The preparation of tissue microarrays (TMA) is carried out starting from formalin-fixed and paraffin-embedded (FFPE) tissue blocks. The FFDE Tissue Blocks Library is exploited to generate glass-slide-supported, tissue microarrays in which micro-sections of cancer tissues will be arrayed together with the healthy tissues from the same patients. More specifically, 10 FFDE blocks for each of the five major human tumors (breast, lung, prostate, colon and ovary) are selected and from each block, cores of healthy and cancer tissues are selected. At the end, each array is constituted by two replicas of 200 tissue samples derived from 50 FFDE blocks. From each block (derived from one tumor patient) four tissue fragments are collected, two from the pathological area (tumour tissue) and two from the healthy area (normal tissue).
The tissue arrays thus generated are screened using immunohistochemistry (IHC) against theYomics library of anti-human protein antibodies with the aim at identifying those proteins selectively expressed in tumor tissues. The presence on the same array of both the disease and the normal tissues from the same patients allows a direct comparison of the IHC data and the selection of potential markers to be further assess in confirmation experiments. Once a specific polyclonal antibody appears to be positive in the high throughput pre-screening analysis, further confirmation by detailed histopathological analysis will be carried out. For TMA preparation a semi-automated tissue array is utilized.
The tissue arrayer takes vertical cores of tissue from a FFPE block and places them at defined intervals in a ‘recipient’ block to form an array. standard wax pathology block has a surface of slightly larger than 3 cm by 2 cm. If tissue cores of 0.6mm diameter are placed in the recipient block with their centers separated by a distance of 1mm, several hundreds cores can be placed in a single TMA. Sections can be cut from the TMA and placed on a single microscope slide for immunohistochemistry. The advantages of TMAs are not limited to time and cost issues.
Despite best efforts, immunohistochemistry is subject to a number of variables which can alter results from slide to slide and day to day. These factors include variations in antibody quality from batch to batch, antibody degradation with time, changes in laboratory conditions, and differences in run-through rates in different slide holders. Hence, for whole sections it is quite possible for sections to show variations in degree of staining due to factors other than true differences in expression of the marker. Many of these factors are reduced by the use of TMAs. All of the sections can be tested on one day with the same reagents. The samples on a single TMA will be exposed to the same conditions. Any slide-to-slide variation can also be detected by including control cores in each TMA, and ensuring that these stain consistently.
Know-how, technologies and infrastructures necessary to carry out this experimental activity are all available at IEO, the Institute that has establish a collaboration with Externautics. In particular, Prof. G. Viale, one of the Company’s founders, is a worldwide leader in the field of pathology and cancer. The quality of the antibodies produced in PRIMM S.r.l. has already been confirmed in Prof. Viale’s laboratories .