Courtesy of Ronald Simon, University of Basel
Barely five years ago, Juha Kononen, then at the National Cancer Institute, presented a straightforward way of constructing tissue microarrays (TMAs): a glass slide covered with as many as 1,000 cores of tissue, measuring from 0.6 mm to 2.0 mm in diameter.1 Suddenly, researchers could analyze gene expression and protein levels on hundreds of tissue samples by processing just one slide, instead of the hundreds previously required. As a result, scientists can better validate molecular markers of both development and disease.
As with many burgeoning fields, new users of TMAs find many options but little guidance in making a purchasing decision.
Though tissues may be created somewhat equal, they certainly aren't by the time they end up in a TMA. In general, TMAs are made using hollow needles to extract tissue cores from formalin-fixed samples. These samples are transferred to a paraffin block, which is sliced to yield hundreds of TMAs.
Collection and handling methods differ among surgeons, pathologists, technicians, hospitals, and companies. If left at room temperature or soaking in formalin too long, RNA can degrade, making the tissue useless for in situ hybridization, Polyak says. And, though existing archived tissue samples could potentially help strengthen the statistical of studies, little information on how these tissues were processed.
A good number of TMA manufacturers are placing their bets on TMAs made from frozen tissues that have not been exposed to chemical fixatives. "For any meaningful genomics procedure, you want to use frozen tissues that haven't suffered any RNA degradation from the fixing," says Steve Turner, CEO of Clinomics Biosciences in Freder-ick, Md. The company owns three patents on producing frozen TMAs and keeping the tissue frozen during delivery.
Polyak agrees: "We couldn't get results when we used [formalin-fixed] tissues for in situ hybridization because of RNA degradation." She uses TMAs made with frozen tissue for examining RNA. Abizar Lakdawalla, technology director at Biogenex in San Ramon, Calif., points out, however, that recent evidence suggests formalin degrades RNAse, the natural enzyme that damages RNA. By fixing tissues immediately after collection, formalin can inhibit RNAse activity.
Simon suggests testing for RNA transcripts of housekeeping genes in the formalin-fixed tissues used in TMAs. "If you have TMAs that are large enough, about two to 3,000 spots, then you can select the spots that were reliable with in situ hybridization," he adds.
Visual inspection of tissues is also important. "As you slice through the paraffin block, it's impossible to predict what's going to be next in the tissue, so there's variability," says Lakdawalla. Before purchasing, request images to ensure that you don't receive TMAs with cores that are necrotic or contain merely connective tissue, blood vessels, or other tissues that don't offer information on the gene and/or protein of interest.
In addition, ask TMA companies about their quality control strategies. Some companies may check every fiftieth TMA as they slice away at the block, and sell the other 49, which are classified into one lot, Lakdawalla says. "There's rarely more than 10% of TMAs that have errors," he adds.
WHAT'S YOUR NUMBER?
The size of each tissue core and the number of cores representing the same tumor within one array are also important considerations. Studies have shown that results from TMA-based experiments correlate with results obtained from experiments using one sample per slide. Most studies show that correlation ceases to improve after using three or four cores from a single tumor sample.
Simon and other researchers at Basel's Institute of Pathology use only one core for every tumor. "We are convinced that the number of cores per tumor has very little influence on the results," Simon says. "We have done several million TMA analyses and have not missed one previously established clinicopathological association in analyzing just one core per tumor."
Various sizes of tissue cores are available. Generally, "0.6 mm is optimal for most tumor tissues," says Kenneth Hillan, vice president of development sciences at Genentech, South San Francisco, Calif. But TMAs with larger cores are best for studying tissue architecture.
Before making a beeline for the TMAs with 1,000 cores, think about your capacity to examine and analyze each core when collecting assay results. The market now offers several advanced automated imaging and analysis hardware and software products. However, many researchers, such as Polyak, are sticking with manual imaging and analysis until the technologies improve further.
Industry and academic leaders are pushing for standardization in collection and processing protocols and improved and expanded collection networks. "We really need [an] initiative to make sure that all patients enrolled in oncology clinical trials ... have their tumors incorporated into TMAs as a matter of course, so we can advance molecular pathology and improve patient care," says Hillan.
In addition, TMA manufacturers are building up their repositories, increasing the variety of tissues and diseases that are available in TMA format. Many companies also offer custom TMAs made from tissues either supplied by the user or obtained from other sources. Easier data sharing is also on the horizon. The Association for Pathology Informatics, in collaboration with industry, academic, and government representatives, has developed an XML-based data-exchange format to enable researchers to share TMA study findings.3
"Because one tissue sample can be made into hundreds of TMAs, they could end up in the hands of different researchers who will make interesting discoveries," says Jules Berman, program director for pathology informatics at the National Cancer Institute. "Using the open-source data-exchange format, all that data can be combined and accrued to greatly enhance the utility of TMAs."
WHAT'S THE NEXT STEP?
Give it a go. For rat and mouse TMAs, try Zyagen Laboratories in San Diego, which offers a wide TMA selection of normal tissues, including those representing various stages of fetal development. Chemicon of Temecula, Calif., and Zymed Laboratories of South San Francisco both offer rabbit TMAs as well as human cancer TMAs.
Otherwise, most of the companies listed below offer human TMAs of normal and diseased tissue, including cancer, and cardiovascular and neurological diseases. Some of these companies will prepare custom arrays from their own or user-supplied samples, too. With slides ranging in price from about $40 to nearly $300, it's virtually risk free to buy a few and see how TMAs make life in the pathology lab much easier.
SELECTED TISSUE MICROARRAY SUPPLIERS
TriStar Technology Group