Liv Furuberg

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

In vitro diagnostic platforms of the future; technological possibilities and challenges

Micro- and nanotechnologies are utilized in the new generation of diagnostic platforms for in vitro analyses. Complex analyses that need a sequence of manual or instrumental process steps in the laboratories can be automated by controlling microfluidic flows of samples and reagents in polymer chips. The biochemistries are adapted to small volume reactions. New and sensitive detection principles will be able to detect down to a few copies of biomarkers per sample. We present examples of platforms analyzing patient samples for proteins and nucleic acids, and some of the challenges involved in volume manufacturing of these platforms.

The MicroActive project : automatic detection of disease-related molecular cell activity

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.

3D Modelling of MEMS Components for Coupled Elastic — Electrostatic Characterization

This paper presents a method for modelling MEMS components whereby the designer can perform precise electrical, mechanical, and coupled electrical and mechanical simulations. Both surface and bulk micromachined components can be modelled at a geometrical level by defining simplified pseudo processes for a commercial CAD tool such as CovertorWare. The procedure for this approach is described with emphasis on the bulk case. A motivating example of a simple comb-finger structure in an accelerometer shows that this method is required to obtain adequate precision. A long thin bulk micromachined mirror array, which is not easily described analytically, was modelled by using a pseudo process and the coupled field problem of its elastic-electrostatic behaviour has been investigated.

Dynamics and structure of displacement fronts in two-dimensional porous media

The dynamics and structure of drainage in 2D porous media are discussed. We restrict the discussion to the extreme cases of slow injection rates, where capillary forces govern the dispacement, and fast injection rates, where viscous forces dominate the process.

Scaling of Overhang Distribution of Invasion Percolation Fronts

We analyse simulations of invasion percolation with a gradient in two dimensions. The fronts of the invaded structures are examined using the overhang size distribution introduced by Hansen et al. [1]. We observe that overhang sizes h are distributed according to a power law: nh ~ h-a where a is measured to be a = 2.3 ± 0.1. The fractal dimension of the external perimeter of invasion percolation clusters is known to be De 1.35. We argue that in general a = Da + 1, where Da is the fractal dimension of the perimeter sampled by particles coming from outside the cluster and moving in straight lines paralles to the gradient and the overhangs.

Growth Patterns and Fronts: Fluid Flow Experiments

Patterns and fronts arise in most fluid flow situations. Waves, clouds, convection patterns and turbulence are well known examples. In porous media the displacement of one fluid by another fluid-leads to many new, often fractal,[1,2] fronts and patterns. The disorder of the porous matrix plays a key role that is not well understood. Depending on the displacement rates, viscosity ratios, miscibility, interfacial tensions and pore geometry a bewildering variety of displacement fronts arise. Lenormand[3] has studied many of the regimes observed under various conditions during two-fluid displacement processes in micromodels of porous media.

Dynamics of Invasion and Dispersion Fronts

The displacement of one fluid by a another fluid in a porous medium is a process of both scientific and practical importance. Depending on the displacement rates, viscosity ratios, irascibility, interfacial tensions and pore geometry a bewildering variety of displacement front behaviors arises. Lenormand1–4 has studied many of the regimes observed under various conditions during two-fluid displacement processes in micromodels of porous media.

Self-Avoiding Walks Between Terminals on Percolation Clusters

Many natural structures exhibit fractal geometry, and much recent interest has been devoted to studying the physical properties of such structures [1]. In the fractal regime, many of these properties depend on the length scale L via power laws, e.g. X(L) ~ L x . Often, one needs an infinite set of exponents, {x}, in order to fully characterize the structure [1]. We find that the various self-avoiding paths connecting two terminals on a percolating cluster scale with the Euclidean distance between the terminals. The different paths scale with different exponents forming a continuous spectrum.