Diagnostics & Imaging Week Contributing Writer
And D&IWs

NORWICH, UK – Recent efforts to optimize metabolomics technology is beginning to bear fruit in the discovery of novel biomarkers for disease. In general, the concentrations of metabolites are amplified relative to the proteins that prompted their formation, making them potentially more sensitive, both in diagnostics and as drug targets, than protein biomarkers.

"Metabolites are downstream from the protein, so you must have an amplification in concentration," Douglas Kell, director of the Manchester Centre for Integrative Systems Biology, said here at the British Association Science Festival last week. "Furthermore, you don't need to know the gene sequence, there are fewer metabolites than proteins, and they are generic, so once you can measure a metabolite, it is the same in every organism – which is not the case for proteins."

It has been estimated that there are about 3,000 metabolites in the human metabolome. The issue is that there is a wide concentration range and it is very difficult to detect metabolites that occur at low concentrations.

Kell's group has developed an automated "robot scientist" that couples gas chromatography separation to mass spectrometry detection, and has optimized and refined the equipment to the point where it can discriminate 1,800 true metabolites.

Using the robot, blood samples from healthy and affected individuals have been analyzed to systematically uncover subsets of metabolites that are markers of particular diseases. For example, examining samples from women affected by pre-eclampsia in pregnancy and those who were not led to the discovery of three metabolites that distinguish sufferers.

"None of the [three metabolites] was visible [with our equipment] when we started; it is only as we improved the process that they were detected," Kell said.

Currently, pregnant women are sifted out as being at risk of pre-eclampsia by measuring blood pressure. "But this is more than a surrogate for blood pressure: You can look in the affected cohort only and follow the development of the disease," Kell said. More recently his group has found further metabolites linked to pre-eclampsia, making the biomarkers potentially even more sensitive.

Kell has discovered metabolites for Huntington's disease and for a number of cardiovascular diseases, also. "Many of these offer the possibility of novel interventions and of prognostic detection of diseases in their earliest stages, before they become life threatening," Kell said.

Researchers track cells, keep them alive

A team from Imperial College London and Cambridge University has devised a method for imaging cells without killing them, making it possible to watch biology in action. Among the real-time processes recorded are the congregating of protein complexes, microvilli responding to changes in osmotic pressure and viruses preparing to infect a cell.

The researches have used their equipment to deliver single molecules to precise locations on the surface of cells. They have "painted" nanoscale pictures by the molecule-by-molecule application of fluorescent DNA and simulated a heart attack in a petri dish by administering a single molecule of a toxin to a cardiomyocyte, prompting it to become arrhythmic.

The technique, called Scanning Ion Conductance Microscopy (SICM) uses a micropipette to deliver a small voltage to the surface of the cell. By scanning the pipette across the cell, it is possible to create an image of the surface. No special preparation is required and the cells can be imaged on standard culture dishes.

In addition, the pipette can be used to perform nanoscale measurements on the cell surface, such as recording a single ion channel, or for the controlled addition of reagents.

"The big advantage of our technique is that it is non-contact," said David Klenerman, of Cambridge University, speaking at the BA Festival of Science here last week. "A lot of structural biology is based on examining dead cells. Now we can see live cells. It is not the highest resolution that has been achieved, but it is the highest achieved on a live cell."

While SICM is not a new technique, the researchers have produced a micropipette that is smaller than any of its predecessors, enabling the resolution to go down to 10 nanometres.

Using fluorescent DNA, Klenerman and colleagues have painted the Cambridge University crest on a cell in lines that are under one micron in width. "This demonstrates very high control of where you put molecules and shows you can use the technique to do assays on individual living cells," Klenerman said.

The team has studied epithelial kidney cells, uncovering the same features that are seen in scanning electron micrographs, but then going on to follow the life history of microvilli, showing them responding to changes in osmotic pressure. Currently, the researchers are applying the technique to visualise ion channels in neurons, and Klenerman said this is leading to the development of a smart patch clamp technique, where the characteristic on/off behaviour of ion channels can be followed live.

The micropipettes can be used also to apply pressure to a cell, pushing it down or sucking it up, without actually making contact. That means that it is possible to probe the mechanical properties of individual cells.

Micropipettes can be constructed with a double barrel, to deliver two different molecules at the same time, and it also is possible to construct them with four and eight barrels.

The technique opens up the possibility of performing cell repairs, but Klenerman said that is some way off.

"We can locally deliver reagents, but at present we are getting information about normal cells and finding out about diseased cells. You could then think about using that information to repair damaged cells."

Imperial College has spun out a company, Ionscope, to sell the equipment. Klenerman told Diagnostic & Imaging Week's sister publication, BioWorld International, that this was suitable for research purposes only at present. "It is far from an established technology: There is some way to go before it could be applied systematically. But we have shown what is possible."

Avitar tests to be used in DRUID Project

Avitar (Canton, Massachusetts), developer of a rapid, on-site, oral fluid-based screening test for drugs-of-abuse, reported that it has been selected to participate in the European Driving Under the Influence of Drugs (DRUID) Project.

Under the DRUID project, oral fluid screening devices will be tested under operational police conditions by police forces in Germany, Spain, Italy, Belgium, Denmark, and Finland.

"Avitar is pleased to be asked to participate in this important project, which is being conducted under the direction of the European Traffic Police Organization (TISPOL)," said Pete Phildius, Avitar's CEO and chairman. "As with workplace random drug testing in the United States, point-of-care [POC] oral fluid-based technology is being recognized as a key element in addressing the growing international drugged driving problem."

Avitar said that non-invasive POC technology, specifically oral fluid-based devices, in combination with applications such as road-side drug testing, is anticipated to be the catalyst for growth. It cited research from Frost & Sullivan saying that by 2009, 33% of all drugs of abuse tests will be performed on the POC format.

CE-mark approvals

• Cyntellect (San Diego) reported that its Laser-Enabled Analysis and Processing (LEAP) System has won CE-marking.

LEAP is an automated instrument that combines high-speed optical imaging of cells, real-time image analysis and high-speed laser manipulation of individual live cells presented in microplate formats. It is designed to enable researchers to rapidly and selectively laser-irradiate specific cells achieving > 99.9% purity. The in situ processing nature means researchers can exploit rare cell populations or cells that are typically refractory to currently available purification technologies (e.g. primary cells, patient cells or delicate cell lines) as well as more robust commonly used cell types.

Cyntellect said that LEAP-based laser manipulation of cells has also been demonstrated to effectively permeabilize cells transiently without inducing cellular toxicity. This approach, called LaserFect, enables introduction of a wide variety of biomolecules including ions, siRNAs, proteins and quantum dots, many of which cannot be transfected using current technologies.

• Pall (East Hills, New York) reported recieving the CE mark for its eBDS system to detect bacterial contamination of red blood cells. The Pall eBDS is a culture-based test used by blood centers to detect bacterial contamination of platelets, the leading infectious cause of sickness and death from a transfusion. Results of a study presented at the International Society of Blood Transfusion (ISBT) 2006 Congress show the efficacy of the system in also detecting bacteria that are commonly found as contaminants of red blood cells.

The study presented to the ISBT tested the Pall eBDS on 662 red blood cell samples that had been inoculated separately with twelve different bacteria strains. Each sample was tested after 48 hours resulting in 100% detection of the contaminating bacteria.

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