Feature: Diagnostics and living to 100

This feature appeared in the March/April 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

A scraping of buccal cells from the lining of the cheek can predict, with a high measure of reliability, one’s risk of developing Alzheimer’s disease, or determine how far they have already progressed towards dementia.

From the ability to predict, comes the prospect of being able to prevent. Dr Michael Fenech, of CSIRO Food and Nutritional Sciences, has employed remarkable new technology called laser scanning cytometry (LSC) to develop a buccal cell test that will inform us how our dietary preferences influence our risk of developing Alzheimer’s disease, and how we might amend our diets to reduce that risk.

Dr Fenech is an internationally respected pioneer of a new field of preventative and diagnostic medicine that has sprung from a stunning technical advance in quantitative imaging cytometry (QIC), CSIRO Food Science and Technology is the first research institution in Australia to acquire a Compucyte iGeneration Laser Scanning Cytometer, through an arrangement with its manufacturer, Boston-based Compucyte Corporation.

Evolving cytometry

Compucyte’s president and chief executive office, Dr Elena Holden, visited Adelaide in January to participate in CSIRO’s first quantitative imaging workshop and symposium on QIC and its application in cytome research, and in measuring oxidative damage to DNA.

CompuCyte invented the first laser scanning cytometer in 1997-98, and has progressively refined and extended the technology and its range of applications over more than 10 years. A decade ago, bioscience researchers hailed QIC as the most significant advance in the technology since Wolfgang Göhde invented the first flow cytometer at the University of Munster in Germany, in 1968.

LSC has evolved rapidly, culminating in Compucyte’s launch of its latest iGeneration device last December. In its latest iteration, Compucyte’s QIC adds a fourth laser to the three used in the previous generation of instruments. The lasers operate at different wavelengths within the visible spectrum, chosen from a palette of six stepped wavelengths between violet (405nm) and orange-red (633nm).

The wavelengths are selected to highlight details of tissues stained with various combinations of fluorescent and chromatic dyes, or to highlight the distribution and concentration of marker proteins, so their distribution and interactions can be visualised.

Holden describes the company’s new iGeneration laser imaging cytometer as the new gold standard in quantitative laser imaging cytometry. Like its predecessors, Compucyte’s iGeneration cytometer frees researchers from the requirement to disaggregate complex, layered tissues into individual cells, which sacrifices information.

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The fourth laser extends the combinatorial possibilities within the still-expanding range of dyes and fluorescent proteins, and the spectrum of laser ‘colours,’ opening new windows into the organisation and dynamics of cell communities, and the internal workings of their component cells.

Holden says one great advantage of QIC over conventional flow cytometry is its ability to image cells in situ in intact tissue samples, and, using Compucyte’s proprietary software, extract a wealth of qualitative and quantitative information.

“A second difference is that, until recently, flow cytometry was ‘blind’: it could count and sort cells, but couldn’t produce images. LSC makes cells very receptive to imaging analysis,” she says.

Shedding light on DNA

QIC is finding applications in tissue and cell research across the life sciences, particularly in cancer and aging research, where it allows researchers to study the erosion of telomeres, the highly repeated DNA sequences that cap chromosomes, centromere dynamics during the cell cycle, and normal and aberrant patterns of protein expression.

QIC can provide information about the identity, and absolute and relative numbers of differentiated cells, and their multipotent precursors.

It yields quantitative information such as the size of cells and organelles like the nucleus and mitochondria, cell phase, the location and concentration of native or fluorescently labelled, hybrid proteins and levels of DNA damage - the prime indicator of cell age and health.

Holden says Compucyte welcomes the opportunity to work with Fenech’s CSIRO group. “Dr Michael Fenech’s name is tremendously well known for his research in nutrition and genetic toxicology,” she says.

“He developed the cytokinesis-block micronucleus assay which is now a gold standard and is required by FDA and respective international agencies as part of genotoxicity testing of new drugs, cosmetics and household chemicals.”

Holden says Fenech’s laboratory has also developed multiple additional markers of cytome health, including an improved technique for studying micronuclei. A micronucleus forms around any chromosome or chromosome fragment that has not been incorporated in a daughter nucleus during cell division. The presence of micronuclei in cells exposed to new drugs, cosmetics or other compounds, is another indicator of genotoxicity.

Holden says that, since January 2009, Fenech’s laboratory has used quantitative imaging cytometry to create a sophisticated panel of biomarkers of cytome health and genotoxicity.

“He needed a technology that allowed him to work with a diverse range of sample types, and provide high-content analyses, high-resolution images, and reliable quantitative data.

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“It’s a very exciting time for us to be collaborating, because nutritional genomics is increasingly recognised as a very important research discipline. DNA damage is now known to be the most fundamental pathology underlying defects in embryonic development, and degenerative diseases that cause aging.

“LSC allows us to monitor the effects of nutritional patterns, and nutritional excesses and deficiencies, which are recognised as major contributors to genome instability. This research is truly revolutionary, and we have very high hopes for its future.”

Reaching 100

Fenech says LSC has allowed his team to study cellular events at a molecular level. Dr Phil Thomas developed the buccal cell diagnostic tests for Alzheimer’s disease as part of his PhD project.

“We established scoring criteria for various biomarkers of cell death, cell damage, and regenerative potential, and applied them in a case-controlled study of Alzheimer’s patients,” says Fenech.

The diagnostic markers included the nucleus/cytoplasm ratio, micronuclei counts and scoring of buccal cell sub-types. Additionally, Dr Wayne Leifert, who is working in Fenech’s team, is currently developing other specific protein biomarker assays on the LSC, including cytokeratin markers, the ratio of Alzheimer’s Precursor Protein (APP) to its neurotoxic cleavage product, beta-amyloid, level of reactive oxygen species (ROS), and oxidative damage to DNA, as part of CSIRO’s Preventative Health Research Flagship.

The team is also measuring two biomarkers of double stranded DNA breaks, the phosphorylated histone-H2AX, and oxidised guanine bases in DNA using LSC. Fenech’s team has also developed a molecular probe to measure telomere length at the level of individual cells.

Telomeres shorten with each round of cell division, throughout an individual’s lifetime, eventually resulting in chromosome replication errors that can lead to cancer or cause inflammatory disorders in tissues with high cell-turnover rates, such as cartilage.

In addition to providing diagnostic information for degenerative disorders like Alzheimer’s disease and Parkinson’s disease, LSC also allows researchers to conduct longitudinal studies of the progression of such disorders, and dissect out the relative contributions of genes and nutritional factors.

“Our goal is to relate cellular events to an individual’s nutritional status, so we can define an optimal concentration and intake of nutrients to reduce DNA damage in that person,” Fenech says.

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The CSIRO DNA Damage laboratory’s findings about the relationship between diet, DNA damage and aging are already being translated into clinical practice, through a world-first preventative medicine clinic, Reach100.

Reach100 is dedicated to equipping patients with the knowledge and tools to ensure that, as they age, they remain as healthy as their age and genetic potential permits. Subjects receive initial consultation, a detailed health questionnaire, a full physical examination and a series of blood tests to assess their dietary status and state of health.

The assessment involves biomarker analysis and a world-first Genome Health Analysis, which measures DNA damage. Subjects are offered co-ordinated specialist appointments if required, detailed results and a Reach100 individualised report.

As its name suggests, Reach100 aims to maximise an individual’s prospects of becoming a centenarian, by monitoring damage to their DNA and recommending dietary and other measures to repair the damage and improve both their health, and the quality of their health.

In Australia, as in other Western nations, the median age of the population is rapidly increasing as the huge cohort of baby boomers born in the post-war years enter their seventh decade.

LSC is a tool for its times. A wealthy but aging population aspires to healthy longevity, as national health budgets sag under the soaring costs of treating age-related diseases like cancer, cardiovascular disease, type 2 diabetes, and osteoarthritis.

The Reach100 venture should become a model for how an ounce of LSC-based preventative medicine can help reduce the burgeoning costs of traditional pharmaceutical and surgical cures.

Reach100 Reach100 is a pioneering clinic dedicated to preventative and anti-aging medicine. It uses the latest diagnostics technology and techniques, such as CSIRO’s Genome Health Analysis test, to deliver a detailed health assessment and DNA analysis which can pre-empt problems before they occur and encourage preventative treatment. www.reach100.com.au