Filter characteristics influencing circulating tumor cell enrichment from whole blood.

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ~25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the syste

Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104:102:1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ~10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC

Unbiased and automated identification of a circulating tumour cell definition that associates with overall survival

Circulating tumour cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC however remains subjective, as they are morphologically heterogeneous. We acquired digital images, using the CellSearch™ system, from blood of 185 castration resistant prostate cancer (CRPC) patients and 68 healthy subjects to define CTC by computer algorithms. Patient survival data was used as the training parameter for the computer to define CTC. The computer-generated CTC definition was validated on a separate CRPC dataset comprising 100 patients. The optimal definition of the computer defined CTC (aCTC) was stricter as compared to the manual CellSearch CTC (mCTC) definition and as a consequence aCTC were less frequent. The computer-generated CTC definition resulted in hazard ratios (HRs) of 2.8 for baseline and 3.9 for follow-up samples, which is comparable to the mCTC definition (baseline HR 2.9, follow-up HR 4.5). Validation resulted in HRs at baseline/follow-up of 3.9/5.4 for computer and 4.8/5.8 for manual definitions. In conclusion, we have defined and validated CTC by clinical outcome using a perfectly reproducing automated algorithm

Is there still a role for nuchal translucency measurement in the changing paradigm of first trimester screening?

Objectives To give an overview of the genetic and structural abnormalities occurring in fetuses with nuchal translucency (NT) measurement exceeding the 95th percentile at first-trimester screening and to investigate which of these abnormalities would be missed if cell-free fetal DNA (cfDNA) were used as a first-tier screening test for chromosomal abnormalities. Methods This is a national study including 1901 pregnancies with NT &gt;= 95th percentile referred to seven university hospitals in the Netherlands between 1 January 2010 and 1 January 2016. All cases with unknown pregnancy outcome were excluded. Results of detailed ultrasound examinations, karyotyping, genotyping, pregnancy and neonatal outcomes, investigation by a clinical geneticist and post-mortem investigations were collected. Results In total, 821 (43%) pregnancies had at least one abnormality. The rate of abnormalities was 21% for fetuses with NT between 95(th) and 99(th) percentile and 62% for fetuses with NT &gt;= 99(th) percentile. Prevalence of single-gene disorders, submicroscopic, chromosomal and structural abnormalities was 2%, 2%, 30% and 9%, respectively. Conclusion Although cfDNA is superior to the combined test, especially for the detection of trisomy 21, 34% of the congenital abnormalities occurring in fetuses with increased NT may remain undetected in the first trimester of pregnancy, unless cfDNA is used in combination with fetal sonographic assessment, including NT measurement.</p

An experimentally-validated numerical model of diffusion and speciation of water in rhyolitic silicate melt

The diffusion of water through silicate melts is a key process in volcanic systems. Diffusion controls the growth of the bubbles that drive volcanic eruptions and determines the evolution of the spatial distribution of dissolved water during and after magma mingling, crystal growth, fracturing and fragmentation, and welding of pyroclasts. Accurate models for water diffusion are therefore essential for forward modelling of eruptive behaviour, and for inverse modelling to reconstruct eruptive and post-eruptive history from the spatial distribution of water in eruptive products. Existing models do not include the kinetics of the homogeneous species reaction that interconverts molecular () and hydroxyl () water; reaction kinetics are important because final species distribution depends on cooling history. Here we develop a flexible 1D numerical model for diffusion and speciation of water in silicate melts. We validate the model against FTIR transects of the spatial distribution of molecular, hydroxyl, and total water across diffusion-couple experiments of haplogranite composition, run at 800–1200°C and 5 kbar. We adopt a stepwise approach to analysing and modelling the data. First, we use the analytical Sauer-Freise method to determine the effective diffusivity of total water as a function of dissolved water concentration and temperature for each experiment and find that the dependence of on is linear for wt.% and exponential for wt.%. Second, we develop a 1D numerical forward model, using the method of lines, to determine a piece-wise function for that is globally-minimized against the entire experimental dataset. Third, we extend this numerical model to account for speciation of water and determine globally-minimized functions for diffusivity of molecular water and the equilibrium constant for the speciation reaction. Our approach includes three key novelties: 1) functions for diffusivities of and , and the speciation reaction, are minimized simultaneously against a large experimental dataset, covering a wide range of water concentration ( wt.%) and temperature (), such that the resulting functions are both mutually-consistent and broadly applicable; 2) the minimization allows rigorous and robust analysis of uncertainties such that the accuracy of the functions is quantified; 3) the model can be straightforwardly used to determine functions for diffusivity and speciation for other melt compositions pending suitable diffusion-couple experiments. The modelling approach is suitable for both forward and inverse modelling of diffusion processes in silicate melts; the model is available as a Matlab script from the electronic supplementary material

Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C0t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes

A validated numerical model for the growth and resorption of bubbles in magma

The rate and timing of bubble growth in magma is an important control on eruption style, determining whether or not magma fragments to produce an explosive eruption. Bubbles nucleate, grow, shrink, and de-nucleate in magma in response to changes in pressure and temperature, and these changes may be recorded in the spatial distribution and speciation of water 'frozen into' the glass in eruptive products. Accurate modelling of growth and resorption is therefore essential both for forward modelling of eruptive processes, and for inverse modelling to reconstruct pre-eruptive history. We present the first experimentally-validated numerical model for bubble growth and resorption in magma. The model includes the kinetics of speciation, allows for arbitrary temperature and pressure pathways, and accounts for the impact of spatial variations in water content on diffusivity and viscosity. We validate the model against three sets of data. (1) Continuous vesicularity-time data collected using optical dilatometry and in-situ synchrotron-source x-ray tomography of natural and synthetic magma during thermally-induced vesiculation and resorption at magmatic temperatures and ambient pressure. This represents approximately isobaric bubble growth and resorption under disequilibrium conditions. (2) Final vesicularity data from decompression experiments at magmatic temperatures and pressures. This represents isothermal, decompression-driven bubble growth from equilibrium to strongly disequilibrium conditions. (3) Speciation data from diffusion-couple experiments on synthetic haplogranites at magmatic temperatures and pressures. The numerical model closely reproduces all experimental data, providing validation against equilibrium and disequilibrium bubble growth/resorption and speciation scenarios. The validated model can be used to predict the growth and resorption of bubbles, and associated changes in magma properties, for arbitrary eruption pathways. It can also be used to reconstruct pressure-temperature-time pathways from textures and volatile contents of eruptive products. This will open up new ways of accessing the dynamics of magma ascent and eruption in unobserved volcanic eruptions

Evaluation of pregnancy and delivery in 13 women who underwent resection of a sacrococcygeal teratoma during early childhood

Sacrococcygeal teratoma resection often brings changes in pelvic anatomy and physiology with possible consequences for defecation, micturition and sexual function. It is unknown, whether these changes have any gynecological and obstetric sequelae. Until now four pregnancies after sacrococcygeal teratoma resection have been described and cesarean section has been suggested to be the method of choice for delivery. We evaluated the pregnancy course and mode of delivery in women previously treated for a sacrococcygeal teratoma. The records of all patients who underwent sacrococcygeal teratoma resection after 1970 in one of the six pediatric surgical centers in the Netherlands were reviewed retrospectively. Women aged 18 years and older were eligible for participation. Patient characteristics, details about the performed operation and tumor histology were retrieved from the records. Consenting participants completed a questionnaire addressing fertility, pregnancy and delivery details. Eighty-nine women were eligible for participation; 20 could not be traced. Informed consent was received from 41, of whom 38 returned the completed questionnaire (92.7%). Thirteen of these 38 women conceived, all but one spontaneously. In total 20 infants were born, 17 by vaginal delivery and 3 by cesarean section, in one necessitated by previous intra-abdominal surgery as a consequence of sacrococcygeal teratoma resection. Conversion to a cesarean section was never necessary. None of the 25 women without offspring reported involuntary childlessness. There are no indications that resection of a sacrococcygeal teratoma in female patients is associated with reduced fertility: spontaneous pregnancy is possible and vaginal delivery is safe for mother and child, irrespective of the sacrococcygeal teratoma classification or tumor histolog