[Evaluation of data on surgical complications after cholecystectomy submitted to a nationwide quality assurance program (BQS) in Germany]. / Qualität der BQS-Dokumentation: Datenevaluation anhand intra- und postoperativer Komplikationen nach Cholezystektomien.

BACKGROUND: The BQS is a nationwide quality assurance program in Germany. The aim was to evaluate the data quality on intra-operative and postoperative complications for cholecystectomy submitted to the BQS. PATIENTS AND METHODS: For 205 patients who underwent cholecystectomy in 2007 complications were retrospectively evaluated and compared with those documented in the BQS database. Additionally the risk factors for complications were documented. RESULTS: A total of 205 patients were included in the study. In 88% of patients the operations were initiated as laparoscopy and the conversion rate was 8.3%. Of the patients 28 suffered from intra-operative or postoperative complications. There were no injuries to the ductus hepatocholedochus (DHC). The most common operation-specific complications were disorders in wound healing (n=7). Multivariate analyses resulted in significant increases in complication rates for ASA status (Odds ratio 3.29, 95% confidence interval 2.12-5.10, p<0.01) and acute cholecystitis (odds ratio 7.71, 9% confidence interval 2.96-20.08, p<0.01). Only 13 patients out of 28 were correctly documented in the BQS database (p<0.01). Sensitivity and specificity for complications were 46 and 98%, respectively. CONCLUSIONS: Less than half of all cases were correctly documented in the BQS database. If documentation inthe BQS database was equally poor for all German surgical departments, neither benchmarking nor general conclusions on quality of surgical care could be drawn from the BQS data.

Non-radioactive oligotyping for HLA-DR1-DRw10 using polymerase chain reaction, digoxigenin-labelled oligonucleotides and chemiluminescence detection.

We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.

Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2'-3'-dideoxy-uridinetriphosphate-labeled oligonucleotide probes.

We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3' end with only one DIG using digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antidigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.

Analysis of biological and biochemical parameters for chromatin and nuclear matrix association of SV40 large T antigen in transformed cells.

We analysed biological and biochemical parameters for the association of the simian virus 40 (SV40) large tumor antigen (large T) with the cellular chromatin and the nuclear matrix in SV40-transformed cells. Nuclear subclasses of large T were isolated by in situ cell fractionation (Staufenbiel & Deppert, 1983) and first analysed for possible biological functions in the maintenance of cellular transformation. Like large T in SV40 wild-type transformants, large T in SV40 tsA mutant (tsA58)-transformed cells, expressing a temperature-dependent phenotype, was present in all nuclear subfractions (nucleoplasm, chromatin and nuclear matrix), when cells were kept at the growth temperature permissive for the expression of the transformed phenotype (32 degrees C). When tsA mutant-transformed cells were shifted to the non-permissive growth temperature (39 degrees C), they reverted to the normal phenotype. Concomitantly, large T lost its ability to associate with the cellular chromatin and the nuclear matrix, indicating that an association of large T with these subcellular structures may be important for the maintenance of cellular transformation. We next analysed the DNA-binding properties (sequence-specific binding to the SV40 origin of replication, ORI) of the nuclear subclasses of SV40 wild-type and of SV40 mutant large T defective in SV40 ORI binding in order to determine the influence of sequence-specific DNA binding on the association of large T with the chromatin and the nuclear matrix. Our detailed analyses show distinct differences in the ability of the various nuclear subclasses of large T to bind to the SV40 ORI, but suggest that the association of large T with the chromatin and the nuclear matrix is mediated by protein-protein interactions rather than by sequence-specific DNA binding.

A new sensitive target-bound DNA binding assay for SV40 large T antigen.

We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI.