A new method for accurate assessment of DNA quality after bisulfite treatment.

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called 'bisulfite treatment', which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies. The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.

Use of optophoresis as an in vitro predictor of cell response to chemotherapy for chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia [CLL] is characterized by active accumulation of clonal CD5+/CD19+/CD23+ B cells. Individualized characterization of patient cell resistance/sensitivity to specific agents can provide important information to guide therapy selection. We have utilized optophoresis, which is a technique for the analysis of the motion of cells within a moving optical gradient field. It detects the broad cellular changes associated with apoptosis based on physical characteristics of the cell, such as morphology, size, refractive index, density, and surface properties. We analyzed peripheral blood samples from 62 CLL patients in the presence of varying concentrations of chemotherapeutic agents. Optophoresis and a more conventional measurement of cell death were utilized. The outcome of ex vivo drug resistance using optophoresis was compared to clinical response in 30 patients for which there was clinical outcome data available. The overall accuracy of optophoresis in reflecting clinical response was 80%. It has advantages over alternative methods of determining chemoresistance including the ability to evaluate very small sample sizes and ability to work in mixed-cell populations. Changes in cell physical characteristics in response to chemotherapy, as measured by optophoresis is an accurate method for predicting chemosensitivity ex vivo in CLL.

Use of moving optical gradient fields for analysis of apoptotic cellular responses in a chronic myeloid leukemia cell model.

To facilitate quantitation of cellular apoptotic responses to various antineoplastic agents, a laser-based technology, Optophoresis, has been developed to provide analysis of cells without any need for labeling or cell processing. Optophoresis is defined as the analysis of the motion of cells, where the motion is either induced or modified by a moving optical gradient field, which produces radiation pressure forces on the cells in an aqueous suspension. Quantitation of the induced motion provides a basis for distinguishing one population of cells from another. One Optophoretic technique, Fast Scan, measures the distribution of distances traversed by a population of cells when exposed to a fast-moving optical gradient. Fast Scan was validated using a cell-based model of chronic myeloid leukemia treated with Gleevec, a specific inhibitor of aberrant Bcr-Abl protein kinase. The Optophoretic measurements were quantitatively comparable to reference assays with regard to drug selectivity and potency and to target specificity, demonstrating the suitability of this technology for pharmaceutical and clinical applications.

Optical forces for noninvasive cellular analysis.

A novel, noninvasive measurement technique for quantitative cellular analysis is presented that utilizes the forces generated by an optical beam to evaluate the physical properties of live cells in suspension. In this analysis, a focused, near-infrared laser line with a high cross-sectional intensity gradient is rapidly scanned across a field of cells, and the interaction of those cells with the beam is monitored. The response of each cell to the laser depends on its size, structure, morphology, composition, and surface membrane properties; therefore, with this technique, cell populations of different type, treatment, or biological state can be compared. To demonstrate the utility of this cell analysis platform, we evaluated the early stages of apoptosis induced in the U937 cancer cell line by the drug camptothecin and compared the results with established reference assays. Measurements on our platform show detection of cellular changes earlier than either of the fluorescence-based Annexin V or caspase assays. Because no labeling or additional cell processing is required and because accurate assays can be performed with a small number of cells, this measurement technique may find suitable applications in cell research, medical diagnostics, and drug discovery.