: A carcinoid syndrome is typically diagnosed in elderly patients, median age at diagnosis was 60 years in two large series. We describe a patient with the carcinoid syndrome at the young age of 15 years. CASE REPORT: The 15-year-old patient's history and findings on physical examination, clinical chemistry, abdominal ultrasound and liver biopsy were typical for a carcinoid with hepatic metastases. The patient's age, however, was very unusual. Because of her youth, we discussed a potentially curative treatment with resection of the primary tumor in the upper jejunum followed by liver transplantation at a second operation. This concept had to be abandoned, when the first operation revealed a tumor in the mesenterium, close to the primary, 5 cm in diameter and around the superior mesenteric artery. This tumor could not be resected. Liver transplantation therefore did not offer this young patient a chance for cure.

[Hemolytic anemia as cause of a marked bilirubin increase in primary biliary cirrhosis]. / Hämolytische Anämie als Ursache eines auffälligen Bilirubinanstieges bei primärer biliärer Zirrhose.

HISTORY AND CLINICAL FINDINGS: 16 years ago a now 53-year-old woman was found to have primary biliary cirrhosis. 5 years later, after bleeding from oesophageal varices, she had a portacaval shunt. For several years she had been taking ursodeoxycholic acid (750 mg daily). Because of steadily increasing jaundice over the past few years she presented for possible liver transplantation. INVESTIGATIONS: There was a discrepancy between the markedly raised serum bilirubin concentration (7.8 mg/dl) and the only slightly raised or normal activities of alkaline phosphatase (247 U/l) and gamma-GT (21 U/l). Further tests confirmed that the patients had not only PBC but also Coombs-negative haemolytic anaemia (haemoglobin 10.7 g/dl, reticulocyte count 122/1000, indirect bilirubin 6.4 mg/dl, haptoglobin not demonstrated, lactate dehydrogenase 316 U/l). She had splenomegaly despite the portacaval shunt. Blood smear revealed spherocytes, but hereditary spherocytosis was not confirmed. TREATMENT AND COURSE: A six-week interruption of taking ursodeoxycholic acid led, as expected, to a rise in the activities of serum alkaline phosphatase and gamma-GT, while haemolysis parameters were not affected. CONCLUSION: Serum bilirubin concentration is a decisive prognostic factor in the course of primary biliary cirrhosis and is therefore of particular relevance for the indication of liver transplantation. The reported case demonstrates the importance of considering other causes of hyperbilirubinaemia.

[Primary sclerosing cholangitis: conventional and quantitative liver function tests during long-term therapy with ursodeoxycholic acid]. / Primär sklerosierende Cholangitis: Konventionelle und quantitative Leberfunktionstests unter einer mehrjährigen Therapie mit Ursodesoxycholsäure.

Primary sclerosing cholangitis, a chronic cholestatic liver disease, frequently leads to an impairment of liver function. In nine men and two women, aged 23 to 57 years, we prospectively studied for three to six years the effect of treatment with ursodeoxycholic acid (UDCA) on liver function. 10 mg UDCA/kg bw significantly reduced serum activities of AP, gamma GT, AST and ALT for several years. After three years of treatment, however, serum concentration of bilirubin was higher than before therapy in eight out of eleven patients (1.8 +/- 0.8 versus 0.9 +/- 0.1 mg/dl; p = 0.01). Likewise, serum concentration of bilirubin was higher in eight out of nine patients after four years of treatment (1.3 +/- 0.3 versus 0.9 +/- 0.1 mg/dl; p = 0.03). In most cases, however, the increase was discrete. Parameters of synthetic liver function (coagulation, serum protein concentration, serum activity of cholinesterase) remained constant in the observation time. Quantitative liver function tests (galactose elimination capacity and indocyanine green half-life) also showed little variation in the observation time. We conclude that UDCA treatment significantly improves serum activities of liver enzymes for several years. Nevertheless, serum bilirubin concentration, believed to be of prognostic value in patients with PSC, seems to rise slowly over time. Serial determinations of galactose elimination capacity and indocyanine green halflife are not superior to conventional liver function tests in the timing of liver transplantation in the individual patient.

Major pitfalls in Doppler investigations. Part II. Low flow velocities and colour Doppler applications.

Many pitfalls result from the limited ability of Doppler instruments to record low flow velocities. These include a misleading resistance or pulsatility index due to diastolic cut-off and taking no signal to equal no flow assuming that no signal means no flow. Comparison of actual flow velocities as measured in an in-vitro system (range: 0.8 to 3.4 cm/s) with the lowest recordable spectral or colour signals in 3 Duplexscanners showed that reduced sensitivity to low flow velocities is not only dependent on the high pass ("wall") filter setting, Doppler frequency or angle of incidence, but also on factors such as vessel diameter, impairing the signal to noise ratio. Characteristic errors of colour flow mapping (misleading vascular anatomy, imitation of pathological findings, erroneous exclusion of flow) are due to partial volume effect, limited temporal and velocity resolution, changing angle of incidence, aliasing and failure to detect low flow velocities.

New method for determination of efficacy of health care personnel hand wash products.

A method of studying the effects of health care personnel hand wash products is described. The fingernail regions of the hands of volunteers are inoculated with a mixture of Escherichia coli and Serratia marcescens, and the areas are dried for a standard time. After routine hand washing, each fingernail region is individually scrubbed with an electric toothbrush which moves longitudinally to the handle into collection fluid contained in a petri dish. The test bacteria in the fluid are then enumerated. (Bacillus subtilis spores may be included as tracers to show degree of physical removal of the procedure.) This method has several advantages over the frequently used glove juice technique. Experimental designs with large numbers of volunteers, multiple sampling sites, and many hand wash products may be performed. Ten sampling sites (fingers) are available, versus the two gloved hands for testing products. (Efficiency is almost 100% in the recovery of spore tracers placed on the fingernails.) Many commercial health care personnel hand wash products containing antimicrobial agents substantive to the skin do not rapidly reduce numbers of inoculated bacteria in the fingernail regions to any greater extent than nonantimicrobial hand washes. Products containing isopropanol or ethanol are very effective in decreasing bacteria in areas around and under the fingernails.

Semiautomated ring carrier to facilitate and expedite disinfectant testing by the AOAC use-dilution method.

The AOAC use-dilution test requires the replicate examination of a number of contaminated metal rings. Ten metal rings may be tested within the 20 min structure of the test. From a statistical and economical viewpoint, it would be highly desirable to increase the number of rings examined in a single test. A semiautomated ring carrier permits testing 200 rings within a 20 min test.

Virus survival on inanimate surfaces.

The persistence of several types of viruses on hard, inanimate surfaces under different relative humidities, temperatures, and types of surfaces was investigated. No differences in survival on glass, vinyl asbestos tile, ceramic tile, and stainless steel were found. Under conditions of low humidity and room temperature, adenovirus, poliovirus, and herpes simplex virus survived for at least 8 weeks. Vaccinia and coxsackie viruses survived for at least 2 weeks but differences due to surfaces found in many environments, in addition to the laboratory, emphasizes the possible role of hard surfaces in the transmission of viruses.