Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

<p>Abstract</p> <p>Background</p> <p>High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL).</p> <p>Results</p> <p>We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 – 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 – 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis.</p> <p>Conclusion</p> <p>This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.</p

Population Structure of Humpback Whales from Their Breeding Grounds in the South Atlantic and Indian Oceans

Although humpback whales are among the best-studied of the large whales, population boundaries in the Southern Hemisphere (SH) have remained largely untested. We assess population structure of SH humpback whales using 1,527 samples collected from whales at fourteen sampling sites within the Southwestern and Southeastern Atlantic, the Southwestern Indian Ocean, and Northern Indian Ocean (Breeding Stocks A, B, C and X, respectively). Evaluation of mtDNA population structure and migration rates was carried out under different statistical frameworks. Using all genetic evidence, the results suggest significant degrees of population structure between all ocean basins, with the Southwestern and Northern Indian Ocean most differentiated from each other. Effective migration rates were highest between the Southeastern Atlantic and the Southwestern Indian Ocean, followed by rates within the Southeastern Atlantic, and the lowest between the Southwestern and Northern Indian Ocean. At finer scales, very low gene flow was detected between the two neighbouring sub-regions in the Southeastern Atlantic, compared to high gene flow for whales within the Southwestern Indian Ocean. Our genetic results support the current management designations proposed by the International Whaling Commission of Breeding Stocks A, B, C, and X as four strongly structured populations. The population structure patterns found in this study are likely to have been influenced by a combination of long-term maternally directed fidelity of migratory destinations, along with other ecological and oceanographic features in the region

Patients with multiple myeloma treated with thalidomide: Evaluation of clinical parameters, cytokines, angiogenic markers, mast cells and marrow CD57+ cytotoxic T cells as predictors of outcome

Background and Objectives: In vitro studies suggest that thalidomide has an immunoregulatory role and alters the marrow microenvironment. We assessed laboratory and clinical parameters in patients with myeloma treated with thalidomide as potential prognostic markers and looked for changes with therapy. Design and Methods: Seventy-five patients with relapsed/refractory myeloma received thalidomide in a phase II trial. Serial samples of platelet-poor plasma and bone marrow were tested for angiogenic cytokines including vascular endothelial growth factor (VEGF), marrow microvessel-density (MVD), mast cells and CD57 cell expression. The effects of these parameters on response rate (RR), progression-free survival (PFS) and overall survival (OS) were analyzed. Results: Elevated baseline VEGF predicted for a superior RR (p=0.018) and PFS. Elevated CD57 cells also predicted superior PFS (p=0.012). MVD did not predict for RR, PFS or OS, but MVD and VEGF fell significantly in responders. Multivariate analysis identified that inferior OS was associated with age >65 years (p=0.017), raised lactate dehydrogenase (p=0.001), raised hepatocyte growth factor levels (p=0.012) and low pre-treatment CD57 cells (

The addition of rituximab to fludarabine and cyclophosphamide chemotherapy results in a significant improvement in overall survival in patients with newly diagnosed mantle cell lymphoma: results of a randomized UK National Cancer Research Institute trial

Mantle cell lymphoma is an incurable and generally aggressive lymphoma that is more common in elderly patients. Whilst a number of different chemotherapeutic regimens are active in this disease, there is no established gold standard therapy. Rituximab has been used widely to good effect in B-cell malignancies but there is no evidence that it improves outcomes when added to chemotherapy in this disease. We performed a randomized, open-label, multicenter study looking at the addition of rituximab to the standard chemotherapy regimen of fludarabine and cyclophosphamide in patients with newly diagnosed mantle cell lymphoma. A total of 370 patients were randomized. With a median follow up of six years, rituximab improved the median progression-free survival from 14.9 to 29.8 months (P&lt;0.001) and overall survival from 37.0 to 44.5 months (P=0.005). This equates to absolute differences of 9.0% and 22.1% for overall and progression-free survival, respectively, at two years. Overall response rates were similar, but complete response rates were significantly higher in the rituximab arm: 52.7% vs. 39.9% (P=0.014). There was no clinically significant additional toxicity observed with the addition of rituximab. Overall, approximately 18% of patients died of non-lymphomatous causes, most commonly infections. The addition of rituximab to fludarabine and cyclophosphamide chemotherapy significantly improves outcomes in patients with mantle cell lymphoma. However, these regimens have significant late toxicity and should be used with caution. This trial has been registered (ISRCTN81133184 and clinicaltrials.gov:00641095) and is supported by the UK National Cancer Research Network.</p

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry : An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and 97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society

Ensuring high quality and efficiency of the worksin the process of constructing the tunnels of in-situ concrete

In the article the author describes the importance of the technological regulations development in the process of constructing various transport constructions: tunnels, subways, bridges and other important objects. In the article the peculiarities of the technological regulations development are fully taken into account; the dependence of the depth of their development and the quality of the concrete constructions, as well as the speed of the objects of transport infrastructure construction, including the examples of building the road tunnels in Moscow. The course of their development is shown with account for the main provisions, which should be included in technological regulations in order to ensure the most complete coverage of the issues arising in engineering, laboratory and Supervisory structure in the process of performing the works. The author proposes new effective materials and technologies of works. In particular, sufficient attention is paid to self-compacting concrete — a new type of concrete, which is able to flow and compact under its own weight, completely filling the formwork even in case of dense reinforcement, while maintaining the homogeneity and having no seals. The application experience of concrete self-sealing in the construction of the metro showed that labor costs for the concrete mixture sealing were 5-6 times reduced, and the speed of laying the concrete increased 2-3 times. When laying self-compacting concrete high-quality surfaces are formed, which do not require additional costs to bring them to the design parameters. In addition, the work shows the parameters of the technological processes and sets various types of works sequence: the article describes the features of formwork, placement and curing of the concrete in terms of year-round construction, shows the importance of thermo physical calculations of concrete hardening and the efficiency of using self-sealing concrete. Sufficient attention is also paid to the methods of quality assurance and to the methods of preventing cracking of various structural elements of a construction, as well as to the safety requirements and ensuring proper protection of the environment

Prognostic indices in diffuse large B-cell lymphoma in the rituximab era:an analysis of the UK National Cancer Research Institute R-CHOP 14 versus 21 phase 3 trial

We compared the International Prognostic Index (IPI), Revised (R)‐IPI and age‐adjusted (aa)‐IPI as prognostic indices for patients with diffuse large B‐cell lymphoma (DLBCL) in the UK National Cancer Research Institute (NCRI) R‐CHOP 14 versus 21 trial (N = 1080). The R‐IPI and aa‐IPI showed no marked improvement compared to the IPI for overall and progression‐free survival, in terms of model fit or discrimination. Similar results were observed in exploratory analyses incorporating the Grupo Español de Linfomas/Transplante de Médula Ósea (GELTAMO)‐IPI, where baseline β2‐microglobulin data were available (N = 655). Although our findings support current use of the IPI, a novel prognostic tool to better delineate a high‐risk DLBCL group in the rituximab era is needed