Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

We report on multicolor fluorescence in situ hybridization protocols for the simultaneous visualization of deletion-prone regions for carrier detection of Duchenne/ Becker (DMD/BMD) muscular dystrophy. Cosmid and yeast artificial chromosome (YAC) clones specific for preferentially deleted subregions of the dystrophin gene were labeled differentially and detected with three different fluorochromes using digital imaging microscopy. This approach allows for an assessment of the carrier status of female relatives even in families where no index patient is available. Cosmid and YAC clones, and different probe-generation protocols are compared with respect to their feasibility for carrier detection. The use of histone-depleted interphase nuclei (Halo-preparations) for deletion mapping is demonstrated and shown to have a resolution power of 5 kb

Mapping of Multiple DNA Gains and Losses in Primary Small Cell Lung Carcinomas by Comparative Genomic Hybridization

Comparative genomic hybridization was applied for a comprehensive screening of under- and overrepresentation of genetic material in 13 autoptic small cell lung cancer specimens. The most abundant genetic changes include DNA losses of chromosome arms 3p, 5q, 10q, 13q, and 17p and DNA gains of 3q, 5p, 8q, and 17q. Amplification sites in these tumors were mapped to 22 chromosome bands. The most frequently involved band was 19q13.1 (4 cases). Bands 1p32, 2p23, 7q11.2, 8q24, and 13q33–34 were involved in two cases each

Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumors by comparative genomic hybridization after universal DNA-amplification

We present a technique which allows the detection and chromosomal localization of DNA sequence copy number changes in solid tumor genomes from frozen sections and paraffin embedded, formalin fixed specimens. Based on comparative genomic hybridization and on universal DNA amplification procedures this technique is possible even if only a few tumor cells are available. We demonstrate the feasibility of this method to visualize complete and partial chromosome gains and losses and gene amplifications In archived solid tumor samples

Acute kidney injury in septua- and octogenarians after cardiac surgery

Background An increasing number of septua- and octogenarians undergo cardiac surgery. Acute kidney injury (AKI) still is a frequent complication after surgery. We examined the incidence of AKI and its impact on 30-day mortality. Methods A retrospective study between 01/2006 and 08/2009 with 299 octogenarians, who were matched for gender and surgical procedure to 299 septuagenarians at a university hospital. Primary endpoint was AKI after surgery as proposed by the RIFLE definition (Risk, Injury, Failure, Loss, End-stage kidney disease). Secondary endpoint was 30-day mortality. Perioperative mortality was predicted with the logistic European System for Cardiac Operative Risk Evaluation (EuroSCORE). Results Octogenarians significantly had a mean higher logistic EuroSCORE compared to septuagenarians (13.2% versus 8.5%; p < 0.001) and a higher proportion of patients with an estimated glomerular filtration rate (eGFR) < 60 ml × min-1 × 1.73 m-2. In contrast, septuagenarians showed a slightly higher median body mass index (28 kg × m-2 versus 26 kg × m-2) and were more frequently active smoker at time of surgery (6.4% versus 1.6%, p < 0.001). Acute kidney injury and failure developed in 21.7% of septuagenarians and in 21.4% of octogenarians, whereas more than 30% of patients were at risk for AKI (30% and 36.3%, respectively). Greater degrees of AKI were associated with a stepwise increase in risk for death, renal replacement therapy and prolonged stays at the intensive care unit and at the hospital in both age groups, but without differences between them. Overall 30-day mortality was 6% in septuagenarians and 7.7% in octogenarians (p = 0.52). The RIFLE classification provided accurate risk assessment for 30-day mortality and fair discriminatory power. Conclusions The RIFLE criteria allow identifying patients with AKI after cardiac surgery. The high incidence of AKI in septua- and octogenarians after cardiac surgery should prompt the use of RIFLE criteria to identify patients at risk and should stimulate institutional measures that target AKI as a quality improvement initiative for patients at advanced age

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in ≥90% of diploid nuclei Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle