• tissue microarrays
• paraffin wax
• quality of wax

Tissue microarray (TMA) technology allows the representation of hundreds of tissue samples on a standard microscope slide. This is achieved by arraying small cores (0.6 mm in diameter) of paraffin wax embedded tissue samples in a recipient wax block. Sections cut from the array can then be assessed by immunohistochemistry or in situ hybridisation, according to standard protocols. TMAs enable the high throughput assessment of the presence and location of expressed genes, saving time, reagents, and clinical material. There have been several reviews on TMA construction and use,¹ but we have recently encountered a technical problem that, as far as we are aware, has not been described in the literature.

Multiple TMAs, each containing 292 cores, were constructed according to standard protocols.² Sections were cut without mishap using the adhesive tape transfer system, and arrays were stored carefully at ambient laboratory temperature for two months. However, when the arrays were then sectioned for a second time, in two of four cases, the wax block sheared off its supporting plastic cassette. Critical analysis of the problem, literature review, and consultation with TMA experts identified several possible causes for this, which are outlined below.

First, it was suggested that when the recipient blocks were made, insufficient wax was poured through the cassette, resulting in weak adhesion between the block and the cassette. However, we ensured that the mould, including the back of the cassette, was filled and that the wax level was not allowed to recede during cooling. Second, rough handling of the arrays could weaken the wax, but we do not believe that this occurred. Third, temperature fluctuations may play a role. Although arrays were constructed and stored at ambient temperature, they were cooled after every 10–15 sections cut, according to standard procedures. We no longer cool arrays during sectioning and quality remains comparable to published studies. Fourth, sections were cut along the length of the array, which may theoretically exert excessive shearing forces on the wax and increase its potential to fracture. Consequently, we now section across the width of the array. Fifth, cores may have been arrayed too deeply into the recipient block, weakening the wax at the cassette surface. Cores are now placed at least 1 mm above the cassette. Sixth, prolonged heating of wax above its melting point may compromise its quality. However, our wax is heated to its melting point of 60°C and kept molten for no longer than one week.

Finally, the quality of wax used to make the recipient block is important. The wax that we had used initially did not contain plasticisers. TMA experts and wax suppliers recommend instead using a pliable wax containing plastic polymers, for example Precision Cut (Thermo Shandon, Runcorn, UK). Moreover, discussions with our wax supplier suggested that the quality of the batch of wax used for the recipient blocks was compromised. Other users of this batch had also reported blocks breaking during sectioning. We concluded this was the principal factor affecting our arrays. In routine histopathology, such fractures are repaired by melting the wax and re–embedding the large tissue samples. This is not possible with TMAs, however, because the hundreds of thin cores would misalign.

To solve this problem we used the following procedure. The broken array was placed into a cooled metal mould, fractured surface uppermost. Using a heating iron, the fractured surface was melted to a depth of 1 mm. A fresh cassette, prewarmed in molten wax, was applied to the array surface and the mould was filled with fresh molten wax. The wax was allowed to set thoroughly and the arrays were successfully sectioned.

In conclusion, TMAs are valuable and delicate resources. To ensure that they can be sectioned on multiple occasions, they need to be carefully constructed and stored. To avoid the problem of TMA shearing we have outlined important precautions, relating especially to the quality of wax in the recipient block. Fractured arrays can be reconstructed using the procedure outlined herein.