Anastomotic Leakage After Stoma Reversal Combined with Incisional Hernia Repair.

BACKGROUND: Stoma reversal in patients with an incisional hernia represents a clinical dilemma, as it remains unknown whether hernia repair should be concomitantly employed. We aimed at examining postoperative complications and mortality in patients undergoing stoma reversal with or without concomitant hernia repair. METHODS: This study included all patients subjected to stoma reversal between 2010 and 2016 at our institution. Patients were grouped according to conductance of concomitant incisional hernia repair or not. The primary outcome was anastomotic leak (AL). Secondary outcomes were surgical site occurrences (SSO), overall surgical complications, 90-day mortality and overall survival. RESULTS: In total, 142 patients were included of whom 18 (13%) underwent concomitant hernia repair. The incidence of AL was significantly higher in patients subjected to concomitant hernia repair (four out of 18 [22.2%]) compared with patients undergoing stoma reversal alone (three out of 124 [2.4%], P = 0.002). Additional variables associated with AL were duration of surgery (P < 0.001) and ischemic heart disease (P = 0.039). Twenty-two patients (15.5%) developed a SSO: eight (44.4%) in the hernia repair group and 14 (11.3%) in the non-hernia repair group (P < 0.001). In the multivariable analysis, concomitant hernia repair remained significantly associated with development of postoperative complications (OR = 5.92, 95% CI = 1.54-25.96, P = 0.012). CONCLUSIONS: Compared with stoma reversal alone, incisional hernia repair concomitant with stoma reversal was associated with a higher incidence of AL and other complications.

Rapid identification of compound mutations in patients with Philadelphia-positive leukaemias by long-range next generation sequencing.

An emerging problem in patients with Philadelphia (Ph)-positive leukaemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukaemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukaemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. Polymerase chain reaction (PCR) amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease.

On the trail of anti-CDE to unexpected highlights of the RHD*weak 4.3 allele in the Upper Austrian population.

BACKGROUND AND OBJECTIVES: The application of a commercial available microcolumn system for ABO/RH determination lead to irregular results in CDE typing of seemingly D- blood samples. In this study, we introduce a comprehensive serological and molecular work-up of a novel haplotype carrying the RHD*weak 4.3 in combination with an aberrant RHCE*ce. MATERIALS AND METHODS: The molecular background was characterized by RHD and RHCE-specific DNA sequencing, RHD cDNA sequencing and RHD zygosity testing. Haplotype-specific extraction and inheritance analysis were initiated to determine the linkage of the polymorphisms. The genetic admixture was studied by whole genome SNP array analysis. Serology was done using commercial available standard techniques and by in-house sera likewise. RESULTS: All samples (n = 29) were shown to harbour an altered RHD(T201R, F223V, P291R) allele known as RHD*weak 4.3 associated with a RHCE*ce(W16C, A36T, L245V) gene formation, expressing C(X) and VS. Both anti-C(X) and anti-V/VS were detected as contaminating antibodies in a commercial available microcolumn system for ABO/RH determination accounting for the positive results in CDE typing. Compared with other population data, the samples were clearly identified as Caucasian. CONCLUSION: The RHD*weak 4.3 allele with markedly reduced antigen D expression was shown to be associated with an altered RHCE gene formation leading to the expression of C(X) and VS. Its frequency was estimated 1 in 854 among apparently D- Upper Austrian blood donors.

Overcoming methodical limits of standard RHD genotyping by next-generation sequencing.

BACKGROUND AND OBJECTIVES: Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence-based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing. MATERIALS AND METHODS: Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing. RESULTS: Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHDψ and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing. CONCLUSION: Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set-up have to be undertaken prior to validation and introduction as a routine service.

The reactivity of N-coordinated amides in metallopeptide frameworks: molecular events in metal-induced pathogenic pathways?

The amino acid derived tertiary amide ligand tert-butoxycarbonyl-(S)-alanine-N,N-bis(picolyl)amide (Boc-(S)-Ala-bpa, 1) has been synthesized as a model for metal-coordinating peptide frameworks. Its reactions with copper(II) and cadmium(II) salts have been studied. Binding of Cu2+ results in amide bond cleavage and formation of [(bpa)(solvent)Cu]2+ complexes. In contrast, the stable, eight-coordinate complex [(Boc-(S)-Ala-bpa)Cd(NO3)2] (5) has been isolated and characterized by X-ray crystallography. An unusual tertiary amide nitrogen coordination is observed in 5; this gives rise to significantly reduced cis-trans isomerization barriers. Possible implications for metal-induced conformational changes in proteins are discussed.