Tissue microarray (TMA)

Tissue microarrays consist of tissue cores from paraffin blocks which are situated in a sequenced fashion to allow multiplex histological analysis. Tissue blocks containing multiple samples were first introduced in 1986 by H. Battifora named ‘multitumor (sausage) tissue block’486. In 1990 this was modified and improved and renamed ‘the checkerboard tissue block’487. The current utilised technique was finalised by J. Kononen in 1998 and it involved the production of a block with similarly sized and shaped tissues, enabling a more precise and dense platform to work on488. Modern microarrays provide a platform enabling collation of hundreds of patient specimens, at different disease stages and sites. Thousands of genes and proteins can then be analysed in a single experiment. Analysis of various tissue pathologies and their gene or protein expression profiles helps in establishing and understanding the diagnostic, prognostic and therapeutic response outcomes of the pathology in question.

The development of endometriosis has been linked to inflammatory, apoptotic and angiogenic pathways. The genes and signalling pathways involved in the above all hold a key into the development of the disease. It is through identification of these genes that novel understanding and therapies are possible.

Due to the stringent criteria used, the number of patients recruited at surgery was low. To create a microarray with adequate numbers of tissues, 200 tissue blocks were retrospectively recruited from the histology database at Barts and the London NHS Trust. Identification of confirmed histological endometriosis was performed by a consultant histopathologist and samples were subdivided into groups according to the site, type and extent of the pathology identified. Malignancies were excluded and all slides were duly coded.

Tissues of interest were identified and confirmed by histopathologists from a haematoxylin and eosin stained section. Criteria relating to the tissues themselves such as hormonal influences, site of specimen biopsy and areas of pathology were recorded. A hollow needle (1 mm2) was used to remove tissue cores of interest from the original biopsy and these cores were then inserted in a recipient paraffin bock in a predefined and precisely spaced array pattern. A microtome was then used to cut sections (5-10 micrometres) in thickness to produce between 100-500 arrays which were utilised for immunostaining. Most endometriotic lesions are histologically heterogenous in nature with the possibility of creating a false representation of the pathology. To minimise this discrepancy, triplicate cores were taken from each sample.

2.7.1 Immunohistochemistry protocol 2.7.1.1 Slide preparation

TMAs were mounted onto adhesive coated slides to minimise compromisation of tissue adherence during heat-induced epitope retrieval especially with fatty or bloody tissue. Slides were labelled with the appropriate information and were incubated at 60ºC for 12 hours prior to processing.

2.7.1.2 De-waxing and de-hydration

To de-wax and de-hydrate the slides, these were placed in de-waxing xylene in the fume cupboard for 5 minutes. They were then transferred into IMS solution for two minutes. Endogenous peroxidise activity was blocked by placing the slides into 2% hydrogen peroxide in IMS solution for 5 minutes. Slides were then transferred into final IMS solution for 2 minutes and were afterwards rinsed in running tap water for 5 minutes.

2.7.1.3 Antigen retrieval

Three litres of vector antigen unmasking solution (50mls of Vector unmasking solution 3300 diluted in 5000mls of distilled water) were placed into the pressure cooker and the temperature was set at 450ºC. Once the solution boiled, the slides were inserted and left within it for 10 minutes. The solution was cooled down with the addition of running ambient temperature tap water for approximately 5 minutes. Preparation of 500mls of wash buffer (Tris-buffered saline with tween (TBS-T)) was performed by mixing one part of wash buffer solution with 9 parts distilled water. (i.e. 50mls wash buffer with 450mls distilled water). Slides were removed from the ‘cooker’ and placed into a trough with wash buffer.

2.7.1.4 Tissue microarray immunohistochemical staining and analysis

For Maspin, E- Cadherin, B-cell lymphoma 2 (Bcl-2)-associated transcription factor (BCLAF) and p53 the SS-enhanced Polymer detection system (Biogenix) was applied using the Dako-autostainer system (Dako). Incubation with optimally diluted primary antibody in BSA and sodium azide (Table 2-4) was performed following antigen retrieval. Staining enhancement was achieved by adding enhancer reagent increasing the antigen location-specific horseradish peroxidase (HRP) activity. Subsequent incubation with chromogen, 3,3’-diaminobenzidine (DAB) produced a brown precipitate at the antigen site. Sections were counterstained with haematoxylin.

The ARIOL imaging system (Genetix, San Jose, CA) was used for TMA antibody staining quantification. Slides were scanned at low resolution (1.25x) followed by high resolution (20x) using the Olympus BX61 microscope with an automated platform (Prior). Training and stain quantification was performed on the high resolution images. Training of the system for detection of stained and unstained cells by the colour intensity and shape of stained regions was performed. Positive staining, as determined by appropriate isotype negative controls, was classified by manually defined hue, saturation and intensity limits. Only epithelial areas were analysed through the use of the manual include/exclude drawing tool (Figure 2-6). The software generated the mean intensity (0-255) of all pixels considered positive, which was subsequently corrected so that the higher the value, the greater the intensity. Results were exported to an Excel sheet and non-parametric statistical analysis was performed on each of the subgroups using a

Mann-Whitney U Test. A P value of <0.05 between the groups was regarded as statistically significant. The resulting data was then plotted graphically.

TABLE 2-4

Antigen Clone Dilution Supplier

Maspin Clone EAW24 1:100 Novocastra

E Cadherin Clone 36B5 1:200 Novocastra

BCLAF Rabbit polyclonal Dilution 1:100 Sigma Prestige

p53 Clone D07 Dilution 1:3000 Dako

FIGURE 2-6

Endometriosis Eutopic uterine

endometrium (case) Eutopic uterine endometrium (control) Maspin E Cadherin BCLAF p53

Representative selection of samples from the created TMA’s stained by the indicated antibodies