Prognostic factors influencing infectious complications after cytoreductive surgery and HIPEC. Results from a tertiary referral center

Background. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) showed promising results in selected patients. High morbidity restrains its wide application. The aim of this study was to report postoperative infectious complications and investigate possible correlations with preoperative nutritional status and other prognostic factors in patients with peritoneal metastases treated with CRS and HIPEC. Methods. For the study we reviewed the clinical records of all patients with peritoneal metastases from different primary cancers and treated by CRS and HIPEC in our Institution from November 2000 to December 2017. Patients were divided according to their nutritional status (SGA) in group A (well-nourished), B/C (mild or severely malnourished). Possible statistical correlations between risk factors and postoperative complications rates have been investigated by univariate and multivariate analysis. Results. Two hundred patients were selected and underwent CRS and HIPEC during the study period. Postoperative complications occurred in 44% of the patients, 35.3% in SGA-A patients and 53% in SGA-B /C patients. Cause of complications was infective in 42, non-infective in 37 and HIPEC related in 9 patients. Infectious complications occurred more frequently in SGA-B /C patients (32.6% vs. 9.8% of SGA-A patients). The most frequent sites of infection were Surgical Site Infections (SSI, 35.7%) and Central Line Associated BloodStream Infections (CLABSI, 26.2%). The most frequent isolated species was Candida (22.8%). ASA score, blood loss, performance status, PCI, large bowel resection, postoperative serum albumin levels and nutritional status correlated with higher risk for postoperative infectious complications. Conclusions. Malnourished patients undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy are more prone to post-operative infectious complications and adequate perioperative nutritional support should be considered, including immune-enhancing nutrition. Sequential monitoring of common sites of infection, antifungal prevention of candidiasis, and careful patient selection should be implemented to reduce complications rate

Hepatitis C Virus Drives the Unconstrained Monoclonal Expansion of VH1–69-Expressing Memory B Cells in Type II Cryoglobulinemia: A Model of Infection-Driven Lymphomagenesis

AbstractChronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the VH1–69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of VH1–69+ cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of VH1–69+ B cells: naive (small, IgMhighIgDhighCD38+CD27−CD21highCD95−CD5−), "early memory" (medium-sized, IgMhighIgDlowCD38−CD27+CD21lowCD95+CD5+), and "late memory" (large-sized, IgMlowIgDlow-negCD38−CD27lowCD21low-negCD5−CD95−). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated VH1–69+ B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal VH1–69+ B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a VH1–69+ natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

Background: Current methods for multiparameter DNA flow cytometry suffer from several Limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. Methods: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degreesC. Results: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. Conclusions: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward. Cytometry 44:120-125, 2001. (C) 2001 Wiley-Liss, Inc

Correlation between terminal restriction fragments and flow-FISH measures in samples over wide range telomere lengths

ObjectivesTerminal restriction fragment (TRF) analysis of human telomeres was used to calibrate flow-fluorescence in situ hybridization (FF) measures of telomere lengths to expand the range of measures and increase power of resolution of our previously published protocol. TRF data used as the gold standard should be obtained by electrophoresis with suitable resolution applied to appropriately isolated genomic DNA. When we considered TRF attained by correct methods, we found our method to be insufficiently accurate, thus we have reviewed our previously published FF protocol to obtain the best coefficient of determination (r(2)) between our experimental results and valid TRF lengths. Materials and methodsUsing human telomere-specific PNA probe, Cy5-OO-(CCCTAA)(3), we measured telomere lengths of continuous cell line and of peripheral blood lymphocytes by FF. We modified hybridization, stringency, negative control handling, stoichiometric DNA staining and telomere fluorescence assessment of the protocol. ResultsWe realized a procedure with increased power of resolution, improved TRF versus FF r(2) values that allowed simultaneous analysis of DNA and telomere duplication. Notwithstanding multiple steps in formamide sampling, recovery was satisfactory. DiscussionThe reviewed FF protocol appeared at least as suitable as the TRF method. Measures obtained by TRF can be affected by chromosome end variability, DNA fragmentation, incomplete digestion and unsuitable electrophoresis. In contrast, the FF technique analyses telomeric sequences confined to preserved nuclei thus overcome most previous limitations. As yet, however, the FF telomere measure cannot be performed together with immunophenotyping and/or generation study by the dye dilution method

Flow Cytometry in Formamide Treated Cells

The use of formamide for the study in flow cytometry of cell cycle phases, by DNA content measurement in human cancer cell lines, was recently published. In this manuscript we verify the possibility of extending the procedure to simultaneous analysis of other parameters. The results obtained, here reported, show that the treatment of samples by formamide is compatible with the simultaneous detection of DNA content and surface phenotypes, with quantification of replicating DNA and with measurement of cells with fractional content of DNA. For each of these three applications we have adapted the procedure in order to gain simple, reproducible and above all advantageous protocols. Regarding the simultaneous analysis of DNA content and phenotyping the use of formamide achieves optimal DNA stoichiometric staining (C.V.<3; G2/G1 ratio = 2 ± 0.05) and sufficient maintenance of physical parameters and membrane fluorescence. In the study of duplicating DNA labeled with click chemistry, our procedure eliminates paraformaldehyde (PFA) fixation improving the DNA stoichiometric staining and allows the use of 7-aminoactinomycin D (7-AAD) preserving the Alexa Fluor 488 quantum efficiency. Concerning the detection of cells with fractional content of DNA, permeabilization and fixation by formamide gives the advantage of resolve on linear scale sub-G1 cells from debris and to allow optimal sample recovery (>90%) which is essential in the study of cell necrobiology. Cells treatment by formamide, suitably modified for different applications, can be used to prepare cell samples for flow cytometry analyses that go far beyond of stoichiometric staining of DNA

OPTIMIZATION OF PCR PERFORMANCE

[No abstract available

Improved Procedure for the Measurement of Telomere Length in Whole Cells by PNA Probe and Flow Cytometry

OBJECTIVES: Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo-PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. MATERIALS AND METHODS: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere-specific PNA probe conjugated with Cy5 fluorochrome, Cy5-OO-(CCCTAA)(3) . After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. RESULTS: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo-PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. CONCLUSION: According to our experience, oligo-PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells