References >> Tissue Microarray

Tissue Microarray Introduction

Tissue microarrays enable the high throughput analysis of a large number of tissue samples that have been collected and archived through the use of paraffin blocks or formalin. Distinct tissue samples from different tissue blocks are punched and relocalized on a new paraffin block.

Traditionally, paraffin blocks have been used to study the histological samples from tissues infected with cancer, tumor or other similar diseases. Tissue microarrays make the analysis of these histological samples easy, by having all the information on a single slide. A paraffin block with an array is prepared and a needle transfers samples from the histological sample blocks into it. The technique was first mentioned in the Journal of Immunological Methods by Wan, Fortuna and Furmanski.

Tissue microarrays are different from DNA microarrays where each spot on an array represents a cloned cDNA or oligonucleotide that binds to the target sequence. With tissue microarrays, each array has patient specific histological samples from cancer infected tissues. The tissue microarray technique is best suited for screening one genetic marker or protein across thousands of samples where as DNA microarrays are best suited to study gene expression across thousands of genes.

Optimal Tissue Utilization Technique

A major problem faced by the pathologists is the chronic shortage of tissues to complete the analysis. When a tissue sample is obtained, it needs to be managed to maximize its research value. Tissue microarrays utilize this scarce resource efficiently. Through sectioning of the tissue samples, a single biopsy sample can be used in multiple assays. Another significant advantage inherent in the technique is that each tissue sample is treated under identical conditions, maintaining experimental uniformity. In addition, only a small amount of reagents are required to analyze an entire cohort of diseases from each slide, thus significantly decreasing the cost of immunohistochemical studies.

Tissue Microarray Preservation

It is an empirical observation that the production of batches of tissue microarray samples may lead to a loss of antigenic properties. This may occur for a relatively smaller storage period of 5-7 days prior to immunostaining. Researchers have reported that the loss of antigenic properties perhaps attributed to oxidation since temperature, pressure or conditions during which a sample was taken seemingly has no affect on the tissue properties. To prevent this loss of antigenic properties, it is suggested that the sectioning should be done without the use of water. It is also advisable to incubate the tissue in xylene and coating in paraffin before storage.

Constructing a Tissue Microarray

Preparation of slides for tissue microarray involves taking a core of the specimen from each donor tissue block. They are arranged on a recipient paraffin block with predefined coordinates. It is a recommended practice to arrange the donor tissue blocks in their order of representation on the TMA slide. This practice avoids confusion at a later stage, for correctly identifying the source of a sample.

The introduction of precision punching instruments which place distinct tissue samples precisely in the recipient block have simplified the TMA manufacturing process.

Depending on the tissue type and the thickness of the donor tissue blocks, 100–200 slides can be sectioned from the recipient block. The resultant TMA slides are then subjected to a variety of in situ tissue analysis including immunohistochemistry and in situ hybridization. By staining one or two master slides, an entire cohort of cases can be analyzed, while maintaining the complete demographic and outcome information for each case.

Advantages of Tissue Microarrays

a) Wider Analysis: For studying altered protein expression, cytogenic liberation, genotypic and phenotypic marker identification, pathologists do a patient by patient analysis. Tissue microarray technique enables them to analyze samples on a much larger level. At a time, one can analyze samples from about 1000 patients. This high throughput nature of the tissue microarray experiments make them a preferred choice for studying cancer biomarker discovery and identification.

b) Better use of Tissue: The initial samples that pathologists submit are not more than 2-5 mm thick. These tissue sections have then to be thinly sliced into tissue sections. The number of sections that can be generated from such small starting amount vary depending upon the initial amount and the care that the technicians take in slicing them. These sections can yield enough tissue for up to 100 arrays. For tissue microarrays, a needle biopsy could yield about 300 times this number (though it depends upon the size of the tumor in the tissue sample). When tissue microarrays are prepared from this array, thinner sections of tissues are sliced. These sections are less than 5 µm thick. Thus, as compared to routine tissue biopsy, the tissue microarray technique provides samples for at least one 100,000 assays (even more in some cases).

c) Uniform Analysis: Any staining technique could be used while preparing tissue microarray slides for image analysis. Tissue microarrays support histochemical staining, fluorescence visualization, in-site hybridization or fluorescence in-situ hybridization. Moreover, reagent concentration, incubation time, temperature, washing conditions and other factors involved in slide building and analysis remain the same since the analysis happens on a single slide.

d) Cost Effective Technique: Tissue microarrays, for a typical cohort analysis, use less reagents and stains while enabling more assays.

Tissue Microarray Data Analysis

Tissue microarrays enable the simultaneous study of tissue specimens from hundreds of patients. Multiple immunostained tissue core images scanned via digital-imaging systems, generate large amounts of data to be analyzed. This data includes biomarker expression, clinico-pathological parameters and survival/recurrence data of patients.

To derive meaningful inferences from this data, rigorous statistical analysis is required. The data is complex, as the covariates are skewed, non-normalized and may be highly correlated. Such complex data analysis needs sophisticated high throughput analysis using appropriate statistical methodologies.

TMA Foresight is a high throughput analytical tool for tissue microarray data analysis. It provides a robust combination of statistical methods, user friendly interface and biologically relevant interpretations. Data preprocessing facility of TMA Foresight allows formatting of raw data into an standard format for further statistical analysis. Cox proportional hazard model is available to study the relationship of independent variables with survival or recurrence time. It identifies the prognostic biomarkers that could impact patient survival. Kaplan-Meier plots provide the facility to generate survival curves for a particular set of patients.

TMA Foresight provides an interactive graphical scale which facilitates grouping of patients based on the variable of interest. Hierarchical clustering of tissue microarray immunostaining data integrated with KM analysis is used to identify prognostically significant biomarkers. Principal component analysis is available for reduction in TMA data dimensionality. Correlation analysis helps in identifying the association between various biomarkers.