In-house Manual Construction of High-Density and High-Quality Tissue Microarrays by Using Homemade Recipient Agarose-Paraffin Blocks

Abstract

Background: Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs.

Methods: Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks.

Results: The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy.

Conclusions: Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks.

Keywords: Paraffin; Pathology; Research; Sepharose; Tissue array analysis.

Fig. 1

Manual construction of a tissue microarray (TMA) with a 2 mm core diameter. (A) A sheet of grid paper is attached to the surface of the blank recipient block made of a paraffinized agarose gel with a double-faced adhesive. (B) A recipient punch made of a modified bone marrow biopsy needle is used to bore an array of holes in the recipient block. (C) After boring the holes, the grid paper is removed. Subsequently, a donor punch made of a disposable skin biopsy punch 2 mm in diameter is used to punch out tissue cores from the donor block and implant them into the pre-punched holes in the recipient block (D). Upon completion of the tissue array (E), the initial TMA is detached from the tissue cassette (F), a misplaced core is replaced with new one (G). The TMA is placed in the embedding mold to be re-melted (H) and re-embedded in paraffin (I).

Fig. 2

Manual construction of a tissue microarray (TMA) with a 1 mm core diameter. (A, B) Tissue punches made of a modified 18 (^*)- and 16 (^**)-gauge needle are used to create holes in the recipient agarose-paraffin block, punch donor tissue cores and implant them into the pre-made holes. Once the TMA is completed (C), the TMA is detached from the cassette (D, E) and placed in the embedding mold in order to be re-melted and re-embedded in paraffin (F-H). The TMA block is trimmed (I) and cut for hematoxylin and eosin (J) and immunohistochemical staining (K, cytokeratin; L, CD20).

Fig. 3

Production of pre-made ready-to-use recipient agarose-paraffin blocks. The recipient block is trimmed to expose the surface of the paraffinized agarose gel (A) and an array of holes with precise spacing is drilled using a mini electric hand drill (B). When the tissue cassette and paraffin encasing the pre-punched or pre-drilled recipient blocks are removed, they can be used as pre-made and ready-to-use recipient blocks (C, 2 mm×108 holes in a 12×9 grid; D, 1 mm×192 holes in a 16×12 grid), which can be used to array tissue cores at a high-density using a self-made donor punch (E, F). (G, H) Complete homogenization between the tissue cores and the recipient block is assured by the re-melting and re-embedding procedure.