Fig. 1Manual 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. 2Manual 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. 3Production 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.
Table 1.Tissue processing protocols used for preparation of the agarose gel to make recipient agarose-paraffin block
Sequence |
Reagent |
For small biopsy specimens
|
For large surgical specimens
|
Time (min) |
°C |
Time (min) |
°C |
Fixation |
Formalin |
44 |
45 |
44 |
45 |
Dehydration |
Ethanol (30%) |
30 |
45 |
30 |
45 |
|
Ethanol (50%) |
30 |
45 |
30 |
45 |
|
Ethanol (70%) |
30 |
45 |
30 |
45 |
|
Ethanol (80%) |
30 |
45 |
30 |
45 |
|
Ethanol (95%) |
30 |
45 |
60 |
45 |
|
Ethanol (100%) |
30 |
45 |
90 |
45 |
Clearing |
Xylene |
45 |
45 |
45 |
45 |
|
Xylene |
45 |
45 |
45 |
45 |
|
Xylene |
90 |
45 |
90 |
45 |
Impregnation |
Paraffin |
60 |
65 |
60 |
65 |
|
Paraffin |
60 |
65 |
60 |
65 |
|
Paraffin |
80 |
65 |
80 |
65 |
Table 2.Overview of in-house manual construction of TMAs using self-made recipient blocks and recipient/donor punches
Recipient block |
Preparation of recipient block |
Extraction of paraffin tissue cores from donor block |
Capacity of TMA block |
Time required for TMA construction |
Spot loss on TMA section |
Reference |
Conventional paraffin |
Boring holes using needle or small screwdriver |
BM biopsy needle |
1 mm, 56 (8 × 7) |
1 hr for 56 cores |
< 1% |
Singh et al. [7] |
1 mm, 108 (12 × 9)a
|
2 mm, 77 (11 × 7)a
|
Conventional paraffin |
Boring holes using mechanical pencil tip |
Mechanical pencil tip |
1 mm, 72 (9 × 8) |
1 hr |
2% |
Shebl et al. [8] |
Conventional paraffin |
Boring holes using mechanical pencil tip |
Mechanical pencil tip |
0.8 mm, 206 (17 × 11) |
NM |
1-2% |
Foda [9] |
Conventional paraffin |
Boring holes guided by dot-grid paper directly attached to the bare paraffin block |
Cannula piercing needle, BM biopsy needle, equipped with retractable stylet |
1 mm, 140 (14 × 10) |
3 hr |
1-2% |
Choi et al. [11] |
2 mm, 70 (10 × 7) |
1.5 hr |
Paraffinized agarose gel (method in this study) |
Boring holes guided by dot-grid paper directly attached to the bare paraffin block |
Cannula piercing needle, BM biopsy needle, equipped with retractable stylet |
1 mm, 220 (20 × 11) |
3 hr |
Nob
|
|
2 mm, 80 (10 × 8) |
1.5 hr |